Alkali salt of substituted alkyl amino sulfonyl triazinyl amino arylamido diazo aryl sulfonyl sulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-07-20 to 2012-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Test item concentration was measured at the start and during the test as follows:

After certain time periods, sample tubes (1 control + 3 sample solutions in buffer) were removed from the incubators/oven and pH of the solutions was measured by a calibrated pH meter with a precision of 0.01.

The test item concentration of the solutions was measured by the previously validated HPLC method. Control samples were subjected to HPLC analysis without dilution, however the other samples were diluted 100 fold with the dilution mixture.

Two injections were made from each sample tube.

At the end of the hydrolysis test, sterility confirmation tests were conducted at each pH. All tests confirmed that the solutions were sterile.

Buffers:

Buffer solution for the preparation of 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.

Preparation of test item solution in pH 9.0 buffer (1 mg/mL):
(Date: 26 July 2012)

For 2 L of pH 9.0 buffer solution: 214 ml 0.2 M Sodium hydroxide and 500 ml 0.2 M Boric acid/Potassium chloride solution were diluted to 2 L with ultrapure water.

3.00 g of test item was dissolved in 3 L of pH 9.0 buffer solution using ultrasonic bath. Nitrogen was bubbled into the solution and the solution was filtered through a 0.22 µm sieve.

This solution was diluted to 100 fold with the dilution mixture (3x25 mL diluted solutions were made). These solutions were used in the hydrolysis test.

Preparation of test item solution in pH 4.0 buffer (1 mg/mL):
(Date: 26 July 2012)

For 2 L of pH 4.0 buffer solution: 500 ml of 0.2 M Potassium-hydrogen-phtalate and 4 ml of 0.2 M sodium hydroxide solution were diluted to 2 L with ultrapure water.

The test item was dissolved in the buffer solution as it was described at pH 9.0.

Preparation of test item solution in pH 7.0 buffer (1 mg/mL):
(Date: 27 July 2012)

For 2 L of pH 4.0 buffer solution: 500 ml of 0.2 M Potassium-hydrogen-phtalate and 295.6 ml of 0.2 M sodium hydroxide solution were diluted to 2 L with ultrapure water.

The test item was dissolved in the buffer solution as it was described at pH 9.0.

Estimation method (if used):

None

Details on test conditions:

Test conditions

Test temperatures: 25 +/- 0.5°C, 37 +/- 0.5°C, 50 +/- 0.5°C

pH: Hydrolysis was examined at pH 4.0, 7.0 and 9.0 in the dark. The pH of each buffer solution was checked with a calibrated pH meter.

Light: The hydrolysis reaction was carried out using a dark thermostat 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.

Sterile conditions: All glassware and other laboratory ware made of plastic material were sterilized before usage. The test item solutions in buffers were filtered through a 0.22 µm membrane filter and filled into glass vials under laminar box to ensure sterile conditions.

Duration:

99.5 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

1 138 mg/L

Duration:

312.5 h

pH:

9

Temp.:

37 °C

Initial conc. measured:

1 138 mg/L

Duration:

768.5 h

pH:

9

Temp.:

25 °C

Initial conc. measured:

1 138 mg/L

Duration:

432 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

1 146 mg/L

Duration:

744 h

pH:

7

Temp.:

37 °C

Initial conc. measured:

1 146 mg/L

Duration:

744 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

1 146 mg/L

Duration:

97 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

998 mg/L

Duration:

310 h

pH:

4

Temp.:

37 °C

Initial conc. measured:

998 mg/L

Duration:

766 h

pH:

4

Temp.:

25 °C

Initial conc. measured:

998 mg/L

Number of replicates:

1 control + 3 sample solutions in buffer

Positive controls:

no

Negative controls:

yes

Statistical methods:

The calibration graph was calculated applying weighted linear regression using 1/concentration as weighting factor

Preliminary study:

The purpose of this study was to evaluate the hydrolysis of Reactive Yellow F01-0555 in solutions of pH 4, pH 7 and pH 9.
 
