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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:
17 January 2012 to 6 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD and EU Guidelines.

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:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: 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 F08-0146
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. At pH 7, 50°C control samples were analysed only at the start and at the end of the experiment.
In the investigations at pH 9, at defined time intervals, the flasks were removed from the thermostat and filled up with acetonitrile. 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 to fit the calibrated range, then they were analysed using a HPLC method.
Buffers:
Buffer solutions:
pH 7.0: 148 ml 0.2 M Sodium hydroxide and 250 ml 0.2 M Potassium dihydrogen phosphate were diluted to 1000 ml with ultra-pure water
pH 9.0: 107 ml 0.2 M Sodium hydroxide, 250 ml 0.2 M Boric acid and 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.

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 6.3 with Phosphoric Acid.

Estimation method (if used):
None
Details on test conditions:
Hydrolysis was examined at two pH values: 7.0 and 9.0 in the dark.

Test temperatures:
pH 7: 25 +/- 0.5°C, 37 +/- 0.5 degrees C and 50 +/- 0.5degrees C
pH 9: 10 +/- 0.5°C, 25 +/- 0.5°C and 37 +/- 0.5 degrees 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 at pH 7 (25 and 37°C). This step was omitted in case of the other, very fats tests.

Test solutions: The test item was dissolved in the buffer solutions. Test item concentration in the buffer solutions was approximately 2-10 µg/ml. The pH of each buffer solution was checked with a calibrated pH meter. In order to ensure sterility test solutions at pH 7 (25 and 37°C) were filtered on 0.22 µm membrane filter. It was impossible in case of the other tests, because of the very fast reaction.

Storage of the solutions: For the experiment at pH 7, solutions were transferred into screw capped tubes. 24 such tubes were prepared and incubated at 25°C, 37°C and 50°C.
In the experiments at pH 9 the hydrolysis occurred very fast. Therefore, aliquots were measured into seven flasks and placed in the thermostats. There was no time to prepare replicates; therefore the whole experiment was repeated twice at 10°C and 25°C and three times at 37°C.

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.

Duration of testopen allclose all
Duration:
118 h
pH:
7
Initial conc. measured:
5.25 mg/L
Duration:
32 h
pH:
7
Initial conc. measured:
5.25 mg/L
Duration:
9 h
pH:
7
Initial conc. measured:
10.32 mg/L
Duration:
12 min
pH:
9
Initial conc. measured:
3.24 mg/L
Duration:
12 min
pH:
9
Initial conc. measured:
2.89 mg/L
Duration:
20 min
pH:
9
Initial conc. measured:
3.28 mg/L
Number of replicates:
Two or three replicate samples were analysed at each time point.
In the experiments at pH 9 the hydrolysis occurred very fast. Therefore, aliquots were measured into seven flasks and placed in the thermostats. There was no time to prepare replicates; therefore the whole experiment was repeated twice at 10°C and 25°C and three times at 37°C.
Positive controls:
no
Negative controls:
yes
Statistical methods:
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.

Results and discussion

Preliminary study:
Summary of the results of the preliminary test:
pH 4:                  Reactive Red F08-0146 proved to be hydrolytically stable
pH 7 and 9:         Significant decomposition was observed.
Test performance:
The test performed in accordance with the parameters specified in the test guidelines.
Transformation products:
yes
Identity of transformation productsopen allclose all
No.:
#1
Reference
Reference substance name:
Unnamed
IUPAC name:
see attachment
No.:
#2
Reference
Reference substance name:
Unnamed
IUPAC name:
see attachment
Total recovery of test substance (in %)open allclose all
% Recovery:
36
pH:
7
Temp.:
25 °C
Duration:
118 h
% Recovery:
33
pH:
7
Temp.:
37 °C
Duration:
32 h
% Recovery:
36
pH:
7
Temp.:
50 °C
Duration:
9 h
% Recovery:
12
pH:
9
Temp.:
10 °C
Duration:
12 min
% Recovery:
12
pH:
9
Temp.:
25 °C
Duration:
12 min
% Recovery:
10
pH:
9
Temp.:
37 °C
Duration:
20 min
Dissipation DT50 of parent compoundopen allclose all
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.009 h-1
DT50:
76 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.987
pH:
7
Temp.:
37 °C
Hydrolysis rate constant:
0.035 h-1
DT50:
20 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.986
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.122 h-1
DT50:
5.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.972
pH:
9
Temp.:
10 °C
Hydrolysis rate constant:
0.176 min-1
DT50:
4 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.998
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.177 min-1
DT50:
4 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.998
pH:
9
Temp.:
37 °C
Hydrolysis rate constant:
0.116 min-1
DT50:
6 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: regression coefficient = 0.976
Other kinetic parameters:
None
Details on results:
According to Sponsor’s experience, this hydrolysis product is most likely DYWJ 5653, for which no reference sample was available . The retention time of an other degradation product (9.2 min) confirms to the retention time of DYWJ 5660. Its amount corresponds to approximately 5-15% of the main hydrolysis product.

