Dihydrogen hexafluorotitanate(2-)

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

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(2-)

Details on test material:

- Name of test material (as cited in study report): Dipotassium hexafluorotitanate
- Molecular weight: 240.05
- Physical state: colourless powder
- Storage condition of test material: stored at 2 to 8°C, with desiccant and protected from light.

Method

Target gene:

not applicable

Species / strain

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.

Controls

Controls 1

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)

Controls 2

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

Controls 3

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

Results and discussion

Test results

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

Applicant's summary and conclusion

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.