Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 20 April 2016 Experimental completion date 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)
EC Number:
300-947-2
EC Name:
O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)
Cas Number:
93964-99-9
Molecular formula:
C8H19N.C8H19O3PS
IUPAC Name:
octan-1-amine dibutyl sulfanylphosphonate
impurity 1
Chemical structure
Reference substance name:
Octylamine
EC Number:
203-916-0
EC Name:
Octylamine
Cas Number:
111-86-4
Molecular formula:
C8H19N
IUPAC Name:
octan-1-amine
Test material form:
liquid
Specific details on test material used for the study:
Identification: X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9
Physical state/Appearance: Amber coloured viscous liquid
Batch: X-019575-00-00
Purity: >95% (treated as 100%)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in the dark

Method

Target gene:
Salmonella tuphimurium - histidine
Escherichia coli - tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 - rat liver homogenate metabolizing system
Test concentrations with justification for top dose:
Experiment 1
Dose selection: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 - determined by the results of Exoeriment 1
5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix: 2 µg/plate for WP2uvrA 3 µg/plate for TA100 5 µg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 Mix: 80 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 Mix: 0.2 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix: 1 µg/plate for TA100 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix: 5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation);



Rationale for test conditions:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1537, TA 98, TA 1535, 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:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.
The maximum dose level of the test item in the first experiment was initially selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level. Consequently the same maximum dose level was used in the second mutation test. The test item induced a slightly stronger toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100 and TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA100, TA1535 and TA98) and at 5000 µg/plate (WP2uvrA and TA1537). The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology (plate incorporation and pre-incubation). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Any other information on results incl. tables

Table1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

68

 

31

 

18

 

19

 

14

 

94

(80)

29

(30)

22

(19)

16

(18)

12

(13)

78

 

30

 

17

 

20

 

13

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

98

 

15

 

28

 

25

 

11

 

92

(94)

18

(19)

25

(25)

8

(16)

10

(9)

93

 

24

 

22

 

16

 

7

 

 

  Table2            Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 09 May 2016

To: 12 May 2016

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

75

67

66

(69)

4.9#

25

25

23

(24)

1.2

18

19

25

(21)

3.8

11

15

20

(15)

4.5

4

11

8

(8)

3.5

1.5 µg

76

74

75

(75)

1.0

30

20

21

(24)

5.5

28

24

16

(23)

6.1

18

14

20

(17)

3.1

6

5

6

(6)

0.6

5 µg

77

78

78

(78)

0.6

30

27

20

(26)

5.1

17

19

20

(19)

1.5

11

22

23

(19)

6.7

7

8

7

(7)

0.6

15 µg

84

76

82

(81)

4.2

25

21

29

(25)

4.0

16

16

13

(15)

1.7

12

16

18

(15)

3.1

7

11

10

(9)

2.1

50 µg

75

73

65

(71)

5.3

28

25

24

(26)

2.1

19

24

18

(20)

3.2

23

16

21

(20)

3.6

10

12

10

(11)

1.2

150 µg

67

82

68

(72)

8.4

25

28

25

(26)

1.7

27

19

17

(21)

5.3

21

20

18

(20)

1.5

9

9

10

(9)

0.6

500 µg

85

76

64

(75)

10.5

28

27

29

(28)

1.0

29

17

15

(20)

7.6

17

16

8

(14)

4.9

5

13

8

(9)

4.0

1500 µg

71

78

57

(69)

10.7

15

23

25

(21)

5.3

22

16

17

(18)

3.2

15

20

25

(20)

5.0

7

10

7

(8)

1.7

5000 µg

56 S

47 S

35 S

(46)

10.5

8 S

8 S

11 S

(9)

1.7

16

29

15

(20)

7.8

12 S

2 S

3 S

(6)

5.5

0 S

1 S

2 S

(1)

1.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

481

696

749

(642)

141.9

1180

1161

1058

(1133)

65.6

1179

1198

1127

(1168)

36.8

143

148

151

(147)

4.0

953

1167

818

(979)

176.0

 

ENNG4NQO9AAS#


ENNG         N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO          4-Nitroquinoline-1-oxide

9AA            9-Aminoacridine

S                Sparse bacterial background lawn

#                Standard deviation

                Table 3         Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 09 May 2016

To: 12 May 2016

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

67

77

66

(70)

6.1#

29

32

33

(31)

2.1

33

27

30

(30)

3.0

24

18

18

(20)

3.5

12

11

4

(9)

4.4

1.5 µg

66

63

67

(65)

2.1

24

25

26

(25)

1.0

23

21

24

(23)

1.5

14

15

15

(15)

0.6

6

6

10

(7)

2.3

5 µg

72

66

65

(68)

3.8

33

25

25

(28)

4.6

18

17

19

(18)

1.0

12

21

19

(17)

4.7

15

10

10

(12)

2.9

15 µg

61

62

69

(64)

4.4

23

24

34

(27)

6.1

19

23

19

(20)

2.3

22

15

17

(18)

3.6

8

9

9

(9)

0.6

50 µg

93

76

74

(81)

10.4

32

28

24

(28)

4.0

17

20

20

(19)

1.7

16

14

8

(13)

4.2

12

10

14

(12)

2.0

150 µg

78

76

70

(75)

4.2

24

31

25

(27)

