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0 YRTPH 3212 9 January 2015 ARTICLE IN PRESS No. of Pages 8, Model 5G
1 Regulatory Toxicology and Pharmacology xxx (2015) xxx-xxx
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology
journal homepage: www.elsevier.com/locate/yrtph
3 The combination of two novel tobacco blends and filter technologies to
4 qi reduce the in vitro genotoxicity and cytotoxicity of prototype cigarettes
7 Q2 Ian Crooks *, Ken Scott, Annette Dalrymple, Debbie Dillon, Clive Meredith
British American Tobacco (Investments) Ltd., GR&D Centre, Regents Park Road, Southampton SO16 8TL, UK
20 21 22
ARTICLE INFO
Article history: Received 31 July 2014 Available online xxxx
Keywords: Genotoxicity Mutagenicity Cytotoxicity In vitro
Tobacco smoke Particulate matter Tobacco sheet substitute Blend treated tobacco Toxicant
Reduced toxicant prototype
ABSTRACT
Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 29
of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emis- 30
sions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend 31
treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to 32
reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and 33
filter technologies were combined into an experimental cigarette and smoked to International Organisa- 34
tion on Standardisation and Health Canada puffing parameters. The resulting particulate matter was sub- 35
jected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma 36
assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes 37
containing toxicant reducing technologies may be developed without observing new additional genotoxic 38
hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mam- 39
malian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There 40
were no significant differences in cytotoxicity relative to the control cigarettes. 41
© 2015 Published by Elsevier Inc. 42
46 1. Introduction
48 Tobacco smoke from a combustible cigarette is a complex and
49 dynamic mixture of more than 6000 chemicals (Perfetti and
Abbreviations: BAT, British American Tobacco; BC, baseline control; BTT, blend treated tobacco; CA, cellulose acetate; CC, commercial control; COM, Committee on Mutagenicity; CFP, Cambridge filter pad; CORESTA, Cooperation Centre for Scientific Research Relative to Tobacco; CR20L, amine-functionalised resin; DIET, Dry Ice Expanded Tobacco; DMEM, Dulbecco's Modified Eagles Medium; DMSO, dimethyl sulfoxide; EC, experimental cigarette; FCS, Foetal Calf Serum; FDA, Food and Drug Administration; GEF, Global Evaluation Factor; GLP, Good Laboratory Practice; HCI, Health Canada Intense; IC50, the concentration causing 50% toxicity in the NRU test; ICH, International Conference on Harmonisation; IOM, Institute of medicine; IPA, isopropyl alcohol; ISO, International Organisation for Standardisation; MLA, mouse lymphoma assay; MNBN, micronucleated binucleate cells; MNvit, in vitro micro-nucleus assay; MRTP, modified risk tobacco product; MSS, mainstream smoke; NFDPM, nicotine free dry particulate matter; NIH, National Institute of Health; NR, no response; NRU, Neutral Red Uptake assay; NS, non-significant; OECD, Organisation for Economic and Cooperative Development; PM, particulate matter; PREP, potentially reduced exposure product; RPMI, Roswell Park Memorial Institute Medium; RTG, relative total growth; S9, post-mitochondrial supernatant; SD, standard deviation; SEM, standard error of the mean ST, split tipping; TFT, trifluoro-thymidine; TSS, tobacco sheet substitute.
* Corresponding author.
E-mail addresses: ian_crooks@bat.com (I. Crooks), ken_scott@bat.com (K. Scott), annette_dalrymple@bat.com (A. Dalrymple), debbie_dillon@bat.com (D. Dillon), clive_meredith@bat.com (C. Meredith).
Rodgman, 2013) and approximately 150 of these have been identi- 50
fied as toxicants (Fowles and Dybing, 2003). The Institute of 51
Medicine (IOM) have reported that smoking related diseases are 52
dose related and epidemiology demonstrates a reduction in disease 53
incidence following smoking cessation (Stratton et al., 2001). 54
Therefore it is postulated that it may be possible to reduce smoking 55
related diseases by reducing toxicant levels in cigarettes (Stratton 56
et al., 2001). Novel cigarettes with reduced toxicant levels have 57
been termed potential reduced-exposure products (PREPs) 58
(Stratton et al., 2001). Strategies for the assessment of modified 59
tobacco products, or PREPs, have been published (Life Sciences 60
Research Office, 2007; World Health Organisation, 2007), with 61
the goal of lowering mainstream smoke (MSS) yields for specific 62
smoke toxicants and biomonitoring populations (Burns et al., 63
2008; Baker, 1999; Hatsukami et al., 2012). 64
The Food and Drug Administration (FDA) have introduced the 65
term 'modified risk tobacco products' (MRTPs). Similar to PREPs, 66
MRTPs are defined as any tobacco product that is sold or distrib- 67
uted for use to reduce harm or the risk of tobacco-related disease 68
and this must be demonstrated by scientific evidence, including 69
in vitro systems, models of disease and biomarkers of exposure. 70
Technologies to reduce toxicant emissions from a cigarette 71
mainly focus on modifying the tobacco blend or the filter. Two 72
blend technologies have been described previously; blend treated 73
tobacco (BTT) by Liu et al., 2011 and tobacco sheet substitute 74
http://dx.doi.org/10.1016/j.yrtph.2015.01.001 0273-2300/® 2015 Published by Elsevier Inc.
