Reactive Blue F08-0170

Hydrolysis

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Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2011 to 25 June 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Details on test material:

Name: Reactive Blue F08-0170

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:

Immediately after preparation of the test solutions and after defined time intervals, samples were taken and analyzed by HPLC.

Buffers:

Buffer pH 4: C6H8O7 / NaOH / NaCl, Fluka, Art.-No.: 33643
Buffer pH 7: KH2PO4 / Na2HPO4, Fluka, Art.-No.: 33646
Buffer pH 9: Na2B4O7 / hydrochloric acid, Fluka, Art.-No.: 33648

Estimation method (if used):

None

Details on test conditions:

pH determination
Description of the method: The determination was done with a pH-meter with single-rod glass electrode after previous calibration.

Sterility test
Description of the method: A plate count tests according to Ph.Eur.6 was performed at the end of the tests.

Hydrolysis at pH = 4

Hydrolysis at 50°C

Preparation of the test solution
0.1054 g of the test item were weighed into a 200 ml volumetric flask and dissolved and diluted to volume with buffer pH 4. This represents a concentration of 0.527 g/L test item (0.0629 g/L VSE/VSE). The stock solution was ultra sonicated for 5 minutes. About 10 ml each were pipetted into 20 ml chromatographic vials. Nitrogen was overlayed over the solution for about 10 seconds to remove the oxygen. The vials were closed and stored at 50°C in a closed dark shaking heat regulator to avoid any photolytic effect. For every test point a new vial was used.
No deviations of the adjusted temperature (50°C ± 0.5°C) were observed.
The pH value was measured at each sampling interval being in the range of 4.00 to 4.08.
At pH 4 only 7 components were monitored. The components VS/VSOH and VSOH/VS were too small and partially interfered with other components. Therefore, these 2 components were not analysed for.

Hydrolysis at pH = 7

Hydrolysis at 50°C (tier 1)
Preparation of the test solution
0.1256 g of the test item were weighed into a 250 ml volumetric flask and dissolved and diluted to volume with buffer pH 7. This represents a concentration of test item of 0.5024 g/L (VSE/VSE: 0.0599 g/L). Nitrogen was passed through the solution for 5 min to remove the oxygen. The solution was stirred for 1 h at room temperature, filtered and 10 ml each were pipetted into 20 ml chromatographic vials. The vials were closed and stored at 50°C in a closed dark shaking heat regulator to avoid any photolytic effect. For every test point a new vial was used.
No deviations of the adjusted temperature (50°C ± 0.5°C) were observed.
The pH value was measured at all sampling times to be in the range of 6.98 – 7.03

Hydrolysis at 20°C (tier 2)

Preparation of the test solution
0.1356 g of the test item were weighed into a 250 ml volumetric flask and dissolved and diluted to volume with buffer pH 7. The solution has a concentration of 0.5424 g/L referring to test item (VSE/VSE: 0.0647 g/L). Nitrogen was passed through the solution for 5 min to remove the oxygen. The solution was stirred for 1 h at room temperature, filtered and 10 ml each were pipetted into 20 ml chromatographic vials. The vials were closed and stored at 20°C in a closed dark shaking heat regulator to avoid any photolytic effect. For every test point a new vial was used.
No deviations of the adjusted temperature (20°C ± 0.5°C) were observed.

Hydrolysis at pH = 9
13.1 Hydrolysis at 50°C (tier 1)
13.1.1 Preparation of the test solution
0.0587 g of the test item were weighed into a 250 ml volumetric flask and dissolved and diluted to volume with buffer pH 9 resulting in a concentration of 0.2348 g/L test item (VSE/VSE: 0.028 g/L) . Nitrogen was passed through the solution for 5 min to remove the oxygen. The solution was stirred for 1 h at room temperature, filtered and 10 ml each were pipetted into 20 ml chromatographic vials. The vials were closed and stored at 50°C in a closed dark shaking heat regulator to avoid any photolytic effect. For every test point a new vial was used.
No deviations of the adjusted temperature (50°C ± 0.5°C) were observed.
The pH value was measured at all sampling times to be in the range of 8.98 – 9.08.

