The in vitro cytotoxicity and genotoxicity of cigarette

Toxicology in Vitro 27 (2013) 1533–1541

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The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields R. Combes ⇑,1, K. Scott, I. Crooks, D. Dillon, C. Meredith, K. McAdam, C. Proctor British American Tobacco, Group Research and Development, Regents Park Road, Southampton SO15 8TL, UK

a r t i c l e

i n f o

Article history: Received 26 October 2012 Accepted 12 March 2013 Available online 27 March 2013 Keywords: Genotoxicity Cytotoxicity In vitro Tobacco smoke Particulate matter Tobacco sheet substitute Blend-treated tobacco Toxicant

a b s t r a c t Tobacco smoke contains more than 5600 constituents, of which approximately 150 are toxicants. This paper describes the activities in the Neutral Red uptake (NRU) assay, the Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT) of Particulate Matter (PM) obtained from experimental cigarettes (ECs), designed to produce reduced levels of toxicants. The designs included tobacco substitute sheet (TSS) containing glycerol, which dilutes toxicants in smoke, or the incorporation of blend-treated (BT) tobacco to reduce the levels of nitrogenous toxicant precursors and some polyphenols. All samples were cytotoxic in the NRU, however TSS reduced PM cytotoxicity in this assay. All PMs were mutagenic in the SAL, MLA and IVMNT. Reductions in bacterial mutagenicity were observed in the SAL, for cigarettes with BT tobacco, compared with their respective controls. The quantitative changes in bacterial mutagenicity could be explained by analytical chemistry data on smoke generated from the ECs used in the study. These observations, and the absence of consistent qualitative differences in the activities of the experimental, control and reference cigarettes, suggest that reduced toxicity cigarettes, as measured by the tests described in this paper, may be developed without introducing any additional cytotoxic or genotoxic hazards, but the impact of this on human health risks remains unknown. Ó 2013 Elsevier Ltd. Open access under CC BY-NC-ND license.

1. Introduction Tobacco smoke is a complex, dynamic, mixture of more than 5600 identified constituents (Perfetti and Rodgman, 2011), of which approximately 150 have been documented as toxicants (Fowles and Dybing, 2003). In recent papers we have described four different individual technological approaches to the reduction of toxicants in cigarette smoke, two of which modified the tobacco blend (McAdam et al., 2011; Liu et al., 2011), and two of which modified the cigarette filter (Branton et al., 2011a, 2011b). This paper describes an assessment of the in vitro toxicities of experimental cigarettes (ECs) incorporating these technologies. The designs include the use of tobacco substitute sheet (TSS), which has the dual function afforded by containing glycerol to dilute toxicants in smoke, and by the provision of a low organic content material in order to reduce the amount of tobacco burnt in the cigarette, and hence smoke yields. Another cigarette includes blend-treated (BT) tobacco to reduce the levels of toxicant precursors in tobacco and hence of toxicants in the tobacco smoke generated from them. TSS is a new material, which, in addition to high levels of glycerol, also contains calcium carbonate (ca. 75%) to minimise the or⇑ Corresponding author. Tel.: +44 7968709236. 1

E-mail address: [email protected] (R. Combes). Independent Consultant, Norwich, UK

0887-2333 Ó 2013 Elsevier Ltd. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.tiv.2013.03.005

ganic content of the TSS and hence smoke yields. The glycerol (ca. 12%) distils into the smoke to dilute the tar, by contributing to the total amount of particulate smoke, measured as nicotine-free dry particulate matter (NFDPM) (also known as ‘tar’). As most cigarettes are designed to meet a specific International Standards Organisation (ISO) NFDPM yield value, incorporation of glycerol into the smoke stream effectively results in a reduced contribution of the tobacco combustion products to the overall NFDPM value, a process termed ‘dilution’. Confirmation of the dilution effect through the incorporation of TSS into cigarettes, has been achieved by showing reductions in a wide range of smoke constituents, after smoking TSS containing cigarettes (McAdam et al., 2011). Cigarettes with BT tobacco have also been developed. BT tobacco has reduced levels of soluble and insoluble proteins, and amino acids in tobacco, which act as toxicant precursors, as well as reduced levels of water soluble polyphenols, such as chlorogenic acid, rutin and scopoletin. The BT process is based on the fact that proteins and amino acids have been reported to be precursors for a number of potentially toxic constituents of tobacco smoke, including 2-aminonaphthalene and 4-aminobiphenyl (Torikaiu et al., 2005) and mutagenic heterocyclic amines (Mizusaki et al., 1977; Matsumoto and Yoshida, 1981; Clapp et al., 1999), the latter being implicated as a primary source of genotoxicity of cigarette smoke condensates (CSCs) (DeMarini, 2004). The BT process is carried out on cut, flue-cured tobacco, and involves the sequential extraction of the tobacco with water,

