Cerium dioxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key 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 guidelines 492 and 437. However, there was no mention to GLP. The study was designed to evaluate eye irritation potential of 20 nanosized materials and 3 micro-scale materials using a 2-tier non-animal testing strategy.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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. 

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 492 (Reconstructed human cornea-like epithelium test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage)

Deviations:

yes

Remarks:

the amounts of solid nano-CeO2 applied differed from those recommended in the guideline (i.e., 50 mg).

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

The test material was submitted to a 2-tier non-animal testing strategy composed of the EpiOcular™ Eye Irritation Test (EpiOcular™-EIT; OECD TG 492) and the Bovine Corneal Opacity and Permeability (BCOP; OECD TG 437) assay including histopathological evaluation of the cornea.

GLP compliance:

not specified

Remarks:

The GLP compliance was not specified in the published article.

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:

- Name of test material: Nanometric cerium dioxide (nano-CeO2 NM-211 and NM-212) - uncoated and produced by precipitation
- Supplier: European Commission’s Joint Research Centre (JRC, Italy)
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance
- Primary particle size (XRD): 10.3 nm for NM-211 and 33 nm for NM-212 (supplier's data)
- Particle size distribution (AUC, LD): The median diameters (D50) of NM-211 and NM-212 agglomerate fractions in water were 2.14 and 0.776 µm, respectively, using LD. AUC indicated that the size interval up to 1000 nm contributed considerably to the total dose (ca. 31.3% of the dose for NM-211 and ca. 66% of the dose for NM-212).
- Stability: Agglomeration
- Specific surface area (BET): 66 m²/g for NM-211 and 28 m²/g for NM-212 (supplier's data)
- Surface charge: No data available
- Isoelectric point: No data available
- Shape (XRD): Cubic for both NM-211 and NM-212 (supplier's data)
- Crystallinity: No data available
- Analytical purity / impurities: > 95% purity for NM-211 and > 99.5% for NM-212 (supplier's data)
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility (AUC combined with ICP-MS / ICP-AES): < 0.1 ppm (Wohlleben W et al., 2013); 0.002 wt.% (Keller J et al., 2014)
- Oxidation degree: No data available
- Surface properties: Non-coated (supplier's data)
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available
- Physical state: Yellowish powder for both NM-211 and NM-212 (supplier's data)

Further explanations on the physico-chemical characterisation of CeO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Test animals / tissue source

Species:

other: not applicable - in vitro tests

Strain:

other: not applicable - in vitro tests

Details on test animals or tissues and environmental conditions:

EpiOcularTM-EIT HUMAN CORNEA-LIKE MODEL
The EpiOcular™-EIT model from MatTek Corp. (USA) and MatTek In Vitro Life Science Laboratories (Slovakia) use the cornea-like non-keratinized tissue construct EpiOcular™ OCL-200 kit that is composed of normal human epidermal keratinocytes obtained from individual donors. The model is cultured in proprietary serum-free culture medium (in Millicells® with 1-cm diameter) which induces corneal differentiation and formation of the organotypic cornea-like model. This three dimensional tissue consists of highly organized cell layers and exhibits barrier properties similar to the normal in vivo corneal epithelium.
On the day of arrival in the laboratory, the EpiOcular™ tissues (0.6 cm² surface area) were transferred to sterile 6-well plates with 1 mL DMEM and pre-conditioned at standard culture conditions (37°C, 5% CO2, 90–95% humidity) in the incubator for 16–24 h. After pre-incubation, the tissues were pre-treated with 20 μL PBS and further incubated at standard culture conditions for 30 min.

BOVINE CORNEA
Bovine corneas (source not given) were mounted in corneal holders. Both the anterior and posterior chambers of these holders were filled to excess with pre-warmed Eagle’s MEM (without phenol red). After equilibration in a vertical position at 32°C for at least 1 h, the medium in both chambers was replaced with fresh pre-warmed medium and the initial corneal opacity was measured.

