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CORESTA Meeting, Smoke Science/Product Technology, 2023, Cancun, STPOST 12 (also presented at TSRC 2023)

Rapid in vitro toxicological screening using ToxTracker to determine the effect of repeated freeze/thaw of combustible cigarette extract

CARMINES E.(2); MISRA M.(2); OH S.(1); HENDRIK G.(3); COFFA B.G.(1,2); ALEKSA K.(1)
(1) Labstat International, Kitchener, ON, Canada; (2) Chemular Inc., Hudson MI, U.S.A.; (3) Toxys Inc., CG Leiden, The Netherlands

Combustible cigarette extracts (CCE) are stored at -80 °C to preserve sample integrity. Sample shipping and repeated freeze/thaw cycles prior to testing may lead to changes in the chemical composition through the formation of ice-water interfaces, phase separation, pH induced changes and chemical degradation may produce inaccurate results in chemistry and in in vitro toxicological assays. We sought to determine the impact of repeated freeze/thaw on CCE at the DNA level using the stem cell based ToxTracker© reporter assay.

Combustible reference cigarettes (1R6F) (n=3) were smoked (Health Canada standard regime). Particulate phase (PP), gas-vapor phase (GVP) and the 1:1 combined phase (PP+GVP)] were collected and evaluated in the ToxTracker assay. Samples were tested within 1 hr of sample generation or after 3 freeze/thaw cycles. Cells were incubated for 24 hr (both +/- S9). Green-fluorescence protein (GFP) reporter gene induction and cytotoxicity were assessed using flow cytometry.

Fresh PP (-S9) samples induced a 35-fold increase in Srxn1 (indicative of oxidative stress) and a 10-fold increase in Ddit3 (indicative of protein damage) at 200 µg/mL; addition of S9 decreased Srxn1 to 12-fold GFP-induction in fresh PP. GFP-induction was not observed in fresh GVP samples. Fresh PP+GVP (+S9), induced Srxn1 and Blvrb (indicative of oxidative stress) 10 and 4-fold, respectively at 200 µg/mL and no effect on Ddit3. Sample freeze and thaw decreased Srxn1 from 35 to 20-fold while Ddit3 increased from 10 to 21-fold (-S9). No significant difference between fresh (+S9) and freeze/thaw (+S9) for PP or PP+GVP samples was observed.

This data indicates that sample freeze/thawing can alter in vitro toxicity results in the ToxTracker assay. A “fresh” sample should be used for the ToxTracker assay to eliminate unintended potential sample manipulation effects. This allows for the test system to be treated with a sample that more closely resembles the chemical profile produced immediately after sample collection and representative of normal smoking behavior.