Safety assessment of cerium oxide nanoparticles: combined repeated-dose toxicity with reproductive/developmental toxicity screening and biodistribution in rats

Abstract Cerium oxide nanoparticles (CeO2 NPs) are widely used in various commercial applications because of their characteristic properties. People can be easily exposed to CeO2 NPs in real life, but the safety assessment of CeO2 NPs has not been fully investigated. Therefore, in this study, we conducted a combined repeated-dose and reproductive/developmental toxicity screening study (OECD testing guideline 422) to investigate the potential hazards on human health, including reproductive/developmental functions, after repeated daily CeO2 NPs oral gavage administration to both males and females. In addition, tissues from parental animals and their pups were collected to analyze the internal accumulation of cerium. CeO2 NPs were orally administered to Sprague-Dawley rats at doses of 0, 100, 300 and 1000 mg/kg during their pre-mating, mating, gestation and early lactation periods. In the general systemic and reproductive/developmental examinations, no marked toxicities were observed in any in-life and terminal observation parameters in this study. In the biodistribution analysis, cerium was not detected in either parental or pup tissues (blood, liver, lungs and kidneys). Repeated oral exposure of CeO2 NPs did not induce marked toxicities affecting general systemic and reproductive/developmental functions up to the dose level of 1000 mg/kg and the CeO2 NPs were not systemically absorbed in parental animals or their pups. This result could be used in risk assessment for humans, and additional toxicity studies with CeO2 NPs will be necessary considering various physicochemical properties and exposure probabilities of these nanoparticles.


Introduction
Nanomaterial development and research have precipitously increased recently, and engineered nano-related items are continuously introduced to the market (Berube et al. 2010). Cerium oxide nanoparticles (CeO 2 NPs) are one of the 13 priority listed representative manufactured nanomaterials to undergo assessments of the environmental safety and human health implications established by the Organization for Economic Co-operation and Development (OECD) Working Party on Manufactured Nanomaterials (OECD 2008). CeO 2 NPs have been reported to have redox activity (Korsvik et al. 2007;Spivak et al. 2012), and their characteristic properties are used in a wide range of industrial areas, including UV-absorbing compounds in sunscreens, solid oxide fuel cells, catalytic convertors for removing toxic gases and diesel fuel catalysts (Cassee et al. 2011;Murray, Tsai, and Barnett 1999;Sun, Li, and Chen 2012;Wu et al. 2010). Extensive usage of CeO 2 NPs in various areas has raised human health concerns because of occupational and environmental exposure. However, the potential adverse effects of CeO 2 NPs have not been widely investigated thus far. health problems. CeO 2 NPs exposure in cyanobacterial and green alga was found to induce cytotoxicity through cell wall and membrane disruption (Rodea-Palomares et al. 2011). In addition, cytotoxicity was also observed in human lung epithelial cells (BEAS-2B) after CeO 2 NPs exposure through increased reactive oxygen species (Park et al. 2008). Moreover, CeO 2 NPs-induced reproductive toxic potentials were also reported in recent studies (Adebayo, Akinloye, and Adaramoye 2018; Preaubert et al. 2016;Pr eaubert et al. 2018). In experimental animal studies, acute inhalation exposure of rats to CeO 2 NPs induced cytotoxicity via oxidative stress in the lungs, and these effects were considered able to aggravate chronic inflammation (Srinivas et al. 2011). Another study showed that exposure of rats to a single intravenous infusion of CeO 2 NPs led to body weight gain impairment, increased spleen weight and hepatic granulomas (Yokel et al. 2012). Moreover, these study results showed that CeO 2 NPs were able to be retained in internal organs, such as the spleen, liver and bone marrow. Consequently, these results support concerns about the adverse effects of long-term exposure to CeO 2 NPs in humans and the environment.
Nevertheless, the potential adverse effects of repeated CeO 2 NPs exposure have not been thoroughly investigated. In particular, developmental and reproductive toxicity studies with CeO 2 NPs have rarely been conducted. In this regard, the current study was conducted to assess the potential general systemic effects as well as developmental and reproductive effects after repeated CeO 2 NPs oral exposure based on an OECD guideline for testing of chemicals (OECD 1996). In addition, we collected parental samples (blood, liver, lungs and kidneys) and F1 pup samples (blood, liver, lungs and kidneys) to analyze the internal systemic distribution of CeO 2 NPs after repeated oral exposure. Oral exposure was selected in this study because OECD guideline recommended the test substance be administered orally by gavage and oral exposure is considered as a general route when exposure routes are varied. In addition, there were not enough toxicity studies with this commonly exposed route of CeO 2 NPs. Therefore, in this study, CeO 2 NPs were orally administered to Sprague-Dawley (SD) rats at doses of 0, 100, 300 and 1000 mg/kg from pre-mating to lactation periods, and then the CeO 2 NPs-induced potential effects on general systemic, developmental and reproductive functions as well as the internal biodistribution of CeO 2 NPs were observed.