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followed by treatment of the tobacco solids and aqueous extract with an aqueous protease enzyme solution, and by further treatment of the resulting solution with adsorbents, followed by reapplication of the remaining soluble materials to the extracted tobacco. The treated tobacco retains the structure of the original tobacco, and is designed to be used with an adsorbent filter, to create a cigarette with a conventional appearance, usage, and smoking experience (Liu et al., 2011). Most cigarette filters contain cellulose acetate, however, adsorbant filters were developed containing CR20L ion-exchange resin. CR20L is an amine-functionalised resin bead material which can be incorporated into cigarette filters (Diainon, Mitsubishi Chemical Corporation, Tokyo). The characterisation and use of CR20L in cigarette filters were described in detail by Branton et al. (2011b). In comparison to filters containing conventional activated carbon, CR20L offers superior reductions for hydrogen cyanide (HCN), formaldehyde and acetaldehyde. Activated carbon is more efficient than CR20L in removing other volatile constituents from the smoke stream. One of the filters in the study contained a polymer-derived activated carbon, with a modified pore structure, to increase adsorption of a range of volatile smoke constituents (Branton et al., 2009). Two of the filters contained both CR20L and polymer-derived activated carbon. Full details of the design and performance of these experimental cigarettes are described elsewhere (McAdam et al., 2012). The results are described of subjecting cigarette smoke particulate matter (PM) samples, generated under ISO machine-smoking conditions (ISO 3308:1977) from reference, control and reduced toxicant cigarettes, to four in vitro toxicity assays. These assays – Neutral Red uptake (NRU), Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and in vitro micronucleus test (IVMNT) – have been accepted by regulatory agencies and the Organisation of Economic Cooperation and Development (OECD) (Aardema et al., 2006; Anon, 2006; Corvi et al., 2008; Honma et al., 1999a; Garriott et al., 2002; Kirsch-Volders et al., 2003; Matsushima et al., 1999; Phelps et al., 2002). The methods were also recommended by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) (Anon, 2010).

tained conventional cigarette materials. TSS6, TSS1 and BT1 were the test cigarettes, to evaluate the new materials. CC1 was the control for TSS1 and BT1. CC6 was the control for TSS6. 2.2. PMs Tobacco preparation, cigarette manufacture and PM production were undertaken at BAT Group Research and Development, Southampton, United Kingdom (UK). To generate cigarette smoke PM, cigarettes were conditioned according to the ISO method, for a minimum of 48 h at 22 ± 1 °C and 60 ± 3% relative humidity (ISO, 1999) and smoked on a RM20CSR smoking machine (BorgwaltKC, Hamburg, Germany) using ISO standard puffing parameters (35 ml puff volume taken over 2 s once per minute) (ISO, 2000). An appropriate number of cigarettes were smoked to obtain approximately 220 mg PM on a 44 mm Cambridge filter pad (Whatman, Maidstone, UK). The pads were weighed before and after smoking to determine the weight of particulate matter deposited, and the PM was then eluted in dimethyl sulphoxide (DMSO) to a concentration of 24 mg/ml and stored protected from light in single-use aliquots at 80 °C. Samples were shipped at 80 °C to an independent laboratory for in vitro tests, where they were stored at 80 °C, and used within 1 month. Crooks et al., 2013 have demonstrated stability of PM when stored at 80 °C for up to 2 years. Test material was tested at different concentrations calculated as lg/ml of PM. At the end of scoring, the concentration data were corrected to give the NFDPM concentration. This was achieved by gas chromatographic analysis of PM from each smoke run to determine nicotine and water contents. The resulting information was used to compute an NFDPM correction factor to convert the concentration of PM tested to NFDPM in lg/ml for each test material. The correction factor was applied to the experimental data to convert the test concentrations from lg PM/ml to lg NFDPM/ml, by using the formula:

NFDPM concentration ¼ ðPM dose NFDPM factorÞ NFDPM concentrations were used to compare the toxicities of the different PMs.

2. Materials and methods 2.1. Cigarettes

2.3. In vitro toxicology testing

Cigarette specifications are detailed in Table 1. 3R4F and M4A were reference cigarettes, providing historical control data. CC1 and CC6 were the control cigarettes, with ISO tar yields of 1 mg and 6 mg per cigarette respectively (ISO, 2000). CC1 and CC6 con-

All in vitro tests were performed in an independent GLP laboratory. Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9) mix provided metabolic activation in the genotoxicity assays.

