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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 23 October 2014 and 01 July 2015.
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
The sample solutions were taken from the waterbath at various times and the pH of each solution recorded.
Buffers:
The test system used sterile buffer solutions at pH’s 4.0, 7.0 and 9.0.
Details on test conditions:
Performance of the Test
Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 100 mg/L in the buffer solutions. The test solutions were split into individual vessels for each data point.
The solutions were shielded from light whilst maintained at the test temperature.


Preliminary Test / Tier 1
Sample solutions at pH 4, 7 and 9* were maintained at 50.0 ± 0.5 °C for a period of at least 48 hours.

Sample solutions at pH 9 [using the alternative, (Tris) pH 9 buffer solution] were maintained at 50.0 ± 0.5 °C for a period of 48 hours.

Tier 2
Results from the Preliminary Test / Tier 1 at pH 9 [using the (Tris) pH 9 buffer solution] showed it was necessary to undertake further testing at pH 9 [using the (Tris) pH 9 buffer solution].


Calculation
The response factors of the standard peak areas (unit peak area per mg/L) were calculated using the following equation:

RF = Rstd / Cstd

where:
RF = response factor for the standard solution
RSTD = peak area for the standard solution
CSTD = concentration for the standard solution (mg/L)

Calculation of the Degree of Hydrolysis
The decrease in concentration (preliminary test) or the degree of hydrolysis was calculated using the formula:

Degree of hydrolysis in % = [ (C0 - Ct) / C0 ] • 100

where:
C0 = concentration of time 0
Ct = concentration of time t

Testing the Reaction for Pseudo First Order
The general rate expression for a first order reaction is:

d(c) / dt = - k • c

or in the integrated form

-1n (C0 / Ct) = -k • t

or

ln ct = (-k) • t – ln c0
(equation of a linear function)

Therefore, the rate constant k is the slope of a plot of ln ct versus t. The data were plotted as ln ct versus t. The linear plots prove that the hydrolysis reaction is pseudo first order. The reaction rate constant k can be calculated by regression analysis or the following equation.

-k = -(1 / t) • 1n (c0 / ct)

The half-life time was calculated from the following equation:

t½ = 0.693/k

where:
c0 = Concentration of the test solution at time 0 [µg/mL]
ct = Concentration of the test solution at time t [µg/mL]
k = First order rate constant [hours-1]
t1/2 = Half-life time [hours]

Evaluation of k at 25 °C
When the rate constants are known for two temperatures, the rate constants at other temperatures can be calculated using the Arrhenius equation:

k = A • e (-E / (R•T))

or

1n k = - (E / R) • (1 / T) + 1n A

A plot of ln k versus 1/T gives a straight line with a slope of -E/R

where:
k = Rate constant, measured at different temperatures
E = Activation energy [kJ/mol]
T = Absolute temperature [K]
R = Gas constant [8.314 J/mol K]
A = Pre-exponential factor

The half-life at 25 °C was interpolated / extrapolated directly from the graph.
Duration:
144 h
Temp.:
50 °C
Initial conc. measured:
100 mg/L
Duration:
48 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
100 mg/L
Duration:
3 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
100 mg/L
Duration:
3 h
pH:
9
Temp.:
30 °C
Initial conc. measured:
100 mg/L
Number of replicates:
Two
Positive controls:
no
Negative controls:
no
Statistical methods:
No data
Preliminary study:
Overall, less than 10 % hydrolysis after 5 days at 50 °C at pH 4 and pH 7, equivalent to a half-life greater than 1 year at 25 °C.
At pH 9, the extent of hydrolysis after 24 hours indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C.
Test performance:
During the preliminary testing, it was noted that the pH 9 injections (using the borate-based buffer solution) were becoming ever-more erroneous. Therefore, it was decided to switch to a (Tris-based) pH 9 buffer solution.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Identification of Hydrolysis Products –Tier 3
An attempt was made to elucidate the structure of each main (m/z) ion found in a “hydrolyzed” test item solution compared to a parent test item solution using high performance liquid chromatography –mass spectrometry (HPLC-MS). Since the use of the ion-pairing reagents is not acceptable in HPLC-MS, the subsequent analysis resulted in the parent test item and the hydrolysis products essentially co-eluting. However, a review of the results, especially the m/z ions for the sample solution indicated that the hydrolysis products could not positively identified.
That said, the Tier 1 / Tier 2 sample chromatograms show that the hydrolysis products elute well before the parent test item; and therefore, must have less affinity for the tetrabutylammonium bromide ion-pair reagent. Therefore, the hydrolysis products must be less negatively charged than the parent. From this, it can be postulated that the hydrolysis products are most likely due to removal of one or both of the terminal sulphooxy- groups, with the central, diazo- group remaining essentially unaffected. This would also be the reason why the hydrolyzed solutions retained a similar, blue color compared to the non-hydrolyzed solutions.
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Key result
pH:
7
Temp.:
25 °C
DT50:
> 1 yr
pH:
9
Temp.:
25 °C
DT50:
0.907 h
Remarks on result:
other: Tier 2 test
Other kinetic parameters:
No data
Details on results:
The estimated half-lives at 25 °C of the test item are:
pH 4 at 50.0 ± 0.5 ºC: Overall, less than 10 % hydrolysis after 5 days at 50 °C, equivalent to a half-life greater than 1 year at 25 °C.
pH 7 at 50.0 ± 0.5 ºC: Overall, less than 10 % hydrolysis after 5 days (24 to 144 hours) at 50 °C, equivalent to a half-life greater than 1 year at 25 °C.
pH 9 (Tris) at 50.0 ± 0.5 ºC: The extent of hydrolysis after 24 hours indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25°C.
pH 9 Arrhenius Data: t½= 0.907 hours