Materials and methods
The current study was conducted in accordance with the OECD Good Laboratory Practice (GLP) regulations (OECD 1998). 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' (OECD 1996). 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 (National Research Council 2011).

CeO 2 NPs and nanoparticle characterization
CeO 2 NPs (CAS No. 1306-38-3) were obtained from Sigma-Aldrich (MO, USA). The primary size and shape of CeO 2 NPs were characterized by transmission electron microscopy (TEM, JEM-2100F, JEOL, Tokyo, Japan). The mean particle size of at least 100 CeO 2 NPs was analyzed using a DigitalMicrograph image analyzer program (Gatan, Inc., CA, USA). The purity of the particles was analyzed using energydispersive X-ray (EDX) (TEM equipped with a silicon drift detector, Oxford Instruments, Abingdon, UK). The particle hydrodynamic diameter and zeta potentials in deionized water (10 mg/ml concentration) were characterized with ELS-8000 (Otsuka Electronics, Osaka, Japan) using the dynamic light scattering (DLS) method. CeO 2 NPs in deionized water were identically sonicated by a Vibra-Cell V R Model VC 505 sonifier (Sonics & Materials, CT, 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. (Seongnam-si, Republic of Korea). Animals were housed as 2 (or one) animal(s) per stainless-steel cage (255 W Â 465L Â 200H mm 3 ). Pregnant and lactating dams were housed individually in a poly-sulfone cage (260 W Â 420L Â 180H mm 3 ) with sterilized Aspen animal bedding (Bio Lab, Cheonansi, Republic of Korea) during the study period. The animal room was maintained under controlled environmental conditions as described previously , 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, MO, 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
The dose level was selected on the basis of the results of a preliminary study with CeO 2 NPs in SD rats (5 animals/sex/group, data not shown). In a preliminary study, animals were daily dosed CeO 2 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). Consequently, there was no test item-related change in all examined parameters, including clinical signs, body weight, food consumption, clinical pathology, macroscopic observation, organ weights, fertility, and cesarean section, at any doses tested. Therefore, 1000 mg/kg, which is the limit dose level, was selected as the high dose. In addition, 300 and 100 mg/kg were selected as the intermediate and low doses, respectively. Vehicle control animals were administered deionized water. CeO 2 NPs were diluted in deionized water and sonicated by the Vibra-Cell V R 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., NJ, USA). CeO 2 NPs were daily administered by oral gavage on each animal. 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. Mated females were weighted 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 period, 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 fertilityrelated data, including mating, fertility, fecundity and pregnancy index, were calculated (Lerman et al. 2009). 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 (Moser 1991;Pierce and Kalivas 2007). 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 was 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, Munich, 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 3000 rpm, 10 min, at room temperature) to obtain plasma. A coagulation test was conducted with an ACL 9000 coagulation analyzer (Instrumentation Laboratory, MA, USA) 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., Tokyo, 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 (approximately 200 mg) for cerium content analysis. Parental animal tissues were collected from 5 individual animals per sex. Pup tissues were collected from at least 5 individual pups and pooled by litter. All collected tissues were stored in a deep freezer (approximately À80 C) until analysis.
For the cerium content analysis, collected samples were thawed and digested in a solution of 1 ml 30% H 2 O 2 and 7 ml 70% HNO 3 with a microwave digestion system (Milestone, Sorisole, Italy). After wet digestion, the cerium content in collected tissues was analyzed with an inductively coupled plasma mass spectrometry (ICP-MS, Elan DRC II, Perkin Elmer, MA, USA) at the Korea Basic Science Institute (Seoul Center, Republic of Korea). The ICP-MS was operated at RF generator power 1300 W and the argon gas flows were at the following setting; plasma, 15 L/min; auxiliary, 1.2 L/min; nebulizer, 0.84 L/min. The limit of detection (LOD) and method quantification limit (MQL) for collected samples were determined to be 0.002647 lg/kg and 0.8911 lg/kg, respectively. The LOD was calculated based on 3 times the standard deviation of nine repeat blank analysis. In addition, the MQL was calculated based on a multiple of total dilution factor and 10 times the standard deviation of nine repeat blank analysis.