Table 1 Composition of different cigarettes smoked to yield PM test products.

a b c d e f g h

PM/cigarette code

Blend

Filter

CC6 TSS6 CC1 TSS1 BT1 M4Ag 3R4Fh

United States (US)-style blend US-style blend (80%)a/TSSb (20%) US-style blend US-style blend (80%)a/TSSb (20%) US-style blenda (25%)/BT tobaccof (75%) Flue-cured blend US-style blend

Cellulose acetatec Two stage filter of cellulose acetatec/modified charcoald (80 mg) Cellulose acetatec Three stage filter of cellulose acetatec/modified charcoald (60 mg)/CR20L (20 mg)e Three stage filter of cellulose acetatec/cavity with modified charcoald (60 mg)/CR20L (20 mg)e Cellulose acetatec Cellulose acetatec

Leaf tobacco. Tobacco substitute sheet. Cellulose acetate as found in conventional cigarettes. Polymer-derived activated carbon to adsorb a broad range of volatile components. Ion exchange resin filter additive to selectively adsorb volatile acidic and carbonyl smoke constituents. Tobacco treated to remove some of the protein and phenols by the method described. M4A is the historical reference cigarette used by British American Tobacco (BAT). 3R4F reference cigarettes used to provide historical control data (originally obtained from the University of Kentucky).

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The NRU cytotoxicity assay was performed as described by Combes et al. (2012). Briefly, Balb/c 3T3 clone A31 mouse fibroblasts cells were maintained in tissue culture in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) foetal calf serum (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 48 h culture, measured by absorbance at 540 nm. Serial dilutions of PM in DMSO were made to determine concentration-dependent inhibition of cell growth. Six wells were used for each concentration of test article, vehicle or positive control; therefore testing was done with six replicates. Two separate assays (experiments) were performed for each test substance. For all experiments, the concentrations (lg/ml) of the test article inducing a 50% inhibition in Neutral Red Uptake (IC50) and the coefficient of variance between values for six replicates per concentration were estimated using validated software (Phototox version 2).This protocol conforms to the guidelines issued by the National Institutes of Health (NIH) (NIH, 2001). A higher IC50 value represents a lower cytotoxicity. IC50 values were considered to be different when their variances did not overlap. The SAL mutagenicity test was performed as described by Combes et al. (2012), using five Salmonella typhimurium strains: TA98, TA100, TA102, TA1535 and TA1537, in the presence and absence of S9. This experimental design used for the Ames test follows CORESTA guidelines for testing tobacco and tobacco products. One pre-incubation test and two plate incorporation tests were performed. A further two plate incorporation tests using strains TA98, TA100 and TA1537 in the presence of S9 were carried out over the linear portion of the dose–response curve. The choice of tester strains was consistent with previous observations on the mutagenicity of tobacco smoke constituents and covered all of the commonly-used strains, including those sensitive to heterocyclic amines, which are believed to contribute to the mutagenic effect of PMs and whose levels in smoke were expected to be reduced by the changes made in tobacco design and composition. All data were analysed using Dunnett’s test for significant differences between solvent control plates and those treated with PM. This method complied with OECD guideline 471 (OECD, 1997a) and International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines (ICH, 1995, 1997). Results are presented as mean revertants/lg NFDPM ± standard error of the mean (SEM) taken from the results of four independent experiments for the plate incorporation assays; and mean revertants/lg NFDPM ± standard deviation (SD) of three replicates for each test concentration (five replicates for vehicle control) for the single pre-incubation experiment. Mutagenic potency was determined from the linear portion(s) slopes of concentration–responses using linear regression analysis. PMs were compared in terms of the slopes of their dose responses. A generalised linear model was used to estimate slopes, modelling the number of revertants using a Poisson distribution with a log link function. Statistically significant differences between slopes were identified by analysis of covariance (ANCOVA). The IVMNT was performed as described by Combes et al. (2012). Briefly, four replicate V79 cell cultures in DMEM supplemented with 10% FCS and 0.52% penicillin/streptomycin, were pulsed with test or control samples for 3 h followed by a 21 h recovery, with and without S9, or for 24 h without S9. Cytochalasin B, formulated in DMSO (0.1 ml), was added to all treatment flasks at time of treatment to give a final concentration of 3 lg/ml. At least 6 dose levels for each PM were scored for cytotoxicity and for micronucleated binucleate cells (MnBn), on duplicate slides. This method complied with OECD guideline 487 (OECD, 2010). PMs were compared in terms of the slopes of their dose responses. A generalised linear model was used to estimate slopes, modelling

