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Hydrolysis

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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.06.2017 - 31.07.2017
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)
Remarks:
TOXI-COOP ZRT 8230 Balatonfüred, Arácsi út 97.
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
Three samples were analysed at the start of the experiments. Two separate tubes were analysed at each analytical occasion of hydrolysis test. One sample was taken from the control vessels.
The whole amount of the vials was transferred to the SPE cartridges. The eluate was analysed by GC.
Buffers:
pH 4.0: 1 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium hydrogen phthalate were diluted to 500 mL with ultra-pure water.
pH 7.0: 74 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium dihydrogen phosphate were diluted to 500 mL with ultra-pure water.
pH 9.0: 54 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Boric acid and Potassium chloride were diluted to 500 mL with ultra-pure water.
The pH of each buffer solution was checked with a calibrated pH meter. The required amount of the buffer solutions was prepared according to the above ratios.
Details on test conditions:
TEST SYSTEM
- Equipment used: screw cap headspace vials
Balances: PG 203 S, Mettler Toledo, No.: 1122082152 BP210D, Sartorius, No.: 60602907
SPE cartridges: Discovery DSC-18, 3mL/500mg, No.: SP8914B
pH meter: InoLab pH Level 2, No.: 03260014
Ultrasonic bath: Branson 3210-E-MT, No.: A700343D
Water purification system: MILLIPORE, DIRECT Q5, No.: F0DA13956K
Thermostat: OMH 400, Heratherm, No.: 41865966
Hot air steriliser: M-53, Binder, No.: 910243
- Temperature: pH 4: 40, 50 and 60 ± 0.5 °C; pH 7: 30, 40 and 50 ± 0.5 °C; pH 9: 10, 20 and 30 ± 0.5 °C
- Lighting: no
- Measures taken to avoid photolytic effects: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects
- Measures to exclude oxygen: Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.

TEST MEDIUM
- Volume used/treatment: 20 mL
- Kind and purity of water: ultra-pure water from water purification system
- Preparation of test medium: 201.65 mg of test item was dissolved in 10 mL of acetonitrile.
- Renewal of test solution: no

OTHER TEST CONDITIONS
- Adjustment of pH: yes, with Sodium hydroxide
Duration:
646 h
pH:
4
Temp.:
40 °C
Initial conc. measured:
187 mg/L
Duration:
525 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
187 mg/L
Duration:
164 h
pH:
4
Temp.:
60 °C
Initial conc. measured:
186 mg/L
Duration:
495.5 h
pH:
7
Temp.:
30 °C
Initial conc. measured:
206 mg/L
Duration:
495.5 h
pH:
7
Temp.:
40 °C
Initial conc. measured:
206 mg/L
Duration:
164 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
187 mg/L
Duration:
215 h
pH:
9
Temp.:
10 °C
Initial conc. measured:
189 mg/L
Duration:
72 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
214 mg/L
Duration:
27 h
pH:
9
Temp.:
30 °C
Initial conc. measured:
233 mg/L
Number of replicates:
3
Positive controls:
no
Negative controls:
no
Statistical methods:
The pseudo-first order rate constant (kobs) for each test temperature was determined from the plots of the natural logarithm of the concentration versus time. Applying the Arrhenius equation the rate constant k and half-life were calculated at 20°C from the plot of ln k versus 1/T.
Preliminary study:
Based on the results of the preliminary test (please refer to IUCLID section 5.1.2) the hydrolysis rate of the test substance was found to be 32% at pH 4, 89% at pH 7 and >99% at pH 9 after 5 days at 50 °C. Based on the results obtained, the test substance was considered as hydrolytically unstable at pH 4, pH 7 and pH 9 under the conditions investigated and the subsequent full test was initiated.
Test performance:
Test solutions were prepared by adding 200 µL of Stock Solution II to 20 mL of the buffer solutions (pH=4, pH=7, pH=9) in screw cap headspace vials. Before vials were closed oxygen was removed by blowing nitrogen into the tubes. Acetonitrile content of the test solutions was 1 % (v/v). Nominal starting concentration test item in the buffer solutions was 0.2 mg/mL around 1.5 mM.

