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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Mai 2016 - 08 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
2004
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)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Radiolabelling:
no
Analytical monitoring:
yes
Remarks:
UPLC-DAD
Details on sampling:
- Sampling intervals for the parent/transformation products: For the preliminary test, samples were taken at test start (0 h) and test end (120 h). For the definitive test, samples were taken at test start (0 h) and at a minimum of 10 spaced points for pH 7 and 9, normally between 10 and 90% of hydrolysis. Quantification of the major transformation products, was carried out, from retained samples of the kinetics part of the study.
- Sampling method: autosampler
- Sampling intervals/times for sterility check: Sterility of the test solutions was checked at test end by colony forming units determination with Water Plate Count Agar from additional samples of the definitive test by incubation at 36 ± 1 °C for 48 h and at 22 ± 1 °C for 72 h. As the test duration was =< 120 h for the test condition at pH 9 and 20, 30 and 50 °C, no determination of CFU was deemed necessary at these test conditions.
- Sample storage conditions before analysis: All samples were analysed as fast as possible to avoid further transformation.
Buffers:
- pH: preliminary test: 4, 7 and 9; final test: 7, 9
- Type of buffer: Buffer solutions were prepared from chemicals with analytical grade or better quality following the composition guidance given in “Küster-Thiel, Rechentafeln fur die Chemische Analytik” and the OECD Guideline No. 111, respectively, by direct weighing of the buffer components. The pH was checked to a precision of at least 0.1 at the test temperatures.
- Composition of buffer:
- Buffer solution pH 4: 0.18 g NaOH and 5.7555 g mono potassium citrate were dissolved in 500 mL double distilled water.
- Buffer solution pH 7: 3.854 g of ammonium acetate were dissolved in 500 ml double distilled water.
- Buffer solution pH 9: 0.426 g NaOH, 1.8638 g KCI and 1.5458 g H3BO3 were dissolved in 500 mL double distilled water.
- Test concentration: 800 mg/L buffer pH 4, 7 and 9 with 1% acetonitrile, respectively.
Estimation method (if used):
The quantity of the transformation products, from retained samples of the kinetics part of the study, were achieved by LC-DAD and calculated via response factor (0.7977 for transformation product #1) of the test item or external standard (transformation product #2).
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 4 mL HPLC vials (test volume: 2 mL)
- Sterilisation method: Buffers were sterilized by filtration through 0.2 µm.
- Lighting: no
- Measures taken to avoid photolytic effects: Photolytic effects were avoided by using opaque water baths.
- Measures to exclude oxygen: Buffers were purged with nitrogen for 5 min.
- Co-solvent: Acetonitrile, < 1 % (v/v)
Duration:
719 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
835 mg/L
Duration:
719 h
pH:
7
Temp.:
30 °C
Initial conc. measured:
835 mg/L
Duration:
600 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
835 mg/L
Duration:
76.3 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
814 mg/L
Duration:
27.9 h
pH:
9
Temp.:
30 °C
Initial conc. measured:
681 mg/L
Duration:
3.68 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
765 mg/L
Number of replicates:
Duplicates per pH value and sampling date, single injected
Preliminary study:
In the preliminary test more than 10 % of the test item was degraded after 120 h at pH 7 and 9.
- pH 4: 4.95% degradation after 120 h
- pH 7: 82.8% degradation after 120 h
- pH 9: 100% degradation after 120h
Transformation products:
yes
No.:
#1
No.:
#2
Details on hydrolysis and appearance of transformation product(s):
- Formation and decline of each transformation product during test: see field "any other information on results incl. tables"
- Pathways for transformation: The presented transformation products confirm the typical ester hydrolysis by cleavage of the ester bond.
- Other: Transformation product #2 was quantified against external standard. The calibration showed a quadratic run of the curve. The quadratic function is acceptable in view of the results. Transformation product #1 was quantified against test item calibration, taking a response factor based on the molecular weights into account. Both transformation products are major transformation products. In combination, precursor ion scans of typical fragment masses (for ionisable analytes) and evaluation of the LC-DAD analysis were performed in order to scan for other possible transformation products. No additional signals were observed.
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
1 536 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
850 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
184 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
22.9 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
7.05 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
0.993 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- For details see field "any other informaiton on results incl. tables"

MINOR TRANSFORMATION PRODUCTS
- No minor transformation products identified.

VALIDITY CRITERIA:

- The regression graphs showed a correlation factor >= 0.8 for each test condition, confirming first order behavior.

- The test temperature did not differ by more than ± 0.5 °C, except at pH 7 and 30 °C.

- The pH values of the buffer solutions were in the range of ± 0.1 pH at test temperature.

- Sensitivity of the analytical method was sufficient to quantify concentrations of the substance at least down to 10% of the applied test item.

DETAILS ON RESULTS:

At pH 4 no significant reduction of the test item concentration was observed in the preliminary test and therefore the test item was considered as hydrolytically stable under this condition and a half-life of > 1 year could be assumed for environmental typical temperatures.

