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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 July 25, 2013 to April 25, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
GLP compliance:
yes (incl. certificate)
Remarks:
according to US EPA and OECD principles of GLP
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals: Samples were collected at Day 0 (test start) and after 5 days.
Buffers:
- pH: Buffer solutions at pH 4, 7 and 9 were used.
- Composition of buffer: The composition of each buffer was as follows:
i) pH 4 Buffer: 4 mL of 0.1 M NaOH and 500 mL of 0.1 M potassium hydrogen phthalate were brought to a total volume of 1 L with reagent water. The final pH of the solution was 4.01.
ii) pH 7 Buffer: 296 mL of 0.1 M NaOH and 500 mL of 0.1 M KH2PO4 were brought to a total volume of 1 L with reagent water. The final pH of the solution was 7.01.
iii) pH 9 Buffer: 500 mL of 0.1 M H3BO3 and 213 mL of 0.1 M NaOH were brought to a total volume of 1 L with reagent water. The pH was analyzed as 8.95 and was adjusted with sodium hydroxide. The final pH of the solution was 9.00.

Prior to their use, all buffer solutions were sterilized by filtering through a 0.45-μm or smaller filter and were autoclaved (at approx. 121°C for approx. 30 minutes). The buffer solutions were stored at room temperature
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 10 and 25 mL volumetric flasks
- Sterilization method: All the glassware were autoclaved at approx. 121°C for approx. 30 minutes.

TEST MEDIUM (preliminary test; tier I)
- Kind and purity of water: Reagent water used in this study was demineralized and purified using a Millipore Milli-Q water purification system comprising of 0.2 μm filter.
- Preparation of test medium: Stock solutions of the test substance were prepared in each of the three buffers (pH: 4, 7 and 9) at approximately 0.9 mg/mL by weighing approximately 36 mg test substance into a 25 mL volumetric flask, correcting for content (62.5%) and bringing the flask to volume with the respective buffer.
- Duplicate, 1 mL aliquots of the above solutions were transferred to 10 mL volumetric flasks, which were brought to volume with 0.2% erythorbic acid. These samples served as the day 0 samples.
- For each buffer, triplicate 1-dram glass amber vials were filled with the above stock solutions and no headspace. The vials were sealed with PTFE lined caps, wrapped in foil and placed in an oven set to 50°C. The pH of each remaining stock solution was measured. After five days of incubation at 50°C, the day 5 samples were removed from incubation and prepared for analysis in the same manner as the day 0 samples.
- At day 0 and 5, quality control (QC) samples were also prepared in each pH buffer at approximately 0.9 mg/mL by weighing approximately 36 mg of the test substance into a 25 mL volumetric flask, correcting for content (62.5%) and bringing the flask to volume the respective buffer. The QC samples were prepared for analysis by transferring 1 mL of each to individual 10 mL volumetric flasks and bringing to volume with 0.2% erythorbic acid.
- All the samples were analyzed by HPLC-UV.
Duration:
0 d
pH:
4
Initial conc. measured:
other: 913 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 908 and 917 µg/mL)
Duration:
0 d
pH:
7
Initial conc. measured:
other: 913 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 907 and 919 µg/mL)
Duration:
0 d
pH:
9
Initial conc. measured:
other: 878 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 881 and 875 µg/mL)
Duration:
5 d
pH:
4
Initial conc. measured:
other: 559 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 567 and 551 µg/mL)
Duration:
5 d
pH:
7
Initial conc. measured:
other: 736 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 741 and 731 µg/mL)
Duration:
5 d
pH:
9
Initial conc. measured:
other: 786 µg/mL (mean of duplicate values of test substance concentration in buffer solution, 763 and 808 µg/mL)
Number of replicates:
Duplicates
Positive controls:
no
Negative controls:
yes
Remarks:
Blank buffer solution (at pH 4) without test substance)
Statistical methods:
No information on statistical method was provided. However, the calculations (including linear regression) were performed using Microsoft Excel (version 2010).
Preliminary study:
- Representative chromatograms of high (102 μg/mL) and low (1.02 μg/mL) Colipa A050 standards were used for calibration. The standard curves used for the quantitation of test substance in the preliminary tests showed linearity, with a correlation coefficient value of >0.999.
- The quality control samples yielded recoveries that ranged from 95.7 to 99.6% of the nominal concentration of Colipa A050 and verified the results of the preliminary test (tier I).
- After five days the percentage of initial test substance concentration recovered in the samples was as follows:
i) At pH 4: 62.1 and 60.4% respectively with C0/Ct values of 1.61 and 1.66 [ln (C0/Ct) values of 0.476 and 0.504]
ii) At pH 7: 81.2 and 80.1%, respectively with C0/Ct values of 1.23 and 1.25 [ln (C0/Ct) values of 0.209 and 0.222]
iii) At pH 9: 86.9 and 92%, respectively with C0/Ct values of 1.15 and 1.09 [ln (C0/Ct) values of 0.140 and 0.083].
- The test substance was unstable at pH 4, 7 and 9. The half-life of test substance was 7, 16 and 31 days at elevated temperature (i.e. 50 ± 0.5°C) and pH 4, 7 and 9, respectively.
- Higher tiered hydrolysis testing was not performed as the Sponsor indicates that degradation was not related to hydrolysis but another mechanism (e.g., oxidation or thermal stability).
Test performance:
No difficulties were encountered that would bias or affect the integrity of the results of this study.
Transformation products:
not measured
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0.099 d-1
DT50:
7 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT90 (at pH 4) = 23.2 days
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.044 d-1
DT50:
16 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT90 (at pH 7) = 52.7 days
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.023 d-1
DT50:
31 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT90 (at pH 9) = 101.7 days
Other kinetic parameters:
Not measured
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: No

