Hemolytic Properties of Fine Particulate Matter (PM2.5) in In Vitro Systems.

Epidemiological studies have suggested that inhalation exposure to particulate matter (PM) air pollution, especially fine particles (i.e., PM2.5 (PM with an aerodynamic diameter of 2.5 microns or less)), is causally associated with cardiovascular health risks. To explore the toxicological mechanisms behind the observed adverse health effects, the hemolytic activity of PM2.5 samples collected during different pollution levels in Beijing was evaluated. The results demonstrated that the hemolysis of PM2.5 ranged from 1.98% to 7.75% and demonstrated a clear dose-response relationship. The exposure toxicity index (TI) is proposed to represent the toxicity potential of PM2.5, which is calculated by the hemolysis percentage of erythrocytes (red blood cells, RBC) multiplied by the mass concentration of PM2.5. In a pollution episode, as the mass concentration increases, TI first increases and then decreases, that is, TI (low pollution levels) < TI (heavy pollution levels) < TI (medium pollution levels). In order to verify the feasibility of the hemolysis method for PM toxicity detection, the hemolytic properties of PM2.5 were compared with the plasmid scission assay (PSA). The hemolysis results had a significant positive correlation with the DNA damage percentages, indicating that the hemolysis assay is feasible for the detection of PM2.5 toxicity, thus providing more corroborating information regarding the risk to human cardiovascular health.

Transparency in Non-Technical Project Summaries to Promote the Three Rs in Respiratory Disease Research.

Non-Technical Project Summaries (NTS) are legal documents that were first introduced by the Directive 2010/63/EU to enhance transparency within scientific animal experimentation. Researchers intending to conduct biological research on animal models must fulfil the NTS requirements by outlining their proposed use of animals and how they plan to implement the Three Rs (replacement, reduction and refinement of animal use) in their experiments. This study outlines a novel systematic analysis approach that enables the assessment of NTS transparency based on the accuracy of reporting of certain Three Rs-specific information. This potentially customisable strategy could help toward the development of practical guidelines for use by Animal Welfare and Ethical Review Bodies (AWERBs) in establishments conducting animal research, in the process of scrutinising NTS during their pre-submission review of proposed licence applications. This could help to identify gaps in reporting of Three Rs-specific information relating to the planned animal experiments, which represents a remarkable step toward achieving greater openness in scientific communication. This study supports the concept that NTS transparency can promote the implementation of non-animal alternatives in fields where this is currently lacking, such as respiratory disease research. Although NTS were originally conceived as informative documents for a lay audience, we can conclude that data in NTS can be successfully used as a basis for systematic analysis. By reviewing the NTS, the experimental limitations of the currently available replacement strategies can also be highlighted, potentially pinpointing where there is a need for future method development.

Microplastic atmospheric dustfall pollution in urban environment: Evidence from the types, distribution, and probable sources in Beijing, China.

Airborne microplastics (MPs) pollution is an environmental problem of increasing concern, due to the ubiquity, persistence and potential toxicity of plastics in the atmosphere. In recent years, most studies on MPs have focused on aquatic and sedimentary environments, but little research has been done on MPs in the urban atmosphere. In this study, a total of ten dustfall samples were collected in a transect from north to south across urban Beijing. The compositions, morphologies, and sizes of the MPs in these dustfall samples were determined by means of Laser Direct Infrared (LDIR) imaging and Field Emission Scanning Electron Microscopy (FESEM). The number concentrations of MPs in the Beijing dustfall samples show an average of 123.6 items/g. The MPs concentrations show different patterns in the central, southern, and northern zones of Beijing. The number concentration of MPs was the highest in the central zone (224.76 items/g), as compared with the southern zone (170.55 items/g), and the northern zone (24.42 items/g). The LDIR analysis revealed nine compositional types of MPs, including Polypropylene (PP), Polyamide (PA), Polystyrene (PS), Polyethylene (PE), Polyethylene Terephthalate (PET), Silicone, Polycarbonate (PC), Polyurethane (PU) and Polyvinylchloride (PVC), among which PP was overall dominant. The PP dominates the MPs in the central zone (76.3%), and the PA dominates the MPs in the southern zone (55.86%), while the northern zone had a diverse combination of MPs types. The morphological types of the individual MPs particle include fragments, pellets, and fibers, among which fragments are dominant (70.9%). FESEM images show the presence of aged MPs in the Beijing atmosphere, which could pose a yet unquantified health risk to Beijing's residents. The average size of the MPs in the Beijing samples is 66.62 µm. Our study revealed that the numbers of fibrous MPs increase with the decrease in size. This pollution therefore needs to be carefully monitored, and methods of decreasing the sources and mitigations developed.