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(TSS) by McAdam et al., 2011. The BTT process is the sequential extraction of tobacco with water followed by protease treatment and adsorbents, resulting in tobacco with reduced amino acid and protein content (Liu et al., 2011). Proteins and amino acids are known precursors for a number of toxicants in smoke, including aromatic and heterocyclic amines that contribute to mutagenicity (Clapp et al., 1999; Matsumoto and Yoshida, 1981; Matsushima et al., 1999; Mizusaki et al., 1977). Therefore the BTT process could potentially reduce the toxicity profile of the cigarette.
TSS contains high levels of glycerol and calcium carbonate and when used as a cigarette blend component it reduces the organic content of tobacco. The glycerol contained in TSS distils into the MSS during the combustion process, diluting the tar, reducing the level of a wide range of smoke constituents and the potential toxicity of cigarettes (McAdam et al., 2011).
Activated charcoal, usually manufactured from coconut shells, is often used in conventional cigarette filters to remove volatile organic compounds (Branton and Bradley, 2011). Polymer derived activated charcoals demonstrate increased adsorption of volatile organic compounds, approximately twice as efficient as standard activated charcoal (Branton et al., 2011a). Another filter technology, CR20L - an amine-functionalised resin bead - has been described previously by Branton et al., 2011b. In comparison to standard activated charcoal, it offers superior reductions in formaldehyde, hydrogen cyanide and acetaldehyde. Polymer derived activated charcoals and CR20L, when contained in the filters of novel cigarettes, may reduce key toxicants that drive mutagenicity and cytotoxicity.
Split tipping (ST), a novel method of filter ventilation, is a gap between two separated sections of tipping paper, exposing an area of the filter. The gap is wrapped with a bespoke paper that is more porous than standard tipping paper, allowing the diffusion of gases and vapours into and out of the filter. ST maintains the ventilation level at higher flow levels generally encountered when consumers smoke cigarettes (Dittrich et al., 2014).
Another important aspect of PREPs and MRTPs is the consumer acceptability of cigarettes that have lower toxicant yields, as they often have a different smoking experience compared to conventional cigarettes. Although the initial acceptability of the experimental cigarette used in this study was lower than the commercial control cigarette, the sensory perception of the experimental cigarette changed over the duration of the study toward a level of acceptability similar to that of the commercial control (Dittrich et al., 2014).
Studies have shown that humans smoke cigarettes with higher intensity when compared to laboratory smoking machines operated to International Organisation for Standardisation (ISO) conditions. Therefore the ISO regime, used as a tobacco industry standard, may not be representative of human smoking (Hatsukami et al., 2012). It has also been reported that MSS constituent yields are different under intense regimes when compared to ISO smoking conditions. As one smoking regime does not represent the smoking behaviour of humans, it is generally considered that the ISO and the more intense Health Canada (HCI) regimes are two extremes of smoking behaviour (St. Charles et al., 2010). Therefore, particulate matter (PM) generated to ISO and HCI smoking regimes were included in this study.
Several guidelines such as those developed by the International Conference on Harmonisation (ICH, 2011) and Committee on Mutagenicity (COM, 2011) recommend a battery of core in vitro genotoxicity assays for the detection of mutagenicity and potential for carcinogenicity. For tobacco products, Health Canada also specifies the Neutral Red Uptake assay (NRU) in addition to the Ames test and the in vitro micronucleus assay (MNvit) (Canada, 2000). Furthermore, the Cooperation Centre for Scientific Research
Table 1
Composition of cigarettes used in the study.
PM code Blend Filter Format
3R4F US-style blend CA King size
M4A Flue-cured blend CA King size
EC 50% BT, 15% TSS, Polymer derived Demi-slim
35% lamina & DIET activated charcoal CR20L,
ST and CA
CC US-style blend CA King size
BC 65% BT feedstock, CA King size
10% DIET, 25% lamina
CA, cellulose acetate; BT, blend treated tobacco; TSS, tobacco sheet substitute; ST, split tipping; DIET, Dry Ice Expanded Tobacco (tobacco that has been expanded with dry ice to increase tobacco filling capacity).
Relative to Tobacco (CORESTA) in vitro Toxicology Task Force recommends the Ames test, the MNvit, the mouse lymphoma assay (MLA) and the NRU (CORESTA, 2004).
This paper describes the in vitro assessment of the genotoxicity and cytotoxicity of the particulate matter generated from experimental cigarettes (ECs) incorporating 50% BTT, 15% TSS, polymer derived activated charcoal, CR20L and split tipping technologies as described above, in a demi-slim format (Table 1) (Dittrich et al., 2014). The blend technologies described have previously been tested singularly (McAdam et al., 2011; Liu et al., 2011; Combes et al., 2012, 2013), but not in combination as described herein. The particulate matter generated from commercial and baseline control cigarettes were included as comparator products. The control cigarettes contained a standard cellulose acetate filter, tipping paper and typical tobacco blends however, the baseline control contained 65% of the BTT feedstock (i.e. the BTT tobacco without water extraction, enzyme and adsorbent treatment). 3R4F and M4A (US style blended and a flue cured product, respectively) were also included for comparison to historical data. The experimental and control cigarettes used in this study had an ISO tar yield of 7 mg per cigarette. Mainstream smoke particulate matter generated under ISO and HCI puffing parameters from reference, experimental and control cigarettes were tested using a battery of in vitro toxicity assays - the Ames test, MLA, MNvit and NRU.