Preliminary Test / Tier 1

The mean peak areas relating to the standard and sample solutions are shown in the following table:

 

Solution

Mean Peak Area

Standard 50.3 mg/L, pH 4

1.0642 x 107

Standard 53.0 mg/L, pH 4

1.1000 x 107

Initial Sample A, pH 4

1.0579 x 107

Initial Sample B, pH 4

1.1546 x 107

Standard 53.6 mg/L, pH 7

1.0086 x 107

Standard 50.3 mg/L, pH 7

8.7894 x 106

Initial Sample A, pH 7

1.1102 x 107

Initial Sample B, pH 7

1.1024 x 107

Standard 50.8 mg/L, pH 9

9.4443 x 106

Standard 54.4 mg/L, pH 9

9.1906 x 106

Initial Sample A, pH 9

1.1939 x 107

Initial Sample B, pH 9

1.1816 x 107

Standard 52.3 mg/L, pH 4

1.0365 x 107

Standard 50.9 mg/L, pH 4

1.1007 x 107

24 Hours Sample A, pH 4

1.1388 x 107

24 Hours Sample B, pH 4

1.1369 x 107

Standard 52.4 mg/L, pH 7

1.0056 x 107

Standard 51.2 mg/L, pH 7

9.7068 x 106

24 Hours Sample A, pH 7

9.6442 x 106

24 Hours Sample B, pH 7

9.5784 x 106

Standard 52.9 mg/L, pH 9

1.0970 x 107

Standard 54.2 mg/L, pH 9

1.1064 x 107

24 Hours Sample A, pH 9

1.0087 x 107

24 Hours Sample B, pH 9

1.0133 x 107

Standard 51.7 mg/L, pH 4

1.1992 x 107

Standard 52.0 mg/L, pH 4

1.1776 x 107

120 Hours Sample A, pH 4

1.1894 x 107

120 Hours Sample B, pH 4

1.1936 x 107

144 Hours Sample A, pH 4

1.2099 x 107

144 Hours Sample B, pH 4

1.2235 x 107

Solution

Mean Peak Area

Standard 51.0 mg/L, pH 7

1.1032 x 107

Standard 53.0 mg/L, pH 7

1.1530 x 107

120 Hours Sample A, pH 7

1.2370 x 107

120 Hours Sample B, pH 7

1.2419 x 107

144 Hours Sample A, pH 7

1.2105 x 107

144 Hours Sample B, pH 7

1.2308 x 107

Standard 52.2 mg/L, pH 9

1.1685 x 107

Standard 51.0 mg/L, pH 9

1.1538 x 107

120 Hours Sample A, pH 9

6.1697 x 106

120 Hours Sample B, pH 9

6.3728 x 106

144 Hours Sample A, pH 9

5.4308 x 106

144 Hours Sample B, pH 9

5.4903 x 106

Standard 25.2 mg/L, pH 9 (Tris)

5.2548 x 106

Standard 26.1 mg/L, pH 9 (Tris)

5.4572 x 106

Initial Sample A, pH 9 (Tris)

3.5719 x 106

Initial Sample B, pH 9 (Tris)

3.4775 x 106

Standard 25.5 mg/L, pH 9 (Tris)

5.3173 x 106

Standard 25.4 mg/L, pH 9 (Tris)

5.2705 x 106

24 Hours Sample A, pH 9 (Tris)

N.D.

24 Hours Sample B, pH 9 (Tris)

N.D.

Standard 25.8 mg/L, pH 9 (Tris)

5.3954 x 106

Standard 25.5 mg/L, pH 9 (Tris)

5.3084 x 106

48 Hours Sample A, pH 9 (Tris)

N.D.

