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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 April 2017 to 28 August 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Sulfates of potassium, sodium and calcium, byproduct from fermentation
Molecular formula:
SO42-, K+, Na+, Ca2+
IUPAC Name:
Sulfates of potassium, sodium and calcium, byproduct from fermentation
Test material form:
solid
Remarks:
light to beige solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: 30 November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle:Test item was soluble at 50 mg/mL concentration using Distilled water.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Distilled water was used as solvent to prepare the stock solution of the test material (50mg/ml). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other:
Remarks:
See attached-table 1
Metabolic activation:
with and without
Metabolic activation system:
Liver extract, S9 fraction from rats which were treated intraperitoneally with phenobarbital and beta- naphthoflavone
Test concentrations with justification for top dose:
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the followingt concentrations were used :
- Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
- Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Test item was soluble at 50 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO and DMF. Distilled water was selected as vehicle (solvent) for the study.
Controls
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (Distilled Water)
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (TA98 without S9); 2-aminoanthracene (for all strains, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)(Inital Assay); preincubation (Confirmatory Assay)
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 minutes
- Exposure duration:48±1 hours at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable.

SELECTION AGENT (mutation assays): not applicable

NUMBER OF REPLICATIONS: triplicate were used

DETERMINATION OF CYTOTOXICITY
- Method: Viability of the bacterial cells was checked.
- Any supplementary information relevant to cytotoxicity: The viability of each testing culture was determined by plating 0.1 mL of the 10E5, 10E6, 10E7 and 10E8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.
Evaluation criteria:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software. The study was considered valid if:

- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:
Statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test resultsopen allclose all
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
The following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
In the preliminary experiment, the number of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
No precipitate was detected on the plates in the preliminary experiment.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See attached table 5

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The viability of the bacterial cells was checked by a plating experiment in each test.

Any other information on results incl. tables

Table 2:Summary Table of the Range Finding Test

 

 

 

Concentrations (µg/plate)

Mean values ofrevertants/ Mutation factor (MF)

Salmonella typhimuriumtester strains

TA98

TA100

-S9

+S9

-S9

+S9

Untreated control

Mean

23.0

33.7

95.3

92.7

MF

0.87

0.98

1.05

0.98

Distilled water control

Mean

-

-

91.3

-

MF

-

-

1.00

-

 

DMSO control

Mean

25.0

25.3

-

96.3

MF

0.95

0.74

-

1.01

Distilled water 100µL control

Mean

26.3

34.3

91.0

95.0

MF

1.00

1.00

1.00

1.00

 

5000

Mean

23.0

21.0

68.3

69.3

MF

0.87

0.61

0.75

0.73

 

2500

Mean

23.3

28.0

61.3

70.3

MF

0.89

0.82

0.67

0.74

 

1000

Mean

28.7

25.0

65.0

76.3

MF

1.09

0.73

0.71

0.80

 

316

Mean

22.3

24.3

78.7

83.3

MF

0.85

0.71

0.86

0.88

 

100

Mean

21.7

26.0

75.0

94.3

MF

0.82

0.76

0.82

0.99

 

31.6

Mean

23.3

24.3

80.0

92.7

MF

0.89

0.71

0.88

0.98

 

10

Mean

22.0

27.0

81.3

96.3

MF

0.84

0.79

0.89

1.01

 

NPD (4mg)

Mean

402.0

-

-

-

MF

16.08

-

-

-

 

2AA (2mg)

Mean

-

2438.7

-

2397.3

MF

-

96.26

-

24.89

 

SAZ (2mg)

Mean

-

-

1201.3

-

MF

-

-

12.65

-


Table 3:Summary Table of the Initial Mutation Test

 

 

 

Concentrations

g/plate)

Mean values ofrevertants/ Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

20.7

30.7

95.3

107.3

8.7

12.0

9.3

8.7

32.0

34.7

MF

0.83

1.08

1.03

1.11

0.59

1.29

0.85

0.81

1.00

0.99

Distilled water control

Mean

-

-

96.3

-

12.3

-

-

-

30.3

-

MF

-

-

1.04

-

0.84

-

-

-

0.95

-

 

DMSO control

Mean

20.3

27.0

-

102.3

-

12.3

9.7

8.3

-

31.0

MF

0.81

0.95

-

1.05

-

1.32

0.88

0.78

-

0.89

Distilled water 100µL control

Mean

25.0

28.3

92.3

97.0

14.7

9.3

11.0

10.7

32.0

35.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

 

5000

Mean

20.7

24.3

79.3

103.7

13.0

9.7

10.0

10.0

31.3

36.3

MF

0.83

0.86

0.86

1.07

0.89

1.04

0.91

0.94

0.98

1.04

 

