Sodium-6-[(3-carboxylatopropanoyl)amino]-2-[(3carboxypropanoyl)amino]hexanoic acid

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

October from 9th to 23rd, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The reliability of the source read-across study was established to be R1: guideline study

Justification for type of information:

Justification for read-across is detailed at section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

adopted 3th April, 2004

Deviations:

yes

Remarks:

Deviations reported in the section "Any other information on material and method"

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: hydrolysis of the test item was followed over 5 days, with one HPLC analysis per day (t1 – t5), at intervals of about 24 h.
- Sampling intervals/times for sterility check: sampling for each series of HPLC analysis was done under sterile conditions (Bunsen burner, fume hood).
- Other observation: 5 ml were transferred with a sterilized pipette tip into a test tube (samples for HPLC do not need to be sterile, but the test vessels must remain sterile). The samples were not filtered, because no signs of precipitation were visible.

Buffers:

- pH 4: mixture of 0.1 M potassium biphthalate and 0.1 N NaOH0.40 ml 0.1 N NaOH + 50 ml 0.1 M potassium biphthalate to3) 100 ml (at 20 °C)
- pH 7: mixture of 0.1 M monopotassium phosphate and 0.1 N NaOH29.63 ml 0.1 N NaOH + 50 ml 0.1 M monopotassium phosphate to 100 ml (at 20 °C)
- pH 9: mixture of 0.1 M H3BO3 in 0.1 M KCl and 0.1 N NaOH21.30 ml 0.1 N NaOH + 50 ml 0.1 M H3BO3 to 100 ml (at 20 °C)

Preparation as described in the OECD guideline 111 for a temperature of 20 °C. For 50 °C the pH had to be adjusted.
The three buffers were sparged with N2 for about 5 min to minimize reaction of the test item with oxygen, and were then placed at 50 ± 0.5 °C for at least 3 hours to allow for temperature equilibration. After checking and if necessary correcting the pH of the three buffers, these were used to prepare test solutions of 50 mg/l test item (set aside 100 ml for the blank solutions).After stirring at least 10 min to ensure full dissolution of the test item, in the 50 °C thermostat cabinet, the pH was adjusted with a precision of 0.1 pH unit.

Details on test conditions:

TEST SYSTEM
- Test vessels: 100 ml vessels with screw cap (Schott, Duran).
- Sterilisation method: the vessels were again taken out of the cabinet, and under sterile conditions (Bunsen burner, hood).100 ml of each test solution and blank solution were filter-sterilized (0.45 μm APES membrane Rapid-Flow, Thermo Scientific) directly into the test and blank vessels.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 50 mg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 50 mg/L

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 50 mg/L

Number of replicates:

Two test vessels (buffer with test item), as well as a blank vessel (buffer without test item), were prepared for each pH value.

Positive controls:

no

Negative controls:

yes

Test performance:

The initially intended loading rate was 50 mg/l test item. The analytically determined start concentrations ranged from 50.0 to 64.5 mg/l, demonstrating the correct dosage of the test item.

Transformation products:

not specified

Details on hydrolysis and appearance of transformation product(s):

At pH 4, the test item hydrolysed readily, whereas at pH 7 and 9, no significant decrease of the test item concentrations were observed over the duration of the test.
For pH 4, the plot of ln(Ct/C0) vs. time gives a linear function. Thus, it can be assumed that the hydrolysis follows a pseudo-first order reaction and the hydrolysis rate constant kobs as well as the half-life (t 0.5) can be calculated.