the proportion of MnBn using a binomial distribution with a probit link function. Statistically significant differences between slopes were identified by ANCOVA. The MLA was performed as described by Combes et al. (2012), using L5178Y thymidine kinase (tk) +/ cells cultured in supplemented Roswell Park Memorial Institute medium (RPMI). In the main experiment 10 treatment concentrations were tested with six vehicle control replicates and four treatment replicates per concentration tested. Overall, there were two independent experiments using a 3 h exposure with S9; and two independent experiments using 3 and 24 h exposures without S9. After a 2 day expression period, cells were grown for 8 days, and then trifluoro-thymidine (TFT) resistant colonies were counted. The method complied with OECD Guideline 476 (OECD, 1997b). Data are plotted as the means of duplicate cultures within each experiment. PMs were compared in terms of the slopes of their dose responses. A generalised linear model was used to estimate slopes, modelling log-transformed mutation frequency (MF) values. Statistically significant differences between slopes were identified by ANCOVA. A test article was considered to be mutagenic in this assay if: (a) the MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF; and (b) the linear trend test was positive. 3. Results 3.1. Neutral Red Uptake Assay All samples were tested in the absence of S9 only, and were cytotoxic, with relatively low intra-experimental variation, but some inter-experimental variation in the results (Table 2). Consistent with historical results, the IC50 values of the two reference PMs were different, with the PM from flue-cured blend (M4A) being more cytotoxic than the PM from the US-style blend (3R4F). CC1 was consistently the most cytotoxic sample, apart from BT1 in one experiment. The TSS1 and TSS6 PMs had higher IC50 values than their respective controls (CC1 and CC6) in both repeat experiments, indicating lower cytotoxicity. BT1 also differed from its control (CC1), but the effect was not reproducible. The IC50 values of PMs from cigarettes with 6 mg ISO tar yields (TSS6 and CC6), were higher than those of PMs from cigarettes with 1 mg tar yields (TSS1 and CC1), in two repeat experiments. 3.2. Salmonella typhimurium reverse mutation assay All samples were mutagenic in the presence of S9, with activity detected in tester strains TA98, TA100 and TA1537 (Figs. 1–5), but were non-mutagenic, in tester strains TA102 and TA1535 (Table 5). The mean numbers of revertants were calculated from the four values of the mean revertants/plate at each concentration, each mean being derived from three replicate plates within each experiment.

Table 2 Responses of PMs in the Neutral Red Uptake Assay (two experiments). PM

3R4F M4A CC6 TSS6 CC1 TSS1 BT1 a

Experiment 1

Experiment 2

IC50 (lg NFDPM/ml)

Variancea

IC50 (lg NFDPM/ml)

Variancea

46.54 36.91 40.07 50.19 28.18 32.23 16.09

2.11 0.82 1.23 1.28 2.06 0.80 2.21

51.64 48.11 43.65 47.28 33.82 38.89 38.88

1.46 0.41 0.43 1.34 0.65 2.08 2.98

Calculated from IC50 values in lg NFDPM/ml.

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Samples were weakly mutagenic in the absence of S9 in tester strains TA98 and TA100, but were non-mutagenic in the other tester strains in the absence of S9 (Table 5). In the responsive strains, the degree of mutagenicity (revertants/lg NFDPM) varied according to PM and tester strain. Consistent with historical data, M4A gave lower mutagenicities than 3R4F in all experiments in TA98 and TA1537, although this was only statistically significant in TA1537 (Table 3). Among the test cigarettes, there was no consistent difference between CC1 and TSS1, or between CC6 and TSS6. In TA98, TA100 and TA1537 the mutagenicities of BT1 were lower than those of CC1 and TSS1 in all of the plate incorporation experiments (Figs. 1–3). The reductions in mutagenicity were statistically significant in TA98 and TA1537 (Table 3). In strains TA98 and TA1537, the pre-incubation mutagenicities of BT1 were lower than CC1 and TSS1 (Figs. 4 and 5). This was also observed with TA100, but only at the non-linear part of the dose response (Fig. 6).

