## 2 Materials and Methods
### Observation Items  
Changes in body weight, thymus, spleen, and submandibular lymph node weights were directly measured using an electronic scale (Precisa Gravimetrics AG, Dietikon, Switzerland).  
Hematological examinations for total white (WBC) and red blood cell (RBC) counts, white blood cell differential count, neutrophil, acidophilus, basic neutrophil and monocyte ratio, hemoglobin level, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count were performed using an automatic complete blood count (CBC) analyzer (Abbott Laboratories, Abbott Park, IL, USA). RBC and WBC counts were measured using electrical impedance and light scattering [39], and packed cell volume (PCV) was measured using the microhematocrit method [40]. MCV was obtained using the formula PCV \(\times\) 10/RBC (10\({}^{6}\)/mm\({}^{3}\)) and MCH was calculated using the formula hemoglobin--Hb (g/100 mL) \(\times\) 10/RBC (10\({}^{6}\)/mm\({}^{3}\)). Whereas MCHC was automatically calculated using an automatic hemocytener via the formula Hb (g/100 mL) \(\times\) 100/PCV.  
IFN-\(\gamma\), TNF-\(\alpha\), IL-1\(\beta\), IL-6, and IL-12 levels in the serum and the spleen, and NK cell activity in the peritoneal cavity and the spleen; changes in NF-\(\kappa\)B, IFN-\(\gamma\), TNF-\(\alpha\), IL-1\(\beta\), IL-6; and IL-12 mRNA expressions in the spleen were determined using real-time RT-PCR in accordance with the method described by [PERSON] [41]. The oligonucleotides used for real-time RT-PCR are listed in Table S2. Splenic and peritoneal NK cell activity was measured using a standard \({}^{51}\)Cr release assay [30].  
Histopathological changes in lymphoid organs; total thymus and cortical thickness, total spleen and white medulla thickness, white medulla number, number of CD3+, CD4+, CD8+, TNF-\(\alpha\)+, IL-1\(\beta\)+, and inducible nitric oxide synthase (iNOS+) immunoreactive cells in spleen tissue; CD3+, CD4+, CD8+, TNF-\(\alpha\)+, IL-1\(\beta\)+, and iNOS+ immune responses in spleen tissue; and the number of cells were determined using the avidin-biotin peroxidase complex (ABC) test method as proposed by [PERSON] et al. [42]. The primary antisera and detection kits for immunohistochemistry used in this study are listed in Table S3.  
The cytotoxicity and splenocytes proliferation activity of the splenocytes harvested on the final day following euthanasia (T-cell proliferation promoter, Concanavalin A (ConA; Sigma-Aldrich, St. Louis, MO, USA) and B-cell proliferation promoter, measurement of proliferation activity of spleen cells by lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA) treatment) were confirmed using an MTT assay [43].  
Anti-mutagenicity was determined through a micronucleus test on the bone marrow smear collected on the final day following euthanasia, including the number of polychromatic erythrocytes (PCE), polychromatic erythrocyte ratio (PCE/PCE + normochromatic erythrocytes--NCE), micronucleated polychromatic erythrocytes (MN), and changes in the number of contained PCE; MNPCE (micronucleated polychromatic erythrocytes), were determined via nuclear staining [44].