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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2011 - August 2012
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:
yes
Remarks:
Main study (Tier 2), pH 9 and at 70°C: the criterion that samples will be incubated until 90% hydroIysis of the test substance is observed, or for 30 days, whichever comes first, was not met.
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling method: duplicate sterile vessels under vacuum were filled with 6 ml test solution and placed in the dark in a temperature controlled environment at 49.9°C ± 0.2°C.
Buffers:
- Acetate buffer pH 4, 0.1 M: solution of 16.7% 0.1 M sodium acetate and 83.3% 0.1 M acetic acid. The buffer contains 0.0009% (w/v) sodium azide.
- Phosphate buffer pH 7, 0.1 M: solution of 0.1 M potassium dihydrogenphosphate adjusted to pH 7 using 10 N sodium hydroxide. The buffer contains
0.0009% (w/v) sodium azide.
- Borate buffer pH 9, 0.1 M: solution of 0.1 M boric acid and 0.1 M potassium chloride adjusted to pH 9 using 10 N sodium hydroxide. The buffer contains 0.0009% (w/v) sodium azide.



Details on test conditions:
The rate of hydrolysis of the test substance as a function of pH was determined at pH values normally found in the environment (pH 4-9).

Preliminary test - Tier 1:
Test substance solutions were prepared in the buffer solutions at a target concentration of 100 mg/L. Each solution was filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman, Dassel, Germany) and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution 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 at 49.9°C ± 0.2°C.
The concentration of the test substance in the test samples was determined immediately after preparation (t=O) and after 5 days. The samples taken at t=5 days were cooled to room temperature using running tap water. The samples were diluted in a 9:1 (v:v) ratio with acetonitrile and analysed.
Blank buffer solutions were treated similarly as the test samples and analysed at t=O.
The pH of each of the test solutions (except for the blanks) was determined at each sampling time.

Main study - Tier 2:
Test samples were prepared and treated similarly as during the preliminary test.
The concentrations of the test substance were determined immediately after preparation (t=O) and at several sampling points after t=O.
The samples not analysed on the sampling day were stored in the freezer. Storage stability under these conditions was determined by the analysis of additional accuracy samples prepared at half the nominal concentration of the test samples. On the day of analysis, the frozen samples were defrosted at room temperature, treated and analysed. The stored samples were found tobe stable if the mean accuracy was in the range 70-110%. Based on the results obtained the samples at pH 7 and pH 9 were stable when stored in the freezer for 30 days. The samples at pH 4 were not stable when stored in the freezer and thus those samples were analysed freshly (results are archived in the raw data).
Blank buffer solutions were treated similarly as the test samples and analysed at t=O.
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.
The study was performed at the following temepratures:
- pH 4: 21.2°C ± 0.4°C 49.8°C ± 0.3°C 59.rc ± 0.2°C
- pH 7: 21.2°C ± 0.4°C 49.8°C ± 0.5°C 59.7°C ± 0.2°C
- pH 9: 21.2°C ± 0.4°C 49.8°C ± 0.5°C 70.0°C ± 0.2°C

