A simplified diagram of the automated countertop cutting assembly is depicted in Figure 1, and a photograph with components labeled the automated countertop cutting inhalation exposure system is presented in Figure 2. Custom exposure system software automatically controlled chamber air flows, particle concentration, and exposure duration. The software also monitored exposure chamber temperature, relative humidity, and CO₂ levels. During exposures, the exposure chamber exhaust airflow was held constant at 30 L/min. This level of airflow was sufficient to maintain CO₂ levels below 2,500 PPM as recommended by NIOSH Morgantown’s internal animal and care use committee. Air entered the top of the exposure chamber either from being drawn through an airtight chamber that housed the automated saw assembly or from a mass flow controller (MCR-50, Alicat Scientific, Tucson AZ) that provided filtered air. Typically, the dilution air flow was set to zero by the exposure system software during exposures until the target exposure time was reached. Immediately before the air entered the exposure chamber it traveled through a cyclone (Model 2000, URG, Chapel Hill, NC), to remove particles with aerodynamic diameters larger than 6 µm.

The aerosol mass concentration inside the exposure chamber was continuously monitored with a Data RAM (DR-40000 Thermo Electron Co.) and gravimetric determinations (37 mm cassettes with 0.45 µm pore-size Teflon filters, (1 L/min sample flow)) were used to calibrate and verify the Data RAM readings during each exposure run. For this study the target concentration was a constant 20 mg/m³ over 4-h long exposures. Single day and four consecutive day exposures were conducted. The custom software automatically determined when to saw and when to stop based on exposure chamber concentration readings. The parameters of this algorithm (S1), length of cut, and error limits were optimized by trial and error over several test exposure runs with an empty exposure chamber. Figure 3 shows a plot of the exposure chamber concentration versus time during one of the inhalation exposure runs. The short duration spike at the end of the exposure was from the dilution air being automatically set to maximum flow. This change in flow would often cause particles to break loose from tubing walls. Even with this spike, using the dilution air cut the time required to reduce the concentration at the end of an exposure run.

Exposure chamber pressure was monitored using a differential pressure transducer (Model 264, Setra Systems, Inc. Boxborough MA). Under typical exposure conditions dilution air was set to zero and the pressure inside the exposure chamber was −6.35 cm-H₂O to ambient. This slight negative pressure would pull air from the saw assembly housing chamber into the exposure chamber via conductive silicone tubing (78.7-mm ID). The temperature and relative humidity inside the exposure chamber were also continuously monitored by using an electronic probe (HMP60, Vaisala Corporation, Helsinki Finland).

A 3D rendering of the automated cutting assembly is shown in Figure 1. Two linear actuators were used (Movopart M75 model, Thomson Linear motion system), driven with stepper motors (Powerpak K3 series, Kollmorgen). A pair of Kollmorgen P6000 stepper drivers were used to interface the custom exposure software with the stepper motors. The first motor (X-axis) moved the countertop away from or in front of the circular saw. The second motor (Y-axis) moved the circular saw to cut through the countertop. Limit switches were used to detect home positions and ensure a safe range of motion. The circular saw (Model number DWE575SB, DeWALT, Towson MA) used anOshlun (SBNF-072560) 18.42 cm diameter 60 teeth, aluminum cutting, professional grade C-1 carbide tip, triple chip grind with 1.59 cm arbor blade. This blade is recommended for cutting solid surface countertops. A 1.75 mm width, the approximate width of the blade, was shaved off the end of the countertop during each cut. This was done to avoid producing large solid sections of cutoff material that could obstruct subsequent cutting. The type of countertop used in our experiments was Dupont Corian light gray in color and measured 76.2-by-38.1-by-1.27 cm.

The automated cutting assembly was housed in a custom airtight 16-gauge stainless steel chamber with a removable clear polycarbonate front door as shown in Figure 2. A silicon gasket and several latches ensured an airtight seal between the door and the chamber. Sound-deadening material was affixed to the outside of the chamber to reduce the noise generated by cutting. Air entered the saw assembly housing chamber on the far-left side by first passing through a HEPA filter. Air with countertop particles exited the cutting assembly chamber from a port located on top of the chamber approximately 0.3 m from the cutting blade. The saw housing chamber also acted as a cart body that held the exposure chamber, mass flow controllers, and all interface electronics. The entire system was on wheels and designed to fit inside an 8-foot walk-in fume hood.

