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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:
1996-12-03 to 1997-02-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study reliable with restrictions - the stability of the test material was missing in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983-05-26
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 1996-02-27
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Potassium hexafluorotitanate (IV) fine powder pure
- Physical state: white granular solid
- Storage condition of test material: ambient temperature (<25°C), stored under artificial light

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from livers of male Sprague-Dawley rats, which had received a single ip. injection of Aroclor 1254.
Test concentrations with justification for top dose:
Preliminary toxicity study.
0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate (with and without S9-mix)
Experiment 2: 0, 50, 150, 500, 1500 and 5000 µg/plate (with and without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: A range of Ames recommended solvents were tested for vehicle suitability with dimethyl sulphoxide producing the best doseable suspension.

The test material was accurately weighed and approximate half-log suspensions in dimethyl sulphoxide prepared by action on a autovortex mixer and sonication for 30 minutes at 30 °C on the day of each experiment.
Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 X 10°-4 microns.

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation; strains TA100 and TA1535

Migrated to IUCLID6: (ENNG); 3 µg/plate for TA100 and 5 µg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation; strain TA1537

Migrated to IUCLID6: (9AA); 80 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine (4NOPD); 5 µg/plate
Remarks:
without metabolic activation; strain TA 1538
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation; strain TA98

Migrated to IUCLID6: (NQO); 0.2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA); 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA 1537, and 0.5 µg/plate for TA1538 and TA98
Remarks:
with metabolic activation; strains TA100, TA1535, TA 1537, TA1538 and TA98
Details on test system and experimental conditions:
Experiment 1 and 2:
METHOD OF APPLICATION: in agar (plate incorporation)
Test material and vehicle controls:
Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar at 45 °C, 0.1 mL of the appropriaty diluted test material or vehicle control and either 0.5 mL of the S9 liver microsome mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.
Positive controls:
- Without activation: a known aliquot (0.1 mL) of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of phosphate buffer was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.
- With activation. a known aliquot (0.1 mL) of 2AA solution was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of S9-mix was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.

DURATION
- Exposure duration: all of the plates were incubated at 37°C for approximately 48 hours.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the frequency of revertant colonies were assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
A preliminary test was carried out to determine the toxicity of the test material to the tester organisms. A mixture of 0.1 mL of bacterial suspension (TA100) , 2 mL of molten, trace histidine supplemented media (histidine/biotin and top agar), 0.1 mL of test material and 0.5 mL phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Five doses of the test material and a vehicle control (dimethyl sulphoxide) were tested in duplicate. In addition, 0.1 mL of the maximum concentration of test material and 2 mL of molten, trace histidine supplemented media were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for revertant colonies using a Domino colony counter and examined for a thinning of the background lawn.
Evaluation criteria:
For a substance to be considered positive in this system, it should have induced a dose-related and statistically significant increase in mutation rate in one or mor strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS and normally Dunnett's method of linear regression is used to evaluate the result. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level with ot without metabolic activation.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No toxicity was exhibited to any of the strains of Salmonella used.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level with ot without metabolic activation.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No toxicity was exhibited to any of the strains of Salmonella used.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100). Please refer to "Any other information on results incl. tables" for more information (Table 1).

NEGATIVE CONTROL:
Results for the negative controls (spontaneous mutation rates) are presented in "Attached background material" below.

POSITIVE CONTROL:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Preliminary toxicity study - Results, number of revertant colonies

Strain

Dose (µg/plate)

0

50

150

500

1500

5000

TA100

110

100

95

101

104

99

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.