48 Hours Sample B, pH 9 (Tris)

N.D.

N.D. = None detected

The test item concentrations at the given time points are shown in the following tables:

 

pH 4 at 50.0 ± 0.5 ºC 

Time (Hours)

Concentration (g/L)

% of mean initial concentration

A

B

A

B

0

0.101

0.110

-

-

24

0.110

0.110

104

104

120

0.104

0.104

98.3

98.7

144

0.106

0.107

100

101

Result: Overall, less than 10 % hydrolysis after 5 days at 50 °C, equivalent to a half-life greater than 1 year at 25 °C.

 

pH 7 at 50.0 ± 0.5 ºC 

Time (Hours)

Concentration (g/L)

% of mean initial concentration

A

B

A

B

0

0.122

0.122

-

-

24

0.101

0.100

82.9

82.3

120

0.108

0.109

88.7

89.0

144

0.106

0.108

86.8

88.3

Result: Overall, less than 10 % hydrolysis after 5 days (24 to 144 hours) at 50 °C, equivalent to a half-life greater than 1 year at 25 °C.

 

pH 9 (Tris) at 50.0 ± 0.5 ºC 

Time (Hours)

Concentration (g/L)

% of mean initial concentration

A

B

A

B

0

6.85 x 10-2

6.67 x 10-2

-

-

24

N.D.

N.D.

-

-

48

N.D.

N.D.

-

-

Result: The extent of hydrolysis after 24 hours indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25°C.

Tier 2

The mean peak areas relating to the standard and sample solutions are shown in the following table:

 

Solution

Mean Peak Area

Standard 25.6 mg/L, pH 9

5.5266 x 106

Standard 25.1 mg/L, pH 9

5.3144 x 106

Initial Sample A, pH 9, 20 ºC

4.2583 x 106

Initial Sample B, pH 9, 20 ºC

4.0556 x 106

0.5 Hours Sample A, pH 9, 20 ºC

3.3053 x 106

0.5 Hours Sample B, pH 9, 20 ºC

3.3381 x 106

1 Hours Sample A, pH 9, 20 ºC

2.5137 x 106

1 Hours Sample B, pH 9, 20 ºC

2.5453 x 106

1.5 Hours Sample A, pH 9, 20 ºC

1.9968 x 106

1.5 Hours Sample B, pH 9, 20 ºC

1.9797 x 106

Standard 25.6 mg/L, pH 9

5.5081 x 106

Standard 25.1 mg/L, pH 9

5.3143 x 106

2 Hours Sample A, pH 9, 20 ºC

1.5383 x 106

2 Hours Sample B, pH 9, 20 ºC

1.5043 x 106

2.5 Hours Sample A, pH 9, 20 ºC

1.1776 x 106

2.5 Hours Sample B, pH 9, 20 ºC

1.1567 x 106

3 Hours Sample A, pH 9, 20 ºC

8.9332 x 105

3 Hours Sample B, pH 9, 20 ºC

8.8168 x 105

Standard 26.2 mg/L, pH 9

5.4647 x 106

Standard 25.2 mg/L, pH 9

5.2767 x 106

Initial Sample A, pH 9, 30 ºC

3.0558 x 106

Initial Sample B, pH 9, 30 ºC

2.9368 x 106

0.5 Hours Sample A, pH 9, 30 ºC

1.6604 x 106

0.5 Hours Sample B, pH 9, 30 ºC

1.6568 x 106

1 Hours Sample A, pH 9, 30 ºC

9.4767 x 105

1 Hours Sample B, pH 9, 30 ºC

9.3029 x 105

1.5 Hours Sample A, pH 9, 30 ºC

5.5273 x 105

1.5 Hours Sample B, pH 9, 30 ºC

5.3451 x 105

Solution

Mean Peak Area

Standard 26.2 mg/L, pH 9

5.4537 x 106

Standard 25.2 mg/L, pH 9

5.2645 x 106

2 Hours Sample A, pH 9, 30 ºC

3.0977 x 105

2 Hours Sample B, pH 9, 30 ºC

3.0801 x 105

2.5 Hours Sample A, pH 9, 30 ºC

1.7780 x 105

2.5 Hours Sample B, pH 9, 30 ºC

1.7628 x 105

3 Hours Sample A, pH 9, 30 ºC

1.0452 x 105

3 Hours Sample B, pH 9, 30 ºC

1.0341 x 105

The test item concentrations at the given time points are shown in the following tables:

 

pH 9 at 20.0 ± 0.5 ºC 

Time (Hours)

Concentration (g/L)

