disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 1992

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

adopted May 12, 1981

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

After incubation 1O-pl aliquots of the test solutions at each pH vatue were analysed without dilution by measuring the signat of SCARLET RN 1165 after HPLC separation of the injected sample solution.

Buffers:

- pH: 4.0, 7.0 & 9.0
- Composition of buffer:
Buffer pH 4, Biphthalate (Art. 5657 - Baker)
Buffer pH 7, Phosphate (Art. 5656 - Baker)
Buffer pH 9, Borate (Art. 7145 - Baker)
The butfer solutions were steritized for 25 minutes in an autoclave. Nitrogen was passed through the butfer solutions for 5 minutes.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: All glassware, which must be inert in the pH range applied, were rinsed with sterile buffer. The hydrolysis was carried out in flasks which were stoppered or sealed with an inert material (e.g. PTFE); Thermostaticalty controlled water bath
- Sterilisation method: The butfer solutions were steritized for 25 minutes in an autoclave. Nitrogen was passed through the butfer solutions for 5 minutes.

TEST MEDIUM
- Preparation of test medium: The test afticle was dissolved in the buffer solutions and incubated at specified temperatures with the water bath kept constant at t 0.1 °C, usually. The concentration of the test article was determined as a function of time at each pH.

Preparation of the Test Solutions

Hydrolysis at 50°C

-pH 4.0, Sample 1 and 2
12.34 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (pH 4.0). Two aliquots of this test solution of approximately 50 ml each were transfeneä into 50 ml Erlenmeyer flasks in order to perform a duplicate test.

-pH 7.0, Sample 3 and 4
10.45 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (pH 7.0). For a duplicate test, two aliquots of this test solution of approximately 50 ml each were transferred in 50 ml Erlenmeyer flasks.

-pH 9.0, Sample 5 and 6
10.57 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (adjusted to pH 9.0). For a duplicate test, two aliquots of this test solution of approximately 50 ml each were transferred in 50 ml Erlenmeyer flasks.

-pH 9.0, Sample 7 and 8
20.62 mg of SCARLET RN 1165 were dissolved in 200 ml buffer solution (pH 9.0). The solution was purged with nitrogen for 2 minutes. Two aliquots of this test solution of approximately 50 ml each were transfened into 50 ml Erlenmeyer flasks in order to perform a duplicate test.

Hydrolysis at 60°C

-pH 9.0, Sample 9 and 10
11.87 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (pH 9.0). The solution was purged with nitrogen for 2 minutes. Two aliquots of this test solution of approximately 50 ml each were transfened into 50 ml Erlenmeyer flasks in order to perform a duplicate test.

-pH 9.0, Sample 15 and 16
11.81 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (pH 9.0). The solution was purged with nitrogen for 2 minutes. Two aliquots of this test solution of approximately 50 ml each were transfened into 50 ml Erlenmeyer flasks in order to perform a duplicate test.

Hydrolysis at 70°C

-pH 9.0, Sample 17 and 18
11.01 mg of SCARLET RN 1165 were dissolved in 100 ml buffer solution (pH 9.0). The solution was purged with nitrogen for 2 minutes. Two aliquots of this test solution of approximately 50 ml each were transfened into 50 ml Erlenmeyer flasks in order to perform a duplicate test.

Samples 11 to 14 were prepared is a similar way, but the chosen sampling times were not suited to establish a degradation curve. Therefore the results are not reported.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

>= 122.5 - <= 123.2 other: µg/mL

Remarks:

preliminary test

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

>= 98.7 - <= 101.4 other: µg/mL

Remarks:

preliminary test

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

>= 104.5 - <= 105.5 other: µg/mL

Remarks:

preliminary test

Duration:

97.5 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

>= 107 - <= 108 other: µg/mL

Duration:

13 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

>= 129 - <= 136 other: µg/mL

Duration:

9 h

pH:

9

Temp.:

70 °C

Initial conc. measured:

>= 117 - <= 124 other: µg/mL

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Statistical methods:

Not used

Transformation products:

not measured

Key result

pH:

4

Temp.:

50 °C

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

7

Temp.:

50 °C

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

100 d

Type:

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

Remarks on result:

other: Isomer I

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

100 d

Type:

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

Remarks on result:

other: Isomer II

Key result

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.017 h-1

DT50:

40.5 h

Type:

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

Remarks on result:

other: Isomer I

Key result

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.009 h-1

DT50:

72 h

Type:

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

Remarks on result:

other: Isomer II

Key result

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.065 h-1

DT50:

