Quantitative differentiation of whole smoke solution‐induced mutagenicity in the mouse lymphoma assay

Abstract

In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to 5 concentrations of each WSS for 4 hr 6 S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD 10 , BMD 50 , BMD 100 , and BMD 200 , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Benchmark Dose Modeling of In Vitro Genotoxicity Data: a Reanalysis

“…A reanalysis of the in vitro genotoxicity data for BMD modeling was performed using the EPA’s Benchmark Dose Software (version 2.7, released on August 18, 2017) in accordance with the BMD technical guidance ( 25 ). Briefly, a continuous model was used in the reanalysis as all response variables can take a continuous range of values ( 22 , 23 ). The BMDS analysis included exponential, Hill, linear, polynomial, and power models.…”

“…In the MLA data, the values of BMDL 10 were lower than the other PoDs, and therefore, the distribution of BMDL 10 values produced the lowest PoD ( 13 ). Among the methods utilized in the studies, including the lowest positive response by the global evaluation factor and mutagenic potency, the BMD approach appeared to be the most reasonable for the quantitative description of genotoxicity data generated from the MLA ( 13 , 23 ).…”

“…In a previous study, six WSSs were generated from two commercial cigarette products [Cigarette type #1 (C1) and Cigarette type #2 (C2)] with two smoke conditions [the International Organization for Standardization (ISO) and Health Canada Intense (HCI) methods] and different numbers of cigarettes (i.e., 20 and 60) ( 23 ). The MFs from these six WSSs in the MLA, in the presence of S9 metabolic activation, were reanalyzed by using the EPA’s BMDS.…”

“…In our laboratory, we have applied PoD and other quantitative metrics to data obtained from the mouse lymphoma assay (MLA) and performed quantitative analyses of the relative mutagenicity of five chemical components representative of different classes of chemicals found in tobacco smoke ( 13 ). More recently, we used BMD modeling to provide a quantitative evaluation of the mutagenicity of four piperidine nitroxides [i.e., 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO] with different substituent groups on the 4-position of the piperidine ring ( 22 ), and six cigarette whole smoke solutions (WSSs) prepared by bubbling machine-generated whole smoke through dimethyl sulfoxide (DMSO) ( 23 ). The findings demonstrated the suitability of BMD modeling for potency ranking of in vitro studies on structurally different chemical agents ( 13 ), structurally similar compounds (i.e., different derivatives of one chemical) ( 22 ), and different complex chemical mixtures ( 23 ).…”

“…BMD modeling is primarily conducted using two software packages, PROAST software, developed by the Netherlands National Institute for Public Health and the Environment (RIVM) ( 24 ), and BMDS, developed by the U.S. EPA ( 25 ). In previous studies, we used the PROAST software to perform quantitative dose-response analyses of the MLA data produced by testing four piperidine nitroxides ( 22 ) and six WSSs ( 23 ). In the present study, we reanalyzed these genotoxicity data from the MLA using the EPA’s BMD software to evaluate the quantitative agreement of the estimates of genotoxic potency.…”