3.3. Mouse lymphoma assay All PMs induced dose-related increases in cytotoxicity and MF, in all treatment conditions (3 h ±S9 and 24 h S9; Table 4), although some dose responses did not exceed the Global Evaluation Factor (GEF). Consistent with historic data, M4A PM gave higher mutagenicity than 3R4F PM. This difference was statistically significant in four of the six experiments (Table 4). Concentration-related increases in MF for each of the pairs of control and treatment PM samples were obtained. Viabilities for all of the concentrations of all of the PMs tested generally exceeded 70% in both experiments, irrespective of treatment parameters. Table 4 presents induction of TFT-resistant colonies by reference, control and test PMs in the form of log MF/lg NFDPM. These figures are from the linear part of the concentration–responses. When the data for the two experiments in each treatment condition were combined, TSS1 and BT1 mutagenicities were lower than that of CC1 PM (Fig. 7), and the reductions were statistically significant (Table 4). TSS6 was also less mutagenic than CC6 (Fig. 8) when treatment was conducted for 3 h in the presence of S9. This was statistically significant in one experiment (Table 4). However, this effect was not seen when S9 was omitted, or when exposure was extended to 24 h in the absence of S9. In the latter case, the response of TSS6 was also lower, but not significantly so, possibly due to high inter-experimental variation.

Fig. 2. Bacterial mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dashes and dots/squares) in TA1537 with S9 (means ± SEM of 4 independent plate incorporation experiments; error bars not visible are contained within the data point; vehicle control mean (±SEM) count: 15.5 (0.83)).

Fig. 3. Bacterial mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in TA100 with S9 (mean ± SEM 4 independent plate incorporation experiments; error bars not visible are contained within the data point; vehicle control mean (±SEM) count: 132.5 (2.78)).

The dose responses included increases in the ratio of small to large colonies, indicating deletions at the tk-locus (Cobb et al., 1989). No large differences in the proportions of small colonies induced were detected between each of the PM samples when tested under the same conditions.

3.4. In vitro micronucleus test

Fig. 1. Bacterial mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in TA98 with S9 (means ± SEM of 4 independent plate incorporation experiments; error bars not visible are contained within the data point; vehicle control mean (±SEM) count: 29.9 (5.94)).

All PMs induced dose-related increases in cytotoxicity in the three treatment conditions, and dose-related increases in the frequency of micronucleated binucleate cells in two treatment conditions (3 h ±S9) (Fig. 9a). Increases in the frequencies of MnBn cells were also observed in the 20 h S9 treatment with all PMs, but

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Fig. 4. Mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in TA98 with S9 (mean ± SD of three replicate plates per dose in a pre-incubation assay (1 experiment); vehicle control mean (±SD) count: 32.6 (9)).

Fig. 6. Mutagenicity of CC1 (continuous/triangles); TSS1 (dashed/diamonds) and BT1 (dots and dashes/squares) in TA100 with S9 (mean ± SD of three replicate plates per dose in a pre-incubation assay (1 experiment); vehicle control mean (±SD) count: 116.8 (21.3)).

Table 4 Results from applying a generalised linear model to estimate slopes for induction of TFT-resistant colonies by reference, control and test PMs. PM source

Slope (log MF/lg NFDPM) 3 h +S9

3R4F M4A CC6 TSS6 CC1 TSS1 BT1 * + ^ ° a

Fig. 5. Mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in TA1537 with S9 (mean ± SD of three replicate plates per dose in a pre-incubation assay (1 experiment); vehicle control mean (±SD) count: 11 (1)).

Table 3 Mutagenicities of PMs in the Salmonella mutagenicity plate incorporation assay with S9. PM source

3R4F M4A CC6 TSS6 CC1 TSS1 BT1

Mean revertants/lg NFDPM (±SEM, n = 4 experiments) TA98 + S9

TA100 + S9

TA1537 + S9

1.8116 1.4300 2.2385 1.9746 2.7576 2.7677 1.4744

0.6103 0.4983 0.5464 0.7379 0.7890 0.7932 0.5612

0.3508 0.2169 0.3398 0.2347 0.3716 0.4186 0.2005

(0.0382) (0.0747) (0.1761) (0.2039) (0.5663) (0.3883) (0.1284)*

(0.0765) (0.1347) (0.0341) (0.0985) (0.1221) (0.1559) (0.1172)

(0.0529) (0.0031)+ (0.0578) (0.0281) (0.0641) (0.0432) (0.0349)*

3 h S9

24 h S9

Expt. 1

Expt. 2

Expt. 1

Expt. 2

Expt. 1

Expt. 2

0.0231* 0.0291 0.0336 0.0202^ Not linear 0.0194° 0.0379

0.0318 0.0332 0.0215 0.0254 0.0217 0.0249a 0.0243

0.0340* 0.0483 0.0257 0.0327 0.0318 0.0216+ 0.0155+

0.0311* 0.0439 0.0311a 0.0275a 0.0418a 0.0301+ 0.0290+,a

0.0455* 0.0654 0.0414 0.0367a 0.0424a 0.0469a 0.0329a

0.0584 0.0706 0.0583 0.0530 0.0522 0.0434 0.0455

Significantly different from M4A (p < 0.05 3 h ±S9, p < 0.01 24 h S9). Significantly different from CC1 (p < 0.05). Significantly different from CC6 (p < 0.01). Significantly different from BT1. GEF not exceeded.