In the course of this preliminary test the test item concentration significantly decreased.

The end/start concentration of Reactive Yellow F01-0555 in the hydrolysis mixture was

-                     20% at pH 4.0,
-                     69% at pH 7.0 and
-                     4% at pH 9.0

after 5 days of incubation at 50 ±1 °C.

Test performance:

The test was considered to be valid - see results of method validation below.

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

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

The hydrolysis reaction of Reactive Yellow F01-0555 follows pseudo-first order kinetics.

IDENTIFICATION OF HYDROLYSIS PRODUCTS

Solutions of the most likely degradation products, DYWJ 5911, DYWJ 5912, DYWJ 5913, DYWJ 5916, DYWJ 5968, DYWJ 5985, and DYWJ 6013 (provided by the Sponsor) were injected in the series of the test samples in order to confirm the identity of the degradation products.

The identification of the possible degradation products is difficult, especially in case of components eluting with retention time less than 2.5 minutes. It is due to the fact that the reference materials are not pure compounds but mixtures of several components.
For the identification of the degradation products, the main peaks corresponding to the main components of the reference substances were considered. The total area of the main peaks considered for identification (2-4 peaks for each reference substance) varied between 66-89% relative to the total peak area of the reference substance chromatograms.
For the identification of the degradation products during hydrolysis, the chromatograms of the last sample taken from the pH 4, 7 and 9 buffers incubated at 50 °C were evaluated. Please see table below under any other information on results for details of the retention times of component peaks and possible degradation products of Reactive Yellow F01-0555 test item in pH4, 7 and 9 buffers.

% Recovery:

10

pH:

9

Temp.:

50 °C

Duration:

99.5 h

% Recovery:

26

pH:

9

Temp.:

37 °C

Duration:

312.5 h

% Recovery:

38

pH:

9

Temp.:

25 °C

Duration:

768.5 h

% Recovery:

41

pH:

7

Temp.:

50 °C

Duration:

432 h

% Recovery:

48

pH:

7

Temp.:

37 °C

Duration:

744 h

% Recovery:

58

pH:

7

Temp.:

25 °C

Duration:

744 h

% Recovery:

21

pH:

4

Temp.:

50 °C

Duration:

97 h

% Recovery:

20

pH:

4

Temp.:

37 °C

Duration:

310 h

% Recovery:

26

pH:

4

Temp.:

25 °C

Duration:

766 h

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.023 h-1

DT50:

30 h

Type:

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

Remarks on result:

other: Slope: 0.02301 Intercept: 0.0551 r2=0.9964

pH:

9

Temp.:

37 °C

Hydrolysis rate constant:

0.004 h-1

DT50:

173 h

Type:

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

Remarks on result:

other: Slope: 0.00400 Intercept: 0.1152 r2=0.9833

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

566 h

Type:

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

Remarks on result:

other: Slope: 0.00122 Intercept: 0.0556 r2=0.9925

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.002 h-1

DT50:

376 h

Type:

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

Remarks on result:

other: Slope: 0.00184 Intercept: 0.1540 r2=0.9405

pH:

7

Temp.:

37 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

777 h

Type:

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

Remarks on result:

other: Slope: 0.00089 Intercept: 0.0766 r2=0.9716

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

985 h

Type:

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

Remarks on result:

other: Slope: 0.00070 Intercept: 0.0449 r2=0.9824

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.017 h-1

DT50:

42 h

Type:

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

Remarks on result:

other: Slope: 0.01659 Intercept: -0.0166 r2=0.9953

pH:

4

Temp.:

37 °C

Hydrolysis rate constant:

0.005 h-1

DT50:

129 h

Type:

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

Remarks on result:

other: Slope: 0.00538 Intercept: -0.0572 r2=0.9972

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.002 h-1

DT50:

374 h

Type:

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

Remarks on result:

other: Slope: 0.00185 Intercept: -0.0572 r2=0.9941

Other kinetic parameters:

None

Details on results:

Please see table below under any other information on results for details of the retention times of component peaks and possible degradation products of Reactive Yellow F01-0555 test item in pH4, 7 and 9 buffers. The structures and molecular formula of the possible degradation products DYWJ5911, DYWJ5916, DYWJ5912 and DYWJ5913 are detailed in the certificate of analysis attached to Section 1.4 of the dossier. The structure and molecular formula of degradation product DYGG 6013 is detailed in the pdf attached under background material below.

