Cerium dioxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin 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:

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

Remarks:

; however, the physico-chemical characterisation of the tested nano-CeO2 was of low quality but at the publication date the scientific community required less physico-chemical endpoints in such articles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: this OECD 471 study was well documented and met the generally accepted scientific principles.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Remarks:

The GLP status was not specified in the article.

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0 (control), 50 to 5000 µg/plate (in triplicate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Nano-CeO2 was not soluble in water or common solvents

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see below in "Details on test system and conditions"

Details on test system and experimental conditions:

POSITIVE CONTROLS
- in the absence of metabolic activation (-S9):
* N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 3 μg/plate for TA100 and 5 μg/plate for TA1535
* 9-Aminoacridine (9AA) at 80 μg/plate for TA1537
* Mitomycin C (MMC) at 0.5 μg/plate for TA102
* 4-Nitroquinoline-1-oxide (4NQO) at 0.2 μg/plate for TA98

- in the presence of metabolic activation (+S9):
* 2-Aminoanthracene (2AA) at 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537
* Benzo(a)pyrene (BP) at 5 μg/plate for TA98
* 1,8-Dihydroxyanthraquinone (DAN) at 10 μg/plate for TA102

DETERMINATION OF CYTOTOXICITY
- Method: growth of the bacteria background lawn

Evaluation criteria:

No data available

Statistics:

No data available

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

- A cream-coloured film was observed at 1500 μg/plate and above with an associated precipitate at 5000 μg/plate. This observation did not, however, prevent the scoring of revertant colonies and confirmed that the samples were tested up to maximal dose level.
- No toxicity of the cerium oxide as no visible reduction in the growth of the bacteria background lawn at any dose level was observed both with and without metabolic activation.
- All of the positive control chemicals used in the study induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

There were no significant increases in the frequency of revertant colonies recorded for any of the strains of Salmonella, at any dose level, either with or without metabolic activation, as detailed in the table below.

Table 1: Summary of mean revertant colonies

Substance	Dose Level (µg/plate)	TA100		TA1535		TA102
		(-S9)	(+S9)	(-S9)	(+S9)	(-S9)	(+S9)
Exp. 1
DMSO	100 µL	118 ± 7	97 ± 13	39 ± 3	32 ± 6	347 ± 29	351 ± 4
Nano-CeO2	50 150 500 1500 5000	100 ± 3 88 ± 13 84 ± 16 101 ± 13 101 ± 4	109 ± 17 115 ± 9 114 ± 20 115 ± 8 113 ± 7	37 ± 2 40 ± 3 33 ± 3 28 ± 4 39 ± 3	29 ± 13 36 ± 1 36 ± 3 32 ± 9 38 ± 2	356 ± 10 360 ± 13 377 ± 12 343 ± 38 343 ± 16	363 ± 25 381 ± 20 350 ± 17 330 ± 17 332 ± 14
Exp. 2
DMSO	100 µL	97 ± 14	132 ± 8	28 ± 6	30 ± 11	394 ± 9	380 ± 25
Nano-CeO2	50 150 500 1500 5000	99 ± 19 98 ± 20 84 ± 6 92 ± 8 95 ± 3	123 ± 17 127 ± 10 126 ± 11 133 ± 7 121 ± 22	32 ± 2 35 ± 6 35 ± 11 36 ± 8 28 ± 5	29 ± 5 40 ± 6 32 ± 4 36 ± 7 29 ± 3	359 ± 28 361 ± 32 382 ± 29 361 ± 43 371 ± 24	397 ± 39 391 ± 32 350 ± 7 313 ± 29 353 ± 36
		TA98		TA1537
		(-S9)	(+S9)	(-S9)	(+S9)
Exp. 1
DMSO	100 µL	28 ± 3	36 ± 7	13 ± 5	22 ± 2
Nano-CeO2	50 150 500 1500 5000	23 ± 5 25 ± 6 26 ± 6 22 ± 3 23 ± 4	36 ± 2 35 ± 6 35 ± 7 35 ± 1 37 ± 4	11 ± 5 12 ± 5 14 ± 5 14 ± 4 12 ± 2	18 ± 4 15 ± 5 17 ± 4 24 ± 2 20 ± 5
Exp. 2
DMSO	100 µL	21 ± 5	38 ± 7	12 ± 10	15 ± 6
Nano-CeO2	50 150 500 1500 5000	21 ± 4 22 ± 4 21 ± 4 19 ± 5 19 ± 3	33 ± 10 32 ± 2 32 ± 5 37 ± 4 26 ± 3	12 ± 1 14 ± 7 14 ± 4 9 ± 3 9 ± 1	13 ± 6 12 ± 4 16 ± 4 12 ± 3 17 ± 2

DETERMINATION OF CYTOTOXICITY

The negative control (polypropylene pipette tips) had a cytotoxicity grade of 0 and the positive control (tin-impregnated PVC strips) showed evidence of a moderate cytotoxicity with a grade 3 score; thus indicating that the assay system was reliable. Both bulk CeO2 and nano-CeO2 showed no evidence of cytotoxicity and each had a cytotoxicity grade of 0.

