Zirconium propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03-04 to 2008-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, scientifically sound GLP study performed according to OECD Guideline 471 and EU Method B.13/14.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Zirconium dioxide

Cas Number:

1314-23-4

Molecular formula:

ZrO2

IUPAC Name:

Zirconium dioxide

Constituent 2

Reference substance name:

Zirconium dioxide

EC Number:

215-227-2

EC Name:

Zirconium dioxide

IUPAC Name:

215-227-2

Details on test material:

- Name of test material (as cited in study report): CC10 Zirconium Oxide
- Molecular formula (if other than submission substance): ZrO2 + HfO2
- Molecular weight (if other than submission substance): 123.22 g/mol
- Physical state: white powder
- Stability under test conditions: not stated
- Storage condition of test material: stored in a closed vessel at room temperature (20 +/- 5 degree C)

Method

Species / strain

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 ug/plate- Experiment one
4998, 2499, and 1250 ug/plate- Experiment two
As the test item wasn't 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

Controls

Controls 1

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 ug for strains TA 97a, TA98 and TA102); Sodium azide in deionised water (without at 6 ug for strains TA100 and TA1535)

Controls 2

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 ug for stain TA98); 2-Aminoanthracene in DMSO (with at 3 ug 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 degree C.
0.1 mL of the appropriate solution of the test item were 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 degree 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 degree 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 degree 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 degree C.


DURATION
- Preincubation period: 20 minutes at 37 degree C
- Exposure duration: 48 hours at 37 degree 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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 97a

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium TA 100

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

Test results 5

Species / strain:

S. typhimurium TA 102

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 didn't 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

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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"

Applicant's summary and conclusion

Conclusions:

CC10 Zirconium Oxide is considered as "not mutagenic under the conditions of the test."