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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-25 to 2012-05-04
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
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals/times for sterility check: The sterility of the prepared buffers was confirmed at the start of the study and the sterility of selected dosed samples was confirmed at the start and end of the study. Sterility was evaluated using 3M PetriFilm Aerobic Count Plates. An aliquot of 0.1-0.5 mL of each dosed samples was pipetted onto the centre of the bottom film and spread using a spreader. Plates were incubated at room temperature for at least 48 hours and then observed for microbial growth. Plates were interpreted as positive, indicating the presence of microbial growth or as negative, indicating the absence of microbial colony formation.

Buffers:
The 0.01 M buffer solutions used during the study were prepared as described below. If necessary, the pH of each buffer was adjusted with 1.0 N sodium hydroxide or 1.0N hydrochloric acid

- pH: 5.0
- Type and final molarity of buffer: acetate
- Composition of buffer: 592 mL of a 0.01 M acetic acid solution (0.576 mL glacial acetic acid solution diluted to 1000 mL with purified reagent water) was combined with 1408 mL of a 0.01 M sodium acetate solution (2.0406 g of sodium acetate trihydrate diluted to 1500 mL with purified water). The pH of the resultany buffer solution was adjusted to 5.01 by the addition of hydrochloric acid.


pH: 7.0
- Type and final molarity of buffer: phosphate
- Composition of buffer: 390 mL of a 0.02 M sodium phosphate solution (1.5599 g sodium phosphate monobasic dihydrate diluted to 500 mL with purified reagent water) was combined with 610 mL of a 0.02 M sodium phosphate dihydrate solution (3.5602 g sodium phosphate dibasic dihydrate diluted to 1000 mL with purified reagent water) and diluted to a final volume of 2000 mL with purified reagent water. The pH of the resultant buffer solution was adjusted to 6.99 by the addition of hydrochloric acid.


pH: 9.0
- Type and final molarity of buffer: botate
- Composition of buffer: 40 mL of a 0.5 M boric acid solution (1.5450 g boric acid diluted to 50 mL with purified reagent water) was diluted to 2000 mL with purified reagent water. The pH of the resultant buffer was adjusted to 9.00 by the addition of 1 N sodium hydroxide and hydrochloric acid.

The pH of each buffer solution was measured using a calibrated Yellow Springs Instrument (YSI) pH 100 meter with a YSI 110-1 pH electrode. The pH of the sterile, nitrogen purged buffer solutions was verified to be 5.0±0.1, 7.0±0.1 and 9.0±0.1 at ambient temperature (approximately 20°C) prior to dosing.
Estimation method (if used):
Rate constants for the test substance at 20°C and 25°C were estimated using the Arrhenius Equation.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: The test apparatus for the hydrolysis testing consisted of 10 mL amber borosilicate glass vials. Prior to testing, the vials and pipettes used to transfer the solutions were autoclaves at 15 psi (approximately 121°C) for 30 minutes to minimise potential for microbial degradation of the test substance. the buffers were autoclaved at 15 psi (approximately 121°C) for 45 minutes. Temperatures were controlled by placing the test samples in individual water baths set to maintain temperature at 15±0.5°C, 25±0.5°C and 35±0.5°C in the dark.

- Measures to exclude oxygen: The buffers were purged with sterile nitrogen for approximately five minutes to exclude oxygen prior to dosing.

Test procedures:
Individual 10 mL amber vials were filled with 2.0 mL of the appropriate buffer (i.e. pH 5.0, 7.0 or 9.0), flushed with a stream of nitrogen, capped with sterile aluminium-Teflon caps, labelled and then equilibrated in 15±0.5°C, 25±0.5°C and 35±0.5°C water baths before dosing. A 20 µL aliquot of the 40 mg/L stock solution was dosed by injecting through a septum using a 25 µL glass syringe to provide a nominal concentration of 0.40 mg/L and timed immediately. Samples were vortex-mixed for 10 seconds and incubated at the equilibrating temperature for various intervals of time before sampling. The percent organic co-solvent in the sample was 0.99%.