2. Materials & methods
2.1. Cigarettes
Cigarette specifications are detailed in Table 1, and also in (Dittrich et al., 2014). 3R4F and M4A were reference cigarettes and are included in all British American Tobacco (BAT) in vitro studies to demonstrate the assay performance was comparable to historic data. A commercial control (CC) and a baseline control (BC) cigarette were included, containing conventional cigarette materials. EC was the experimental cigarette containing the blend and filter technologies. 3R4F were supplied by the University of Kentucky and all other cigarettes were manufactured by BAT.
2.2. PM generation
Cigarettes were conditioned to ISO 3402 (ISO, 1999) and smoked on a Borgwaldt RM200s (Borgwaldt-KC, Hamburg, Germany) machine using ISO 3308 (ISO, 2000b, 35 mL puff volume, of 2 s duration, every 60 s, 0% vent blocking) and Health Canada (Canada, 2000, 55 mL puff volume, of 2 s duration, every 30 s, 100% vent blocking) puffing parameters. Approximately 6220 mg PM was collected onto each 44 mm Cambridge filter pad (CFP) (Whatman, Maidstone, UK). Pads were weighed pre and post smoking, to determine the mass of PM deposited. 1/4 of each pad
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was subsequently placed into isopropyl alcohol (IPA) (Sigma-Aldrich, Poole, UK) for nicotine, water and nicotine free dry partic-ulate matter (NFDPM) determination (ISO, 2000a). PM from the 3/4 pad was eluted with anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Poole, UK) under vacuum to a final stock concentration of 24 mg/mL. PM extracts were stored in single-use aliquots immediately at -80 °C. Samples were transported on dry ice to an independent Good Laboratory Practice (GLP) accredited laboratory for in vitro testing, where they were stored immediately at -80 °C and used within 12 months. Crooks et al., 2013 have demonstrated stability of PM when stored at -80 °C for up to 2 years. Samples were blind tested using a suitable coding system.
2.3.3. MNvit
Four replicate V79 (University of Swansea, UK) cell cultures in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% Foetal Calf Serum (FCS) and 0.52% penicillin/streptomycin were treated with PM for 3 h followed by a 21 h recovery ±S9, or treated for 24 h -S9 with no recovery period. Cytochalasin B formulated in DMSO, was added to all treatment flasks at the time of treatment at a final concentration of 3 ig/mL. At least four concentrations were tested for each PM and scored for cytotoxicity and micronucleated binucleate cells (MNBN) on quadruplicate slides and 2000 cells per slide were analysed. This method complied with OECD Test Guideline 487 (OECD, 2010).
2.3. In vitro toxicology testing
Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9) mix (Moltox™, North Carolina, USA) provided metabolic activation in the genotoxicity assays, where specified. Appropriate positive and vehicle controls were included in all experiments, as recommended by the relevant guidelines. All chemicals were obtained from Sigma-Aldrich, Poole, UK, unless otherwise stated.
2.3.1. Ames test
Five tester strains of Salmonella typhimurium (TA98, TA1535, TA1537 (obtained from UK National Collection of Type Cultures), TA100 and TA102 (derived from Covance Laboratories Inc., Vienna, USA)) were used in the presence and absence of S9 in accordance with Organisation for Economic and Cooperative Development (OECD) Test Guideline 471 (OECD, 1997a). At least eight PM concentrations per cigarette type were used per experiment. Four independent repeat plate incorporation tests and one pre-incubation test were performed. With tester strains TA98, TA100 and TA1537 +S9, five, four and ten replicate plates, respectively, were used per concentration in all experiments as recommended by Scott et al., 2013. The remaining strains and treatment conditions used three replicate plates.
2.3.2. MLA
The MLA was performed using L5178Y tk+/- cells (Burroughs Wellcome Co., London, UK) cultured in supplemented Roswell Park Memorial Institute Medium (RPMI). Ten concentrations were tested with four replicates cultures per concentration. Two independent repeat experiments were performed, each using a 3 h +S9, 3 and 24 h -S9 exposures. After a 2 days expression period, cells were grown for 8 days and triflouro-thymidine (TFT) resistant colonies were counted and the percent relative total growth (RTG) determined. PMs that exceeded the Global Evaluation Factor (GEF) (plus vehicle control) were considered mutagenic. This method complied with OECD Test Guideline 476 (OECD, 1997b).
2.3.4. NRU
Balb/c 3T3 clone A31 mouse fibroblasts (European Collection of Cell Cultures) were maintained in DMEM supplemented with 10% (v/v) FCS, 4 mM L-glutamine and penicillin/streptomycin (1% v/v). Cytotoxicity was expressed as a reduction in the uptake of Neutral Red dye into the lysosomes of cells after a 24 h culture, measured by absorbance at 450 nm. A minimum of six concentrations were tested for each PM, to determine concentration-dependent inhibition of cell growth. Six wells per concentration were used for each PM, vehicle or positive control (sodium dodecyl sulphate). This protocol conforms to the guidelines issued by the National Institute of Health (NIH, 2001). Four independent repeat experiments were performed.
2.3.5. Statistical analysis
The concentration-response of each PM in the genotoxicity assays were assessed for significance above the vehicle control. The Ames test was analysed with Dunnett's test; the MLA used weighted regression (Robinson et al., 1989); and the MNvit used Fisher's Exact Test (Richardson et al., 1989).
