Iodomethane

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2000 to 18 July 2001

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)

Version / remarks:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

PREPARATION OF FORTIFICATION SOLUTIONS
Fortification solutions of [14C]test material used for test 1 and 2 were prepared in buffered water (OPTIMA grade) at a concentration that did not exceed the 14.2 mg/mL (at 25 °C) solubility limit of the test material. The concentration and radiochemical purity of the treatment solution was determined by LSC and HPLC-RAD, respectively, prior to application to the test system. All 14C-labeled materials and standards were stored at < 10 °C. Both fortification solutions were sterilised by filtration through sterile, non-pyrogenic Cameo 25NS 0.22-μm nylon syringe filter within a sterile field. The sterile fortification solutions were dispensed into pre-sterilised 10-mL Wheaton amber serum bottles. Enough solution was processed to fill the serum bottle to the top and nearly eliminate the available headspace. The bottles where then immediately sealed using 20-mm aluminium crimp-caps with PTFE coated, butyl rubber septa and then refrigerated at < 10 °C while not in use.

WATER SOLUBILITY AND JUSTIFICATION OF TEST SOLUTION CONCENTRATION
The water solubility of the test material is approximately 14.2 mg/mL at 25 °C. To limit the exposure to personnel involved in the laboratory portion of this study, the target concentration for the test solution was 25 mg/L, which is well within the solubility range in 0.01 M buffered water. The test material concentration used ( < 0.2 mM) was also less than the maximum allowable levels (half the water solubility or 0.0 IM, whichever is less) described in the appropriate regulatory guidelines.

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

PREPARATION OF HYDROLYSIS SAMPLES
- Preparation of the test systems for tests 1 and 2 were performed within a sterile field. Five 10 μL aliquots of the cold fortification solution were withdrawn through the septa using a pre-cleaned (triplicate rinses of acetone, methanol, and acetonitrile) 10 μL Hamilton gas-tight syringe and analysed by LSC. Immediately after use, the fortification solution was returned to the refrigerator. After the concentration of the fortification solution was determined, the three sterilised buffer solutions were removed from their temporary storage location in the freezer and each treated with 3060 or 2830 μL of the fortification solution for test 1 or 2, respectively. The fortified test solutions were then capped, shaken and returned to the freezer. Each buffered test solution was then removed separately, and rapidly dispensed by pouring into each of the appropriately labelled pre-sterilised 10 mL amber serum bottles. Efforts were made to completely fill each of the serum bottles to the top to minimise any headspace area. Once filled each bottle was immediately sealed using a 20 mm aluminium crimp-cap with PTFE coated, butyl rubber septa. Exactly 20 test systems were prepared for each pH for both test 1 and 2. Test systems were then placed in a water bath set at 50 ± 0.1 °C for test 1 and at 25 ± 1 °C for test 2. Duplicate samples were then analysed in random order at the appropriate sampling intervals.

SAMPLING INTERVALS
- Test 1: 0, 1, 2, 3, 4, 5, 6 and 7 days
- Test 2: 0, 3, 7,1 4, 21, 28 and 30 days

MATERIAL BALANCE
- The material balance was determined for each test system at the appropriate sampling interval by counting the radioactivity in triplicate 100 μL aliquots of the test solution. The test solution concentration for each pH to be used for calculating mass balance was determined from the average of triplicate 100 μL aliquots taken from the two samples analysed at time 0 (average of six total aliquots). These time 0 average values for each pH were then set at 100 %. The material balance should be within the range of 90 to 110 % for any given test system.

STORAGE CONDITIONS AND STABILITY OF SAMPLES
- All samples were analysed within 5 days after sampling. therefore storage stability was not an issue for this study. After analysis, all samples were
stored in a refrigerator at < 10 °C.

