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Hydrolysis

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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07 June, 1996 to 19 July, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
Cited as Directive 84/449/EEC, C.10
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: 50.2% of test substance in water.
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
At each sampling time 750 µL of sample was taken and added to 750 µL mobile phase. The samples were stored in the freezer until analyses. The temperature of the thermostatic water bath was checked at least at the start and end of the tests and at each sampling time.
Buffers:
pH=4: 100 mL of 0.5 M potassium hydrogen phtalate and 0.8 mL 0.5 M NaOH made up to 1 L with deionized water
pH=7: 100 mL of 0.5 M potassium hydrogen phosphate and 5.92 mL 5 M NaOH made up to 1 L with deionized water
pH=9: 100 mL of 0.5 M boric acid, 100 mL of 0.5 M potassium chloride and 100 mL 4.32 M NaOH made up to 1 L with deionized water
Details on test conditions:
Glassware: Air-tight glass vessels
Other equipment: Thermostatic water bath, thermometer, pH meter.
Method of sterilization: All glassware and buffers were sterilized by autoclaving.
Duration:
8 350 min
pH:
4
Temp.:
20 °C
Initial conc. measured:
ca. 4.606 mg/L
Duration:
8 350 min
pH:
7
Temp.:
20 °C
Initial conc. measured:
ca. 5.455 mg/L
Duration:
8 350 min
pH:
9
Temp.:
20 °C
Initial conc. measured:
ca. 2.948 mg/L
Number of replicates:
2
Transformation products:
not specified
Key result
pH:
4
Temp.:
20 °C
DT50:
>= 1 yr
Key result
pH:
7
Temp.:
20 °C
DT50:
>= 1 yr
Key result
pH:
9
Temp.:
20 °C
DT50:
>= 1 yr
Details on results:
- After 5 days at 50°C, less than 10% decrease in the test substance concentration was measured at all three pH values. It was therefore concluded that test substance was hydrolytically stable with a half-life equal to or greater than one year at pH 4, 7 and 9 at ambient temperatures. A minor decrease of test substance at all three tested pH values was observed, which was probably due to inaccuracies of the test method and/or the analytical method.
- The results of this study with C12-16 chains of the test substance were representative for the whole range of products. All substances have the same molecular structure, similar very high solubility and are completely discosiated in water (in cationic form). All products are only on the market as solutions in water, and are known to be stable for many years.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the test substance was hydrolytically stable with a half-life equal to or greater than one year at pH 4, 7 and 9 at ambient temperature.
Executive summary:

A study was performed to determine the hydrolysis of the test substance according to EU Method C.7, in compliance with GLP. The hydrolytic stability was investigated in buffer solutions of pH 4, 7 and 9 in a water bath at 50ºC for 5 days. Samples were taken at t0 and at regular intervals afterwards to determine half-life time (t½). If t½ is less than 2.4 h, or if less than 10% of the test substance is hydrolysed in 5 d, no further tests need to be performed at that pH. After 5 d at 50°C, less than 10% decrease in test substance concentration was measured at all three pH values. Under the study conditions, the test substance was hydrolytically stable with a half-life equal to or greater than one year at pH 4, 7 and 9 at ambient temperature (Geurts, 1996).

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 July, 1987 to 12 August, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EPA Guideline Subdivision N 161-1 (Hydrolysis)
Deviations:
no
GLP compliance:
yes
Remarks:
US EPA GLP
Specific details on test material used for the study:
- Physical state: Non-radiolabelled = viscous, light yellow/amber liquid; radiolabelled = viscous, light yellow/amber liquid
- Analytical purity: 30% w/w a.s. in aqueous solution
- Lot/batch No.: Non-radiolabelled = 05-6K; radiolabelled = not specified
- Radiochemical purity (if radiolabelling): 98.4%
- Specific activity (if radiolabelling): 3.84 x 10E7 dpm/mL
- Stability under test conditions: The non-radiolabelled a.s., ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Under refrigeration
Radiolabelling:
yes
Analytical monitoring:
yes
Buffers:
Preparation of buffer solutions for the definitive study:
- The pH 5 buffer was prepared by adding 14.8 mL of 0.2 M acetic acid (11.55 mL of acetic acid to 1000 mL of water) to 35.2 mL of 0.2 M sodium acetate (16.4 g of sodium acetate in 1000 mL water). The solution was diluted to 200 mL with water.
- The pH 7 (TRIS) buffer was prepared by adding 378 mL of 0.2 M HCl (16.7 mL of HCl to 983.3 mL of water) to 375 mL of 0.2 M tris (hydroxymethyl) aminomethane (24.2 g of tris in 1000 mL water). The solution was diluted to 1500 mL with water.
- The pH 7 (HEPES) buffer was prepared by diluting 50 mL of 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic cid (HEPES) (2.383 g of HEPES in 1000 mL of water) to a volume of 200 mL and then adding 0.2 M KOH (12.9 g in 1000 mL water) until a pH of 7 was reached.
- The pH 9 buffer was prepared by adding 50 mL of a 0.2 M boric acid (12.4 g of boric acid in 1000 mL of water) to 59 mL of a 0.2 M borax solution (76.3 g borax in 1000 mL water). The solution was diluted to 200 mL with water.

