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Hydrolysis

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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-08 to 2009-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Buffers:
Due to the low water solubility of Aldimine 1, the test item was applied as acetone solution into different buffer solutions.
pH 4.0:
1 mg/mL stock solution was diluted with dried, sterile acetone to 100 µg/mL. 1 mL of the tempered buffer solution (pH = 4) was measured into a 25 mL volumetric flask and 1 mL of the 100 µg/mL solution was added to the flask. In order to exclude oxygen, the flask was flushed out with nitrogen. The flasks were closed and placed in a dark thermostat.
Nominal start concentration was 50 µg/mL. The final concentration of solvent (acetone) was 50 %. pH was checked at the start and at the end of the test.
pH 7.0:
1 mg/mL stock solution was diluted with dried, sterile acetone to 100 µg/mL. 80 mL of this solution was added to 720 mL of the tempered buffer solution (pH = 7). Solutions were transferred into 20 ml vials and placed in dark thermostats.
Nominal start concentration was 10 µg/mL. The final concentration of solvent (acetone) was 10 %. pH was checked at each analytical occasion.
pH 9.0:
7.5 mL of the 1 mg/mL stock solution was made up to 750 mL with the tempered buffer solution (pH = 9). Solutions were transferred into 20 mL vialsand placed in dark thermostats.
Nominal start concentration was 10 µg/mL. The final concentration of solvent (acetone) was 1 %. pH was checked at each analytical occasion.
Details on test conditions:
pH: The hydrolysis was examined at three different pH values 4.0, 7.0 and 9.0. Sterile buffer solutions were prepared. The pH of each buffer solution was checked with a calibrated pH meter.
Temperature: 15 ± 1.0 °C, 25 ± 0.5 °C and at 35 ± 0.5 °C
Light and oxygen: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.
HPLC: Thermo-Finnigan Surveyor MS Pump, Serial No.: 54075
Thermo-Finnigan Surveyor Autosampler, Serial No.: 55097
Mass
Spectrometer: LCQ Duo, Serial No.: 00360 equipped with electrospray (ESI) LC interface
Balances: BP 221S Sartorius, No.: 11809117
L2200P Sartorius, No.: 38100037
Ultrasonic
bath: Elmasonic S 300 H, No.: 010890105
pH meter: OP – 211/1, Radelkis No.: 6431
Thermostats: LP 132, No.: 870595
B6 Heraus, No.: 97113066
Climate-controlled chamber, No.: 6
Hot Air
Steriliser: ATP line FED, WTB Binder, No.: 9110-0035
Autoclave: Labor Mim 5403, No.: 356(31-881316)
Water
purification
system: MILLIPORE, DIRECT Q3, FOMNO 7334I
Vials: 20 ml headspace–vial, clear glass,
with septum and screw cap, La-Pha-Pack
Duration:
25 min
pH:
4
Temp.:
15 °C
Initial conc. measured:
52.6 other: µg/mL
Duration:
25 min
pH:
4
Temp.:
25 °C
Initial conc. measured:
56.1 other: µg/mL
Duration:
15 min
pH:
4
Temp.:
35 °C
Initial conc. measured:
38.7 other: µg/mL
Duration:
330 min
pH:
7
Temp.:
15 °C
Initial conc. measured:
8.16 other: µg/mL
Duration:
300 min
pH:
7
Temp.:
25 °C
Initial conc. measured:
6.38 other: µg/mL
Duration:
270 min
pH:
7
Temp.:
35 °C
Initial conc. measured:
6.69 other: µg/mL
Duration:
187 h
pH:
9
Temp.:
15 °C
Initial conc. measured:
8.04 other: µg/mL
Duration:
187 h
pH:
9
Temp.:
25 °C
Initial conc. measured:
7.38 other: µg/mL
Duration:
190 h
Initial conc. measured:
8.8 other: µg/mL
Number of replicates:
Two replicate samples were analysed at each time point, except for pH 4 test, where we have three replicate data points.
Positive controls:
no
Negative controls:
yes
Statistical methods:
The chromatograms were evaluated with the help of “LaChrom” chromatogram processor software. Calculations were carried out using “EXCEL for Windows". The calibration curves were constructed with “STATISTICA for Windows" using weighted linear regression. The factor was 1/concentration.
Preliminary study:
As Aldimine 1 proved to be unstable during analytical method validation trials, the preliminary test is not required.
Test performance:
no remarks
Transformation products:
yes
No.:
#1
No.:
#2
Details on hydrolysis and appearance of transformation product(s):
The degradation products following hydrolysis are 2,2-dimethyl-3-oxopropyl dodecanoate (CAS # 102985-93-3) and 5-amino-1,3,3-trimethyl-cyclohexanemethylamine (CAS # 2855-13-2).
% Recovery:
0.9
pH:
4
Temp.:
15 °C
Duration:
25 min
% Recovery:
1.7
pH:
4
Temp.:
25 °C
Duration:
25 min
% Recovery:
0.5
pH:
4
Temp.:
35 °C
Duration:
25 min
% Recovery:
12
pH:
7
Temp.:
15 °C
Duration:
330 min
% Recovery:
13
pH:
7
Temp.:
25 °C
Duration:
300 min
% Recovery:
11
pH:
7
Temp.:
35 °C
Duration:
270 min
% Recovery:
28
pH:
9
Temp.:
15 °C
Duration:
187 h
% Recovery:
20
pH:
9
Temp.:
25 °C
Duration:
187 h
% Recovery:
10
pH:
9
Temp.:
35 °C
Duration:
190 h
Key result
pH:
4
Temp.:
15 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
25 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
35 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
99 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
35 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
111 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
99 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
105 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
85 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
62 h
Type:
(pseudo-)first order (= half-life)
Details on results:
Calibration
The calibration series was measured every day. The following concentrations were applied:
0.01 - 0.02 - 0.05 - 0.1 - 0.2 - 0.3 - 0.4 and 0.5 µg Aldimine 1 /mL. The stock solution was diluted with dried acetone