An airtight 55.9 by 55.9 by 50.8-cm (L × W × H) exposure chamber was constructed out of 16-gauge stainless steel with a clear polycarbonate door. A stainless-steel cage rack that could hold up to 36 mice in individual cage partitions was used to house the mice during exposures. The cage rack rested on top of cage support beams which were 0.95 cm outside diameter stainless steel tubes with small holes (3.3 mm diameter) drilled into the undersides. The tubes were connected to house vacuum through a mass flow controller (MCRW-50-DS, Alicat Scientific, Tucson AZ). Each hole was placed at the center of each cage partition such that aerosols would be drawn to each animal’s breathing space. Air entered from the top center of the chamber through a dispersion nozzle to aid aerosol mixing. This type of chamber, the Cube 150, was previously described by Goldsmith et al. (2011). The Cube 150 was previously tested for concentration homogeneity within the animal cage partitions using aerosol and was reported to have less than 5% variation between all animal locations within the chamber. The exposure chamber air flow rate was 30 L/min. Aerosol homogeneity in the exposure chamber is achieved through several methods. First, the inlet nozzle is designed to disperse aerosol evenly throughout the chamber, second, exhaust ports placed below each individual animal cage draw air evenly through the breathing space of each animal. Additionally, during exposures that span multiple days, animal location within the chamber is randomized each day.

Aerodynamic particle size distribution (count based) data were collected (APS Model 3321, TSI Inc., Shoreview, MN) while cutting at several different saw travel speeds. The samples were taken inside the inhalation exposure chamber a few cm above the animal cages, and exposure chamber concentration was held constant at 20 mg/m³ during the sample collection. Each sample lasted 5 min. The sample flow rate for the APS was 5 L/min, and its detectable size range is from 0.5 to 20 µm. This data were collected to determine if travel saw speed had a significant effect on the respirable particle size distribution. A second aerodynamic particle sizing instrument with a smaller size detection range (0.07 to 10 µm), but a smaller total number of size bins, was also used to collect data (ELPI Model ELPI classic, Dekati Ltd., Finland). A 5-min sample was taken from inside the exposure chamber with the ELPI at a cutting speed of 2.5 mm/sec. The ELPI’s sample flow was 10 L/min. Mass-based aerodynamic particle size distribution was determined in the exposure chamber by using a Micro-Orifice Uniform Deposit Impactor (MOUDI, MSP Model 110 R, MSP Corporation, Shoreview, MN). Cutting speed for this sample was 2.5 mm/sec, and its sample flow was 10 L/min. The countertop cutting particles inside the exposure chamber were sampled at 1 L/min onto polycarbonate filters (Whatman, Clinton, PA) and imaged with a scanning electron microscope (FE-SEM; Hitachi, S-4800, Tokyo, Japan). All of these sizing samples were taken without animals inside the chamber while the system ran in the same manner as an exposure run.

Male C57BL/6J mice (4 weeks old, 20–25 g) were purchased from The Jackson Laboratory (Bar Harbor, ME), housed in ventilated polycarbonate cages, and acclimated for at least 7 days before the study began. The animals were provided HEPA-filtered air, irradiated Teklad 2918 diet (Harlan, Madison WI), a combination of ALPHA-dri and Teklad sani-chips as bedding, and tap water ad libitum. Mice were housed in two per cage with a 12-h light-dark cycle, and the facility was maintained at 22 ± 2 °C and 40–60% humidity. The study protocol was reviewed and approved by the CDC-Morgantown Institutional Animal Care and Use Committee. The CDC-Morgantown Institutional Animal Care and Use Committee is accredited by AAALAC International.

