Cerium dioxide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013 - 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2020

Materials and methods

Objective of study:

absorption

distribution

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD test guideline 453

Version / remarks:

An organ burden study with nano CeO2 was performed as part of a carcinogenicity study performed according to OECD test guideline no. 453.

Principles of method if other than guideline:

The CeO2 burden for lung, lung-associated lymph nodes, and major non-pulmonary organs, blood, and feces, was determined in a chronic whole-body inhalation study in female Wistar rats undertaken according to OECD TG453. Female rats were whole body exposed to nano CeO2 NM-212, 6 h per day for 5 days per week for a 104 weeks with the following concentrations: 0, 0.1, 0.3, 1.0, and 3.0 mg/m3. Then, animals were sacrificed after 3, 12, 24 months. Different spectroscopy methods (ICP-MS, ion-beam microscopy) were used for the quantification of organ burden and for visualization of NP distribution patterns in tissues.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cerium dioxide

EC Number:

215-150-4

EC Name:

Cerium dioxide

Cas Number:

1306-38-3

Molecular formula:

CeO2

IUPAC Name:

cerium dioxide

Test material form:

solid: nanoform

Details on test material:

Remark: It should be noted that the information on CeO2 characterisation as reported in the article of Tentschert et al; (2020) differ for some parameters to those reported in the articles of Keller et al (2015) and XX (carcinogeicity study to be reported when available) while both articles are related to the same study (OECD 453 study performed with nanoCeO2).

Information as reported in the article:
This study used CeO2 NPs (NM-212), which were also part of the ‘Nanomaterial Testing Sponsorship Program’ of the OECD (2007). The NP characterization was done by the European Commission Joint Research Center (JRC) Nanomaterial Repository (Ispra, Italy).
- State of aggregation: yes. The results indicate an agglomerated micron-sized aerosol, which varied with concentration (1.4–2.3 mm) - See MMAD below.
- Primary particle size : 28.4 nm
- Particle size distribution: no data
- Mass median aerodynamic diameter (MMAD): 2.3, 1.7, 1.5, 1.4 µm at the targeted concentrations of 0,1, 0.3, 1.0, 3.0 mg nano CeO2/m3, respectively.
- Geometric standard deviation (GSD): 2.4, 2.3, 2.3, 2.1 at the targeted concentrations of 0,1, 0.3, 1.0, 3.0 mg nano CeO2/m3, respectively.
- Shape of particles: not specified
- Surface area of particles: 27.2m2/g (mean BET surface area)
- Crystal structure: not specified
- Coating: no
- Surface properties: not specified
- Density: not specified
- Moisture content: not specified
- Residual solvent: not specified
- Activation: not specified
- Stabilisation: not specified
- Water solubility: <1 µg/L,
- Purity: > 99.5 %,
- Other: Information provided by Singh et al. (2014) and Fh-IME Schmallenberg.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany).
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified in this article
- Housing: The animals were maintained in groups of up to five animals in polysulfon cages (TECNIPLAST, Germany) with access to wooden gnawing blocks.
- Diet (e.g. ad libitum): ad libitum. GLP certified feed (Kliba laboratory diet, Provimi Kliba SA, Kaiseraugst, Basel, Switzerland).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified in this article

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified in this article
- Humidity (%): not specified in this article
- Air changes (per hr): not specified in this article
- Photoperiod (hrs dark / hrs light):not specified in this article

IN-LIFE DATES: From: To: not specified in this article

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

unchanged (no vehicle)

Details on exposure:

For details, see Keller et al. (2015) in IUCLID scetion 7.5.2

Duration and frequency of treatment / exposure:

The animals were exposed to CeO2 aerosols for a total of 104 weeks (2 years). Exposure to CeO2 NPs was 5 days a week for 6 h per day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

Measured concentrations : 0,1 +/- 0,1 mg/m3 +/- SD

Doses / concentrations 3

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

Measured concentrations : 0,3 +/- 0,1 mg/m3 +/- SD

Doses / concentrations 4

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

Measured concentrations : 1,0 +/- 0,1 mg/m3 +/- SD

Doses / concentrations 5

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

Measured concentrations : 3,0 +/- 0,4 mg/m3 +/- SD

No. of animals per sex per dose / concentration:

For the 3 and 12 months’ time point analyses, three animals per dose group were sacrificed for burden analyses. For the 24 months’ time point analyses four animals per dose group were used for this study.

