The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays

Abstract

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM storage at -80 °C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference cigarettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored versus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.

Development, qualification, validation and application of the Ames test using a VITROCELL® VC10® smoke exposure system

“…Cigarette whole smoke is made up of both a particulate fraction (total particulate matter (TPM)) and a vapor phase component. This whole smoke mixture, consisting of more than 7000 chemicals [ 9 ], makes testing by standard methods extremely difficult, and to date, most testing has focussed on testing TPM using standard methodology in several toxicological endpoints [ [10] , [11] , [12] ]. These endpoints include the Ames reverse mutation test, the in vitro micronucleus assay (IVMN), the neutral red uptake assay (NRU) and the Mouse Lymphoma Assay (MLA) [ 11 , [13] , [14] , [15] ].…”

Characterisation of a Vitrocell® VC 10 in vitrosmoke exposure system using dose tools and biological analysis

“…Assessment of tobacco smoke in vitro has traditionally focused on the particulate phase captured on a Cambridge filter pad and eluted in DMSO [14] or bubbled through cell culture media or PBS [27]. However, these techniques do not capture the full extent of the vapour phase of cigarette smoke and semi-volatiles which not only make up the majority fraction of tobacco smoke, but include reactive chemicals with known toxicological properties [7].…”

“…This is partly because traditional exposure techniques tend to focus on the particulate phase of cigarette smoke [13,14] and not the complete aerosol. Traditional techniques include capturing the particulate fraction on a Cambridge filter pad and eluting in dimethyl sulphoxide (DMSO) or bubbling the smoke aerosol through cell culture media or phosphate buffered saline (PBS) to obtain a soluble fraction.…”

A Device for measuring the in-situ response of Human Bronchial Epithelial Cells to airborne environmental agents

“…Several approaches have been developed to study the biological effect of aerosols on airway epithelium 7,8 . In some in vitro studies, the particulate matter is collected on filters and suspended/dissolved in a liquid, where it is exposed to the cells for biological responses 9–11 , a strategy more suitable for cells in liquid culture. However, cells cultured on an air-liquid interface, which emulates in vivo exposures, can be exposed to particles in an exposure chamber 8,12,13 .…”