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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 November, 2017 to 18 January, 2018
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)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Preparation of Stock Solutions
26.7mg of test item was weighed, dissolved and made up to 25 mL with methanol to obtain the stock solution with the concentration of 1068 mg/L.
The stock solution was diluted by methanol to obtain a series of standard solutions od 12.5 mg/L, 25 mg/L ,100 mg/L and 200 mg/L to make calibration curve.

Preparation of the test item solutions
11.6 mg of test item was weighed, dissolved and made up with buffer solution (pH4.0) to obtain a stock solution of 116 mg/L; 10.0 mg of test item was weighed, dissolved and made up with buffer solution (pH7.0) to obtain a stock solution of 100 mg/L; 10.5 mg of test item was weighed, dissolved and made up with buffer solutions (pH9.0) to obtain a stock solution of 105 mg/L.
Buffers:
Preparation of buffer solutions
0.1M monopotassium phosphate: 3.4022g of monopotassium phosphate were weighed, dissolved and made up to 250 mL with ultrapure water.
0.1M sodium hydroxide: 1.0012g of sodium hydroxide were weighed, dissolved and made up to 250 mL with ultrapure water.
0.1M potassium chloride: 1.8608g of potassium chloride were weighed, dissolved and made up to 250 mL with ultrapure water.
0.1M boric acid: 1.5450g of boric acid were weighed, dissolved and made up to 250 mL with 0.1M potassium chloride.
0.1M potassium acid phthalate: 5.1052g of potassium acid phthalate were weighed, dissolved and made up to 250 mL with ultrapure water.
Buffer solution (pH4.0): 0.800 mL 0.1M sodium hydroxide and 100mL 0.1M potassium acid phthalate was transferred, dissolved and made up to 200mL with ultrapure water.
Buffer solution (pH7.0): 59.26 mL 0.1M sodium hydroxide and 100mL 0.1M monopotassium phthalate was transferred, dissolved and made up to 200mL with ultrapure water.
Buffer solution (pH9.0): 42.60mL 0.1M sodium hydroxide and 100mL 0.1M boric acid was transferred, dissolved and made up to 200mL with ultrapure water.
The buffer solution is accurately adjusted to pH4.0, pH7.0, pH9.0 with 0.1 mol/L of hydrochloric acid or 0.1mol/L of sodium hydroxide which is tested by pH meter.
All buffer solutions will be filtered by 0.45 µm hydrophilic PTFE filter and sterilized for 15 minutes at 121℃ before the first use. If it is not freshly sterilized, bubbling nitrogen for 5 minutes before use.
Estimation method (if used):
Method Validation
Limit of detection and Limit of Quantification was 0.04mg/L and 0.125 mg/L, respectively.

Specificity
Blank sample is used to prove specificity and response of blank sample at retention time of test item should be less than 30% of LOQ (Limit of Quantification).

Linearity
Linearity of detector response is checked by analysis of standards and plotting a calibration graph of peak area versus concentration. The coefficient of correlation will be calculated (R^2 should be >0.99).
After the calibration curve was made, the standard solution was analyzed to check the calibration curve each day before sample analysis. If the deviation between the measured concentration and the theoretical concentration of the standard solution is less than 10%, the calibration curve can be used; otherwise the calibration curve will be remade.

Accuracy (Recovery)
234.2µL and 705µL of 1068 mg/L stock solution were added to 5 mL buffer solutions (pH4.0, pH7.0 and pH9.0) to obtain the solution with the concentration of 50 mg/L and 150 mg/L, set 5 replicates and one blank sample.
The averaged recovery should be in the range of 90~110%, and the relative standard deviation was less than 10%. A maximum of 1 outlier may be discarded at each fortification level. Where more than one outlier has been identified additional determinations must be included.

