Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: negative with and without metabolic activation

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- histidine gene (for S.typhimurium strains)
- tryptophan gene (for E.coli strains)
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
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
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.
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
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
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
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
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

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

in-vitro Genotoxicity:

Ames test

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.

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.

Justification for classification or non-classification

According to Regulation (EU) No 1272/2008 (CLP) ammonium hexafluorozirconate has not to be classified regarding genetic toxicity.