Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-05-17 to 2013-07-18

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

/ EC No. 440/2008 Method C.7

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Not specified

IUPAC Name:

Not specified

Details on test material:

- Name of test material (as cited in study report): Reactive Golden Yellow HF-RN 1331
- Molecular formula: C17H11F2Na2O7S2 (as sodium salt) C17H13F2N7O7S2 (as free acid)
- Physical state: Solid, red to orange powder
- Analytical purity: 100 %
- Purity test date: 2012-10-08
- Lot/batch No.: Kilo 10 & 12
- Expiration date of the lot/batch: 2014-01-17
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container; since 2013-03-01 additionally under nitrogen

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
In the preliminary test, samples were taken at test start (0 h) and at test end (120 h).
In the advanced test, samples were taken at test start and at 11 spaced points, normally between 10 and 90 % of hydrolysis, at each test temperature at pH 9. All samples were analysed immediately (max. 30 min for preparation and analyses) via HPLC-DAD.

Buffers:

Sterile buffer solutions at pH 4, 7 and 9

Buffer solution pH 4 45 mL of 0.1 mol/L NaOH were mixed with 250 mL 0.1 mol/L monopotassium citrate and diluted to 500 mL with double distilled water.

Buffer solution pH 7 148.15 mL of 0.1 mol/L NaOH were mixed with 250 mL 0.1 mol/L KH2PO4 and diluted to 500 mL with double distilled water.

Buffer solution pH 9 106.5 mL of 0.1 mol/L NaOH were mixed with 250 mL 0.1 mol/L H3BO3 in 0.1 mol/L KCl and diluted to 500 mL with double distilled water.

Buffers were prepared from chemicals with analytical grade or better quality. Buffers were purged with nitrogen for 5 min. Then the pH was checked to a precision of at least 0.1 at the required temperature, adjusted if necessary and sterilised by filtration through 0.2 µm.

Reason for the selection Buffer systems were selected according to the guideline. The buffer systems were suitable for their pH values.



Details
Chemical Origin Batch number Purity [%]
NaOH VWR 12L110007 99.4
H3BO3 VWR 11E300005 100
KCl VWR 12D030022 99.7
KH2PO4 VWR 12E160023 100
KH2 Citrate FLUKA BCBH3957V ≥ 98
Double distilled water ROTH 173199587 1)
422192602
1) conductivity: < 2.0 μS/cm

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: HPLC vials, volume 4 mL
- Measures taken to avoid photolytic effects: Photolytic effects were avoided by using opaque water baths.
- Measures to exclude oxygen:
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No
TEST MEDIUM
- Volume used/treatment: 4 mL
- Preparation of test medium: The test item was applied once at test start by direct weighing. The test solutions were sterilised by filtration through 0.20 µm sterile membrane filters into the test containers.
After Application the test containers were sealed and transferred into the thermostat. The time between test item application and transfer to thermostat/analysis did not exceed 30 min.

- Incubation: preliminary test: 2013-05-17 to 2013-05-22
advanced test: 2013-06-10 to 2013-07-10

- Temperatures:
Preliminary test: 50  0.5 °C
Advanced test: 20, 30 and 50  0.5 °C

- Sterility: For pH 9 the sterility of the test solutions was checked by colony forming units (CFU)-determination with Water Plate Count Agar. The CFU were determined from additional samples at test end by incubation at 36 ± 1 °C for 48 hours and at 22 ± 1 °C for 72 hours.

- Renewal of test solution: None

Duration of test

Duration of test 1

Duration:

714 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

1.05 g/L

Duration of test 2

Duration:

714 h

pH:

9

Temp.:

30 °C

Initial conc. measured:

1.05 g/L

Duration of test 3

Duration:

208 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

1.05 g/L

Number of replicates:

Duplicates, single injected

Positive controls:

no

Negative controls:

yes

Remarks:

buffer solutions (pH 4, 7 and 9)

Results and discussion

Test performance:

CHRONOLOGICAL
TEST DESCRIPTION
- Method validation
- Preparation of the sterile test solutions (experimental starting)
- Thermostatisation of the test solutions
- Analysis of samples
- Calculations


                     

Transformation products:

no

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

No significant hydrolysis was observed at pH 4 and 7. Therefore the test item was considered as hydrolytically stable under this condition and a half-life of > 1 year could be assumed for environmental typical temperatures.

