The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter

Abstract

In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from different cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recommends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity.

Comparative In Vitro Toxicity Profile of Electronic and Tobacco Cigarettes, Smokeless Tobacco and Nicotine Replacement Therapy Products: E-Liquids, Extracts and Collected Aerosols

“…The Ames test, also known as the bacterial reverse mutation assay, is widely used for the determination of a compound’s ability to induce mutations and has been shown to have a high predictive value with rodent carcinogenicity tests [ 28 ].…”

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

“…In the absence of S9, each tester strain was tested in triplicate plates. In the presence of S9, triplicate plates were used for strains TA1535 and TA102, quadruplicate plates for TA100, quintuplicate plates for TA98 and ten plates per concentration were treated for strain TA1537 as recommended by Scott et al [44]. At least 6 concentrations were tested in each treatment condition and experiment.…”

“…Each experiment was composed of two 3 h exposure treatments, one in the presence and one in the absence of S9 (Aroclor 1254-induced rat liver post-mitochondrial supernatant mix), and one 24 h exposure treatment in the absence of S9. At least 9 concentrations were tested per GVP sample, with four replicate cultures per concentration as recommended by Scott et al [44]. Duplicate cultures were used for the positive controls.…”

“…Statistical comparative analyses of flavoured and unflavoured GVP mutagenic activity in the mouse lymphoma assay were performed as per Scott et al [44] recommendations for each independent experiment, providing that for each comparison both GVP test items were mutagenic and provided a concentration related response.…”

The mutagenic assessment of mainstream cigarette smoke using the Ames assay: A multi-strain approach

“…The Ames assay has been used to determine the mutagenicity of complex chemical mixtures [4] and as such has been used to assess the mutagenicity of cigarette smoke, which has been demonstrated to have clear mutagenic activity [5]. The assay is relatively S. typhimurium TA98 Frameshift (hisD3052) [2] pKM101 bio, rfa, uvrB [2] Positive [18][19][20][21] Positive [22][23][24][25] S. typhimurium TA100 Mis-sense (hisG46) [2] pKM101 bio, rfa, uvrB [2] Positive [18,20,21,26,27] Positive [23][24][25] Negative [22] S. typhimurium TA1535 c Mis-sense (hisG46) [2] bio, rfa, uvrB [2] Negative [18,27,28] Negative [24] S. typhimurium TA1537 c Mis-sense (hisC3076) [2] bio, rfa, uvrB [2] Positive [18,20,21,28] Negative [22,29] Negative [24] E. coli WP2 uvrA pKM101 Mis-sense (trpE56) [30] pKM101 uvrA [30] N/A Positive [24] Negative [25] S. typhimurium TA97 c Frameshift (hisD6610) [31] pKM101, rfa, uvrB [31] N/A N/A S. t...…”