Table of Contents

Abstract

Introduction

Materials and Methods

1.1 Materials

1.1.1 Test System

1.1.2 Main Reagents

1.2 Methods

1.2.1 Test Substance Preparation

1.2.2 Dose Selection Basis

1.2.3 Exposure Method

1.2.4 Microscopic Examination

1.2.5 Result Evaluation

1.3 Statistical Analysis

Results

2.1 Cell Precipitation

2.2 Cytotoxicity

2.3 Chromosome Examination

Discussion

References

Abstract

Objective: To evaluate the in vitro chromosomal aberration genotoxicity of e-cigarettes under Good Laboratory Practice (GLP) conditions.

Methods: Following the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2(R1) guidelines and the Organization for Economic Co-operation and Development (OECD) Test Guideline 473 for chromosomal aberration tests, Chinese hamster lung fibroblast cells (CHL) were used. The final exposure concentrations of VAPEPIE 40000 e-liquid were set at 409.4, 136.5, 45.5, and 15.2 μg/mL. Solvent control, cyclophosphamide, and mitomycin C positive control groups were included. Cells were exposed for approximately 4 hours (with or without metabolic activation system, S9) and 24 hours (without S9). After exposure, cells were harvested, stained with trypan blue for counting, subjected to hypotonic treatment, fixation, slide preparation, and Giemsa staining. One thousand metaphase cells were observed, and at least 300 well-spread metaphases were analyzed for aberration types. Fisher's exact test was used for statistical comparisons.

Results: No test substance precipitation was observed under any exposure conditions. Relative population doubling (%) was >50% in all groups, with mitotic index and mitotic suppression rates within normal ranges, indicating no significant cytotoxicity. No meaningful increase in CHL cell chromosomal aberration rates was observed.

Conclusion: Under the tested conditions, the e-cigarette did not cause a statistically significant increase in CHL cell chromosomal structural aberration rates, yielding a negative result.

Introduction

E-cigarettes, as novel tobacco products, appeal to younger demographics due to their design, aesthetics, and sensory experience. They are commonly perceived as less harmful than traditional cigarettes, particularly among adolescents and young adults. E-cigarettes and other electronic nicotine delivery systems typically contain nicotine derived from tobacco or its substitutes, along with flavorings, propylene glycol, glycerol, polycyclic aromatic hydrocarbons, phenols, and other components. Long-term exposure to certain e-cigarette aerosol constituents may lead to respiratory complications in the short term, such as asthma, chronic obstructive pulmonary disease, and inflammation. Exposure of normal epithelial cells and head and neck squamous cell carcinoma cell lines to e-cigarette vapor significantly reduces cell viability, clonogenic survival, and increases apoptosis and necrosis rates. Previous studies have analyzed differences in compounds between e-cigarettes and traditional cigarettes, as well as health risk assessments, identifying some e-cigarette components as toxic or potentially carcinogenic/mutagenic. Current genotoxicity research on e-cigarettes is relatively limited or incomplete. Potential genotoxicity from long-term repeated exposure requires analysis under robust experimental standards, referencing ICH S2(R1) guidelines on genotoxicity testing and data interpretation for pharmaceuticals and OECD Test Guideline 473 for in vitro mammalian chromosomal aberration tests. Building on existing research, this study further investigates the genotoxicity of e-cigarette liquids.

Materials and Methods

1.1 Materials

1.1.1 Test System

Chinese hamster lung fibroblast cell line (CHL) was obtained from Zhejiang Meisen Cell Technology Co., Ltd. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with various amino acids and glucose (10% fetal bovine serum, 1% penicillin-streptomycin) at (37 ± 2)°C and (5 ± 0.5)% CO₂. The modal chromosome number is 25.