One vial from each pH value was used for pH and sterility analysis at the start of the study. One vial from each pH/temperature combination was used for pH and sterility analysis at the end of the study. Duplicate samples were analysed at various intervals during the tests to determine the concentration of 2,4-hexadienoic acid, 3-(trimethoxysilyl)propyl)ester in the test samples. sampling intervals for the definitive study are shown in Table 1.

At each sampling interval, each sample vial was immediately diluted with 8 mL of 99:0.1 acetonitrile:sodium hydroxide, acetonitrile or 99:0.1 acetonitrile:hydrochloric acid (pH 5.0, 7.0 or 9.0 respectively) and vortex-mixed for 30 seconds simultaneously. The diluent was added usinga 10 ml graduated syringe to quench hydrolysis. Aliquots (approximately 1.5 mL) were then transferred to autosampler vials and analysed by HPLC-UV. The remaining sample was capped and stored frozen.
Duration:
80 min
pH:
5
Temp.:
15 °C
Initial conc. measured:
400 µg/L
Duration:
40 min
pH:
5
Temp.:
25 °C
Initial conc. measured:
400 µg/L
Duration:
40 min
pH:
5
Temp.:
35 °C
Initial conc. measured:
400 µg/L
Duration:
48 h
pH:
7
Temp.:
15 °C
Initial conc. measured:
400 µg/L
Duration:
24 h
pH:
7
Temp.:
25 °C
Initial conc. measured:
400 µg/L
Duration:
8 h
pH:
7
Temp.:
35 °C
Initial conc. measured:
400 µg/L
Duration:
60 min
pH:
9
Temp.:
15 °C
Initial conc. measured:
400 µg/L
Duration:
40 min
pH:
9
Temp.:
25 °C
Initial conc. measured:
400 µg/L
Duration:
15 min
pH:
9
Temp.:
35 °C
Initial conc. measured:
400 µg/L
Number of replicates:
Duplicate
Positive controls:
no
Negative controls:
no
Preliminary study:
A preliminary study was not performed because the test substance is known to be hydrolytically unstable at all pH conditions.
Transformation products:
no
Key result
pH:
5
Temp.:
15 °C
Hydrolysis rate constant:
0.047 min-1
DT50:
15 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9985, DT90 = 49 min
Key result
pH:
5
Temp.:
25 °C
Hydrolysis rate constant:
0.072 min-1
DT50:
9.6 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9951, DT90= 32 min
Key result
pH:
5
Temp.:
35 °C
Hydrolysis rate constant:
0.114 min-1
DT50:
6.1 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9986, DT90= 20 min
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.07 h-1
DT50:
10 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9903, DT90= 33 h
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.146 h-1
DT50:
4.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9947, DT90= 16 h
Key result
pH:
7
Temp.:
35 °C
Hydrolysis rate constant:
0.304 h-1
DT50:
2.3 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9944, DT90= 7.6 h
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.048 min-1
DT50:
14 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9936, DT90= 48 min
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.116 min-1
DT50:
6 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9986, DT90= 20 min
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
0.218 min-1
DT50:
3.2 min
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2= 0.9977, DT90= 11 min
Details on results:
The test substance is known to be hydrolytically unstable at pH 5, pH 7 and pH 9. At pH 5.0, the hydrolysis half-lives were 15 minutes, 9.6 minutes and 6.1 minutes at 15°C, 25°C and 35°C respectively.

At pH 7.0, the half-lives are 10 hours, 4.7 hours and 2.3 hours at 15°C, 25°C and 35°C respectively. At pH 9.0, the half-lives are 14 minutes, 6.0 minutes and 3.2 minutes at 15°C, 25°C and 35°C respectively.

The calculated half-lives based on Arrhenius Equation at 25°C were 9.5 minutes at pH 5, 4.7 hours at pH 7 and 6.5 minutes at pH 9. At 20°C, the calculated half-lives are 12 minuets at pH 5, 6.9 hours at pH 7 and 9.5 minutes at pH 9.

