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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2020 - 4 March 2020
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)
Version / remarks:
April 13 2004
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test material was stored in a dark storage place at room temperature.
- Stability under storage conditions: stable.
- Solubility in dimethylsulfoxide: ≤ 439 g/L
- Solubility in acetone: ≤ 0.03 g/L
- Water solubility: ≤ 310 g/L
Radiolabelling:
not specified
Analytical monitoring:
yes
Details on sampling:
Tier 1
- Number of test repetitions: 1 at initiation and 2 after 5 days.
- Analytical method: HPLC.
- Light condition: test vessel was covered with aluminium foil.

Tier 2
- Number of repetitions: 1 at initiation and 2 at each measurement point.
- Analytical method: HPLC.
- Light condition: test vessel was covered with aluminium foil.

Tier 3
- Analytical method: LC-MS.
Buffers:
Three buffer solutions were prepared.
- pH of buffer solution 4.0
- Preparation method: The solution was prepared by mixing sodium hydroxide solution (4.5 mL, 1 mol/L) and potassium dihydrogen citrate solution (50 mL, 0.5 mol/L), and then filling up to 500 mL with purified water. The pH was then adjusted to pH 4.0 with 1 mol/L hydrochloric acid.

- pH of buffer solution 7.0
- Preparation method: The solution was prepared by mixing sodium hydroxide solution (14.8 mL, 1 mol/L) and potassium dihydrogen citrate solution (50 mL, 0.5 mol/L), and then filling up to 500 mL with purified water.

- pH of buffer solution 9.0
- Preparation method: The solution was prepared by mixing sodium hydroxide solution (10.7 mL, 1 mol/L) and potassium chloride (50 mL, 0.5 mol/L) and boric acid solution (50 mL, 0.5 mol/L), and then filling up to 500 mL with purified water.
Details on test conditions:
TEST EQUIPMENT
- Dry thermo unit, dry thermo bath, thermostatic water bath and multi function water quality meter.
- Test vessel: 10 mL test tube with glass stopper.

TEST CONDITIONS
Tier 1 (pH 7.0):
- Test concentration: 145 mg/L
- Volume of test solution: 10 mL
- Test temperature: 50 ± 0.5 °C
- Test duration: 5 days.
- Measurement point: At initiation and after 5 days.
- Number of test repetitions: At initiation: 1. After 5 days: 2.
- Light conditions: Test vessel was covered with aluminium foil.
Tier 2 and 3 (pH 4.0 and pH 9.0):
- Test concentration: 145 mg/L
- Volume of test solution: 10 mL
- Test temperature (pH 4.0): 50 ± 0.5 °C, 60 ± 0.5 °C, 70 ± 0.5 °C
- Test duration (pH 4.0): 34 days, 24 days, 10 days.
- Test temperature (pH 9.0): 50 ± 0.5 °C, 60 ± 0.5 °C, 70 ± 0.5 °C
- Test duration (pH 9.0): 18 days, 4 days, 2 days.
- Measurement point: At initiation and 6 points in test duration.
- Number of test repetitions: At initiation: 1. At each measurement point: 2.
- Light conditions: Test vessel was covered with aluminium foil.

TEST MATERIAL SOLUTION
- The test material (200.14 mg) was dissolved in purified water with ultrasonic irradiation and then filled up to 10 mL with purified water to obtain 14500 mg/L test material solution.

TEST PROCEDURE
- Glassware sterilised with ethanol.
- The test material solution (2 mL, 29.0 mg as test material) was added to a 200 mL volumetric flask and filled up with each buffer solution.
- The solution was aerated for 5 minutes with helium gas after filtration with a sterile filter (0.22 μm).
- This solution (10 mL) was poured into respective test vessels which were then sealed.
- At initiation one test solution was pretreated and analysed.
- Remaining test solutions were warmed at test temperature.
- The test solutions were analysed by cooling to room temperature before being pretreated.

CONFIRMATION OF STERILE CONDITIONS
The sterile conditions were confirmed for one of the test solutions at the final measurement point of each test temperature at pH 4.0 and pH 9.0 according to the plate culture method using agar medium.
Specifically, the mixture of Tryptic Soy Agar medium (4 g) and purified water (100 mL) was sterilised with autoclave at 121°C for 15 minutes to prepare an agar medium, and then the agar medium was maintained at about 50°C in a water bath. The test solution (1 mL) and the agar media (ca 15 mL) were mixed in a sterilised petri dish, before being cooled to room temperature. once solidified, the petri dish was made reverse and maintained at 25 ± 1°C for > 5 days in an incubator. The colony count on the agar medium was recorded.

