Lung tumor susceptible male A/J mice, age 5–7 weeks, were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in an AAALAC-accredited, specific pathogen-free, environmentally controlled facility. Mice were housed two per cage in ventilated cages and provided HEPA-filtered air under a controlled light cycle (12 h light/12 h dark). Animals were acclimated to the animal facility for 1 week and allowed access to a conventional diet (6% Irradiated NIH-31 Diet, Harlan Teklad, Madison, WI) and filtered tap water ad libitum. All animal studies were approved by the Centers for Disease Control-Morgantown Institutional Animal Care and Use Committee and applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Mice were weight- and age-matched and organized into three groups (n = 80/group; sham, Fe₂O₃, and CaCrO₄). Mice were exposed by oropharyngeal aspiration, as previously described (Falcone et al., 2018b; Rao et al., 2003), for a period of 26 weeks at a frequency of one time per week (Fig. 1, panel A). Two hundred six mice remained after the exposure period. Body weights were recorded weekly throughout the entire 70 week experimental protocol.

Doses for the CaCrO₄ and Fe₂O₃ were 100 μg (cumulative 2.6 mg) and 1 mg (cumulative 26 mg) per exposure, respectively. The relevancy of the Fe₂O₃ dose was approximated using previous estimates for mouse studies of welding fumes (Erdely et al., 2011). Using 5 mg/m³, the NIOSH and American Conference of Governmental Industrial Hygienists (ACGIH) exposure limits for iron oxide, a ventilation rate of 20 L/min, exposure for 8 h/day and an alveolar deposition of 16 %, the daily estimated alveolar deposition in a human would be 7.7 mg (Raabe et al., 1988). Using surface area (102 m² for a human and 0.05 m² for a mouse) the equivalent in a mouse would be 3.77 μg/day. The mouse was given 26 mg over 26 weeks representing 27.6 years of human exposure working 8 h/day at the exposure limit of 5 mg/m³. Therefore, the exposure dose is roughly equivalent to a working lifetime. The positive control of CaCrO₄ was 1/10 the dose of Fe₂O₃ or 2.6 mg cumulative dose. This was expected to provide a positive result based on previous Cr studies (Beaver et al., 2009; Steinhoff et al., 1986; Zeidler-Erdely et al., 2008), consistent with the fact the exposure limit of hexavalent Cr [Cr(VI)] is 1/1000, or 5 μg/m³. The metal oxide suspensions were prepared in 50 μL USP-grade calcium and magnesium-free phosphate buffered saline (PBS) and sham animals were exposed to 50 μL of the vehicle using the same exposure regime. CaCrO₄ (product number CDS001277; 156.07 g/mol) and Fe₂O₃ (product number 310050; 159.69 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO). Scanning electron microscopy images of the particles are shown in Fig. 1, panel B and C. Additional characterization including specific surface area, hydrodynamic diameter, and zeta potential was done previously (Falcone et al., 2018b).

Animals were sacrificed at 70 weeks after the first exposure by an overdose of sodium pentobarbital euthanasia solution (Fatal Plus; 100–300 mg/kg intraperitoneal; 390 mg/mL; Henry Schein; Dublin, Ohio) then weighed. Once unresponsive to a toe pinch, mice were euthanized by exsanguination and the abdomen and thoracic cavity were opened and examined for any abnormalities. Grossly observed lung tumors were counted at sacrifice as described previously (Falcone et al., 2017). Gross lung images were taken using an Olympus DP21 digital camera (Olympus America; San Jose, CA). For histopathology analysis, formalin fixed whole lung tissue were embedded in paraffin then a 5 μm standardized section was cut. Slides were stained with hematoxylin and eosin and interpreted by a contracted board-certified veterinary pathologist. Histopathologic lesions were classified using standard published INHAND terminology (Renne et al., 2009). Neoplastic findings were recorded as present and the number of tumors/section were recorded and non-neoplastic histopathologic findings were graded and recorded using the grading scale derived from Mann et al. (2012). Non-neoplastic findings were scored on the following severity scale: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Foreign material and mineralization were only recorded as either present or not present.

