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Environmental fate & pathways

Hydrolysis

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Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 May 2012 to 18 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study with GLP certificate conducted in accordance with OECD Guideline.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318
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:
Test solutions: The test item was dissolved in the buffer solutions. Test item concentration in the buffer solutions was approximately 100 µg/ml. The pH of each buffer solution was checked with a calibrated pH meter. In order to ensure sterility test solutions were filtered on 0.22 µm membrane filter.

Sampling: The reaction solutions were analysed at the start of the test and after suitable reaction periods. At each analytical occasion three tubes of test solution and 1 tube of control buffer were removed from the thermostat and analysed.

Analysis of the samples: Samples were diluted with eluent buffer to fit the calibrated range, then they were analysed with the HPLC method described below.

Sterility confirmation: At the end of the experiments 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
Buffers:
Buffer solution for the eluent: 45 mM Sodium dihydrogen phosphate with 5 mM Disodium hydrogen phosphate.

Buffer solutions:
pH 4.0: 4 ml 0.2 M Sodium hydroxide and 500 ml 0.2 M Potassium hydrogen phtalate were diluted to 2000 ml with ultra-pure water
pH 7.0: 295.6 ml 0.2 M Sodium hydroxide and 500 ml 0.2 M Potassium dihydrogen phosphate were diluted to 2000 ml with ultra-pure water
pH 9.0: 214 ml 0.2 M Sodium hydroxide, 500 ml 0.2 M Boric acid and Potassium chloride were diluted to 2000 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.

Estimation method (if used):
None
Details on test conditions:
TEST CONDITIONS

pH: Hydrolysis was examined at three pH values: 4.0, 7.0 and 9.0 in the dark.
Buffer solutions:
pH 4.0 :4 ml 0.2 M Sodium hydroxide and 500 ml 0.2 M Potassium hydrogen phtalate were diluted to 2000 ml with ultra-pure water
pH 7.0: 295.6 ml 0.2 M Sodium hydroxide and 500 ml 0.2 M Potassium dihydrogen phosphate were diluted to 2000 ml with ultra-pure water
pH 9.0: 214 ml 0.2 M Sodium hydroxide, 500 ml 0.2 M Boric acid and Potassium chloride were diluted to 2000 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: 25 +/- 0.5°C, 37 +/- 0.5°C and 50 +/- 0.5°C
Light: The hydrolysis reaction was carried out using dark thermostats to avoid photolytic effects.
Oxygen: In order to exclude oxygen, nitrogen was bubbled into the water for five minutes before the preparation of the solutions.
Duration of testopen allclose all
Duration:
192 h
pH:
4
Initial conc. measured:
103 500 µg/L
Duration:
71 h
pH:
4
Initial conc. measured:
113 000 µg/L
Duration:
23 h
pH:
4
Initial conc. measured:
107 000 µg/L
Duration:
289 h
pH:
7
Initial conc. measured:
114 000 µg/L
Duration:
71 h
pH:
7
Initial conc. measured:
127 000 µg/L
Duration:
23 h
pH:
7
Initial conc. measured:
109 200 µg/L
Duration:
5 h
pH:
9
Initial conc. measured:
99 800 µg/L
Duration:
2 h
pH:
9
Initial conc. measured:
113 200 µg/L
Duration:
1.75 h
pH:
9
Initial conc. measured:
108 900 µg/L
Number of replicates:
At each analytical occasion three tubes of test solution and 1 tube of control buffer were removed from the thermostat and analysed
Positive controls:
no
Negative controls:
yes
Statistical methods:
Evaluation: The chromatograms were evaluated with the help of “LaChrom Chromatogram Processor".
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.
The rate constant (kobs) for each pH value and both temperatures of the tests were determined from the plots of the logarithms of the concentration versus time.

