Cerium dioxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

0ECD 1996

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio, Inc. (Republic of Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 1 or 2 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.
- Diet (e.g. ad libitum): The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum.
- Water (e.g. ad libitum): The water was irradiated by UV light and filtered prior to provide ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): 10–20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light-dark cycle

IN-LIFE DATES: The experimental phase of this study was conducted in 2015.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks.
- Proof of pregnancy: Mating was confirmed by the presence of sperm in the vaginal smear and/or the vaginal plug, and this was considered GD 0.
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

- 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).

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control : deionized water (vehicle)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of the results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group). 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).
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, and 300 and 100 mg/kg were determined to be the intermediate and low doses, respectively.

- Rationale for animal assignment (if not random):
Healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study. 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).

- Fasting period before blood sampling for clinical biochemistry: Yes, approximately 16 hours (overnight) prior to sacrifice

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical examinations including mortality and general clinical signs were examined twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were examined once weekly during the study period

BODY WEIGHT: Yes
- Time schedule for examinations: Animal body weights were measured twice weekly during the pre-mating and mating periods. Mated females were weightedon days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in the same days of the body weight measurement except for the mating and was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified.

OTHER:
> FOB :
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).

> Hematogoly and chemical chemistry :
- Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
- Blood for clinical pathology were collected from the caudal vena cava from 5 randomly
selected animals/sex/group. 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).

Oestrous cyclicity (parental animals):

Oestrous cyclicity was measured.

Sperm parameters (parental animals):

All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed and processed for histopathological analyses.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
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.

GROSS NECROPSY
All animals were subjected to macroscopic observations.

HISTOPATHOLOGY / ORGAN WEIGHTS
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.

Postmortem examinations (offspring):

After parturition, pup mortality and general clinical signs were examined once daily. Pup external abnormalities were recorded. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter.

Statistics:

Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study (Lee et al. 2019). 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. 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.

Reproductive indices:

> 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.
> 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.

Offspring viability indices:

> 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.

Clinical signs:

no effects observed

Description (incidence and severity):

Observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No CeO2 NPs-related dead or moribund animals were observed during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in body weight or weight gain during the study (see Figure 1 in "Attached background material")

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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 but no alteration were seen afterward. No effect of the treatment was seen in the other treated groups in males and in all groups of treated females as compared to the controls animals (see Table 1 in "Any other information on results incl. tables").vThis finding was considered by the authors to be incidental since it was transient and did not have a dose response.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. However, 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. Other hematology values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 2 in "Any other information on results incl. tables"). Thus, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. However, 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 but the other clinical chemistry values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 3 in "Any other information on results incl. tables"). The authors have considered the increase in GGT in 1000 mg/kg dose-group males as incidental since there were no correlated changes in organ weights and histopathological examinations.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys (Grade 1) was observed. No such effect were seen in females. No other effect in the examined organs were seen in the treated groups of male and females animals as compared to the controls. The increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental by the autors since it also occurred sporadically in normal animals, did not have an obvious dose response and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO2 NPs-related changes in other examinations for general systemic effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

> FOB :
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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No estrus cycle abnormalities was observed in this study.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in fertility results with precoital time. Mating index, Fertility index and Fecundidity index were all equal to 100 in all groups of males. Mating index, Fertility index and Pregnancy index were also found all equal to 100 whatever the dose level groups in females. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods (see Tables 6 and 7 in "Any other information on results incl. tables").

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic and reproductive effect.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

In general clinical signs and external examination of F1 pups at necropsy, there were no treatment-related changes among the treatment and control animals.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect of the treatment was seen on the perinatal death and the viability index (see Table 7 in "Any other information on results incl. tables").

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No overt effect of the treatment was seen on pup body weight. An increase in F1 male and female pup covariate-adjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4 for males and PND 0 for females) was observed at 1000 mg/kg. (see Table 8 in "Any other information on results incl. tables"). Since there were no concurrent changes in maternal body weight, litter size or gestation length and no concurrent estrus cycle abnormalities and histopathological changes in reproductive organs, therefore, the increased pup body weight was not considered treatment-related.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No pup with external abnormalities was found in this study (see Table 7 in "Any other information on results incl. tables").

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effect observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

> Tissue distribution of cerium:

Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO2 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.

> Tables

Table 1: Food consumption of CeO2 NPs-treated males and females during the study period.

CeO2 NPs (mg/kg bw/day)	0	100	300	1000
Males
Pre-mating day 1–4 (g)	30.6 ± 1.9	29.3 ± 0.8	28.6 ± 1.5*	30.5 ± 1.6
Pre-mating day 4–8 (g)	31.3 ± 2.2	30.4 ± 1.2	30.2 ± 1.6	31.2 ± 1.4
Pre-mating day 8–11 (g)	31.4 ± 2.3	31.5 ± 1.5	30.5 ± 2.3	32.2 ± 1.2
Pre-mating day 11–14 (g)	32.3 ± 2.0	32.6 ± 1.8	31.4 ± 2.4	32.7 ± 1.1
Total period (g, pre-mating day 1–14)	31.4 ± 2.0	30.9 ± 1.1	30.2 ± 1.9	31.7 ± 1.0
Females
Pre-mating day 1–4 (g)	21.1 ± 1.6	20.2 ± 1.1	21.3 ± 1.1	21.2 ± 1.6
Pre-mating day 4–8 (g)	21.8 ± 1.2	21.4 ± 0.8	22.7 ± 1.2	22.2 ± 1.6
Pre-mating day 8–11 (g)	21.9 ± 1.7	22.3 ± 1.4	23.0 ± 2.2	22.1 ± 1.9
Pre-mating day 11–14 (g)	23.0 ± 1.5	23.0 ± 1.5	23.7 ± 0.8	23.3 ± 1.9
Gestation day 0–7 (g)	25.6 ± 1.7	26.0 ± 2.6	25.7 ± 2.2	26.8 ± 1.4
Gestation day 7–14 (g)	25.5 ± 1.5	25.9 ± 2.3	25.9 ± 2.3	25.8 ± 2.0
Gestation day 14–20 (g)	29.8 ± 2.1	29.2 ± 1.9	29.3 ± 1.9	31.5 ± 2.0
Post-natal day 0–4 (g)	35.8 ± 5.0	36.8 ± 3.2	35.9 ± 6.1	40.1 ± 4.1
Total period (g, pre-mating day 1 to lactation day 4)	25.0 ± 1.1	25.0 ± 1.4	25.4 ± 1.2	25.9 ± 1.3

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=12, mean ± SD).

 

Table 2: Hematology results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
RBC (10^6/µL)	9.1 ± 0.4	9.1 ± 0.3	8.9 ± 0.5	8.9 ± 0.1	7.8 ± 0.5	7.7 ± 0.3	7.9 ± 0.5	8.1 ± 0.2
HGB (g/dL)	16.6 ± 0.6	16.9 ± 0.4	16.8 ± 0.6	16.8 ± 0.6	14.9 ± 0.9	14.8 ± 0.3	14.8 ± 0.9	15.3 ± 0.5
HCT (%)	51.0 ± 2.5	51.4 ± 2.0	50.7 ± 2.1	50.4 ± 2.1	46.0 ± 2.6	45.6 ± 1.4	45.5 ± 2.7	47.0 ± 1.5
MCV (fL	56.1 ± 1.7	56.2 ± 0.9	57.2 ± 1.8	56.9 ± 1.9	59.4 ± 0.9	59.4 ± 1.2	57.9 ± 2.0	58.2 ± 1.2
MCH (pg)	18.3 ± 0.4	18.5 ± 0.3	18.9 ± 0.7	18.9 ± 0.5	19.3 ± 0.2	19.3 ± 0.5	18.8 ± 0.7	18.9 ± 0.3
MCHC (g/dL)	32.5 ± 0.4	33.0 ± 0.7	33.1 ± 0.6	33.3 ± 0.4	32.5 ± 0.5	32.5 ± 0.5	32.5 ± 0.4	32.4 ± 0.4
PLT (10^3/µL)	1130.8 ± 149.7	1024.2 ± 133.7	940.4 ± 41.3	1101.6 ± 84.8	1351.0 ± 167.3	1213.2 ± 174.2	1258.4 ± 222.7	1305.0 ± 253.0
RET (%)	2.5 ± 0.5	2.5 ± 0.3	2.5 ± 0.3	2.5 ± 0.6	5.8 ± 1.3	6.4 ± 1.7	6.3 ± 1.4	6.2 ± 1.7
RETA (10^9/µL	223.8 ± 41.4	226.0 ± 22.2	223.8 ± 31.6	218.4 ± 48.7	448.3 ± 78.3	492.2 ± 142.9	489.7 ± 77.8	502.9 ± 136.4
WBC(10^3/µL)	10.6 ± 3.3	11.6 ± 4.1	12.4 ± 1.0	11.0 ± 2.2	9.5 ± 2.5	9.8 ± 2.4	11.2 ± 1.1	12.8 ± 3.2
NEU (%)	14.8 ± 6.9	11.4 ± 1.7	12.1 ± 3.0	13.4 ± 2.5	12.2 ± 5.0	13.3 ± 2.2	12.8 ± 3.2	13.2 ± 2.8
NEUA(10^3/µL)	1.5 ± 0.7	1.4 ± 0.7	1.5 ± 0.5	1.5 ± 0.5	1.2 ± 0.7	1.3 ± 0.3	1.4 ± 0.2	1.7 ± 0.8
LYM (%)	81.3 ± 6.7	84.7 ± 2.1	83.2 ± 3.6	81.7 ± 2.0	81.5 ± 5.2	80.5 ± 2.0	80.7 ± 4.0	80.3 ± 3.2
LYMA(10^3/µL)	8.7 ± 2.8	9.8 ± 3.3	10.3 ± 0.7	9.0 ± 1.7	7.7 ± 2.0	7.9 ± 2.1	9.1 ± 1.4	10.2 ± 2.3
EOS (%)	0.8 ± 0.2	0.9 ± 0.2	0.9 ± 0.3	1.0 ± 0.4	0.8 ± 0.4	0.6 ± 0.2	0.7 ± 0.3	0.7 ± 0.3
EOSA (10^3/µL)	0.09 ± 0.05	0.10 ± 0.03	0.12 ± 0.05	0.11 ± 0.04	0.07 ± 0.02	0.06 ± 0.04	0.07 ± 0.02	0.09 ± 0.04
MON (%)	1.9 ± 0.6	1.9 ± 0.6	2.3 ± 0.7	2.5 ± 0.6	4.3 ± 0.7	4.0 ± 1.2	4.6 ± 0.9	4.6 ± 0.8
MONA (10^3/µL)	0.2 ± 0.1	0.3 ± 0.2	0.3 ± 0.1	0.3 ± 0.0	0.4 ± 0.1	0.4 ± 0.1	0.5 ± 0.1	0.6 ± 0.2
BAS (%)	0.6 ± 0.1	0.5 ± 0.1	0.7 ± 0.1	0.6 ± 0.2	0.5 ± 0.1	0.5 ± 0.2	0.4 ± 0.1	0.4 ± 0.1
BASA (10^3/µL)	0.06 ± 0.02	0.06 ± 0.03	0.09 ± 0.01	0.06 ± 0.01	0.04 ± 0.01	0.05 ± 0.03	0.05 ± 0.01	0.05 ± 0.02
LUC (%)	0.7 ± 0.3	0.6 ± 0.1	0.7 ± 0.1	0.8 ± 0.1	0.8 ± 0.3	1.1 ± 0.1	0.8 ± 0.3	0.8 ± 0.2
LUCA (10^3/µL)	0.07 ± 0.05	0.07 ± 0.04	0.09 ± 0.02	0.09 ± 0.01	0.07 ± 0.01	0.10 ± 0.03	0.09 ± 0.03	0.10 ± 0.03
PT (sec)	14.0 ± 1.7	13.7 ± 0.8	16.0 ± 2.7	13.6 ± 0.7	13.6 ± 1.2	15.5 ± 0.3*	15.3 ± 0.9	14.4 ± 1.5
APTT (sec)	17.5 ± 0.7	18.0 ± 0.8	18.3 ± 0.9	17.8 ± 1.0	6.1 ± 0.7	15.6 ± 1.2	14.8 ± 1.0	15.0 ± 0.9

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=5, mean ± SD).

