CORESTA Congress, Kunming, 2018, Smoke Science/Product Technology Groups, STPOST 43

In vitro micronucleus (MN) assay using TK6 cells: review of historical positive and negative control data

STANKOWSKI L.F. Jr(1); HURTADO S.B.(1); WELLS M.M.(1); KWOK V.Y.(1); DOSHI U.(2); COFFA B.G.(2); LEE K.M.(2)
(1) Charles River Laboratories, Genetic and In Vitro Toxicology, Skokie, IL, U.S.A.; (2) Altria Client Services LLC, Research, Development & Regulatory Affairs, Richmond, VA, U.S.A.

TK6 human lymphoblastoid cells are widely used in the in vitro MN assay as a part of regulatory genotoxicity testing for pharmaceuticals, but have seen relatively limited use in testing of other test materials, including tobacco-related products. As an established cell line, they do not exhibit the donor to donor variability observed in primary cells such as human lymphocytes. Unlike other routinely used p53-deficient cells (CHO, V79 and L5178Y), TK6 cells are p53-proficient, capable of DNA repair and normal cell cycle regulation, and generally have lower spontaneous MN frequencies and a lower rate of false or misleading positive results. We have used TK6 cells extensively to test a variety of e-liquids and e-vapor condensates, and total particulate matter (TPM) from combusted cigarettes. Here we present historical positive and negative control data, compiled from these OECD TG487- and GLP-compliant studies. The average negative control values (%MN) for the three treatments ranged from 0.93-1.03 with comparable ranges between different vehicles (negative controls). The known positive controls (two clastogens and one aneugen), run concurrently, gave consistent and robust responses in %MN induction and ranged from 6.36-10.53. The consistency of results is critical since OECD guidelines place emphasis on the use of historical controls in evaluating individual assay acceptability and the biological relevance of results, and demonstrate the utility of this test system for evaluating tobacco- and ENDS-related products.