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The concentrations used in the 2-year study were chosen on the basis of results from a preliminary 28 day study (Keller et al., 2014) summarized in IUCLID Section 7.5.2.
- Exposure conditions during the study: The animals were exposed whilst being in wire cages located in a stainless-steel whole-body inhalation chamber (V=2.8m3 or V=1.4m3). The aerosols entered the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. Characterization, generation, and monitoring of aerosols, animal whole-body exposure, and all clinical pathology were performed at BASF SE Ludwigshafen, Germany. For more information, see Keller (2015) as summarized in IUCLID scetion 7.5.2.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY
Organ burden, distribution and localisation of nano CeO2 were analysed in this study:
- Tissues and body fluids sampled : brain, olfactory bulbs, lungs, kidneys, liver, heart, spleen, and small intestine (jejunum). Feces were taken directly from the intestine of the sacrificed animals of the appropriate dose group. Additionally, bone, bone marrow, and blood samples were taken at the same time points. For the lymph nodes, a distinction was made between tracheobronchial, mediastinal, and mesenteric lymph nodes. The tracheobronchial lymph nodes were analyzed after 3, 12, and 24 months of exposure, while the mesenteric lymph nodes were removed at 12 and 24 month time points and the mediastinal at 24 months.
- Time and frequency of sampling: Animals were sacrificed at 3, 12, and 24 months.
- Other: For the 3 and 12 months’ time point, three animals per dose group were sacrificed, for the 24 months’ time point four animals per dose group were used.

Statistics:

Data from CeO2 contents of each test group from different time points were used in a two-way ANOVA for calculating dependencies between organ burden and exposure concentration, as well as between organ burden and time. Results were statistically significant at p ≤ 0.05. Dunnett’s test was used to compare CeO2 tissue contents between the control group and each test group. For calculations the software R3.03 was used.
Outliers for each organ within each test group were identified using the Grubbs test.(Grubbs, 1950). Deposition of CeO2 of each organ and test group was illustrated applying software REGRESSA.
Results of tissue-related CeO2 contents are presented as mean value ± standard deviation (SD) for liver, kidneys and spleen (n = 3 each) on the last day of exposure (except spleen dose group #1, n = 2). All other data points reflect the CeO2 values of one organ and time point per animal.
The measured elemental tissue cerium contents were converted to CeO2, because CeO2 NPs were previously assumed to remain nanoparticles (NPs) even after distribution and deposition.

Results and discussion

Preliminary studies:

Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:

> Lung burden:
During the 2-year inhalation study, the lung burden was evaluated after 3, 12, and 24 months. After 24 months of exposure, the maximum CeO2 lung burden (5.88mg CeO2/lung) with a mean value of 4.4 ± 1.0 mg/lung was found associated with the highest aerosol concentration of Group 4. The lung burden (in µg/lung +/- SD) of each test groups after 3, 12 and 24 month of exposure were as follow (see also Table 1 below in "Any other information on results incl. tables):
Control group: 1.23 ± 0.63, 1.56 ± 0.63, 1.35 ± 036 µg/lung at 3, 12 and 24 months of exposure, respectively.
0,1 mg/m3 group (Group # 1): 19.92 ± 2.13, 49.22 ± 11.46, 78.02 ± 34.91 µg/lung at 3, 12 and 24 months of exposure, respectively.
0,3 mg/m3 group (Group # 2): 48.47 ± 5.65, 227.83 ± 27.71, 348.4 ± 54.25 µg/lung at 3, 12 and 24 months of exposure, respectively.
1 mg/m3 group (Group # 3): 258.14 ± 24.94, 924.36 ± 99.96, 1450.07 ± 275.03 µg/lung at 3, 12 and 24 months of exposure, respectively.
3 mg/m3 group (Group # 4): 772.03 ± 126.45, 2603.02 ± 337, 4411.94 ± 1038.55 µg/lung at 3, 12 and 24 months of exposure, respectively.