All water used to prepare buffers was Millipore Milli-Q purified, filtered through a 0.2 µm filter and sterilized in autoclave. The pH of each buffer was measured with a Corning Model 140 pH meter. The buffers were then sterilized for 1 h at 250 degF (120 degC) 15 psi.
Details on test conditions:
- Preliminary study:
Determination of an approximate degradation rate; setting of the definitive study sample schedule; evaluation of the adsorption of test containers and of the stability of samples under the storage conditions scheduled during the study. All quantitative radiotracer data for the preliminary study was obtained by radio-thin layer chromatography plate scanner.
- Main study:
1. Preparation of the solution:
The test samples were prepared by placing 1.86 mL of the primary stock (698 ug/mL in deionised water) into each of four Erlenmeyer flasks along with 130 mL of the appropriate buffer. A 1 mL aliquot of acetonitrile was added to each flask to inhibit glass adsorption and promote dissolution of the test compound. 10 mL of the resulting solutions were places in silanized amber vials. Duplicate vials of each solution for each sample point were prepared (48 samples total), and places in a box in a Norlake environmental chamber set at 25±1°C. Samples were prepared at pH 5, 7 (HEPES), 7 (tris) and 9.
2. Treatment of the samples:
- The samples were assayed at Day 0, 1, 3, 7, 14, 22 and 30 for total 14C-activity.
- Duplicate 100 µL aliquots from each vial were analysed by LSC. 100 uL aliquots were spotted on silica gel TLC plates. The TLC plates were then analyzed. The pH was measured with Universal pH indicator papers. The D30 sample's pH was measured also with a Corning pH meter 140.
- In the samples that showed <90% mass accountability, the containers were rinsed with 5 mL of methanol. Duplicate 100 uL aliquots from each vial were analyzed by LSC.
- The concentration of the test substance in the samples was determined by multiplying the total 14C-activity found into the sample, expressed as the test substance equivalents in ug/g, by the fraction that was determined to be the test substance by TLC. The concentration found at each time point was divided by that found at time zero to give percent of time zero.
- The degradation rate of the test substance was calculated assuming first order kinetics. The natural logarithm of the percent of time zero concentration was plotted versus timer and linear regression analysis of equation 1 was used to determine the slope of the line, which is k, the rate constant. The half-life was then calculated by equation 2.
First order kinetics calculations for the hydrolysis of the test substance:
Equation 1: y = mx+b
Where: y = dependent variable
x = independent variable
b = a constant
m= slope of the line
Equation 2: t1/2 = ln2/k
where: t1/2 = half life of the test compound
K= reaction rate constant
Duration:
30 d
Temp.:
25 °C
Initial conc. measured:
10 µg/L
Number of replicates:
Two
Preliminary study:
The preliminary study conducted with buffered solutions of the test substance indicated that the test substance was stable and a 30-d hydrolysis period would be appropriate to evaluate its hydrolysis rate.
- The test substance was found to adsorb onto the glass. Therefore the glass for the definitive study was silanized. Acetonitrile (1%) was used as a co-solvent to reduce the adsorption to the glass-ware.
- It was also determined that samples were stable when stored in either the refrigerator or the freezer.
Transformation products:
no
Details on hydrolysis and appearance of transformation product(s):
- The measured pH values in the four buffer systems indicate that the systems were properly prepared and were stable for the duration of the study. The observed pH for the test substance hydrolysis samples during the 30-d study period ranged from 5 to 5.01, 7 to 7.02, 6.96 to 7 and 9 to 9.02 for pH 5, 7(H), 7(t) and 9 respectively.
- The data for the hydrolysis evaluations in the four buffer systems demonstrate that no significant degradation of the test substance occurred over the pH range of 5 to 9 at 25°C. The mass balance of respective pH hydrolysis samples are presented below under 'total recovery of the test substance'. The overall mean 14C-activity accountability for this study was 96.3%.

