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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-08 to 2016-03-16
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:
OECD Guideline 111 for Testing of Chemicals (2004)
Deviations:
yes
Remarks:
Due to the fast hydrolysis at pH 4 and 9, less than 6 injections (samplings) could be performed until the LOQ was reached. This deviation is considered to have no impact on quality and integrity of the study.
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
Due to the fast hydrolysis at pH 4 and 9, less than 6 injections (samplings) could be performed until the LOQ was reached. This deviation is considered to have no impact on quality and integrity of the study.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
One sample per temperature condition and pH value was prepared at test start. Single injections were made at test start (0 h) and subsequently at a minimum of 12 spaced points, if possible, by the auto sampler. The temperature of the sample compartment was checked every minute.
Buffers:
- pH: 4
- Composition of buffer: 0.18 g of NaOH and 5.7555 g of mono potassium citrate were dissolved in 500 mL double distilled water


- pH: 7
- Composition of buffer: 0.7358 g of NaOH and 4.3012 g of KH2PO4 were dissolved in 500 mL double distilled water


- pH: 9
- Type and final molarity of buffer: 0.426 g NaOH, 1.8638 g KCl and 1.5458 g H3BO3 were dissolved in 500 mL double distilled water

The details of the buffer are shown in Table 1
Details on test conditions:
Sterile buffer solutions were prepared from chemicals with analytical grade or better quality following the composition guidance given in “KÜSTER-THIEL, Rechentafeln für 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 pHs were checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilised by filtration through 0.2 µm via sterile vacuum filtration system.

Experimental procedure

Test concentration/solution: 50 µg/L (pH value 4, 7 and 9)
Co-solvent: Acetonitrile, = 1% (v/v)
Control: Buffer solutions (pH value 4, 7 and 9)


Test method

Test container: HPLC vials, polypropylene, volume: 1.5 mL. Test container suitability, regarding test item adsorption, was successfully verified during analytical method development, showing no adsorption tendency of 3-trimethoxysilylpropyl methacrylate to the test container walls

Test volume: 1 mL
Application: Via stock solution the test item was dissolved in acetonitrile. Buffer solutions were spiked to a test item concentration of 50 µg/L and filled into the test containers. After the vials were sealed they were transferred into the temperature controlled sample compartment. The time between test item application and start of analysis did not exceed 30 min

Incubation time: The test was conducted for at least two half live periods, optimally until 90% degradation is reached or 30 days, whichever was shorter

Temperature: 16.5, 20 and 25 ± 0.5°C
Light: Photolytic effects were precluded by avoidance of direct sunlight within the sample compartment
Sterility: No sterility check was deemed necessary due to the short incubation times

Equipment:
Balance, KERN
pH-Meter, Lab850, SI ANALYTICS
Pipettes, THERMO LABSYSTEMS + THERMO SCIENTIFIC
Direct displacement pipette, GILSON
Sterile vacuum filtration system 0.2 µm, SARSTEDT
Thermometer, 175-T3and temperature sensing device, TESTO
Standard laboratory equipment
Duration:
1 d
pH:
4
Temp.:
16.5 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-10
Duration:
1 d
pH:
4
Temp.:
20 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-09
Duration:
1 d
pH:
4
Temp.:
25 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-11
Duration:
2 d
pH:
7
Temp.:
16.5 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-10 to 2016-03-11
Duration:
1 d
pH:
7
Temp.:
20 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-09
Duration:
1 d
pH:
7
Temp.:
25 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-16
Duration:
1 d
pH:
9
Temp.:
16.5 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-10
Duration:
1 d
pH:
9
Temp.:
20 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-09
Duration:
1 d
pH:
9
Temp.:
25 °C
Initial conc. measured:
50 µg/L
Remarks:
2016-03-11
Number of replicates:
At least 12 subsequent samples for one test container, if possible
Positive controls:
no
Negative controls:
no
Preliminary study:
Preliminary study was not conducted
Transformation products:
not measured
% Recovery:
>= 49 - <= 57
pH:
4
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 3.50 µg/L (1 x LOQ)
% Recovery:
>= 48 - <= 52
pH:
4
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 35.0 µg/L (10 x LOQ)
% Recovery:
>= 77 - <= 83
pH:
7
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 3.5 µg/L (1 x LOQ)
% Recovery:
>= 80 - <= 88
pH:
7
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 35.0 µg/L (10 x LOQ)
% Recovery:
>= 64 - <= 79
pH:
9
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 3.50 µg/L (1 x LOQ)
% Recovery:
>= 68 - <= 77
pH:
9
Remarks on result:
other: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile. Fortified concentrations: 35.0 µg/L (10 x LOQ)
Key result
pH:
4
Temp.:
16.5 °C
Hydrolysis rate constant:
0.012 s-1
DT50:
0.017 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0.011 s-1
DT50:
0.018 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.012 s-1
DT50:
0.015 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
16.5 °C
Hydrolysis rate constant:
0 s-1
DT50:
2.52 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Confidence interval: 2.41 to 2.63 h
Key result
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
1.87 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Confidence interval: 1.75 to 1.99 h
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0 s-1
DT50:
1.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Confidence interval:1.56 to 1.86 h
Key result
pH:
9
Temp.:
16.5 °C
Hydrolysis rate constant:
0.002 s-1
DT50:
0.088 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Confidence interval: 0.0790 to 0.0972 h
Key result
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0.003 s-1
DT50:
0.068 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Confidence interval: 0.0545 to 0.0799 h
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.004 s-1
DT50:
0.049 h
Type:
(pseudo-)first order (= half-life)
Other kinetic parameters:
For the test conditions pH 4 at 16.5, 20 and 25 °C and pH 9 at 25 °C no calculation of confidence interval could be done due to the low number of data points.