 

Table 3: Clinical chemistry results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
GLU (mg/dL)	158.9 ± 20.3	149.2 ± 28.8	157.6 ± 45.1	148.5 ± 29.1	131.2 ± 22.4	116.0 ± 15.1	108.8 ± 10.7	139.1 ± 17.1
BUN (mg/dL)	15.1 ± 1.6	13.7 ± 1.5	14.3 ± 1.7	13.6 ± 1.8	24.1 ± 4.7	19.4 ± 3.4	21.2 ± 4.1	20.3 ± 3.6
CREA (mg/dL)	0.49 ± 0.04	0.48 ± 0.03	0.47 ± 0.04	0.47 ± 0.06	0.57 ± 0.08	0.51 ± 0.03	0.54 ± 0.04	0.56 ± 0.03
TP (g/dL)	6.6 ± 0.3	6.7 ± 0.2	6.6 ± 0.2	6.6 ± 0.4	7.0 ±0.4	7.0 ± 0.4	7.0 ± 0.2	7.1 ± 0.3
ALB (g/dL)	4.3 ± 0.2	4.2 ± 0.1	4.2 ± 0.1	4.2 ± 0.2	4.5 ±0.2	4.6 ± 0.3	4.5 ± 0.1	4.6 ± 0.1
A/G (ratio)	1.8 ± 0.2	1.7 ± 0.1	1.8 ± 0.1	1.8 ± 0.1	1.8 ±0.1	2.0 ± 0.2	1.9 ± 0.1	1.9 ± 0.1
AST (IU/L)	149.4 ± 21.9	137.5 ± 34.5	129.9 ± 4.3	153.9 ± 18.5	142.7 ± 44.3	129.1 ± 23.1	135.6 ± 17.7	121.3 ± 8.0
ALT (IU/L)	33.4 ± 5.7	28.6 ± 5.6	32.5 ± 3.0	31.5 ± 3.9	40.2 ± 8.0	41.5 ± 8.1	45.1 ± 5.2	39.8 ± 2.6
TBIL (mg/dL)	0.14 ± 0.02	0.12 ± 0.01	0.15 ± 0.02	0.13 ± 0.02	0.13 ± 0.02	0.12 ± 0.02	0.13 ± 0.02	0.12 ± 0.02
GGT (IU/L)	0.41 ± 0.23	0.65 ± 0.13	0.60 ± 0.12	0.92 ± 0.18**	0.59 ± 0.22	0.78 ± 0.10	0.67 ± 0.27	0.71 ± 0.38
ALP (IU/L)	581.6 ± 179.2	510.3 ± 110.0	467.4 ± 57.3	514.4 ± 66.4	332.3 ± 111.0	293.1 ± 58.0	232.7 ± 31.7	267.1 ± 83.9
TCHO (mg/dL)	70.8 ± 26.9	64.2 ± 19.5	60.0 ± 8.9	68.4 ± 10.2	49.8 ± 16.0	58.0 ± 16.5	53.8 ± 10.8	55.2 ± 12.8
TG (mg/dL)	31.8 ± 9.3	34.2 ± 11.8	23.9 ± 10.0	21.4 ± 10.9	38.9 ± 22.5	36.9 ± 13.1	41.7 ± 14.9	54.7 ± 25.5
Ca (mg/dL)	11.1 ± 0.3	11.3 ± 0.4	11.2 ± 0.2	11.0 ± 0.7	11.7 ± 0.4	11.8 ± 0.5	11.7 ± 0.3	11.9 ± 0.2
IP (mg/dL)	10.2 ± 0.5	10.3 ± 0.6	10.5 ± 0.4	10.3 ± 0.7	9.9 ± 0.8	10.2 ± 0.1	10.3 ± 1.2	9.8 ± 0.7
K (mmol/L)	8.3 ± 0.9	7.5 ± 0.9	8.4 ± 0.9	7.8 ± 1.6	8.6 ± 0.5	8.3 ± 0.5	8.2 ± 1.1	8.2 ± 1.7
CK (IU/L)	715.6 ± 197.6	584.2 ± 203.2	483.6 ± 79.4	652.0 ± 218.0	611.8 ± 331.8	518.0 ± 112.9	570.6 ± 129.1	448.8 ± 130.3
PL (mg/dL)	102.4 ± 25.0	93.6 ± 22.7	92.0 ± 7.3	99.2 ± 10.4	106.8 ± 26.9	116.8 ± 24.8	108.6 ± 14.8	115.8 ± 19.1
Na (mmol/L)	148.0 ± 0.7	147.8 ± 1.9	147.4 ± 1.1	147.8 ± 1.6	144.8 ± 1.3	145.0 ± 1.9	145.0 ± 1.4	146.0 ± 1.6
Cl (mmol/L)	102.4 ± 1.7	101.6 ± 0.6	102.6 ± 1.3	102.4 ± 0.9	101.4 ± 0.6	101.2 ± 1.5	101.6 ± 1.1	102.2 ± 2.4

**: Represent a significant difference at the p<0.01 level compared to the vehicle control (n=5, mean ± SD).

 

Table 4: Absolute and relative organ weights of CeO2 NPs-treated males during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight^a(g)	(g)	427.0 ± 38.7	439.2 ± 23.7	436.4 ± 36.0	448.9 ± 28.6
	N	12	12	12	12
Adrenal glands	Absolute (g)	0.06 ± 0.01	0.07 ± 0.01	0.06 ± 0.01	0.07 ± 0.00
	Relative^b(%)	0.016 ± 0.003	0.016 ± 0.002	0.016 ± 0.002	0.015 ± 0.001
	N	5	5	5	5
Brain	Absolute (g)	1.97 ± 0.12	1.99 ± 0.07	2.03 ± 0.09	2.00 ± 0.12
	Relative (%)	0.484 ± 0.068	0.460 ± 0.023	0.497 ± 0.030	0.461 ± 0.013
	N	5	5	5	5
Heart	Absolute (g)	1.25 ± 0.12	1.29 ± 0.14	1.33 ± 0.13	1.32 ± 0.11
	Relative (%)	0.304 ± 0.023	0.300 ± 0.030	0.325 ± 0.024	0.303 ± 0.028
	N	5	5	5	5
Kidneys	Absolute (g)	3.25 ± 0.19	3.40 ± 0.15	3.50 ± 0.33	3.47 ± 0.42
	Relative (%)	0.794 ± 0.071	0.788 ± 0.050	0.854 ± 0.047	0.797 ± 0.070
	N	5	5	5	5
Liver	Absolute (g)	11.71 ± 2.07	12.74 ± 0.71	12.12 ± 2.09	12.51 ± 1.41
	Relative (%)	2.825 ± 0.217	2.950 ± 0.1063	2.940 ± 0.246	2.870 ± 0.197
	N	5	5	5	5
Pituitary gland	Absolute (g)	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00
	Relative (%)	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000
	N	5	5	5	5
Prostate	Absolute (g)	0.62 ± 0.12	0.59 ± 0.17	0.66 ± 0.13	0.65 ± 0.09
	Relative (%)	0.144 ± 0.026	0.135 ± 0.041	0.151 ± 0.031	0.146 ± 0.018
	N	12	12	12	12
Spleen	Absolute (g)	0.66 ± 0.13	0.67 ± 0.15	0.65 ± 0.13	0.69 ± 0.08
	Relative (%)	0.158 ± 0.013	0.155 ± 0.033	0.158 ± 0.020	0.158 ± 0.013
	N	5	5	5	5
Thymus	Absolute (g)	0.34 ± 0.02	0.39 ± 0.08	0.35 ± 0.11	0.32 ± 0.06
	Relative (%)	0.083 ± 0.010	0.090 ± 0.018	0.084 ± 0.019	0.072 ± 0.011
	N	5	5	5	5
Lungs	Absolute (g)	1.50 ± 0.13	1.58 ± 0.12	1.57 ± 0.17	1.62 ± 0.07
	Relative (%)	0.367 ± 0.041	0.367 ± 0.031	0.383 ± 0.021	0.374 ± 0.021
	N	5	5	5	5
Right testis	Absolute (g)	1.70 ± 0.17	1.74 ± 0.13	1.69 ± 0.11	1.65 ± 0.12
	Relative (%)	0.400 ± 0.049	0.396 ± 0.031	0.391 ± 0.051	0.369 ± 0.028
	N	12	12	12	12
Left testis	Absolute (g)	1.73 ± 0.16	1.74 ± 0.12	1.71 ± 0.11	1.67 ± 0.12
	Relative (%)	0.407 ± 0.051	0.397 ± 0.026	0.394 ± 0.044	0.372 ± 0.030
	N	12	12	12	12
Right epididymis	Absolute (g)	0.66 ± 0.06	0.69 ± 0.06	0.68 ± 0.06	0.66 ± 0.07
	Relative (%)	0.155 ± 0.017	0.158 ± 0.016	0.156 ± 0.021	0.148 ± 0.021
	N	12	12	12	12
Left epididymis	Absolute (g)	0.67 ± 0.07	0.68 ± 0.06	0.66 ± 0.06	0.65 ± 0.06
	Relative (%)	0.157 ± 0.019	0.156 ± 0.014	0.152 ± 0.019	0.145 ± 0.018
	N	12	12	12	12
Seminal vesicles with coagulating glands	Absolute (g)	1.68 ± 0.21	1.65 ± 0.29	1.68 ± 0.22	1.69 ± 0.21
	Relative (%)	0.396 ± 0.047	0.378 ± 0.072	0.388 ± 0.057	0.377 ± 0.048
	N	12	12	12	12

N=5 or 12, mean ± SD.

a: Terminal body weight were measured immediately before necropsy.

b: Organ weight/terminal body weight ratio.