Oxidative potential and water-soluble heavy metals of size-segregated airborne particles in haze and non-haze episodes: Impact of the "Comprehensive Action Plan" in China.

Air pollution is a major environmental health challenge in megacities, and as such a Comprehensive Action Plan (CAP) was issued in 2017 for Beijing, the capital city of China. Here we investigated the size-segregated airborne particles collected after the implementation of the CAP, intending to understand the change of oxidative potential and water-soluble heavy metal (WSHM) levels in 'haze' and 'non-haze' days. The DNA damage and the levels of WSHM were analyzed by Plasmid Scission Assay (PSA) and High-Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS) techniques. The PM mass concentration was higher in the fine particle size (0.43-2.1 µm) during haze days, except for the samples affected by mineral dust. The particle-induced DNA damage caused by fine sized particles (0.43-2.1 µm) exceeded that caused by the coarse sized particles (4.7-10 µm). The DNA damage from haze day particles significantly exceeded those collected on non-haze days. Prior to the instigation of the CAP, the highest value of DNA damage decreased, and DNA damage was seen in the finer size (0.43-1.1 µm). The Pearson correlation coefficient between the concentrations of water-soluble Pb, Cr, Cd and Zn were positively correlated with DNA damage, suggesting that these WSHM had significant oxidative potential. The mass concentrations of water-soluble trace elements (WSTE) and individual heavy metals were enriched in the finer particles between 0.43 µm to 1.1 µm, implying that smaller sized particles posed higher health risks. In contrast, the significant reduction in the mass concentration of water-soluble Cd and Zn, and the decrease of the maximum and average values of DNA damage after the CAP, demonstrated its effectiveness in restricting coal-burning emissions. These results have demonstrated that the Beijing CAP policy has been successful in reducing the toxicity of 'respirable' ambient particles.

COVID-19 mortality and exposure to airborne PM2.5: A lag time correlation.

COVID-19 has escalated into one of the most serious crises in the 21st Century. Given the rapid spread of SARS-CoV-2 and its high mortality rate, here we investigate the impact and relationship of airborne PM2.5 to COVID-19 mortality. Previous studies have indicated that PM2.5 has a positive relationship with the spread of COVID-19. To gain insights into the delayed effect of PM2.5 concentration (µgm-3) on mortality, we focused on the role of PM2.5 in Wuhan City in China and COVID-19 during the period December 27, 2019 to April 7, 2020. We also considered the possible impact of various meteorological factors such as temperature, precipitation, wind speed, atmospheric pressure and precipitation on pollutant levels. The results from the Pearson's correlation coefficient analyses reveal that the population exposed to higher levels of PM2.5 pollution are susceptible to COVID-19 mortality with a lag time of >18 days. By establishing a generalized additive model, the delayed effect of PM2.5 on the death toll of COVID-19 was verified. A negative correction was identified between temperature and number of COVID-19 deaths, whereas atmospheric pressure exhibits a positive correlation with deaths, both with a significant lag effect. The results from our study suggest that these epidemiological relationships may contribute to the understanding of the COVID-19 pandemic and provide insights for public health strategies.

The chemical composition and toxicological effects of fine particulate matter (PM2.5) emitted from different cooking styles.