Statistical analysis
Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study ). Statistical analysis was performed using the Pristima System or Statistical Analysis Systems (SAS Institute, NC, USA), and the level of significance was taken when p < 0.05 or p < 0.01. The litter was used as a statistical unit for litter data. Pup body weight was analyzed using one-way analysis of covariance (ANCOVA), and the litter size was used as the covariate.

Physicochemical properties of CeO 2 NPs
The physicochemical properties of CeO 2 NPs, including analyses of primary size, shape, purity and hydrodynamic size with zeta potential, are summarized in Table 1. TEM analysis showed the majority of the CeO 2 NPs had a polyhedral shape with the average primary size of approximately 14 nm. The hydrodynamic size in deionized water obtained by DLS was approximately 76 nm and this result indicated that CeO 2 NPs formed larger agglomerates in deionized water. In addition, the data of zeta potential represented that CeO 2 NPs were considerably stable in deionized water.

General systemic observations
All animals survived to the scheduled sacrifice. Furthermore, observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups (data not shown).
Body weights during the study period are presented in Figure 1. There were no treatment-related changes in body weight or weight gain during the study. Food consumption during the study period is presented in Table 2. In male rats of the 300 mg/kg dose group, food consumption during the pre-mating day 1-4 was significantly lower (93% of control) than in vehicle control animals.
In functional observations including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, there were no treatment-related changes during the study (data not shown).
In macroscopic observations, there were no treatment-related changes among the treatment and control animals (data not shown). Hematology results are presented in Table 3. In female rats of the 100 mg/kg dose group, the PT value was significantly higher (1.14-fold over control) than the respective level in the vehicle control animals. Clinical chemistry results are presented in Table 4. In male rats of the 1000 mg/kg dose group, the GGT value was significantly higher (2.24-fold over control) than the respective level in the vehicle control animals. Other hematology and clinical  Tables 5  and 6. There were no treatment-related changes in organ weights among the treatment and control animals.
Histopathological examination results are presented in Table 7. In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys was observed.

Developmental and reproductive observations
Fertility results with precoital time are presented in Table 8. There were no treatment-related changes in fertility results with precoital time. Reproductive and litter findings are presented in Table 9. There were no treatment-related changes in reproductive and litter finding parameters during the gestation and lactation periods. In general clinical signs and external examination of F1 pups at necropsy, there were no treatment-related changes among the treatment and control animals (data not shown). F1 pup body weights are presented in Table 10. An increase in F1 male and female pup covariateadjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4) was observed at 1000 mg/kg.

Tissue distribution of cerium
Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO 2 NPs were not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.

