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Environmental fate & pathways

Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September - 21 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study, conducted to recognised guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Radiolabelling:
no
Analytical monitoring:
no
Details on sampling:
Not applicable - screening test; substance was sampled and analysed after 5 days.
Buffers:
Buffer solutions:
pH 4.0: 5.11 g Potassium hydrogen phthalate was measured into a 500 ml volumetric flask and 1 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.
pH 7.0: 3.40 g Potassium dihydrogen phosphate was measured into a 500 ml volumetric flask and 73.9 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.
pH 9.0: 1.55 g Boric acid and 1.87 g Potassium chloride were measured into a 500 ml volumetric flask and 53.5 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.

These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water.
The pH of each buffer solution was checked with a calibrated pH meter.
Estimation method (if used):
Not used.
Details on test conditions:
PRINCIPLE OF THE TEST

Sterile aqueous buffer solutions of different pH values are treated with the test substance and incubated in the dark for 5 days. Concentration of the test substance is determined at the start and at the end of incubation.

APPARATUS

HPLC system:
Merck-Hitachi LaChrom HPLC system:
D-7000 Interface, No.: 1231-056
L-7100 HPLC pump, No.: 1272-039
L-7200 Autosampler, No.: 1273-016
L-7400 UV Detector, No.: 1260-088
Balances:
BP221S Sartorius, No.: 11809117
L2200P Sartorius, No.: 38100037
Thermostat: LP 132, No.: 870595
pH meter: METTLER TOLEDO S20 Easy Seven, No.: 1231025119
Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I
Hot Air Steriliser: ATP line FED, WTB Binder, No.: 9110-0035
Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105
Centrifuge: Heraeus Biofuge, No.: 272369
Laminar air flow instrument: CAPTAIR®bio, No.: 1222001


HPLC Conditions:

Detector: UV at 210 nm
Column: ZORBAX SB-C8; 250x4.6 mm; 5 µm No.: USSE010939
Mobil Phase: Acetonitrile : Water : Tetrahydrofuran = 70 : 20 : 10
Flow: 1 ml/min.
Injection volume: 10 *l

Results of the method validation are detailed in Table 1 below:

TEST CONDITIONS

Test temperature: 50 ¿ 1.0 °C

pH: Hydrolysis was examined at three different pH values: 4.0, 7.0 and 9.0 in the dark.

Buffer solutions:
pH 4.0: 5.11 g Potassium hydrogen phthalate was measured into a 500 ml volumetric flask and 1 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.
pH 7.0: 3.40 g Potassium dihydrogen phosphate was measured into a 500 ml volumetric flask and 73.9 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.
pH 9.0: 1.55 g Boric acid and 1.87 g Potassium chloride were measured into a 500 ml volumetric flask and 53.5 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.

These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water.
The pH of each buffer solution was checked with a calibrated pH meter.

Light: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects.

Oxygen: Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.


PERFORMANCE OF THE TEST

Test solutions: Kronitex TXP was dissolved in acetone and spiked in sterile buffer solutions. The pH of each buffer solution was checked with a calibrated pH meter.

Storage of the solutions: Solutions were transferred into 25 ml screw cap tubes. From each test solution five tubes were prepared. From control samples two test tubes were prepared.
The tubes were thermostated at 50 °C +/- 1 deg C.

Sampling: The reaction solutions were analysed at the start of the test and after five days with five replicate samples each.

Analysis of the samples: Samples were diluted with eluent, then they were analysed with the above presented HPLC method.
Duration:
5 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
9.6 other: µg/ml
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
8.8 other: µg/ml
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
9.7 other: µg/ml
Number of replicates:
5
Positive controls:
no
Negative controls:
no
Statistical methods:
Evaluation: The chromatograms were evaluated with the help of “LaChrom Chromatogram Processor".
Calculations were carried out using “EXCEL for Windows".
Preliminary study:
In the course of the preliminary test the observed hydrolysis of Kronitex TXP was less than 10 per cent after 5 days at a temperature of 50 °C at pH 4, 7 and 9. Therefore Kronitex TXP is considered to be hydrolytically stable under these circumstances and normally no additional testing is required.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Not applicable.
% Recovery:
100
pH:
4.09
Temp.:
50 °C
Duration:
5 d
% Recovery:
97
pH:
7.07
Temp.:
50 °C
Duration:
5 d
% Recovery:
100
pH:
9.09
Temp.:
50 °C
Duration:
5 d
Key result
pH:
4
Temp.:
50 °C
DT50:
> 1 yr
Key result
pH:
7
Temp.:
50 °C
DT50:
> 1 yr
Key result
pH:
9
Temp.:
50 °C
DT50:
> 1 yr
Other kinetic parameters:
None
Details on results:
Calibration
The calibration series was prepared in eluent using the test item. It was measured at both analytical occasions. Concentrations of the calibration samples were 0.1, 0.2, 0.5, 2, 5, 10, 20 and 50 µg / ml. Parameters of the resulting equations are given in Table 2 below.

Results of the hydrolysis test
The hydrolysis test was performed at 50 +/- 0.5 deg C, at pH 4, 7 and 9. Measured concentrations and pH values are summarised in Table 3 below.

Table 2.: Regression data

Analytical occasion

Intercept

Slope

Correlation Coefficient.

16 September 2010

2414

26610

1.000

21 September2010

-427

27660

1.000

 

Table 3.: Measured data

pH

Sampling time, hour

Concentration of Test Item , µg/ml

Measured pH

Results of the separate test vessels

Mean withthe 95% confidence intervals

End/Start, %

4

0

(Start)

Control buffer

-

4.02

9.7

9.6 ± 0.12

-

4.09

9.5

9.7

9.6

9.5

120

Control buffer

-

4.02

9.5

9.6 ± 0.18

100

4.10

9.7

9.6

9.7

9.4

7

0

(Start)

Control buffer

-

7.02

9.0

9.1 ± 0.13

-

7.04

9.1

9.2

9.2

9.0

120

Control buffer

-

7.01

9.4

8.8 ± 1.77

97

7.07

8.3

8.4

*

9.3

9

0

(Start)

Control buffer

-

9.02

9.7

9.8 ± 0.08

-

9.06

9.7

9.8

9.7

9.8

120

Control buffer

-

9.02

10.7

9.7 ± 0.94

100

9.09

8.8

10.2

9.3

9.6

* sample preparation error

 

Validity criteria fulfilled:
yes
Conclusions:
At pH 4, 7 and 9, Kronitex TXP proved to be hydrolytically stable.
Executive summary:

At pH 4, 7 and 9, Kronitex TXP proved to be hydrolytically stable. The substance is not considered to achieve removal / degradation by hydrolysis.

Description of key information

Hydrolysis as a function of pH

Key value for chemical safety assessment

Half-life for hydrolysis:
1 yr
at the temperature of:
50 °C

Additional information

The substance was stable to hydrolysis at pH 4,7 and 9 at 50 °C. The half life at all pH's is considered to be > 1 year.