Table 5 Results of Induction of micronuclei in cultured V79 cells by reference, control and test PMs. PM source

3R4F M4A CC6 TSS6 CC1 TSS1 BT1

log MnBn/lg NFDPM 3 h S9

3 h +S9

24 h S9

0.00880* 0.01234 0.00791+ 0.01184 0.00605# 0.00572# 0.00943

0.00676* 0.01180 0.00329 0.00370 0.00730# 0.00636# 0.01060

0.00617* 0.01811 Not linear 0.00523 0.01987# 0.00419a 0.00675

* Significantly different from CC1 (TA98 p < 0.01; TA1537 p < 0.05) and TSS1 (TA98 and TA1537 p < 0.01). + Significantly different (p < 0.05) from 3R4F.

* Significantly different from M4A (3 h S9, p < 0.01; 3 h +S9 and 24 h S9, p < 0.001). + Significantly different from TSS6 (p < 0.001); TSS1 (TA98 p < 0.01, TA1537 p < 0.05). # Significantly different from BT1 (3 h S9, p < 0.01; 3 h +S9, p < 0.05 for CC1 and p < 0.001 for TSS1; 24 h S9, p < 0.001). a Negative slope excluded from statistical comparisons.

they were not always dose-related. The most sensitive treatment was 3 h without S9, where up to 5-fold inductions of MnBn cells were observed (Fig. 9b).

Consistent with historical data, M4A PM induced more MnBn cells than PM from 3R4F (Table 5). The differences were small, but statistically significant in all three treatments.

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Fig. 7. Mutagenicity of CC1 (continuous/triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in the MLA; exposed for 3 h without S9 (means (±SEM) from 2 independent experiments; vehicle control (67.44 ± 1.89) and GEF combined = 193.44 mutants/106 surviving cells.

Fig. 9b. Responses of CC1 (continuous/inverted triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in the IVMNT; 3 h incubation without S9 (mean ± SD of four replicate slides per dose; 1 experiment; vehicle control: 0.78 ± 0.16; % viabilities at top concentrations tested were: 43% (CC1); 55% (TSS1); and 15% (BT1)).

4. Discussion

Fig. 8. Mutagenicity of CC6 (continuous/triangles) and TSS6 (dashes/squares) in the MLA; 3 h treatment with S9 (mean ± SEM of 2 independent experiments; vehicle control (78.75 ± 6.08) and GEF combined = 204.75 mutants/106 survivors).

Fig. 9a. Responses of CC1 (continuous/inverted triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in the IVMNT; 3 h incubation with S9 (mean ± SD of four replicate slides per dose; 1 experiment; vehicle control: 1.01 ± 0.46; % viabilities at top concentrations tested were: 49% (CC1); 26% (TSS1); and 34% (BT1)).

Some statistically significant differences between control and test PMs were also detected (Table 5). The slope of BT1 PM‘s dose response was greater than those of CC1 and TSS1 in the 3 h treatments, but less than CC1 in the 20 h treatment (Figs. 9a–9c). The slope of TSS6 PM’s dose response was greater than that of CC6 PM in the 3 h S9 treatment (Fig. 10). Given the variability observed within experiments, the shape of the dose responses, the size of the possible differences and the levels of toxicity observed at the higher doses, these statistical differences are not likely to be biologically significant.