3.8

17

30

27

(25)

6.8

24

16

25

(22)

4.9

7

17

14

(13)

5.1

500 µg

70

80

82

(77)

6.4

27

25

25

(26)

1.2

27

14

24

(22)

6.8

24

22

27

(24)

2.5

8

9

6

(8)

1.5

1500 µg

71

71

80

(74)

5.2

28

25

14

(22)

7.4

23

20

21

(21)

1.5

17

26

19

(21)

4.7

13

12

10

(12)

1.5

5000 µg

25 S

42 S

41 S

(36)

9.5

9 S

4 S

4 S

(6)

2.9

18

26

23

(22)

4.0

10 S

5 S

14 S

(10)

4.5

0 S

6 S

7 S

(4)

3.8

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1273

1226

952

(1150)

173.4

187

195

246

(209)

32.0

285

277

218

(260)

36.6

131

125

161

(139)

19.3

239

289

292

(273)

29.8

  Table4            Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 13 May 2016

To: 16 May 2016

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

102

91

92

(95)

6.1#

18

29

21

(23)

5.7

23

31

27

(27)

4.0

21

17

16

(18)

2.6

23

26

15

(21)

5.7

5 µg

93

116

99

(103)

11.9

19

21

20

(20)

1.0

21

31

14

(22)

8.5

15

15

15

(15)

0.0

16

26

21

(21)

5.0

15 µg

88

81

90

(86)

4.7

16

19

23

(19)

3.5

26

28

22

(25)

3.1

15

23

27

(22)

6.1

13

18

20

(17)

3.6

50 µg

91

85

92

(89)

3.8

18

16

22

(19)

3.1

37

40

25

(34)

7.9

11

18

20

(16)

4.7

17

24

21

(21)

3.5

150 µg

95

89

92

(92)

3.0

18

18

21

(19)

1.7

23

22

14

(20)

4.9

18

15

10

(14)

4.0

14

17

14

(15)

1.7

500 µg

69

76

79

(75)

5.1

17 S

13 S

12 S

(14)

2.6

22

28

16

(22)

6.0

12

18

15

(15)

3.0

4 S

12 S

9 S

(8)

4.0

1500 µg

63 S

51 S

57 S

(57)

6.0

8 S

13 S

13 S

(11)

2.9

24

12

9

(15)

7.9

16 S

9 S

4 S

(10)

6.0

6 S

3 S

6 S

(5)

1.7

5000 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

18 S

16 S

10 S

(15)

4.2

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

508

518

504

(510)

7.2

1178

1227

1218

(1208)

26.1

724

569

594

(629)

83.2

200

152

212

(188)

31.7

157

216

181

(185)

29.7

 

ENNG4NQO9AASV#


ENNG         N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO          4-Nitroquinoline-1-oxide

9AA            9-Aminoacridine

S                Sparse bacterial background lawn

V               Very weak bacterial background lawn

#                Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 13 May 2016

To: 16 May 2016

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

85

84

86

(85)

1.0#

26

24

15

(22)

5.9

27

25

26

(26)

1.0

19

31

18

(23)

7.2

17

13

14

(15)

2.1

5 µg

106

75

92

(91)

15.5

18

20

29

(22)

5.9

27

18

23

(23)

4.5

18

20

31

(23)

7.0

24

13

17

(18)

5.6

15 µg

76

101

81

(86)

13.2

19

17

32

(23)

8.1

31

23

22

(25)

4.9

18

29

21

(23)

5.7

13

5

7

(8)

4.2

50 µg

83

100

86

(90)

9.1

19

13

22

(18)

4.6

26

26

33

(28)

4.0

14

19

27

(20)

6.6

16

16

19

(17)

1.7

150 µg

92

89

92

(91)

1.7

21

25

23

(23)

2.0

31

28

22

(27)

4.6

20

22

30

(24)

5.3

12

8

12

(11)

2.3

500 µg

94

114

97

(102)

10.8

32

19

20

(24)

7.2

22

27

26

(25)

2.6

24

30

24

(26)

3.5

10

12

17

(13)

3.6

1500 µg

79 S

75 S

82 S

(79)

3.5

11 S

16 S

10 S

(12)

3.2

26

19

22

(22)

3.5

25 S

31 S

24 S

(27)

3.8

11

19

16

(15)

4.0

5000 µg

45 S

55 S

52 S

(51)

5.1

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

404

498

472

(458)

48.5

174

180

180

(178)

3.5

199

225

207

(210)

13.3

108

92

111

(104)

10.2

170

159

177

(169)

9.1

 

BP2AASV#


BP           Benzo(a)pyrene

2AA        2-Aminoanthracene

S                    Sparse bacterial background lawn

V                   Very weak bacterial background lawn

#            Standard deviation

Applicant's summary and conclusion

Conclusions:
X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9 was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The second experiment was performed at a later date (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was initially selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of theSalmonellatester strains at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted toEscherichia colistrain WP2uvrAat any test item dose level. Consequently the same maximum dose level was used in the second mutation test. The test item induced a slightly stronger toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100 and TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA100, TA1535 and TA98) and at 5000 µg/plate (WP2uvrAand TA1537). The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology (plate incorporation and pre-incubation). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Conclusion

X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9 was considered to be non-mutagenic under the conditions of this test.

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