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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 august 1996 - 3 november 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
May 1981
Deviations:
yes
Remarks:
Hydrolysis tested at pH1 and pH5 instead of pH4. This is considered to not have influence on the overall interpretation of the study.
Qualifier:
according to
Guideline:
EPA Guideline Subdivision N 161-1 (Hydrolysis)
Version / remarks:
October 1982
Qualifier:
according to
Guideline:
other: Pruefung des Verhaltens von Pflanzenbehandlungsmitteln im Wasser, Merkblatt Nr. 55, Teil I and II; Biologische Bundesanstalt fuer Land-und Forstwirtschaft, Bundesrepublik Deutschland
Version / remarks:
October 1980
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Remarks:
14C-labelled at guanidine group (position 4 of oxadiazine ring)
Analytical monitoring:
yes
Details on sampling:
STORAGE AND MAINTENANCE OF SAMPLES
The volatile collection (Preliminary Incubations Only), pH measurement, radioassay, and thin layer chromatography analyses were performed in that order immediately after removal from the incubator. The high performance liquid chromatography of Replicate 1 sample was performed simultaneously with the TLC analyses. The Replicate 2 HPLC assay was performed immediately following the Replicate 1 HPLC assay. Analysis times were recorded on specially prepared forms. Samples were stored at freezer temperatures following analysis. The temperature record of the freezer was printed from the Environmental Monitoring System and were properly stored.

SAMPLE ANALYSIS
The incubation samples were placed in the incubator until the designated time for harvest. Samples from preliminary incubations were purged to collect volatiles at the last sampling time only. Samples from incubations in pH 9 buffered solutions were acidified at the time of collection (or after the pH was checked immediately following volatile collection) to stop further degradation of this base labile compound. The sample volume was adjusted to include the added volume of the acid for calculation of the recovery from incubation. Each sample was evaluated by measuring the pH value, by radioassay, and by TLC analysis. HPLC analyses were performed on random samples from this experiment. Samples from the pH9 incubation were analyzed every 15 minutes on the first day. Each HPLC assay required one hour run time plus the time for reequilibration of the column between runs. Even with use of two HPLC systems, not all sampling times could be immediately assayed at the time of collection.
Buffers:
BUFFERS
- pH 1: Hydrochloric Acid Buffer (0.1M HCl)
- pH 5: Sodium Acetate Buffer (0.01M)
- pH 7: Phosphate buffer (0.01M)
- pH 9: Sodium (tetra) Borate Buffer (0.01M)

BUFFER PREPARATIONS IN THE PRELIMINARY INCUBATIONS AT 60 °C
- pH 1: Approximately 125 ml were sterilized by filtration, sealed and placed in an incubator at 60°C. Approximately 0.0050g of the radiolabeled dosing compound was dissolved with approximately 4 ml of acetonitrile. Calculations determined that 844 μl of the dose stock solution added to approximately 90 ml of the buffer solution and brought to a total volume of 100 ml with additional buffer would equal a final test item concentration of approximately 10 ppm. The solution was thoroughly mixed by shaking for approximately 25 to 30 seconds. The Time 0 Replicates 1 and 2 were removed for immediate assay by TLC and HPLC. The remaining solution was sterilized by filtration using a 250 ml sterile Nalgene filter assembly with a 0.2 μm filter. The sterile solution was immediately transferred aseptically into a sterile 125 ml Erlenmeyer glass flask. The 5 ml (Predose) sample was removed and six aliquots were radioassayed. While the Predose sample was radioassayed, the remaining dosed buffered solution was dispensed into silylated 8 ml sterile amber vials, 5 ml per vial, using a precalibrated Manostat Minipet. Headspace was left to facilitate volatile assay on Day 5. The vials were sealed, labeled, and immediately placed into an incubator preset and stabilized at 60°C. The last filled vial was retained as the Postdose sample. Six aliquots of this Postdose sample were radioassayed. The Postdose sample was also assayed by TLC and HPLC. Three plate count agar plates were streaked with 50 μl each from both the Predose and the Postdose samples. The agar plates were marked and placed in the Constant Temperature room at 25°c. The average of the Predose and Postdose radioassay values was used to calculate the incubation recoveries.
- pH 5: Approximately 125 ml of buffer solution were sterilized by filtration, sealed, and placed in an incubator at 60°C. Approximately 0.0050g of the radiolabeled dosing compound was dissolved with approximately 4 ml of acetonitrile. Calculations determined that 830 μl of the dose stock solution added to approximately 90 ml buffered solution brought to a. total volume of 100 ml with additional buffer would equal a final test item concentration of approximately 10 ppm. The average of the Predose and Postdose values was used to calculate the incubation recoveries.
- pH 7: Approximately 125 ml of buffer solution were sterilized by filtration, sealed, and stored in an incubator at 60°C. Approximately 1.0 mg of the test material was, dissolved in 0.9 ml of acetonitrile. It was calculated that 0.765 ml of the stock solution brought to a 100 ml volume with the buffer solution would yield a final test item concentration of approximately 10 ppm. The dosed buffer solution was prepared, sterilized by filtration, transferred to a sterile glass flask, and the samples and sterility plates were prepared. The average of the Predose and Postdose values was used to calculate the incubation recoveries.
- ph 9: Approximately 125 ml of buffer solution were sterilized by filtration, sealed and placed in an incubator at 60°c. Approximately 1.0 mg of the test material were dissolved in 0.950 ml of acetonitrile. Calculations indicated 0.713 ml of the stock solution brought to 100 ml with the buffer solution would equal approximately 10 ppm. The dosed buffer solution was prepared and sterilized by filtration. Since the test substance is labile at pH 9, it was necessary to acidify the samples immediately after collection. Therefore, after the pH of the sample was determined, between 100 μL to 125 μL of 1M HCl (predetermined volume) was added to each replicate, the sample was vigorously shaken and the pH was determined. The 1M HCl was used to adjust samples to approximately pH 2 and to terminate pH 9 degradation. This pH adjustment at the time of collection was used for all final incubations done at pH 9. The average of the Predose and Postdose radioassay values were used to calculate the incubation recoveries.

