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Toxicological information

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:
2010-11-16 to 2010-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
236-675-5
EC Name:
Titanium dioxide
Cas Number:
13463-67-7
Molecular formula:
O2Ti
IUPAC Name:
dioxotitanium
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): H-29865
- Physical state: white solid
- Stability under test conditions: the test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Chemical reactivity delta b: 1.5
- pH: 5.7

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from male Sprague-Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Toxicity ,mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg/plate
Mutagenicity test: 333, 667, 1000, 3333 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: based on the solubility of the test substance and compatibility with the target cells.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control: with metabolic activation: dissolved in DMSO; concentration: 2.5 µg/plate; strain: TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive control without metabolic activation: dissolved in DMSO; concentration: 1.0 µg/plate; strain WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: acridine mutagen ICR-191
Remarks:
Positive control without metabolic activation: dissolved in sterile water; concentration: 2.0 µg/plate; strain: TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: dissolved in sterile water; concentration: 2.0 µg/plate; strain: TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control: with metabolic activation: dissolved in DMSO; concnetration: 2.5 and 25.0 µg/plate; strain: TA100, TA1535, TA1537 and WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Positive control: without metabolic activation: dissolved in DMSO; concentation: 1.0 µg/plate; strain: TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
In the non-activated assays, 0.5 mL of sham mix and 100 µL of vehicle, test substance dilution, or positive control were added to pre-heated (45 - 48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain.
In the S9-activated assays, 100 µL of the vehicle, test substance dilution, or positive control were added to pre-heated (45 - 48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain and 0.5 mL of S9 mix.
All mixtures were vortexed and overlaid onto the surface of minimum glucose agar plates. After the overlay solidified, the plates were inverted and incubated for approximately 48 - 51 hours at 37 ± 2°C. Plates that were not evaluated immediatley following the incubation period were stored at approximately 4°C. All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.
Dose levels for the mutagenicity test were chosen from the toxicity-mutation test results.

SCORING
The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered as signs of toxicity.
Revertant colonies were counted with an automated colony counter (Sorcerer, Perceptive Instruments Ltd., Sulfolk, United Kingdom). Plates that could not be accurately counted automatically were counted manually.

CRITERIA FOR DETERMINATION OF A VALID TEST
1. Tester strain integrity
To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrA and uvrB mutations, all tester strains cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the pKM101 plasmid, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin.

2. Tester strain culture density
To endure that appropriate numbers of bacteria are plated, all testser strain culture densities must be apporximately 10^9 cells per milliliter.

3. Negative control values
The tester strain cultures must exhibit a charactersitic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions. The acceptable ranges for the mean values of negative controls are as follows:
- TA98: 8 - 60
- TA100: 60 - 240
- TA1535: 4 - 45
- TA1537: 2 - 25
- WP2uvrA: 5 -60

4. Positive control values
EAch mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative (vehicle)control value for each tester strain.

5. Toxicity
A minimum of 3 non-toxic scorable dose levels are required to validate the study. A dose level is considered toxic if it causes:
- A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibits a dose-dependent drop in the revertant count or
- A reduction in the background lawn
in the event that less than 3 non-toxic dose levels are achieved, the affected portion of the test will be repeated with an appropriate change in dose levels.
Evaluation criteria:
Criteria for a positive response:
1. Strains TA1535 and TA1537
Data will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

2. Strains TA98, TA100 and WP2uvrA
Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
Statistics:
For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: the test substance formed a milky white suspension in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

TOXICITY-MUTATION TEST
No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. A >50% reduction in mean number of revertants was observed at: 3333 µg/plate for TA98 with S9 activation, 66.7 µg/plate for TA1535 with S9 activation, 3333 µg/plate for TA1537 with S9 activation, 667 µg/plate for TA1535 without S9 activation, and 3333 µg/plate for TA1537 without S9 activation. These reductions occurred at intermediate dose levels with no dose related correlation and are not considered to be biologically relevant. Test substance precipitation was observed starting at 100 µg/plate in the non-activated test system and at all dose levels in the activated testing system with the exception of TA100 with S9 activation where precipitation was observed starting at 333 µg/plate.

MUTAGENICITY TEST
No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed at all dose levels (333 to 5000 µg/plate) with all tester strains both in the absence and presence of S9 activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

The test substance showed no evidence of mutagenicity in the bacterial reverse mutation test either in the absence or presence of Aroclor-induced rat liver S9.