reaction mass of cerium(IV)oxide, praseodymium(III,IV)oxide and zirconium (IV)oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria

The endpoint was covered using the results of Ames tests performed with zirconium dioxide (LAUS, 2008), praseodymium(III,IV) oxide (Haddouk, 2007a) and the reaction mass of cerium dioxide and zirconium dioxide (Haddouk, 2007b). These three substances tested negative with and without metabolic activation under the conditions of the respective tests. The outcome of this comparison is in line with the assumption that the addition of cerium dioxide and praseodymium(III,IV) oxide to the reaction mass does not alter the unhazardous properties of zirconium dioxide. Therefore, and according to the read across strategy, it was considered justified to cover the higher endpoints on genetic toxicity using studies for zirconium dioxide alone (i.e., the most dominant constituent of the reaction mass).

In vitro cytogenicity in mammalian cells

An in vitro chromosome aberration study performed with the read across substance zirconium dioxide according to OECD guideline 473 was used for endpoint coverage (NOTOX, 2010a). Zirconium dioxide tested negative in cultured peripheral human lymphocytes with and without metabolic activation.

In vitro gene mutation in mammalian cells

A mouse lymphoma assay performed with the read across substance zirconium dioxide according to OECD guideline 476 was used for endpoint coverage (NOTOX, 2010b). Zirconium dioxide tested negative in mouse lymphoma L5178Y cells with and without metabolic activation.

Based on the results of these studies, it could be concluded that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is not genotoxic and does not need to be classified for this endpoint under the CLP Regulation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-04-19 to 2010-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

In the dose range finding study/first cytogenetic assay during incubation period, temperature was outside the range of 37.0±1.0°C as specified in the protocol with a minimum of 31.3°C for approx 1.5 hour. This deviation had no effects on the results

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

See section 'Any other information on materials and methods incl. tables'

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany) (S9 fraction)

Test concentrations with justification for top dose:

Dose range finding test/first cytogenetic assay: at 3 h exposure time: 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix; at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix
Second cytogenicity test: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9 mix (second cytogenetic assay)
- Expression time (cells in growth medium): after 3 h exposure, the cells exposed to zirconium dioxide in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44-46 h; the cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): see above

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium) - during the last 2.5-3 h of the culture period
STAIN (for cytogenetic assays): Cell cultures were centrifuged for 5 min at 1300 rpm (365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. in case the number of aberrant cells, gaps excluded, was > or = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. The highest concentration analysed was based on the solubility of zirconium dioxide in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: no

OTHER: Test substance preparation: Zirconium dioxide was suspended in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany) at concentrations of 0.3 mg/mL and above. the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dioxide was dissolved in dimethyl sulfoxide at concentrations of 0.1 mg/mL and below. Zirconium dioxide concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v)

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-side, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
X²=[(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]
where b = the total number of aberrant cells in the control cultures, d = the total number of non aberrant cells in the control cultures, n0 = the total number of cells scored in the control cultures, a = the total number of aberrant cells in treated cultures to be compared with the control, c = the total number of non aberrant cells in treated cultures to be compared with the control, n1 = the total number of cells scored in the treated cultures, N = sum of n0 and n1
If P [X² > [(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence interval.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Remarks:

all strains/cell types tested

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Remarks:

all strains/cell types tested

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Zirconium dioxide was tested in the absence and presence of 1.8% (v/v) S9-fraction. Lymphocytes (0.4 mL blood of a healthy male donor + 5 mL or 4.8 mL culture medium + (+ or - S9) + 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of zirconium dioxide for 3h, 24h, and 48h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of zirconium dioxide in the culture medium at the 3h exposure time. At a concentration of 100 µg/mL zirconium dioxide precipitated in the culture medium. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included. At the 24h and 48h exposure time, zirconium dioxide was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 h exposure to zirconium dioxide in the absence or presence fo S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 and 48h fixation time).Cytotoxicity of zirconium dioxide in the lymphocyte cultures cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was ommited. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the mutation frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Results:

Both in the absence and presence of S9-mix zirconium dioxide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of zirconium dioxide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that zirconium dioxide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of this test.