PMs were compared according to the recommendations of Scott et al., 2013. For the genotoxicity assays, the linear portion of the concentration response curve was identified by fitting a generalised linear model, using adaptations to accommodate the lognormal, binomial and Poisson distributions of the MLA, MNvit and Ames test, respectively. The slopes of the two PMs being compared were tested for parallelism and if significant, there was a statistical difference in slopes. If non-significant, the magnitudes of the two PMs were compared. If linearity was not identified, pair-wise comparisons were made using t-tests at each common concentration. All tests were performed with two sided risk.
For the NRU, the PM concentration causing 50% toxicity (IC50) was calculated by fitting a four parameter logistic model to the percentage survival. Pairwise comparisons of the log-transformed IC50 estimates were made using a two-sample t-test.
0 J-,-,-,-,-,- 0 -I-,-,-,-,-.-,-
0 50 100 150 200 250 300 0 50 100 1 60 200 260 300
PM Concentration ((ig/plate) PM Concentration (pg/plate)
Fig. 1. Bacterial mutagenicity of particulate matter generated from the experimental cigarette (♦), commercial (□) and baseline (A) controls in TA98 in the presence of S9 at the ISO (A) and HCI (B) regimes. Data presented as the mean of four independent plate incorporation experiments ± SEM.
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3. Results
3.1. Reference products
The particulate matter generated at ISO and HCI puffing parameters demonstrated that the flue cured product, M4A, was less mutagenic than the American-blended product, 3R4F in the Ames test. In the MLA, MNvit and NRU, M4A was more active than 3R4F. This data was consistent with the historical datasets for these products generated under ISO conditions.
In strain TA1537 +S9, variable results were obtained. The partic-ulate matter generated from the experimental cigarette generated under the HCI regime showed overall reduced mutagenicity compared to the commercial control generated under the same conditions and equivalent mutagenic activity relative to the baseline control (Fig. 3B and Table 2). The particulate matter generated to the ISO regime demonstrated that the experimental cigarette par-ticulate matter was more mutagenic than the baseline control (Fig. 3A and Table 2). However, relative to the commercial control, the two products were equivalent (Fig. 3A and Table 2).
3.2. Ames test
3.3. MLA
In the Ames test, all particulate matters were non-mutagenic in all tester strains -S9, and in strain TA1535 +S9. Some increases in revertants in tester strain TA102 +S9 were observed, but this was only seen in one experiment with no more than a 1.4-fold increase in revertants above the vehicle control and did not demonstrate concentration related increases. Consistent and reproducible mutagenic activity was detected in strains TA98, TA100 and TA1537, all in the presence of S9 only, with the exception of the commercial and baseline control particulate matters generated under the Health Canada regime in strain TA100 +S9 in one experiment, where the particulate matter concentration revertant colonies did not exceed those of the vehicle control; this data was excluded from comparative analysis.
In tester strain TA98 in the presence of S9, the experimental cigarette was less mutagenic than the commercial control particulate matter generated to ISO, however the mutagenicity of the experimental cigarette was equivalent to the baseline control (Fig. 1A and Table 2). At the HCI regime, the experimental cigarette was less mutagenic than the commercial and baseline controls (Fig. 1B and Table 2).
In TA100 in the presence of S9, the experimental cigarette was less mutagenic than the commercial and baseline controls at both smoking regimes (Fig. 2 and Table 2).
All particulate matters in all treatment conditions induced concentration related increases in cytotoxicity to approximately 20% RTG. In addition, statistically significant increases in mutation frequency were observed and all particulate matters exceeded the GEF plus vehicle control.
The experimental cigarette was less genotoxic relative to the commercial control in the 3 h treatments both in the absence and presence of S9 (Figs. 4A, 5A and Table 3) at the ISO regime. However, there was no difference between the experimental cigarette and the commercial control particulate matters generated to the HCI regimes (Figs. 4A, 5A and Table 3).
Comparison with the baseline control particulate matter generated to the ISO regime, the experimental cigarette was of equivalent genotoxic potential in the 3 h treatments in the presence and absence of S9 (Figs. 4A, 5A and Table 3). However, under the HCI regime, the experimental cigarette was less genotoxic than the baseline control in the 3 h -S9 (Fig. 4B) and +S9 treatments (Table 3).
In the 24 h treatment, the experimental cigarette was of equivalent mutagenicity to the commercial control under the ISO (Fig. 5B and Table 3) and HCI regimes. Relative to the baseline control, the experimental cigarette was less mutagenic at the ISO regime (Fig. 5Band Table 3). At the HCI regime, there were no
Table 2
Relative mutagenicity of the experimental cigarette (EC) compared to the commercial (CC) and baseline (BC) controls at the ISO and HCI smoking regimes in tester stains TA98, TA100 andTA1537 +S9 for each individual experiment. Grey boxes indicate where product A (i.e. the experimental cigarette) was less active than product B (i.e. the commercial or baseline control). White boxes indicate where product A was more active than product B.