SPE tubes were conditioned with 3 mL of tetrahydrofuran, then 3 mL methanol and 3 mL ultrapure water. Then 20 mL of the test solution (the whole amount of the vial) was loaded to the packing together with 0.5 mL Internal Standard Stock Solution. Then it was dried. The cartridges were washed with 1 mL ultrapure water. The test item was eluted with 5 mL of acetonitrile. The eluate was analysed by GC.
Transformation products:
not measured
% Recovery:
51
pH:
4
Temp.:
40 °C
Duration:
646 h
% Recovery:
27
pH:
4
Temp.:
50 °C
Duration:
525 h
% Recovery:
21
pH:
4
Temp.:
60 °C
Duration:
164 h
% Recovery:
27
pH:
7
Temp.:
30 °C
Duration:
495.5 h
% Recovery:
3
pH:
7
Temp.:
40 °C
Duration:
495.5 h
% Recovery:
6
pH:
7
Temp.:
50 °C
Duration:
164 h
% Recovery:
15
pH:
9
Temp.:
10 °C
Duration:
215 h
% Recovery:
21
pH:
9
Temp.:
20
Duration:
44.1 h
% Recovery:
15
pH:
9
Temp.:
30
Duration:
27 h
Key result
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0 h-1
DT50:
8 122 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
784 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0.022 h-1
DT50:
32 h
Type:
(pseudo-)first order (= half-life)
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Transformation products were not investigated

Measured data at pH 4

 

Temperature

Sampling time (hours)

Concentration
(mg/L)
n=2

Hydrolysis
(%)

Measured pH

40 °C

Control buffer

-

-

4.00

Start

187 (n=3)

-

4.01

22

184

2

4.02

120

158

16

4.00

166

158

15

4.03

222

140

26

4.02

239

141

25

4.01

311

134

28

4.04

484

105

44

4.00

646

95

49

4.01

50 °C

Control buffer

-

-

4.02

Start

187 (n=3)

-

3.98

22

181

4

4.01

70

154

18

3.99

120

131

30

4.00

166

116

38

4.02

239

93.5

50

4.01

311

80.9

57

4.00

381

70.4

62

3.98

525

51.2

73

4.04

60 °C

Control buffer

-

-

4.03

Start

186 (n=3)

-

4.02

3

188

0

4.02

20

166

11

4.02

29

139

25

4.01

52

121

35

4.02

75

95.3

49

4.03

92

83.2

55

4.02

164

39.8

79

4.01

 

Measured data at pH 7

 

Temperature

Sampling time (days)

Concentration
(mg/L)
n=2

Hydrolysis
(%)

Measured pH

30 °C

Control buffer

-

-

7.04

Start

206 (n=3)

-

7.02

15.5

190

8

7.04

47.5

178

14

7.00

88.5

163

21

6.97

160.5

140

32

7.03

230.5

104

49

7.04

333.5

92.9

55

7.04

423.5

71.2

65

7.03

495.5

56.3

73

7.00

40 °C

Control buffer

-

-

7.02

Start

206 (n=3)

-

7.02

15.5

182

12

7.00

47.5

148

28

7.02

71.5

121

41

6.99

88.5

112

46

7.02

160.5

68.7

67

7.02

230.5

45.4

78

7.01

333.5

14.1

93

7.04

423.5

9.65

95

7.03

495.5

6.72

97

6.99

50 °C

Control buffer

-

-

7.04

Start

187

-

7.00

3

188

0

7.02

20

137

27

7.00

29

114

39

7.01

51.5

84.1

55

7.02

75

52.0

72

7.02

92

38.6

79

7.02

114

28.7

85

7.01

164

11.1

94

7.02

 

Measured data at pH 9

 

Temperature

Sampling time (hours)

Concentration
(mg/L)
n=2

Hydrolysis
(%)

Measured pH

10 °C

Control buffer

-

-

9.03

Start

189 (n=3)

-

9.02

46

135

29

8.99

76

112

41

9.01

94

101

47

9.01

144

76.7

59

9.00

175

63.3

66

9.01

215

47.1

75

9.02

20 °C

Control buffer

-

-

9.00

Start

214

-

9.03

7

192

10

8.99

21

128

40

9.03

31

106

50

9.01

48

73.0

66

8.99

56

63.2

70

9.00

72

44.1

79

9.01

30 °C

Control buffer

-

-

8.98

Start

233

-

9.00

1.5

202

14

9.01

3

171

26

9.02

4.5

160

31

8.98

6

150

36

9.03

7.5

130

44

9.01

11.5

103

56

9.01

22

50.6

78

9.02

27

34.2

85

8.99

 