Buffers:

- Aqueous buffer solutions of pH 4.0, 7.0, and 9.0 were prepared at concentrations of 0.01 M using the appropriate buffering compounds in OPTIMA grade water. These buffer solution concentrations were below the 0.02 M maximum allowable level, necessary to minimize buffer catalysis. The pH 4.0 buffer was made by combining solutions of 0.01 M acetic acid with 0.01 M sodium acetate until a pH of 4.0 was obtained. The pH 7.0 buffer was made by adjusting the pH of a 0.01 M TRIS solution to 7.0 with a 1:1 solution of HCI in water. Finally, the pH 9.0 buffer was made by combining solutions of 0.01 M boric acid with 0.01 M sodium borate until a pH of 9.0 was obtained.
- Each of the three buffer solutions were sterilised by filtration through a sterile 500 mL 0.2 μm nylon filter system with a polystyrene reservoir and bottle within a sterile field. Four-hundred mL of each sterilised buffer was then transferred aseptically to a separate presterilised amber bottle with a Teflon lined lid. All glassware, caps, and septa coming in contact with the sterilised buffer solution during the study were sterilized by autoclaving at ≥ 120 °C for approximately 30 minutes. All of the pre-sterilised glassware and caps were maintained within the sterile field until test system preparation was completed. The sealed sterilised buffer solutions were then refrigerated (< 10 °C) after preparation and then placed in the freezer (< -4 °C) for approximately 20 minutes prior to and immediately following fortification.

Details on test conditions:

Test were carried out in a water bath set at 50 °C (Test 1) or 25 °C (Test 2).

Duration:

7 d

pH:

4

Temp.:

50

Initial conc. measured:

21.7 mg/L

Duration:

7 d

pH:

7

Temp.:

50

Initial conc. measured:

22.4 mg/L

Duration:

7 d

pH:

9

Temp.:

50

Initial conc. measured:

21.7 mg/L

Duration:

30 d

pH:

4

Temp.:

25

Initial conc. measured:

22.7 mg/L

Duration:

30 d

pH:

7

Temp.:

25

Initial conc. measured:

22.7 mg/L

Duration:

30 d

pH:

9

Temp.:

25

Initial conc. measured:

23.1 mg/L

Number of replicates:

Duplicate test systems for each pH and temperature and timepoint.

Positive controls:

yes

Remarks:

[14C] Methanol

Statistical methods:

First-order rate constants determined by plotting the ln of the average % applied versus time.

Test performance:

FORTIFICATION SOLUTION PURITY AND CONCENTRATION
-The concentration of the fortification solutions used for test 1 and 2 were 3.3 g/L (140.4 μCi/mL) and 3.5 g/L (151.7 μCi/mL) respectively. Both fortification solution concentrations were below the water solubility of the test material. The radiochemical purity of the test 1 and 2 fortification solutions was 97.3 and 98.9 %, respectively, exceeding the required 95 % minimal level.

VERIFICATION OF STERILITY
No microbial growth was observed in the test 1 samples. Microbial growth did occur in one replicate at time O in test 2. Since no degradation occurred in the sample when compared to the other time 0 replicate and no other samples throughout the study had microbial growth, the positive result was believed to be a result of contamination on the plate used.

INCUBATION CONDITIONS
The incubation temperatures were maintained between 49.8 and 49.6 °C for test 1 and between 24.8 and 25.1 °C for test 2. For both tests, the incubation temperatures did not fall out of their acceptable ranges (50 ± 0.5 °C for test 1 and 25 ± 0.5 °C for test 2).

PH DETERMINATION OF THE TEST SOLUTIONS
In test 1, the pH 4, 7, and 9 test solutions were maintained between 3.99 and 4.03, 6.95 and 7.05, and 9.01 and 9.06 pH units, respectively. In test 2, the pH 4, 7, and 9 test solutions were maintained between 4.01 and 4.07, 6.96 and 7.03, and 8.97 and 9.03 pH units, respectively. Overall, the pH values were kept within ± 0.1 pH unit throughout the experimental period .

Transformation products:

yes

No.:

#1

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

MS and HPLC co-chromatography confimed major metabolite is methanol.