Test system

Vehicle:

other: undiluted for EpiOcular™ assay or diluted in water for BCOP assay (see below in "Details on study design")

Controls:

other: see below in "Details on study design"

Amount / concentration applied:

- OECD 492 – EpiOcular™-EIT HUMAN CORNEA-LIKE MODEL
NM-211: 61 mg (corresponding to 50 μL bulk volume)
NM-212: 28 mg (corresponding to 50 μL bulk volume)

- OECD 437 - BCOP
NM-211 and NM-212: 20% (w/v) (corresponding to 750 μL bulk volume)

Duration of treatment / exposure:

- OECD 492 – EpiOcular™-EIT HUMAN CORNEA-LIKE MODEL
Exposure period: 90 min (variant 1), or 6 h (variant 2)
Post-exposure period: 12 min + 18 h (variant 1) or 25 min + 18 h (variant 2)

- OECD 437 - BCOP
Exposure period: 4 h

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

- OECD 492 – EpiOcular™-EIT HUMAN CORNEA-LIKE MODEL
2 tissues per treatment group

- OECD 437 - BCOP
3 corneas per treatment group

Details on study design:

In the 2-tier "bottom up" testing strategy suggested by the authors, the EpiOcular™-EIT was performed in Tier 1 to distinguish "Category 1 or 2" from "Non-category" substances. In Tier 2, the BCOP assay was conducted to identify "Category 1" substances from within the set of "Category 1 or 2" substances identified in Tier 1. All substances that were not identified as "Category 1" in the BCOP assay in Tier 2 were classified as "Category 2".

OECD 492 - EpiOcularTM-EIT HUMAN CORNEA-LIKE MODEL
Controls (see in Table 2): highly deionized water (50 µL) was used as negative control (NC) and methyl acetate (purity > 98 %; 50 µL) as positive control (PC).
Furthermore, 3 organic pigments that fall under the EU recommendation on the definition of nanomaterials representing three different chemical classes, i.e. Pigment Red 57:1, Pigment Yellow 95, and Pigment Black 32, were included (commercial grades from BASF SE). According to Kolle SN et al., these pigments were selected based upon availability of physico-chemical characterisation data and Good Laboratory Practice-compliant animal data. To date, no organic pigment has been classified as an eye irritant. In addition, non-nanosized talc (from a local retailer), the historical negative control for in vivo eye irritation testing, was added to the spectrum of test materials. The 3 organic pigments and talc were applied undiluted at amounts corresponding to 50 μL bulk volume: i.e., 12 mg of Pigment red 57:1, 9 mg of Pigment Yellow 95, 8 mg of Pigment Black 32, and 16 mg of talc.

The EpiOcular™-EIT was performed in two variants. Both NM-211 and NM-212 were submitted to protocol variant 1, i.e. as described by the supplier MatTek and Harbell et al. (2009). The three organic pigments and talc were assessed in accordance with variant 2, i.e. as described in the OECD TG 492. To ensure data comparability, the dry-powder NM-211 and NM-212 were additionally submitted to variant 2. The two test protocol variants differed in respect to the exposure and post-exposure immersion periods laid down for solid test materials.

The test materials were applied undiluted at amounts enabling to cover the entire tissue surfaces (i.e., 50 μL bulk volume). Using a sharp spoon or pipette, the dry-powder items were applied to cover the entire tissue surface. Two tissues were treated with either the test materials, the NC or the PC. After test material application, the tissues were placed into the incubator for the following exposure periods: 90 min (variant 1), or 6 h (variant 2). To remove the test materials, the tissues were washed with sterile PBS and immediately immersed into 12-well plates, pre-filled with 5 mL pre-warmed medium per well to remove test material residuals. After 12 min (variant 1) or 25 min (variant 2), each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL pre-warmed medium per well (post-exposure immersion). Subsequently, the tissues were incubated at standard culture conditions (post-exposure incubation) for 18 h. During the post-exposure immersion and incubation periods, weak cytotoxic effects might reverse, and more pronounced effects might increase.