For all aerosol concentrations tested (Groups #1 to #4), a linear increase in lung burden was observed over time (R2 values ranging from 0.96 to 0.99). No difference in the curve shape between the lowest and the highest dose group was observed. The highest dose group of 3 mg/m3 (m =172) showed approximately a threefold higher slope than the next lower exposure concentration (dose Group #3; m =56). The same relationship was found between the other dose groups, except dose Group #1 with a slightly lower m value as expected (m =3). This indicates a proportional relationship for exposure dose versus time and amongst, at least, for the aerosol exposure Groups #2–#4. The predicted and measured lung burden was found to match reasonably well with however, some underestimation of measured lung burden values, especially after the 12-month time point, according to the authors.
A retention half-time of 164 days was calculated for dose Group #3, which was higher than expected. The calculated retention half-time for Group #4 is 200, which would mean the overload condition of the lung is reached (see also Table 1 in "Any other information on results incl. tables). Based on the aerosol characteristics measured during NP exposure, a prediction of the deposited alveolar fraction was generated using the MPPD model. For the four aerosol concentrations (#1–#4), an alveolar deposition fraction of around 12% was determined (see also Table 1 in "Any other information on results incl. tables).

Details on distribution in tissues:

> Lymph nodes burden:
As lymph nodes play a key role in lung clearance, three different lymph node regions were investigated to measure their CeO2 load. The lung associated lymph nodes (LALN), comprising the tracheobronchial plus the mediastinal lymph nodes and the mesenteric lymph nodes were analyzed individually for all 4 dose groups (see Table 3 in "Any other information on results incl. Tables"). The NP contents of LALN reached a level of 2400 ± 570 µg/organ CeO2 in Group # 4 at 24 months of exposure. These results show that the LALN carry the highest CeO2 burden from all investigated organs besides the lung, for all dose groups at all studied time points, whereas CeO2 values of the mesenteric lymph nodes are more than 10 000 times lower (<80 ng of total CeO2 in all mesenteric lymph nodes collected).
Visaulisation of CeO2 NP in lung tissue samples using ToF-SIMS, ligt microscopy and mass spectrometry showed NP agglomerates within the lung sections of the animals group # 4 at 24-month time point. Tissue sections of the 2-year study revealed an inhomogeneity throughout the whole lung. NP concentrations in different lung tissue areas varied rather widely. The particles were found mainly in macrophages. In addition, the alveolar septum contained some smaller NP agglomerates. Large NP agglomerates are concentrated in the bronchus-associated lymphoid tissue (BALT) with smaller agglomerates distributed throughout the remaining lung tissue.
Very large agglomerates (category 10: >200 mm2) of CeO2 NP or a fraction of very small agglomerates (category 1: 1–1.49 mm2) were found in the test group # 4 (3.0 mg/m3) after 24 months of exposure. The small and medium-sized agglomerates are either significantly less abundant or completely absent. In the the next lower dose (group # 3, 1 mg/m3), after 24 months, all observed NP agglomerates were smaller than 200 mm2. The highest number of agglomerates was observed in category 6 (6.1–12.17 mm2).