% Recovery:

>= 93.4 - <= 105.6

pH:

4

Temp.:

50 °C

Duration:

>= 0 - <= 7 d

% Recovery:

>= 94.2 - <= 103.6

pH:

7

Temp.:

50 °C

Duration:

>= 0 - <= 7 d

% Recovery:

>= 91.9 - <= 102.9

pH:

9

Temp.:

50 °C

Duration:

>= 0 - <= 7 d

% Recovery:

>= 91.3 - <= 102.6

pH:

4

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

% Recovery:

>= 91.7 - <= 102.7

pH:

7

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

% Recovery:

>= 92.6 - <= 102.7

pH:

9

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Key result

pH:

4

Temp.:

50 °C

DT50:

3.3 d

Type:

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

Key result

pH:

7

Temp.:

50 °C

DT50:

2.3 d

Type:

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

Key result

pH:

9

Temp.:

50 °C

DT50:

3 d

Type:

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

Key result

pH:

4

Temp.:

25 °C

DT50:

104.7 d

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

93.9 d

Type:

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

Key result

pH:

9

Temp.:

25 °C

DT50:

108.8 d

Type:

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

Key result

pH:

4

Temp.:

20 °C

DT50:

223.9 d

Type:

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

Key result

pH:

7

Temp.:

20 °C

DT50:

212.7 d

Type:

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

Key result

pH:

9

Temp.:

20 °C

DT50:

240.8 d

Type:

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

Other kinetic parameters:

The adjusted DT50 of the pH 7 samples were 3.2 days, 112.8 days and 247.1 days for 50, 25 and 20 °C respectively.

Details on results:

TEST SOLUTION CONCENTRATION
- The target concentrations of the test solutions for test 1 and 2 were both calculated to be 25.0 mg/L (1074 μCi/L). Due to volatilisation of the test material during test system preparation, the concentration of the test solutions within each pH gradually decreased, as they were dispensed into their individual test vessels. The actual concentration of the test solutions used for the determination of mass balance was taken from the average of the concentration of the time 0 samples for each pH. Randomisation of the test systems, specifically the averaged time 0 samples, provided a test solution concentration that was representative of all of the samples within each pH. For test 1 pH 4, 7, and 9, the test solution concentrations were 21.7 mg/L (0.93 μCi/mL), 22.4 mg/L (0.96 μCi/mL), and 21. 7 mg/L (0.93 μCi/mL), respectively. For test 2 pH 4, 7, and 9, the concentrations were 22.7 mg/L (0.98 μCi/mL), 22.7 mg/L (0.97 μCi/mL), and 23.1 mg/L (0.99 Ci/mL), respectively.

MATERIAL BALANCE
- Test 1 (50 °C) Material Balance: The material balance for the individual pH 4 samples ranged from 93.4 to 105.6 %, with an average value of 101.0 %. Next, the material balance for pH 7 samples ranged from 94.2 to 103.6 %, with an average value of 100.0 %. Finally, the material balance for pH 9 samples ranged from 91.9 to 102.9 %, with an average value of 99.0 %. Overall, the material balance for individual replicates in test 1 ranged from 91.9 to 105.6 %, with an average value of 100.0 %, indicating that acceptable material balance was maintained to draw conclusions regarding chemical degradation in the test system.
- Test 2 (25 °C) Material Balance: The material balance for the individual pH 4 samples ranged from 91.3 to 102.6 % with and average value of 98.3 %. Next, the material balance for pH 7 samples ranged from 91.7 to 102.7 %, with an average value of 97.6 %. Finally, the material balance for pH 9 samples ranged from 92.6 to 102.7 %, with an average value of 98.0 %. Overall, the material balance for individual replicates in test 2 ranged from 91.3 to 102.7 % with an average value of 98.0 %, indicating that acceptable material balance was maintained to draw conclusions regarding chemical degradation in the test system.