Hydrolysis at 20°C (tier 2)
13.2.1 Preparation of the test solution
0.1405 g of the test item were weighed into a 250 ml volumetric flask and dissolved and diluted to volume with buffer pH 9 resulting in a concentration of test item of 0.562 g/L (VSE/VSE: 0.067 g/L). Nitrogen was passed through the solution for 5 min to remove the oxygen. The solution was stirred for 1 h at room temperature, filtered and 10 ml each were pipetted into 20 ml chromatographic vials. The vials were closed and stored at 20°C in a closed dark shaking heat regulator to avoid any photolytic effect. For every test point a new vial was used.
No deviations of the adjusted temperature (20°C ± 0.5°C) were observed.
The pH value was measured at all sampling times to be in the range of 8.99 – 9.04

The pH value was measured at all sampling times to be in the range of 7.00 – 7.06

Duration of test

Duration of test 1

Duration:

5 d

pH:

4

Initial conc. measured:

0.213 other: g/L, initial concentration for sum of 7 components, see results for initial concentrations of individual components

Duration of test 2

Duration:

5 d

pH:

7

Initial conc. measured:

0.262 other: g/L, initial concentration for sum of 9 components, see results for initial concentrations of individual components

Duration of test 3

Duration:

29.8 d

pH:

7

Initial conc. measured:

0.268 other: g/L, initial concentration for sum of 9 components, see results for initial concentrations of individual components

Duration of test 4

Duration:

6.2 d

pH:

9

Initial conc. measured:

0.116 other: g/L, initial concentration for sum of 9 components, see results for initial concentrations of individual components

Duration of test 5

Duration:

30 d

pH:

9

Initial conc. measured:

0.192 other: g/L, initial concentration for sum of 9 components, see results for initial concentrations of individual components

Number of replicates:

For every test point a new vial was used.

Positive controls:

no

Negative controls:

no

Statistical methods:

The logarithms of the concentrations are plotted against time and the slope of the resulting straight line (assuming first-order or pseudo-first order behavior) gives the rate constant from the formula.
kobs = - slope x 2.303 (if log10 is used).
When the rate constants are known for two or more temperatures, the rate constants at other temperatures can be calculated using the Arrhenius equation:

Results and discussion

Preliminary study:

In the course of the preliminary test different rates of decomposition of Reactive Blue F08-0170 were observed after 5 days at a temperature of 50°C at pH 4, 7 and 9. See study endpoint Hydrolysis.002 for details.

Test performance:

The recovery and the repeatability were checked during the hydrolysis study by analyzing a solution of the test item in water on every day of application (system suitability test). At pH 7 (50°C) no control of recovery was performed but the other data show that the method is working properly. The recovery is between 90% und 110% and meet the requirements of OECD 111.
With a satisfying mean recovery of 100.1% the recovery of the determination is demonstrated. The relative standard deviation of 2.4% is satisfying taking the instability of the test item and the length of the test period of approx. 3 month into account.
Chromatograms show that the method is sensitive enough to determine hydrolysis rates of greater than 90%.

Transformation products:

yes

Identity of transformation products

Identity of transformation products 1

No.:

#1

Identity of transformation products 2

No.:

#2

Identity of transformation products 3

No.:

#3

Identity of transformation products 4

No.:

#4

Identity of transformation products 5

No.:

#5

Identity of transformation products 6

No.:

#6

Identity of transformation products 7

No.:

#7

Identity of transformation products 8

No.:

#8

Identity of transformation products 9

No.:

#9

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

Whereas the components of the test item remained stable at pH 4 (50°C), degradation and formation of components was observed at pH 7 and 9. Generally, the products formed at pH 7 and pH 9 are the same ones. The reaction kinetics is slightly different as at pH 9 the degradation is quicker and more progressed than at pH 7.

Consequently, the primary reactions for hydrolysis are understood as elimination of the sulphate groups yielding the respective vinylsulfones. In a second step, the vinylsulfon moieties are hydrolysed by addition of water forming the respective alcohol as described in the scheme attached below under background material.

The Cu-free components or hydrolysis products behave in the same way as the Cu-containing ones. Elimination of Cu from the complexes has not been observed.

Total recovery of test substance (in %)

% Recovery:

ca. 70

pH:

9

Temp.:

20 °C

Duration:

30 d

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

7

Temp.:

20 °C

Type:

other: The data show that the total substance (sum of 9 components) is rather stable but a small decay might be suspected.

Remarks on result:

other: However, for the calculation and extrapolation of an exact half-life time t/1/2 at 20°C, the data are not robust enough due to scattering.