Table 1: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 24 h and 48 h continuous exposure time in the dose range finding test.

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
	Absolute	Percentage of control
Without metabolic activation (-S9 -mix)
24 h exposure time, 24 h fixation time
Control a)	36	100
1	33	92
3	32	89
10	31	86
33	36	100
100 b)	34	94
333 c)	38	106
1000 c)	38	106
48 h exposure time, 48 h fixation time
Control a)	42	100
1	44	105
3	44	105
10	42	100
33	39	93
100 b)	42	100
333 c)	44	105
1000 c)	44	105

a) Dimethyl sulfoxide

b) Zirconium dioxide precipitated in the culture medium

c) Zirconium dioxide precipitated heavily in the culture medium which would interfere with the scoring of chromosome aberrations

Table 2: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 3 h exposure time in the dose range finding test (first cytogenetic assay)

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
Without metabolic activation (-S9 -mix)	Absolute	Percentage of control
3 h exposure time, 24 h fixation time
Control b)	46 - 51	100
10	48 - 50	101
33	47 - 49	99
100	51 - 53	107
MMC-C; 0.5 µg/mL	38 - 33	73
With metabolic activation (+ S9 -mix)
Control b)	54 -54	100
10	50 - 49	92
33	55 - 54	101
100 c)	50 - 53	95
CP; 10 µg/mL	21 - 28	45

a) Duplicate cultures

b) Dimethyl sulfoxide

c) Zirconium dioxide precipitated in the culture medium

Table 3: Mitotic index of human lymphocyte cultures treated with zirconium dioxide in the second cytogenetic assay

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
	Absolute	Percentage of control
Without metabolic activation (-S9 -mix)
24 h exposure time, 24 h fixation time
Control b)	65 -68	100
10	63 - 69	99
33	60 65	94
100 c)	58 -61	89
MMC-C; 0.2 µg/mL	31 - 35	50
48 h exposure time, 48 h fixation time
Control b)	71 - 68	100
10	65 - 69	96
33	68 - 66	96
100 c)	62 - 60	88
MMC-C; 0.1 µg/mL	53 - 55	78
With metabolic activation (+S9 -mix)
3 h exposure time, 48 h fixation time
Control b)	75 - 77	100
10	72 - 76	97
33	79 - 79	104
100	78 - 75	101
CP; 10 µg/mL	28 - 25	d)

a) Duplicate cultures

b) Dimethyl sulfoxide

d) Zirconium dioxide precipitated in the culture medium

e) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Finally, it is concluded that this test is valid and that zirconium dioxide is not clastogenic in human lymphocytes under the experimental conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), Prinses Beatrixlaan 2, 2595 AL Den Haag, Postbus 19508, 2500,CM Den Haag, The Netherlands

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

thymidine-kinase (TK) locus L5178Y

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and beta-naphtoflavone

Test concentrations with justification for top dose:

0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; CP was dissolved in Hanks' balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at < or = -15°C in the dark and one sample was thawed immediately before use.

Details on test system and experimental conditions:

In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 or 12 days (TFT selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours (MTT staining)

SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: for the determination of mutation frequency a total number of 9.6 x 1E05 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 1E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Type and identity of media: horse serum was inactivated by incubiation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modificiation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- State of the suspension/solution according to the concentration: at a concentration of 0.12 mg/mL and higher zirconium dioxide was suspended in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmdstadt, Germany). At a concentration of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dixoide concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v).