 

Table 5: Absolute and relative organ weights of CeO2 NPs-treated females during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight	(g)	314.5 ± 19.9	316.7 ± 24.1	311.0 ± 23.7	316.8 ± 19.8
Adrenal glands	Absolute (g)	0.08 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0.01
	Relative (%)	0.028 ± 0.003	0.024 ± 0.003	0.027 ± 0.003	0.027 ± 0.002
Brain	Absolute (g)	1.94 ± 0.09	1.93 ± 0.08	2.02 ± 0.08	1.96 ± 0.05
	Relative (%)	0.652 ± 0.036	0.650 ± 0.022	0.692 ± 0.028	0.654 ± 0.030
Heart	Absolute (g)	1.00 ± 0.06	0.95 ± 0.04	0.97 ± 0.07	1.04 ± 0.06
	Relative (%)	0.334 ± 0.013	0.321 ± 0.017	0.334 ± 0.026	0.346 ± 0.019
Kidneys	Absolute (g)	2.28 ± 0.19	2.23 ± 0.21	2.14 ± 0.14	2.23 ± 0.07
	Relative (%)	0.763 ± 0.028	0.750 ± 0.051	0.734 ± 0.036	0.744 ± 0.027
Liver	Absolute (g)	10.52 ± 1.21	10.40 ± 0.87	10.19 ± 0.30	10.91 ± 0.73
	Relative (%)	3.522 ± 0.266	3.503 ± 0.277	3.495 ± 0.169	3.638 ± 0.259
Pituitary gland	Absolute (g)	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
	Relative (%)	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.000
Spleen	Absolute (g)	0.62 ± 0.07	0.69 ± 0.09	0.65 ± 0.08	0.70 ± 0.12
	Relative (%)	0.209 ± 0.021	0.231 ± 0.029	0.223 ± 0.032	0.232 ± 0.043
Thymus	Absolute (g)	0.31 ± 0.03	0.31 ± 0.05	0.31 ± 0.08	0.38 ± 0.12
	Relative (%)	0.104 ± 0.013	0.104 ± 0.021	0.105 ± 0.027	0.125 ± 0.041
Lungs	Absolute (g)	1.34 ± 0.04	1.29 ± 0.06	1.42 ± 0.05	1.36 ± 0.11
	Relative (%)	0.451 ± 0.013	0.435 ± 0.018	0.486 ± 0.022	0.453 ± 0.034
Uterus	Absolute (g)	0.78 ± 0.12	0.78 ± 0.09	0.83 ± 0.12	0.73 ± 0.06
	Relative (%)	0.260 ± 0.039	0.262 ± 0.030	0.284 ± 0.035	0.243 ± 0.026
Right ovary	Absolute (g)	0.07 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
	Relative (%)	0.021 ± 0.002	0.021 ± 0.004	0.019 ± 0.003	0.019 ± 0.004
Left Ovary	Absolute (g)	0.07 ± 0.02	0.06 ± 0.01	0.06 ± 0.01	0.05 ± 0.01
	Relative (%)	0.023 ± 0.006	0.020 ± 0.002	0.020 ± 0.005	0.018 ± 0.002

N=5, mean ± SD.

 

Table 6: Fertility with precoital time results of CeO2 NPs treated males during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Males
Mating index^a	100	100	100	100
Fertility index^b	100	100	100	100
Fecundity index^c	100	100	100	100
Females
Mating index^d	100	100	100	100
Fertility index^e	100	100	100	100
Pregnancy index^f	100	100	100	100
Precoital Time (day)	3.3 ± 3.6	1.8 ± 0.6	2.2 ± 1.1	1.9 ± 1.1

 

N=12, mean ± SD.

a: (No. of males with evidence of mating/No. of males paired) x 100.

b: (No. of males impregnating a female/No. of males paired) x 100.

c: (No. of males impregnating a female/No. of males with evidence of mating) x 100.

d: (No. of females with evidence of mating/No. of females paired) x 100.

e: (No. of pregnant females/No. of females paired) x 100.

f: (No. of pregnant females/No. of females with evidence of mating) x100.

 

 

Table 7: Reproductive and litter findings results of CeO2 NPs-treated females during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Gestation period (day)	21.5 ± 0.4	21.5 ± 0.3	21.6 ± 0.3	21.8 ± 0.4
Corpora lutea (N)	17.8 ± 2.8	16.5 ± 2.6	16.3 ± 1.7	17.4 ± 2.0
Implantations (N)	15.3 ± 3.1	15.3 ± 2.4	14.8 ± 2.1	15.4 ± 2.0
Pups born (N)	14.8 ± 3.1	14.4 ± 2.4	13.9 ± 2.0	14.8 ± 2.1
Perinatal death (N)	0.08 ± 0.29	0.00 ± 0.00	0.00 ± 0.00	0.25 ± 0.45
Unaccounted-for sites^a(%)	2.8 ± 4.5	5.5 ± 5.8	6.2 ± 4.5	3.9 ± 4.5
Sex ratio^b(%)	106.9 ± 62.6	93.9 ± 28.2	105.3 ± 55.5	130.7 ± 66.8
Live litter size (N) PND 0 PND 4	14.8 ± 3.1 14.8 ± 3.1	14.4 ± 2.4 14.3 ± 2.4	13.9 ± 2.0 13.8 ± 2.0	14.6 ± 2.1 14.4 ± 2.0
Viability index^c(%)	100.0	99.4 ± 1.9	98.9 ± 2.6	99.0 ± 2.4
Delivery index^d(%)	100.0	100.0	100.0	100.0
Pups with external abnormalities	0	0	0	0

 

N = 12, mean ± SD.

a: (No. of implantation sites/litter) - (No. of live pups at birth/litter)/No. of implantation sites/litter x 100.

b: (No. of male pups on PND 0/litter)/(No. of female pups on PND 0/litter) x 100.

c: (No. of live pups on PND 4/litter)/(No. of live pups at birth/litter) x 100.

d: (No. of dams with live pups)/(No. of pregnant dams) x 100.

 

 

Table 8: F1 pups body weights of CeO2 NPs-treated parental animals during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
F1 male pups
PND 0
Body weight (g)	6.5 ± 0.4	6.7 ± 0.3	6.8 ± 0.6	7.0 ± 0.5
Covariate-adjusted mean (g)	6.5	6.7	6.8	7.0*
PND 4
Body weight (g)	10.1 ± 0.8	10.7 ± 1.1	11.0 ± 1.1	11.0 ± 1.2
Covariate-adjusted mean (g)	10.1	10.6	10.9	11.2*
F1 female pups
PND 0
Body weight (g)	6.2 ± 0.4	6.3 ± 0.3	6.4 ± 0.6	6.7 ± 0.6
Covariate-adjusted mean (g)	6.2	6.3	6.4	6.7*
PND 4
Body weight (g)	9.5 ± 0.9	10.0 ± 1.0	10.4 ± 1.2	10.6 ± 1.4
Covariate-adjusted mean (g)	9.6	10.1	10.4	10.5

 

N =12, mean ± SD.

*Represent a significant difference at the p<0.05 level compared to the vehicle control.

Conclusions:

In conclusion, under the experimental conditions of this study design, there were no CeO2 NPs related adverse effects in terms of general systemic signs as well as development and reproduction, at doses up to 1000 mg/kg bw/d. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.

Executive summary:

In a reproduction/developmental toxicity screening study, the effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD Guideline no 422 and was compliant to GLP. The study was thus considered as a key study of reliablility 1 according to Klimisch score.

Groups of 12 males and 12 females SD rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg/day nano CeO2 in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effect of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily.

Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).

 

No unscheduled death or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study.

No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time was found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.

 

Tissue distribution analysis of cerium in parental and pup tissues revealed that nano CeO2 was 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.

 

The authors concluded that, under the experimental conditions of this study, no nano CeO2 related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 422

Version / remarks:

OECD 1996

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio, Inc. (Republic of Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 1 or 2 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.
- Diet (e.g. ad libitum): The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum.
- Water (e.g. ad libitum): The water was irradiated by UV light and filtered prior to provide ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): 10–20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light-dark cycle

IN-LIFE DATES: The experimental phase of this study was conducted in 2015.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks.
- Proof of pregnancy: Mating was confirmed by the presence of sperm in the vaginal smear and/or the vaginal plug, and this was considered GD 0.
- After successful mating each pregnant female was caged (how): individually

Duration of treatment / exposure:

- 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).

Frequency of treatment:

daily

Duration of test:

38 days in males and 41 days in females

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control : deionized water (vehicle)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of the results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group). 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 29days).
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, and 300 and 100 mg/kg were determined to be the intermediate and low doses, respectively.

- Rationale for animal assignment (if not random):
Healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study. 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).

- Fasting period before blood sampling for clinical biochemistry: Yes, approximately 16 hours (overnight) prior to sacrifice

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical examinations including mortality and general clinical signs were examined twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were examined once weekly during the study period

BODY WEIGHT: Yes
- Time schedule for examinations: Animal body weights were measured twice weekly during the premating and mating periods. Mated females were weightedon days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in the same days of the body weight measurement except for the mating and was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified.

OTHER:
> FOB :
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).

> HEMATOLOGY and CLINICAL CHEMISTRY :
- Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
- Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. 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 3,000rpm, 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).

POSTMORTEM EXAMINATIONS (PARENTAL ANIMALS):
>SACRIFICE
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.

>GROSS NECROPSY
All animals were subjected to macroscopic observations.

> HISTOPATHOLOGY / ORGAN WEIGHTS
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.

POSTMORTEM EXAMINATIONS (OFFSPRINGS):
After parturition, pup mortality and general clinical signs were examined once daily. Pup external abnormalities were recorded. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Not specified
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes for external abnormalities
- Soft tissue examinations: No
- Skeletal examinations: No
- Other : number and sex of pups, stillbirths, live births, postnatal mortality, general clinical signs, presence of gross anomalies, weight gain.

Statistics:

Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study (Lee et al. 2019). 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. 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.

Indices:

Reproductive indices:
> 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.
> 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.

Offspring viability indices:
> 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.

Historical control data:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No CeO2 NPs-related dead or moribund animals were observed during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in body weight or weight gain during the study (see Figure 1 in "Attached background material")

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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 but no alteration were seen afterward. No effect of the treatment was seen in the other treated groups in males and in all groups of treated females as compared to the controls animals (see Table 1 in "Any other information on results incl. tables"). This finding was considered by the authors to be incidental since it wa transient and did not have a dose response.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. 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. Other hematology values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 2 in "Any other information on results incl. tables"). Thus, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. 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 but the other clinical chemistry values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 3 in "Any other information on results incl. tables"). The authors have considered the increase in GGT in 1000 mg/kg dose-group males as incidental since there were no correlated changes in organ weights and histopathological examinations.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in organ weights among the treatment and control animals (see Tables 4 and 5 in "Any other information on results incl. tables").

Gross pathological findings:

no effects observed

Description (incidence and severity):

In macroscopic observations, there were no treatment-related changes among the treatment and control animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys (Grade 1) was observed. No such effect were seen in females. No other effect in the examined organs were seen in the treated groups of male and females animals as compared to the controls. The increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental by the autors since it also occurred sporadically in normal animals, did not have an obvious dose response and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO2 NPs-related changes in other examinations for general systemic effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

> FOB:
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.

Number of abortions:

no effects observed

Description (incidence and severity):

No aborption occured during the study.

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The number of dams with live pups was equal to the number of pregnant dams (see Table 7 in "Any other information on results incl. tables").

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in delivery index, viability index and perinatal death and the number of pups borns was similar in all groups (see Table 7 in "Any other information on results incl. tables").

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No change of pregnancy duration due to treatment was observed in this study (see Tables 6 and 7 in "Any other information on results incl. tables").

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No change in number of pregnant due to treatment was observed in this study (see Tables 6 and 7 in "Any other information on results incl. tables"). Pregnancy index was 100 in all dose-level groups.

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in fertility results with precoital time. Mating index, Fertility index and Fecundidity index were all equal to 100 in all groups of males. Mating index, Fertility index and Pregnancy index were also found all equal to 100 whatever the dose level groups in females.
There were no treatment-related changes in reproductive (estrus cycle abnormalities, gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods (see Tables 6 and 7 in "Any other information on results incl. tables").

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No systemic effect observed.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No overt effect of the treatment was seen on pup body weight. An increase in F1 male and female pup covariate-adjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4 for males and PND 0 for females) was observed at 1000 mg/kg. (see Table 8 in "Any other information on results incl. tables"). Since there were no concurrent changes in maternal body weight, litter size or gestation length and no concurrent estrus cycle abnormalities and histopathological changes in reproductive organs, therefore, the increased pup body weight was not considered treatment-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No effect of the treatement was observed on the number of pup borned and on the perinatal death (see Table 7 in "Any other information on results incl. tables")

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of the treatment on the sex ratio (see Table 7 in "Any other information on results incl. tables").

