trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July 2006 to 25 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch: TZ 5604 BOP 01/06
Purity: Content of organic part (Na-salt): approx. 78 %
Oligomers: 13 %
Main component: approx. 48 %
Appearance: Solid, orange powder
Expiration date: February 01, 2011
Storage: At room temperature at about 20 °C in a desiccator.

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Before and after incubation, 10 µL aliquots of the test solutions at each pH value were analysed without dilution by measuring the UV signal of FAT 40826/A after HPLC separation of the injected sample solution.

Buffers:

Buffer pH 4, Biphthalate
Buffer pH 7, Phosphate
Buffer pH 9, Borate

Estimation method (if used):

The half-life period at 25 °C can be estimated using the Arrhenius equation.

Details on test conditions:

The test item was dissolved in an aqueous solution at a specific pH-value (pH 4.0, pH 7.0 and pH 9.0) and was incubated at a specific temperature with a maximum deviation of ± 0.5 °C and then kept constant within ± 0.1 °C . The concentration of the test item was determined as a function of time, using a suitable analytical method. In case of decreasing concentrations, the logarithms of the concentrations are plotted against time (log10 (ct). If the plot is a straight line, the reaction is considered to be of (pseudo-) first order. The rate constant and the half live time is then calculated using the slope. Otherwise, the rate constant can be estimated by the use of the Arrhenius relationship.

Preparation of test substances:

Hydrolysis at 50 °C:
- pH 4.0, Preliminary Test: 10.94 mg of FAT 40826/A were dissolved, by the aid of sonification, in 100 ml buffer solution (pH 4.0) to prepare a test solution of 109.4 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 4.0, Main Test: 11.98 mg of FAT 40826/A were dissolved in 100 ml buffer solution (pH 4.0) to prepare a test solution of 119.8 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 7.0, Preliminary test: 10.77 mg of FAT 40826/A were dissolved, by the aid of sonification, in 100 ml buffer solution (pH 7.0) to prepare a test solution of 107.7 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 7.0, Main Test: 11.22 mg of FAT 40826/A were dissolved in 100 ml buffer solution (pH 7.0) to prepare a test solution of 112.2 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 9.0, Preliminary and main test: 11.22 mg of FAT 40826/A were dissolved, by the aid of sonification, in 100 ml buffer solution (pH 9.0) to prepare a test solution of 112.2 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.

Hydrolysis at 60 °C:
- pH 4.0, Main Test: 17.62 mg of FAT 40826/A were dissolved in 100 ml buffer solution (pH 4.0) to prepare a test solution of 176.2 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 9.0, Main test: 16.46 mg of FAT 40826/A were dissolved, by the aid of sonification, in 100 ml buffer solution (pH 9.0) to prepare a test solution of 164.6 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.

Hydrolysis at 70 °C:
- pH 4.0, Main Test: 19.68 mg of FAT 40826/A were dissolved in 100 ml buffer solution (pH 4.0) to prepare a test solution of 196.82 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
- pH 9.0, Main test: 11.14 mg of FAT 40826/A were dissolved, by the aid of sonification, in 100 ml buffer solution (pH 9.0) to prepare a test solution of 111.4 µg/ml. Two aliquots of this test solution of approximately 50 ml each were transferred into 50 ml Erlenmeyer flasks in order to perform a duplicate test. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.

Number of replicates:

2

Preliminary study:

The results of the preliminary test show, that FAT 40826/A is not stable at pH 4.0. Therefore, the test at 50 °C was repeated in the range of 20-70 % of degradation. The results of the preliminary test show, that FAT 40826/A is not stable at pH 4.0. The results of pH 7.0 showed no significant degradation of FAT 40826/A at 50 °C. According to the EEC Directive 92/69, Section C.7, it can be concluded, that the estimated half-life time is higher than one year under representative environmental conditions (25 °C). Therefore, FAT 40826/A can be considered to be hydrolytically stable at pH 7.0 and no further testing was necessary at this pH-value. The results of the preliminary test show, that FAT 40826/A is not stable at pH 9.0. Therefore, the test at 50 °C was expanded to the range of 20 % to 70 % degradation.

Transformation products:

not measured

Details on hydrolysis and appearance of transformation product(s):

no data

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.023 h-1

DT50:

31 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 0.997

pH:

4

Temp.:

60 °C

Hydrolysis rate constant:

0.051 h-1

DT50:

14 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 0.999

pH:

4

Temp.:

70 °C

Hydrolysis rate constant:

0.101 h-1

DT50:

7 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 1.000

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.005 h-1

DT50:

137 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 0.997

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.026 h-1

DT50:

26 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 0.998

pH:

9

Temp.:

70 °C

Hydrolysis rate constant:

0.091 h-1

DT50:

8 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 0.998

Details on results:

The half-life time of the hydrolysis reaction of FAT 40826/A at 25 °C and pH 4.0 was calculated to be 254 hours (11 days).
The half-life time of the hydrolysis reaction of FAT 40826/A at 25 °C and pH 9.0 was calculated to be 8331 hours (347 days).

pH 4.0

The results of the preliminary test show, that FAT 40826/A is not stable at pH 4.0. Therefore, the test at 50 °C was repeated in the range of 20 % to 70 % degradation.