Results of the Hydrolysis Tests
Hydrolysis rate at pH 4 was above 90 % at the first analysis, therefore t1/2 is considered to be less than 5 minutes at each of the three temperatures.
At pH 7 the reaction lasted for about five hours, the calculated t1/2 is 86 – 111 minutes for the different temperatures.
At pH 9 the reaction proved to be slower, it lasted for eight days. The calculated t1/2 is 62 - 105 hours depending on the applied temperature.

Measured data at pH 4

Temperature,

pH

Sampling time, min

Measured concentration, µg/ml

(mean of two or three)

Hydrolysis rate, %

Measured pH

15 °C, pH 4

Start

Control

-

4.00

Start

Control with acetone

-

6.23

Start

52.6

-

6.25

5

1.19

97.7

-

10

0.77

98.5

-

15

0.82

98.4

-

20

0.66

98.7

-

25

0.47

99.1

6.25

25 °C, pH 4

Start

Control

-

4.01

Start

Control with acetone

-

6.26

Start

56.1

-

6.26

5

2.78

95.0

-

10

2.38

95.8

-

15

1.57

97.2

-

20

0.82

98.5

-

25

0.97

98.3

6.24

35 °C, pH 4

Start

Control

-

4.00

Start

Control with acetone

-

6.22

Start

38.7

-

6.24

5

0.99

97.4

-

10

0.42

98.9

-

15

0.19

99.5

6.25

Measured data at pH 7

Temperature,

pH

Sampling time, min

Measured concentration, µg/ml

(mean of two)

Hydrolysis rate, %

Measured pH

15 °C, pH 7

Start

Control

-

7.00

Start

Control with acetone

-

7.22

Start

8.16

-

7.22

60

5.13

37

7.20

90

4.40

46

7.22

120

3.25

60

7.19

150

2.89

65

7.21

180

2.55

69

7.20

210

2.13

74

7.20

240

1.94

76

7.19

270

1.39

83

7.20

300

1.10

86

7.22

330

1.00

88

7.21

25 °C, pH 7 

Start

Control

-

7.01

Start

Control with acetone

-

7.20

Start

6.38

-

7.23

20

4.87

24

7.21

40

4.79

25

7.21

60

4.44

30

7.21

120

3.61

43

7.21

150

2.19

66

7.20

180

2.12

67

7.21

210

1.27

80

7.22

240

1.02

84

7.21

270

1.04

84

7.21

300

0.81

87

7.20

35 °C, pH 7

Start

Control

-

7.01

Start

Control with acetone

-

7.21

Start

6.69

-

7.20

30

5.04

25

7.20

60

4.21

37

7.21

90

3.57

47

7.22

120

2.69

60

7.22

150

2.25

66

7.22

180

1.85

72

7.21

210

1.63

76

7.22

240

0.80

88

7.22

270

0.72

89

7.22

Measured data at pH 9

Temperature,

pH

Sampling time, hours

Measured concentration, µg/ml

(mean of two)

