Cerium dioxide

Endpoint summary

Description of key information

> Oral route:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, GLP) performed with nanoCeO2:

* NOAEL (parent) = 1000 mg/kg bw/day (no significant effect observed for the systemic toxicity and the reproductive performances)

* NOEL (developmental) = 1000 mg/kg bw/day (no significant effect observed on pups)

> Inhalation route (1 year) - provisional pending the publication of the carcinogenicity study done by BASF

* Systemic effect: NOAEC (male/female rodent) > 3 mg/m3

* Local effect (respiratory tract): NOAEC (male/female rodent) ≥ will be defined later when the carcinogenicity study will be available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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:

OECD 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 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

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.Aapproximately 16 hours (overnight) prior to sacrifice

- Post-exposure recovery period in satellite groups: no

Positive control:

no

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.

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").

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

Key result

Critical 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Studies performed according to OECD guidelines and in compliance with GLP, meeting REACH and CLP requirements.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was well documented and performed according to the OECD guideline 412 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

The test was performed only with female rats.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610 x 435 x 215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: At the concentration of 0.5 mg/m3: 0.9 µm / 2.5 (MMAD / mean GSD)
At the concentration of 5 mg/m3: 1.9 µm / 2.9 (MMAD / mean GSD)
At the concentration of 25 mg/m3: 2.2 µm / 2.4 (MMAD / mean GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ; The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

25 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest aerosol concentration was 25 mg/m3, which was expected to cause biological effects and should lead to lung overload at least for 20 exposures. The mid and low aerosol concentrations were 5 and 0.5 mg/m3. The low aerosol concentration with an expected lung burden far below the overload condition should not lead to any adverse effects. The mid aerosol concentration, which was spaced 10-fold higher than the low concentration, was expected to cause some biological effects.
- Rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Yes, 129-day post-exposure observation
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations of the animals were recorded for each animal at least three times per day on exposure days and once a day during the pre-exposure and post-exposure periods. Signs and findings were recorded for each animal. During exposure, examination was possible only on a group basis.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0), and twice weekly until killing or twice within the 5 exposure days.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH, Germany))
- Animals fasted: Yes- How many animals: 5 rats per test group
- Parameters checked: Red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell as well as differential blood cell counts with a hematology analyser

CLINICAL CHEMISTRY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Animals fasted: Yes
- How many animals: 5 rats per test group
- Parameters checked (in serum): rat haptoglobin and rat γ2-macroglobulin by ELISA; enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and other blood parameters of clinical chemistry using an automatic analyser

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* BRONCHOALVEOLAR LAVAGE
Twenty-four hours and 35 days after the end of exposure, five animals per test group were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anaesthesia. The lungs of the animals were lavaged in situ twice with saline solution. A total of 11 mL bronchoalveolar lavage fluid (BALF) was obtained per animal for analysis. Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with a hematology analyser. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes, and atypical cells were performed on Wright-stained cytocentrifuge slide preparations. The differential cell count was evaluated manually by counting at least 400 BALF cells per sample. Using a reaction rate analyser, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) were measured.

* INFLAMMATORY MEDIATORS IN BALF AND SERUM
Cytokines and chemokines in BALF and serum were measured. The parameters comprised various cytokines, chemokines, adhesion molecules, matrix metalloproteinases, acute-phase proteins, signal proteins of apoptosis, or cell proliferation: monocyte chemoattractant protein-1 level (MCP-1), cytokine-induced polymorphonuclear neutrophil chemoattractant-1 level (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF) and osteopontin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy and histopathology were performed after 2 and 34 days after the end of exposure (4 weeks of exposure). In general, 5 animals per test group were investigated for pathological examination. However, 10 animals were examined for pathological examination of the respiratory tract and all gross lesions. At necropsy, animals were exsanguinated by opening of the abdominal great vessels under deep pentobarbital anesthesia. All organs were preserved according to OECD TG No. 412. Following organs were weighed: adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, and thyroid glands. The lungs were instilled with and fixed in 10 % neutral-buffered formalin (NBF). Once fixed, the lungs were transferred to 70 % ethanol. All other organs were fixed in 10 % NBF. All the organs and tissues described in the OECD TG No. 412 were trimmed. After paraplast-embedding, the blocks were cut at 2- to 3-μm thickness, mounted on glass slides and stained with hematoxylin and eosin. Extrapulmonary organs and the respiratory tract compromising nasal cavity (four levels), larynx (three levels), trachea (transverse and longitudinal with carina), lung (five lobes), and mediastinal and tracheobronchial lymph nodes were assessed by light microscopy. For the lungs, whole histopathological examination was performed in animals of all test groups. For all other tissues, only the animals of the control and high concentration group of nano-CeO2 were initially examined. When changes were observed in the high concentration group, respective organs and tissues of the animals exposed to low and intermediate aerosol concentrations were also examined by light microscopy. All histopathological examinations were performed by a well-experienced board-certified veterinarian toxicopathologist followed by an internal pathology peer review.

Other examinations:

ORGAN BURDEN
Cerium (Ce) content was determined at 7 time points over 129 days of post-exposure period. Ce content in the lungs, lung-associated lymph nodes, and liver of either 3 or 5 animals per test group were examined. 1 and 35 days after the end of exposure, the lavaged lungs and aliquots of BALF of 5 animals per group were used for the determination of lung burden. This examination method likely caused a loss of the test material during preparation and handling of the lungs. Furthermore, lung burdens were measured 2 days after the end of exposure using the left half lungs of 5 animals/test group, only. On the basis of the availability of total lung weights, lung burdens were calculated up from the half lung burden values with the corresponding weight of the half lungs. Lung burden of the remaining time points was determined using the whole (not lavaged) lung.
After digestion with mixed acid, samples of each lung or lymph node were dissolved in sulphuric acid and ammonium sulphate. 140Ce content in the obtained solution was analysed by inductively coupled plasma mass spectrometry (ICP-MS) or by inductively coupled plasma optical emission spectrometry (ICP-OES) with a wavelength of 419 nm. The limit of detection for Ce was 0.3 μg. The amounts of CeO2 in the respective tissues were calculated by measuring elemental Ce with ICP-MS.

Statistics:

For body weight changes, Dunnett’s test was used for a comparison of each test group with the control group test. Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analysed by non-parametric one-way analysis using the Kruskal–Wallis test (two-sided). If the resulting p value was ≤ 0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by non-parametric one-way analysis using the two-sided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

the animals exposed for 4 weeks to NM-212 showed no clinical signs or findings compared to the control animals [data not shown]

Mortality:

no mortality observed

Description (incidence):

the animals exposed for 4 weeks to NM-212 showed no clinical signs or findings compared to the control animals [data not shown]

Body weight and weight changes:

no effects observed

Description (incidence and severity):

4 weeks of inhalation exposure to NM-212 did not affect the body weight development of the animals [data not shown]

Food consumption and compound intake (if feeding study):

not specified

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):

haematological parameters were not affected in rats exposed to NM-212 [see Table 4 in "any other information on results" below]

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

clinical chemistry parameters and acute phase protein levels were not affected in rats exposed to NM-212 [data not shown]

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative lung weights were significantly increased at aerosol concentrations of 25 mg/m3 NM-212 (+30 and 29 %, respectively) 2 days after the end of the exposure and were still significantly elevated (+16 and 20 %) 34 days after the end of the exposure. Two days after the end of the exposure, absolute and relative lung weights of animals exposed to 5 mg/m3 were increased significantly by +13 and 10 %, respectively. They returned to control levels within the following 34 days. No effects on organ weights were observed after inhalation of 0.5 mg/m3.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two days after the end of exposure, mediastinal lymph nodes of 2 animals (out of 10) were enlarged at aerosol concentrations of 25 mg/m3 NM-212. Thirty four days after the end of exposure, the incidence of animals with enlarged, yellow white-coloured mediastinal lymph nodes increased from 2 to 8 (out of 10) per group at aerosol concentrations of 25 mg/m3 NM-212. Mediastinal lymph nodes of animals exposed to 5 mg/m3 were firstly enlarged 34 days after the end of exposure (2/10 rats). All other extrapulmonary organs including the tracheobronchial lymph nodes revealed no macroscopical findings after inhalation exposure to nano-CeO2.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

* LUNGS
Two days after the end of the exposure, alveolar macrophages were observed in the lungs of all NM-212-exposed animals, regardless of the aerosol concentration. Eosinophilic granular material and small particles were distributed in the alveoli of all animals exposed to 5 and 25 mg/m3 NM-212. The occurrence of alveolar histiocytosis and of eosinophilic granular material was correlated with increased lung weights in animals exposed to 5 and 25 mg/m3 NM-212. In bronchus-associated lymphoid tissue (BALT), single, small macrophage aggregates with particles occurred in 5/10 animals exposed to 25 mg/m3 NM-212 but none was seen in lower doses. In addition, single or a few amber-like coloured particles occurred in BALT, in the extracellular compartment, without any macrophage activation at aerosol concentrations of 5 (8/10 rats) and 25 mg/m3 (5/10 rats). Thirty four days after the end of exposure, alveolar histiocytosis and eosinophilic granular material with particles were still observed at concentrations of 5 and 25 mg/m3 NM-212, but a tendency towards regression could be seen for the parameter "eosinophilic granular material with particles", notably at 5 mg/m3 (2/10 rats). At 0.5 mg/m3, in contrast, amber-like coloured particles could only be noted within single histiocyte (10/10 rats). In 1 animal of 5 mg/m3 and in 5 out of 10 animals exposed to 25 mg/m3 NM-212, a multifocal granulomatous inflammation appeared 129 days after the end of exposure. In BALT, single or few amber-coloured particles at 0.5, 5, and 25 mg/m3 NM-212 as well as an increasing number of animals with macrophage aggregates with particles at 5 and 25 mg/m3 were still observed. All compound-related findings after exposure of 5 and 25 mg/m3 NM-212 were correlated with increased lung weights in these test groups.

* LUNG-ASSOCIATED LYMPH NODES
Two days after the end of exposure to 25 mg/m3 NM-212, multifocal macrophage aggregates with particles were observed in the mediastinal (4/10 rats) as well as in the tracheobronchial lymph nodes (8/10 rats). A lympho-reticulocellular hyperplasia was present in both lymph nodes, mostly seen in animals from the group of 25 mg/m3 (4 to 7/10 rats). The hyperplasia of the mediastinal lymph nodes was correlated with their corresponding macroscopic enlargement after exposure to 25 mg/m3 NM-212. Thirty four days after the end of exposure, the number of animals with macrophage aggregates (incidence and grading) in both lymph nodes was higher compared to the animals examined 2 days after the end of the exposure. Nearly all other findings were still present 34 days after the end of the exposure.

* UPPER RESPIRATORY TRACT (data not shown)
Two days after the end of the exposure to 5 and 25 mg/m3 NM-212, amber-like coloured particles occurred similarly in the dorsal area of the larynx (level III). Animals exposed to 25 mg/m3 showed particles in the carina of the trachea. At aerosol concentrations of 25 mg/m3 NM-212, single amber-like coloured particles were firstly found in the nasal-associated lymphoid tissue (NALT), inside single macrophages or in the extracellular compartment. These findings were still present 34 days after the end of exposure.

* EXTRAPULMONARY ORGANS (data not shown)
Histological examination of extrapulmonary organs (e.g., liver, spleen, and kidneys) did not show any substance-related morphological changes in animals exposed to 0.5, 5, and 25 mg/m3 NM-212 in this short-term study with 4 weeks of exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

OTHER:
BRONCHOALVEOLAR LAVAGE / INFLAMMATORY MEDIATORS IN BALF AND SERUM
Four weeks of inhalation exposure to 5 and 25 mg/m3 NM-212 resulted in an increase in total cells in BALF due to increases in polymorph nuclear neutrophils, lymphocytes, and monocytes in BALF (see Table 5 in "any other information on results" below). Consistent with these findings, several other parameters including the examined cell mediators were increased. Thirty five days after the end of exposure, some of the BALF parameters returned to control levels, whereas several of them were still significantly increased at 5 and 25 mg/m3 (e.g., total cells, lymphocytes, neutrophils; GGT, LDH, ALP; MCP-1, CINC-1). Except for a significant increase in total protein levels in BALF, no other clinical pathology parameters displayed significant change in animals exposed to 0.5 mg/m3 NM-212.

ORGAN BURDEN
Inhalation exposure of 0.5, 5, or 25 mg/m3 NM-212 resulted in mean lung burdens of 0.04, 0.52, or 2.62 mg 1 day after the end of exposure. Two days after the end of exposure, higher lung burdens of the left lungs were measured but these data were disregarded for half-time calculations. At aerosol concentrations of 0.5 mg/m3 NM-212, a retention half-time of 40 days was determined. Higher aerosol concentrations of 25 mg/m3 NM-212 resulted, however, in a much longer half-time above 200 days. The Ce burden in the lung-associated lymph nodes (tracheobronchial and mediastinal lymph nodes) was 10 μg, 3 days after the end of the exposure to 25 mg/m3 NM-212 and increased to 350 μg, 129 days after the end of exposure. After the exposure to 25 mg/m3 NM-212, Ce was also detected in the liver (1.56 and 1.93 μg) 3 and 65 days after the end of the exposure, respectively.

Dose descriptor:

other: NOAEC (systemic)

Effect level:

> 25 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: - No NOAEC was determined in the publication, thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects

Dose descriptor:

other: NOAEC (local)

Effect level:

> 0.5 - < 5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

other: Mediastinal and tracheo-bronchial lymph nodes.

Treatment related:

yes

Dose response relationship:

yes

Table 4: Mean clinical pathology parameters in blood after exposure to nano-CeO2

	Control	NM-212
Target conc. [mg/m3]	0	0.5	5	25
Measured conc. (mg/m³) + SD	0	0.5 ± 0.2	5.3 ± 0.9	25.9 ± 6.0
Blood cells
Neutrophils [giga/L] + SD Time point 1 (a) Time point 2 (b)	1.19 ± 0.95 0.72 ± 0.26	0.73 ± 0.24 0.71 ± 0.24	0.83 ± 0.33 0.85 ± 0.17	1.18 ± 0.32 1.20 ± 0.37
Lymphocytes [giga/L] + SD Time point 1 (a) Time point 2 (b)	3.67 ± 1.13 2.86 ± 0.73	3.47 ± 0.72 3.65 ± 1.14	3.01 ± 0.53 2.95 ± 0.88	2.65 ± 0.62 3.26 ± 0.63
Neutrophils [%] + SD Time point 1 (a) Time point 2 (b)	22.2 ± 14.5 19.4 ± 7.8	16.4 ± 3.8 15.9 ± 5.2	20.0 ± 4.4 21.8 ± 3.6	29.9 ± 6.2 25.6 ± 3.9
Lymphocytes [%] + SD Time point 1 (a) Time point 2 (b)	72.3 ± 14.2 75.7 ± 8.6	78.2 ± 4.3 79.3 ± 6.6	75.2 ± 5.5 73.4 ± 3.6	66.1 ± 5.8 70.2 ± 4.0

* statistically significant, p ≤ 0.05; ** statistically significant, p ≤ 0.01. 

(a) time point 1 is 2 days after the end of exposure; (b) time point 2 is 35 days after the end of exposure

SD: standard deviation

Table 5: Clinical pathology parameters in BALF of the short-term study with 4 weeks of exposure

	Control	NM-212
Target conc. [mg/m3]	0	0.5	5	25
Measured conc. (mg/m³) + SD	0	0.5 ± 0.2	5.3 ± 0.9	25.9 ± 6.0
BALF cell counts (cn/μL)
Total cells Time point 1 (a) Time point 2 (b)	76.42 ± 23.97 75.29 ± 14.10	75.10 ± 21.51 62.23 ± 11.59	133.44* ± 48.40 97.44 ± 34.23	296.90** ± 124.92 220.50** ± 105.27
Neutrophils (PMN) Time point 1 (a) Time point 2 (b)	0.85 ± 0.35 2.44 ± 1.01	1.75 ± 1.13 2.52 ± 1.37	65.66** ± 50.23 41.70** ± 22.0	222.29** ± 99.25 161.69** ± 87.82
Lymphocytes Time point 1 (a) Time point 2 (b)	0.55 ± 0.50 1.65 ± 1.13	0.63 ± 0.74 0.77 ± 0.49	5.41** ± 3.21 7.43* ± 9.02	8.93** ± 6.04 10.89** ± 3.48
Macrophages Time point 1 (a) Time point 2 (b)	74.94 ± 23. 79 71.14 ± 13.31	72.71 ± 21.16 58.78 ± 11.04	60.25 ± 20.82 47.58 ± 16.48	59.32 ± 26.28 45.17 ± 25.33
Monocytes Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.06 ± 0.09	0.00 ± 0.00 0.04 ± 0.08	1.65* ± 1.90 0.68* ± 0.43	3.95* ± 4.65 2.46* ± 3.03
Eosinophils Time point 1 (a) Time point 2 (b)	0.09 ± 0.12 0.00 ± 0.00	0.00 ± 0.00 0.12 ± 0.11	0.00 ± 0.00 0.05 ± 0.11	0.34 ± 0.49 0.00 ± 0.00
Atypical cells Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.48 ± 0.72 0.00 ± 0.00	2.07* ± 1.71 0.29 ± 0.40
Total protein/enzymes
Total protein (mg/L) Time point 1 (a) Time point 2 (b)	60 ± 4 81 ± 23	83** ± 5 60 ± 22	94** ± 21 98 ± 49	245** ± 77 175** ± 98
GGT (nkat/L) Time point 1 (a) Time point 2 (b)	37 ± 17 42 ± 12	51 ± 11 44 ± 20	111** ± 21 83** ± 30	149** ± 31 123** ± 30
LDH (μkat/L) Time point 1 (a) Time point 2 (b)	0.51 ± 0.18 0.58 ± 0.08	0.55 ± 0.15 0.50 ± 0.20	1.08** ± 0.37 0.84* ± 0.22	2.28** ± 0.52 1.88** ± 1.20
ALP (μkat/L) Time point 1 (a) Time point 2 (b)	0.83 ± 0.16 0.70 ± 0.09	0.84 ± 0.35 0.67 ± 0.08	1.16** ± 0.10 1.05** ± 0.16	1.53** ± 0.21 1.09** ± 0.25
NAG (nkat/L) Time point 1 (a) Time point 2 (b)	45 ± 5 47 ± 8	55 ± 15 38 ± 6	53* ± 8 47 ± 7	86* ± 26 71 ± 35
Cell mediators (pg/mL)
MCP-1 Time point 1 (a) Time point 2 (b)	14.0 ± 0.0 17.3 ± 2.6	19.6 ± 11.0 15.4 ± 3.1	559.4** ± 444.4 492.5** ± 553.1	3587.2** ± 281.0 1854.2** ± 1184.0
CINC-1/IL-8 Time point 1 (a) Time point 2 (b)	104.2 ± 26.7 158.8 ± 38.1	103.8 ± 14.0 133.9 ± 45.3	506.7** ± 195.9 449.4** ± 226.7	1190.9** ± 294.9 831.0** ± 497.1
M-CSF Time point 1 (a) Time point 2 (b)	26 ± 17 46 ± 26	22 ± 12 55 ± 29	27 ± 18 41 ± 14	48 ± 29 53 ± 12
Osteopontin Time point 1 (a) Time point 2 (b)	391.44 ± 187.39 337.36 ± 282.91	288.80 ± 110.90 284.40 ± 292.66	755.44* ± 206.21 1003.18* ± 434.20	592.14 ± 336.47 838.48 ± 529.45

* Statistically significant, p < 0.05

** Statistically significant, p < 0.01; n = 5; SD standard deviation

(a) Time point 1 is 1 day after the end of exposure

(b) Time point 2 is 35 days after the end of exposure

Conclusions:

Inhaled nano-CeO2 (NM-212) induced loco-regional effects manifested by a significant pulmonary inflammation and granulomatous alterations of the lung, mainly at 25 mg/m3. However, no systemic toxicity occurred. The inflammatory responses observed were typical of poorly soluble particles.

Executive summary:

Keller J et al. (2014) assessed the lung deposition and clearance kinetics as well as the inhalation toxicity of nanometric cerium dioxide (nano-CeO2) in a subacute toxicity study with 4 weeks of inhalation exposure, according to OECD guideline 412 and in compliance with GLP.

A commercial nano-CeO2 (NM-212), from Umicore, of 40 nm was used in this study. These globular nanoparticles agglomerated and aggregated (3 - 150 µm). Moreover, the crystalline nano-CeO2 displayed a specific surface area of 27 to 30 m²/g, a zeta potential of +42 mV (at pH 7), an isoelectric point greater than pH 10, an extremely low solubility (Ce < 0.001 to 0.02 wt%) and a significant photocatalytic activity. The oxidation degree of NM-212 was determined: 14% Ce(III) and 86% Ce(IV). The nano-CeO2 MMAD ranged between 0.9 and 2.2 µm depending on the concentration tested. Although nano-CeO2 was described as pure (99.3%) and uncoated, the surface chemistry showed that organic contaminants (ester and alkyl groups) were present at 0.7% on nano-CeO2 surface.

Female Wistar rats (10/group) were administered NM-212, by whole-body inhalation exposure, at aerosol concentrations of 0 (control), 0.5, 5, and 25 mg/m3  for 6 h/day on 5 consecutive days/week for 4 weeks with a post-exposure period of 129 days. Control animals were exposed to conditioned air. Pulmonary responses were studied by analysing the bronchoalveolar lavage fluid (BALF) and blood, and by performing a histopathological analysis of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

According to the authors, a 28-day inhalation exposure to nano-CeO2 NM-212 substance-related adverse effects was limited to the lung. No clinical sign and no effect in the body weight development were observed in the treated animals as compared to controls. No altered blood parameter could be detected after 4 weeks of inhalation exposure. A whole panel of extrapulmonary organs and tissues was examined histologically and very low cerium contents were detected in the liver at two time points and at the concentration of 25 mg/m3 NM-212 (which is a general finding for inhaled nanoparticles) without any related morphological abnormalities. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low CeO2 concentrations found in extrapulmonary tissues.

An aerosol concentration of 0.5 mg/m3 did not cause inflammatory response in the lung. With a lung burden of 41 μg/lung after 4 weeks of exposure, inhaled NM-212 at 0. 5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. This was in the range of physiological retention half-times of poorly soluble particles being between 60 and 70 days. Higher aerosol concentration impaired this clearance: an aerosol concentration of 25 mg/m3 elicited a lung burden of 2.62 mg/lung resulting in a retarded retention half-time above 200 days. At the mid concentration of 5 mg/m3, the lung burdens at 3 time points indicated a retarded retention half-time as the lung burden (0.5 mg/lung) stayed at a constant level during 4 weeks. The lung burden decreased from 2.62 to 1.8 mg, 129 days after the exposure to 25 mg/m3 for 4 weeks. In pathology, lung weights were found increased following exposures to 5 and 25 mg/m3 NM-212. By light microscopy, CeO2 particles were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. CeO2 was not detected within alveolar epithelial cells. Moreover, alveolar histiocytosis was observed and had even progressed to a multifocal granulomatous inflammation within 4 weeks after the end of exposure to 5 and 25 mg/m3 nano-CeO2. The combination of moderate alveolar histiocytosis with particles and the presence of eosinophilic material, potentially precursors of granulomatous inflammation, were considered to be adverse. In contrast, at 0.5 mg/m3, the histopathological findings (i.e., alveolar histiocytosis and particles, either free or within macrophages) reflected an expected physiological response.

Nano-CeO2-related adverse effects were limited to the lung after inhalation exposure. The reported pulmonary inflammation was assessed by the changes in BALF parameters (e.g., neutrophils and proinflammatory cytokines) and histopathological findings (alveolar histiocytosis and granulomatous inflammation). Pulmonary inflammation was only observed at concentrations of 5 and 25 mg/m3. However, BALF parameters showed a regression during the post-exposure period. Histopathological findings, in contrast, progressed to granulomatous inflammation after the end of exposure at concentrations of 5 mg/m3 nano-CeO2 and above. It has to be noted that the inflammatory response in lungs based on the increase in neutrophil counts in BALF was lower after 4 weeks compared to 5 days of exposure (see in the previous ESR). The decay in neutrophil numbers after 4 weeks was by far slower than after 5 days, suggesting that inflammation developing at lower dose rate was longer lasting and more persistent.

A smaller fraction of the particles was transferred to the lung-associated lymph nodes. Indeed, in lung-associated lymph nodes, 0.35 mg CeO2 was found 129 days after exposure to 25 mg/m3 NM-212 for 4 weeks. Moderate macrophage aggregates with particles in the lung-associated lymph nodes, combined with lympho-reticulocellular hyperplasia, were considered to be adverse. Demonstrated by histological evaluations and confirmed by measured Ce lymph node burdens, the lymphatic clearance of inhaled CeO2 via the lymphatic vessels from the pulmonary region to the lung-associated lymph nodes was around 13% of the initial retained burden after the end of the exposure. Most of the CeO2 nanoparticles were presumably cleared by mucociliary clearance and subsequent faecal excretion, which was not evaluated in this work.

According to the authors, the mass lung burden of 41 μg, achieved at 0.5 mg/m3, was well below the overload threshold proposed by Morrow, while the lung burden of 2.62 mg, achieved at 25 mg/m3, was above it (Morrow, 1988). At the highest concentration, a strong pulmonary inflammation was apparent. The mid concentration of 5 mg/m3 NM-212 elicited pulmonary inflammation at a constant lung burden of around 0.52 mg, which was slightly below or at the border of the overload threshold. Based on calculations, volumetric lung overload could only be assumed after 4 weeks exposure to 25 mg/m3. Impaired lung clearance (which is one of the consequences of lung overload conditions) was, however, already observed after inhalation of 5 mg/m3 CeO2. And thus, the concentration of 5 mg/m3 was the lowest aerosol concentration at which the early as well as the later inflammation response was observed, even though lung burdens were different at the onset of the 2 phases.

Based on the results of this study, the NOAEC for systemic toxicity is set as > 25 mg/m3 and the NOAEC for local toxicity (pulmonary tract) can be established at > 0.5 mg/m3 - < 5 mg/m3.

In conclusion, inhaled nano-CeO2 (NM-212) induced loco-regional effects manifested by a significant pulmonary inflammation and granulomatous alterations of the lung, mainly at 25 mg/m3. However, no systemic toxicity occurred. The inflammatory responses observed were typical of poorly soluble particles. Moreover, it has to be noted that both concentrations which induced pulmonary effects corresponded to or were at the limit of overload conditions. Thus, the biological responses observed might be specific to the rat and could not be extrapolated to human. 

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Interim results (13 and 52 weeks) of the 2-year inhalation carcinogenicity study are reported in this rubust study summary. When the full report of the carcinogenicity study will be available, this summary will maybe need to be revised.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Interim results of the long-term study after 13 and 52 weeks of inhalation exposure to nano-
CeO2 are presented in this summary

Deviations:

yes

Remarks:

Only female rats were used in the study.

Principles of method if other than guideline:

The results reported in this summary are interim results of the 2-year study where some parameters have been evaluated after 13 and 52 weeks of exposure to CeO2 NM-212. Any further information that would become available after the release of the carcinogenicity study will be added to this summary.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610 x 435 x 215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

At the concentration of 0.1 mg/m3: 2.3 µm / 2.4 (MMAD / GSD)
At the concentration of 0.3 mg/m3: 1.7 µm / 2.3 (MMAD / GSD)
At the concentration of 1 mg/m3: 1.5 µm / 2.3 (MMAD / GSD)
At the concentration of 3 mg/m3: 1.4 µm / 2.1 (MMAD / GSD)

Presented mean values of > 14 measurements (MMAD, GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.
The target concentrations were met and maintained well throughout the studies. Particle size distribution demonstrated that all particles were in
the respirable range for rats. The particles were largely agglomerated.

Duration of treatment / exposure:

13 and 52 weeks

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0.1 mg/m³ air (nominal)

Dose / conc.:

0.1 mg/m³ air (analytical)

Remarks:

0.1 +/- 0.1 mg/m3 air

Dose / conc.:

0.3 mg/m³ air (nominal)

Dose / conc.:

0.3 mg/m³ air (analytical)

Remarks:

0.3 +/- 0.1 mg/m3

Dose / conc.:

1 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (analytical)

Remarks:

1.0 +/- 0.1 mg/m3

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

3.0 +/- 0.4 mg/m3

No. of animals per sex per dose:

5 animals/group examined for the lung and lymph node burdens, bronchoalveolar lavage analyses and hematology and acute phase proteins in blood.
10 animals/group examined for histopathological analyses.

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the lung burdens and toxicological findings after short-term exposure (see Keller and al., 2014(a) & (b)), aerosol concentrations of 0.1, 0.3, 1 and 3 mg/m³ nano-CeO2 were selected for a long-term inhalation study with up to two years of exposure. The long-term inhalation study with nano-CeO2 (NM-2012) was initiated in 2013 and performed within the framework of the EU project “NanoREG” (Teunenbroek et al. 2013). The study was performed according to OECD test guideline No. 453, under GLP and with a relevant aerosol concentration range (OECD 2009).
The results reported in this summary are interim results of the 2-year study where the following parameters have been evaluated after 13 and 52 weeks of exposure to CeO2 NM-212:
- Organ burden
- BALF cytology, cell mediators, protein and enzyme activities
- Hemtology according to OECD TG 412; acute phase proteins

Observations and examinations performed and frequency:

see below (Other examinations)

Sacrifice and pathology:

see below (Other examinations)

Other examinations:

LUNG AND LYMPH NODE BURDENS:
Directly after 13 and 52 weeks of exposure, the lavaged lungs, aliquots of lavage fluids and tracheobronchial and mediastinal lymph nodes of five animals per group were used to determine lung burden.
Each tissue sample was dried and sulfuric acid was added. The sample was then washed and acid was vaporized at 500°C for 15 min. Sulfuric and nitric acid were added to the residue. Then a mixture of nitric acid, sulfuric acid and perchloric acid ( 2:1:1 v/v/v) was added and the solution was heated to oxidize organic matter. After evaporation, the residue was dissolved in concentrated sulfuric acid. The resulting solution was analyzed for Ce content by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7500C (Agilent, Frankfurt, Germany). The limit of detection for Ce is 0.3 μg per tissue sample.

BRONCHOALVEOLAR LAVAGE
Bronchoalveolar lavage fluids were assessed for 5 animals per group after 13 and 52 weeks of exposure. The animals were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anesthesia. The lungs of the animals were lavaged in situ twice with 6 mL (22 mL/ kg body weight) of 9 % (w/v) saline solution.
- Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with an ADVIA® 120 (Siemens Diagnostics, Fernwald, Germany) hematology analyzer. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes were performed on Wright-stained cytocentrifuge slide preparations (Warheit and Hartsky 1993). The differential cell count was evaluated manually by counting at least 400 BALF cells per sample.
- Using a Hitachi 917 (Roche Diagnostics, Mannheim, Germany) reaction rate analyzer, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β- glucosaminidase (NAG) were measured. and acute phase proteins haptoglobin (HAPT) and alpha 2-macroglobin (A2M).
- Cytokines and chemokines in BALF and serum were measured at Rules-based Medicine Inc., Austin, TX, USA, with xMAP technology (Luminex Corp., Austin, TX, USA) using ELISA methods.The measured parameters comprised rat monocyte chemoattractant protein-1 (MCP-1), rat cytokine-induced polymorphonuclear neutrophil chemoattractant-1 (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF), rodent osteopontin.

HEMATOLOGY AND ACUTE PHASE PROTEINS IN BLOOD
Blood sampling of five fasted rats per test group was performed in the morning by retro-orbital venous plexus puncture under isoflurane anesthesia. Neutrophils counts was measured using a hematology analyzer (ADIVA) and the and the acute phase proteins haptoglobin (HAPT) and alpha 2-macroglobin (A2M) were determined in the serum using Elisa methods.

HISTOPATHOLOGY INVESTIGATIONS
Following sacrifice of the animals after 12 months of exposure, all organs and tissues were fixed and stored in 4% buffered formaldehyde. The lungs were transferred to 70% ethanol following a 24-48h fixation time in formaldehyde. All organs/tissues according to Table 1 (any other information on materials and methods) were trimmed, dehydrated, embedded in paraffin wax and sectioned at a nominal thickness of 3-4 µm. Bones were decalcified prior to trimming. All sections were stained with hematoxylin and eosin (H&E). An additional section of the left lung lobe from all animals was stained with Masson trichrome for assessment of fibrotic changes. Histologic alterations were described, wherever possible, according to their distribution (focal, multifocal, diffuse), severity (grades) and morphologic character. The grades were used for a grading system that takes into consideration either the severity or the number or the size of a microscopic finding (see Table 2, any other information on materials and methods). The severity of each lesion was graded on a scale of very slight to very severe, indicating the approximate fraction of the organ/tissue or organ structure to be involved.

Statistics:

Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analyzed by nonparametric one-way analysis using the Kruskal–Wallis test (twosided). If the resulting p value was ≤0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by nonparametric one-way analysis using the twosided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.
The statistical analysis of the histopathology data was performed with the Provantis system using a Chi-squared and 2-sided Fisher's Exact test.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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):

Neutrophils counts was measured in blood of 5 animals per groups 13 and 52 weeks after expsoure to nanoCeO2 MN-212. No effect of treatment was seen on mean neutrophil levels at any tested concentration and time points (see Table 3 in section: Any information on results including table).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The acute phase proteins haptoglobin (HAPT) and α2-macroglobulin (A2M) were determined in the serum of 5 rats after expsoure to nanoCeO2 NM-212.
Thirteen and 52 weeks of inhalation exposure to nano-CeO2 did not affect clinical chemistry parameters in blood at any tested concentration apart from increased HAPT and A2M levels in serum after 52 weeks of exposure to 3 mg/m³ CeO2 (see Table 3 in section: Any information on results including table). A dose-dependent incease was observed for A2M after 52 weeks of exposure using the median values rather than the means (median values of 10.94 and 11.64 for 1 and 3 mg/m³, respectively). After 13 weeks, the acute phase protein A2M was only significantly increased at 1 mg/m³, but had returned to near control values by 52 weeks. The change was not dose-dependent and regarded as incidental (see Table 3 in section: Any information on results including table).

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MACROSCOPIC FINDINGS
After 12 months of exposure to CeO2, the animals were sacrificed and the organs examined for gross lesions. The tracheobronchial and mediastinal lymphnodes of all 10 animals of the highest test concentration (3 mg/m³) revealed a white-beige to white-yellow discoloration and were moderately enlarged. The same findings were observed in 9 (discoloration) and 3 animals (enlarged) respectively, of the 1 mg/m3 test group. Few animals at test concentrations of 0.1, 1.0 and 3.0 mg/m³ showed a single focus in the lungs.

All other findings were single observations or equally distributed over the test groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY OF THE RESPIRATORY TRACT
Exposure-related microscopic changes were exclusively observed in the respiratory tract and included re-active/adaptive changes such as accumulation of particle-laden macrophages in the nasal cavity, larynx, lungs, tracheo- bronchial and mediastinal lymph nodes.

Nasal cavity:
In the nasal cavity, the incidence of age-related intra-epithelial eosinophilic globules was increased in the 3 mg/m³ high-dose CeO2 exposure group as compared to the control group and associated with minimal inflammatory cell infiltration.
Indeed, the presence of (multi)focal intracytoplasmic eosinophilic globules within the olfactory epithelium was increased in incidence and grade in the 3 mg/m3 CeO2 exposure group (5/10 very slight, 3/10 slight, 1/10 moderate) as compared to the 1 mg/m3 CeO2 exposure group (3/10 very slight, 1/10 slight), the 0.3 mg/m3 CeO2 exposure group (1/10 very slight, 1/10 slight), the 0.1 mg/m3 CeO2 exposure group (2/10 very slight, 1/10 slight) and the clean air control 5/10 very slight, 1/10 slight). A similar trend was observed for eosinophilic globules in the respiratory epithelium: The incidence in the control group was 5/10 (all very slight), in the 0.1 mg/m3 CeO2 exposure group 3/10 (all very slight), in the 0.3 mg/m3 CeO2 exposure group 1/10 (very slight) and in the 1 mg/m3 CeO2 exposure group 3/10 (all very slight) whereas 9/10 (7/10 very slight, 2/10 slight) females in 3 mg/m3 CeO2 exposure group were affected.
Although the difference between the control and CeO2 high-dose test group was statistically not significant, the increase in incidence and severity of this change in both types of epithelium is considered to be exposure-related.
The same is true for (multi)focal very slight subepithelial (mixed) inflammatory cell infiltration which occurred in 3/10, 3/10, 2/10, 4/10 and 7/10 females of group control, 0.1mg/m3 CeO2, 0.3 mg/m3 CeO2, 1mg/m3 CeO2 and 3 mg/m3 CeO2, respectively.

Further exposure-related findings such as (multi)focal very slight accumulation of particle-laden macrophages within the NALT (nasal mucosa-associated lymphoid tissue) were diagnosed in 1/10, 0/10, 4/10 and 10/10 animals of test group 0.1mg/m3, 0.3 mg/m3, 1mg/m3 and 3 mg/m3 CeO2, respectively. Moreover, multifocal very slight amounts of intraepithelial (intracytoplasmic) particles were observed in all animals of the 3 mg/m3 CeO2 group. Occasional particles were seen also in epithelial cells of the submucosal glands (Bowman’s glands).

Incidental findings in the nasal cavity which were considered to be unrelated to particle exposure included dilatation of submucosal glands, mucous cell hyperplasia, subepithelial mononuclear cell infiltration and subepithelial mineralization and were seen in up to 3/10 animals in all test groups.

Larynx:
In 4/10 animals of the 3 mg/m3 CeO2 exposure test group, (multi)focal subepithelial accumulation of particle-laden macrophages (3/10 very slight, 1/10 slight) was observed.

Spontaneous findings included very slight to slight subepithelial mononuclear cell infiltration in up to 5/10 animals as well as very slight to slight dilatation of submucosal glands in 2/10 females of groups exposed to 0.1mg/m3, 0.3 mg/m3 and 3 mg/m3 CeO2.

Lungs:
The adverse and non-adverse histopathological findings observed in the lungs are summarized in Table 6 and 7 (See section: Any information on results including table). Non-adverse findings consisted of accumulation of particle-laden macrophages in the alveolar/interstitial areas and in the BALT as well as particle-laden syncytial giant cells in the BALT. In addition, bronchiolo-alveolar hyperplasia of the bronchiolar type graded no more than “very slight” (grade 1) or “slight” (grade 2) was considered as a non-adverse finding. Adverse effects in the lungs included dose-dependent alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation and interstitial fibrosis, alveolar lipoproteinosis and cholesterol granuloma(s). Except alveolar lipoproteinosis and cholesterol granuloma(s), all changes were seen at dose-dependent incidences and severity grades in all CeO2 exposure test groups.

Reactive/adapatative (= non adverse) pulmonary finfings:
(Multi)focal alveolar/interstitial accumulation of particle-laden macrophages was observed dose-dependently in 10/10 females each of the 0.1 mg/m3 (8/10 very slight, 2/10 slight), 0.3 mg/m3 (8/10 very slight, 2/10 slight), 1 mg/m3 (1/10 very slight, 9/10 slight) and 3 mg/m3 (7/10 slight, 3/10 moderate) CeO2 groups. Deposits of particle-laden macrophages were present not only in alveoli but also in interstitial (intraseptal, peribronchiolar and perivascular) compartments. In addition, agglomerates of CeO2 particles were lying freely within alveoli at very slight to slight degrees in 3/10 animals of 0,1 mg/m3 group and in 10/10 females each of 0,3-3 mg/m3 CeO2 groups. (Multi)focal aggregates of particle-laden macrophages were also observed dose-dependently within the bronchus-associated lymphoid tissue (BALT) at incidences of 10/10 each in 0.1 mg/m3 (all very slight), 0.3 mg/m3 (8/10 very slight, 2/10 slight), 1 mg/m3 (8/10 slight, 2/10 moderate) and 3 mg/m3 CeO2 groups (1/10 slight, 7/10 moderate, 2/10 severe). Syncytial giant cells - mainly particle-laden - were present in the BALT of 3/10, 9/10 and 10/10 females of test groups 0.3, 1 and 3 mg/m3 CeO2 groups, respectively. The amount of the intracellular particle-load in both single-nucleated macrophages and multinucleated giant cells corresponded well to the used CeO2 exposure dose.
(Multi)focal bronchiolo-alveolar hyperplasia of the bronchiolar type (Synonym: alveolar bron-chiolization) was observed in a single animal of the 0,1 mg/m3 CeO2 group (very slight) and in 2/10 (all very slight), 10/10 (all very slight) and 10/10 (9/10 very slight, 1/10 slight) females of the 0,3, 1 and 3 mg/m3 CeO2 groups, respectively.

Adverse changes:
(Multi)focal alveolar/interstitial (mixed) inflammatory cell infiltration occurred in a single control animal (very slight) as a spontaneous finding, in 4/10 females at 0.1 mg/m³ (all very slight) and in 10/10 animals each at 0.3 mg/m³ (9/10 very slight, 1/10 slight), 1 mg/m³ (7/10 very slight, 3/10 slight) and 3 mg/m³ (4/10 very slight, 6/10 slight). In the 0.3, 1 and 3 mg/m³ exposure groups, the difference to the control was statistically significant.
Multifocal alveolar/interstitial granulomatous inflammation was observed in 1/10 females at 0.1 mg/m³ (very slight), in 3/10 females at 0.3 mg/m³ (all very slight) and at significantly increased incidences in 10/10 animals each at 1 mg/m³ (7/10 very slight, 3/10 slight) and 3 mg/m³ (4/10 very slight, 6/10 slight). The term ‘granulomatous inflammation’ was used only, if (mixed) inflammatory cell infiltration, syncytial giant cells and interstitial fibrosis were present in conjunction to form a granuloma-like focal lesion.
(Multi)focal very slight interstitial fibrosis (mainly intraseptal) was diagnosed in 3/10, 4/10, 10/10 and 10/10 females at 0.1, 0.3, 1 and 3 mg/m³, respectively. At 1 and 3 mg/m³, the difference to the control group was statistically significant. Alveolar lipoproteinosis was observed in 4/10 animals ((2/10 very slight, 1/10 slight, 1/10 severe) of the 3 mg/m³ high-dose CeO2 exposure group only and cholesterol granulomas occurred in a single female each of the 1 and 3 mg/m3 CeO2 exposure groups (very slight and slight, respectively).

Incidental pulmonary findings occurring in single animals of different exposure groups as well as in control group consisted of focal very slight osseous metaplasia, focal very slight neuroendocrine cell hyperplasia and focal very slight hair granuloma. In addition, 4/10 control animals revealed focal very slight alveolar macrophage aggregation. All these findings were considered to be unrelated to particle exposure.

Tracheobronchial and mediastinal lymph nodes:
The lymph nodes at both sites showed a dose-dependent (multi)focal very slight to severe accumulation of particle-laden macrophages.
Regarding the tracheobronchial lymph node, the incidences were 8/8 (all very slight) at 0.1 mg/m³, 9/9 (1/9 very slight, 7/9 slight, 1/9 moderate) at 0.3 mg/m³ and 10/10 at 1 mg/m³ (2/10 slight, 8/10 moderate) and 3 mg/m³ (5/10 moderate, 5/10 severe). In addition, particle-laden syncytial (multinucleated) giant cells were present in the tracheobronchial lymph node of 1/8, 6/9, 10/10 and 10/10 females at 0.1, 0.3, 1 and 3 mg/m³, respectively.
In the mediastinal lymph nodes, the incidences of (multi)focal accumulation of particle-laden macrophages were 6/10 (all very slight) at 0.1 mg/m³, 10/10 (all very slight) at 0.3 mg/m³, 9/9 (3/9 slight, 6/9 moderate) at 1 mg/m³ and 10/10 (5/10 moderate, 5/10 severe) at 3 mg/m³ CeO2, while syncytial giant cells were only observed in 9/9 and 10/10 females of groups at 1 and 3 mg/m³CeO2, respectively.

Remaining organs of the respiratory tract:
Within the remaining organs of the respiratory tract such as trachea and nasopharynx no lesions were detected in any investigated group.

HISTOPATHOLOGY OF THE OTHER ORGANS
Several sporadic neoplastic and non-neoplastic findings were observed in the other organs examined histopathologically. These occurred either incidentally or were similar in distribution pattern and severity in control rats compared to the CeO2 high-dose test group. Sporadic findings in the other CeO2 exposure groups were recorded only as correlates of macroscopic findings. All of the observed findings were considered to be without any relation to CeO2 exposure:
- A total number of 6 neoplasms were observed: an adenoma of the pars distalis in the pituitary gland of single females each of control, 0,1 and 0,3 mg/m3 CeO2 groups, a sebaceous adenoma and a lipoma of the skin/subcutaneous tissue in single animals in the 0,3 mg/m3 CeO2 group, and an endometrial stromal polyp of the uterus in a female control animal.
- Findings such as epithelial degeneration (incidence up to 6/10 rats per test group) and interstitial inflammation (incidence up to 7/10 rats per test group) of the Harderian glands are most likely considered to be related to the blood sampling procedure. Further common spontaneous findings included (multi)focal very slight intratubular mineralization of the kidneys (incidence up to 8/10 rats per test group), (multi)focal very slight mononuclear cell infiltration of the liver (incidence up to 7/10 rats per test group), chondromucinous degeneration of sternebral cartilage (incidence up to 7/10 rats per test group), epithelial hyperplasia (incl. hyperplasia of the type ‘epithelial tubules and cords’) at incidences of up to 8/10 rats per test group in the thymus and acinar cell hypertrophy of the salivary glands (incidence up to 4/10 rats per test group). Estrous cycle-depending luminal dilatation of the uterus, C-cell hyperplasia of the thyroids, and parasites (nematodes) in the rectum, colon and/or cecum were observed in up to 3/10 animals per test group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

After 12 month of inhalation exposure neither neoplastic nor pre-neoplastic treatment-related findings were seen in the lungs of CeO2-exposed animals.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ANALYSES OF THE BRONCHOALVEOLAR FLUIDS:
Bronchoalveolar lavage fluids were assessed for 5 animals per group after 13 and 52 weeks of exposure to CeO2 NM-212.
The assessed parameters were: total cell count, differential cell count (macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes), total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β- glucosaminidase (NAG) and cytokines and chemokines (rat monocyte chemoattractant protein-1 (MCP-1), rat cytokine-induced polymorphonuclear neutrophil chemoattractant-1 (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF), rodent steopontin).
All the results are summarised in Table 5 (see below: Any other information on results including tables)

According to the authors, after 13 weeks, exposure to CeO2 induced a dose-dependent increase in neutrophil cell counts from 1 mg/m3 and a statistically significant increase in total cells at all concentrations. Lymphocytes and monocytes were significantly increased only at 3 mg/m³. Macrophage numbers were statistically significantly increased at the lower concentrations (0.1 and 0.3 mg/m³) but not at the highest concentrations. The enzymes GGT, LDH and ALP were statistically significantly increased by aerosol concentrations of 1mg/m³ and above. Total protein and NAG were increased only at 3 mg/m³. Cell mediators (MCP-1,CINC-1 and Osteopontin) were statistically significant increased at 1 mg/m³ and above. A high variability in the measures is noted for these parameters. No increase was observed for M-CSF whatever the concentration used.