BUFFER PREPARATIONS OF FINAL INCUBATIONS
The radiolabeled material was added to the buffer solution for a final concentration of approximately 10 ppm. Two samples, Time 0 replicates 1 and 2, were immediately removed, radioassayed and assayed by TLC and HPLC. The remaining bulk solution was sterilized by filtration, then transferred aseptically to a sterile glass flask in a sterile hood. Samples for incubation were dispensed, 5 ml per vial, into 8 ml silylated, sterile amber vials using a sterile Manostat precalibrated dispenser. The vials were sealed, then were immediately placed in an incubator which had been preset and stabilized at the appropriate temperature. The first and last vials filled were retained as Predose and Postdose, respectively. These two samples were radioassayed and the average DPM/ml values of the two were used to calculate the percent recovery of each sample. Three agar plates were also streaked from each of these samples and incubated for one week to insure that the samples were sterile at the time of dosing. The Postdose sample was also evaluated by HPLC and TLC.
- pH 7 at 25°C: Approximately 3 mg of the radiolabeled material were dissolved in 2 ml acetonitrile as a stock solution and the solution was radioassayed. The dosed solution was prepared by adding 1.3 ml
of the stock solution to the buffer solution for a final volume of 150 ml.
- pH 7 at 40°C: Approximately 5 mg of the solid dosing compound were dissolved in 4.0 ml acetonitrile as a stock solution and the solution was radioassayed. The buffered solution was dosed by adding 1.275 ml of the stock solution to approximately 100 ml of the buffered solution and bringing it to a final volume of 150 ml with additional buffer.
- pH 7 at 60°C: It was placed in the 60°C incubator prior to dosing. 4 ml acetonitrile were added to the vial containing the 5 mg of radiolabeled dosing material for stock solution and the solution was radioassayed. The buffer solution was prepared by adding 1.278 ml of the stock solution to approximately 110 ml of the prepared buffer, then adding sufficient buffer to achieve a final volume of 150 ml.
- pH 9 at 25°C: The buffer solution was preconditioned to 25 °C prior to adding the radiolabeled material. To prepare a stock solution approximately 5 mg of the radiolabeled material were dissolved in 4 ml acetonitrile and radioassayed. The buffer solution was dosed by adding 1.68 ml of the stock solution to approximately 150 ml of the buffer, then adding sufficient buffer to achieve a final volume of 200 ml. Two 5 ml samples, Time 0 replicates 1 and 2, were removed. Immediately, a predetermined volume of 115 μl of 1M HCl, was added to each of these samples to prevent further degradation of the test material. The remaining bulk sample was sterilized by filtration, then was dispensed into 8 ml vials. The samples were incubated at 25°C in the constant temperature room.
- pH 9 at 40°C: The buffer solution was preconditioned to 40°C prior to adding the radiolabeled material. Four ml acetonitrile were added to 5 mg dosing material to prepare a stock solution, which was radioassayed. The dosed buffer solution was prepared by adding 810 μl of the stock solution to approximately 98 ml of the buffer solution, then adding sufficient additional buffer to achieve a final volume of 100 ml. The two Time 0 replicates were removed and acidified with 100 μl of 1M HCl immediately after mixing the dosed buffer solution. The pH of the Postdose sample was also adjusted at the time of collection to prevent further degradation of the radiolablled material. Each sample was acidified immediately after harvest.
- pH 9 at 60°C: The buffer solution was preconditioned to 60°C. Prior to adding the radiolabeled material the buffer solution was transferred to a 60°C water bath in a sterile hood. The stock solution was prepared by dissolving 5 mg of test material in 4 ml acetonitrile. Approximately 0.845 ml of the of the dosed stock solution was added to buffered solution to achieve a final volume of 100 ml. The two Time 0 replicates were removed and acidified with approximately 120 μl and 125 μl of 1M HCl immediately after mixing the dosed buffered solution. The pH of the Postdose sample was also adjusted as other samples were at the time of collection to prevent further degradation of the radiolabelled materials.
Details on test conditions:
PH MEASUREMENTS
A pH meter equipped with an Acc-pHast glass body electrode was used for pH measurements. A two point calibration was performed using certified pH 4, pH 7, and/or pH 10 buffered solutions to confirm the meter's calibration and performance. The pH value of each prepared buffered solution to be dosed with the test compound was rechecked just prior to dosing. Each sample pH value was measured immediately at collection and the value recorded on a specially prepared form. The pH meter was also used to check the pH of the mobile phases used for HPLC.