Any other information on results incl. tables

Measured data at pH 7

Temperature

Sampling time, hour

Measured concentration,

µg/ml (mean of three)

Hydrolysis rate, %

Measured pH

25°C

Start

5.25

-

7.02

24

4.58

13

7.02

31

4.15

21

7.02

46

3.64

31

7.03

70

3.10

41

7.03

94

2.21

58

7.02

118

1.87

64

7.02

37°C

Start

5.25

-

7.02

5

5.00

5

7.01

10

4.09

22

7.03

20

2.81

47

7.01

22

2.73

48

7.02

25

2.36

55

7.02

29

2.10

40

7.03

32

1.72

67

7.02

50°C

Start

10.32

-

7.01

2

9.58

7

7.01

3

8.67

16

7.02

4

7.40

28

7.01

5

6.41

38

7.03

6

5.87

43

7.03

7

4.83

53

7.02

8

4.25

59

7.00

9

3.71

64

7.01

Measured data at pH 9

Temperature

Sampling time, min

Measured concentration,

µg/ml

Hydrolysis rate, %

Measured pH

10°C

Start

3.24*

-

9.06

2

2.34*

28

-

4

1.63*

50

-

6

1.18*

64

-

8

0.86*

73

-

10

0.57*

82

-

12

0.38*

88

9.05

25°C

Start

2.89*

-

9.02

2

1.92*

34

-

4

1.45*

50

-

6

1.06*

63

-

8

0.69*

76

-

10

0.49*

83

-

12

0.34*

88

9.03

37°C

Start

3.28**

-

9.01

2

2.50**

24

-

4

1.79**

45

-

6

1.17**

64

-

10

0.78**

76

-

15

0.51**

84

-

20

0.32**

90

9.00

*    Mean of two experiments

**  Mean of three experiments

Calibration

The calibration series was prepared in acetonitrile : water (6:4). It was measured 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 the table below.

 

 Regression data

Analytical occasion

Intercept

Slope

Correlation Coefficient.

02 February 2012

-634

8200

0.999

03 February 2012

-588

8394

1.000

04 February 2012

-598

8557

0.999

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Summary of the results:

pH Temperature t1/2
7 25°C 76 h
37°C 20 h
50°C 5.7 h
9 10°C < 10 min
25°C < 10 min
37°C < 10 min

According to Sponsor’s experience, this hydrolysis product is most likely DYWJ 5653, for which no reference sample was available . The retention time of an other degradation product (9.2 min) confirms to the retention time of DYWJ 5660. Its amount corresponds to approximately 5-15% of the main hydrolysis product.
Executive summary:

In the course of the hydrolysis preliminary test(10/285-336ANE) performed at 50°C, Reactive Red F08‑0146 proved to be hydrolytically stable at pH 4; but significant decomposition was observed at pH7 and 9. Therefore the purpose of this study was to perform the hydrolysis main test and evaluate the abiotic degradation of Reactive Red F08-0146 at pH7 and 9 at different temperatures.

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.).

Summary of the results:

 

pH

Temperature

t1/2

7

25°C

76 h

37°C

20 h

50°C

5.7 h

9

10°C

< 10 min

25°C

< 10 min

37°C

< 10 min

According to Sponsor’s experience, this hydrolysis product is most likely DYWJ 5653, for which no reference sample was available . The retention time of an other degradation product (9.2 min) confirms to the retention time of DYWJ 5660. Its amount corresponds to approximately 5-15% of the main hydrolysis product.