Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay

doi:10.1016/j.fct.2004.01.009

Food and Chemical Toxicology

Volume 42, Issue 5, May 2004, Pages 851-863

https://doi.org/10.1016/j.fct.2004.01.009 Get rights and content

Abstract

Numerous chemical and toxicological studies indicate that smoke from ECLIPSE^®, a cigarette that primarily heats rather than burns tobacco, is simplified and reduced in specific chemicals believed to be associated with smoking-related diseases, and demonstrates reduced smoke toxicity and biological activity in vitro when compared to conventional tobacco burning cigarettes. These data led to the hypothesis that cigarette smoke condensate (CSC) from ECLIPSE^® should have lower tumorigenicity than 1R4F condensate in the SENCAR mouse dermal tumor promotion assay. Female SENCAR mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with ECLIPSE^® or 1R4F CSC. Dermal application of 10, 20, or 40 mg ECLIPSE^® or 1R4F CSC three times/week for 29 weeks did not alter body weights, survival or other indicators of subchronic toxicity. In DMBA-initiated mice, there were significant increases in both the number of microscopically confirmed tumor-bearing animals and total number of microscopically confirmed dermal tumors at all 1R4F CSC doses and the high-dose ECLIPSE^® CSC. However, the number of ECLIPSE^® tumor-bearing animals were reduced 83%, 93% and 67% at the low-, mid- and high-doses, respectively, compared to the 1R4F. Similarly, the total number of dermal tumors was reduced 91%, 94% and 87% at the low-, mid- and high-dose, respectively, compared to the 1R4F CSC. ECLIPSE^® CSC demonstrated dramatic reductions in dermal tumor promotion potential compared to 1R4F CSC.

Introduction

The ECLIPSE^® cigarette is based upon a novel cigarette design that primarily heats tobacco instead of burning tobacco. A diagram of the configuration of the ECLIPSE^® cigarette is presented in Fig. 1. Basically, the ECLIPSE^® design uses a small carbon heat source that when burned produces energy to vaporize tobacco flavors and glycerol from a two-part tobacco rod. The tobacco rod is not consumed during smoking. Condensation of the tobacco flavors and glycerol produces a smoke aerosol, which exits the cigarette through the mouthpiece (http://www.ECLIPSE.RJRT.com).

Only a small amount of tobacco is burned when ECLIPSE^® cigarettes are smoked, therefore the concentrations of many of the chemical compounds in the mainstream smoke are substantially reduced compared to the mainstream smoke of tobacco burning cigarettes with similar “tar” delivery. The total particulate matter (TPM) of ECLIPSE^® cigarettes are reported to be about 75% glycerol and water and about 25% “tar” and nicotine while 1R4F research cigarettes are reported to consist of 20% glycerol and water and 80% “tar” and nicotine. “Tar” is the total particulate matter in the particulate phase of the mainstream smoke, minus water and nicotine, which is retained on a Cambridge filter pad when smoke passes through it. Specific components of the “tar” from ECLIPSE^® cigarettes, including benzo[a]pyrene, catechol, 4-aminobiphenyl, 2-aminonaphthalene, free radicals and tobacco specific nitrosamines, among others, were reduced 77–99% compared to the “tar” from the 1R4F cigarette (ECLIPSE Expert Panel, 2000), while furfural was the only measured compound that increased.

Reduced and simplified smoke chemistry would be expected to produce reduced biological activity. A number of studies support this hypothesis and have been reviewed by an expert panel (ECLIPSE Expert Panel, 2000). Highlighting some of these supporting studies, cigarette smoke condensate (CSC) from earlier ECLIPSE^® prototypes was markedly less genotoxic in the Ames bacterial mutagenicity assay, the sister chromatid exchange assay and the chromosomal aberration assay than CSC from 1R4F (full-flavor low-tar) and 1R5F (ultra-low-tar) cigarettes (Bombick et al., 1998). When evaluated for cytotoxicity with the neutral red and lactate dehydrogenase (LDH) release assays, CSC from an earlier ECLIPSE^® prototype was less cytotoxic than 1R4F and 1R5F CSC (Bombick et al., 1998).

In whole animal studies, Brown et al. (1997) demonstrated that smoke from an earlier ECLIPSE prototype produced few, if any, increases in lung and heart DNA adducts, compared to untreated controls, when mice were exposed for 1 h/day, 5 days/wk for 4 weeks. In contrast, smoke from the 1R4F cigarette produced an exposure-dependent increase in DNA adducts in the lungs and heart of mice under an identical exposure regimen.

In human studies, urine voided by smokers is more mutagenic in the Ames mutagenicity assay than urine voided by nonsmokers (IARC, 1986, Doolittle et al., 1990). Studies employing a double crossover design demonstrated a 72–79% reduction in the mutagenicity of urine from smokers of an earlier ECLIPSE^® prototype cigarette as compared to when the same smokers were smoking their usual brand of tobacco-burning cigarette (Smith et al., 1996).

These data lead to the hypothesis that CSC from the ECLIPSE^® cigarette should demonstrate significantly less activity in the SENCAR mouse dermal tumor promotion assay than CSC from tobacco-burning cigarettes. This assay is the most relevant method available to study the tumor promoting potential of cigarette smoke. To test this hypothesis, SENCAR mice were initiated with a single dermal administration of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with CSC from either the 1R4F or ECLIPSE^® cigarette. The study design was based upon a protocol developed for comparing the activities of CSCs from different cigarettes using the SENCAR mouse dermal tumor promotion assay (Meckley et al., 2004).

