Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 07 July 2006; Experiment completion date - 09 August 2006; Study completion date - 24 January 2007.

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:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Standard Solutions
31.74 mg of FAT 40827/A was dissolved in 100 ml of water to prepare a stock solution of 317.4 µg/ml FAT 40827/A. Calculated volumes of this stock solution were diluted with water to obtain several standard solutions. Six of these solutions in the range from 6.348 µg/ml to 126.96 µg/ml FAT 40827/A were used to calibrate the HPLC-system.
During and at the end of the test the stability and accuracy of the calibration solution was confirmed by preparing and analysing new calibration solutions.

Sample Preparation
Before and after incubation, 10 µl aliquots of the test solutions at each pH value were analysed without dilution by measuring the UV signal of FAT 40827/A after HPLC separation of the injected sample solution

Buffers:

Buffer pH 4, Biphthalate: Baker Art No. 5657
Buffer pH 7, Phosphate: Baker Art No. 5656
Buffer pH 9, Borate: Baker Art No. 7145

Estimation method (if used):

HPLC

Details on test conditions:

Glassware: All glassware, which must be inert in the pH range applied, was rinsed with sterile buffer. The hydrolysis was carried out in flasks, which were stoppered or sealed with an inert material. The test vessels were labelled with the following information: RCC study number and the additional information necessary to assure unmistakable identification.

Water Baths: Thermostatically controlled water baths: Julabo SW22 from Julabo

Balance: Mettier XP205DR

The test item was dissolved in an aqueous solution at a specific pH-value (pH 4.0, pH 7.0 and pH 9.0) and was incubated at a specific temperature with a maximum deviation of ± 0.5 °C and then kept constant within ± 0.1 °C.

Duration:

48 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

>= 173.8 - <= 174.9 µg/L

Duration:

58 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

>= 184.1 - <= 185.6 µg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

>= 99.5 - <= 106.1 µg/L

Number of replicates:

Two samples were determined for each pH value at different temperature.

Positive controls:

no

Negative controls:

no

Statistical methods:

Injected samples were quantified by peak area with reference to the calibration curve. The latter was obtained by correlation of the peak area of the analytical standards with their corresponding concentration. The correlation was performed using a least squares fit of a linear function. Therefore, the rate constant k is the slope of a plot of In ct versus t.

Preliminary study:

Test item was found to be stable at pH 7.0 and 50 °C. Therefore no further testing was performed at this pH-value.

Test item was not stable at pH 4.0 and at pH 9.0, further testing was performed at 50 °C, 60 °C and 70 °C in the buffered test solution at pH 4.0 and at
pH 9.0.

Test performance:

The retention time of test item was at about 25.6 minutes under the HPLC-conditions.

Transformation products:

not measured

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.003 h-1

DT50:

206 h

Type:

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

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

Remarks on result:

other: The hydrolysis rate constant was not reported.

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

867 h

Type:

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

pH	Temperature (°C)	Reaction rate constant K (1/hours)	Reaction rate constant K (1/s)	Half-life time (hours)
4.0	25	3.37×10^-3	9.36×10^-7	206
	50	2.53×10^-2	7.02×10^-6	27
	60	4.91×10^-2	1.36×10^-5	14
	70	1.01×10^-1	2.82×10^-5	7
9.0	25	7.99×10^-4	2.22×10^-7	867
	50	1.94×10^-2	5.39×10^-6	36
	60	5.54×10^-2	1.54×10^-5	13
	70	1.75×10^-1	4.86×10^-5	4

Validity criteria fulfilled:

yes

Conclusions:

Test item at pH 4.0 has a half-life time of 206 hours (9 days) at 25 °C, its half-life time is longer than one year at 25 °C and pH 7.0, and at pH 9.0 has a half-life time of 867 hours (36 days) at 25 °C.

Executive summary:

The test was performed according to OECD Guideline for Testing of Chemicals No. 111 (2004) and EEC Directive 92/69, Section C.7 (1992) to evaluate the hydrolysis of test item as a function of pH. The test item was dissolved in aqueous buffer solution, and the temperature and pH were checked at pH 4, 7 and 9, respectively. The decrease of the concentration with time was determined by HPLC. The rate constant and the half-life period were calculated using the slope of the line (the logarithms of the concentration against time). The test results showed that the test substance at pH 4 has a half-life period of 206 hours (9 days) at 25 ℃, at pH 7 is stable with a half-life of more than one year at 25 ℃, and at pH 9 has a half-life period of 867 hours (36 days) at 25 ℃.

Description of key information

The test substance at pH 4 has a half-life period of 206 hours (9 days) at 25 ℃, at pH 7 is stable with a half-life of more than one year at 25 ℃, and at pH 9 has a half-life period of 867 hours (36 days) at 25 ℃.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

The test was performed according to OECD Guideline for Testing of Chemicals No. 111 (2004) and EEC Directive 92/69, Section C.7 (1992) to evaluate the hydrolysis of test item as a function of pH. The test item was dissolved in aqueous buffer solution, and the temperature and pH were checked at pH 4, 7 and 9, respectively. The decrease of the concentration with time was determined by HPLC. The rate constant and the half-life period were calculated using the slope of the line (the logarithms of the concentration against time). The test results showed that the test substance at pH 4 has a half-life period of 206 hours (9 days) at 25℃, at pH 7 is stable with a half-life of more than one year at 25 ℃, and at pH 9 has a half-life period of 867 hours (36 days) at 25 ℃.