ldentification of hydrolysis products- Tier 3:
Research to investigate the identity or nature and rates of formation and decline of hydrolysis products was not required since the studies were conducted according to the REACH testing guidelines provided in Annex VII and VIII (volume 1-100 tons/year).
Duration:
720 h
pH:
4
Temp.:
20 °C
Initial conc. measured:
105 mg/L
Duration:
265.9 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
106 mg/L
Duration:
71.5 h
pH:
4
Temp.:
60 °C
Initial conc. measured:
>= 102 - <= 103 mg/L
Duration:
720 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
>= 107 - <= 112 mg/L
Duration:
476.3 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
>= 104 - <= 105 mg/L
Duration:
315.6 h
pH:
7
Temp.:
60 °C
Initial conc. measured:
>= 104 - <= 109 mg/L
Duration:
720 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
>= 112 - <= 113 mg/L
Duration:
720 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
109 mg/L
Duration:
433.8 h
pH:
9
Temp.:
70 °C
Initial conc. measured:
>= 117 - <= 118 mg/L
Number of replicates:
Preliminary test (Tier 1) and Main test (Tier 2): 2 replicates per pH
Positive controls:
no
Negative controls:
no
Statistical methods:
All logarithms of the relative concentrations were correlated with time using linear regression analysis. The statistical parameters are: slope [1/h], intercept, and coefficent of correlation.
Preliminary study:
Preliminary test- Tier 1
At pH 4, pH 7 and pH 9 a degree of hydrolysis of >/= 10% after 5 days was observed. According to the guideline, the higher Tier test was required to determine the half-life time of the test substance.
No test substance was detected in the blank buffer solutions.
The mean recoveries of the test substance containing buffer solutions at t=O and at pH 4 and pH 7 fell within the acceptable range of 70-110% for non-labelled chemicals (based on experience in our Iabaratory the range of 90-110% as used for radiolabeIIed substances can typically not be reached for non-labelled substances). lt demonstrated that the analytical method was adequate to support the hydro Iysis study on the test substance. The mean recovery of the buffer solutions at t=O and at pH 9 was 113%. This mean recoverywas accepted since it was only slightly higher than the given criterion.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Not applicable.
Key result
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0 h-1
DT50:
680 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.581
Key result
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0 h-1
DT50:
257 d
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
50 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.571
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
33 d
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.006 h-1
DT50:
5.2 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.987
Key result
pH:
7
Temp.:
60 °C
Hydrolysis rate constant:
0.02 h-1
DT50:
35 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.999
Key result
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0 h-1
DT50:
62 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.641
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
55 d
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.001
DT50:
21 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.957
Key result
pH:
9
Temp.:
70 °C
Hydrolysis rate constant:
0.012
DT50:
58 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Coefficient of correlation = 0.990
Details on results:
pH 4:
- No test substance was detected in the blank buffer solutions.
- The mean recoveries of the test substance containing buffer solutions at t=O fell within the acceptable range of 70-110% for non-labelled chemicals. lt demonstrated that the analytical method was adequate to support the hydrolysis study on the test substance.
- For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The half-life times of the test substance were determined according to the model for pseudo-first order reactions.
- Alllogarithms of the relative concentrations were correlated with time using linear regression analysis.
- The rate constant (kobs) and half-life time of the test substance at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25°C: kobs = 1.12 x 10E-04 1/h; t1/2 = 257 days.

pH 7:
- No test substance was detected in the blank buffer solutions.
- The mean recoveries of the test substance containing buffer solutions at t=O and at 50°C and 60°C fell within the acceptable range of 70-110% for non-labelled chemicals. lt demonstrated that the analytical method was adequate to support the hydrolysis study on the test substance. The mean recovery of the buffer solutions at t=O and at 20°C was 112%. This mean recovery was accepted since it was only slightly higher than the given criterion.
- For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The half-life times of the test substance were determined according to the model for pseudo-first order reactions.
- At 20°C and 50°C, all logarithms of the relative concentrations were correlated with time using linear regression analysis. At 60°C, the samples taken after 55.5 hours were excluded from the regression curve, because the logarithms of the relative concentrations did not fit in the linear curve. This was possibly caused by fact that the hydrolysis reaction reached an equilibrium.
- The rate constant (kobs) and half-life time of the test substance at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25°C.

pH 9:
- No test substance was detected in the blank buffer solutions.
- The mean recoveries of the test substance containing buffer solutions at t=O and at 20°C fell within the acceptable range of 70-110% for non-labelled chemicals. lt demonstrated that the analytical method was adequate to support the hydrolysis study on the test substance. The mean recovery of the buffer solutions at t=O and at 20°C and 70°C was 112% and 111% respectively. This mean recovery was accepted since it was only slightly higher than the given criterion.
- For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The half-life times of the test substance were determined according to the model for pseudo-first order reactions.
- At 20°C and 50°C, all logarithms of the relative concentrations were correlated with time using linear regression analysis. At 70°C, the samples taken after 97.6 hours were excluded from the regression curve, because the logarithms of the relative concentrations did not fit in the linear curve. This was possibly caused by fact that the hydrolysis reaction reached an equilibrium.
- The rate constant (kobs) and half-life time of the test substance at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25°C.

1. Specificity:

The chromatogram of the test substance solution showed two test substance peaks. The sum of the area of these peaks was used as response in the calculations.

The chromategram of the blank sample showed no peak at the retention time of the test substance. Since no interferences were detected, the specificity requirements were met and the analytical method was found to be specific for the test substance.

2. Linearity:

The calibration line was constructed using all data. There was a linear relationship between response and test substance concentration in the range of 0.0701 - 110 mg/L (in end solution). Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range 85-115% the calibration line was accepted.