After 52 weeks of exposure to aerosol concentrations of 0.3 mg/m³ CeO2 and above, lymphocytes and neutrophils counts were still elevated, but total cell counts were only increased at 3 mg/m³. Increase in monocyte counts was obserserved at 1 and 3 mg/m3 and no increase as compared to controls was observed for macrophages and eosinophils counts whatever the CeO2 concentrations used. Exposure of 0.1 mg/m³ CeO2 caused no changes in BALF cell numbers. Total protein and the enzyme LDH were significantly increased at 1 and 3 mg/m³. ALP and NAG levels were not affected at any aerosol concentration whereas GGT were significantly increased at all concentrations. MCP-1 and CINC-1 levels were still elevated but to a lower extent compared with 13 weeks of exposure. No change was observed for M-CSF and osteopontin whatever the CeO2 concentration used.

it should be noted that a high variability was noted in all the measured parameters. But individual data were not reported in the report.

LUNG and LYMPH NODE BURDENS:
Directly after 13 and 52 weeks of exposure, the lavaged lungs, aliquots of lavage fluids and tracheobronchial and mediastinal lymph nodes of five animals per group were used to determine lung burden. The amounts of CeO2 in lungs and lymph nodes were estimated by measuring Ce with ICP-MS and extrapolating to CeO2 (assuming that it was still particulate). The results at the low (0.1 mg/m3) and high (3 mg/m3) concentrations are presented in the Table 4 (see section: Any other information on results including tables).
During the exposure period, lung burdens increased with longer exposure duration. The lung burden of CeO2 after 13 weeks was nearly doubled after 52 weeks of exposure at aerosol concentrations of 0.1 and 3 mg/m³. Only a slight translocation of the nanoparticles to the tracheobronchial and mediastinal lymph nodes was observed (1.9 % of the CeO2 lung burden).

Details on results:

Although statistically not significant, some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 low-dose CeO2 exposure group. Thus, a NOAEC (no observed adverse effect concentration) could not be established for the lungs after 12 months of exposure to the present CeO2 nanoparticle concentrations.

Dose descriptor:

NOAEC

Remarks:

Systemic effects (52 weeks)

Effect level:

3 mg/m³ air

Sex:

female

Basis for effect level:

other: no adverse systemic effects occurred

Remarks on result:

other:

Remarks:

Provisional conclusion pending the publication of the carcinogenicity study done by BASF.

Dose descriptor:

NOAEC

Remarks:

Local effects (52 weeks)

Effect level:

< 0.1 mg/m³ air

Sex:

female

Basis for effect level:

other: Some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 group

Remarks on result:

other:

Remarks:

Provisional conclusion pending the publication of the carcinogenicity study done by BASF.

Critical effects observed:

not specified

Table 3: Mean neutrophil levels and acute phase proteins in blood after 13 and 52 weeks of exposure to CeO2 MN-212

Test concentrations [mg/m3]		Control	0.1	0.3	1	3
Neutrophils (giga/L) Mean value+/- SD	13 w	0.63+/-0.25	0.95+/-0.16	0.78+/-0.10	0.93+/-0.30	1.09+/-0.35
	52 w	0.67+/-0.17	0.79+/-0.19	0.70+/-0.17	1.02+/-0.20	0.97+/-0.29
Neutrophils (%) Mean value+/- SD	13 w	15.2+/-4.60	23.4+/-4.3	19.8+/-5.0	22.4+/-7.6	25.4+/-9.8
	52 w	26.7+/-5.80	33.8+/-5.8	28.1+/-4.5	32.6+/-7.1	32.3+/-4.8
Acute phase proteins (µg/mL)
HAPT Mean value+/- SD	13 w	204.00+/-47.01	223.60+/-67.11	228.60+/-55.86	192.20+/-51.45	229.20+/-58.14
	52 w	139.60+/-74.26	215.40+/-54.45	184.20+/-73.96	173.60+/-37.35	251.40*+/-63.06
A2M Mean value+/- SD	13 w	14.55+/-5.90	18.18+/-4.22	20.69+/-4.55	22.66*+/-7.06	19.65+/-4.45
	52 w	8.86+/-2.14	9.52+/-2.17	9.88+/-4.48	12.39+/-3.98	12.19*+/-1.61

w: weeks of treatment;statistically significant, p < 0.05; ** statistically significant, p < 0.01; n=5; SD: standard deviation, HAPT: haptoglobin, A2M:α2-macroglobulin; blood were collected directly after 13 weeks of exposure and one day after 52 weeks of exposure.

 

Table 4: Overview of organ burden analysis after 13 and 52 weeks of exposure to CeO2 NM-212

Weeks of exposure	13		52
Study day	94		367
Time point	One day after last exposure
Number of animals	5		5
Test Concentrations [mg/m3]	0.1	3	0.1	3
Lung burden [μg] ±SD	11.96+/-2.82		42.07+/-11.05
Lung burden [mg] ±SD		1.39+/-0.16		2.61+/-0.52
Tracheobronchial lymph node burden [μg] ±SD	ND	11.93+/-14.21	ND	ND
Mediastinal lymph node burden [μg] ±SD	ND	13.78+/-15.85	ND	ND

ND: not determined, SD: standard deviation, n=5; burden is mg or μg per lung; lavaged lungs and corresponding aliquots were used for determination of lung burden by ICP-MS.

 

Table 5: Bronchoalveolar lavage fluid analysis after 13 and 52 weeks of exposure to CeO2 NM-212 

Test concentrations [mg/m3]		Control	0.1	0.3	1	3
Mean value+/- SD
Total cells (counts/μL)	13 w	66.3±22.04	115.22**±23.72	98.70*±30.49	117.51*±43.99	163.33**±57.29
	52 w	54.27±20.27	59.95±26.52	60.97±20.18	68.56±28.69	96.06*±29.29
Neutrophils (counts/μL)	13 w	1.71±0.66	0.53±0.58	2.85±3.45	18.56**±11.68	72.59**±54.65
	52 w	0.58±0.69	1.05±0.99	4.82**±3.94	16.07**±5.17	31.97**±14.23
Lymphocytes (counts/μL)	13 w	0.55±0.42	0.27±0.26	0.38±0.63	2.21±2.23	5.43**±3.52
	52 w	0.56±0.36	1.30±1.12	9.79*±18.06	6.69**±2.16	9.72**±5.97
Macrophages (counts/μL)	13 w	63.99±22.36	114.37**±24.06	95.43*±27.12	96.30±46.85	83.52±15.72
	52 w	53.01±19.77	57.49±25.85	46.10±12.59	45.33±26.17	53.57±12.31
Monocytes (counts/μL)	13 w	0.00±0.00	0.00±0.00	0.00±0.00	0.13±0.30	0.94**±0.67
	52 w	0.04±0.06	0.04±0.06	0.16±0.18	0.29*±0.24	0.43*±0.40
Eosinophils (counts/μL)	13 w	0.04±0.08	0.00±0.00	0.04±0.08	0.13±0.20	0.25±0.39
	52 w	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.05±0.10
Total protein (mg/L)	13 w	52 ±10	57±9	60±23	60±18	84*±26
	52 w	31±8	33±12	67±73	61**±11	73**±28
GGT (nkat/L)	13 w	41±7	40±5	53±13	80**±18	106**±26
	52 w	26±4	37**±6	38*±9	70**±23	65**±14
LDH (μkat/L)	13 w	0.38±0.06	0.43±0.03	0.52±0.16	0.61**±0.18	0.95**±0.33
	52 w	0.35±0.04	0.37±0.08	0.44±0.13	0.66**±0.14	0.80**±0.19
ALP (μkat/L)	13 w	0.51±0.10	0.45±0.06	0.58±0.09	0.73*±0.17	0.84**±0.19
	52 w	0.44±0.12	0.54±0.10	0.48±0.13	0.58±0.19	0.50±0.06
NAG (nkat/L)	13 w	35±7	35±5	37±13	43±9	51**±8
	52 w	37±7	42±14	38±	47±9	47±12
MCP-1 (pg/mL)	13 w	24.2±8.4	19.1±7.5	30.8±26.4	111.4**±100.1	620**±613.5
	52 w	15.3±8.2	17.9±7.1	49.3*±37.6	169.4**±113.9	378.2**±245.5
CINC-1/IL-8 (pg/mL)	13 w	93.7±18.7	60.6±38.9	77.7±38.9		280.6*±190.8
	52 w	82.4±25.4	68.8±37.1	69.7±26.5	132.9*±49.1	180.6*±114.8
M-CSF (pg/mL)	13 w	14±0	14±0	14±0	14±0	14±0
	52 w	21±12	18±3	18±4	27±12	25±15
Osteopontin (pg/mL)	13 w	49.02±40.86	37.40±32.69	118.49±133.12	136.87*±72.89	300.41*±253.01
	52 w	162.79±98.61	166.71±187.14	135.32±61.53	175.72±88.75	228.80±217.96

* statistically significant, p < 0.05; ** statistically significant, p < 0.01; n=5; mean values and standard deviation (SD); lungs were lavaged one day after last exposure.

Table 6: Summary of incidences of lung changes related to CeO2 exposure

Test concentrations [mg/m3]	Control	0.1	0.3	1	3
Lung / No. of animals	10	10	10	10	10
Accumulation, Particle-Laden Macrophages, Alveolar/Interstitial, Grade 1-3	0	10*	10*	10*	10*	non-adverse lesion
Accumulation, Particle-Laden Macrophages, BALT, Grade 1-4	0	10*	10*	10*	10*
Giant Cells, Syncytial, BALT, Present, no grade	0	0	3	9*	10*
Hyperplasia, Bronchiolo-Alveolar, Bronchiolar type Grade 1-2	0	1	2	10*	10*
Infiltration, Inflammatory Cells, Alveolar/ Interstitial, Grade 1-2	1	4	10*	10*	10*	adverse lesion
Infiltration, Granulometous, Alveolar/ Interstitial, Grade 1-2	0	1	3	10*	10*
Fibrosis, Interstitial, Grade 1	0	3	4	10*	10*
Lipoproteinosis, Alveolar, Grade 1-4	0	0	0	0	4
Granuloma, Cholesterol, Grade 1-2	0	0	0	1	1

*= p < 0.001,Chi-Quadrat/Fisher-Test, two-sided

Table 7: Summary of grade of lesions of lung changes related to CeO2 exposure

Test concentrations [mg/m3]	Control	0.1	0.3	1	3
Lungs / No. of animals	10	10	10	10	10
Accumulation, Particle-Laden Macrophages, Alveolar/Interstitial	0	1.2*	1.2*	1.7*	2.9*
Accumulation, Particle-Laden Macrophages, BALT	0	1*	1.2*	2.2*	3*
Giant Cells, Syncytial, BALT	0	0	0.3	0.9*	1*
Hyperplasia, Bronchiolo-Alveolar, Bronchiolar type	0	0.1	0.2	1*	1.1*
Infiltration, Inflammatory Cells, Alveolar/ Interstitial	0.1	0.4	1.1*	1.3*	1.6*
Infiltration, Granulometous, Alveolar/ Interstitial	0	0.1	0.3	1.3*	1.6*
Fibrosis, Interstitial	0	0.3	0.4	1*	1*
Lipoproteinosis, Alveolar	0	0	0	0	0.8
Granuloma, Cholesterol	0	0	0	0.1	0.2

*= p < 0.001,Chi²-test/Fisher-Test, two-sided

Conclusions:

In the present inhalation study which reports interim results of a 2-year study otained after 13 and 52 weeks of exposure of females rats to nano-CeO2 (NM-212), high biopersistence of the nanoparticles with the induction of a local lung inflammation was observed. However nano-CeO2 showed a low bioavailability and elicited no or only minimal systemic effects. Thus, nano-CeO2 was considered as a poorly soluble with an inherent toxicity in the lung in this study. The local no observed adverse effect concentrations in the lung (NOAEC) of CeO2 - based on BALF in female rats after 13 and 52 weeks of exposure was found to be 0.3 and 0.1 mg/m³, respectively. However, from the results of the histopathological analyses of animals after 52 weeks of exposure to CeO2, it was concluded that the local no observed adverse effect concentrations in the lung (NOAEC) could not be established after exposure to the present CeO2 nanoparticle concentrations. In the absence of systemic effects, the overall systemic NOAEC was considered to be 3 mg/m3.

Executive summary:

This is a provisional discussion pending the publication of the carcinogenicity study done by BASF.

 

This work described the interim results of a carcinogenicity study done in the rats (performed according to OECD test guideline No. 453, under GLP, and with a relevant aerosol concentration range (OECD 2009)), observed after 13 and 52 weeks of inhalation exposure to nano-CeO2 NM-212. The aim of this interim observations was to investigate the lung deposition and clearance of inhaled nanomaterials, and the resulting effects on the rat organism at different time points.

 

Female Wistar rats inhaled nano-CeO2 by whole-body exposure, 6 hours per day, 5 days per week for a total of two years. The tested aerosol concentrations were 0.1, 0.3, 1 and 3 mg/m³ CeO2. The Interim results after 13 and 52 weeks of exposure presented in this summary included results collected from 5 females/group on lung retention and clearance kinetics based on lung and associated lymph node burdens, and pulmonary effects based on bronchoalveolar lavage fluid (BALF) analyses including total protein concentrations, total and differential cell counts, enzyme activities (LDH, ALP, GGT, NAG), acute phase proteins (HAPT nad A2M) and cytokines (MCP-1, CIN-1/IL-8 and M-CSF and rodent osteopontin). Systemic effects were evaluated by analysis of blood including neutrophils counts and acute phase proteins HAPT and A2M. Histopathological analyses were done after 12 weeks of exposure on 10 animals/group.

 

According to the author, during the exposure period, lung burdens increased with longer exposure duration. The lung burden of CeO2 after 13 weeks was nearly doubled after 52 weeks of exposure at aerosol concentrations of 0.1 and 3 mg/m3. However, only a slight translocation of the nanoparticles to the tracheobronchial and mediastinal lymph nodes was observed (below 2 % of the initial lung burden after 13 weeks) indicating that, at least at this lung burden range, they play no major role for particle clearance.

 

Inhalation exposure to nano-CeO2 for 13 and 52 weeks elicited no or only minimal systemic effects: no change in mean neutrophil levels were observed in the blood of rats at any concentrations and time points slight increase of acute phase protein levels (haptoglobin andα2-macroglobulin) in serum was observed after 52 weeks of exposure to 3 mg/m3 CeO2.

 

Local inflammation in the lung was observed by increases in BAL neutrophils, lymphocytes, monocytes, enzyme cativities and cell mediator levels at the highest tested concentrations (1 and 3 mg/m3) after 13 weeks of exposure. BAL changes (cell counts, enzyme activities, total protein and cell mediator levels) after 52 weeks of exposure to 3 mg/m³ CeO2 were comparable to those after 13 weeks or slightly reduced. Minor changes in BAL were observed at lower aerosol concentrations of 1 mg/m³. Exposure to 0.3 mg/m³ elicited no BAL changes after 13 weeks and only minor changes after 52 weeks (neutrophils, GGT, MCP-1). In animals exposed to lower aerosol concentrations (0.3 mg/m3) rather than in those exposed to higher, BAL lymphocytes were higher increased than neutrophils after 52 weeks of exposure indicating a later phase of inflammation.

 

Histopathology findings related to CeO2 exposure were exclusively observed in the respiratory tract and included non-adverse reactive/adaptive changes such as accumulation of particle-laden macrophages in the nasal cavity, larynx, lungs, tracheobronchial and mediastinal lymph nodes. Adverse effects in the lungs included dose-dependent alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation and interstitial fibrosis. Alveolar lipoproteinosis was observed in the 3 mg/m³ high-dose CeO2 exposure group only and cholesterol granulomas occurred in a single female each of the 1 and 3 mg/m³ CeO2 exposure groups. Although statistically not significant, some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 low-dose CeO2 exposure group. After 12 month of inhalation exposure neither neoplastic nor pre-neoplastic treatment-related findings were seen in the lungs of CeO2-exposed animals. In the histopathological analyses of the other organs, all the findings were considered to be without any relation to CeO2 exposure.

 

The local no observed adverse effect concentrations in the lung (NOAEC) of CeO2 - based on BALF in female rats after 13 and 52 weeks of exposure - was found to be 0.3 and 0.1 mg/m³, respectively. However, from the results of the histopathological analyses of animals after 52 weeks of exposure to CeO2, it was concluded that the local no observed adverse effect concentrations in the lung (NOAEC) could not be established for the lungs after 12 months of exposure to the present CeO2 nanoparticle concentrations. In the absence of systemic effects, the overall systemic NOAEC is 3 mg/m3.

 

In summary, inhaled CeO2 showed high biopersistence in the lungs inducing a local inflammation but no systemic effect. Thus CeO2 was considered as a poorly soluble with an inherent toxicity in the lung in the rats.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: :

Remarks:

the study was well documented and performed according to OECD guideline 412. However, there was no mention to GLP and a detailed description on the generation of the test atmosphere was missing. Furthermore, results were described too briefly or not shown, and presented a very high variability, as indicated by elevated standard deviations (SD); this made it difficult to draw a definite conclusion.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced selection of organs for pathological examination

GLP compliance:

not specified

Remarks:

The GLP status was not specified in the article.

Limit test:

no

Species:

rat

Strain:

other: Wistar WU (Crl:[WI] WU BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Germany)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 203 g for males and 150 g for females (average body weights [bw])
- Fasting period before study: No
- Housing: In macromolon cages, 5 animals/cage separated by group and by sex, bedding of wood shavings (Lignocel, Type 3/4) and strips of paper (Enviro-dri)
- Diet: Ad libitum, except during exposure and the fasting period (overnight) before sacrifice; commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, Special Diets Services, UK)
- Water: Ad libitum, except during exposure and the fasting period (overnight) before sacrifice
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 10 air changes per hour
- Photoperiod: Artificial light on a 12 hour cycle

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: clean humidified compressed air

Remarks on MMAD:

MMAD / GSD: MMAD / GSD determined using APS (µm)
NM-211: 1.02 ± 0.04 / 1.82
NM-212: 1.17 ± 0.34 / 2.07
NM-213: 1.40 ± 0.11 / 1.64

MMAD / GSD determined using SMPS (µm)
NM-211: 0.276 ± 0.037 / 1.48
NM-212: 0.366 ± 0.058 / 1.56
NM-213: 0.464 ± 0.058 / 1.32

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus, method of holding animals in test chamber, source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Test atmospheres of CeO2 powders were generated using a dust feeder, a venturi (Fox Valve Development corp., Dover, USA) and a jet mill. The test atmosphere was directed to the top inlet of the exposure unit.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate: No data available
- Method of particle size determination: Particle size distributions were measured daily by a single particle mass spectrometer (SMPS) and an aerodynamic particle sizer (APS) and weekly by a 11-stage cascade impactor.
- Treatment of exhaust air: The test atmosphere was exhausted at the bottom of the exposure unit.

TEST ATMOSPHERE
- Brief description of analytical method used: Particle size distributions were measured daily by a single particle mass spectrometer (SMPS) and an aerodynamic particle sizer (APS), and weekly by a cascade impactor (see in "any other information on materials and methods" below for details).
- Samples taken from breathing zone: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the test material in the atmosphere were measured at least 6 times daily by gravimetric analysis.
Mass concentrations were monitored for at least 2 h per day with a condensation particle counter (CPC).
(see in "any other information on materials and methods" below for details)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Low dose group: 40 min/day, 5 days/week (except on public holidays)
Mid dose group: 2 hours/day, 5 days/week (except on public holidays)
High dose group: 6 hours/day, 5 days/week (except on public holidays)

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

for different daily exposure durations (see in "any other information on materials and methods" below for details)
basis: nominal micro-CeO2 (NM-213) conc. for a 6-h exposure

No. of animals per sex per dose:

5 males and 5 females per dose (see table 1 in "any other information on materials and methods" below for details)

Control animals:

other: Yes, stream of clean humidified compressed air

Details on study design:

- Dose selection rationale, rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Recovery groups were included consisting of control and 6-h exposure (high dose) groups of 5 males and 5 females kept for a recovery period of 28 days before sacrifice
- Section schedule rationale: No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (no detail). If necessary, animals were handled to detect signs of general toxicity.

DETAILED CLINICAL OBSERVATIONS: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded 1 day before the start of exposure (day -1), prior to exposure on the first day (day 0), weekly thereafter, and on the day before overnight fasting and on the scheduled sacrifice date.

FOOD CONSUMPTION: Yes
- Measured per cage by weighing the feeders

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were taken from the abdominal aorta of sacrificed rats (K3-EDTA was added as anticoagulant to one portion).
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight before sacrifice)
- How many animals: 10 (5 males and 5 females)
- Parameters checked in each sample: Haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Remaining blood was collected from the abdominal aorta of sacrificed rats in heparinized tubes and plasma was prepared by centrifugation.
- Animals fasted: Yes (overnight before sacrifice)
- How many animals: 10 (5 males and 5 females)
- Parameters checked in plasma samples using an auto-analyser: Alkaline phosphatase activity (ALP), bilirubin total, aspartate aminotransferase activity (ASAT), cholesterol, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, fasting glucose

URINALYSIS: Yes
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked: Creatinine, hydroxydeoxyguanosine

NEUROBEHAVIOURAL EXAMINATION: No data available

OTHER: BRONCHOALVEOLAR LAVAGE
At necropsy, the right half of the lungs were lavaged twice with saline (26.7 mL/kg bw). Two aliquots of bronchoalveolar lavage fluid (BALF) were collected and centrifuged.
* The supernatant from the first lung lavage was used for biochemical determinations: total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (γ-GT) and superoxide dismutase (SOD) were determined using an auto-analyser. In addition, malondialdehyde (MDA) in BALF was analysed by derivatizing MDA with thiobarbituric acid, extracting the formed product with n-butanol, and measuring the MDA-derivate using high performance liquid chromatography (HPLC) with fluorescence detection (FLD).
* Cell pellets that originated from centrifugation were resuspended in saline. Total white blood cell numbers were counted using an automated haematology analyser. The percentages of viable cells were determined using an acridine orange/ethidium bromide staining method in combination with fluorescent microscopic evaluation. Differential white blood cells were determined by counting 200 cells under the microscope.

FIBRINOLYSIS
Von Willebrand Factor (vWF) and plasminogen activator inhibitor 1 (PAI-1) were analysed in the serum using a multiplex analysis immunoassay. Fibrinogen was analysed using a highly sensitive two-site enzyme linked immunoassay (ELISA).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (n = 5)
HISTOPATHOLOGY: Yes (n = 5)
The weight of organs (adrenals, brain, kidney, lungs, liver, and spleen) was determined prior to preservation in formaldehyde (by infusion for lungs). For histopathological examination the above tissues as well as the larynx, tracheobronchial lymph nodes, nasopharyngeal tissues (6 levels) and trachea (2 levels including 1 longitudinal section through the carina and 1 transverse section) of the high dose group and of the controls were embedded in paraffin wax, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) for examination of gross abnormalities.

Statistics:

Body weight data, clinical pathology data, and organ weights were analysed using one-way analysis of variance (ANOVA), after checking homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data were not normally distributed, the data were stepwise log or rank transformed prior to the ANOVA. Intergroup comparisons with the control group were made by Dunnett's multiple comparison post-hoc test. P < 0.05 was accepted as statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

No exposure related signs of toxicity or behaviour effects or mortality were observed during the 4-week exposure period and recovery period for all three CeO2 (data not shown).

Mortality:

no mortality observed

Description (incidence):

No exposure related signs of toxicity or behaviour effects or mortality were observed during the 4-week exposure period and recovery period for all three CeO2 (data not shown).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Cerium exposure did not influence body weight (data not shown).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Cerium exposure did not influence food consumption (data not shown).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigation and clinical chemistry at necropsy revealed no treatment related changes for NM-213 particles. NM-212 exposure was associated with a slight treatment-related increase in the number of neutrophils in the mid and high dose male groups and in the high dose female group. This elevation was still observed in male animals at the end of the recovery period. NM-211 exposure also induced a slight but significant treatment-related increase in the neutrophil number in females from low and mid dose groups only and none in the high dose group at the end of exposure and in recovery group. No other treatment-related changes were observed in haematological investigations (data not shown).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Due to absence of treatment related changes in clinical chemistry at the end of the exposure period (data not shown), thus, no determination was performed in the recovery group at the end of the recovery period.
Parameters for fibrinolysis did not reveal treatment related changes for all materials (data not shown).

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

According to the authors, urinalysis did not reveal any changes in hydroxydeoxyguanosine as a parameter for DNA damage repair (data not shown). Thus, oxidative stress could not be demonstrated.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in organ weights, except for a treatment-related increase in absolute and relative lung weights for all three materials.
After exposure to the NM-213 material, treatment-related increase in absolute and relative lung weights was observed in females directly after treatment and in the recovery groups. In males, there is only a dose-related effect in the recovery groups.
For NM-212 and NM-211 particle exposures, the increase in lung weight after exposure was reduced but still significant after a 28-day period of recovery in both females and males (see also section "Any other information on results incl. tables").

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observation at necropsy revealed pale tracheobronchial lymph nodes (unilateral) in the recovery groups, after exposure to NM-213 (all high dose animals), to NM-212 (5 males and 3 females) and NM-211 (1 male and 3 females), in comparison with the control groups. For all CeO2 materials, this was not observed in the exposed animals sacrificed one day after the end of the exposures.
No micro or macroscopic changes were detected in the liver (data not shown).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination at the end of the treatment period revealed aggregates of brown/green particles and/or macrophages containing these particles in the lungs and the draining (tracheobronchial) lymph nodes of animals exposed to the high dose of all types of CeO2. These aggregates were also found in the trachea of some animals exposed to the high dose of NM-213 and NM-212 (no data available for NM-211). At the end of the recovery period, macrophages with brown/greenish particles/aggregates still persisted in the alveoli and in the tracheobronchial lymph nodes. Of note that brown pigmented macrophages were also found in tracheobronchial lymp nodes of some control females at the end of the exposure period (3 to 5) as well as in males (1 or 2) and females (4 or 5) of the control group at the end of the recovery period in the experiment conducted with NM-211, NM-212, and NM-213 CeO2. In addition, increased septal cellularity was observed in NM-212 and NM-211exposed animals, suggesting that a tissue reaction was on-going. There was no data available on the occurrence of septal cellularity in NM-213 exposed animals.
In conclusion, following inhalation, CeO2 nanomaterial deposited in several parts of the airways (lungs, trachea, tracheobronchial lymph nodes) as was noticed by brown/green material in the macrophages.
No micro or macroscopic changes were detected in the liver (data not shown).

Histopathological findings: neoplastic:

not examined

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 10.8 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 (test mat.)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 19.9 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 55 mg/m³ air (nominal)

Based on:

other: bulk CeO2 NM-213 (test mat.)

Sex:

male/female

Basis for effect level:

other: - No NOAEC was determined in the publication thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects - Effect level corresponding to 653 000 particles/m3; 0.205 m²/m3 as surface concentration

Dose descriptor:

other: NOEC/NOAEC (local)

Based on:

other: nano-CeO2 NM-211 and NM-212, and bulk CeO2 NM-213 (test mat.)

Sex:

male/female

Basis for effect level:

other: - No NOEC/NOAEC was determined in the publicationand no NOEC/NOAEC could be set by the registrant due to the very high variability and the recovery, at least partial, of BALF results and to the brevity of histopathology data.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified.

Critical effects observed:

not specified

- DEPOSITION OF CeO2 MATERIALS:

Using MPPD modelling, the total deposited dose at the end of the 28-day exposure period was estimated at 0.83, 1.54 and 4.24 mg for NM-211, NM-212 and NM-213, respectively. Based on the density of CeO2 of 7.65 g/cm3, a threshold for overload is expected to be at 11.4 mg (6% of the volume increase of lung macrophages due to phagocytized material => volumetric lung burden of 1.5 x 10E9 - 1.5 x 10E10 µm3). Thus, the authors concluded that it was unlikely the study was performed under overload conditions.

- EFFECTS OF CeO2 PARTICLE EXPOSURE ON LUNG WEIGHT:

After exposure to nano-sized NM-212, significant increases in absolute (abs.) and relative (rel.) lung weight were observed in males from the mid dose (0.44 and 1.53 g/kg bw, respectively) and high dose groups (0.50 and 1.74 g/kg bw, respectively), when compared to respective controls (0.36 and 1.23 g/kg bw, respectively). Moreover, abs. and rel. lung weights were significantly elevated in the recovery group (0.47 and 1.30 g/kg bw, respectively, vs. 0.43 and 1.17 g/kg bw for controls). Similar effects were seen in females, with the exception of a significant increase of abs. and rel. lung weights also seen in the low dose group (0.32 and 1.81 g/kg bw), when compared with controls (0.28 and 1.66 g/kg bw).

For nano-sized NM-211, significant increases in abs. and rel. lung weight were observed in males and females from mid dose (0.45 and 1.68 g/kg bw for males and 0.35 and 2.15 g/kg bw for females, respectively) and high dose groups (0.58 and 2.09 g/kg bw for males and 0.45 and 2.62 g/kg bw for females, respectively) as compared to controls (0.38 and 1.35 for males and 0.30 and 1.71 g/kg bw for females, respectively). Moreover the elevations in abs. and rel. lung weight were still significant after a 28-day period of recovery in both males and females.

After exposure to micro-sized NM-213, there was an increase in abs. and rel. lung weights observed in female directly after treatment in the mid (0.34 and 2.00 g/kg bw, respectively) and high dose groups (0.39 and 2.38 g/kg bw, respectively) as compared to controls (0.28 and 1.68 g/kg bw, respectively) but not in recovery (high dose) group. In males, there was only an increase in rel. lung weight in the recovery (high dose) group (1.41 g/kg bw) when compared to controls (1.20 g/kg bw).

For the other organs evaluated, there was no effect of treatments on organ weights.

- BRONCHOALVEOLAR LAVAGE / LUNG TOXICITY:

A statistically significant increase in total cell counts in BALF was observed only in the high dose and recovery groups of females for NM-212, in the mid dose and high dose groups of females as well as the high dose group of males for NM-211 and in male and female high dose group and only in female high dose group for NM-213.

The percentage of viable cells showed no treatment-related change; hence the absolute number of viable cells was also statistically significantly higher in the high dose groups (data not shown).

In control animals (regardless of the sex), the macrophages accounted for > 98% of the total number of cells recovered in BALF. The neutrophils, lymphocytes, and eosinophils were present in only very low numbers and varied between 0 and 1.4% of the total number of cells for both sexes.

A significant increase in absolute numbers of macrophages was observed in the high dose groups of females exposed to NM-212, in males of high dose group and in females of recovery group for NM-213 and in both sexes from the high dose group for NM-211. All animals except females exposed to NM-213 and from the high recovery group returned to control level at the end of the recovery period

Regardless of the particle type and of the dose administered, the absolute number of neutrophils was significantly increased in all groups of males and females, as well as in the recovery groups.

At last, the absolute number of lymphocytes was significantly elevated in mid and high dose groups of males and females exposed to NM-211 and NM-213 and in the recovery groups of males and females exposed to NM-211 and only in females for NM-213. In contrast, regarding NM-212 the parameter increased only in males from mid and high groups and in females from the recovery groups.

It is noticeable that, whatever the particles and the cell types considered, a decrease of the effects was observed in both males and female recovery groups.

It has to be noted that the different parameters studied showed very high variability, as indicated by standard deviations (SD). Thus, the significance of the observed differences could be questionable.

When expressing the cell counts as relative numbers (%), the relative number of macrophages significantly decreased as a function of the administered concentration in both males and females exposed to the 3 types of CeO2, while the percentages of neutrophils and lymphocytes increased in the same animals. Globally, a similar trend was found in the recovery groups of males and females, when compared to their controls.

Regarding the clinical/biochemical parameters measured in BALF (ALP, GGT, LDH, NAG, protein), all CeO2 induced significant increases in males and females (combined data), but the elevations seemed more important in rats exposed to NM-211 > NM-212 and > NM-213. These effects were still present after the 28-day recovery period though a noticeable decrease was observed for all parameters. Furthermore, the authors stated that the levels of SOD and MDA in BALF were not significantly affected by any exposure (data not shown).

According to the authors, the increased neutrophil content and elevated biochemical parameters in BALF supported by increased lung weight for NM-213, NM-212 and NM-211 indicated that exposure to CeO2 resulted in inflammation in the lungs at all dose levels.

- EXPOSURE METRIC:

Benchmark concentrations (BMCs) were ranked for the three most prominent lung toxicity parameters for which changes were detected: LDH, total protein, and neutrophil levels in the BALF. When ranked on a mass concentration basis, NM-211 was associated with the smallest BMC and therefore the highest toxicity (lowest concentration at which a 100% increase over controls was found). This was followed by NM-212 while micro-sized NM-213 revealed the lowest toxicity. There was a significant difference in the level of neutrophils between males and females for NM-212 and NM-211. However, the mass-toxicity ranking of the materials remained the same when data was separated for sex of animal.

When the BMC was expressed as particle number, the ranking was found to change. NM-213 and NM-211 were found to have similar toxicity in terms of LDH and total protein in BALF, both of which were ranked lower than NM-212. Based on the level of neutrophils, NM-213 was the most inflammogenic.

When the dose was expressed as surface concentration, NM-213 remained the most toxic material with the lowest BMC for all three parameters. NM-211 was the next most toxic material, followed by NM-212, opposite to when expressed as mass concentration.

Conclusions:

Regardless of the size of the CeO2 tested, repeated inhalation exposures of male/female rats induced moderate pulmonary responses with little quantitative differences but without any mortality, clinical or pathological signs, or systemic effect. Moreover, there was little evidence for a dominant predictive exposure metric for the observed effects.

Executive summary:

Gosens I et al. (2013) assessed the hazard of two nano-scale (NM-211 and NM-212) and one microscale (NM-213) cerium dioxide (CeO2) materials in 28-day inhalation toxicity studies in rats, according to the OECD 412 guideline but with some deviations. This study was conducted within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials.

The 3 tested CeO2 were highly pure (≥ 99.5%) and had a negligible solubility in water at neutral pH. NM-211 had a primary size (nominal) of 5-10 nm, NM-212 of 40 nm and NM-213 of < 5 µm. Although the primary particle size of all materials differed considerably, the mass median aerodynamic diameter was surprisingly similar because all materials were aggregated when aerosolised in air. The specific surface areas differed between the tested CeO2: 63.95 m²/g for NM-211, 27.15 m²/g for NM-212 and 3.73 m²/g for NM-213. Moreover, NM-211 showed the highest number concentration in the test atmosphere (1.75 x 10E6 particles/cm3) and the smallest mass concentration (10.79 mg/m3), while NM-212 had a number concentration of up to 1 x 10E6 particles/cm3 and a mass concentration of up to 19.9 mg/m3. NM-213 had a number concentration of 0.68 x 10E6 particles/cm3 and a mass concentration of up to 55 mg/m3.

Male and female Wistar rats (5/sex/dose) were nose-only exposed 40 min to 6 h/day for 5 days/week during 4 weeks to 0 (control), 1.2 - 10.8 mg/m3 of NM-211, 2.5 - 19.9 mg/m3 of NM-212 and 5.9 - 55 mg/m3 of NM-213. Control animals were exposed to a stream of clean humidified compressed air. Cage side observations were performed. Mortality, body weight, and food consumption were also monitored. Moreover, at the end of the exposure period, haematological parameters, clinical chemistry in blood and bronchoalveolar lavage fluid (BALF), inflammatory cell counts in BALF, urinalysis, fibrinolysis, and gross/histo-pathology were carried out to determine the local (pulmonary) and systemic effects of the CeO2 particles. A recovery group was included in this study: males and females exposed to NM-211, NM-212 and NM-213 (high dose) 5 days/week for 4 weeks were kept for a recovery period of 28 days before sacrifice.

There were no exposure-related signs of toxicity, behaviour effects or mortality during the 4-week exposure period and recovery period.

All materials were efficiently deposited in lungs of animals: 0.83, 1.54 mg and 4.24 mg for NM-211, NM-212 and NM-213, respectively; the particle exposures increased the lung weight.

According to the authors, all materials induced a dose-dependent pulmonary inflammation and lung cell damage, but without any gross or histopathological changes immediately after exposure. Inflammation was still present after the 28-day recovery period, albeit at a lower level. Nevertheless, although significant, the parameters used (e.g., BALF cell counts) to draw these conclusions showed a high variability which could indicate that the inflammatory effects observed might be even more modest or almost negligible in some cases.

In contrast, there was no evidence of systemic toxicity or other haematological changes and no macroscopic or microscopic change in the liver following exposure to any of the 3 tested CeO2. According to the authors, these data suggested that the adversity of the effects could be classified as minimal.

When exposure levels were expressed as mass concentration, NM-211 was the most potent material, while based on surface area concentration pulmonary inflammation/damage was induced in a lesser extent by both nano-CeO2 than by the micrometric counterpart. Particles were equipotent based on particle number concentrations.

In conclusion, similar moderate pulmonary toxicity profiles including inflammation were observed for both nanomaterials and the micrometric counterpart and systemic effects were virtually absent. These similar patterns probably resulted from the equivalent aerodynamic diameters found for all materials and there was little evidence for a dominant predictive exposure metric for the observed effects.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Although the study was performed according to OECD guideline 413, there are no information on GLP compliance. Furthermore, histopathological analyses was performed only on the control and high dose (3 mg/m3 CeO2) groups. Further, results of all parameters that should be studied in an OECD 413 study are either briefly reported (e.g clinical signs, body weight gain) or absent (e.g. organ weight, macroscopic examination). Therefore, no NOEC/LOAEC can be derived for systemic and/or local effects in this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

The test was performed only with female rats / All analyses usually performed with OECD 413 were not performed (ex: only histopathological analysis of respiratory tract was performed. Further, only control and high dose groups animals were analysed).

GLP compliance:

not specified

Remarks:

The GLP compliant status was not specified in this published article.

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 10 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups of two animals in Makrolon polycarbonate cages Type IV
- Diet: Ad libitum (“V1534”, sniff Spezialdiäten GmbH, Soest, Germany)
- Water: Ad libitum
- Acclimation period: Subsequent to 1 week of acclimatization rats were habituated to nose-only tubes for 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 40 to 70%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: high-pressurized, high velocity pressurized air

Remarks on MMAD:

MMAD / GSD:
At the concentration of 0.1 mg/m3: 0.71 µm / 3.59 (MMAD / mean GSD)
At the concentration of 0.3 mg/m3: 0.63 µm / 3.83 (MMAD / mean GSD)
At the concentration of 1.0 mg/m3: 0.68 µm / 4.23 (MMAD / mean GSD)
At the concentration of 3.0 mg/m3: 0.79 µm / 3.50 (MMAD / mean GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a nose-only inhalation system.
- Source and rate of air, method of conditioning air: The test material was located in reservoirs on a rotating disc and sucked into the air flow system.
- System of generating particulates/aerosols: Aerosols were generated by dry powder dispersion using a high-pressurized, high velocity pressurized air dispersion nozzle developed at the Fraunhofer Institute for Toxicology and Experimental Medicine ITEM (Germany). Different nanoparticle concentrations were achieved by adjusting the feed rate via rotational speed regulation.
- Temperature, humidity, pressure in air chamber: No data
- Air flow rate: No data
- Air change rate: No data
- Method of particle size determination: Information provided by Sigh C et al., 2014 and Fh-IME Schmallenberg.
- Treatment of exhaust air: No data

TEST ATMOSPHERE
- Brief description of analytical method used: See below in “Details on analytical verification of doses or concentrations”
- Samples taken from breathing zone: No data

VEHICLE
- Justification for use and choice of vehicle: Clean air
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
Control group animals were provided with clean air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aerosol concentrations were continuously recorded by a light scattering aerosol photometer (Fraunhofer ITEM, Hannover, Germany) and compared to additional filter sample analysis.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0 mg/m³ air

Remarks:

(control)

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

0.12 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

0.33 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

1.06 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

3.04 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

No. of animals per sex per dose:

Not reported (only a total of 576 animals is mentioned).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose range selected should cover specific conditions of absent inflammation in combination with absent lung overload (0.1 and 0.3 mg/m3), inflammation and no overload (1.0 mg/m3) as well as inflammation and overload (3.0 mg/m3).
- Rationale for animal assignment, rationale for selecting satellite groups: Animals were randomly assignated.
- Post-exposure recovery period in satellite groups: Yes, up to 1-, 28- and 90-day post-exposure observation.
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The health condition of animals was checked daily. Broad inspection for clinical abnormalities outside of the cage were done once a week.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed after one and 28 days of exposure as well as after one, 28 and 90 days post-exposure period. On exposure days, clinical observations were done before, after and if necessary during exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were checked once a week

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded weekly for a representative subgroup of ten animals from each dose group.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was recorded weekly for a representative subgroup of ten animals from each dose group.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken by puncture of the retrobulbar venous plexus at post-exposure day one. Since small change (increase of 5-10% neutrophil level in CeO2 treatment) were observed at this time point, no further analysis at a later time point was performed.
- Anaesthetic used for blood collection: Yes, slight isoflurane anesthesia.
- Animals fasted: No data - How many animals: 10 rats per test group
- Parameters checked: Full blood analysis parameters were recorded according to OECD TG 413 requirements.

CLINICAL CHEMISTRY: Yes,
- Time schedule for collection of blood: Blood was taken by puncture of the retrobulbar venous plexus at post-exposure day one. Since no change were observed at this time point, no further analysis at a later time point was performed.
- Animals fasted: No data
- How many animals: 10 rats per test group
- Parameters checked (in serum): Full clinical chemistry parameters were recorded according to OECD TG 413 requirements.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
* LUNG BURDEN
In order to determine the lung retention of CeO2, five animals of all dose groups were examined at all days of sacrifices. Explanted lungs of exposed animals were separated and the right lobes were used for analysis of lung burden. The isotopes 140Ce/142Ce in organ samples were quantified via inductively coupled plasma mass spectrometry (ICP-MS) using a quadrupole ICP-MS system (X-Serie II, Thermo Fisher Scientific). Sample preparation included lyophilisation of shredded tissue for at least 6 h (0.37 mbar). Organ weights were recorded prior and subsequently to freezedrying. For removal of organic material samples were further processed by plasma ashing (cool plasma conditions, 400 W, 1 mbar O2, 24 h) and subsequent microwave digestion (H2SO4, 96%, supra quality, max. 500 W).

* BRONCHOALVEOLAR LAVAGE ANALYSIS
Bronchoalveolar lavages (BAL) of rat lungs were performed in five animals of each dose group at all five time points. The method is based on Henderson RF et al., 1987 with minor modifications. Lungs were lavaged twice using 4 mL 0.9% NaCl. The following parameters were determined from collected lavage fluids: total cell count, differential cell count (macrophages, neutrophils, eosinophils and lymphocytes), biochemical mediators (lactic dehydrogenase, ß-glucuronidase and total protein), as well as cytokine levels. Total cell counts were measured using a counting chamber (Fuchs-Rosenthal). Differential cell counts were prepared by centrifugation of BAL fluid on cytoslides and subsequent Giemsa staining. Biochemical indicators were determined in the supernatant of centrifuged BAL fluid according to routine clinical chemistry protocols.

Sacrifice and pathology:

Animals were killed by carbon dioxide overdose and subsequent exsanguination. All organs and tissues were preserved and wet weights were recorded according to OECD TG 413.

Histopathological examinations of respiratory organs were performed at all implemented days of sacrifice in ten animals of the clean air control and 3.0 mg/m3 CeO2 respectively. Left lung lobes including bronchi as well as mediastinal and tracheobronchial lung-associated lymph nodes, trachea, pharynx and nasal cavities including nasal mucosa associated lymphoid tissue were investigated.

All respiratory tract organs were fixed in formalin (10%) for 24 h and trimmed according to Ruehl-Fehlert C et al., 2003, Kittel B et al., 2004 and Morawietz G et al., 2004. The left lung lobe was inflated with formalin (10%) at 20 cm water pressure prior to formalin fixation. After trimming tissues were embedded in paraffin, sectioned, and hematoxylin and eosin (HE) stained for analysis Additionally, Masson trichrome staining of the lung was done for detection of connective tissue production.

Other examinations:

*IMMUNOHISTOCHEMISTRY: See in the RSS: "Nano-CeO2 - Gen. Tox in vivo Rat DNA damage - V4 2017Schw"
To investigate the underlying mechanism of the detected histopathological changes, immunohistochemical staining of lung tissue for markers related to genotoxicity, proliferation and apoptosis were applied. Four markers were selected to determine possible particle-related genotoxicity (Histon γ-H2AX and Hydroxy-2′-deoxyguanosine (8-OHdG)), proliferation (Ki67), and apoptosis (cleaved caspase-3).

