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Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 November 2011 to 1 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU and OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
Name: Reactive Orange F08-0314
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
Sampling: The reaction solutions were analysed at the start of the test and after suitable reaction periods. During the two slower experiments (pH 7 – 25 and 37°C) at each analytical occasion three tubes of test solution and 1 tube of control buffer were removed from the thermostat and analysed.
In the other experiments, at defined time intervals, aliquots were taken from the three tubes of test solution for analysis and then the experiment continued with the same tubes. Control samples were taken and analysed at the start and at the end of the experiment.

Analysis of the samples: Samples were diluted with acetonitrile : water : phosphoric acid (200:800:0.1) to fit the calibrated range, then they were analysed with the HPLC method detailed below.

Sterility confirmation: At the end of the experiments at pH 7 – 25 and 37°C, three replicate samples of the test solutions were submitted for sterility confirmation. The replicate samples were combined before the sterility testing. Samples were investigated using liquid culture media and the inoculated tubes were incubated at 30°C for seven days. After the incubation period the tubes were evaluated for the growth of microorganisms.
Growth of microorganisms was not detected

In the other experiments sterility confirmation test was disregarded because of the fast reaction.
Buffers:
Buffer solutions:
pH 7.0: 443 ml 0.2 M Sodium hydroxide and 20.4 g Potassium dihydrogen phosphate were diluted to 3000 ml with ultra-pure water
pH 9.0: 107 ml 0.2 M Sodium hydroxide, 3.09 g Boric acid and 3.73 g Potassium chloride were diluted to 1000 ml with ultra-pure water
These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water.

Buffer solution for the eluent:
0.02 M Sodium dihydrogen phosphate with
0.005 M Disodium hydrogen phosphate
pH of this solution was adjusted to 5.0 with Phosphoric Acid.

Estimation method (if used):
None
Details on test conditions:
pH: Hydrolysis was examined at two pH values: 7.0 and 9.0 in the dark.
Buffer solutions:
pH 7.0: 443 ml 0.2 M Sodium hydroxide and 20.4 g Potassium dihydrogen phosphate were diluted to 3000 ml with ultra-pure water
pH 9.0: 107 ml 0.2 M Sodium hydroxide, 3.09 g Boric acid and 3.73 g Potassium chloride were diluted to 1000 ml with ultra-pure water
These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water.
The pH of each buffer solution was checked with a calibrated pH meter.
Test temperatures: pH 7: 25 +/- 0.5°C, 37 +/- 0.5°C and 50 +/- 0.5°C
pH 9: 10 +/- 0.5°C, 25 +/- 0.5°C and 37 +/- 0.5°C
Light: The hydrolysis reaction was carried out using dark thermostats to avoid photolytic effects.
Oxygen: Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.

The test item was dissolved in the buffer solutions. Test item concentration in the buffer solutions was 0.5 mg/ml. The pH of each buffer solution was checked with a calibrated pH meter. The test solutions were filtered on 0.22 µm membrane filter to ensure sterility

Storage of the solutions: For the experiment at pH 7, solutions were transferred into 25 ml tubes. 33 such tubes were prepared and incubated at 25 and 37°C. In all other experiments (pH 7 – 50°C, and all temperatures at pH 9) the hydrolysis occurred very fast. Due to this fact, it was only possible to prepare three replicate tubes. (There was no time to prepare more replicates.)
Duration of testopen allclose all
Duration:
192 h
pH:
7
Initial conc. measured:
510 mg/L
Duration:
192 h
pH:
7
Initial conc. measured:
518 mg/L
Duration:
192 h
pH:
7
Initial conc. measured:
505 mg/L
Duration:
24 h
pH:
9
Initial conc. measured:
476 mg/L
Duration:
5 h
pH:
9
Initial conc. measured:
466 mg/L
Duration:
1.5 h
pH:
9
Initial conc. measured:
497 mg/L
Number of replicates:
Three replicate samples were analysed at each time point.
Positive controls:
no
Negative controls:
no
Statistical methods:
Calculations were carried out using “EXCEL for Windows". The calibration curves were constructed with “STATISTICA for Windows" using weighted linear regression. The factor was 1/concentration.

Results and discussion

Preliminary study:
In the course of the hydrolysis preliminary test (10/296-336ANE) Reactive Orange F08-0314 proved to be hydrolytically stable at pH 4; but significant decomposition was observed at pH 7 and 9. Therefore the purpose of this study was to perform the hydrolysis main test and evaluate the abiotic degradation of Reactive Orange F08 0314 at pH 7 and 9.
Test performance:
The test performed in accordance with the parameters as specified by the guideline.
Transformation products:
yes
Identity of transformation productsopen allclose all
Details on hydrolysis and appearance of transformation product(s):
At the end of the study, solutions of the most likely degradation products, DYWJ5330 and DYWJ5362 (provided by the Sponsor) were also injected in the series of the test samples in order to confirm the identity of the degradation products.
Deviation of the retention time of the main hydrolysis product from the retention time of DYWJ 5362 (0.3 min) is within the variability of the HPLC method. Retention time of the second highest degradation product confirms to the retention time of DYWJ5330.