Two separate exposures were conducted for this study. In the first exposure, following acclimatization, the animals (n = 6) were exposed by whole-body inhalation to HEPA- and carbon-filtered air in the exposure chamber with the saw running but not cutting, or an average concentration of 20 mg/m³ SSC-emissions, for 4 h. Immediately following exposure or 24 h-post, the mice were euthanized following an intraperitoneal injection of 100–300mg sodium pentobarbital/kg body weight (Fort Dodge Animal Health; Fort Dodge, IA). In the second exposure, the animals were randomly divided into two groups (n = 9 per group) and exposed by whole-body inhalation to HEPA- and carbon-filtered air in the exposure chamber with the saw running but not cutting, or an average concentration of 20 mg/m³SSC-emissions, for 4 h per day for 4 days. Animals from the second exposure were euthanized at 1- and 56-days post-exposure. Upon euthanasia following both exposures, bodyweight was measured and whole blood was collected via cardiac puncture and transferred to a vacutainer containing EDTA (Becton-Dickinson; Franklin Lakes, NJ) for whole blood hematological measurements. Each group was divided into subgroups, with the right lungs (n = 4)frozen for biochemical analysis and the left lung fixed for histopathology. Bronchoalveolar lavage was performed on the remainder (n = 5, BALF collection further described below).

Tissue Al concentrations were determined by Galbraith Laboratories, Inc. (Knoxville, TN), using inductively coupled plasma (ICPor ICP–atomic emission spectrometry (AES)). Briefly, duplicate samples of 150–300 mg of lung tissues were digested in concentrated nitric acid with the addition of hydrogen peroxide and heating in a digestion block and analyzed using ICP–AES (Galbraith method ME-70) using a PerkinElmer Optima 4300 (PerkinElmer, Waltham, MA). The instrument quantitation limit for the analysis was 0.0154 mg/L.

Bronchoalveolar lavage of the whole lung was performed by inserting a cannula into the trachea and gently flushing with Ca²⁺ and Mg²⁺ free phosphate-buffered saline (PBS), pH 7.4. The initial whole lung lavage was performed twice using 0.6 mL PBS for a collection volume of 1.2 mL, which was processed for cytokine and LDH activity analysis. Three subsequent lavages were performed using a 1 mL aliquot per lavage for a collection volume of 3 mL, which was utilized for cell differential quantification. BALF cells were collected through centrifugation (1,500 × g, 5 min, 4 °C) of the first and subsequent lavages, cell pellets were combined, washed with 1 mL PBS, and centrifuged (1,500 × g, 5 min, 4 °C). BALF cells were re-suspended with 250 µL PBS and total cell counts were determined using a Coulter Counter (Multisizer 4, Beckman Coulter, Brea CA). Cytospins of the BALF cells were generated using a Shandon Cytospin 4 (Thermo Fisher; Waltham, MA) and stained with modified Wright stain (Thermo Fisher). Cytospins were analyzed for cell differentials using an Olympus AX70 light microscope (Tokyo, Japan) coupled with Olympus DP73 camera, and analyzed using the Olympus CellSens Dimension program. Two-hundred cells per slide were counted.

Total protein and LDH activity were evaluated in the first acellular fraction of BALF. Analyses were conducted on the Synergy H1 Microplate Reader (BioTek; Winooski, VT) utilizing the Pierce BCA Protein Assay Kit (Fisher Scientific; Waltham, MA) for protein quantification and the Lactate Dehydrogenase Reagent Set (Pointe Scientific; Lincoln Park, MI) for LDH activity determination.

The levels of pro- and anti-inflammatory cytokines in lung BALF were measured using a V-PLEX Pro-inflammatory Mouse panel(MSD; Meso Scale Discovery, Rockville, MD) according to the manufacturer protocol. IL1-β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p7- tumor necrosis factor alpha, (TNFα), interferon (IFN)-γ, and neutrophil-activating protein 3 (KC/GRO), were measured from undiluted BALF. Data were acquired using a QuickPlex SQ 120 plate reader (MSD).

Lung and nasal cavity were fixed in 10% neutral buffered formalin (NBF), embedded in paraffin, cut at 5 mm and stained with Hematoxylin and Eosin (H&E) for histopathological evaluation. Additional sections of lung were stained with Picrosirius Red. All glass slides were submitted to Experimental Pathology Laboratories (EPL), Inc., Durham, NC for examination by a board-certified pathologist using bright field and polarizing light microscopy (for visualizing particles). Findings were graded from one to five (1 = minimal; 2 = mild; 3 = moderate; 4 = marked; and 5 = severe), depending upon severity. A description of the scoring system can be found in S11.