Rate constants and half-lives based on the measured data

pH

Temperature

Slope

kobs, 1/h

t1/2

Regression Coefficient

4

40°C

-0.00109

0.00109

639 h

0.984

50°C

-0.00251

0.00251

276 h

0.992

60°C

-0.00946

0.00946

73.3 h

0.996

7

30°C

-0.00251

0.00251

276 h

0.992

40°C

-0.00718

0.00718

96.6 h

0.991

50°C

-0.01723

0.01723

40.2 h

0.999

9

10°C

-0.00624

0.00624

111 h

0.997

20°C

-0.02206

0.02206

31.4 h

0.997

30°C

-0.06860

0.06860

10.1 h

0.998

Estimation of the half lives of the reaction at 20°C

Temperature

1/T

pH 4

pH 7

pH 9

°C

(1/K)

kobs

ln k-

t1/2, h

kobs

ln k

t1/2, h

kobs

ln k

t1/2, h

10

0.00353

-

-

-

-

-

-

0.0062

-5.08

111

20

0.00341

-

-

-

-

-

-

0.0221

-3.81

31.4

30

0.00330

-

-

-

0.0025

-5.99

276

0.0686

-2.68

10.1

40

0.00319

0.0011

-6.83

639

0.0072

-4.94

96.6

-

-

-

50

0.00309

0.0025

-5.99

276

0.0172

-4.06

40.2

-

-

-

60

0.00300

0.0095

-4.66

73.3

-

-

-

-

-

-

20 (calculated)

 

8.5·10-5

-9.37

8122

0.00088

-7.03

784

0.0217

-3.83

32

Constant

29.1

25.2

31.3

X coefficient

-11263

-9438

-10294

R Squared

0.979

0.999

1.000

Validity criteria fulfilled:
yes
Conclusions:
The calculated half lives of the hydrolysis reactions at pH 4, pH 7 and pH 9 are as follows:
t 1/2 at pH4 and 20 degree C: 8122 h
t 1/2 at ph7 and 20 degree C: 784 h
t 1/2 at pH9 and 20 degree C: 32 h
Executive summary:

A study was conducted according to Regulation (EC) No 440/2008 Part C.7 and OECD test guideline 111 to determine the hydrolysis of the test substance as a function of pH. The tier 1 test according to guideline was conducted and revealed that the test item is hydrolytically not stable at pH 4, 7 and 9. Thus, the subsequent Tier 2 test for the determination of the hydrolysis half-life was conducted. Hydrolysis was examined at three different pH values 4, 7 and 9 at three different temperatures: pH 4: 40, 50 and 60 ± 0.5 °C; pH 7: 30, 40 and 50 ± 0.5 °C; pH 9: 10, 20 and 30 ± 0.5 °C . The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Three samples were analysed at the start of the experiments. Two separate tubes were analysed at each analytical occasion of hydrolysis test. One sample was taken from the control vessels. Samples are diluted or concentrated on SPE cartridges and then analysed by a GC – FID method in the presence of benzonitrile internal standard. The pseudo-first order rate constant (kobs) for each test temperature was determined from the plots of the natural logarithm of the concentration versus time. Applying the Arrhenius equation the rate constant k and half-life were calculated at 20°C for pH 4= 8.5E-5 h-1 and 8122 h respectively, for pH 7 = 8.8E-4 and 784 h respectively and for pH 9 = 0.0217 h-1 and 32 h respectively.

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-02 till 2017-4
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)
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test item was dissolved in acetonitrile.
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals: at start and at the end of test (5 days)
- Sampling method: grab samples
- Sampling times for pH measurements: at start and at the end of the test
Buffers:
- pH: 4
- Composition of buffer: 1 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium hydrogen phthalate were diluted to 500 mL with ultra-pure water.

- pH: 7
- Composition of buffer: 74 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium dihydrogen phosphate were diluted to 500 mL with ultra-pure water.

- pH: 4
- Composition of buffer: 54 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Boric acid and Potassium chloride were diluted to 500 mL with ultra-pure water.
Details on test conditions:
TEST SYSTEM
- Equipment used: screw cap headspace vials
Balances: PG 203 S, Mettler Toledo, No.: 1122082152 BP210D, Sartorius, No.: 60602907
SPE cartridges: Discovery DSC-18, 3mL/500mg, No.: SP8914B
pH meter: InoLab pH Level 2, No.: 03260014
Ultrasonic bath: Branson 3210-E-MT, No.: A700343D
Water purification system: MILLIPORE, DIRECT Q5, No.: F0DA13956K
Thermostat: OMH 400, Heratherm, No.: 41865966
Hot air steriliser: M-53, Binder, No.: 910243
- Temperature: 50 ° ± 0.5 °C
- Lighting: no
- Measures taken to avoid photolytic effects: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects
- Measures to exclude oxygen: Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.