Results of the Method Validation (11/174-316AN)

Selectivity	No interference was observed.
Reinjection repeatability (7 injections)	Coefficient of Variation ≤ 0.6%
Linear range	0.5 - 50 µg/mL
Limit of Quantification	0.5 µg/mL
Recovery fromaqueous test media	100%, 103%    (1, 100 g/L in ultrapure water) 85%, 103%      (pH 4, 7 buffer solutions) 107%               (pH 9 buffer solutions)
Stability of the samples	At least 29 hours in the autosampler
Stock solution stability	At least 6 days at 5±3°C

Retention time of component peaks and possible degradation products of Reactive Yellow F01-0555 test item in pH 4, 7 and 9 buffers

Retention time of component peaks	Possible degradation product
pH 9, 50 °C
~1.3 min	DYWJ5913 (DYWJ5912)*
~1.8 min	DYGG6013
~2.8 min	no specific reference substance can be identified
~4.0 min	DYWJ5911
~6.6 min	Specific reference substance cannot be identified
~6.9 min	DYWJ5916
~8.7 min	Reactive Yellow F01-0555
pH 7, 50 °C
~1.3 min	DYWJ5913 (DYWJ5912)*
~1.8 min	DYGG6013
~6.7 min	Specific reference substance cannot be identified
~6.9 min	DYWJ5916
~8.7 min	Reactive Yellow F01-0555
pH 4, 50 °C
~1.3 min	DYWJ5912 (DYWJ5913)*
~1.8 min	DYGG6013
~3.25min	Reactive Yellow F01-0555
~4.0 min	DYWJ5911

 

*The reference substance in brackets might be a degradation product during the hydrolysis reaction, but it cannot be determined precisely.

Validity criteria fulfilled:

yes

Conclusions:

The hydrolysis reaction of Reactive Yellow F01-0555 follows pseudo-first order kinetics.

The half-life time of the test item varies with the pH: at the same temperature
t0.5 (pH 9.0) < t0.5 (pH 4.0) < t0.5 (pH 7.0).
The degradation products were identified.

Executive summary:

The hydrolysis reaction of Reactive Yellow F01-0555 follows pseudo-first order kinetics.

 

The half-life time of the test item varies with the pH: at the same temperature

t_0.5(pH 9.0) <t_0.5(pH 4.0) <t_0.5(pH 7.0).

 

The results of rate constant, half-life time and activation energy calculation are summarized below:

 

Test condition	kobs,1/h	t0.5	E activation energy
pH 9.0; 50°C	0.02301	30 h (1.25 days)	94006 J/mol
pH 9.0; 37°C	0.00400	173 h (7.2 days)
pH 9.0; 25°C	0.00122	566 h (23.6 days)
pH 7.0; 50°C	0.00184	376 h (15.7 days)	30837 J/mol
pH 7.0; 37°C	0.00089	777 h (32.4 days)
pH 7.0; 25°C	0.00070	985 h (41 days)
pH 4.0; 50°C	0.01659	42 h (1.75 days)	70247 J/mol
pH 4.0; 37°C	0.00538	129 h (5.4 days)
pH 4.0; 25°C	0.00185	374 h (15.6 days)

The chromatograms of the reference substances were evaluated and compared to the chromatograms of the last samples taken from the incubated test item solutions in buffers. The degradation products were identified 

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2012-03-12 to 2012-06-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in accordance with OECD and EU Guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The reaction solutions were analysed at the start and at the end of the test.

pH measurement
Before the start of the experiment, the pH of the control samples and the buffer solutions containing the test item was measured.
At the end of the hydrolysis experiment, the pH of the solutions in each vials was measured and recorded

At the start of the hydrolysis experiment, one sample was withdrawn from each control vial and five samples were taken from each of the three buffer solutions containing the test item prior to the addition of the solutions into vials.
At the end of the hydrolysis experiment, one sample was withdrawn from each vial.