Hydrolysis test temperature:

The temperature of the samples was maintained at 15±0.5°C, 25±0.5°C and 35±0.5°C for the duration of the study as shown in Table 2, Table 3 and Table 4.

Table 2: Temperature measurements of the water bath recorded during the 15±0.5°C Tier 2 hydrolysis tests at pH 5.0, 7.0 and 9.0

Date

Temperature (°C)

minimum*

current**

Maximum*

2012-04-26

15.1

15.1

15.1

2012-04-27

15.1

15.2

15.3

2012-04-28

15.2

15.3

15.3

2012-04-30

15.2

15.3

15.4

2012-04-30

15.2

15.3

15.3

2012-05-01

15.2

15.3

15.4

 

Table 3: Table 2: Temperature measurements of the water bath recorded during the 25±0.5°C Tier 2 hydrolysis tests at pH 5.0, 7.0 and 9.0

Date

Temperature (°C)

minimum*

current**

Maximum*

2012-04-25

25.0

25.0

25.0

2012-04-26

25.0

25.1

25.1

2012-04-30

25.1

25.1

25.1

2012-04-30

25.1

25.1

25.2

2012-05-01

24.7

24.7

25.2

 

Table 4: Temperature measurements of the water bath recorded during the 35±0.5°C Tier 2 hydrolysis tests at pH 5.0, 7.0 and 9.0

Date

Temperature (°C)

minimum*

current**

Maximum*

2012-04-26

Not applicable

35.0

Not applicable

2012-04-26

35.0

35.1

35.1

2012-04-27

35.0

35.0

35.1

2012-04-30

34.9

35.0

35.1

2012-04-30

35.0

35.0

35.1

2012-05-01

35.0

35.0

35.1

*Minimum and maximum temperatures reflect the temperature fluctuation between data recorded

**Current temperature reflects the temperature at the time of recording

Verification of sterility and pH:

The sterility of the samples was maintained at 15±0.5°C, 25±0.5°C and 35±0.5°C hydrolysis samples at Day 0 and hydrolysis samples at study termination were checked for each hydrolysis condition. All samples were confirmed to be sterile using 3M Petrifil Aerobic Count Plates as shown in Table 5.

The pH of the sterile buffer samples prior to dosing was 5.06, 6.85 and 8.96. The pH of the sterile buffer samples after dosing was 5.04, 6.93 and 8.96. The pH for the hydrolysis samples analysed during the Tier 2 hydrolyssi testing in all the test temperatures ranged from 5.00 - 5.03, 6.92 - 6.94 and 8.91 -8.96 at pH 5.0, 7.0 and 9.0 respectively, see Table 5.

Table 5: pH measurements recorded and sterility evaluation during hydrolysis testing

Buffers

pH values*

Day 0 before dosing

Day 0 after dosing

Termination**

15°C

25°C

35°C

pH 5

5.04

5.04

5.00

5.03

5.02

pH 7

6.95

6.93

6.93

6.94

6.92

pH 9

8.96

8.96

8.91

8.91

8.96

 

Buffers

Sterility evaluation

Temperature (°C)

Before dosing

Experimental end

pH 5

15

Negative

Negative

 

25

 

35

pH 7

15

Negative

Negative

 

25

 

35

pH 9

15

Negative

Negative

 

25

 

35

*All pH measurements were conducted at approximately 20°C, ambient temperature of the laboratory

**Temperatures listed for the purpose of identification of the test system

Sterile pH 5.0 aqueous buffer:

The mean measured concentration of the test substance at 15°C decreased from 103.1% at time 0 to 15.8% nominal at 40 minutes and was <LOQ at 80 minutes.

At 25°C, the mean measured concentration of the test substance decreased from 103.1% of nominal at time 0 to 23.9% of nominal at 20 minutes and was <LOQ at 40 minutes.

At 35°C, the mean measured concentration of the test substance decreased from 103.1% of nominal at time 0 to 10.3% of nominal at 20 minutes and was <LOQ at 40 minutes.

Sterile pH 7.0 aqueous buffer:

The mean measured concentration of the test substance at 15°C decreased from 104.1% at time 0 to 34.8% nominal at 16 hours and was <LOQ at 48 hours.