For the test conditions pH 7 at 16.5, 20 and 25 °C and pH 9 at 16.5 and 20 °C the ln concentration vs. time plots have regression graphs with slopes significantly non zero. Therefore first order reaction kinetics was applied for data computation.

Method validation:

Linearity: The analytical system gave a linear response for the test item in the range of 2 – 50 µg/L. The coefficients of determination (r2) of all calibration curves were > 0.992.

System quantification limit: The system quantification limit was fixed at 2 µg/L for the analytical standard verified by an S/N of 68

LOQ: The limit of quantification of the analytical method (LOQ) was fixed at 3.5 µg/L. The 1 x LOQ as well as the 10 x LOQ were checked by means of accuracy.

The determination was prepared with 5% (v/v) acetonitrile. A second determination was prepared with concentration at 1 x LOQ with 1% (v/v) acetonitrile only at pH 7, due to the minor recovery rates at the first determination.

Accuracy and precision: For pH 7 and 9 sufficient accuracy (mean recovery > 70 %, Tables 5 and 6) could be demonstrated. For the pH 4 test system only low recoveries at approx. 50% (Table 4) could be achieved due to hydrolysis, so accuracy could not be demonstrated. Nevertheless, as the hydrolysis reaction was expected and as sufficient recoveries could be demonstrated for the other test systems, the method was deemed suitable also for the pH 4 test condition. In addition to this, it was necessary to increase the co-solvent amount for method validation purpose to 5 % acetonitrile in order to slow down the hydrolysis reaction to achieve the minimum time frame for sample preparation.

The corresponding coefficients of variation were = 7.3% except showing a sufficient precision of the analytical method.

Table 4: Recovery Rates of Fortified Samples at pH 4 with 5% (v/v) Acetonitrile

Fortified concentrations: 3.50 µg/L (1 x LOQ) and 35.0 µg/L (10 x LOQ)

 

3-trimethoxysilylpropylmethacrylate

 

1 xLOQ

10 xLOQ

Replicate

Calc. conc.

RR

Calc.conc.

RR

 

[µg/L]

[%]

[µg/L]

[%]

1

1.8441

53

17.3

49

2

1.7141

49

16.9

48

3

1.7771

51

18.0

52

4

1.8301

52

17.0

49

5

1.9911

57

17.4

50

Mean

1.83

52

17.3

50

SD

0.09

 

0.4

 

CV [%]

5.0

 

2.3

 

 

Table 5: Recovery Rates of Fortified Samples at pH 7 with 5% (v/v) Acetonitrile

Fortified concentrations: 3.50 µg/L (1 x LOQ) and 35.0 µg/L (10 x LOQ)

 

3-trimethoxysilylpropylmethacrylate

 

1 xLOQ

10 xLOQ

Replicate

Calc. conc.

RR

Calc.conc.

RR

 

[µg/L]

[%]

[µg/L]

[%]

1

2.71

77

30.7

88

2

2.73

78

29.6

84

3

2.91

83

27.9

80

4

2.79

80

29.3

84

5

2.78

80

30.6

87

Mean

2.78

80

30

85

SD

0.08

 

1

 

CV [%]

2.9

 

3.5

 

 

Table 6: Recovery Rates of Fortified Samples at pH 9 with 5% (v/v) Acetonitrile

Fortified concentrations: 3.50 µg/L (1 x LOQ) and 35.0 µg/L (10 x LOQ)

 

3-trimethoxysilylpropylmethacrylate

 

1 xLOQ

10 xLOQ

Replicate

Calc. conc.

RR

Calc.conc.