CALCULATION FOR RESIDUAL PERCENTAGE OF TEST MATERIAL
- Tier 1:
- % = concentration of test material after 5 days (mg/L) divided by concentration of test material at initiation (mg/L) multiplied by 100.
- In case that the residual percentage of the test material is larger than 90%, the test material is judged to be hydrolytically stable.
- Tier 2:
- A regression equation was obtained by plotting logarithm of the test material concentration at each time (log C) versus time (t).
- Slope (a) and intercept (c) were calculated at pH 4.0 and pH 9.0.
- Regression equation: logC = at + b
- Hydrolysis rate constant of test material and the half life were calculated using k = -2.303 x a and t(1/2) = 0.693 / k. Where k is the hydrolysis rate constant and t(1/2) is the half life.
- Logarithm values of logk were plotted versus 1/T. The hydrolysis rate and the half life at 25 °C were calculated by extrapolation from the regression equation.
Duration:
5 d
pH:
7.02
Temp.:
50 °C
Initial conc. measured:
145 mg/L
Duration:
34 d
pH:
4.01
Temp.:
50 °C
Initial conc. measured:
144 mg/L
Duration:
24 d
pH:
4.03
Temp.:
60 °C
Initial conc. measured:
145 mg/L
Duration:
10 d
pH:
4.03
Temp.:
70 °C
Initial conc. measured:
145 mg/L
Duration:
18 d
pH:
8.99
Temp.:
50 °C
Initial conc. measured:
144 mg/L
Duration:
4 d
pH:
9.01
Temp.:
60 °C
Initial conc. measured:
145 mg/L
Duration:
2 d
pH:
9.01
Temp.:
70 °C
Initial conc. measured:
145 mg/L
Number of replicates:
Tier 1:
- At initiation: 1. After 5 days: 2.
Tier 2 and 3:
- At initiation: 1. At each measurement point: 2.
Positive controls:
no
Negative controls:
no
Preliminary study:
- The test material was not hydrolysed at pH 7.0, it was hydrolysed at pH 4.0 and pH 9.0 therefore the Tier 1 test was performed at pH 7.0 and Tier 2 and 3 tests were performed at pH 4.0 and pH 9.0.
Transformation products:
not specified
Remarks:
Hydrolysis products were observed - not further specified (unknown identities)
Details on hydrolysis and appearance of transformation product(s):
- Retention time 11.3 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0/805.9 [Mt -3Na + 2H]^-
- Retention time 11.3 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0 [Mt -3Na + 2H]^-
- Retention time 11.4 – 11.5 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 803.9/805.9 [Mt -3Na + 2H]^-
- Retention time 11.4 – 11.5 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.4 [Mt -3Na + H]^2-, 803.9/806.0 [Mt -3Na + 2H]^-
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0 h-1
DT50:
256 d
Type:
not specified
Remarks on result:
other: Peak 1
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0 h-1
DT50:
309 d
Type:
not specified
Remarks on result:
other: Peak 2
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.002 h-1
DT50:
187 d
Type:
not specified
Remarks on result:
other: Peak 1
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.002 h-1
DT50:
196 d
Type:
not specified
Remarks on result:
other: Peak 2
Details on results:
- The colony count was determined to be zero at pH 4.0 and pH 9.0, at test temperatures of 50, 60 and 70 °C.
- The residual percentage of test material at pH 7.0 was larger than 90% in Tier 1 therefore the test material is hydrolytically stable at pH 7.0.
- At pH 4.0 and pH 9.0 the hydrolysis rate constant and half life at 25 °C are considered to be acceptable as the regression equation was confirmed to be a straight. As the colony was not counted after the test it was considered that sterile conditions were maintained therefore, microorganism was not affected by the production of hydrolysis products.

Hydrolysis rate constant and half life of test material.

Test solution

Hydrolysis rate constant (hour^-1)

Half life (hour)

pH

Peak

Test Temperature (°C)

Measured Value

Average

Measured Value

Average

4.0

1

50

1.10 x 10^-3

1.11 x 10^-3

628

624

 

1.12 x 10^-3

619

60

3.06 x 10^-3

2.99 x 10^-3

227

232

2.93 x 10^-3

237

70

5.24 x 10^-3

5.64 x 10^-3

132

124

6.03 x 10^-3

115

2

50

1.00 x 10^-3

1.01 x 10^-3

692

688

1.01 x 10^-3

684

60

2.79 x 10^-3

2.73 x 10^-3

248

254

2.67 x 10^-3

259

70

5.00 x 10^-3

5.42 x 10^-3

139

129

5.85 x 10^-3

118

9.0

1

50

3.47 x 10^-3

3.46 x 10^-3

200

200

3.45 x 10^-3

201

60

1.51 x 10^-2

1.53 x 10^-2

45.9

45.2

1.56 x 10^-2

44.5

70

3.25 x 10^-2

3.22 x 10^-2

21.4

21.5

3.20 x 10^-2

21.7

2

50

3.17 x 10^-3

3.14 x 10^-3

219

221

3.12 x 10^-3

222

60

1.42 x 10^-2

1.44 x 10^-2

48.9

48.2

1.46 x 10^-2

47.5

70

2.86 x 10^-2

2.85 x 10^-2

24.2

24.3

2.84 x 10^-2

24.4

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study the half life at pH 4.0 was 256 days for peak one and 309 days for peak 2. The hydrolysis rate constant was 1.13 x 10^-4 hour^-1 and 3.09 x 10^-4 hour^-1 respectively. The half life at pH 9.0 was 187 days for peak 1 and 196 days for peak 2. The hydrolysis rate constant was 1.75 x 10^-3 hour^-1 for both peaks.
Executive summary:

The hydrolytic stability of the test material was investigated in accordance with the standardised guidelines OECD 111 under GLP conditions.

The preliminary test concluded that the test material was hydrolytically stable at pH 7.0. The test material was not hydrolytically stable at pH 4.0 and pH 9.0.

The residual percentage of test material at pH 7.0 was larger than 90% in Tier 1 therefore the test material is hydrolytically stable at pH 7.0.

At pH 4.0 and pH 9.0 the hydrolysis rate constant and half life at 25 °C were considered to be acceptable as the regression equation was confirmed to be a straight. As the colony was not counted after the test it was considered that sterile conditions were maintained therefore, microorganism was not affected by the production of hydrolysis products.

The hydrolysis product of the test material was identified using LC-MS. One of the test solutions at pH 4.0 (70 °C) and one of the test solutions at pH 9.0 (50 °C) were used as the test solutions for qualitative analysis.

At a retention time of 11.3 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0/805.9 [Mt -3Na + 2H]^-

At a retention time of 11.3 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0 [Mt -3Na + 2H]^-

At a retention time of 11.4 – 11.5 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 803.9/805.9 [Mt -3Na + 2H]^-

At a retention time of 11.4 – 11.5 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.4 [Mt -3Na + H]^2-, 803.9/806.0 [Mt -3Na + 2H]^-

Under the conditions of the study the half life at pH 4.0 was 256 days for peak one and 309 days for peak 2. The hydrolysis rate constant was 1.13 x 10^-4 hour^-1 and 3.09 x 10^-4 hour^-1 respectively. The half life at pH 9.0 was 187 days for peak 1 and 196 days for peak 2. The hydrolysis rate constant was 1.75 x 10^-3 hour^-1 for both peaks.

Description of key information

Under the conditions of the study the half life at pH 4.0 was 256 days for peak one and 309 days for peak 2. The hydrolysis rate constant was 1.13 x 10^-4 hour^-1 and 3.09 x 10^-4 hour^-1 respectively. The half life at pH 9.0 was 187 days for peak 1 and 196 days for peak 2. The hydrolysis rate constant was 1.75 x 10^-3 hour^-1 for both peaks.

Key value for chemical safety assessment

Additional information

The hydrolytic stability of the test material was investigated in accordance with the standardised guidelines OECD 111 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The preliminary test concluded that the test material was hydrolytically stable at pH 7.0. The test material was not hydrolytically stable at pH 4.0 and pH 9.0.

The residual percentage of test material at pH 7.0 was larger than 90% in Tier 1 therefore the test material is hydrolytically stable at pH 7.0.

At pH 4.0 and pH 9.0 the hydrolysis rate constant and half life at 25 °C were considered to be acceptable as the regression equation was confirmed to be a straight. As the colony was not counted after the test it was considered that sterile conditions were maintained therefore, microorganism was not affected by the production of hydrolysis products.

The hydrolysis product of the test material was identified using LC-MS. One of the test solutions at pH 4.0 (70 °C) and one of the test solutions at pH 9.0 (50 °C) were used as the test solutions for qualitative analysis.

At a retention time of 11.3 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0/805.9 [Mt -3Na + 2H]^-

At a retention time of 11.3 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 804.0 [Mt -3Na + 2H]^-

At a retention time of 11.4 – 11.5 minutes, pH 4.0 a hydrolysis product was observed with molecular weight [Ions]: 402.5 [Mt -3Na + H]^2-, 803.9/805.9 [Mt -3Na + 2H]^-

At a retention time of 11.4 – 11.5 minutes, pH 9.0 a hydrolysis product was observed with molecular weight [Ions]: 402.4 [Mt -3Na + H]^2-, 803.9/806.0 [Mt -3Na + 2H]^-

Under the conditions of the study the half life at pH 4.0 was 256 days for peak one and 309 days for peak 2. The hydrolysis rate constant was 1.13 x 10^-4 hour^-1 and 3.09 x 10^-4 hour^-1 respectively. The half life at pH 9.0 was 187 days for peak 1 and 196 days for peak 2. The hydrolysis rate constant was 1.75 x 10^-3 hour^-1 for both peaks.