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of the treatment on litter size and weight (see Tables 7 and 8 in "Any other information on results incl. tables").

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect of the treatment on postnatal survival at PND4 (see Table 7 in "Any other information on results incl. tables").

External malformations:

no effects observed

Description (incidence and severity):

No pup with external abnormalities was found in this study (see Table 7 in "Any other information on results incl. tables").

Skeletal malformations:

not examined

Visceral malformations:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No peri- and post natal effect were observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

> Tissue distribution of cerium:

Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO2 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.

> Tables:

Table 1: Food consumption of CeO2 NPs-treated males and females during the study period.

CeO2 NPs (mg/kg bw/day)	0	100	300	1000
Males
Pre-mating day 1–4 (g)	30.6 ± 1.9	29.3 ± 0.8	28.6 ± 1.5*	30.5 ± 1.6
Pre-mating day 4–8 (g)	31.3 ± 2.2	30.4 ± 1.2	30.2 ± 1.6	31.2 ± 1.4
Pre-mating day 8–11 (g)	31.4 ± 2.3	31.5 ± 1.5	30.5 ± 2.3	32.2 ± 1.2
Pre-mating day 11–14 (g)	32.3 ± 2.0	32.6 ± 1.8	31.4 ± 2.4	32.7 ± 1.1
Total period (g, pre-mating day 1–14)	31.4 ± 2.0	30.9 ± 1.1	30.2 ± 1.9	31.7 ± 1.0
Females
Pre-mating day 1–4 (g)	21.1 ± 1.6	20.2 ± 1.1	21.3 ± 1.1	21.2 ± 1.6
Pre-mating day 4–8 (g)	21.8 ± 1.2	21.4 ± 0.8	22.7 ± 1.2	22.2 ± 1.6
Pre-mating day 8–11 (g)	21.9 ± 1.7	22.3 ± 1.4	23.0 ± 2.2	22.1 ± 1.9
Pre-mating day 11–14 (g)	23.0 ± 1.5	23.0 ± 1.5	23.7 ± 0.8	23.3 ± 1.9
Gestation day 0–7 (g)	25.6 ± 1.7	26.0 ± 2.6	25.7 ± 2.2	26.8 ± 1.4
Gestation day 7–14 (g)	25.5 ± 1.5	25.9 ± 2.3	25.9 ± 2.3	25.8 ± 2.0
Gestation day 14–20 (g)	29.8 ± 2.1	29.2 ± 1.9	29.3 ± 1.9	31.5 ± 2.0
Post-natal day 0–4 (g)	35.8 ± 5.0	36.8 ± 3.2	35.9 ± 6.1	40.1 ± 4.1
Total period (g, pre-mating day 1 to lactation day 4)	25.0 ± 1.1	25.0 ± 1.4	25.4 ± 1.2	25.9 ± 1.3

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=12, mean ± SD).

 

Table 2: Hematology results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
RBC (10^6/µL)	9.1 ± 0.4	9.1 ± 0.3	8.9 ± 0.5	8.9 ± 0.1	7.8 ± 0.5	7.7 ± 0.3	7.9 ± 0.5	8.1 ± 0.2
HGB (g/dL)	16.6 ± 0.6	16.9 ± 0.4	16.8 ± 0.6	16.8 ± 0.6	14.9 ± 0.9	14.8 ± 0.3	14.8 ± 0.9	15.3 ± 0.5
HCT (%)	51.0 ± 2.5	51.4 ± 2.0	50.7 ± 2.1	50.4 ± 2.1	46.0 ± 2.6	45.6 ± 1.4	45.5 ± 2.7	47.0 ± 1.5
MCV (fL	56.1 ± 1.7	56.2 ± 0.9	57.2 ± 1.8	56.9 ± 1.9	59.4 ± 0.9	59.4 ± 1.2	57.9 ± 2.0	58.2 ± 1.2
MCH (pg)	18.3 ± 0.4	18.5 ± 0.3	18.9 ± 0.7	18.9 ± 0.5	19.3 ± 0.2	19.3 ± 0.5	18.8 ± 0.7	18.9 ± 0.3
MCHC (g/dL)	32.5 ± 0.4	33.0 ± 0.7	33.1 ± 0.6	33.3 ± 0.4	32.5 ± 0.5	32.5 ± 0.5	32.5 ± 0.4	32.4 ± 0.4
PLT (10^3/µL)	1130.8 ± 149.7	1024.2 ± 133.7	940.4 ± 41.3	1101.6 ± 84.8	1351.0 ± 167.3	1213.2 ± 174.2	1258.4 ± 222.7	1305.0 ± 253.0
RET (%)	2.5 ± 0.5	2.5 ± 0.3	2.5 ± 0.3	2.5 ± 0.6	5.8 ± 1.3	6.4 ± 1.7	6.3 ± 1.4	6.2 ± 1.7
RETA (10^9/µL	223.8 ± 41.4	226.0 ± 22.2	223.8 ± 31.6	218.4 ± 48.7	448.3 ± 78.3	492.2 ± 142.9	489.7 ± 77.8	502.9 ± 136.4
WBC(10^3/µL)	10.6 ± 3.3	11.6 ± 4.1	12.4 ± 1.0	11.0 ± 2.2	9.5 ± 2.5	9.8 ± 2.4	11.2 ± 1.1	12.8 ± 3.2
NEU (%)	14.8 ± 6.9	11.4 ± 1.7	12.1 ± 3.0	13.4 ± 2.5	12.2 ± 5.0	13.3 ± 2.2	12.8 ± 3.2	13.2 ± 2.8
NEUA(10^3/µL)	1.5 ± 0.7	1.4 ± 0.7	1.5 ± 0.5	1.5 ± 0.5	1.2 ± 0.7	1.3 ± 0.3	1.4 ± 0.2	1.7 ± 0.8
LYM (%)	81.3 ± 6.7	84.7 ± 2.1	83.2 ± 3.6	81.7 ± 2.0	81.5 ± 5.2	80.5 ± 2.0	80.7 ± 4.0	80.3 ± 3.2
LYMA(10^3/µL)	8.7 ± 2.8	9.8 ± 3.3	10.3 ± 0.7	9.0 ± 1.7	7.7 ± 2.0	7.9 ± 2.1	9.1 ± 1.4	10.2 ± 2.3
EOS (%)	0.8 ± 0.2	0.9 ± 0.2	0.9 ± 0.3	1.0 ± 0.4	0.8 ± 0.4	0.6 ± 0.2	0.7 ± 0.3	0.7 ± 0.3
EOSA (10^3/µL)	0.09 ± 0.05	0.10 ± 0.03	0.12 ± 0.05	0.11 ± 0.04	0.07 ± 0.02	0.06 ± 0.04	0.07 ± 0.02	0.09 ± 0.04
MON (%)	1.9 ± 0.6	1.9 ± 0.6	2.3 ± 0.7	2.5 ± 0.6	4.3 ± 0.7	4.0 ± 1.2	4.6 ± 0.9	4.6 ± 0.8
MONA (10^3/µL)	0.2 ± 0.1	0.3 ± 0.2	0.3 ± 0.1	0.3 ± 0.0	0.4 ± 0.1	0.4 ± 0.1	0.5 ± 0.1	0.6 ± 0.2
BAS (%)	0.6 ± 0.1	0.5 ± 0.1	0.7 ± 0.1	0.6 ± 0.2	0.5 ± 0.1	0.5 ± 0.2	0.4 ± 0.1	0.4 ± 0.1
BASA (10^3/µL)	0.06 ± 0.02	0.06 ± 0.03	0.09 ± 0.01	0.06 ± 0.01	0.04 ± 0.01	0.05 ± 0.03	0.05 ± 0.01	0.05 ± 0.02
LUC (%)	0.7 ± 0.3	0.6 ± 0.1	0.7 ± 0.1	0.8 ± 0.1	0.8 ± 0.3	1.1 ± 0.1	0.8 ± 0.3	0.8 ± 0.2
LUCA (10^3/µL)	0.07 ± 0.05	0.07 ± 0.04	0.09 ± 0.02	0.09 ± 0.01	0.07 ± 0.01	0.10 ± 0.03	0.09 ± 0.03	0.10 ± 0.03
PT (sec)	14.0 ± 1.7	13.7 ± 0.8	16.0 ± 2.7	13.6 ± 0.7	13.6 ± 1.2	15.5 ± 0.3*	15.3 ± 0.9	14.4 ± 1.5
APTT (sec)	17.5 ± 0.7	18.0 ± 0.8	18.3 ± 0.9	17.8 ± 1.0	6.1 ± 0.7	15.6 ± 1.2	14.8 ± 1.0	15.0 ± 0.9

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=5, mean ± SD).

 

Table 3: Clinical chemistry results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
GLU (mg/dL)	158.9 ± 20.3	149.2 ± 28.8	157.6 ± 45.1	148.5 ± 29.1	131.2 ± 22.4	116.0 ± 15.1	108.8 ± 10.7	139.1 ± 17.1
BUN (mg/dL)	15.1 ± 1.6	13.7 ± 1.5	14.3 ± 1.7	13.6 ± 1.8	24.1 ± 4.7	19.4 ± 3.4	21.2 ± 4.1	20.3 ± 3.6
CREA (mg/dL)	0.49 ± 0.04	0.48 ± 0.03	0.47 ± 0.04	0.47 ± 0.06	0.57 ± 0.08	0.51 ± 0.03	0.54 ± 0.04	0.56 ± 0.03
TP (g/dL)	6.6 ± 0.3	6.7 ± 0.2	6.6 ± 0.2	6.6 ± 0.4	7.0 ±0.4	7.0 ± 0.4	7.0 ± 0.2	7.1 ± 0.3
ALB (g/dL)	4.3 ± 0.2	4.2 ± 0.1	4.2 ± 0.1	4.2 ± 0.2	4.5 ±0.2	4.6 ± 0.3	4.5 ± 0.1	4.6 ± 0.1
A/G (ratio)	1.8 ± 0.2	1.7 ± 0.1	1.8 ± 0.1	1.8 ± 0.1	1.8 ±0.1	2.0 ± 0.2	1.9 ± 0.1	1.9 ± 0.1
AST (IU/L)	149.4 ± 21.9	137.5 ± 34.5	129.9 ± 4.3	153.9 ± 18.5	142.7 ± 44.3	129.1 ± 23.1	135.6 ± 17.7	121.3 ± 8.0
ALT (IU/L)	33.4 ± 5.7	28.6 ± 5.6	32.5 ± 3.0	31.5 ± 3.9	40.2 ± 8.0	41.5 ± 8.1	45.1 ± 5.2	39.8 ± 2.6
TBIL (mg/dL)	0.14 ± 0.02	0.12 ± 0.01	0.15 ± 0.02	0.13 ± 0.02	0.13 ± 0.02	0.12 ± 0.02	0.13 ± 0.02	0.12 ± 0.02
GGT (IU/L)	0.41 ± 0.23	0.65 ± 0.13	0.60 ± 0.12	0.92 ± 0.18**	0.59 ± 0.22	0.78 ± 0.10	0.67 ± 0.27	0.71 ± 0.38
ALP (IU/L)	581.6 ± 179.2	510.3 ± 110.0	467.4 ± 57.3	514.4 ± 66.4	332.3 ± 111.0	293.1 ± 58.0	232.7 ± 31.7	267.1 ± 83.9
TCHO (mg/dL)	70.8 ± 26.9	64.2 ± 19.5	60.0 ± 8.9	68.4 ± 10.2	49.8 ± 16.0	58.0 ± 16.5	53.8 ± 10.8	55.2 ± 12.8
TG (mg/dL)	31.8 ± 9.3	34.2 ± 11.8	23.9 ± 10.0	21.4 ± 10.9	38.9 ± 22.5	36.9 ± 13.1	41.7 ± 14.9	54.7 ± 25.5
Ca (mg/dL)	11.1 ± 0.3	11.3 ± 0.4	11.2 ± 0.2	11.0 ± 0.7	11.7 ± 0.4	11.8 ± 0.5	11.7 ± 0.3	11.9 ± 0.2
IP (mg/dL)	10.2 ± 0.5	10.3 ± 0.6	10.5 ± 0.4	10.3 ± 0.7	9.9 ± 0.8	10.2 ± 0.1	10.3 ± 1.2	9.8 ± 0.7
K (mmol/L)	8.3 ± 0.9	7.5 ± 0.9	8.4 ± 0.9	7.8 ± 1.6	8.6 ± 0.5	8.3 ± 0.5	8.2 ± 1.1	8.2 ± 1.7
CK (IU/L)	715.6 ± 197.6	584.2 ± 203.2	483.6 ± 79.4	652.0 ± 218.0	611.8 ± 331.8	518.0 ± 112.9	570.6 ± 129.1	448.8 ± 130.3
PL (mg/dL)	102.4 ± 25.0	93.6 ± 22.7	92.0 ± 7.3	99.2 ± 10.4	106.8 ± 26.9	116.8 ± 24.8	108.6 ± 14.8	115.8 ± 19.1
Na (mmol/L)	148.0 ± 0.7	147.8 ± 1.9	147.4 ± 1.1	147.8 ± 1.6	144.8 ± 1.3	145.0 ± 1.9	145.0 ± 1.4	146.0 ± 1.6
Cl (mmol/L)	102.4 ± 1.7	101.6 ± 0.6	102.6 ± 1.3	102.4 ± 0.9	101.4 ± 0.6	101.2 ± 1.5	101.6 ± 1.1	102.2 ± 2.4

