Ammonium hexafluorozirconate

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:

31.03.2017 - 21.04.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Ammonium hexafluorozirconate

EC Number:

240-970-4

EC Name:

Ammonium hexafluorozirconate

Cas Number:

16919-31-6

Molecular formula:

F6Zr.2H4N

IUPAC Name:

ammonium hexafluorozirconate

Test material form:

solid: particulate/powder

Method

Target gene:

- histidine gene (for S.typhimurium strains)
- tryptophan gene (for E.coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1st Run (Plate incorporation): 50, 150, 500, 1500, 5000 µg/plate
2nd Run (Pre-incubation): 78, 156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino anthracene, 4-Nitro-1,2-phenylene Diamine,

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2 (plate-incorporation and pre-incubation method)

PREPARATION:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO) and ethanol.
Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock solution containing 50 g/L (nominal) of the test item in demin. water was prepared. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
The following nominal concentrations were prepared for the second experiment: 5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate, 156 μg/plate and 78 μg/plate.

PLATE INCORPORATION METHOD:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
• 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

PRE-INCUBATION METHOD:
The following materials were gently vortexed in a test tube and incubated at 37 ± 1°C for 20 minutes:
• 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

EVALUATION:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Mean Revertants First Experiment

Strain induction		TA 97a		TA 98		TA 100		TA 102		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	64	84	9	13	78	87	193	249	8	9
	SD	7.5	4.6	2.5	1.0	5.2	3.5	9.0	23.7	0.6	2.1
DMSO	Mean	66	78	9	10	77	82	224	263	9	8
	SD	6.1	17.6	2.1	1.5	10.8	2.1	12.0	67.4	1.5	1.5
Positive Controls*	Mean	712	535	475	66	340	709	813	1397	243	54
	SD	82.7	183.8	103.7	7.2	47.2	56.2	54.3	116.0	30.6	8.1
5000 µg/plate	Mean	73	92	7	8	50	77	157	195	7	8
	SD	7.1	11.4	1.0	1.0	1.5	8.1	28.4	16.7	0.6	2.1
1500 µg/plate	Mean	57	81	8	13	61	65	199	227	11	9
	SD	8.7	12.8	0.6	5.3	7.2	4.9	6.1	23.4	1.5	3.2
500 µg/plate	Mean	66	83	8	12	69	85	221	203	13	11
	SD	6.7	10.7	1.0	1.2	10.4	2.1	24.4	8.3	2.9	2.9
150 µg/plate	Mean	56	73	10	13	82	76	215	260	14	8
	SD	7.5	10.2	3.1	2.6	7.6	2.6	50.6	58.1	2.1	1.5
50 µg/plate	Mean	57	65	10	12	64	76	279	243	12	8
	SD	10.6	11.4	3.0	4.2	16.2	11.0	38.0	10.1	2.5	1.5

* Different positive controls were used, see section positive control

 Mean Revertants Second Experiment

Strain induction		TA 97a		TA 98		TA 100		TA 102		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	84	86	12	14	105	83	260	259	13	9
	SD	8.3	17.1	0.0	4.0	5.2	6.0	67.7	74.0	2.9	2.9
DMSO	Mean	84	85	11	13	89	99	367	305	11	10
	SD	5.9	9.8	2.9	2.0	3.5	11.7	11.5	85.7	0.0	2.3
Positive Controls*	Mean	600	400	336	59	544	597	1283	1243	72	77
	SD	93.6	68.2	114.5	12.1	221.8	47.7	374.1	339.5	10.6	41.1
5000 µg/plate	Mean	0	0	13	16	72	91	246	282	15	7
	SD	0.0	0.0	4.9	3.5	2.9	9.0	31.4	57.2	2.5	1.5
2500 µg/plate	Mean	102	97	12	15	95	100	245	265	15	14
	SD	2.1	3.1	2.1	0.6	6.1	14.0	51.3	34.1	2.1	3.8
1250 µg/plate	Mean	80	83	11	17	110	92	275	284	12	12
	SD	3.8	20.0	1.7	0.6	5.3	16.9	65.2	56.4	3.0	1.2
625 µg/plate	Mean	86	86	13	16	93	108	267	251	16	8
	SD	13.6	4.0	3.5	4.2	18.5	21.6	19.7	37.8	3.1	1.2
312 µg/plate	Mean	99	102	13	13	93	93	337	334	14	12
	SD	15.3	10.1	2.3	2.3	8.3	4.4	23.4	48.5	3.0	1.0
625 µg/plate	Mean	88	100	11	17	79	81	353	245	14	13
	SD	3.5	10.1	1.5	3.5	7.2	11.0	9.2	30.3	2.9	2.5
312 µg/plate	Mean	96	86	11	13	77	88	349	269	13	12
	SD	8.7	13.6	0.6	3.6	1.5	1.7	15.1	71.0	2.0	2.1

 * Different positive controls were used, see section positive control

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Ammonium hexafluorocirconate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activa-tion under the experimental conditions in this study.

Executive summary:

The study was performed to investigate the mutagenic potential of the test substance in a bacterial reverse mutation assay. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Ammonium hexafluorocirconate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the first experiment, the test item (dissolved in demin. water) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in any tested strain, in the presence and the absence of metabolic activation. Based on the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations. In the bacteria strain TA97a, the bacterial background lawn was not present at the highest concentrations (5000 μg/plate) and no bacteria growth was observed. No signs of toxicity were observed towards the other four bacteria strains. The results of this experiments showed, that the test item caused no increase in the num-ber of revertants in any bacteria strain compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.