Results and discussion

Preliminary study:
In the course of the preliminary test significant decomposition of REACTIVE RED F03-0318 was observed at pH 4, 7 and 9; less than 2% of the original concentration was measured after 120 hours. See Hydrolysis supporting study for details.
Test performance:
The test was considered to be valid - see results of method validation below.
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, DYKJ 5305, DYDJ 5310, DYDJ 5312 and DYDJ 5313 (provided by the Sponsor) were also injected in the series of the test samples in order to confirm the identity of the degradation products.
At pH 7 and pH 9 retention time of the main hydrolysis product (11.0 min) significantly deviates from the retention time of the above standards. Its amount corresponds to approximately 50-77% of the main hydrolysis product.
According to Sponsor’s experience and according to the Certificate of Analysis, this hydrolysis product is most likely the vinylated form of the dye, for which no reference sample was available. This compound is the intended hydrolysis product and the virtual dyeing component during the dyeing process, which takes place at alkaline pH at about 60°C.
Main component of the provided standards elutes at different retention times: DYDJ 5313 (7.4 min), DYDJ 5310 (8.6 min), DYKJ 5305 (8.9 min) DYDJ 5312 (12.1 min). Therefore, identity of the main hydrolysis product was not supported by this experiment.
However, the retention time of a main hydrolysis product at pH 4 confirms to the retention time of DYDJ 5310. Its amount corresponds to approximately 65-74% of the main hydrolysis product.
The structures of the main hydrolysis products are attached under background material.
Total recovery of test substance (in %)open allclose all
% Recovery:
21
pH:
4
Temp.:
25 °C
Duration:
192 h
% Recovery:
11
pH:
4
Temp.:
37 °C
Duration:
71 h
% Recovery:
24
pH:
4
Temp.:
50 °C
Duration:
23 h
% Recovery:
34
pH:
7
Temp.:
25 °C
Duration:
289 h
% Recovery:
15
pH:
7
Temp.:
37 °C
Duration:
71 h
% Recovery:
14
pH:
7
Temp.:
50 °C
Duration:
23 h
% Recovery:
28
pH:
9
Temp.:
25 °C
Duration:
5 h
% Recovery:
15
pH:
9
Temp.:
37 °C
Duration:
2 h
% Recovery:
12
pH:
9
Temp.:
50 °C
Duration:
1.75 h
Dissipation DT50 of parent compoundopen allclose all
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.009 h-1
DT50:
79 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.995
pH:
4
Temp.:
37 °C
Hydrolysis rate constant:
0.031 h-1
DT50:
22 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.995
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0.064 h-1
DT50:
11 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.988
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.004 h-1
DT50:
176 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.989
pH:
7
Temp.:
37 °C
Hydrolysis rate constant:
0.028 h-1
DT50:
25 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.994
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.088 h-1
DT50:
8 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.975
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.259 h-1
DT50:
2.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.999
pH:
9
Temp.:
37 °C
Hydrolysis rate constant:
0.976 h-1
DT50:
0.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.963
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
1.213 h-1
DT50:
0.6 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Regression coefficient = 0.930
Other kinetic parameters:
None
Details on results:
See below for results of the method validation.

At the end of the study, solutions of the most likely degradation products, DYKJ 5305, DYDJ 5310, DYDJ 5312 and DYDJ 5313 (provided by the Sponsor) were also injected in the series of the test samples in order to confirm the identity of the degradation products.
At pH 7 and pH 9 retention time of the main hydrolysis product (11.0 min) significantly deviates from the retention time of the above standards. Its amount corresponds to approximately 50-77% of the main hydrolysis product.
According to Sponsor’s experience and according to the Certificate of Analysis, this hydrolysis product is most likely the vinylated form of the dye, for which no reference sample was available. This compound is the intended hydrolysis product and the virtual dyeing component during the dyeing process, which takes place at alkaline pH at about 60°C.
Main component of the provided standards elutes at different retention times: DYDJ 5313 (7.4 min), DYDJ 5310 (8.6 min), DYKJ 5305 (8.9 min) DYDJ 5312 (12.1 min). Therefore, identity of the main hydrolysis product was not supported by this experiment.
However, the retention time of a main hydrolysis product at pH 4 confirms to the retention time of DYDJ 5310. Its amount corresponds to approximately 65-74% of the main hydrolysis product.


Any other information on results incl. tables

Table 1.: Results of the Method Validation (11/008-316AN)

Selectivity

No interfering component was observed

Reinjection repeatability
(11 injections)

Coefficient of Variation<2%

Linear range

0.1 - 10 µg/ml

Limit of Quantification

0.1µg/ml

Recovery frombuffer solutions

94 – 103%

Stability of the samples

At least 22 hours in the autosampler

Stock solution stability

At least 14 days at 5±3°C, in the dark

The calibration series was prepared in eluent buffer.It wasmeasured at each analytical occasion.Concentrations of the calibration samples were 0.1, 0.2, 0.5, 1, 2, 5 and 10 µg/ml. Parameters of three representative equations are given in Table 2.

 

Table 2: Regression data

Analytical occasion

Intercept

Slope

Correlation Coefficient.

04 June 2012

-187

10044

1.000

06 June 2012

-211

10346

1.000

07 June 2012

-52

9980

1.000

08 June 2012

-79

9519

1.000

09 June 2012

-121

10390

1.000

10 June 2012

-227

9665

1.000

11 June 2012

-59

9443

1.000

12 June 2012

74

10271

1.000

13 June 2012

53

9908

1.000

14 June 2012

55

9433

1.000

18 June 2012

334

10051

0.999

Table 3: Measured data at pH 4

Temperature

Sampling time, hour

Measured concentration,

µg/ml (mean of three)

Hydrolysis rate, %

Measured pH

25°C

Start

103.5

-

4.03

24

89.9

13

4.03

29

85.1

18

4.02

46

77.2

25

4.03

72

59.2

43

4.05

96

49.9

52

4.05

120

40.1

61

4.05

144

30.3

71

4.05

192

21.5

79

4.05

37°C

Start

113

-

4.01

10

94.0

17

4.02

22

57.7

49

4.03

26

50.2

56

4.03

34

43.0

62

4.03

46

26.9

76

 