1.1.2 Main Reagents

VAPEPIE 40000 e-cigarette cartridge (Green Fan Yingying Waterfall Smoke, hereinafter referred to as VAPEPIE 40000; batch number: SHVD02ADY; form: liquid; manufacturer: Shenzhen RELX Technology Co., Ltd.; main components: glycerol, propylene glycol, tobacco extract, nicotine, benzoic acid; total nicotine per cartridge: 38.9 mg; volume: 1.9 mL; concentration: 18 mg/mL, compliant with GB 47100-2022). Phosphate-buffered saline (PBS; batch number: 2305001; from Solarbio). Cyclophosphamide (CPA; batch number: C22HY05122504A; from Accustandard, USA). Mitomycin C (MMC; batch number: 130438-201103; from China National Institutes for Food and Drug Control). Colchicine (batch number: 113972; from MedChemExpress). Rat liver S9 (batch number: 23FS007B) and rat liver S9 activation system (batch number: CHI20230417) from Qi's Biotechnology Co., Ltd.

1.2 Methods

1.2.1 Test Substance Preparation

Under sterile conditions, the required volume of VAPEPIE 40000 e-cigarette liquid was aspirated, filtered through a 0.45 μm filter for sterilization, and diluted with PBS to the desired concentrations. Lower concentrations were prepared by diluting higher concentration samples.

1.2.2 Dose Selection Basis

According to ICH S2(R1) guidelines and OECD Test Guideline 473, the maximum concentration for chromosomal aberration tests is recommended as 0.5 mg/mL when not limited by solubility or cytotoxicity, with at least three analyzable concentrations. The highest preparable concentration was 20.47 mg/mL, leading to a final maximum exposure of 0.4094 mg/mL. Preliminary tests at 409.4 μg/mL showed no precipitation or cytotoxicity, with good cell growth (approximately 80% confluence). Thus, doses were set at 409.4, 136.5, 45.5, and 15.2 μg/mL, alongside solvent control, positive control 1 (MMC), and positive control 2 (CPA). See Table 1.

1.2.3 Exposure Method

CHL cells in exponential growth phase were washed with PBS, trypsinized, and resuspended. Cells were seeded into 50 mL flasks at approximately 5 × 10⁴ cells/mL and cultured for 24–48 hours in a CO₂ incubator.

Without metabolic activation: Original medium was discarded; solvent control received 4.9 mL DMEM + 0.1 mL PBS; positive control received4.9 mL DMEM + 0.1 mL MMC; test groups received 4.9 mL DMEM + 0.1 mL test substance (duplicate flasks per dose). Incubated for ~4 hours, then washed twice with PBS, replenished with 5 mL medium, and cultured to ~24 hours total. Colchicine (20 μg/mL, 0.1 mL) was added ~4 hours before harvest.

With metabolic activation: Original medium discarded; solvent control received 4.4 mL DMEM + 0.5 mL S9 mix + 0.1 mL PBS; positive control received 4.4 mL DMEM + 0.5 mL S9 mix + 0.1 mL CPA; test groups received 4.4 mL DMEM + 0.5 mL S9 mix + 0.1 mL test substance (duplicates). Incubated for ~4 hours, washed twice with PBS, replenished with 5 mL medium, and cultured to ~24 hours. Colchicine added as above.

Supernatant was collected, cells washed with PBS, trypsinized, centrifuged at 1000 r/min (r=1.90 cm) for 10 min, supernatant discarded, and cells stained with trypan blue for counting. Relative population doubling and population doubling were calculated, followed by hypotonic treatment, fixation, slide preparation, staining, and examination.

1.2.4 Microscopic Examination

One thousand cells per group were examined to record metaphase cells and calculate mitotic index (%) and mitotic suppression rate. At least 300 well-spread metaphases per group were scored for chromosomal aberration rates. Structural aberrations were recorded (gaps noted separately but not included in rates); numerical aberrations like polyploidy and endoreduplication were recorded separately but not in rates.
Mitotic index (%) = (Number of mitotic cells / Total cells (1000)) × 100%

Mitotic suppression rate (%) = [(Solvent control mitotic index - Dose group mitotic index) / Solvent control mitotic index] × 100%

Chromosomal aberration rate (%) = (Number of aberrant cells / Total metaphases) × 100%

1.2.5 Result Evaluation

Results described cytotoxicity and precipitation per dose. Aberration rates were expressed as percentages. A positive result required a significant increase in aberration rate at least one concentration, with dose-dependency and exceeding historical negative control ranges. Equivocal results warranted further study or repeat tests. Biological relevance was prioritized; statistics aided evaluation but were not sole criteria. Increased polyploidy suggested mitotic inhibition or numerical aberrations; increased endoreduplication indicated cell cycle effects.

