4,4'-methylenebis[2,6-diethylaniline]

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - May 2015

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The reaction solutions were analysed at the start of the test and after suitable reaction periods. Each analytical occasion three tubes of test solution and 1 tube of control buffer were removed from the thermostat and analysed. At the end of the test three tubes were submitted for sterility confirmation.
Samples were directly injected and analysed with the HPLC method mentioned below.

Buffers:

Full testing was needed at pH 4 only. Preparation of the buffer solution: 250 mL 0.2 M Potassium hydrogen phthalate and 2 mL 0.2 M Sodium hydroxide were diluted to 1000 mL with ultra-pure water. The pH was checked with a calibrated pH meter. Buffer solution was sterilised by filtering on a 0.22 μm Steritop filter unit.

Estimation method (if used):

not applicable

Details on test conditions:

- Test temperatures: 30 ± 0.5 °C, 40 ± 0.5 °C and 50 ± 0.5 °C. :
- Light: The reaction was carried out using dark thermostats to avoid photolytic effects.
- Oxygen: Nitrogen was bubbled into the water for five minutes before the preparation of the solution in order to exclude oxygen.

Test solution:
750 mL sterile solutions were prepared. The test item was applied as acetonitrile solution into the buffer solution. Acetonitrile content of the test solutions was 0.04%. Nominal test item concentration in the buffer solutions was 0.4 μg/mL. The pH of each test solution was checked with a calibrated pH meter. :
Test solution was transferred into 20 mL headspace–vials under sterile circumstances, under a laminar flow hood. The tubes were entirely filled with the solution.

Storage of the solutions:
33 tubes of test solution and 15 tubes of control buffer were placed in each of the three thermostats (set to 30, 40 and 50 °C).
The reaction solutions were analysed at the start of the test and after suitable reaction periods. Each analytical occasion three tubes of test solution and 1 tube of control buffer were removed from the thermostat and analysed.

Sampling:
At the end of the test three tubes were submitted for sterility confirmation.
Samples were directly injected and analysed with the above presented HPLC method.

Analysis of the samples:
At the end of the experiments three replicate samples of the test solutions from each temperature were submitted for sterility confirmation. Samples were investigated by plating experiment on Columbia blood agar and nutrient agar plates. These agar plates were incubated at 37°C, for 48 hours, thereafter evaluated. No microbial growth was obtained, the plates remained clear. In parallel liquid culture media were inoculated with the above test solutions and cultured at 37°C for 48 hours. After the incubation period the tubes were evaluated for the growth of micro-organisms.

Sterility confirmation:
Growth of microorganisms was not detected.

Evaluation:
The chromatograms were evaluated with the help of “EZChrom Elite Client/Server” Chromatogram Processor. The calculations were performed using “Microsoft Office Excel”. Weighted linear regression was computed with the help of PASW Statistics 18.0.0. The factor was 1/concentration.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.374 ng/L

Remarks:

pretest

Duration:

30 d

pH:

4

Temp.:

30 °C

Initial conc. measured:

0.428 ng/L

Remarks:

main test

Duration:

26 d

pH:

4

Temp.:

40 °C

Initial conc. measured:

0.385 ng/L

Remarks:

main test

Duration:

14 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.37 ng/L

Remarks:

main test

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.372 ng/L

Remarks:

pretest only

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.38 ng/L

Remarks:

pretest only

Number of replicates:

33 tubes of test solution and 15 tubes of control buffer were placed in each of the three thermostats (set to 30, 40 and 50 °C).

Positive controls:

no

Negative controls:

yes

Remarks:

buffer solutions

Statistical methods:

The calculations were performed using “Microsoft Office Excel”. Weighted linear regression was computed with the help of PASW Statistics 18.0.0. The factor was 1/concentration.

Preliminary study:

In the course of the preliminary test LZ 596 proved to be hydrolytically stable at pH 7 and 9.
Significant decomposition of LZ 596 was observed after 5 days at a temperature of 50 °C at pH 4. Therefore LZ 596 is considered to be hydrolytically unstable and was further tested at pH 4 at temperatures of 30°C, 40°C and 50°C.

Test performance:

5 days

Transformation products:

not measured

Remarks:

Identification of the degradation products was not feasible because of the low water solubility of the test item.

% Recovery:

20

pH:

4

Temp.:

50 °C

Duration:

5 d

Remarks on result:

other: pretest

% Recovery:

27

pH:

4

Temp.:

30 °C

Duration:

30 d

Remarks on result:

other: main test

% Recovery:

9

pH:

4

Temp.:

40 °C

Duration:

26 d

Remarks on result:

other: main test

% Recovery:

8

pH:

4

Temp.:

50 °C

Duration:

14 d

Remarks on result:

other: main test

% Recovery:

90

pH:

7

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

92

pH:

9

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

576 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: main test; calculated value

Key result

pH:

4

Temp.:

30 °C

Hydrolysis rate constant:

0.002 h-1

DT50:

392 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: main test

Key result

pH:

4

Temp.:

40 °C

Hydrolysis rate constant:

0.004 h-1

DT50:

185 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: main test

Key result

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.007 h-1

DT50:

94 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: main test

Key result

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

9

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

Hydrolysis of the test item was investigated at three different pH values.
- pH 4: 30 °C: half life: 392 h (16 days)
40 °C: half life: 185 h (7.7 days)
50 °C: half life: 94 h (3.9 days)
- pH 7 and 9: LZ 596 proved to be hydrolytically stable in the course of the preliminary test (698-111-0167).

Results with reference substance:

no reference substance tested

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to be hydrolytically stable in the course of the preliminary test at 50°C and at pH 7 and 9. The extrapolated half-life times at these pH- values at 25°C are therefore > one year (> 8760 hours). At pH 4, significant degradation occurred in the pretest at pH 4 and 50°C, requiring further testing at 30, 40 and 50°C at this pH value. In the main study, t1/2 at pH 4 and 25°C was determined to be 576 hours. Identification of the degradation products was not feasible because of the low water solubility of the test item.

Executive summary:

Hydrolysis of the test item was investigated at three different pH values according to OECD test guideline no. 111. The test item was found to be stable in the pretest at pH 7 and 9 at 50°C; no further testing was required at these pH-values. At ph 4, the substance showed significant degradation in the pretest at 50°C, therefore a main test was necessary at this pH at temperatures of 30, 40 and 50°C. pH 4: 30 °C: half life: 392 h (16 days) 40 °C: half life: 185 h (7.7 days) 50 °C: half life: 94 h (3.9 days) pH 7 and 9: LZ 596 proved to be hydrolytically stable in the course of the preliminary test. Identification of the degradation products was not feasible because of the low water solubility of the test item.

Description of key information

The test item was found to be hydrolytically stable in the course of the preliminary test at 50°C at pH 7 and 9. The extrapolated half-life times at these pH-avlues at 25°C are therefore > one year (> 8760 hours). At pH 4, significant degradation occured in the pretest at pH 4 and 50°C, requiring further testing at 30, 40 and 50°C at this pH value. In the main study, t1/2 at pH 4 and 25°C was determined to be 576 hours. Identification of the degradation products was not feasible because of the low water solubility of the test item.

Key value for chemical safety assessment

Half-life for hydrolysis:

576 h

at the temperature of:

25 °C

Additional information

Source: GLP-report