disodium 4-hydroxy-3-[2-[4-({4-[2-(1-hydroxy-4-sulfonatonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-05-17 to 2013-05-22

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:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

EC Number:

700-924-0

Molecular formula:

C41H30N4Na2O10S2

IUPAC Name:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
Samples were taken at 0 h and after 120 h. All samples were diluted with acetonitrile: HPLC water (50:50) and analysed. All test item containing samples were analysed immediately (max. 30 minutes until start of analyses) via HPLC with UV (DAD) detection.
The bath temperature was checked automatically once in an hour and manually once per working day.



- Sample storage conditions before analysis: The samples were diluted with acetonitrile:HPLC water (50:50) at a total dilution factor of 4 and analysed.

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 KH2-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, 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, 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 test temperature, adjusted if necessary and sterilised by filtration through 0.2 µm.


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


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 422192602 1)

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: 50 mL
- Preparation of test medium: 50 mg of the test item was weighed and filled up to a volume of 50 mL with the respective sterile buffer. Afterwards the solutions were sterilised by filtration through 0.20 µm sterile membrane filters into the test containers. The test containers were sealed and transferred into the thermostat. The time between test item application and transfer to laboratory incubator/analysis did not exceed 30 minutes.
- Incubation: 120 hours
- Temperatures: 50 ± 0.5 °C
- Sterility: The solutions were sterilised by filtration through 0.20 µm sterile membrane filters into the test containers.

- Renewal of test solution: None

Duration of test

Duration of test 1

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

1.09 mg/L

Duration of test 2

Duration:

599 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

1.01 mg/L

Duration of test 3

Duration:

338 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.9 mg/L

Number of replicates:

Two replicates at each sampling interval

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):

The test item was found to be stable in the preliminary test at pH 4, 7 and 9 at 50 °C (Table 1). Reaction rate constants and half-lives could not be calculated due to the fact that the test item undergoes no significant hydrolysis. According to the guideline a half life of > 1 year under environmental conditions can be assumed.

Details on results:

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

pH-Value of the Test System (50 °C)
measured at test temperature on day of preparation (before sterilisation)

Intended pH-value Measured pH-value at 50 °C
4.0 ± 0.1 4.02
7.0 ± 0.1 7.00
9.0 ± 0.1 8.96

The temperature was in good agreement with the nominal range throughout the test. The mean temperature was 49.9 °C with a minimum of 49.6 °C and a maximum of 50.0 °C during the study.

Any other information on results incl. tables

pH-Value of the Test System (50 °C)

measured at test temperature on day of preparation (before sterilisation)

Intended pH-value	Measured pH-value at 50 °C
4.0 ± 0.1	4.02
7.0 ± 0.1	7.00
9.0 ± 0.1	8.96

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to be stable in the preliminary test at pH 4, 7 and 9 at 50 °C (Table 1). Reaction rate constants and half-lives could not be calculated due to the fact that the test item undergoes no significant hydrolysis. According to the guideline a half life of > 1 year under environmental conditions can be assumed.

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 from 2013-05-17 to 2013-05-22 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

The study was conducted with test item concentrations of 1000 mg/L in buffer solutions at pH 4, 7 and 9 at a test temperature of 50 °C (preliminary test).

Samples were taken at test start (0 hours) and test end (120 hours) and analysed via HPLC with UV (DAD) detection on a reversed phase column using an external standard. Buffer solutions were analysed at test start and test end and indicated no interference with the test item. The analytical method for determination of the test item was validated and tested with satisfactory results in regard to linearity, accuracy, precision and specificity.

Degradation was calculated as the percentage loss of the test item over the time. In the preliminary test, the test item was found to be stable at pH 4, 7 and 9, respectively. No further testing was deemed necessary as less than 10 % of the applied test item was transformed after 120 hours (5 days) at a temperature of 50 °C and each of the three pH values. Reaction rate constants and half-lives could not be calculated because the test item undergoes no significant hydrolysis. With respect to the guidelines a half life of > 1 year could be assumed for environmental typical temperature conditions.

Degradation [%] of the test item at 50 °C after 120 Hours

Hydrolysis Time [hours]	Degradation [%]
	pH 4	pH 7	pH 9
120	7.34	0.99	6.67