DISTRIBUTION OF RADIOACTIVE RESIDUES
- Distribution of the Applied Radioactivity in Test 1 (50 °C): In the pH 4 test systems, the percent of applied radioactivity as the test material decreased from average values of 96.6 % at time 0 to 22.2 % by 7 days. Hydrolysis product methanol correspondingly increased from average values of 2.8 % at time 0 to 76.4 % by 7 days. Other unknown metabolites ranged from 0.0 to 1.4 % of the total applied radioactivity, with an average value of 0.9 %. Next, in the pH 7 test systems, the test material decreased from average values of 96.2 % at time 0 to 10.87% by day 7, with averaged increases in methanol from 2.9 % at time 0 to 81.0 % at day 7. One other compound gradually increased from an average of 0.0 % at time 0 to 7.0 % by day 6, but then decreased to an average of 4.3 % by day 7. This compound resulted from the direct reaction of the test material with the TRIS buffer (TRIS artefact), and was not a hydrolysis product. Additional experiments were performed to show that over time at 50 °C, this artefact will slowly degrade to methanol. The percent of applied radioactivity as the test material was further adjusted to subtract out the effects of its reaction with the TRIS buffer, to determine a more accurate hydrolysis half-life. All other unknown metabolites ranged from 0.0 to 1.8 % of the total applied radioactivity with an average value of 1.1 %. Finally, in the pH 9 test systems the test material decreased, with average values of 95.8 % at time 0 to 16.7 % by 7 days. Hydrolysis product methanol correspondingly increased from average values of 2.8 % at time 0 to 78.0% by 7 days. Other unknown metabolites ranged from 0.5 to 1.6 % of the total applied radioactivity, with an average value of 1.0 %.
- Distribution of the Applied Radioactivity in Test 2 (25 °C): In the pH 4 test systems, the percent of applied radioactivity as the test material decreased from average values of 97.8 % at time 0 to 78.6 % by 30 days. Hydrolysis product methanol correspondingly increased from average values of 0.9 % at time 0 to 16.3 % by 28 days, where it remained relatively constant until day 30. Other unknown metabolites ranged from 0.0 to 0.4 % of the total applied radioactivity. Next, in the pH 7 test systems, the test material decreased from average values of 97.5 % at time 0 to 78.9 % by day 30, with averaged increases in methanol from 0.9 % at time 0 to 17 .5 % at day 30. One other compound gradually increased from an average of 0.0 % at time 0 to 2.7 % by day 28, but then slightly decreased to an average of 2.5 % by day 30. This compound resulted from the direct reaction of the test material with the TRIS buffer, and was not a hydrolysis product. Additional experiments were performed to show that over time this artefact will slowly degrade to methanol. The percent of applied radioactivity as the test material was further adjusted to subtract out the effects of its reaction with the TRIS buffer, to determine a more accurate hydrolysis half-life. All other unknown metabolites ranged from 0.0 to 0.4 % of the total applied radioactivity. Finally, in the pH 9 test systems the test material decreased, with average values of 97.4 % at time 0 to 80.5 % by 30 days. Hydrolysis product methanol correspondingly increased from average values of 1.0 % at time 0 to 18.4 % by 30 days. Other unknown metabolites ranged from 0.0 to 0.5 % of the total applied radioactivity.