Evaluation criteria:

The global evaluation factor (GEF) has been defined as the mean of the negative/solvent mutation frequency distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more then mutation frequency (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) non of the tested concentrations reaches a mutation frequency of mutation frequency (controls) + 126; b) the results are confirmed in an independent repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

all strains/cell types tested

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

first and second experiment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Zirconium dioxide precipitated in the exposure medium at concentration of 100 µg/mL and above. Zirconium dioxide was tested beyond the limit of solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. After 3 hours of treatment: both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment with various concentrations of Zirconium dioxide, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

The growth rate over the two-day expression period for cultured treated with DMSO was between 20 and 28 (3 hours treatment) and 40 and 50 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 26- and 14-fold for MMS in the absence of S9-mix, and by 19-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Experiment 1: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (3 hours treatment)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	53	31	20
SC2	100	113	100	100	51	31	19
0.03	112	101	87	98	50	23	25
0.1	105	110	95	100	54	29	23
0.3	110	94	81	90	54	26	26
1	117	111	96	113	50	21	28
3	112	101	87	97	49	25	22
10	106	98	85	90	58	34	23
33	102	97	84	85	58	30	27
100 (1)	103	105	91	94	52	29	22
MMS	66	57	49	32	1334	804	318

With 8% (v/v) metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	88	100	100	54	32	21
SC2	100	89	100	100	53	29	23
0.03	100	102	116	116	53	34	18
0.1	99	83	94	93	54	38	15
0.3	99	79	90	89	59	32	26
1	100	81	92	93	67	33	33
3	92	74	83	77	78	47	29
10	99	86	98	97	60	31	27
33	92	90	102	94	56	33	21
100 (1)	100	77	87	87	61	32	28
CP	53	72	82	44	1000	674	191

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = DMSO; MMS = Methylmethanesulfonate; CP = cyclophosphamide

(1) zirconium dioxide precipitated in the exposure medium

Experiment 2: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (24 hours)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	57	32	23
SC2	100	104	100	100	63	36	25
0.03	120	88	79	95	72	43	27
0.1	127	107	96	122	66	34	29
0.3	137	120	108	148	50	29	20
1	127	111	100	128	54	34	18
3	139	110	99	138	55	37	17
10	140	91	82	115	80	48	29
33	138	115	103	143	69	41	25
100 (1)	153	97	87	133	54	38	15
MMS	119	77	69	83	815	564	157

With 12% (v/v) metabolic activation:

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	111	100	100	67	40	25
SC2	100	80	100	100	85	44	37
0.03	107	77	80	86	85	57	26
0.1	97	86	90	87	86	45	37
0.3	99	102	107	105	64	34	28
1	97	107	111	108	69	43	24
3	99	97	101	100	75	53	20
10	90	99	104	93	77	54	20
33	89	107	111	99	94	49	40
100 (1)	91	102	107	97	71	45	24
CP	42	54	56	24	1422	832	355

(1) = Zirconium dioxide precipitated in the exposure medium

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamid (1) = Zirconium dioxide precipitated in the exposure medium

Conclusions:

Interpretation of results: negative with and without metabolic activation

In conclusion, zirconium dioxide is not mutagenic in the TK mutation test system under the specified experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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)

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

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 nor precipitates, but tested up to recommended limit 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

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

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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-03-04 to 2008-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate provided by Rheinlandpfalz

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

4998, 1499, 500, and 50 µg/plate - Experiment one
4998, 2499, and 1250 µg/plate - Experiment two
As the test item was not soluble in any suitable solvent, a stock suspension containing 50 g/L was prepared and diluted as necessary.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine in DMSO (without at 80 µg for strains TA 97a, TA98 and TA102); Sodium azide in deionised water (without at 6 µg for strains TA100 and TA1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo-a-pyrene; 2-Amino-anthracene in DMSO (with at 40 µg for stain TA98); 2-Aminoanthracene in DMSO (with at 3 µg for strains TA97a, TA100, TA102 and TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION:

- In agar (plate incorporation) - Experiment one
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated (size of pores was 0.2 µm) into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.

- Pre-incubation - Experiment two
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37 degrees C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agar was added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.