Strain & Treatment Condition Experiment EC ISO (A) v CC ISO (B) EC ISO (A) v BC ISO (B) EC HCl (A) v CC HCl (B) EC HCl (A) v BC HCl (B)
1 NS p S 0.001 p á 0.001 p á 0.001
2 p S 0.001 p S 0.05 p á 0.01 p ü 0.05
TA98 +S9 3 p < 0.01 NS p < 0.001 p < 0.001
4 p < 0.01 p < 0.001 p < 0.01 p < 0.001
Pre-incubation p S 0.001 p S 0.001 p <S 0.001 NS
1 p s 0.001 p S 0.001 p á 0.001 p <S 0.001
2 NS p á 0.05 p S 0.001 p S 0.001
TA100 +S9 3 p £ 0.001 p S 0.001 p £ 0.01 p <; 0.001
4 p S 0.001 p S 0.05 NR NR
Pre-incubation p < 0.001 p < 0.001 p < 0.05 p < 0.001
1 p s 0.001 p á 0.001 p S 0.001 p S 0.001
2 NS p S 0.01 p S 0.001 NS
TA1537 +S9 3 p s 0.001 p s 0.001 p ¿ 0.001 p ¿ 0.001
4 p S 0.001 p S 0.001 p S 0.001 p <; 0.001
Pre-incubation p s 0.001 NS p á 0.001 p s 0.01
P value indicates level of statistical significance; NS, not significant; NR, no response was obtained.
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Fig. 2. Bacterial mutagenicity of particulate matter generated from the experimental cigarette (♦), commercial (□) and baseline (A) controls in TA100 in the presence of S9 at the ISO (A) and HCI (B) smoking regimes. Data presented as the mean of four (ISO) and three (HCI) independent plate incorporation tests ± SEM.
Fig. 3. Bacterial mutagenicity of the particulate matter generated from experimental cigarette (♦), commercial (□) and baseline (A) controls in TA1537 +S9 at the ISO (A) and HCI (B) regime. Data presented as the mean of four independent plate incorporation tests ± SEM.
Fig. 4. Genotoxicity (mutation frequency per 106 viable cells) of particulate matter generated to the ISO (A) and HCI (B) smoking regimes from the experimental cigarette (♦), commercial (□) and baseline (A) controls as assessed in the mouse lymphoma assay for 3 h -S9. Data shown is the mean of two independent experiments ± SEM.
Fig. 5. Genotoxicity (mutation frequency per 106 viable cells) of particulate matter generated to the ISO smoking regime from the experimental cigarette (♦), commercial (□) and baseline (A) controls as assessed in the mouse lymphoma assay for 3 h +S9 (A) and 24 h -S9 (B). Data shown is the mean of two independent experiments ± SEM.
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Table 3
Relative genotoxicity of the experimental cigarette (EC) compared to the commercial (CC) and baseline (BC) controls at the ISO and HCI smoking regimes in the mouse lymphoma assay for each individual experiment. Grey boxes indicate where product A (i.e. the experimental cigarette) was less active than product B (i.e. the commercial or baseline control). White boxes indicate where product A was more active than product B.
Treatment Experiment EC ISO (A) v CC ISO (B) EC ISO (A) v BC ISO (B) EC HCI (A) v CC HCI (B) EC HCI (A) v BC HCI (B)
3h -S9 1 p < 0.05 p< 0.001 NS p< 0.001
2 p< 0.001 p < 0.05 NS p < 0.05
3h +S9 1 p< 0.001 p<0.01 p< 0.001 p< 0.001
2 p< 0.001 p<0.01 p< 0.001 p< 0.001
24h -S9 1 NS NS p< 0.001 NS
2 NS p<0.01 NS p < 0.05
P value indicates level of statistical significance; NS, not significant.
Fig. 6. Genotoxicity of particulate matter generated to the ISO (A) and HCI (B) smoking regimes from the experimental cigarette (♦), commercial (□) and baseline (A) controls as assessed in the in vitro micronucleus test for 24 with V79 cells for hours -S9. Micronucleated binucleate cells are per 2000 cells analysed. Data shown is the mean of four replicate cultures ± SD.
Table 4
Relative genotoxicity of the experimental cigarette (EC) compared to the commercial (CC) and baseline (BC) controls at the ISO and HCI smoking regimes in the in vitro micronucleus test. Grey boxes indicate where product A (i.e. the experimental cigarette) was less active than product B (i.e. the commercial or baseline control). White boxes indicate where product A was more active than product B.
Treatment Experiment EC ISO (A) v CC ISO (B) EC ISO (A) v BC ISO (B) EC HCI (A) v CC HCI (B) EC HCI (A) v BC HCI (B)
3h -S9 1 p<0.01 p<0.01 p<0.01 NS
3h +S9 1 p < 0.05 p< 0.001 p < 0.05 p< 0.001
24h-S9 1 NS p< 0.001 p< 0.001 p< 0.001
P value indicates level of statistical significance; NS, not significant.
differences between the experimental and baseline cigarettes (Table 3).
3.4. MNvit
In the MNvit, all particulate matters in all treatment conditions induced concentration related increases in cytotoxicity to approximately 60%. In addition, statistically significant linear trends were observed in induced MNBN frequency.
Under all treatment conditions, the experimental cigarette particulate matter generated to ISO conditions was less genotoxic than the commercial and baseline controls (Fig. 6A and Table 4). The only exception to this was observed in the 3 h +S9 treatment, where the experimental cigarette was more mutagenic than the commercial control (Table 4). However, under the HCI regime, in the 3 h +S9 and 24 h -S9 treatment conditions, the experimental cigarette was less active than the commercial and baseline controls (Fig. 6B and Table 4). In the 3 h -S9 treatment, the experimental
Table 5
IC50 (ig/mL) values obtained in the NRU assay from the experimental cigarette (EC), commercial (CC) and baseline (BC) controls at the ISO and HCI regimes.