Table 1: Content of Major Transformation Products and Mass Balance at pH 7 and 9 and 20, 30 and 50 °C.

pH 7
20 °C 30°C 50°C
Hydrolysis time [h] Transformation product #1 [mg/L] Transformation product #2 [mg/L] Parent Substance [mg/L] MB [%] Hydrolysis time [h] Transformation product #1 [mg/L] Transformation product #2 [mg/L] Parent Substance [mg/L] MB [%] Hydrolysis time [h] Transformation product #1 [mg/L] Transformation product #2 [mg/L] Parent Substance [mg/L] MB [%]
0 0 0 846 100 0 0 0 846 100 0 0 0 846 100
5.45 0 0 877 104 5.63 0 0 797 94 2.05 0 0 833 98
23.3 0 0 890 105 23.1 8.06 0 811 96 5.77 14.4 0 801 95
75.8 8.86 0 862 102 76.3 29.5 0 758 90 22.8 53.9 13.9 713 90
166 22.9 0 846 100 166 53.7 14.5 730 92 30.9 73.8 24.3 687 91
239 29.2 17.5 847 107 239 84.3 23.8 710 93 76.6 142 41.7 544 81
336 40.3 19.5 844 108 335 106 24 623 83 165 208 51.3 359 63
407 47 18.4 683 88 407 109 26.6 565 78 239 247 63.4 227 52
506 57.2 17.8 653 84 506 145 35.6 538 78 335 282 69.4 209 53
599 84.6 18.7 705 91 599 132 35.6 458 68 406 289 77.9 148 49
719 68.7 30.1 680 92 719 153 40.6 478 73 600 339 78.9 80.2 41

pH 9

20 °C

30°C

50°C

Hydrolysis time [h]

Transformation product #1 [mg/L]

Transformation product #2 [mg/L]

Parent Substance [mg/L]

MB [%]

Hydrolysis time [h]

Transformation product #1 [mg/L]

Transformation product #2 [mg/L]

Parent Substance [mg/L]

MB [%]

Hydrolysis time [h]

Transformation product #1 [mg/L]

Transformation product #2 [mg/L]

Parent Substance [mg/L]

MB [%]

0

0

0

724

100

0

0

0

716

100

0

0

0

724

100

5.45

39.1

19.9

727

110

5.63

16

8.5

775

112

2.05

58

31.8

642

104

23.3

86.7

49.2

675

116

23.1

47.5

26.2

688

109

5.77

107

62.1

584

110

75.8

205

127

455

123

76.3

72.9

53.5

589

112

22.8

141

90

482

109

166

230

140

431

125

166

113

69.1

566

112

30.9

178

109

385

105

239

233

153

302

114

239

143

89.1

490

140

76.6

228

144

268

105

336

226

143

351

116

335

176

99.1

422

106

165

248

154

220

103

407

247

152

270

109

407

193

123

377

189

239

162

151

177

96

506

234

153

358

122

506

211

126

299

102

335

292

174

124

99

599

293

182

174

110

599

319

195

70.7

103

406

314

192

117

107

719

362

225

70.6

116

719

329

207

37.8

104

600

343

196

34.7

97

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The test item showed a slow hydrolysis rate (t1/2 > 30 d) for pH 7 at 20 and 30 °C, a moderate hydrolysis rate (2.4 h =< t1/2 =< 30 d) for pH 7 at 50 °C and for pH 9 at 20 and 30 °C and a fast hydrolysis rate at pH 9 at 50 °C, with the higher half-lives at acidic conditions. At pH 4 no significant reduction of the test item concentration was observed in the preliminary test and therefore the test item was considered as hydrolytically stable under this condition and a half-life of > 1 year was assumed for environmental typical temperatures. Two major transformation products were found confirming the typical ester hydrolysis by cleavage of the ester bond.
Executive summary:

The hydrolysis potential of the substance as a function of pH was determined according to the OECD Guideline 111, the Council Regulation (EC) No. 440/2008, Method C.7 and GLP. The hydrolysis determination was performed via LC-DAD on a reversed phase analytical column using the test item as external standard. Based on the results of a preliminary test the definitive test was conducted with a test item concentration of 800 mg/L in buffer solutions of pH 7 and 9 containing 1% acetonitrile at temperatures of 20, 30 and 50 °C, respectively. Samples were taken at test start and at 9 to 10 spaced points until test end.

The substance has a slow hydrolysis rate (t1/2 > 30 d) at pH 7 and 20 - 30 °C, a moderate hydrolysis rate (2.4 h ≤ t1/2 ≤ 30 d) at pH 7 and 50 °C as well as at pH 9 and 20 - 30 °C while a fast hydrolysis rate was found at pH 9 and 50 °C (t1/2 = 12. h). At pH 4 no significant reduction of the test item concentration was observed and the test item was considered as hydrolytically stable at this pH and environmental typical temperatures. Two major transformation products were determined confirming the typical ester hydrolysis by cleavage of the ester bond.

Description of key information

Half-life 1536 h at pH 7 and 20°C

Key value for chemical safety assessment

Half-life for hydrolysis:
1 536 h
at the temperature of:
20 °C

Additional information

The hydrolysis potential of the substance as a function of pH was determined according to the OECD Guideline 111, the Council Regulation (EC) No. 440/2008, Method C.7 and GLP. The hydrolysis determination was performed via LC-DAD on a reversed phase analytical column using the test item as external standard. Based on the results of a preliminary test the definitive test was conducted with a test item concentration of 800 mg/L in buffer solutions of pH 7 and 9 containing 1% acetonitrile at temperatures of 20, 30 and 50 °C, respectively. Samples were taken at test start and at 9 to 10 spaced points until test end.

The substance is characterized by a slow hydrolysis rate (t1/2 > 30 d) at pH 7 and 20 - 30 °C, a moderate hydrolysis rate (2.4 h ≤ t1/2 ≤ 30 d) at pH 7 and 50 °C as well as at pH 9 and 20 - 30 °C while a fast hydrolysis rate was found at pH 9 and 50 °C (t1/2 = 12. h). At pH 4 no significant reduction of the test item concentration was observed and the test item was considered as hydrolytically stable at this pH and environmental typical temperatures. Two major transformation products were determined confirming the typical ester hydrolysis by cleavage of the ester bond.