Tissue destruction was determined by formazan reduction after incubation with the tetrazolium salt 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT), which reflected impaired mitochondrial dehydrogenase activity. Upon completion of the post-exposure period, the assay medium was replaced by 0.3 mL MTT solution. After incubating the tissues for 3 h, the tissues were washed with PBS to terminate the MTT incubation. The produced formazan was extracted by incubating the tissues in isopropanol at room temperature overnight or on a plate shaker for at least 2 h. The optical density of the formazan extracts was determined spectrophotometrically at a wavelength of 570 nm (OD570). For each microtitre plate, blank values were established from 4 wells filled with isopropanol.

For all test materials, pre-tests as described in the OECD TG 492 were performed that precluded the test materials’ ability to directly reduce MTT.

Tissue OD570 values were calculated by subtracting the mean blank value of the respective microtitre plate from the measured tissue OD570 value, and mean OD570 values were calculated for the two tissues of each treatment group. The quotient of the mean OD570 values of the test material-treated tissues and those of the NC (i.e., the mean relative tissue viability) was determined to evaluate whether or not a test material was an irritant:
* Mean relative tissue viabilities ≤ 60 % indicated "irritancy to the eye";
* Mean relative tissue viabilities > 60 % indicated "no irritancy to the eye".

- Acceptance criteria: in case one of the following acceptance criteria (AC) as described in the OECD TG 492 was not met, repetition of the EpiOcular™-EIT was considered.
* AC for the NC: the OD570 of the NC reflected the laboratory-specific tissue viability under the specific conditions of the assay. It was considered acceptable if the mean OD570 of the NC was ≥ 0.8 and ≤ 2.5 and the historical in-house mean at the respective time of testing was met (variant 1: OD570 of NC = 1.361 ± 0.138; variant 2: OD570 of NC = 1.650 ± 0.159).
* AC for the PC: in-house, the PC methyl acetate usually elicited relative tissue viabilities of approx. 25% (historical in-house means at the time of testing in accordance with variant 1: OD570 of PC = 0.318 ± 0.119; variant 2: OD570 of PC = 0.396 ± 0.098). In addition to these historical means, all relative tissue viability values < 50 % were considered acceptable.
* AC for tissue variability: the relative inter-tissue variability (ITV%) between the two tissues of a treatment group was considered acceptable if it was ≤ 20 %.

OECD 437 - BCOP
Controls (see in Table 2): highly deionized water was used as NC. Imidazole 20 % (w/v) dissolved in highly deionized water was used as PC. Moreover, the 3 organic pigments, and talc were applied undiluted (120, 80, 48, 45, and 40 mg, for talc and the three organic pigments Pigment Red 57:1, Pigment Yellow 95, and Pigment Black 32, respectively).

The BCOP assay was conducted according to OECD TG 437.
Bovine corneas were mounted in corneal holders. Both the anterior and posterior chambers of these holders were filled to excess with pre-warmed Eagle’s MEM (without phenol red). After equilibration in a vertical position at 32 °C for at least 1 h, the medium in both chambers was replaced with fresh pre-warmed medium and the initial corneal opacity was measured.

NM-211 and NM-212 powders were suspended in highly deionized water to achieve final concentrations of 20% (w/v) suspension and then stirred with a magnetic stirrer. All test material suspensions were applied immediately after preparation, and also during test material application, the preparations were stirred with a magnetic stirrer to ensure continued homogeneity. Generally, each treatment group (NC, PC, or test material) consisted of 3 corneas. Before application of the test materials, the medium in the anterior chamber was removed:
* For the NC, the anterior chambers were filled with 750 μL highly deionized water and, for the PC, with 750 μL of the 20% (w/v) imidazole solution.
* For NM-211 and NM-212, 750 μL of the 20% (w/v) test material preparation was applied directly to the epithelial surface of the cornea (i.e., using the "open chamber method").
* Talc and the three organic pigments Pigment Red 57:1, Pigment Yellow 95, and Pigment Black 32 were applied undiluted.