DEGRADATION KINETICS
- Degradation of the test material at pH 4, 7, and 9 in Test 1 (50 °C): The linear regression equation slopes of the best fit line through each set of data, determined by least squares analysis, was -0.208 (R^2 = 0.993), -0.300 (R^2 = 0.979), and -0.234 (R^2 = 0.970), for pH 4, 7, and 9, respectively. Since all of the correlation coefficients are ≥ 0.97, there is a high level of confidence in these slopes and consequently the calculated half-life values. The experimental half-lives of the test material at pH 4, 7, and 9 were directly calculated to be 3.3, 2.3, and 3.0 days, respectively. The rate of hydrolysis for the pH 7-test systems was further adjusted to subtract out the reaction of test material with TRIS. The first-order rate constant for the formation of the TRIS artefact was determined from day 1 to 5 by plotting the In of the % ROI of the artefact in each chromatogram versus time. Days 1 to 5 were used for this analysis because they follow the same general trend. Results from days 6 and 7 begin to plateau off and decline, and were therefore not used for the analysis. Next, the rate constant from this data was multiplied by the sampling day and added to the value for the y-intercept (1.05) to determine the amount of TRIS artefact that was theoretically formed at each interval. These theoretical amounts increase from 3.5 % at day 1 to 10.8 % at day 7, and indicate the original percent of test material that reacted with TRIS, which then further slowly degraded to methanol. By multiplying the theoretical % ROI values by the percent recovered radioactivity in the sample (100% maximum), the theoretical percent of applied radioactivity as the TRIS artefact was determined. This % of applied radioactivity as TRIS artefact was then added back to the percent of applied radioactivity as the test material remaining to estimate the amount of non-hydrolysed test material remaining in each test system. The first-order rate constants for the theoretical amount of non-hydrolysed test material remaining was determined by plotting the natural logarithm (In) of the average percent of applied radioactivity versus time. The linear regression equation slopes of the best fit line through the data, determined by least squares analysis, was -0.215 (R^2 = 0.977). Since the correlation coefficient was ≥ 0.97, there is a high level of confidence in the slope and consequently the calculated half-life value. The adjusted experimental half-life of the test material at pH 7 was calculated to be 3.2 days, which now falls between the half-lives for the pH 4 and 9 samples.
- Degradation of the test material at pH 4, 7, and 9 in Test 2 (25 °C): The first-order rate constants for the test material at pH 4, 7, and 9 at 25 °C were determined by plotting the natural logarithm (In) of the average percent of applied radioactivity versus time. The linear regression equation slopes of the best fit line through each set of data, determined by least squares analysis, was -0.0066 (R^2 = 0.885), -0.0074 (R2 = 0.879), and -0.0064 (R2 = 0.899), for pH 4, 7, and 9, respectively. Since all of the correlation coefficients were ≥ 0.87, there is a high level of confidence in these slopes and consequently the calculated half-life values. The experimental half-lives of the test material at pH 4, 7, and 9 were calculated to be 104.7, 93.9, and 108.8 days, respectively. The rate of hydrolysis for the pH 7 test systems was further adjusted to subtract out the reaction of the test material with TRIS. The first-order rate constant for the formation of the TRIS artefact was determined from day 7 to day 30 by plotting the In of the average percent of applied radioactivity of the artefact versus time. Days 7 to 30 were used for this analysis because they maintained a good correlation coefficient (R2 = 0.927), showing that the data over this time was following the same general trend. Results from time 0 and day 3 were both 0, and were therefore not used for the analysis. Next, the rate constant from the data was multiplied by the sampling day and added to the value for they-intercept (-0.95) to determine the amount of TRIS artefact that was theoretically formed at each interval. These theoretical amounts increase from 0.61 % at day 7 to 3.05 % at day 30, and indicate the original percent of test material that reacted with TRIS, which then further slowly degraded to methanol. Since both time 0 and day 3 had no TRIS artefact present, their values were not adjusted. By multiplying these theoretical amounts by the percent recovered radioactivity (100 % maximum) and adding them back to the percent test material remaining at each respective interval, the amount of non-hydrolysed test material remaining in each test system could be estimated. The first-order rate constants for the theoretical amount of non-hydrolysed test material remaining was determined by plotting the natural logarithm (ln) of the average percent of applied radioactivity versus time. The linear regression equation slopes of the best fit line through the data, determined by least squares analysis, was -0.0061 (R^2 = 0.826). Since the correlation coefficient was ≥ 0.82, there is a high level of confidence in the slope and consequently the calculated half-life value. The adjusted experimental half-life of the test material at pH 7 was calculated to be 112.8 days.
- Extrapolation of Degradation Kinetics and Half-Lives at 20 °C: The half-lives and the rate constants for the hydrolysis of the test material at 20 °C were determined for each pH value via an Arrhenius-plot. The extrapolated rate constants (Kh) for pH 4, 7, and 9 at 20 °C are 0.0031, 0.0033, and 0.0029, respectively. The calculated half-lives for pH 4, 7, and 9 at 20 °C are 223.9, 212.7, and 240.8 days, respectively. The adjusted rate constant for pH 7 was determined to be 0.0028 giving a half-life at 20 °C of 247.1 days.

