disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)naphthalene-2,7-disulfonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 15,2010-September 20, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Remarks:

The test was ordered and performed in a GLP laboratory, with the same quality standards

Analytical monitoring:

yes

Buffers:

The test was carried out at three pH values: 4.0, 7.0 and 9.0. The actual pH values at given temperatures were measured using a calibrated pH meter.
Buffer solutions were prepared in accordance with the Method C.7 – Degradation – Abiotic Degradation: Hydrolysis as a Function of pH, Directive 92/69/EEC. Published in OJ No L 142/518 A, May 2008, appendix 3: Buffer Systems – Clark and Lubs:

Buffer pH 4:
10.2 g of Potassium hydrogen phthalate were weighed into a 1000mL volumetric flask and Potassium hydrogen phthalate was dissolved in ca 800 mL water. After it 4 mL of 0.1M NaOH were added and the volumetric flask was filled up to its volume with water.
pH = 4.04 (23.1 °C)

Buffer pH 7:
6.8 g of KH2PO4 were weighed into a 1000mL volumetric flask and KH2PO4 was dissolved in ca 800 mL water. After it 296 mL of 0.1M NaOH were added and the volumetric flask was filled up to its volume with water.
pH = 7.00 (23.1 °C)

Buffer pH 9:
3.09 g of H3BO3 and 3.73 g of KCl were weighed into a 1000mL volumetric flask and substances were dissolved in ca 800 mL water. After it 213 mL of 0.1M NaOH were added and the volumetric flask was filled up to its volume with water.
pH = 9.00 (23.0 °C)

Details on test conditions:

Instruments and equipment
Liquid chromatograph with UV detection, Shimadzu LC20AC
Analytical Balance Mettler AT 261, capable of weighing to the nearest 0.01 mg (preparation of sample solution and calibration solutions)
Analytical Balance Mettler PB 3002DR, capable of weighing to the nearest 0.01 mg (preparation of buffers and mobile phases)
pH meter HANNA instruments Model 213, capable of measuring 0.01 pH units (HANNA Instruments)
Thermometer TESTO 925 (calibration certificate No. 929/2010)
Thermostat Julabo F34
Ultrasonic Bath with timer SONOREX RK 510 (Bandelin)
DEMIWA 10 Rosa – system for preparation of deionised water (Watek)
Standard laboratory glassware


3.3. Reagents and standards
Deionised water
NaOH p.a. (>98%), Lach-Ner
Potassium hydrogen phthalate puriss., p.a. (>99.9%), Sigma Aldrich
KH2PO4 p.a. (>99 %), Lach-Ner
H3BO3 p.a. (>99.5%), Lachema
KCl p.a. (min 99 %), Litolab
CH3COONH4 p.a. (>99 %), Penta
Buffer solutions CertiPUR (pH 4.01/pH 7.00/pH 9.00), Cat. No. 199005, Merck
Buffer solution CertPUR (pH 9.18), Cat. No. 107203, Merck
Methanol, gradient grade, Lab-Scan


0.1M NaOH: 4.0 g of NaOH were weighed into a 1000mL volumetric flask, dissolved in and diluted to volume with deionised water.

3.5. Samples preparation
All solutions, i.e. deionised water and buffers used for preparation of the samples in hydrolysis testing were before testing bubbled by nitrogen and sterilized. All laboratory glassware used for preparation of the samples in hydrolysis testing was before testing sterilized.
About 200 mg of the test substance was weighed into a 100 ml volumetric flask, added ca 50 ml of the deionised water. The mixture was sonicated for 5 minutes and the volumetric flask was filled up to volume with the deionised water.
10 ml of this mixture was pipetted to 100 ml volumetric flask (six times). After the volumetric flasks (two flasks for each buffer) were filled up to its volume with the appropriate buffer, wrapped into an aluminium foil and tightly closed with a stopper.
The volumetric flask was kept in the thermostatic bath during one whole test. For analyses required volumes were only withdrawn from the volumetric flask. The withdrawn samples were quickly cooled to room temperature (ca 25 °C) without delay and analysed.


3.7. Temperature determination
The actual temperature of the thermostatic bath was daily controlled during the whole test. A calibrated thermometer was used and a temperature correction was taken into account via information from the calibration list,
i.e. 50,0°C (Etalon-measurement standard) ~ 49.7°C (Thermometer)


3.8. pH determination
The actual pH value of each buffer was measured. The pH-meter calibrated to buffer solutions CertiPUR was used.

Duration:

5 d

pH:

4.08

Initial conc. measured:

ca. 205.46 mg/L

Duration:

5 d

pH:

6.91

Initial conc. measured:

ca. 204.5 mg/L

Duration:

5 d

pH:

8.8

Initial conc. measured:

ca. 203.86 mg/L

Number of replicates:

2

Preliminary study:

Preliminary Test (Tier 1)
The preliminary test is performed at 50 +/- 0.5 °C and pH 4.0, 7.0, and 9.0. If less than 10% of hydrolysis is observed after 5 days (t0.525°C > 1 year)*, the test substance is considered hydrolytically stable and, normally, no additional testing is required. If more than 10% of hydrolysis is observed within 5 days the test continues with Tier 2. If the substance is known to be unstable at environmentally relevant temperatures, the preliminary test is not required and the test starts with Tier 2 immediately. *) 10% hydrolysis of a test substance at 50 °C corresponds to half-life of approx.. 30 days which corresponds to a value of approx. 1 year at 25 °C.

Transformation products:

no

% Recovery:

ca. 97

St. dev.:

0.3

pH:

4.08

Temp.:

50 °C

Duration:

ca. 5 d

% Recovery:

ca. 96.15

St. dev.:

0.4

pH:

6.91

Temp.:

50 °C

Duration:

ca. 5 d

% Recovery:

ca. 92.35

St. dev.:

0.3

pH:

8.8

Temp.:

50 °C

Duration:

ca. 5 d

pH:

4.08

Temp.:

25 °C

DT50:

> 1 yr

pH:

6.91

Temp.:

25 °C

DT50:

> 1 yr

pH:

8.8

Temp.:

25 °C

DT50:

> 1 yr

Validity criteria fulfilled:

yes

Conclusions:

The preliminary test at all pH (4.08, 6.91 and 8.80) showed that less than 10% of the test substance was hydrolysed within 5 days period. The half-life time of hydrolysis can therefore be expected greater than 1 year at 25°C and further testing is not required.

Description of key information

Not hydrolysable

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

A single screening study (Jana Netusilova, 2011) was performed on Acid Black 210 at different pH showing less than 10% of the test substance hydrolysing within 5 days period.