Dipotassium titanate(2-)

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:

02 March 2011 - 25 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

This study was performed in accordance with the guideline set forth in "Standards for Mutagenicity Tests with Microorganisms" (Notification No. 77, September 1, 1988 & Notification No. 67, June 2, 1997 & Notification No. 120, December 25, 2000, Ministry of Labour, Japan) and "Revision of the form for report of results of mutagenicity test using microorganisms" (Notification No.653, September 29, 1997, Labour Standards Bureau, Ministry of Labour, Japan). And it was conducted in compliance with the "Standards to be observed by Mutagenicity Testing Institutions(Notification No.76, September 1, 1988 & Notification No. 13, March 29, 2000 & Notification No. 120, December 25, 2000, Ministry of Labour, Japan)”

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium titanate(2-)

EC Number:

261-919-2

EC Name:

Dipotassium titanate(2-)

Cas Number:

59766-31-3

Molecular formula:

K2O17Ti8

IUPAC Name:

dipotassium titanate(2-)

Test material form:

solid: crystalline

Details on test material:

Appearance: White acicular crystal

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose range finder: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate
Main study: 20, 39, 78, 156 and 313 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3 -(5-nitro-2-furyl)acrylamide (AF-2)

Remarks:

without S9; 0.01 µg/plate in DMSO for TA100 and WPsuvrA; 0.1 µg/plate in DMSO for TA98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 0.5 µg/plate in distilled water for TA1535

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl (ICR-191)

Remarks:

without S9; 1.0 µg/plate in DMSO for TA1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9; 5.0 µg/plate in DMSO for TA98, TA100 and TA1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9; 2.0 µg/plate in DMSO for TA1535 and 10 µg/plate in DMSO for WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (pre-incubation method)
For the tests without metabolic activation, 0.5mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test substance solution. For the tests with metabolic activation, 0.5mL of the S9 mix were added to each tube instead of the 0.1M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37°C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of a minimal glucose agar plate.

DURATION
- Exposure duration: 20 minute pre-incubation + 48 hour incubation

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 2.6-5.7x10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

In the dose-finding test and the main test if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and a dose-response and reproducibility were also observed, the test substance was to be judged positive.

Statistics:

Statistical analysis was not done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 313 µg/plate and higher (with and without S9) in the dose range finder. In the main study precipitation was observed at 156 and 313 μg/plate without metabolic activation, and at 313 μg/plate with metabolic activation.
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- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 313 µg/plate

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to Japanese guidelines for screening mutagenicity testing of chemicals, TISMO-D was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to Japanese guidelines for screening mutagenicity testing of chemicals and in accordance with GLP principles. All bacterial strains showed negative responses up to and including 5000 µg/plate in the dose-range finding test and up to and including 313 µg/plate in the main test, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity of the test substance was observed. Precipitation was observed at dose levels of 313 µg/plate and higher (with and without S9) in the dose range finder and at 156 and 313 μg/plate without metabolic activation and at 313 μg/plate with metabolic activation in the main 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.

Based on the results of this study it is concluded that TISMO-D is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.