The current study was conducted in accordance with the OECD Good Laboratory Practice (GLP) regulations.[13] The study design was conducted in general accordance with OECD testing guideline 422, namely, “Combined repeated-dose toxicity study with the reproductive/developmental toxicity screening test”.[12] The experimental phase of this study was conducted in 2015, and animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) in accordance with the Animal Protection Act and the Guide for the Care and Use of Laboratory Animals.[14]
CeO 2 NPs and Nanoparticle Characterization
CeO2 NPs (CAS No. 1306-38-3) were obtained from Sigma-Aldrich (USA). The primary size and shape of CeO2 NPs were characterized by transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan). The mean particle size of at least 100 CeO2 NPs was analyzed using a DigitalMicrograph image analyzer program (Gatan, Inc., USA). The purity of the particles was analyzed using energy-dispersive X-ray (EDX) (TEM equipped with a silicon drift detector, Oxford Instruments, UK). The particle hydrodynamic diameter and zeta potentials in vehicle (10 mg/ml concentration) were characterized with ELS-8000 (Otsuka Electronics, Japan) using the dynamic light scattering (DLS) method. CeO2 NPs in deionized water were identically sonicated by a Vibra-Cell® Model VC 505 sonifier (Sonics & Materials, USA) as dose formulation preparation for oral gavage administration.
Animals and Maintenance
Specific pathogen free naïve male and female SD rats (7 weeks of age) were obtained from Orient Bio, Inc. (Republic of Korea). Animals were housed as 2 (or one) animal(s) per stainless-steel cage (255W × 465L × 200H mm3). Pregnant and lactating dams were housed individually in a poly-sulfone cage (260W × 420L × 180H mm3) with sterilized Aspen animal bedding (Bio Lab, Republic of Korea) during the study period. The animal room was controlled environmental conditions as described previously,[15] with a temperature range of 22 ± 3 °C, a relative humidity range of 50 ± 20%, a 12 hours light-dark cycle and a ventilation range of 10–20 air changes per hour. The water was irradiated by UV light and filtered prior to provide ad libitum. The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum. All animals received an acclimation period of 5 days to become accustomed to the laboratory environment. Then, healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study.
Dose Selection and Experimental Group
In the previous results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group) were used to select the dose levels of this study (data not shown). Animals were daily dosed CeO2 NPs with 100, 300 and 1000 mg/kg dose levels for two weeks prior to mating, and dosing was continued through final sacrifice in males (total 28 days) and through gestation day (GD) 15 in females (total of at least 29 days). In a preliminary study, there was no test item-related changes in all examined parameters at any doses tested. Therefore, 1000 mg/kg, which is the limit dose level, was determined to the high dose, and 300 and 100 mg/kg were determined to the intermediate and low doses. Vehicle control animals were administered deionized water. CeO2 NPs were diluted in deionized water and sonicated by the Vibra-Cell® sonifier with a 13 mm probe at 25% amplitude for 8 min. Dose formulations were mixed by a stirrer during the dosing, and dosing volume was 10 ml/kg.
Twelve male and twelve female SD rats were divided to each of the groups to have a similar mean body weight using the Pristima system (Xybion Medical System Co., USA). CeO2 NPs were daily administered by oral gavage in a volume of 10 mL on each animals. Males were administered during a 2-week premating period and during mating and up to the final sacrifice in males (total of 38 days). Females were administered during a 2-week premating period and during mating, gestation and up to lactation day (LD) 4(total of at least 41 days).
In-life Observations
Clinical examinations including mortality and general clinical signs were examined twice daily. In addition, detailed clinical signs were examined once weekly during the study period. Animal body weights were measured twice weekly during the pre-mating and mating periods, on days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation. Food consumption was also measured in the same days except for the mating and was calculated as g/animal/day. During mating, males and females were mated on a one-to-one basis for a period of up to 2 weeks. Mating was confirmed by the presence of sperm in the vaginal smear and/or the vaginal plug, and this was considered GD 0. Based on these mating results, the number of days the animals were confirmed to mate (precoital time) and fertility-related data, including mating, fertility, fecundity and pregnancy index, were calculated. Functional observations of animals, including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, were conducted with 6 animals/sex/group before necropsy. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups, live pup body weight and pup external abnormalities were recorded. After parturition, pup mortality and general clinical signs were examined once daily. Based on the parturition and pup mortality results, the delivery index (% of dams with live pups among pregnant dams) and viability index (% of survival pups on post-natal day 4 after birth) were calculated. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter.