DURATION
- Pre-incubation period: 20 minutes at 37 degrees C
- Exposure duration: 48 hours at 37 degrees C - Both experiments

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: at least 10^9 cells/mL correlating to 100 colonies / plate

Evaluation criteria:

A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >/= 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).

- Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.

- No toxicity was observed

Remarks on result:

other: all strains/cell types tested

Mean Revertants First Experiment:

Strain		97a		98		100		102		1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	139	109	13	11	152	185	212	192	15	16
	sd	58.2	11.6	1.7	3.8	25.1	31.1	22.0	58.2	3.6	5.7
DMSO	Mean	136	155	8	10	206	178	204	221	18	15
	sd	16.8	49.5	4.7	3.4	30.2	34.9	12.4	73.3	2.4	5.4
Pos Contr	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	7.36	6.46	125.1	100.1	6.59	5.62	4.91	4.53	66.73	66.73
4998 µg/pl.	Mean	171	100	10	9	129	183	198	209	19	14
	sd	28	8	3	1	11	17	18	68	6	2
	f(I)	1.23	0.92	0.77	0.82	0.85	0.99	0.93	1.09	1.27	0.88
1499 µg/pl.	Mean	156	146	12	9	170	160	198	178	15	18
	sd	17	29	3	3	19	30	32	54	4	3
	f(I)	1.12	1.36	0.92	0.82	1.12	0.86	0.93	0.93	1.00	1.13
500 µg/pl.	Mean	139	133	16	8	160	152	157	176	13	15
	sd	43	10	4	2	25	59	31	51	2	4
	f(I)	1.00	1.22	1.23	0.73	1.05	0.82	0.74	0.92	0.87	0.94
150 µg/pl.	Mean	134	113	10	9	152	178	209	168	15	12
	sd	41	7	4	2	17	37	50	45	4	4
	f(I)	0.96	1.04	0.77	0.82	1.00	0.96	0.99	0.88	1.00	0.75
50 µg/pl.	Mean	135	145	10	6	145	127	218	200	17	20
	sd	42	34	4	2	11	26	18	54	2	5
	f(I)	0.97	1.33	0.77	0.55	0.95	0.69	1.03	1.04	1.13	1.25

In this table ">1000" is represented by "1001"

Mean Revertants Second Experiment:

Strain		97a		98		100		102		1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	99	118	5	12	160	151	156	137	15	13
	sd	54.9	13.6	1.7	3.9	23.5	20.1	14.0	25.0	1.8	4.9
DMSO	Mean	152	115	7	12	142	133	167	164	8	11
	sd	9.6	6.1	0.8	3.4	17.0	1.9	8.6	31.0	2.1	2.2
Pos.Contr.	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	6.59	8.70	143.0	83.42	6.26	7.53	5.99	6.10	66.73	91.00
4998 µg/pl.	Mean	126	115	8	11	112	187	141	156	10	15
	sd	35	45	4	5	10	46	15	27	3	5
	f(I)	1.27	0.97	1.60	0.92	0.70	1.24	0.90	1.14	0.67	1.15
2499 µg/pl.	Mean	123	142	8	7	150	130	185	143	12	9
	sd	39	24	4	6	14	47	13	46	6	3
	f(I)	1.24	1.20	1.60	0.58	0.94	0.86	1.19	1.04	0.80	0.69
1250 µg/pl.	Mean	145	149	10	9	164	157	204	182	15	12
	sd	10	10	5	1	5	16	9	33	5	2
	f(I)	1.46	1.26	2.00	0.75	1.03	1.04	1.31	1.33	1.00	0.92

In this table "> 1000" is represented by "1001"

Conclusions:

Interpretation of results: negative

CC10 zirconium oxide is considered as "not mutagenic under the conditions of the test."