**: Represent a significant difference at the p<0.01 level compared to the vehicle control (n=5, mean ± SD).

 

Table 4: Absolute and relative organ weights of CeO2 NPs-treated males during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight^a(g)	(g)	427.0 ± 38.7	439.2 ± 23.7	436.4 ± 36.0	448.9 ± 28.6
	N	12	12	12	12
Adrenal glands	Absolute (g)	0.06 ± 0.01	0.07 ± 0.01	0.06 ± 0.01	0.07 ± 0.00
	Relative^b(%)	0.016 ± 0.003	0.016 ± 0.002	0.016 ± 0.002	0.015 ± 0.001
	N	5	5	5	5
Brain	Absolute (g)	1.97 ± 0.12	1.99 ± 0.07	2.03 ± 0.09	2.00 ± 0.12
	Relative (%)	0.484 ± 0.068	0.460 ± 0.023	0.497 ± 0.030	0.461 ± 0.013
	N	5	5	5	5
Heart	Absolute (g)	1.25 ± 0.12	1.29 ± 0.14	1.33 ± 0.13	1.32 ± 0.11
	Relative (%)	0.304 ± 0.023	0.300 ± 0.030	0.325 ± 0.024	0.303 ± 0.028
	N	5	5	5	5
Kidneys	Absolute (g)	3.25 ± 0.19	3.40 ± 0.15	3.50 ± 0.33	3.47 ± 0.42
	Relative (%)	0.794 ± 0.071	0.788 ± 0.050	0.854 ± 0.047	0.797 ± 0.070
	N	5	5	5	5
Liver	Absolute (g)	11.71 ± 2.07	12.74 ± 0.71	12.12 ± 2.09	12.51 ± 1.41
	Relative (%)	2.825 ± 0.217	2.950 ± 0.1063	2.940 ± 0.246	2.870 ± 0.197
	N	5	5	5	5
Pituitary gland	Absolute (g)	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00
	Relative (%)	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000
	N	5	5	5	5
Prostate	Absolute (g)	0.62 ± 0.12	0.59 ± 0.17	0.66 ± 0.13	0.65 ± 0.09
	Relative (%)	0.144 ± 0.026	0.135 ± 0.041	0.151 ± 0.031	0.146 ± 0.018
	N	12	12	12	12
Spleen	Absolute (g)	0.66 ± 0.13	0.67 ± 0.15	0.65 ± 0.13	0.69 ± 0.08
	Relative (%)	0.158 ± 0.013	0.155 ± 0.033	0.158 ± 0.020	0.158 ± 0.013
	N	5	5	5	5
Thymus	Absolute (g)	0.34 ± 0.02	0.39 ± 0.08	0.35 ± 0.11	0.32 ± 0.06
	Relative (%)	0.083 ± 0.010	0.090 ± 0.018	0.084 ± 0.019	0.072 ± 0.011
	N	5	5	5	5
Lungs	Absolute (g)	1.50 ± 0.13	1.58 ± 0.12	1.57 ± 0.17	1.62 ± 0.07
	Relative (%)	0.367 ± 0.041	0.367 ± 0.031	0.383 ± 0.021	0.374 ± 0.021
	N	5	5	5	5
Right testis	Absolute (g)	1.70 ± 0.17	1.74 ± 0.13	1.69 ± 0.11	1.65 ± 0.12
	Relative (%)	0.400 ± 0.049	0.396 ± 0.031	0.391 ± 0.051	0.369 ± 0.028
	N	12	12	12	12
Left testis	Absolute (g)	1.73 ± 0.16	1.74 ± 0.12	1.71 ± 0.11	1.67 ± 0.12
	Relative (%)	0.407 ± 0.051	0.397 ± 0.026	0.394 ± 0.044	0.372 ± 0.030
	N	12	12	12	12
Right epididymis	Absolute (g)	0.66 ± 0.06	0.69 ± 0.06	0.68 ± 0.06	0.66 ± 0.07
	Relative (%)	0.155 ± 0.017	0.158 ± 0.016	0.156 ± 0.021	0.148 ± 0.021
	N	12	12	12	12
Left epididymis	Absolute (g)	0.67 ± 0.07	0.68 ± 0.06	0.66 ± 0.06	0.65 ± 0.06
	Relative (%)	0.157 ± 0.019	0.156 ± 0.014	0.152 ± 0.019	0.145 ± 0.018
	N	12	12	12	12
Seminal vesicles with coagulating glands	Absolute (g)	1.68 ± 0.21	1.65 ± 0.29	1.68 ± 0.22	1.69 ± 0.21
	Relative (%)	0.396 ± 0.047	0.378 ± 0.072	0.388 ± 0.057	0.377 ± 0.048
	N	12	12	12	12

N=5 or 12, mean ± SD.

a: Terminal body weight were measured immediately before necropsy.

b: Organ weight/terminal body weight ratio.

 

Table 5: Absolute and relative organ weights of CeO2 NPs-treated females during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight	(g)	314.5 ± 19.9	316.7 ± 24.1	311.0 ± 23.7	316.8 ± 19.8
Adrenal glands	Absolute (g)	0.08 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0.01
	Relative (%)	0.028 ± 0.003	0.024 ± 0.003	0.027 ± 0.003	0.027 ± 0.002
Brain	Absolute (g)	1.94 ± 0.09	1.93 ± 0.08	2.02 ± 0.08	1.96 ± 0.05
	Relative (%)	0.652 ± 0.036	0.650 ± 0.022	0.692 ± 0.028	0.654 ± 0.030
Heart	Absolute (g)	1.00 ± 0.06	0.95 ± 0.04	0.97 ± 0.07	1.04 ± 0.06
	Relative (%)	0.334 ± 0.013	0.321 ± 0.017	0.334 ± 0.026	0.346 ± 0.019
Kidneys	Absolute (g)	2.28 ± 0.19	2.23 ± 0.21	2.14 ± 0.14	2.23 ± 0.07
	Relative (%)	0.763 ± 0.028	0.750 ± 0.051	0.734 ± 0.036	0.744 ± 0.027
Liver	Absolute (g)	10.52 ± 1.21	10.40 ± 0.87	10.19 ± 0.30	10.91 ± 0.73
	Relative (%)	3.522 ± 0.266	3.503 ± 0.277	3.495 ± 0.169	3.638 ± 0.259
Pituitary gland	Absolute (g)	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
	Relative (%)	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.000
Spleen	Absolute (g)	0.62 ± 0.07	0.69 ± 0.09	0.65 ± 0.08	0.70 ± 0.12
	Relative (%)	0.209 ± 0.021	0.231 ± 0.029	0.223 ± 0.032	0.232 ± 0.043
Thymus	Absolute (g)	0.31 ± 0.03	0.31 ± 0.05	0.31 ± 0.08	0.38 ± 0.12
	Relative (%)	0.104 ± 0.013	0.104 ± 0.021	0.105 ± 0.027	0.125 ± 0.041
Lungs	Absolute (g)	1.34 ± 0.04	1.29 ± 0.06	1.42 ± 0.05	1.36 ± 0.11
	Relative (%)	0.451 ± 0.013	0.435 ± 0.018	0.486 ± 0.022	0.453 ± 0.034
Uterus	Absolute (g)	0.78 ± 0.12	0.78 ± 0.09	0.83 ± 0.12	0.73 ± 0.06
	Relative (%)	0.260 ± 0.039	0.262 ± 0.030	0.284 ± 0.035	0.243 ± 0.026
Right ovary	Absolute (g)	0.07 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
	Relative (%)	0.021 ± 0.002	0.021 ± 0.004	0.019 ± 0.003	0.019 ± 0.004
Left Ovary	Absolute (g)	0.07 ± 0.02	0.06 ± 0.01	0.06 ± 0.01	0.05 ± 0.01
	Relative (%)	0.023 ± 0.006	0.020 ± 0.002	0.020 ± 0.005	0.018 ± 0.002

N=5, mean ± SD.

 

Table 6: Fertility with precoital time results of CeO2 NPs treated males during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Males
Mating index^a	100	100	100	100
Fertility index^b	100	100	100	100
Fecundity index^c	100	100	100	100
Females
Mating index^d	100	100	100	100
Fertility index^e	100	100	100	100
Pregnancy index^f	100	100	100	100
Precoital Time (day)	3.3 ± 3.6	1.8 ± 0.6	2.2 ± 1.1	1.9 ± 1.1

 

N=12, mean ± SD.

a: (No. of males with evidence of mating/No. of males paired) x 100.

b: (No. of males impregnating a female/No. of males paired) x 100.

c: (No. of males impregnating a female/No. of males with evidence of mating) x 100.

d: (No. of females with evidence of mating/No. of females paired) x 100.

e: (No. of pregnant females/No. of females paired) x 100.

f: (No. of pregnant females/No. of females with evidence of mating) x100.

