Dipotassium hexafluorotitanate

Genetic toxicity: in vitro

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium hexafluorotitanate

EC Number:

240-969-9

EC Name:

Dipotassium hexafluorotitanate

Cas Number:

16919-27-0

Molecular formula:

F6Ti.2K

IUPAC Name:

dipotassium hexafluorotitanate

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

Storage Conditions: Room temperature, protected from light

Method

Target gene:

his

Species / strain

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).

Controls

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

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.