Reference 5

Endpoint:

genetic toxicity in vitro, other

Remarks:

read across from in vitro gene mutation studies in bacteria and in vitro cytogenicity and gene mutation studies in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Read across based on studies performed with zirconium dioxide (in vitro gene mutation in bacteria, in vitro gene mutation in mammalian cells, in vitro cytogenicity in mammalian cells), praseodymium(III,IV) oxide (in vitro gene mutation in bacteria) and the reaction mass of cerium dioxide and zirconium dioxide (in vitro gene mutation in bacteria). The read across justification document is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: read across conclusion

Remarks on result:

other: The reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is concluded not to be genotoxic.

Remarks:

Conclusion based on the results of a series of read across studies: Ames tests with zirconium dioxide (LAUS, 2008), praseodymium(III,IV) oxide (Haddouk, 2007a) and the reaction mass of cerium dioxide and zirconium dioxide (Haddouk, 2007b), an in vitro cytogenicity study in mammalian cells with zirconium dioxide (NOTOX, 2010a) and an in vitro gene mutation study in mammalian cells with zirconium dioxide (NOTOX, 2010b).

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 May 2007 - 17 December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa mutation; uvrB mutation (except TA102); addition of plasmid pKM101 to TA98, TA100 and TA102.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver post-mitochondrial 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 and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material is insoluble in most vehicles - formed homogeneous suspension to the naked eye in DMSO. Suspended at the concentration of 100 mg/mL.
- Volume of vehicle/solvent in the medium: 0.05 mL per 2.60 mL medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

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 and third test with S9 mix, which were 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 TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 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.

Species / strain:

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 nor precipitates, but tested up to recommended limit 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 material to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 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 obtained in the study were in the range of the historical control data observed in the laboratory. The study was therefore considered valid.

RESULTS
In the first experiment, no noteworthy toxicity was induced in any of the five tester strains with and without S9 mix. In the second experiment with S9 mix (pre-incubation method) with the TA 98 strain, an up to 3.3-fold increase in the number of revertants was noted, without any clear evidence of a dose-relationship. A third experiment was performed with this strain under the same experimental conditions in order to check the reliability of these slight increases. No confirmation of the increase in the number of revertants was observed in this third experiment. The test material did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the remaining tester strains.

Remarks on result:

other: all strains/cell types tested

Table 2: 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*	28	22	No	5	8	No	25	38	No
312.5	31	29	No (Mp)	7	10	No (Mp)	24	32	No (Mp)
625	38	35	No (Mp)	8	9	No (Mp)	26	25	No (Mp)
1250	32	24	No (Sp/Mp)	9	8	No (Mp)	27	29	No (Mp)
2500	36	15	No (Sp)	10	9	No (Sp)	18	29	No (Sp)
5000	34	19	No (Sp)	4	6	No (Sp)	16	29	No (Sp)
Positive control	668	188	No	352	128	No	161	1287	No

 

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	120	138	No	388	480	No
312.5	118	141	No (Mp)	330	405	No (Mp)
625	123	129	No (Mp)	449	378	No (Mp)
1250	104	115	No (Mp)	405	380	No (Mp)
2500	113	123	No (Sp)	422	406	No (Sp)
5000	126	109	No (Sp)	497	208	No (Sp)
Positive control	604	567	No	2129	3775	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

 

Table 3: 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*	11	7	No	5	7	No	25	21	No
312.5	12	11	No (Mp)	3	6	No (Mp)	29	44	No (Mp)
625	10	17	No (Mp)	5	10	No (Mp)	40	33	No (Mp)
1250	10	18	No (Sp/Mp)	4	10	No (Sp/Mp)	29	39	No (Sp/Mp)
2500	15	13	No (Sp)	8	4	No (Sp)	37	69	No (Sp)
5000	9	13	No (Sp)	5	6	No (Sp)	21	21	No (Sp)
Positive control	593	153	No	1443	148	No	303	1377	No

 

	TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	127	136	No	341	551	No
312.5	123	150	No (Mp)	275	454	No (Mp)
625	140	164	No (Mp)	362	569	No (Mp)
1250	131	135	No (Sp/Mp)	338	508	No (Sp/Mp)
2500	152	108	No (Sp)	344	523	No (Sp)
5000	146	101	No (Sp)	279	233	No (Sp)
Positive control	655	712	No	1907	1682	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