Table 2: Cytotoxicity results

Test material	Mean cytotoxicity / reactivity grade
Culture medium (vehicle control)	0
Polypropylene pipette tips (negative control)	0
Tin-impregnated PVC strips (positive control)	3
Nano-CeO2	0
Bulk CeO2	0

Conclusions:

Nano-CeO2 was non-mutagenic to Salmonella typhimurium strains in this Ames test with and without metabolic activation at concentrations between 50 and 5000 µg/plate

Executive summary:

Park B et al. (2007, 2008) investigated the genotoxic potential of nanometric cerium dioxide (nano-CeO2) in vitro.

A commercial nano-CeO2 from Oxonica was used in this study. These crystalline nanoparticles displayed a primary particle size of 9 nm and a specific surface area of 94.7 m²/g. The analysis of analytical purity showed that nano-CeO2 and its surface were composed of Ce and O. Moreover, nano-CeO2 was commercialised as aqueous slurry at an approximate concentration of 8%w/w.

The genotoxic potential of nano-CeO2 was determined by performing an Ames test, according to OECD guideline 471. Five strains of Salmonella typhimurium (i.e., TA 1535, TA 1537, TA 98, TA 100, TA 102) were treated with 50 to 5000 µg/plate of nano-CeO2 suspended in dimethyl sulfoxide (DMSO), in the presence or absence of metabolic activation.

All of the positive control chemicals used in the study induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The test showed no significant increases in the frequency of revertant colonies for any of the S. typhimurium strains, at any nano-CeO2 dose level, either with or without metabolic activation in any of the experiments. Moreover, nano-CeO2 induced no cytotoxicity as no visible reduction in the growth of the bacteria background lawn at any dose level was observed both with and without metabolic activation. A cream-coloured film was observed at 1500μg/plate and above with an associated precipitate at 5000 μg/plate.

Therefore, nano-CeO2 showed no mutagenic activity in this bacterial reverse mutation (Ames) test up to the limit concentration of 5000 µg/plate with or without metabolic activation.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2007

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

EpiDerm "EPI-200" human epidermal model skin irritation test (as developed in MatTek Corporation, Ashland, MA, USA).

GLP compliance:

not specified

Remarks:

The GLP compliance status was not specified in this 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:

other: nanometric and micrometric materials in suspension

Details on test material:

- Name of test material: Nanometric cerium dioxide (nano-CeO2) received as aqueous slurry at an approximate concentration of 8%w/w; micrometric CeO2 (bulk)
- Supplier: Energenics Europe Ltd. (Envirox™) for nano-CeO2 and Aldrich for bulk CeO2
- Substance type: Monoconstituent substance
- Substance form: Nanoparticulate substance for nano-CeO2 and microparticulate substance for bulk CeO2
- Primary particle size (derived from BET): Mean particle size of 9 nm for nano-CeO2; 320 nm for bulk CeO2 (< 5 µm according to the supplier)
- Particle size distribution: No data available
- Stability: No data available
- Specific surface area (BET): 94.7 m²/g for nano-CeO2; 2.64 m²/g for bulk CeO2
- Surface charge: No data available
- Isoelectric point: No data available
- Shape: No data available
- Crystallinity (XRD): Cerianite for both nano-CeO2 and bulk CeO2
- Analytical purity: Nano-CeO2 and bulk CeO2 composed of Ce and O (EDX); 99.9% purity for bulk CeO2 (supplier’s data)
- Impurities: No data available
- Number density of nano-CeO2 in the suspension: No data available
- Cerium content in nano-CeO2 suspension: Aqueous slurry at an approximate concentration of 8%w/w nano ceria (as-received, according to the supplier)
- Solubility: No data available
- Oxidation degree: Cerium(IV) oxide for bulk CeO2 (supplier’s data)
- Surface properties (XPS): Surface chemistry of nano-CeO2 and bulk CeO2 => Ce, O
- 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".