Statistics:

Evaluation of body weights, food and water consumption as well as hematology data was done applying ANOVA with Dunnett post-hoc comparison. Bronchoalveolar lavage parameters and immunohistochemistry marker levels were statistically evaluated using Kruskal-Wallis-ANOVA with Mann-Whitney U-Test as post-hoc analysis. Histopathological findings were analyzed by a two-tailed Fisher test.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs due to particle exposure were not observed.

Mortality:

no mortality observed

Description (incidence):

All animals were in good physical conditions up to sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weights were detected (data not shown).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant changes in food consumption were detected (data not shown).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No significant changes in water consumption were detected (data not shown).

Ophthalmological findings:

not examined

Haematological findings:

not specified

Description (incidence and severity):

Hematological parameters were measured after end of nanoparticle exposure (day 90 + 1rec). Since small changes ( increase of 5-10% neutrophil level in CeO2 treatment) were observed at this time point, no further analysis at a later time point was performed. Compared to control levels, in the mid (1.0 mg/m3) and high (3.0 mg/m3) dose group of CeO2 the ratio between segmented neutrophils and lymphocytes shifted in favor of increasing neutrophil numbers. The only significant value was measured for neutrophil levels in the CeO2 mid dose group. Further blood parameters measured did not display any significant changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical markers measured did not display any significant changes.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Respiratory organs of rats exposed to clean air and 3.0 mg/m3 cerium oxide were examined histopathologically at all 5 days of sacrifice. Table 3 (see section "Any other information on results incl. tables") presents an overview of the most prominent findings with mean grades of severity, separately calculated for all groups and time points considered. After 6 h exposure to 3 mg/m3 CeO2 nanoparticles already caused significant accumulations of particle-laden macrophages in the alveolar space and bronchus-associated lymphoid tissue (BALT). The amount of macrophages increased up to the end of post-exposure with translocation to the lung associated lymph nodes (LALN) detected from day 28 of exposure. These findings were accompanied by alveolar and interstitial inflammatory cell infiltrations and very slight bronchiolo-alveolar hyperplasia. Free particles (agglomerates) were detected in the alveolar space after end of exposure, mainly in areas of macrophages containing particulate matter. The authors indicate that such accumulations often originate from degrading macrophages. All described pathological conditions remained persistent during 90-day post-exposure. In addition to that, signs of interstitial fibrosis were detected (mean grade of severity were 0.2, 0.3 and 0.7 at day 90+1, 90+28 and 90+90 post exposure period, respectively) that was significant at d90+90 post exposure recovery only.

The authors reported also alveolar/interstitial foci of macrophages and inflammatory cells. Those infiltrations mainly consisted of lymphocytes and were often located next to bronchioles. Some foci further showed development of a granulomatous inflammation but the number of animals concerned was not specified. Accumulations of particle-laden macrophages, with syncytial giant cell formation were additionally found in BALT and LALN. Detailled overview of histopathological findings with grade and incidence is reported in Table 4 (see section "Any other information on results incl. tables"). The presence of particle-laden macrophages indicated its migration from the alveolar space to lymphoid tissue for clearance of phagocytosed material. Foci of bronchiolo-alveolar hyperplasia of the bronchiolar type (syn.: alveolar bronchiolization) occurred at very slight (minimal) grade in lungs from 28 days of exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Description (incidence and severity):

*LUNG BURDEN:
The lung burden was predicted based on the deposited alveolar fraction calculated using the "multiple path particle dosimetry (MPPD) model" and expected first order elimination with half-times of 70 days (0.1, 0.3, 1.0 mg/m3) or 200 days (3.0 mg/m3). The lung burden analysis showed a substance deposition that is concentration dependent (see Table 2 in "Any other information on results incl. tables"). The lower CeO2 dose groups (0.1, 0.3 mg/m3) as well as the mid and high (1.0, 3.0 mg/m3), respectively showed similar development of lung burdens (deposition fraction: 10.5%, 11.5%, 10.9% and 9.6%, respectively). But at higher CeO2 concentrations, higher deposition rates have been detected with reduced elimination, especially for 3.0 mg/m3 CeO2 (half-time >200 days). Nevertheless, particle elimination was visible in all treatment groups after end of exposure and lung burdens clearly tended to recover during the post-exposure period. Ubiquitous Ce levels were detected in the clean air control groups also. The authors claimed that predicted values for Ce deposition during 90 days exposure were quite close to the measured lung retention. The calculated deposition fraction as well as the expected non-overload or overload conditions after exposure to 0.1 and 0.3 or 3.0 mg/m3 nanoparticles, respectively match quite well. However, differences between predicted (higher predicted values) and measured values increased over time.

*BRONCHOALVEOLAR LAVAGES:
The BALF analysis showed a statistically significant time- and concentration-dependent increase of inflammatory cells, especially neutrophils (PMN) and lymphocytes (LYMPH) along with a slight increase total protein (TP), lactate dehydrogenase (LDH) and ß-glucuronidase (GL) levels in the CeO2 high dose group. Increased occured from 28 and 90 days of exposure for the concentration 1 and 3 mg/m3, respectively. Respective parameters decreased during post-exposure but did not reach controls levels until the end of the study. It should be note that a high variability was observed in these parameters, more particularly at the time point 90 days exposure + 1 days recovery in the highest dose-group.

*IMMUNOHISTOCHEMISTRY:
See in the RSS: "Nano-CeO2 - Gen. Tox in vivo Rat DNA damage - V4 2017Schw"

Remarks on result:

not determinable

Remarks:

As the study reported mainly histological evaluation of lungs of animals from the control and the highest concentration tested in the study (3 mg/m3 nanoCeO2), a definition of NO(A)EC/LO(A)EC for systemic and local effects is not possible.

Table 2. Lung burden and clearance half-times of exposed rats

	Lung burden (µg/lung +/- SD)					Clearance t1/2 (days)
	Day 1	Day 28	Day 90 + 1 rec.	Day 90 + 28 rec.	Day 90 + 90 rec.
Clean air	1.2 +/- 1.0	0.6 +/- 0.2	1.8 +/- 0.8	0.8 +/- 0.5	1.3 +/- 1.3	-
0.1 mg/m3 CeO2	2.5 +/- 0.8	12.0 +/- 2.9	33.1 +/- 1.4	24.7 +/- 6.1	13.2 +/- 3.2	67
0.3 mg/m3 CeO2	5.4 +/- 1.9	33.5 +/- 2.8	99.2 +/- 10.1	85.1 +/- 18.2	41.9 +/- 8.8	69
1.0 mg/m3 CeO2	19.6 +/- 5.6	152 +/- 37.4	476 +/- 74.0	366 +/- 24.7	263 +/- 15.4	108
3.0 mg/m3 CeO2	21.0 1.0	391 +/- 92.3	1280 +/- 82.5	1285 +/- 69.9	1013 +/- 243	224

 

Table 3. Summary of significant histopathological findings after exposure to 3.0 mg/m³ CeO2

CeO2	Lung	d1	d28	d90 + 1	d90 + 28	d90 + 90
	Accumulation, particle-laden macrophages, alv./interst.	*** 1.0	*** 1.0	*** 1.0	*** 1.9	*** 1.9
	Accumulation, particle-laden macrophages, BALT	* 0.5	*** 1.0	*** 1.4	*** 1.7	*** 1.5
	Hyperplasia, bronchiolo-alveolar	0.0	0.4	* 0.6	*** 0.9	* 0.5
	Infiltration of inflammatory cells, alv./interst.	0.0	0.2	*** 1.4	*** 1.5	*** 1.5
	Particles, alveolar	0.0	0.0	*** 1.0	*** 1.0	*** 1.0
	Fibrosis, interstitial	0.0	0.0	0.2	0.3	** 0.7
	Lung-associated lymph nodes (LALN)
	Accumulation, particle-laden macrophages, mediastinal Inn.	0.0	** 0.7	*** 1.9	*** 2.8	*** 3.1
	Accumulation, particle-laden macrophages, tracheobr. Inn.	0.0	*** 1.0	*** 2.7	*** 2.7	*** 3.0
	Nasal cavity
	Accumulation of particle-laden macrophages, NALT	0.0	* 0.5	*** 1.0	* 0.6	*** 1.0
	Globules, eosinophilic, olfactory epithelial	0.0	0.0	0.2	0.5	0.2
	Globules, eosinophilic, respiratory epithelial	0.0	0.0	0.2	0.1	0.2
	Hyperplasia, mucous cell	0.0	0.0	0.0	0.0	0.0
	Infiltration, inflammatory cell, subepithelial	0.0	0.0	0.0	0.2	0.0

 

BALT = bronchus-associated lymphoid tissue; NALT = nasal mucosa-associated lymphoid tissue; Values are presented as mean grade of severity: 0 = none, 1 = very slight, 2 = slight, 3 = moderate, 4 = severe (color gradient from green to red indicates increasing severity); n = 9-10; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. clean air control; Group Factor Chi-Squared and Fisher's Exact two sided/Pearson two sided

 

Table 4: Detailled overview of histopathological findings with grade and incidence of effects per animal from control (clean air) and 3.0 mg/m3 CeO2 groups.

 

Histoptahological findings		Incidence
		Day 1		Day 28		Day 90 + 1 day of recovery		Day 90 + 28 days of recovery		Day 90 + 90 days of recovery
		Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2
Lung
Accumulation, particle-laden macrophages, alveolar/interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Very slight	0	10	0	10	0	10	0	1	0	1
	slight	0	0	0	0	0	0	0	9	0	9
Accumulation, particle-laden macrophages, BALT*	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	5	0	10	0	6	0	5	0	6
	slight	0	0	0	0	0	4	0	3	0	3
	moderate	0	0	0	0	0	0	0	2	0	1
Hyperplasia, bronchiolo-alveolar	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	4	0	4	0	7	0	5
	slight	0	0	0	0	0	1	0	1	0	0
Infiltration of inflammatory cells, alveolar/interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	2	0	6	0	5	0	5
	slight	0	0	0	0	0	4	0	5	0	5
Particles, alveolar	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	10	0	10	0	10
Giant cells, syncytial, BALT*	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	2	0	5	0	2
Fibrosis, interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	2	0	3	0	7
Lung-associated lymph nodes
Accumulation, particle-laden macrophages, mediastinal lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	7	0	2	0	0	0	0
	slight	0	0	0	0	0	6	0	2	0	0
	moderate	0	0	0	0	0	1	0	8	0	9
	severe	0	0	0	0	0	0	0	0	0	1
Accumulation, particle-laden macrophages, tracheobronchial lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	10	0	0	0	0	0	0
	slight	0	0	0	0	0	3	0	0	0	0
	moderate	0	0	0	0	0	6	0	8	0	10
	severe	0	0	0	0	0	0	0	1	0	0
Giant cells, syncytial, mediastinal lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	4	0	7	0	10
Giant cells, syncytial, tracheobronchial lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	8	0	8	0	10
Nasal cavity
Accumulation of particle-laden macrophages, NALT*	10	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	5	0	10	0	6	0	10
Globules, eosinophilic, olfactory epithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	1	2	2	3	1	2
	slight	0	0	0	0	0	0	0	1	0	0
Globules, eosinophilic, respiratory epithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	1	2	0	1	2	2
	slight	0	0	0	0	0	0	0	0	0	0
Hyperplasia, mucous cell	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	0	0	0	0	0
	slight	0	0	0	0	0	0	0	0	0	0
Infiltration, inflammatory cell, subepithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	0	0	0	0	0
	slight	0	0	0	0	0	0	0	1	0	0

 

*BALT bronchus-associated lymphoid tissue, NALT nasal mucosa-associated lymphoid tissue

Conclusions:

All animals appeared in a good physical conditions up to sacrifice (based on body weight, food and water consumption) and no clinical signs due to particle exposure were observed. However, inflammatory reactions were seen in the lung at 1.0 and 3.0 mg/m3 CeO2 exposure and a lung overload at 3 mg/m3 nano CeO2 exposure with a shift overtime to from non-adverse to adverse findings with development of interstitial fibrosis although the grade was minimal.
However, no NO(A)EC/LO(A)EC for systemic or local effect can be derived in this study due to low number of pramaters examined and reported in this study.

Executive summary:

Schwotzer D.et al. (2017) investigated potential health effects of cerium oxide NM-212 nanoparticles to rats in a subchronic inhalation toxicity study. The study was performed according to OECD TG 413 but there is no mention to GLP compliance. Further, several parameters that should be analysed according to this guideline were briefly reported (e.g. clinical signs, body weight) or not reported (e.g. organ weight, FOB, macroscopic analyses). In addition, histopathological analyses were porformed on respiratory tract only and solely in the control and high dose (3m/m3 CeO2) groups. For this reason, the study was awarded a reliability score of 2 (Klimisch, 1997) and was used as a supporting study.

Female Wistar rats were exposed nose only to nanoCeO2 NM-212 (primary particle size: 28,4 nm) at 0 (clean air control), 0.1, 0.3, 1.0 and 3.0 mg/m3 (with a MMAD ranging from 0.63 to 0.79 µm) 6h/day, 5 days/week for 1, 28 or 90 days followed by 1-, 28- or 90-day recovery periods. The dose range of this study was selected to cover specific conditions of absent inflammation in combination with absent lung overload (0.1 and 0.3 mgm3), inflammation and no overload (1 mg/m3) as well as inflammation and overload (3 mg/m3).

The health condition of animals was check daily. Broad inspection for clinical abnormalities outside of the cage, body weight, food and water consumption were recorded once a week. Heamatology and clinical chemistry were done at post-exposure day 1.Lung burden were analysed in 5 animals of all dose groups at all days of sacrifices. Bronchoalveolar lavage analyses (total cell count, differential cell count, biochemical mediators, cytokines) were done in 5 animals of all dose groups at the 5 time points. Histopathological examinations of respiratory organs (left lung lobe, bronchi, mediastinal and tracheobronchial associated lymph nodes, trachea, pharynx, nasal cavities and nasal mucosa associated lymphoid tissue) were performed at all days of sacrifice in ten animals of the clean air control and the 3.0 mg/m3 CeO2.

 

According to the authors, all animals were in good physical conditions up to sacrifice. No significant changes in body weights, food and water consumption were reported and clinical signs due to particle exposure were not observed. But no qualitative or quantitative results were shown.

Blood and biochemical parameters (not specified) measured at post exposure day 1 did not display any significant changes excepted for neutrophil levels in the CeO2 mid dose group. Compared to control levels, in the mid (1.0 mg/m3) and high (3.0 mg/m3) dose group of CeO2 the ratio between segmented neutrophils and lymphocytes shifted in favor of increasing neutrophil numbers.

 

The BALF analyses done after 1 and 28 day of exposure and day 1, 28 and 90 post exposure showed a time- and concentration-dependent increase of the inflammatory cells PMN (significant from 28 days of exposure to 3 mg/m3 CeO2) and lymphocytes (significant from Day 1 post exposure at 1 and 3 mg/m3 CeO2). However, high variability was observed in the results, more particularly at 1days post exposure. PMN but not lymphocytes cells number tend to decrease during the post-exposure period. A slight increase of total protein (TP), lactate dehydrogenase (LDH) and beta-glucuronidase (GL) levels were also observed in the CeO2 high dose group from day 28 of exposure to day 1 post exposure period, then a decrease of all these parameters was observed afterward.

 

The lung burden analysis showed that the substance deposition was concentration dependent and particle elimination was visible in all treatment groups after end of exposure. But at higher CeO2 concentrations, higher deposition rates have been detected with reduced elimination especially for 3.0 mg/m3 CeO2. Thus, corresponding calculated clearance half-time values were 67, 69, 108 and 224 days after 0.1, 0.3, 1.0 and 3.0 nanoCeO2 mg/m3, respectively. The authors concluded that the overload/inflammation hypothesis were achieved for the low dose levels (0.1 and 0.3 mg/m3) and the high dose level (3.0 mg/m3). Clearance half-times were below the expected mean value of 70 days for the low doses levels while for the 3 mg/m3 exposure group, the half-life was > 200 days for particle clearance reflecting a present overload situation. For the 1 mg/m3CeO2 dose, clearance half-time was calculated to be 108 days and signs of inflammation were present at this dose and thus, the expected situation of lung inflammation at non-overload was not clearly achieved. The authors calculated a deposition fraction of about 10 % of the initial nanoparticle concentration for all exposure doses.

 

Histopathology analyses, performed only on respiratory organs (lungs, lung-associated lymph nodes and nasal cavity) of the rats from control group (clean air) and from the high dose group only (3 mg/m3 CeO2) after 1 day and 28 days of exposure and at Day 1, 28 and 90 days of post-exposure showed that after the first exposure to CeO2 nanoparticles, very slight but significant accumulations of particle-laden macrophages in the alveolar space and bronchus-associated lymphoid tissue (BALT) were observed. The amount of macrophages increased in few animals from slight to moderate up to the end of the post-exposure period with very slight to moderate translocation to the lung associated lymph nodes (LALN) detected from day 28 to the end of the recovery that indicated clearance of the phagocytosed material. Very slight to slight alveolar and interstitial inflammatory cell infiltrations and very slight bronchiolo-alveolar hyperplasia were observed after 28 days of exposure to high dose of nanoCeO2 up to the end of the recovery period. Free particles (agglomerates) were detected in the alveolar space after end of exposure, mainly in areas of macrophages containing particulate matter. Such accumulations often originate from degrading macrophages. Furthermore, during the recovery period, very slight interstitial fibrosis was observed which was significant at the 90 day-post exposure time point. No pathological change was observed in the nasal cavity of exposed rats.

Acccording to the authors, this series of effects observed at the highest dose level illustrates the consequence of particle overload (impaired macrophage activity and particle elimination leads to translocation of particles to the interstitium, causing local interstitial effects like inflammatory cell infiltrations associated with fibrotic lesions although observed at a very slight grade.

 

Overall, animals appeared in a good physical conditions up to sacrifice. However, inflammatory reactions were seen in the lung at 1.0 and 3.0 mg/m3 CeO2 exposure based on bronchoalveolar lavages and a lung overload at 3 mg/m3 nano CeO2 exposure with a shift overtime to from non-adverse to adverse findings with development of interstitial fibrosis although the grade was minimal.

However, no NO(A)EC/LO(A)EC for systemic or local effect can be derived in this study due to the low number of parameters examined and reported in this study.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focused on the lung effect of nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

This study was performed in order to assess the influence of redox activity by modifications of cerium dioxide nanoparticles (nano-CeO2) via zirconium doping on the distribution, pulmonary and cardiovascular effects in mice following sub-acute inhalation. This study further explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Only results observed with nano CeO2 are reported in this summary. Mice from each strain were exposed to the nanoparticles during 4 weeks and the effects were evaluated 4 weeks after the last exposure day (i.e day 56).
Experiments were conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable local and EU regulations.

GLP compliance:

not specified

Remarks:

The GLP compliant status was not specified in this published article.

Limit test:

no

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

3 mice models were used in this study to explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice model, a well established model for the study of the vascular disease atherosclerosis (Coleman et al., 2006). ApoE-/- mouse were used to study hematology, pulmonary and cardiovascular effects of the nanoparticles.
- Alzheimer’s disease mouse model (5xFAD), were included to study neurological effects (not included in the article and should be published in another article) and the hematology and pulmonary effects of the nanoparticles.
- C57BL/6J mouse - (non-genetically modified) strain, was used as the background strain of the disease mouse models and was used to study biodistribution, hematology and pulmonary effectsof the nanoparticles.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and source:
* Specific Pathogen free (SPF) ApoE−/− mice come from Taconic, Danemark
* 5xFAD and wild type (WT) cross bread C57BL/6J littermates mice come from Jackson Laboratories.
- Age at study initiation:
* ApoE−/− mice: 10-12 weeks
* 5xFAD and C57BL/6J mice :8-11 weeks
- Weight at study initiation, fasting period before study: No data available
- Housing: macrolon cages
- Diet: Ad libitum, The ApoE-/- mice were fed a commercially available rodent Western (high fat) diet (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), starting at the first day of the exposure period until the end of the experiment. The other mice were fed a standard commercially available rodent diet (SMR-A, ABdiets, Woerden, The Netherlands).
- Water: Ad libitum, except during the exposure periods
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2 °C
- Humidity: 40 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 280 nm

Geometric standard deviation (GSD):

1.55

Remarks on MMAD:

The Count Median Diameter (CMD) and the Mass Median Diameter (MMD) were estimated using the Aerosol Instrument Manager Software (Release Version 9.0.0.0, 15:32:53, Nov 11 2010 from TSI Inc., St Pauls, MN, USA), assuming spherical aggregation around primary particles of 4.7 ± 1.4 nm.

Details on inhalation exposure:

Approximately one week before the 4-week exposure period, 20 samples of the nano-CeO2 (one for each day) with a concentration of 1 mg/mL were prepared from the stock dispersions (20 mg/mL), by diluting with ultrapure water to the desired concentration. Stock and sample dispersions were sonicated for 5 minutes in an ultrasonic bath (Branson CPX2800, 40 kHz, 110W) before use to re-disperse any possible agglomerates.
Freshly generated aerosols of NPs were generated using a spray nozzle technique, diluted with pressurized clean particle-free air, and heated to 24-25°C.
Exposure was controlled based on stable particle number counts, mass concentrations, temperature and relative humidity, measured continuously using a condensation particle counter (CPC 3022A from TSI Inc., St. Paul, MN, USA), a tempered element oscillating microbalance (TEOM series1400A from Rupprecht & Patashnick, NY, USA) and M-170 Measurement Indicator (Vaisala M170, Vaisala Oyj, Helsinki, Sweden), respectively, during each exposure period.
During the 3 hour exposure periods to the nanoparticles, the control groups were exposed to filtered air under the same conditions (nose-only tubes) for the same amount of time.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was characterized at least twice during each exposure session using an optical particle sizer (OPS 3330, TSI Inc., St. Paul, MN, USA) and a scanning mobility particle sizer (SMPS 3936 from TSI Inc.,St. Paul, MN, USA). In addition, aerosols were collected on polycarbonate filters for scanning electron microscopy (SEM) analysis. The SEM samples were prepared by placing a small piece of the filter on the SEM stub and coating it with platinum, and visualizing with an XL30 Environmental SEM-FEG microscope (Philips XL30 ESEMFEG).
The total mass concentration generated over the 3-hour exposure period was determined by gravimetric analysis of pre-weighed and post-weighed polytetrafluoroethylene (PTFE) filters (Teflo R2PJO47, Pall corporation, Port Washington, New York, USA) using a micro-balance (Mettler MC or ME-5 microbalance, Mettler-Toledo LLC, Columbus, OH, USA).

Duration of treatment / exposure:

4 weeks of exposure. Tthe effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

To explore the (patho)physiological effects of nanoparticle on multiple organ systems, three different mouse models were exposed:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice are a well established model for the study of the vascular disease atherosclerosis (Coleman et al.,2006), a disease characterized by the build-up of lipid- and inflammatory cell-rich plaques within arteries, which underlies the majority of cardiovascular diseases. The 4-week exposure protocol was integrated into an 8-week high-fat feeding regime that has been shown to generate complex atherosclerotic plaques with many of the hallmarks of the human disease in specific arterial locations (Cassee et al., 2012, Miller et al., 2013).
ApoE−/− mice were used to study hematology, pulmonary and cardiovascular effects.
- The 5xFAD mice are an Alzheimer’s disease mouse model. Although these mice were included to study neurological effects that will be published in a separate paper, the hematology and pulmonary effects were also studied within the same animals and reported in this paper.
- C57BL/6J mice were used as the background (non-genetically modified) strain of the disease mouse models and used to study biodistribution, hematology and pulmonary effects.
Hematology, pulmonary and cardiovascular effects were assessed 4 weeks post exposure. This period was included, firstly, to provide the extra four weeks necessary for mice to develop sufficient plaque formation in arteries and secondly, to investigate the persistency of the pulmonary and cardiovascular effects.
One group of each mouse model was exposed nose-only for four weeks to 4 mg/m3 nano-CeO2 for 5 days/week for 3 hours/day and one control group to clean air under the same experimental conditions (nose-only tube, 3h). Effects were assessed 4 weeks post the final exposure (56 days after the initial exposure).
The number of animals per group was different for each of the mouse models, to provide sufficient statistical power to detect differences between exposed and control animals in the most important effect parameter of each mouse model. Based on previous experiments, 8 ApoE-/- mice per group were expected to be sufficient to detect statistically significant differences in atherosclerotic plaque size and 5 C57BL/6J mice per group were expected to be sufficient to detect statistically significant differences in the number of neutrophils in the bronchoalveolar lavage fluid (BALF). However, 10 C57BL/6J mice per group were included, since 10 C57BL/6J mice per group and 16 5xFAD mice per group were needed to provide sufficient statistical power in the neurological study.

Positive control:

no

Observations and examinations performed and frequency:

Animals were monitored by cage-side observations and, if necessary, handled to detect signs of compromised health. The body weight of each animal was recorded one day before the start of exposure (day -1), prior to exposure on the first day and weekly thereafter.

Hematology, neurological (results published in a separate article which was not avaible at the time of the dossier preparation), pulmonary and cardiovascular effects were assessed 4 weeks after the last exposure of the mice (i.e. 56 days after the initial exposure).

- QUANTIFICATION OF CERIUM IN TISSUES
During necropsy, organs from half of the C57BL/6J mice per group were obtained to evaluate the distribution of the nano CeO2 throughout the body. Liver, spleen, kidneys, heart and right (exposed mice) or left (control mice) lung, were weighed and immediately frozen in liquid nitrogen for determination of the Ce concentrations. To allow measurement of multiple parameters within the same animal, different parts of the lungs were selected for the exposed compared to the control animals. From the exposed groups the right lung was used for quantification of Cerium, because the left lung was needed for histopathological examination. From the control group the left lung was used for quantification of Cerium, because the right lung was needed for bronchoalveolar lavage. The organs were digested by acidification of each sample with 2 mL nitric acid for 12h. Hydrogen fluoride (0.2 mL) was added, followed by microwave heating for 45 min up to 185°C, and maintained for a further 20 min. Boric acid (2 mL) was added to neutralize the hydrogen fluoride, and the samples were re-heated for 20 min to 160 °C, and maintained for 10 min. Once cooled, samples were filtered with a 450 nm syringe filter, diluted with 10 mL deionized water and stored at room temperature (RT). The presence of Cerium in the lungs, liver, spleen, kidneys and heart was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NexION 300X instrument operated in standard mode for Cerium. The isotope measured was 140Ce using 115In and 159Tb as internal standards. Calibration standards (0 – 100 μg/L) were prepared from VWR 1000 mg/L stock solutions. Quantities are expressed as µg/g organ tissue.

- HEMATOLOGY
Animals were anaesthetized with a mixture of ketamine and xylazine. Two blood samples were taken by eye extraction. The first sample was collected in a K3-EDTA tube (Minicollect K3EDTA, 1 mL, 450474 Greiner Bio-One) for hematological parameters as determined in a blood auto analyzer (ADVIA 2120 Hematology System, Siemens Healthineers) within 3 hours after collection. The second sample was collected in a serum tube and stored at -20°C for further analysis. Total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cells counts were meaured.

- BRONCHOALVEOLAR LAVAGE
Lung lavage was performed at necropsy. A cannula was placed in the trachea and the diaphragm opened to decrease the amount of air inside the lungs. For the control animals and all ApoE-/- animals, the right lung half was rinsed twice with approximately 0.5 mL (26.7 mL per kg body weight) of physiological saline solution, after ligation of the left lung. The injected volume was inserted and recovered 3 times, after which the lavage liquid was collected and stored on ice for less than 2 hours. To allow necropsy of the planned number of animals within one day, both lungs were lavaged for 10 of the 16 exposed 5xFAD mice and 5 of the 10 exposed C57BL/6J mice, using the same procedure, but approximately 0.8 mL (40 mL per kg body weight) of physiological saline solution.
BALF was centrifuged at 400g for 10 minutes at 4°C. The supernatant was divided into two separate aliquots of 125 µL for total protein (TP; an indicator for acute lung injury), lactate dehydrogenase (LDH; an indicator of cytotoxicity), gammaglutamyl transferase (GGT; an indicator of lung cell damage) and alkaline phosphatase (ALP; an indicator of type II cell damage) measurements using an autoanalyser (LX20- Pro, Beckman-Coulter, Woerden, the Netherlands). The cell pellet was scored for the presence of erythrocytes, re-suspended in 500 μL phosphate-buffered saline (PBS) and kept on ice. Cell counts were determined in the re-suspended pellet using a Coulter counter (Beckman-Coulter, Live Sciences). Cell concentrations were determined using a single sample using at least 150 µL of the re-suspended cells. Cytospins (Cytospin 3, Thermo-Shandon) were prepared and stained using May-Grunwald and Giemsa stain, and cell differentiation was performed by counting 400 cells per slide.

Sacrifice and pathology:

HISTOPATHOLOGY
- Lung tissue:
For the majority of the animals, the right lung was removed after the collection of the BALF and immediately frozen in liquid nitrogen and stored at - 80°C for further analysis. The left lung was removed and, after weighing, cannulated and infused with formaldehyde for 1 hour at a pressure of 20 cm H2O. Lungs were processed for histopathology; embedded in paraffin wax, sectioned at 2-4 µm intervals and stained with hematoxylin and eosin for histopathological examination.
Histopathological changes were described according to distribution, severity and morphological characteristics. The morphological characteristics of chronic inflammation include for example the presence of lymphocytes and macrophages in the lung tissue, while acute inflammation is characterized by the presence of polymorphoneclear neutrophils (PMNs).
Severity scores were assigned as follows: Grade 1 Minimal/very few/very small; Grade 2 Slight/few/small; Grade 3 Moderate/moderate number/moderate size; Grade 4 Marked/many/large; Grade 5 Massive/extensive number/extensive size.

- Assessment of atherosclerosis:
Arteries (brachiocephalic, aortic arch, thoracic aorta) were isolated from ApoE-/- mice. Atherosclerosis was quantified, as previously published (Miller et al., 2013). Briefly, brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 µm intervals, beginning at the first section of the artery with a fully intact media. Sections were stained with Masson’s Trichrome. The cross-sectional area of the plaque was measured and standardized to the medial area. A single mean value for atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery. A single section from each artery (the section exhibiting the largest plaque in cross-section) was chosen for mac-2 immunohistochemistry for macrophage-derived cells. A rat anti-mouse primary antibody was used (1/12000; CL8942AP, VH Bio, Gateshead, UK) with rat IgG (1/12000; I-400, Vector Labs, Peterborough, UK) as a negative control, followed by a goat anti-rat IgG biotinylated secondary antibody (BA-9400, Vector Labs). The area of positive staining was expressed as a proportion of the total plaque area.

- Other organs:
The spleen, liver, heart and kidneys were removed, weighed and stored in 4% formaldehyde for pathological analysis if required based on macroscopic findings.

Statistics:

Statistical analyses were performed using GraphPad Prism v7.00 (GraphPad Software, San Diego, California, USA). Ordinary one-way analysis of variance (ANOVA) analyses including all experimental groups were performed followed by a Tukey’s posthoc multiple comparisons test. A p-value ≤0.05 was considered statistically significant.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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):

Total and differential white blood cell (lymphocytes, neutrophils, eosinophils and monocytes) counts were measured 4 weeks after the last day of exposure (day 56). Results are the mean of 8 to 16 animals. ApoE-/- mice had more neutrophils compared to exposed and control C57BL/6J and 5xFAD mice (p<0.05 in Tukey’s post-hoc test following one-way ANOVA). No statistically significant differences were observed in the total white blood cell counts (data not shown) or differential (white blood cell counts of the exposed groups compared to the controls in blood from all strains of mice.

Clinical biochemistry findings:

not examined

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):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Although the authors used the 5xFAD mice, an Alzheimer’s disease mouse model, to study neurological effects of exposure to nanoparticles, they said that the results will be published in a separate paper that was not published at the time of the preparation of this dossier.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP but not in the other strains (see table 1 in "Any information on results incl. tables").
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the NP exposed animals but not in the control groups

Other effects:

no effects observed

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE (BALF)
- Total and differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytesin) from BALF measured 4 weeks after the last exposure day (mean of 5 to 16 animals) in the 3 mice strains: No statistically significant differences were observed in the total cell counts (data not shown) or differential cell counts in BALF of the exposed groups compared to the controls for any strain of mouse.

- Total protein, Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transpeptidase (GGT) levels in bronchoalveolar lavage fluid (BALF) 4 weeks after exposure (mean of 5 to 16 mice from the each tested strains):
Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels between the exposed and control groups.

No constitutive differences in differential cell counts or protein levels were observed between the different mice strains.

EFFECTS OF NANO CeO2 ON ATHEROSCLEROSIS IN THE BRACHIOCEPHALIC ARTERY OFApoE-/- MICE AFTER INHALATION
ApoE-/- mice exhibited regions of dense plaques in the aortic arch and branch points of large arteries. Plaques were composed of fibroblastic matrix, smooth muscle cells, lipid cavities and cholesterol crystals. Atherosclerotic burden was quantified in the brachiocephalic artery, with control (air-exposed) mice having a mean plaque size of 94 ± 9% (standardized to the area of the vascular media). Exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries (p=0.62; One-way ANOVA) and in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

Dose descriptor:

NOAEC

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

Highest concentration tested in this study.

Sex:

female

Basis for effect level:

other: No major toxicological effect

Critical effects observed:

no

Estimated deposition of inhaled nano-CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1020 µg. The total deposited dose (estimated using the MPPD model) was found to be 618 µg. The deposited fraction per region, estimated using the MPPD model, was found to be 48.8 % for the head, 3.5 % for the tracheobronchial region and 8.0 % for the alveolar region of the inhaled mass dose. The retained dose in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, was found to be 16.2 µg.

Quantification of cerium in tissues (en µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

The highest concentrations of Cerium were found in the lung (~ 67 µg/g), followed by much lower concentrations in the heart (~2.2 µg/g), spleen (~1.1 µg/g), kidneys (~0.3 µg/g) and liver (~ 0.2 µg/g), respectively. As would be expected, significantly higher levels of Ce were observed in the lungs of exposed mice compared to the controls (~1.1 µg/g in control lung). In most of the other organs, the cerium concentrations were not statistically signicantly different from the background concentrations measured in the control animals (heart (~2.8 µg/g), spleen (~1.0 µg/g), kidneys (~1.3 µg/g) and liver (~ 0.05 µg/g)).

Tha authors indicated that background levels of Ce detected in various organs of animals exposed to clean air (controls) might be caused by Ce contamination of the drinking water, food and/or bedding of the animals as previously found by others (Yokel et al. 2012, 2013, see the section 7.1 Toxicokinetics, metabolism and distribution of this dossier)).

The authors said that, based on the estimated retained dose of nano CeO2 in the lung (16.2 µg, see Table 3) using MPPD model and the average lung weight (158 mg), the expected Ce concentration in the lung was approximately 103 µg/g while the measured Ce concentration in the lungs of the nano CeO2 exposed mice was slightly lower (67 µg/g) but in the same order of magnitude. They concluded that the difference between the predicted and measured concentrations may reflect the lung clearance rate used by the MPPD model compared to the actual lung clearance rate in vivo.

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages	Particle loaded alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal	Minimal	Slight
C57BL/6J	Control	60% (3/5)a	-	-	-	-
	Nano CeO2	80% (4/5)	-	20% (1/5)	-	-
ApoE-/-	Control	25% (2/8)	-	-	-	-
	Nano CeO2	38% (3/8)	-	-	13% (1/8)	75% (6/8)
5xFAD	Control	69% (11/16)	-	6% (1/16)	-	-
	Nano CeO2	83% (5/6)	-	17% (1/6)	-	-

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

The authors concluded that nanoCeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease..

Executive summary:

In this subacute inhalation study, Dekkers et al. (2015) explored the (patho)physiological effects of nano CeO2 exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. As this study was performed using a test procedure in agreement with generally accepted scientific standards but with some limitations (incomplete data on substance characterisation and on systemic toxicity), the study was awared a reliability code of 2 according to Klimisch criteria and was used as weight of evidence.

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m3 nano CeO2 (primary particle size of 4.7 nm) for 3h/day, 5 days/week for 4 weeks.

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs were done and the tissue distribution of cerium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of nano-CeO2 on the artherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

According to the authors, no statistically significant differences were observed in the total or differential white blood cell counts in the blood and in the BALF of the exposed groups compared to the controls in all strains of mice. Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels in BALF between the exposed and control groups. Furthermore, no treatment related effects on organ weights were observed and no macroscopic findings related to treatment were observed. In lungs, the histopathological analyses showed an increased incidence, described as modest by the authors, in minimal chronic bronchoalveolar or alveolar inflammation observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in most of the ApoE-/- mice exposed to nano CeO2 NP but not in the other stains. In the bronchoalveolar lavage fluid, particle loaded macrophages were seen in all the exposed animals but not in the control groups. In tissues, background Ce concentrations were measured in the control animals and significantly higher levels of Ce were only observed in the lungs and the liver of exposed mice as compared to the controls but not in the other organs.

In ApoE-/- mice, exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries and in the proportion of plaque staining positive for mac-2. The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concuded that in this subacute inhalation study, nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

No NOAEL was derived in this study by the authors. However, according to the results and the conclusion of the authors (nano CeO2 has a low biological activity in their mouse models), it can be concluded that the NOAEC for lung toxicity is > 4 mg/m3, the only dose tested in this study.

Reference 6

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

other: standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: :

Remarks:

the study was well documented and performed according to generally accepted scientific principles and in compliance with GLP. However, given the experimental conditions applied (e.g., 5-day exposure duration), this study should be viewed as a preliminary work.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Repeated exposure of rats to an aerosol of nanometric cerium dioxide (nano-CeO2) for 5 consecutive days (6 h/day) through whole-body inhalation route
- Pulmonary inflammation: Analysis of the bronchoalveolar lavage fluid, lung tissue homogenates and blood samples (i.e., cytokine profiles)
- Tissue injury: Histopathological examination of all respiratory tract and extrapulmonary tissues using optical microscopy
- Cerium (Ce) content in the lung, lung-associated mediastinal lymph nodes and extrapulmonary organs

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610x435x215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: - NM-211
At the concentration of 0.5 mg/m3: 1.6 µm / 2.1
At the concentration of 25 mg/m3: 1.3 µm / 2.1

- NM-212
At the concentration of 0.5 mg/m3: 1.4 µm / 2.3
At the concentration of 5 mg/m3: 1.2 µm / 2.1
At the concentration of 25 mg/m3: 1.0 µm / 2.5

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle, purity of vehicle: Not applicable

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

25 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest aerosol concentration was 25 mg/m3, which was expected to cause biological effects and should lead to lung overload at least for 20 exposures. The mid and low aerosol concentrations were 5 and 0.5 mg/m3. The low aerosol concentration with an expected lung burden far below the overload condition should not lead to any adverse effects. The mid aerosol concentration, which was spaced 10-fold higher than the low concentration, was expected to cause some biological effects.
- Rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Yes, up to 24-day post-exposure observation
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations of the animals were recorded for each animal at least three times per day on exposure days and once a day during the pre-exposure and post-exposure periods. Signs and findings were recorded for each animal. During exposure, examination was possible only on a group basis.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0), and twice weekly until killing or twice within the 5 exposure days.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH, Germany))
- Animals fasted: Yes- How many animals: 5 rats per test group
- Parameters checked: Red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell as well as differential blood cell counts with a hematology analyser

CLINICAL CHEMISTRY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Animals fasted: Yes
- How many animals: 5 rats per test group
- Parameters checked (in serum): rat haptoglobin and rat γ2-macroglobulin by ELISA; enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and other blood parameters of clinical chemistry using an automatic analyser

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* BRONCHOALVEOLAR LAVAGE
Twenty-four hours and 35 days after the end of exposure, five animals per test group were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anaesthesia. The lungs of the animals were lavaged in situ twice with saline solution. A total of 11 mL bronchoalveolar lavage fluid (BALF) was obtained per animal for analysis. Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with a hematology analyser. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes, and atypical cells were performed on Wright-stained cytocentrifuge slide preparations. The differential cell count was evaluated manually by counting at least 400 BALF cells per sample. Using a reaction rate analyser, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) were measured.

* INFLAMMATORY MEDIATORS IN BALF AND SERUM
Cytokines and chemokines in BALF and serum were measured. The parameters comprised various cytokines, chemokines, adhesion molecules, matrix metalloproteinases, acute-phase proteins, signal proteins of apoptosis, or cell proliferation: monocyte chemoattractant protein-1 level (MCP-1), cytokine-induced polymorphonuclear neutrophil chemoattractant-1 level (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF) and osteopontin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy and histopathology were performed after 5 days of exposure and 21 days after the end of exposure (5 days of exposure). In general, 5 animals per test group were investigated for pathological examination. At necropsy, animals were exsanguinated by opening of the abdominal great vessels under deep pentobarbital anaesthesia. All organs were preserved according to OECD TG No. 412. Following organs were weighed: adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, and thyroid glands. The lungs were instilled with and fixed in 10 % neutral-buffered formalin (NBF). Once fixed, the lungs were transferred to 70 % ethanol. All other organs were fixed in 10 % NBF. All the organs and tissues described in the OECD TG No. 412 were trimmed. After paraplast-embedding, the blocks were cut at 2- to 3-μm thickness, mounted on glass slides and stained with hematoxylin and eosin. Extrapulmonary organs and the respiratory tract compromising nasal cavity (four levels), larynx (three levels), trachea (transverse and longitudinal with carina), lung (five lobes), and mediastinal and tracheobronchial lymph nodes were assessed by light microscopy. For the lungs, whole histopathological examination was performed in animals of all test groups. For all other tissues, only the animals of the control and high concentration group of nano-CeO2 were initially examined. When changes were observed in the high concentration group, respective organs and tissues of the animals exposed to low and intermediate aerosol concentrations were also examined by light microscopy. All histopathological examinations were performed by a well-experienced board-certified veterinarian toxicopathologist followed by an internal pathology peer review.

Other examinations:

ORGAN BURDEN
Lung burden of the two different nano-CeO2 was evaluated twice, immediately after 5 days of exposure and after 21 days after the exposure end. Cerium (Ce) content in the lungs, lung-associated lymph nodes, and liver of either 3 or 5 animals per test group were examined. After digestion with mixed acid, samples of each lung or lymph node were dissolved in sulphuric acid and ammonium sulphate. 140Ce content in the obtained solution was analysed by inductively coupled plasma mass spectrometry (ICP-MS) or by inductively coupled plasma optical emission spectrometry (ICP-OES) with a wavelength of 419 nm. The limit of detection for Ce was 0.3 μg. The amounts of CeO2 in the respective tissues were calculated by measuring elemental Ce with ICP-MS.

Statistics:

For body weight changes, Dunnett’s test was used for a comparison of each test group with the control group test. Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analysed by non-parametric one-way analysis using the Kruskal–Wallis test (two-sided). If the resulting p value was ≤0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by non-parametric one-way analysis using the two-sided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

The animals exposed for 5 days to NM-211 and NM-212 showed no clinical signs or findings compared to the control animals (data not shown).

Mortality:

no mortality observed

Description (incidence):

The animals exposed for 5 days to NM-211 and NM-212 showed no clinical signs or findings compared to the control animals (data not shown).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Five days of inhalation exposure to either NM-212 or NM-211 did not affect the body weight development of the animals (data not shown).

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:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative neutrophil counts in blood were increased 3 days after the end of exposure to 25 mg/m3 NM-212 and NM-211, whereas relative lymphocyte counts were decreased (see in Table 5 in "any other information on results" below). The changes were in a concentration-related manner. Twenty four days after the end of exposure, all parameters had returned to near control values. No other blood parameters were affected.

Clinical biochemistry findings:

not specified

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 5 days of exposure, no increase in lung weights was observed after exposure to NM-212. An aerosol concentration of 25 mg/m3 NM-211, however, resulted in significant increases in absolute and relative lung weights (+20 and 24 %, respectively). This increase in lung weight was no longer present 21 days after the end of exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

After 5 days of exposure, the macroscopic finding enlarged mediastinal lymph nodes were found in individual animals of all test groups. Twenty one days after the end of the exposure, enlarged mediastinal lymph nodes were observed in almost all animals exposed to Ceria NM-211 or NM-212. The tracheobronchial lymph nodes and all other examined organs did not show any macroscopic findings.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

* LUNGS
After 5 days of exposure, single or accumulated macrophages were located in the lumen of the alveoli and a few macrophages occurred also in the alveolar wall and ducts. They were distributed multifocally in all lobes over the whole lung. Most of them were loaded with amber-like coloured particles of different sizes (below 1 μm diameter). These particles were considered to represent agglomerated or aggregated nano-CeO2 (“alveolar histiocytosis with particles”). Alveolar histiocytosis and free eosinophilic granular material with particles, interpreted as remnants of destroyed macrophages, were found in the lung of all animals exposed to 25 mg/m3 NM-211 and NM-212. At aerosol concentrations of 0.5 mg/m3 NM-211, 0.5 and 5 mg/m3 NM-212, amber-coloured particles within single histiocyte in the alveoli were noted in all animals. In the bronchus-associated lymphoid tissue (BALT), particles were detected free or in single macrophages, mostly at 5 mg/m3 nano-CeO2 and above. Findings regressed but were still present 21 days after the end of exposure. The finding “eosinophilic granular material with particles” was no longer visible after the post-exposure period. Particles were detected free or in single macrophages in BALT, even at the lowest concentration of 0.5 mg/m3 NM-211. Only one animal exposed to 25 mg/m3 NM-211 showed macrophage aggregates in BALT 21 days after the end of exposure.