1581

Mean

19.3

25.3

95.7

113.0

10.7

10.3

9.7

9.0

30.7

36.7

MF

0.77

0.89

1.04

1.16

0.73

1.11

0.88

0.84

0.96

1.05

 

500

Mean

20.7

25.3

98.0

150.0

11.3

9.7

11.3

12.7

30.0

31.7

MF

0.83

0.89

1.06

1.55

0.77

1.04

1.03

1.19

0.94

0.90

 

158.1

Mean

20.7

23.3

82.7

114.3

10.7

11.3

13.0

13.0

45.0

59.3

MF

0.83

0.82

0.90

1.18

0.73

1.21

1.18

1.22

1.41

1.70

 

50

Mean

21.0

24.0

87.0

100.7

12.3

10.0

12.0

11.0

39.7

39.3

MF

0.84

0.85

0.94

1.04

0.84

1.07

1.09

1.03

1.24

1.12

 

15.81

Mean

21.3

21.0

79.0

100.0

13.3

9.3

9.7

11.7

38.0

40.7

MF

0.85

0.74

0.86

1.03

0.91

1.00

0.88

1.09

1.19

1.16

 

NPD (4mg)

Mean

414.0

-

-

-

-

-

-

-

-

-

MF

20.36

-

-

-

-

-

-

-

-

-

 

2AA (2mg)

Mean

-

2431.3

-

2361.3

-

208.7

-

194.7

-

-

MF

-

90.05

-

23.07

-

16.92

-

23.36

-

-

 

2AA (50mg)

Mean

-

-

-

-

-

-

-

-

-

193.7

MF

-

-

-

-

-

-

-

-

-

6.25

 

SAZ (2mg)

Mean

-

-

1192.0

-

1213.3

-

-

-

-

-

MF

-

-

12.37

-

98.38

-

-

-

-

-

 

9AA (50mg)

Mean

-

-

-

-

-

-

372.0

-

-

-

MF

-

-

-

-

-

-

38.48

-

-

-

 

MMS (2mL)

Mean

-

-

-

-

-

-

-

-

1034.0

-

MF

-

-

-

-

-

-

-

-

34.09

-


Table 4:Summary Table of the Confirmatory Mutation Test

 

 

 

Concentrations

g/plate)

Mean values ofrevertants/ Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

25.3

30.0

99.3

96.0

12.7

11.3

12.0

13.7

56.7

57.3

MF

0.84

1.03

0.99

0.96

1.09

0.97

0.90

0.98

0.98

0.99

Distilled water control

Mean

--

--

98.3

--

11.7

--

--

--

58.7

--

MF

--

--

0.98

--

1.00

--

--

--

1.02

--

 

DMSO control

Mean

25.3

28.3

--

95.7

--

11.7

12.3

11.7

--

55.3

MF

0.84

0.98

--

0.96

--

1.00

0.93

0.83

--

0.95

Distilled water 100µL control

Mean

30.0

29.0

100.0

99.7

11.7

11.7

13.3

14.0

57.7

58.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

 

5000

Mean

24.3

39.7

107.0

107.0

13.7

13.3

14.0

11.3

55.0

53.3

MF

0.81

1.37

1.07

1.07

1.17

1.14

1.05

0.81

0.95

0.92

 

1581

Mean

26.3

40.0

98.0

99.3

10.3

13.0

12.7

13.3

53.3

56.7

MF

0.88

1.38

0.98

1.00

0.89

1.11

0.95

0.95

0.92

0.98

 

500

Mean

33.3

30.3

96.0

106.0

10.7

15.3

12.7

12.7

58.0

53.3

MF

1.11

1.05

0.96

1.06

0.91

1.31

0.95

0.90

1.01

0.92

 

158.1

Mean

27.7

31.0

101.3

106.3

12.3

12.7

10.7

11.7

55.3

62.3

MF

0.92

1.07

1.01

1.07

1.06

1.09

0.80

0.83

0.96

1.07

 

50

Mean

28.7

29.7

99.7

102.3

14.7

12.0

12.7

12.0

54.7

54.0

MF

0.96

1.02

1.00

1.03

1.26

1.03

0.95

0.86

0.95

0.93

 

15.81

Mean

30.7

29.3

95.7

114.3

12.3

11.0

10.3

13.3

56.0

58.7

MF

1.02

1.01

0.96

1.15

1.06

0.94

0.78

0.95

0.97

1.01

 

5

Mean

31.7

30.3

92.0

109.3

10.0

9.7

9.7

12.7

55.0

55.0

MF

1.06

1.05

0.92

1.10

0.86

0.83

0.73

0.90

0.95

0.95

 