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

not specified

Details on test system:

EpiDermTM "EPI-200" HUMAN EPIDERMAL MODEL
The EpiDerm™ EPI-200 human epidermal model from MatTek Corporation (USA) was used in this study for assessment of the safety of Envirox™ since the skin is a potential route of exposure during the formulation and use of the nano-CeO2 containing diesel fuel additive when accidental skin contamination might reasonably be expected to occur.

1% (w/v) Triton X-100 and 1% of 20% (w/v) SLS were used as positive control and standard reference material, respectively.

Control samples:

yes, concurrent positive control

Amount/concentration applied:

50 mg of test substance applied

Duration of treatment / exposure:

Exposure times of 960, 1200 and 1440 minutes

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: mean irritation potential (MIP)

Value:

< 0.01

Remarks on result:

other: for nano-CeO2

Remarks:

- Basis: mean calculated as ET50 (reference material) / ET50 (test material). - Reversibility: no data.

Results 2

Irritation / corrosion parameter:

other: MIP

Value:

0.003

Remarks on result:

other: for bulk CeO2

Remarks:

- Reversibility: no data.

Any other information on results incl. tables

- EpiDermTM “EPI-200” HUMAN EPIDERMAL MODEL

Nano-CeO2 and bulk CeO2 did not directly reduce MTT. The ET50 values for nano-CeO2, 1% (w/v) TritonX-100 the positive control and 1% of 20% (w/v) SLS, a standard reference material, were 1517.18, 260.10 and 20.68 minutes respectively. The bulk CeO2 was assayed at a later time than nano-CeO2 and the corresponding ET50 values for bulk CeO2, the positive control and the reference material were > 1440, 410.60 and 46.22 minutes respectively.

A mean irritation potential (MIP) score was calculated as follows: MIP = ET50 (reference material) / ET50 (test material).

The MIP for both the nanometric and bulk CeO2 was calculated and determined as 0.01 for nano-CeO2 and 0.03 for bulk CeO2. Since both nanometric and bulk CeO2 had MIP values which were < 0.8 neither was considered to have the potential to be an in vivo skin irritant.

 

Test material	Exposure time (min)	Mean OD450	% viability	ET50 min	MIP
Negative control	960 1440	1.685	100
Nano-CeO2	960 1200 1440	2.010 1.138 1.089	119.29 67.54 64.53	1517.18	< 0.01
Bulk CeO2	960 1200 1440	1.994 1.975 1.729	92.87 97.36 86.23	> 1440	0.003
Positive control (1% TX100)	240 360 480	1.035 0.212 0.095	61.42 12.58 5.64	260.10	< 0.18
Reference material 20% SLS	15 30 60 120	0.983 0.710 0.273 0.100	58.34 42.14 16.20 5.93	20.68	1.00

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since nano-CeO2 and bulk CeO2 had a mean irritation potential (MIP) value lower than 0.8, both materials were not considered to have the potential to be an in vivo skin irritant in this in vitro EpiDerm test.

Executive summary:

Park B et al. (2007, 2008) investigated the skin irritating potential of nanometric cerium dioxide (nano-CeO2) and micrometric CeO2 (bulk CeO2) in vitro.

A commercial nano-CeO2 from Energenics Europe Ltd. (EnviroxTM) was used in this study. The nanoparticles displayed a mean primary size of 9 nm and a specific surface area of 94.7 m²/g. A bulk CeO2 was also added to the study. This micrometric material provided by Aldrich had a mean primary size of 320 nm and a specific surface area of 2.64 m²/g. The surface chemistry of both crystalline materials was evaluated and demonstrated that the surface of nanometric and bulk CeO2 was composed of Ce and O.

The EpiDerm™ EPI-200 human epidermal model from MatTek Corporation (USA) was used in this study for assessment of skin irritation. Fifty milligrams of test substance was applied to the human epidermal model for 960, 1200 and 1440 minutes. The irritation assessment was based on the measurement of cellular viability using the MTT assay. A mean irritation potential (MIP) score was calculated as follows: MIP = ET50 (reference material) / ET50 (test material) where ET50 was the exposure time at which relative value of optical density was 50% of the negative control and the reference material was SLS (1% of 20% (w/v)).  

Nano-CeO2 exposure induced variations of cellular viability: 119.3% at 960 minutes, 67.5% at 1200 minutes, 64.5% at 1440 minutes. Exposure to bulk CeO2 caused no or only few changes in cell viability: 92.87% at 960 minutes, 97.36% at 1200 minutes, 86.23% at 1440 minutes. As nanoparticles and bulk material had a MIP value lower than 0.8 (i.e., < 0.01 for nano-CeO2 and 0.003 for bulk CeO2), both nano- and micro-CeO2 were not considered to have the potential to be an in vivo skin irritant based on this in vitro EpiDerm test.