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CORESTA Meeting, Smoke Science/Product Technology, 2023, Cancun, STPOST 10

In vitro micronucleus in V79 cells using Microflow - treatment at the air liquid interface with aerosol from three different nicotine containing products

ROTHWELL E.; SMITH G.; MARTIN S.; HOLLINGS M.
Labcorp Early Development Laboratories Ltd, Harrogate, U.K.

The in vitro micronucleus assay is a standard genetic toxicology test. Historically this has been run with a slide scoring endpoint which is labour intensive and low throughput for testing multiple products. Flow cytometer analysis can be performed, with the use of additional stains, to measure micronucleus induction.

This process was used for analysis of samples treated at the air-liquid interface (ALI) with whole aerosols. A Vitrocell® VC10® smoking robot and high throughput dilution system was utilised to generate aerosols from a 1R6F Kentucky Reference cigarettes (ISO 20768 for 12 min), commercially available heated tobacco product (HTP) (modified ISO 20768 for 42 min) and electronic nicotine delivery system (ENDS) (ISO 20778 for 180 min). Whole aerosol was tested at varying concentrations and diluted with the addition of clean air.

V79 cells grown on 24 mm Transwells™ were placed at the ALI within the Vitrocell High Throughput system, where they were treated with these aerosols. Liquid traps were placed in each airflow concentration and analysed for carbonyls and nicotine.

Endpoints were measured using the Litron™ MicroFlow kit. Cytotoxicity was measured by RPD, additionally the use of Cell Sorting Set-up Beads (Invitrogen™) during flow cytometry assessment was performed.

Results showed increases in micronucleus (MN) induction across aerosol concentrations for combustible cigarette. HTP and ENDS products showed low MN induction up until the increase of cytotoxicity where the MN induction increased.

This study demonstrated that micronuclei can be detected at the ALI using V79 cells treated with varying aerosols.