Dissipation DT50 of parent compound 2

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

212 h

Type:

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

Remarks on result:

other: For component #1 (VSE/VSE)

Dissipation DT50 of parent compound 3

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

870 h

Type:

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

Remarks on result:

other: For component #2 (VSOH/VSE)

Dissipation DT50 of parent compound 4

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

84 h

Type:

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

Remarks on result:

other: For component #7 (VSE/VSE-Cu-free)

Dissipation DT50 of parent compound 5

pH:

9

Temp.:

20 °C

DT50:

2 mo

Type:

other: Estimations performed using the highest and the lowest concentration show a decline of about 30% within 30 days.

Remarks on result:

other: Using these data, an estimated half-life was derived.

Dissipation DT50 of parent compound 6

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

3.6 h

Type:

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

Remarks on result:

other: For component #1 (VSE/VSE)

Dissipation DT50 of parent compound 7

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

14 h

Type:

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

Remarks on result:

other: For component #2 (VSOH/VSE)

Dissipation DT50 of parent compound 8

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

2.6 h

Type:

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

Remarks on result:

other: For component #7 (VSE/VSE-Cu-free)

Dissipation DT50 of parent compound 9

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.001 s-1

DT50:

0.3 h

Type:

other: Only 2 data

Remarks on result:

other: For component #8 (VS/VSE-Cu-free)

Other kinetic parameters:

None

Details on results:

- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

See pdfs attached under background material for details of results.

Any other information on results incl. tables

For those components which show a first order kinetics, the following hydrolysis rate constants and half-life times at 25°C were calculated from the experimental data using the Arrhenius function.

Half-life time and hydrolysis rate constant at pH = 4

The test item is found to be stable at pH 4 and 50°C. Therefore it can be assumed that the test item is also stable at 25°C and no half-life time and hydrolysis rate were calculated.

Half-life time and hydrolysis rate constant at pH = 7

Half-life times and hydrolysis rate constants for individual components:

At 50 degrees C and pH 7:

Component	Half-life t(1/2) (h)	Rate constant k [s^-1]	Remarks kinetics
VSE/VSE	3.0	6.39359E-05	1st order
VSOH/VSE	28	6.83942E-06	1st order
VS/VSE	-*	-*	Higher order
VS/VS	-*	-*	Higher order
VSOH/VS	-*	-*	Higher order
VS/VSOH	-*	-*	Higher order
VSE/VSE Cu-free	5.8	3.31263E-05	1st order
VS/VS Cu-free	-*	-*	Higher order
VS/VSE Cu-free	-*	-*	Higher order

* Determination of half-life and rate constant not possible

At 20 degrees C and pH 7:

Component	Half-life (1/2) (h)	Rate constant k [s^-1]	Remarks kinetics
VSE/VSE	212	9.08760E-07	1st order
VSOH/VSE	870	2.21286E-07	1st order
VS/VSE	-*	-*	Higher order
VS/VS	-*	-*	Higher order
VSOH/VS	-*	-*	Higher order
VS/VSOH	-*	-*	Higher order
VSE/VSE-Cu-free	84	2.28893E-06	1st order
VS/VS Cu-free	-*	-*	Higher order
VS/VSE-Cu-free	-*	-*	Higher order

* Determination of half-life and rate constant not possible

Overall half-life time and hydrolysis rate constant for the test item at pH 7 and at 20 degrees C:

The hydrolysis behaviour of the 9 components originating from the test item is described above. The data show that the total substance (sum of 9 components) is rather stable but a small decay might be suspected. However, for the calculation and extrapolation of an exact half-life time t/1/2 at 20°C, the data are not robust enough due to scattering.

Half-life time and hydrolysis rate constant at pH = 9

At 50 degrees C and pH 9:

NLP-Fraction	Half-life	Rate constant	Remarks
	t(1/2) (h)	k [s^-1]	kinetics
VSE/VSE	0.1	1.54598E-03	Only 2 data
VSOH/VSE	0.4	5.02847E-04	Only 3 data
VS/VSE	0.5	3.93247E-04	1st order
VS/VS	-*	-*	Higher order
VSOH/VS	-*	-*	Higher order
VS/VSOH	-*	-*	Higher order
VSE/VSE-Cu-free	-*	-*	Higher order
VS/VS Cu-free	-*	-*	Higher order
VS/VSE-Cu-free	-*	-*	Higher order

* Determination of half-life and rate constant not possible

At 20 degrees C and pH 9:

NLP-Fraction	Half-life	Rate constant	Remarks
	(1/2) (h)	k [s^-1]	kinetics
VSE/VSE	3.6	5.36338E-05	1st order
VSOH/VSE	14	1.37662E-05	1st order
VS/VSE	-*	-*	Higher order
VS/VS	-*	-*	Higher order
VSOH/VS	-*	-*	Higher order
VS/VSOH	-*	-*	Higher order
VSE/VSE-Cu-free	2.6	7.34426E-05	1st order
VS/VS Cu-free	-*	-*	Higher order
VS/VSE-Cu-free	0.3	6.32529E-04	Only 2 data