Statistical parameters: - Slope: 4.16 x 10E04

- Intercept: 6.22 x 10E02

- Weighting factor: 1/concentration²

- r: 0.9990

An additional calibration curve was prepared and analysed to perform the research on the stability of stock solutions. The curve was similar to the validation curve and fell within the criteria ranges.

3. Accuracy and repeatability:

Since the mean accuracy at each concentration Ievel fell in the criterion 70-110% and the coefficient of variation was </= 20% the analytical method was accepted for the analysis of the test substance in phosphate buffer pH 7, 0.1 M in the target concentration range of 0.1 - 100 mg/L.

4. Limit of quantification:

The Iimit of quantification (LOQ) was assessed at 0.1 mg/L in phosphate buffer pH 7, 0.1 M.

5. Stability of the analytical system and end solutions:

Since the coefficient of variation at both concentration Ievels was </= 20% the analytical system and end solutions

were stable over at least a 5.11 hour time interval.

Additional data on the stability of the analytical system and end solutions was obtained with the analysis of the stability of stock solutions (see paragraph 6.). The results were similar to the validation results and fell within the criteria range.

6. Stability of stock solutions:

The coefficient of variation on the response factors of the calibration solutions prepared with fresh and stored stock solutions was 4.1 %. Since the value was </= 10% the stock solutions were stable when stored at room temperature for at least 7 days.

7. Storage stability of samples:

Since the mean accuracy of the frozen samples fell in the criterion 70-110% the samples were stable when stored in the freezer.

8. Conclusion

The analytical method was validated for the following parameters:

- Specificity: specific

- Linearity: r = 0.9990

- Accuracy: 86 and 110%

- Repeatability: 1.7and 1.0%

- Limit of quantification: 0.1 mg/L

- Stability analytical system and end solutions: stable

- Stability stock solutions: stable

- Storage stability: stable

Validity criteria fulfilled:
not specified
Conclusions:
Fastest hydrolysis of TDI-Urone could be observed at pH 7 with a hydrolysis half-life of 50 days at 20 °C.
Executive summary:

Hydrolysis of non-radiolabelled TDI-Urone at approximetaly 100 mg/L was studied in the dark at 20, 50, and 60/70 ºC in sterile aqueous buffered solutions at pH 4 (acetate buffer), pH 7 (phosphate buffer) and pH 9 (borate buffer) for up to 720 hours (30 days). The experiment was conducted in accordance with the OECD Guideline 111, and in compliance with the OECD-GLP standards.

Samples were analyzed at defined time intervals using HPLC-UV. Transformation products were not identified as this is not a requirement for a REACH Annex VII/VIII registration.

The half-life times of the test substance (main test) were:

 pH 4     pH 7    pH 9   
 Temperature [°C]  t 1/2  Temperature [°C]  t 1/2  Temperature [°C]  t 1/2
 20  680 d  20  50 d  20  62 d
 25  257 d  25  33 d  25  55 d
 50  75 h  50  5.2 d  50  21 d
 60  19 h  60  35 h  70  58 h

This study is classified acceptable and satisfies the guideline requirement for hydrolysis study.

Description of key information

TDI-Urone hydrolyses relatively slowly at pH 4 (t1/2 = 257 d at 25 °C). T1/2 of hydrolysis is faster at pH 7 and 9 with half-lives of 33 d and 55 d, respectively, at 25 °C. 

Key value for chemical safety assessment

Half-life for hydrolysis:
257 d
at the temperature of:
25 °C

Additional information

Hydrolysis of non-radiolabelled TDI-urone at approximately 100 mg/L was studied in the dark at 20, 50, and 60/70 ºC in sterile aqueous buffered solutions at pH 4 (acetate buffer), pH 7 (phosphate buffer) and pH 9 (borate buffer) for up to 720 hours (30 days). The experiment was conducted in accordance with the OECD guideline 111, and in compliance with the OECD GLP standards.

Samples were analysed at defined time intervals using HPLC-UV. Transformation products were not identified as this is not a requirement for a REACH Annex VII/VIII registration.

The half-life times of the test substance (main test) were:

 pH 4    pH 7    pH 9  
 Temperature [°C]  t 1/2  Temperature [°C]  t 1/2  Temperature [°C]  t 1/2
 20  680 d  20  50 d  20  62 d
 25  257 d  25  33 d  25  55 d
 50  75 h  50  5.2 d  50  21 d
 60  19 h  60  35 h  70  58 h

This study is classified acceptable and satisfies the guideline requirement for hydrolysis study.