Carbamic acid, [(1S)-2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-, phenylmethyl ester, dihydrochloride

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

No further testing after the preliminary test.

Principles of method if other than guideline:

As in the preliminary test at 50 °C stabiltiy was confirmed for pH 4 and 7 only values where more than 10 % hydrolytic degradation was observed an additional test at only one elevated temperature (80°C) was conducted in order to extrapolate to the rate constant at 25 °C. Experience has shown that this procedure leads to a good and sufficiently exact determination of the rate constants.

GLP compliance:

no

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

At each sampling time, one or two vials from each of the hydrolysis solutions were removed from the thermostatic bath and aliquots of each vessel were analyzed by HPLC. The pH values of the test solutions were measured at least at the start and at different sampling times.

Buffers:

BUFFER SOLUTIONS:
- 0.05 M acetate buffer solution pH 4: 0.657 g (0.008 mol sodium acetate p.a. Merck (M = 82.03 g/mol)) and 2.4 mL (0.042 mol acetic acid 100 % p.a. Merck (M = 60.05 g/mol, density = 1.05 g/mL)) were dissolved to 1000 mL with double distilled water. The pH value was adjusted to 4 by
adding acetic acid.
- 0.05 M phosphate buffer solution pH 7: 4.34 g (0.031 mol disodiumhydrogen phosphate p. a. Merck (M = 141.96 g/mol)) and 2.64 g (0.019 mol potassium dihydrogen phosphate p. a. Merck (M = 136.09 g/mol)) were dissolved to 1000 mL with double distilled water. The pH value was adjusted to 7 by dilute phosphoric acid (10% v/v).
- 0.05 M borate buffer solution pH 9: 2.52 g (0.0125 mol disodium tetraberate p. a. Merck (M = 201.22 g/mol)) and 46 mL (0.0046 mol hydrochloric acid Titrisol Merck (0.1 M)) were dissolved to 1000 mL with double distilled water.
- buffer solutions for calibration of pH meter: standard buffer solutions pH 4, 7 and 9, Merck

As the pH value of borate buffer solutions decreases with increasing temperature, the pH value had tobe adjusted to 9.20 at 25 °C in order to obtain a pH value of 9.00 at 50 oc. For the experiment at 80 °C, the pH value had tobe adjusted to 9.44 at 25 °C. The pH value was adjusted with 0.1 M sodium hydroxide solution

Details on test conditions:

PREPARATION OF THE TEST SOLUTIONS:
- Test solutions with concentrations of approx. 100 mg/L in 0.05 M buffer solution pH 4, 7 and 9 with 1 % acetonitrile were prepared in the corresponding buffer solution; all buffer solutions had been purged with nitrogen to remove dissolved oxygen
- Experiments were conducted using sterilized equipment and buffer solutions.

PROCEDURES FOR INCUBATION AND SAMPLING:
- The vials were incubated in a thermostatic bath at 50 or 80°C and the temperature was kept constant within ± 0.1 °C.
- Prior to incubation the initial concentration of the test substance was measured on the test solutions.
- During the incubation of the test solutions in the thermostatic bath possible photolytic degradation of the test substance was prevented by exclusion of light from the hydrolysis solutions by using a thermostatic bath made of stainless steel with a metal cover.
- At each sampling time, one or two vials from each of the hydrolysis solutions were removed from the thermostatic bath and aliquots of each vessel were analyzed by HPLC.
- The pH values of the test solutions were measured at least at the start and at different sampling times.

APPARATUS AND MATERIALS:
- Analytical balance AT 250 (Mettler)
- Water bath with thermostattype DC3 I W19 (Haake) or thermostated metalblock TR-L-288 (Liebisch)
- Calibrated digital thermometertype 950 (testoterm), connected to a computer, registration of temperature by "Comfort software V.2.5" (testoterm)
- pH meter 766 (Knick) with calibrated glass electrode (Mettler Toledo or Knick)
- HPLC system

MATERIALS:
- acetonitrile p. a. (Merck)
- double distilled water (silica apparatus)
- methanol p. a. (Merck)

Duration:

168.1 h

pH:

4

Temp.:

50

Initial conc. measured:

88.59 mg/L

Duration:

736.7 h

pH:

7

Temp.:

50

Initial conc. measured:

114.27 mg/L

Duration:

216 h

pH:

9

Temp.:

50

Initial conc. measured:

107.42 mg/L

Duration:

74.1 h

pH:

7

Temp.:

80

Initial conc. measured:

106.16 mg/L

Duration:

24 h

pH:

9

Temp.:

80

Initial conc. measured:

92.73 mg/L

Preliminary study:

The results of the preliminary test showed that the test substance is hydrolytically stable at pH 4 (less than 10 % degradation within 6 days at 50 °C). According to the EC test guideline, the test substance can therefore be considered hydrolytically stable at pH4 (t1/2 > 1 year at 25 °C) and no further
testing was necessary. At pH 7 and 9, more than 10 % degradation were observed within 5 days at 50 °C. Therefore hydrolysis experiments at an additional, elevated temperature (80 °C) were conducted at pH 7 and 9.

Test performance:

No further testing was carried out after the preliminary test.

Transformation products:

yes

No.:

#1

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

Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl) contains an ester group which is cleaved at pH values >= 7. The primary products are the free alcohol, benzyl alcohol, and the free acid. However, this free acid is an instable
carbaminic acid which is further hydrolyzed under decarboxylation to give the corresponding amine. At pH 7 the carbaminic acid seemed to be relatively stable. The products could be identified in the HPLC chromatograms of the hydrolysis solutions in peaks at approx. 2 and 3 min increasing with hydrolysis time. At pH 9 from the beginning only the final hydrolysis product and traces of the primary hydrolysis product were observed.

Remarks on result:

hydrolytically stable based on preliminary test

Validity criteria fulfilled:

yes

Conclusions:

Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl) is hydrolytically stable with half-lives at 25 °C of higher than 1 year at pH 4 and 7. The half-life of 80.4 d at 25 °C was determined for pH 9.

Executive summary:

According to EU testing guideline C.7 with the exception of some deviations the hydrolytical behavior was experimentally investigated by the results of a hydrolysis study with carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl) in aqueous solution buffered to pH values of 4, 7 and 9. After the preliminary test at 50 °C was conducted, for pH values where more than 10 % hydrolytic degradation was observed an additional test at only one elevated temperature (80°C) was conducted in order to extrapolate to the rate constant at 25 °C. Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl) is hydrolytically stable with half-lives at 25 °C of higher than 1 year at pH 4 and 7. The half-life of 80.4 d at 25 °C was determined for pH 9.

Description of key information

Carbamic acid [(1S)-,2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-,phenylmethyl ester, dihydrochloride (9Cl) is hydrolytically stable (half-lives of higher than 1 year at pH 4 and 7 at 25 °C ). A half-life of 80.4 d at 25 °C  was determined for pH 9. 

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

Should read >1 year. The half-life at pH7 was taken for assessment.