 

 

Table 7: Reproductive and litter findings results of CeO2 NPs-treated females during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Gestation period (day)	21.5 ± 0.4	21.5 ± 0.3	21.6 ± 0.3	21.8 ± 0.4
Corpora lutea (N)	17.8 ± 2.8	16.5 ± 2.6	16.3 ± 1.7	17.4 ± 2.0
Implantations (N)	15.3 ± 3.1	15.3 ± 2.4	14.8 ± 2.1	15.4 ± 2.0
Pups born (N)	14.8 ± 3.1	14.4 ± 2.4	13.9 ± 2.0	14.8 ± 2.1
Perinatal death (N)	0.08 ± 0.29	0.00 ± 0.00	0.00 ± 0.00	0.25 ± 0.45
Unaccounted-for sites^a(%)	2.8 ± 4.5	5.5 ± 5.8	6.2 ± 4.5	3.9 ± 4.5
Sex ratio^b(%)	106.9 ± 62.6	93.9 ± 28.2	105.3 ± 55.5	130.7 ± 66.8
Live litter size (N) PND 0 PND 4	14.8 ± 3.1 14.8 ± 3.1	14.4 ± 2.4 14.3 ± 2.4	13.9 ± 2.0 13.8 ± 2.0	14.6 ± 2.1 14.4 ± 2.0
Viability index^c(%)	100.0	99.4 ± 1.9	98.9 ± 2.6	99.0 ± 2.4
Delivery index^d(%)	100.0	100.0	100.0	100.0
Pups with external abnormalities	0	0	0	0

 

N = 12, mean ± SD.

a: (No. of implantation sites/litter) - (No. of live pups at birth/litter)/No. of implantation sites/litter x 100.

b: (No. of male pups on PND 0/litter)/(No. of female pups on PND 0/litter) x 100.

c: (No. of live pups on PND 4/litter)/(No. of live pups at birth/litter) x 100.

d: (No. of dams with live pups)/(No. of pregnant dams) x 100.

 

Table 8: F1 pups body weights of CeO2 NPs-treated parental animals during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
F1 male pups
PND 0
Body weight (g)	6.5 ± 0.4	6.7 ± 0.3	6.8 ± 0.6	7.0 ± 0.5
Covariate-adjusted mean (g)	6.5	6.7	6.8	7.0*
PND 4
Body weight (g)	10.1 ± 0.8	10.7 ± 1.1	11.0 ± 1.1	11.0 ± 1.2
Covariate-adjusted mean (g)	10.1	10.6	10.9	11.2*
F1 female pups
PND 0
Body weight (g)	6.2 ± 0.4	6.3 ± 0.3	6.4 ± 0.6	6.7 ± 0.6
Covariate-adjusted mean (g)	6.2	6.3	6.4	6.7*
PND 4
Body weight (g)	9.5 ± 0.9	10.0 ± 1.0	10.4 ± 1.2	10.6 ± 1.4
Covariate-adjusted mean (g)	9.6	10.1	10.4	10.5

 

N =12, mean ± SD.

*Represent a significant difference at the p<0.05 level compared to the vehicle control.

Conclusions:

In conclusion, under the experimental conditions of this study design, there were no CeO2 NPs related adverse effects in terms of general systemic signs as well as development and reproduction, at doses up to 1000 mg/kg bw/d. Therefore, the NOAEL for maternal toxicity can be set at 1000 mg/kd bw/day and the NOAEL for peri and post natal developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.

Executive summary:

In a reproduction/developmental toxicity screening study, the effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD Guideline no 422 and was compliant to GLP. The study was thus considered as a key study of reliablility 1 according to Klimisch score.

Groups of 12 males and 12 females SD rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg/day nano CeO2 in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effect of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily.

Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).

No unscheduled death or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study.

No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time was found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.

Tissue distribution analysis of cerium in parental and pup tissues revealed that nano CeO2 was 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.

The authors concluded that, under the experimental conditions of this study, no nano CeO2 related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and therefore, the maternal toxicity can de set at 1000 mg/kg bw/ay and the NOAEL for peri and post natal developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD 1996

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cerium dioxide

EC Number:

215-150-4

EC Name:

Cerium dioxide

Cas Number:

1306-38-3

Molecular formula:

CeO2

IUPAC Name:

cerium dioxide

Test material form:

solid: nanoform

Details on test material:

- State of aggregation: according to the authors, CeO2 NPs formed larger agglomerates in deionised water.
- Particle size distribution: not specifed
- Mass median aerodynamic diameter (MMAD): not applicable
- Geometric standard deviation (GSD): not applicable
- Shape of particles (TEM): the majority of the CeO2 NPs had a polyhedral shape.
- Surface area of particles: not specified
- Crystal structure: not specified
- Coating: none
- Surface properties: not specified
- Density: not specified
- Moisture content: not specified
- Residual solvent: not specified
- Activation: not specified
- Stabilisation: not specified
- Other:
> Primary Particle size (TEM): 14.2 ± 5.0 nm
> Hydrodynamic size (DLS) : 76.3 nm in deionised water
> Zeta potential (DLS): 47.37 mV in deionised water
> Supplier: Sigma-Aldrich (USA).
> Purity (EDX): 100 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio, Inc. (Republic of Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 1 or 2 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.
- Diet (e.g. ad libitum): The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum.
- Water (e.g. ad libitum): The water was irradiated by UV light and filtered prior to provide ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): 10–20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light-dark cycle

IN-LIFE DATES: The experimental phase of this study was conducted in 2015.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

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.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

- 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).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control : deionized water (vehicle)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of the results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group). 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).
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, and 300 and 100 mg/kg were determined to be the intermediate and low doses, respectively.

- Rationale for animal assignment (if not random):
Healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study. 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).

- Fasting period before blood sampling for clinical biochemistry: Yes.Aapproximately 16 hours (overnight) prior to sacrifice

- Post-exposure recovery period in satellite groups: no

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical examinations including mortality and general clinical signs were examined twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were examined once weekly during the study period

BODY WEIGHT: Yes
- Time schedule for examinations: Animal body weights were measured twice weekly during the premating and mating periods. Mated females were weightedon days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in the same days of the body weight measurement except for the mating and was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
- Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. 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).

CLINICAL CHEMISTRY: Yes
- 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).

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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).

IMMUNOLOGY: Not specified

OTHER:
The following parameters related to the reproduction were assessed during the study :
> Oestrous cyclicity was measured.
> All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed and processed for histopathological analyses.
> Reproductives indices:
Based on the 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 ca lculated.
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.
> Offspring vialbility indices:
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. Pup number, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain were also recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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. All animals were subjected to macroscopic observations.

HISTOPATHOLOGY: Yes
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.

Other examinations:

Tissue colection 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 -80 °C) until analysis.
For the cerium content analysis, collected samples were thawed and digested in a solution of 1ml 30% H2O2 and 7ml 70% HNO3 with a microwave digestion system. After wet digestion, the cerium content in collected tissues was analyzed with an inductively coupled plasma mass spectrometry (ICP-MS) at the Korea Basic Science Institute (Seoul Center, Republic of Korea). The limit of detection (LOD) and method quantification limit (MQL) for collected samples were determined to be 0.002647 μg/kg and 0.8911 μg/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.

Statistics:

Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study (Lee et al. 2019). 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. 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups.

Mortality:

no mortality observed

Description (incidence):

No CeO2 NPs-related dead or moribund animals were observed during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in body weight or weight gain during the study (see Figure 1 in "Any other information on results incl. tables")

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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 but no alteration were seen afterward. No effect of the treatment was seen in the other treated groups in males and in all groups of treated females as compared to the controls animals (see Table 1 in "Any other information on results incl. tables"). This finding was considered by the authors to be incidental since it was transient and did not have a dose response.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. However, 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. Other hematology values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 2 in "Any other information on results incl. tables"). Thus, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No overt effect of the treatment was observed as compared to the controls. However, 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 but the other clinical chemistry values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 3 in "Any other information on results incl. tables"). The authors have considered the increase in GGT in 1000 mg/kg dose-group males as incidental since there were no correlated changes in organ weights and histopathological examinations.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in organ weights among the treatment and control animals (see Tables 4 and 5 in "Any other information on results incl. tables").

Gross pathological findings:

no effects observed

Description (incidence and severity):

In macroscopic observations, there were no treatment-related changes among the treatment and control animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys (Grade 1) was observed. No such effect were seen in females. No other effect in the examined organs were seen in the treated groups of male and females animals as compared to the controls. The increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental by the autors since it also occurred sporadically in normal animals, did not have an obvious dose response and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO2 NPs-related changes in other examinations for general systemic effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

> Reproductive function / performances of parents:
There were no treatment-related changes in fertility results with precoital time. Mating index, Fertility index and Fecundidity index were all equal to 100 in all groups of males. Mating index, Fertility index and Pregnancy index were also found all equal to 100 whatever the dose level groups in females. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantati on sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods (see Tables 6 and 7 in "Any other information on results incl. tables").

> Pup examinations:
- Clinical signs: In general clinical signs and external examination of F1 pups at necropsy, there were no treatment-related changes among the treatment and control animals.
- Mortality: No effect of the treatment was seen on the perinatal death and the viability index (see Table 7 in "Any other information on results incl. tables").
- Body weight and weight changes: No overt effect of the treatment was seen on pup body weight. An increase in F1 male and female pup covariate-adjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4 for males and PND 0 for females) was observed at 1000 mg/kg. (see Table 8 in "Any other information on results incl. tables"). Since there were no concurrent changes in maternal body weight, litter size or gestation length and no concurrent estrus cycle abnormalities and histopathological changes in reproductive organs, therefore, the increased pup body weight was not considered treatment-related.
- External abnormalities: No pup with external abnormalities was found in this study (see Table 7 in "Any other information on results incl. tables").

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicity observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

> Tissue distribution of cerium:

Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO2 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.

> Tables

Table 1: Food consumption of CeO2 NPs-treated males and females during the study period.

CeO2 NPs (mg/kg bw/day)	0	100	300	1000
Males
Pre-mating day 1–4 (g)	30.6 ± 1.9	29.3 ± 0.8	28.6 ± 1.5*	30.5 ± 1.6
Pre-mating day 4–8 (g)	31.3 ± 2.2	30.4 ± 1.2	30.2 ± 1.6	31.2 ± 1.4
Pre-mating day 8–11 (g)	31.4 ± 2.3	31.5 ± 1.5	30.5 ± 2.3	32.2 ± 1.2
Pre-mating day 11–14 (g)	32.3 ± 2.0	32.6 ± 1.8	31.4 ± 2.4	32.7 ± 1.1
Total period (g, pre-mating day 1–14)	31.4 ± 2.0	30.9 ± 1.1	30.2 ± 1.9	31.7 ± 1.0
Females
Pre-mating day 1–4 (g)	21.1 ± 1.6	20.2 ± 1.1	21.3 ± 1.1	21.2 ± 1.6
Pre-mating day 4–8 (g)	21.8 ± 1.2	21.4 ± 0.8	22.7 ± 1.2	22.2 ± 1.6
Pre-mating day 8–11 (g)	21.9 ± 1.7	22.3 ± 1.4	23.0 ± 2.2	22.1 ± 1.9
Pre-mating day 11–14 (g)	23.0 ± 1.5	23.0 ± 1.5	23.7 ± 0.8	23.3 ± 1.9
Gestation day 0–7 (g)	25.6 ± 1.7	26.0 ± 2.6	25.7 ± 2.2	26.8 ± 1.4
Gestation day 7–14 (g)	25.5 ± 1.5	25.9 ± 2.3	25.9 ± 2.3	25.8 ± 2.0
Gestation day 14–20 (g)	29.8 ± 2.1	29.2 ± 1.9	29.3 ± 1.9	31.5 ± 2.0
Post-natal day 0–4 (g)	35.8 ± 5.0	36.8 ± 3.2	35.9 ± 6.1	40.1 ± 4.1
Total period (g, pre-mating day 1 to lactation day 4)	25.0 ± 1.1	25.0 ± 1.4	25.4 ± 1.2	25.9 ± 1.3

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=12, mean ± SD).