WEIGHT MEASUREMENT
All weight measurements were made on Mettler balances. The calibration of each balance was checked before each use for accuracy by weighing a class "S" weight of a size similar to that of the sample to be weighed. Each balance deviated less than 1% from the calibration weight each time it was used.

NON-RADIOLABELED REFERENCE STANDARDS
Subsamples of these reference standards were prepared. The desired quantities were weighed in tared vials and a measured volume of an appropriate solvent was added. Consistent Rf values of the standards assured that there was no degradation. All reference standards and subsamples of the reference standards were stored in a freezer.

STERILIZATION AND STERILITY TESTING
All supplies used to prepare the sterile samples were sterilized by autoclave, sterile filtering, or were purchased in a sterile condition. In addition, a water:ethanol 70:30 solution was used to spray the bioflow hood, gloves, etc. An alcohol burner flame was also used to sterilize the glass rods used to streak the agar plates. Difco Plate Count agar was prepared, sterilized, and poured aseptically into petri dishes. These plates were stored up to 30 days in a refrigerator or at room temperature before use. Sterility of the test solutions was tested both before and after the dosing procedure and once a week during the incubation period of each phase of the study. Triplicate plates were streaked with approximately 50 μL of each sample tested. The plates were incubated at 25°C and monitored for 7 days for growth. A daily record was kept for each plate.

PURITY CONFIRMATION OF DOSING COMPOUND
The radiopurity of the radiolabeled compound used in preparing the dosed buffered solutions was reassayed within 24 hours of the initiation of each phase of the study. Either a small subsample of the compound was weighed into a small vial and diluted with acetonitrile or the entire batch for each study phase was diluted prior to the assay. TLC evaluation was performed using several plates, both glass and plastic-backed, due to the variation of recovery values from the various plates. The radiochemical purity of the parent compound was >96% for each batch utilized in the study.

INCUBATION PROCEDURE
Incubators were equilibrated at the appropriate temperature for each experiment prior to use. All samples were incubated in silylated, amber vials, and were placed in the incubators immediately after dosing. Those samples placed in the constant temperature room were also covered with foil to insure that the incubation period was carried out under dark conditions. The temperature of each incubator was monitored every 20 minutes by computers interfaced to the Environmental Monitoring System. A visual check of the incubator thermometer and/or thermograph was made at least once each working day while samples were in each incubator. Thermographs produced a trace which recorded the temperature for each incubation. Each sample was removed from its incubator immediately prior to analysis. Sterility testing was performed on selected incubated samples throughout the incubation term.

VOLATILE COLLECTION AND ASSAY
Volatiles were collected from the last sampling time of each preliminary incubation except for pH 9 at 60°c in which the 24 hour Replicate 2 was used instead of Replicate 1 because the Replicate 1 sample was inadvertently opened before collecting volatiles. New series of traps were prepared using 20 ml scintillation vials with open lids and septa. The six vials were connected in a series with either stainless steel cannulas or with Peek tubing. A negative airflow was used to draw the sample gases through the solutions in the traps. The sequence of vials connected to the sample were as follows: empty trap, ethylene glycol (10 ml), empty trap, KOH (10%) trap 1 (15 ml), KOH (10%) trap 2 (15 ml) and empty trap. This series was connected to a manifold and vacuum pump. The cannulas or tubing were placed above the liquid surface in vials on the outlet end of the trapping solutions and to the bottom of the vials at the inlet end. Each sample was flushed for 1 hour (Preliminary Incubations) through the trapping apparatus. Each trap was assayed; ethylene glycol in 10 ml Ready Safe or ScintiSafe with approximately 50 μl methanol, KOH in 15 ml Ready Gel or ScintiSafe Gel with 5 ml water. For the Final incubations there were no collection of volatiles since data from the preliminary studies indicated <1% of the total radiolabel was attributed to the volatile fraction.