The results are summarised qualitatively in Table 6. These data broadly agree with observations made by previous authors using PMs from a variety of tobaccos, ever since the original observations of Kier et al. (1974). In summary, all PMs were genotoxic in a variety of assays (Baker et al., 2004; Clive et al., 1997; Cobb et al., 1989; COM, 2009; Combes et al., 2012; DeMarini et al., 2008; DeMarini 2004; Guo et al., 2011; McAdam et al., 2011; Mitchell et al., 1981; Richter et al., 2010; Rickert et al., 2011; Rickert et al., 2007; Roemer et al., 2004, 2002; Sato et al., 1977). Cobb et al. (1989) detected the induction of deletions at the tk locus, which is compatible with our current observations and those made earlier (Combes et al., 2012) that small colonies were induced by all of the PM samples, with no discernible quantitative differences. However, Putnam et al. (2002) did not find a clear concentrationrelated increase in cytotoxicity induced by PM in the NRU assay, and Roemer et al. (2002) observed mutagenicity in Ames strains TA98 and TA100 without S9. Also, on this occasion, extended treatments were no more genotoxic than 3 h treatments in both the MLA and the IVMNT, which contrasts with our previous observations with PMs obtained from a variety of cigarette types (Combes et al., 2012) and published suggestions for including a 20 h treatment regimen in protocols for the assay (Anon, 2004; Honma et al., 1999b; Clements, 2000; Moore et al., 2007). TSS has previously been shown to reduce the cytotoxicity and genotoxicity of PM (McAdam et al., 2011), when included at more than 30%. These authors found that, in the SAL assay, cigarettes containing 60% TSS, with cellulose acetate filters gave significantly (p < 0.05) less mutagenicity than their respective controls when tested in TA98, TA100 and TA1537 with S9, while those containing 30% TSS showed no statistical differences. Interestingly, in the present experiments, the PMs from cigarettes containing 60% TSS with a dual carbon filter were less genotoxic than their controls in the IVMNT at all of the concentrations tested, whereas those with 30% TSS were not significantly different. In addition, no significant differences for any of the PMs that McAdam et al. (2011) used in their study were detected in the MLA in our experiments, when concentrations of the PMs were corrected for NFDPM content. Our findings, that the SAL and IVMNT genotoxic potential of TSS1 and TSS6 were not significantly different from their respective controls (CC1 and CC6), are compatible with the data obtained by McAdam et al. (2011), since the ECs in the present work only contained 20% TSS, as opposed to those used earlier with higher proportions of TSS. However, we also observed a reduction in NRU cytotoxicity at a lower level of TSS than in McAdam et al. (2011), and a small reduction in MLA genotoxicity without S9 mix.


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Fig. 9c. Responses of CC1 (continuous/inverted triangles); TSS1 (dashes/diamonds) and BT1 (dots and dashes/squares) in the IVMNT; 24 h incubation without S9 (mean ± SD of four replicate slides per dose; 1 experiment; vehicle control: 1.61 ± 0.6; % viabilities at top concentrations tested were: 47% (CC1); 36% (TSS1); and 28% (BT1)).

Fig. 10. Responses of CC6 (continuous/diamonds) and TSS6 (dashes/squares) in the IVMNT; 3 h incubation without S9 (mean ± SD of four replicate slides per dose; vehicle control 0.78 ± 0.16). % viabilities at top concentrations tested were: 40% (CC6); and 46% (TSS6)).

BT1 PM was less mutagenic in the SAL assay than PM from the control cigarette, CC1. It is likely that the reduction of mutagenicity was because BT1 contained blend-treated tobacco. BT tobacco did not affect NRU cytotoxicity or IVMNT genotoxicity but a small reduction in MLA genotoxicity was observed. The reduction of bacterial mutagenicity, achieved with BT tobacco, as shown by the BT1 results, has been reported elsewhere (Combes et al., 2012). These authors observed consistent and statistically significant, reduced mutagenic potency (revertants/ lg NFDPM) in TA98 with S9, of PMs obtained from cigarettes made with 80% BT tobacco, made at a different time, but using the same process as employed to produce BT1 cigarettes used in this paper, to reduce the level of toxicants in mainstream cigarette smoke (MS). Moreover, the cigarettes containing BT tobacco, that were shown by Combes et al. (2012) to exhibit reduced mutagenicity compared with their controls, were constructed with the same charcoal/resin filters. Interestingly, consistent and significant reductions in mutagenicity (seen with the ECs used in the earlier experiments) were detected only in TA98, with the maximum decrease being 30% of the control. In the present study, the maximum decrease was 87%, also in TA98, but with reductions in mutagenicity also being detected in TA100 and TA1537. It is noteworthy that the removal of protein from tobacco is reported to generate PMs that, when tested with S9, exhibit mutagenicity that is reduced by up to 80% in TA98 and by up to 50% in TA100 (Clapp et al.,1999). The considerable reduction in protein nitrogen content, resulting from the use of BT tobacco, would be expected to decrease bacterial mutagenicity (Mizusaki et al., 1977), since it is a precursor of