Extrapulmonary distribution (see Table 3 in "Any other information on results incl. Tables"):
The absolute CeO2 concentrations in the control groups for each time point (3, 12 and 24 months of exposure) were very low. The average values ranged from 0.001 to 0.04 ng/mg for tissue and from 0.0001 mg/mg to 0.02 mg/mg for feces.
The cerium blood concentrations for all dose groups were below or close to the limit of detection (0.04 mg/ mL) and thus negligible.
Generally, the highest extra-pulmonary organ burden was detected in Group #4 and was lower in Groups 3, 2, and 1 (see also Table 3 in "Any other information on results incl. tables). Besides the LALN, CeO2 burden was highest in liver (~12 µg CeO2/organ, in group 4 at time point 24 months) with the following ranking liver > kidneys, spleen >>> heart > brain > olfactory bulb after 24 months. The ratios of liver to lung burdens are low with a peak at the 12-month time point followed by a leveling off or reduction for all investigated exposure concentrations. For brain and heart no significant changes in CeO2 organ burden compared to control levels were detected after 24 months of exposure. This holds also true for the Ce-concentrations measured in blood. Relatively high amounts of Ce was found in femur bones (~1800 +/- 400 ng/g) and bone marrow (350 +/- 110 ng/g) in animals of group # 4 after 24 month of exposure in comparison to all other extrapulmonary organs, except LALNs. The burden of the skeleton in the same animal group was calculated to be 44.9 µg/skeleton (calculated from the CeO2 concentration in femur tissue and the assumption of a homogenous distribution and a skeleton weight corresponding to 6 % of the body weight of the rat). According to the authors, under these assumptions, around 1.1% of the lung burden (4412 µg/lung) would be retained in the skeleton for the group #4. In comparison, 1.2% of the lung burden was found in all investigated extra-pulmonary organs excluding lymph nodes and skeleton.

Details on excretion:

From the results reported in the section "details on distribution in tissues" and in Table 3 in "Any other information on results incl. tables, the authors concluded that, of all investigated organs excluding the lung, lymph nodes in close proximity of the lung carry the highest NP burden resulting from particles deposited in the alveolar region indicating the primary excretory route.
A clearance via fecal excretion was also observed since an increase of CeO2 concentrations (as µg/g of feces) was observed in all treated groups between 3 and 12 months followed by a decrease between month 12 and 24 (see Table 3 in "Any other information on results incl. tables). When calculated by day, considereing 4 g/day of feces, fecal excretion reached 64 to 180 µg/day in the group #4 during the study.

Any other information on results incl. tables

Chemical entity of Ce:

ToF-SIMS imaging mass spectrometry was used to answer the question in which chemical entity Ce was present in the femur sections of exposed rats ( group #4, 24 months of exposure). Via mass spectrometry, the authors could clearly identify and distinguish CeO+ ions and cerium phosphate ions from other ions like calcium phosphate. Large sized particule agglomerates from CeO2 and cerium phosphate were located in distinctive areas of the femur bone tissue sections. Whilst cerium phosphate locates predominantly in the trabeculae of the spongy bone section, the CeO2 NP agglomerates locate close to the bone marrow. The authors suggests that this indicates that the CeO2 NP might enter the bone through the bone marrow canals and afterward mineralize as cerium phosphate within areas where calcium phosphate is already present.

Tables:

Table 1:Lung burden - Predicted lung burden, clearance half-times and deposition fraction of exposed rats

Exposure time	1day	3 months	12 months	24 months
Aerosol concentrations	Measured lung burden (µg/lung) ± SD				Predicted clearance t1/2 (days)*
Control	-	1.23 ± 0.63	1.56 ± 0.63	1.35 ± 036	-
0.1 mg/m3	-	19.92 ± 2.13	49.22 ± 11.46	78.02 ± 34.91	86
0.3 mg/m3	-	48.47 ± 5.65	227.83 ± 27.71	348.4 ± 54.25	114
1.0 mg/m3	-	258.14 ± 24.94	924.36 ± 99.96	1450.07 ± 275.03	164
3.0 mg/m3	-	772.03 ± 126.45	2603.02 ± 337	4411.94 ± 1038.55	200
	Predicted lung burden (µg/lung)*				Deposition fraction (%)*
0.1 mg/m3	0.85	31.48	59.62	64.49	11.8
0.3 mg/m3	3.15	108.44	205.36	222.13	14.6
1.0 mg/m3	9.72	415.54	1170.92	1658.14	13.5
3.0 mg/m3	17.7	753.78	2124.03	3007.83	8.2

 

*Calculated with MPPD model.