CONFIRMATION OF THE TEST MATERIAL BY GC-MS
- The identity was confirmed by GC-MS in the EI mode. In both spectra, the m/z ion at 142 corresponds to the molecular ion (M+) of the test material. The m/z ion at 144 represents the molecular ion containing one 14C-atom (M+ + 2). The fragment ion at 127 was due to the loss of CH3. from M+ during the ionisation process.

IDENTIFICA T/ON OF HYDROLYSIS PRODUCT METHANOL
- Co-Chromatography of Collected Methanol and Standard: The identity of metabolite methanol was confirmed by co-chromatography. The results of this study show that by co-injecting the standard and sample together that there is an increase in the percentage peak area of methanol, thus confirming that the two peaks are from the same compound.
- Comparison Mass Spectra of Methanol Standard and Isolated [14C]Methanol: The identity of methanol was confirmed GC-MS. In both spectra, the m/z ion at 32 corresponds to the M+ of methanol. In the spectra of isolated methanol, m/z ion at 34 represents the molecular ion containing one 14C-atom (M+ + 2). The m/z ions at 31 and 29 are due to the loss of one and three hydrogen atoms, respectively. Finally, the m/z ion at 15 is CH3+ resulting from the loss of .OH.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the Half-lives of the test material in sterile aqueous buffer solutions are: 223.9, 212.7 (247.1 adjusted) and 240.8 days at 20 °C; 104.7, 93.9 (112.8 adjusted) and 108.8 days at 25 °C and 3.3, 2.3 (3.2 adjusted) and 3.0 days at 50 °C for pHs 4, 7 and 9 respectively.

Executive summary:

The hydrolysis of the test material was investigated in accordance with the standardised guidelines OECD 111 and EPA 161-1, under GLP conditions.

A hydrolysis study was conducted with [14C] labelled test material at a concentrations ranging from 21.7 to 23.1 mg/L in sterile aqueous buffer solutions of pH 4, 7, and 9, in the dark at both 50 and 25 °C. For the 50 °C test, duplicate test systems for pH 4, 7, and 9 were measured at time 0 and days 1, 2, 3, 4, 5, 6, and 7. The material balance ranged from 91.9 to 105.6 %, with an average value of 100.0 %. The experimental half-lives for pH 4, 7, and 9 were calculated to be 3.3, 2.3, and 3.0 days, respectively. Methanol was the major degradate, reaching an average of 76.4, 81.0, 78.0 % of the total applied radioactivity in the pH 4, 7 and 9 test systems, respectively, by 7 days. A minor artefact from the reaction of the test material with the pH 7 TRIS buffer resulted in the slightly lower half-life. The half-life value was adjusted to add back in the theoretical amount of test material that reacted with TRIS to get a better idea of the direct hydrolysis half-life. The adjusted half-life value for pH 7 was calculated to be 3.2 days. Tests were also performed using acetate and phosphate as a buffer at pH 7 in place of TRIS, but results showed that acetate could not buffer the system well enough at pH 7 and phosphate also produced artefacts. TRIS was determined to be the best choice for the pH 7 buffer since it buffers well at that pH and produced only one minor artefact.

For the 25 °C test, duplicate test systems for pH 4, 7, and 9 were measured at time 0 and days 3, 7, 14, 21, 28, and 30. The overall material balance for test 2 ranged from 91.3 to 102.7 % with an average value of 98.0 %. The experimental half-lives of the test material at pH 4, 7, and 9, were calculated to be 104.7. 93.9, and 108.8 days, respectively. Methanol was the major degradate, reaching an average of 16.3. 17.5, and 18.4 % (maximum) of the total applied radioactivity in the pH 4, 7, and 9 test systems, respectively. The TRIS buffer artefact was also present in the 25 °C study. The adjusted half-life for the pH 7 buffer was 112.8 days.