1.3 Statistical Analysis

Using Stata/IC 15.1 software, aberration rates were expressed as percentages. Fisher's exact test compared positive controls vs. solvent control, and test doses vs. solvent control. If overall significance (P ≤ 0.05), pairwise comparisons were performed between positive/solvent and test/solvent groups.

Results

2.1 Cell Precipitation

Compared to solvent control, under 4-hour exposure with or without S9, and 24-hour without S9, no test substance-related precipitation was observed in VAPEPIE 40000 groups at any dose during medium change or harvest.

2.2 Cytotoxicity

With S9 (4 hours): Solvent control mitotic index = 4.30%; positive control = 4.30%, suppression rate = 0%. No significant cytotoxicity in test groups vs. solvent. See Table 2.

Without S9 (4 hours): Solvent control = 4.00%; positive control = 4.50%, suppression rate = -12.50%. No significant cytotoxicity. See Table 3.

Without S9 (24 hours): Solvent control = 3.90%; positive control = 3.40%, suppression rate = 12.82%. No significant cytotoxicity.

2.3 Chromosome Examination

Under all conditions (4 hours with/without S9; 24 hours without S9), solvent control aberration rates were within normal background. Positive controls showed significantly higher rates (P < 0.05), validating the system. VAPEPIE at 15.2, 45.5, 136.5, and 409.4 μg/mL showed no significant changes in CHL structural aberration rates vs. solvent (all P > 0.05), remaining within normal ranges with no meaningful increases.

Discussion

Under the tested conditions, VAPEPIE 40000 at 15.2, 45.5, 136.5, and 409.4 μg/mL did not cause meaningful increases in CHL chromosomal structural aberration rates during 4-hour exposures with/without S9 or 24-hour without S9, resulting in a negative outcome.
E-cigarettes deliver nicotine and tobacco extracts via electrical heating or physical atomization, reducing combustion-related harmful products like tobacco-specific nitrosamines. Cigarette smoke is a complex mixture with bioactive substances from combustion, but e-cigarette components are controlled per national standards. Toxicological evaluations of e-cigarette components (e.g., nicotine, propylene glycol, glycerol, flavors) in 90-day rat exposures indicate minimal harm to cells, animals, and humans. Mutation assays on three e-cigarette smoke extracts showed no significant mutagenicity in transgenic mouse or human fibroblasts. Replicated studies on cytotoxicity, mutagenicity, and genotoxicity (bacterial reverse mutation, in vitro micronucleus) indicated mild or no toxicity for e-cigarettes, consistent with prior findings. In vitro mammalian cell micronucleus tests with e-cigarette liquids were negative with/without metabolic activation. Oral mucosal micronucleus rates in 55 e-cigarette users were lower than non-smokers.

Comet, micronucleus, and trypan blue assays on 33 e-liquids showed DNA damage, chromosomal breaks, and cell death at 1% concentration, with some positive only with S9. Four e-liquid additives induced mutations and chromosomal damage in three cases. Exposure to nicotine-containing and nicotine-free e-vapor extracts induced DNA breaks, increased cell death, and reduced clonogenicity in epithelial and squamous cell lines, independent of nicotine. Inconsistent results prevent definitive conclusions; short-term in vitro studies may not fully capture long-term human exposure to toxic/carcinogenic aerosols. Variability in e-cigarette toxicity studies (device evolution, exposure models, doses) hinders comparisons, allowing only qualitative health risk conclusions. With increasing market diversity, further GLP-compliant genotoxicity studies on e-cigarette components are needed for rapid detection methods.