 

 

Table2: Organ weights of the tissue, hard bone, bone marrow and feces collected during the study

Time points

3 months

12 months

24 months

CeO2[mg/m³] exposure groups

0

0.1

0.3

1

3

0

0.1

0.3

1

3

0

0.1

0.3

1

3

Organ weights and feces (g) +/- SD

lung

0,86 +/- 0.03

0.90 +/- 0.09

0.95 +/- 0.10

0.90 +/- 0.03

1.06 +/- 0.04

1.02 +/-0.09

1.05 +/- 0.09

1.00 +/- 0.01

1.09 +/- 0.02

1.17+/- 0.03

1.11 +/-0.04

1.12 +/- 0.05

1.12 +/-0.08

1.19 +/- 0.05

1.33 +/-0.17

olf.bulb

0.08 +/- 0.01

0.08 +/- 0.01

0.09 +/- 0.01

0.09 +/- 0.01

0.08 +/- 0.01

0.07 +/- 0.02

0.07 +/- 0.01

0.06 +/- 0.01

0.06 +/- 0.01

0.07 +/- 0.01

0.08 +/- 0.00

0.62 +/- 0.35

0.83 +/- 0.09

0.87 +/- 0.04

0.42 +/- 0.43

Brain

1.80 +/- 0.05

1.80 +/- 0.09

1.91 +/- 0.12

1.83 +/- 0.02

1.92 +/- 0.05

2.00 +/- 0.10

1.93 +/- 0.02

1.91 +/- 0.09

1.90 +/- 0.09

2.03 +/- 0.05

2.00 +/- 0.04

2.02 +/- 0.01

1.93 +/- 0.05

2.01 +/- 0.06

1.94 +/- 0.08

Lymph nodes (lung)