At 25°C, the mean measured concentration of the test substance decreased from 104.1% of nominal at time 0 to 10.6% of nominal at 16 hours and was <LOQ at 24 hours.

At 35°C, the mean measured concentration of the test substance decreased from 104.1% of nominal at time 0 to 9.6% at 8 hours.

Sterile pH 9.0 aqueous buffer:

The mean measured concentration of the test substance at 15°C decreased from 97.5% at time 0 to 13.3% nominal at 40 minutes and was <LOQ at 60 minutes.

At 25°C, the mean measured concentration of the test substance decreased from 97.5% of nominal at time 0 to 9.6% of nominal at 16 hours and was <LOQ at 40 minutes.

At 35°C, the mean measured concentration of the test substance decreased from 97.5% of nominal at time 0 to 11.4% of nominal at 10 minutes and was <LOQ at 15 minutes.

Rate constants and half-lifes

A first-order linear regression analysis was performed on the 15±0.5°C, 25±0.5°C and 35±0.5°C data for the sterile pH 5.0, pH 7.0 and pH 9.0 samples to determine the rates of hydrolysis. The half-lives are summarised in Table 6.

Table 6: Hydrolysis half-lives abd rate constants

 Buffers  Temperature (

°C)

 First order rate constant (k)  DT50  DT90  R(exp.)2 (a)  Time range (b)
 pH 5  15  0.0467  15 mins  49 mins  0.9985  0 to 40 mins
 pH 5  25  0.0721  9.6 mins  32 mins  0.9951  0 to 20 mins
 pH 5  35  0.1142  6.1 mins  20 mins  0.9986  0 to 20 mins
 pH 7  15  0.0695  10 hours  33 hours  0.9903  0 to 16 hours
 pH 7  25  0.1464  4.7 hours  16 hours  0.9947  0 to 16 hours
 pH 7  35  0.3045  2.3 hours  7.6 hours  0.9944  0 to 8 hours
 pH 9  15  0.0483  14 mins  48 mins  0.9936  0 to 40 mins
 pH 9  25  0.1155  6.0 mins  20 mins  0.9986  0 to 20 mins
 pH 9  35  0.2181  3.2 mins  11 mins  0.9977  0 to 10 mins

(a): Square of linear correlation coefficient.

(b): Data range used for first order rate constant, correlation coefficient and half-life calculation.

Energy of activation:

Rate constants for the test substance at 25 and 20°C were estimated using the Arrhenius Equation. The results are

summarized in Table 7:

Table 7: Arrhenius plot data

 Buffers  1/T (a)  ln k  Linear regression analysis  25°C   25°C  25°C    25 °C  20°C    20°C    20°C   20°C
        (slope and y-intercept)  ln k  k  DT50  DT90  ln k  k  DT50  DT90
 pH 5 (b)  0.0035  -3.0632 Slope -E/R = -3962.18Intercept lnA = 10.68E = 32.94 -2.618   0.0730  9.5  32  -2.844  0.0582  12  40
0.0034  -2.6291                  
   0.0032  -2.1702                  
 pH 7 (c)  0.0035  -2.6658 Slope -E/R = -6554.40Intercept lnA = 20.07E = 54.49  -1.920   0.1466  4.7  16  -2.295  0.1007  6.9  23
 0.0034  -1.9211                  
   0.0032  -1.1890                  
 pH 9 (b)  0.0035  -3.0308 Slope -E/R = -6704.02Intercept lnA =  20.26E = 55.74 -2.232   0.1073  6.5  21 -2.616   0.0731  9.5  31
 0.0034  -2.1588                  
 0.0032  -1.5299                  

Arrhenius equation ln k = -E/RT + ln A

k = rate constant, measured at different temperatures (pH 5 and 9 are in 1/minutes and pH 7 are in 1/hours

E = activation energy (KJ/mol)

T = absolute temperature [K]

R = gas constant [8.314J/mol*K)

(a) 1/T = Temperature, 1/K

(b) The half-life (DT50) and DT90 values are in minutes,

(c) The half-life (DT50) and D90 values are in hours.

