In Vitro Testing of an Ethanol Collection Method Combining Particulate and Gas-Vapor Phase Components: Bacterial Reversion Mutation (Ames) Assay
Health Canada (HC) guidelines (T-502) for the collection and testing of cigarette smoke are used frequently for in vitro testing. Although the guidelines allow for the collection and testing of the particulate phase (PP), the gas-vapor (GVP) phase, and a combination of both (PP+GVP), this method has several limitations. The PP is collected in DMSO and can be tested for cytotoxicity (NRU assay), mutagenicity (Ames assay), and clastogenicity (MN assay), while the GVP phase is collected in PBS and only tested using the NRU assay. PBS has limited trapping efficiency of volatile or non-water-soluble compounds and must be used within 60 minutes of collection. These limitations could be overcome with a method that allows collection of the PP and GVP together in a solvent providing enhanced trapping and stability of GVP components. We have tested the use of ethanol to collect PP and GVP components using the Ames assay (HC T-501 guideline). Reference 3R4F cigarettes were used. Five strains of Salmonella bacteria (TA98, TA100, TA102, TA1535 and TA1537) were tested in the absence and presence of S9. Results showed that the ethanol condensate collecting PP+GVP together resulted in increased bacterial lawn cytotoxicity in TA98, TA100, TA1535 and TA1537. DMSO-extracted PP showed toxicity in TA98, TA100, TA1537 while PP+GVP in PBS only showed toxicity in TA1537. With metabolic activation, TPM+GVP in ethanol induced an 18-fold and 11-fold increase in the number of revertants in TA98 and TA1537 respectively. In comparison, DMSO extracted PP induced a 16-fold and 8-fold increase, while TPM+GVP in PBS induced a 14-fold and 6-fold increase in the same strains. The ethanol method allows for combined collection of PP+GVP components yielding a single whole-smoke extract significantly increasing the mutagenic and cytotoxic effects observed in the Ames assay. NRU, MN, and chemistry results are presented separately.
Sanjay K. Bharti, Bhagyalaxmi Sukka-Ganesh, Mariano J. Scian, and Gene I. Gillman
Enthalpy Analytical, LLC, Henrico, VA 23228