The corneas were incubated in a horizontal position at 32°C for 4 h as prescribed for non-surfactant solids in the OECD TG. Upon completion of the incubation period, the NC, PC and test materials were removed from the anterior chamber with a syringe, and the respective epithelia were washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

The final corneal opacity was measured, and the opacity change per cornea was calculated by subtracting the initial from the final opacity value. Subsequently, the mean opacity change of the NC was subtracted thereby providing the corrected opacity change. Test results were provided as means of all corrected opacity changes per treatment group.
To determine corneal permeability, the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL) and incubated in a horizontal position for 90 min at 32°C. The amount of sodium fluorescein that permeated through the corneas was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtitre plate and the optical density value (OD490) was determined subtracting the mean blank OD490 (blank = Eagle’s MEM without phenol red) from the OD490 of each cornea. Corrected OD490 values were calculated by subtracting the mean OD490 values of the corresponding NC. Final test results were calculated as means of all corrected OD490 values per treatment group.
The In Vitro Irritancy Score (IVIS) was calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation (SD) was determined: IVIS = mean opacity value + (15 x mean permeability value). An IVIS > 55 indicated a risk of serious damage to the eyes.

- Acceptance criteria: in case one of the following ACs laid down in OECD TG 437 was not met, repetition of the BCOP assay was considered.
* AC for the NC: the NC responses should be lower than the established upper limits for background opacity and permeability values for the respective NC.
* AC for the PC: the IVIS calculated for the PC should not lie outside the two-fold range of the SDs of the historical mean (i.e., 88.0–147.1 for imidazole).
* AC for the treatment groups: at least 2 of the 3 corneas per treatment group should provide predictions that coincided with the mean of all 3 corneas, and none of the corneas should provide a discordant prediction of 10 IVIS units above or below the cut-off threshold of 55.

HISTOPATHOLOGICAL EVALUATION
For histopathological evaluation by light microscopy, the corneas were fixed in 10% neutral buffered formalin for at least 24 h and trimmed along the whole diameter (2 stripes of 3–4 mm width). They were histotechnically processed with a standard method for light microscopy and stained with Hematoxylin and Eosin. Histopathological findings were assessed in the epithelium based on the depth of injury by using a standard semi-quantitative grading system (from 1 to 5) that is related to the extent of affected cell layers beginning from the corneal surface (squamous cell layer) down to the basal cell layer. In the stroma, tissue swelling and keratocyte changes were evaluated. These findings were summarized in a so-called Histopathological Score of Irritation (HSI) assigned for each cornea ranging from 0 = no irritation to IV = severe irritation. HSI IV was assessed as "severe irritation"; HSI I, II and III were overall assessed as "non-severe irritation"; HSI 0 was regarded as no irritation.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

EpiOcular test - Variant 1 - MN-211

Run / experiment:

Run 1

Value:

81

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

EpiOcular Test - Variant 1 - NM-212

Run / experiment:

Run 1

Value:

81

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

EpiOcular test - Variant 2 - NM-211

Run / experiment:

Run 1

Value:

129

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Remarks:

EpiOcular Test - Variant 2 - NM-211

Run / experiment:

Run 2

Value:

109

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

in vitro irritation score

Remarks:

EpiOcular test - Variant 2 - NM-212

Run / experiment:

Run 1

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

cornea opacity score

Remarks:

BCOP - MN-211

Run / experiment:

Run 1

Value:

18

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Remarks:

BCOP - NM-212

Run / experiment:

Run 1

Value:

17.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

OTHER INFORMATION :

> OECD 492 - EpiOcularTM-EIT HUMAN CORNEA-LIKE MODEL (see in Table 3)
The acceptance criteria (ACs) for the NC and the PC were always met in the EpiOcular™-EIT performed in accordance with either the protocol variants 1 and 2.