Discussion and conclusion
CeO 2 NPs have been used in a wide range of industrial areas as a consequence of their characteristic properties. However, their potential toxicological effects in human health are not well investigated. An in vitro study with CeO 2 NPs showed that they did not induce genotoxicities, including DNA and chromosomal damage, in human lens epithelial cells (Pierscionek et al. 2010). However, other in vitro studies imply the opposite results with respect to CeO 2 NPs toxicity. CeO 2 NPs exposure in human lung cancer cells yielded elevated oxidative stress and cell membrane damage (Lin et al. 2006). In addition, CeO 2 NPs exposure in human hepatocellular carcinoma SMMC-7721 cells induced damage and apoptosis via oxidative stress and the activation of MAPK signaling pathways (Cheng et al. 2013). The genotoxic potential of CeO 2 NPs was also reported in previous papers (Franchi et al. 2015; K€ onen-Adıg€ uzel and Ergene 2018).
In vivo studies, such as acute oral gavage toxicity studies with CeO 2 NPs (30 nm; 100 and 5000 mg/kg) in rats revealed that CeO 2 NPs did not have overt toxicities (Park, Park, and Park 2009). Another acute oral gavage exposure study of CeO 2 NPs (<25 nm; 30 and 300 mg/kg) in rats reported that no significant toxicities were observed with respect to general clinical signs, clinical pathology parameters and histopathological examinations (Park et al. 2018). Repeated-dose oral gavage exposure of mice to CeO 2 NPs (3-5 nm; weekly 0.5 mg/kg for 2 or 25.0 ± 1.1 25.0 ± 1.4 25.4 ± 1.2 25.9 ± 1.3 Ã Represent a significant difference at the p < 0.05 level compared to the vehicle control (n ¼ 12, mean ± SD). 5 weeks) did not induce overt toxicities (Hirst et al. 2013). Conversely, another repeated-dose oral gavage exposure study with CeO 2 NPs (<25 nm; 30, 300 and 600 mg/kg for 28 days) in rats revealed that prolonged exposure to high concentrations has the potential to cause biochemical alterations, genetic damage and histological changes in the liver, spleen and brain (Kumari, Kumari, and Grover 2014). In addition, single oral gavage exposure to CeO 2 NPs (4 ± 1 nm; 0.14 mg to 2.57 mg/kg based on the representative animal weight) induced the disruption of microvascular smooth muscle signaling (Minarchick et al. 2015). These inconsistent and insufficient toxicity results obtained with CeO 2 NPs indicate the necessity of standard toxicity studies that are acceptable by the regulatory authorities of various countries.
Combined repeated-dose toxicity with reproductive/developmental toxicity screening tests are required by OECD test guidelines for testing 16.1 ± 0.7 15.6 ± 1.2 14.8 ± 1.0 15.0 ± 0.9 Ã Represent a significant difference at the p < 0.05 level compared to the vehicle control (n ¼ 5, mean ± SD). chemicals that are reviewed in light of scientific progress by the various regulatory authorities (OECD 1996). This toxicity study is needed for industrial chemicals with a production or import level of more than 10 tonnage per year according to the European Registration, Evaluation, Authorization and Restriction of Chemicals (EU REACH) legislation (Beekhuijzen et al. 2014). This study design is able to assess various developmental and reproductive toxicity endpoints as well as general toxicity endpoints in subacute toxicity studies. Thus far, since controversial toxicity results have been obtained with CeO 2 NPs in previous in vitro and in vivo studies, the combined repeated-dose toxicity with reproductive/developmental toxicity screening test could be considered one of the most appropriate toxicity studies to evaluate the potential effects of CeO 2 NPs on general functions as well as developmental and reproductive functions. In addition, we also analyzed the internal systemic distribution of CeO 2 NPs in major organs (blood, liver, lungs and kidneys) of parental animals and their pups after repeated oral exposure.