RR

 

[µg/L]

[%]

[µg/L]

[%]

1

2.51

72

25.0

71

2

2.68

76

23.9

68

3

2.78

79

24.2

69

4

2.24

64

24.3

69

5

2.47

71

26.9

77

Mean

2.5

72

25

71

SD

0.2

 

1

 

CV [%]

7.3

 

4.4

 

 

Table 7: Recovery Rates of Fortified Samples at pH 7 with 1% (v/v) Acetonitrile

Fortified concentrations: 3.50 µg/L (1 x LOQ)

 

3-trimethoxysilylpropylmethacrylate

 

1 xLOQ

Replicate

Calc. conc.

RR

 

[µg/L]

[%]

1

 0.7371

21

2

 0.6931

20

3

 0.9631

28

4

 0.1921

5

5

 0.7841

22

Mean

 0.7

19

SD

 0.3

 

CV [%]

38.3

 

 

Calc. Conc. = Calculated concentration

RR = Recovery rate regarding to the fortified concentration of the test item

SD = Standard deviation

CV = Coefficient of variation

1) = Value below calibration range

Specificity: For each sample, two transitions of the same precursor (ion one quantifier used for evaluation and one secondary used for confirmation of the analyte identity) were measured, confirming the test item identity. No significant signal was observed for the blank samples, confirming the specificity of the analytical method.

pH check:

Table 8: pH-Value of the Test Systems

measured before start of hydrolysis

Intended pH-value

Measured pH-value
at 20 °C

Measured pH-value
at 30 °C

Measured pH-value
at 50 °C

4.0 ± 0.1

4.03

4.02

4.03

7.0 ± 0.1

7.00

7.01

7.01

9.0 ± 0.1

9.04

9.04

9.04

 

Temperature monitoring

Table 9: Incubation Temperature measured every minute

pH value

Intended Temperature

Measured Temperature

Mean ± SD

Min. / Max.

4

16.5 ± 0.5

16.5 ± 0.0

16.5 / 16.5

20.0 ± 0.5

20.3 ± 0.0

20.3 / 20.3

25.0 ± 0.5

24.91

-

7

16.5 ± 0.5

16.3 ± 0.2

16.1 / 16.7

20.0 ± 0.5

20.1 ± 0.2

19.8 / 20.3

25.0 ± 0.5

24.7 ± 0.1

24.6 / 24.9

9

16.5 ± 0.5

16.6 ± 0.04

16.5 / 16.6

20.0 ± 0.5

20.4 ± 0.05

20.3 / 20.4

25.0 ± 0.5

24.91

-

 

SD = Standard deviation

1)= Value taken from manually documentation

The additional manually taken values confirm the results of the automated temperature recording of the datalogger.

Validity criteria fulfilled:
yes
Conclusions:
Hydrolysis half-lives of 0.018 h at pH 4, 1.87 h at pH 7, and 0.068 h at pH 9 and 20°C were determined for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.
Executive summary:

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 3-trimethoxysilylpropylmethacrylate (batch number:15CTEC010) from 2016-03-08 to 2016-03-16 at the test facility in 31157 Sarstedt, Germany.

Analyses of the test item, 3-trimethoxysilylpropylmethacrylate were performed via LC-MS/MS using the test item as the external standard. The analytical method was validated with satisfactory results regarding linearity, accuracy, precision and specificity.

The test was conducted with a nominal test item concentration of 50 µg/L in buffer solution of pH 4, pH 7 and pH 9 at temperatures of 16.5, 20 and 25°C. Up to 12 subsequent samples were taken 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 order reaction kinetics model.

The test item showed a hydrolysis rate (t1/2= 2.4 h) at all test conditions except for pH 7 at 16.5 °C. For pH 7 at 16.5 °C a hydrolysis (t1/2= 2.4 h but = 30 d) was observed.

Due to the fast hydrolysis at pH 4 and pH 9, it was not possible to obtain the minimum number of six samples, as recommended by the guideline. Nevertheless, the statistical evaluation at these and the other test conditions indicate that the selected first order reaction kinetics model is applicable and that the obtained reaction rate constants and half-lives are plausible. In addition, for the test conditions pH 4 at 16.5, 20 and 25°C, it was not possible to obtain a valid analytical result except for the starting analysis. Nevertheless, the first sample, although below the LOQ, could be evaluated and was used to estimate the reaction rate constants and half-lives for these test conditions.

Description of key information

Hydrolysis half-lives: 0.018 h at pH 4, 1.87 h at pH 7, and 0.068 h at pH 9 and 20°C (OECD 111)

Key value for chemical safety assessment

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

Additional information

The registration substance, 3-trimethoxysilylpropyl methacrylate contains two types of hydrolysable groups: three trimethoxy groups (which react to form the corresponding silanols) and one methacrylate ester group (which has the potential to react to form the corresponding alcohol plus methacrylic acid). The chemical safety assessment of the substance assumes that these reaction processes will proceed independently based on their rates under the relevant conditions.

