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Environmental fate & pathways

Hydrolysis

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Link to relevant study record(s)

Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-17 to 2015-03-05
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:
2004
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: samples at pH 4 and 7 were taken at test start and at a minimum of 9 spaced points (between 10 and 90% of hydrolysis)
Buffers:
- pH: 4
- Composition of buffer: 0.18 g of NaOH and 5.7555 g of mono potassium citrate, dissolved in 500 mL double distilled water

- pH: 7
- Composition of buffer: 0. 7358 g of NaOH and 4.3012 g of KH2P04 were dissolved in 500 mL double distilled water

- pH: 9
- Composition of buffer: 0.426 g NaOH, 1.8638 g KCI and 1.5458 g H3B03 were dissolved in 500 mL double distilled water
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: HPLC vials, volume 1.5 mL
- Sterilisation method: Buffers were sterilised by filtration through 0.2 µm, no sterilisation for pH 7 at 30 °C and 50 °C and for pH 9 due to the quick hydrolysis. Check for sterility by determination of colony forming units.
- Measures taken to avoid photolytic effects: use of opaque water baths

TEST MEDIUM
- Volume: 0.75 mL
- Preparation of test medium: Buffer solutions were prepared from chemicals with analytical grade or better quality following the composition guidance given in "KOSTER-THIEL, Rechentafeln for die Chemische Analytik" and the OECD Guideline No. 111, respectively, by direct weighing of the buffer components. Buffers were purged with nitrogen for 5 min and then the pH was checked to a precision of at least 0.1 at the test temperatures.
- Identity and concentration of co-solvent: Acetonitrile, ≤ 1% (v/v)
Duration:
742 h
pH:
4
Temp.:
20 °C
Duration:
742 h
pH:
4
Temp.:
30 °C
Duration:
742 h
pH:
4
Temp.:
50 °C
Duration:
289 h
pH:
7
Temp.:
20 °C
Duration:
99.4 h
pH:
7
Temp.:
30 °C
Duration:
22.2 h
pH:
7
Temp.:
50 °C
Duration:
1 411 min
pH:
9
Temp.:
10 °C
Duration:
210 min
pH:
9
Temp.:
20 °C
Duration:
89 min
pH:
9
Temp.:
30 °C
Number of replicates:
Duplicates per pH value and sampling date, single injected
Positive controls:
no
Negative controls:
no
Preliminary study:
In the preliminary test more than 10% of the test item degraded after 120 hours at pH 4.
Degradation at 120 h and pH 4 15.1%, pH 7 100% and pH 9 100%.
Therefore the main test has to be done on all three pH's.
Test performance:
Linearity: The analytical system gave linear response for the test item in the range of 0.5 – 25 mg 1,6-Bis- (maleinimido)-2,2,4-trimethylhexan/L. The coefficients of determination (r2) of all calibration curves were ≥ 0.992 for 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan.
Limit of Quantification (LOQ): For preliminary test and advanced test the same limit of quantification was reached. The system quantification limit was fixed at 0.5 mg/L for the test item. For the advanced test the limit of detection was fixed at 0.15 mg/L and confirmed by signal-to-noise ratio of 5.
The limit of quantification of the analytical method (LOQ) was fixed at 1.5 mg/L for 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan.
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
No.:
#6
No.:
#7
No.:
#8
Details on hydrolysis and appearance of transformation product(s):
Transformation product signals were evaluated from the LC-DAD chromatograms of the definitive study. Major transformation products (≥ 10 % of the initial peak area of the parent molecule) were observed at retention time (RT; given in minutes) 0.48 for the pH 4 test medium and at RT 1.11 and 1.49 for the pH 7 and 9 test media. One further transformation product was postulated based on chemical deliberations and could be qualitatively verified at RT 0.28 (co-elution with buffer components) in the pH 4 test medium. Identification was performed via LC-MS/MS by postulation of possible transformation products based on molecule and molecule adduct signals and verification of the postulates by MS/MS experiments.