* LUNG-ASSOCIATED LYMPH NODES
After 5 days of exposure to 25 mg/m3 NM-211 and NM-212, comparable to the finding of particles in BALT of the lung, amber-like coloured particles were seen partly within macrophages or extracellularly in the lymphoid tissue, without any macrophage activation or aggregation (2/5 to 5/5 rats for NM-212; 3/5 to 4/5 rats for NM-211). Twenty one days after the end of exposure to 25 mg/m3, findings in mediastinal and the tracheobronchial lymph nodes progressed: multifocal macrophage aggregates with amber-like coloured particles were noted in animals exposed to NM-211 (2 to 3/5 rats) and NM-212 (4/5 rats). In both lymph nodes, lympho-reticulocellular hyperplasia was observed after 5 days of exposure and 21 days after the end of exposure to NM-211 and NM-212.

* UPPER RESPIRATORY TRACT (data not shown)
After 5 days of exposure to 25 mg/m3 NM-211 and NM-212, extracellular, amber-like coloured particles with diameters up to 1.5 μm were found in the lamina propria mucosae of the dorsal area of the larynx (level III). At aerosol concentrations of 25 mg/m3 NM-211 and NM-212, resembling amber-like coloured particles were detected within the subepithelial tissue in the carina of the trachea. Twenty one days after the end of exposure, findings in the larynx were only observed for animals exposed to NM-212, whereas particles in the carina (trachea) were still present in animals exposed to NM-211 and NM-212.

* EXTRAPULMONARY ORGANS
Extrapulmonary organs of animals exposed for 5 days to NM-211 and NM-212 were not examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Details on results:

BRONCHOALVEOLAR LAVAGE AND INFLAMMATORY MEDIATORS IN BALF AND SERUM
BALF analysis of 5 animals per test group was obtained 3 and 24 days after the end of exposure. The resulting BALF parameters are presented in Table 6.
In animals exposed to NM-212, the majority of BALF parameters were increased at aerosol concentrations of 5 and 25 mg/m3. At 0.5 mg/m3, the neutrophil counts and CINC-1 levels were both statistically increased and were slightly above the historical control range. With NM-211, but not NM-212, MCP-1 and M-CSF were increased at aerosol concentrations of 0.5 mg/m3 and above. Twenty four days after the end of exposure, a full recovery was observed at aerosol concentrations of 0.5 mg/m3 and a partial recovery at aerosol concentrations of 5 and 25 mg/m3. The recovery of animals exposed to 25 mg/m3 NM-211 seemed to be slower than those exposed to NM-212.

ORGAN BURDEN
Exposure to 0.5 mg/m3 NM-212 resulted in a lung burden of 0.011 mg/lung, directly after 5 days of exposure and decreased to 0.006 mg/lung 21 days after the end of the exposure, whereas exposure to 5 and 25 mg/m3 yielded higher lung burdens (0.1 and 0.53 mg/lungs, respectively) with only little decrease (0.088 and 0.4 mg/lung, respectively) within 21 days after the end of the exposure. Lung burdens of NM-211 were around 2-fold lower compared to those of NM-212. Cerium content in the lung-associated lymph nodes at aerosol concentrations of 25 mg/m3 increased from 1.7 to 5 μg for NM-212 and from 1.4 μg to 3 μg for NM-211 in the short-term study with 5 days of exposure.

Dose descriptor:

other: NOAEC (systemic)

Effect level:

> 25 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 and NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: - No NOAEC was determined in the publication, thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects

Dose descriptor:

other: NOAEC (local)

Effect level:

> 0.5 - < 5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 and NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Table 4: Mean clinical pathology parameters in blood after exposure to nano-CeO2

	Control	NM-212			NM-211
Target conc. [mg/m3]	0	0.5	5	25	0.5	25
Measured conc. (mg/m³) +SD	0	0.48 ± 0.0	5.2 ± 1.1	25.6 ± 6.0	0.45 ± 0.1	25.8 ± 1.7
Blood cells
Neutrophils [giga/L] + SD Time point 1 (a) Time point 2 (b)	0.55 ± 0.21 0.64 ± 0.19	0.52 ± 0.07 0.58 ± 0.02	0.92* ± 0.22 0.59 ± 0.13	1.08** ± 0.14 0.64 ± 0.10	0.67 ± 0.16 0.73 ± 0.26	1.14* ± 0.30 0.75 ± 0.23
Lymphocytes [giga/L] + SD Time point 1 (a) Time point 2 (b)	3.16 ± 1.09 3.00 ± 0.66	2.55 ± 0.72 3.12 ± 1.27	3.77 ± 1.16 4.85** ± 0.57	2.90 ± 0.56 3.15 ± 0.51	3.12 ± 0.85 2.24 ± 0.39	3.34 ± 1.09 3.19 ± 0.40
Neutrophils [%] + SD Time point 1 (a) Time point 2 (b)	14.1 ± 0.9 17.3 ± 6.8	17.0 ± 5.3 15.3 ± 6.4	19.9 ± 8.0 10.4** ± 1.9	26.2** ± 4.5 16.5 ± 3.8	17.4 ± 5.0 22.1 ± 7.4	25.0** ± 7.8 18.3 ± 6.0
Lymphocytes [%] + SD Time point 1 (a) Time point 2 (b)	81.6 ± 1.4 78.3 ± 7.1	78.4 ± 5.5 80.5 ± 7.9	76.0 ± 8.5 85.6 ± 2.4	69.3** ± 5.7 79.7 ± 4.4	77.6 ± 4.7 72.9 ± 7.6	70.5* ± 8.3 78.1 ± 5.9

* statistically significant, p ≤ 0.05; ** statistically significant, p ≤ 0.01. 

(a) time point 1 is 3 days after the end of exposure; (b) time point 2 is 24 days after the end of exposure

SD: standard deviation

 

Table 5: Clinical pathology parameters in BALF of the short-term study with 5 days of exposure

	Control	NM-212			NM-211
Target conc. [mg/m3]	0	0.5	5	25	0.5	25
Measured conc. (mg/m3) + SD	0	0.48 ± 0.0	5.2 ± 1.1	25.6 ± 6.0	0.45 ± 0.1	25.8 ± 1.70
BALF cell counts (cn/μL)
Total cells Time point 1 (a) Time point 2 (b)	63.73 ± 1.03 53.09 ± 13.33	69.76 ± 26.15 44.20 ± 2.45	98.24** ± 16.38 43.98 ± 17.70	337.35** ± 125.24 46.71 ± 8.16	71.32 ± 22.69 47.01 ± 11.34	374.98** ± 77.84 81.20 ± 38.87
Neutrophils (PMN) Time point 1 (a) Time point 2 (b)	0.82 ± 0.80 0.69 ± 0.38	3.70** ± 2.53 1.34 ± 2.01	49.19** ± 17.68 5.15 ± 9.91	272.30** ± 106.62 4.63** ± 3.83	4.41* ± 3.38 2.27** ± 0.56	297.21** ± 59.23 25.57** ± 33.95
Lymphocytes Time point 1 (a) Time point 2 (b)	0.22 ± 0.32 0.32 ± 0.33	0.17 ± 0.22 0.11 ± 0.10	1.57* ± 0.97 0.43 ± 0.31	9.20** ± 4.51 1.03 ± 0.95	0.39 ± 0.34 0.51 ± 0.25	19.14** ± 15.13 2.74** ± 2.17
Macrophages Time point 1 (a) Time point 2 (b)	62.65 ± 11.56 52.05 ± 13.00	75.72 ± 15.10 42.32 ± 3.66	46.83 ± 23.88 38.34 ± 11.92	51.21 ± 24.76 40.93 ± 6.31	66.47 ± 22.13 44.23 ± 11.53	52.99 ± 42.89 52.61 ± 15.51
Monocytes Time point 1 (a) Time point 2 (b)	0.04 ± 0.08 0.00 ± 0.00	0.0 ± 0.00 0.00 ± 0.00	0.52* ± 0.37 0.03 ± 0.07	3.50** ± 1.86 0.07 ± 0.11	0.05 ± 0.11 0.00 ± 0.00	4.40** ± 2.68 0.28 ± 0.34
Eosinophils Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.03 ± 0.06	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.03 ± 0.06	0.39 ± 0.54 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00
Atypical cells Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.12 ± 0.17 0.00 ± 0.00	0.76 ± 0.73 0.05 ± 0.07	0.00 ± 0.00 0.00 ± 0.00	1.25* ± 0.85 0.00 ± 0.00
Total protein/enzymes
Total protein (mg/L) Time point 1 (a) Time point 2 (b)	39 ± 15 97 ± 122	47 ± 16 59 ± 28	75* ± 23 51 ± 16	198** ± 52 60* ± 9	44 ± 7 92 ± 91	207** ± 54 82 ± 14
GGT (nkat/L) Time point 1 (a) Time point 2 (b)	20 ± 15 30 ± 7	36 ± 20 28 ± 17	106** ± 26 33 ± 14	125** ± 15 61** ± 16	37 ± 26 25 ± 10	128** ± 16 87** ± 20
LDH (μkat/L) Time point 1 (a) Time point 2 (b)	0.46 ± 0.09 0.36 ± 0.14	0.58 ± 0.17 0.47 ± 0.25	0.89** ± 0.25 25 0.42 ± 0.04	2.17** ± 0.21 0.72** ± 0.20	0.67 0.58 ± 0.40	2.60** ± 0.31 1.08** ± 0.32
ALP (μkat/L) Time point 1 (a) Time point 2 (b)	0.43 ± 0.10	0.53 ± 0.08 1.0	1.31** ± 0.53 1.5*	1.56** ± 0.15 1.9**	0.55* ± 0.09 0.51 ± 0.20	1.56** ± 0.35 0.87** ± 0.12
NAG (nkat/L) Time point 1 (a) Time point 2 (b)	46 ± 9 41 ± 10	47 ± 11 37 ± 12	57 ± 12 39 ± 4	74** ± 7 46 ± 7	51 ± 19 46 ± 7	94** ± 16 59* ± 10
Cell mediators (pg/mL)
MCP-1 Time point 1 (a) Time point 2 (b)	14.7 ± 0.6 48.2 ± 59.1	19.1 ± 5.5 22.8 ± 12.5	101.1** ± 31.3 26.7 ± 10.8	1342.9** ± 530.0 144.1* ± 82.8	17.5* ± 1.6 30.6 ± 26.0	1581.0** ± 771.3 329.2** ± 290.5
CINC-1/IL-8 Time point 1 (a) Time point 2 (b)	59.8 ± 17.7 88.3 ± 25.7	83.4* ± 24.6 87.5 ± 24.7	348.6** ± 185.4 94.3 ± 35.7	322.4** ± 93.9 155.1** ± 13.5	88.3* ± 21.1 99.7 ± 39.4	436.0** ± 228.3 254.3** ± 70.4
M-CSF Time point 1 (a) Time point 2 (b)	41 ± 17 45 ± 19	52 ± 23 48 ± 5	34 ± 25 71* ± 13	91** ± 28 49 ± 3	61* ± 16 51 ± 12	114** ± 48 60 ± 24
Osteopontin Time point 1 (a) Time point 2 (b)	172.28 ± 48.04 320.38 ± 177.64	194.81 ± 131.24 191.47 ± 116.09	301.40 ± 238.96 247.44 ± 189.46	849.04** ± 386.44 495.92 ± 251.93	134.32 ± 82.72 111.92 ± 80.28	1116.02** ± 653.35 398.72 ± 140.88

* Statistically significant, p < 0.05

** Statistically significant, p < 0.01; n = 5; SD standard deviation

(a) Time point 1 is 3 days after the end of exposure

(b) Time point 2 is 24 days after the end of exposure

Conclusions:

Inhaled nano-CeO2 (NM-211 and NM-212) induced loco-regional effects manifested by a significant but transient pulmonary inflammation at 5 and 25 mg/m3. However, no systemic toxicity occurred.

Executive summary:

Keller J et al. (2014) assessed the lung deposition and clearance kinetics as well as the inhalation toxicity of two nanometric cerium dioxide (nano-CeO2) in a short-term study with a 5-day inhalation exposure (i.e., standard short-term inhalation study, STIS), in compliance with GLP. The study was performed, within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials and was funded via the European Commission’s 7th Framework Programme project NanoMILE.

Both nano-CeO2 (NM-211 and NM-212) were extensively characterised (see in the table below). Compared to NM-212, NM-211 had considerably smaller primary particles, larger specific surface, significantly fewer organic contaminations on the surface, and reduced photocatalytic activity.

 

Physico-chemical parameters	Results		Methods
	NM-211	NM-212
Supplier	Antaria	Umicore
Primary particle size	4 to 15 nm with a D50 of 8.2 nm	40 nm	Transmission electron microscopy (TEM)
Particle size distribution	No data 5, 70 nm and ca. 10 µm	3000 to 150 000 nm 35 nm and ca. 7 µm	Scanning EM (SEM) Hg porosimetry
Stability	Agglomeration / aggregation	Agglomeration / aggregation	-
Specific surface area	53 m²/g 33 m²/g	27 m²/g 30 m²/g	Brunauer, Emmett, Teller (BET) Hg porosimetry
Surface charge (zeta potential at pH7)	+16 mV	+42 mV	Electrophoretic mobility
Isoelectric point	pH 8.3	> pH 10 (always cationic)	Electrophoretic mobility
Shape	Globular	Globular	TEM, X-ray diffraction (XRD)
Crystallinity	Cerianite cubic, with a crystalline size of 12.5 nm	Cerianite cubic, with a crystalline size of 40 nm	XRD
Analytical purity	98.4%	99.3%	Thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS)
Impurities	1.6% contaminations, thereof small amounts of nitrates and alkyls found on the particle surface	0.7% organic contaminations, identified as ester + alkyl groups, found mostly on the particle surface (i.e., a very thin and homogeneous layer around the purely inorganic nano-CeO2)	TGA, XPS
Solubility	< 0.001 wt% regardless of the medium in which nanoparticles were suspended	< 0.001 wt% to 0.02% depending on the medium in which nanoparticles were suspended	ICP-MS
Oxidation degree	22% Ce(III) and 78% Ce(IV)	14% Ce(III) and 86% Ce(IV)	XPS
Surface properties (atom %)	O 57.2 Ce 28.7 C 14.1	C 79.9 (C-C 62.6; C-O 7.0; C=O 3.5; COOH 6.9) O 17.7 Ce 2.4	XPS
Description	Yellowish white powder synthesised by precipitation	Yellowish white powder synthesised by precipitation	-
Agglomerate density	0.6 g/m3	2.0 g/m3	Hg porosimetry
D50 and average agglomerate number (AAN), in water	D50 = 2839 nm, AAN = 346	D50 = 432 nm, AAN = 11	Centrifugation
Photocatalytic activity	0.0005 ± 0.0002	0.01 ± 0.005	Photon efficiency

Female Wistar rats (10/group) inhaled nano-CeO2 aerosol at concentrations of 0 (control), 0.5, 5, and 25 mg/m3 by whole-body exposure for 6 h/day on 5 consecutive days with a post-exposure period of 24 days. Control animals were exposed to conditioned air. Biological pulmonary effects of nano-CeO2 were studied by analysis of bronchoalveolar lavage fluid (BALF) and blood, and histopathology of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

With lung burdens being about 0.004 to 0.53 mg/lung after 5 days of exposure, inhaled nano-CeO2 at 0.5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. At aerosol concentrations higher than 0.5 mg/m3, this clearance was impaired resulting in a half-time above 200 days (i.e., at 25 mg/m3). In pathological examinations, an increase of lung weights was observed only in rats exposed to 25 mg/m3 NM-211 for 5 days. By light microscopy, both nano-CeO2 were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. The nanoparticles, either NM-211 or NM-212, were not detected within alveolar epithelial cells. At the lowest concentration of 0.5 mg/m3, the histopathological findings were alveolar histiocytosis and particles, either free or within macrophages; according to the authors, this reflected an expected physiological response.

Substance-related adverse effects after inhalation exposure to both nano-CeO2 were limited to the lung. Thus, after 5 days of nano-CeO2 inhalation, the responses observed were mainly local pulmonary effects. Exposure to 25 mg/m3 for 5 days (retained lung burden of 0.53 mg) resulted in higher absolute and relative neutrophil cell counts in the blood, without an increase in total cell counts for both NM-211 and NM-212. This slight neutrophilia was detected directly after the end of exposure and was no longer present after 3 weeks of post-exposure. Considering the pronounced inflammation in the lung at 25 mg/m3, the neutrophilia in blood was considered to be secondary to the local effects (systemic acute-phase response). No other blood parameters were affected. Furthermore, a whole panel of extrapulmonary organs and tissues was examined histologically as required by OECD test guideline 412. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low extrapulmonary tissue CeO2 concentrations in this study.

Compared to NM-212, NM-211 elicited higher increases in lymphocytes, cell mediators (e.g., MCP-1), and neutrophils in BALF at 25 mg/m3 with a slower recovery during the post-exposure period. Moreover, significant increase in lung weights was noted in animals exposed to 25 mg/m3 NM-211, but not NM-212.

In the present study, the larger specific surface area of NM-211 seemed to contribute to the higher biological activity as compared to NM-212. Thus, the surface area of the particles provided a dose metrics with the best correlation of nano-CeO2 inflammatory responses; hence, the inflammation appeared to be directed by the particle surface rather than mass or volume in the lung.

No NOAEC was defined in the study. However, from the observed results, a NOAEC for local effect (pulmonary tract) could be set at > 0.5 mg/m3 - < 5 mg/m3 and the NOAEC (systemic effect) was > 25 mg/m3 for both tested nano-CeO2.

In conclusion, inhaled nano-CeO2 (NM-211 and NM-212) induced loco-regional effects manifested by a significant but transient pulmonary inflammation at 5 and 25 mg/m3. However, no systemic toxicity occurred.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Several studies, including 2 OECD 412, 1 OECD 413 studies and interim results (13 and 52 weeks) of the 2-year inhalation carcinogenicity study, conducted on nano-CeO2 with various physico-chemical properties were available and used in a weight-of-evidence approach to assess the repeated dose toxicity potential of the test substance. These studies were awarded a reliability score of 1 to 3 (Klimisch, 1997) and were thus considered sufficient for the assessment of nano-CeO2 repeated dose toxicity via inhalation route, therefore the overall quality of the dataset is considered to be acceptable.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was well documented and performed according to the OECD guideline 412 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

The test was performed only with female rats.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610 x 435 x 215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: At the concentration of 0.5 mg/m3: 0.9 µm / 2.5 (MMAD / mean GSD)
At the concentration of 5 mg/m3: 1.9 µm / 2.9 (MMAD / mean GSD)
At the concentration of 25 mg/m3: 2.2 µm / 2.4 (MMAD / mean GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ; The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

25 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest aerosol concentration was 25 mg/m3, which was expected to cause biological effects and should lead to lung overload at least for 20 exposures. The mid and low aerosol concentrations were 5 and 0.5 mg/m3. The low aerosol concentration with an expected lung burden far below the overload condition should not lead to any adverse effects. The mid aerosol concentration, which was spaced 10-fold higher than the low concentration, was expected to cause some biological effects.
- Rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Yes, 129-day post-exposure observation
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations of the animals were recorded for each animal at least three times per day on exposure days and once a day during the pre-exposure and post-exposure periods. Signs and findings were recorded for each animal. During exposure, examination was possible only on a group basis.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0), and twice weekly until killing or twice within the 5 exposure days.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH, Germany))
- Animals fasted: Yes- How many animals: 5 rats per test group
- Parameters checked: Red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell as well as differential blood cell counts with a hematology analyser

CLINICAL CHEMISTRY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Animals fasted: Yes
- How many animals: 5 rats per test group
- Parameters checked (in serum): rat haptoglobin and rat γ2-macroglobulin by ELISA; enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and other blood parameters of clinical chemistry using an automatic analyser

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* BRONCHOALVEOLAR LAVAGE
Twenty-four hours and 35 days after the end of exposure, five animals per test group were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anaesthesia. The lungs of the animals were lavaged in situ twice with saline solution. A total of 11 mL bronchoalveolar lavage fluid (BALF) was obtained per animal for analysis. Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with a hematology analyser. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes, and atypical cells were performed on Wright-stained cytocentrifuge slide preparations. The differential cell count was evaluated manually by counting at least 400 BALF cells per sample. Using a reaction rate analyser, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) were measured.

* INFLAMMATORY MEDIATORS IN BALF AND SERUM
Cytokines and chemokines in BALF and serum were measured. The parameters comprised various cytokines, chemokines, adhesion molecules, matrix metalloproteinases, acute-phase proteins, signal proteins of apoptosis, or cell proliferation: monocyte chemoattractant protein-1 level (MCP-1), cytokine-induced polymorphonuclear neutrophil chemoattractant-1 level (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF) and osteopontin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy and histopathology were performed after 2 and 34 days after the end of exposure (4 weeks of exposure). In general, 5 animals per test group were investigated for pathological examination. However, 10 animals were examined for pathological examination of the respiratory tract and all gross lesions. At necropsy, animals were exsanguinated by opening of the abdominal great vessels under deep pentobarbital anesthesia. All organs were preserved according to OECD TG No. 412. Following organs were weighed: adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, and thyroid glands. The lungs were instilled with and fixed in 10 % neutral-buffered formalin (NBF). Once fixed, the lungs were transferred to 70 % ethanol. All other organs were fixed in 10 % NBF. All the organs and tissues described in the OECD TG No. 412 were trimmed. After paraplast-embedding, the blocks were cut at 2- to 3-μm thickness, mounted on glass slides and stained with hematoxylin and eosin. Extrapulmonary organs and the respiratory tract compromising nasal cavity (four levels), larynx (three levels), trachea (transverse and longitudinal with carina), lung (five lobes), and mediastinal and tracheobronchial lymph nodes were assessed by light microscopy. For the lungs, whole histopathological examination was performed in animals of all test groups. For all other tissues, only the animals of the control and high concentration group of nano-CeO2 were initially examined. When changes were observed in the high concentration group, respective organs and tissues of the animals exposed to low and intermediate aerosol concentrations were also examined by light microscopy. All histopathological examinations were performed by a well-experienced board-certified veterinarian toxicopathologist followed by an internal pathology peer review.

Other examinations:

ORGAN BURDEN
Cerium (Ce) content was determined at 7 time points over 129 days of post-exposure period. Ce content in the lungs, lung-associated lymph nodes, and liver of either 3 or 5 animals per test group were examined. 1 and 35 days after the end of exposure, the lavaged lungs and aliquots of BALF of 5 animals per group were used for the determination of lung burden. This examination method likely caused a loss of the test material during preparation and handling of the lungs. Furthermore, lung burdens were measured 2 days after the end of exposure using the left half lungs of 5 animals/test group, only. On the basis of the availability of total lung weights, lung burdens were calculated up from the half lung burden values with the corresponding weight of the half lungs. Lung burden of the remaining time points was determined using the whole (not lavaged) lung.
After digestion with mixed acid, samples of each lung or lymph node were dissolved in sulphuric acid and ammonium sulphate. 140Ce content in the obtained solution was analysed by inductively coupled plasma mass spectrometry (ICP-MS) or by inductively coupled plasma optical emission spectrometry (ICP-OES) with a wavelength of 419 nm. The limit of detection for Ce was 0.3 μg. The amounts of CeO2 in the respective tissues were calculated by measuring elemental Ce with ICP-MS.

Statistics:

For body weight changes, Dunnett’s test was used for a comparison of each test group with the control group test. Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analysed by non-parametric one-way analysis using the Kruskal–Wallis test (two-sided). If the resulting p value was ≤ 0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by non-parametric one-way analysis using the two-sided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

the animals exposed for 4 weeks to NM-212 showed no clinical signs or findings compared to the control animals [data not shown]

Mortality:

no mortality observed

Description (incidence):

the animals exposed for 4 weeks to NM-212 showed no clinical signs or findings compared to the control animals [data not shown]

Body weight and weight changes:

no effects observed

Description (incidence and severity):

4 weeks of inhalation exposure to NM-212 did not affect the body weight development of the animals [data not shown]

Food consumption and compound intake (if feeding study):

not specified

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):

haematological parameters were not affected in rats exposed to NM-212 [see Table 4 in "any other information on results" below]

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

clinical chemistry parameters and acute phase protein levels were not affected in rats exposed to NM-212 [data not shown]

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative lung weights were significantly increased at aerosol concentrations of 25 mg/m3 NM-212 (+30 and 29 %, respectively) 2 days after the end of the exposure and were still significantly elevated (+16 and 20 %) 34 days after the end of the exposure. Two days after the end of the exposure, absolute and relative lung weights of animals exposed to 5 mg/m3 were increased significantly by +13 and 10 %, respectively. They returned to control levels within the following 34 days. No effects on organ weights were observed after inhalation of 0.5 mg/m3.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two days after the end of exposure, mediastinal lymph nodes of 2 animals (out of 10) were enlarged at aerosol concentrations of 25 mg/m3 NM-212. Thirty four days after the end of exposure, the incidence of animals with enlarged, yellow white-coloured mediastinal lymph nodes increased from 2 to 8 (out of 10) per group at aerosol concentrations of 25 mg/m3 NM-212. Mediastinal lymph nodes of animals exposed to 5 mg/m3 were firstly enlarged 34 days after the end of exposure (2/10 rats). All other extrapulmonary organs including the tracheobronchial lymph nodes revealed no macroscopical findings after inhalation exposure to nano-CeO2.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

* LUNGS
Two days after the end of the exposure, alveolar macrophages were observed in the lungs of all NM-212-exposed animals, regardless of the aerosol concentration. Eosinophilic granular material and small particles were distributed in the alveoli of all animals exposed to 5 and 25 mg/m3 NM-212. The occurrence of alveolar histiocytosis and of eosinophilic granular material was correlated with increased lung weights in animals exposed to 5 and 25 mg/m3 NM-212. In bronchus-associated lymphoid tissue (BALT), single, small macrophage aggregates with particles occurred in 5/10 animals exposed to 25 mg/m3 NM-212 but none was seen in lower doses. In addition, single or a few amber-like coloured particles occurred in BALT, in the extracellular compartment, without any macrophage activation at aerosol concentrations of 5 (8/10 rats) and 25 mg/m3 (5/10 rats). Thirty four days after the end of exposure, alveolar histiocytosis and eosinophilic granular material with particles were still observed at concentrations of 5 and 25 mg/m3 NM-212, but a tendency towards regression could be seen for the parameter "eosinophilic granular material with particles", notably at 5 mg/m3 (2/10 rats). At 0.5 mg/m3, in contrast, amber-like coloured particles could only be noted within single histiocyte (10/10 rats). In 1 animal of 5 mg/m3 and in 5 out of 10 animals exposed to 25 mg/m3 NM-212, a multifocal granulomatous inflammation appeared 129 days after the end of exposure. In BALT, single or few amber-coloured particles at 0.5, 5, and 25 mg/m3 NM-212 as well as an increasing number of animals with macrophage aggregates with particles at 5 and 25 mg/m3 were still observed. All compound-related findings after exposure of 5 and 25 mg/m3 NM-212 were correlated with increased lung weights in these test groups.

* LUNG-ASSOCIATED LYMPH NODES
Two days after the end of exposure to 25 mg/m3 NM-212, multifocal macrophage aggregates with particles were observed in the mediastinal (4/10 rats) as well as in the tracheobronchial lymph nodes (8/10 rats). A lympho-reticulocellular hyperplasia was present in both lymph nodes, mostly seen in animals from the group of 25 mg/m3 (4 to 7/10 rats). The hyperplasia of the mediastinal lymph nodes was correlated with their corresponding macroscopic enlargement after exposure to 25 mg/m3 NM-212. Thirty four days after the end of exposure, the number of animals with macrophage aggregates (incidence and grading) in both lymph nodes was higher compared to the animals examined 2 days after the end of the exposure. Nearly all other findings were still present 34 days after the end of the exposure.

* UPPER RESPIRATORY TRACT (data not shown)
Two days after the end of the exposure to 5 and 25 mg/m3 NM-212, amber-like coloured particles occurred similarly in the dorsal area of the larynx (level III). Animals exposed to 25 mg/m3 showed particles in the carina of the trachea. At aerosol concentrations of 25 mg/m3 NM-212, single amber-like coloured particles were firstly found in the nasal-associated lymphoid tissue (NALT), inside single macrophages or in the extracellular compartment. These findings were still present 34 days after the end of exposure.

* EXTRAPULMONARY ORGANS (data not shown)
Histological examination of extrapulmonary organs (e.g., liver, spleen, and kidneys) did not show any substance-related morphological changes in animals exposed to 0.5, 5, and 25 mg/m3 NM-212 in this short-term study with 4 weeks of exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

OTHER:
BRONCHOALVEOLAR LAVAGE / INFLAMMATORY MEDIATORS IN BALF AND SERUM
Four weeks of inhalation exposure to 5 and 25 mg/m3 NM-212 resulted in an increase in total cells in BALF due to increases in polymorph nuclear neutrophils, lymphocytes, and monocytes in BALF (see Table 5 in "any other information on results" below). Consistent with these findings, several other parameters including the examined cell mediators were increased. Thirty five days after the end of exposure, some of the BALF parameters returned to control levels, whereas several of them were still significantly increased at 5 and 25 mg/m3 (e.g., total cells, lymphocytes, neutrophils; GGT, LDH, ALP; MCP-1, CINC-1). Except for a significant increase in total protein levels in BALF, no other clinical pathology parameters displayed significant change in animals exposed to 0.5 mg/m3 NM-212.

ORGAN BURDEN
Inhalation exposure of 0.5, 5, or 25 mg/m3 NM-212 resulted in mean lung burdens of 0.04, 0.52, or 2.62 mg 1 day after the end of exposure. Two days after the end of exposure, higher lung burdens of the left lungs were measured but these data were disregarded for half-time calculations. At aerosol concentrations of 0.5 mg/m3 NM-212, a retention half-time of 40 days was determined. Higher aerosol concentrations of 25 mg/m3 NM-212 resulted, however, in a much longer half-time above 200 days. The Ce burden in the lung-associated lymph nodes (tracheobronchial and mediastinal lymph nodes) was 10 μg, 3 days after the end of the exposure to 25 mg/m3 NM-212 and increased to 350 μg, 129 days after the end of exposure. After the exposure to 25 mg/m3 NM-212, Ce was also detected in the liver (1.56 and 1.93 μg) 3 and 65 days after the end of the exposure, respectively.

Dose descriptor:

other: NOAEC (systemic)

Effect level:

> 25 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: - No NOAEC was determined in the publication, thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects

Dose descriptor:

other: NOAEC (local)

Effect level:

> 0.5 - < 5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

other: Mediastinal and tracheo-bronchial lymph nodes.

Treatment related:

yes

Dose response relationship:

yes

Table 4: Mean clinical pathology parameters in blood after exposure to nano-CeO2

	Control	NM-212
Target conc. [mg/m3]	0	0.5	5	25
Measured conc. (mg/m³) + SD	0	0.5 ± 0.2	5.3 ± 0.9	25.9 ± 6.0
Blood cells
Neutrophils [giga/L] + SD Time point 1 (a) Time point 2 (b)	1.19 ± 0.95 0.72 ± 0.26	0.73 ± 0.24 0.71 ± 0.24	0.83 ± 0.33 0.85 ± 0.17	1.18 ± 0.32 1.20 ± 0.37
Lymphocytes [giga/L] + SD Time point 1 (a) Time point 2 (b)	3.67 ± 1.13 2.86 ± 0.73	3.47 ± 0.72 3.65 ± 1.14	3.01 ± 0.53 2.95 ± 0.88	2.65 ± 0.62 3.26 ± 0.63
Neutrophils [%] + SD Time point 1 (a) Time point 2 (b)	22.2 ± 14.5 19.4 ± 7.8	16.4 ± 3.8 15.9 ± 5.2	20.0 ± 4.4 21.8 ± 3.6	29.9 ± 6.2 25.6 ± 3.9
Lymphocytes [%] + SD Time point 1 (a) Time point 2 (b)	72.3 ± 14.2 75.7 ± 8.6	78.2 ± 4.3 79.3 ± 6.6	75.2 ± 5.5 73.4 ± 3.6	66.1 ± 5.8 70.2 ± 4.0

* statistically significant, p ≤ 0.05; ** statistically significant, p ≤ 0.01. 

(a) time point 1 is 2 days after the end of exposure; (b) time point 2 is 35 days after the end of exposure

SD: standard deviation

Table 5: Clinical pathology parameters in BALF of the short-term study with 4 weeks of exposure

	Control	NM-212
Target conc. [mg/m3]	0	0.5	5	25
Measured conc. (mg/m³) + SD	0	0.5 ± 0.2	5.3 ± 0.9	25.9 ± 6.0
BALF cell counts (cn/μL)
Total cells Time point 1 (a) Time point 2 (b)	76.42 ± 23.97 75.29 ± 14.10	75.10 ± 21.51 62.23 ± 11.59	133.44* ± 48.40 97.44 ± 34.23	296.90** ± 124.92 220.50** ± 105.27
Neutrophils (PMN) Time point 1 (a) Time point 2 (b)	0.85 ± 0.35 2.44 ± 1.01	1.75 ± 1.13 2.52 ± 1.37	65.66** ± 50.23 41.70** ± 22.0	222.29** ± 99.25 161.69** ± 87.82
Lymphocytes Time point 1 (a) Time point 2 (b)	0.55 ± 0.50 1.65 ± 1.13	0.63 ± 0.74 0.77 ± 0.49	5.41** ± 3.21 7.43* ± 9.02	8.93** ± 6.04 10.89** ± 3.48
Macrophages Time point 1 (a) Time point 2 (b)	74.94 ± 23. 79 71.14 ± 13.31	72.71 ± 21.16 58.78 ± 11.04	60.25 ± 20.82 47.58 ± 16.48	59.32 ± 26.28 45.17 ± 25.33
Monocytes Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.06 ± 0.09	0.00 ± 0.00 0.04 ± 0.08	1.65* ± 1.90 0.68* ± 0.43	3.95* ± 4.65 2.46* ± 3.03
Eosinophils Time point 1 (a) Time point 2 (b)	0.09 ± 0.12 0.00 ± 0.00	0.00 ± 0.00 0.12 ± 0.11	0.00 ± 0.00 0.05 ± 0.11	0.34 ± 0.49 0.00 ± 0.00
Atypical cells Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.48 ± 0.72 0.00 ± 0.00	2.07* ± 1.71 0.29 ± 0.40
Total protein/enzymes
Total protein (mg/L) Time point 1 (a) Time point 2 (b)	60 ± 4 81 ± 23	83** ± 5 60 ± 22	94** ± 21 98 ± 49	245** ± 77 175** ± 98
GGT (nkat/L) Time point 1 (a) Time point 2 (b)	37 ± 17 42 ± 12	51 ± 11 44 ± 20	111** ± 21 83** ± 30	149** ± 31 123** ± 30
LDH (μkat/L) Time point 1 (a) Time point 2 (b)	0.51 ± 0.18 0.58 ± 0.08	0.55 ± 0.15 0.50 ± 0.20	1.08** ± 0.37 0.84* ± 0.22	2.28** ± 0.52 1.88** ± 1.20
ALP (μkat/L) Time point 1 (a) Time point 2 (b)	0.83 ± 0.16 0.70 ± 0.09	0.84 ± 0.35 0.67 ± 0.08	1.16** ± 0.10 1.05** ± 0.16	1.53** ± 0.21 1.09** ± 0.25
NAG (nkat/L) Time point 1 (a) Time point 2 (b)	45 ± 5 47 ± 8	55 ± 15 38 ± 6	53* ± 8 47 ± 7	86* ± 26 71 ± 35
Cell mediators (pg/mL)
MCP-1 Time point 1 (a) Time point 2 (b)	14.0 ± 0.0 17.3 ± 2.6	19.6 ± 11.0 15.4 ± 3.1	559.4** ± 444.4 492.5** ± 553.1	3587.2** ± 281.0 1854.2** ± 1184.0
CINC-1/IL-8 Time point 1 (a) Time point 2 (b)	104.2 ± 26.7 158.8 ± 38.1	103.8 ± 14.0 133.9 ± 45.3	506.7** ± 195.9 449.4** ± 226.7	1190.9** ± 294.9 831.0** ± 497.1
M-CSF Time point 1 (a) Time point 2 (b)	26 ± 17 46 ± 26	22 ± 12 55 ± 29	27 ± 18 41 ± 14	48 ± 29 53 ± 12
Osteopontin Time point 1 (a) Time point 2 (b)	391.44 ± 187.39 337.36 ± 282.91	288.80 ± 110.90 284.40 ± 292.66	755.44* ± 206.21 1003.18* ± 434.20	592.14 ± 336.47 838.48 ± 529.45

* Statistically significant, p < 0.05

** Statistically significant, p < 0.01; n = 5; SD standard deviation

(a) Time point 1 is 1 day after the end of exposure

(b) Time point 2 is 35 days after the end of exposure

Conclusions:

Inhaled nano-CeO2 (NM-212) induced loco-regional effects manifested by a significant pulmonary inflammation and granulomatous alterations of the lung, mainly at 25 mg/m3. However, no systemic toxicity occurred. The inflammatory responses observed were typical of poorly soluble particles.

Executive summary:

Keller J et al. (2014) assessed the lung deposition and clearance kinetics as well as the inhalation toxicity of nanometric cerium dioxide (nano-CeO2) in a subacute toxicity study with 4 weeks of inhalation exposure, according to OECD guideline 412 and in compliance with GLP.

A commercial nano-CeO2 (NM-212), from Umicore, of 40 nm was used in this study. These globular nanoparticles agglomerated and aggregated (3 - 150 µm). Moreover, the crystalline nano-CeO2 displayed a specific surface area of 27 to 30 m²/g, a zeta potential of +42 mV (at pH 7), an isoelectric point greater than pH 10, an extremely low solubility (Ce < 0.001 to 0.02 wt%) and a significant photocatalytic activity. The oxidation degree of NM-212 was determined: 14% Ce(III) and 86% Ce(IV). The nano-CeO2 MMAD ranged between 0.9 and 2.2 µm depending on the concentration tested. Although nano-CeO2 was described as pure (99.3%) and uncoated, the surface chemistry showed that organic contaminants (ester and alkyl groups) were present at 0.7% on nano-CeO2 surface.

Female Wistar rats (10/group) were administered NM-212, by whole-body inhalation exposure, at aerosol concentrations of 0 (control), 0.5, 5, and 25 mg/m3  for 6 h/day on 5 consecutive days/week for 4 weeks with a post-exposure period of 129 days. Control animals were exposed to conditioned air. Pulmonary responses were studied by analysing the bronchoalveolar lavage fluid (BALF) and blood, and by performing a histopathological analysis of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

According to the authors, a 28-day inhalation exposure to nano-CeO2 NM-212 substance-related adverse effects was limited to the lung. No clinical sign and no effect in the body weight development were observed in the treated animals as compared to controls. No altered blood parameter could be detected after 4 weeks of inhalation exposure. A whole panel of extrapulmonary organs and tissues was examined histologically and very low cerium contents were detected in the liver at two time points and at the concentration of 25 mg/m3 NM-212 (which is a general finding for inhaled nanoparticles) without any related morphological abnormalities. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low CeO2 concentrations found in extrapulmonary tissues.

An aerosol concentration of 0.5 mg/m3 did not cause inflammatory response in the lung. With a lung burden of 41 μg/lung after 4 weeks of exposure, inhaled NM-212 at 0. 5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. This was in the range of physiological retention half-times of poorly soluble particles being between 60 and 70 days. Higher aerosol concentration impaired this clearance: an aerosol concentration of 25 mg/m3 elicited a lung burden of 2.62 mg/lung resulting in a retarded retention half-time above 200 days. At the mid concentration of 5 mg/m3, the lung burdens at 3 time points indicated a retarded retention half-time as the lung burden (0.5 mg/lung) stayed at a constant level during 4 weeks. The lung burden decreased from 2.62 to 1.8 mg, 129 days after the exposure to 25 mg/m3 for 4 weeks. In pathology, lung weights were found increased following exposures to 5 and 25 mg/m3 NM-212. By light microscopy, CeO2 particles were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. CeO2 was not detected within alveolar epithelial cells. Moreover, alveolar histiocytosis was observed and had even progressed to a multifocal granulomatous inflammation within 4 weeks after the end of exposure to 5 and 25 mg/m3 nano-CeO2. The combination of moderate alveolar histiocytosis with particles and the presence of eosinophilic material, potentially precursors of granulomatous inflammation, were considered to be adverse. In contrast, at 0.5 mg/m3, the histopathological findings (i.e., alveolar histiocytosis and particles, either free or within macrophages) reflected an expected physiological response.

Nano-CeO2-related adverse effects were limited to the lung after inhalation exposure. The reported pulmonary inflammation was assessed by the changes in BALF parameters (e.g., neutrophils and proinflammatory cytokines) and histopathological findings (alveolar histiocytosis and granulomatous inflammation). Pulmonary inflammation was only observed at concentrations of 5 and 25 mg/m3. However, BALF parameters showed a regression during the post-exposure period. Histopathological findings, in contrast, progressed to granulomatous inflammation after the end of exposure at concentrations of 5 mg/m3 nano-CeO2 and above. It has to be noted that the inflammatory response in lungs based on the increase in neutrophil counts in BALF was lower after 4 weeks compared to 5 days of exposure (see in the previous ESR). The decay in neutrophil numbers after 4 weeks was by far slower than after 5 days, suggesting that inflammation developing at lower dose rate was longer lasting and more persistent.

A smaller fraction of the particles was transferred to the lung-associated lymph nodes. Indeed, in lung-associated lymph nodes, 0.35 mg CeO2 was found 129 days after exposure to 25 mg/m3 NM-212 for 4 weeks. Moderate macrophage aggregates with particles in the lung-associated lymph nodes, combined with lympho-reticulocellular hyperplasia, were considered to be adverse. Demonstrated by histological evaluations and confirmed by measured Ce lymph node burdens, the lymphatic clearance of inhaled CeO2 via the lymphatic vessels from the pulmonary region to the lung-associated lymph nodes was around 13% of the initial retained burden after the end of the exposure. Most of the CeO2 nanoparticles were presumably cleared by mucociliary clearance and subsequent faecal excretion, which was not evaluated in this work.

According to the authors, the mass lung burden of 41 μg, achieved at 0.5 mg/m3, was well below the overload threshold proposed by Morrow, while the lung burden of 2.62 mg, achieved at 25 mg/m3, was above it (Morrow, 1988). At the highest concentration, a strong pulmonary inflammation was apparent. The mid concentration of 5 mg/m3 NM-212 elicited pulmonary inflammation at a constant lung burden of around 0.52 mg, which was slightly below or at the border of the overload threshold. Based on calculations, volumetric lung overload could only be assumed after 4 weeks exposure to 25 mg/m3. Impaired lung clearance (which is one of the consequences of lung overload conditions) was, however, already observed after inhalation of 5 mg/m3 CeO2. And thus, the concentration of 5 mg/m3 was the lowest aerosol concentration at which the early as well as the later inflammation response was observed, even though lung burdens were different at the onset of the 2 phases.

Based on the results of this study, the NOAEC for systemic toxicity is set as > 25 mg/m3 and the NOAEC for local toxicity (pulmonary tract) can be established at > 0.5 mg/m3 - < 5 mg/m3.

In conclusion, inhaled nano-CeO2 (NM-212) induced loco-regional effects manifested by a significant pulmonary inflammation and granulomatous alterations of the lung, mainly at 25 mg/m3. However, no systemic toxicity occurred. The inflammatory responses observed were typical of poorly soluble particles. Moreover, it has to be noted that both concentrations which induced pulmonary effects corresponded to or were at the limit of overload conditions. Thus, the biological responses observed might be specific to the rat and could not be extrapolated to human. 

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Interim results (13 and 52 weeks) of the 2-year inhalation carcinogenicity study are reported in this rubust study summary. When the full report of the carcinogenicity study will be available, this summary will maybe need to be revised.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Interim results of the long-term study after 13 and 52 weeks of inhalation exposure to nano-
CeO2 are presented in this summary

Deviations:

yes

Remarks:

Only female rats were used in the study.

Principles of method if other than guideline:

The results reported in this summary are interim results of the 2-year study where some parameters have been evaluated after 13 and 52 weeks of exposure to CeO2 NM-212. Any further information that would become available after the release of the carcinogenicity study will be added to this summary.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610 x 435 x 215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

At the concentration of 0.1 mg/m3: 2.3 µm / 2.4 (MMAD / GSD)
At the concentration of 0.3 mg/m3: 1.7 µm / 2.3 (MMAD / GSD)
At the concentration of 1 mg/m3: 1.5 µm / 2.3 (MMAD / GSD)
At the concentration of 3 mg/m3: 1.4 µm / 2.1 (MMAD / GSD)

Presented mean values of > 14 measurements (MMAD, GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.
The target concentrations were met and maintained well throughout the studies. Particle size distribution demonstrated that all particles were in
the respirable range for rats. The particles were largely agglomerated.

Duration of treatment / exposure:

13 and 52 weeks

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0.1 mg/m³ air (nominal)

Dose / conc.:

0.1 mg/m³ air (analytical)

Remarks:

0.1 +/- 0.1 mg/m3 air

Dose / conc.:

0.3 mg/m³ air (nominal)

Dose / conc.:

0.3 mg/m³ air (analytical)

Remarks:

0.3 +/- 0.1 mg/m3

Dose / conc.:

1 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (analytical)

Remarks:

1.0 +/- 0.1 mg/m3

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

3.0 +/- 0.4 mg/m3

No. of animals per sex per dose:

5 animals/group examined for the lung and lymph node burdens, bronchoalveolar lavage analyses and hematology and acute phase proteins in blood.
10 animals/group examined for histopathological analyses.

