In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT).

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

Performance of the in vitro transgene mutation assay in MutaMouse FE1 cells: Evaluation of nine misleading ("False") positive chemicals.

The screening of chemicals for the protection of human health and the environment requires the assessment of genetic toxicity. However, existing, internationally-accepted in vitro mammalian genotoxicity tests have been criticized for their low specificity (i.e. high frequency of "false" or "misleading" positive results for compounds that are negative in vivo). An in vitro transgene mutation assay has been established that uses a metabolically competent cell line derived from MutaMouse lung (i.e. FE1 cells). Mutation scoring employs the well-characterized lacZ positive selection system, and the assay is proposed as an alternative in vitro assessment tool. In this study, the performance of the FE1 cell assay was evaluated by examining responses to nine non-DNA-reactive chemicals that previously elicited misleading positive results in other mammalian cell genotoxicity assays. FE1 cells were exposed to concentrations up to approximately 10 mM and/or concentrations that yielded approximately 80-90% cytotoxicity (as measured by relative increase in cell count). The assay demonstrated excellent specificity; exposures to the chemicals examined did not yield any positive responses even when tested in the presence of an exogenous metabolic activation system (i.e. S9) or with an extended sampling time. These results indicate that the FE1 cell mutagenicity assay is an effective and practical alternative to traditional mammalian cell gene mutation assays. The development and validation of effective in vitro tools such as the MutaMouse FE1 cell assay will contribute to international efforts to reduce, refine, and replace experimental animals for toxicity assessment. Environ. Mol. Mutagen. 58:582-591, 2017. © 2017 Wiley Periodicals, Inc.

Mutagenicity of smoke condensates from Canadian cigarettes with different design features.

There is currently limited knowledge regarding the impact of different cigarette designs on the toxicological properties of cigarette smoke condensate (CSC). This study used the Salmonella Mutagenicity Assay to examine the mutagenic activity of mainstream CSCs from 11 commercial Canadian cigarette brands with different design features or tobacco blend. The brands were selected to include design features that are common for cigarettes sold in the Canadian market, as well as cigarettes with alternate filters (charcoal or MicroBlue™), the super slim design, and cigarettes containing mixed blends of different tobacco types. CSCs were obtained using the International Organization for Standardization (ISO) and Health Canada Intense (HCI) smoking regimes, and mutagenic activity was assessed using Salmonella strains TA98, YG1041 and YG5185. Comparisons of the commercial brands to the Kentucky 3R4F, the Canadian Monitor 8 reference and a Canadian best seller revealed no significant reduction in CSC mutagenicity for cigarettes with alternate filters. However, the super slim design did afford some reduction in mutagenic potency. Nevertheless, since the study did not attempt to evaluate the impact of the cigarette designs on human health at the individual or population level, the super slim cigarettes cannot be considered 'reduced-harm' cigarettes.

A global toxicogenomic analysis investigating the mechanistic differences between tobacco and marijuana smoke condensates in vitro.

Like tobacco smoking, habitual marijuana smoking causes numerous adverse pulmonary effects. However, the mechanisms of action involved, especially as compared to tobacco smoke, are still unclear. To uncover putative modes of action, this study employed a toxicogenomics approach to compare the toxicological pathways perturbed following exposure to marijuana and tobacco smoke condensate in vitro. Condensates of mainstream smoke from hand-rolled tobacco and marijuana cigarettes were similarly prepared using identical smoking conditions. Murine lung epithelial cells were exposed to low, medium and high concentrations of the smoke condensates for 6h. RNA was extracted immediately or after a 4h recovery period and hybridized to mouse whole genome microarrays. Tobacco smoke condensate (TSC) exposure was associated with changes in xenobiotic metabolism, oxidative stress, inflammation, and DNA damage response. These same pathways were also significantly affected following marijuana smoke condensate (MSC) exposure. Although the effects of the condensates were largely similar, dose-response analysis indicates that the MSC is substantially more potent than TSC. In addition, steroid biosynthesis, apoptosis, and inflammation pathways were more significantly affected following MSC exposure, whereas M phase cell cycle pathways were more significantly affected following TSC exposure. MSC exposure also appeared to elicit more severe oxidative stress than TSC exposure, which may account for the greater cytotoxicity of MSC. This study shows that in general MSC impacts many of the same molecular processes as TSC. However, subtle pathway differences can provide insight into the differential toxicities of the two complex mixtures.

Genetic toxicology and toxicogenomic analysis of three cigarette smoke condensates in vitro reveals few differences among full-flavor, blonde, and light products.