Whole blood collected from each mouse (n = 9/group)was transferred to a tube containing EDTA as an anticoagulant. Complete blood count (CBC) tests were performed post-exposure to evaluate hematological parameters, which included peripheral erythrocyte and leukocyte counts, leukocyte differentials (percent lymphocytes, neutrophils, monocytes, basophils, and eosinophils), platelet counts, mean platelet volume (MPV), hemoglobin levels, hematocrit, mean corpuscular hemoglobin (MCH) and hemoglobin concentration (MCHC), red blood cell distribution width (RDV), reticulocyte counts, mean platelet volume (MCV) using a ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc., Westbrook, ME).

The right lung from each non-lavaged animal was homogenized with a Bead Mill 24 Homogenizer (Fisher Scientific International, Inc.; Hampton, NH) for 2 min at 4 °C in 1 mL cold PBS (pH 7.4) containing protease inhibitor cocktails and EDTA (Halt Protease Inhibitor Cocktails, Thermo Scientific, Waltham, MA). Data were acquired using a Synergy H1 Microplate Reader (BioTek, Winooski, VT). Tissue superoxide dismutase (SOD) activity was measured using ab65354 Superoxide Dismutase (SOD) Activity Assay Kit (Colorimetric) (Abcam, Cambridge, United Kingdom) and antioxidant capacity was measuring using Total Antioxidant Capacity (TAC) Assay Kit (ab65329). The values for each sample were normalized to sample total protein content determined using the Pierce BCA Protein Assay Kit (Fisher Scientific International, Inc., Hampton, NH).

Two-way ANOVA and subsequent t-tests on the main and interaction effects were performed. If one or more of the necessary ANOVA assumptions were violated, then a Kruskal Wallis test as an omnibus test and a Wilcoxon test on the main and interaction effects was used. For endpoints where some values fell below the assay lower limit of quantification (LLOQ), those values were replaced by the given LLOQ value divided by the square root of two.

The aerodynamic particle sizing data taken (APS Model 3321) from inside the exposure chamber, while cutting at several different saw travel speeds (0.5, 1, 2 and 4 mm/s), is shown in Figure 4(A). Saw travel speeds ranging from 0.5 mm/s to 4 mm/s did not have a substantial effect on the exposure chamber particle size. The count median aerodynamic diameter (CMAD) was 830 nm. The ELPI instrument was also used to collect particle size data inside the exposure chamber to determine if there was a second mode of particles less than 500 nm. The cutting speed during the ELPI data collection was 2.5 mm/s. These data are given in Figure 4(B). The ELPI software indicated a CMAD of 820 nm, with particles ranging in aerodynamic size from 0.1 to 5 µm.

The APS and ELPI both provide count-based size distributions. A micro-orifice uniform deposit impactor (MOUDI) was used to determine the mass-based aerodynamic particle size distribution during typical exposure run conditions, 2.5 mm/s cutting speed and 20 mg/m³ concentration. These data are plotted in Figure 5. The mass median aerodynamic diameter was 1.78 µm with a geometric standard deviation of 2.75. The red line in the plot is a log-normal distribution curve fitted to the data.

Scanning electron microscope (SEM) images of particles collected from the exposure chamber are shown in Figure 6. The small dark circles on the surface are pores in the filter material and the particles are deposited on top. The particles seem to have sharp edges and complex shapes, with rectangular being the most common. Physical particle sizes ranged from 0.2 to 4 µm in geometric diameter, with 1 to 2 µm being the most common. When single particles are inspected closely, they seem to be made up of thin sheet-like layers. SEM energy dispersive X-ray analysis (EDX) was used to verify the particles contained Al as expected (not presented).

The aerosol mass concentration inside the exposure chamber was continuously monitored with a Data RAM (DR-40000 Thermo Electron Co.) and gravimetric determinations (37 mm cassettes with 0.45 µm pore-size Teflon filters, 1 L/min. sample flow) were used to calibrate and verify the Data RAM readings during each exposure run. For this study the target concentration was a steady 20 mg/m³ over 4-h long exposures. Single day and four consecutive day exposures were conducted. There were two separate single day exposures and the gravimetric data over these two days yielded an average concentration of 19.9 mg/m³ with a standard deviation of 1.5 mg/m³. For the 4-day exposures, the average daily concentration was 20.1 mg/m³ with a standard deviation of 1.2 mg/m³.