NPD (4mg)

Mean

401.3

--

--

--

--

--

--

--

--

--

MF

15.84

--

--

--

--

--

--

--

--

--

 

2AA (2mg)

Mean

--

2486.7

--

2416.0

--

228.0

--

203.3

--

--

MF

--

87.76

--

25.25

--

19.54

--

17.43

--

--

 

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

275.0

MF

--

--

--

--

--

--

--

--

--

4.97

 

SAZ (2mg)

Mean

--

--

1192.0

--

1185.3

--

--

--

--

--

MF

--

--

12.12

--

98.78

--

--

--

--

--

 

9AA (50mg)

Mean

--

--

--

--

--

--

400.7

--

--

--

MF

--

--

--

--

--

--

32.49

--

--

--

 

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1018.7

--

MF

--

--

--

--

--

--

--

--

17.36

--


Table 5 : Historical Control Data (Period of 2011-2015)


Untreated control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

22.7

103.4

11.4

6.9

33.7

30.0

111.3

11.3

8.7

39.8

St. dev.

5.8

22.9

5.0

3.2

10.3

6.8

20.6

3.7

3.6

10.2

Range

9-46

54-210

1-46

1-24

11-82

10-56

65-204

1-35

1-28

16-89

n

1108

1093

1101

1107

1104

1108

1095

1100

1110

1101

DMSO control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

21.5

98.6

11.6

7.1

32.6

29.1

109.4

11.3

8.7

39.0

St. dev.

5.8

22.3

4.9

3.3

10.1

7.1

21.8

3.6

3.6

10.1

Range

6-55

40-217

1-43

1-25

7-81

11-67

53-229

2-27

1-29

9-85

n

1183

1173

1179

1185

1182

1182

1170

1178

1185

1176

Distilled water control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

23.3

103.0

11.5

7.5

34.7

30.9

112.1

11.1

9.1

41.0

St. dev.

6.3

24.2

4.9

3.2

10.3

7.3

23.0

3.5

3.4

10.2

Range

11-45

45-215

2-47

2-24

12-84

10-53

64-222

3-39

1-20

17-91

n

201

1092

1101

201

1122

201

1101

1107

201

1116

DMF control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

20.3

89.2

11.1

6.8

35.3

28.4

99.6

10.9

7.9

38.6

St. dev.

5.6

18.0

4.8

3.0

12.5

7.0

19.2

3.5

3.0

10.2

Range

8-38

54-152

1-34

1-19

16-99

13-49

60-156

3-20

1-23

17-76

n

198

198

198

195

192

198

198

198

195

192

Acetone control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

22.6

97.6

11.9

7.2

35.9

29.3

108.9

11.2

8.4

41.7

St. dev.

5.2

16.1

6.0

2.9

9.8

6.8

14.5

3.4

3.1

9.1

Range

11-39

62-160

4-49

1-17

17-62

15-52

66-177

4-22

2-19

17-69

n

224

225

225

225

222

225

225

225

225

225

Positive reference control data

 

without metabolic activation (-S9 Mix)

with metabolic activation (+S9 Mix)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Mean

351.9

1263.9

1184.3

466.2

1054.1

2425.9

2445.4

238.0

223.1

267.3

St. dev.

124.9

211.5

217.0

186.0

144.8

346.2

316.9

150.5

61.0

123.4

Range

152-2336

880-2120

208-2440

149-2104

516-1708

312-4918

1192-5240

101-2216

117-838

125-2512

n

1108

1095

1101

1107

1107

1108

1095

1104

1110

1101

TA98:Salmonella typhimuriumTA98, TA100:Salmonella typhimuriumTA100, TA1535:Salmonella typhimuriumTA1535, TA1537:Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2uvrA;n: numberof cases

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the test item Sulfates of potassium, sodium and calcium, by- product from fermentation (Batch Number: EXP LIberica Lote: AI16503047) has no mutagenic activity on the growth of the bacterial strains used in this study.
Executive summary:

The purpose of this GLP study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/beta-naphthoflavone- induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test  item  was  dissolved  in  Distilled water at a concentration of 50 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations  in  the  Initial  Mutation  Test  were  5000,  1581,  500,  158.1,  50  and 15.81 μg/plate. In  the Confirmatory Mutation Test, they were 5000, 1581, 500, 158.1,  50, 15.81 and 5 μg/plate.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Tests and in the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Under the experimental conditions of the study, the test item Sulfates of potassium, sodium and calcium, by- product from fermentation (Batch Number: EXP LIberica Lote: AI16503047) has no mutagenic activity on the growth of the bacterial strains used in this study.

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