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Genetic toxicity: in vitro

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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:
18 July 2012 to 30 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is conducted in compliance with Good Laboratory Practice (GLP) and according to OECD guideline 471 (1997)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Titanium tetra(octanolate), branched and linear
EC Number:
281-199-3
EC Name:
Titanium tetra(octanolate), branched and linear
Cas Number:
83897-91-0
Molecular formula:
C32H68O4Ti, molecular weight range not applicable (UVCB substance)
IUPAC Name:
titanium tetra(octanolate), branched and linear
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Titanium tetra(octanolate) branched and linear
- Physical state: clear colourless to pale yellow liquid
- Lot/batch No.: 304-300164/000004
- 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
- Hygroscopic: Yes, store in well-sealed container
- Reactivity: Reactive to moisture
- Test substance handling: No specific handling required

Method

Target gene:
histidin locus in selected strains
Species / strainopen allclose all
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 deletion of uvrB gene; presence of R factor plasmid pKM101 in strains TA98 and TA100 to further increase the sensitivity of these strains to some mutagens
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:
Dose range finding test: Titanium tetra(octanolate), branched and linear was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
main study: Based on the range finding test 3330, 5000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
tetrahydrofuran
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate
Positive control substance:
sodium azide
Remarks:
for strain TA 1535: without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
2-nitrofluorene
Remarks:
for strain TA 98: without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
650 µg/plate
Positive control substance:
methylmethanesulfonate
Remarks:
for strain TA 100: without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate
Positive control substance:
other: ICR 191
Remarks:
for strain TA 1537: without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for strain WP2uvrA: without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
DURATION
- Exposure duration: 48±4 h
- Temperature: 37 ± 2°C

NUMBER OF REPLICATIONS: Three


DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the revertant colonies per plate and/or disappearance of the bacterial background lawn was considered as an indication of cytotoxicity

Evaluation criteria:
Criteria for a 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) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain
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.

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.
Statistics:
no data

Results and discussion

Test results
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards

RANGE-FINDING/SCREENING STUDIES: Substance was tested in tester strain TA100 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. The individual data are presented in Table 3 (refer section attahced background meterial)

ADDITIONAL INFORMATION ON CYTOTOXICITY: To determine the toxicity of substance the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. A slight decrease in the bacterial background lawn was observed at a concentration of 5000 µg/plate in the absence of S9-mix. In addition, a slight to moderate decrease in the bacterial background lawn was observed at concentrations of 3330 and 5000 µg/plate in the presence of S9-mix.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Refer to the section "Attached background material" for data tables.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Titanium tetra(octanolate), branched and linear is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 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. Although in the first mutation experiment an up to 4 times increase in the number of revertants was observed in tester strain WP2uvrA in the presence of S9-mix, this increase was not dose dependent and not confirmed in two independent repeat experiments. Therefore this increase was considered not biologically relevant.

Based on the results of this study it is concluded that Titanium tetra(octanolate), branched and linear is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Titanium tetra(octanolate), branched and linearwas 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 in the microbial assays 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.

This study was regarded reliable with one restriction; the study does not include full range of recommended strains. However, the study performed according to OECD 471 guideline. Thus, the result of this study is used as a key value in hazard assessment.

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