% Recovery:
94.6
St. dev.:
4.65
pH:
5
Temp.:
25 °C
Duration:
30 d
% Recovery:
96.8
St. dev.:
3.47
pH:
7
Temp.:
25 °C
Duration:
30 d
% Recovery:
94.4
St. dev.:
4.88
pH:
7
Temp.:
25 °C
Duration:
30 d
% Recovery:
99.5
St. dev.:
3.49
pH:
9
Temp.:
25 °C
Duration:
30 d
Key result
Remarks on result:
other: no significant degradation of the test substance was detected during the 30-d evaluation period
Other kinetic parameters:
- An accurate estimated of half-life for the hydrolysis could not be determined since no significant degradation of the test substance was detected during the 30-d evaluation period.
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study, the test substance was determined to be hydrolytically stable in the pH range of 5 to 9 at 25°C with a very small amount of degradation during the 30-day study period.
Executive summary:

A study was conducted to determine the hydrolysis of the test substance according to US EPA Guideline Subdivision N 161-1, in compliance with GLP. The hydrolysis as a function of pH at 25°C was investigated. Hydrolysis rate constants and the half-lives for degradation of the test substance were determined in aqueous buffered solutions of pH 5, 7 and 9 at 25±1°C. Two pH 7 buffers were employed during the study to evaluate buffer catalysis of the degradation process. All experiments were conducted for a 30 d period at a nominal test concentration of 10 µg/mL. The measured pH values in the four buffer systems indicate that they were properly prepared and stable for the duration of the study. The results show that no significant degradation of the test substance occurred over the pH range of 5 to 9 at 25°C. The mean total recovery was 96.3%. Further, an accurate estimate of half-life for the hydrolysis could not be determined since no significant degradation of the test substance was detected during the 30 day evaluation period. Under the conditions of the study, the test substance was determined to be hydrolytically stable in the pH range of 5 to 9 at 25°C with a very small amount of degradation during the 30 d study period (Carpenter, 1988).

Description of key information

Key value for chemical safety assessment

Half-life for hydrolysis:
1 yr
at the temperature of:
20 °C

Additional information

Study 1. A study was performed to determine the hydrolysis of the test substance according to EU Method C.7, in compliance with GLP. The hydrolytic stability was investigated in buffer solutions of pH 4, 7 and 9 in a water bath at 50ºC for 5 days. Samples were taken at t0 and at regular intervals afterwards to determine half-life time (t½). If t½ is less than 2.4 h, or if less than 10% of the test substance is hydrolysed in 5 d, no further tests need to be performed at that pH. After 5 d at 50°C, less than 10% decrease in test substance concentration was measured at all three pH values. Under the study conditions, the test substance was hydrolytically stable with a half-life equal to or greater than one year at pH 4, 7 and 9 at ambient temperature (Geurts, 1996).

Study 2. A study was conducted to determine the hydrolysis of the test substance according to US EPA Guideline Subdivision N 161-1, in compliance with GLP. The hydrolysis as a function of pH at 25°C was investigated. Hydrolysis rate constants and the half-lives for degradation of the test substance were determined in aqueous buffered solutions of pH 5, 7 and 9 at 25±1°C. Two pH 7 buffers were employed during the study to evaluate buffer catalysis of the degradation process. All experiments were conducted for a 30 d period at a nominal test concentration of 10 µg/mL. The measured pH values in the four buffer systems indicate that they were properly prepared and stable for the duration of the study. The results show that no significant degradation of the test substance occurred over the pH range of 5 to 9 at 25°C. The mean total recovery was 96.3%. Further, an accurate estimate of half-life for the hydrolysis could not be determined since no significant degradation of the test substance was detected during the 30 day evaluation period. Under the conditions of the study, the test substance was determined to be hydrolytically stable in the pH range of 5 to 9 at 25°C with a very small amount of degradation during the 30 d study period (Carpenter, 1988).