Section snippets

Cigarettes

The Reference cigarette used in this study was the 1R4F research cigarette from the Tobacco and Health Research Institute, University of Kentucky (Lexington, KY). This cigarette is an air-diluted, filtered cigarette containing a blend of tobacco and represents an average full-flavor low-“tar” American cigarette (Sullivan, 1984, Diana and Vaught, 1990). The Reference cigarettes were stored at 4 °C until use. They were conditioned to the ambient laboratory environment (17.8–26.1 °C and 30–50%

Analysis of mainstream smoke and CSC from cigarettes

Table 2 provides a comparison of the mainstream smoke analyses for the Reference and Test cigarettes used in this study. Total Particulate Matter (TPM) from the Test cigarette was 47% of that from the Reference cigarette, while nicotine and carbon monoxide from the Test cigarette were 21% and 67% of that from the Reference, respectively. These data are similar to those previously published (Borgerding et al., 1997; http://www.ECLIPSE.RJRT.com) for earlier ECLIPSE^® prototype cigarettes.

Table 3

Discussion

The major objective of this study was to compare the dermal tumor promoting potential of ECLIPSE^® CSC to CSC from the 1R4F cigarette. Since the early studies of Wynder (1953), rodent dermal tumor initiation/promotion assays have been used to study CSC. These early studies indicated CSC was a weak tumorigen, while studies by Van Duuren (1966) indicated CSC could promote DMBA-initiated tumorigenesis in the mouse dermal initiation/promotion assay. Since that time, the mouse dermal assay has been

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Numerous skin painting studies with SENCAR mice, using DMBA as initiator and CSC as promoter, demonstrated dose-related, consistent, and reproducible tumorigenic responses (Gaworski et al., 1999; Meckley et al., 2004a). The methodological difference from reported studies of the present study is how to collect CSCs from cigarettes; CSCs were collected under Health Canada Intensive Regimen (CIR) in this study, in contrast to the International Organization for Standardization method (International Organization for Standardization, 1991; Meckley et al., 2004b; Werley et al., 2008). The intent of our investigation was to compare dermal tumor promotion activity as well as irritating property of CSC collected from Test cigarettes to that from reference cigarettes using a standard initiation-promotion design using SENCAR mice.
Test cigarette (prototype “heated” cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30 mg tar/application, three times per week for 30 weeks. Test groups showed a clearly longer latency at lower doses (⩽15 mg), but the difference was less clear at higher doses (⩾22.5 mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.
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The mouse dermal assay has long been used to assess the dermal tumorigenicity of cigarette smoke condensate (CSC). This mouse skin model has been developed for use in carcinogenicity testing utilizing the SENCAR mouse as the standard strain. Though the model has limitations, it remains as the most relevant method available to study the dermal tumor promoting potential of mainstream cigarette smoke. In the typical SENCAR mouse CSC bioassay, CSC is applied for 29 weeks following the application of a tumor initiator such as 7,12-dimethylbenz[a]anthracene (DMBA). Several endpoints are considered for analysis including: the percentage of animals with at least one mass, latency, and number of masses per animal. In this paper, a relatively straightforward analytic model and procedure is presented for analyzing the time course of the incidence of masses. The procedure considered here takes advantage of Bayesian statistical techniques, which provide powerful methods for model fitting and simulation. Two datasets are analyzed to illustrate how the model fits the data, how well the model may perform in predicting data from such trials, and how the model may be used as a decision tool when comparing the dermal tumorigenicity of cigarette smoke condensate from multiple cigarette types. The analysis presented here was developed as a statistical decision tool for differentiating between two or more prototype products based on the dermal tumorigenicity.
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Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay

Abstract

Introduction

Section snippets

Cigarettes

Analysis of mainstream smoke and CSC from cigarettes

Discussion

Food and Chemical Toxicology

Food and Chemical Toxicology

Toxicology

Mutation Research

The premalignant nature of mouse skin papillomas: histopathologic, cytogenetic and biochemical evidence

Mouse skin assay of condensate from smoking products other than tobacco

Journal of the National Cancer Institute

Carcinogenic activity of smoke condensate from cigarettes with ammonium sulfamate treated paper

Cancer

Cigarette smoke composition. Part 2. Method for determining major components in smoke of cigarettes that heat instead of burn tobacco

Journal of the Association of Analytical Chemists

Chemical and biological studies of a new cigarette which primarily heats tobacco Part 1, Chemical composition of mainstream smoke

Food and Chemical Toxicology

Comparative study of DNA adduct formation in mice following inhalation of smoke from cigarettes that burn or primarily heat tobacco

Environmental Molecular Mutagenesis

Research Cigarettes

Zeitschrift fur Krebsforschung und Klinische Onkologie

Experimental investigations on the tumorigenic activity of cigarette smoke condensate on mouse skin. VII. Comparative studies of condensates from different modified cigarettes

Zeitschrift fur Krebsforschung und Klinische Onkologie

A safer cigarette? A comparative study. A consensus report

Inhalation Toxicology

Reconstituted tobacco-smoking and health possibilities

Journal of the National Cancer Institute

Skin bioassay in tobacco carcinogenesis

Progress in Experimental Tumor Research