Silver nanoparticles (CAS No. 7440-22-4) were purchased from NAMATECH, Ltd. (Daejeon, Korea), and were at least 99.98% pure. Count median diameter and geometric standard deviation of silver nanoparticles in 0.5% aqueous carboxymethylcellulose (CMC, Sigma USA) analyzed by transmission electron microscopy were 56 nm and 1.46, respectively (Figure 1).
Transmission Electron Microscopy
The filters on which the silver nanoparticles in the 0.5% CMC were filtered were coated with carbon, mounted on an electron microscope grid (200 mesh, Veco, Eerbeek, Holland), and visualized under a transmission electron microscope (TEM, Hitachi 7100). The diameters of 400 randomly selected particles were measured at 50,000 × magnification, and the silver particles were analyzed using an energy-dispersive x-ray analyzer (EDX-200, Horiba, Japan) at an accelerating voltage of 75 kV.
Animals and Conditions
Four-week-old male and female, specific-pathogen free (SPF) Fisher 344 rats were purchased from Japan SLC Inc. (Japan) and acclimated for 7 days before starting the experiments. During the acclimation and experimental periods, the rats were housed in polycarbonate cages (maximum of 3 rats per cage) in a room with controlled temperature (22.2 ± 1.7°C) and humidity (48.4 ± 6.0%), and a 12-h light/dark cycle. The rats were fed a rodent diet (Harlan Teklad, USA) and filtered water ad libitum. The rats were divided into 4 groups (10 rats in each group): vehicle control (0.5% carboxymethylcellulose, CMC), low-dose group (30 mg/kg/day), middle-dose group (125 mg/kg/day), and high-dose group (500 mg/kg/day). When the rats reached five weeks of age, they were exposed to silver nanoparticles following OECD test guideline 408  by gavage for 13 weeks of repeated oral administration (dosing volumes were 10 ml/kg). Dose levels were selected based on previous observations in a 28-day oral toxicity study by Kim et al. . The study was conducted under OECD Good Laboratory Practices.
Clinical Chemistry and Hematology
At the conclusion of the 13-wk experiment, the rats were 18 wks old. Before necropsy, food was withheld for 24 h and the rats were anesthetized with CO2 gas. Blood was then drawn from the abdominal aorta, collected in heparinized vacutainers, and analyzed for ALB (albumin), ALP (alkaline phosphatase), Ca (calcium), CHO (cholesterol), CRE (creatinine), gamma-GT (gamma-glutamyl transpeptidase), GLU (glucose), GOT (glutamic oxaloacetic transaminase), GPT (glutamic pyruvic transminase), IP (inorganic phosphorus), LDH (lactate dehydrogenase), MG (magnesium), TP (total protein), UA (uric acid), BUN (blood urea nitrogen), TBIL (total bilirubin), CK (creatine phosphokinase), Na (sodium), K (potassium), Cl (chloride), TG (triglyceride), and A/G (ratio of albumin to globulin) using a biochemical blood analyzer (Hitachi 7180, Hitachi, Japan). The blood was also analyzed for the WBC (white blood cell count), RBC (red blood cell count), Hb (hemoglobin concentration), HTC (hematocrits), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width), PLT (platelet count), MPV (mean platelet volume), NE# (number of neutrophils), NE% (percent of neutrophils), LY# (number of lymphocytes), LY% percent of lymphocytes), MO# (number of monocytes), MO% (percent of monocytes), EO# (number of eosinophils), EO% (percent of eosinophils), BA# (number of basophils), and BA% (percent of basophils) using a blood cell counter (Hemavet 0950, CDC Tech., USA).
Organ Weights and Histopathology
After collecting blood samples, the rats were killed by cervical dislocation. Adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, esophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain were removed carefully, weighed, and fixed in a 10% formalin solution containing neutral phosphate-buffered saline. Thereafter, the organs were embedded in paraffin, stained with hematoxylin and eosin, and examined under light microscopy.
Determination of Tissue Silver
Tissues were digested with conc. nitric acid by using a microwave digestion system (MARS 230/60, CEM). The concentration of silver in digested fluid was analyzed with a flameless method using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace (Perkin Elmer 5100ZL, Zeeman Furnace Module, USA) based on the NIOSH 7300 method . The concentration of silver in the tissue was expressed as μg/g wet weight.
Statistical analysis was performed with SPSS (Version 12). Statistical evaluation was performed by analysis of two-tailed Student's t-test or analysis of variance (ANOVA) following multiple comparison tests with Duncan's method. The level of statistical significance was set at p < 0.05