ISO HCI
EC 53.86 60.04
CC 54.24 68.91
BC 50.43 60.24
cigarette was equivalent to the commercial and baseline controls 360
(Table 4). 361
3.5. NRU 362
In the Neutral Red Uptake assay, although there were some 363
small differences in the calculated IC50 values, there were no signif- 364
icant differences between the experimental cigarette and the base- 365
line and commercial controls at either smoking regime (Table 5). 366
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367 4. Discussion
368 The potential of BTT, TSS and filter additives to reduce the
369 mutagenicity and genotoxicity of the particulate matter from an
370 experimental cigarette was investigated. These blend technologies
371 have previously been described and tested in vitro individually
372 however, they have not been tested in combination. The filter addi-
373 tives will also reduce the level of volatile compounds in the main-
374 stream smoke of the experimental cigarette, which is further
375 enhanced by the TSS diluent technology (McAdam et al., 2011).
376 The cigarette smoke particulate matter tested was used as it has
377 a history of use, is easily collected, multiple doses can be tested and
378 produces consistent results (Wan et al., 2009). Other exposure
379 methods are available that are more physiologically relevant and
380 also take into consideration the gas (vapour) phase of the main-
381 stream smoke aerosol, rather than the particulate in isolation.
382 However, these whole smoke exposure systems are being devel-
383 oped and optimised for the toxicological assessment of cigarette
384 smoke aerosols and are currently not standardised (Aufderheide
385 et al., 2003; Thorne and Adamson, 2013; Wan et al., 2009).
386 The results demonstrate that in the Ames test with tester
387 strains TA98 and TA100 +S9 and the in vitro micronucleus test,
388 the experimental cigarette containing the toxicant reducing tech-
389 nologies was less mutagenic than the commercial and baseline
390 control at both smoking regimes, in the majority of comparisons
391 made. The differences were generally small, ranging between 8%
392 and 30% in the Ames test and 6-33% in the MNvit relative to the
393 commercial or baseline control responses. There were some
394 instances, particularly in the Ames test with tester strain TA1537
395 and the MLA where, although reduced mutagenicity and genotox-
396 icity of the experimental cigarette was sometimes observed, inter-
397 experimental variation was large or the differences were not of sta-
398 tistical significance (Tables 2-4). The variability observed in
399 TA1537 (Table 2) could be attributed to tester strain characteristics
400 between the two strains that detect frame-shift mutagens, TA98
401 and TA1537. Strain TA98 contains a plasmid (pKM101) that codes
402 for error prone DNA repair mechanisms (Maron and Ames, 1983;
403 McMahon et al., 1979), whereas TA1537 has normal DNA repair
404 mechanisms. This potentially helps explain enhanced differentia-
405 tion between experimental cigarettes containing toxicant reducing
406 technologies in TA98, compared to TA1537.
407 Furthermore, the variability could be due to the robust statisti-
408 cal methods applied to this study (Scott et al., 2013), including the
409 increase in replicates used per concentration, which was designed
410 to enhance the discriminatory ability of the assays. However,
411 reductions in the MLA and MNvit were not expected, based on
412 the inclusion levels of BTT and TSS in the experimental cigarette
413 described here. Compared to the previous individually tested blend
414 technologies (Combes et al., 2012, 2013; McAdam et al., 2011; Liu
415 et al., 2011), the inclusion level of BTT and TSS appear to be at the
416 threshold to observe biological effects. This is consistent with pre-
417 viously published data, where Combes et al., 2012 concluded that
418 experimental cigarettes containing 40% BTT were not different to
419 Q3 their respective controls. In Combes et al. (2011), experimental cig-
420 arettes containing 20% TSS had small reductions in the NRU and
421 MLA in the absence of S9, but did not affect the Ames or MNvit.
422 However, in this study, the combination of BTT and TSS are
423 included at levels where in previous publications no differences
424 were observed (Combes et al., 2012, 2013; McAdam et al., 2011;
425 Liu et al., 2011), appear to have effectively reduced mutagenicity
426 in some Ames strains, and in the MLA and MNvit, without affecting
427 cytotoxicity as assessed by the NRU.
