An Evaluation of Cigarette Smoke Aerosols in Vitro Using a Modified Ames Methodology and the Balb/c 3t3 Neutral Red Uptake Cytotoxicity Assay Michael Hollings, Amber Woodhams, Adam Seymour, Mark Ballantyne and Julie Clements; Covance Laboratories Ltd., Harrogate, UK

References 1. Kilford, J., Thorne, D., Payne, R., Dalrymple, A., Clements, J., Meredith, C., Dillon, D. A method for assessment of

the genotoxicity of mainstream cigarette-smoke by use of the bacterial reverse-mutation assay and an aerosol-based exposure system. Mutation Research 769 (2014) 20-28

2.Thorne, D., Kilford, J., Hollings, M., Dalrymple, A., Ballantyne, M., Meredith, C., Dillon, D. The mutagenic assessment of mainstream cigarette smoke using the Ames assay: A multi-strain approach. Mutation Research 782 (2015) 9–17

3.Thorne, D., Kilford, J. Payne, R., Haswell, L., Dalrymple, A., Meredith, C., Dillon, D., Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system. BMC Research Notes 2014, 7:367

4.Adamson, J., Thorne, D., Dalrymple, A., Dillon, D., Meredith, C. Cigarette smoke deposition in a Vitrocell® exposure module: real-time quantification in vitro using quartz crystal microbalances. Chemistry Central Journal 2013; 7:15

Introduction Cigarette smoke is a complex aerosol consisting of two fractions; the particulate phase constituting between 4-9% and the significantly larger vapour phase constituting the remaining >90%. Historically the tobacco industry has tested the particulate phase (TPM) by trapping the particulate matter on glass fibre pads and eluting it in dimethyl sulfoxide, and the vapour phase (GVP) by capturing the remaining vapour phase in a combination of matrices, prior to testing them independently in the in vitro genotoxicity battery. The challenge now for the tobacco industry is developing air/liquid interface (ALI) exposure systems to allow assessment of whole smoke. Whole smoke exposures take into account the synergistic interactions that may be taking place between the vapour and particulate phases. The negative aspect of TPM is that the complete vapour phase cannot be captured in a single solvent matrix and testing can only be performed in solution. Whole smoke exposures aim to increase the biological relevance of any results and enable better comparison to human exposure conditions. It is therefore important to develop systems that enable exposure of both the particulate and vapour phase of whole smoke at the air/liquid interface. Aim The aim of this study is to assess previously published methodologies1-3 for Ames and cytotoxicity assays for their capabilities to differentiate cigarettes of different smoke yields. Whole Smoke Generation A Vitrocell® VC 10 Smoking Robot (Vitrocell® Systems, Waldkirch, Germany) was used to generate smoke aerosols from 3R4F, 1R5F (University of Kentucky, USA) and CORESTA Monitor 7 (CM7) cigarettes(supplied by Borgwaldt GmbH, Germany) before being directed to the exposure surface. All cigarettes were conditioned to ISO 3402:199 prior to smoking to ISO 3308:2012 (2 second puff, 35mL, 60 second interval with a 5mL vacuum). Where cigarette puff numbers differed, experiments were matched for exposure time. For all experiments quartz crystal microbalances (QCMs) were included for a quantitative measure of deposited mass3.

NRU: Balb/c 3t3 mouse fibroblasts were cultured on 24mm Transwells® (Corning Inc.) for 24 hours until the confluence was >90%. Exposures were conducted for 184 min at the ALI as detailed in Thorne et al. 2014, prior to cytotoxicity assessment via the neutral red uptake assay. Experiments were conducted over 2 days to complete an 8 airflow dose range and experiments were conducted in duplicate.

Ames: S. typhimurium strains TA98 and TA100 were selected due to their sensitivity to tobacco combustion products especially nitroarenes and aromatic amines. Exposures were conducted using the modified Ames methodology as detailed in Kilford et al. 2014 for 24 min in the presence and absence of a metabolic activation system. Cultures were exposed in triplicate and experiments were duplicated to confirm consistency of results.

Conclusions ▶ Concentration related increases >2 fold observed in TA98 and TA100 in the

presence of metabolic activation system, indicating all smoke aerosols tested cause both frame-shift mutations and base pair substitutions.

▶ TA98 is more responsive and therefore more sensitive than TA100 to whole smoke aerosol.

▶ Increases in deposition correlate with increases in revertant number; CM7 at 1L/min is the exception due to the presence of toxicity at this dose.

▶ Cytotoxicity was observed in all smoke aerosols, with varying IC50 values obtained for the three products. CM7 is more toxic than 3R4F, and subsequently 3R4F is more toxic than 1R5F.

▶ Previously published methodologies are suitable in differentiating between combustible products of varying yield.

Figure 1. Percentage relative cell survival in a comparison study of 3R4F, 1R5F and CM7. Cells were exposed to diluted smoke and to an ISO regime for 184 min. Inter-experimental variation is observed in cells at an average relative survival <50%. IC50 values have been calculated and expressed in L/min.

Figure 2. Average mean revertants from exposure to diluted mainstream cigarette smoke generated from 3R4F, 1R5F and CM7 cigarettes in two strains of S. typhimurium (TA98 and TA100) at diluting airflows of 12, 8, 4 and 1L/min, with a constant vacuum of 5mL/min in the presence of S-9 metabolic activation. Positive responses >2-fold were observed in TA98 and TA100 in the presence of metabolic activation.

IC50 (L/min) 3R4F 6.5 1R5F 3.1 CM7 10.5

Figure 4. Whole smoke deposition data and mean revertants from exposure to 3R4F, 1R5F and CM7 for TA98 and TA100. Mean revertants data is highlighted in red, with mean deposition (ng/cm2) in black.

Diluting Air (L/min)

TA98 TA100 + S-9 - S-9 + S-9 - S-9

3R4F

Air - 4.9 - 4.7 - 17.2 - 15.9 12 -4.1 6.4 16.9 4.5 -21.0 18.0 -17.6 19.8 8 69.7 11.7 42.3 4.0 47.3 26.7 88.3 14.2 4 488.5 32.7 593.7 5.8 404.9 38.9 711.1 20.0 1 3375.3 58.7 3818.4 6.3 1437.9 32.5 3668.2 22.7

1R5F

Air - 5.2 - 3.0 - 21.9 - 21.5 12 19.5 5.2 256.6 3.3 -10.4 19.5 -2130.0 20.4 8 -25.8 7.4 -35.1 4.5 -2.1 24.2 16.3 17.5 4 54.0 7.7 -8.3 4.5 59.9 29.9 45.4 16.2 1 374.2 26.5 223.5 5.2 770.8 51.0 486.3 25.0

CM7

Air - 5.2 - 4.2 - 22.0 - 17.5 12 106.3 9.7 33.4 4.3 81.5 24.2 -131.3 17.5 8 209.4 15.9 -210.9 4.9 200.1 23.2 -295.7 22.4 4 925.2 36.7 955.3 3.2 2919.2 45.0 2100.8 24.3 1 11924.8 26.7 8096.5 4.4 15163.9 54.0 13759.9 20.0

Image supplied by Vitrocell® Systems GmbH

Strain TA98 TA100 Presence of metabolising agent

1R5F Positive Positive 3R4F Positive Positive CM7 Positive Positive

Absence of metabolising agent 1R5F Negative Negative 3R4F Negative Negative CM7 Negative Negative

Figure 3. Whole smoke dose-response. Average mean revertants from exposure to diluted cigarette smoke (Log Deposition ng/cm2)generated from 3R4F, 1R5F and CM7 for TA98 [A] and TA100 [B].

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Presented at TSRC 2016