Reaction mass of cerium dioxide and lanthanum oxide and lanthanum fluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

08 November 2005 - 03 January 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP OECD TG 476-compliant study, using a main constituent of the reaction mass for a read-across purpose

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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

Method

Target gene:

HPRT locus of V79 Chinese Hamster cells

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal Essential Medium supplemented with 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphtoflavone-induced rat liver S9 fraction

Test concentrations with justification for top dose:

Experiment I:
- 4 hours without S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL
- 4 hours wIth S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL

Experiment II:
- 24 hours without S9: 28.1, 56.3, 112.5, 225, 450 and 1800 µg/mL
- 4 hours with S9: 14.1, 28.1, 56.3, 112.5, 225 and 1800 µg/mL

Vehicle / solvent:

- Solvent used: deionized water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

yes

Remarks:

untreated cells

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: without S9

Controls 2

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

yes

Remarks:

untreated cells

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: not applicable
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): approx. 7 days
- Selection time (if incubation with a selection agent): not specified
- Fixation time (start of exposure up to fixation or harvest of cells): not specified


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: duplicate cultures in 2 independent experiments


NUMBER OF CELLS EVALUATED: 5x10E2 (cytotoxicity, cloning efficiency I) - 1.5x10E6 (mutant frequency)


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency I (cytotoxicity); cloning efficiency II (cell viability)


OTHER EXAMINATIONS:
- Mutant colonies per 10E6 cells = mean number of mutant colonies per flask found after plating in 6-thioguanine containing medium x 10E6 divided by the number of cells survived
- Induction factor = mutant colonies per 10E6 cells / mutant colonies per 10E6 cells of the corresponding solvent control

Evaluation criteria:

The assay is considered acceptable if:
- the numbers of mutant colonies per 10E6 cells in negative and/or solvent controls fall with the test facility historical data range
- the positive control substances produce a significant increase in mutant colony frequencies
- the cloning efficiency II value of the negative and/or solvent controls exceed 0.5

A test substance is considered as positive if it induces either a concentration-related increase in the mutation frequency or a reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency).

Statistics:

Since the distribution of mutant cells does not follow known statistical models, no adequate statistical method was available

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation was observed by the naked eye in all parts of pre-experiment at 112.5 µg/mL and above. In the first main experiment (4-hour exposure), precipitation was seen at 225 µg/mL and above with or without S9. In the second main experiment, precipitation was observed at 225 µg/mL and above in the absence of S9 (24-hour exposure) and at 112.5 µg/mL and above in the presence of S9 (4-hour exposure).


RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments, using same experimental conditions as described for the main experiments. In the pre-test, the colony forming ability of approximately 500 single cells after exposure to the test substance was observed and compared to the controls.
The highest concentration used in the pre-test was chosen with regard to the purity (99.8 %) and the molecular weight of the test item (172.12 g/mol). Test item concentrations between 14.1 and 1800 µg/mL (approximately 10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. No relevant toxic effect (relative cloning efficiency at or below 50 %) occurred up to the high-est concentration of both treatment periods with and without metabolic activation. Precipitation was noted at 112.5 µg/mL and above in the absence and presence of metabolic activation at both treatment intervals (4 and 24 hours). There was no relevant shift of osmolarity and pH values of the medium even at the maximum concentration of the test item.


COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments (with or without S9), the range of the negative and solvent controls was from 2.1 to 12.3 mutants per 10E6 cells. The range of the cells exposed to the test substance was from 0.9 to 26.3 mutants per 10E6 cells. However all experimental points remained within the historical control data range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary result table:

