A comparison of cigarette smoke test matrices and their responsiveness in the mouse lymphoma assay: A case study.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

In vitro mutagenicity of gas-vapour phase extracts from flavoured and unflavoured heated tobacco products.

The in vitro mutagenic and genotoxic potential of Heated Tobacco Products (HTPs) has already been studied with the particulate phase and reported previously. This study has been designed to complement the in vitro assessment of the HTP and to determine whether the inclusion of potential flavourings would alter the in vitro response by testing the other phase of the aerosol, the gas-vapour phase (GVP). Both flavoured and unflavoured Neostik GVP samples did not show any sign of mutagenic activity in the Ames test but induced a mutagenic response in the mouse lymphoma assay (MLA), however, these responses were significantly less than those of the reference cigarette, 3R4F. The results demonstrated that GVP emissions of this HTP did not induce either new qualitative or quantitative mutagenic hazards compared to 3R4F, as assessed by the Ames test (no new responsive strains) and MLA (a lower mutagenic response), respectively. A statistical comparative analysis of the responses showed that the addition of flavourings that may thermally decompose under the conditions of use did not add to the in vitro baseline responses of the unflavoured Neostik.