0.11 +/- 0.11

0.17 +/- 0.02

0.118 +/- 0.04

0.18 +/- 0.06

0.38 +/- 0.12

0.02 +/- 0.00

0.03 +/- 0.01

0.03 +/- 0.01

0.06 +/- 0.00

0.07 +/- 0.01

-*

-

-

-

-

Tracheobronchial lymph nodes

-

-

-

-

-

-

-

-

-

-

0.01 +/- 0.00

0.00 +/- 0.00

0.01 +/- 0.00

0.02 +/- 0.00

0.03 +/- 0.02

Mediastinal lymph nodes

-

-

-

-

-

-

-

-

-

-

0.02 +/- 0.00

0.02 +/- 0.00

0.02 +/- 0.01

0.04 +/- 0.03

0.06 +/- 0.04

Mesenteric lymph node

-

-

-

-

-

0.27 +/- 0.11

0.28 +/- 0.11

0.25 +/- 0.07

0.19 +/- 0.01

0.22 +/- 0.03

0.29 +/- 0.08

0.17 +/- 0.05

0.24 +/- 0.13

0.18 +/- 0.01

0.17 +/- 0.08

Jejunum

0.75 +/- 0.14

0.83 +/-0.09

1.05+/- 0.14

0.80+/- 0.04

0.85 +/- 0.05

1.20 +/- 0.14

0.95 +/-0.19

0.80+/- 0.21

1.00+/- 0.33

1.14+/- 0.46

0.50 +/- 0.27

0.46 +/- 0.05

0.57 +/- 0.20

0.47 +/- 0.12

0.49 +/- 0.26

Liver

6.71 +/- 0.39

7.97 +/- 0.56

7.23 +/- 0.38

7.66+/- 0.58

8.36+/- 0.61

7.55 +/- 0.88

8.21 +/- 0.93

7.51+/- 0.92

8.30+/- 0.56

8.08+/- 0.70

9.10 +/- 0.21

10.21 +/- 1.23

9.20+/- 0.79

8.23+/- 0.61

8.61+/- 2.28

Kidneys

1.33 +/- 0.09

1.41 +/- 0.09

1.49+/- 0.01

1.40 +/- 0.02

1.53 +/- 0.03

1.60 +/- 0.22

1.74 +/- 0.17

1.59+/- 0.11

1.61 +/- 0.13

1.74 +/- 0.04

2.20 +/- 0.01

2.02 +/- 0.13

2.09+/- 0.16

1.91 +/- 0.11

2.15 +/- 0.60

Spleen

0.51 +/- 0.05

0.51 +/- 0.05

0.57 +/- 0.02

0.57 +/- 0.02

0.61 +/- 0.07

0.62 +/- 0.06

0.64 +/- 0.13

0.59 +/- 0.10

0.69 +/- 0.27

0.74 +/- 0.03

0.72 +/- 0.03

0.94 +/- 0.28

0.86 +/- 0.16

0.72 +/- 0.14

0.72 +/- 0.05

Heart

0.65 +/- 0.03

0.67 +/- 0.05

0.76 +/- 0.05

0.71 +/- 0.04

0.76 +/- 0.03

0.79 +/- 0.07

0.84 +/- 0.04

0.78 +/- 0.04

0.79 +/- 0.08

0.78 +/- 0.03

1.14 +/- 0.00

0.95 +/- 0.08

1.02 +/- 0.09

0.94 +/- 0.09

0.97 +/- 0.10

Femura

-

-

-

-

-

-

-

-

-

-

2.58 +/- 0.74

2.15 +/- 0.54

2.01 +/- 0.30

1.67 +/- 0.09

2.19 +/- 0.21

Bone marrow

0.03 +/- 0.01

0.03 +/- 0.00

0.02 +/- 0.01

0.02 +/- 0.01

0.02 +/- 0.01

0.03 +/- 0.01

0.02 +/- 0.01

0.02 +/- 0.01

0.02 +/- 0.01

0.02 +/- 0.01

0.04 +/- 0.03

0.04 +/- 0.01

0.05 +/- 0.02

0.04 +/- 0.01

0.04 +/- 0.02

Blood

-

-

-

-

-

-

-

-

-

-

1.87 +/- 0.01

1.90 +/- 0.08

1.85 +/- 0.09

1.94 +/- 0.05

1.94 +/- 0.07

Feces

1.27 +/- 0.43

2.08 +/- 0.52

1.25 +/- 0.53

1.60 +/- 0.95

1.67 +/- 0.41

1.41 +/- 0.23

1.46 +/- 0.90

1.42 +/- 0.29

1.61 +/- 0.26

1.17 +/- 0.44

1.81 +/- 0.21

2.51 +/- 0.81

2.66 +/- 0.44

1.73 +/- 0.49

1.37 +/- 0.72

 

*: - = not measured

 

Table 3: CeO₂organs burden

Months of exposure

3

12

24

CeO2[mg/m³] exposure groups

0.1

0.3

1

3

0.1

0.3

1

3

0.1

0.3

1

3

Organ burdens (µg or ng/organ or ng/g of tissue)

Lung-associated lymph node (LALN) µgCeO2/organ(mean +/- SD)

0.01 +/- 0.01

0.11 +/- 0.01

1.33 +/- 0.85

30.39 +/- 0.00

0.61 +/- 0.50

18.55 +/- 5.93

165.68 +/- 45.11

646.67 +/- 144.43

0.33 +/- 0.17

12.59 +/- 7.65

572.45 +/- 140.71

2372.15 +/-  569.25

Mesenteric lymph node ngCeO2/organ(mean +/- SD)

-*

-

-

-

2.17 +/- 0.10

4.03 +/- 1.52

16.26 +/- 7.29

48.36 +/- 10.44

2.06 +/- 0.59

7.88 +/- 4.63

39.26 +/- 23.57

72.93 +/- 19.96

Liver µgCeO2/organ (mean +/- SD)

0.23 +/- 0.06

0.33 +/- 0.11

1.22 +/- 0.08

2.01 +/- 0.42

0.59 +/- 0.32

1.38 +/- 0.28

4.58 +/- 0.37

4.78 +/- 0.65

0.30 +/- 0.08

2.63 +/- 0.39

3.05 +/- 1.06

11.71 +/- 3.38

Speen ngCeO2/organ (mean +/- SD)

