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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

bacterial reverse mutation test: negative

bacterial reverse mutation test (TA 102): negative

in vitro mammalian cell gene mutation assay: negative

in vitro mammalian cell micronucleus test: positive

in vivo micronucleus test in rats: negative

Overall conclusion on the genetic toxicity of dipotassium hexafluorotitanate: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-25 - 2020-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP, additional information for strain TA102, this however completely reliable. Study is not sufficient on its own but supports results of preliminary study.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, adopted July 21, 1997.
Deviations:
yes
Remarks:
Only strain TA102 tested
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The S. typhimurium tester strain was obtained from Dr. Bruce Ames, University of California, Berkeley. The tester strain may also be obtained from Molecular Toxicology Inc. (Moltox).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 4173, Exp. Date: 04 Dec 2021; Lot No. 4201, Exp. Date: 29 Jan 2022) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use as indicated below:
Component Final Concentration
(3-nicotinamide-adenine dinucleotide phosphate 4 mM
Glucose-6-phosphate 5 mM
Potassium chloride 33 mM
Magnesium chloride 8 mM
Phosphate Buffer (pH 7.4) 100 mM
S9 homogenate 10% (v/v)
The Sham mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.
Frequency and Route of Administration
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).
Test concentrations with justification for top dose:
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500, and 5000 μg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile Water for Injection was the vehicle of choice based on information provided by the Sponsor.
- Justification for choice of solvent/vehicle: The sponsor has indicated that the test substance is soluble in water at 12.8 g/L (at 20°C).
Untreated negative controls:
no
Remarks:
solvent controls
Negative solvent / vehicle controls:
yes
Remarks:
sterile water for injection
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: sterigmatocystin
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate)
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 100 µL of 0.3x10E9 cells per milliliter
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none
- Exposure duration/duration of treatment: 48-7h
- Harvest time after the end of treatment (sampling/recovery times): 48-72h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition (reduction of revertant count)
- Any supplementary information relevant to cytotoxicity: The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA102 was exposed to the vehicle alone, positive controls and eight dose levels of the test substance, in duplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.
Rationale for test conditions:
As indicated in the guideline / results from prelliminary cytotoxicity and precipitation testing
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strain TA102
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 102
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:
valid
Remarks:
vehicle
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not available
- Data on osmolality: not available
- Possibility of evaporation from medium: no
- Water solubility: not exceeded
- Precipitation and time of the determination: no precipitation
- Other confounding effects: none known

RANGE-FINDING/SCREENING STUDIES (if applicable):
Initial Toxicity-Mutation Assay
No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain TA102 in the absence of S9 activation.
No positive mutagenic responses were observed in either the presence or absence of S9 activation.
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500, and 5000 µg per plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data , colony counts:
MMC, -S9: 1656, 1552, 1475
water, -S9: 388, 382, 366
STM, +S9: 1879, 1857, 1626
water, +S9: 334, 347, 340

Ames test:
- Signs of toxicity
Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain TA102 in the absence of S9 activation.
- Mean number of revertant colonies per plate and standard deviation : None over background

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)

Activation
Control None Rat Liver
Mean SD Min Max 95% CL Mean SD Min Max 95% CL
Neg 322 58 193 459 206-438 361 55 213 492 251-471
Pos 1777 536 661 2964 1990 549 532 3865
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control
Conclusions:
The test was conducted to complete the available results of a previous Ames test with only four of the five tester strains (or individual strains of a group of tester strains). So, there was only strain TA 102 tested, which is in this context however sufficient. Otherwise the test was conducted according to the most recent OECD guideline 471 under GLP. The results can be hence considered reliable and sufficient to complete the ones of the former study. The test revealed negative reults for genotoxicity, both with and without metabolic activation, which is consistent with the results of the previous study. It so can be concluded that the test item does not need to be considered genotoxic in bacteria.
Executive summary:

The test substance, potassium hexafluorotitanate (K2TiF6), was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of tester strain of Salmonella typhimurium TA102 in the presence and absence of an exogenous metabolic activation system. Sterile Water for Injection was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500, and 5000 μg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA102 in the absence of S9 activation. No positive mutagenic responses were observed in either the presence or absence of S9 activation.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500, and 5000 μg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA102 in the absence of S9 activation. No positive mutagenic responses were observed in either the presence or absence of S9 activation.

