Validation of an in vitro test battery to evaluate smokeless tobacco products
Evaluating potential cytotoxicity and genotoxicity of smokeless tobacco products poses unique challenges for in vitro testing. Each product is a complex mixture of vegetable matter, and may include a packaging component as part of the final product. Therefore, such products are evaluated in a manner similar to packaging products or medical devices – incubated with various fluids to extract leachable components which are subsequently applied to the test system. Also, individual extract constituents may be dilute and/or innocuous, requiring large dose volumes to detect any effect. Here we report validating sample preparation, and basic processing and handling parameters, for an in vitro test battery including the bacterial reverse mutation (Ames) assay, in vitro micronucleus in TK6 cells, and neutral red uptake (NRU) cytotoxicity assay. Two commercial smokeless tobacco products (one flavored, one unflavored) were shredded and sieved, and then sonicated and extracted in artificial saliva according to published procedures. Initial results in an Ames screen (using tester strains TA98 and TA100 ±S9, pre-incubation treatment, and dose volumes up to 200 µL/plate) revealed: moderate to severe cytotoxicity for the flavored product at a dose of 200 µL/plate; discoloration of the plates at the highest dose levels (without interference with automated scoring); and a lack of mutagenicity for both extracts under these conditions. Preliminary results using nicotine as a surrogate marker indicate that the extracts are stable for at least one month at -70 °C. Additional experiments in the full assays, and to qualify other flavor markers or tobacco constituents, are in progress.