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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February 2010 to 11 May 2010
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)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products: beginning, 2.4 and 120 hours
- Sampling method:Before and after incubation, aliquots of the test solutions at each pH value and temperature were analyzed without dilution by measuring the UV/VIS signal of FAT 40850/A TE after HPLC separation of the injected sample solution.
- Sampling methods for the volatile compounds, if any: not applicable
- Sampling intervals/times for pH measurements: no data
- Sampling intervals/times for sterility check: no data
- Sample storage conditions before analysis: not applicable
- Other observation, if any (e.g.: precipitation, color change etc.): no observations reported
Buffers:
Buffer pH 4, Biphthalate Fluka Art. No. 82566
Buffer pH 7, Phosphate Fluka Art. No. 82571
Buffer pH 9, Borate Fluka Art. No. 82574
The buffer solutions were sterilized for 25 minutes in an autoclave prior to first use. Nitrogen was passed through the buffer solutions for at least 5 minutes except when freshly sterilized.
Estimation method (if used):
Arrhenius equation
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used:50mL Erlenmeyer flask, stoppered or sealed with an inert material
- Sterilisation method:All glassware, which must be inert in the pH range applied, was rinsed with sterile buffer. The buffer solutions were sterilized for 25 minutes in an autoclave
- Lighting: no data
- Measures taken to avoid photolytic effects:not applicable
- Measures to exclude oxygen:Nitrogen was passed through the buffer solutions for 5 minutes except when freshly sterilized.
- Details on test procedure for unstable compounds:Due to the instability of the test item found in the preliminary test at 50.0 °C, further hydrolysis tests were performed at 50 °C, 60 °C and 70 °C in the buffered test solution at pH 4.0 and pH 9.0. At pH 7.0 the test item was found to be stable at 50 °C.
- Details of traps for volatile, if any: not applicable
- If no traps were used, is the test system closed/open: closed
- Is there any indication of the test material adsorbing to the walls of the test apparatus? no observations reported

TEST MEDIUM
- Volume used/treatment: 50 ml
- Kind and purity of water: Millyy-Q supply
- Preparation of test medium: For example: 39.72 mg of FAT 40850/A TE were dissolved in 100 mL of water to prepare a stock solution of 397.2 μg/mL FAT 40850/A TE. Calculated volumes of this stock solution were diluted water to obtain ten standard solutions in the range from 0.397 μg/mL to 119.2 μg/mL FAT 40850/A TE.
- Renewal of test solution:Further sets of standard solutions were prepared accordingly. These solutions were prepared for calibration and also measured in respect to the calibration curve to verify the HPLC-system response during the test.
- Identity and concentration of co-solvent: not applicable
OTHER TEST CONDITIONS
- Adjustment of pH: solutions buffered to pH 4.0, 7.0 and 9.0
- Dissolved oxygen: No data reported. To reduce the oxygen in the solution, nitrogen was passed through for 5 minutes.
Duration:
265.53 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
2 835 856 - 2 836 075 other: peak area
Duration:
47.2 h
pH:
4
Temp.:
60 °C
Initial conc. measured:
1 541 556 - 1 545 665 other: peak area
Duration:
25.22 h
pH:
4
Temp.:
70 °C
Initial conc. measured:
1 735 620 - 1 744 004 other: peak area
Duration:
265.53 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
3 034 173 - 3 116 864 other: peak area
Duration:
47.2 h
pH:
9
Temp.:
60 °C
Initial conc. measured:
1 745 642 - 1 768 115 other: peak area
Duration:
96.1 h
pH:
9
Temp.:
70 °C
Initial conc. measured:
2 312 077 - 2 318 888 other: peak area
Number of replicates:
2
Positive controls:
no
Negative controls:
no
Preliminary study:
Incubation Time at 50.0 °C ± 0.1 °C
0 hours 2.4 hours 5 days
A A B A B
pH 4.0 2835856 2834147 0 1076345 62
2836075 2859976 -1 1083932 62
pH 7.0 2566132 2533608 1 2453174 4
2517971 2521052 0 2484823 1
pH 9.0 3116864 3047156 2 1222250 61
3034173 3011864 1 1204060 60
A: Peak Area measured [counts]
B: Hydrolyze Reaction [%]

The results obtained in the preliminary test at pH 7.0, showed no significant degradation of FAT 40850/A TE at 50 °C. According 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 environmental conditions (25 °C). Therefore, FAT 40850/A TE can be considered to be hydrolytically stable at pH 7.0 and no further testing was necessary at this pH-value.
Test performance:
Results Hydrolysis at 50°C

pH 7.0
The results obtained in the preliminary test at pH 7.0, showed no significant degradation of FAT 40850/A TE at 50 °C. According 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 environmental conditions (25 °C). Therefore, FAT 40850/A TE can be considered to be hydrolytically stable at pH 7.0 and no further testing was necessary at this pH-value.

pH 4.0 and 9.0
The results of the preliminary test showed, that the main compound of FAT 40850/A TE forming the main test item peak is not stable at pH 4.0 and pH 9.0. Therefore the test at 50 °C was prolonged in the range of 10% to 90% degradation.