Animals that survived to 70 weeks and were without tumors elsewhere besides the lung were included in the final analyses. Statistical analyses were done using JMP version 12.4 and SAS version 9.4 for Windows (SAS Institute; Cary NC). Histopathological findings using the graded scale were analyzed using nonparametric Kruskal Wallis tests followed by Wilcoxon Rank Sum tests for pair-wise comparisons. Lung tumor incidence was analyzed using a Chi-square test in SAS ‘Proc Freq,’ utilizing Fishers Exact Test, while tumor multiplicity was analyzed using Poisson regression in SAS ‘Proc Genmod’. In cases where overdispersion existed, a negative binomial regression was performed. Body weight measures were analyzed using repeated measures analysis of variance with time as the repeated factor. Additionally, body weight data were evaluated using analysis of covariance with time as the covariate to compare the slopes of the growth curves. For all analyses, a p-value of < 0.05 was set as the criterion for significance.

During the post-exposure period 13 sham, 13 CaCrO₄, and 8 Fe₂O₃ mice were either euthanized because of ongoing morbidities or found dead. Morbidities included abscesses, pelvic/abdominal/spinal masses, and gastrointestinal bleeds. By 70 weeks, 172 mice remained. The final dataset for the gross tumor counts and body weight analysis includes 156 animals. Eleven mice were removed due to tumors found elsewhere besides the lung and five sham mice were removed because of inadvertent dosing with CaCrO₄. Body weight analysis of the slopes of the growth curves revealed that the growth rate increase over time was significantly lower in the CaCrO₄ and Fe₂O₃ groups compared to sham (Fig. 2).

Gross images of CaCrO₄- and Fe₂O₃-exposed (panel A and B, respectively) mouse lungs 24 h post-fixation are shown in Fig. 3. The black arrows indicate lung tumors which were opaque and had white coloration. Fe₂O₃-exposed lungs were consistently red in color with black deposits throughout while particle deposition was only occasionally visible upon gross exam in the CaCrO₄-exposed lungs. At 70 weeks, oropharyngeal aspiration exposure to CaCrO₄ or Fe₂O₃ significantly increased gross-observed lung tumor multiplicity (average tumor number/mouse lung ± SE) compared to sham (Table 1). Multiplicity in the CaCrO₄ and Fe₂O₃ groups were 9.63 ± 0.55 and 3.35 ± 0.30, respectively, compared to 2.31 ± 0.19 in the sham animals. As expected, gross tumor incidence was not different among the groups and was 90, 94, and 100 % in the Fe₂O₃, sham, and CaCrO₄ groups, respectively, at 77–79 weeks of age. Grossly observed background tumor frequency in the A/J mouse, as reported in the literature, can range from 31 to 40% between 43–53 weeks of age and continue to increase to near 100 % by approximately 2 years of age (Curtin et al., 2004; Groch et al., 1997; Witschi et al., 2004). The spontaneous tumor rate in this model is a known limitation and a less reliable indicator of carcinogenicity compared to lung multiplicity. Therefore, multiplicity remains the primary indicator of a positive tumorigenic response in the A/J mouse (Shimkin and Stoner, 1975; Witschi, 2005).

It was shown by histopathological evaluation (Table 2) that the predominant neoplastic lesions observed in the lungs were bronchiolo-alveolar adenomas and carcinomas (BAA and BAC, respectively). These tumor types were the most prevalent in the CaCrO₄ group (30 tumors in 47 mice), followed by the Fe₂O₃ (21 tumors in 46 mice), and least prevalent in the sham group (11 tumors in 38 mice). In the shams, BAA were generally 3–5 mm in diameter, well-circumscribed, round, expansile, and densely cellular neoplasms comprised of tubules and papillary structures lined by uniform cuboidal neoplastic epithelial cells. BAC were typically > 5 mm in diameter, well- to poorly-circumscribed, irregularly shaped, expansile, and densely cellular neoplasms composed of larger, pleomorphic, more mitotically active neoplastic epithelial cells arranged in tubules, trabeculae, papillary structures, and/or less defined, solid areas. In both exposed groups, BAA were similar to those described in the sham animals. BAC were also similar to those of the sham group, but were sometimes larger (15–20 mm diameter) with areas of necrosis and scirrhous reaction (Fig. 3, panels C&D).