All samples were subjected to HPLC analysis to determine the Reactive Yellow F01-0555 content of the samples. Control samples were measured directly without dilution. The samples containing the test item were diluted to 100 fold using the dilution mixture.

Buffers:

Buffer solution for the preparation of 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.

pH 4.0 buffer solution: Preparation of 1000 mL solution: 0.2 M Potassium hydrogen phthalate (250 mL) with 0.2 M Sodium-hydroxide (2 mL) diluted with sterile ultrapure water.
pH 7.0 buffer solution: Preparation of 1000 mL solution: 0.2 M Potassium hydrogen phosphate (250 mL) with 0.2 M Sodium-hydroxide (147.8 mL) diluted with sterile ultrapure water.
pH 9.0 buffer solution: Preparation of 1000 mL solution: 0.2 M Boric acid (250 mL) with 0.2 M Sodium-hydroxide (107 mL) diluted with sterile ultrapure water.

Estimation method (if used):

None

Details on test conditions:

Test conditions

Test temperature: 50 +/- 1 °C

pH: Hydrolysis was examined at pH 4.0, 7.0 and 9.0 in the dark. The pH of each buffer solution was checked with a calibrated pH meter.

Light: The hydrolysis reaction was carried out using a dark thermostat 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.

Sterile conditions: Before the test, all glass and plastic labware were sterilized. For the test procedure, sterile solutions were used.

Test procedure

250 mL, approximately 1 mg/mL sterile solutions were prepared with Reactive Yellow F01-0555 in buffer solutions of pH 4.0, pH 7.0 and pH 9.0. Nitrogen was bubbled into the solutions, and the solutions were filtered through 0.22 μm membrane filter.

The hydrolysis experiments were carried out in screw cap vials in 5 replicates at each pH. Alltogether 18 vials were prepared: 3x5 vials contained the test item in buffer solutions of pH 4.0, 7.0 and 9.0, while 3x1 vial contained the buffer solutions of pH 4.0, 7.0 and 9.0 without the test item (control). The same test conditions and procedure were applied to all test vials.

Duration:

120 h

pH:

4

Initial conc. measured:

955.1 mg/L

Duration:

120 h

pH:

7

Initial conc. measured:

1 060.5 mg/L

Duration:

120 h

pH:

9

Initial conc. measured:

1 059.4 mg/L

Number of replicates:

The hydrolysis experiments were carried out in screw cap vials in 5 replicates at each pH. Alltogether 18 vials were prepared: 3x5 vials contained the test item in buffer solutions of pH 4.0, 7.0 and 9.0, while 3x1 vial contained the buffer solutions of pH 4.0, 7.0 and 9.0 without the test item (control).

Positive controls:

no

Negative controls:

yes

Statistical methods:

The chromatograms were evaluated using “LaChrom Chromatogram Processor". Calculations were carried out using “EXCEL for Windows".

Preliminary study:

Not applicable

Test performance:

The test performed in accordance with the parameters specified in the test guidelines.

Transformation products:

not measured

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

The end/start concentration of Reactive Yellow F01-0555 in the hydrolysis mixture was

- 20% at pH 4.0,
- 69% at pH 7.0 and
- 4% at pH 9.0

after 5 days of incubation at 50 +/- 1 °C. See tables below for details.

Other kinetic parameters:

None

Details on results:

See tables below.

Results of the hyrdolysis experiment

 

Measured concentrations and pH values are summarized in Table 3.

