담배연기 발생, 배출 특성 분석 및 독성 평가를 위한 통합적 방법론 개발과 적용 = Development and application of integrated methodologies for cigarette smoke generation, emission characterization, and toxicological assessment

국문 초록 (Abstract)

본 논문에서는 재현성과 신뢰성을 갖춘 담배 독성 평가 체계를 구축하기 위해, 담배 배출물 응축 시료를 활용한 통합적 평가 방법론을 제시하고자 하였다. 먼저, 일반 궐련 담배(3R4F)와 3종의 heated tobacco products (HTPs; HTP-A, -B, and -C)로부터 담배 응축물을 확보하고, 응축물에 함유된 organic compounds (OCs)에 대한 정성 및 정량 분석을 수행하였다. 또한, 담배 연기 배출물과의 hazard index (HI)를 비교 평가하여 독성학적 유효성을 검증함으로써, 담배 응축물 기반 독성 평가가 재현성과 신뢰성을 갖출 수 있음을 확인하였다.
이어, 담배 응축물 대량 확보와 담배 연구의 접근성과 효율성을 높이기 위해 automated system for generation and collection of smoke (ASGCS)을 개발하였다. ASGCS는 Health Canada Intense (HCI) 흡연 조건에서 10%미만의 낮은 연기 누출률을 기록하였으며, 3R4F 30개비로부터 채취된 total particulate matter (TPM)농도는 21.2±0.2 mg cig–1로 기존 담배 연기 채취 장비보다 약 19.1% 높은 채취율을 나타냈다. WCSC에 함유된 니코틴과 total volatile organic compound (TVOC) 농도는 각각 1.25±0.01mg cig–1, 2,276 μg cig–1로, 담배 연기 대비 약 65%–135%의 높은 회수율을 기록하였으며, 세포 독성 평가 결과 WCSC는 용량 의존적으로 세포 생존율을 감소시켰으며, ASGCS로 확보한 담배 응축물이 독성 평가용 시료로서 신뢰성을 확보하였음을 검증하였다.
마지막으로, 담배 연기의 주요 성분인 니코틴을 대상으로 채취 과정에서 발생할 수 있는 손실률을 평가하였다. 니코틴 액상 표준시료와 3R4F 담배 연기를 Cambridge filter pad (CFP)에 채취한 후, 다양한 빛 노출 조건에서 시간에 따른 니코틴 손실률을 정량적으로 평가한 결과, 암조건에서는 48시간 후 1.6%, 가시광선에서는 9%, 자외선에서는 16.9% 감소하였다. 특히, 자외선 조건에서는 니코틴 손실이 시간에 따라 일정하게 발생하였으며, 상대회수율은 시간당 -0.335%로 나타났고, R2=0.9465로 높은 선형성을 보였다. 이를 통해, 니코틴이 보관 시간과 빛 조건에 따라 광분해 영향을 받는다는 것을 실험적으로 검증하였다. 이를 통해 니코틴의 정확한 정량 분석을 위해 시료는 빛 차단 조건에서 보관하고, 전처리를 신속히 수행해야 함을 확인하였다.
본 연구는 담배 응축물의 화학적 특성 규명과 독성학적 유효성 검증, ASGCS 개발 및 WCSC 확보, 니코틴 손실 평가를 통합함으로써, 담배 응축물 기반 독성 평가의 표준화 및 신뢰성 있는 통합 평가 체계를 확립하였다. 이러한 연구 결과는 담배 유해성 평가의 정확성과 재현성을 높이고, 향후 정책 수립과 규제 연구의 기초자료로 활용될 수 있을 것으로 기대한다.

다국어 초록 (Multilingual Abstract)