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the lung burdens and toxicological findings after short-term exposure (see Keller and al., 2014(a) & (b)), aerosol concentrations of 0.1, 0.3, 1 and 3 mg/m³ nano-CeO2 were selected for a long-term inhalation study with up to two years of exposure. The long-term inhalation study with nano-CeO2 (NM-2012) was initiated in 2013 and performed within the framework of the EU project “NanoREG” (Teunenbroek et al. 2013). The study was performed according to OECD test guideline No. 453, under GLP and with a relevant aerosol concentration range (OECD 2009).
The results reported in this summary are interim results of the 2-year study where the following parameters have been evaluated after 13 and 52 weeks of exposure to CeO2 NM-212:
- Organ burden
- BALF cytology, cell mediators, protein and enzyme activities
- Hemtology according to OECD TG 412; acute phase proteins

Observations and examinations performed and frequency:

see below (Other examinations)

Sacrifice and pathology:

see below (Other examinations)

Other examinations:

LUNG AND LYMPH NODE BURDENS:
Directly after 13 and 52 weeks of exposure, the lavaged lungs, aliquots of lavage fluids and tracheobronchial and mediastinal lymph nodes of five animals per group were used to determine lung burden.
Each tissue sample was dried and sulfuric acid was added. The sample was then washed and acid was vaporized at 500°C for 15 min. Sulfuric and nitric acid were added to the residue. Then a mixture of nitric acid, sulfuric acid and perchloric acid ( 2:1:1 v/v/v) was added and the solution was heated to oxidize organic matter. After evaporation, the residue was dissolved in concentrated sulfuric acid. The resulting solution was analyzed for Ce content by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7500C (Agilent, Frankfurt, Germany). The limit of detection for Ce is 0.3 μg per tissue sample.

BRONCHOALVEOLAR LAVAGE
Bronchoalveolar lavage fluids were assessed for 5 animals per group after 13 and 52 weeks of exposure. The animals were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anesthesia. The lungs of the animals were lavaged in situ twice with 6 mL (22 mL/ kg body weight) of 9 % (w/v) saline solution.
- Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with an ADVIA® 120 (Siemens Diagnostics, Fernwald, Germany) hematology analyzer. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes were performed on Wright-stained cytocentrifuge slide preparations (Warheit and Hartsky 1993). The differential cell count was evaluated manually by counting at least 400 BALF cells per sample.
- Using a Hitachi 917 (Roche Diagnostics, Mannheim, Germany) reaction rate analyzer, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β- glucosaminidase (NAG) were measured. and acute phase proteins haptoglobin (HAPT) and alpha 2-macroglobin (A2M).
- Cytokines and chemokines in BALF and serum were measured at Rules-based Medicine Inc., Austin, TX, USA, with xMAP technology (Luminex Corp., Austin, TX, USA) using ELISA methods.The measured parameters comprised rat monocyte chemoattractant protein-1 (MCP-1), rat cytokine-induced polymorphonuclear neutrophil chemoattractant-1 (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF), rodent osteopontin.

HEMATOLOGY AND ACUTE PHASE PROTEINS IN BLOOD
Blood sampling of five fasted rats per test group was performed in the morning by retro-orbital venous plexus puncture under isoflurane anesthesia. Neutrophils counts was measured using a hematology analyzer (ADIVA) and the and the acute phase proteins haptoglobin (HAPT) and alpha 2-macroglobin (A2M) were determined in the serum using Elisa methods.

HISTOPATHOLOGY INVESTIGATIONS
Following sacrifice of the animals after 12 months of exposure, all organs and tissues were fixed and stored in 4% buffered formaldehyde. The lungs were transferred to 70% ethanol following a 24-48h fixation time in formaldehyde. All organs/tissues according to Table 1 (any other information on materials and methods) were trimmed, dehydrated, embedded in paraffin wax and sectioned at a nominal thickness of 3-4 µm. Bones were decalcified prior to trimming. All sections were stained with hematoxylin and eosin (H&E). An additional section of the left lung lobe from all animals was stained with Masson trichrome for assessment of fibrotic changes. Histologic alterations were described, wherever possible, according to their distribution (focal, multifocal, diffuse), severity (grades) and morphologic character. The grades were used for a grading system that takes into consideration either the severity or the number or the size of a microscopic finding (see Table 2, any other information on materials and methods). The severity of each lesion was graded on a scale of very slight to very severe, indicating the approximate fraction of the organ/tissue or organ structure to be involved.

Statistics:

Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analyzed by nonparametric one-way analysis using the Kruskal–Wallis test (twosided). If the resulting p value was ≤0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by nonparametric one-way analysis using the twosided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.
The statistical analysis of the histopathology data was performed with the Provantis system using a Chi-squared and 2-sided Fisher's Exact test.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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):

Neutrophils counts was measured in blood of 5 animals per groups 13 and 52 weeks after expsoure to nanoCeO2 MN-212. No effect of treatment was seen on mean neutrophil levels at any tested concentration and time points (see Table 3 in section: Any information on results including table).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The acute phase proteins haptoglobin (HAPT) and α2-macroglobulin (A2M) were determined in the serum of 5 rats after expsoure to nanoCeO2 NM-212.
Thirteen and 52 weeks of inhalation exposure to nano-CeO2 did not affect clinical chemistry parameters in blood at any tested concentration apart from increased HAPT and A2M levels in serum after 52 weeks of exposure to 3 mg/m³ CeO2 (see Table 3 in section: Any information on results including table). A dose-dependent incease was observed for A2M after 52 weeks of exposure using the median values rather than the means (median values of 10.94 and 11.64 for 1 and 3 mg/m³, respectively). After 13 weeks, the acute phase protein A2M was only significantly increased at 1 mg/m³, but had returned to near control values by 52 weeks. The change was not dose-dependent and regarded as incidental (see Table 3 in section: Any information on results including table).

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MACROSCOPIC FINDINGS
After 12 months of exposure to CeO2, the animals were sacrificed and the organs examined for gross lesions. The tracheobronchial and mediastinal lymphnodes of all 10 animals of the highest test concentration (3 mg/m³) revealed a white-beige to white-yellow discoloration and were moderately enlarged. The same findings were observed in 9 (discoloration) and 3 animals (enlarged) respectively, of the 1 mg/m3 test group. Few animals at test concentrations of 0.1, 1.0 and 3.0 mg/m³ showed a single focus in the lungs.

All other findings were single observations or equally distributed over the test groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY OF THE RESPIRATORY TRACT
Exposure-related microscopic changes were exclusively observed in the respiratory tract and included re-active/adaptive changes such as accumulation of particle-laden macrophages in the nasal cavity, larynx, lungs, tracheo- bronchial and mediastinal lymph nodes.

Nasal cavity:
In the nasal cavity, the incidence of age-related intra-epithelial eosinophilic globules was increased in the 3 mg/m³ high-dose CeO2 exposure group as compared to the control group and associated with minimal inflammatory cell infiltration.
Indeed, the presence of (multi)focal intracytoplasmic eosinophilic globules within the olfactory epithelium was increased in incidence and grade in the 3 mg/m3 CeO2 exposure group (5/10 very slight, 3/10 slight, 1/10 moderate) as compared to the 1 mg/m3 CeO2 exposure group (3/10 very slight, 1/10 slight), the 0.3 mg/m3 CeO2 exposure group (1/10 very slight, 1/10 slight), the 0.1 mg/m3 CeO2 exposure group (2/10 very slight, 1/10 slight) and the clean air control 5/10 very slight, 1/10 slight). A similar trend was observed for eosinophilic globules in the respiratory epithelium: The incidence in the control group was 5/10 (all very slight), in the 0.1 mg/m3 CeO2 exposure group 3/10 (all very slight), in the 0.3 mg/m3 CeO2 exposure group 1/10 (very slight) and in the 1 mg/m3 CeO2 exposure group 3/10 (all very slight) whereas 9/10 (7/10 very slight, 2/10 slight) females in 3 mg/m3 CeO2 exposure group were affected.
Although the difference between the control and CeO2 high-dose test group was statistically not significant, the increase in incidence and severity of this change in both types of epithelium is considered to be exposure-related.
The same is true for (multi)focal very slight subepithelial (mixed) inflammatory cell infiltration which occurred in 3/10, 3/10, 2/10, 4/10 and 7/10 females of group control, 0.1mg/m3 CeO2, 0.3 mg/m3 CeO2, 1mg/m3 CeO2 and 3 mg/m3 CeO2, respectively.

Further exposure-related findings such as (multi)focal very slight accumulation of particle-laden macrophages within the NALT (nasal mucosa-associated lymphoid tissue) were diagnosed in 1/10, 0/10, 4/10 and 10/10 animals of test group 0.1mg/m3, 0.3 mg/m3, 1mg/m3 and 3 mg/m3 CeO2, respectively. Moreover, multifocal very slight amounts of intraepithelial (intracytoplasmic) particles were observed in all animals of the 3 mg/m3 CeO2 group. Occasional particles were seen also in epithelial cells of the submucosal glands (Bowman’s glands).

Incidental findings in the nasal cavity which were considered to be unrelated to particle exposure included dilatation of submucosal glands, mucous cell hyperplasia, subepithelial mononuclear cell infiltration and subepithelial mineralization and were seen in up to 3/10 animals in all test groups.

Larynx:
In 4/10 animals of the 3 mg/m3 CeO2 exposure test group, (multi)focal subepithelial accumulation of particle-laden macrophages (3/10 very slight, 1/10 slight) was observed.

Spontaneous findings included very slight to slight subepithelial mononuclear cell infiltration in up to 5/10 animals as well as very slight to slight dilatation of submucosal glands in 2/10 females of groups exposed to 0.1mg/m3, 0.3 mg/m3 and 3 mg/m3 CeO2.

Lungs:
The adverse and non-adverse histopathological findings observed in the lungs are summarized in Table 6 and 7 (See section: Any information on results including table). Non-adverse findings consisted of accumulation of particle-laden macrophages in the alveolar/interstitial areas and in the BALT as well as particle-laden syncytial giant cells in the BALT. In addition, bronchiolo-alveolar hyperplasia of the bronchiolar type graded no more than “very slight” (grade 1) or “slight” (grade 2) was considered as a non-adverse finding. Adverse effects in the lungs included dose-dependent alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation and interstitial fibrosis, alveolar lipoproteinosis and cholesterol granuloma(s). Except alveolar lipoproteinosis and cholesterol granuloma(s), all changes were seen at dose-dependent incidences and severity grades in all CeO2 exposure test groups.

Reactive/adapatative (= non adverse) pulmonary finfings:
(Multi)focal alveolar/interstitial accumulation of particle-laden macrophages was observed dose-dependently in 10/10 females each of the 0.1 mg/m3 (8/10 very slight, 2/10 slight), 0.3 mg/m3 (8/10 very slight, 2/10 slight), 1 mg/m3 (1/10 very slight, 9/10 slight) and 3 mg/m3 (7/10 slight, 3/10 moderate) CeO2 groups. Deposits of particle-laden macrophages were present not only in alveoli but also in interstitial (intraseptal, peribronchiolar and perivascular) compartments. In addition, agglomerates of CeO2 particles were lying freely within alveoli at very slight to slight degrees in 3/10 animals of 0,1 mg/m3 group and in 10/10 females each of 0,3-3 mg/m3 CeO2 groups. (Multi)focal aggregates of particle-laden macrophages were also observed dose-dependently within the bronchus-associated lymphoid tissue (BALT) at incidences of 10/10 each in 0.1 mg/m3 (all very slight), 0.3 mg/m3 (8/10 very slight, 2/10 slight), 1 mg/m3 (8/10 slight, 2/10 moderate) and 3 mg/m3 CeO2 groups (1/10 slight, 7/10 moderate, 2/10 severe). Syncytial giant cells - mainly particle-laden - were present in the BALT of 3/10, 9/10 and 10/10 females of test groups 0.3, 1 and 3 mg/m3 CeO2 groups, respectively. The amount of the intracellular particle-load in both single-nucleated macrophages and multinucleated giant cells corresponded well to the used CeO2 exposure dose.
(Multi)focal bronchiolo-alveolar hyperplasia of the bronchiolar type (Synonym: alveolar bron-chiolization) was observed in a single animal of the 0,1 mg/m3 CeO2 group (very slight) and in 2/10 (all very slight), 10/10 (all very slight) and 10/10 (9/10 very slight, 1/10 slight) females of the 0,3, 1 and 3 mg/m3 CeO2 groups, respectively.

Adverse changes:
(Multi)focal alveolar/interstitial (mixed) inflammatory cell infiltration occurred in a single control animal (very slight) as a spontaneous finding, in 4/10 females at 0.1 mg/m³ (all very slight) and in 10/10 animals each at 0.3 mg/m³ (9/10 very slight, 1/10 slight), 1 mg/m³ (7/10 very slight, 3/10 slight) and 3 mg/m³ (4/10 very slight, 6/10 slight). In the 0.3, 1 and 3 mg/m³ exposure groups, the difference to the control was statistically significant.
Multifocal alveolar/interstitial granulomatous inflammation was observed in 1/10 females at 0.1 mg/m³ (very slight), in 3/10 females at 0.3 mg/m³ (all very slight) and at significantly increased incidences in 10/10 animals each at 1 mg/m³ (7/10 very slight, 3/10 slight) and 3 mg/m³ (4/10 very slight, 6/10 slight). The term ‘granulomatous inflammation’ was used only, if (mixed) inflammatory cell infiltration, syncytial giant cells and interstitial fibrosis were present in conjunction to form a granuloma-like focal lesion.
(Multi)focal very slight interstitial fibrosis (mainly intraseptal) was diagnosed in 3/10, 4/10, 10/10 and 10/10 females at 0.1, 0.3, 1 and 3 mg/m³, respectively. At 1 and 3 mg/m³, the difference to the control group was statistically significant. Alveolar lipoproteinosis was observed in 4/10 animals ((2/10 very slight, 1/10 slight, 1/10 severe) of the 3 mg/m³ high-dose CeO2 exposure group only and cholesterol granulomas occurred in a single female each of the 1 and 3 mg/m3 CeO2 exposure groups (very slight and slight, respectively).

Incidental pulmonary findings occurring in single animals of different exposure groups as well as in control group consisted of focal very slight osseous metaplasia, focal very slight neuroendocrine cell hyperplasia and focal very slight hair granuloma. In addition, 4/10 control animals revealed focal very slight alveolar macrophage aggregation. All these findings were considered to be unrelated to particle exposure.

Tracheobronchial and mediastinal lymph nodes:
The lymph nodes at both sites showed a dose-dependent (multi)focal very slight to severe accumulation of particle-laden macrophages.
Regarding the tracheobronchial lymph node, the incidences were 8/8 (all very slight) at 0.1 mg/m³, 9/9 (1/9 very slight, 7/9 slight, 1/9 moderate) at 0.3 mg/m³ and 10/10 at 1 mg/m³ (2/10 slight, 8/10 moderate) and 3 mg/m³ (5/10 moderate, 5/10 severe). In addition, particle-laden syncytial (multinucleated) giant cells were present in the tracheobronchial lymph node of 1/8, 6/9, 10/10 and 10/10 females at 0.1, 0.3, 1 and 3 mg/m³, respectively.
In the mediastinal lymph nodes, the incidences of (multi)focal accumulation of particle-laden macrophages were 6/10 (all very slight) at 0.1 mg/m³, 10/10 (all very slight) at 0.3 mg/m³, 9/9 (3/9 slight, 6/9 moderate) at 1 mg/m³ and 10/10 (5/10 moderate, 5/10 severe) at 3 mg/m³ CeO2, while syncytial giant cells were only observed in 9/9 and 10/10 females of groups at 1 and 3 mg/m³CeO2, respectively.

Remaining organs of the respiratory tract:
Within the remaining organs of the respiratory tract such as trachea and nasopharynx no lesions were detected in any investigated group.

HISTOPATHOLOGY OF THE OTHER ORGANS
Several sporadic neoplastic and non-neoplastic findings were observed in the other organs examined histopathologically. These occurred either incidentally or were similar in distribution pattern and severity in control rats compared to the CeO2 high-dose test group. Sporadic findings in the other CeO2 exposure groups were recorded only as correlates of macroscopic findings. All of the observed findings were considered to be without any relation to CeO2 exposure:
- A total number of 6 neoplasms were observed: an adenoma of the pars distalis in the pituitary gland of single females each of control, 0,1 and 0,3 mg/m3 CeO2 groups, a sebaceous adenoma and a lipoma of the skin/subcutaneous tissue in single animals in the 0,3 mg/m3 CeO2 group, and an endometrial stromal polyp of the uterus in a female control animal.
- Findings such as epithelial degeneration (incidence up to 6/10 rats per test group) and interstitial inflammation (incidence up to 7/10 rats per test group) of the Harderian glands are most likely considered to be related to the blood sampling procedure. Further common spontaneous findings included (multi)focal very slight intratubular mineralization of the kidneys (incidence up to 8/10 rats per test group), (multi)focal very slight mononuclear cell infiltration of the liver (incidence up to 7/10 rats per test group), chondromucinous degeneration of sternebral cartilage (incidence up to 7/10 rats per test group), epithelial hyperplasia (incl. hyperplasia of the type ‘epithelial tubules and cords’) at incidences of up to 8/10 rats per test group in the thymus and acinar cell hypertrophy of the salivary glands (incidence up to 4/10 rats per test group). Estrous cycle-depending luminal dilatation of the uterus, C-cell hyperplasia of the thyroids, and parasites (nematodes) in the rectum, colon and/or cecum were observed in up to 3/10 animals per test group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

After 12 month of inhalation exposure neither neoplastic nor pre-neoplastic treatment-related findings were seen in the lungs of CeO2-exposed animals.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ANALYSES OF THE BRONCHOALVEOLAR FLUIDS:
Bronchoalveolar lavage fluids were assessed for 5 animals per group after 13 and 52 weeks of exposure to CeO2 NM-212.
The assessed parameters were: total cell count, differential cell count (macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes), total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β- glucosaminidase (NAG) and cytokines and chemokines (rat monocyte chemoattractant protein-1 (MCP-1), rat cytokine-induced polymorphonuclear neutrophil chemoattractant-1 (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF), rodent steopontin).
All the results are summarised in Table 5 (see below: Any other information on results including tables)

According to the authors, after 13 weeks, exposure to CeO2 induced a dose-dependent increase in neutrophil cell counts from 1 mg/m3 and a statistically significant increase in total cells at all concentrations. Lymphocytes and monocytes were significantly increased only at 3 mg/m³. Macrophage numbers were statistically significantly increased at the lower concentrations (0.1 and 0.3 mg/m³) but not at the highest concentrations. The enzymes GGT, LDH and ALP were statistically significantly increased by aerosol concentrations of 1mg/m³ and above. Total protein and NAG were increased only at 3 mg/m³. Cell mediators (MCP-1,CINC-1 and Osteopontin) were statistically significant increased at 1 mg/m³ and above. A high variability in the measures is noted for these parameters. No increase was observed for M-CSF whatever the concentration used.

After 52 weeks of exposure to aerosol concentrations of 0.3 mg/m³ CeO2 and above, lymphocytes and neutrophils counts were still elevated, but total cell counts were only increased at 3 mg/m³. Increase in monocyte counts was obserserved at 1 and 3 mg/m3 and no increase as compared to controls was observed for macrophages and eosinophils counts whatever the CeO2 concentrations used. Exposure of 0.1 mg/m³ CeO2 caused no changes in BALF cell numbers. Total protein and the enzyme LDH were significantly increased at 1 and 3 mg/m³. ALP and NAG levels were not affected at any aerosol concentration whereas GGT were significantly increased at all concentrations. MCP-1 and CINC-1 levels were still elevated but to a lower extent compared with 13 weeks of exposure. No change was observed for M-CSF and osteopontin whatever the CeO2 concentration used.

it should be noted that a high variability was noted in all the measured parameters. But individual data were not reported in the report.

LUNG and LYMPH NODE BURDENS:
Directly after 13 and 52 weeks of exposure, the lavaged lungs, aliquots of lavage fluids and tracheobronchial and mediastinal lymph nodes of five animals per group were used to determine lung burden. The amounts of CeO2 in lungs and lymph nodes were estimated by measuring Ce with ICP-MS and extrapolating to CeO2 (assuming that it was still particulate). The results at the low (0.1 mg/m3) and high (3 mg/m3) concentrations are presented in the Table 4 (see section: Any other information on results including tables).
During the exposure period, lung burdens increased with longer exposure duration. The lung burden of CeO2 after 13 weeks was nearly doubled after 52 weeks of exposure at aerosol concentrations of 0.1 and 3 mg/m³. Only a slight translocation of the nanoparticles to the tracheobronchial and mediastinal lymph nodes was observed (1.9 % of the CeO2 lung burden).

Details on results:

Although statistically not significant, some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 low-dose CeO2 exposure group. Thus, a NOAEC (no observed adverse effect concentration) could not be established for the lungs after 12 months of exposure to the present CeO2 nanoparticle concentrations.

Dose descriptor:

NOAEC

Remarks:

Systemic effects (52 weeks)

Effect level:

3 mg/m³ air

Sex:

female

Basis for effect level:

other: no adverse systemic effects occurred

Remarks on result:

other:

Remarks:

Provisional conclusion pending the publication of the carcinogenicity study done by BASF.

Dose descriptor:

NOAEC

Remarks:

Local effects (52 weeks)

Effect level:

< 0.1 mg/m³ air

Sex:

female

Basis for effect level:

other: Some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 group

Remarks on result:

other:

Remarks:

Provisional conclusion pending the publication of the carcinogenicity study done by BASF.

Critical effects observed:

not specified

Table 3: Mean neutrophil levels and acute phase proteins in blood after 13 and 52 weeks of exposure to CeO2 MN-212

Test concentrations [mg/m3]		Control	0.1	0.3	1	3
Neutrophils (giga/L) Mean value+/- SD	13 w	0.63+/-0.25	0.95+/-0.16	0.78+/-0.10	0.93+/-0.30	1.09+/-0.35
	52 w	0.67+/-0.17	0.79+/-0.19	0.70+/-0.17	1.02+/-0.20	0.97+/-0.29
Neutrophils (%) Mean value+/- SD	13 w	15.2+/-4.60	23.4+/-4.3	19.8+/-5.0	22.4+/-7.6	25.4+/-9.8
	52 w	26.7+/-5.80	33.8+/-5.8	28.1+/-4.5	32.6+/-7.1	32.3+/-4.8
Acute phase proteins (µg/mL)
HAPT Mean value+/- SD	13 w	204.00+/-47.01	223.60+/-67.11	228.60+/-55.86	192.20+/-51.45	229.20+/-58.14
	52 w	139.60+/-74.26	215.40+/-54.45	184.20+/-73.96	173.60+/-37.35	251.40*+/-63.06
A2M Mean value+/- SD	13 w	14.55+/-5.90	18.18+/-4.22	20.69+/-4.55	22.66*+/-7.06	19.65+/-4.45
	52 w	8.86+/-2.14	9.52+/-2.17	9.88+/-4.48	12.39+/-3.98	12.19*+/-1.61

w: weeks of treatment;statistically significant, p < 0.05; ** statistically significant, p < 0.01; n=5; SD: standard deviation, HAPT: haptoglobin, A2M:α2-macroglobulin; blood were collected directly after 13 weeks of exposure and one day after 52 weeks of exposure.

 

Table 4: Overview of organ burden analysis after 13 and 52 weeks of exposure to CeO2 NM-212

Weeks of exposure	13		52
Study day	94		367
Time point	One day after last exposure
Number of animals	5		5
Test Concentrations [mg/m3]	0.1	3	0.1	3
Lung burden [μg] ±SD	11.96+/-2.82		42.07+/-11.05
Lung burden [mg] ±SD		1.39+/-0.16		2.61+/-0.52
Tracheobronchial lymph node burden [μg] ±SD	ND	11.93+/-14.21	ND	ND
Mediastinal lymph node burden [μg] ±SD	ND	13.78+/-15.85	ND	ND

ND: not determined, SD: standard deviation, n=5; burden is mg or μg per lung; lavaged lungs and corresponding aliquots were used for determination of lung burden by ICP-MS.

 

Table 5: Bronchoalveolar lavage fluid analysis after 13 and 52 weeks of exposure to CeO2 NM-212 

Test concentrations [mg/m3]		Control	0.1	0.3	1	3
Mean value+/- SD
Total cells (counts/μL)	13 w	66.3±22.04	115.22**±23.72	98.70*±30.49	117.51*±43.99	163.33**±57.29
	52 w	54.27±20.27	59.95±26.52	60.97±20.18	68.56±28.69	96.06*±29.29
Neutrophils (counts/μL)	13 w	1.71±0.66	0.53±0.58	2.85±3.45	18.56**±11.68	72.59**±54.65
	52 w	0.58±0.69	1.05±0.99	4.82**±3.94	16.07**±5.17	31.97**±14.23
Lymphocytes (counts/μL)	13 w	0.55±0.42	0.27±0.26	0.38±0.63	2.21±2.23	5.43**±3.52
	52 w	0.56±0.36	1.30±1.12	9.79*±18.06	6.69**±2.16	9.72**±5.97
Macrophages (counts/μL)	13 w	63.99±22.36	114.37**±24.06	95.43*±27.12	96.30±46.85	83.52±15.72
	52 w	53.01±19.77	57.49±25.85	46.10±12.59	45.33±26.17	53.57±12.31
Monocytes (counts/μL)	13 w	0.00±0.00	0.00±0.00	0.00±0.00	0.13±0.30	0.94**±0.67
	52 w	0.04±0.06	0.04±0.06	0.16±0.18	0.29*±0.24	0.43*±0.40
Eosinophils (counts/μL)	13 w	0.04±0.08	0.00±0.00	0.04±0.08	0.13±0.20	0.25±0.39
	52 w	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.05±0.10
Total protein (mg/L)	13 w	52 ±10	57±9	60±23	60±18	84*±26
	52 w	31±8	33±12	67±73	61**±11	73**±28
GGT (nkat/L)	13 w	41±7	40±5	53±13	80**±18	106**±26
	52 w	26±4	37**±6	38*±9	70**±23	65**±14
LDH (μkat/L)	13 w	0.38±0.06	0.43±0.03	0.52±0.16	0.61**±0.18	0.95**±0.33
	52 w	0.35±0.04	0.37±0.08	0.44±0.13	0.66**±0.14	0.80**±0.19
ALP (μkat/L)	13 w	0.51±0.10	0.45±0.06	0.58±0.09	0.73*±0.17	0.84**±0.19
	52 w	0.44±0.12	0.54±0.10	0.48±0.13	0.58±0.19	0.50±0.06
NAG (nkat/L)	13 w	35±7	35±5	37±13	43±9	51**±8
	52 w	37±7	42±14	38±	47±9	47±12
MCP-1 (pg/mL)	13 w	24.2±8.4	19.1±7.5	30.8±26.4	111.4**±100.1	620**±613.5
	52 w	15.3±8.2	17.9±7.1	49.3*±37.6	169.4**±113.9	378.2**±245.5
CINC-1/IL-8 (pg/mL)	13 w	93.7±18.7	60.6±38.9	77.7±38.9		280.6*±190.8
	52 w	82.4±25.4	68.8±37.1	69.7±26.5	132.9*±49.1	180.6*±114.8
M-CSF (pg/mL)	13 w	14±0	14±0	14±0	14±0	14±0
	52 w	21±12	18±3	18±4	27±12	25±15
Osteopontin (pg/mL)	13 w	49.02±40.86	37.40±32.69	118.49±133.12	136.87*±72.89	300.41*±253.01
	52 w	162.79±98.61	166.71±187.14	135.32±61.53	175.72±88.75	228.80±217.96

* statistically significant, p < 0.05; ** statistically significant, p < 0.01; n=5; mean values and standard deviation (SD); lungs were lavaged one day after last exposure.

Table 6: Summary of incidences of lung changes related to CeO2 exposure

Test concentrations [mg/m3]	Control	0.1	0.3	1	3
Lung / No. of animals	10	10	10	10	10
Accumulation, Particle-Laden Macrophages, Alveolar/Interstitial, Grade 1-3	0	10*	10*	10*	10*	non-adverse lesion
Accumulation, Particle-Laden Macrophages, BALT, Grade 1-4	0	10*	10*	10*	10*
Giant Cells, Syncytial, BALT, Present, no grade	0	0	3	9*	10*
Hyperplasia, Bronchiolo-Alveolar, Bronchiolar type Grade 1-2	0	1	2	10*	10*
Infiltration, Inflammatory Cells, Alveolar/ Interstitial, Grade 1-2	1	4	10*	10*	10*	adverse lesion
Infiltration, Granulometous, Alveolar/ Interstitial, Grade 1-2	0	1	3	10*	10*
Fibrosis, Interstitial, Grade 1	0	3	4	10*	10*
Lipoproteinosis, Alveolar, Grade 1-4	0	0	0	0	4
Granuloma, Cholesterol, Grade 1-2	0	0	0	1	1

*= p < 0.001,Chi-Quadrat/Fisher-Test, two-sided

Table 7: Summary of grade of lesions of lung changes related to CeO2 exposure

Test concentrations [mg/m3]	Control	0.1	0.3	1	3
Lungs / No. of animals	10	10	10	10	10
Accumulation, Particle-Laden Macrophages, Alveolar/Interstitial	0	1.2*	1.2*	1.7*	2.9*
Accumulation, Particle-Laden Macrophages, BALT	0	1*	1.2*	2.2*	3*
Giant Cells, Syncytial, BALT	0	0	0.3	0.9*	1*
Hyperplasia, Bronchiolo-Alveolar, Bronchiolar type	0	0.1	0.2	1*	1.1*
Infiltration, Inflammatory Cells, Alveolar/ Interstitial	0.1	0.4	1.1*	1.3*	1.6*
Infiltration, Granulometous, Alveolar/ Interstitial	0	0.1	0.3	1.3*	1.6*
Fibrosis, Interstitial	0	0.3	0.4	1*	1*
Lipoproteinosis, Alveolar	0	0	0	0	0.8
Granuloma, Cholesterol	0	0	0	0.1	0.2

*= p < 0.001,Chi²-test/Fisher-Test, two-sided

Conclusions:

In the present inhalation study which reports interim results of a 2-year study otained after 13 and 52 weeks of exposure of females rats to nano-CeO2 (NM-212), high biopersistence of the nanoparticles with the induction of a local lung inflammation was observed. However nano-CeO2 showed a low bioavailability and elicited no or only minimal systemic effects. Thus, nano-CeO2 was considered as a poorly soluble with an inherent toxicity in the lung in this study. The local no observed adverse effect concentrations in the lung (NOAEC) of CeO2 - based on BALF in female rats after 13 and 52 weeks of exposure was found to be 0.3 and 0.1 mg/m³, respectively. However, from the results of the histopathological analyses of animals after 52 weeks of exposure to CeO2, it was concluded that the local no observed adverse effect concentrations in the lung (NOAEC) could not be established after exposure to the present CeO2 nanoparticle concentrations. In the absence of systemic effects, the overall systemic NOAEC was considered to be 3 mg/m3.

Executive summary:

This is a provisional discussion pending the publication of the carcinogenicity study done by BASF.

 

This work described the interim results of a carcinogenicity study done in the rats (performed according to OECD test guideline No. 453, under GLP, and with a relevant aerosol concentration range (OECD 2009)), observed after 13 and 52 weeks of inhalation exposure to nano-CeO2 NM-212. The aim of this interim observations was to investigate the lung deposition and clearance of inhaled nanomaterials, and the resulting effects on the rat organism at different time points.

 

Female Wistar rats inhaled nano-CeO2 by whole-body exposure, 6 hours per day, 5 days per week for a total of two years. The tested aerosol concentrations were 0.1, 0.3, 1 and 3 mg/m³ CeO2. The Interim results after 13 and 52 weeks of exposure presented in this summary included results collected from 5 females/group on lung retention and clearance kinetics based on lung and associated lymph node burdens, and pulmonary effects based on bronchoalveolar lavage fluid (BALF) analyses including total protein concentrations, total and differential cell counts, enzyme activities (LDH, ALP, GGT, NAG), acute phase proteins (HAPT nad A2M) and cytokines (MCP-1, CIN-1/IL-8 and M-CSF and rodent osteopontin). Systemic effects were evaluated by analysis of blood including neutrophils counts and acute phase proteins HAPT and A2M. Histopathological analyses were done after 12 weeks of exposure on 10 animals/group.

 

According to the author, during the exposure period, lung burdens increased with longer exposure duration. The lung burden of CeO2 after 13 weeks was nearly doubled after 52 weeks of exposure at aerosol concentrations of 0.1 and 3 mg/m3. However, only a slight translocation of the nanoparticles to the tracheobronchial and mediastinal lymph nodes was observed (below 2 % of the initial lung burden after 13 weeks) indicating that, at least at this lung burden range, they play no major role for particle clearance.

 

Inhalation exposure to nano-CeO2 for 13 and 52 weeks elicited no or only minimal systemic effects: no change in mean neutrophil levels were observed in the blood of rats at any concentrations and time points slight increase of acute phase protein levels (haptoglobin andα2-macroglobulin) in serum was observed after 52 weeks of exposure to 3 mg/m3 CeO2.

 

Local inflammation in the lung was observed by increases in BAL neutrophils, lymphocytes, monocytes, enzyme cativities and cell mediator levels at the highest tested concentrations (1 and 3 mg/m3) after 13 weeks of exposure. BAL changes (cell counts, enzyme activities, total protein and cell mediator levels) after 52 weeks of exposure to 3 mg/m³ CeO2 were comparable to those after 13 weeks or slightly reduced. Minor changes in BAL were observed at lower aerosol concentrations of 1 mg/m³. Exposure to 0.3 mg/m³ elicited no BAL changes after 13 weeks and only minor changes after 52 weeks (neutrophils, GGT, MCP-1). In animals exposed to lower aerosol concentrations (0.3 mg/m3) rather than in those exposed to higher, BAL lymphocytes were higher increased than neutrophils after 52 weeks of exposure indicating a later phase of inflammation.

 

Histopathology findings related to CeO2 exposure were exclusively observed in the respiratory tract and included non-adverse reactive/adaptive changes such as accumulation of particle-laden macrophages in the nasal cavity, larynx, lungs, tracheobronchial and mediastinal lymph nodes. Adverse effects in the lungs included dose-dependent alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation and interstitial fibrosis. Alveolar lipoproteinosis was observed in the 3 mg/m³ high-dose CeO2 exposure group only and cholesterol granulomas occurred in a single female each of the 1 and 3 mg/m³ CeO2 exposure groups. Although statistically not significant, some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 low-dose CeO2 exposure group. After 12 month of inhalation exposure neither neoplastic nor pre-neoplastic treatment-related findings were seen in the lungs of CeO2-exposed animals. In the histopathological analyses of the other organs, all the findings were considered to be without any relation to CeO2 exposure.

 

The local no observed adverse effect concentrations in the lung (NOAEC) of CeO2 - based on BALF in female rats after 13 and 52 weeks of exposure - was found to be 0.3 and 0.1 mg/m³, respectively. However, from the results of the histopathological analyses of animals after 52 weeks of exposure to CeO2, it was concluded that the local no observed adverse effect concentrations in the lung (NOAEC) could not be established for the lungs after 12 months of exposure to the present CeO2 nanoparticle concentrations. In the absence of systemic effects, the overall systemic NOAEC is 3 mg/m3.

 

In summary, inhaled CeO2 showed high biopersistence in the lungs inducing a local inflammation but no systemic effect. Thus CeO2 was considered as a poorly soluble with an inherent toxicity in the lung in the rats.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: :

Remarks:

the study was well documented and performed according to OECD guideline 412. However, there was no mention to GLP and a detailed description on the generation of the test atmosphere was missing. Furthermore, results were described too briefly or not shown, and presented a very high variability, as indicated by elevated standard deviations (SD); this made it difficult to draw a definite conclusion.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced selection of organs for pathological examination

GLP compliance:

not specified

Remarks:

The GLP status was not specified in the article.

Limit test:

no

Species:

rat

Strain:

other: Wistar WU (Crl:[WI] WU BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Germany)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 203 g for males and 150 g for females (average body weights [bw])
- Fasting period before study: No
- Housing: In macromolon cages, 5 animals/cage separated by group and by sex, bedding of wood shavings (Lignocel, Type 3/4) and strips of paper (Enviro-dri)
- Diet: Ad libitum, except during exposure and the fasting period (overnight) before sacrifice; commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, Special Diets Services, UK)
- Water: Ad libitum, except during exposure and the fasting period (overnight) before sacrifice
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: 10 air changes per hour
- Photoperiod: Artificial light on a 12 hour cycle

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: clean humidified compressed air

Remarks on MMAD:

MMAD / GSD: MMAD / GSD determined using APS (µm)
NM-211: 1.02 ± 0.04 / 1.82
NM-212: 1.17 ± 0.34 / 2.07
NM-213: 1.40 ± 0.11 / 1.64

MMAD / GSD determined using SMPS (µm)
NM-211: 0.276 ± 0.037 / 1.48
NM-212: 0.366 ± 0.058 / 1.56
NM-213: 0.464 ± 0.058 / 1.32

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus, method of holding animals in test chamber, source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Test atmospheres of CeO2 powders were generated using a dust feeder, a venturi (Fox Valve Development corp., Dover, USA) and a jet mill. The test atmosphere was directed to the top inlet of the exposure unit.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate: No data available
- Method of particle size determination: Particle size distributions were measured daily by a single particle mass spectrometer (SMPS) and an aerodynamic particle sizer (APS) and weekly by a 11-stage cascade impactor.
- Treatment of exhaust air: The test atmosphere was exhausted at the bottom of the exposure unit.

TEST ATMOSPHERE
- Brief description of analytical method used: Particle size distributions were measured daily by a single particle mass spectrometer (SMPS) and an aerodynamic particle sizer (APS), and weekly by a cascade impactor (see in "any other information on materials and methods" below for details).
- Samples taken from breathing zone: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the test material in the atmosphere were measured at least 6 times daily by gravimetric analysis.
Mass concentrations were monitored for at least 2 h per day with a condensation particle counter (CPC).
(see in "any other information on materials and methods" below for details)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Low dose group: 40 min/day, 5 days/week (except on public holidays)
Mid dose group: 2 hours/day, 5 days/week (except on public holidays)
High dose group: 6 hours/day, 5 days/week (except on public holidays)

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

for different daily exposure durations (see in "any other information on materials and methods" below for details)
basis: nominal micro-CeO2 (NM-213) conc. for a 6-h exposure

No. of animals per sex per dose:

5 males and 5 females per dose (see table 1 in "any other information on materials and methods" below for details)

Control animals:

other: Yes, stream of clean humidified compressed air

Details on study design:

- Dose selection rationale, rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Recovery groups were included consisting of control and 6-h exposure (high dose) groups of 5 males and 5 females kept for a recovery period of 28 days before sacrifice
- Section schedule rationale: No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (no detail). If necessary, animals were handled to detect signs of general toxicity.

DETAILED CLINICAL OBSERVATIONS: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded 1 day before the start of exposure (day -1), prior to exposure on the first day (day 0), weekly thereafter, and on the day before overnight fasting and on the scheduled sacrifice date.

FOOD CONSUMPTION: Yes
- Measured per cage by weighing the feeders

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were taken from the abdominal aorta of sacrificed rats (K3-EDTA was added as anticoagulant to one portion).
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight before sacrifice)
- How many animals: 10 (5 males and 5 females)
- Parameters checked in each sample: Haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Remaining blood was collected from the abdominal aorta of sacrificed rats in heparinized tubes and plasma was prepared by centrifugation.
- Animals fasted: Yes (overnight before sacrifice)
- How many animals: 10 (5 males and 5 females)
- Parameters checked in plasma samples using an auto-analyser: Alkaline phosphatase activity (ALP), bilirubin total, aspartate aminotransferase activity (ASAT), cholesterol, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, fasting glucose

URINALYSIS: Yes
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked: Creatinine, hydroxydeoxyguanosine

NEUROBEHAVIOURAL EXAMINATION: No data available

OTHER: BRONCHOALVEOLAR LAVAGE
At necropsy, the right half of the lungs were lavaged twice with saline (26.7 mL/kg bw). Two aliquots of bronchoalveolar lavage fluid (BALF) were collected and centrifuged.
* The supernatant from the first lung lavage was used for biochemical determinations: total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (γ-GT) and superoxide dismutase (SOD) were determined using an auto-analyser. In addition, malondialdehyde (MDA) in BALF was analysed by derivatizing MDA with thiobarbituric acid, extracting the formed product with n-butanol, and measuring the MDA-derivate using high performance liquid chromatography (HPLC) with fluorescence detection (FLD).
* Cell pellets that originated from centrifugation were resuspended in saline. Total white blood cell numbers were counted using an automated haematology analyser. The percentages of viable cells were determined using an acridine orange/ethidium bromide staining method in combination with fluorescent microscopic evaluation. Differential white blood cells were determined by counting 200 cells under the microscope.

FIBRINOLYSIS
Von Willebrand Factor (vWF) and plasminogen activator inhibitor 1 (PAI-1) were analysed in the serum using a multiplex analysis immunoassay. Fibrinogen was analysed using a highly sensitive two-site enzyme linked immunoassay (ELISA).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (n = 5)
HISTOPATHOLOGY: Yes (n = 5)
The weight of organs (adrenals, brain, kidney, lungs, liver, and spleen) was determined prior to preservation in formaldehyde (by infusion for lungs). For histopathological examination the above tissues as well as the larynx, tracheobronchial lymph nodes, nasopharyngeal tissues (6 levels) and trachea (2 levels including 1 longitudinal section through the carina and 1 transverse section) of the high dose group and of the controls were embedded in paraffin wax, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) for examination of gross abnormalities.

Statistics:

Body weight data, clinical pathology data, and organ weights were analysed using one-way analysis of variance (ANOVA), after checking homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data were not normally distributed, the data were stepwise log or rank transformed prior to the ANOVA. Intergroup comparisons with the control group were made by Dunnett's multiple comparison post-hoc test. P < 0.05 was accepted as statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

No exposure related signs of toxicity or behaviour effects or mortality were observed during the 4-week exposure period and recovery period for all three CeO2 (data not shown).

Mortality:

no mortality observed

Description (incidence):

No exposure related signs of toxicity or behaviour effects or mortality were observed during the 4-week exposure period and recovery period for all three CeO2 (data not shown).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Cerium exposure did not influence body weight (data not shown).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Cerium exposure did not influence food consumption (data not shown).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigation and clinical chemistry at necropsy revealed no treatment related changes for NM-213 particles. NM-212 exposure was associated with a slight treatment-related increase in the number of neutrophils in the mid and high dose male groups and in the high dose female group. This elevation was still observed in male animals at the end of the recovery period. NM-211 exposure also induced a slight but significant treatment-related increase in the neutrophil number in females from low and mid dose groups only and none in the high dose group at the end of exposure and in recovery group. No other treatment-related changes were observed in haematological investigations (data not shown).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Due to absence of treatment related changes in clinical chemistry at the end of the exposure period (data not shown), thus, no determination was performed in the recovery group at the end of the recovery period.
Parameters for fibrinolysis did not reveal treatment related changes for all materials (data not shown).

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

According to the authors, urinalysis did not reveal any changes in hydroxydeoxyguanosine as a parameter for DNA damage repair (data not shown). Thus, oxidative stress could not be demonstrated.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in organ weights, except for a treatment-related increase in absolute and relative lung weights for all three materials.
After exposure to the NM-213 material, treatment-related increase in absolute and relative lung weights was observed in females directly after treatment and in the recovery groups. In males, there is only a dose-related effect in the recovery groups.
For NM-212 and NM-211 particle exposures, the increase in lung weight after exposure was reduced but still significant after a 28-day period of recovery in both females and males (see also section "Any other information on results incl. tables").

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observation at necropsy revealed pale tracheobronchial lymph nodes (unilateral) in the recovery groups, after exposure to NM-213 (all high dose animals), to NM-212 (5 males and 3 females) and NM-211 (1 male and 3 females), in comparison with the control groups. For all CeO2 materials, this was not observed in the exposed animals sacrificed one day after the end of the exposures.
No micro or macroscopic changes were detected in the liver (data not shown).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination at the end of the treatment period revealed aggregates of brown/green particles and/or macrophages containing these particles in the lungs and the draining (tracheobronchial) lymph nodes of animals exposed to the high dose of all types of CeO2. These aggregates were also found in the trachea of some animals exposed to the high dose of NM-213 and NM-212 (no data available for NM-211). At the end of the recovery period, macrophages with brown/greenish particles/aggregates still persisted in the alveoli and in the tracheobronchial lymph nodes. Of note that brown pigmented macrophages were also found in tracheobronchial lymp nodes of some control females at the end of the exposure period (3 to 5) as well as in males (1 or 2) and females (4 or 5) of the control group at the end of the recovery period in the experiment conducted with NM-211, NM-212, and NM-213 CeO2. In addition, increased septal cellularity was observed in NM-212 and NM-211exposed animals, suggesting that a tissue reaction was on-going. There was no data available on the occurrence of septal cellularity in NM-213 exposed animals.
In conclusion, following inhalation, CeO2 nanomaterial deposited in several parts of the airways (lungs, trachea, tracheobronchial lymph nodes) as was noticed by brown/green material in the macrophages.
No micro or macroscopic changes were detected in the liver (data not shown).

Histopathological findings: neoplastic:

not examined

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 10.8 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 (test mat.)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 19.9 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-212 (test mat.)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

other: NOAEC (systemic)

Effect level:

>= 55 mg/m³ air (nominal)

Based on:

other: bulk CeO2 NM-213 (test mat.)

Sex:

male/female

Basis for effect level:

other: - No NOAEC was determined in the publication thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects - Effect level corresponding to 653 000 particles/m3; 0.205 m²/m3 as surface concentration

Dose descriptor:

other: NOEC/NOAEC (local)

Based on:

other: nano-CeO2 NM-211 and NM-212, and bulk CeO2 NM-213 (test mat.)

Sex:

male/female

Basis for effect level:

other: - No NOEC/NOAEC was determined in the publicationand no NOEC/NOAEC could be set by the registrant due to the very high variability and the recovery, at least partial, of BALF results and to the brevity of histopathology data.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified.