nitrosamines and heterocyclic amines in tobacco smoke. Tobacco treatment, should result in the generation (on smoking) of lower levels of aromatic amines and heterocyclic amines (protein pyrolysate products), which are considered to be the main cause of mutagenicity in the Salmonella assay (Van Duuren et al., 1960; DeMarini, 2004). Moreover, the spectrum of activity seen in the Salmonella tester strains is consistent with previous conclusions that the mutagenicity of tobacco combustion products is primarily due to the generation of heterocyclic amines (DeMarini, 2004; Felton et al., 1995; Sugimura et al., 2004; Mortelmans and Zeiger, 2000). In addition, the spectrum of changes in mutagenicity seen with the tester strains, being more pronounced in TA98 and TA1537 (both sensitive to frame-shift mutagens), than in TA100, is consistent with the removal from tobacco of heterocyclic amine precursors. A different approach to investigate the effect of changing cigarette composition on toxicity is to add various substances to tobacco and then to determine smoke chemistry and in vitro responses (Gaworski et al., 2011). When three heterocyclic amines, 2-acetylpyridine (AP), 2,3-diethylpyrazine (DEP) and 2,3,5,6-tetramethylpyrazine (TMP) were separately added to cigarette tobacco, at low, intermediate and high concentrations, and smoked, some minor changes in smoke chemistry were detected with DEP only, while there were no consistent effects on toxicity of either the resulting gas–vapour or particulate phases of smoke, as measured by in vitro cytotoxicity and bacterial mutagenicity (Coggins et al., 2011a). While this would seem to contradict our data showing reduced mutagenicity with BT cigarettes, it should be noted that none of these chemicals is considered to be mutagenic (EFSA, 2008; Müller and Rappert, 2010; John, 1982), and presumably the smoking of tobacco containing them did not result in the generation of new, mutagenic heterocyclic amines. BT tobacco is also characterised by having reduced levels of polyphenols, but these were not tested by addition to tobacco (Gaworski et al., 2011). However, it is noteworthy that, in another publication (Coggins et al., 2011b), cytotoxicity was reduced by the highest inclusion of triacetin into tobacco for both gas–vapour and particulate phases of smoke, and mutagenicity was reduced by the middle and high inclusion levels of triacetin into tobacco. A similar smoke-dilution effect was also observed with glycerol and the cytotoxicity data we obtained with TSS tobacco, which contains high levels of glycerol. TSS cigarettes also contain high levels of calcium carbonate, a substance that was unfortunately not tested by Coggins and colleagues. In conclusion, the data indicate that no consistent qualitative differences were detected in the four in vitro tests, three of them

1540


Table 6 Qualitative summary of in vitro data. PM

In vitro test SAL

IVMNT +S9

S9 TA102/TA1535/TA1537

TA98/TA100

TA102/TA1535

TA98/TA100/TA1537

MLA

NRU

S9

+S9

S9

+S9

S9

3R4F

+

+

+

+

+

+

+

M4A

+

+

+

+

+

+

+

CC6

+

+

+

+

+

+

+

TSS6

+

+

+

+

+

+

+

CC1

+

+

+

+

+

+

+

TSS1

+

+

+

+

+

+

+

BT1

+

+

+

+

+

+

+

: Inactive; +: active.

genotoxicity assays, between PM samples obtained by individually machine-smoking control and test cigarettes containing different tobacco blends and filters, some of which contained tobacco substituted sheet or BT tobacco to reduce levels of smoke toxicants and their precursors. In addition, BT tobacco reduced mutagenicity in the SAL, with strains TA98, TA100 and TA1537 in the presence of S9. These observations are consistent with the hypothesis that reducing tobacco protein content would reduce bacterial mutagenicity, without introducing any new genotoxic or cytotoxic hazard. Further toxicity testing is warranted to investigate the effects of the tobacco treatment in more detail, and to add to the data already obtained. Overall, the data show that the pre-incubation assay was more sensitive than plate-incorporation to the effects of changing cigarette design. This result could not have been predicted at the start of the experiments and the main object of the study was to see how changing tobacco design and composition might affect overall genotoxicity profiles. Hence, it was felt important to use a variety of Ames tester strains and protocols and other genotoxicity assays. However, any further comparisons of the cigarettes used might well focus on use of the pre-incubation protocol. It is also noteworthy that preliminary short-term clinical studies have demonstrated greater reductions in a range of biomarkers of exposure to other potential toxicants, including acrolein and crotonaldehyde after smoking BT1 and TSS1 cigarettes, when each is compared with the effects seen following smoking their respective control CC1 cigarettes (Shepperd et al., 2009; Shepperd et al., 2011). Conflict of interest The authors are employees of BAT, except for Dr. R. Combes who acts as a consultant to BAT, and who was paid for his contribution to this manuscript. BAT funded this research as part of its harm reduction programme. The Authors declare that no financial or personal conflicts of interest exist with regard to the submission of the manuscript entitled ‘‘The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields’’. Acknowledgement The in vitro assays were performed by Covance Laboratories. References Aardema, M.J., Snyder, R.D., Spicer, C., Divi, K., Morita, T., Mauthe, R.J., et al., 2006. SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells. Mutat. Res. 607, 61–87.

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