This study aimed to establish a reproducible and reliable tobacco toxicity assessment system by developing an integrated evaluation methodology using condensed tobacco emission samples. Tobacco condensates were obtained from conventional cigarettes (3R4F) and three types of heated tobacco products (HTPs; HTP-A, -B, and -C), and qualitative and quantitative analyses of the organic compounds (OCs) in the condensates were performed. The toxicological validity of the condensates was further confirmed by comparing the hazard index (HI) with that of tobacco smoke emissions, demonstrating that tobacco condensate-based toxicity assessment possesses both reproducibility and reliability.
To enable large-scale acquisition of tobacco condensates and improve research accessibility and efficiency, an automated system for the generation and collection of smoke (ASGCS) was developed. Under Health Canada Intense (HCI) smoking conditions, the ASGCS exhibited a smoke leakage rate below 10%. Total particulate matter (TPM) collected from 30 3R4F cigarettes was 21.2±0.2 mg cig–1, showing approximately 19.1% higher collection efficiency than conventional smoke collection equipment. Nicotine and total volatile organic compound (TVOC) concentrations in the whole cigarette smoke condensate (WCSC) were 1.25±0.01 mg cig–1 and 2,276 μg cig–1, respectively, corresponding to recovery rates 65%–135% higher than those of cigarette smoke. Cytotoxicity assessment revealed dose-dependent reductions in cell viability, confirming that WCSC obtained via ASGCS provides reliable samples for toxicity evaluation.
Finally, nicotine loss during the collection process was quantitatively evaluated. Liquid nicotine standards and 3R4F tobacco smoke were collected onto Cambridge filter pads (CFP), and nicotine loss was measured over time under various light conditions. After 48 hours, nicotine concentrations decreased by 1.6% in the dark, 9% under visible light, and 16.9% under ultraviolet (UV) radiation. Under UV exposure, nicotine loss occurred at a constant rate, with a relative recovery of −0.335% h–1 and high linearity (R² = 0.9465), experimentally verifying the photodegradation of nicotine depending on storage time and light conditions. These results indicate that accurate quantitative analysis of nicotine requires prompt sample pretreatment and storage under light-blocking conditions.
By integrating chemical characterization, toxicological validation, ASGCS development, WCSC acquisition, and nicotine loss evaluation, this study established a standardized and reliable integrated assessment system for tobacco condensate-based toxicity evaluation. The findings are expected to improve the standardization and reproducibility of tobacco toxicity assessment and provide foundational data for future policy-making and regulatory research.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• Chapter 2. Toxicological validity assessment of tobacco emission condensates 9
• 2.1. Background 9
• 2.2. General experiment and result outline 12
• 2.3. Materials and methods 13
• 2.3.1. Preparation and analysis of tobacco condensates (WCSC and HTPAC) 13
• 2.3.1.1. Generation of tobacco emissions (from 3R4F cigarette smoke and HTP aerosols) 16
• 2.3.1.2. Collection, pretreatment, and analysis for tobacco condensates (WCSC and HTPAC) 17
• 2.3.2. Preparation of working standards for calibration curve acquisition 18
• 2.3.3. Instrumental analysis 22
• 2.3.4. Quantitative determination of OC concentrations in tobacco condensates (WCSC and HTPAC) 24
• 2.3.5. Preliminary tobacco emission data for comparative analysis with tobacco condensate 27
• 2.3.6. Gathering toxicity data for OCs 28
• 2.4. Results and discussion 29
• 2.4.1. Qualitative and quantitative assessment of OCs in tobacco condensates 29
• 2.4.2. Comparative analysis of OC concentrations in tobacco emissions and condensates 38
• 2.4.3. Hazard assessment of tobacco products using tobacco emission and condensate data 50
• 2.4.4. Challenges and considerations in the quantitative hazard assessment of tobacco emission and condensate 54
• Chapter 3. Development of an automated system for cigarette smoke generation and collection with chemical and biological evaluation 57
• 3.1. Background 57
• 3.2. General experiment and result outline 59
• 3.3. Materials and methods 61
• 3.3.1. Structure of ASGCS (cigarette smoke generation and collection system) 61
• 3.3.2. Verification of cigarette smoke generation and collection accuracy in ASGCS 64
• 3.3.2.1. Evaluation of cigarette smoke leakage error 65
• 3.3.2.2. Evaluation of cigarette smoke puff duration and interval errors 67
• 3.3.3. Evaluation of the transfer rates of VOCs in WCSC obtained via ASGCS (nicotine and TVOC) 68
• 3.3.3.1. Preparation of WCSC 68
• 3.3.3.2. Preparation of working standards (nicotine and toluene) 71
• 3.3.3.3. Instrumental analysis conditions 73
• 3.3.3.4. Calibration and quality control (QC) results 75
• 3.3.4. In vitro toxicity assessment of WCSC obtained via ASGCS 77
• 3.3.4.1. Cell culture 77
• 3.3.4.2. Cell viability and IC50 77
• 3.4. Results and discussion 79
• 3.4.1. Accuracy evaluation for cigarette smoke generation and collection in ASGCS 79
• 3.4.1.1. Cigarette smoke leakage error (%): from generation to collection 79
• 3.4.1.2. Cigarette smoke puff duration and interval errors 82
• 3.4.2. Evaluation of VOC transfer rates in WCSC 83
• 3.4.2.1. Analysis of VOCs in WCSC 83
• 3.4.2.2. Evaluation of nicotine and TVOC transfer rates in WCSC 87
• 3.4.3. In vitro toxicity assessment of WCSC 92
• 3.4.3.1. Decrease in cell viability after WCSC exposure 92
• 3.4.3.2. IC50 of WCSC on WT MEF cells 97
• 3.4.4. Industrial and academic significance of this study 99
• 3.4.4.1. Academic contributions to environmental chemistry and safety 99
• 3.4.4.2. Industrial Applications of ASGCS 100
• Chapter 4. Assessment of nicotine stability in cigarette smoke under various storage conditions 101
• 4.1. Background 101
• 4.2. General experiment and result outline 103
• 4.3. Materials and methods 105
• 4.3.1. Experimental schemes 105
• 4.3.1.1. Nicotine standard (Experimental stage 1) 111
• 4.3.1.2. Cigarette smoke (Experimental stage 2) 111
• 4.3.2. Preparation of liquid nicotine standards 113
• 4.3.3. Instrumental system 115
• 4.3.4. Calibration analysis of the nicotine standards 117
• 4.4. Results and discussion 119
• 4.4.1. Assessment of nicotine recovery under light exposure conditions using nicotine solutions (Experimental stage 1) 119
• 4.4.2. Nicotine recovery assessment in cigarette smoke under UV radiation exposure conditions (Experimental stage 2) 123
• 4.4.3. Comparison of nicotine recovery in nicotine solution and cigarette smoke under different light conditions 126
• 4.4.4. Study significance, limitations, and further research requirements 129
• Chapter 5. Conclusions 130
• References 133