Terminal Observations
All surviving males on the day after final dosing and females on LD 5 were humanely sacrificed with isoflurane. Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice. Blood for hematology was placed into tubes containing potassium salt of ethylenediaminetetraacetic acid (EDTA) and then analyzed with an ADVIA2120i hematology analyzer (Siemens, Germany) for the following parameters: total red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), platelet count (PLT), reticulocyte count, total white blood cell count (WBC) and WBC differential count (absolute and relative counts of neutrophils [NEU], lymphocytes [LYM], monocytes [MON], basophils [BAS] and eosinophils [EOS]). Blood for coagulation was put into tubes containing 3.2% sodium citrate and centrifuged (approximately 3,000 rpm, 10 min, at room temperature) to obtain plasma. A coagulation test was conducted with an ACL 9000 coagulation analyzer (Instrumentation Laboratory, Italy) for the following parameters: activated partial thromboplastin time (APTT) and prothrombin time (PT). Blood samples for clinical chemistry were placed into tubes without anticoagulant and kept at room temperature for a minimum of 90 min and then centrifuged (approximately 3000 rpm, 10 min, at room temperature) to obtain serum. Clinical chemistry analysis was conducted with a Toshiba 200 FR NEO chemistry analyzer (Toshiba Co., Japan) for the following parameters: glucose (GLU), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), total cholesterol (TCHO), triglyceride (TG), albumin/globulin ratio (A/G), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), phospholipid (PL), creatine phosphokinase (CK), sodium (Na), inorganic phosphorus (IP), calcium (Ca), potassium (K) and chloride (Cl). After blood collection for clinical pathology, all animals were subjected to macroscopic observations, and the following organs were examined and preserved in 10% neutral buffered formalin or an appropriate fixative for histopathology: ovaries, testes, uterus with cervix, brain, stomach, ileum, duodenum, jejunum, colon, cecum, rectum, liver, kidneys, adrenal glands, spinal cord (cervical, thoracic, lumbar), prostate, epididymides, seminal vesicles with coagulation glands, thyroid with parathyroid glands, trachea, lungs with bronchi, mesenteric lymph nodes, mandibular lymph nodes, urinary bladder, femur with marrow, sciatic nerve, spleen, heart, thymus and abnormal lesions. All reproductive organs and the other organs from 5 animals per sex in each group were further processed to slides and stained with hematoxylin and eosin for histopathological examinations. Kidneys were also examined in the low- and intermediate-dose groups to further investigate the treatment-related changes. All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed, and the following organs were weighed from 5 animals per sex in each group: liver, kidneys brain, pituitary gland, heart, thymus, spleen, ovaries, adrenal glands, lungs and uterus with cervix. Paired reproductive organs were weighed separately.
Tissue Collection and Cerium Analysis
Parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected and weighed (approximately 200 mg) for cerium content analysis. Pup tissues were collected from at least 5 individual pups and pooled by litter. All collected tissues were stored in a deep freezer (approximately − 80 °C) until analysis. Cerium content analysis was conducted according to our previous study.[16] Briefly, thawed tissues were digested in a solution of 1 ml 30% H2O2 and 7 ml 70% HNO3 with a microwave digestion system (Milestone, Italy). After wet digestion, the tissue concentrations of cerium were analyzed with an inductively coupled plasma atomic emission spectroscopy (ICP-AES) equipped with an Ultima 2 apparatus (Horiba Jobin, France).
Statistical Analysis
Statistical analyses were conducted based on the general statistical method used in this types of toxicology study and our previous study.[17] Statistical analysis was performed using the Pristima System or Statistical Analysis Systems (SAS Institute, USA), and the level of significance was taken when p < 0.05 or p < 0.01. Litter data were statistically evaluated using the statistical unit as a litter. Pup body weight was analyzed using one-way analysis of covariance (ANCOVA), and the litter size was used as the covariate.