53

23.5

79

4.02

71

12.9

89

4.03

50°C

Start

107

-

4.05

4

89.9

16

4.04

6

81.2

24

4.03

8

75.0

30

4.03

10

60.6

43

4.03

12

54.3

49

4.02

14

49.2

54

4.03

23

25.4

76

4.02

Table 4: Measured data at pH 7

Temperature

Sampling time, hour

Measured concentration,

µg/ml

Hydrolysis rate, %

Measured pH

25°C

Start

114

-

6.97

46

100

12

7.03

72

87.9

23

7.02

96

82.0

28

7.05

144

65.4

43

7.04

192

59.9

48

7.04

289

36.9

68

7.04

37°C

Start

127

-

7.02

10

105

18

7.03

22

75.9

40

7.03

26

58.4

54

7.03

34

52.5

59

7.03

46

35.8

72

7.05

53

30.9

76

7.02

71

18.7

85

7.03

50°C

Start

109.2

-

7.02

4

90.7

17

7.01

6

78.7

28

7.03

8

71.6

34

7.02

10

56.8

48

7.02

12

44.8

59

7.02

14

40.3

63

7.04

23

14.9

86

7.03

Table 5: Measured data at pH 9

Temperature

Sampling time, hour

Measured concentration,

µg/ml

Hydrolysis rate, %

Measured pH

25°C

Start

99.8

-

9.05

1

79.0

21

9.04

2

60.8

39

9.05

2.5

54.3

46

9.04

3

46.4

54

9.05

3.5

40.9

59

9.05

4

36.3

64

9.05

4.5

31.7

68

9.02

5

27.6

72

9.04

37°C

Start

113.2

-

9.06

0.5

87.6

23

9.05

0.75

74.5

34

9.06

1

58.1

49

9.05

1.25

47.5

58

9.06

1.5

32.1

72

9.05

1.75

23.4

79

9.07

2

17.0

85

9.05

50°C

Start

108.9

-

8.97

0.25

90.2

17

8.96

0.75

65.6

40

8.94

1

50.8

53

8.96

1.25

35.0

68

8.98

1.5

16.9

84

8.96

1.75

13.4

88

8.97

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
At the end of the study, solutions of the most likely degradation products, DYDJ 5305, DYDJ 5310, DYDJ 5312 and DYDJ 5313 (provided by the Sponsor) were also injected in the series of the test samples in order to confirm the identity of the degradation products.
In the course of the hydrolysis preliminary test (11/008-336ANE) performed at 50°C, significant decomposition was observed at pH 4,7 and 9. Therefore the purpose of this study was to perform the hydrolysis main test and evaluate the abiotic degradation of Reactive Red F03-0318 at pH 4,7 and 9 at different temperatures.
At pH 7 and pH 9 retention time of the main hydrolysis product (11.0 min) significantly deviates from the retention time of the above standards. Its amount corresponds to approximately 50-77% of the main hydrolysis product. Main component of the provided standards elutes at different retention times: DYDJ 5313 (7.4 min), DYDJ 5310 (8.6 min), DYDJ 5305 (8.9 min) DYDJ 5312 (12.1 min). Therefore, identity of the main hydrolysis product was not supported by this experiment. However, the retention time of a main hydrolysis product at pH 4 confirms to the retention time of DYDJ 5310. Its amount corresponds to approximately 65-74% of the main hydrolysis product.
Executive summary:

In the course of the hydrolysis preliminary test (11/008-336ANE) performed at 50°C, significant decomposition was observed at pH 4,7 and 9. Therefore the purpose of this study was to perform the hydrolysis main test and evaluate the abiotic degradation of Reactive Red F03-0318 at pH 4,7 and 9 at different temperatures.

Rate constants and half-lives based on the measured data

pH

Temperature

Slope

kobs

t1/2

regression coefficient

4

25°C

-0.0038

0.0088

79 h

0.995

37°C

-0.0135

0.0311

22 h

0.995

50°C

-0.0277

0.0637

11 h

0.988

7

25°C

-0.0017

0.0039

176 h

0.989

37°C

-0.0120

0.0276

25 h

0.994

50°C

-0.0384

0.0885

8 h

0.975

9

25°C

-0.1122

0.2585

2.7 h

0.999

37°C

-0.4239

0.9764

0.7 h

0.963

50°C

-0.5268

1.2133

0.6 h

0.930

 

At the end of the study, solutions of the most likely degradation products, DYDJ 5305, DYDJ 5310, DYDJ 5312 and DYDJ 5313 (provided by the Sponsor) were also injected in the series of the test samples in order to confirm the identity of the degradation products.

At pH 7 and pH 9 retention time of the main hydrolysis product (11.0 min) significantly deviates from the retention time of the above standards. Its amount corresponds to approximately 50-77% of the main hydrolysis product. Main component of the provided standards elutes at different retention times: DYDJ 5313 (7.4 min), DYDJ 5310 (8.6 min), DYDJ 5305 (8.9 min) DYDJ 5312 (12.1 min). Therefore, identity of the main hydrolysis product was not supported by this experiment. However, the retention time of a main hydrolysis product at pH 4 confirms to the retention time of DYDJ 5310. Its amount corresponds to approximately 65-74% of the main hydrolysis product.