Table 4: Third experiment (preincubation with S9 mix) - Mean revertant colony count

	TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)
0*	-	21	No
312.5	-	16	No (Mp)
625	-	22	No (Mp)
1250	-	21	No (Sp)
2500	-	23	No (Sp)
5000	-	23	No (Sp)
Positive control	-	1342	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

MA : Metabolic activation

 

Conclusions:

Under the experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1. Information on praseodymium(III,IV) oxide

Bacterial reverse mutation test:

The mutagenic potential of praseodymium(III,IV) oxide was evaluated using an Ames test performed according to OECD guideline 471 and in compliance with GLP. Five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were used in this study and exposed to a concentration series of the test item in the presence and absence of metabolic activation (Haddouk, 2007a; Klimisch 1). Under the conditions of this study, praseodymium(III,IV) oxide did not show any mutagenic activity in the Salmonella strains tested, both in the absence and presence of metabolic activation.

2. Information on the reaction mass of cerium dioxide and zirconium dioxide

Bacterial reverse mutation test:

Haddouk (2007b; Klimisch 1) performed a bacterial reverse mutation study according to OECD guideline 471 and EU Method B13/14) study. The study was performed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102, exposing them to 312.5 to 5000 µg/plate with and without metabolic activation. The reaction mass of cerium dioxide and zirconium dioxide did not show any mutagenic activity with or without metabolic activation under the conditions of the test.

3. Information on zirconium dioxide

Bacterial reverse mutation test:

LAUS (2008) performed a bacterial reverse mutation study according to OECD guideline 471 and EU method B13/14. Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to 50 to 4998 µg/plate with and without metabolic activation in two independent experiments. Vehicle and positive controls were valid. Zirconium dioxide did not induce mutation with and without metabolic activation and no cytotoxicity was observed.

In vitro cytogenicity in mammalian cells:

NOTOX (2010a) performed a chromosome aberration test according to OECD guideline 473. Cultured peripheral human lymphocytes were exposed for 3 hours to 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix (dose range finding test/first cytogenetic assay); at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix. A second cytogenicity test was performed as follows: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 48 h fixation time). Vehicle and positive control substances were tested simultaneously and considered valid. Zirconium dioxide tested negative with and without metabolic activation. No cytotoxicity was observed.

In vitro gene mutation in mammalian cells:

NOTOX B.V. (2010b) performed a mouse lymphoma test according to OECD guideline 476. Mouse lymphoma L5178Y cells were exposed to 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL zirconium dioxide with and without metabolic activation. In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Zirconium dioxide tested negative in both experiments with and without metabolic activation. No cytotoxicity was observed and positive and vehicle controls were considered valid.

4. Conclusion on the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide

Based on the fact that Ames tests performed with zirconium dioxide, praseodymium(III,IV) oxide and the reaction mass of cerium dioxide and zirconium dioxide all yielded negative results with and without metabolic activation, it was concluded that a similar test with the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide would also be negative. Since a comparison of basic toxicological endpoints (Annex VII) among these three substances demonstrates that they are equally unhazardous, it was considered justified to cover higher endpoints by data available for zirconium dioxide alone (i.e. the most dominant constituent in the reaction mass). Therefore the endpoints on in vitro cytogenicity and gene mutation in mammalian cells were covered by studies performed with zirconium dioxide. Based on the results of these studies (all negative, in the absence and presence of metabolic activation), it was concluded that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is not genotoxic and does not need to be classified for this endpoint under the CLP Regulation.

Justification for classification or non-classification

Based on the available studies for zirconium dioxide, praseodymium(III,IV) oxide, and the reaction mass of cerium dioxide and zirconium dioxide, it could be concluded that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide does not need to be classified for genotoxicity under the CLP Regulation.