TiO2 particles
TiO2 nanoparticles (AEROXIDEⓇ TiO2 P25, KRISS CRM 301–03-001, anatase/rutile (80/20), 99.9%; average primary particle size range 14–21 nm) were purchased from Evonik Industries AG (Essen, Germany). TiO2 nanoparticles were in the form of a white hydrophilic powder without surface modification and were stored at room temperature. TiO2 nanoparticle suspensions were prepared by dispersing the particles in 5 mM sodium phosphate buffer (pH 8.0; Sigma-Aldrich Corp., St. Louis, MO, USA) followed by sonication (Branson Ultrasonics 450D; Thermo Fisher Scientific, Waltham, MA, USA) at 50 W for 17.5 min in an ice water bath to prevent the suspensions from overheating.
Particle characterization
The hydrodynamic diameters of the particles in 5 mM sodium phosphate buffer were measured by DLS using a Zetasizer Nano ZS90 (Malvern Panalytical Ltd., Malvern, UK) and were confirmed by TEM using the FEI Tecnai F30, operated at an acceleration voltage of 300 kV. The pH value of the prepared vehicle was measured with a pH meter (Orion Star A210; Thermo Fisher Scientific, Waltham, MA, USA) and the pH range was confirmed to be 8.05–8.11.
Animals
Pathogen-free SD rats [Crl:CD(Sprague-Dawley)] were purchased from Orient Bio Inc. (Seongnam, Korea). Healthy young adult animals (males and non-pregnant females) were acclimated and closely monitored for 6 days after arrival in the SPF animal facility area, and were randomly assigned to the control and treatment groups. Only animals with the best appearance were selected for subsequent testing. The body weights of the male and female rats were 210–232 g and 157–185 g, respectively, at the time of the first administration in the repeated-dose 28-day experiment. The body weights of the male and female rats were 185–207 g and 149–183 g, respectively, at the time of the first administration in the repeated-dose 90-day experiment. Rats were housed two per cage in an environmentally controlled room at 22.9 ± 0.5 °C and relative humidity of 54.3 ± 4.2%. The room air was replaced 10–15× per hour. Lighting was set to a 12-h light/dark cycle (on at 08 h00 and off at 20 h00).
Experimental design
One hundred and forty healthy adult SD rats were used in this study (Table S8). For the repeated-dose 28-day experiment, the animals were randomly divided into four groups, each consisting of five animals per sex. For the repeated-dose 90-day experiment, the animals were randomly assigned to four groups. Each group consisted of 10–15 animals per sex. One group was administered 5 mM sodium phosphate buffer by gavage and served as the vehicle control group (G1). The three remaining groups received one of three agglomerated/aggregated TiO2 P25 dosages by gavage (250 mg kg− 1 d− 1 (G2), 500 mg kg− 1 d− 1 (G3), and 1000 mg kg− 1 d− 1 (G4)). The dosing volume was 10 mL kg− 1 body weight. The agglomerated/aggregated TiO2 P25 were administered every morning for either 28 days or 90 days.
This study was performed in compliance with Good Laboratory Practices (GLP) and the OECD Guidelines No. 407 and 408 and was approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Conformity Laboratories.
Clinical observations, body weight, and food consumption
Detailed clinical observations were made once on all surviving animals before the onset of administration. Functional observations were conducted during the last week of treatment for the 90-day treated study and during the last week of observation for the recovery study. Functional observations were performed within 6 h after administration. Individual animal weights were recorded at acquisition and grouping, before administration, once weekly during the study, and before necropsy. Food consumption was measured immediately before the first administration and once weekly during the study. To calculate daily food intake, the food ration in each cage was weighed the day before the body weight measurement, and orts (leftover food) were measured on the day of the body weight measurement. Food consumption per animal was recalculated according to the average individual consumption (g rat− 1 d− 1), and water intake was measured immediately before the first administration and once weekly during the study. The measurement and calculation of the water intake per animal were consistent with those for the food consumption.
Hematology and clinical biochemistry
All animals were fasted overnight before necropsy, but water was provided ad libitum. At necropsy, the rats were anesthetized with isoflurane. Blood samples were extracted from the abdominal aorta using a syringe and collected in EDTA-K2 tubes (Microtainer®; Becton, Dickinson, and Company, Franklin Lakes, NJ, USA), 3.2% sodium citrate tubes (Vacuette®; Greiner Bio-One, Kremsmünster, Austria), and serum-separating tubes (Insepack®; Sekisui Diagnostics, Lexington, MA, USA). In certain cases, blood samples were collected from the jugular vein to measure the methemoglobin concentration within 6 h after administration on the last day of treatment. For the recovery study, the blood samples were drawn from the abdominal aorta at necropsy and stored in heparin tubes (20–30 IU mL-l). Blood collected in the EDTA-K2 tube was analyzed with a hematology analyzer (Advia 2120; Siemens Limited, Dublin, Ireland). Methemoglobin concentrations were determined with a blood gas analyzer (GEM Premier 4000; Instrumentation Laboratory Company, Bedford, MA, USA). Erythrocytes with Heinz bodies were counted after supravital staining. Blood biochemistry was analyzed with a biochemistry analyzer (Hitachi 7180; Hitachi, Chiyoda, Tokyo, Japan). Serum was isolated and collected by centrifugation in a serum-separating tube at 3000 rpm for 10 min.
Necropsy and histopathology
After administration, necropsies were conducted on all surviving animals, and complete post-mortem examinations were performed on all organs. All organs were harvested, and some organs were weighed immediately after extraction (Table S9). Excised organs were fixed in 10% neutral phosphate-buffered formalin. Testes and epididymis were fixed in Bouin’s solution, and eyes were fixed in Davidson’s solution. Bilateral organs were fixed and organs with macroscopically abnormal lesions were preserved. Thin sections were made from all the preserved organs and tissues of the vehicle control and high-dose groups, mounted on histology slides, and examined histopathologically by hematoxylin and eosin (H&E) staining.
Statistical analysis
Statistical differences among the vehicle control and dosing groups were analyzed by parametric or nonparametric multiple comparison methods. Differences were considered statistically significant at P < 0.05. The incidence rate was represented as a percentage. Statistical analysis was performed with SPSS for Windows v. 12.0 (IBM Corp., Armonk, NY, USA) and in compliance with the standard operating procedures of the testing facility.