RT0.48 was identified to be a mixture of 1-(6-amino-2,4,4-trimethylhexyl)-1H-pyrrole-2,5-dione and 1-(6-amino-2,2,4-trimethylhexyl)-1H-pyrrole-2,5-dione (#1 and #2, direct transformation products of the test item), which transformed into 2,2,4-trimethylhexane-1,6-diamine and 2,4,4-trimethylhexane-1,6-diamine (RT0.28, #4 and #3).
RT1.49 was identified to be a mixture of (2Z)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-3,3,5-trimethylhexyl]amino}-4-oxobut-2-enoic acid and (2Z)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-3,5,5-trimethylhexyl]amino}-4-oxobut-2-enoic acid (#7 and #8, direct transformation products of the test item), which transformed into (2Z)-3-({6-[(2E)-3-carboxyprop-2-enamido]-3,3,5-trimethylhexyl}carbamoyl)prop-2-enoic acid and (2Z)-3-({6-[(2E)-3-carboxyprop-2-enamido]-3,5,5-trimethylhexyl}carbamoyl)prop-2-enoic acid (RT 1.11, #5 and #6).

Mass balance was calculated for the test item and transformation product signals by evaluation of the LC-DAD chromatograms. Transformation products were quantified against the test item as standard. For RT 0.48 in the pH 4 test medium a response factor of two was applied due to the total elimination of one of two chromophoric group. The mass balance was calculated to be 58.5 – 95.3% at pH4, 57.6 – 67.6% at pH 7 and 56.7 – 72.3% at pH 9.
% Recovery:
95.3
pH:
4
Temp.:
20 °C
Duration:
742 h
% Recovery:
89.4
pH:
4
Temp.:
30 °C
Duration:
742 h
% Recovery:
58.5
pH:
4
Temp.:
50 °C
Duration:
742 h
% Recovery:
67.6
pH:
7
Temp.:
20 °C
Duration:
289 h
% Recovery:
63.6
pH:
7
Temp.:
30 °C
Duration:
99.4 h
% Recovery:
57.6
pH:
7
Temp.:
50 °C
Duration:
22.2 h
% Recovery:
56.7
pH:
9
Temp.:
10 °C
Duration:
1 411 min
% Recovery:
72.3
pH:
9
Temp.:
20 °C
Duration:
210 min
% Recovery:
65.2
pH:
9
Temp.:
30 °C
Duration:
89 min
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
4 593 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: half-life extrapolated, range confidence interval 4593 to 5979 h (p = 95%)
pH:
4
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
2 109 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: half-life extrapolated, range confidence interval 2110 to 2868 h (p = 95%)
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
360 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 360 to 373 h (p = 95%)
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
86.4 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 86.4 to 88.6 h (p = 95%)
pH:
7
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
26.8 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 26.8 to 27.0 h (p = 95%)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
3.38 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 3.38 to 3.50 h (p = 95%)
pH:
9
Temp.:
10 °C
Hydrolysis rate constant:
0 s-1
DT50:
174 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 174 to 182 min (p = 95%)
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
52.5 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 52.5 to 54.8 min (p = 95%)
pH:
9
Temp.:
30 °C
Hydrolysis rate constant:
0.001 s-1
DT50:
17.3 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: range confidence interval 17.3 to 17.7 min (p = 95%)
Details on results:
TEST CONDITIONS
Deviations from Guideline: None
At pH 4, samples at sampling point 9 and 10 could not be analysed within 5 % of the total run time due to a technical malfunction of the analytical system. Samples were taken, diluted and frozen until analysis. For 30 °C the temperature was out of the temperature range for 1 % (about 7 - 8 h) of the total run time (742 h) due to a technical malfunction. These deviations are considered to have no impact on quality and integrity of the study. Sterility was checked by CFU-determination. No significant colony forming units were found, confirming the sterility of the test solutions.