* Test results, variant 1 (90-min treatment)
After washing, test material residues were observed on the EpiOcular™ tissues treated with CeO2 NM-211 and NM-212. However, since none of the test materials was able to reduce MTT directly, it was concluded that these residues did not interfere with the MTT assay.
For both NM-211 and NM-212, mean tissue viabilities above 60% relative to the NC were calculated. Hence, none of the tested nano-CeO2 revealed eye irritation potential in the EpiOcular™-EIT under the chosen test conditions (i.e., indicating likelihood of "neither Category 1 nor 2").

* Test results, variant 2 (6-hour treatment)
After the 1st test run conducted in accordance with the EpiOcular™-EIT variant 2, mean relative tissue viabilities > 60% (indicating likelihood of "neither Category 1 nor 2") with concordant satisfactory ITV% were determined for CeO2 NM-212. Also for the three organic pigments and talc the mean relative tissue viabilities consistently exceeded 60%.
Due to failure to meet the AC for tissue variability or high optical density values recorded in single measurements of the 1st test run, two test runs each were conducted to evaluate the eye irritating potential of CeO2 NM-211. For NM-211, all individual and mean relative tissue variability values ranged above 90%. Therefore, all of these findings were assessed as indicating likelihood of "neither Category 1 nor 2".

> OECD 437 - BCOP (see in Table 4)
The AC for the NC was always met. Due to high opacity scores, one PC did not meet the AC for the PC. However, since all other ACs were met and the test material results were unambiguous, the study was assessed as being valid.

According to the authors, as determined by AUC and / or LD as relevant, NM-211 and NM-212 suspended in water (i.e., as prepared for the BCOP assay) were predominantly present as agglomerates around 1 μm diameter. Dispersed fractions of the total dose with diameters up to 1 μm that exceeded 50% of the total dose (i.e., indicating higher dispersibility) were recorded for CeO2 NM-212. Lower than 50% of the total dose, but still noteworthy fractions of small agglomerates below 100 nm were further recorded for CeO2 NM-211.

None of the tested nano-CeO2 induced serious eye damage in the BCOP as assessed by the respective IVIS that were far below the cut-off value of 55. Likewise, the three organic pigments and talc did not induce serious eye damage in the BCOP, with all IVIS being 0.

> HISTOPATHOLOGICAL EVALUATION (see in Table 5)
Minimal findings (mostly minimal multifocal or diffuse desquamation) were observed for both NM-211 and NM-212, and an HSI of I (minimal) was assigned.

Any other information on results incl. tables

Table 3: Mean tissue viability of the negative (NC), positive controls (PC), CeO2 NM-211, NM-212, three organic pigments, and talc applied in the EpiOcular™ MTT eye irritation assay

Test substance	Protocol "variant 1"	Protocol "variant 2"
	Run 1	Run 1	Run 2	Run 4
Mean OD570 of NC	1.411	1.680	-	-
Mean NC% (and ITV%)	100 (7)	100 (4)	-	-
Mean relative PC (and ITV%)	21 (11)	25 (4)	-	-
Mean relative NM-211 (and ITV%)	81 (22)a	129 (3)b	109 (8)	-
Mean relative NM-212 (and ITV%)	81 (0)	105 (16)	-	-
Mean relative talc (and ITV%)	-	-	-	98 (10)
Mean relative Pigment Red 57:1 (and ITV%)	-	-	-	98 (0)
Mean relative Pigment Yellow 95 (and ITV%)	-	-	-	91 (8)
Mean relative Pigment Black 32 (and ITV%)	-	-	-	88 (18)

The mean tissue viability of 2 tissues per test group was expressed relative to the corresponding negative control value further indicating (in brackets) the ITV%

Abbreviations: ITV%: Relative inter-tissue variability; OD: Optical density

a: Since the ITV of the two tissues was > 20 %, the corresponding acceptance criterion was not met. However, since all other acceptance criteria were met and due to the non-ambiguous result recorded for the test substance, the test was considered valid despite this deviation and therefore was not repeated.

b: Repeat of test run due to high optical density value (> 130 % relative to the NC) of one of the measurements

Footnote to Table 3:

Highly deionized water was used as NC and methyl acetate as PC.