The obtained data for FAT 40850/A TE at pH 4.0 and 50.0 °C (main test) are plotted at ln ct versus t. The linear plots prove that the hydrolysis reaction is of pseudo first order in the range from 10% to 90% hydrolysis at pH 4.0.

The obtained data for FAT 40850/A TE at pH 9.0 and 50.0 °C (main test) are plotted at ln ct versus t. The linear plots prove that the hydrolysis reaction is of pseudo first order in the range from 10% to 90% hydrolysis at pH 9.0

The reaction rate constant k for pH 4.0 and pH 9.0 was calculated by regression analysis.

The half-life time of the hydrolysis reaction at pH 4.0 and 50 °C was calculated to be (Main Peak): 131 hours (5.5 days)

The half-life time of the hydrolysis reaction at pH 9.0 and 50 °C was calculated to be (Main Peak): 94 hours (3.9 days)
Transformation products:
not measured
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0 h-1
DT50:
2 611 h
Type:
other: calculated with the Arrhenius equation
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0.005 h-1
DT50:
131 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
60 °C
Hydrolysis rate constant:
0.031 h-1
DT50:
22 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
70 °C
Hydrolysis rate constant:
0.048 h-1
DT50:
14 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
25 °C
DT50:
1 yr
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.002 h-1
DT50:
426 h
Type:
other: calculated with the Arrhenius equation
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.007 h-1
DT50:
94 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
60 °C
Hydrolysis rate constant:
0.027 h-1
DT50:
25 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
70
Hydrolysis rate constant:
0.024 h-1
DT50:
29 h
Type:
(pseudo-)first order (= half-life)
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): none

MAJOR TRANSFORMATION PRODUCTS
no data
FAT 40850/A TE showed several peaks in the HPLC-chromatogram. To determine the hydrolysis behavior, the degradation of the main peak was followed.

MINOR TRANSFORMATION PRODUCTS
no data

MINERALISATION (distinguish between dark and irradiated samples)
- % of applied radioactivity present as CO2 at end of study: not applicable

INDICATION OF UNSTABLE TRANSFORMATION PRODUCTS: no data

VOLATILIZATION (at end of study)
not applicable

UNIDENTIFIED RADIOACTIVITY (at end of study)
not applicable

PATHWAYS OF HYDROLYSIS
not applicable

SUPPLEMENTARY EXPERIMENT (if any): RESULTS: no data
Validity criteria fulfilled:
yes
Conclusions:
The main peak of FAT 40850/A TE at pH 4.0 has a half-life time of 2611 hours (109 days) at 25 °C.
The half-life time is longer than one year at 25 °C.
The main peak of FAT 40850/A TE at pH 9.0 has a half-life time of 426 hours (18 days) at 25 °C.
Executive summary:

The purpose of this study was to determine the hydrolysis behaviour of FAT 40850/A TE in aqueous buffered solutions adjusted to pH 4.0, pH 7.0 and pH 9.0. The study followed the protocol given in Council Regulation (EC) No. 440/2008, 30 May 2008, Part C, Methods for the determination of Ecotoxicity, C.7 “Abiotic Degradation: Hydrolysis as a Function of pH” and OECD Guideline for Testing of Chemicals No. 111, “Hydrolysis as a Function of pH"; adopted April 13, 2004 (only at pH 4.0, pH 7.0 and pH 9.0).

The hydrolysis of the test item was estimated at 50.0 °C ± 0.1 °C at each of pH 4.0, pH 7.0 and pH 9.0 in a preliminary test. FAT 40850/A TE was found to be stable at pH 7.0 and 50 °C. Therefore no further testing was performed at this pH value. FAT 40850/A TE showed several peaks in the chromatogram. The main peak, which was used for quantification was not stable at pH 4.0 and pH 9.0, therefore further testing was performed at elevated temperatures in order to calculate the rate constant (k25) and the half-life time of the hydrolysis at pH 4.0 and pH 9.0 at 25 °C.