These results indicate potassium hexafluorotitanate (K2TiF6) was negative for the ability to induce reverse mutations at selected loci of Salmonella typhimurium (TA102) in the presence and absence of an exogenous metabolic activation system.

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.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983-05-26
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 1996-02-27
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
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.

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; 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; 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; 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.
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

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

Conclusions:
Interpretation of results:
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-07 to 2012-01-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to the laboratory were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- Cytotoxicity Range-Finder Experiment: 50, 100, 200, 400, 800 and 1600 µg/mL (with and without metabolic activation)
- Experiment 1: 50, 100, 200, 225, 250, 275, 300, 325, 350, 375, 400 and 500 µg/mL (with and without metabolic activation)
- Experiment 2: 50, 100, 200, 250, 275, 300, 325, 350, 375, 400 and 500 µg/mL (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Preliminary solubility data indicated that dipotassium hexafluorotitanate was soluble in sterile water for irrigation (purified water) at a concentration of approximately 16 mg/mL. The solubility limit in culture medium was less than 1600 μg/mL, as indicated by precipitation at this concentration 20 hours after test article addition.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive controls: all solutions were prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). Positive controls, if not used immediately, were stored as frozen aliquots at <-50°C, protected from light. 0.15 and 0.20 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive controls: all solutions were prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). Positive controls, if not used immediately, were stored as frozen aliquots at <-50°C, protected from light. 2.00 and 3.00 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (RPMI 1640 media)

DETERMINATION OF CYTOTOXICITY
A maximum concentration of 1600 μg/mL was selected for the cytotoxicity Range-Finder Experiment (50, 100, 200, 400, 800 and 1600 µg/mL; tested in absence and presence of S-9) in order that treatments were performed up to a precipitating concentration. Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.
Single cultures only were used and positive controls were not included. At least 10^7 cells in a volume of 17 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle or test article was added. S-9 mix or 150 mM KCl was added. The final treatment volume was 20 mL.
Following 3 hour incubation period at 37±1°C with gentle agitation, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

MUTATION ASSAYS (Experiment 1 and 2):
- Treatment of cell cultures: at least 10^7 cells in a volume of 17 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle, test article or 0.2 mL positive control solution (plus 1.8 mL purified water) was added. S-9 mix or 150 mM KCl was added. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1ºC with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 20 mL RPMI 10. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival.
- Plating for survival: following adjustment of the cultures to 2 x 10^5 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.
- Expression period: cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of not exceeding 1 x 10^6 cells/mL and, where possible, retaining at least 6 x 10^6 cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures, as can be seen in table 1 (please refer to "Any other information on materials and methods incl. tables" below), were selected to be plated for viability and 6TG resistance.
- Plating for viability: at the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 to 8 days). Wells containing viable clones were identified by eye using background illumination and counted.
- Plating for 6TG resistance: at the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (13 to 14 days) and wells containing clones were identified as above and counted.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
1. The mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value).
2. At least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in mutant frequency.
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. The effects described above were reproducible.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: in absence and presence of S-9 concentrations 375 to 500 µg/mL; Experiment 2: in absence of S-9 concentrations 325 to 500 µg/mL and in presence of S-9 concentrations 350 to 500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes in pH (>1 pH unit) were observed at the highest concentration tested under both treatment conditions (800 μg/mL), compared to the concurrent vehicle controls.
- Effects of osmolality: no marked changes in osmolality (>50 mOsm/kg) were observed at the highest concentration tested under both treatment conditions (800 μg/mL), compared to the concurrent vehicle controls.
- Water solubility: preliminary solubility data indicated that dipotassium hexafluorotitanate was soluble in sterile water for irrigation (purified water) at a concentration of approximately 16 mg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 50 to 1600 μg/mL (limited by solubility in primary vehicle, purified water). Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations in the absence and presence of S-9 (800 and 1600 μg/mL). The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and the higher concentration was discarded. The highest concentration to provide >10% RS was 200 μg/mL, which gave 65% and 109% RS in the absence and presence of S-9, respectively. The % RS values are shown in table 2 (please refer to "Any other information on results incl. tables" below).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 twelve concentrations, ranging from 50 to 500 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest three concentrations tested in the absence and presence of S-9 (375 to 500 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentration analysed was 350 μg/mL, which gave 13% and 18% RS in the absence and presence of S-9, respectively.
In Experiment 2 eleven concentrations, ranging from 50 to 500 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest five concentrations in the absence of S-9 (325 to 500 μg/mL) and the highest four concentrations in the presence of S-9 (350 to 500 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 300 μg/mL in the absence of S-9 and 325 μg/mL in the presence of S-9, both of which gave 12% RS

