Zirconium, tetrakis(isobutyrato)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other:

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st November-15th December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(isobutyrato)zirconium

EC Number:

619-635-5

Cas Number:

12567-73-6

Molecular formula:

Hill formula: C16H28O8Zr CAS formula: C16H28O8Zr

IUPAC Name:

Tetrakis(isobutyrato)zirconium

Constituent 2

Reference substance name:

Zirconium, tetrakis(isobutyrato)- (8CI)

IUPAC Name:

Zirconium, tetrakis(isobutyrato)- (8CI)

Details on test material:

- Name of test material (as cited in study report): Zirkonium-tetrakis(isobutyrato)
- Physical state: solid
- Analytical purity: 97.4 % (complexometric titration)
- Impurities (identity and concentrations): By-products:
toluene: ca. 2.1 %.
iso-butyric acid: ca. 0.5 %.
- Lot/batch No.: 02/05 (HK1005)
- Expiration date of the lot/batch: September 2006
- Storage condition of test material: Ambient temperature. Tightly closed. Storage under a nitrogen atmosphere, as the substance reacts with water.

Method

Target gene:

His

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

microsomal fraction of homogenised livers of female Sprague Dawley rats treated once with 500 mg/kg of Aroclor 1254

Test concentrations with justification for top dose:

test substance (1st experiment): 5000, 1667, 556, 185 and 62 μg/plate (3 samples)
test substance (2nd experiment): 1667, 556, 185, 62 and 21 μg/plate (3 samples)
control (DMSO): 100 μl (6 samples)
positive control: from 1 to 50 μg/plate, depending on the positive control (3 samples)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not enough soluble in water. DMSO is a common
vehicle for the Ames test.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA98, TA100, TA1535, with S9

Controls 2

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

TA97a, with S9

Controls 3

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxy-anthraquinone

Remarks:

TA102, with S9

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S9

Controls 5

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535, without S9

Controls 6

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine

Remarks:

TA97a, without S9

Controls 7

Positive controls:

yes

Positive control substance:

other: t-Butyl-hydroperoxide

Remarks:

TA102, without S9

Controls 8

Untreated negative controls:

yes

Remarks:

Dimethylsulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: The results were verified by a second, independent experiment. Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted, for the control groups six-fold repetitions were run.

NUMBER OF CELLS EVALUATED: 2 to 3 x 109 cells per mL

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: A reduced bacterial background lawn (mottled instead of homogeneous), microcolonies of bacteria instead of a homogeneous background lawn, no background lawn, clearly reduced numbers of revertant colonies.

Evaluation criteria:

Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the historic data of the Ames test.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Test substance:

Toxicity: results of the test substance did not show toxicity up to 1667 μg/plate. At 5000 μg/plate the bacterial background could not be distinguished from the precipitate.

Solubility: a turbidity was visible when the test substance was mixed with the agar at the 5000, 1667 and 556 μg/plate samples. When the colonies were counted, a precipitate impeded the counting of the colonies at 5000 μg/plate. At 1667 and 556 μg/plate a slight precipitate was still observed in the plates with metabolisation, but the colony counting and the determination of toxicity was not affected.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

According to the results obtained in this study, Zirkonium-tetrakis(isobutyrato) is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolic activation.

Executive summary:

Genetic toxicity: key study in Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535, according to OECD guideline 471 and directive 2000/32/EC, part B.13/14.

Zirkonium-tetrakis(isobutyrato) was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). In the first experiment the following concentrations were tested: 62, 185, 556, 1667 and 5000 μg per plate without external metabolisation, and 62, 185, 556, 1667 and 5000 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system. In the second experiment the following concentrations were tested: 21, 62, 185, 556 and 1667 μg per plate without external metabolisation, and 21, 62, 185, 556 and 1667 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system. The test was performed according to the "direct plate incorporation method". Negative and positive controls were included. An independent repetition of the experiment was performed.

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Results of the test substance did not show toxicity up to 1667 μg/plate. At 5000 μg/plate the bacterial background could not be distinguished from the precipitate. Solubility: A turbidity was visible when the test substance was mixed with the agar at the 5000, 1667 and 556 μg/plate samples. When the colonies were counted, a precipitate impeded the counting of the colonies at 5000 μg/plate. At 1667 and 556 μg/plate a slight precipitate was still observed in the plates with metabolisation, but the colony counting and the determination of toxicity was not affected.

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values was obtained. Metabolic activation did not change these results.

According to the results obtained in this study, Zirkonium-tetrakis(isobutyrato) is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to the limit of toxicity.