Precision
The recovery solution of 50 mg/L and 150 mg/L in different media was injected 5 times for repeatability test. The relative standard deviation of repeatability should be not more than 5%. A maximum of 1 outlier may be discarded at each fortification level. Where more than one outlier has been identified additional determinations must be included.
Duration:
0 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
111.474 mg/L
Duration:
5 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
111.644 mg/L
Duration:
0 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
104.426 mg/L
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
103.811 mg/L
Duration:
0 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
112.46 mg/L
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
113.673 mg/L
Number of replicates:
5
Preliminary study:
Pretreatment of Samples
Samples will be analyzed directly after sampling.
Test performance:
Method validation
The analytical method has been validated. The results are as follows:
Limit of detection (LOD): The limit of detection of the method was 0.04 mg/L.
Limit of quantification (LOQ): The limit of quantification of the method was 0.125 mg/L.
1. Specificity
No peak appears at the corresponding retention time of test item in chromatogram of blank sample.
2. Linearity
The standard curve equation is:
y=30180.3x+759.270, R=0.9999645 (12.5 mg/L-200mg/L)
Linearity of test item was good in the concentration range of 12.5-200 mg/L. Therefore, the calibration curve could be used for precise quantification.
After the calibration curve was made, the standard solution with the concentration of 50 mg/L was analyzed. The deviation was 0.4%, which satisfied the criteria if <10% (Table 1).
3. Accuracy (Recovery)
At the concentration of 50 mg/L and 150 mg/L, the averaged recovery of test item in buffer solution of pH4.0 was in the range of 103.1% to 100.9%; the relative standard deviation was in the range of 0.6% to 0.9%. The average recovery of test item in buffer solution of pH7.0 was in the range of 102.5% to 101.6%; the relative standard deviation was in the range of 0.5% to 0.6%. The averaged recovery of test item in buffer solution of pH9.0 was in the range of 102.7% to 101.8%; the relative standard deviation was in the range of 102.7% to 101.8%; the relative standard deviation was in the range of 0.8% to 0.4% (Table 2 – Table 4).
4. Precision
The results of precision analysis showed: The recovery sample of test item at the concentration of 5 mg/L and 150 mg/L in different media were injected five times and the relative standard deviations (RSD) were in the range of 0.2%-0.6%, the relative standard deviation were less than 5%, which meet the test requirement (Table 5).
Transformation products:
no
Details on hydrolysis and appearance of transformation product(s):
At the beginning of the test, the mean initial concentration of test item in buffer solution (pH4.0, pH7.0 and pH9.0) were 111.474 mg/L, 104.426 mg/L and 112.460 mg/L.
The results of hydrolysis at the temperature of 50℃.
After 5 days, the mean concentration of test item in buffer solution (pH4.0, pH7.0 and pH9.0) were 111.644 mg/L, 103.811 mg/L and 113.673 mg/L. The respective hydrolysis rate in buffer solution of pH4.0, pH7.0 and pH9.0 were -0.2%, 0.6% and -1.1%, which were all less than 10% of hydrolysis observed after 5 days (Table 6).
% Recovery:
>= 100
St. dev.:
0.6
pH:
4
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
50mg/L
% Recovery:
>= 99.5
St. dev.:
0.9
pH:
4
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
150mg/L
% Recovery:
>= 100
St. dev.:
0.5
pH:
7
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
50mg/L
% Recovery:
>= 100
St. dev.:
0.6
pH:
7
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
150 mg/L
% Recovery:
>= 100
St. dev.:
0.8
pH:
9
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
50mg/L
% Recovery:
>= 100
St. dev.:
0.4
pH:
9
Temp.:
50 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
150mg/L
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
25 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
25 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
Details on results:
1. Measurement of pH
The pH value of buffer solution of pH4.0, pH7.0 and pH9.0 were 4.05, 7.01 and 9.03, respectively.
2. Temperature monitoring
Temperature meets the test requirement. The temperature variation was within the range of ±0.5℃.

Table 1. Verification Result of Calibration Curve

Nominal Concentration Date of Analysis Measured Concentration (mg/L) Deviation
50 2017/12/5 49.817 0.40%

Note: Deviation% = | determined concentration of standard solution - theoretical concentration of standard solution | / theoretical concentration of standard solution x 100%

Table 2. Accuracy result of test item in buffer solution of pH4.0

Fortification level (mg/L) Replicates Measured Concentration (mg/L) Recovery Mean RSD
50 1 51.503 103.0% 103.1% 0.6%
2 51.061 102.1%
3 51.688 103.4%
4 51.827 103.7%
5 51.611 103.2%
150 1 149.221 99.5% 100.9% 0.9%
2 152.143 101.4%
3 151.511 101.0%
4 152.699 101.8%
5 151.386 100.9%

Table 3. Accuracy result of test item in buffer solution of pH7.0

Fortification level (mg/L) Replicates Measured Concentration (mg/L) Recovery Mean RSD
50 1 51.351 102.7% 102.5% 0.5%
2 50.848 101.7%
3 51.261 102.5%
4 51.316 102.6%
5 51.482 103.0%
150 1 150.951 100.5% 101.6% 0.6%
2 152.316 101.5%
3 152.698 101.8%
4 152.601 101.7%
5 153.673 102.4%

