N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 August 1991 to 30 January 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Version / remarks:

26 June, 1991

GLP compliance:

yes

Specific details on test material used for the study:

14C-MGK 264
Specific activity was 18.4 mCi/mmol (148,000 dpm/μg)
Radiochemical purity was 98.5 %
Batch number was JB3163

Radiolabelling:

yes

Remarks:

14C

Analytical monitoring:

yes

Details on sampling:

Duplicate 100-μL aliquots of each sample were analyzed by LSC for 14C-activity to determine the mass balance and the HPLC column recovery.
The pH of each sample was measured using a Corning Model 140 pH meter.
Each sample was characterized by using the previously described HPLC method. The injection volume of each sample ws 100 μL.

Buffers:

A 0.1-M stock of pH 5 buffer was prepared by adding 150 mL of 0.2 M acetic acid (11.55 mL of glacial acetic acid to 1000 mL of water) to 350 mL of 0.2 M sodium acetate (16.4 g of sodium acetate in 1000 mL water). The solution was diluted to 1000 mL with water. A 1:10 dilution was made by taking a 20-mL portion of the stock solution and diluting with 200 mL of water to make a 0.01 M pH 5 buffer solution.

A 0.1-M stock of pH 7 (TRIS) buffer was prepared by combining 250 mL of 0.2 M HCl (16.7 mL of I2 M HCl to 1000 mL water) with 235 mL of 0.2 M tris (hydroxymethyl aminomethane) (24.2 g of TRIS in 1000 mL water). The solution was diluted to 1000 mL with water and adjusted to pH 7 with 1.0 N KOH. A 1: 10 dilution was made by taking a 20-mL portion of the stock solution and diluting with 200 mL of water to make a 0.01-M pH 7 TRIS buffer solution.

A 0.01-M stock of pH 7 (HEPES) buffer was prepared by combining 1000 mL of 0.01 M N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (2.383 g of HEPES in 1000 mL water) with 0.2 N KOH (12.9 g in 1000 mL water) until a pH of 7 was reached.

A 0.1-M stock of pH 9 buffer was prepared by adding 250 mL of a 0.2-M boric acid solution (12.4 g of boric acid in 1000 mL of water) to 300 mL of a 0.2-M borax solution (76.3 g borax in 1000 mL water). The solution was diluted to 1000 mL with water. A 1:10 dilution was made by taking a 20-mL portion of the stock solution and diluting with 200 mL of water to make a 0.01 M pH 9 buffer solution.

Details on test conditions:

Ten borosilicate glass culture tubes were completely filled to ensure zero head space for each test solution. The culture tubes were sealed with Teflon-lined screw caps. Each sample was placed in a Norlake™ environmental chamber maintained under dark conditions at 25.5 ± 0.6°C. One of the 10 samples of each test solution was used for the time 0 sample point.

Duration:

30 d

pH:

5

Temp.:

25 °C

Initial conc. measured:

1 other: µg/ml

Remarks:

Nominal concentration

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

1 other: µg/ml

Remarks:

Nominal Concentration

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

1 other: µg/ml

Remarks:

Nominal Concentration

Number of replicates:

10 samples per pH

Positive controls:

no

Negative controls:

no

Statistical methods:

First-Order Kinetics Calculations for the Hydrolysis of 14C-MGK 264
The concentration of 14C-MGK 264 in the test sample was determined by multiplying the total 14C-activity found in the sample, expressed as 14CMGK 264 equivalents in μg/mL, by the fraction that HPLC determined to be 14C-MGK 264. The concentrations found at each time point were divided by the values found at time 0 to give a percent of time 0.

The degradation rate of 14C-MGK 264 was calculated assuming first-order kinetics. The natural logarithm of the percent of time 0 concentration was plotted versus time and linear regression analysis of equation 2 determined the slope of the line. The absolute value of the slope of the line yields k, the rate constant. The half-life was then calculated by equation 1. (t1/2 = 1n2/k)

Transformation products:

not specified

% Recovery:

>= 100 - < 105.2

St. dev.:

2.2

pH:

5

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

>= 87.2 - < 103.2

St. dev.:

5.1

pH:

7

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

TRIS buffer

% Recovery:

>= 94.4 - <= 107.4

St. dev.:

4.4

pH:

7

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

HEPES Buffer

% Recovery:

>= 93.6 - <= 103.6

St. dev.:

3.3

pH:

9

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

5

Temp.:

25 °C

Type:

other: calculated assuming first-order kinetics

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Within a 95 % confidence interval, the slope is not significantly different than 0, yielding an infinite t1/2.

Key result

pH:

7

Temp.:

25 °C

Type:

other: calculated assuming first-order kinetics

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

TRIS Buffer: Within a 95 % confidence interval, the slope is not significantly different than 0, yielding an infinite t1/2.

Key result

pH:

7

Temp.:

25 °C

Type:

other: calculated assuming first-order kinetics

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

HEPES Buffer: Within a 95 % confidence interval, the slope is not significantly different than 0, yielding an infinite t1/2.