Details on results:

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


Temperature of the Test System
measured hourly

Intended Temperature Measured Temperature
Preliminary Test Advanced Test
Mean ± SD Mean ± SD min. / max.
20.0 ± 0.5 n.a. 20.1 ± 0.04 20.4 / 20.5
30.0 ± 0.5 30.1 ± 2 29.9 / 62.41
50.0 ± 0.5 49.9 ± 0.07 50.0 ± 0.03 49.9 / 50.1
SD = Standard deviation
n.a. = not applicable, only tested at 50 °C in preliminary testing

1) Due to a technical malfunction, the test temperature exceeded the nominal range of 30 ± 0.5 °C for less than two hours. Compared to the total incubation time of 714 h and the calculated half life of 968 h, this deviation could be regarded as negligible and a significant influence on the study results could be precluded.

Any other information on results incl. tables

pH-Value of the Test Systems

measured before start of hydrolysis

Test	Intended pH-value	Measured pH-value at 20 °C	Measured pH-value at 30 °C	Measured pH-value at 50 °C
Preliminary	4.0 ± 0.1	n.a.		4.02
	7.0 ± 0.1			7.00
	9.0 ± 0.1			8.96
Advanced	9.0 ± 0.1	8.93	8.93	8.93

                      n.a.: not applicable, only tested at 50 °C in preliminary testing

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item Reactive Golden Yellow HF-RN 1331 showed no hydrolysis for pH values of 4 and 7.

The test item Reactive Golden Yellow HF-RN 1331 showed only a slow hydrolysis at pH 9 and the temperatures of 20 and 30 °C and a moderate hydrolysis the elevated temperature of 50 °C.

Executive summary:

Hydrolysis as a function of pH was determined according to OECD Guideline No. 111 and Council Regulation (EC) No. 440/2008, Method C.7 for the test item Reactive Golden Yellow HF-RN 1331 (batch number: Kilo10&12) from 2013-05-17 to 2013-07-18 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

Analyses of the test item were performed via HPLC-DAD on a reversed phase column using an external standard. The method was validated with satisfactory results in regard to linearity, accuracy, precision and specificity.

The preliminary test was conducted with a test item concentration of 1000 mg/L in buffer solutions at pH 4, 7 and 9 and 50 °C. For pH 9 the advanced test was performed, as a significant reduction (> 10 %) of the test item concentration was observed in the preliminary test. At pH 4 and 7 no significant elimination was observed and therefore the test item was considered as hydrolytically stable under this condition and a half-life of > 1 year could be assumed for environmental typical temperatures.

The advanced test was conducted with a test item concentration of 1000 mg/L in buffer solutions at pH  9 and temperatures of 20, 30 and 50 °C, respectively. Samples were taken at test start (0 h) and at 11 spaced points until test end.

At all pH 9 test conditions a significant transformation of the test item was observed.

 

Buffer solutions were analysed at test start and test end and there was no analytical interference with the test item.

Reaction rate constants and half-lives for the pH 9 test condition were calculated from the analysed concentrations and are presented in Table 1. The test item Reactive Golden Yellow HF-RN 1331 showed only a slow hydrolysis at pH 9 and the temperatures of 20 and 30 °C and a moderate hydrolysis the elevated temperature of 50 °C.

Reaction Rate Constants and Half-Lives of Reactive Golden Yellow HF-RN 1331

	pH 9
	20 °C	30 °C	50 °C
Reaction rate constant kobs [1/s]	4.11 x 10^-8	1.99 x 10^-7	3.25 x 10^-6
Half-life T½ [h]	4685	968	59.2
Half-life T½ [d]	195	40.3	2.47
Slope of regression graph	significantly non zero