Critical effects observed:

not specified

- DEPOSITION OF CeO2 MATERIALS:

Using MPPD modelling, the total deposited dose at the end of the 28-day exposure period was estimated at 0.83, 1.54 and 4.24 mg for NM-211, NM-212 and NM-213, respectively. Based on the density of CeO2 of 7.65 g/cm3, a threshold for overload is expected to be at 11.4 mg (6% of the volume increase of lung macrophages due to phagocytized material => volumetric lung burden of 1.5 x 10E9 - 1.5 x 10E10 µm3). Thus, the authors concluded that it was unlikely the study was performed under overload conditions.

- EFFECTS OF CeO2 PARTICLE EXPOSURE ON LUNG WEIGHT:

After exposure to nano-sized NM-212, significant increases in absolute (abs.) and relative (rel.) lung weight were observed in males from the mid dose (0.44 and 1.53 g/kg bw, respectively) and high dose groups (0.50 and 1.74 g/kg bw, respectively), when compared to respective controls (0.36 and 1.23 g/kg bw, respectively). Moreover, abs. and rel. lung weights were significantly elevated in the recovery group (0.47 and 1.30 g/kg bw, respectively, vs. 0.43 and 1.17 g/kg bw for controls). Similar effects were seen in females, with the exception of a significant increase of abs. and rel. lung weights also seen in the low dose group (0.32 and 1.81 g/kg bw), when compared with controls (0.28 and 1.66 g/kg bw).

For nano-sized NM-211, significant increases in abs. and rel. lung weight were observed in males and females from mid dose (0.45 and 1.68 g/kg bw for males and 0.35 and 2.15 g/kg bw for females, respectively) and high dose groups (0.58 and 2.09 g/kg bw for males and 0.45 and 2.62 g/kg bw for females, respectively) as compared to controls (0.38 and 1.35 for males and 0.30 and 1.71 g/kg bw for females, respectively). Moreover the elevations in abs. and rel. lung weight were still significant after a 28-day period of recovery in both males and females.

After exposure to micro-sized NM-213, there was an increase in abs. and rel. lung weights observed in female directly after treatment in the mid (0.34 and 2.00 g/kg bw, respectively) and high dose groups (0.39 and 2.38 g/kg bw, respectively) as compared to controls (0.28 and 1.68 g/kg bw, respectively) but not in recovery (high dose) group. In males, there was only an increase in rel. lung weight in the recovery (high dose) group (1.41 g/kg bw) when compared to controls (1.20 g/kg bw).

For the other organs evaluated, there was no effect of treatments on organ weights.

- BRONCHOALVEOLAR LAVAGE / LUNG TOXICITY:

A statistically significant increase in total cell counts in BALF was observed only in the high dose and recovery groups of females for NM-212, in the mid dose and high dose groups of females as well as the high dose group of males for NM-211 and in male and female high dose group and only in female high dose group for NM-213.

The percentage of viable cells showed no treatment-related change; hence the absolute number of viable cells was also statistically significantly higher in the high dose groups (data not shown).

In control animals (regardless of the sex), the macrophages accounted for > 98% of the total number of cells recovered in BALF. The neutrophils, lymphocytes, and eosinophils were present in only very low numbers and varied between 0 and 1.4% of the total number of cells for both sexes.

A significant increase in absolute numbers of macrophages was observed in the high dose groups of females exposed to NM-212, in males of high dose group and in females of recovery group for NM-213 and in both sexes from the high dose group for NM-211. All animals except females exposed to NM-213 and from the high recovery group returned to control level at the end of the recovery period

Regardless of the particle type and of the dose administered, the absolute number of neutrophils was significantly increased in all groups of males and females, as well as in the recovery groups.

At last, the absolute number of lymphocytes was significantly elevated in mid and high dose groups of males and females exposed to NM-211 and NM-213 and in the recovery groups of males and females exposed to NM-211 and only in females for NM-213. In contrast, regarding NM-212 the parameter increased only in males from mid and high groups and in females from the recovery groups.

It is noticeable that, whatever the particles and the cell types considered, a decrease of the effects was observed in both males and female recovery groups.

It has to be noted that the different parameters studied showed very high variability, as indicated by standard deviations (SD). Thus, the significance of the observed differences could be questionable.

When expressing the cell counts as relative numbers (%), the relative number of macrophages significantly decreased as a function of the administered concentration in both males and females exposed to the 3 types of CeO2, while the percentages of neutrophils and lymphocytes increased in the same animals. Globally, a similar trend was found in the recovery groups of males and females, when compared to their controls.

Regarding the clinical/biochemical parameters measured in BALF (ALP, GGT, LDH, NAG, protein), all CeO2 induced significant increases in males and females (combined data), but the elevations seemed more important in rats exposed to NM-211 > NM-212 and > NM-213. These effects were still present after the 28-day recovery period though a noticeable decrease was observed for all parameters. Furthermore, the authors stated that the levels of SOD and MDA in BALF were not significantly affected by any exposure (data not shown).

According to the authors, the increased neutrophil content and elevated biochemical parameters in BALF supported by increased lung weight for NM-213, NM-212 and NM-211 indicated that exposure to CeO2 resulted in inflammation in the lungs at all dose levels.

- EXPOSURE METRIC:

Benchmark concentrations (BMCs) were ranked for the three most prominent lung toxicity parameters for which changes were detected: LDH, total protein, and neutrophil levels in the BALF. When ranked on a mass concentration basis, NM-211 was associated with the smallest BMC and therefore the highest toxicity (lowest concentration at which a 100% increase over controls was found). This was followed by NM-212 while micro-sized NM-213 revealed the lowest toxicity. There was a significant difference in the level of neutrophils between males and females for NM-212 and NM-211. However, the mass-toxicity ranking of the materials remained the same when data was separated for sex of animal.

When the BMC was expressed as particle number, the ranking was found to change. NM-213 and NM-211 were found to have similar toxicity in terms of LDH and total protein in BALF, both of which were ranked lower than NM-212. Based on the level of neutrophils, NM-213 was the most inflammogenic.

When the dose was expressed as surface concentration, NM-213 remained the most toxic material with the lowest BMC for all three parameters. NM-211 was the next most toxic material, followed by NM-212, opposite to when expressed as mass concentration.

Conclusions:

Regardless of the size of the CeO2 tested, repeated inhalation exposures of male/female rats induced moderate pulmonary responses with little quantitative differences but without any mortality, clinical or pathological signs, or systemic effect. Moreover, there was little evidence for a dominant predictive exposure metric for the observed effects.

Executive summary:

Gosens I et al. (2013) assessed the hazard of two nano-scale (NM-211 and NM-212) and one microscale (NM-213) cerium dioxide (CeO2) materials in 28-day inhalation toxicity studies in rats, according to the OECD 412 guideline but with some deviations. This study was conducted within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials.

The 3 tested CeO2 were highly pure (≥ 99.5%) and had a negligible solubility in water at neutral pH. NM-211 had a primary size (nominal) of 5-10 nm, NM-212 of 40 nm and NM-213 of < 5 µm. Although the primary particle size of all materials differed considerably, the mass median aerodynamic diameter was surprisingly similar because all materials were aggregated when aerosolised in air. The specific surface areas differed between the tested CeO2: 63.95 m²/g for NM-211, 27.15 m²/g for NM-212 and 3.73 m²/g for NM-213. Moreover, NM-211 showed the highest number concentration in the test atmosphere (1.75 x 10E6 particles/cm3) and the smallest mass concentration (10.79 mg/m3), while NM-212 had a number concentration of up to 1 x 10E6 particles/cm3 and a mass concentration of up to 19.9 mg/m3. NM-213 had a number concentration of 0.68 x 10E6 particles/cm3 and a mass concentration of up to 55 mg/m3.

Male and female Wistar rats (5/sex/dose) were nose-only exposed 40 min to 6 h/day for 5 days/week during 4 weeks to 0 (control), 1.2 - 10.8 mg/m3 of NM-211, 2.5 - 19.9 mg/m3 of NM-212 and 5.9 - 55 mg/m3 of NM-213. Control animals were exposed to a stream of clean humidified compressed air. Cage side observations were performed. Mortality, body weight, and food consumption were also monitored. Moreover, at the end of the exposure period, haematological parameters, clinical chemistry in blood and bronchoalveolar lavage fluid (BALF), inflammatory cell counts in BALF, urinalysis, fibrinolysis, and gross/histo-pathology were carried out to determine the local (pulmonary) and systemic effects of the CeO2 particles. A recovery group was included in this study: males and females exposed to NM-211, NM-212 and NM-213 (high dose) 5 days/week for 4 weeks were kept for a recovery period of 28 days before sacrifice.

There were no exposure-related signs of toxicity, behaviour effects or mortality during the 4-week exposure period and recovery period.

All materials were efficiently deposited in lungs of animals: 0.83, 1.54 mg and 4.24 mg for NM-211, NM-212 and NM-213, respectively; the particle exposures increased the lung weight.

According to the authors, all materials induced a dose-dependent pulmonary inflammation and lung cell damage, but without any gross or histopathological changes immediately after exposure. Inflammation was still present after the 28-day recovery period, albeit at a lower level. Nevertheless, although significant, the parameters used (e.g., BALF cell counts) to draw these conclusions showed a high variability which could indicate that the inflammatory effects observed might be even more modest or almost negligible in some cases.

In contrast, there was no evidence of systemic toxicity or other haematological changes and no macroscopic or microscopic change in the liver following exposure to any of the 3 tested CeO2. According to the authors, these data suggested that the adversity of the effects could be classified as minimal.

When exposure levels were expressed as mass concentration, NM-211 was the most potent material, while based on surface area concentration pulmonary inflammation/damage was induced in a lesser extent by both nano-CeO2 than by the micrometric counterpart. Particles were equipotent based on particle number concentrations.

In conclusion, similar moderate pulmonary toxicity profiles including inflammation were observed for both nanomaterials and the micrometric counterpart and systemic effects were virtually absent. These similar patterns probably resulted from the equivalent aerodynamic diameters found for all materials and there was little evidence for a dominant predictive exposure metric for the observed effects.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Although the study was performed according to OECD guideline 413, there are no information on GLP compliance. Furthermore, histopathological analyses was performed only on the control and high dose (3 mg/m3 CeO2) groups. Further, results of all parameters that should be studied in an OECD 413 study are either briefly reported (e.g clinical signs, body weight gain) or absent (e.g. organ weight, macroscopic examination). Therefore, no NOEC/LOAEC can be derived for systemic and/or local effects in this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

The test was performed only with female rats / All analyses usually performed with OECD 413 were not performed (ex: only histopathological analysis of respiratory tract was performed. Further, only control and high dose groups animals were analysed).

GLP compliance:

not specified

Remarks:

The GLP compliant status was not specified in this published article.

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 10 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups of two animals in Makrolon polycarbonate cages Type IV
- Diet: Ad libitum (“V1534”, sniff Spezialdiäten GmbH, Soest, Germany)
- Water: Ad libitum
- Acclimation period: Subsequent to 1 week of acclimatization rats were habituated to nose-only tubes for 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 40 to 70%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: high-pressurized, high velocity pressurized air

Remarks on MMAD:

MMAD / GSD:
At the concentration of 0.1 mg/m3: 0.71 µm / 3.59 (MMAD / mean GSD)
At the concentration of 0.3 mg/m3: 0.63 µm / 3.83 (MMAD / mean GSD)
At the concentration of 1.0 mg/m3: 0.68 µm / 4.23 (MMAD / mean GSD)
At the concentration of 3.0 mg/m3: 0.79 µm / 3.50 (MMAD / mean GSD)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a nose-only inhalation system.
- Source and rate of air, method of conditioning air: The test material was located in reservoirs on a rotating disc and sucked into the air flow system.
- System of generating particulates/aerosols: Aerosols were generated by dry powder dispersion using a high-pressurized, high velocity pressurized air dispersion nozzle developed at the Fraunhofer Institute for Toxicology and Experimental Medicine ITEM (Germany). Different nanoparticle concentrations were achieved by adjusting the feed rate via rotational speed regulation.
- Temperature, humidity, pressure in air chamber: No data
- Air flow rate: No data
- Air change rate: No data
- Method of particle size determination: Information provided by Sigh C et al., 2014 and Fh-IME Schmallenberg.
- Treatment of exhaust air: No data

TEST ATMOSPHERE
- Brief description of analytical method used: See below in “Details on analytical verification of doses or concentrations”
- Samples taken from breathing zone: No data

VEHICLE
- Justification for use and choice of vehicle: Clean air
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle: Not applicable
- Purity of vehicle: No data available

OTHER
Control group animals were provided with clean air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aerosol concentrations were continuously recorded by a light scattering aerosol photometer (Fraunhofer ITEM, Hannover, Germany) and compared to additional filter sample analysis.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours per day / 5 days per week

Dose / conc.:

0 mg/m³ air

Remarks:

(control)

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

0.12 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

0.33 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

1.06 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

3.04 mg/m³ air (analytical)

Remarks:

basis: measured nano-CeO2 conc.

No. of animals per sex per dose:

Not reported (only a total of 576 animals is mentioned).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose range selected should cover specific conditions of absent inflammation in combination with absent lung overload (0.1 and 0.3 mg/m3), inflammation and no overload (1.0 mg/m3) as well as inflammation and overload (3.0 mg/m3).
- Rationale for animal assignment, rationale for selecting satellite groups: Animals were randomly assignated.
- Post-exposure recovery period in satellite groups: Yes, up to 1-, 28- and 90-day post-exposure observation.
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The health condition of animals was checked daily. Broad inspection for clinical abnormalities outside of the cage were done once a week.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed after one and 28 days of exposure as well as after one, 28 and 90 days post-exposure period. On exposure days, clinical observations were done before, after and if necessary during exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were checked once a week

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded weekly for a representative subgroup of ten animals from each dose group.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was recorded weekly for a representative subgroup of ten animals from each dose group.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken by puncture of the retrobulbar venous plexus at post-exposure day one. Since small change (increase of 5-10% neutrophil level in CeO2 treatment) were observed at this time point, no further analysis at a later time point was performed.
- Anaesthetic used for blood collection: Yes, slight isoflurane anesthesia.
- Animals fasted: No data - How many animals: 10 rats per test group
- Parameters checked: Full blood analysis parameters were recorded according to OECD TG 413 requirements.

CLINICAL CHEMISTRY: Yes,
- Time schedule for collection of blood: Blood was taken by puncture of the retrobulbar venous plexus at post-exposure day one. Since no change were observed at this time point, no further analysis at a later time point was performed.
- Animals fasted: No data
- How many animals: 10 rats per test group
- Parameters checked (in serum): Full clinical chemistry parameters were recorded according to OECD TG 413 requirements.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
* LUNG BURDEN
In order to determine the lung retention of CeO2, five animals of all dose groups were examined at all days of sacrifices. Explanted lungs of exposed animals were separated and the right lobes were used for analysis of lung burden. The isotopes 140Ce/142Ce in organ samples were quantified via inductively coupled plasma mass spectrometry (ICP-MS) using a quadrupole ICP-MS system (X-Serie II, Thermo Fisher Scientific). Sample preparation included lyophilisation of shredded tissue for at least 6 h (0.37 mbar). Organ weights were recorded prior and subsequently to freezedrying. For removal of organic material samples were further processed by plasma ashing (cool plasma conditions, 400 W, 1 mbar O2, 24 h) and subsequent microwave digestion (H2SO4, 96%, supra quality, max. 500 W).

* BRONCHOALVEOLAR LAVAGE ANALYSIS
Bronchoalveolar lavages (BAL) of rat lungs were performed in five animals of each dose group at all five time points. The method is based on Henderson RF et al., 1987 with minor modifications. Lungs were lavaged twice using 4 mL 0.9% NaCl. The following parameters were determined from collected lavage fluids: total cell count, differential cell count (macrophages, neutrophils, eosinophils and lymphocytes), biochemical mediators (lactic dehydrogenase, ß-glucuronidase and total protein), as well as cytokine levels. Total cell counts were measured using a counting chamber (Fuchs-Rosenthal). Differential cell counts were prepared by centrifugation of BAL fluid on cytoslides and subsequent Giemsa staining. Biochemical indicators were determined in the supernatant of centrifuged BAL fluid according to routine clinical chemistry protocols.

Sacrifice and pathology:

Animals were killed by carbon dioxide overdose and subsequent exsanguination. All organs and tissues were preserved and wet weights were recorded according to OECD TG 413.

Histopathological examinations of respiratory organs were performed at all implemented days of sacrifice in ten animals of the clean air control and 3.0 mg/m3 CeO2 respectively. Left lung lobes including bronchi as well as mediastinal and tracheobronchial lung-associated lymph nodes, trachea, pharynx and nasal cavities including nasal mucosa associated lymphoid tissue were investigated.

All respiratory tract organs were fixed in formalin (10%) for 24 h and trimmed according to Ruehl-Fehlert C et al., 2003, Kittel B et al., 2004 and Morawietz G et al., 2004. The left lung lobe was inflated with formalin (10%) at 20 cm water pressure prior to formalin fixation. After trimming tissues were embedded in paraffin, sectioned, and hematoxylin and eosin (HE) stained for analysis Additionally, Masson trichrome staining of the lung was done for detection of connective tissue production.

Other examinations:

*IMMUNOHISTOCHEMISTRY: See in the RSS: "Nano-CeO2 - Gen. Tox in vivo Rat DNA damage - V4 2017Schw"
To investigate the underlying mechanism of the detected histopathological changes, immunohistochemical staining of lung tissue for markers related to genotoxicity, proliferation and apoptosis were applied. Four markers were selected to determine possible particle-related genotoxicity (Histon γ-H2AX and Hydroxy-2′-deoxyguanosine (8-OHdG)), proliferation (Ki67), and apoptosis (cleaved caspase-3).

Statistics:

Evaluation of body weights, food and water consumption as well as hematology data was done applying ANOVA with Dunnett post-hoc comparison. Bronchoalveolar lavage parameters and immunohistochemistry marker levels were statistically evaluated using Kruskal-Wallis-ANOVA with Mann-Whitney U-Test as post-hoc analysis. Histopathological findings were analyzed by a two-tailed Fisher test.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs due to particle exposure were not observed.

Mortality:

no mortality observed

Description (incidence):

All animals were in good physical conditions up to sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weights were detected (data not shown).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant changes in food consumption were detected (data not shown).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No significant changes in water consumption were detected (data not shown).

Ophthalmological findings:

not examined

Haematological findings:

not specified

Description (incidence and severity):

Hematological parameters were measured after end of nanoparticle exposure (day 90 + 1rec). Since small changes ( increase of 5-10% neutrophil level in CeO2 treatment) were observed at this time point, no further analysis at a later time point was performed. Compared to control levels, in the mid (1.0 mg/m3) and high (3.0 mg/m3) dose group of CeO2 the ratio between segmented neutrophils and lymphocytes shifted in favor of increasing neutrophil numbers. The only significant value was measured for neutrophil levels in the CeO2 mid dose group. Further blood parameters measured did not display any significant changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical markers measured did not display any significant changes.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Respiratory organs of rats exposed to clean air and 3.0 mg/m3 cerium oxide were examined histopathologically at all 5 days of sacrifice. Table 3 (see section "Any other information on results incl. tables") presents an overview of the most prominent findings with mean grades of severity, separately calculated for all groups and time points considered. After 6 h exposure to 3 mg/m3 CeO2 nanoparticles already caused significant accumulations of particle-laden macrophages in the alveolar space and bronchus-associated lymphoid tissue (BALT). The amount of macrophages increased up to the end of post-exposure with translocation to the lung associated lymph nodes (LALN) detected from day 28 of exposure. These findings were accompanied by alveolar and interstitial inflammatory cell infiltrations and very slight bronchiolo-alveolar hyperplasia. Free particles (agglomerates) were detected in the alveolar space after end of exposure, mainly in areas of macrophages containing particulate matter. The authors indicate that such accumulations often originate from degrading macrophages. All described pathological conditions remained persistent during 90-day post-exposure. In addition to that, signs of interstitial fibrosis were detected (mean grade of severity were 0.2, 0.3 and 0.7 at day 90+1, 90+28 and 90+90 post exposure period, respectively) that was significant at d90+90 post exposure recovery only.

The authors reported also alveolar/interstitial foci of macrophages and inflammatory cells. Those infiltrations mainly consisted of lymphocytes and were often located next to bronchioles. Some foci further showed development of a granulomatous inflammation but the number of animals concerned was not specified. Accumulations of particle-laden macrophages, with syncytial giant cell formation were additionally found in BALT and LALN. Detailled overview of histopathological findings with grade and incidence is reported in Table 4 (see section "Any other information on results incl. tables"). The presence of particle-laden macrophages indicated its migration from the alveolar space to lymphoid tissue for clearance of phagocytosed material. Foci of bronchiolo-alveolar hyperplasia of the bronchiolar type (syn.: alveolar bronchiolization) occurred at very slight (minimal) grade in lungs from 28 days of exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Description (incidence and severity):

*LUNG BURDEN:
The lung burden was predicted based on the deposited alveolar fraction calculated using the "multiple path particle dosimetry (MPPD) model" and expected first order elimination with half-times of 70 days (0.1, 0.3, 1.0 mg/m3) or 200 days (3.0 mg/m3). The lung burden analysis showed a substance deposition that is concentration dependent (see Table 2 in "Any other information on results incl. tables"). The lower CeO2 dose groups (0.1, 0.3 mg/m3) as well as the mid and high (1.0, 3.0 mg/m3), respectively showed similar development of lung burdens (deposition fraction: 10.5%, 11.5%, 10.9% and 9.6%, respectively). But at higher CeO2 concentrations, higher deposition rates have been detected with reduced elimination, especially for 3.0 mg/m3 CeO2 (half-time >200 days). Nevertheless, particle elimination was visible in all treatment groups after end of exposure and lung burdens clearly tended to recover during the post-exposure period. Ubiquitous Ce levels were detected in the clean air control groups also. The authors claimed that predicted values for Ce deposition during 90 days exposure were quite close to the measured lung retention. The calculated deposition fraction as well as the expected non-overload or overload conditions after exposure to 0.1 and 0.3 or 3.0 mg/m3 nanoparticles, respectively match quite well. However, differences between predicted (higher predicted values) and measured values increased over time.

*BRONCHOALVEOLAR LAVAGES:
The BALF analysis showed a statistically significant time- and concentration-dependent increase of inflammatory cells, especially neutrophils (PMN) and lymphocytes (LYMPH) along with a slight increase total protein (TP), lactate dehydrogenase (LDH) and ß-glucuronidase (GL) levels in the CeO2 high dose group. Increased occured from 28 and 90 days of exposure for the concentration 1 and 3 mg/m3, respectively. Respective parameters decreased during post-exposure but did not reach controls levels until the end of the study. It should be note that a high variability was observed in these parameters, more particularly at the time point 90 days exposure + 1 days recovery in the highest dose-group.

*IMMUNOHISTOCHEMISTRY:
See in the RSS: "Nano-CeO2 - Gen. Tox in vivo Rat DNA damage - V4 2017Schw"

Remarks on result:

not determinable

Remarks:

As the study reported mainly histological evaluation of lungs of animals from the control and the highest concentration tested in the study (3 mg/m3 nanoCeO2), a definition of NO(A)EC/LO(A)EC for systemic and local effects is not possible.

Table 2. Lung burden and clearance half-times of exposed rats

	Lung burden (µg/lung +/- SD)					Clearance t1/2 (days)
	Day 1	Day 28	Day 90 + 1 rec.	Day 90 + 28 rec.	Day 90 + 90 rec.
Clean air	1.2 +/- 1.0	0.6 +/- 0.2	1.8 +/- 0.8	0.8 +/- 0.5	1.3 +/- 1.3	-
0.1 mg/m3 CeO2	2.5 +/- 0.8	12.0 +/- 2.9	33.1 +/- 1.4	24.7 +/- 6.1	13.2 +/- 3.2	67
0.3 mg/m3 CeO2	5.4 +/- 1.9	33.5 +/- 2.8	99.2 +/- 10.1	85.1 +/- 18.2	41.9 +/- 8.8	69
1.0 mg/m3 CeO2	19.6 +/- 5.6	152 +/- 37.4	476 +/- 74.0	366 +/- 24.7	263 +/- 15.4	108
3.0 mg/m3 CeO2	21.0 1.0	391 +/- 92.3	1280 +/- 82.5	1285 +/- 69.9	1013 +/- 243	224

 

Table 3. Summary of significant histopathological findings after exposure to 3.0 mg/m³ CeO2

CeO2	Lung	d1	d28	d90 + 1	d90 + 28	d90 + 90
	Accumulation, particle-laden macrophages, alv./interst.	*** 1.0	*** 1.0	*** 1.0	*** 1.9	*** 1.9
	Accumulation, particle-laden macrophages, BALT	* 0.5	*** 1.0	*** 1.4	*** 1.7	*** 1.5
	Hyperplasia, bronchiolo-alveolar	0.0	0.4	* 0.6	*** 0.9	* 0.5
	Infiltration of inflammatory cells, alv./interst.	0.0	0.2	*** 1.4	*** 1.5	*** 1.5
	Particles, alveolar	0.0	0.0	*** 1.0	*** 1.0	*** 1.0
	Fibrosis, interstitial	0.0	0.0	0.2	0.3	** 0.7
	Lung-associated lymph nodes (LALN)
	Accumulation, particle-laden macrophages, mediastinal Inn.	0.0	** 0.7	*** 1.9	*** 2.8	*** 3.1
	Accumulation, particle-laden macrophages, tracheobr. Inn.	0.0	*** 1.0	*** 2.7	*** 2.7	*** 3.0
	Nasal cavity
	Accumulation of particle-laden macrophages, NALT	0.0	* 0.5	*** 1.0	* 0.6	*** 1.0
	Globules, eosinophilic, olfactory epithelial	0.0	0.0	0.2	0.5	0.2
	Globules, eosinophilic, respiratory epithelial	0.0	0.0	0.2	0.1	0.2
	Hyperplasia, mucous cell	0.0	0.0	0.0	0.0	0.0
	Infiltration, inflammatory cell, subepithelial	0.0	0.0	0.0	0.2	0.0

 

BALT = bronchus-associated lymphoid tissue; NALT = nasal mucosa-associated lymphoid tissue; Values are presented as mean grade of severity: 0 = none, 1 = very slight, 2 = slight, 3 = moderate, 4 = severe (color gradient from green to red indicates increasing severity); n = 9-10; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. clean air control; Group Factor Chi-Squared and Fisher's Exact two sided/Pearson two sided

 

Table 4: Detailled overview of histopathological findings with grade and incidence of effects per animal from control (clean air) and 3.0 mg/m3 CeO2 groups.

 

Histoptahological findings		Incidence
		Day 1		Day 28		Day 90 + 1 day of recovery		Day 90 + 28 days of recovery		Day 90 + 90 days of recovery
		Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2	Ctrl.	CeO2
Lung
Accumulation, particle-laden macrophages, alveolar/interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Very slight	0	10	0	10	0	10	0	1	0	1
	slight	0	0	0	0	0	0	0	9	0	9
Accumulation, particle-laden macrophages, BALT*	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	5	0	10	0	6	0	5	0	6
	slight	0	0	0	0	0	4	0	3	0	3
	moderate	0	0	0	0	0	0	0	2	0	1
Hyperplasia, bronchiolo-alveolar	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	4	0	4	0	7	0	5
	slight	0	0	0	0	0	1	0	1	0	0
Infiltration of inflammatory cells, alveolar/interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	2	0	6	0	5	0	5
	slight	0	0	0	0	0	4	0	5	0	5
Particles, alveolar	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	10	0	10	0	10
Giant cells, syncytial, BALT*	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	2	0	5	0	2
Fibrosis, interstitial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	2	0	3	0	7
Lung-associated lymph nodes
Accumulation, particle-laden macrophages, mediastinal lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	7	0	2	0	0	0	0
	slight	0	0	0	0	0	6	0	2	0	0
	moderate	0	0	0	0	0	1	0	8	0	9
	severe	0	0	0	0	0	0	0	0	0	1
Accumulation, particle-laden macrophages, tracheobronchial lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	10	0	0	0	0	0	0
	slight	0	0	0	0	0	3	0	0	0	0
	moderate	0	0	0	0	0	6	0	8	0	10
	severe	0	0	0	0	0	0	0	1	0	0
Giant cells, syncytial, mediastinal lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	4	0	7	0	10
Giant cells, syncytial, tracheobronchial lnn.	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	Present, no grade	0	0	0	0	0	8	0	8	0	10
Nasal cavity
Accumulation of particle-laden macrophages, NALT*	10	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	5	0	10	0	6	0	10
Globules, eosinophilic, olfactory epithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	1	2	2	3	1	2
	slight	0	0	0	0	0	0	0	1	0	0
Globules, eosinophilic, respiratory epithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	1	2	0	1	2	2
	slight	0	0	0	0	0	0	0	0	0	0
Hyperplasia, mucous cell	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	0	0	0	0	0
	slight	0	0	0	0	0	0	0	0	0	0
Infiltration, inflammatory cell, subepithelial	N° of rats examined	10	10	10	10	10	10	10	10	10	10
	very slight	0	0	0	0	0	0	0	0	0	0
	slight	0	0	0	0	0	0	0	1	0	0

 

*BALT bronchus-associated lymphoid tissue, NALT nasal mucosa-associated lymphoid tissue

Conclusions:

All animals appeared in a good physical conditions up to sacrifice (based on body weight, food and water consumption) and no clinical signs due to particle exposure were observed. However, inflammatory reactions were seen in the lung at 1.0 and 3.0 mg/m3 CeO2 exposure and a lung overload at 3 mg/m3 nano CeO2 exposure with a shift overtime to from non-adverse to adverse findings with development of interstitial fibrosis although the grade was minimal.
However, no NO(A)EC/LO(A)EC for systemic or local effect can be derived in this study due to low number of pramaters examined and reported in this study.

Executive summary:

Schwotzer D.et al. (2017) investigated potential health effects of cerium oxide NM-212 nanoparticles to rats in a subchronic inhalation toxicity study. The study was performed according to OECD TG 413 but there is no mention to GLP compliance. Further, several parameters that should be analysed according to this guideline were briefly reported (e.g. clinical signs, body weight) or not reported (e.g. organ weight, FOB, macroscopic analyses). In addition, histopathological analyses were porformed on respiratory tract only and solely in the control and high dose (3m/m3 CeO2) groups. For this reason, the study was awarded a reliability score of 2 (Klimisch, 1997) and was used as a supporting study.

Female Wistar rats were exposed nose only to nanoCeO2 NM-212 (primary particle size: 28,4 nm) at 0 (clean air control), 0.1, 0.3, 1.0 and 3.0 mg/m3 (with a MMAD ranging from 0.63 to 0.79 µm) 6h/day, 5 days/week for 1, 28 or 90 days followed by 1-, 28- or 90-day recovery periods. The dose range of this study was selected to cover specific conditions of absent inflammation in combination with absent lung overload (0.1 and 0.3 mgm3), inflammation and no overload (1 mg/m3) as well as inflammation and overload (3 mg/m3).

The health condition of animals was check daily. Broad inspection for clinical abnormalities outside of the cage, body weight, food and water consumption were recorded once a week. Heamatology and clinical chemistry were done at post-exposure day 1.Lung burden were analysed in 5 animals of all dose groups at all days of sacrifices. Bronchoalveolar lavage analyses (total cell count, differential cell count, biochemical mediators, cytokines) were done in 5 animals of all dose groups at the 5 time points. Histopathological examinations of respiratory organs (left lung lobe, bronchi, mediastinal and tracheobronchial associated lymph nodes, trachea, pharynx, nasal cavities and nasal mucosa associated lymphoid tissue) were performed at all days of sacrifice in ten animals of the clean air control and the 3.0 mg/m3 CeO2.

 

According to the authors, all animals were in good physical conditions up to sacrifice. No significant changes in body weights, food and water consumption were reported and clinical signs due to particle exposure were not observed. But no qualitative or quantitative results were shown.

Blood and biochemical parameters (not specified) measured at post exposure day 1 did not display any significant changes excepted for neutrophil levels in the CeO2 mid dose group. Compared to control levels, in the mid (1.0 mg/m3) and high (3.0 mg/m3) dose group of CeO2 the ratio between segmented neutrophils and lymphocytes shifted in favor of increasing neutrophil numbers.

 

The BALF analyses done after 1 and 28 day of exposure and day 1, 28 and 90 post exposure showed a time- and concentration-dependent increase of the inflammatory cells PMN (significant from 28 days of exposure to 3 mg/m3 CeO2) and lymphocytes (significant from Day 1 post exposure at 1 and 3 mg/m3 CeO2). However, high variability was observed in the results, more particularly at 1days post exposure. PMN but not lymphocytes cells number tend to decrease during the post-exposure period. A slight increase of total protein (TP), lactate dehydrogenase (LDH) and beta-glucuronidase (GL) levels were also observed in the CeO2 high dose group from day 28 of exposure to day 1 post exposure period, then a decrease of all these parameters was observed afterward.

 

The lung burden analysis showed that the substance deposition was concentration dependent and particle elimination was visible in all treatment groups after end of exposure. But at higher CeO2 concentrations, higher deposition rates have been detected with reduced elimination especially for 3.0 mg/m3 CeO2. Thus, corresponding calculated clearance half-time values were 67, 69, 108 and 224 days after 0.1, 0.3, 1.0 and 3.0 nanoCeO2 mg/m3, respectively. The authors concluded that the overload/inflammation hypothesis were achieved for the low dose levels (0.1 and 0.3 mg/m3) and the high dose level (3.0 mg/m3). Clearance half-times were below the expected mean value of 70 days for the low doses levels while for the 3 mg/m3 exposure group, the half-life was > 200 days for particle clearance reflecting a present overload situation. For the 1 mg/m3CeO2 dose, clearance half-time was calculated to be 108 days and signs of inflammation were present at this dose and thus, the expected situation of lung inflammation at non-overload was not clearly achieved. The authors calculated a deposition fraction of about 10 % of the initial nanoparticle concentration for all exposure doses.

 

Histopathology analyses, performed only on respiratory organs (lungs, lung-associated lymph nodes and nasal cavity) of the rats from control group (clean air) and from the high dose group only (3 mg/m3 CeO2) after 1 day and 28 days of exposure and at Day 1, 28 and 90 days of post-exposure showed that after the first exposure to CeO2 nanoparticles, very slight but significant accumulations of particle-laden macrophages in the alveolar space and bronchus-associated lymphoid tissue (BALT) were observed. The amount of macrophages increased in few animals from slight to moderate up to the end of the post-exposure period with very slight to moderate translocation to the lung associated lymph nodes (LALN) detected from day 28 to the end of the recovery that indicated clearance of the phagocytosed material. Very slight to slight alveolar and interstitial inflammatory cell infiltrations and very slight bronchiolo-alveolar hyperplasia were observed after 28 days of exposure to high dose of nanoCeO2 up to the end of the recovery period. Free particles (agglomerates) were detected in the alveolar space after end of exposure, mainly in areas of macrophages containing particulate matter. Such accumulations often originate from degrading macrophages. Furthermore, during the recovery period, very slight interstitial fibrosis was observed which was significant at the 90 day-post exposure time point. No pathological change was observed in the nasal cavity of exposed rats.

Acccording to the authors, this series of effects observed at the highest dose level illustrates the consequence of particle overload (impaired macrophage activity and particle elimination leads to translocation of particles to the interstitium, causing local interstitial effects like inflammatory cell infiltrations associated with fibrotic lesions although observed at a very slight grade.

 

Overall, animals appeared in a good physical conditions up to sacrifice. However, inflammatory reactions were seen in the lung at 1.0 and 3.0 mg/m3 CeO2 exposure based on bronchoalveolar lavages and a lung overload at 3 mg/m3 nano CeO2 exposure with a shift overtime to from non-adverse to adverse findings with development of interstitial fibrosis although the grade was minimal.

However, no NO(A)EC/LO(A)EC for systemic or local effect can be derived in this study due to the low number of parameters examined and reported in this study.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focused on the lung effect of nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

This study was performed in order to assess the influence of redox activity by modifications of cerium dioxide nanoparticles (nano-CeO2) via zirconium doping on the distribution, pulmonary and cardiovascular effects in mice following sub-acute inhalation. This study further explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Only results observed with nano CeO2 are reported in this summary. Mice from each strain were exposed to the nanoparticles during 4 weeks and the effects were evaluated 4 weeks after the last exposure day (i.e day 56).
Experiments were conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable local and EU regulations.

GLP compliance:

not specified

Remarks:

The GLP compliant status was not specified in this published article.

Limit test:

no

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

3 mice models were used in this study to explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice model, a well established model for the study of the vascular disease atherosclerosis (Coleman et al., 2006). ApoE-/- mouse were used to study hematology, pulmonary and cardiovascular effects of the nanoparticles.
- Alzheimer’s disease mouse model (5xFAD), were included to study neurological effects (not included in the article and should be published in another article) and the hematology and pulmonary effects of the nanoparticles.
- C57BL/6J mouse - (non-genetically modified) strain, was used as the background strain of the disease mouse models and was used to study biodistribution, hematology and pulmonary effectsof the nanoparticles.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and source:
* Specific Pathogen free (SPF) ApoE−/− mice come from Taconic, Danemark
* 5xFAD and wild type (WT) cross bread C57BL/6J littermates mice come from Jackson Laboratories.
- Age at study initiation:
* ApoE−/− mice: 10-12 weeks
* 5xFAD and C57BL/6J mice :8-11 weeks
- Weight at study initiation, fasting period before study: No data available
- Housing: macrolon cages
- Diet: Ad libitum, The ApoE-/- mice were fed a commercially available rodent Western (high fat) diet (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), starting at the first day of the exposure period until the end of the experiment. The other mice were fed a standard commercially available rodent diet (SMR-A, ABdiets, Woerden, The Netherlands).
- Water: Ad libitum, except during the exposure periods
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2 °C
- Humidity: 40 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 280 nm

Geometric standard deviation (GSD):

1.55

Remarks on MMAD:

The Count Median Diameter (CMD) and the Mass Median Diameter (MMD) were estimated using the Aerosol Instrument Manager Software (Release Version 9.0.0.0, 15:32:53, Nov 11 2010 from TSI Inc., St Pauls, MN, USA), assuming spherical aggregation around primary particles of 4.7 ± 1.4 nm.

Details on inhalation exposure:

Approximately one week before the 4-week exposure period, 20 samples of the nano-CeO2 (one for each day) with a concentration of 1 mg/mL were prepared from the stock dispersions (20 mg/mL), by diluting with ultrapure water to the desired concentration. Stock and sample dispersions were sonicated for 5 minutes in an ultrasonic bath (Branson CPX2800, 40 kHz, 110W) before use to re-disperse any possible agglomerates.
Freshly generated aerosols of NPs were generated using a spray nozzle technique, diluted with pressurized clean particle-free air, and heated to 24-25°C.
Exposure was controlled based on stable particle number counts, mass concentrations, temperature and relative humidity, measured continuously using a condensation particle counter (CPC 3022A from TSI Inc., St. Paul, MN, USA), a tempered element oscillating microbalance (TEOM series1400A from Rupprecht & Patashnick, NY, USA) and M-170 Measurement Indicator (Vaisala M170, Vaisala Oyj, Helsinki, Sweden), respectively, during each exposure period.
During the 3 hour exposure periods to the nanoparticles, the control groups were exposed to filtered air under the same conditions (nose-only tubes) for the same amount of time.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was characterized at least twice during each exposure session using an optical particle sizer (OPS 3330, TSI Inc., St. Paul, MN, USA) and a scanning mobility particle sizer (SMPS 3936 from TSI Inc.,St. Paul, MN, USA). In addition, aerosols were collected on polycarbonate filters for scanning electron microscopy (SEM) analysis. The SEM samples were prepared by placing a small piece of the filter on the SEM stub and coating it with platinum, and visualizing with an XL30 Environmental SEM-FEG microscope (Philips XL30 ESEMFEG).
The total mass concentration generated over the 3-hour exposure period was determined by gravimetric analysis of pre-weighed and post-weighed polytetrafluoroethylene (PTFE) filters (Teflo R2PJO47, Pall corporation, Port Washington, New York, USA) using a micro-balance (Mettler MC or ME-5 microbalance, Mettler-Toledo LLC, Columbus, OH, USA).

Duration of treatment / exposure:

4 weeks of exposure. Tthe effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

To explore the (patho)physiological effects of nanoparticle on multiple organ systems, three different mouse models were exposed:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice are a well established model for the study of the vascular disease atherosclerosis (Coleman et al.,2006), a disease characterized by the build-up of lipid- and inflammatory cell-rich plaques within arteries, which underlies the majority of cardiovascular diseases. The 4-week exposure protocol was integrated into an 8-week high-fat feeding regime that has been shown to generate complex atherosclerotic plaques with many of the hallmarks of the human disease in specific arterial locations (Cassee et al., 2012, Miller et al., 2013).
ApoE−/− mice were used to study hematology, pulmonary and cardiovascular effects.
- The 5xFAD mice are an Alzheimer’s disease mouse model. Although these mice were included to study neurological effects that will be published in a separate paper, the hematology and pulmonary effects were also studied within the same animals and reported in this paper.
- C57BL/6J mice were used as the background (non-genetically modified) strain of the disease mouse models and used to study biodistribution, hematology and pulmonary effects.
Hematology, pulmonary and cardiovascular effects were assessed 4 weeks post exposure. This period was included, firstly, to provide the extra four weeks necessary for mice to develop sufficient plaque formation in arteries and secondly, to investigate the persistency of the pulmonary and cardiovascular effects.
One group of each mouse model was exposed nose-only for four weeks to 4 mg/m3 nano-CeO2 for 5 days/week for 3 hours/day and one control group to clean air under the same experimental conditions (nose-only tube, 3h). Effects were assessed 4 weeks post the final exposure (56 days after the initial exposure).
The number of animals per group was different for each of the mouse models, to provide sufficient statistical power to detect differences between exposed and control animals in the most important effect parameter of each mouse model. Based on previous experiments, 8 ApoE-/- mice per group were expected to be sufficient to detect statistically significant differences in atherosclerotic plaque size and 5 C57BL/6J mice per group were expected to be sufficient to detect statistically significant differences in the number of neutrophils in the bronchoalveolar lavage fluid (BALF). However, 10 C57BL/6J mice per group were included, since 10 C57BL/6J mice per group and 16 5xFAD mice per group were needed to provide sufficient statistical power in the neurological study.

Positive control:

no

Observations and examinations performed and frequency:

Animals were monitored by cage-side observations and, if necessary, handled to detect signs of compromised health. The body weight of each animal was recorded one day before the start of exposure (day -1), prior to exposure on the first day and weekly thereafter.

Hematology, neurological (results published in a separate article which was not avaible at the time of the dossier preparation), pulmonary and cardiovascular effects were assessed 4 weeks after the last exposure of the mice (i.e. 56 days after the initial exposure).

- QUANTIFICATION OF CERIUM IN TISSUES
During necropsy, organs from half of the C57BL/6J mice per group were obtained to evaluate the distribution of the nano CeO2 throughout the body. Liver, spleen, kidneys, heart and right (exposed mice) or left (control mice) lung, were weighed and immediately frozen in liquid nitrogen for determination of the Ce concentrations. To allow measurement of multiple parameters within the same animal, different parts of the lungs were selected for the exposed compared to the control animals. From the exposed groups the right lung was used for quantification of Cerium, because the left lung was needed for histopathological examination. From the control group the left lung was used for quantification of Cerium, because the right lung was needed for bronchoalveolar lavage. The organs were digested by acidification of each sample with 2 mL nitric acid for 12h. Hydrogen fluoride (0.2 mL) was added, followed by microwave heating for 45 min up to 185°C, and maintained for a further 20 min. Boric acid (2 mL) was added to neutralize the hydrogen fluoride, and the samples were re-heated for 20 min to 160 °C, and maintained for 10 min. Once cooled, samples were filtered with a 450 nm syringe filter, diluted with 10 mL deionized water and stored at room temperature (RT). The presence of Cerium in the lungs, liver, spleen, kidneys and heart was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NexION 300X instrument operated in standard mode for Cerium. The isotope measured was 140Ce using 115In and 159Tb as internal standards. Calibration standards (0 – 100 μg/L) were prepared from VWR 1000 mg/L stock solutions. Quantities are expressed as µg/g organ tissue.

- HEMATOLOGY
Animals were anaesthetized with a mixture of ketamine and xylazine. Two blood samples were taken by eye extraction. The first sample was collected in a K3-EDTA tube (Minicollect K3EDTA, 1 mL, 450474 Greiner Bio-One) for hematological parameters as determined in a blood auto analyzer (ADVIA 2120 Hematology System, Siemens Healthineers) within 3 hours after collection. The second sample was collected in a serum tube and stored at -20°C for further analysis. Total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cells counts were meaured.

- BRONCHOALVEOLAR LAVAGE
Lung lavage was performed at necropsy. A cannula was placed in the trachea and the diaphragm opened to decrease the amount of air inside the lungs. For the control animals and all ApoE-/- animals, the right lung half was rinsed twice with approximately 0.5 mL (26.7 mL per kg body weight) of physiological saline solution, after ligation of the left lung. The injected volume was inserted and recovered 3 times, after which the lavage liquid was collected and stored on ice for less than 2 hours. To allow necropsy of the planned number of animals within one day, both lungs were lavaged for 10 of the 16 exposed 5xFAD mice and 5 of the 10 exposed C57BL/6J mice, using the same procedure, but approximately 0.8 mL (40 mL per kg body weight) of physiological saline solution.
BALF was centrifuged at 400g for 10 minutes at 4°C. The supernatant was divided into two separate aliquots of 125 µL for total protein (TP; an indicator for acute lung injury), lactate dehydrogenase (LDH; an indicator of cytotoxicity), gammaglutamyl transferase (GGT; an indicator of lung cell damage) and alkaline phosphatase (ALP; an indicator of type II cell damage) measurements using an autoanalyser (LX20- Pro, Beckman-Coulter, Woerden, the Netherlands). The cell pellet was scored for the presence of erythrocytes, re-suspended in 500 μL phosphate-buffered saline (PBS) and kept on ice. Cell counts were determined in the re-suspended pellet using a Coulter counter (Beckman-Coulter, Live Sciences). Cell concentrations were determined using a single sample using at least 150 µL of the re-suspended cells. Cytospins (Cytospin 3, Thermo-Shandon) were prepared and stained using May-Grunwald and Giemsa stain, and cell differentiation was performed by counting 400 cells per slide.