MUTATION (Experiment 1 and 2):
In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Dipotassium hexafluorotitanate at any concentration analysed in the absence and presence of S-9. A weak, statistically significant linear trend was observed in the absence of S-9 in Experiment 1, but in the absence of any significant increases in mutant frequency at any concentration tested this observation was not considered biologically relevant. There were no statistically significant linear trends in the absence of S-9 in Experiment 2 or in the presence of S-9 in Experiments 1 and 2.
Remarks on result:
other: all strains/cell types tested

Table 2: RS Values - Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

50

94

118

100

106

118

200

65

109

400

1

0

800 PP

0

0

1600 PP

NP

NP

%RTG = Percentage relative survival

PP = Precipitation observed following treatment incubation period

NP = Not plated for survival due to precipitation

Conclusions:
Interpretation of results:
negative

It is concluded that dipotassium hexafluorotitanate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-11 to 2011-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
other: OECD 487 in vitro Mammalian Cell Micronucleus test (adopted 2010-07-22)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: mammalian (human; females)
Details on mammalian cell type (if applicable):
- blood from two healthy, non-smoking female volunteers (age: 25 and 26 years) from a panel of donors at the laboratory was used for each experiment in this study
- no volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals.
- all volunteers are non-smokers and are not heavy drinkers of alcohol.
- donors were not taking any form of medication (contraceptive pill excluded).
- the measured cell cycle time of the donors used at the laboratory falls within the range 13 +/- 2 hours.
- for each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for 48 hours and rocked continuously.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Range-finder:
- 5.805 to 1600 (3 hours treatment + 21 hours recovery; with and without metabolic activation (S9))
- 5.805 to 1600 (24 hours treatment + 24 hours recovery; without metabolic activation (S9))
Micronucleus experiment:
- 100.0 to 750.0 (3 hours treatment + 21 hours recovery; without metabolic activation)
- 100.0 to 1000 (3 hours treatment + 21 hours recovery; with metabolic activation)
- 25.00 to 400.0 (24 hours treatment + 24 hours recovery; without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile purified water
Preliminary solubility data indicated that dipotassium hexafluorotitanate was soluble in water for irrigation (purified water) at concentrations of approximately 16 mg/mL. The solubility limit in culture medium was approximately 1600 μg/mL, as indicated by a lack of any visible precipitation at this concentration immediately upon test article addition but visible following a period of approximately 20 hours after test article addition incubated at 37 ± 1°C.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control: Mitomycin C was dissolved in purified water immediately prior to use and was used for the pulse treatment. 0.60 and 0.80 µg/mL (without metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive controls: Cyclophosphamide was dissolved in anhydrous analytical grade DMSO, frozen (<-50°C) and thawed immediately prior to use and diluted accordingly. The positive control substance was used for the pulse treatment. 6.25
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Vinblastine; 0.02, 0.03 and 0.04 µg/mL (without metabolic activation)
Remarks:
Vinblastine was dissolved in purified water immediately prior to use and used for the continuous 24 + 24 hour treatment.
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
A maximum concentration of 1600 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the limit of solubility in the primary solvent. Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article or vehicle controls (1 mL per culture) as can be seen in Table 1 (please refer to "Any other information on materials and methods incl. tables" below).
Positive control treatments were not included.
The final culture volume was 10 mL.
Cultures were incubated at 37 ± 1°C for the designated exposure time.
Osmolality measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment. pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment (all treatment conditions) and Experiment 1 (3+21 hour ± S-9 treatment conditions only).

MICRONUCLEUS EXPERIMENT
Immediately prior to treatment, all positive control cultures had 0.9 mL culture medium added to give a final pre-treatment volume of 9.4 mL.
S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle or positive controls (1.0 mL per culture for test article and vehicle control or 0.1 mL for positive control cultures) as can be seen in Table 1 (please refer to "Any other information on materials and methods incl. tables" below). . The final culture volume was 10 mL. Cultures were incubated at 37 ± 1°C for the designated exposure time.
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 ± 1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. Cyto-B, formulated in DMSO, was added to post wash-off culture medium to give a final concentration of 6 μg/mL per culture.

pH measurements on post-treatment incubation medium were taken in the Micronucleus Experiment (3+21 hour ± S-9 treatment conditions only).