The mass, chemical composition and toxicological properties of fine particulates (PM2.5) emitted from cooking activities in three Hong Kong based restaurants and two simulated cooking experiments were characterized. Extracts from the PM2.5 samples elicited significant biological activities [cell viability, generation of reactive oxygen species (ROS), DNA damage and inflammation effect (TNF-α)] in a dose-dependent manner. The composition of PAHs, oxygenated PAHs (OPAHs) and azaarenes (AZAs) mixtures differed between samples. The concentration ranges of the Σ30PAHs, Σ17OPAHs and Σ4AZAs and Σ7Carbonyls in the samples were 9627-23,452 pg m-3, 503-3700 pg m-3, 33-263 pg m-3 and 158 - 5328 ng m-3, respectively. Cell viability caused by extracts from the samples was positively correlated to the concentration of benzo[a]anthracene, indeno[1,2,3-cd]pyrene and 1,4-naphthoquinone in the PM2.5 extracts. Cellular ROS production (upon exposure to extracts) was positively correlated with the concentrations of PM2.5, decaldehyde, acridine, Σ17OPAHs and 7 individual OPAHs. TNF-α showed significant positive correlations with the concentrations of most chemical species (elemental carbon, 16 individual PAHs including benzo[a]pyrene, Σ30PAHs, SO42-, Ca2+, Ca, Na, K, Ti, Cr, Mn, Fe, Cu and Zn). The concentrations of Al, Ti, Mn, Σ30PAHs and 8 individual PAHs including benzo[a]pyrene in the samples were positively correlated with DNA damage caused by extracts from the samples. This study demonstrates that inhalation of PM2.5 emitted from cooking could result in adverse human health effects.

Multiple relationships between aerosol and COVID-19: A framework for global studies.

COVID-19 (Corona Virus Disease 2019) is a severe respiratory syndrome currently causing a human global pandemic. The original virus, along with newer variants, is highly transmissible. Aerosols are a multiphase system consisting of the atmosphere with suspended solid and liquid particles, which can carry toxic and harmful substances; especially the liquid components. The degree to which aerosols can carry the virus and cause COVID-19 disease is of significant research importance. In this study, we have discussed aerosol transmission as the pathway of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), and the aerosol pollution reduction as a consequence of the COVID-19 lockdown. The aerosol transmission routes of the SARS-CoV-2 can be further subdivided into proximal human-exhaled aerosol transmission and potentially more distal ambient aerosol transmission. The human-exhaled aerosol transmission is a direct dispersion of the SARS-CoV-2. The ambient aerosol transmission is an indirect dispersion of the SARS-CoV-2 in which the aerosol acts as a carrier to spread the virus. This indirect dispersion can also stimulate the up-regulation of the expression of SARS-CoV-2 receptor ACE-2 (Angiotensin Converting Enzyme 2) and protease TMPRSS2 (Transmembrane Serine Protease 2), thereby increasing the incidence and mortality of COVID-19. From the aerosol quality data around the World, it can be seen that often atmospheric pollution has significantly decreased due to factors such as the reduction of traffic, industry, cooking and coal-burning emissions during the COVID-19 lockdown. The airborne transmission potential of SARS-CoV-2, the infectivity of the virus in ambient aerosols, and the reduction of aerosol pollution levels due to the lockdowns are crucial research subjects.

The oxidative capacity of indoor source combustion derived particulate matter and resulting respiratory toxicity.

Indoor air pollution sources with emissions of fine particles (PM2.5), including environmental tobacco smoke (ETS) and incense smoke (IS) deteriorate indoor air quality and may cause respiratory diseases in humans. This study characterized the emission factors (EFs) of five types of tobacco and incense in Hong Kong using an environmental chamber. Human alveolar epithelial cells (A549) were exposed to PM2.5 collected from different indoor sources to determine their cytotoxicity. The PM2.5 EF of ETS (109.7±36.5 mg/g) was higher than IS (97.1±87.3 mg/g). The EFs of total polycyclic aromatic hydrocarbons (PAHs) and carbonyls for IS were higher than ETS, and these two combustion sources showed similar distributions of individual PAHs and carbonyls. Oxidative damage and inflammatory responses (i.e. DNA damage, 8-hydroxy-desoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6)) of A549 cells was triggered by exposure to PM2.5 generated from ETS and IS. Different indoor sources showed different responses to oxidative stress and inflammations due to the accumulation effects of mixed organic compounds. High molecular weight PAHs from incense combustion showed higher correlations with DNA damage markers, and most of the PAHs from indoor sources demonstrated significant correlations with inflammation. Exposure to anthropogenic produced combustion emissions such as ETS and IS results in significant risks (e.g. lung cancer) to the alveolar epithelium within the distal human respiratory tract, of which incense emissions posed a higher cytotoxicity.