Hydrolysis of the trimethoxysilane groups:

The hydrolysis of the submission substance has been determined in accordance with OECD 111 Test Guideline and in compliance with GLP. The measured hydrolysis half-life values are:

pH 4: 0.0165 h at 16.5°C, 0.018 h at 20°C, 0.0155 h at 25°C

pH 7: 2.52 h at 16.5°C, 1.87 h at 20°C, 1.7 h at 25°C

pH 9: 0.0883 h at 16.5°C, 0.068 h at 20°C, 0.0491 h at 25°C

The results are considered to be reliable and are used for assessment purposes.

The key study is supported by two other reliable hydrolysis rates that are available for the registered substance. Hydrolysis half-lives at 20-25°C of 0.3 h at pH 4, 0.3 h at pH 5, 4 h at pH 7 and 0.1 h at pH 9 were predicted for the substance using validated QSAR estimation methods. Similarly, hydrolysis half-lives of 3 h at 25°C, 4 h at 20°C and pH 7 were determined in a preliminary study conducted in accordance with OECD Test Guideline 111 and in compliance with GLP. At pH 4 and pH 9, the test substance was observed to undergo rapid hydrolysis and as such half-lives at these pHs was estimated to be < 1 hour.

In a secondary source to which reliability could not be assigned, a hydrolysis half-life of 121.6 mins (approximately 2 hours) at pH 4.5 and 25°C was reported for the substance. The available data are in agreement that the substance undergoes rapid hydrolytic degradation in contact with water.

As the hydrolysis reaction may be acid or base catalysed, the rate of reaction is expected to be slowest at around pH 7 and increase as the pH is raised or lowered. For an acid-base catalysed reaction in buffered solution, the measured rate constant is a linear combination of terms describing contributions from the uncatalyzed reaction as well as catalysis by hydronium, hydroxide, and general acids or bases.

kobs = k0 + kH3O+[H3O+] + kOH-[OH-] + ka[acid] + kb[base]

At extremes of pH and under standard hydrolysis test conditions, it is reasonable to suggest that the rate of hydrolysis is dominated by either the hydronium or hydroxide catalysed mechanism.

Therefore, at low pH:

kobs ˜ kH3O+[H3O+]

At pH 4 [H3O+] = 10-4 mol dm-3 and at pH 2 [H3O+] = 10-2 mol dm-3; therefore, kobs at pH 2 should be approximately 100 times greater than kobs at pH 4.

The half-life of a substance at pH 2 is calculated based on:

t1/2(pH 2) = t1/2(pH 4) / 100

The calculated half-life of the substance at pH 2 and 20°C is therefore 0.00018 h (approximately 1 second). However, it is not appropriate or necessary to attempt to predict accurately when the half-life is less than 5-10 seconds. The half-life is therefore reported as 5 seconds as a worst case.

Reaction rate increases with temperature therefore hydrolysis will be faster at physiologically relevant temperatures compared to standard laboratory conditions. Under ideal conditions, hydrolysis rate can be recalculated according to the equation:

DT50(XºC) = DT50(T) * e(0.08.(T-X))

Where T = temperature for which data are available and X = target temperature.

Thus, for 3-trimethoxysilylpropyl methacrylate the hydrolysis half-life at 37.5ºC and pH 7 (relevant for lungs and blood) is approximately 0.46 hours. At 37.5ºC and pH 2 (relevant for conditions in the stomach following oral exposure), it is not appropriate to apply any further correction for temperature to the limit value and the hydrolysis half-life is therefore approximately 5 seconds.

At 37.5ºC and pH 5.5 (relevant for dermal exposure), the hydrolysis half-life will be in between the half-lives at pH 4 and pH 7 at 37.5ºC.

The hydrolysis products in this case are 3-(trihydroxysilyl)propyl methacrylate and methanol.

Note on hydrolysis of the methacrylate ester group:

Very limited evidence of the hydrolysis of the methacrylate ester group is available. The hydrolysis half-life of methyl methacrylate (a much more soluble substance) was reported to be >1 year at 25°C and pH 7 and 14.4 days at 25°C and pH 9 (WHO CICAD summary citing Howard, 1989). However, in the case of the registration substance, 3-trimethoxysilylpropyl methacrylate, the reaction may be slower and for the purposes of chemical safety assessment, in the absence of clear evidence it is considered that the methacrylate ester group reaction is not significant for the chemical safety assessment at any relevant pH. Therefore, where the chemical safety assessment considers hydrolysis products, these are identified as 3-(trihydroxysilyl)propyl methacrylate and methanol. The same assumptions are made for read-across substance containing methacrylate groups. This is a conservative position in the absence of clear evidence, as the methacrylate structural feature is likely to be more toxic than the corresponding alcohol.

Reference:

Howard P (1989) Handbook of environmental fate and exposure data for organic chemicals. Vol. 1. Chelsea, MI, Lewis Publishers Inc., pp. 402–407