The experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (Number 21–01-0170). Thirty-five C57BL/6NCrlOri male mice (6-week-old) were obtained from Orient Bio Inc. (Seongnam, Korea). The mice were housed in polycarbonate cages (135W × 280L × 145H mm) in an environmentally controlled animal facility with 22 ± 3 °C controlled temperature, 50 ± 10% relative humidity, 150–300 Lux light intensity, and a 12 h light/dark cycle. The air ventilation system in the animal room was operational 10–20 times/h.

Based on the most recently measured body weight (BW), mice were randomly assigned to the vehicle control group, magnesium nitrate, Kathon, and Proclin 200 exposed group using the Pristima System (Version 6.4; Xybion Medical System Co., Lawrenceville, NJ, USA), a toxicology data management program. The study consisted of five groups defined by their specific exposure profile (each group, n = 7): a vehicle control group, two magnesium nitrate exposed groups of 3% and 22.6% concentrations, a Kathon and a Proclin 200 exposed groups of 1.14 mg active ingredient (a.i.)/kg dose.

The constituents of CMIT/MIT-containing products are shown in Table 1. The concentrated stock solution of Kathon CG (1.519% CMIT/MIT, 22.6% magnesium nitrate from DOW Chemical Com., Midland, MI, USA) and Proclin 200 (1.5% CMIT/MIT, 3% magnesium nitrate from Sigma-Aldrich, St. Louis, MO, USA) was diluted in saline to create equivalent doses of 1.14 mg a.i./kg, which were intratracheally instilled using a modified automatic video instillator (Doobae System, Seoul, Korea) at 50 μL/head. The dose of magnesium nitrate was calculated based on the amount of magnesium nitrate contained therein after the dilution of Kathon CG and Proclin 200. All substances including saline, magnesium nitrate, Kathon CG, and Proclin 200 were administered six times on days 1, 4, 6, 8, 11, and 13. Before the intratracheal instillation (ITI), the mice were exposed to 2.5% isoflurane and instillation was performed immediately.

Twenty-four hours after the last ITI dosing, the mice were euthanized by continuous exposure to an overdose of isoflurane until one minute after they stopped breathing. Weight of the left lung was recorded, and the lung samples were fixed in 10% formalin or stored in a -70 °C deep freezer until analysis.

After the mice were anesthetized, the left lungs were ligated, and the right lungs were gently lavaged thrice via a tracheal tube using phosphate-buffered saline (PBS, Thermo Fisher Scientific Inc., Waltham, MA, USA). Using a NucleoCounter (NC-250; ChemoMetec, Gydevang, Denmark), the total cells of the collected BALF were counted. For differential cell counts, BALF cell smears were prepared using Cytospin (Thermo Fisher Scientific Inc.) and stained with Diff-Quik solution (Dade Diagnostics, Aguada, Puerto Rico). The different cell types were counted (n = 200/slide). BALF was immediately centrifuged at 452 rcf for 5 min, and the collected supernatant was stored at – 70 ℃ until the cytokine levels were measured through enzyme-linked immunosorbent assay (ELISA). Interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor-alpha (TNF-α) were quantified through ELISA using commercial kits (Thermo Fisher Scientific Inc.), according to the manufacturer’s protocol.

The left lung tissues were fixed in 10% neutral-buffered formalin and staining was performed at Korean Pathology Technical Center (KP&T, Cheongju, Korea). Briefly, the paraffin-embedded tissue blocks were cut into 4 μm sections and stained with Hematoxylin & Eosin (H&E), Periodic acid-Schiff (PAS), and Masson’s trichrome (MT) (Sigma-Aldrich, St. Louis, MO, USA) solutions. Stained sections were analyzed under a light microscope (Axio Imager M1; Carl Zeiss, Oberkochen, Germany). Each successive field was individually assessed to determine the severity of inflammatory cell infiltration, eosinophils infiltration to perivascular/alveolar areas, mucus production, and goblet cell hyperplasia. The lesions were estimated by an experienced histopathologist using a blinded, scoring system outlined previously [11, 12]. The degree of histological observations was graded on a semi-quantitative scale of 0 to 4 depending on the degree–0 grade indicated that the tissue was normal or showed no changes and grades were assigned as 1 (minimal), 2 (slight), 3 (moderate), or 4 (severe) based on an increasing severity or complexity of changes.

All data were presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software (v8, GraphPad Software Inc., San Diego, CA, USA). Statistical multiple comparisons were performed by one-way analysis of variance followed by Dunnett’s test. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.