In the tests using the protocol "variant 1", run 1 included the testing of CeO2 NM-211 and NM-212.

In the tests using the protocol "variant 2", run 1 included the tests of CeO2 NM-211 and NM-212, run 2 the 2nd testing of CeO2 NM-211, and run 4 the testing of the three organic pigments and talc.

Table 4: Mean opacity and permeability values and corresponding in vitro irritation score (IVIS) of the negative (NC) and positive controls (PC), CeO2 NM-211, NM-212, three organic pigments, and talc used in the BCOP assay

Test substance and parameter	Run 3	Run 7	Run 8
NC Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	9.1 ± 6.0 0.02 ± 0.01 9.5 ± 5.9	8.1 ± 4.9 0.01 ± 0.00 8.2 ± 4.9	7.3 ± 3.3 0.00 ± 0.00 7.3 ± 3.3
PC Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	67.8 ± 9.4 3.4 ± 0.9 118.7 ± 8.8	83.0 ± 11.1 1.3 ± 0.2 102.4 ± 10.1	70.2 ± 25.4 3.0 ± 0.5 115.1 ± 33.3
NM-211 Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	18.0 ± 9.9 0.03 ± 0.04 18.5 ± 10.5	- - -	- - -
NM-212 Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	17.2 ± 5.4 -0.02 ± 0.01 16.9 ± 5.3	- - -	- - -
Talc Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	- - -	0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0	- - -
Pigment Red 57:1 Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	- - -	0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0	- - -
Pigment Yellow 95 Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	- - -	0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0	- - -
Pigment Black 32 Mean opacity value ± SD Mean permeability value ± SD IVIS ± SD	- - -	0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0	0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Footnote to Table 4:

Highly deionized water was used as NC and imidazole (20% (w/v) highly deionized water) as PC.

Run 3 included the tests of CeO2 NM-211 and NM-212, run 7 the testing of the three organic pigments and talc, and run 8 the testing of Pigment Red 57:1.

Table 5: Histopathological evaluation of the bovine corneas incubated with NM-211 or NM-212

Test material	Test run	HSI	Histopathological findings
CeO2 NM-211	1	I	Multifocal vacuolation in the squamous and wing cell layers; multifocal desquamation and brown granules on the epithelial surface
CeO2 NM-212	1	I	Multifocal desquamation and brown granules on the epithelial surface

HSI histopathological score of irritation (HSI) of 0 = no findings, I = minimal, II = mild, III = moderate, IV = severe

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, both tested nano-CeO2 did not elicit eye irritation in either the EpiOcular™-EIT or the BCOP assay pointing thus to the low eye irritation potential of either NM-211 or NM-212.

Executive summary:

Kolle SN et al. investigated the in vitro eye irritation potential of nanosized cerium dioxide (nano-CeO2). The study was conducted according to OECD guidelines 492 and 437. However, there was no mention to GLP. The study was designed to evaluate eye irritation potential of 20 nanosized materials and 3 micro-scale materials using a 2 -tier EpiOcular™ Eye Irritation Test (EpiOcular™-EIT) and BCOP testing strategy including histopathology of the bovine corneas. The study was performed, within the context of the OECD Sponsorship Program for the Testing of Manufactured Nanomaterials.

