Dipotassium hexafluorozirconate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-12-03 to 1997-02-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study reliable with restrictions - the stability of the test material was missing in the study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium hexafluorozirconate

EC Number:

240-985-6

EC Name:

Dipotassium hexafluorozirconate

Cas Number:

16923-95-8

Molecular formula:

F6Zr.2K

IUPAC Name:

dipotassium hexafluorozirconate

Details on test material:

- Name of test material (as cited in study report): POTASSIUM HEXAFLUOROZIRCONATE
- Physical state: white crystalline solid
- Storage condition of test material: ambient temperature <25°C under artificial light

Method

Target gene:

not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 2

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: sterile distilled water

The test material was accurately weighed and approximate half-log dilutions in sterile distilled water prepared by action on a autovortex mixer and sonication for 20 minutes at 30 °C on the day of each experiment. The test formulations were kept warm and regularly sonicated throughout the dosing period to maintain solubility.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation; strains TA100 and TA1535

Migrated to IUCLID6: (ENNG); 3 µg/plate for TA100 and 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation; strain TA1537

Migrated to IUCLID6: (9AA); 80 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine (4NOPD); 5 µg/plate

Remarks:

without metabolic activation; strain TA1538

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation; strain TA98

Migrated to IUCLID6: (4NQO); 0.2 µg/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA); 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537

Remarks:

with metabolic activation; strains TA100, TA1535 and TA1537

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 appropriately 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 (sterile distilled water) 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 test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS and normally Dunnett's method of linear regression is used to evaluate the result. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test results

Test results 1

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 or 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

Test results 2

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 or without metabolic activation.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

No toxicity was exhibited to any of the strains of Salmonella used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100). Please refer to "Any other information on results incl. tables" for more information (Table 1).

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

POSITIVE CONTROL:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Preliminary toxicity study - Results

Strain	Dose (µg/plate)
	0	50	150	500	1500	5000
TA100	98	113	108	111	91	87

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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