428 These results are consistent with the BTT process removing
429 more than half of the protein nitrogen, and more than 40% of the
430 total polyphenols (Liu et al., 2011). The considerable reduction in
protein nitrogen resulted in the generation of lower levels of aro- 431
matic and heterocyclic amine protein products generated on smok- 432
ing, considered to be the main contributor to mutagenicity (Clapp 433
et al., 1999; Matsumoto and Yoshida, 1981; Matsushima et al., 434
1999; Mizusaki et al., 1977). The addition of the TSS to effectively 435
dilute the MSS, has further enhanced the reductions achieved by 436
BTT (McAdam et al., 2011). Further testing on the 'whole smoke' 437
aerosol could be warranted when exposure techniques become 438
standardised and widely used. This would allow the in vitro toxico- 439
logical evaluation of the smoke aerosol in its native form, including 440
the gas (vapour) phase, rather than the particulate matter in isola- 441
tion (Ritter et al., 2004; Thorne et al., 2013). This is of critical 442
importance, as filter additives do not affect particulate matter col- 443
lected on a CFP (Baker, 1999) and therefore the vapour phase 444
effects were not assessed in this study. 445
In conclusion, these data indicate that combining BTT and TSS 446
have the potential to reduce the mutagenicity and genotoxicity 447
of the particulate matter derived from the experimental cigarette, 448
without affecting its cytotoxic potential. Furthermore, the incorpo- 449
ration of these technologies do not add to the genotoxic potential, 450
nor are new genotoxic hazards observed, as assessed by the assays 451
used herein. While in vitro tests alone cannot predict risk, they con- 452
tribute to a weight of evidence paradigm for the risk assessment of 453
reduced toxicant prototype cigarettes. Together with smoke com- 454
position, biomarkers of exposure and biological effect, urine muta- 455
genicity and smoking behaviour data, in vitro genotoxicity and 456
cytotoxicity studies can help to evaluate the biological significance 457
of exposure to tobacco smoke toxicants. 458
Conflicts of interest statement 459
This work was funded by British American Tobacco (BAT) and 460
all authors are employees of BAT. 461
Uncited references 462
Thorne et al. (2014). Q4 463
Acknowledgments 464
The authors thank Research Support Services, BAT for producing 465
the particulate matter and Covance Laboratories, UK for conduct- 466
ing the in vitro testing and providing the statistical analysis. 467
References 468
Aufderheide, M., Knebel, J.W., Ritter, D., 2003. An improved in vitro model for 469
testing the pulmonary toxicity of complex mixtures such as cigarette smoke. 470
Exp. Toxicol. Pathol. 55, 51-57. 471
Baker, R.R., 1999. Smoke chemistry. In: Davis, D.L., Nielson, M.T. (Eds.), Tobacco 472
Production, Chemistry and Technology. Blackwell Science, Malden, 473
Massachusetts, pp. 398-439. 474
Branton, P., Bradley, R.H., 2011. Effects of active carbon pore size distributions on 475
adsorption of toxic organic compounds. Adsorption 17, 293-301. 476
Branton, P., McAdam, K.G., Duke, M.G., Liu, C., Curle, M., Mola, M., Proctor, C.J., 477
Bradley, R.H., 2011a. Use of classical adsorption theory to understand the 478
dynamic filtration of volatile toxicants in cigarette smoke by active carbons. 479
Adsorption Sci. Technol. 29 (2), 117-138. 480
Branton, P.J., McAdam, K.G., Winter, D., Liu, C., Duke, M.G., Proctor, C.J., 2011b. 481
Reduction of aldehydes and hydrogen cyanide in mainstream cigarette smoke 482
using an amine functionalised ion exchange resin. Chem. Cent. J. 5,15, <http:// 483
journal.chemistrycentral.com/content/pdf/1752-153X-5-15.pdf>. 484
Burns, D., Dybing, E., Gray, N., Hecht, S., Anderson, C., Sanner, T., O'Connor, R., 485
Djordjevic, M., Dresler, C., Hainaut, P., Jarvis, M., Opperhuizen, A., Straif, K., 2008. 486
Mandated lowering of toxicants in cigarette smoke: a description of the World 487
Health Organisation TobReg proposal. Tob. Control 17, 132-141. 488
Canada, 2000. Tobacco reporting regulations. T-11.5/SOR/2000-272. 489
Clapp, W.L., Fagg, B.S., Smith, C.J., 1999. Reduction in Ames Salmonella mutagenicity 490
of mainstream cigarette smoke condensate by tobacco protein removal. Mutat. 491
Res. 446, 167-174. 492
YRTPH 3212 ARTICLE IN PRESS No. of Pages 8, Model 5G
9 January 2015
I. Crooks et al. /Regulatory Toxicology and Pharmacology xxx (2015) xxx-xxx
COM, 2011. Guidance on a strategy for genotoxicity testing of chemical substances. <http://www.iacom.org.uk/guidstate/documents/COMGuidanceFINAL2.pdf> (accessed July 2012).
Combes, R., Scott, K., Dillon, D., Meredith, C., McAdam, K., Proctor, C., 2012. The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter. Toxicol. In Vitro 26,1022-1029.
Combes, R., Scott, K., Crooks, I., Dillon, D., Meredith, C., McAdam, K., Proctor, C., 2013. The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields. Toxicol. In Vitro 27,1533-1541.
CORESTA, 2004. In vitro toxicology task force: the rationale and strategy for conducting in vitro toxicology testing of tobacco smoke. <http://www.coresta. org/Reports/IVT_TF_Rationale-IVT-Testing-Tob.-Smoke_Report_Jun04.pdf>.
Crooks, I., Dillon, D.M., Scott, J.K., Ballantyne, M., Meredith, C., 2013. The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays. Regul. Toxicol. Pharm. 65, 196-200.
Dittrich, D.J., Fieblekorn, R., Bevan, M.J., Rushforth, D., Murphy, J.J., Ashley, M., McAdam, K.G., Liu, C., Proctor, C., 2014. Approaches for the design of reduced toxicant emission cigarettes. SpringerPlus 3, 374.
Fowles, J., Dybing, E., 2003. Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob. Control 12, 424-430.
Hatsukami, D.K., Biener, L., Leischow, S.J., Zeller, M.R., 2012. Tobacco and nicotine product testing. Nicotine Tob. Res. 14 (1), 7-17.
ICH, 2011. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use. <http://www.ich.org/products/ guidelines/safety/safety-single/article/guidance-on-genotoxicity-testing-anddata-interpretation-for-pharmaceuticals-intended-for-human-use.html> (accessed July 2012).
ISO, 1999. Tobacco and tobacco products. Atmosphere for conditioning and testing. ISO 3402:1999. Available from: <http://www.iso.org/iso/iso_catalogue/ catalogue_tc/catalogue_detail.htm?csnumber=28324>.
ISO, 2000. Tobacco and tobacco products - routine analytical cigarette-smoking machine - definitions and standard conditions. ISO 3308:2000, 23pp. Available from: <http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail. htm?csnumber=28325>.