Two commercial nano-CeO2 (NM-211 and NM-212) provided by JRC (Italy) were used in this study. The nanomaterials were physico-chemically characterised by Kolle SN et al.:

 

Parameters	Results		Methods
	NM-211	NM-212
Supplier	JRC	JRC	-
Primary particle size	10.3 nm	33 nm	X-ray diffraction (XRD)
Particle size distribution	D50 of agglomerate fractions = 2.14 µm in water	D50 of agglomerate fractions = 0.776 µm in water	Laser diffraction (LD)
Stability	Agglomeration	Agglomeration	-
Specific surface area	66 m²/g	28 m²/g	Brunauer-Emmett-Teller (BET) technique
Surface charge (zeta potential at pH 7)	No data	No data	-
Isoelectric point	No data	No data	-
Shape	Cubic	Cubic	XRD
Crystallinity	No data	No data	-
Analytical purity	> 95%	> 99.5%	Supplier’s data
Impurities	No data	No data	-
Solubility	< 0.1 ppm in water and DMEM + FCS (< 0.001% dissolution) or 0.002 wt% (Wohlleben W et al., 2013; Keller J et al., 2014)	< 0.1 ppm in water and DMEM + FCS (< 0.001% dissolution) or up to 0.02% depending on the medium (Wohlleben W et al., 2013; Keller J et al., 2014)	Analytical ultracentrifugation (AUC) combined with inductively coupled plasma-mass spectrometry or atomic emission spectroscopy (ICP-MS / ICP-AES)
Oxidation degree	No data	No data	-
Surface properties (atom %)	Non-coated	Non-coated	Supplier’s data
Physical state	Yellowish powder	Yellowish powder	Supplier’s data

The EpiOcular™-EIT model from MatTek Corp. (USA) uses the cornea-like non-keratinized tissue construct EpiOcular™ that is composed of normal human epidermal keratinocytes obtained from individual donors. The test was performed in two variants. Both NM-211 and NM-212 were submitted to protocol variant 1, i.e. as described by the supplier MatTek and Harbell et al. (2009), and protocol variant 2, i.e. as described in the OECD TG 492.

The test materials were applied undiluted at amounts enabling to cover the entire tissue surfaces (i.e., 61 mg for NM-211 and 28 mg for NM-212, corresponding to 50 μL bulk volume). Two tissues were treated with either the test materials, the negative control (NC) highly deionized water (50 µL), or the positive control (PC) methyl acetate (purity > 98 %; 50 µL). After test material application, the tissues were placed into the incubator for 90 min (variant 1), or 6 h (variant 2). After being washed, the tissues were immersed for 12 min (variant 1) or 25 min (variant 2) into 12-well plates, pre-filled with pre-warmed medium per well to remove test material residuals. Then, each tissue was dried and transferred to fresh 6-well plates filled with pre-warmed medium per well for 18-h incubation at standard culture conditions.

Upon completion of the post-exposure period, the assay medium was replaced by MTT solution for 3 h, and then the tissues were washed with PBS and incubated with isopropanol for at least 2 h to extract the produced formazan. The optical density of the formazan extracts was determined spectrophotometrically at a wavelength of 570 nm (OD570). Mean relative tissue viabilities ≤ 60 % indicated "irritancy to the eye"; mean relative tissue viabilities > 60 % indicated "no irritancy to the eye".

For all test materials, pre-tests as described in the OECD TG 492 were performed that precluded the test materials’ ability to directly reduce MTT.

The BCOP assay was conducted according to OECD TG 437. Bovine corneas were mounted in corneal holders. Both the anterior and posterior chambers of these holders were filled with Eagle’s MEM (without phenol red). After equilibration in a vertical position at 32°C for at least 1 h, the medium in both chambers was replaced with fresh medium and the initial corneal opacity was measured.

