Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 1996 to 13 June 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

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)

Specific details on test material used for the study:

Name: FAT 40'549/A
Batch No: TV 1
Colour: yellow-brown
Aggregate State at room temp.: solid
Stability in Solvent: in water: 48 hours
Stability at storage conditions: stable
Molecular Weight: 1138 g/mol (free acid)
Storage: at room temperature
Expiration Date: January 01, 2001

Radiolabelling:

no

Analytical monitoring:

yes

Buffers:

pH 4: 0.40 vol% 0.1 M NaOH
50.00 vol% 0.1 M potassium biphthalate
49.60 vol% double distilled water
pH 7: 29.63 vol%0.1M NaOH
50.00 vol% 0.1 M monopotassium phosphate
20.37 vol% double distilled water
pH 9: 21.30 vol% 0.1 M NaOH
50.00 vol% 0.1 M boric acid
28.70 vol% double distilled water

The pH of each buffer solution was checked with a calibrated pH meter before and after dissolving the test article at the required temperature to a precision of at least ± 0.1. As far as necessary the pH value of the test solutions was adjusted to the desired pH value with either 0.1 M boric acid or 0 1 M NaOH.

The buffer solutions were sterilised. If necessary, nitrogen was bubbled for about two minutes into the buffer solutions before the preparation of the test solutions to reduce their oxygen content.

Estimation method (if used):

HPLC

Details on test conditions:

The degradation of the main product of FAT 40'549/A is described by a vinylation reaction in a first step and a following hydrolysis reaction of the vinylated main product in a second step. Therefore each reaction step was investigated separately in this study to estimate the abiotic degradation of the main product of FAT 40'549/A at 25 °C as a function of pH. The vinylation of the main product was observed after dissolving the test article in the representative buffer solutions and following the hydrolysis procedure to determine the rate constant for the vinylation.
To determine the rate constant of the hydrolysis reaction, the hydrolysis of the vinylated main product was observed following the hydrolysis procedures after a pre-treatment of the test article where the main product was vinylated nearly completely.

DETERMINATION OF THE VINYLATION:
The tests were performed at 50 ± 0.1 °C at pH 4, pH 7 and pH 9 in duplicate to determine the vinylation reaction of the main product of FAT 40549/A as a function of pH.
Further vinylation tests were performed at 70 °C and 80 °C in the buffered test solution at pH 4 and at 25 °C in the buffered test solution at pH 7, due to the instability found of the main product in the vinylation test at 50 °C.

DETERMINATION OF THE HYDROLYSIS:
The hydrolysis tests were performed at 50 ± 0.1 °C at pH 4.0, pH 7.0 and pH 9.0, respectively each in duplicate. Aliquots of each test solution were analysed in time intervals using the analytical method as described later in this report.
Further hydrolysis tests were performed at 70 °C and 80 °C in the buffered test solution at pH 4.0 and at 60 °C and 70 °C in the aqueous buffered test solution at pH 9, due to the instability of the vinylated main product of FAT 40549/A found in the hydrolysis test at 50 °C.

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

107.5 µg/L

Remarks:

Mean of duplicate reading (104 and 111 µg/L)

Duration:

120 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

98.7 µg/L

Duration:

120 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

84.55 µg/L

Remarks:

Mean of duplicate reading (91.6 and 77.5 µg/L)

Number of replicates:

Duplicate

Positive controls:

no

Negative controls:

no

Statistical methods:

The obtained linear plots of the samples prove that the hydrolysis reaction is pseudo first order. The reaction rate constant k was calculated by regression analysis.

Preliminary study:

VINYLATION:
The analytical results of the sample solution of FAT 40'549/A at pH 9.0 indicate that the main product of FAT 40'549/A already starts to vinylate immediately after dissolving in the buffer solution pH 9.0. The vinylation of the main product of FAT 40'549/A at pH 9.0 was determined to be > 50% after 2.4 hours incubation at 50.0 °C ± 0.1 °C. According to the guidelines, the half-life time of the main product of FAT 40'549/A at pH 9.0 was estimated to be shorter than one day at 25 °C and therefore no further testing was performed at pH 9.0.
The half-life times of FAT 40'549/A at pH 4.0 and at pH 7.0 were estimated to be shorter than one year, but longer than one day at 25 °C. Therefore, according to the guidelines advanced testing of FAT 40'549/A was necessary to determine the rate constant (k2s) of the vinylation reaction at 25 °C in this pH range.

HYDROLYSIS:
The hydrolysis of the vinylated main product of FAT 40549/A at pH 7.0 was determined to be < 3 % at 50.0 °C ± 0.1 °C. According to the guidelines, the half-life times of the vinylated main product of FAT 40'549/A at pH 7.0 was estimated to be more than one year at 25 °C and therefore no further testing was performed at pH 7.0.
The results at pH 4.0 and pH 9.0 show that the vinylated main product of FAT 40'549/A is not stable at this pH values.
In conclusion, the half-life times of FAT 40'549/A at pH 4.0 and pH 9.0 were estimated to be shorter than one year, but longer than one day at 25 °C. Therefore, according to the guidelines advanced testing of FAT 40'549/A was necessary to determine the rate constant (k2s) of the hydrolysis reaction of the vinylated main product of FAT 40'549/A at pH 4.0 and pH 9.0.

