Lithium potassium titanium oxide

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:

26 June-08 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium Potassium Titanium Oxide

EC Number:

445-620-6

Cas Number:

39318-30-4

Molecular formula:

Hill Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95) CAS Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95)

IUPAC Name:

Lithium Potassium Titanium Oxide

Details on test material:

Batch: 2D82A
White powder, crystal structure
Expiry date: 31 May 2003
Test substance storage: At room temperature in the dark

Method

Target gene:

An histidine-requiring gene in S. typhimurium and a tryptophan-requiring gene in E.coli.

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:

S9 liver homogenate of rat treated with Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding on TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (with and without 5% metabolic activation).

Mutation assay: first experiment on TA1535, TA1537 and TA98; second experiment on TA1535, TA1537, TA98, TA100 and WP2uvrA.
Concentration range: 10- 33-100-333-1000 µg/plate (with and without metabolic activation: 5% in experiment 1; 10% in experiment 2).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO). The test substance was suspended in dimethyl sulfoxide. Treated with ultrasonic waves.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix: 5 µg/plate in Saline for TA1535

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix: 60 µg/plate in Saline for TA1537

Controls 3

Positive controls:

yes

Positive control substance:

other: daunomycine 4 µg/plate in Saline for TA98

Remarks:

without S9 mix

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix: 650 µg/plate in DMSO for TA100

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix: 10 µg/plate in DMSO for WP2 uvr A

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

other: 2-aminoanthracene (1, 2.5 or 5 µg/plate in DMSO) for all tester strains

Remarks:

with S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In agar (plate incorporation)
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
The revertant colonies were counted automatically or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS:
Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.
In the first and second experiment 5% (v/v) and 10% (v/v) S9-mix was used, respectively.

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:
Precipitation of the test substance.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a)The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
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)It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b)The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
Strain Minimum value Maximum value Mean ± 3 x S.D
TA1535 -S9-mix 3 25 12 ± 12
+S9-mix 3 25 13 ± 12
TA1537 -S9-mix 3 26 6 ± 9
+S9-mix 3 28 7 ± 10
TA98 - S9-mix 12 45 18 ± 17
+S9-mix 12 54 24 ± 22
TA100 -S9-mix 56 180 96 ± 80
+S9-mix 59 184 94 ± 73
WP2uvrA -S9-mix 4 30 12 ± 14
+S9-mix 4 30 12 ± 14
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
Strain Minimum value Maximum value Mean ± 3 x S.D
TA1535 -S9-mix 91 993 284 ± 575
+S9-mix 60 1028 222 ± 302
TA1537 -S9-mix 77 1897 348 ± 539
+S9-mix 73 1794 339 ± 583
TA98 -S9-mix 100 1855 592 ± 931
+S9-mix 169 2597 783 ± 1126
TA100 -S9-mix 279 2070 810 ± 779
+S9-mix 190 2753 908 ± 1260
WP2uvrA -S9-mix 70 1827 561 ± 887
+S9-mix 62 933 233 ± 434
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Results and discussion

Test results

Test results 1

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

Remarks:

at 1000 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Remarks:

at 1000 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
Precipitation of TERRACESS L on the plates was observed at the end of the incubation period at concentrations of 1000 µg/plate.

TOXICITY:
No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

Any other information on results incl. tables

The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

TERRACESS L is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without S9 mix tested, in the concentration range 10 up to and including 1000 µg/plate.