statistical significance in 300 mg/kg dose group males, this finding was considered to be incidental since it was transient and did not have a doseresponse. As for hematology, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response. Regarding clinical chemistry, a statistically significant increase in GGT in 1000 mg/kg dose-group males was also considered to be incidental since there were no correlated changes in organ weights and histopathological examinations. In the histopathological examinations, the increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental since it also occurred sporadically in normal animals, did not have an obvious doseresponse and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO 2 NPs-related changes in other examinations for general systemic effects. In developmental and reproductive observations after repeated CeO 2 NPs oral exposure, there were no CeO 2 NPs-related changes in any of the parameters except the body weights of the F1 pups. The covariate-adjusted body weights of the pups increased in both males and females during PND 0 and 4 at the 1000 mg/kg. A change in pup growth compared with that of the concurrent control is one of the endpoints that often indicates toxicity in a reproductive toxicity study (Hood 2011). It may be secondarily influenced by gestation length, maternal nutritional status and litter size (Carney et al. 2004;Romero et al. 1992). However, there were no concurrent changes in maternal body weight, litter size or gestation length in this study. It has also been reported that test substances with endocrine modes of action cause increased pup body weights. However, these endocrine effects were not clear in this study since there were no concurrent mode of action-related changes, including estrus cycle abnormalities and histopathological changes in reproductive organs (ECHA 2017). In addition, biodistribution results indicated that CeO 2 NPs were not systemically observed in parental animals or their pups. Therefore, an increased pup body weight was not considered treatment-related, although the mechanism of CeO 2 NPs induced changes in the body weight of pups is not clear. Additional endpoint evaluations with an extended postnatal period were considered necessary to clearly elucidate the potential toxicological effects of CeO 2 NPs on the development of pups.
A biodistribution analysis after CeO 2 NPs exposure has been conducted in a few previous studies. Single-and 28-day repeated inhalation exposure to CeO 2 NPs (<5000, 40 and 5-10 nm; 55.00, 19.95 and 10.79 mg/m 3 ) resulted in their distributed to the lungs, liver, kidneys, spleen, brain, testes and epididymides (Geraets et al. 2012). Hirst et al. (2013) also reported that CeO 2 NPs (3-5 nm; weekly 0.5 mg/kg for 2 or 5 weeks) were deposited in the spleen, liver, lungs and kidneys after intravenous and intraperitoneal administration. However, CeO 2 NPs were not considered to be deposited in internal organs after oral exposure. Most of the orally exposed CeO 2 NPs were excreted through feces within 24 hours. Other oral exposure studies with CeO 2 NPs (<25 nm; 30 and 300 mg/kg for 1 day and 6.6 nm; 1 mg/animal for 1 day) suggested that they could hardly be absorbed in the gastrointestinal tract, and most of the orally exposed CeO 2 NPs were excreted in feces within a few days (He et al. 2010;Park et al. 2018). Our results also confirmed these previous studies showing that CeO 2 NPs were not deposited in parental internal organs after repeated oral exposure. In addition, we confirmed that CeO 2 NPs did not accumulate in the internal organs of the pups.
A lack of marked toxicity of CeO 2 NPs was observed in this study, but this finding does not mean that CeO 2 NPs are safe for humans and the environment. The toxicity of nanoparticles can be changed depending on their physicochemical properties, such as particle size, chemical composition and surface structure (Nel et al. 2006). In fact, sizedependent toxicity and internal distribution differences of nanoparticles have been reported in previous studies (M at e et al. 2016;Song et al. 2012;Wang et al. 2007). Furthermore, nanoparticles exposure in humans and the environment can occur through various exposure routes and scenarios considering the wide range of CeO 2 NPs application (Hansen et al. 2008;Hendren et al. 2011). Therefore, additional toxicity studies of CeO 2 NPs would be necessary considering their various physicochemical properties and exposure scenarios.
In conclusion, under the experimental conditions of this study design, there were no CeO 2 NPsrelated adverse effects in terms of general systemic Grades: 1 (minimal), 2 (slight), 3 (moderate), 4 (marked), 5 (severe), -: Not examined, (n ¼ 5). signs as well as development and reproduction, at doses up to 1000 mg/kg. In addition, CeO 2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure. These results in this study could be used for the risk assessment of repeated CeO 2 NPs exposure in humans, and further toxicity studies will be necessary to understand the potential risk to humans considering the various physicochemical properties and exposure scenarios of CeO 2 NPs.