The decrease of the FAT 40826/A concentration at pH 4.0 and 50 °C (main test) for each duplicate sample. The linear plots prove that the hydrolysis reaction is of pseudo first order in the range from 20 % to 70 % hydrolysis at pH 4.0. The reaction rate constant k for pH 4.0 was calculated by regression analysis. The half-life time of FAT 40826/A at 50.0 °C and pH 4.0 was calculated to be 30 hours.

 

pH 7.0

The results of pH 7.0 showed no significant degradation of FAT 40826/A at 50 °C. According to the EEC Directive 92/69, Section C.7, it can be concluded, that the estimated half-life time is higher than one year under representative environmental conditions (25 °C). Therefore, FAT 40826/A can be considered to be hydrolytically stable at pH 7.0 and no further testing was necessary at this pH-value.

 

pH 9.0

The results of the preliminary test show, that FAT 40826/A is not stable at pH 9.0. Therefore, the test at 50 °C was expanded to the range of 20 % to 70 % degradation. The linear plots prove that the hydrolysis reaction is of pseudo first order in the range from 20 % to 70 % hydrolysis at pH 9.0. The reaction rate constant k for pH 9.0 was calculated by regression analysis. The half-life time of FAT 40826/A at 50.0 °C and pH 9.0 was calculated to be 137 hours (= 6 days).

 

HYDROLYSIS AT pH 4.0 AND DIFFERENT TEMPERATURES

Calculation of the Half-Life Time

The further hydrolysis tests at pH 4.0 were performed at 60 °C and 70 °C, each in duplicate. Each buffered test solution was analysed in time intervals. The obtained linear plot of each sample prove that the hydrolysis reaction is pseudo first order at 60 °C and 70 °C.

The half-life time at pH 4.0 and 60 °C was calculated to be 14 hours.

The half-life time at pH 4.0 and 70 °C was calculated to be 7 hours.

 

Evaluation of the Half-Life Time at 25 °C

The half-life time of the hydrolysis reaction of FAT 40826/A at 25 °C and pH 4.0 was calculated to be 254 hours (11 days).

 

HYDROLYSIS AT pH 9.0 AND DIFFERENT TEMPERATURES

Calculation of the Half-Life Time at pH 9.0

The further hydrolysis tests at pH 9.0 were performed at 60 °C and 70 °C, each in duplicate. Each buffered test solution was analysed in time intervals. The obtained linear plot of each sample prove that the hydrolysis reaction is pseudo first order at 60 °C and 70 °C. The reaction rate constant k was calculated for both temperatures by regression analysis. The half-life time at pH 9.0 and 60 °C was calculated to be 26 hours. The half-life time at pH 9.0 and 70 °C was calculated to be 8 hours.

 

Evaluation of the Half-Life Time at 25 °C

The half-life time of the hydrolysis reaction of FAT 40826/A at 25 °C and pH 9.0 was calculated to be 8331 hours (347 days).

Validity criteria fulfilled:

yes

Conclusions:

The test substance is stable at pH 7 and ambient temperatures
The half·life time of the hydrolysis reaction of the test substance at 25 °C and pH 4.0 was calculated to be 254 hours (11 days).
The half-life time of the hydrolysis reaction of the test substance at 25 °C and pH 9.0 was calculated to be 8331 hours (347 days).

Executive summary:

A hydrolysis test was performed following the OECD 111 guideline and EEC C7 guideline under GLP. The hydrolysis of the test item was performed at 50.2 °C ± 0.0 °C at each of pH 4.0, pH 7.0 and pH 9.0. The test substance was found to be stable at pH 7.0 and 50 °C. Therefore, no further testing was performed at this pH-value. The test substance was not stable at pH 4.0 and at pH 9.0, further testing was performed at elevated temperatures in order to calculate the rate constant (k25) and the half-life time of the hydrolysis at pH 7.0 and pH 9.0 at 25 °C. At pH 4.0, the test substance has a half-life time of 254 hours (11 days) at 25 °C. The test substance is stable at pH 7.0, its half-life lime is longer than one year at 25 °C. At pH 9.0, the test substance has a half-life time of 8331 hours (347 days) at 25 °C.

Description of key information

The test substance is hydrolytically stable at pH 7.

Key value for chemical safety assessment

Half-life for hydrolysis:

11 d

at the temperature of:

25 °C

Additional information

A hydrolysis test was performed following the OECD 111 guideline and EEC C7 guideline under GLP. The hydrolysis of the test item was performed at 50.2 °C ± 0.0 °C at each of pH 4.0, pH 7.0 and pH 9.0. The test substance was found to be stable at pH 7.0 and 50 °C . Therefore, no further testing was performed at this pH-value. The test substance was not stable at pH 4.0 and at pH 9.0, further testing was performed at elevated temperatures in order to calculate the rate constant (k25) and the half-life time of the hydrolysis at pH 7.0 and pH 9.0 at 25 °C. At pH 4.0, the test substance has a half-life time of 254 hours (11 days) at 25 °C. The test substance is stable at pH 7.0, its half-life lime is longer than one year at 25 °C. At pH 9.0, the test substance has a half-life time of 8331 hours (347 days) at 25 °C.