A New Look at the Purported Health Benefits of Commercial and Natural Clays.

Clays attributed to have medicinal properties have been used since prehistoric times and are still used today as complementary medicines, which has given rise to unregulated "bioceutical" clays to treat skin conditions. Recently, clays with antibacterial characteristics have been proposed as alternatives to antibiotics, potentially overcoming modern day antibiotic resistance. Clays with suggested antibacterial properties were examined to establish their effects on common wound-infecting bacteria. Geochemical, microscopical, and toxicological characterization of clay particulates, their suspensions and filtered leachates was performed on THP-1 and HaCaT cell lines. Cytoskeletal toxicity, cell proliferation/viability (MTT assays), and migration (scratch wounds) were further evaluated. Clays were assayed for antibacterial efficacy using minimum inhibitory concentration assays. All clays possessed a mineral content with antibacterial potential; however, clay leachates contained insufficient ions to have any antibacterial effects. All clay leachates displayed toxicity towards THP-1 monocytes, while clay suspensions showed less toxicity, suggesting immunogenicity. Reduced clay cytotoxicity on HaCaTs was shown, as many leachates stimulated wound-healing responses. The "Green" clay exhibited antibacterial effects and only in suspension, which was lost upon neutralization. pH and its interaction with clay particle surface charge is more significant than previously understood to emphasize dangers of unregulated marketing and unsubstantiated bioceutical claims.

Particulate matter and SARS-CoV-2: A possible model of COVID-19 transmission.

Coronavirus disease 2019 (COVID-19), an acute respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly developed into a pandemic throughout the world. This disease is a highly infectious novel coronavirus and can affect people of all ages. Previous reports observed that particulate matter (PM) provided a platform for intermixing with viruses (i.e., influenza). However, the role of PM in SARS-CoV-2 transmission remains unclear. In this paper, we propose that PM plays a direct role as a "carrier" of SARS-CoV-2. SARS-CoV-2 is reported to have a high affinity for the angiotensin-converting enzyme 2 (ACE2) receptor. Indirectly, exposure to PM increases ACE2 expression in the lungs which facilitates SARS-CoV-2 viral adhesion. Thus, the high risk of SARS-CoV-2 in heavily polluted regions can be explained by upregulation of ACE2 caused by PM. PM could be both a direct and indirect transmission model for SARS-CoV-2 infection.

The role of airborne particles and environmental considerations in the transmission of SARS-CoV-2.

Corona Virus Disease 2019 (COVID-19) caused by the novel coronavirus, results in an acute respiratory condition coronavirus 2 (SARS-CoV-2) and is highly infectious. The recent spread of this virus has caused a global pandemic. Currently, the transmission routes of SARS-CoV-2 are being established, especially the role of environmental transmission. Here we review the environmental transmission routes and persistence of SARS-CoV-2. Recent studies have established that the transmission of this virus may occur, amongst others, in the air, water, soil, cold-chain, biota, and surface contact. It has also been found that the survival potential of the SARS-CoV-2 virus is dependent on different environmental conditions and pollution. Potentially important pathways include aerosol and fecal matter. Particulate matter may also be a carrier for SARS-CoV-2. Since microscopic particles can be easily absorbed by humans, more attention must be focused on the dissemination of these particles. These considerations are required to evolve a theoretical platform for epidemic control and to minimize the global threat from future epidemics.

Particle-induced oxidative damage by indoor size-segregated particulate matter from coal-burning homes in the Xuanwei lung cancer epidemic area, Yunnan Province, China.

Size-segregated samples of airborne particulate matter were collected at the coal-burning homes of the Hutou high lung cancer epidemic village and a comparison site Xize village of the Xuanwei County, Yuanan Province, by an Anderson Cascade Impact Sampler in winter and spring to study the toxicological characteristics of different-sized particles. The DNA damage caused by the water-soluble fractions of these size-segregated particles was analyzed by the plasmid scission assay, and the trace element compositions were determined by Inductively Coupled Plasma Mass Spectrometry. The DNA damage rate from the airborne particles in the high lung cancer incidence area was higher than that in Xize village. The different-sized particles have highly varying DNA damage rates, with the values being greater in the small size range than in the large size range. The particle-induced DNA damage rates had a significantly positive correlation with total water-soluble trace elements. Further analysis of the individual elements indicated that the water-soluble heavy metals Zn, Cu, Cd, Rb, Cs, and Sb had a positive correlation with the particle-induced DNA damage, implying that these water-soluble heavy metals played an important role in the DNA damage. The Sr had a negative correlation with the particle-induced DNA damage, suggesting that the water-soluble Sr might counter DNA damage. The mass concentrations of the total and individual water-soluble trace elements were mostly enriched in the small particle size ranges, thus implying the indoor airborne particles in the small size ranges would have a higher health risk.