 

Table 2: Hematology results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
RBC (10^6/µL)	9.1 ± 0.4	9.1 ± 0.3	8.9 ± 0.5	8.9 ± 0.1	7.8 ± 0.5	7.7 ± 0.3	7.9 ± 0.5	8.1 ± 0.2
HGB (g/dL)	16.6 ± 0.6	16.9 ± 0.4	16.8 ± 0.6	16.8 ± 0.6	14.9 ± 0.9	14.8 ± 0.3	14.8 ± 0.9	15.3 ± 0.5
HCT (%)	51.0 ± 2.5	51.4 ± 2.0	50.7 ± 2.1	50.4 ± 2.1	46.0 ± 2.6	45.6 ± 1.4	45.5 ± 2.7	47.0 ± 1.5
MCV (fL	56.1 ± 1.7	56.2 ± 0.9	57.2 ± 1.8	56.9 ± 1.9	59.4 ± 0.9	59.4 ± 1.2	57.9 ± 2.0	58.2 ± 1.2
MCH (pg)	18.3 ± 0.4	18.5 ± 0.3	18.9 ± 0.7	18.9 ± 0.5	19.3 ± 0.2	19.3 ± 0.5	18.8 ± 0.7	18.9 ± 0.3
MCHC (g/dL)	32.5 ± 0.4	33.0 ± 0.7	33.1 ± 0.6	33.3 ± 0.4	32.5 ± 0.5	32.5 ± 0.5	32.5 ± 0.4	32.4 ± 0.4
PLT (10^3/µL)	1130.8 ± 149.7	1024.2 ± 133.7	940.4 ± 41.3	1101.6 ± 84.8	1351.0 ± 167.3	1213.2 ± 174.2	1258.4 ± 222.7	1305.0 ± 253.0
RET (%)	2.5 ± 0.5	2.5 ± 0.3	2.5 ± 0.3	2.5 ± 0.6	5.8 ± 1.3	6.4 ± 1.7	6.3 ± 1.4	6.2 ± 1.7
RETA (10^9/µL	223.8 ± 41.4	226.0 ± 22.2	223.8 ± 31.6	218.4 ± 48.7	448.3 ± 78.3	492.2 ± 142.9	489.7 ± 77.8	502.9 ± 136.4
WBC(10^3/µL)	10.6 ± 3.3	11.6 ± 4.1	12.4 ± 1.0	11.0 ± 2.2	9.5 ± 2.5	9.8 ± 2.4	11.2 ± 1.1	12.8 ± 3.2
NEU (%)	14.8 ± 6.9	11.4 ± 1.7	12.1 ± 3.0	13.4 ± 2.5	12.2 ± 5.0	13.3 ± 2.2	12.8 ± 3.2	13.2 ± 2.8
NEUA(10^3/µL)	1.5 ± 0.7	1.4 ± 0.7	1.5 ± 0.5	1.5 ± 0.5	1.2 ± 0.7	1.3 ± 0.3	1.4 ± 0.2	1.7 ± 0.8
LYM (%)	81.3 ± 6.7	84.7 ± 2.1	83.2 ± 3.6	81.7 ± 2.0	81.5 ± 5.2	80.5 ± 2.0	80.7 ± 4.0	80.3 ± 3.2
LYMA(10^3/µL)	8.7 ± 2.8	9.8 ± 3.3	10.3 ± 0.7	9.0 ± 1.7	7.7 ± 2.0	7.9 ± 2.1	9.1 ± 1.4	10.2 ± 2.3
EOS (%)	0.8 ± 0.2	0.9 ± 0.2	0.9 ± 0.3	1.0 ± 0.4	0.8 ± 0.4	0.6 ± 0.2	0.7 ± 0.3	0.7 ± 0.3
EOSA (10^3/µL)	0.09 ± 0.05	0.10 ± 0.03	0.12 ± 0.05	0.11 ± 0.04	0.07 ± 0.02	0.06 ± 0.04	0.07 ± 0.02	0.09 ± 0.04
MON (%)	1.9 ± 0.6	1.9 ± 0.6	2.3 ± 0.7	2.5 ± 0.6	4.3 ± 0.7	4.0 ± 1.2	4.6 ± 0.9	4.6 ± 0.8
MONA (10^3/µL)	0.2 ± 0.1	0.3 ± 0.2	0.3 ± 0.1	0.3 ± 0.0	0.4 ± 0.1	0.4 ± 0.1	0.5 ± 0.1	0.6 ± 0.2
BAS (%)	0.6 ± 0.1	0.5 ± 0.1	0.7 ± 0.1	0.6 ± 0.2	0.5 ± 0.1	0.5 ± 0.2	0.4 ± 0.1	0.4 ± 0.1
BASA (10^3/µL)	0.06 ± 0.02	0.06 ± 0.03	0.09 ± 0.01	0.06 ± 0.01	0.04 ± 0.01	0.05 ± 0.03	0.05 ± 0.01	0.05 ± 0.02
LUC (%)	0.7 ± 0.3	0.6 ± 0.1	0.7 ± 0.1	0.8 ± 0.1	0.8 ± 0.3	1.1 ± 0.1	0.8 ± 0.3	0.8 ± 0.2
LUCA (10^3/µL)	0.07 ± 0.05	0.07 ± 0.04	0.09 ± 0.02	0.09 ± 0.01	0.07 ± 0.01	0.10 ± 0.03	0.09 ± 0.03	0.10 ± 0.03
PT (sec)	14.0 ± 1.7	13.7 ± 0.8	16.0 ± 2.7	13.6 ± 0.7	13.6 ± 1.2	15.5 ± 0.3*	15.3 ± 0.9	14.4 ± 1.5
APTT (sec)	17.5 ± 0.7	18.0 ± 0.8	18.3 ± 0.9	17.8 ± 1.0	6.1 ± 0.7	15.6 ± 1.2	14.8 ± 1.0	15.0 ± 0.9

*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=5, mean ± SD).

 

Table 3: Clinical chemistry results of CeO2 NPs-treated males and females during the study period.

	Males				Females
CeO2 NPs (mg/kg)	0	100	300	1000	0	100	300	1000
GLU (mg/dL)	158.9 ± 20.3	149.2 ± 28.8	157.6 ± 45.1	148.5 ± 29.1	131.2 ± 22.4	116.0 ± 15.1	108.8 ± 10.7	139.1 ± 17.1
BUN (mg/dL)	15.1 ± 1.6	13.7 ± 1.5	14.3 ± 1.7	13.6 ± 1.8	24.1 ± 4.7	19.4 ± 3.4	21.2 ± 4.1	20.3 ± 3.6
CREA (mg/dL)	0.49 ± 0.04	0.48 ± 0.03	0.47 ± 0.04	0.47 ± 0.06	0.57 ± 0.08	0.51 ± 0.03	0.54 ± 0.04	0.56 ± 0.03
TP (g/dL)	6.6 ± 0.3	6.7 ± 0.2	6.6 ± 0.2	6.6 ± 0.4	7.0 ±0.4	7.0 ± 0.4	7.0 ± 0.2	7.1 ± 0.3
ALB (g/dL)	4.3 ± 0.2	4.2 ± 0.1	4.2 ± 0.1	4.2 ± 0.2	4.5 ±0.2	4.6 ± 0.3	4.5 ± 0.1	4.6 ± 0.1
A/G (ratio)	1.8 ± 0.2	1.7 ± 0.1	1.8 ± 0.1	1.8 ± 0.1	1.8 ±0.1	2.0 ± 0.2	1.9 ± 0.1	1.9 ± 0.1
AST (IU/L)	149.4 ± 21.9	137.5 ± 34.5	129.9 ± 4.3	153.9 ± 18.5	142.7 ± 44.3	129.1 ± 23.1	135.6 ± 17.7	121.3 ± 8.0
ALT (IU/L)	33.4 ± 5.7	28.6 ± 5.6	32.5 ± 3.0	31.5 ± 3.9	40.2 ± 8.0	41.5 ± 8.1	45.1 ± 5.2	39.8 ± 2.6
TBIL (mg/dL)	0.14 ± 0.02	0.12 ± 0.01	0.15 ± 0.02	0.13 ± 0.02	0.13 ± 0.02	0.12 ± 0.02	0.13 ± 0.02	0.12 ± 0.02
GGT (IU/L)	0.41 ± 0.23	0.65 ± 0.13	0.60 ± 0.12	0.92 ± 0.18**	0.59 ± 0.22	0.78 ± 0.10	0.67 ± 0.27	0.71 ± 0.38
ALP (IU/L)	581.6 ± 179.2	510.3 ± 110.0	467.4 ± 57.3	514.4 ± 66.4	332.3 ± 111.0	293.1 ± 58.0	232.7 ± 31.7	267.1 ± 83.9
TCHO (mg/dL)	70.8 ± 26.9	64.2 ± 19.5	60.0 ± 8.9	68.4 ± 10.2	49.8 ± 16.0	58.0 ± 16.5	53.8 ± 10.8	55.2 ± 12.8
TG (mg/dL)	31.8 ± 9.3	34.2 ± 11.8	23.9 ± 10.0	21.4 ± 10.9	38.9 ± 22.5	36.9 ± 13.1	41.7 ± 14.9	54.7 ± 25.5
Ca (mg/dL)	11.1 ± 0.3	11.3 ± 0.4	11.2 ± 0.2	11.0 ± 0.7	11.7 ± 0.4	11.8 ± 0.5	11.7 ± 0.3	11.9 ± 0.2
IP (mg/dL)	10.2 ± 0.5	10.3 ± 0.6	10.5 ± 0.4	10.3 ± 0.7	9.9 ± 0.8	10.2 ± 0.1	10.3 ± 1.2	9.8 ± 0.7
K (mmol/L)	8.3 ± 0.9	7.5 ± 0.9	8.4 ± 0.9	7.8 ± 1.6	8.6 ± 0.5	8.3 ± 0.5	8.2 ± 1.1	8.2 ± 1.7
CK (IU/L)	715.6 ± 197.6	584.2 ± 203.2	483.6 ± 79.4	652.0 ± 218.0	611.8 ± 331.8	518.0 ± 112.9	570.6 ± 129.1	448.8 ± 130.3
PL (mg/dL)	102.4 ± 25.0	93.6 ± 22.7	92.0 ± 7.3	99.2 ± 10.4	106.8 ± 26.9	116.8 ± 24.8	108.6 ± 14.8	115.8 ± 19.1
Na (mmol/L)	148.0 ± 0.7	147.8 ± 1.9	147.4 ± 1.1	147.8 ± 1.6	144.8 ± 1.3	145.0 ± 1.9	145.0 ± 1.4	146.0 ± 1.6
Cl (mmol/L)	102.4 ± 1.7	101.6 ± 0.6	102.6 ± 1.3	102.4 ± 0.9	101.4 ± 0.6	101.2 ± 1.5	101.6 ± 1.1	102.2 ± 2.4

**: Represent a significant difference at the p<0.01 level compared to the vehicle control (n=5, mean ± SD).

 