Hydrolysis rate, %

Measured pH

15 °C, pH 9

Start

Control

-

9.01

Start

8.04

-

9.01

19

6.72

16

9.00

24

6.83

15

9.00

41

5.78

28

9.01

67

4.45

45

9.00

74

4.45

45

8.99

118

3.27

59

9.00

163

2.87

64

9.00

187

2.22

72

9.00

25 °C, pH 9

Start

Control

-

9.00

Start

7.38

-

9.00

19

6.58

11

9.00

24

5.16

30

9.00

41

4.53

39

9.01

67

3.79

49

9.00

74

3.60

51

9.01

118

2.76

63

9.00

163

1.91

74

9.00

187

1.46

80

9.01

35 °C, pH 9

Start

Control

-

9.01

Start

8.80

-

9.01

21

6.68

28

9.00

26

6.32

32

9.01

30

6.20

33

9.01

45

6.03

35

9.00

70

4.08

56

8.99

77.5

3.79

59

9.00

121.5

2.40

74

9.01

166

1.56

83

9.01

190

0.98

90

9.00

 

Rate constants and half-lives based on the measured data

pH

Temperature

Slope

kobs

t1/2

4

15 °C

-

-

< 5 min

25 °C

-

-

< 5 min

35 °C

-

-

< 5 min

7

15 °C

-0.006229

0.006229

111 min

25 °C

-0.006970

0.006970

99min

35 °C

-0.008033

0.008033

86 min

9

15 °C

-0.006583

0.006583

105 hours

25 °C

-0.008150

0.008150

85 hours

35 °C

-0.011115

0.011115

62 hours
Validity criteria fulfilled:
yes
Conclusions:
Hydrolysis was most rapid in acidic environment (pH 4) and high temperature compared to basic environment (pH 9) and low temperature:
pH, t1/2 at 15 °C 25 °C 35 °C
4, < 5 min < 5 min < 5 min
7, 111 min 99 min 86 min
9, 105 hours 85 hours 62 hours
The degradation products following hydrolysis are 2,2-dimethyl-3-oxopropyl dodecanoate (CAS # 102985-93-3) and 5-amino-1,3,3-trimethyl-cyclohexanemethylamine (CAS # 2855-13-2).
Executive summary:

Abiotic degradation of Aldimine 1 was assessed in solutions of different pH values. Hydrolysis was most rapid in acidic environment (pH 4) and high temperature compared to basic environment (pH 9) and low temperature:

pH, t1/2 at 15 °C 25 °C 35 °C

4, < 5 min < 5 min < 5 min

7, 111 min 99 min 86 min

9, 105 hours 85 hours 62 hours

Due to the small number of microorganisms present in the test mixtures (5 - 25 microorganisms/mL test mixture), there was a very low possibility of microbial decomposition. Based on the above results a potential for chemical decomposition (hydrolysis) is indicated.

The degradation products following hydrolysis are 2,2-dimethyl-3-oxopropyl dodecanoate (CAS # 102985-93-3) and 5-amino-1,3,3-trimethyl-cyclohexanemethylamine (CAS # 2855-13-2).

Endpoint:
hydrolysis
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structural analogue substance SIKA Hardener LI is used in a read-across approach to assess the hydrolysis behaviour of SIKA Hardener LH. Both sharing structural similarities and uses. On contact with water, both SIKA Hardener LH and SIKA Hardener LI rapidly hydrolyses to a common compund (2,2-dimethyl-3-oxopropyl dodecanoate) and a non-common compound (diamine).

2. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarities and comparable hydrolysis reaction resulting in one common and one non-common compound, the applied read-across apporach is suitable and reliable for fulfilling the REACH requirements of Annex VIII, section 9.2.2. This read-across approach is justified by the supporting study conducted with the target substance SIKA Hardener LH showing fast hydrolysis in acid environment as also observed in the GLP and Guideline compliant stud conducted with the source substance SIKA Hardener LI.
Reason / purpose for cross-reference:
read-across source
Transformation products:
yes
No.:
#1
No.:
#2
% Recovery:
0.9
pH:
4
Temp.:
15 °C
Duration:
25 min
% Recovery:
1.7
pH:
4
Temp.:
25 °C
Duration:
25 min
% Recovery:
0.5
pH:
4
Temp.:
35 °C
Duration:
25 min
% Recovery:
12
pH:
7
Temp.:
15 °C
Duration:
330 min
% Recovery:
13
pH:
7
Temp.:
25 °C
Duration:
300 min
% Recovery:
11
pH:
7
Temp.:
35 °C
Duration:
270 min
% Recovery:
28
pH:
9
Temp.:
15 °C
Duration:
187 h
% Recovery:
20
pH:
9
Temp.:
25 °C
Duration:
187 h
% Recovery:
10
pH:
9
Temp.:
35 °C
Duration:
190 h
Key result
pH:
4
Temp.:
15 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
25 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
35 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
99 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
35 °C
DT50:
< 5 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
111 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.01 min-1
DT50:
99 min
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
105 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
85 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
62 h
Type:
(pseudo-)first order (= half-life)
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of the analysis was to perform a hydrolysis test to evaluate the abiotic degradation of SIKA Hardener LH in water and if hydrolysis is acid catalysed.
GLP compliance:
no
Buffers:
1mM NH4HCOO
1 % acetic acid in water
Details on test conditions:
short term-test :
Samples were dissolved in water free methanole with 1mM NH4HCOO (100 µg/mL) and were contacted with water or 1% acetic acid in water

test a) Hydrolysis with acetic water in changing mixing ratio in carrier flow (water / organic solvent)
test b) hydrolysis with bidest. water in changing mixing ratio water / organic solvent)

Long-term-test:
samples were dissolved in water free acetonitile (100µg/mL) and were contacted with water or 1% acetic acid in water

test c) Hydrolysis with acetic water contacted sample as a function of time
test d) Hydrolysis with bidest water contacted sample as a function of time
test e) Hydrolysis with slightly carbonated water contacted sample as a function of time
Number of replicates:
1
Positive controls:
no
Negative controls:
no
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Hydrolysis time
a) in acetous water (1 % acetic acid): < 2 sec
b) in bidest. water: >> 2 sec
c) in acetous water as a function of time: < 10 min
d) in bidest. water as a function of time: 22 h
e) in slightly carbonated water as a function of time: no data
Validity criteria fulfilled:
yes
Conclusions:
Hydrolysis of SIKA Hardener LH was very fast and most rapid in acidic environment (acetous water, < 2 sec) compared to basic environment (bidest. water, >> 2 sec).
Executive summary:

The purpose of the analysis was to perform a hydrolysis test to evaluate the abiotic degradation of SIKA Hardener LH in water and if hydrolysis is acid catalysed. The substance was identified by flow injection via syring pump 12 µL/min with a FIMS-MS. The substance was tested in a short term test and in a long-term test. In the short-term test the samples were dissolved in water free methanole with 1 mM NH4HCOO (100 µg/mL) and were contacted with water or 1 % acetic acid in water. In the long term test samples were dissolved in water free acetonitile (100 µg/mL) and were contacted with water or 1 % acetic acid in water. The following results were received:

a) in acetous water (1 % acetic acid): < 2 sec

b) in bidest. water: >> 2 sec

c) in acetous water as a function of time: < 10 min

d) in bidest. water as a function of time: 22 h

Description of key information

Based on the applied read-across approach with structural analogue SIKA Hardener LI and the supporting hydrolysis study with the target substance SIKA Hardener LH, fast hydrolysis was observed, which was most rapid in acidic environment compared to basic environment.

Key value for chemical safety assessment

Half-life for hydrolysis:
0.19 d
at the temperature of:
12 °C

Additional information

To assess the hydrolysis behaviour of SIKA Hardener LH a read-across approach was applied using data from the structural analogue substance SIKA Hardener LI, with both substances sharing comparable hydrolytic properties. SIKA Hardener LI was assessed in an abiotic degradation study according to EU-method C.7 and OECD guideline no. 111. Hydrolysis of SIKA Hardener LI was most rapid in acidic environment (pH 4) and high temperature compared to basic environment (pH 9) and low temperature. At pH 7 and 12 °C (285.2 K) the degradation half-life was 0.19 days and rate constant 3.56 days (85.44 h).

As shown in a supporting study, hydrolysis of SIKA Hardener LH was very fast and most rapid in acidic environment (acetous water, < 2 sec) compared to basic environment (bidest. water, >> 2 sec). This result supported and justified the applied read-across approach.