ISO, 2000a. Cigarettes - determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine. ISO 4387:2000. Available from: <http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail. htm?csnumber=28323>.
Life Sciences Research Office (LSRO), 2007. In: St. Hillaire, C.L. (Ed.), Scientific methods to evaluate potential risk -reduced tobacco products, LSRO Press, Bethesda.
Liu, C., DeGrandpre, Y., McAdam, K., Porter, A., Griffiths, A., Proctor, C., 2011. The use of a novel tobacco treatment process to reduce toxicants yields in cigarette smoke. Food Chem. Toxicol. 49 (9), 1904-1917.
Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215.
Matsumoto, T., Yoshida, D., 1981. Determination of mutagens, amino-alpha-carbolines in grilled foods and cigarette smoke condensate. Cancer Lett. 12, 105-110.
Matsushima, T., Hayashi, M., Matsuoka, A., Ishidate Jr., M., Miura, K.F., Shimizu, H., Suzuki, Y., Morimoto, K., Ogura, H., Mure, K., Koshi, K., Sofuni, T., 1999. Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU). Mutagenesis 14, 569-580.
McAdam, K.G., Gregg, E.O., Liu, C., Dittrich, D.J., Duke, M.G., Proctor, C.J., 2011. The use of a novel tobacco-substitute sheet and smoke dilution to reduce toxicant yields in cigarette smoke. Food Chem. Toxicol. 49, 1684-1696.
McMahon, R.E., Cline, J.C., Thompson, C.Z., 1979. Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res. 39, 682-693.
Mizusaki, S., Okamotoa, H., Akiyamaa, A., Fukuhara, Y., 1977. Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate. Mutat. Res. 48, 319-325.
NIH, 2001. Guidance document on using in vitro data to estimate starting doses for acute toxicity. Publication 01-4500. National Institute of Environmental Health Sciences, National Institute of Health, US Department of Health and Human Services, Public Health Service, Washington, D.C.
OECD, 1997a. Test No. 471: bacterial reverse mutation test, OECD guidelines for the testing of chemicals, Section 4: health effects, OECD Publishing. Doi: http://dx. doi.org/10.1787/9789264071247-en.
OECD, 1997b. Test No, 476. In vitro mammalian cell gene mutation test, OECD Guidelines for the testing of chemicals, Section 4: health effects, OECD Publishing. Doi: http://dx.doi.org/10.1787/9789264071322-en.
OECD, 2010. Test No. 487. In vitro mammalian cell micronucleus test, OECD guidelines for the testing of chemicals, Section 4: health effects, OECD Publishing. Doi: http://dx.doi.org/10.1787/9789264091016-en.
Perfetti, T.A., Rodgman, A., 2013. The Chemical Components of Tobacco and Tobacco Smoke, second ed. CRC Press.
Richardson, C., Williams, D.A., Allen, J.A., Amphlett, G., Chanter, D.O., Phillips, B., 1989. Analysis of data from in vitro cytogenetic assays. In: Kirkland, D.J. (Ed.), Statistical Evaluation of Mutagenicity Test Data, UKEMS Guidelines Subcommittee Report, Part (III). Cambridge University Press, pp. 141-154.
Ritter, D., Knebel, J.W., Aufderheide, M., 2004. Comparative assessment of toxicities of mainstream smoke from commercial cigarettes. Inhalation Toxicol. 16, 691700.
Robinson, W.D., Green, M.H.L., Cole, J., Garner, R.C., Healy, M.J.R., Gatehouse, D., 1989. Statistical evaluation of bacterial/mammalian fluctuation tests. In: Kirkland, D.J. (Ed.), Statistical Evaluation of Mutagenicity Test Data. Cambridge University Press, pp. 102-140.
Scott, K., Saul, J., Crooks, I., Camacho, O.M., Dillon, D., Meredith, C., 2013. The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter. Toxicol. In Vitro 27 (4), 1312-1319.
St. Charles, F.K., Kabbani, A.A., Borgerding, M.F., 2010. Estimating tar and nicotine exposure: human smoking versus machine generated smoke yields. Regul. Toxicol. Pharm. 56, 100-110.
Stratton, K., Shetty, P., Wallace, R., Bondurant, S. (Eds.), 2001. Clearing the Smoke Assessing the Science Base for Tobacco Harm Reduction. National Academy Press, Washington, D.C..
Thorne, D., Kilford, J., Payne, R., Adamson, J., Scott, K., Dalrymple, A., Meredith, C., Dillon, D., 2013. Characterisation of a Vitrocell VC-10 in vitro smoke exposure system using dose tools and biological analysis. Chem. Central 7,146-156.
Thorne, D., Adamson, J., 2013. A review of in vitro cigarette smoke exposure systems. Exp. Toxicol. Pathol. 65,1183-1193.
Thorne, D., Kilford, J., Payne, R., Haswell, L., Dalrymple, A., Meredith, C., Dillon, D., 2014. Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system. BMC Res Notes 7, 367-375.
World Health Organisation (WHO), 2007. The scientific basis of tobacco product regulation: Report of a WHO Study Group. WHO Technical Report Series 945, Geneva.
Wan, J., Johnson, M., Schilz, J., Djordjevic, M.V., Rice, J.R., Shields, P.G., 2009. Evaluation of in vitro assays for assessing the toxicity of cigarette smoke and smokeless tobacco. Cancer Epidemiol. Biomarkers Prev. 18, 2363-3304.
