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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2015 - 05 September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
(2008)
Deviations:
yes
Remarks:
Many recoveries were not minimal 70% (53-74%), due to the nature of the test substance.
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
(2004)
Qualifier:
according to
Guideline:
EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)
Version / remarks:
(2008)
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Tier 1 preliminary test: Samples for analysis were taken immediately after preparation (t=0) and after 5 days. The samples taken at t=5 days were cooled to room temperature using running tap water.
- Tier 2 main study: Samples for analysis were taken immediately after preparation (t=0) and at several sampling points after t=0.
- Pretreatment: Immediately after sampling or sampling/cooling, subsamples of 750 µL were diluted in a 1:1 (v:v) ratio with Tetrahydrofuran (THF) and analysed. If necessary, the samples were further diluted with 50/50 (v/v) THF/buffer pH 4, pH 7 or pH 9 to obtain concentrations within the calibration range.
Buffers:
- Acetate buffer pH 4, 0.01 M: aqueous solution of 16.7% (v/v) 0.01 M sodium acetate (Merck, Germany) and 83.3% (v/v) 0.01 M acetic acid (Merck, Germany).
- Phosphate buffer pH 7, 0.01 M: aqueous solution of 0.01 M potassium di-hydrogenphosphate (Merck, Germany) adjusted to pH 7 using 1 N sodium hydroxide (Merck, Germany).
- Borate buffer pH 9, 0.01 M: aqueous solution of 0.01 M boric acid (Merck, Germany) and 0.01 M potassium chloride (Merck, Germany) adjusted to pH 9 using 1 N sodium hydroxide.
- The buffers contain 0.0009% (w/v) sodium azide (Merck, Germany).

Details on test conditions:
TIER 1: preliminary test:
- At pH 4, 7 and 9 and 50.1 ± 0.3°C
- Test substance solutions were prepared in the buffer solutions at a target concentration of 5000 mg/L
- The pH of each of the test solutions (except for the blanks) was determined at each sampling time.
- Actual pH's: 4.1-4.2, 7.0 and 8.7-8.8

TIER 2: Main study:
- At pH 7 and 9
- Three different temperatures/pH (see below for the temperatures)
- Test samples at a target concentration of about 2500 mg/L were prepared in buffer solution at pH 7 and pH 9
- The two-times lower target concentration was selected in order to try to improve the recovery of the test samples compared to nominal concentrations.
- The pH of each of the test solutions (except for the blanks) was determined at least at the beginning and at the end of the test.
- Actual pH's: 7.0-7.1 and 8.9-9.1

TEST SYSTEM
- The test substance solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman, Germany) and transferred into sterile vessels.
- To exclude oxygen, nitrogen gas was purged through the solutions for 5 minutes.
- For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment.
- Blank buffer solutions were treated similarly as the test samples and analysed at t=0.
No test substance was detected in the blank buffer solutions.
Duration:
736.9 h
pH:
7
Initial conc. measured:
>= 1 795 - <= 1 846 mg/L
Duration:
735.43 h
pH:
7
Initial conc. measured:
>= 1 549 - <= 1 628 mg/L
Duration:
734.75 h
pH:
7
Initial conc. measured:
>= 1 656 - <= 1 715 mg/L
Duration:
736.43 h
pH:
9
Initial conc. measured:
>= 1 812 - <= 1 866 mg/L
Duration:
735.43 h
pH:
9
Initial conc. measured:
>= 1 704 - <= 1 729 mg/L
Duration:
734.75 h
pH:
9
Initial conc. measured:
>= 1 768 - <= 1 792 mg/L
Number of replicates:
Two
Positive controls:
no
Negative controls:
no
Preliminary study:
- A degree of hydrolysis of < 10% was observed at pH 4 after 5 days (mean of 1.6%)
- A degree of hydrolysis of ≥ 10% was observed at pH 7 and pH 9 after 5 days (means of 19 and 48%, respectively)
- The mean recoveries of the test samples at pH 4, pH 7 and pH 9 were 56%, 53% and 63% to nominal concentration, respectively, at t=0. A possible reason could be that a part of the test substance (possibly highly molecular weight compounds) were filtrated from the test solutions (see below for more details).
- According to the guideline, the higher Tier test was required to determine the half-life time of the test substance at pH 7 and pH 9
Test performance:
RECOVERIES (Tier 2)
- The recovery in the table below is the mean of duplicate test samples.
- Recovery in the table below is the concentration analysed at t=0 for the test sample, relative to the nominal concentration.
- The mean recoveries fell slightly outside the acceptable range of 70-110% (61%-73%). The possible reason for this could be that a part of the test substance (possibly highly molecular weight compounds) were filtrated from the test solutions. It is required to filtrate the solutions in order to obtain sterilised conditions. The tests were performed using test samples at about 2500 mg/L, which was the lowest possible test concentration taking into that 90% hydrolysis should be determined. The polymeric content of the test samples was considered to be representative for the test substance.
Transformation products:
not measured
% Recovery:
70
pH:
7
Temp.:
20 °C
% Recovery:
61
pH:
7
Temp.:
50 °C
% Recovery:
63
pH:
7
Temp.:
60 °C
% Recovery:
73
pH:
9
Temp.:
20 °C
% Recovery:
70
pH:
9
Temp.:
50 °C
% Recovery:
69
pH:
9
Temp.:
60 °C
Key result
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Type:
not specified
Key result
pH:
7
Temp.:
25 °C
DT50:
> 1 yr
Type:
not specified
Key result
pH:
9
Temp.:
25 °C
DT50:
> 1 yr
Type:
not specified
Details on results:
Main study (pH 7):
- At 20°C it was found that the test substance was stable for the testing period of 30.7 days (i.e. limited degradation was observed).
- At 50°C, the content of the test substance decreased with increasing time. However, between the time points of 207.33 and 332.1 hours the degree of hydrolysis based on mean values was 8.5%. The time difference between these data points was 124.8 hours (5.2 days). Since the degradation of the test substance at 50°C for 5 days was < 10% it was concluded that the half-life time of the compound at 25°C is > 1 year.
- The behaviour of the test substance at 60°C was similar to the test at 50°C.
- It was concluded that the test substance was stable.