Validity criteria fulfilled:
yes
Conclusions:
Hydrolysis half-lives of 9.6 minutes at pH 5, 4.7 hours at pH 7 and 6 minutes at pH 9 and 25°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:

The hydrolysis half-lives of the substance was determined in accordance with OECD 111 Test Guideline and in compliance with GLP.

Description of key information

Hydrolysis half-life: 9.6 minutes at pH 5, 4.7 hours at pH 7, 6 minutes at pH 9 and 25°C (OECD 111)

Key value for chemical safety assessment

Half-life for hydrolysis:
4.7 h
at the temperature of:
25 °C

Additional information

The hydrolysis half-lives of the substance have been measured in accordance with OECD Test Guideline 111 and in compliance with GLP.

The hydrolysis half-lives of the substance are:

pH 5.0 = 15 minutes at 15°C, 9.6 minutes at 25°C and 6.1 minutes at 35°C

pH 7.0 = 10 hours at 15°C, 4.7 hours at 25°C and 2.3 hours at 35°C

pH 9.0 = 14 minutes at 15°C, 6 minutes at 25°C and 3.2 minutes at 35°C

The hydrolysis half-lives stated above are for the removal of the parent substance. The hydrolysis study did not identify the intermediate or final products, although several peaks that can be assumed to be intermediate/final hydrolysis products are observed in the HPLC chromatograms. These products must retain the double bond and/or carbonyl functionality of the parent in order to be observed by the UV-detector.

Trimethoxysilanes are known to hydrolyse rapidly to trisilanols by sequential loss of the three methoxy groups (Spivack et al., 1997). The reaction can be catalysed by hydronium or hydroxide ions; the rate is slowest at around neutral pH and faster under acid or base conditions (Spivack et al., 1997).

The ester group in the side-chain of the registration substance could undergo hydrolytic degradation, but is expected to be relatively stable compared to the trimethoxy group at all pHs. Simple aliphatic acid esters (including those with double bonds adjacent to the carbonyl group) have half-lives at pH 7 and 25°C of days to years; only halogenated esters or others with strongly electron withdrawing groups hydrolyse rapidly (Mabey and Mill, 1978). For example, 2 -propenoic acid ethyl ester has a half-life at pH 7 and 25°C of 3.5 years (Mabey and Mill, 1978).

Therefore, the assessment of environmental hazards and exposure is based on the properties of 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate and methanol. Abiotic degradation of the side-chain may be a mechanism for removal of 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate from the environment.

As the hydrolysis reaction may be acid or base catalysed, the rate of reaction is expected to be slowest at 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. This is supported by studies for various organosilicon compounds in which calculation of kH3O+ and kOH- from the experimental results at pH 4 and 9, respectively, resulted in reasonable estimates of the half-life at pH 7.

Therefore, at low pH:

kobs≈kH3O+[H3O+]

 

The half-life at pH 2 may be estimated from t1/2(pH 2) = t1/2(pH 5) / 1000 = 0.00016 hours (0.6 seconds). However, it is not appropriate or necessary to attempt to predict accurately when the half-life is less than 5-10. As a worst-case it can therefore be considered that the half-life for the substance at pH 2 and 25°C is approximately 5 seconds.

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) x e(0.08.(T-X))

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

Thus, for 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate, the hydrolysis half-life at 37.5ºC and pH 7 (relevant for lungs and blood) is 1.7 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.

The products of hydrolysis are 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate and methanol.

References:

Mabey W and Mill T (1978). Critical Review of Hydrolysis of Organic Compounds in Water Under Environmental Conditions. J. Phys. Chem. Ref. Data, Vol. 7, No. 2, 1978.

Spivack J L, Pohl E R, Kochs P (1997). Organoalkoxysilanes, Organosilanols, and Organosiloxanols. The Handbook of Environmental Chemistry Vol. 3 Part H. Organosilicon Materials (ed. By G Chandra) Springer-Verlag Berlin Heidelberg.