Transformation products:

no

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

pH:

9

Temp.:

25 °C

DT50:

81 d

Type:

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

Details on results:

The half-life time of the vinylation reaction of FAT 40'549/A at pH 4.0 and 70 °C was calculated to be 85 hours.
The half-life time of the vinylation reaction of FAT 40'549/A at pH 4.0 and 80 °C was calculated to be 42 hours.
The half-life time of the vinylation reaction of FAT 40'549/A at 25 °C and pH 4.0 was estimated to be about 1.2 years using the Arrhenius equation.
The half-life time of the observed vinylation reaction of FAT 40'549/A at 25 °C and pH 7.0 was determined to be 8.9 days.
The half-life times of the hydrolysis reaction observed of the vinylated main product of FAT 40'549/A pH 4.0 was determined to be about 87 hours at 70 °C and to be about 40 hours at 80 °C.
The half-life times of the hydrolysis reaction observed of the vinylated main product of FAT 40'549/A pH 9.0 was determined to be about 18 hours at 60 °C and to be about 5.0 hours at 70 °C.
The half-life times of the hydrolysis reaction of the vinylated main product of FAT 40'549/A at 25 °C were estimated to be about 313 days at pH 4.0 and to be 81 days at pH 9.0.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the vinylation reaction (t1/2 (25 °C) is <24 hours) of the main product of FAT 40549/A and the following hydrolysis reaction (t1/2 (25 °C) = 81 days) of the vinylated main product formed was estimated to be fast at 25 °C and pH 9.

Furthermore, the main product of FAT 40549/A was considered to be stable at pH 4, since the half-life time for the vinylation reaction was estimated to be more than 1 year at 25 °C.

At pH 7, a relative slow vinylation (t1/2 (25 °C) = 9.8 days) of the main product of FAT 40549/A was determined at 25 °C and pH 7, but the vinylated main product formed is considered to be stable at this pH range (t1/2 (25 °C) > 1 year.)

Executive summary:

The determination of the abiotic degradation of FAT 40549/A was performed according to the OECD guideline no. 111 : "Hydrolysis as a function of pH" (1981) and the EEC directive 92/69, C7: "Abiotic degradation: hydrolysis as a function of pH" (1992).

The vinylation reaction of the main product of FAT 40549/A was determined at pH 4.0, pH 7.0 and pH 9.0 in the first section of this study. Due to the vinylation reaction found of the main product of FAT 40'549/A at 50 °C, further vinylation tests at pH 4.0 were performed at 60 °C and 70 °C and further vinylation tests at pH 7.0 were performed at 25 °C.

In the second section, the hydrolysis reaction of the vinylated main product of FAT 40549/A was observed at pH 4.0, pH 7.0 and pH 9.0. Due to the instability found of the vinylated main product of FAT 40'549/A at 50 °C, further hydrolysis tests at pH 4.0 were performed at 70 °C and 80 °C and further hydrolysis tests at pH 9.0 were performed at 60 °C and 70 °C.

In conclusion, the vinylation reaction (t_1/2(25 °C) < 24 hours) of the main product of FAT 40549/A and the following hydrolysis reaction (t_1/2(25 °C) = 81 days) of the vinylated main product formed was estimated to be fast at 25 °C and pH 9.

Furthermore, the main product of FAT 40549/A was considered to be stable at pH 4, since the half-life time for the vinylation reaction was estimated to be more than 1 year at 25 °C.

At pH 7, a relative slow vinylation (t_1/2(25 °C) = 9.8 days) of the main product of FAT 40549/A was determined at 25 °C and pH 7, but the vinylated main product formed is considered to be stable at this pH range (t_1/2(25 °C) > 1 year).

Description of key information

The vinylation reaction (t1/2 (25 °C) < 24 hours) of the main product of FAT 40'549/A and the following hydrolysis reaction (t1/2 (25 °C) = 81 days) of the vinylated main product formed was estimated to be fast at 25 °C and pH 9.
The main product of FAT 40'549/A was considered to be stable at pH 4, since the half-life time for the vinylation reaction was estimated to be more than 1 year at 25 °C.
At pH 7, a relative slow vinylation (t1/2 (25 °C) = 9.8 days) of the main product of FAT 40'549/A was determined at 25 °C and pH 7, but the vinylated main product formed is considered to be stable at this pH range (t1/2 (25 °C) > 1 year.)

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

The determination of the abiotic degradation of FAT 40549/A was performed according to the OECD guideline no. 111 : "Hydrolysis as a function of pH" (1981) and the EEC directive 92/69, C7: "Abiotic degradation: hydrolysis as a function of pH" (1992).

The vinylation reaction of the main product of FAT 40549/A was determined at pH 4.0, pH 7.0 and pH 9.0 in the first section of this study. Due to the vinylation reaction found of the main product of FAT 40'549/A at 50 °C, further vinylation tests at pH 4.0 were performed at 60 °C and 70 °C and further vinylation tests at pH 7.0 were performed at 25 °C.

In the second section, the hydrolysis reaction of the vinylated main product of FAT 40549/A was observed at pH 4.0, pH 7.0 and pH 9.0. Due to the instability found of the vinylated main product of FAT 40'549/A at 50 °C, further hydrolysis tests at pH 4.0 were performed at 70 °C and 80 °C and further hydrolysis tests at pH 9.0 were performed at 60 °C and 70 °C.

In conclusion, the vinylation reaction (t_1/2(25 °C)<24 hours) of the main product of FAT 40549/A and the following hydrolysis reaction (t_1/2(25 °C) = 81 days) of the vinylated main product formed was estimated to be fast at 25 °C and pH 9.

Furthermore, the main product of FAT 40549/A was considered to be stable at pH 4, since the half-life time for the vinylation reaction was estimated to be more than 1 year at 25 °C.

At pH 7, a relative slow vinylation (t_1/2(25 °C) = 9.8 days) of the main product of FAT 40549/A was determined at 25 °C and pH 7, but the vinylated main product formed is considered to be stable at this pH range (t_1/2(25 °C) > 1 year).