% Recovery:

ca. 21.3

pH:

4

Temp.:

50 °C

Duration:

ca. 5 d

% Recovery:

ca. 97.4

pH:

7

Temp.:

50 °C

Duration:

ca. 5 d

% Recovery:

ca. 100

pH:

9

Temp.:

50 °C

Duration:

ca. 5 d

Details on results:

For all pH levels, the pH measurements after each sampling showed that the pH of the test solutions remained constant at ± 0.1 unit of the target value.
The temperature could not be held at the required 50 ± 0.5 °C. The temperature ranged from 49.2 to 52.8 °C with a mean of 51.0 °C. Considering that no hydrolysis was observed for pH 7 and 9 and that the hydrolysis at pH 4 followed the expected kinetics of a pseudo first-order reaction, this slight variation seems not to have had a significant effect on the outcome of this study.

Determined hydrolysis rate contants and half-lives for test item

	Kobs	Lower CI	Upper CI	Half-Life	Lower CI	Upper CI
ph 4	0.301/d	0.325	0.276	2.30 d	2.13	2.51
ph 7	no hydrolysis
ph 9	no hydrolysis

Test item concentration of individual replicates and mean; percentage of hydrolysis. In(Ct/C0) used for the calculations of the rate constants.

pH 4 and 50 °C

Time (d)	Replicate A (mg/l)	Replicate B (mg/l)	Mean (mg/l)	% hydrolysis	In(Ct/C0)
0	64.5	57.9	61.2	0.0	0.000
1	42.4	44.1	43.3	29.3	-0.347
2	34.3	32.4	33.4	45.5	-0.607
3	26.0	25.8	25.9	57.7	-0.860
4	17.9	19.2	18.5	69.7	-1.195
5	12.6	13.5	13.0	78.7	-1.546

pH 7 and 50 °C

Time (d)	Replicate A (mg/l)	Replicate B (mg/l)	Mean (mg/l)	% hydrolysis	In(Ct/C0)
0	52.0	51.5	51.7	0.0	0.000
1	50.6	51.3	50.9	1.6	-0.016
2	52.5	51.7	52.1	-0.7	0.007
3	51.5	51.4	51.5	0.5	-0.005
4	51.4	50.5	50.9	1.6	-0.016
5	50.7	50.1	50.4	2.6	-0.027

pH 9 and 50 °C

Time (d)	Replicate A (mg/l)	Replicate B (mg/l)	Mean (mg/l)	% hydrolysis	In(Ct/C0)
0	50.1	50.0	50.1	0.0	0.000
1	51.2	50.9	51.1	-2.0	0.020
2	50.2	49.9	50.1	0.0	0.000
3	50.0	49.8	49.9	0.3	-0.003
4	49.7	50.1	49.9	0.3	-0.003
5	50.0	50.1	50.1	0.0	0.000

Validity criteria fulfilled:

not applicable

Conclusions:

Hydrolysis of the test item was only observed at pH 4, with a half-life of 2.30 days. At pH 7 and 9, no significant decrease of the test item concentrations was found over the duration of the test.

Executive summary:

Method

The hydrolysis of the substance was investigated in a preliminary test (Tier 1) according to TG OECD 111 by incubating solutions of about 50 mg test item/l at three environmentally relevant pH values (pH 4, 7 and 9) and 50 °C over five days.

Observations

The results obtained under the test conditions are summarized in the table below, which gives the determined hydrolysis rate constants (kobs) and calculated half-lifes including 95 % confidence intervals (CI):

	Kobs	Lower CI	Upper CI	Half-Life	Lower CI	Upper CI
ph 4	0.301/d	0.325	0.276	2.30 d	2.13	2.51
ph 7	no hydrolysis
ph 9	no hydrolysis

Results

Hydrolysis of the test item was only observed at pH 4, with a half-life of 2.30 days. At pH 7 and 9, no significant decrease of the test item concentrations was found over the duration of the test.

According to the guideline, a second hydrolysis study (Tier 2) has to be conducted at the pH values where the test item hydrolyses and at different temperatures. In accordance with the sponsor and since the test item is readily biodegradable, no Tier 2 study was conducted.

Description of key information

Hydrolysis of test item was only observed at pH 4, with an half-life of 2.30 days. At pH values of 7 and 9 no hydrolysis was observed.