% of mean initial concentration

A

B

A

B

0

7.96 x 10-2

7.58 x 10-2

-

-

0.5

6.81 x 10-2

6.24 x 10-2

79.5

80.3

1

4.70 x 10-2

4.76 x 10-2

60.5

61.2

1.5

3.73 x 10-2

3.70 x 10-2

48.0

47.6

2

2.88 x 10-2

2.82 x 10-2

37.1

36.2

2.5

2.21 x 10-2

2.17 x 10-2

28.4

27.9

3

1.67 x 10-2

1.65 x 10-2

21.5

21.2

Result: Slope = -0.224

kobs= 0.516 hour-1

t½= 1.34 hours

pH 9 at 30.0 ± 0.5 ºC 

Time (Hours)

Concentration (g/L)

% of mean initial concentration

A

B

A

B

0

5.84 x 10-2

5.62 x 10-2

-

-

0.5

3.18 x 10-2

3.17 x 10-2

55.4

55.3

1

1.81 x 10-2

1.78 x 10-2

31.6

31.0

1.5

1.06 x 10-2

1.02 x 10-2

18.4

17.8

2

5.94 x 10-3

5.90 x 10-3

10.4

10.3

2.5

3.41 x 10-3

3.38 x 10-3

5.95

5.90

3

2.00 x 10-3

1.98 x 10-3

3.50

3.46

Result: Slope = -0.486

kobs= 1.12 hour-1

t½ = 0.620 hours

The Arrhenius plot was constructed using the data shown in the following tables: 

pH 9 Arrhenius Data 

T (ºC)

T (K)

kobs(hr-1)

Ln kobs

20

293.15

3.41 x 10-3

0.516

-0.662

30

303.15

3.30 x 10-3

1.12

0.112

From the graph of the above data, the rate constant and half-life at 25 °C have been estimated to be as follows:

kobs= 0.764 hour-1
t½= 0.907 hours

Validation

The linearity of the detector response with respect to concentration was assessed over the nominal concentration range of 10 to 200 mg/L. The results were satisfactory with correlation coefficients (r) of ≥ 0.999 being obtained.

Validity criteria fulfilled:
yes
Conclusions:
The estimated half-lives at 25 °C of the test item are:
At pH 4 the estimated half life at 25 °C was > 1 year.
At pH 7 the estimated half life at 25 °C was > 1 year.
At pH 9 the estimated half life at 25 °C was 0.907 hours (rate constant 0.764 hr-1).
Executive summary:

The test item was assessed for hydrolytic stability according to EU Method C.7 and OECD Guideline 111. Tier 2 testing began at 50 °C, with time points at 0, 1, 2, 3, 4 and 5 hours. However, no test item was detected in the 1 hour samples onwards. Therefore, it was decided to perform Tier 2 testing at 20 °C and 30 °C only (to allow interpolation to 25 °C). As the half-life at pH 9 at 25 °C was interpolated from the Tier 2 results at 20 °C and 30 °C, it was considered that testing at a third temperature was unnecessary. The kinetics of the study has been determined to be consistent with that of a pseudo-first order reaction as the graphs of log10 concentration versus time are straight lines. No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions. The estimated half-lives at 25 °C are shown in the following table:

 

pH

Rate constant (hr-1)

Estimated half-life at 25 °C

4

-

>1 year

7

-

>1 year

9

0.764

0.907 hours

 

Description of key information

The estimated half-lives at 25 °C are:
At pH 4 the estimated half-life at 25 °C was > 1 year.
At pH 7 the estimated half-life at 25 °C was > 1 year.
At pH 9 the estimated half-life at 25 °C was 0.907 hours (rate constant 0.764 hr-1).

Key value for chemical safety assessment

Half-life for hydrolysis:
1 yr
at the temperature of:
25 °C

Additional information

The test item was assessed for hydrolytic stability according to EU Method C.7 and OECD Guideline 111. Tier 2 testing began at 50 °C, with time points at 0, 1, 2, 3, 4 and 5 hours. However, no test item was detected in the 1 hour samples onwards. Therefore, it was decided to perform Tier 2 testing at 20 °C and 30 °C only (to allow interpolation to 25 °C). As the half-life at pH 9 at 25 °C was interpolated from the Tier 2 results at 20 °C and 30 °C, it was considered that testing at a third temperature was unnecessary. The kinetics of the study has been determined to be consistent with that of a pseudo-first order reaction as the graphs of log10 concentration versus time are straight lines. No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions. The estimated half-lives at 25 °C are shown in the following table:

 

pH

Rate constant (hr-1)

Estimated half-life at 25 °C

4

-

>1 year

7

-

>1 year

9

0.764

0.907 hours