ICP-AES analysis of frozen whole lung tissue indicated that the mean Al concentration in the control animals was 2.33 ± 0.26 µg/g wet weight (Figure 7). At 0 h post-exposure the concentration was 19.13 ± 5.03 µg/g. At 24 h post-exposure, the Al concentration had decreased to 15.17 ± 2.26 µg/g, although this value was not significantly different from the 0-h concentration. Because elemental Al only comprises roughly 30% of the SSC composite (Qi et al. 2016), the actual lung burden can be estimated at 64 and 51 µg/g for the 0- and 24-h timepoints, respectively. The average lung weight for the 0 h exposure group was 205 mg, yielding an average of 13.2 µg of SSC lung deposition per animal, per 4-h day of exposure.

H&E-stained tissue sections of left lung (LL) and nasal cavity (T1, T2, T3, and T4) were examined for routine histopathology using bright field microscopy and with polarized light to screen for birefringent particle deposition. Tissues from four animals from each post-exposure time point were examined. Filtered air-exposed mice were used as controls. Microscopic abnormalities were not detected in lung or nasal cavity of any animals examined, at any timepoint, suggesting that SSC exposure did not cause acute toxicity or an inflammatory response in lung or nasal cavity tissue. In addition, test article was not observed in lung or nasal cavity of exposed mice using either brightfield or polarizing light microscopy. Given that the SSC particles generated were small (1 µm diameter) and colorless, it is not unexpected that they could not be visualized with routine light microscopy and the relatively low amount of material deposited likely resulted in very few particles per histology slide to observe. Representative histopathologic micrographs are presented in Supplemental Figures 2–5.

A CBC assay is a valuable tool in murine toxicology studies. It evaluates hematological parameters, assessing hematotoxic effects, organ function, coagulopathy, and overall health. This comprehensive assessment helps understand the toxic potential of various substances. A small decrease in the percentage of circulating monocytes and eosinophil number was observed compared to controls at 56 days post-exposure. Migration to the lungs in response to pulmonary injury could lead to a transient decrease in monocyte and eosinophil peripheral blood count; however, the lack of observed inflammation in the lung makes this unlikely. It is possible that there may be effects of the SSC exposure on hematopoiesis or cell survival that could contribute to the observed change. The results from the CBC analysis are listed in Table 1.

Lung tissue TAC and SOD activity are critical markers of oxidative stress, a common mechanism of pulmonary toxicity. TAC reflects the total antioxidant capacity, while SOD specifically neutralizes superoxide radicals. Elevated LDH levels in BALF indicate tissue damage, often associated with pulmonary inflammation. BALF total protein serves as a marker of alveolar-capillary barrier dysfunction, a hallmark of lung injury. No changes in lung tissue TAC, SOD activity, or BALF LDH activity were observed, however BALF total protein content was elevated compared to control in the Day 1 animals (Figure 8).

The measurement of BALF cytokines provides crucial insights into the immunological response to lung injury. IFN-γ, IL-1β, IL-6, and TNF-α are pro-inflammatory cytokines involved in initiating and amplifying the inflammatory response. IL-2, IL-4, IL-5, and IL-10 are associated with adaptive immune responses, including T-cell activation and differentiation. IL-12p70 is a key regulator of Th1 responses, while KC/GRO is a chemokine involved in neutrophil recruitment.IL-2, IL-4, IL-10, and IL-12p70 were below the assay lower limit of quantification, and no differences were no differences in any other BALF cytokine at either time point.

Assessing total cells, macrophages, PMNs, and lymphocytes in the BLAF provides information about the pulmonary inflammatory response to particlate inhalation. Total cell counts indicate the overall extent of inflammation, while differential cell counts reveal specific immune cell populations involved. Macrophages are key players in initiating and resolving inflammation, PMNs are recruited for phagocytosis of debris and pathogens, and lymphocytes, including T cells and B cells, contribute to adaptive immune responses. No changes at any timepoint were detected in BALF cell counts (Figure 9).