The mass spectral and HPLC co-chromatographic data confirmed that the major metabolite was methanol.

Under the conditions of this study, the half-lives of the test material in sterile aqueous buffer solutions are: 223.9, 212.7 (247.1 adjusted) and 240.8 days at 20 °C; 104.7, 93.9 (112.8 adjusted) and 108.8 days at 25 °C and 3.3, 2.3 (3.2 adjusted) and 3.0 days at 50 °C for pHs 4, 7 and 9 respectively.

Description of key information

Under the conditions of this study, the half-lives of the test material in sterile aqueous buffer solutions are: 223.9, 212.7 (247.1 adjusted) and 240.8 days at 20 °C; 104.7, 93.9 (112.8 adjusted) and 108.8 days at 25 °C and 3.3, 2.3 (3.2 adjusted) and 3.0 days at 50 °C for pHs 4, 7 and 9 respectively.

Key value for chemical safety assessment

Half-life for hydrolysis:

247.1 d

at the temperature of:

20 °C

Additional information

The hydrolysis of the test material was investigated in accordance with the standardised guidelines OECD 111 and EPA 161-1, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A hydrolysis study was conducted with [14C] labelled test material at a concentrations ranging from 21.7 to 23.1 mg/L in sterile aqueous buffer solutions of pH 4, 7, and 9, in the dark at both 50 and 25 °C. For the 50 °C test, duplicate test systems for pH 4, 7, and 9 were measured at time 0 and days 1, 2, 3, 4, 5, 6, and 7. The material balance ranged from 91.9 to 105.6 %, with an average value of 100.0 %. The experimental half-lives for pH 4, 7, and 9 were calculated to be 3.3, 2.3, and 3.0 days, respectively. Methanol was the major degradate, reaching an average of 76.4, 81.0, 78.0 % of the total applied radioactivity in the pH 4, 7 and 9 test systems, respectively, by 7 days. A minor artefact from the reaction of the test material with the pH 7 TRIS buffer resulted in the slightly lower half-life. The half-life value was adjusted to add back in the theoretical amount of test material that reacted with TRIS to get a better idea of the direct hydrolysis half-life. The adjusted half-life value for pH 7 was calculated to be 3.2 days. Tests were also performed using acetate and phosphate as a buffer at pH 7 in place of TRIS, but results showed that acetate could not buffer the system well enough at pH 7 and phosphate also produced artefacts. TRIS was determined to be the best choice for the pH 7 buffer since it buffers well at that pH and produced only one minor artefact.

For the 25 °C test, duplicate test systems for pH 4, 7, and 9 were measured at time 0 and days 3, 7, 14, 21, 28, and 30. The overall material balance for test 2 ranged from 91.3 to 102.7 % with an average value of 98.0 %. The experimental half-lives of the test material at pH 4, 7, and 9, were calculated to be 104.7. 93.9, and 108.8 days, respectively. Methanol was the major degradate, reaching an average of 16.3. 17.5, and 18.4 % (maximum) of the total applied radioactivity in the pH 4, 7, and 9 test systems, respectively. The TRIS buffer artefact was also present in the 25 °C study. The adjusted half-life for the pH 7 buffer was 112.8 days.

The mass spectral and HPLC co-chromatographic data confirmed that the major metabolite was methanol.

Under the conditions of this study, the half-lives of the test material in sterile aqueous buffer solutions are: 223.9, 212.7 (247.1 adjusted) and 240.8 days at 20 °C; 104.7, 93.9 (112.8 adjusted) and 108.8 days at 25 °C and 3.3, 2.3 (3.2 adjusted) and 3.0 days at 50 °C for pHs 4, 7 and 9 respectively.