Key result

pH:

9

Temp.:

25 °C

Type:

other: calculated assuming first-order kinetics

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Within a 95 % confidence interval, the slope is not significantly different than 0, yielding an infinite t1/2.

Other kinetic parameters:

Within a 95 % confidence interval, the slope is not significantly different than 0, yielding an infinite t1/2 for any of the buffer solutions tested.

A 30-day study was conducted with MGK 264 at a nominal test concentration of 1.0μg/mL.Test solutions were prepared with four aqueous buffer solutions: pH 5, 10.0 mM acetate buffer; pH 7, 10.0 mM (hydroxymethyl) aminomethane {TRIS); pH 7, 10.0 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); and pH 9, 10.0 mM borate buffer. The test samples were analyzed by LSC and HPLC after 0, 1, 3, 7, 9, 14, 21, and 30 days of hydrolysis. The data presented in Tables II-VI show the results of the LSC/HPLC analysis of MGK 264. The degradation rates for each test solution were calculated assuming first-order kinetics (Tables III-VI, Figures 3-6).

 

No significant degradation of parent compound was observed in any of the buffered test systems during the 30-day study. The rate constants (slope of degradation rate curves) were not significantly different than O; therefore, the half-lives were defined as infinite. Confirmational analysis by mass spectrometry was performed on the day-30 pH 7 HEPES sample. The results from the confirmational analysis confirm MGK 264 remains stable (Figures 11-14).

 

The pH of all samples was measured and is shown in Table I. No significant changes were observed in any of the sample pH measurements.

 

The sterility of the test samples was verified by conducting total plate count analysis for bacterial and fungal populations on the initial buffer solutions and the 30-day samples. The total 14C-activity in the samples (μg/mL as parent equivalents) was used to determine the 14C-mass balance. The overall 14C-mass balance indicated no significant loss of 14C-activity from any of the test samples. The mean 14C-mass balance for the pH 5, pH 7 {TRIS), pH 7 (HEPES), and pH 9 buffers were 102.7%, 97.8%, 103.5%, and 101.1 %, respectively (TableII).

Validity criteria fulfilled:

not specified

Conclusions:

MGK 264 is stable in aqueous buffered solutions at pH 5, 7, and 9 when maintained under sterile conditions at 25.5 + 0.6 °c. Also, the stability of MGK 264 is not affected by buffer type at pH 7.

Executive summary:

A hydrolysis study was conducted with MGK 264 at a nominal test concentration of 1.0 μg/mL in four aqueous buffer solutions: pH5,10.0 mM acetate buffer; pH 7, 10.0 mM Tris (hydroxymethyl) aminomethane (TRIS); pH 7, 10.0 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES); and pH 9, 10.0 mM borate buffer. To determine the percent

 

MGK 264 in each test sample, high performance liquid chromatography was used. Liquid scintillation counting was used to determine the total amount of radioactivity present at each sample point.

 

The data generated during this study prove that14C-MGK 264 in aqueous solution over the pH range of 5-9 at 25°C for 30 days does not hydrolyze. Also, 14C-MGK 264 is not affected by buffer type at pH 7. Because of the lack of degradation, the rate constants were determined to be statistically not significantly different from 0, yielding infinite half-lives.

 

Confirmational analysis by mass spectrometry was performed on the day-30 pH 7 (HEPES) sample. The results from the confirmational analysis confirm MGK 264 remains stable. The overall 14C-mass balance indicated no significant loss of 14C-activity from any of the test sample. The mean14C-mass balance for the pH5,pH 7 (TRIS), pH 7 (HEPES), and pH 9 buffers were 102.7%, 97.8%, 103.5%, and 101.1 %, respectively.

Description of key information

A hydrolysis study was conducted with MGK 264 at a nominal test concentration of 1.0 μg/mL in four aqueous buffer solutions: pH5,10.0 mM acetate buffer; pH 7, 10.0 mM Tris (hydroxymethyl) aminomethane (TRIS); pH 7, 10.0 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES); and pH 9, 10.0 mM borate buffer. To determine the percent MGK 264 in each test sample, high performance liquid chromatography was used. Liquid scintillation counting was used to determine the total amount of radioactivity present at each sample point.

 

The data generated during this study show that [14C]-MGK 264 in aqueous solution over the pH range of 5 -9 at 25°C for 30 days does not hydrolyze. Also, [14C]-MGK 264 is not affected by buffer type at pH 7. Due to the lack of degradation, the rate constants were determined to be statistically not significantly different from 0, yielding infinite half-lives.

 

Confirmational analysis by mass spectrometry was performed on the day 30 pH 7 (HEPES) sample. The results from the confirmational analysis confirm MGK 264 remains stable. The overall [14C]-mass balance indicated no significant loss of [14C]-activity from any of the test sample. The mean [14C]-mass balance for the pH5, pH 7 (TRIS), pH 7 (HEPES), and pH 9 buffers were 102.7%, 97.8%, 103.5%, and 101.1 %, respectively.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C