Sacrifice and pathology:

HISTOPATHOLOGY
- Lung tissue:
For the majority of the animals, the right lung was removed after the collection of the BALF and immediately frozen in liquid nitrogen and stored at - 80°C for further analysis. The left lung was removed and, after weighing, cannulated and infused with formaldehyde for 1 hour at a pressure of 20 cm H2O. Lungs were processed for histopathology; embedded in paraffin wax, sectioned at 2-4 µm intervals and stained with hematoxylin and eosin for histopathological examination.
Histopathological changes were described according to distribution, severity and morphological characteristics. The morphological characteristics of chronic inflammation include for example the presence of lymphocytes and macrophages in the lung tissue, while acute inflammation is characterized by the presence of polymorphoneclear neutrophils (PMNs).
Severity scores were assigned as follows: Grade 1 Minimal/very few/very small; Grade 2 Slight/few/small; Grade 3 Moderate/moderate number/moderate size; Grade 4 Marked/many/large; Grade 5 Massive/extensive number/extensive size.

- Assessment of atherosclerosis:
Arteries (brachiocephalic, aortic arch, thoracic aorta) were isolated from ApoE-/- mice. Atherosclerosis was quantified, as previously published (Miller et al., 2013). Briefly, brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 µm intervals, beginning at the first section of the artery with a fully intact media. Sections were stained with Masson’s Trichrome. The cross-sectional area of the plaque was measured and standardized to the medial area. A single mean value for atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery. A single section from each artery (the section exhibiting the largest plaque in cross-section) was chosen for mac-2 immunohistochemistry for macrophage-derived cells. A rat anti-mouse primary antibody was used (1/12000; CL8942AP, VH Bio, Gateshead, UK) with rat IgG (1/12000; I-400, Vector Labs, Peterborough, UK) as a negative control, followed by a goat anti-rat IgG biotinylated secondary antibody (BA-9400, Vector Labs). The area of positive staining was expressed as a proportion of the total plaque area.

- Other organs:
The spleen, liver, heart and kidneys were removed, weighed and stored in 4% formaldehyde for pathological analysis if required based on macroscopic findings.

Statistics:

Statistical analyses were performed using GraphPad Prism v7.00 (GraphPad Software, San Diego, California, USA). Ordinary one-way analysis of variance (ANOVA) analyses including all experimental groups were performed followed by a Tukey’s posthoc multiple comparisons test. A p-value ≤0.05 was considered statistically significant.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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):

Total and differential white blood cell (lymphocytes, neutrophils, eosinophils and monocytes) counts were measured 4 weeks after the last day of exposure (day 56). Results are the mean of 8 to 16 animals. ApoE-/- mice had more neutrophils compared to exposed and control C57BL/6J and 5xFAD mice (p<0.05 in Tukey’s post-hoc test following one-way ANOVA). No statistically significant differences were observed in the total white blood cell counts (data not shown) or differential (white blood cell counts of the exposed groups compared to the controls in blood from all strains of mice.

Clinical biochemistry findings:

not examined

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):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Although the authors used the 5xFAD mice, an Alzheimer’s disease mouse model, to study neurological effects of exposure to nanoparticles, they said that the results will be published in a separate paper that was not published at the time of the preparation of this dossier.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP but not in the other strains (see table 1 in "Any information on results incl. tables").
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the NP exposed animals but not in the control groups

Other effects:

no effects observed

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE (BALF)
- Total and differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytesin) from BALF measured 4 weeks after the last exposure day (mean of 5 to 16 animals) in the 3 mice strains: No statistically significant differences were observed in the total cell counts (data not shown) or differential cell counts in BALF of the exposed groups compared to the controls for any strain of mouse.

- Total protein, Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transpeptidase (GGT) levels in bronchoalveolar lavage fluid (BALF) 4 weeks after exposure (mean of 5 to 16 mice from the each tested strains):
Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels between the exposed and control groups.

No constitutive differences in differential cell counts or protein levels were observed between the different mice strains.

EFFECTS OF NANO CeO2 ON ATHEROSCLEROSIS IN THE BRACHIOCEPHALIC ARTERY OFApoE-/- MICE AFTER INHALATION
ApoE-/- mice exhibited regions of dense plaques in the aortic arch and branch points of large arteries. Plaques were composed of fibroblastic matrix, smooth muscle cells, lipid cavities and cholesterol crystals. Atherosclerotic burden was quantified in the brachiocephalic artery, with control (air-exposed) mice having a mean plaque size of 94 ± 9% (standardized to the area of the vascular media). Exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries (p=0.62; One-way ANOVA) and in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

Dose descriptor:

NOAEC

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

Highest concentration tested in this study.

Sex:

female

Basis for effect level:

other: No major toxicological effect

Critical effects observed:

no

Estimated deposition of inhaled nano-CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1020 µg. The total deposited dose (estimated using the MPPD model) was found to be 618 µg. The deposited fraction per region, estimated using the MPPD model, was found to be 48.8 % for the head, 3.5 % for the tracheobronchial region and 8.0 % for the alveolar region of the inhaled mass dose. The retained dose in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, was found to be 16.2 µg.

Quantification of cerium in tissues (en µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

The highest concentrations of Cerium were found in the lung (~ 67 µg/g), followed by much lower concentrations in the heart (~2.2 µg/g), spleen (~1.1 µg/g), kidneys (~0.3 µg/g) and liver (~ 0.2 µg/g), respectively. As would be expected, significantly higher levels of Ce were observed in the lungs of exposed mice compared to the controls (~1.1 µg/g in control lung). In most of the other organs, the cerium concentrations were not statistically signicantly different from the background concentrations measured in the control animals (heart (~2.8 µg/g), spleen (~1.0 µg/g), kidneys (~1.3 µg/g) and liver (~ 0.05 µg/g)).

Tha authors indicated that background levels of Ce detected in various organs of animals exposed to clean air (controls) might be caused by Ce contamination of the drinking water, food and/or bedding of the animals as previously found by others (Yokel et al. 2012, 2013, see the section 7.1 Toxicokinetics, metabolism and distribution of this dossier)).

The authors said that, based on the estimated retained dose of nano CeO2 in the lung (16.2 µg, see Table 3) using MPPD model and the average lung weight (158 mg), the expected Ce concentration in the lung was approximately 103 µg/g while the measured Ce concentration in the lungs of the nano CeO2 exposed mice was slightly lower (67 µg/g) but in the same order of magnitude. They concluded that the difference between the predicted and measured concentrations may reflect the lung clearance rate used by the MPPD model compared to the actual lung clearance rate in vivo.

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages	Particle loaded alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal	Minimal	Slight
C57BL/6J	Control	60% (3/5)a	-	-	-	-
	Nano CeO2	80% (4/5)	-	20% (1/5)	-	-
ApoE-/-	Control	25% (2/8)	-	-	-	-
	Nano CeO2	38% (3/8)	-	-	13% (1/8)	75% (6/8)
5xFAD	Control	69% (11/16)	-	6% (1/16)	-	-
	Nano CeO2	83% (5/6)	-	17% (1/6)	-	-

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

The authors concluded that nanoCeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease..

Executive summary:

In this subacute inhalation study, Dekkers et al. (2015) explored the (patho)physiological effects of nano CeO2 exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. As this study was performed using a test procedure in agreement with generally accepted scientific standards but with some limitations (incomplete data on substance characterisation and on systemic toxicity), the study was awared a reliability code of 2 according to Klimisch criteria and was used as weight of evidence.

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m3 nano CeO2 (primary particle size of 4.7 nm) for 3h/day, 5 days/week for 4 weeks.

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs were done and the tissue distribution of cerium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of nano-CeO2 on the artherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

According to the authors, no statistically significant differences were observed in the total or differential white blood cell counts in the blood and in the BALF of the exposed groups compared to the controls in all strains of mice. Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels in BALF between the exposed and control groups. Furthermore, no treatment related effects on organ weights were observed and no macroscopic findings related to treatment were observed. In lungs, the histopathological analyses showed an increased incidence, described as modest by the authors, in minimal chronic bronchoalveolar or alveolar inflammation observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in most of the ApoE-/- mice exposed to nano CeO2 NP but not in the other stains. In the bronchoalveolar lavage fluid, particle loaded macrophages were seen in all the exposed animals but not in the control groups. In tissues, background Ce concentrations were measured in the control animals and significantly higher levels of Ce were only observed in the lungs and the liver of exposed mice as compared to the controls but not in the other organs.

In ApoE-/- mice, exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries and in the proportion of plaque staining positive for mac-2. The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concuded that in this subacute inhalation study, nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

No NOAEL was derived in this study by the authors. However, according to the results and the conclusion of the authors (nano CeO2 has a low biological activity in their mouse models), it can be concluded that the NOAEC for lung toxicity is > 4 mg/m3, the only dose tested in this study.

Reference 6

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

other: standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: :

Remarks:

the study was well documented and performed according to generally accepted scientific principles and in compliance with GLP. However, given the experimental conditions applied (e.g., 5-day exposure duration), this study should be viewed as a preliminary work.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Repeated exposure of rats to an aerosol of nanometric cerium dioxide (nano-CeO2) for 5 consecutive days (6 h/day) through whole-body inhalation route
- Pulmonary inflammation: Analysis of the bronchoalveolar lavage fluid, lung tissue homogenates and blood samples (i.e., cytokine profiles)
- Tissue injury: Histopathological examination of all respiratory tract and extrapulmonary tissues using optical microscopy
- Cerium (Ce) content in the lung, lung-associated mediastinal lymph nodes and extrapulmonary organs

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI(Han) (i.e., Wistar)
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: < 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: In groups up to five animals in a polysulfone cage (H-Temp (PSU), TECNIPLAST, Germany) with a floor area of about 2065 cm² (610x435x215 mm) wooden gnawing blocks
- Diet: Ad libitum, GLP-certified feed (Kliba laboratory diet, Provimi Kliba SA, Switzerland), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: To adapt to the exposure conditions, the animals were acclimatized to fresh air under the study flow conditions in whole-body
inhalation chambers for 2 days before the start of the exposure period. Up to 2 animals/cage were exposed in wire cages, type DKIII (BECKER & Co., Germany) in a whole-body chamber.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: - NM-211
At the concentration of 0.5 mg/m3: 1.6 µm / 2.1
At the concentration of 25 mg/m3: 1.3 µm / 2.1

- NM-212
At the concentration of 0.5 mg/m3: 1.4 µm / 2.3
At the concentration of 5 mg/m3: 1.2 µm / 2.1
At the concentration of 25 mg/m3: 1.0 µm / 2.5

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed in wire cages that were located in a stainless steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3).
- Method of holding animals in test chamber: None
- Source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Nano-CeO2 aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany) using compressed air (1.5 m3/h). The so generated dust aerosol was diluted by conditioned air passed into whole-body inhalation chambers. The desired concentrations were achieved by varying the feeding speed of the substance pellet and by varying the rotating speed of the brush. Based on the data of a comprehensive technical trial, the aerosol concentrations within the chambers were considered to be homogenous (data not shown).
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: 54.5 m3/h
- Air change rate: 20 air changes/h
- Method of particle size determination: Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple personal cascade impactor (USA). In addition, light-scattering aerosol spectrometer (WELAS® 2000, Palas, Germany) was used to measure particles from 0.24 to 10 μm. To measure particles in the submicrometer range, scanning mobility particle sizer (SMPS 5.400, Grimm Aerosoltechnik, Germany) was used.
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: Generated aerosols were continuously monitored by scattered light photometers (VisGuard, Sigrist).
- Samples taken from breathing zone: No data available

VEHICLE
- Justification for use and choice of vehicle: No data available
- Composition of vehicle: Not applicable
- Concentration of test material in vehicle: See below in “Concentrations”
- Lot/batch no. of vehicle, purity of vehicle: Not applicable

OTHER
For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reached the control animals. For the treated animals, the amount of exhaust air was higher than the supply air (negative pressure) to prevent the contamination of the laboratory as a result of potential leakages from the inhalation chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle concentrations in the inhalation atmospheres were analysed by gravimetric measurement of air filter samples.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

25 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest aerosol concentration was 25 mg/m3, which was expected to cause biological effects and should lead to lung overload at least for 20 exposures. The mid and low aerosol concentrations were 5 and 0.5 mg/m3. The low aerosol concentration with an expected lung burden far below the overload condition should not lead to any adverse effects. The mid aerosol concentration, which was spaced 10-fold higher than the low concentration, was expected to cause some biological effects.
- Rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Yes, up to 24-day post-exposure observation
- Section schedule rationale: The post-exposure period and the examination time points were scheduled to address the progression or regression of the biological effects, with their correlation to lung burden and lung clearance kinetics.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations of the animals were recorded for each animal at least three times per day on exposure days and once a day during the pre-exposure and post-exposure periods. Signs and findings were recorded for each animal. During exposure, examination was possible only on a group basis.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0), and twice weekly until killing or twice within the 5 exposure days.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH, Germany))
- Animals fasted: Yes- How many animals: 5 rats per test group
- Parameters checked: Red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell as well as differential blood cell counts with a hematology analyser

CLINICAL CHEMISTRY: Yes, the extent of the examination was according to the data requirements of OECD test guideline 412.
- Time schedule for collection of blood: Blood sampling was performed in the morning by retro-orbital venous plexus puncture under anaesthesia.
- Animals fasted: Yes
- How many animals: 5 rats per test group
- Parameters checked (in serum): rat haptoglobin and rat γ2-macroglobulin by ELISA; enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and other blood parameters of clinical chemistry using an automatic analyser

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* BRONCHOALVEOLAR LAVAGE
Twenty-four hours and 35 days after the end of exposure, five animals per test group were killed by exsanguination from the aorta abdominals and vena cava under pentobarbital anaesthesia. The lungs of the animals were lavaged in situ twice with saline solution. A total of 11 mL bronchoalveolar lavage fluid (BALF) was obtained per animal for analysis. Aliquots of the BALF were used for the determinations of total protein concentration, total cell count, differential cell count, and activity of the enzymes. Total BALF cell counts were determined with a hematology analyser. Counts of macrophages, polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, monocytes, and atypical cells were performed on Wright-stained cytocentrifuge slide preparations. The differential cell count was evaluated manually by counting at least 400 BALF cells per sample. Using a reaction rate analyser, levels of BALF total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) were measured.

* INFLAMMATORY MEDIATORS IN BALF AND SERUM
Cytokines and chemokines in BALF and serum were measured. The parameters comprised various cytokines, chemokines, adhesion molecules, matrix metalloproteinases, acute-phase proteins, signal proteins of apoptosis, or cell proliferation: monocyte chemoattractant protein-1 level (MCP-1), cytokine-induced polymorphonuclear neutrophil chemoattractant-1 level (CINC-1/IL-8), macrophage colony-stimulating factor (M-CSF) and osteopontin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy and histopathology were performed after 5 days of exposure and 21 days after the end of exposure (5 days of exposure). In general, 5 animals per test group were investigated for pathological examination. At necropsy, animals were exsanguinated by opening of the abdominal great vessels under deep pentobarbital anaesthesia. All organs were preserved according to OECD TG No. 412. Following organs were weighed: adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, and thyroid glands. The lungs were instilled with and fixed in 10 % neutral-buffered formalin (NBF). Once fixed, the lungs were transferred to 70 % ethanol. All other organs were fixed in 10 % NBF. All the organs and tissues described in the OECD TG No. 412 were trimmed. After paraplast-embedding, the blocks were cut at 2- to 3-μm thickness, mounted on glass slides and stained with hematoxylin and eosin. Extrapulmonary organs and the respiratory tract compromising nasal cavity (four levels), larynx (three levels), trachea (transverse and longitudinal with carina), lung (five lobes), and mediastinal and tracheobronchial lymph nodes were assessed by light microscopy. For the lungs, whole histopathological examination was performed in animals of all test groups. For all other tissues, only the animals of the control and high concentration group of nano-CeO2 were initially examined. When changes were observed in the high concentration group, respective organs and tissues of the animals exposed to low and intermediate aerosol concentrations were also examined by light microscopy. All histopathological examinations were performed by a well-experienced board-certified veterinarian toxicopathologist followed by an internal pathology peer review.

Other examinations:

ORGAN BURDEN
Lung burden of the two different nano-CeO2 was evaluated twice, immediately after 5 days of exposure and after 21 days after the exposure end. Cerium (Ce) content in the lungs, lung-associated lymph nodes, and liver of either 3 or 5 animals per test group were examined. After digestion with mixed acid, samples of each lung or lymph node were dissolved in sulphuric acid and ammonium sulphate. 140Ce content in the obtained solution was analysed by inductively coupled plasma mass spectrometry (ICP-MS) or by inductively coupled plasma optical emission spectrometry (ICP-OES) with a wavelength of 419 nm. The limit of detection for Ce was 0.3 μg. The amounts of CeO2 in the respective tissues were calculated by measuring elemental Ce with ICP-MS.

Statistics:

For body weight changes, Dunnett’s test was used for a comparison of each test group with the control group test. Clinical pathology parameters (BALF cytology, enzyme data, and BALF and serum cell mediator data) were analysed by non-parametric one-way analysis using the Kruskal–Wallis test (two-sided). If the resulting p value was ≤0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U test (both two-sided) (p ≤ 0.05 for statistical significance). Comparison of organ weights among test groups was performed by non-parametric one-way analysis using the two-sided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians in case of p ≤ 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

The animals exposed for 5 days to NM-211 and NM-212 showed no clinical signs or findings compared to the control animals (data not shown).

Mortality:

no mortality observed

Description (incidence):

The animals exposed for 5 days to NM-211 and NM-212 showed no clinical signs or findings compared to the control animals (data not shown).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Five days of inhalation exposure to either NM-212 or NM-211 did not affect the body weight development of the animals (data not shown).

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:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative neutrophil counts in blood were increased 3 days after the end of exposure to 25 mg/m3 NM-212 and NM-211, whereas relative lymphocyte counts were decreased (see in Table 5 in "any other information on results" below). The changes were in a concentration-related manner. Twenty four days after the end of exposure, all parameters had returned to near control values. No other blood parameters were affected.

Clinical biochemistry findings:

not specified

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 5 days of exposure, no increase in lung weights was observed after exposure to NM-212. An aerosol concentration of 25 mg/m3 NM-211, however, resulted in significant increases in absolute and relative lung weights (+20 and 24 %, respectively). This increase in lung weight was no longer present 21 days after the end of exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

After 5 days of exposure, the macroscopic finding enlarged mediastinal lymph nodes were found in individual animals of all test groups. Twenty one days after the end of the exposure, enlarged mediastinal lymph nodes were observed in almost all animals exposed to Ceria NM-211 or NM-212. The tracheobronchial lymph nodes and all other examined organs did not show any macroscopic findings.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

* LUNGS
After 5 days of exposure, single or accumulated macrophages were located in the lumen of the alveoli and a few macrophages occurred also in the alveolar wall and ducts. They were distributed multifocally in all lobes over the whole lung. Most of them were loaded with amber-like coloured particles of different sizes (below 1 μm diameter). These particles were considered to represent agglomerated or aggregated nano-CeO2 (“alveolar histiocytosis with particles”). Alveolar histiocytosis and free eosinophilic granular material with particles, interpreted as remnants of destroyed macrophages, were found in the lung of all animals exposed to 25 mg/m3 NM-211 and NM-212. At aerosol concentrations of 0.5 mg/m3 NM-211, 0.5 and 5 mg/m3 NM-212, amber-coloured particles within single histiocyte in the alveoli were noted in all animals. In the bronchus-associated lymphoid tissue (BALT), particles were detected free or in single macrophages, mostly at 5 mg/m3 nano-CeO2 and above. Findings regressed but were still present 21 days after the end of exposure. The finding “eosinophilic granular material with particles” was no longer visible after the post-exposure period. Particles were detected free or in single macrophages in BALT, even at the lowest concentration of 0.5 mg/m3 NM-211. Only one animal exposed to 25 mg/m3 NM-211 showed macrophage aggregates in BALT 21 days after the end of exposure.

* LUNG-ASSOCIATED LYMPH NODES
After 5 days of exposure to 25 mg/m3 NM-211 and NM-212, comparable to the finding of particles in BALT of the lung, amber-like coloured particles were seen partly within macrophages or extracellularly in the lymphoid tissue, without any macrophage activation or aggregation (2/5 to 5/5 rats for NM-212; 3/5 to 4/5 rats for NM-211). Twenty one days after the end of exposure to 25 mg/m3, findings in mediastinal and the tracheobronchial lymph nodes progressed: multifocal macrophage aggregates with amber-like coloured particles were noted in animals exposed to NM-211 (2 to 3/5 rats) and NM-212 (4/5 rats). In both lymph nodes, lympho-reticulocellular hyperplasia was observed after 5 days of exposure and 21 days after the end of exposure to NM-211 and NM-212.

* UPPER RESPIRATORY TRACT (data not shown)
After 5 days of exposure to 25 mg/m3 NM-211 and NM-212, extracellular, amber-like coloured particles with diameters up to 1.5 μm were found in the lamina propria mucosae of the dorsal area of the larynx (level III). At aerosol concentrations of 25 mg/m3 NM-211 and NM-212, resembling amber-like coloured particles were detected within the subepithelial tissue in the carina of the trachea. Twenty one days after the end of exposure, findings in the larynx were only observed for animals exposed to NM-212, whereas particles in the carina (trachea) were still present in animals exposed to NM-211 and NM-212.

* EXTRAPULMONARY ORGANS
Extrapulmonary organs of animals exposed for 5 days to NM-211 and NM-212 were not examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Details on results:

BRONCHOALVEOLAR LAVAGE AND INFLAMMATORY MEDIATORS IN BALF AND SERUM
BALF analysis of 5 animals per test group was obtained 3 and 24 days after the end of exposure. The resulting BALF parameters are presented in Table 6.
In animals exposed to NM-212, the majority of BALF parameters were increased at aerosol concentrations of 5 and 25 mg/m3. At 0.5 mg/m3, the neutrophil counts and CINC-1 levels were both statistically increased and were slightly above the historical control range. With NM-211, but not NM-212, MCP-1 and M-CSF were increased at aerosol concentrations of 0.5 mg/m3 and above. Twenty four days after the end of exposure, a full recovery was observed at aerosol concentrations of 0.5 mg/m3 and a partial recovery at aerosol concentrations of 5 and 25 mg/m3. The recovery of animals exposed to 25 mg/m3 NM-211 seemed to be slower than those exposed to NM-212.

ORGAN BURDEN
Exposure to 0.5 mg/m3 NM-212 resulted in a lung burden of 0.011 mg/lung, directly after 5 days of exposure and decreased to 0.006 mg/lung 21 days after the end of the exposure, whereas exposure to 5 and 25 mg/m3 yielded higher lung burdens (0.1 and 0.53 mg/lungs, respectively) with only little decrease (0.088 and 0.4 mg/lung, respectively) within 21 days after the end of the exposure. Lung burdens of NM-211 were around 2-fold lower compared to those of NM-212. Cerium content in the lung-associated lymph nodes at aerosol concentrations of 25 mg/m3 increased from 1.7 to 5 μg for NM-212 and from 1.4 μg to 3 μg for NM-211 in the short-term study with 5 days of exposure.

Dose descriptor:

other: NOAEC (systemic)

Effect level:

> 25 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 and NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: - No NOAEC was determined in the publication, thus it was set by the registrant from data available in the publication. - Basis: no overall systemic effects

Dose descriptor:

other: NOAEC (local)

Effect level:

> 0.5 - < 5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 NM-211 and NM-212 (test mat.)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Table 4: Mean clinical pathology parameters in blood after exposure to nano-CeO2

	Control	NM-212			NM-211
Target conc. [mg/m3]	0	0.5	5	25	0.5	25
Measured conc. (mg/m³) +SD	0	0.48 ± 0.0	5.2 ± 1.1	25.6 ± 6.0	0.45 ± 0.1	25.8 ± 1.7
Blood cells
Neutrophils [giga/L] + SD Time point 1 (a) Time point 2 (b)	0.55 ± 0.21 0.64 ± 0.19	0.52 ± 0.07 0.58 ± 0.02	0.92* ± 0.22 0.59 ± 0.13	1.08** ± 0.14 0.64 ± 0.10	0.67 ± 0.16 0.73 ± 0.26	1.14* ± 0.30 0.75 ± 0.23
Lymphocytes [giga/L] + SD Time point 1 (a) Time point 2 (b)	3.16 ± 1.09 3.00 ± 0.66	2.55 ± 0.72 3.12 ± 1.27	3.77 ± 1.16 4.85** ± 0.57	2.90 ± 0.56 3.15 ± 0.51	3.12 ± 0.85 2.24 ± 0.39	3.34 ± 1.09 3.19 ± 0.40
Neutrophils [%] + SD Time point 1 (a) Time point 2 (b)	14.1 ± 0.9 17.3 ± 6.8	17.0 ± 5.3 15.3 ± 6.4	19.9 ± 8.0 10.4** ± 1.9	26.2** ± 4.5 16.5 ± 3.8	17.4 ± 5.0 22.1 ± 7.4	25.0** ± 7.8 18.3 ± 6.0
Lymphocytes [%] + SD Time point 1 (a) Time point 2 (b)	81.6 ± 1.4 78.3 ± 7.1	78.4 ± 5.5 80.5 ± 7.9	76.0 ± 8.5 85.6 ± 2.4	69.3** ± 5.7 79.7 ± 4.4	77.6 ± 4.7 72.9 ± 7.6	70.5* ± 8.3 78.1 ± 5.9

* statistically significant, p ≤ 0.05; ** statistically significant, p ≤ 0.01. 

(a) time point 1 is 3 days after the end of exposure; (b) time point 2 is 24 days after the end of exposure

SD: standard deviation

 

Table 5: Clinical pathology parameters in BALF of the short-term study with 5 days of exposure

	Control	NM-212			NM-211
Target conc. [mg/m3]	0	0.5	5	25	0.5	25
Measured conc. (mg/m3) + SD	0	0.48 ± 0.0	5.2 ± 1.1	25.6 ± 6.0	0.45 ± 0.1	25.8 ± 1.70
BALF cell counts (cn/μL)
Total cells Time point 1 (a) Time point 2 (b)	63.73 ± 1.03 53.09 ± 13.33	69.76 ± 26.15 44.20 ± 2.45	98.24** ± 16.38 43.98 ± 17.70	337.35** ± 125.24 46.71 ± 8.16	71.32 ± 22.69 47.01 ± 11.34	374.98** ± 77.84 81.20 ± 38.87
Neutrophils (PMN) Time point 1 (a) Time point 2 (b)	0.82 ± 0.80 0.69 ± 0.38	3.70** ± 2.53 1.34 ± 2.01	49.19** ± 17.68 5.15 ± 9.91	272.30** ± 106.62 4.63** ± 3.83	4.41* ± 3.38 2.27** ± 0.56	297.21** ± 59.23 25.57** ± 33.95
Lymphocytes Time point 1 (a) Time point 2 (b)	0.22 ± 0.32 0.32 ± 0.33	0.17 ± 0.22 0.11 ± 0.10	1.57* ± 0.97 0.43 ± 0.31	9.20** ± 4.51 1.03 ± 0.95	0.39 ± 0.34 0.51 ± 0.25	19.14** ± 15.13 2.74** ± 2.17
Macrophages Time point 1 (a) Time point 2 (b)	62.65 ± 11.56 52.05 ± 13.00	75.72 ± 15.10 42.32 ± 3.66	46.83 ± 23.88 38.34 ± 11.92	51.21 ± 24.76 40.93 ± 6.31	66.47 ± 22.13 44.23 ± 11.53	52.99 ± 42.89 52.61 ± 15.51
Monocytes Time point 1 (a) Time point 2 (b)	0.04 ± 0.08 0.00 ± 0.00	0.0 ± 0.00 0.00 ± 0.00	0.52* ± 0.37 0.03 ± 0.07	3.50** ± 1.86 0.07 ± 0.11	0.05 ± 0.11 0.00 ± 0.00	4.40** ± 2.68 0.28 ± 0.34
Eosinophils Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.03 ± 0.06	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.03 ± 0.06	0.39 ± 0.54 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00
Atypical cells Time point 1 (a) Time point 2 (b)	0.00 ± 0.00 0.00 ± 0.00	0.00 ± 0.00 0.00 ± 0.00	0.12 ± 0.17 0.00 ± 0.00	0.76 ± 0.73 0.05 ± 0.07	0.00 ± 0.00 0.00 ± 0.00	1.25* ± 0.85 0.00 ± 0.00
Total protein/enzymes
Total protein (mg/L) Time point 1 (a) Time point 2 (b)	39 ± 15 97 ± 122	47 ± 16 59 ± 28	75* ± 23 51 ± 16	198** ± 52 60* ± 9	44 ± 7 92 ± 91	207** ± 54 82 ± 14
GGT (nkat/L) Time point 1 (a) Time point 2 (b)	20 ± 15 30 ± 7	36 ± 20 28 ± 17	106** ± 26 33 ± 14	125** ± 15 61** ± 16	37 ± 26 25 ± 10	128** ± 16 87** ± 20
LDH (μkat/L) Time point 1 (a) Time point 2 (b)	0.46 ± 0.09 0.36 ± 0.14	0.58 ± 0.17 0.47 ± 0.25	0.89** ± 0.25 25 0.42 ± 0.04	2.17** ± 0.21 0.72** ± 0.20	0.67 0.58 ± 0.40	2.60** ± 0.31 1.08** ± 0.32
ALP (μkat/L) Time point 1 (a) Time point 2 (b)	0.43 ± 0.10	0.53 ± 0.08 1.0	1.31** ± 0.53 1.5*	1.56** ± 0.15 1.9**	0.55* ± 0.09 0.51 ± 0.20	1.56** ± 0.35 0.87** ± 0.12
NAG (nkat/L) Time point 1 (a) Time point 2 (b)	46 ± 9 41 ± 10	47 ± 11 37 ± 12	57 ± 12 39 ± 4	74** ± 7 46 ± 7	51 ± 19 46 ± 7	94** ± 16 59* ± 10
Cell mediators (pg/mL)
MCP-1 Time point 1 (a) Time point 2 (b)	14.7 ± 0.6 48.2 ± 59.1	19.1 ± 5.5 22.8 ± 12.5	101.1** ± 31.3 26.7 ± 10.8	1342.9** ± 530.0 144.1* ± 82.8	17.5* ± 1.6 30.6 ± 26.0	1581.0** ± 771.3 329.2** ± 290.5
CINC-1/IL-8 Time point 1 (a) Time point 2 (b)	59.8 ± 17.7 88.3 ± 25.7	83.4* ± 24.6 87.5 ± 24.7	348.6** ± 185.4 94.3 ± 35.7	322.4** ± 93.9 155.1** ± 13.5	88.3* ± 21.1 99.7 ± 39.4	436.0** ± 228.3 254.3** ± 70.4
M-CSF Time point 1 (a) Time point 2 (b)	41 ± 17 45 ± 19	52 ± 23 48 ± 5	34 ± 25 71* ± 13	91** ± 28 49 ± 3	61* ± 16 51 ± 12	114** ± 48 60 ± 24
Osteopontin Time point 1 (a) Time point 2 (b)	172.28 ± 48.04 320.38 ± 177.64	194.81 ± 131.24 191.47 ± 116.09	301.40 ± 238.96 247.44 ± 189.46	849.04** ± 386.44 495.92 ± 251.93	134.32 ± 82.72 111.92 ± 80.28	1116.02** ± 653.35 398.72 ± 140.88

* Statistically significant, p < 0.05

** Statistically significant, p < 0.01; n = 5; SD standard deviation

(a) Time point 1 is 3 days after the end of exposure

(b) Time point 2 is 24 days after the end of exposure

Conclusions:

Inhaled nano-CeO2 (NM-211 and NM-212) induced loco-regional effects manifested by a significant but transient pulmonary inflammation at 5 and 25 mg/m3. However, no systemic toxicity occurred.

Executive summary:

Keller J et al. (2014) assessed the lung deposition and clearance kinetics as well as the inhalation toxicity of two nanometric cerium dioxide (nano-CeO2) in a short-term study with a 5-day inhalation exposure (i.e., standard short-term inhalation study, STIS), in compliance with GLP. The study was performed, within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials and was funded via the European Commission’s 7th Framework Programme project NanoMILE.

Both nano-CeO2 (NM-211 and NM-212) were extensively characterised (see in the table below). Compared to NM-212, NM-211 had considerably smaller primary particles, larger specific surface, significantly fewer organic contaminations on the surface, and reduced photocatalytic activity.

 

Physico-chemical parameters	Results		Methods
	NM-211	NM-212
Supplier	Antaria	Umicore
Primary particle size	4 to 15 nm with a D50 of 8.2 nm	40 nm	Transmission electron microscopy (TEM)
Particle size distribution	No data 5, 70 nm and ca. 10 µm	3000 to 150 000 nm 35 nm and ca. 7 µm	Scanning EM (SEM) Hg porosimetry
Stability	Agglomeration / aggregation	Agglomeration / aggregation	-
Specific surface area	53 m²/g 33 m²/g	27 m²/g 30 m²/g	Brunauer, Emmett, Teller (BET) Hg porosimetry
Surface charge (zeta potential at pH7)	+16 mV	+42 mV	Electrophoretic mobility
Isoelectric point	pH 8.3	> pH 10 (always cationic)	Electrophoretic mobility
Shape	Globular	Globular	TEM, X-ray diffraction (XRD)
Crystallinity	Cerianite cubic, with a crystalline size of 12.5 nm	Cerianite cubic, with a crystalline size of 40 nm	XRD
Analytical purity	98.4%	99.3%	Thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS)
Impurities	1.6% contaminations, thereof small amounts of nitrates and alkyls found on the particle surface	0.7% organic contaminations, identified as ester + alkyl groups, found mostly on the particle surface (i.e., a very thin and homogeneous layer around the purely inorganic nano-CeO2)	TGA, XPS
Solubility	< 0.001 wt% regardless of the medium in which nanoparticles were suspended	< 0.001 wt% to 0.02% depending on the medium in which nanoparticles were suspended	ICP-MS
Oxidation degree	22% Ce(III) and 78% Ce(IV)	14% Ce(III) and 86% Ce(IV)	XPS
Surface properties (atom %)	O 57.2 Ce 28.7 C 14.1	C 79.9 (C-C 62.6; C-O 7.0; C=O 3.5; COOH 6.9) O 17.7 Ce 2.4	XPS
Description	Yellowish white powder synthesised by precipitation	Yellowish white powder synthesised by precipitation	-
Agglomerate density	0.6 g/m3	2.0 g/m3	Hg porosimetry
D50 and average agglomerate number (AAN), in water	D50 = 2839 nm, AAN = 346	D50 = 432 nm, AAN = 11	Centrifugation
Photocatalytic activity	0.0005 ± 0.0002	0.01 ± 0.005	Photon efficiency

Female Wistar rats (10/group) inhaled nano-CeO2 aerosol at concentrations of 0 (control), 0.5, 5, and 25 mg/m3 by whole-body exposure for 6 h/day on 5 consecutive days with a post-exposure period of 24 days. Control animals were exposed to conditioned air. Biological pulmonary effects of nano-CeO2 were studied by analysis of bronchoalveolar lavage fluid (BALF) and blood, and histopathology of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

With lung burdens being about 0.004 to 0.53 mg/lung after 5 days of exposure, inhaled nano-CeO2 at 0.5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. At aerosol concentrations higher than 0.5 mg/m3, this clearance was impaired resulting in a half-time above 200 days (i.e., at 25 mg/m3). In pathological examinations, an increase of lung weights was observed only in rats exposed to 25 mg/m3 NM-211 for 5 days. By light microscopy, both nano-CeO2 were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. The nanoparticles, either NM-211 or NM-212, were not detected within alveolar epithelial cells. At the lowest concentration of 0.5 mg/m3, the histopathological findings were alveolar histiocytosis and particles, either free or within macrophages; according to the authors, this reflected an expected physiological response.

Substance-related adverse effects after inhalation exposure to both nano-CeO2 were limited to the lung. Thus, after 5 days of nano-CeO2 inhalation, the responses observed were mainly local pulmonary effects. Exposure to 25 mg/m3 for 5 days (retained lung burden of 0.53 mg) resulted in higher absolute and relative neutrophil cell counts in the blood, without an increase in total cell counts for both NM-211 and NM-212. This slight neutrophilia was detected directly after the end of exposure and was no longer present after 3 weeks of post-exposure. Considering the pronounced inflammation in the lung at 25 mg/m3, the neutrophilia in blood was considered to be secondary to the local effects (systemic acute-phase response). No other blood parameters were affected. Furthermore, a whole panel of extrapulmonary organs and tissues was examined histologically as required by OECD test guideline 412. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low extrapulmonary tissue CeO2 concentrations in this study.

Compared to NM-212, NM-211 elicited higher increases in lymphocytes, cell mediators (e.g., MCP-1), and neutrophils in BALF at 25 mg/m3 with a slower recovery during the post-exposure period. Moreover, significant increase in lung weights was noted in animals exposed to 25 mg/m3 NM-211, but not NM-212.

In the present study, the larger specific surface area of NM-211 seemed to contribute to the higher biological activity as compared to NM-212. Thus, the surface area of the particles provided a dose metrics with the best correlation of nano-CeO2 inflammatory responses; hence, the inflammation appeared to be directed by the particle surface rather than mass or volume in the lung.

No NOAEC was defined in the study. However, from the observed results, a NOAEC for local effect (pulmonary tract) could be set at > 0.5 mg/m3 - < 5 mg/m3 and the NOAEC (systemic effect) was > 25 mg/m3 for both tested nano-CeO2.

In conclusion, inhaled nano-CeO2 (NM-211 and NM-212) induced loco-regional effects manifested by a significant but transient pulmonary inflammation at 5 and 25 mg/m3. However, no systemic toxicity occurred.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

Several studies, including two OECD 412, one OECD 413 studies and interim results (13 and 52 weeks) of the 2-year inhalation carcinogenicity study, conducted on nano-CeO2 with various physico-chemical properties were available and used in a weight-of-evidence approach to assess the repeated dose toxicity potential of the test substance. These studies were awarded a reliability score of 1 to 3 (Klimisch, 1997) and were thus considered sufficient for the assessment of nano-CeO2 repeated dose toxicity via inhalation route, therefore the overall quality of the dataset is considered to be acceptable.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

This is a provisional discussion pending the publication of the carcinogenicity study done by BASF. The dossier will be further updated in view of the CoRAP evaluation of cerium dioxide planned in 2021.

To date, several in vivo studies on the repeated toxicity of nano-CeO2 are available (Aalapati S et al., 2014; Dekkers et al, 2017 and 2018, Demokritou P et al., 2013; Gosens I et al., 2014; Keller J et al., 2014 and 2015/ Schaudien D. et al 2019; Kumari etal., 2014; Landsiedel R et al., 2014; Lee J. et al, 2020; Ma J et al., 2015; Morimoto et al. 2015; Poma A et al., 2014; Rocca A et al., 2015; Schwotzer D. et al, 2017); these studies were all conducted on rodents exposed to the nanoparticles by oral, inhalation, intratracheal and intraperitoneal route.

Almost all studies displayed a physico-chemical characterisation of nano-CeO2. Among other things, authors attempted to characterise the agglomeration / aggregation state of nanoparticles in the inhalation studies. However, it has to be noted that there was no clear definition of the terms “agglomeration” and “aggregation” in any of these studies, thus both terms were used in an indistinguishable manner. The most reliable studies were reported and discussed below.

REPEATED DOSE TOXICITY: ORAL

The effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated in a reliable combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, following daily oral administration by gavage to Sprague-Dawley rats (Lee J. et al, 2020). 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 of a polyhedral nano CeO2 (mean particle size of about 14 nm) 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.

REPEATED DOSE TOXICITY: INHALATION

Ten published studies evaluated the effects of nano-CeO2 after short term/repeated inhalation exposures in rats and mice models. These studies were performed with nano CeO2 presenting various physico-chemical properties (Aalapati S et al., 2014; Dekkers et al., 2017 and 2018, Demokritou P et al., 2013; Gosens I et al., 2014; Keller J et al., 2014 and 2015; Landsiedel R et al., 2014; Morimoto et al., 2015; Schwotzer D. et al, 2017); these publications are thus considered all together in a weight-of-evidence approach to assess the subacute/chronic toxicity potential of nano-CeO2 by inhalation route.

The characterisation of physico-chemical properties of the CeO2 particles tested in these studies are summarised in Table 2 for comparison.

Table 2: Physico-chemical characteristics available on the CeO2 particles tested by inhalation route

Physico-chemical parameters	Demokritou P et al. (2013)	Demokritou P et al. (2013)	Landsiedel R et al. (2014)	Schwotzer D et al. (2017)
	Non coated nano-CeO2	SiO2-coated nano-CeO2	-	MN-212
Supplier	None (in-house synthesis)	Antaria	Nanostructured and Amorphous Materials Inc.	JRC (Ispra, Italy)
Synthesis method	Flame spray pyrolysis method	Flame spray pyrolysis method	No data	No data
Primary particle size	12.8 nm	19.2 nm	10 to 200 nm	28.4 nm
Particle size distribution	241 nm	214 nm	(i) > 10 µm as dry powder (ii) 90 nm in water (iii) 54 nm in DMEM medium with FCS	No data
Mass median aerodynamic diameter (MMAD)	281 nm	341 nm	0.6 to 0.9 µm	0.63 to 0.79 µm
Stability	Agglomeration / aggregation	Agglomeration / aggregation	Agglomeration	No data
Specific surface area	61 m²/g	50 m²/g	33 to 34 m²/g	27.2 m²/g (Mean BET surface area)
Surface charge (zeta potential)	-10.8 mV	-10.8 mV	+6 mV at pH 7	No data
Isoelectric point	No data	No data	pH 7.5	No data
Shape	No data	No data	Globular	No data
Crystallinity	Crystalline	Crystalline	Crystalline (cerianite / cubic) with a crystal size of 36 nm	No data
Purity / impurities	No data	No data	No data	> 99.5 %
Solubility	No data	No data	< 0.001% dissolution in water and DMEM + FCS (Ce < 0.1 ppm)	< 1 µg/L (water solubility)
Oxidation degree	No data	No data	No data	No data
Surface properties	No data	2-4 nm SiO2 coating layer	Surface chemistry: O = 61% Ce = 16% Al = 9% Zr = 5%	No data
Photocatalytic activity	No data	No data	No data	No data
Physico-chemical parameters	Gosens I et al. (2014) and Keller J et al. (2014a and b)	Gosens I et al. (2014) and Keller J et al.(2014 a and b; 2015), Keller J 2015/Schaudien et al. (2019)	Gosens I et al. (2014)	Aalapati Srinivas et al. (2014)
	NM-211	NM-212	NM-213	-
Supplier	Umicore	Sigma-Aldrich	No data	None (in-house synthesis)
Synthesis method	No data	No data	No data	Sol-gel method
Primary particle size	8.2 nm	40 nm	< 5 µm	45 nm
Particle size distribution	5 nm to 10 µm	35 nm to 150 µm	No data	344 nm (wet state)
MMAD	1.02 to 1.6 µm	0.90 to 2.2 µm	1.4 µm	1.4 µm
Stability	Agglomeration / aggregation	Agglomeration / aggregation	Agglomeration / aggregation	Agglomeration / aggregation in wet state
Specific surface area	33 to 63.95 m²/g	27.15 to 30 m²/g	3.73 m²/g	56 m²/g
Surface charge (zeta potential)	+16 mV at pH 7	+42 mV at pH 7	No data	-47.4 mV (wet state)
Isoelectric point	pH 8.3	> pH 10	No data	No data
Shape	Globular	Globular	No data	No data
Crystallinity	Crystalline (cerianite / cubic) with a crystal size of 12.5 nm	Crystalline (cerianite / cubic) with a crystal size of 40 nm	No data	Crystalline (cubic)
Purity / impurities	98.4% purity Impurities: 1.6% nitrates and alkyls at particle surface	99.3% purity Impurities: 0.7% esters and alkyls at particle surface	99.5% purity	94.82% purity
Solubility	< 0.001 wt% regardless of the medium	< 0.001 wt% to 0.02% depending on the medium	Negligible in water at neutral pH	No data
Oxidation degree	22% Ce3+ and 78% Ce4+	14% Ce3+ and 86% Ce4+	No data	No data
Surface properties	Surface chemistry: O = 57.2% C = 14.1% Ce = 28.7%	Surface chemistry: C = 79.9% (C-C 62.6%; C-O 7.0%; C=O 3.5%; COOH 6.9%) O = 17.7% Ce = 2.4%	No data	No data
Photocatalytic activity	0.0005	0.01	No data	No data

RATS

Demokritou P et al. (2013) evaluated the toxicological implications of inhaled nano-CeO2 using an intact rat model. There was no mention to either guidelines or GLP. The study was well described and met the generally accepted scientific principles. However, given the experimental conditions applied (e.g., 4-day exposure duration), and observations mainly restrict to the respiratory tract, this study should be viewed as a preliminary work. Thus, it was awarded a reliability score of 2 (Klimisch, 1997) and was flagged as supporting study.

Uncoated and SiO2-coated nano-CeO2 were in-house synthesised and then physico-chemically characterised (see in Table 2 above).