Table 4: Absolute and relative organ weights of CeO2 NPs-treated males during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight^a(g)	(g)	427.0 ± 38.7	439.2 ± 23.7	436.4 ± 36.0	448.9 ± 28.6
	N	12	12	12	12
Adrenal glands	Absolute (g)	0.06 ± 0.01	0.07 ± 0.01	0.06 ± 0.01	0.07 ± 0.00
	Relative^b(%)	0.016 ± 0.003	0.016 ± 0.002	0.016 ± 0.002	0.015 ± 0.001
	N	5	5	5	5
Brain	Absolute (g)	1.97 ± 0.12	1.99 ± 0.07	2.03 ± 0.09	2.00 ± 0.12
	Relative (%)	0.484 ± 0.068	0.460 ± 0.023	0.497 ± 0.030	0.461 ± 0.013
	N	5	5	5	5
Heart	Absolute (g)	1.25 ± 0.12	1.29 ± 0.14	1.33 ± 0.13	1.32 ± 0.11
	Relative (%)	0.304 ± 0.023	0.300 ± 0.030	0.325 ± 0.024	0.303 ± 0.028
	N	5	5	5	5
Kidneys	Absolute (g)	3.25 ± 0.19	3.40 ± 0.15	3.50 ± 0.33	3.47 ± 0.42
	Relative (%)	0.794 ± 0.071	0.788 ± 0.050	0.854 ± 0.047	0.797 ± 0.070
	N	5	5	5	5
Liver	Absolute (g)	11.71 ± 2.07	12.74 ± 0.71	12.12 ± 2.09	12.51 ± 1.41
	Relative (%)	2.825 ± 0.217	2.950 ± 0.1063	2.940 ± 0.246	2.870 ± 0.197
	N	5	5	5	5
Pituitary gland	Absolute (g)	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00
	Relative (%)	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000	0.003 ± 0.000
	N	5	5	5	5
Prostate	Absolute (g)	0.62 ± 0.12	0.59 ± 0.17	0.66 ± 0.13	0.65 ± 0.09
	Relative (%)	0.144 ± 0.026	0.135 ± 0.041	0.151 ± 0.031	0.146 ± 0.018
	N	12	12	12	12
Spleen	Absolute (g)	0.66 ± 0.13	0.67 ± 0.15	0.65 ± 0.13	0.69 ± 0.08
	Relative (%)	0.158 ± 0.013	0.155 ± 0.033	0.158 ± 0.020	0.158 ± 0.013
	N	5	5	5	5
Thymus	Absolute (g)	0.34 ± 0.02	0.39 ± 0.08	0.35 ± 0.11	0.32 ± 0.06
	Relative (%)	0.083 ± 0.010	0.090 ± 0.018	0.084 ± 0.019	0.072 ± 0.011
	N	5	5	5	5
Lungs	Absolute (g)	1.50 ± 0.13	1.58 ± 0.12	1.57 ± 0.17	1.62 ± 0.07
	Relative (%)	0.367 ± 0.041	0.367 ± 0.031	0.383 ± 0.021	0.374 ± 0.021
	N	5	5	5	5
Right testis	Absolute (g)	1.70 ± 0.17	1.74 ± 0.13	1.69 ± 0.11	1.65 ± 0.12
	Relative (%)	0.400 ± 0.049	0.396 ± 0.031	0.391 ± 0.051	0.369 ± 0.028
	N	12	12	12	12
Left testis	Absolute (g)	1.73 ± 0.16	1.74 ± 0.12	1.71 ± 0.11	1.67 ± 0.12
	Relative (%)	0.407 ± 0.051	0.397 ± 0.026	0.394 ± 0.044	0.372 ± 0.030
	N	12	12	12	12
Right epididymis	Absolute (g)	0.66 ± 0.06	0.69 ± 0.06	0.68 ± 0.06	0.66 ± 0.07
	Relative (%)	0.155 ± 0.017	0.158 ± 0.016	0.156 ± 0.021	0.148 ± 0.021
	N	12	12	12	12
Left epididymis	Absolute (g)	0.67 ± 0.07	0.68 ± 0.06	0.66 ± 0.06	0.65 ± 0.06
	Relative (%)	0.157 ± 0.019	0.156 ± 0.014	0.152 ± 0.019	0.145 ± 0.018
	N	12	12	12	12
Seminal vesicles with coagulating glands	Absolute (g)	1.68 ± 0.21	1.65 ± 0.29	1.68 ± 0.22	1.69 ± 0.21
	Relative (%)	0.396 ± 0.047	0.378 ± 0.072	0.388 ± 0.057	0.377 ± 0.048
	N	12	12	12	12

N=5 or 12, mean ± SD.

a: Terminal body weight were measured immediately before necropsy.

b: Organ weight/terminal body weight ratio.

 

Table 5: Absolute and relative organ weights of CeO2 NPs-treated females during the study period.

CeO2 NPs (mg/kg)		0	100	300	1000
Terminal body weight	(g)	314.5 ± 19.9	316.7 ± 24.1	311.0 ± 23.7	316.8 ± 19.8
Adrenal glands	Absolute (g)	0.08 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0.01
	Relative (%)	0.028 ± 0.003	0.024 ± 0.003	0.027 ± 0.003	0.027 ± 0.002
Brain	Absolute (g)	1.94 ± 0.09	1.93 ± 0.08	2.02 ± 0.08	1.96 ± 0.05
	Relative (%)	0.652 ± 0.036	0.650 ± 0.022	0.692 ± 0.028	0.654 ± 0.030
Heart	Absolute (g)	1.00 ± 0.06	0.95 ± 0.04	0.97 ± 0.07	1.04 ± 0.06
	Relative (%)	0.334 ± 0.013	0.321 ± 0.017	0.334 ± 0.026	0.346 ± 0.019
Kidneys	Absolute (g)	2.28 ± 0.19	2.23 ± 0.21	2.14 ± 0.14	2.23 ± 0.07
	Relative (%)	0.763 ± 0.028	0.750 ± 0.051	0.734 ± 0.036	0.744 ± 0.027
Liver	Absolute (g)	10.52 ± 1.21	10.40 ± 0.87	10.19 ± 0.30	10.91 ± 0.73
	Relative (%)	3.522 ± 0.266	3.503 ± 0.277	3.495 ± 0.169	3.638 ± 0.259
Pituitary gland	Absolute (g)	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
	Relative (%)	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.000
Spleen	Absolute (g)	0.62 ± 0.07	0.69 ± 0.09	0.65 ± 0.08	0.70 ± 0.12
	Relative (%)	0.209 ± 0.021	0.231 ± 0.029	0.223 ± 0.032	0.232 ± 0.043
Thymus	Absolute (g)	0.31 ± 0.03	0.31 ± 0.05	0.31 ± 0.08	0.38 ± 0.12
	Relative (%)	0.104 ± 0.013	0.104 ± 0.021	0.105 ± 0.027	0.125 ± 0.041
Lungs	Absolute (g)	1.34 ± 0.04	1.29 ± 0.06	1.42 ± 0.05	1.36 ± 0.11
	Relative (%)	0.451 ± 0.013	0.435 ± 0.018	0.486 ± 0.022	0.453 ± 0.034
Uterus	Absolute (g)	0.78 ± 0.12	0.78 ± 0.09	0.83 ± 0.12	0.73 ± 0.06
	Relative (%)	0.260 ± 0.039	0.262 ± 0.030	0.284 ± 0.035	0.243 ± 0.026
Right ovary	Absolute (g)	0.07 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
	Relative (%)	0.021 ± 0.002	0.021 ± 0.004	0.019 ± 0.003	0.019 ± 0.004
Left Ovary	Absolute (g)	0.07 ± 0.02	0.06 ± 0.01	0.06 ± 0.01	0.05 ± 0.01
	Relative (%)	0.023 ± 0.006	0.020 ± 0.002	0.020 ± 0.005	0.018 ± 0.002

N=5, mean ± SD.

 

Table 6: Fertility with precoital time results of CeO2 NPs treated males during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Males
Mating index^a	100	100	100	100
Fertility index^b	100	100	100	100
Fecundity index^c	100	100	100	100
Females
Mating index^d	100	100	100	100
Fertility index^e	100	100	100	100
Pregnancy index^f	100	100	100	100
Precoital Time (day)	3.3 ± 3.6	1.8 ± 0.6	2.2 ± 1.1	1.9 ± 1.1

 

N=12, mean ± SD.

a: (No. of males with evidence of mating/No. of males paired) x 100.

b: (No. of males impregnating a female/No. of males paired) x 100.

c: (No. of males impregnating a female/No. of males with evidence of mating) x 100.

d: (No. of females with evidence of mating/No. of females paired) x 100.

e: (No. of pregnant females/No. of females paired) x 100.

f: (No. of pregnant females/No. of females with evidence of mating) x100.

 

 

Table 7: Reproductive and litter findings results of CeO2 NPs-treated females during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
Gestation period (day)	21.5 ± 0.4	21.5 ± 0.3	21.6 ± 0.3	21.8 ± 0.4
Corpora lutea (N)	17.8 ± 2.8	16.5 ± 2.6	16.3 ± 1.7	17.4 ± 2.0
Implantations (N)	15.3 ± 3.1	15.3 ± 2.4	14.8 ± 2.1	15.4 ± 2.0
Pups born (N)	14.8 ± 3.1	14.4 ± 2.4	13.9 ± 2.0	14.8 ± 2.1
Perinatal death (N)	0.08 ± 0.29	0.00 ± 0.00	0.00 ± 0.00	0.25 ± 0.45
Unaccounted-for sites^a(%)	2.8 ± 4.5	5.5 ± 5.8	6.2 ± 4.5	3.9 ± 4.5
Sex ratio^b(%)	106.9 ± 62.6	93.9 ± 28.2	105.3 ± 55.5	130.7 ± 66.8
Live litter size (N) PND 0 PND 4	14.8 ± 3.1 14.8 ± 3.1	14.4 ± 2.4 14.3 ± 2.4	13.9 ± 2.0 13.8 ± 2.0	14.6 ± 2.1 14.4 ± 2.0
Viability index^c(%)	100.0	99.4 ± 1.9	98.9 ± 2.6	99.0 ± 2.4
Delivery index^d(%)	100.0	100.0	100.0	100.0
Pups with external abnormalities	0	0	0	0

 

N = 12, mean ± SD.

a: (No. of implantation sites/litter) - (No. of live pups at birth/litter)/No. of implantation sites/litter x 100.

b: (No. of male pups on PND 0/litter)/(No. of female pups on PND 0/litter) x 100.

c: (No. of live pups on PND 4/litter)/(No. of live pups at birth/litter) x 100.

d: (No. of dams with live pups)/(No. of pregnant dams) x 100.

 

Table 8: F1 pups body weights of CeO2 NPs-treated parental animals during the study period.

 

CeO2 NPs (mg/kg)	0	100	300	1000
F1 male pups
PND 0
Body weight (g)	6.5 ± 0.4	6.7 ± 0.3	6.8 ± 0.6	7.0 ± 0.5
Covariate-adjusted mean (g)	6.5	6.7	6.8	7.0*
PND 4
Body weight (g)	10.1 ± 0.8	10.7 ± 1.1	11.0 ± 1.1	11.0 ± 1.2
Covariate-adjusted mean (g)	10.1	10.6	10.9	11.2*
F1 female pups
PND 0
Body weight (g)	6.2 ± 0.4	6.3 ± 0.3	6.4 ± 0.6	6.7 ± 0.6
Covariate-adjusted mean (g)	6.2	6.3	6.4	6.7*
PND 4
Body weight (g)	9.5 ± 0.9	10.0 ± 1.0	10.4 ± 1.2	10.6 ± 1.4
Covariate-adjusted mean (g)	9.6	10.1	10.4	10.5

 

N =12, mean ± SD.

*Represent a significant difference at the p<0.05 level compared to the vehicle control.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the experimental conditions of this study design, there were no CeO2 NPs related adverse effects in terms of general systemic signs as well as development and reproduction, at doses up to 1000 mg/kg bw/d. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.

Executive summary:

In a reproduction/developmental toxicity screening study, the effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD Guideline no 422

and was compliant to GLP. The study was thus considered as a key study of reliablility 1 according to Klimisch score.

Groups of 12 males and 12 females SD rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg/day nano CeO2 in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effect of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily.

Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).

No unscheduled death or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study. No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time was found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.

Tissue distribution analysis of cerium in parental and pup tissues revealed that nano CeO2 was 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.

The authors concluded that, under the experimental conditions of this study, no nano CeO2 related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.