lambda4-cerium(4+) zirconium(4+) tetraoxidandiide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 21-MAY-2007 to 23-NOV-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of cerium dioxide and zirconium dioxide

EC Number:

909-709-8

Molecular formula:

(Ce, Zr)O2

IUPAC Name:

Reaction mass of cerium dioxide and zirconium dioxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Actalys Lisa
- Substance type: multi-constituent substance
- Physical state: solid
- Further information on test material confidential.

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate for the three experiments, with or without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: homogeneous suspension to the naked eye
- Volume of vehicle/solvent in the medium: 0.05 mL per 2.60 mL medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA1535 and TA100 without S9 mix, 1 µg/plate); 9-aminoacridine (TA1537 without S9 mix, 50 µg/plate); 2-nitrofluorene (TA98 without S9 mix, 0.5 µg/plate); mitomycin C (TA102 without S9 mix, 0.5 µg/plate)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine (TA1535, TA1537 and TA98 with S9 mix, 2 µg/plate; TA102 with S9 mix, 10 µg/plate); benzo(a)pyrene (TA100 with S9 mix, 5 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method

DURATION
- Pre-incubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATES: three plates/dose-level

OTHER: SCORING METHOD: automated

Evaluation criteria:

A reproducible 2-fold increase (for the TA98, TA100 and TA102 strains) or 3-fold increase (for the TA1535 and TA1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-response was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.

Statistics:

not concerned

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 312.5 µg/plate (the precipitate did not interfere with the scoring).

RANGE-FINDING/SCREENING STUDIES
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA98, TA100 and TA102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 100 µg/plate. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data were in the range of the historical control data observed in the laboratory.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: First experiment (direct plate incorporation) - Mean revertant colony counts

	TA 1535			TA 1537			TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	30	21	No	9	13	No	26	23	No
312.5	27	28	No (Mp)	5	11	No (Mp)	36	22	No (Mp)
625	32	20	No (Mp)	9	11	No (Mp)	38	39	No (Mp)
1250	32	26	No (Mp)	9	7	No (Mp)	31	27	No (Mp)
2500	29	19	No (Sp)	10	8	No (Sp)	49	27	No (Sp)
5000	21	23	No (Sp)	8	6	No (Sp)	50	52	No (Sp)
Positive control	541	195	No	386	103	No	183	1641	No

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	143	157	No	415	440	No
312.5	133	127	No (Mp)	368	619	No (Mp)
625	153	111	No (Mp)	569	710	No (Mp)
1250	195	133	No (Mp)	485	586	No (Mp)
2500	138	90	No (Sp)	513	563	No (Sp)
5000	141	129	No (Sp)	570	587	No (Sp)
Positive control	515	364	No	2249	2795	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

Table 2: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count

	TA 1535			TA 1537			TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	18	15	No	8	6	No	28	34	No
312.5	20	15	No (Mp)	5	9	No (Mp)	53	43	No (Mp)
625	17	11	No (Mp)	5	6	No (Mp)	32	36	No (Mp)
1250	18	14	No (Mp)	6	10	No (Mp)	49	44	No (Mp)
2500	19	14	No (Sp)	4	5	No (Sp)	26	25	No (Sp)
5000	9	10	No (Sp)	6	8	No (Sp)	37	42	No (Sp)
Positive control	551	154	No	847	131	No	218	1122	No

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	130	107	No	440	558	No
312.5	153	131	No (Mp)	437	588	No (Mp)
625	149	135	No (Mp)	455	442	No (Mp)
1250	131	136	No (Mp)	512	436	No (Mp)
2500	127	102	No (Sp)	469	531	No (Sp)
5000	149	118	No (Sp)	477	452	No (Sp)
Positive control	639	771	No	1992	3017	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

Table 3: Third experiment (direct plate incorporation with S9 mix) - Mean revertant colony count

	TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)
0*	-	32	No
312.5	-	37	No (Mp)
625	-	40	No (Mp)
1250	-	37	No (Mp)
2500	-	34	No (Sp)
5000	-	40	No (Sp)
Positive control	-	1650	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the test, the reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity in the bacterial mutation test with Salmonella typhimurium.

Executive summary:

The objective of this study was to evaluate the potential of the reaction mass of cerium dioxide and zirconium dioxide to induce reverse gene mutations in Salmonella typhimurium.

The study was performed according to international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

The test item was tested in two independent experiments, with and without a metabolic activation system, i.e. S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor1254. A third experiment was performed with S9 mix.

Salmonella typhimurium TA1535, TA1537, TA98, TA100 and TA102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

Solvent control (DMSO) and positive controls were used.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The selected treatment-levels ranged from 312.5 to 5000 µg/plate, either with or without S9 mix.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.

The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.

Under the experimental conditions of the test, the reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.