Male Sprague-Dawley rats (6/group) were exposed (whole body) to particle-free environment (control) or 2.7 mg/m3 of uncoated CeO2 or SiO2-coated CeO2 for 2 h/day during 4 days. Twenty four hours after the last exposure, animals were sacrificed; pulmonary inflammation (alveolar macrophage (AM) and polymorphonuclear neutrophil (PMN) cell counts) and injury (LDH), air/capillary damage (albumin) were evaluated in bronchoalveolar lavage fluid (BALF) from treated and control rats.

There were no data on mortality, clinical signs or body weight changes that might have occurred during the experiments. However, the results showed lung injury and inflammation by an increase in PMN number and LDH levels in the BALF of uncoated CeO2-exposed rats. However, no increase in AM count or albumin level was observed. In contrast, exposure to SiO2-coated CeO2 did not induce any pulmonary toxicity in treated animals. Indeed, no change was observed on the 4 parameters. However, intratracheal instillation of uncoated nano-CeO2 and SiO2-coated nano-CeO2 demonstrated that both nanomaterials were present in AMs collected from CeO2 and SiO2-coated CeO2 exposed animals, respectively. In their review published in 2014, Yokel RA et al. cited the study of Demokritou P et al. (2013) and mentioned additional data which were not presented by Demokritou P et al. According to Yokel RA et al. (2014), PMN and LDH levels returned to control levels 84 days post-exposure; there was no further data detailed in the review.

In conclusion, according to the authors, inhalation of a nano-CeO2 aerosol induced a pulmonary toxicity in rats, whereas SiO2-encapsulated nano-CeO2 did not.

No No-Observed-Adverse-Effect-Concentration (NOAEC) was established by the authors in this study.

As there was no data available on the potential systemic effects of nano-CeO2 with or without a surface coating, the No-Observed-Effect-Concentration (NOEC) or NOAEC for systemic effects could not be determined. However, based on the increase in PMN number and LDH level as measured in BALF 24 hours after the 4-day exposure described above, NOAEC for local effects in the lungs of male rats could be extrapolated from the results: the local NOAEC for uncoated nano-CeO2 was lower than 2.7 mg/m3, while the one for SiO2-coated nano-CeO2 was higher than or equal to 2.7 mg/m3.

Landsiedel R et al. (2014) assessed the hazard of nano-CeO2 by performing a short-term inhalation study (STIS). The study was well described and met generally accepted scientific principles. However, given the experimental conditions applied (i.e., exposure duration, detailed analyses focused mainly to evaluate lung effects), this study should be viewed as a preliminary work; it was awarded a reliability score of 2 (Klimisch, 1997) and was flagged as weight of evidence.

Nano-CeO2 of 10 to 200 nm was used in this study. The physico-chemical characterisation of the tested nano-CeO2 is detailed in Table 2 above.

Male Wistar rats (3 to 6/group) were exposed (nose/head only) to nano-CeO2 at 0 (control), 0.5, 2.5 and 10 mg/m3 for 6 h/day on 5 consecutive days with a 21-day post-exposure period of observation. Control animals were exposed to conditioned air. Blood was collected at the end of exposure and recovery periods; haematology and clinical chemistry were then performed. BALF and histopathological sections of the entire respiratory tract were examined to determine the lung inflammation and injury. Pulmonary deposition and clearance of nano-CeO2 were also assessed.

There were no data on mortality/morbidity, body weight, or food consumption and then no systemic NOAEC could be derived in this study.

Inhaled nano-CeO2 was found in the lung, in alveolar macrophages, and more rarely in the draining lymph nodes; but none was found in extrapulmonary organs according to the authors. Nano-CeO2 induced a transient pulmonary inflammation which was concentration-dependent. Most effects were at least partially reversible during the post-exposure period. The histopathological analysis of lungs revealed no signs of beginning granulomatous changes or fibrosis. The authors derived a NOAEC < 0.5 mg/m3 based on changes of all cytological and biochemical parameters in BALF, particles in macrophages, partial regression of BALF effects and mild diffuse or multifocal alveolar histiocytosis remaining. Therefore, Landsiedel R et al. considered nano-CeO2 in a group of materials having a higher toxic potency to lung although a recovery was visible during the post-exposure period. However, only few of these changes (PMN, lymphocyte counts and M-CSL level slightly increased in BALF) were seen at the concentration of 0.5 mg/m3 at the end of exposure and recovery occurred for all these parameters; but some mild multifocal alveolar histiocytosis and particles in macrophages were also observed in animals in recovery group at this concentration. However, according to Keller J et al., (2014), this reflected an expected physiological response. And thus it could be considered that NOAEC was lower or equal to 0.5 mg/m3 in this study. In conclusion, inhaled nano-CeO2 induced only loco-regional effects manifested by a transient pulmonary inflammation. However, no systemic toxicity occurred.

Furthermore, Keller J et al. (2014a) assessed the lung deposition and clearance kinetics as well as the inhalation toxicity of nano-CeO2 NM-211 and NM-212 in a short-term study with a 5-day inhalation exposure (i.e., standard short-term inhalation study, STIS). The study was well documented and performed according to generally accepted scientific principles and in compliance with GLP. Given the experimental conditions applied, this study should be viewed as a preliminary work. The publication was awarded a reliability score of 2 (Klimisch, 1997) and was flagged as weight of evidence.

The tested substances NM-211 and NM-212 were selected and tested in the context of the OECD sponsorship programme for safety testing of manufactured nanomaterials. The physico-chemical characterisation done by Gosens I et al. (2014) (see below) was further completed in the study of Keller J et al. (2014) (see above in Table 2). Compared to NM-212, NM-211 had considerably smaller primary particles, larger specific surface, significantly fewer organic contaminations on the surface, and reduced photocatalytic activity. 

Female Wistar rats (10/group) inhaled nano-CeO2 aerosol at concentrations of 0 (control), 0.5, 5, and 25 mg/m3 by whole-body exposure for 6 h/day on 5 consecutive days with a post-exposure period of 24 days. Control animals were exposed to conditioned air. Biological pulmonary effects of nano-CeO2 were studied by analysis of BALF and blood, and histopathology of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

Treatment with both nano-CeO2 did not affect the body weight development of the animals and induced no clinical signs or finding as compared to the controls.

With lung burdens being about 0.006 or 0.53 mg/lung for NM-211 and NM-212, respectively, after 5 days of exposure, inhaled nano-CeO2 at 0.5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. At aerosol concentrations higher than 0.5 mg/m3, this clearance was impaired resulting in a half-time above 200 days (i.e., at 25 mg/m3). In pathological examinations, an increase of lung weights was observed only in rats exposed to 25 mg/m3 NM-211 for 5 days. By light microscopy, both nano-CeO2 were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. The nanomaterials, either NM-211 or NM-212, were not detected within alveolar epithelial cells. At the lowest concentration of 0.5 mg/m3, the histopathological findings were alveolar histiocytosis and particles, either free or within macrophages; according to the authors, this reflected an expected physiological response.

Substance-related adverse effects after inhalation exposure to nano-CeO2 were limited to the lung. Thus, after 5 days of nano-CeO2 inhalation, the responses observed were mainly local pulmonary effects. Exposure to 25 mg/m3 for 5 days (retained lung burden of 0.53 mg) resulted in higher absolute and relative neutrophil cell counts in the blood, without an increase in total cell counts for both NM-211 and NM-212. This slight neutrophilia was detected directly at the end of exposure and was no longer present at 3 weeks post-exposure. Considering the pronounced inflammation in the lung at 25 mg/m3, the neutrophilia in blood was considered to be secondary to the local effects (systemic acute-phase response). No other blood parameters were affected. Furthermore, a whole panel of extrapulmonary organs and tissues was examined histologically as required by OECD guideline 412. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low extrapulmonary CeO2 concentrations described in this study.

Compared to NM-212, NM-211 elicited higher increases in lymphocytes, cell mediators (e.g., MCP-1), and neutrophils in BALF at 25 mg/m3 with a slower recovery during the post-exposure period. Moreover, significant increase in lung weights was noted in animals exposed to 25 mg/m3 NM-211, but not NM-212.

In the present study, the larger specific surface area of NM-211 seemed to contribute to the higher biological activity as compared to NM-212. Thus, the surface area of particles provided a dose metrics with the best correlation to nano-CeO2 inflammatory responses according to the authors; hence, the inflammation appeared to be directed by particle surface rather than mass or volume in the lung.

In conclusion, inhaled nano-CeO2 (NM-211 and NM-212) induced loco-regional effects manifested by a significant but transient pulmonary inflammation at 5 and 25 mg/m3 with impaired lung clearance.

However, no systemic toxicity occurred. The inflammatory response observed was typical of poorly soluble particles with significant biopersistence.

No NOEC or NOAEC for systemic toxicity or local effects was proposed by the authors of this study. Based on few data available on the systemic effects of nano-CeO2, the NOAEC for systemic toxicity was determined to be greater than 25 mg/m3 for both nano-CeO2 tested. However, based on the loco-regional effects described in this study, a NOAEC for local effects in the lungs could be extrapolated as higher or equal than 0.5 mg/m3 but smaller than 5 mg/m3 for both NM-211 and NM-212.

The toxicological impact of NM-212 was further assessed by Keller J et al. (2014b). The study was performed according to OECD guideline 412 and in compliance with GLP. Thus, the publication was awarded a Klimisch score of 1 and was flagged as weight of evidence.

Female Wistar rats (10/group) were administered NM-212, by whole-body inhalation exposure, at aerosol concentrations of 0 (control), 0.5, 5, and 25 mg/m3for 6 h/day on 5 days/week for 4 weeks with a post-exposure period of 129 days. Control animals were exposed to conditioned air. Pulmonary responses were studied by analysing the BALF and blood, and by performing a histopathological analysis of respiratory tract. Biokinetics were assessed by the determination of lung and lung-associated lymph node burdens at different time points.

No clinical sign and no effect in the body weight development were observed in the treated animals as compared to controls and according to the authors, substance-related adverse effects after 28-d inhalation exposure to nano-CeO2 NM-212 was limited to the lung. No altered blood parameter could be detected after 4 weeks of inhalation exposure. A whole panel of extrapulmonary organs and tissues was histologically examined; very low cerium contents were detected in the liver at two time points and at the concentration of 25 mg/m3 NM-212 (which is a general finding for inhaled nanoparticles) without any related morphological abnormalities. None of the other extrapulmonary organs showed any morphological abnormalities. The absence of systemic effects was consistent with the very low CeO2 concentrations in extrapulmonary tissues.

An aerosol concentration of 0.5 mg/m3 did not cause inflammatory response in the lung. With a lung burden of 41 μg/lung after 4 weeks of exposure, inhaled NM-212 at 0.5 mg/m3 was deposited in the lung and cleared with a half-time of 40 days. This was in the range of physiological retention half-times of poorly soluble particles between 60 and 70 days. Higher aerosol concentration impaired this clearance: a higher aerosol concentration of 25 mg/m3 elicited a lung burden of 2.62 mg/lung resulting in a retarded retention half-time above 200 days. At the mid concentration of 5 mg/m3, the lung burdens at 3 time points indicated a retarded retention half-time as the lung burden (0.5 mg/lung) stayed at a constant level during 4 weeks. The lung burden decreased from 2.62 to 1.8 mg, 129 days after the exposure to 25 mg/m3 for 4 weeks. In pathology, lung weights were found increased following exposures to 5 and 25 mg/m3 NM-212. By light microscopy, CeO2 particles were primarily seen extracellularly and intra-alveolar or engulfed by alveolar macrophages. CeO2 was not detected within alveolar epithelial cells. Moreover, alveolar histiocytosis was observed and had even progressed to a multifocal granulomatous inflammation within 4 weeks after the end of exposure to 5 and 25 mg/m3 nano-CeO2. The combination of moderate alveolar histiocytosis with particles and the presence of eosinophilic material, potentially precursors of granulomatous inflammation, were considered to be adverse. In contrast, at 0.5 mg/m3, the histopathological findings alveolar histiocytosis and particles, either free or within macrophages, reflected and expected physiological response.

Nano-CeO2-related adverse effects were limited to the lung after inhalation exposure. The reported pulmonary inflammation was assessed by the changes in BALF parameters (e.g., neutrophils and pro-inflammatory cytokines) and histopathological findings (alveolar histiocytosis and granulomatous inflammation). Pulmonary inflammation was only observed at concentrations of 5 and 25 mg/m3. However, BALF parameters showed a regression during the post-exposure period. Histopathological findings, in contrast, progressed to granulomatous inflammation after the end of exposure at concentrations of 5 mg/m3 nano-CeO2 and above. It has to be noted that the inflammatory response in lungs based on the increase in neutrophil counts in BALF was lower after 4 weeks compared to 5 days of exposure (see in the previous ESR from the same authors). The decay in neutrophil numbers after 4 weeks was by far slower than after 5 days, suggesting that inflammation developing at lower dose rate was longer lasting and more persistent.

A smaller fraction of the particles was transferred to the lung-associated lymph nodes. Indeed, in the lung-associated lymph nodes, 0.35 mg CeO2 was found 129 days after exposure to 25 mg/m3 NM-212 for 4 weeks. Moderate macrophage aggregates with particles in the lung-associated lymph nodes, combined with lympho-reticulocellular hyperplasia, were considered to be adverse. Demonstrated by histological evaluations and confirmed by measured Ce lymph node burdens, the lymphatic clearance of inhaled CeO2 via the lymphatic vessels from the pulmonary region to the lung-associated lymph nodes was around 13% of the initial retained burden after the end of the exposure. Most of the CeO2 nanoparticles were presumably cleared by mucociliary clearance and subsequent faecal excretion, which was not evaluated in this work.

According to the authors, the mass lung burden of 41 μg, achieved at 0.5 mg/m3, was well below the overload threshold proposed by Morrow, while the lung burden of 2.62 mg, achieved at 25 mg/m3, was above it (Morrow, 1988). At the highest concentration, a strong pulmonary inflammation was apparent. The mid concentration of 5 mg/m3 NM-212 elicited pulmonary inflammation at a constant lung burden of around 0.52 mg, which was slightly below or at the border of the overload threshold. Based on calculations, volumetric lung overload could only be assumed after 4 weeks exposure to 25 mg/m3. Impaired lung clearance (which is one of the consequences of lung overload conditions) was, however, already observed after inhalation of 5 mg/m3 CeO2. And thus, 5 mg/m3 was the lowest aerosol concentration at which the early as well as the later inflammation response was observed, even though lung burdens were different at the onset of the 2 phases.

In conclusion, inhaled nano-CeO2 (NM-212) induced loco-regional effects manifested by a significant pulmonary inflammation and granulomatous alterations of the lung, mainly at 25 mg/m3. However, no systemic toxicity occurred. The inflammatory responses observed were typical of poorly soluble particles with significant biopersistence. Moreover, it has to be noted that both concentrations which induced pulmonary effects corresponded to or were at the limit of overload conditions.

As the authors stated that substance-related adverse effects after 28-d inhalation exposure to nano-CeO2 NM-212 was limited to the lung and based on the above mentioned results, a NOAEC for systemic toxicity could be set at > 25 mg/m3 and a NOAEC for loco regional effects (based on pulmonary inflammation and histopathological changes in lung associated lumph nodes) would be established at > 0.5 mg/m3 but < 5 mg/m3.

Then, Gosens I et al. (2014) assessed the hazard of two nano-scale (NM-211 and NM-212) and one micro-scale (NM-213) CeO2 in a 28-day inhalation toxicity study in rats. The study was performed, within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials, according to the OECD guideline 412 but with some deviations and restrictions. The study was thus awarded a reliability score of 2 (Klimisch, 1997) and was flagged as supporting study due to missing information in the protocol as well as in the results.

The three CeO2 used in this study were characterised (see above in Table 2). Although the primary particle size of all materials differed considerably, the mass median aerodynamic diameter was surprisingly similar because all materials were aggregated when aerosolised in air. NM-211 showed the highest number concentration in the test atmosphere (up to 1.75 x 10E6 particles/cm3) and the smallest mass concentration (up to 10.8 mg/m3), while NM-212 had a number concentration of up to 1 x 10E6 particles/cm3 and a mass concentration of up to 19.9 mg/m3. NM-213 had a number concentration of 0.68 x 106 particles/cm3 and a mass concentration of up to 55 mg/m3.

Male and female Wistar rats (5/sex/group) were nose-only exposed from 40 min to 6 h/day for 5 days/week during 4 weeks to reach dose equivalent to 0 (control), 1.2, 3.5 and 10.8 mg/m3 of NM-211, 0 (control), 2.5, 6.7 and 19.9 mg/m3 of NM-212, and 0 (control), 5.9, 18.4 and 55 mg/m3 of NM-213. Control animals were exposed to a stream of clean humidified compressed air. Cage-side observations were performed. Mortality, body weight, and food consumption were also monitored. Moreover, at the end of exposure period, haematological parameters, clinical chemistry in blood and BALF, inflammatory cell counts in BALF, urinalysis, fibrinolysis, and gross pathology and histopathology analysis were carried out to determine the local (pulmonary) and systemic effects of nano-CeO2. A recovery group was included in this study: males and females exposed to NM-211, NM-212, or MN-213 (high dose) 5 days/week for 4 weeks were kept for a recovery period of 28 days before sacrifice.

There were no exposure-related signs of toxicity, behaviour effects or mortality during the 4-week exposure period and recovery period.

All materials were efficiently deposited in lungs of animals: 0.83, 1.54 and 4.24 mg for NM-211, NM-212 and NM-213, respectively. The particle exposures increased lung weights.

According to the authors, all materials induced a dose-dependent pulmonary inflammation and lung cell damage (as measured in the BALF), but without any histopathological change in lungs and liver immediately after exposure. Inflammation was still present at the end of the 28-day recovery period, albeit at a lower level. In contrast, there was no evidence of systemic toxicity or other haematological changes following exposure to all CeO2 tested. According to the authors, these data suggested that the adversity of the observed effects could be classified as minimal.

Nevertheless, although significant, the parameters used (e.g., BALF cell counts) to draw these conclusions showed a high variability which could indicate that the inflammatory effects observed might be even more modest or almost negligible in some cases.

When exposure levels were expressed as mass concentration, NM-211 was the most potent material, while based on surface area concentration pulmonary inflammation/damage was induced in a lesser extent by both nano-CeO2 than by the micrometric counterpart. Particles were equipotent based on particle number concentrations.

In conclusion, similar moderate pulmonary toxicity profiles including inflammation were observed for both nanomaterials and the micrometric counterpart and systemic effects were virtually absent. These similar patterns probably resulted from the equivalent aerodynamic diameters found for all materials and there was little evidence for a dominant predictive exposure metric for the observed effects.

No NOEC or NOAEC for systemic or local toxicity were determined in this study by the authors. However, based on the absence of systemic effects from all CeO2 tested, a NOAEC for systemic toxicity could be set for both nanomaterials and the micrometric form above the highest dose tested and thus ≥ 10.8 mg/m3 for NM-211, ≥ 19.9 mg/m3 for NM-212 and ≥ 55 mg/m3 for NM-213, for both male and female rats.

Regarding local toxicity, no NOAEC could be set by the registrant due to the very high variability and the recovery, at least partial, of BALF results and to the brevity of histopathology data.

Schwotzer D.et al.(2017) investigated potential health effects of cerium oxide NM-212 nanoparticles to rats in a subchronic inhalation toxicity study. The study was performed according to OECD TG 413 but there is no mention to GLP compliance. Further, several parameters that should be analysed according to this guideline were briefly reported (e.g. clinical signs, body weight) or not reported (e.g. organ weight, FOB, macroscopic analyses). In addition, histopathological analyses were porformed on respiratory tract only and solely in the control and high dose (3m/m3 CeO2) groups. For this reason, the study was awarded a reliability score of 2 (Klimisch, 1997) and was used as a supporting study.

Female Wistar rats were exposed (nose only) to aerosol of nanoCeO2 NM-212 at 0 (clean air control), 0.1, 0.3, 1.0 and 3.0 mg/m3 (see table 2 above for detailed PC characterization) with a MMAD ranging from 0.63 to 0.79 µm for 6h/day, 5 days/week for 1, 28 or 90 days followed by a 1-, 28- or 90-day recovery period. According to the authors, animals were check daily for health conditions. Inspection for clinical abnormalities, body weight gain, food and water consumption were recorded once a week. Heamatology and clinical chemistry were done at post-exposure day 1 only since no or small changes were observed at this time point. Lung burden and bronchoalveolar lavage analyses (total cell count, differential cell count, biochemical mediators, and cytokines) were analysed in all dose groups at all days of sacrifices. Histopathological examinations of respiratory organs (left lung lobe, bronchi, mediastinal and tracheobronchial associated lymph nodes, trachea, pharynx, nasal cavities and nasal mucosa associated lymphoid tissue) were performed at all days of sacrifice in animals of the clean air control and the 3.0 mg/m3 CeO2 groups only.

According to the authors, all animals were in good physical conditions up to sacrifice. No significant changes in body weights, food and water consumption were detected. Clinical signs due to particle exposure were not observed but data were shown.

The lung burden analysis showed that the substance deposition was concentration dependent and particle elimination was visible in all treatment groups after end of exposure. But at higher CeO2 concentrations, higher deposition rates have been detected with reduced elimination especially for 3.0 mg/m3 CeO2. Corresponding calculated clearance half-time values were 67, 69, 108 and 224 days after 0.1, 0.3, 1 and 3 nanoCeO2 mg/m3. The authors calculated a deposition fraction of about 10 % of the initial nanoparticle concentration for all exposure doses.

Blood and biochemical parameters (not specified) measured at post exposure day 1 did not display any significant changes excepted for neutrophil levels in the CeO2 mid dose groups.

The BALF analyses done after 1 and 28 day of exposure and day 1, 28 and 90 post exposure showed a time- and concentration-dependent increase of the inflammatory cells PMN (significant from 28 days of exposure to 3 mg/m3 CeO2) and lymphocytes (significant from Day 1 post exposure at 1 and 3 mg/m3 CeO2) with a slight decrease of PMN during the post-exposure period. However, high variability was observed in the results, more particularly at 1 day post exposure. A slight increase of total protein (TP), lactate dehydrogenase (LDH) and ß-glucuronidase (GL) levels were also observed in the CeO2 high dose group from day 28 of exposure to day 1 post exposure but recovery occurred during post exposure period.

Histopathology analyses, done on respiratory tract of the control group (clean air) and the high dose group (3 mg/m3 CeO2) only, have shown a very slight but significant accumulation of particle-laden macrophages in the alveolar space and bronchus-associated lymphoid tissue (BALT) after the first exposure to CeO2 nanoparticles. The amount of macrophages increased from slight to moderate up to the end of the post-exposure period with very slight to moderate translocation to the lung associated lymph nodes (LALN) detected from day 28 to the end of the recovery. This indicated clearance of the phagocytosed material. Very slight to slight alveolar and interstitial inflammatory cell infiltrations and very slight bronchiolo-alveolar hyperplasia were observed after 28 days of exposure to the highest dose of nanoCeO2 up to the end of the recovery period. Free particles (agglomerates) were detected in the alveolar space after end of exposure, mainly in areas of macrophages containing particulate matter. Such accumulations often originate from degrading macrophages. Furthermore, during the recovery period, very slight interstitial fibrosis was observed which was significant at the 90 day-post exposure time point. No pathological change was observed in the nasal cavity of exposed rats. The authors reported that this serie of effects illustrates the consequence of particle overload.

In the study, the authors selected the dose range to cover specific conditions of absent inflammation in combination with absent lung overload (0.1 and 0.3 mg/m3), inflammation and no overload (1.0 mg/m3) as well as inflammation and overload (3.0 mg/m3). The authors concluded that analysis of lung burden suggested that the respective conditions were achieved for the low dose levels (0.1 and 0.3 mg/m3; for which the clearance half-times were below the expected mean value of 70 days), and the high dose level (3 mg/m3) for which the half-life was > 200 days for particle clearance reflecting an overload situation. For the 1 mg/m3 CeO2 dose, the clearance half-time was calculated to be 108 days and signs of inflammation were present at this dose and thus, the expected situation of lung inflammation at non-overload was not clearly achieved.

The authors concluded that all together, these results indicated a present inflammatory reaction in the lung at 1.0 and 3.0 mg/m3 CeO2 exposure and a lung overload at 3 mg/m3 nano CeO2 exposure with a shift overtime to from non-adverse to adverse findings with development of interstitial fibrosis although the grade was minimal. The authors extrapolated a NOAEL_BMDLat 0.41 mg/m3 based on PMN data using the US-EPA benchmark software.

No NOAEC for systemic and/or local effects have been derived by the authors in this study. These values cannot be derived from the information of the study due to the low numbers of parameters examined and reported in this study and the absence of histopathological investigation other than those performed in the respiratory tract of animals of the high dose group only.

Keller J. (2015) and Schaudien D. and collaborators (2019) presented interim results of a carcinogenicity study done in the rats (performed according to OECD test guideline No. 453 ander under GLP) observed after 13 and 52 weeks of inhalation exposure to nano-CeO2 NM-212. Despite only partial results were presented in these available reports, the study was awarded a reliability score of 1 (Klimisch, 1997) and was used as a Weight of Evidence, waiting for the release of the full carcinocenity study results. The aim of this interim observations was to investigate the lung deposition and the clearance of inhaled nanomaterials, and the resulting effects on the rat organism at different time points.

In this study, Female Wistar rats inhaled nano-CeO2 by whole-body exposure, 6 hours per day, 5 days per week for a total of two years. The tested aerosol concentrations were 0.1, 0.3, 1 and 3 mg/m³ CeO2 (see table 2 above for further CeO2 physico-chemical charcateristics). The Interim results after 13 and 52 weeks of exposure presented in this summary included results collected from 5 females/group on lung retention and clearance kinetics based on lung and associated lymph node burdens, and pulmonary effects based on bronchoalveolar lavage fluid (BALF) analyses including total protein concentrations, total and differential cell counts, enzyme activities (LDH, ALP, GGT, NAG), acute phase proteins (HAPT and A2M) and cytokines (MCP-1, CIN-1/IL-8 and M-CSF and rodent osteopontin). Systemic effects were evaluated by analysis of blood including neutrophils counts and acute phase proteins HAPT and A2M. In addition, histopathological analyses were done after 12 weeks of exposure on 10 animals/group.

 

According to the author, during the exposure period, lung burdens increased with longer exposure duration. The lung burden of CeO2 after 13 weeks was nearly doubled after 52 weeks of exposure at aerosol concentrations of 0.1 and 3 mg/m3. However, only a slight translocation of the nanoparticles to the tracheobronchial and mediastinal lymph nodes was observed (below 2 % of the initial lung burden after 13 weeks) indicating that, at least at this lung burden range, they play no major role for particle clearance.

Inhalation exposure to nano-CeO2 for 13 and 52 weeks elicited no or only minimal systemic effects: no change in mean neutrophil levels were observed in the blood of rats at any concentrations and time points slight increase of acute phase protein levels (haptoglobin andα2-macroglobulin) in serum was observed after 52 weeks of exposure to 3 mg/m3 CeO2.

Local inflammation in the lung was observed by increases in BAL neutrophils, lymphocytes, monocytes, enzyme cativities and cell mediator levels at the highest tested concentrations (1 and 3 mg/m3) after 13 weeks of exposure. BAL changes (cell counts, enzyme activities, total protein and cell mediator levels) after 52 weeks of exposure to 3 mg/m³ CeO2 were comparable to those after 13 weeks or slightly reduced. Minor changes in BAL were observed at lower aerosol concentrations of 1 mg/m³. Exposure to 0.3 mg/m³ elicited no BAL changes after 13 weeks and only minor changes after 52 weeks (neutrophils, GGT, MCP-1). In animals exposed to lower aerosol concentrations (0.3 mg/m3) rather than in those exposed to higher, BAL lymphocytes were higher increased than neutrophils after 52 weeks of exposure indicating a later phase of inflammation.

 

Histopathology findings related to CeO2 exposure were exclusively observed in the respiratory tract and included non-adverse reactive/adaptive changes such as accumulation of particle-laden macrophages in the nasal cavity, larynx, lungs, tracheobronchial and mediastinal lymph nodes. Adverse effects in the lungs included dose-dependent alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation and interstitial fibrosis. Alveolar lipoproteinosis was observed in the 3 mg/m³ high-dose CeO2 exposure group only and cholesterol granulomas occurred in a single female each of the 1 and 3 mg/m³ CeO2 exposure groups. Although statistically not significant, some adverse effects such as alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, and interstitial fibrosis have already been observed in the 0.1 mg/m3 low-dose CeO2 exposure group. After 12 month of inhalation exposure neither neoplastic nor pre-neoplastic treatment-related findings were seen in the lungs of CeO2-exposed animals. In the histopathological analyses of the other organs, all the findings were considered to be without any relation to CeO2 exposure.

 

The local no observed adverse effect concentrations in the lung (NOAEC) of CeO2 - based on BALF in female rats after 13 and 52 weeks of exposure - was found to be 0.3 and 0.1 mg/m³, respectively. However, from the results of the histopathological analyses of animals after 52 weeks of exposure to CeO2, it was concluded that the local no observed adverse effect concentrations in the lung (NOAEC) could not be established for the lungs after 12 months of exposure to the present CeO2 nanoparticle concentrations. In the absence of systemic effects, the overall systemic NOAEC is 3 mg/m3.

 

In summary, inhaled CeO2 showed high biopersistence in the lungs inducing a local inflammation but no systemic effect. Thus CeO2 was considered as a poorly soluble with an inherent toxicity in the lung in the rats.

MICE

In this subacute inhalation study, Dekkers et al. (2017) explored the (patho)physiological effects of nano CeO2 exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. As this study was performed using a test procedure in agreement with generally accepted scientific standards but with some limitations (incomplete data on substance characterisation and on systemic toxicity), the study was awared a reliability code of 2 according to Klimisch criteria and was used as weight of evidence.

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m3 nano CeO2 (primary particle size of 4.7 nm) for 3h/day, 5 days/week for 4 weeks.

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs were done and the tissue distribution of cerium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of nano-CeO2 on the artherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

According to the authors, no statistically significant differences were observed in the total or differential white blood cell counts in the blood and in the BALF of the exposed groups compared to the controls in all strains of mice. Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels in BALF between the exposed and control groups. Furthermore, no treatment related effects on organ weights were observed and no macroscopic findings related to treatment were observed. In lungs, the histopathological analyses showed an increased incidence, described as modest by the authors, in minimal chronic bronchoalveolar or alveolar inflammation observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in most of the ApoE-/- mice exposed to nano CeO2 NP but not in the other stains. In the bronchoalveolar lavage fluid, particle loaded macrophages were seen in all the exposed animals but not in the control groups. In tissues, background Ce concentrations were measured in the control animals and significantly higher levels of Ce were only observed in the lungs and the liver of exposed mice as compared to the controls but not in the other organs.

In ApoE-/- mice, exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries and in the proportion of plaque staining positive for mac-2. The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concuded that in this subacute inhalation study, nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

No NOAEL was derived in this study by the authors. However, according to the results and the conclusion of the authors (nano CeO2 has a low biological activity in their mouse models), it can be concluded that the NOAEL for lung toxicity is > 4 mg/m3, the only dose tested in this study.

Aalapati Srinivas et al. (2014) investigated the toxicity and bio-accumulation of nano-CeO2 in mice through nose-only inhalation route. The study was well described but there were some restrictions regarding the experimental protocol applied (e.g., no control group, numerous missing parameters found in repeated dose exposure study) and there was no mention to guidelines or GLP. Thus, the publication was awarded a Klimisch score of 3 and was flagged as disregarded study.

The commercial nano-CeO2 used in this study was characterised (see in Table 2 above). It has to be noted that, compared to the other nano-CeO2 used in the abovementioned studies, the nano-CeO2 tested by Aalapati S et al. displayed a lower purity (94.8%vs. 99.9% according to the supplier and > 98% for other nano-CeO2). However, the authors did not identify the impurities which might have lowered the nano-CeO2 purity.

Male CD-1 mice (12/group) were daily exposed, by nose-only inhalation, to 2 mg/m3 nano-CeO2 for 7, 14 and 28 days followed by 14 or 28 days of recovery. The following endpoints were assessed at the end of exposure and recovery periods: markers of lung injury and pro-inflammatory cytokines (interleukin [IL]-1β, tumour necrosis factor [TNF]-α, IL-6 and macrophage inflammatory protein [MIP]-2) in BALF, oxidative markers malondialdehyde (MDA) and GSH in lung tissues, nano-CeO2 biodistribution in lung, liver, kidney, heart, brain, and histopathology of pulmonary and extrapulmonary tissues. There was no animal treated with filtered air or vehicle for 0 to 28 days; thus, no control group was included in this study. For the authors, the controls animals were the Day-0 animals group.

There were no data on mortality/morbidity, body weight, or food consumption.

BALF analysis revealed the induction of pulmonary inflammation, as evidenced by an increase in the neutrophil influx and a significant secretion of pro-inflammatory cytokines which led to generation of oxidative stress and cytotoxicity, as demonstrated by MDA induction, GSH depletion and increased LDH and protein levels in BALF. The histopathological examination revealed that inhaled nano-CeO2 was located all over the pulmonary parenchyma, inducing a severe, chronic and active inflammatory response characterised by necrosis, proteinosis, fibrosis and well-formed discrete granulomas in the pulmonary tissue and tubular degeneration leading to coagulative necrosis in kidneys. Inductively coupled plasma spectroscopy showed a significant bio-accumulation of nanoparticles in pulmonary and extra-pulmonary tissues, even one month after the end of inhalation exposure.

The authors concluded that inhalation exposure of nano-CeO2 could induce pulmonary and extra-pulmonary toxicity in their rodent model.

No NOAEC (systemic or loco-regional) were defined in this study by the authors. However, based on the poor information on systemic toxicity no NOAEC for systemic toxicity could be deduced from this study. But, based on the loco-regional effects described in the lungs, a NOAEC for pulmonary local effects could be viewed as to be lower than 2 mg/m3.

Nevertheless, these conclusions were mainly based on the histopathological observations which could not be evaluated by peers since only few photos were available and no pictures of control animals were displayed in the publication. Moreover, similar exposures were performed in rats (Gosens Iet al., 2014; Keller Jet al., 2014a and 2014b; Landsiedel R et al., 2014) and macroscopic/microscopic observations performed on lung tissues showed less severe and chronic damages. The rodent species chosen might be an explanation for these differences: mice and rats can display discrepancies regarding the biological responses induced by metallic engineered nanoparticles (Sharma HS et al., 2012). Further, the CD-1 strain might not be a suitable strain for inhalation toxicity studies based on their genetic background (Chia R et al., 2005); Manenti G et al. (2003) demonstrated that CD-1 mice displayed a high susceptibility to spontaneous and chemically induced lung tumorigenesis. In addition, the lack of accurate control groups (i.e., mice exposed to air for time periods similar to that performed for treated rodents) and the absence of general systemic parameters such as clinical signs, or body weight for instance, made it impossible to interpret the results. The historical data provided by the authors were not sufficient for an interpretation of results, and these data were not displayed for all studied parameters.

General discussion on repeated dose toxicity by inhalation

Seven studies evaluated the effects of nano-CeO2 inhalation in rats and two were performed in mice. Three studies (Gosens I et al., 2014, Keller J et al., 2014) used the same nano-CeO2 (NM-211 and/or NM-212) that were selected in the OECD program (OECD Sponsorship Program for the Testing of Manufactured Nanomaterials). Primary particle size of the tested nano-CeO2 ranged from 8.2 to 45 nm (see in Table 2 above). It has to be noted that all measured MMAD were higher than 100 nm and were close or in the micrometre range; the tested nano-CeO2 all agglomerated and/or aggregated in the test atmosphere. Additionally, Gosens I et al. (2014) included a micro-scale CeO2 (NM-213; < 5 µm) for comparison. From the results described above, it can be observed that physico-chemical characteristics such as primary particle size (8 nm to < 5 µm), specific surface area (3.7 to 63.9 m²/g), or surface charge (-47 to +42 mV) seem to have no or few impact on the toxicological responses induced by nano-CeO2 in rodents.

Overall, the seven studies conducted in rats demonstrated that, in all cases and regardless of the administered concentration (0.5 up to 25 mg/m3) and the exposure duration (4 to 28 days), repeated inhalation of nano-CeO2 (at concentrations ≥ 0.5 mg/m3) mainly induced no overt systemic toxicity based on the absence of mortality, clinical signs, clinical chemistry change, and histopathological alterations of extrapulmonary organs and as well as on no or few but transient haematological changes. Instead, inhaled nano-CeO2 induced loco-regional effects to the lungs of exposed male and female rats: cell infiltration and change in levels of enzyme activities and inflammatory mediators in BALF; accumulation of particle-loaded macrophages (histiocytosis) in lungs and transfer of a small particle fraction to lung-associated lymph nodes. According to Keller J et al. (2014), the effects observed at the concentration of 0.5 mg/m3 were non adverse, probably adaptive, while the effects described in lungs at higher concentrations were considered as adverse. Indeed, the authors observed that nano-CeO2 concentrations higher than 0.5 mg/m3 impaired the lung clearance. Keller J et al. (2014) concluded from their observations that the inflammation appeared to be directed by particle surface rather than mass or volume in the lung. In contrast, Gosens I et al. (2014) tested the same nanomaterials than Keller J et al. (2014) plus a micro-scale CeO2 and found little evidence for a dominant predictive exposure metric when the observed effects were expressed as mass concentration, surface concentration or particle number. The inflammatory responses observed in rats could regress, at least partially, in studies that included relevant recovery periods.

In mice, no consistent results were observed in the 2 studies considered.

Indeed, Dekkers et al, (2017) concuded that in her subacute inhalation study, nano CeO2 at 4 mg/m3 up to 4 weeks has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

In contrast, in the study of Aalaapati S et al (2014), repeated inhalation of nano-CeO2 caused loco-regional effects in lung tissues (inflammation, oxidative stress, chronic-active bronchiolo-alveolar inflammation, necrosis, proteinosis, fibrosis, and well-formed discrete granulomas), as well as systemic effects (i.e., degenerative changes in the kidney). The results displayed by Aalapati S et al. (2014) in mice suggested that either surface properties of nano-CeO2 (e.g., purity and/or surface charge) or the animal model (mouse vs. rat) could have impacted its toxicological properties. Indeed, the study was conducted on a nano-CeO2 being 94.8% pure and displaying a highly negative surface charge, thus having potential surface impurities and/or coating. Aalapati S et al. described the most severe effects in the lungs of male mice, notably pulmonary necrosis, proteinosis, and fibrosis. The pulmonary effects occurring in mice exposed for up to 4 weeks to 2 mg nano-CeO2/m3 were more severe than those observed in rats exposed for 4 weeks to nano-CeO2 at concentrations up to 25 mg/m3.

At concentrations of 2 mg/m3 and above, it cannot be excluded that the effects observed in exposed rodents were the result of impairment of lung clearance corresponding to the overload phenomenon. Indeed, according to the ECETOC document on overload (2013) and the recent publication of Keller J et al. (2014), a lung overload is expected to occur at respirable particle concentrations ≥ 3 mg/m3 (Muhle H et al., 1988; cited in ECETOC documentation) and more particularly at nano-CeO2 concentrations ≥ 5 mg/m3 (Keller J et al., 2014). If the effects described in the study of Aalapati S et al. and the five other publications resulted from a lung overload phenomenon, these biological response should be taken into consideration with precaution regarding their relevance to human, considering the difference in respiratory tracts between rodents and human. In ECHA R7a guidance “appendix R7-1”, the rat species is indeed described as being more sensitive to lung overload when compared to human (Valberg PAet al., 2009). Nevertheless, the data displayed in the different studies suggested that pulmonary injury and inflammation might be transient, implying a possible recovery over time; but the post-exposure periods tested in the different studies seemed to be too short to accurately assess the recovery. Indeed, Yokel RA et al. (2014) demonstrated that a suitable recovery period should last at least 90 days. The recovery periods lasted 1 to 28 days in the different studies detailed above.

Moreover, Aalapati S et al. described an extrapulmonary distribution of nano-CeO2 and systemic impairments (i.e., tubular degeneration in kidneys), while the five other publications documented no or rare distribution in extrapulmonary organs, no systemic toxicity and/or no mortality or morbidity. As abovementioned, it could not be excluded that the species and strain chosen by Aalapati S et al. might have impacted the biological effects observed. Nevertheless, as no control groups were included in this study and since the results were rather compared only to historical data, it was difficult to assess the reliability of the effects described by Aalapati S et al. (2014). Furthermore, even i.v. injection of high doses of nano-CeO2 did not lead to similar changes in rat kidney and heart (Yokel R et al., 2012; see in section 7.1.1 Basic toxicokinetics for details).

In conclusion, these studies demonstrated that a subacute inhalation exposure in rodents to nano-CeO2 (up to 25 mg/m3) induced no mortality and no or rare systemic toxicity. However, the tested nanomaterials caused loco-regional effects in rodent lungs from the concentration level of 0.5 mg/m3 which induced an impaired clearance corresponding to a lung overload. Thus, the obtained results might not be suitable for the assessment of nano-CeO2 hazard in human.

REPEATED DOSE TOXICITY: OTHER ROUTES

Additional in vivo studies are available on nano-CeO2 administered by intratracheal (Ma J et al., 2015) and intraperitoneal (i.p.) routes (Poma A et al., 2014; Rocca A et al., 2015).

Ma J et al. (2015) evaluated the pulmonary responses to nano-CeO2 in a time- and dose-dependent manner, in rats exposed by intratracheal instillation. The authors also investigated the possibility to inhibit nanoparticle toxicity by encapsulating nano-CeO2 with an amorphous nanothin SiO2 layer. The publication was awarded a Klimisch score of 2 and was flagged as supporting study. Male Sprague-Dawley rats (5 to 6/group) were exposed once by intratracheal instillation to 0, 0.15, 1, or 3.5 mg/kg bw of uncoated nano-CeO2 (CeO2), SiO2-coated nano-CeO2 (aSiO2/CeO2), or SiO2 coating (aSiO2). Animals were sacrificed at days 1, 3, 10, 28 and/or 84 post-exposure. Morphological analysis of lung showed an increased inflammation, surfactant and collagen fibres after CeO2 (high dose at 3.5 mg/kg) treatment at 28 days post-exposure. aSiO2 coating significantly reduced CeO2-induced inflammatory responses in the airspace and appeared to attenuate fibrosis.

Poma A et al. (2014) investigated the effects of nano-CeO2 (0, 50, 500, 5000 µg/kg bw) in male and female CD-1 mice (3/sex/dose) treated daily via i.p. route for a period of 14 days. The publication was awarded a Klimisch score of 3 and was flagged as supporting study. The authors concluded that nano-CeO2 exposure resulted in inducing no adverse toxic effects but proved to be able to be uptaken by liver and to reach lungs by hematic flux. Except possible haematological changes and histological observations (which suggested an inflammation in liver and kidneys according to the authors), there was no adverse effect observed in exposed mice of either sex, regardless of the dose administered.

In addition, Rocca A et al. (2015) investigated the antioxidant effects of nano-CeO2 as a potential pharmaceutical approach for the treatment of obesity. The publication was awarded a Klimisch score of 3 and was flagged as supporting study. The authors evaluated the impact of nano-CeO2 (0 or 0.5 mg/kg bw) following repeated i.p. administration to male Wistar rats (6/group) injected twice a week for 6 weeks. Nano-CeO2 at 0.5 mg/kg bw/d did not have appreciable toxic effects in i.p. injected rats, but instead efficiently contributed in reducing the weight gain, as well as insulin, leptin, glucose and triglycerides concentration with respect to the control group. The histopathological analysis of tissues demonstrated that adipose tissue, kidney, liver and spleen displayed no alterations of tissue architecture. The authors concluded that nano-CeO2 was well tolerated by rats; and nano-CeO2 interfered with the adipogenesis due to its ROS scavenger ability, inhibiting both genes transcription and phenotype development typical of terminally differentiated adipocytes.

Overall, studies of Poma A et al. (2014) and Rocca A et al. (2015) demonstrated that, regardless of the experimental conditions applied and the physico-chemical properties of tested nano-CeO2, the i.p. administration of nano-CeO2 induced some few haematological changes and a potential accumulation in lungs and reticulo-endothelial tissues such as liver. However, i.p. exposure caused no systemic adverse effects. On the contrary, Rocca A et al. showed that nano-CeO2 could have beneficial effects in rodents receiving the nanoparticles in suspension via i.p route. Nevertheless, in our regulatory context, these results are difficult to use for the assessment of nano-CeO2 chronic hazard.

REPEATED DOSE TOXICITY: DERMAL

There is no data available on the repeated dermal toxicity of nano-CeO2. CeO2 is an insoluble inorganic expected to be poorly absorbed via dermal exposure; and it is established that intact skin greatly restricts and, in most cases, prevents nanoparticle uptake. Nanomaterials dermally applied do not penetrate deeply the intact skin, at least not as far as the stratum (Yokel RA and MacPhail C, 2011), from which nanomaterials could enter lymphatic or blood circulation. As a consequence, nano-CeO2 dermally applied will not or poorly translocate from skin to blood; thus a systemic toxicity of nano-CeO2 is not expected.

In addition, based on the data on repeated dose toxicity of nano-CeO2 already available by oral and inhalation routes and in accordance with column 2 adaptation of REACH Annex VIII (section 8.6.1), the repeated dose toxicity study via the dermal route does not need to be conducted if the study does not appear to be scientifically necessary. Nine repeated toxicity studies (1 by oral route and 8 by inhalation route) are available on nano-CeO2. Moreover, really limited skin absorption of nano-CeO2 is anticipated due to its physico-chemical properties (e.g., inorganic substance, extremely low solubility, and aggregation / agglomeration state). Furthermore, oral and inhalation exposure routes are the most appropriate to assess the subacute/chronic toxicity hazard presented by the substance based on its physico-chemical characteristics and use pattern.

Justification for classification or non-classification

No classification is proposed according to GHS criteria at the time being as the repeated dose toxicity section is provisional waiting for further results.