HARVESTING
At the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 37 ± 1°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

SLIDE PREPARATION
Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.

SELECTION OF CONCENTRATION FOR MICRONUCLEUS EXPERIMENT
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
The RI, which indicates the relative number of nuclei compared to controls was determined using the formulae below:
RI = (number binucleate cells + 2(number multinucleate cells))/total number of cells in treated cultures
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures/RI of vehicle controls) x 100
Cytotoxicity (%) is expressed as (100 – Relative RI).
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.
A suitable range of concentrations was selected for the Micronucleus Experiment based on these toxicity data.

SELETION OF CONCENTRATIONS FOR MICRONUCLEUS ANALYSIS (MICRONUCLEUS EXPERIMENT ONLY)
Slides were examined, uncoded, for proportions of mono-, bi- and multinucleate cells to a minimum of 500 cells per culture.
The highest concentration for micronucleus analysis was one at which approximately 55 ± 5% reduction in RI had occurred. Analysis of slides from highly cytotoxic concentrations was avoided. Opaque media was observed at the end of treatment but was not clear detailing whether that it was observed following 3+21 hour treatment in the absence or presence of S-9 or both. To ensure that an appropriate highest concentration for analysis was selected, selection was primarily based on cytotoxicity whilst taking precipitation into consideration.
Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
For each treatment regime, two vehicle control cultures were analysed for micronuclei. Positive control concentrations, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, were analysed.

SLIDE ANALYSIS
Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips. One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was noted.
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
Micronucleus analysis was not conducted on slides generated from the Range-Finder treatments.

ACCETANCE CRITERIA
The assay was to be considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen.
2. The frequency of MNBN cells in vehicle controls fell within the current historical vehicle control (normal) ranges.
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal ranges.
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in negative control cultures at the time of harvest.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al, 1990).

*Reference
Scott D, Dean B J, Danford N D Kirkland D J (1990) Metaphase chromosome aberration assays in vitro. Basic Mutagenicity Tests; UKEMS recommended procedures. Kirkland D J (Ed) pp 62-86
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion
test (Richardson et al, 1989)*.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test (Richardson et al, 1989)*. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

*Reference
Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D O and Phillips B (1989) Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154
Species / strain:
lymphocytes: mammalian (human; females)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
please refer to "Attached background material" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to "Attached background material" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Cytotoxicity Range-Finder Experiment: a significant decrease in pH (greater than 1 pH unit) was observed at 1600 μg/mL. However, as concentrations tested in the Micronucleus Experiment were below this concentration, this observation was considered to not affect study interpretation.
Micronucleus Experiment: yellow medium was observed at the end of treatment in the Micronucleus Experiment. Therefore, pH readings were taken to confirm that there were no marked changes in pH to affect data interpretation.

- Effects of osmolality:
Cytotoxicity Range-Finder Experiment: no marked changes in osmolality were observed compared to the concurrent vehicle controls.
- Water solubility: preliminary solubility data indicated that dipotassium hexafluorotitanate was soluble in water for irrigation (purified water) at concentrations of approximately 16 mg/mL.

RANGE-FINDING/SCREENING STUDIES:
The results of the RI determinations from the cytotoxicity Range-Finder Experiment can be seen in the attachment below (please refer to "Attached background material" below)

MICRONUCLEUS EXPERIMENT:
The results of the RI determinations from the Micronucleus Experiment can be seen in the attachment below (please refer to "Attached background material" below).
A summary of the number of cells containing micronuclei is given in Appendix 1 (please refer to "Attached background material" below).

VALIDITY OF STUDY
The data in Appendix 1, Appendix 2, Appendix 3 and Table 13 to Table 15 indicate that (pleaser refer to "Attached background material" below):
1) The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures (Appendix 2).
2) The frequency of MNBN cells in vehicle controls fell within the normal range (Appendix 3).
3) The positive control chemicals induced statistically significant increases in the proportion of MNBN cells (Appendix 1). Both replicate cultures of the positive control concentration analysed under each treatment condition exceeded the normal ranges.
4) A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in negative control cultures at the time of harvest (Table 13 to Table 15).

