Validation of the VITROCELL Mammalian 48 Exposure Module Using In Vitro Cytotoxicity via the Neutral Red Uptake Assay in Chinese Hamster Ovary Cells Following Exposure to Whole Smoke from Combustible Cigarettes

December 15, 2023

Brian M. Keyser1, Robert Leverette1, Michael Hollings2, Emma Rothwell2, Randy A. Weidman1, Carlton J. Bequette1, and Kristen Jordan1
1Scientific & Regulatory Affairs, RAI Services Company, Winston-Salem, North Carolina, USA.
2Labcorp Early Development Laboratories Ltd., Harrogate, United Kingdom.

Introduction: The Vitrocell® Mammalian 48 exposure module offers a high throughput platform for air-liquid interface (ALI) exposures that mimic human exposure conditions for assessing the potential cytotoxicity of a test article. The aim of this study was to evaluate the in vitro cytotoxic response of whole smoke in Chinese hamster ovary (CHO) cells, using the Neutral Red Uptake Assay across two modules. The secondary aim was to further characterize the system utilizing three dosimetry techniques.

Methods: Whole smoke generated from 3R4F Kentucky Reference cigarettes was examined over a diluting airflow range 10 to 1 L/min. Cigarettes were smoked according to both standard and intense International Organisation for Standardisation (ISO) smoking regimens. Aerosol photometers (using area under curve, AUC), fluorescence of anhydrous dimethyl sulfoxide (DMSO), and nicotine quantification. Photometer AUC, particulate deposition (measured by fluorescence), and nicotine determination were used to characterize the modules.

Results and Discussion: Dosimetry measures (AUC, fluorescence, nicotine) demonstrated consistent aerosol delivery across rows within and between the Vitrocell® Mammalian 48 exposure modules. Cytotoxicity was achieved using the intense ISO smoking regimen with IC50 calculated as 5.6 ± 2.3 and 3.8 ± 1.6 L/min, respectively for both modules, with no statistical difference observed between the modules (p > 0.05). We have also demonstrated that the cytotoxic response with ISO smoking regimens is consistent with previously published data using Vitrocell® 12/4 modules.

Conclusion: The data in this study support the validation and characterization of the Vitrocell® Mammalian 48 module for whole smoke exposure at the ALI for cytotoxicity assessment with the Neutral Red Uptake assay.

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