Human airway construct model is suitable for studying transcriptome changes associated with indoor air particulate matter toxicity.

In vitro models mimicking the human respiratory system are essential when investigating the toxicological effects of inhaled indoor air particulate matter (PM). We present a pulmonary cell culture model for studying indoor air PM toxicity. We exposed normal human bronchial epithelial cells, grown on semi-permeable cell culture membranes, to four doses of indoor air PM in the air-liquid interface. We analyzed the chemokine interleukin-8 concentration from the cell culture medium, protein concentration from the apical wash, measured tissue electrical resistance, and imaged airway constructs using light and transmission electron microscopy. We sequenced RNA using a targeted RNA toxicology panel for 386 genes associated with toxicological responses. PM was collected from a non-complaint residential environment over 1 week. Sample collection was concomitant with monitoring size-segregated PM counts and determination of microbial levels and diversity. PM exposure was not acutely toxic for the cells, and we observed up-regulation of 34 genes and down-regulation of 17 genes when compared to blank sampler control exposure. The five most up-regulated genes were related to immunotoxicity. Despite indications of incomplete cell differentiation, this model enabled the comparison of a toxicological transcriptome associated with indoor air PM exposure.

The effects of particle-induced oxidative damage from exposure to airborne fine particulate matter components in the vicinity of landfill sites on Hong Kong.

The physical, chemical and bioreactivity characteristics of fine particulate matter (PM2.5) collected near (<1 km) two landfill sites and downwind urban sites were investigated. The PM2.5 concentrations were significantly higher in winter than summer. Diurnal variations of PM2.5 were recorded at both landfill sites. Soot aggregate particles were identified near the landfill sites, which indicated that combustion pollution due to landfill activities was a significant source. High correlation coefficients (r) implied several inorganic elements and water-soluble inorganic ions (vanadium (V), copper (Cu), chloride (Cl-), nitrate (NO3-), sodium (Na) and potassium (K)) were positively associated with wind flow from the landfill sites. Nevertheless, no significant correlations were also identified between these components against DNA damage. Significant associations were observed between DNA damage and some heavy metals such as cadmium (Cd) and lead (Pb), and total Polycyclic Aromatic Hydrocarbons (PAHs) during the summer. The insignificant associations of DNA damage under increased wind frequency from landfills suggested that the PM2.5 loading from sources such as regional sources was possibly an important contributing factor for DNA damage. This outcome warrants the further development of effective and source-specific landfill management regulations for particulate matter production control to the city.

Prolonged systemic inflammation and damage to the vascular endothelium following intratracheal instillation of air pollution nanoparticles in rats.

BACKGROUND: Exposure to air pollution is associated with cardiovascular disease, including increased morbidity and mortality rates. OBJECTIVE: The aim of this investigation was to assess the effect, in rats, of intratracheal instillation of particulate air pollution on biomarkers of leucocyte activation and vascular endothelial damage. METHODS: Air pollution particles (PM10) were instilled into rats, and blood samples were taken three days and six weeks post instillation. Plasma neutrophil elastase and Von Willebrand factor were measured by ELISA. RESULTS: Plasma neutrophil elastase increased from 175±44 ng/ml at baseline to 288±26 ng/ml 3 days post instillation (p = 0.038). vWF increased from 0.160±0.015 IU/ml at baseline to 0.224±0.015 IU/ml at 3 days post and 0.208±0.01 IU/ml at 6 weeks post (p = 0.006, ANOVA). sICAM-1 increased from 17.75±0.70 ng/ml at baseline to 19.03±0.33 ng/ml at 3 days post and 21.72±1.16 ng/ml at 6 weeks post (p = 0.009, ANOVA). CONCLUSION: Instillation caused prolonged systemic inflammation, activation of blood leucocytes and damage to the vascular endothelium.