ANALYSIS OF DATA
Treatment of cells with dipotassium hexafluorotitanate for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls for the highest two concentrations analysed (400.0 and 550.0 μg/mL inducing 26% and 48% cytotoxicity, respectively) (Appendix 1 and Appendix 2; pleaser refer to "Attached background material" below). The MNBN cell frequency of both treated cultures at these concentrations exceeded the normal range (Appendix 3; pleaser refer to "Attached background material" below ). A concentration-related increase in the mean MNBN cell frequency was observed at cytotoxicity levels not exceeding the recommended value of 60%.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls for the highest two concentrations analysed (400.0 and 600.0 μg/mL inducing 29 and 51% cytotoxicity, respectively) (Appendix 1 and Appendix 2; pleaser refer to "Attached background material" below ). The MNBN cell frequency of both treated cultures at these concentrations and a single culture at 200.0 μg/mL exceeded the normal range (Appendix 3; pleaser refer to "Attached background material" below). A concentration-related increase in the mean MNBN cell frequency was observed at cytotoxicity levels not exceeding the recommended value of 60%.

The highest concentrations for micronucleus analysis following 3+21 hour treatment in the absence and presence of S-9 (550 and 600 μg/mL, respectively) were selected based on cytotoxicity, as these concentrations reduced the RI by approximately 55 ± 5%. Although opaque media was noted, it was unclear to which treatment condition it referred. This could have reduced the highest concentrations selected for micronucleus analysis to 400 μg/mL for the appropriate treatment condition. However, as statistically significant (p ≤ 0.01) increases in the frequencies of MNBN cells at 400 μg/mL following 3+21 hour treatment in the absence and presence of S-9 were observed, limiting the highest concentrations selected based on cytotoxicity rather than precipitation did not affected the conclusions drawn from these data.

Treatment of cells for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls for all analysed (Appendix 1 and Appendix 2; pleaser refer to "Attached background material" below). The MNBN cell frequency of all treated cultures exceeded the normal range (Appendix 3; pleaser refer to "Attached background material" below ). A concentration-related increase in the mean MNBN cell frequency was observed at cytotoxicity levels not exceeding the recommended value of 60%.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
positive

It is concluded that dipotassium hexafluorotitanate induced micronuclei in cultured human peripheral blood lymphocytes when tested up to cytotoxic concentrations, in both the absence and presence of S-9.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

bacterial reverse mutation test: negative

bacterial reverse mutation test (TA 102): negative

in vitro mammalian cell gene mutation assay: negative

in vitro mammalian cell micronucleus test: positive

in vivo micronucleus test in rats: negative

Overall conclusion on the genetic toxicity of dipotassium hexafluorotitanate: negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-20 to 2012-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2008-05-31
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Type of assay:
micronucleus assay
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 31 - 32 days; females: 32 - 33 days
- Weight at study initiation: males: 88 - 122 g; females: 83 - 101 g
- Assigned to test groups randomly: yes
- Fasting period before study: feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.
- Housing: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The animals were kept in groups of 2 - 3 by sex in MAKROLON cages (type III plus).
- Diet (ad libitum, except for fasting period before administration): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Drinking water
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used:0.8% aqueous hydroxypropylmethylcellulose (Methocel; batch no. 11 A 27-N27, Fagron GmbH & Co., 22885 Barsbüttel, Germany)

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended to the appropriate concentrations in 0.8% aqueous hydroxypropylmethylcellulose . The administration volume was 20 mL/kg b.w.
Duration of treatment / exposure:
single dose application
Frequency of treatment:
once
Post exposure period:
24 hours or 48 hours after the last treatment
Remarks:
Doses / Concentrations:
31, 62 and 125 mg potassium hexafluoro titanate
Basis:
actual ingested
No. of animals per sex per dose:
5 males/5 females per sampling interval and group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (batch no. 068K1131, SIGMA-ALDRICH CHEMIE GmbH, 82024 Taufkirchen, Germany)
- Route of administration: intraperitoneal
- Vehicle: 0.9% NaCl solution (batch no. 12013451; B. Braun Melsungen AG, 34212 Melsungen, Germany)
- Dose level: 27 mg/kg bw
- Administration volume: 20 mL/kg b.w.
Tissues and cell types examined:
The slides were coded and randomised before microscopic analysis. Two thousand polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 1000 erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION (RANGE FINDER):
The dose levels had been selected based on a preliminary oral acute toxicity study employing one rat (Crl: CD(SD)) per sex and dose. Five dose levels of 31, 62, 125, 250 and 500 mg potassium hexafluoro titanate/kg b.w. were tested by gavage. The administration volume was 20 mL/kg bw and the vehicle was 0.8% aqueous hydroxypropylmethylcellulose. The animals were observed for symptoms for a period of 3 days.