Table 4. Accuracy result of test item in buffer solution of pH9.0

Fortification level (mg/L) Replicates Measured Concentration (mg/L) Recovery Mean RSD
50 1 50.778 101.6% 102.7% 0.8%
2 51.328 102.7%
3 51.719 103.4%
4 51.718 103.4%
5 51.312 102.6%
150 1 152.387 101.6% 101.8% 0.4%
2 152.915 101.9%
3 153.343 102.2%
4 151.926 101.3%
5 153.187 102.1%

Table 5. Precision result of test item in different buffer solution

Medium Fortification level (mg/L) Replicates Measured Concentration (mg/L) Mean RSD
pH4.0 50 1 51.688 51.558 0.6%
2 51.520
3 51.881
4 51.036
5 51.665
150 1 151.511 151.171 0.4%
2 150.478
3 151.270
4 152.005
5 150.591
pH7.0 50 1 51.261 51.378 0.5%
2 51.175
3 51.550
4 51.727
5 51.177
150 1 152.698 152.75 0.2%
2 152.224
3 152.626
4 153.165
5 153.038
pH9.0 50 1 51.719 51.754 0.5%
2 52.219
3 51.451
4 51.736
5 51.643
150 1 153.343 152.961 0.3%
2 152.500
3 152.542
4 153.673
5 152.747

Table 6. Hydrolysis result of test item in different buffer solution at 50℃

Time (d) pH of buffer solution Concentration (mg/L) Mean of Concentration (mg/L) Hydrolysis rate
0 4.0 110.827 111.474 -
112.120
5 111.690 111.644 -0.2%
111.597
0 7.0 104.071 104.426 -
104.781
5 103.820 103.811 0.6%
103.801
0 9.0 112.218 112.460 -
112.702
5 114.547 113.673 -1.1%
112.798

Note: Hydrolysis rate % = (1-Mean of determined concentration of 5d / Mean of determined concentration of 0d)x100%

Validity criteria fulfilled:
yes
Conclusions:
At the temperature of 50℃, the respective hydrolysis rate after 5 days in buffer solution of pH4.0, pH7.0 and pH9.0 were -0.2%, 0.6% and -1.1%, which were all less than 10% of hydrolysis observed after 5 days (T1/2 at 25℃>1 year), the test item is considered hydrolytically stable and no additional testing is required.
Executive summary:

Test method:

According to OECD111, the stability of the test item in different pH buffer solution and temperature condition were studies.

Test results:

At the concentration of 50mg/L and 150 mg/L, the averaged recoveries of test item in buffer solution of pH4.0 were 103.1% and 100.9%, the relative standard deviations were 0.6% and 0.9%; the relative standard deviations were 0.5% and 0.6%; the averaged recovery of test item in buffer solution of pH9.0 were 102.7% and 101.8%, the relative standard deviations were 0.8% and 0.4%.

The mean initial concentration C0 (0d) of the test item in buffer solution of pH4.0, pH7.0 and pH9.0 were 111.474 mg/L, 104.426 mg/L and 112.460 mg/L.

At the temperature of 50, the respective hydrolysis rate after 5 days in buffer solution of pH4.0, pH7.0 and pH9.0 were -0.2%, 0.6% and -1.1%. There was less than 10 percent of hydrolysis observed after 5 days (T1/2 at 25> 1 year), the test item is considered hydrolytically stable and no additional testing is required.

Conclusion:

At the temperature of 50, the respective hydro lysis rate after 5 days in buffer solution of pH4.0, pH7.0 and pH9.0 were -0.2%, 0.6% and -1.1%, which were all less than 10% if hydrolysis observed after 5 days (T1/2 at 25> 1 year), the test item is considered hydrolytically stable and no additional testing is required.

Description of key information

At the temperature of 50, the respective hydro lysis rate after 5 days in buffer solution of pH4.0, pH7.0 and pH9.0 were -0.2%, 0.6% and -1.1%, which were all less than 10% if hydrolysis observed after 5 days (T1/2at 25> 1 year), CJ303 is considered hydrolytically stable and no additional testing is required (OECD 111).

Key value for chemical safety assessment

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

Additional information