11 h

Type:

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

Remarks on result:

other: Isomer I

Key result

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.04

DT50:

17 h

Type:

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

Remarks on result:

other: Isomer II

Key result

pH:

9

Temp.:

70 °C

Hydrolysis rate constant:

0.24 h-1

DT50:

3 h

Type:

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

Remarks on result:

other: Isomer I

Key result

pH:

9

Temp.:

70 °C

Hydrolysis rate constant:

0.14 h-1

DT50:

5 h

Type:

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

Remarks on result:

other: Isomer II

Details on results:

HYDROLYSIS AT 50.0 °C

pH 9.0
The results show, that SCARLET RN 1165 is not stable at pH 9.0. Therefore the test at 50 °C was continued until about 80% of SCARLET RN 1165 were hydrolysed. The linear plots prove that the hydrolysis reaction of both isomers of SCARLET RN 1165 is of pseudo first oder in the range from 20% to 70% hydrolysis at pH 9.0. The half-life time of SCARLET RN 1165 at 50.0 C and pH 9.0 was calculated to be 40.5 and 74 hours, respectively, for the two isomers.

pH 7.0 and pH 4.0
The results of pH 7.0 and pH 4.0 showed no significant degradation of SCARLET RN 1165 at 50°C. Aocording 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 environmentral conditions (25 °C). Therefore, SCARLET RN 1165 can be considered to be hydrolytically stable at pH 7.0 and pH 4.0, and no further testing was necessary at these pH-values.

HYDROLYSIS AT DIFFERENT TEMPERATURES
The hydrolysis reaction at pH 9.0 is relatively slow. Therefore, the further hydrolysis tests at this pH were performed at 60°C and 70 °C, each in duplicate. The test solutions were analysed in time intervals. The values represent rounded results which were obtained by calculation using the exact raw data. 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 11 and 17 hours, respectively, for the two isomers. The half-life time at pH 9.0 and 70 oC was calculated to be 3 and 5 hours, respectively for the two isomers.

EVALUATION OF THE HALF-LIFE TIME AT 25 °C

The half-life time of the hydrolysis reaction of SCARLET RN 1165 at 25 °C and pH 9.0 was calculated to be 50 and 101 days, respectively, for the two isomers.

Results with reference substance:

none

Validity criteria fulfilled:

yes

Conclusions:

In the pre-test, SCARLET RN 1165 is stable at pH 4.0 and pH 7.0 at 50 °C. Its half-life time is longer than one year at 25°C.
In the pre-test, SCARLET RN 1165 is not stable at pH 9.0 at 50 °C. Reaction rate contsants and laf-life time were therefore measured for both isomers at pH 9.0 at 25; 50; 60; 70°C.

Executive summary:

SCARLET RN 1165 consists of two isomers. Both isomers were studied separately. The hydrolysis pre-test was performed at 50.0°C +/- 0.1°C at each of pH 4.0, pH 7.0, and pH 9.0. As SCARLET RN 1165 was not stable at pH 9.0, further testing was perfonned at elevated temperatures in order to calculate the astivation energy and the rate constant (k25 and the half-life time of the hydrolysis at pH 9.0 and 25 °C. SCARLET RN 1165 was found to be stable at pH 7.0 and pH 4.0 at 50 °C. Therefore no further testing was performed at these pH-values. The results are summarized below (calculated using the Arrhenius equation):

lsomer I

pH	Temperature [°C]	Reaction rate constant k [1/h]	Half-life time t 1/2
9.0	25	0.00058	50 days
	50	0.017	40.5 hours
	60	0.065	11 hours
	70	0.24	3 hours

lsomer II

pH	Temperature [°C]	Reaction rate constant k [1/h]	Half-life time t 1/2
9.0	25	0.00029	100 days
	50	0.0093	72 hours
	60	0.040	17 hours
	70	0.14	5 hours

SCARLET RN 1165 is stable at pH 4.0. Its half-life time is longer than one year at 25 °C.

SCARLET RN 1165 is stabte at pH 7.0. lts half-life time is longer than one year at 25 °C.

Description of key information

In the pre-test, SCARLET RN 1165 is stable at pH 4.0 and pH 7.0 at 50 °C. Its half-life time is longer than one year at 25°C.

In the pre-test, SCARLET RN 1165 is not stable at pH 9.0 at 50 °C. Reaction rate contsants and laf-life time were therefore measured for both isomers at pH 9.0 at 25; 50; 60; 70°C.