0.93 +/- 0.35

1.75 +/- 0.72

4.37 +/- 2.25

48.51 +/- 4.11

5.86 +/- 5.44

20.93 +/- 9.33

155.31 +/- 34.26

169.57 +/- 14.25

19.77 +/- 4.18

75.89 +/- 42.59

281.59+/- 113.19

793.70 +/-  115.20

/Kidneys ngCeO/organ (mean +/- SD)

9.19 +/- 3.80

26.30 +/- 2.12

16.62 +/- 3.86

39.11 +/- 7.77

31.53 +/- 3.03

50.87 +/- 1.76

118.80 +/- 21.17

217.69 +/- 41.34

53.93 +/- 7.64

211.85 +/- 75.73

317.84 +/- 86.10

854.46 +/- 386.64

Brain ngCeO2/organ(mean +/- SD)

0.42 +/- 0.59

0.21 +/- 0.18

1.71 +/- 0.33

3.37 +/- 2.04

1.06 +/- 0.22

0.98 +/- 0.09

2.33 +/- 0.14

4.42 +/- 0.30

6.21 +/- 5.18

2.17 +/- 0.60

5.48 +/- 1.48

13.14 +/- 8.65

Heart ngCeO2/organ (mean +/- SD)

0.63 +/- 0.29

0.72 +/- 0.09

1.60 +/- 0.36

4.23 +/- 1.32

1.75 +/- 0.17

3.64 +/- 0.60

7.37 +/- 0.81

11.87 +/- 3.50

8.68 +/- 5.05

11.08 +/- 2.77

30.53 +/- 17.90

58.22 +/- 16.38

Olfactory bulb ngCeO2/g tissue(mean +/- SD)

0.16 +/- 0.16

0.18 +/- 0.02

0.40 +/- 0.06

2.52 +/- 1.78

0.22 +/- 0.05

0.42 +/- 0.02

1.00 +/- 0.39

1.79 +/- 0.32

0.42 +/- 0.11

1.29 +/- 0.16

2.11 +/- 0.75

8.98 +/- 5.07

Small intestine (jejunum) ngCeO2/g tissue(mean +/- SD)

19.43 +/- 7.85

36.82 +/- 24.06

67.73 +/- 6.11

154.48+/- 60.17

9.15 +/- 1.08

22.30 +/- 0.69

28.84 +/- 9.09

174.07 +/- 183.51

3.87 +/- 1.76

11.89 +/-  6.26

39.06 +/- 14.12

167.15 +/- 123.62

Bone marrow ngCeO2/g tissue(mean +/- SD)

11.19 +/- 4.04

5.95 +/- 2.66

8.01 +/- 5.68

31.28 +/- 11.51

29.36 +/- 14.73

30.28 +/- 10.61

99.08 +/- 50.79

213.40 +/- 25.77

26.46 +/- 14.91

70.46 +/- 14.59

228.44 +/- 105.58

349.30 +/- 108.22

µgCeO2/Skeleton**  (mean +/- SD)

-

-

-

-

-

-

-

-

3.83 +/- 1.15

9.77  +/- 2.09

24.22 +/-  4.97

44.89 +/- 8.57

µgCeO2/g of feces***  (mean +/- SD)

~ 1 +/- 0.2

~ 1 +/- 0.0

~ 6 +/- 1

~ 28 +/- 0.0

~ 3.5 +/- 1

~ 5 +/- 1.5

~ 18 +/- 10

~ 46 +/- 14

~ 1 +/- 0.0

~ 3 +/- 0.5

~ 12.5 +/- 5

~ 16 +/- 6

 

*: - = not measured

**: CeO₂bone (femur) burden (extrapolated based on the assumption that 6% BW of the rat equal the whole skeleton of the rat (Trevos 2006).

***: approximate concentrations deduced from the graph included in the article since exact values were not included under a table format.