Oxidative capacity and hemolytic activity of settled dust from moisture-damaged schools.

Exposure to moisture-damaged indoor environments is associated with adverse respiratory health effects, but responsible factors remain unidentified. In order to explore possible mechanisms behind these effects, the oxidative capacity and hemolytic activity of settled dust samples (n = 25) collected from moisture-damaged and non-damaged schools in Spain, the Netherlands, and Finland were evaluated and matched against the microbial content of the sample. Oxidative capacity was determined with plasmid scission assay and hemolytic activity by assessing the damage to isolated human red blood cells. The microbial content of the samples was measured with quantitative PCR assays for selected microbial groups and by analyzing the cell wall markers ergosterol, muramic acid, endotoxins, and glucans. The moisture observations in the schools were associated with some of the microbial components in the dust, and microbial determinants grouped together increased the oxidative capacity. Oxidative capacity was also affected by particle concentration and country of origin. Two out of 14 studied dust samples from moisture-damaged schools demonstrated some hemolytic activity. The results indicate that the microbial component connected with moisture damage is associated with increased oxidative stress and that hemolysis should be studied further as one possible mechanism contributing to the adverse health effects of moisture-damaged buildings.

The 2017 Lush Prize Awards.

This year saw eighteen prizewinning projects from eleven countries, and a new award in recognition of the work of Andrew Tyler.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

An In Vitro Versus In Vivo Toxicogenomic Investigation of Prenatal Exposures to Tobacco Smoke.

Approximately 1 million women smoke during pregnancy despite evidence demonstrating serious juvenile and/or adult diseases being linked to early-life exposure to cigarette smoke. Susceptibility could be determined by factors in previous generations, that is, prenatal or "maternal" exposures to toxins. Prenatal exposure to airborne pollutants such as mainstream cigarette smoke has been shown to induce early-life insults (i.e., gene changes) in Offspring that serve as biomarkers for disease later in life. In this investigation, we have evaluated genome-wide changes in the lungs of mouse Dams and their juvenile Offspring exposed prenatally to mainstream cigarette smoke. An additional lung model was tested alongside the murine model, as a means to find an alternative in vitro, human tissue-based replacement for the use of animals in medical research. Our toxicogenomic and bio-informatic results indicated that in utero exposure altered the genetic patterns of the fetus, which could put them at greater risk for developing a range of chronic illnesses in later life. The genes altered in the in vitro, cell culture model were reflected in the murine model of prenatal exposure to mainstream cigarette smoke. The use of alternative in vitro models derived from human medical waste tissues could be viable options to achieve human endpoint data and conduct research that meets the remits for scientists to undertake the 3Rs practices.

Alternative approaches for acute inhalation toxicity testing to address global regulatory and non-regulatory data requirements: An international workshop report.

Inhalation toxicity testing, which provides the basis for hazard labeling and risk management of chemicals with potential exposure to the respiratory tract, has traditionally been conducted using animals. Significant research efforts have been directed at the development of mechanistically based, non-animal testing approaches that hold promise to provide human-relevant data and an enhanced understanding of toxicity mechanisms. A September 2016 workshop, "Alternative Approaches for Acute Inhalation Toxicity Testing to Address Global Regulatory and Non-Regulatory Data Requirements", explored current testing requirements and ongoing efforts to achieve global regulatory acceptance for non-animal testing approaches. The importance of using integrated approaches that combine existing data with in vitro and/or computational approaches to generate new data was discussed. Approaches were also proposed to develop a strategy for identifying and overcoming obstacles to replacing animal tests. Attendees noted the importance of dosimetry considerations and of understanding mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Recommendations were made to (1) develop a database of existing acute inhalation toxicity data; (2) prepare a state-of-the-science review of dosimetry determinants, mechanisms of toxicity, and existing approaches to assess acute inhalation toxicity; (3) identify and optimize in silico models; and (4) develop a decision tree/testing strategy, considering physicochemical properties and dosimetry, and conduct proof-of-concept testing. Working groups have been established to implement these recommendations.