	Concentration (µg/mL)	S9 mix	Relative cloning efficiency I (%)	Relative cloning efficiency II (%)	Mutant colonies per 10E6 cells	Induction factor	Relative cloning efficiency I (%)	Relative cloning efficiency II (%)	Mutant colonies per 10E6 cells	Induction factor
Experiment I /4 htreatment			Culture I				Culture II
Negative control	0	-	100.0	100.0	5.7	-	100.0	100.0	7.2	-
Solvent control (water)	0	-	100.0	100.0	12.3	1.0	100.0	100.0	7.7	1.0
Positive control (EMS)	300.0	-	30.3	80.2	137.5	24.0	30.1	86.9	67.3	9.4
Test item	28.1	-	103.6	culture discontinued*			95.8	culture discontinued*
Test item	56.3	-	105.2	97.7	3.7	0.3	102.7	98.1	6.7	0.9
Test item	112.5	-	89.3	89.3	5.4	0.4	91.4	95.8	6.9	0.9
Test item	225.0 (p)	-	101.4	82.1	7.2	0.6	97.5	92.3	10.6	1.4
Test item	450.0 (p)	-	94.2	102.0	5.6	0.5	83.6	98.5	7.9	1.0
Test item	1800.0 (p)	-	103.0	84.9	5.4	0.4	100.6	98.8	7.1	0.9
Negative control	0	+	100.0	100.0	2.1	-	100.0	100.0	4.9	-
Solvent control (water)	0	+	100.0	100.0	4.2	1.0	100.0	100.0	9.6	1.0
Positive control (DMBA)	2.0	+	8.6	80.3	480.6	229.8	8.7	52.1	840.7	172.1
Test item	28.1	+	94.0	culture discontinued*			85.0	culture discontinued*
Test item	56.3	+	92.2	88.7	9.7	2.3	86.6	95.8	9.5	1.0
Test item	112.5	+	93.1	94.5	0.9	0.2	91.7	90.0	8.3	0.9
Test item	225.0 (p)	+	90.3	98.0	3.2	0.8	91.8	109.7	10.5	1.1
Test item	450.0 (p)	+	96.1	97.8	2.6	0.6	91.3	111.9	8.1	0.8
Test item	1800.0 (p)	+	97.7	97.4	7.0	1.7	91.8	103.5	8.3	0.9
Experiment II / 24 h treatment			Culture I				Culture II
Negative control	0	-	100.0	100.0	6.7	-	100.0	100.0	5.3	-
Solvent control (water)	0	-	100.0	100.0	8.0	1.0	100.0	100.0	4.7	1.0
Positive control (EMS)	150.0	-	68.8	82.4	205.0	25.8	70.7	78.1	156.1	33.0
Test item	28.1	-	133.0	89.7	7.5	0.9	106.0	120.0	7.7	1.6
Test item	56.3	-	150.7	97.8	4.6	0.6	110.9	112.0	4.1	0.9
Test item	112.5	-	137.3	98.3	3.7	0.5	106.3	110.6	6.1	1.3
Test item	225.0 (p)	-	138.7	89.4	5.0	0.6	98.1	112.9	9.9	2.1
Test item	450.0 (p)	-	135.3	22.0	26.3	3.3	103.1	128.6	5.5	1.2
Test item	1800.0 (p)	-	140.3	culture discontinued*			100.9	culture discontinued*
Experiment II /4 htreatment
Negative control	0	+	100.0	100.0	5.7	-	100.0	100.0	8.4	-
Solvent control (water)	0	+	100.0	100.0	3.0	1.0	100.0	100.0	5.2	1.0
Positive control (DMBA)	2.0	+	24.1	80.2	510.3	172.2	77.8	55.8	1034.3	200.6
Test item	14.1	+	96.4	64.3	8.1	2.7	89.0	66.1	3.4	0.7
Test item	28.1	+	93.4	73.2	4.5	1.5	88.9	72.8	8.5	1.7
Test item	56.3	+	91.5	73.6	3.1	1.1	82.2	81.6	5.5	1.1
Test item	112.5 (p)	+	102.4	74.1	6.8	2.3	85.2	74.2	2.3	0.4
Test item	225.0 (p)	+	112.2	32.9	15.7	5.3	88.6	95.4	4.1	0.8
Test item	1800.0 (p)	+	107.5	culture discontinued*			76.0	culture discontinued*

(p) Precipitation visible to unaided eye

* Since a minimum of 4 analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with or without metabolic activation

No evidence of gene mutations at the HPRT locus in V79 cells up to the concentration of 1800 µg/mL with or without S9

Executive summary:

The potential of Cerium Oxide to induce gene mutations at the HPRT locus in Chinese Hamster V79 cells was tested. The assay was performed in two independent experiments, each using duplicate cultures. In the first experiment, the cells were exposed to the test substance suspended in deionized water at concentrations ranging from 28.1 to 1800 µg/mL (equivalent to10 mM) for 4 hours with or without metabolic activation (S9). In the second experiment, cell exposure was 24 hours at concentrations ranging from 28.1 to 1800 µg/mL in the absence of S9 and 4 hours at concentrations ranging from 14.1 to 1800 µg/mL in the presence of S9. The cells were evaluated for mutant frequency at selected test concentrations. Positive controls consisted of 1.2 or 2.4 mM ethylmethane sulfonate and 7.7 µM 7,12-dimethylbenz(a)anthracene without and with S9, respectively.

 

Precipitation was observed from 225 µg/mL up to the maximum concentration with and without S9 (4 h treatment) in the first main experiment and without S9 mix in the second experiment (24 h treatment). It was also observed from 112.5 µg/mL up to the maximum concentration in the second experiment with S9 (4 h treatment). No relevant cytotoxic effects indicated by relative cloning efficiency lower than 50% were observed up to 1800 µg/mL in both main assays with or without S9. No significant and reproducible dose-dependent increases in the mutation frequency were observed in both main experiments.

 

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

 

Therefore, Cerium Oxide did not induce gene mutations at the HPRT locus in V79 cells up to the concentration of 1800 µg/mL with or without S9.

 

This study is classified as acceptable. It satisfies the OECD 476 guideline requirements on In vitro Mammalian Cell Gene Mutation Test.