 

Applicant's summary and conclusion

Conclusions:

After exposure by inhalation to 0.1, 0,3, 1 and 3 mgm3 nano CeO2 for 24 month in an OECD 453 study, the CeO2 burden in the organs analysed was found with the following ranking: lungs > lymph node > hard bone > liver > bone marrow > kidneys, spleen >>> heart > brain > olfactory bulb. According to the authors, estimated alveolar deposition fraction was found to be around 12 % and 1.2% of the lung burden was found in all investigated extra-pulmonary organs excluding lymph nodes and skeleton and around 1.1% of the lung burden would be retained in the skeleton for the group #4.

Executive summary:

The CeO2 organ burden, distribution and localisation was determined in a chronic whole-body inhalation study in female Wistar rats performed according to OECD TG 453 and in compliance with GLP. Groups of female rats were whole body exposed to nano CeO2 NM-212, 6 h per day for 5 days per week for a 104 weeks with the following concentrations: 0 (control- air), 0.1, 0.3, 1.0, and 3.0 mg/m3. The CeO2 burden was measured in lung, lung-associated lymph nodes, brain, olfactory bulbs, kidneys, liver, heart, spleen, small intestine (jejunum), bone (femur), bone marrow, blood and in feces in 3 to 4 animals/groups after 3, 12, 24 months of exposure. Quantification of CeO2 organ burden and visualization of nanoparticule distribution in tissues were performed using ICP-MS and ion-beam microscopy. Experimentally quantified lung burdens were compared with predictions of the deposited alveolar burden using the software an MPPD model.

The lung and the lung associated lumph nodes (LALNs) were found to contain the highest burden of CeO2. After 24 months of exposure, the mean CeO2 lung burden was found to be 1 ± 0,3, 78 ± 35, 348 ± 54, 1450 ± 275 and 4412 ± 1030 µg/lung in groups exposed to 0, 0.1, 0.3, 1.0, and 3.0 mg/m3, respectively. For all aerosol concentrations tested, a linear increase in lung burden was observed over time. The alveolar deposition fraction was estimated using MMPD model around 12% for all exposed groups. The nanoCeO2 contents of the lung associated lymph nodes reach of level of 2372 ± 560 µg CeO2/organ in the high dose concentration group after 24 months of exposure whereas the CeO2 content in mesenteric lymph nodes in the same animal group was = 73 +/- 20 ng/organ. Inhomogenous nanoparticle agglomerates were foung throughout the whole lung. Large NP agglomerates (several up to > 200 mm²) are concentrated in the bronchus-associated lymphoid tissue (BALT) with smaller agglomerates distributed throughout the remaining lung tissue. The particles were found mainly in macrophages. In addition, the alveolar septum contained some smaller NP agglomerates.

The content of CeO2 in all extrapulmonary organ was very low according to the authors. Besides the lung associated lymph nodes, CeO2 burden was highest in liver (12 +/- 3 µg/organ) and relatively high amounts of Ce was found also in femur bones (~1.8 +/- 0.4 µg/g) and bone marrow (~0.350 +/- 0.1 µg/g) in animals of group 4 after 24 month of exposure in comparison to all other extrapulmonary organs, except LALNs. The burden of the skeleton (calculated from CeO2 concentration in femur tissue) in the same animal group was calculated to be 44.9 µg/skeleton. CeO2 burden in other organs were negligible. The following ranking was proposed by the authors lungs > lymph node > hard bone > liver > bone marrow > kidneys, spleen >>> heart > brain > olfactory bulb. According to the authors, under these assumptions, around 1.1% of the lung burden would be retained in the skeleton for the group 4. In comparison, 1.2% of the lung burden was found in all investigated extra-pulmonary organs excluding lymph nodes and skeleton.

According to the accumulation rates, the authors concluded that, with a low accumulation rate, the liver can be regarded as a depot, whereas kidneys, the skeleton and bone marrow seem to be dumps due to steadily increasing nanoparticle burden over time.

It should be noted that in all examined extrapulmonary organs and histopathological examinations of the rats of this study, there was no detection of any adverse effects and no indications of systemic toxicity were found in any of the examined organs (see Keller et al, 2015 and Schaudien et al, 2019, in IUCLID section 7.5.2.