Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Further characterization of the 9q31 microdeletion phenotype; delineation of a common region of overlap containing ZNF462.

BACKGROUND: Loss of function variants and whole gene deletions of ZNF462 has been associated with a novel phenotype of developmental delay/intellectual disability and distinctive facial features. Over two dozen cases have been reported to date and the condition is now known as Weiss-Kruszka syndrome (OMIM# 618619). There are several older reports in the literature and DECIPER detailing individuals with interstitial deletions of 9q31 involving the ZNF462 gene. Many of the characteristic facial features described in these microdeletion cases are similar to those who have been diagnosed with Weiss-Kruszka syndrome. METHODS: We describe three additional patients with overlapping 9q31 deletions and compare the phenotypes of the microdeletion cases reported in the literature to Weiss-Kruszka syndrome. RESULTS: Phenotypic overlap was observed between patients with 9q31 deletions and Weiss-Kruszka syndrome. Several additional features were noted in 9q31 deletion patients, including hearing loss, small head circumference, palate abnormalities and short stature. CONCLUSIONS: The common region of overlap of microdeletion cases implicates ZNF462 as the main driver of the recognizable 9q31 microdeletion phenotype. The observation of additional features in patients with 9q31 microdeletions that are not reported in Weiss-Kruszka syndrome further suggests that other genes from the 9q31 region likely act synergistically with ZNF462 to affect phenotypic expression.

The impacts of trail infrastructure on vegetation and soils: Current literature and future directions.

Reflecting the popularity of nature-based activities such as hiking and mountain biking, there are thousands of kilometres of recreational trails worldwide traversing a range of natural areas. These trails have environmental impacts on soils and vegetation, but where has there been research, what impacts have been found and how were they measured? Using a systematic quantitative literature review methodology, we assessed the impacts of trails on vegetation and soils, highlighting what is known, but also key knowledge gaps. Of the 59 original research papers identified on this topic that have been published in English language peer-reviewed academic journals, most were for research conducted in protected areas (71%), with few from developing countries (17%) or threatened ecosystems (14%). The research is concentrated in a few habitats and biodiversity hotspots, mainly temperate woodland, alpine grassland and Mediterranean habitats, often in the USA (32%) or Australia (20%). Most examined formal trails, with just 15% examining informal trails and 11% assessing both types. Nearly all papers report the results of observational surveys (90%), collecting quantitative data (66%) with 24% using geographic information systems. There was an emphasis on assessing trail impacts at a local scale, either on the trail itself and/or over short gradients away from the trail edge. Many assessed changes in composition and to some degree, structure, of vegetation and soils with the most common impacts documented including reduced vegetation cover, changes in plant species composition, trail widening, soil loss and soil compaction. There were 14 papers assessing how these local impacts can accumulate at the landscape scale. Few papers assessed differences in impacts among trails (7 papers), changes in impacts over time (4), species-specific responses (3) and only one assessed effects on plant community functioning. This review provides evidence that there are key research gaps including assessing informal trails, comparing trail types, landscape and temporal scale impacts, functional responses and impacts on threatened ecosystems/species. A more diverse geographic spread of research is also required including in regions experiencing rapid growth in tourism and recreation.

Recreational trails as a source of negative impacts on the persistence of keystone species and facilitation.