NM-211 and NM-212 powders were suspended in highly deionized water to achieve final concentrations of 20% (w/v) suspension (corresponding to 750 μL bulk volume) and then stirred with a magnetic stirrer. All test material suspensions were applied immediately after preparation. Three corneas were treated with either the test materials, NC, or PC. The medium in the anterior chamber was removed. The anterior chambers were filled with 750 μL highly deionized water (NC), or 750 μL of the 20% (w/v) imidazole solution in deionized water (PC). For NM-211 and NM-212, 750 μL of the 20% (w/v) test material preparation was applied directly to the epithelial surface of the cornea (i.e., using the "open chamber method"). The corneas were incubated in a horizontal position at 32°C for 4 h as prescribed for non-surfactant solids in the OECD TG. Upon completion of the incubation period, the NC, PC and test materials were removed from the anterior chamber, the respective epithelia were washed with EMEM, and both chambers were then refilled with fresh EMEM.

The final corneal opacity was measured, and the opacity change per cornea was calculated. To determine corneal permeability, the medium in the anterior chamber was replaced by sodium fluorescein solution (5 mg/mL) and incubated in a horizontal position for 90 min at 32°C. The amount of sodium fluorescein that permeated through the corneas was measured spectrophotometrically on 3 aliquots per cornea. The In Vitro Irritancy Score (IVIS) was calculated per treated cornea and then per treatment group was determined: IVIS = mean opacity value + (15 x mean permeability value). An IVIS > 55 indicated a risk of serious damage to the eyes.

Then, a histopathological evaluation of fixed corneas was performed using a standard method for light microscopy and a staining with Hematoxylin and Eosin. Histopathological findings were assessed in the epithelium (based on the depth of injury), and in the stroma. These findings were summarized in a so-called Histopathological Score of Irritation (HSI) assigned for each cornea ranging from 0 = no irritation to IV = severe irritation. HSI IV was assessed as "severe irritation"; HSI I, II and III were overall assessed as "non-severe irritation"; HSI 0 was regarded as no irritation.

Using the protocol "variant 1", the authors described that test material residues were observed on the EpiOcular™ tissues treated with CeO2 NM-211 and NM-212 after washing. However, since none of the test materials was able to reduce MTT directly, it was concluded that these residues did not interfere with the MTT assay. For both NM-211 and NM-212, mean tissue viabilities above 60% relative to the NC were calculated. Hence, none of the tested nano-CeO2 revealed eye irritation potential in the EpiOcular™-EIT under the chosen test conditions (i.e., indicating likelihood of "neither Category 1 nor 2").

Applying protocol "variant 2", mean relative tissue viabilities above 60% were determined for CeO2 NM-212 in the 1st test run conducted. Due to failure to meet the acceptance criteria for tissue variability or high optical density values recorded in single measurements of the 1st test run, two test runs each were conducted to evaluate the eye irritating potential of CeO2 NM-211. All individual and mean relative tissue variability values ranged above 90%. Therefore, all of these findings were assessed as indicating likelihood of "neither Category 1 nor 2".

According to the authors, NM-211 and NM-212 suspended in water (i.e., as prepared for the BCOP assay) were predominantly present as agglomerates around 1 μm diameter. Dispersed fractions of the total dose with diameters up to 1 μm that exceeded 50% of the total dose (i.e., indicating higher dispersibility) were recorded for CeO2 NM-212. Lower than 50% of the total dose, but still noteworthy fractions of small agglomerates below 100 nm were further recorded for CeO2 NM-211.

None of the tested nano-CeO2 induced serious eye damage in the BCOP as assessed by the respective IVIS that were far below the cut-off value of 55 (i.e., 18.5 and 16.9 for NM-211 and NM-212, respectively).

In addition, minimal findings (mostly minimal multifocal or diffuse desquamation) were observed for both NM-211 and NM-212, and an HSI of I (minimal) was assigned.

In conclusion, both tested nano-CeO2 did not elicit eye irritation in either the EpiOcular™-EIT or the BCOP assay pointing thus to the low eye irritation potential of either NM-211 or NM-212. According to Kolle SN et al., these results were consistent with available in vivo data for bulk material of the same composition.