Main study (pH 9):
- At 20°C it was found that the test substance decreased with increasing time.
- At 50°C, the content of the test substance decreased quickly between t=0 and t=187 hours. However, between the time points of 207.33 and 332.1 hours the degree of hydrolysis based on mean values was 1.4%. The time difference between these data points was 124.8 hours (5.2 days). Since the degradation of the test substance at 50°C for 5 days was < 10% it was concluded that the half-life time of the compound at 25°C is > 1 year.
- The behaviour of the test substance at 60°C was similar to the test at 50°C.
- It was concluded that the test substance was stable.

Half-life times at 25°C (pH 4, 7 and 9):

 

pH

Temperature

Half-life

4

25°C

> 1 year

7

25°C

> 1 year

9

25°C

> 1 year

Validity criteria fulfilled:
yes
Remarks:
Although recoveries were < 70%, the validity criteria were considered fulfilled. The substance is an UVCB with constituents that have a molecular weight ranging from 89 to 20000 g/mole. Part of the substance was probably filtrated from the test solutions.
Conclusions:
The substance is hydrolytically stable at pH 4, 7 and 9. A half-life time of > 1 year at 25°C was determined at pH values normally found in the environment (pH 4-9) in a GLP-compliant study according to OECD 111.
Executive summary:

The rate of hydrolysis of the substance at pH values normally found in the environment (pH 4-9) was determined in a GLP-compliant study according to EC C.7, OECD 111 and EPA OPPTS 835.2120 guidelines. At pH 4 and 50 °C, in the Tier 1 test, a degree of < 10% of hydrolysis was observed after 5 days. Therefore it was concluded that the substance is hydrolytically stable at pH 4. In the Tier 2 test, hydrolysis of the substance at pH 7 and pH 9, at three different temperatures, was determined. Based on the results of the Tier 2 test it was concluded that the substance is also hydrolytically stable at pH 7 and 9. Therefore, a half-life time of > 1 year at 25°C was determined at pH values normally found in the environment (pH 4-9).

Description of key information

The substance is hydrolytically stable. A half-life time of > 1 year at 25°C was determined at pH values normally found in the environment (pH 4-9).

Key value for chemical safety assessment

Additional information

The rate of hydrolysis of the substance at pH values normally found in the environment (pH 4-9) was determined in a GLP-compliant study according to EC C.7, OECD 111 and EPA OPPTS 835.2120 guidelines. At pH 4 and 50 °C, in the Tier 1 test, a degree of < 10% of hydrolysis was observed after 5 days. Therefore it was concluded that the substance is hydrolytically stable at pH 4. In the Tier 2 test, hydrolysis of the substance at pH 7 and pH 9, at three different temperatures, was determined. Based on the results of the Tier 2 test it was concluded that the substance is also hydrolytically stable at pH 7 and 9. Therefore it was concluded that the substance is hydrolytically stable at pH values normally found in the environment (pH 4-9).