Table 1: Hydrolysis of Colipa A050 at pH: 4 (study# 92467)

Sample

Concentration of sample from HPLC (μg/mL)

Percent of initial concentration

C0/Ct

ln C0/Ct

At day 0

Replicate 1

908

-

-

-

Replicate 2

917

-

-

-

Mean of replicates

913

100

1.00

0.000

At day 5

Replicate 1

567

62.1

1.61

0.476

Replicate 2

551

60.4

1.66

0.504

Table 2: Hydrolysis of Colipa A050 at pH: 7 (study# 92467)

Sample

Concentration of sample from HPLC (μg/mL)

Percent of initial concentration

C0/Ct

ln C0/Ct

At day 0

Replicate 1

907

-

-

-

Replicate 2

919

-

-

-

Mean of replicates

913

100

1.00

0.000

At day 5

Replicate 1

741

81.2

1.23

0.209

Replicate 2

731

80.1

1.25

0.222

Table 3: Hydrolysis of Colipa A050 at pH: 9 (study# 92467)

Sample

Concentration of sample from HPLC (μg/mL)

Percent of initial concentration

C0/Ct

ln C0/Ct

At day 0

Replicate 1

881

-

-

-

Replicate 2

875

-

-

-

Mean of replicates

878

100

1.00

0.000

At day 5

Replicate 1

763

86.9

1.15

0.140

Replicate 2

808

92

1.09

0.083

Validity criteria fulfilled:
yes
Conclusions:
N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was unstable under the test conditions with half-life of 7, 16 and 31 days at elevated temperature (i.e. 50 ± 0.5°C) and pH 4, 7 and 9, respectively.
Executive summary:

This study of N,N-bis(2 -hydroxyethyl)-p-phenylenediamine sulphate was conducted following OECD Guideline 111 (Determination of Hydrolysis as a Function of pH).

A preliminary hydrolysis test (tier I) was conducted with buffer solutions of pH 4, 7 and 9 at temperature of 50 ± 0.5°C.

Test solutions were prepared by dissolving appropriate amounts of test substance in appropriate buffer (sterile) and diluting to the mark with that same buffer. Test solutions were taken in10 mL volumetric flasks which were brought to volume with 0.2% erythorbic acid. Sample were transferred to glass amber vials which were sealed with PTFE lined caps, wrapped in foil and placed in an oven set at 50°C. Samples in duplicate were collected at day 0 (test start) and after 5 days.The concentration of the test substance was determined by means of High Performance Liquid Chromatography using UV detector (at λ=254 nm). The quality control samples were also prepared and analysed in the same manner.

The quality control samples yielded recoveries that ranged from 95.7 to 99.6% of the nominal concentration of Colipa A050 and verified the results of the preliminary test (tier I).

After five days the percentage of initial test substance concentration recovered in the samples was as follows:

At pH 4: 62.1 and 60.4% respectively with C0/Ct values of 1.61 and 1.66

At pH 7: 81.2 and 80.1%, respectively with C0/Ct values of 1.23 and 1.25

At pH 9: 86.9 and 92%, respectively with C0/Ct values of 1.15 and 1.09

Higher tiered hydrolysis testing was not performed as the Sponsor indicates that degradation was not related to hydrolysis but another mechanism (e.g., oxidation or thermal stability).

In conclusion, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was unstable under the test conditions with half-life of 7, 16 and 31 days at elevated temperature (i.e. 50 ± 0.5°C) and pH 4, 7 and 9, respectively.

This hydrolysis test is classified as acceptable, and satisfies the guideline requirements of the OECD guideline 111.

Description of key information

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was unstable under the test conditions with half-life of 7, 16 and 31 days at elevated temperature (i.e. 50 ± 0.5°C) and pH 4, 7 and 9, respectively. At environmental pH (pH 7) the half-life was recalculated at 12°C in accordance with the ECHA guidance R16 and this calculated value is 334.5 days.

Key value for chemical safety assessment

Half-life for hydrolysis:
334.5 d
at the temperature of:
12 °C

Additional information

The hydrolysis study of N,N-bis(2 -hydroxyethyl)-p-phenylenediamine sulphate was conducted following OECD Guideline 111 (Determination of Hydrolysis as a Function of pH). A preliminary hydrolysis test (tier I) was conducted with buffer solutions of pH 4, 7 and 9 at temperature of 50 ± 0.5°C.

The concentration of the test substance was determined by means of High Performance Liquid Chromatography using UV detector (at λ=254 nm).

The quality control samples yielded recoveries that ranged from 95.7 to 99.6% of the nominal concentration and verified the results of the preliminary test (tier I).

After five days the percentage of initial test substance concentration recovered in the samples was 62.1% and 60.4% at pH4, 81.2% and 80.1% at pH7 and 86.9% and 92% at pH9.

Higher tiered hydrolysis testing was not performed as the Sponsor indicates that degradation was not related to hydrolysis but another mechanism (e.g., oxidation or thermal stability).

In conclusion, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulphate was unstable under the test conditions with half-life of 7, 16 and 31 days at elevated temperature (i.e. 50 ± 0.5°C) and pH 4, 7 and 9, respectively.

At environmental pH (pH 7) the half-life was recalculated at 12°C in accordance with the ECHA guidance R16 and this calculated value is 334.5 days.