TRANSFORMATION PRODUCTS: At pH4, 20°C:
- transformation product #1 and #2
- concentration range (both together): no signal - 1.3 micromol / L (measured), corresponding to 0-4.8% of content

At pH4, 30°C:
- transformation product #1 and #2
- concentration range (both together): no signal - 3.65 micromol / L (measured), corresponding to 0-13.5% of content

At pH4, 50 °C:
- transformation product #1 and #2
- concentration range (both together): no signal - 9.57 micromol / L (measured), corresponding to 0-35.4% of content
- maximum concentration at 675 h hydrolysis time

At pH7, 20 °C:
- transformation product #5 and #6
- concentration range (both together): no signal - 11.4 micromol / L (measured), corresponding to 0-20.8% of content

At pH7, 30 °C:
- transformation product #5 and #6
- concentration range (both together): no signal - 11.8 micromol / L (measured), corresponding to 0-21.2% of content

At pH7, 50 °C:
- transformation product #5 and #6
- concentration range (both together): no signal - 21.6 micromol / L (measured), corresponding to 0-37.7% of content

At pH7, 20 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -22.3 micromol / L (measured), corresponding to 0-40.8% of content
- maximum concentration at 193 h hydrolysis time

At pH7, 30 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -22.2 micromol / L (measured), corresponding to 0-39.9% of content
- maximum concentration at 48.1 h hydrolysis time

At pH7, 50 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -23.9 micromol / L (measured), corresponding to 0-41.8% of content
- maximum concentration at 6.37 h hydrolysis time

At pH9, 10 °C:
- transformation product #5 and #6
- concentration range (both together): no signal -21.8 micromol / L (measured), corresponding to 0-40.2% of content

At pH9, 20 °C:
- transformation product #5 and #6
- concentration range (both together): no signal -15.9 micromol / L (measured), corresponding to 0-28.0% of content

At pH9, 30 °C:
- transformation product #5 and #6
- concentration range (both together): no signal -18.2 micromol / L (measured), corresponding to 0-35.2% of content

At pH9, 10 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -25.1 micromol / L (measured), corresponding to 0-46.3% of content
- maximum concentration at 424 min hydrolysis time

At pH9, 20 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -24.9 micromol / L (measured), corresponding to 0-43.8% of content
- maximum concentration at 118 min hydrolysis time

At pH9, 10 °C:
- transformation product #7 and #8
- concentration range (both together): no signal -22.3 micromol / L (measured), corresponding to 0-43.3% of content
- maximum concentration at 35 min hydrolysis time

PATHWAYS OF HYDROLYSIS
- Figures of chemical structures attached: Yes

The results of hydrolysis are presented in the tables below:

Hydrolysis Results for the test item 1,6 -Bis-(maleinimido)-2,2,4 -trimethylhexan at pH4 and 20 °C

Hydrolysis Time [h]

Concentration [mg/L]

Ln Concentration

0.00

34.7

3.55

77.3

34.5

3.54

125

34.5

3.54

167

34.2

3.53

246

33.8

3.52

311

33.8

3.52

407

33.2

3.50

485

32.8

3.49

576

32.3

3.47

675

31.7

3.46

742

30.9

3.43

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH4 and 30 °C 

HydrolysisTime[h]

Concentration[mg/L]

LnConcentration

0.00

34.7

3.55

77.1

34.0

3.53

125

33.6

3.51

167

33.1

3.50

246

32.3

3.47

311

31.6

3.45

407

30.7

3.42

485

30.3

3.41

576

29.3

3.38

675

28.9

3.36

742

26.1

3.26

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH4 and 50 °C 

Hydrolysis Time [h]

Concentration [mg/L]

Ln Concentration

0.00

34.7

3.55

76.9

29.9

3.40

124

27.2

3.30

167

25.0

3.22

246

21.3

3.06

310

18.7

2.93

407

15.6

2.75

484

13.5

2.61

576

10.9

2.39

675

9.70

2.27

742

8.34

2.12

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH7 and 20 °C 

HydrolysisTime[h]

Concentration[mg/L]

Ln Concentration

0.00

34.8

3.55

5.90

32.9

3.49

23.7

28.6

3.35

30.4

27.0

3.29

103

14.9

2.70

121

12.9

2.56

144

10.6

2.36

167

8.82

2.18

193

7.30

1.99

264

4.18

1.43

289

3.44

1.23

 

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethyl hexan at pH7 and 30 °C

Hydrolysis Time [h]

Concentration [mg/L]

Ln Concentration

0.000

35.5

3.57

0.750

34.6

3.54

1.10

34.5

3.54

5.28

31.0

3.43

6.10

30.2

3.41

22.2

20.0

2.99

25.0

18.6

2.92

28.7

16.9

2.83

48.1

10.2

2.32

71.0

5.65

1.73

99.4

2.73

1.00

 