Tumor multiplicity and incidence was statistically significant for BAC, as well as BAA, in the CaCrO₄ group when analyzed individually and in combination. While gross-observed tumor multiplicity reached statistical significance in the Fe₂O₃ group, the histopathological analysis for BAC and BAA + BAC did not. Fe₂O₃ caused a 59 % increase in BAA + BAC and a doubling of BAC (p = 0.085), however, which indicates a trend toward an increase in the malignant tumor type in this group compared to sham. It should be noted that the sample size evaluated microscopically was less than the gross-observed because selected lungs were flash-frozen for later analysis at sacrifice. This likely decreased the overall statistical power to detect a significant difference for the histopathological evaluation of the Fe₂O₃ group. Incidences of cystic keratinizing epithelioma, nonkeratinizing epithelioma (2 CaCrO₄ mice) and lymphoma (1 sham mouse) were isolated and not significantly different (data not shown).

Similar to tumor multiplicity and incidence, inflammation and injury in the lung followed the same pattern with the CaCrO₄ group the most severe followed by Fe₂O₃ (Table 3). No associated lung injury with PBS exposure was found in the sham group. In the CaCrO₄- and Fe₂O₃-exposed groups, the inflammation was significantly increased compared to the sham group and was typically associated with the hyperplastic or neoplastic lesions, but was also associated with foreign material, particularly in the Fe₂O₃ group. In the CaCrO₄ group, inflammation was chronic-active and ranged from minimal to marked in severity and was characterized by numerous macrophages, viable and degenerate neutrophils, multinucleated giant cells, and fewer lymphocytes and plasma cells within alveoli and occasionally bronchioles. Chronic-active inflammation was occasionally associated with basophilic spicules or globules of foreign material (e.g., CaCrO₄) that was found in 17 % of the lungs. In the Fe₂O₃ group, chronic alveolar inflammation ranged from minimal to moderate in severity and was characterized predominantly by heavily black pigment-laden macrophages (e.g. Fe₂O₃) with fewer numbers of neutrophils, lymphocytes, and plasma cells. Fe₂O₃, reported as foreign material characterized by abundant extracellular and intracellular black granular pigment, was observed in 46 of 46 lungs (100 %) at 70 weeks. In comparison, the chronic alveolar inflammation in the sham group, likely due to the spontaneous lung tumors, was minimal in severity and characterized predominantly by macrophages with variably abundant eosinophilic cytoplasm.

Type II pneumocyte hyperplasia was another significant non-neoplastic finding in the CaCrO₄ group (Table 3). This hyperplasia was peribronchiolar to subpleural in distribution, ranged from minimal to marked in severity, and was characterized by a single layer of cuboidal epithelial cells (type II pneumocytes) lining the alveolar septae. Other predominant findings were bronchiectasis and mineralization. Bronchiectasis ranged from minimal to moderate in severity and was characterized by dilation of the bronchial/iolar lumens occasionally associated with intraluminal mucopurulent exudate. Mineralization, indicative of repeated tissue damage, which was characterized by mineral within bronchial/iolar or alveolar walls and/or associated with chronic-active inflammation.

In the Fe₂O₃ group, significant non-neoplastic findings included hyperplasia of the mucosa-associated lymphoid tissue (MALT) and atelectasis (Table 3). Increased MALT hyperplasia, which ranged from minimal to moderate in severity, was likely a reactive lymphoid response to the exposure. The atelectasis was likely associated with increased inflammation, damage to the pulmonary parenchyma and interference with gas exchange leading to alveolar collapse. Overall, the increased bronchiectasis, mineralization, and atelectasis were presumably reactive responses to CaCrO₄ and/or Fe₂O₃ exposure and/or increased numbers of lung tumors.