Hiking trails, which are among the most common forms of infrastructure created for nature-based tourism, can alter key ecological processes. Trails can damage plants that facilitate the establishment and growth of other species leading to changes in community and functional composition. This can be a particular concern in harsh alpine ecosystems where plant communities are often dominated by one or two keystone species that provide shelter to a suite of beneficiary species. We analysed how a hiking trail affects interspecific facilitation by a dominant trampling-sensitive nurse shrub in the highest National Park in Australia. First we assessed the effects of the trail on the abundance, size and density of the nurse shrub at different distances from the trail. We then compared species richness and composition between areas in, and out, of the nurse shrub's canopy at different distances from the trail. To better understand why some species may benefit from facilitation and any effects of the trail on the quality of facilitation we compared functional composition between quadrats using community trait weighted means calculated by combining plant composition with species functional traits (canopy height, leaf area, % dry weight of leaves and specific leaf area). The abundance, size and density of nurse shrubs was lower on the trail edges than further away, particularly on the leeward edge, where there was more bare ground and less shrub cover. There were differences in species richness, cover, composition and functional composition in and outside the nurse shrub canopy. The shrubs appeared to facilitate species with more competitive, but less stress tolerant traits (e.g. taller plants with leaves that were larger, had high specific leaf area and low dry matter content). However, despite reductions in nurse shrubs near the trail, where they do exist, they appear to provide the same 'quality' of facilitation as nurse shrubs further away. However, longer-term effects may be occurring as the loss of nurse shrubs alters the wind profile of the ridgeline and therefore succession. The use of a steel mesh walkway along the trail may facilitate the regeneration of nurse shrubs and other plants that require protection from wind. Our results highlight the importance of diversifying recreation ecology research to assess how trails affect important ecological processes.

Differences in the impacts of formal and informal recreational trails on urban forest loss and tree structure.

Recreational trails are one of the most common types of infrastructure used for nature-based activities such as hiking and mountain biking worldwide. Depending on their design, location, construction, maintenance and use, these trails differ in their environmental impacts. There are few studies, however, comparing the impacts of different trail types including between formal management-created trails and informal visitor-created trails. Although both types of trails can be found in remote natural areas, dense networks of them often occur in forests close to cities where they experience intense visitor use. To assess the relative impacts of different recreational trails in urban forests, we compared the condition of the trail surface, loss of forest strata and changes in tree structure caused by seven types of trails (total network 46.1 km) traversing 17 remnants of an endangered urban forest in Australia. After mapping and classifying all trails, we assessed their impact on the forest condition at 125 sites (15 sites per trail type, plus 15 control sites within undisturbed forest). On the trail sites, the condition of the trail surface, distance from the trail edge to four forest strata (litter, understory, midstorey and tree cover) and structure of the tree-line were assessed. Informal trails generally had poorer surface conditions and were poorly-designed and located. Per site, formal and informal trails resulted in similar loss of forest strata, with wider trails resulting in greater loss of forest. Because there were more informal trails, however, they accounted for the greatest cumulative forest loss. Structural impacts varied, with the widest informal trails and all formal hardened trails resulting in similar reductions in canopy cover and tree density but an increase in saplings. These structural impacts are likely a function of the unregulated and intense use of large informal trails, and disturbance from the construction and maintenance of formal trails. The results demonstrate that different types of recreational trails vary in the type and range of impacts they cause to forests. They highlight the importance of careful consideration towards management options when dealing with trail networks especially in areas of high conservation value.

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

Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell(®) VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0, 4.0, 8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (µg/cm(2)). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.

Tourism and recreation listed as a threat for a wide diversity of vascular plants: a continental scale review.

Tourism and recreation are diverse and popular activities. They may also contribute to the risk of extinction for some plants because of the range and severity of their impacts, including in protected areas: but which species, where and how? To evaluate the extent to which tourism and recreation may be threatening process for plants, we conducted a continental level review of listed threats to endangered vascular plants using data from Australia. Of the 659 vascular plant species listed as critically endangered or endangered by the Australian Government, tourism and recreation were listed as a threat(s) for 42%. This is more than those listed as threatened by climate change (26%) and close to the proportion listed as threatened by altered fire regimes (47%). There are plant species with tourism and recreation listed threats in all States and Territories and from all but one bioregion in Australia. Although more than 45 plant families have species with tourism and recreation listed as threats, orchids were the most common species listed as at risk from these threats (90 species). The most common types of threats listed were visitors collecting plants in protected areas (113 species), trampling by hikers and others (84 species), damage from recreational vehicles (59 species) and road infrastructure (39 species). Despite the frequency with which tourism and recreation were listed as threats in Australia, research quantifying these threats and methods to ameliorate their impacts are still limited. Although this lack of information contributes to the challenge of managing tourism and recreation, impacts from visitors will often be easier to manage within natural areas than those from larger scale threats such as altered fire regimes and climate change.