Total recovery of test substance (in %)open allclose all
% Recovery:
21
pH:
7
Temp.:
25 °C
Duration:
192 h
% Recovery:
14
pH:
7
Temp.:
37 °C
Duration:
192 h
% Recovery:
14
pH:
7
Temp.:
50 °C
Duration:
192 h
% Recovery:
9
pH:
9
Temp.:
10 °C
Duration:
24 h
% Recovery:
3
pH:
9
Temp.:
25 °C
Duration:
5 h
% Recovery:
3
pH:
9
Temp.:
37 °C
Duration:
1.5 h
Dissipation DT50 of parent compoundopen allclose all
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.008 h-1
DT50:
87 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
37 °C
Hydrolysis rate constant:
0.04 h-1
DT50:
18 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.163 h-1
DT50:
4.3 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
10 °C
Hydrolysis rate constant:
0.1 h-1
DT50:
7 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.749 h-1
DT50:
56 min
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
37 °C
Hydrolysis rate constant:
0.04 h-1
DT50:
17 min
Type:
(pseudo-)first order (= half-life)
Other kinetic parameters:
None
Details on results:
See tables under any other information.

Any other information on results incl. tables

Table 1.: Measured data at pH 7

Temperature

Sampling time, hour

Measured concentration,

µg/ml (mean of three)

Hydrolysis rate, %

Measured pH

25°C

Start

510

 

6.94

48

350

31

6.91

58

330

35

6.91

71

285

44

6.90

80

264

48

6.90

95.5

239

53

6.89

143

167

67

6.89

166

138

73

6.88

192

110

79

6.90

37°C

Start

518

-

6.90

8

422

19

6.90

10

402

22

6.90

12

377

27

6.91

14

354

32

6.90

23.5

230

56

6.92

30

181

65

6.89

36

137

73

6.88

51.5

73

86

6.86

50°C

Start

505

-

6.90

1

477

5

-

2

418

17

-

3

363

28

-

4

322

36

-

5

261

48

-

6

222

56

-

7

196

61

-

8

163

68

-

12

72

86

6.84

Table 2.: Measured data at pH 9

Temperature

Sampling timer

Measured concentration,

µg/ml (mean of three)

Hydrolysis rate, %

Measured pH

10°C

Start

476

-

8.99

2 h

385

19

-

4 h

309

35

-

6 h

256

46

-

8 h

210

56

-

10 h

173

64

-

12 h

142

70

-

14 h

117

75

-

24 h

43

91

8.96

25°C

Start

466

-

8.97

0.5 h

344

26

-

1 h

240

48

-

1.5 h

168

64

-

2 h

118

75

-

2.5 h

79

83

-

3 h

51

89

-

3.5 h

38

92

-

4 h

26

94

-

4.5 h

15

97

-

5 h

12

97

8.94

37°C

Start

497

-

8.98

10 min

393

21

-

20 min

297

40

-

30 min

218

56

-

40 min

147

70

-

50 min

98

80

-

60 min

59

88

-

70 min

38

92

-

80 min

23

95

-

90 min

15

97

8.96

 

In order to calculate the half lives of the reactions the log-transformed data of Reactive Orange F08-0314 concentrations against time were plotted. A line was fitted on the measured data and the rate constant and the half-live were obtained from its slope, according to equations 2 and 3. Results are summarised in the table below:

 

Table 3.: Rate constants and half-lives based on the measured data

pH

Temperature

Slope

kobs

t1/2

regression coefficient

7

25°C

-0.007949

0.007949

87 h

0.999

37°C

-0.03954

0.03954

18 h

0.993

50°C

-0.1628

0.1628

4.3 h

0.986

9

10°C

-0.09968

0.09968

7.0 h

1.000

25°C

-0.7491

0.7491

56 min

0.998

37°C

-0.04001

0.04001

17 min

0.988

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Two hydrolysis products of Reactive Orange F08-0314 were identified: DYWJ5330 and DYWJ5362 (IUPAC names see attachment).
Executive summary:

In the course of the hydrolysis preliminary test (10/296-336ANE) Reactive Orange F08-0314 proved to be hydrolytically stable at pH 4; but significant decomposition was observed at pH 7 and 9. Therefore the purpose of this study was to perform the hydrolysis main test and evaluate the abiotic degradation of Reactive Orange F08‑0314 at pH 7 and 9.

This study has been performed in accordance with the study plan,OECD Guidelines for Testing of Chemicals (No. 111),Commission Regulation: Methods for the Determination of Ecotoxicity (C.7.)and the Principles of Good Laboratory Practice (Hungarian GLP Regulations: 9/2001. (III. 30.) EüM-FVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENV/MC/CHEM (98) 17.).

Two hydrolysis products of Reactive Orange F08-0314 were identified: DYWJ5330 and DYWJ5362.

Summary of the results:

 

pH

Temperature

t1/2

7

25°C

87 h

37°C

18 h

50°C

4.3 h

9

10°C

7.0 h

25°C

56 min

37°C

17 min