TREATMENT AND SAMPLING TIMES:
The dose levels should cover a range from the maximum toxicity to little or none. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. The highest dose may also be defined as the dose that produces some indication of toxicity of the bone marrow (e.g. reduction of the proportion of immature erythrocytes among total erythrocytes in the bone marrow) for solutions the maximum feasible application volume.
For the main study, the following sampling times were used: 24 hours for all groups and 48 hours after administration for the vehicle control and the high dose group. The experiment consisted of the following groups, as can been seen in table 1 (please refer to "Any other information on materials and methods incl. tables").

DETAILS OF SLIDE PREPARATION:
The rats were sacrificed at the indicated time points. After having removed some of the muscles, the femurs were excised below the knee and at the iliac joint. The bone marrow was flushed out with calf serum and centrifuged at 850 x g for 3 to 5 minutes. The supernatant was removed and the sediment resuspended in a drop of calf serum by using a Pasteur Pipette. Then a smear of 30 to 60 mm length was prepared.
Once dry, the preparations were immediately fixed in methanol for 5 minutes. Cells were stained for 6 minutes using filtered Mayers Haemaleum. The slides were rinsed with cold tap water for 5 minutes and then further stained in 0.5% w/v ethanolic eosin solution for 1 minute. The slides were again left to air-dry before being dipped in xylene and mounted.

ACCEPTANCE CRITERIA:
The micronucleus test was considered valid if
i) the heterogeneity chi-square test provided evidence of acceptable variability between animals within a group
ii) the incidence of micronucleated PCE in the vehicle control groups fell within or close to the historical vehicle control range
iii) at least 5 analysable animals per sex of each group at each kill were available for analysis
iv) the positive reference chemical (CPA) induced clear and statistically significant increases in the frequencies of micronucleated PCE.
Evaluation criteria:
The test chemical was considered as clearly positive in this assay if:
i) a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time
ii) the frequency of micronucleated PCE at such a point exceeded the historical control range
iii) corroborating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times, or dose response profiles.
Statistics:
After completion of scoring and decoding of slides, the ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequencies of micronucleated PCE/1000 were also determined.
PCE/NCE ratios were determined in order to evaluate possible bone marrow toxicity.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971)* as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity test data).
The PCE/NCE ratios and frequencies of micronucleated PCE in the vehicle control animals were compared with historical control ranges to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity chi-square test.
The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in the vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p ≤ 0.05 were accepted as significant.

* References:
- COLQUHOUN, D. Lectures on Biostatistics, Clarendon Press, Oxford (1971)

Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Please refer to "Additional information on results" below
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Treatment with 31, 62 or 125 mg potassium hexafluoro titanate/kg bw, p.o. revealed slight signs of toxicity. Dose levels of 250 or 500 mg/kg b.w. caused slight signs of toxicity or slight to moderate signs of toxicity and death of all treated animals (death 24 hours after administration or death 1 hour after administration).
Hence, three ascending doses of 31, 62 and 125 mg potassium hexafluoro titanate/kg b.w. were employed.

RESULTS OF DEFINITIVE STUDY
- Clinical signs:
the animals treated with 31 or 62 mg potassium hexafluoro titanate/kg b.w., p.o. revealed pilo-erection 60 or 30 minutes to 3 hours after administration, respectively. Treatment with 125 mg potassium hexafluoro titanate/kg b.w., p.o. caused slightly reduced motility, slight ataxia and pilo-erection 30 minutes to 3 hours and slightly reduced muscle tone and slight dyspnoea 30 to 60 minutes after administration (sacrifice after 24 or 48 hours).
Immediately after sacrifice, bone marrow smears were prepared.
- Micronucleus assay:
No test item-related increase of micronucleated polychromatic erythrocytes was observed in the treated groups as compared to the corresponding vehicle control group at the two sampling times )please refer to table 1 in "Any oterh information on results incl. tables" below). Systemic exposure was demonstrated by signs of little to maximum tolerable toxicity.
The positive reference item group which received cyclophosphamide (27 mg/kg b.w., i.p.) exhibited a significant increase in the number of micronucleated polychromatic erythrocytes.