The results are summarised below (the values typed in italics were calculated using the Arrhenius equation):

pH

Temperature

Reaction rate constant k

Reaction rate constant k

Half-life time t 1/2

 

[°C]

[1/hours]

[1/s]

[hours]

4.0

25

2.65 x 10-4

7.37 x 10-8

 2611

50

5.29 x 10-3*

1.47 x 10-6

 131

60

3.10 x 10-2*

8.61 x 10-6

 22

70

4.81 x 10-2*

1.34 x 10-5

 14

9.0

25

1.63 x 10-3

4.51 x 10-7

 426

50

7.37 x 10-3*

2.05 x 10-6

 94

60

2.74 x 10-2*

7.62 x 10-6

 25

70

2.44 x 10-2*

6.79 x 10-6

 29

* The tabulated value is the mean of two samples.

FAT 40850/A TE is stable at pH 7.0. Its half-life time is longer than one year at 25 °C. The test item has a half-life time of 2611 hours (109 days) at 25 °C and pH 4.0 and the half-life time at pH of 9.0 and 25 °C was determined to be 426 hours (18 days).

Description of key information

Based on the read across data generated from the hydrolysis study with Reactive Red 286 FAT 40850/A, the main peak and half life of target substance Reactive Red 024:1 is also considered as stable at pH 7.0. Its half-life time is longer than one year at 25 °C. The test item has a half-life time of 2611 hours (109 days) at 25 °C and pH 4.0 and the half-life time at pH of 9.0 and 25 °C was determined to be 426 hours (18 days).

Key value for chemical safety assessment

Half-life for hydrolysis:
1 yr
at the temperature of:
20 °C

Additional information

Data on hydrolysis study was not available for the target substance Reactive Red 024:1. To fill the data gaps, read across approach is adapted using similar substance Reactive Red 286 (FAT 40850). Read-across is claimed basis of structural relationship of the target and the source chemicals. Read across substance have been investigatedto determine the hydrolysis behaviour of FAT 40850/A TE in aqueous buffered solutions adjusted to pH 4.0, pH 7.0 and pH 9.0. The study followed the protocol given in Council Regulation (EC) No. 440/2008, 30 May 2008, Part C, Methods for the determination of Ecotoxicity, C.7 “Abiotic Degradation: Hydrolysis as a Function of pH” and OECD Guideline for Testing of Chemicals No. 111, “Hydrolysis as a Function of pH"; adopted April 13, 2004 (only at pH 4.0, pH 7.0 and pH 9.0).

The hydrolysis of the test item was estimated at 50.0 °C ± 0.1 °C at each of pH 4.0, pH 7.0 and pH 9.0 in a preliminary test. FAT 40850/A TE was found to be stable at pH 7.0 and 50 °C. Therefore no further testing was performed at this pH value. FAT 40850/A TE showed several peaks in the chromatogram. The main peak, which was used for quantification was not stable at pH 4.0 and pH 9.0, therefore further testing was performed at elevated temperatures in order to calculate the rate constant (k25) and the half-life time of the hydrolysis at pH 4.0 and pH 9.0 at 25 °C.

The results are summarised below (the values typed in italics were calculated using the Arrhenius equation):

pH

Temperature

Reaction rate constant k

Reaction rate constant k

Half-life time t 1/2

 

[°C]

[1/hours]

[1/s]

[hours]

4.0

25

2.65 x 10-4

7.37 x 10-8

 2611

50

5.29 x 10-3*

1.47 x 10-6

 131

60

3.10 x 10-2*

8.61 x 10-6

 22

70

4.81 x 10-2*

1.34 x 10-5

 14

9.0

25

1.63 x 10-3

4.51 x 10-7

 426

50

7.37 x 10-3*

2.05 x 10-6

 94

60

2.74 x 10-2*

7.62 x 10-6

 25

70

2.44 x 10-2*

6.79 x 10-6

 29

* The tabulated value is the mean of two samples.

Based on the read across data generated from the 28-day repeated dose oral toxicity with FAT 40850/A, the main peak and half life of target substance Reactive Red 024:1 is also considered as below.

Test substance is stable at pH 7.0. Its half-life time is longer than one year at 25 °C. The test item has a half-life time of 2611 hours (109 days) at 25 °C and pH 4.0 and the half-life time at pH of 9.0 and 25 °C was determined to be 426 hours (18 days).