The in vitro toxicology of Swedish snus.

Three commercial brands of Swedish snus (SWS), an experimental SWS, and the 2S3 reference moist snuff were each tested in four in vitro toxicology assays. These assays were: Salmonella reverse mutation, mouse lymphoma, in vitro micronucleus, and cytotoxicity. Water extractions of each of the 5 products were tested using several different concentrations; the experimental SWS was also extracted using dimethyl sulfoxide (DMSO). Extraction procedures were verified by nicotine determinations. Results for SWS in the mutagenicity assays were broadly negative: there were occasional positive responses, but these were effectively at the highest concentration only (concentrations well above those suggested by regulatory guidelines), and were often associated with cytotoxicity. The 2S3 reference was unequivocally positive in one of the three conditions of the micronucleus assay (MNA), at the highest concentration only. Positive controls produced the expected responses in each assay. The SWS data are contrasted with data reported for combusted tobacco in the form of cigarettes, where strongly positive responses have been routinely reported for mutagenicity and cytotoxicity. These negative findings in a laboratory setting concur with the large amount of epidemiological data from Sweden, data showing that SWS are associated with considerably lower carcinogenic potential when compared with cigarettes.

Further investigations into the genotoxicity of 2,6-xylidine and one of its key metabolites.

The metabolite of several amide anaesthetics, 2,6-xylidine, is a possible human (Group 2B) carcinogen and induced nasal tumours in rats after dietary administration. However, published papers on the genotoxicity of 2,6-xylidine in vitro have given inconsistent results. It has been proposed that the genotoxicity of 2,6-xylidine is dependent on its metabolism to a key metabolite dimethylphenyl N-hydroxylamine (DMHA), which would then be further converted to form a reactive nitrenium ion by phase 2 (mainly acetylation) metabolism. In order to study whether the inconsistent results could be explained by different systems having different potential for DMHA to be formed and to induce genotoxicity in vitro, we have tested 2,6-xylidine in conventional Ames bacteria, and strains engineered to overexpress acetyltransferase, in the presence of different concentrations of induced rat liver and human liver S9. All tests gave consistently negative results. The formation of DMHA by induced rat liver S9 and human S9 was clearly shown to occur, and to be concentration- and time-dependent. The potential inhibitory effects of the solvent DMSO were also studied, but it was clearly not responsible for the negative results with 2,6-xylidine. Thus, whatever is the mode of action of 2,6-xylidine carcinogenicity in rodents, it has proven impossible to detect mutagenic effects in Ames tests with numerous variations of metabolic conditions, or even using acetyltransferase overexpressing strains of bacteria.

MNT and MutaMouse studies to define the in vivo dose response relations of the genotoxicity of EMS and ENU.

Although there are a multitude of in vitro and in vivo studies on the genotoxic activity of EMS, no lifetime carcinogenicity studies, repeat dose mutation data or exposure analysis are available to serve as a solid basis for risk assessment for human exposure cases. The present studies were undertaken to investigate whether a threshold for mutagenic and clastogenic activity in vivo could be established, using the bone marrow micronucleus (MNT) and MutaMouse test systems, in the hope to provide reassurance to the patients that their accidental exposure to EMS at doses up to 0.055 mg/kg did not carry a toxicological risk. Dose levels ranging from 1.25 to 260 mg/kg/day were applied orally for up to 28 days. As a reference we included ENU at doses of 1.1-22 mg/kg/day. Our studies showed that daily doses of up to 25mg/kg/day (bone marrow, GI tract) and 50 mg/kg/day (liver) did not induce mutations in the lacZ gene in the three organs tested. Doses up to 80mg/kg/day (7-day dosing regime) did not induce micronuclei in mouse bone marrow. The genotoxic activity of EMS became apparent only at higher dose levels. Dose fractionation of EMS (28 times 12.5mg/kg versus a single high dose 350 mg/kg) provided further evidence for the thresholded dose response of EMS and showed that no cumulation of gene mutations below a threshold was occurring. In contrast, for ENU no threshold was apparent and dose fractionation indicated full additivity of individual dose effects.

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites.

para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.