TEST MEDIUM
- Volume used/treatment: 20 mL
- Kind and purity of water: ultra-pure water from water purification system
- Preparation of test medium: 201.65 mg of test item was dissolved in 10 mL of acetonitrile.
- Renewal of test solution: no

OTHER TEST CONDITIONS
- Adjustment of pH: yes, with Sodium hydroxide
Duration:
5 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
> 197 - < 212 mg/L
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
> 213 - < 222 mg/L
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
> 188 - < 219 mg/L
Number of replicates:
6
Positive controls:
no
Negative controls:
no
Statistical methods:
The chromatograms were evaluated with the help of TotalChrom™ chromatography software. Ratio of the peak areas (the test item and the internal standard) was calculated and used for the computations. Calculations were carried out using “EXCEL for Windows".
Preliminary study:
pH 4: 32 % hydrolysis
pH 7: 89 % hydrolysis
pH 9: >99 % hydrolysis
Transformation products:
no
% Recovery:
68
St. dev.:
2.9
pH:
4
Temp.:
50 °C
Duration:
5 d
% Recovery:
11
St. dev.:
0.46
pH:
7
Temp.:
50 °C
Duration:
5 d
% Recovery:
< 0.6
pH:
9
Temp.:
50 °C
Duration:
5 d
Remarks on result:
other: below limit of quantification
Details on results:
TEST CONDITIONS
- pH and temperature maintained throughout the study: Yes


At pH4:
- Range of maximum concentrations at end of study period:
At the end of the study period, the concentrations were 131 - 147 mg/L.

At pH7:
- Range of maximum concentrations at end of study period:
At the end of the study period, the concentrations were 22 - 25 mg/L.

At pH9:
- Range of maximum concentrations at end of study period:
At the end of the study period, the concentrations were below the LOQ of 1.3 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the preliminary test the hydrolysis rate of test item was found to be 32% at pH 4, 89% at pH 7 and >99% at pH 9 after 5 days at 50 °C. Based on the results obtained, test item can be considered as hydrolytically unstable at pH 4, pH 7 and pH 9 under the conditions investigated.
Executive summary:

The hydrolysis of the test item was assessed in accordance with the OECD guideline 111 and the EU method C.7 in a preliminary test. The hydrolysis of the test item in different aqueous buffer solutions (pH = 4, 7 and 9) at 50 °C was investigated using GC. The content of test item in the different pH buffer samples was determined at the beginning and the end of the test periods. Six samples were analysed at the start of the study. Based on the measured concentration the hydrolysis of the test item was determined to be 32 % at pH=4; 89 % at pH=7 and >99 % at pH=9. Based on the results obtained, test item can be considered as hydrolytically unstable at pH 4, pH 7 and pH 9 under the conditions investigated. No observations were made which case doubt on the validity of the study results.

Description of key information

The rate constant k and half-life were determined at 20°C for pH 4= 8.5E-5 h-1 and 8122 h respectively, for pH 7 = 8.8E-4 and 784 h respectively and for pH 9 = 0.0217 h-1 and 32 h respectively.

Key value for chemical safety assessment

Half-life for hydrolysis:
784 h
at the temperature of:
20 °C

Additional information

Key study

A study was conducted according to Regulation (EC) No 440/2008 Part C.7 and OECD test guideline 111 to determine the hydrolysis of the test substance as a function of pH. The tier 1 test according to guideline was conducted and revealed that the test item is hydrolytically not stable at pH 4, 7 and 9. Thus, the subsequent Tier 2 test for the determination of the hydrolysis half-life was conducted. Hydrolysis was examined at three different pH values 4, 7 and 9 at three different temperatures: pH 4: 40, 50 and 60 ± 0.5 °C; pH 7: 30, 40 and 50 ± 0.5 °C; pH 9: 10, 20 and 30 ± 0.5 °C . The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Three samples were analysed at the start of the experiments. Two separate tubes were analysed at each analytical occasion of hydrolysis test. One sample was taken from the control vessels. Samples are diluted or concentrated on SPE cartridges and then analysed by a GC – FID method in the presence of benzonitrile internal standard. The pseudo-first order rate constant (kobs) for each test temperature was determined from the plots of the natural logarithm of the concentration versus time. Applying the Arrhenius equation the rate constant k and half-life were calculated at 20°C for pH 4= 8.5E-5 h-1 and 8122 h respectively, for pH 7 = 8.8E-4 and 784 h respectively and for pH 9 = 0.0217 h-1 and 32 h respectively.

Supporting study

The hydrolysis of the test item was assessed in accordance with the OECD guideline 111 and the EU method C.7 in a preliminary test. The hydrolysis of the test item in different aqueous buffer solutions (pH = 4, 7 and 9) at 50 °C was investigated using GC. The content of test item in the different pH buffer samples was determined at the beginning and the end of the test periods. Six samples were analysed at the start of the study. Based on the measured concentration the hydrolysis of the test item was determined to be 32 % at pH=4; 89 % at pH=7 and >99 % at pH=9. Based on the results obtained, test item can be considered as hydrolytically unstable at pH 4, pH 7 and pH 9 under the conditions investigated. No observations were made which case doubt on the validity of the study results.