VALIDITY OF STUDY:
All criteria for validity were met. The data of the positive control were heterogeneous, however, this significance is scientifically not relevant as the data were clearly positive.

STATISTICAL ANALYSIS OF DATA:
The numbers of micronucleated PCE was not influenced by the treatment with the test item. Neither the PCE/NCE ratios nor the numbers of micronucleated PCE were influenced by administration of the test item. No statistical significance was reached after statistical analysis by chi2 test:
The numbers of micronucleated PCE of the negative control and treatment groups were similar to those seen in historical controls.
In all of the cyclophosphamide-treated rats, the numbers of micronucleated PCE significantly exceeded those seen in the vehicle control groups, such that the group mean frequency for both sexes combined (20.5/1000) was approximately 34 times greater than the group mean frequency seen in the concurrent vehicle control.

Table 1 Summarised data of the mutagenicity study

Potassium hexafluoro titanate [mg/kg bw, p.o.]

Sampling time (h)

No. of polychromatic erythrocytes scored per group*

Ratio

PCE/NCE**

Micronucleated polychromatic erythrocytes

 

Mean frequency per 1000 PCE

Significance

Males

Females

Mean*

Males

Females

Mean*

0

24

20000

0.91

0.85

0.88

0.5

0.6

0.6

-

31

24

20000

0.90

0.75

0.83

0.9

0.4

0.7

n.s.

62

24

20000

0.88

0.99

0.94

0.9

0.2

0.6

n.s.

125

24

20000

0.96

0.96

0.96

0.3

0.6

0.5

n.s.

0

48

20000

0.96

0.94

0.95

0.5

0.7

0.6

-

125

48

20000

0.91

0.93

0.92

0.6

0.6

0.6

n.s.

Cyclophosphamide

27 mg/kg bw, i.p.

24

20000

0.53

0.52

0.53

24.6

16.4

20.5

s.

* = males and females combined

** = per 1000 counted cells

s. = significant at p ≤ 0.05 for increases compared to the control

n.s. = not significant at p≤ 0.05 for increases compared to the control

p.o. = per oral

i.p. = intraperitoneal

PCE = ploychromatic erythrocytes

NCE = normochromatic erythrocytes

Conclusions:
Interpretation of results: negative
Potassium hexafluoro titanate tested up to the maximum tolerated dose of 125 mg potassium hexafluoro titanate/kg bw by oral administration showed no mutagenic properties in the rat bone marrow micronucleus study at the two tested sampling times of 24 hours and 48 hours. Systemic exposure was demonstrated by signs of little to maximum tolerable toxicity.
In the same system, cyclophosphamide induced significant increases in micronucleus frequency, at a decrease of the PCE:NCE ratio of 40%.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

The following GLP and guideline conform GLP studies are considered as key studies and will be used for classification:

The in vitro mammalian cell gene mutation test by Lloyd (2012) according to OECD 476 has shown that dipotassium hexafluorotitanate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells up to the highest test concentration, limited by cytotoxic effects.

Testing in bacteria reverse mutation assays, although of limited relevance for metals (HERAG, 2007), also yielded negative results (Thompson, 1997) up to the limit concentration of 5000 µg/plate.

Negative as well as positive results were obtained in clastogenicity assays: Dipotassium hexafluorotitanate appears to induce micronuclei in vitro in cultured human peripheral blood lymphocytes, but only at high concentrations and via mechanisms that appear to lack physiological relevance (Watters, 2012), and is not mirrored in corresponding in vivo (RL=1) GLP-conform micronucleus test in the bone marrow of rats exposure via the oral route (Flügge, 2012). Availability of the substance was demonstrated by signs of general toxicity, thus exposure of the target organ was ensured.

In conclusion, based upon the evaluation of all available studies, genotoxicity is not an endpoint appropriate to be carried forward to risk characterisation.

 

References:

HERAG (2007) Fact sheet 05 - Mutagenicity. EBRC Consulting GmbH / Hannover /Germany.August 2007. [www.herag.net]

Justification for classification or non-classification

Based upon the evaluation of all available studies, the classification of dipotassium hexafluorotitanate with respect to mutagenic potential is not appropriate. Thus, accordingto Directive EEC 67/548 and to EC Regulation No. 1272/2008, dipotassium hexafluorotitanate is not considered to have a mutagenic potential, hence, no classification or labelling is required.