 

Table 3.: Measured data during hydrolysis experiment of Reactive Yellow F01-0555

Sampling time, h	Test item concentration, µg/mL		End/Start concentration	Measured pH
	Results of the separate test vessels	Mean withthe 95% confidence intervals
pH=4.0
0 (Start)	Control buffer		-	4.00
	946.3	955.1 ± 16.0	-	4.05
	939.6
	973.0
	960.0
	956.6
120 (End)	Control buffer		-	4.02
	186.1	188.3 ± 2.9	20%	4.00
	192.0			4.01
	189.2			4.02
	186.9			4.02
	187.5			4.02
pH=7.0
0 (Start)	Control buffer		-	7.00
	1067.2	1060.5 ± 30.9	-	7.01
	1035.9
	1074.2
	1034.2
	1091.2
120 (End)	Control buffer		-	7.01
	744.6	729.5 ± 18.4	69%	7.00
	741.5			7.00
	719.0			7.00
	732.5			6.99
	709.8			6.99
pH=9.0
0 (Start)	Control buffer		-	9.00
	1070.8	1059.4± 23.7	-	8.98
	1068.2
	1066.3
	1066.3
	1025.5
120 (End)	Control buffer		-	9.00
	43.2	41.6 ± 2.6	4%	9.00
	39.6			9.00
	42.3			9.00
	43.6			9.00
	39.0			9.00

The chromatogram of the hydrolysis solution of pH 9.0 showed that one of the two main components eluting with 3.23 minutes retention time (ester form of the dye) is not present in the hydrolysis solution at the start of the hydrolysis experiment (Chromatogram 5). It is due to the transformation of this component into the other main component eluting with 8.63 retention time (vinyl form of the dye). This reaction is very fast: during the course of the analytical method validation, this transformation took less than three minutes, as this is the intended reaction of the dye during the dyeing process, which takes place at alkaline pH at about 60°C The raw data of the analytical method validation contains evidence for this observation.

 

At pH 7.0 and 4.0 such phenomena was not observed.

Validity criteria fulfilled:

yes

Conclusions:

In the course of this preliminary test the test item concentration significantly decreased.

The end/start concentration of Reactive Yellow F01-0555 in the hydrolysis mixture was

- 20% at pH 4.0,
- 69% at pH 7.0 and
- 4% at pH 9.0

after 5 days of incubation at 50 +/- 1 °C.


Since the concentration decrease was more than 10% at each pH, further testing should be implemented at pH 4.0, pH 7.0 and pH 9.0 to evaluate the hydrolysis property of Reactive Yellow F01-0555.

Executive summary:

The purpose of this study was to evaluate the hydrolysis of Reactive Yellow F01-0555 in solutions of pH 4, pH 7 and pH 9.

 

In the course of this preliminary test the test item concentration significantly decreased.

The end/start concentration of Reactive Yellow F01-0555 in the hydrolysis mixture was

-  20% at pH 4.0,

-  69% at pH 7.0 and

-   4% at pH 9.0

after 5 days of incubation at 50 ± 1 °C.

Since the concentration decrease was more than 10% at each pH, further testing should be implemented at pH 4.0, pH 7.0 and pH 9.0 to evaluate the hydrolysis property of Reactive Yellow F01-0555.

Description of key information

The results of the Tier 2 hydrolysis test showed that the substance undergoes hydrolysis at environmentally relevant pH’s (t1/2 = 374 hours (15.6 days) at pH 4, 985 hours (41 days) at pH 7, and 566 hours (23.6 days) at pH 9, at 25°C). The hydrolysis products were identified.

Key value for chemical safety assessment

Half-life for hydrolysis:

41 d

at the temperature of:

25 °C

Additional information

In the course of the hydrolysis preliminary test performed at 50°C, significant decomposition was observed at pH 4, 7 and 9. The results of the Tier 2 hydrolysis test showed that the substance undergoes hydrolysis at environmentally relevant pH’s (t1/2 = 374 hours (15.6 days) at pH 4, 985 hours (41 days) at pH 7, and 566 hours (23.6 days) at pH 9, at 25°C). It should be noted that use of the substance as a dyestuff will take place at elevated temperatures within alkaline pH solutions that promote the dyeing process.