Titanium tetrabutanolate

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:

12 July 2012 - 05 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Titanium tetrabutanolate

EC Number:

227-006-8

EC Name:

Titanium tetrabutanolate

Cas Number:

5593-70-4

Molecular formula:

C16H36O4Ti

IUPAC Name:

titanium(4+) tetrakis(butan-1-olate)

Details on test material:

- Name of test material (as cited in study report): Titanium tetrabutanolate
- Physical state: Clear light yellow liquid
- Analytical purity: 95-100%
- Lot/batch No.: 2198
- Expiration date of the lot/batch: 21 May 2014
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Gene of histidine in Salmonella typhimurium and gene of tryptophan in Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa w all mutation and delation of uvr B gene: presence of R factor plasmid pKM101 in strains TA98 and TA 100 to further increase the sensitivity of these strains to some mutagens

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat-liver homogenate activation system (S-9)

Test concentrations with justification for top dose:

Titanium tetrabutanolate was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Based on the results of the dose range finding test, Titanium tetrabutanolate was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

the test subsatane was disolved in hexane. Test substance concentrations were used within 2.5 hrs after preparation.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

used for assay w ith metabolic activation for all tester strains

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate

Positive control substance:

sodium azide

Remarks:

used for assay w ith metabolic activation for strain TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate

Positive control substance:

other: ICR-191

Remarks:

used for assay w ith metabolic activation for strain TA 1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

used for assay w ith metabolic activation for strain TA 98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

650 µg/plate

Positive control substance:

methylmethanesulfonate

Remarks:

used for assay w ith metabolic activation for strain TA100

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

used for assay w ith metabolic activation for strain WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hrs
-Temperature: 37.0 ± 1.0 degrees

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of titanium tetrabutanolate, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

Criteria for valid assay:
The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No data

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:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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

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:

valid

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance on the plates eas observed at the strat and at the end of the incubation eriod at concentrations of 1000 µg/plate and upwards

RANGE-FINDING/SCREENING STUDIES: Titanium Tetrabutanolate was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA: In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In an independent repeat of the assay with additional parameters, Titanium Tetrabutanolate was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Titanium Tetrabutanolate precipitated on the plates at test substance concentrations of 333 and 1000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Dose range finding study:

Titanium tetrabutanolate was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. 

This dose range finding test is reported as a part of the first experiment of the mutation test Table.1(refer the section attached background material)

Mutation assay:

Based on the results of the dose range finding test, Titanium tetrabutanolate was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown inTable1and Table2 (refer the section attached background material)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that titanium tetrabutanolate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Titanium tetrabutanolate was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2uvrA in concentrations up to 5000 µg per plate. The compound was not mutagenic either in the presence or absence of a liver microsomal system, i.e., it did not induce a significant increase over the spontaneous mutation frequency. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The study was conducted in compliance with Good Laboratory Practice (GLP) and according to OECD guideline 471 (1997). This study was regarded reliable without restriction and the study result is used as a key information in hazard assesment.