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH7 and 50 °C 

Hydrolysis Time [h]

Concentration [mg/L]

Ln Concentration

0.000

36.5

3.60

0.350

35.0

3.55

0.683

32.5

3.48

1.12

30.2

3.41

1.75

26.1

3.26

2.72

21.9

3.09

3.57

18.5

2.92

4.82

14.0

2.64

6.37

10.1

2.31

7.98

7.26

1.98

22.2

0.368*

-1.00*

*)=<LOQ, not included in calculations

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH9 and 10 °C

Hydrolysis Time [min]

Concentration [mg/L]

Ln Concentration

0.00

34.5

3.54

4.00

33.0

3.50

10.0

32.2

3.47

16.0

31.7

3.46

30.0

30.4

3.42

48.0

27.7

3.32

73.0

25.0

3.22

100

22.2

3.10

143

18.6

2.92

244

12.4

2.52

424

6.36

1.85

1411

0.127*

-2.06*

*)=<LOQ, not included in calculations

 

Hydrolysis Results for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan at pH9 and 20 °C 

Hydrolysis Time [min]

Concentration [mg/L]

Ln Concentration

0.00

36.3

3.59

3.00

35.3

3.56

5.00

33.7

3.52

8.00

32.3

3.47

15.0

29.4

3.38

24.0

27.0

3.30

36.0

21.8

3.08

50.0

18.2

2.90

70.0

14.0

2.64

118

7.41

2.00

210

2.30

0.833

 

Hydrolysis Results for the test item 1,6 -Bis-(maleinimido)-2,2,4 -trimethylhexan at pH9 and 30 °C 

Hydrolysis Time [min]

Concentration [mg/L]

Ln Concentration

0.00

33.0

3.50

1.00

31.1

3.44

2.00

30.1

3.40

4.00

27.9

3.33

7.00

24.4

3.19

12.0

20.0

3.00

18.0

15.7

2.75

25.0

11.9

2.48

35.0

8.09

2.09

50.0

4.39

1.48

89.0

0.680*

-0.386*

*)=<LOQ, not included in calculations

 

Validity criteria fulfilled:
yes
Conclusions:
The test item showed a fast hydrolysis (DT50 ≤ 2.4 h) at pH 9 at 20 and 30 °C as well as a moderate hydrolysis (2.4 h ≥ DT50 ≤ 30 d) for the test conditions: pH 4 at 50 °C; pH 7 at 20, 30, 50 °C; pH 9 at 10 °C. At pH 4 at 20 and 30 °C, only a slow hydrolysis (DT50 > 30 d) of the test item was observed.

Description of key information

The test item showed a fast hydrolysis (DT50 ≤ 2.4 h) at pH 9 at 20 and 30 °C as well as a moderate hydrolysis (2.4 h ≥ DT50 ≤ 30 d) for the test conditions: pH 4 at 50 °C; pH 7 at 20, 30, 50 °C; pH 9 at 10 °C. At pH 4 at 20 and 30 °C, only a slow hydrolysis (DT50 > 30 d) of the test item was observed.

Key value for chemical safety assessment

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

Additional information

Hydrolysis as a function of pH was determined according to OECD Guideline No. 111 and Council Regulation (EC) No. 440/2008, Method C.7 for the test item 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan (batch number: 0493122001) from 2014-10-16 to 2015-01-15. Analyses of the test item was performed via LC-DAD on a reversed phase column using an external standard. Two chromatographic methods were employed, as the analytical system had to be changed for the advanced testing. The analytical methods were validated with satisfactory results with regard to linearity, accuracy, precision and specificity.

