Cerium dioxide

Additional ecotoxological information

Administrative data

Endpoint:

additional ecotoxicological information

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Exposure of primary trout hepatocytes to nano-CeO2 or bulk CeO2 suspensions
- Cytotoxicity: Lactate dehydrogenase (LDH) assay

GLP compliance:

not specified

Type of study / information:

Cytotoxicity of nanometric cerium dioxide (nano-CeO2) and micrometric CeO2 (bulk) in primary trout hepatocytes

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 oxide (nano-CeO2)
- Supplier: None (in-house synthesis using wet chemistry method)
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance
- Primary particle size (SEM, XRD): Average diameter of 16-22 nm (data from De Marzi et al., 2013)
- Particle size distribution (SEM): Presence of ~40-nm clusters (data from De Marzi et al., 2013)
- Stability: Slight agglomeration/aggregation based on SEM observations (data from De Marzi et al., 2013)
- Specific surface area: No data available
- Surface charge (DLS): Zeta potential of -46.2 mV in water at pH 7.4 (data from De Marzi et al., 2013)
- Isoelectric point: No data available
- Shape (SEM): No data available
- Crystallinity (XRD): No data available
- Analytical purity / impurities: No data available
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility: No data available
- Oxidation degree: No data available
- Surface properties (SEM, FTIR): Smoothed and non-porous surface; detection of Ce-O bond, O-H bond in water adsorbed on the sample surface and residual surfactant (CH2) (data from De Marzi et al., 2013)
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available

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

Specific details on test material used for the study:

- Name of test material: Nanometric cerium dioxide (nano-CeO2)
- Supplier: Sigma-Aldrich
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance / nanomaterial in suspension
- Primary particle size (XRD): 11.5 ± 0.2 nm (< 25 nm according to the supplier)
- Particle size distribution (AFM, DLS, HRTEM): From 14.9 to 411 nm in Caco-2 medium (see in Table 1 below)
- Stability: Aggregation based on particle size distribution
- Specific surface area (BET): 60 m²/g
- Surface charge (electrophoretic mobility): Zeta potential of -7 mV in Caco-2 cell medium
- Isoelectric point: No data available
- Shape: No data available
- Crystallinity: No data available
- Analytical purity / impurities: No impurities detected for nano-CeO2 powder
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility: Essentially insoluble in solution
- Oxidation degree (EELS): 62% Ce(III) and 38% Ce(IV) as a raw powder; 100% Ce(IV) in Caco-2 cell medium
- Surface properties: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: no data available

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

A non-nano (bulk) CeO2 was also tested in this study:
- Supplier: Sigma
- Substance type: Monoconstituent substance
- Substance form: Microparticulate substance / bulk material in suspension
- Primary particle size (XRD): > 1000 nm (< 5 µm according to the supplier)
- Particle size distribution (AFM, DLS, HRTEM): From 62.1 to 1666 nm in Caco-2 medium (see in Table 1 below)
- Stability: Aggregation based on particle size distribution
- Specific surface area (BET): 0.4 m²/g
- Surface charge(zeta potentiometry): Zeta potential of -5 mV in Caco-2 cell medium
- Isoelectric point: No data available
- Shape: No data available
- Crystallinity: No data available
- Analytical purity / impurities: No data available
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: No data available
- Solubility (UF-ICP-MS): Essentially insoluble
- Oxidation degree (EELS): Mainly Ce(IV) with a very thin layer of Ce(III) on the surface (1 to 10 nm) as a raw powder; no data in Caco-2 cell medium
- Surface properties: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available

Results and discussion

Any other information on results incl. tables

- CYTOTOXICTY ASSESSMENT:

Treatment of primary trout hepatocytes with CeO2 particles of either size for 24 h did not cause significant LDH release up to and including the concentration of 1000 mg/mL. Thus, this result demonstrated that CeO2 induced no alteration of membrane integrity and consequently no cytotoxicity, despite evidence of direct spatial contact and even uptake.

Applicant's summary and conclusion

Conclusions:

Nano-CeO2 and bulk CeO2 did not cause cytotoxicity in the primary fish hepatocytes.

Executive summary:

Gaiser BK et al. (2012) performed an in vitro test to assess the cytotoxicity of nanometric cerium dioxide (nano-CeO2) and micrometric CeO2 (bulk) to primary trout hepatocytes.

Primary trout hepatocytes were cultured in Sigma Medium 199 supplemented with 344 mg/L NaHCO3, 500 mg/L CaCl2 x 2H2O, 10 % foetal calf serum (FCS), 834 mg/L N-(2-Hydroxyethyl)piperazine-N’-(2 -ethane sulfonic acid), 344 mg/L NaHCO3, 500 g/L CaCl2 and 100 U/mL penicillin/0.1 mg/mL streptomycin, and incubated at 12°C. For the purpose of the experiment, hepatocytes were seeded at 100 000 cells/well in a 96-well plate, incubated overnight, and treated with particles concentrations ranging from 0 to 1000 mg/L for 24 h in the appropriate medium. Plates were centrifuged at 25 g to remove cell debris and particles, and cytotoxicity was measured immediately as supernatant lactate dehydrogenase content. Indeed, the release of this molecule is indicative of the alteration of membrane integrity.

In parallel, nano-CeO2 and bulk CeO2 were characterised for several physico-chemical parameters. However, the results obtained in cell culture medium of primary trout hepatocytes are not provided in the publication as the authors indicated that these data were not obviously different from those obtained on another cell medium tested in the publication: 

 

Parameters		Results		Methods
		Nano-CeO2	Bulk CeO2
Primary particle size (raw powder)		< 25 nm 11.5 ± 0.2 nm	< 5 µm > 1000 nm	Supplier’s data XRD
Particle size distribution	in Caco-2 cell medium supplemented with 10% FCS	14.9 ± 7.4 nm 411 ± 33 nm 14.9 ± 6.3 nm	62.1 ± 48.4 nm 1666 ± 1554 nm 387 ± 82 nm	AFM DLS HRTEM
	in Daphnia magna medium	3958 ± 1955 nm	2729 ± 1858 nm	DLS
Specific surface area (raw powder)		60 m²/g	0.4 m²/g	BET
Surface charge	in Caco-2 cell medium supplemented with 10% FCS	Zeta potential of -7 mV	-5 mV	Zeta potentiometry charge
	in Daphnia magna medium	Zeta potential of -10 mV	-4 mV	Zeta potentiometry charge
Dissolution		Essentially insoluble in Caco-2 cell and D. magna media	Essentially insoluble in Caco-2 cell and D. magna media	UF-ICP-MS
Surface speciation / oxidation degree	raw powder	62% Ce(III) and 38%Ce(IV)	Mainly Ce(IV) with a very thin layer of Ce(III) on the surface (1 to 10 nm)	EELS / XPS
	in Caco-2 cell medium	100% Ce(IV)	No data	EDX / XPS
Purity (raw powder)		No impurities detected	No data	EDX / XPS

Treatment of primary trout hepatocytes with CeO2 of either size for 24 h did not cause significant release of lactate dehydrogenase up to and including the concentration of 1000 mg/L; thus implying no alteration of membrane integrity and consequently no cytotoxicity.