Cigarette smoking leads to various detrimental health outcomes. Tobacco companies produce different brands of cigarettes that are marketed as reduced harm tobacco products. Early examples included "light" cigarettes, which differ from regular cigarettes due to filter ventilation and/or differences in chemical constituents. In order to establish baseline similarities and differences among different tobacco brands available in Canada, the present study examined the cytotoxicity, mutagenicity, clastogenicity, and gene expression profiles of cigarette smoke condensate (CSC) from three tobacco products, encompassing a full-flavor, blonde, and "light" variety. Using the Salmonella mutagenicity assay, we confirmed that the three CSCs are mutagenic, and that the potency is related to the presence of aromatic amines. Using the Muta™Mouse FE1 cell line we determined that the CSCs were clastogenic and cytotoxic, but nonmutagenic, and the results showed few differences in potencies among the three brands. There were no clear brand-specific changes in gene expression; each brand yielded highly similar expression profiles within a time point and concentration. The molecular pathways and biological functions affected by exposure included xenobiotic metabolism, oxidative stress, DNA damage response, cell cycle arrest and apoptosis, as well as inflammation. Thus, there was no appreciable difference in toxicity or gene expression profiles between regular brands and products marketed as "light," and hence no evidence of reduced harm. The work establishes baseline CSC cytotoxicity, mutagenicity, and expression profiles that can be used as a point of reference for comparison with data generated for products marketed as reduced harm and/or modified risk tobacco products.

The genotoxicity of mainstream and sidestream marijuana and tobacco smoke condensates.

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.

Mutagenic and carcinogenic hazards of settled house dust. I: Polycyclic aromatic hydrocarbon content and excess lifetime cancer risk from preschool exposure.

Settled house dust (SHD) may be a significant source of children's indoor exposure to hazardous substances including polycyclic aromatic hydrocarbons (PAHs). In this study, organic extracts of sieved vacuum cleaner dust from 51 homes were examined for the presence of 13 PAHs via GC/MS. PAHs were found in all samples with levels of total PAHs ranging between 1.5 and 325 microg g(-1). The PAH concentrations in the SHD were correlated with information contained in corresponding household questionnaires. Analyses showed levels of PAHs to be negatively associated with noncombustion activities such as vacuum cleaning frequency. A risk assessment was conducted to evaluate the excess lifetime cancer risks posed to preschool aged children who ingested PAHs in SHD. The assessment revealed that exposure to PAHs at levels found in 90% of the homes (< 40 microg g(-1)) would result in excess cancer risks that are considered acceptable (i.e., 1-100 x 10(-6)). However, exposure to higher levels of PAHs found in five homes yielded risks that could be higher than 1 x 10(-4).

Mutagenic and carcinogenic hazards of settled house dust. II: Salmonella mutagenicity.

Settled house dust (SHD) is a complex mixture that contains numerous chemical contaminants. Very little is known about the hazards of SHD as compared to other complex matrices such as air and soil. In this study, the mutagenic hazards associated with the extracts of sieved dust from 52 homes were examined using the Salmonella Mutagenicity Test. All of the SHD samples displayed mutagenic activity and the mean mutagenic potencies ranged from 2300to 23 600 revertants per gram. Testing with various Salmonella strains revealed a predominance of frameshift mutagens in the dust samples. Analyses showed that polycyclic aromatic hydrocarbons (PAHs) were likely responsible for a quarter of the mutagenic activity of the SHD samples. In an effort to identify factors that influenced dust mutagenicity, the relationships between SHD mutagenicity and household activities were investigated. Mutagenicity was positively correlated with parameters such as the time since last vacuuming (r2 = 0.11, p < 0.05) and the number of people living in the home (r2 = 0.11-0.43, p < 0.05). However, the causative factors responsible for these relationships remain unclear.

The mutagenic hazards of settled house dust: a review.

Given the large proportion of time people spend indoors, the potential health risks posed by chemical contaminants in the indoor environment are of concern. Research suggests that settled house dust (SHD) may be a significant source for indoor exposure to hazardous substances including polycyclic aromatic hydrocarbons (PAHs). Here, we summarize the literature on the mutagenic hazards of SHD and the presence of PAHs in dust. We assess the extent to which PAHs are estimated to contribute to the mutagenicity of SHD, and evaluate the carcinogenic risks associated with exposures to PAHs in SHD. Research demonstrates that SHD has a Salmonella TA98 mutagenic potency of 1000-7000 revertants/g, and contains between 0.5 and 500 microg/g of PAHs. Although they only account for a small proportion of the variability, analyses of pooled datasets suggest that cigarette smoking and an urban location contribute to higher levels of PAHs. Despite their presence, our calculations show that PAHs likely account for less than 25% of the overall mutagenic potency of dust. Nevertheless, carcinogenic PAHs in dust can pose potential health risks, particularly for children who play and crawl on dusty floors, and exhibit hand-to-mouth behaviour. Risk assessment calculations performed in this study reveal that the excess cancer risks from non-dietary ingestion of carcinogenic PAHs in SHD by preschool aged children is generally in the range of what is considered acceptable (1 x 10(-6) to 2 x 10(-6)). Substantially elevated risk estimates in the range 1.5 x 10(-4) to 2.5 x 10(-4) correspond only to situations where the PAH content is at or beyond the 95th percentile, and the risk estimates are adjusted for enhanced susceptibility at early life stages. Analyses of SHD and its contaminants provide an indication of indoor pollution and present important information for human exposure assessments.