* Determination of half-life and rate constant not possible

Overall half-life time and hydrolysis rate constant

The area-% values of the half-life time and hydrolysis rate constant for the test item at pH = 9 and 20°C do not follow a 1st order kinetics and thus an exact half-life could not be calculated. Estimations performed using the highest and the lowest concentration show a decline of about 30% within 30 days. Using these data, an estimated half-life was derived.

Half-life time (t/1/2): about 2 months (estimated)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The stability of the test item is found to decrease with increasing pH-value.
Hydrolysis at pH 4:
The test item is found to be stable at pH 4 and 50°C. Therefore it can be assumed that the test item is also stable at 20°C and no half-life time and hydrolysis rate were calculated.
Hydrolysis at pH 7 and 9
Whereas the components of the test item remained stable at pH 4 (50°C), degradation and formation of components was observed at pH 7 and 9. Generally, the products formed at pH 7 and pH 9 are the same ones. The reaction kinetics is slightly different as at pH 9 the degradation is quicker and more progressed than at pH 7.

Executive summary:

For Reactive Blue F08-0170 the hydrolysis behavior was investigated at pH = 4, 7 and 9 using a combined HPLC/MS/UV method for analysis. Quantitation of the educts and hydrolysis products was performed using the HPLC/UV method. HPLC/MS was used to elucidate and to confirm the structure of the components over the whole hydrolysis period.

Reactive Blue F08-0170 is a complex mixture of components with different molecular weight. In total nine components could be identified and their behaviour was monitored. These 9 components represent about 75% of the organic part of the substance (HPLC peak area) at time 0.

VSE/VSE 19.55 %

VSOH/VSE 11.34 %

VS/VSE 27.48 %

VS/VS 4.40 %

VS/VSOH 2.66 %

VSOH/VS 1.36 %

VSE/VSE Cu-free (DYEJ 6302) 3.23 %

VS/VSE Cu free (DYEJ 6621) 3.91 %

VS/VS Cu-free (DYEJ 6630) 1.52 %

Identity of components and hydrolysis products was ascertained using structural information from the sponsor, by comparison with enriched fractions of individual components and by monitoring of LC/MS-ESI-spectra.

The stability of the test item is found to decrease with increasing pH-value.

Hydrolysis at pH 4:

The test item is found to be stable at pH 4 and 50°C. Therefore it can be assumed that the test item is also stable at 20°C and no half-life time and hydrolysis rate were calculated.

Hydrolysis at pH 7 and 9

Whereas the components of the test item remained stable at pH 4 (50°C), degradation and formation of components was observed at pH 7 and 9. Generally, the products formed at pH 7 and pH 9 are the same ones. The reaction kinetics is slightly different as at pH 9 the degradation is quicker and more progressed than at pH 7.

Behaviour of components during hydrolysis at pH 7 and 9. (In brackets, the half-life at 20°C is given where scientifically justified):

pH 7			pH9
Decreasing	Increasing and decreasing	Increasing	Decreasing	Increasing and decreasing	Increasing
VSE/VSE	VS/VSE	VS/VS	VSE/VSE	VS/VS
(t1/2=204h)			(t1/2=3,6h)
VSE/VSE Cu-	VS/VSE Cu-	VS/VS Cu-	VSE/VSE	VS/VS
free	free	free	Cu-free	Cu-free
(t1/2=123h)			(t1/2=2,6h)
VSOH/VSE		VS/VSOH	VSOH/VSE	VS/VSE
(t1/2=815h)			(t1/2=14h)
		VSOH/VS	VS/VSE Cu- free (t1/2=0,3h)	VS/VSOH
				VSOH/VS
Sum of 9 components stable			Sum of 9 components (t1/2 ca. 2 months)

It was recognised that a few components hydrolyse and disappear. Other components (some of them are already present at time 0) first increase with time, which however may later decrease. Hydrolysis rate constants and the half-life times could only be calculated for components which react in a 1st order kinetics, i.e. decrease constantly.

According to these data, the primary reactions for hydrolysis are understood as elimination of the sulphate groups yielding the respective vinylsulfones. In a second step, the vinylsulfon moieties are hydrolysed by addition of water forming the respective alcohol as described in the scheme attached under background material.