The preliminary test was conducted with a test item concentration of 35 mg/L in buffer solutions at pH 4, 7 and 9 and 50 °C. For all pH values the advanced test was performed, as a significant reduction (> 10 %) of the test item concentration was observed in the preliminary test. The advanced test was conducted with a test item concentration of 35 mg/L in buffer solutions of pH 4 and 7 at temperatures of 20, 30 and 50 °C and at temperatures of 10, 20 and 30 °C for pH 9. Samples were taken at test start (0 h) and at 10 spaced points until test end. Buffer solutions were analysed at test start and test end and there was no analytical interference with the test item. Reaction rate constants and half-lives were calculated from the analysed samples based on a first respectively pseudo first order reaction kinetic model. The test item showed a fast hydrolysis (T1/2=< 2.4 h) at pH 9 at 20 and 30 °C as well as a moderate hydrolysis (2.4 h >= T1/2=< 30 d) for the test conditions: pH 4 at 50 °C; pH 7 at 20, 30, 50 °C; pH 9 at 10 °C. At pH 4 at 20 and 30 °C, only a slow hydrolysis (T1/2 > 30 d) of the test item was observed.

Transformation product signals were evaluated from the LC-DAD chromatograms of the definitive study. Major transformation products (≥ 10 % of the initial peak area of the parent molecule) were observed at retention time (RT; given in minutes) 0.48 (#1 and 2) for the pH 4 test medium and at RT 1.11 (#5 and 6) and 1.49 (#7 and 8) for the pH 7 and 9 test media. One further transformation product was postulated based on chemical deliberations and could be qualitatively verified at RT 0.28 (#3 and 4) (co-elution with buffer components) in the pH 4 test medium. Identification was performed via LC-MS/MS by postulation of possible transformation products based on molecule and molecule adduct signals and verification of the postulates by MS/MS experiments.

Identity and transformation pathways of the hydrolysis products were identified as follows:

RT0.48 was identified to be a mixture of 1-(6-amino-2,4,4-trimethylhexyl)-1H-pyrrole-2,5-dione and 1-(6-amino-2,2,4-trimethylhexyl)-1H-pyrrole-2,5-dione (#1 and #2, direct transformation products of the test item), which transformed into 2,2,4-trimethylhexane-1,6-diamine and 2,4,4-trimethylhexane-1,6-diamine (RT0.28, #4 and #3).

RT1.49 was identified to be a mixture of (2Z)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-3,3,5-trimethylhexyl]amino}-4-oxobut-2-enoic acid and (2Z)-4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-3,5,5-trimethylhexyl]amino}-4-oxobut-2-enoic acid (#7 and #8, direct transformation products of the test item), which transformed into (2Z)-3-({6-[(2E)-3-carboxyprop-2-enamido]-3,3,5-trimethylhexyl}carbamoyl)prop-2-enoic acid and (2Z)-3-({6-[(2E)-3-carboxyprop-2-enamido]-3,5,5-trimethylhexyl}carbamoyl)prop-2-enoic acid (RT 1.11, #5 and #6).

Mass balance was calculated for the test item and transformation product signals by evaluation of the LC-DAD chromatograms. Transformation products were quantified against the test item as standard. For RT 0.48 in the pH 4 test medium a response factor of two was applied due to the total elimination of one of two chromophoric group. The mass balance was calculated to be 58.5 – 95.3 % at pH4, 57.6 – 67.6 % at pH 7 and 56.7 – 72.3 % at pH 9

Reaction Rate Constants and Half-Lives of 1,6-Bis-(maleinimido)-2,2,4-trimethylhexan

 

 

pH4

pH7

pH9

20°C

30°C

50°C

20°C

30°C

50°C

10°C

20°C

30°C

Reaction rate constant kobs[1/s]

 

4.19x 10-8

 

9.13x 10-8

 

5.35x 10-7

 

2.23x 10-6

 

7.17x 10-6

 

5.69x 10-5

 

6.64x 10-5

 

2.20x 10-4

 

6.67x 10-4

Half-life T½

[min]

 

-

 

174

 

52.5

 

17.3

Half-life T½

[h]

 

45931

 

21091

 

360

 

86.4

 

26.8

 

3.38

 

2.90

 

0.875

 

0.289

Half-life T½

[d]

 

1911

 

87.91

 

15.0

 

3.60

 

1.12

 

0.141

 

-

Number of data points

 

11

 

11

 

11

 

11

 

11

 

10

 

11

 

11

 

10

Slope of regression

graph

 

 

Significantly non-zero

1)  = T½ extrapolated