PREPARATION OF SAMPLES FOR MASS SPECTRAL ANALYSIS
Based on the histogram resulting from radioassay of the preparative HPLC vials peaks of interest were identified and the respective vials were combined. Several vials were selected for isolation of M1 and M2. The following procedure was followed for each set of vials: The contents from each respective set of vials were sonicated in the Heat Systems XL Ultrasonicator then transferred to a graduated cylinder. Approximately 1 mL of water was added to each vial. The vials were sonicated as previously outlined and the contents were added to the cylinder. A second rinse of approximately 1 mL of acetonitrile was also added to the cylinder. An aliquot of each isolated component was concentrated to approximately 200 μL under nitrogen and submitted for mass spectral analysis.
Duration:
5 d
pH:
1
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Remarks:
Preliminary test
Duration:
5 d
pH:
5
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Remarks:
Preliminary test
Duration:
5 d
pH:
7
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Remarks:
Preliminary test
Duration:
24 h
pH:
9
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Remarks:
Preliminary test
Duration:
30 d
pH:
7
Temp.:
25 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
7
Temp.:
40 °C
Initial conc. measured:
10 mg/L
Duration:
20 d
pH:
7
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
9
Temp.:
25 °C
Initial conc. measured:
10 mg/L
Duration:
4 d
pH:
9
Temp.:
40 °C
Initial conc. measured:
10 mg/L
Duration:
5.5 h
pH:
9
Temp.:
60 °C
Initial conc. measured:
10 mg/L
Number of replicates:
2 per time point measurement
Positive controls:
no
Negative controls:
no
Statistical methods:
Not reported
Preliminary study:
An overview of the results of the preliminary study and the final studies are presented in 'Any other information on restuls incl. tables'.
- pH 1: The 5 day study showed >90% parent remained at the end of the 5 days. Assay of the volatiles yielded 0.01% of total dose on Day 5. The half-life was calculated as 63.92 days with a correlation coefficient of -0.9790. The slope was -0.0108 per day. The compound was stable at pH 1, therefore no further testing was necessary.
- pH 5: This preliminary study showed little or no breakdown of the parent compound. The volatiles accounted for 0.03% of the total radiolabel balance in the preliminary incubation. The compound was stable for 5 days at pH 5 at 60°C. No further testing was necessary at elevated temperatures. In a previous study, A 30 day hydrolysis test was conducted with the 2-14C-Thiazolyl radiolabelled test substance at the pH 5 at 25°C. Because it was shown in that the substance was stable at pH 5, 25°C, a 30 day
study under these conditions was not conducted for the 14C-guanidine radiolabelled test substance.
- pH 7: This preliminary.evaluation demonstrated that, based on TLC results, 72.65% of the parent compound remained after 5 days. The volatiles accounted for approximately 0.43% of the total radiolabel balance. The half-life was calculated as 12.04 days with a correlation coefficient of -0.9989. The slope was -0.0576 per day. Two 30 day studies were conducted for pH 7, one each for 25°C and 40°C. A 20 day study was conducted at 60°C.
- pH 9: This preliminary evaluation was conducted for only 24 hours because the compound was extremely labile under these conditions. The percent of total dose of parent remaining by the last timepoint was 0.67%. Collected volatiles accounted for 0.07% of the total dose by 24 hours. The half-life was 3.12 hours (0.13 days) with a correlation coefficient of -0.9995. The slope was -0.2223 per hour. Final incubations were conducted for pH 9 at 25°C, 40°C, and 60°C.
Test performance:
Not reported
Transformation products:
yes
No.:
#1
No.:
#2
Details on hydrolysis and appearance of transformation product(s):
PATTERN OF FORMATION AND DECLINE OF DEGRADATES
The two degradates that accounted for >10% of the total radiolabel were identified as: M1 and M2.

IDENTIFICATION OF RADIOACTIVE FRACTIONS
- M1: M1 was isolated from Day 23 Replicate 1 sample of the pH 9 at 25°C Final Incubation experiment by preparative scale HPLC. The isolate coeluted by TLC with the reference standard M1 in Solvent Systems I and II. The mass spectral fragmentation pattern of the sample was consistent with the fragmentation pattern of the M1 reference standard. Significant fragments were m/z 248 (M+l), m/z 175 (248 - CH20CH2NCH3), m/z 132
(248 - cleavage between guanidine amine and methylene bridge between rings), and m/z 129 (248 - cleavage between methylene bridge carbon and thiazolyl ring).
- M2: M2 was isolated from Day 23 replicate 1 at pH 9, 25°C. The mass spectral fragmentation pattern of the component was consistent with the fragmentation pattern of reference standard M2. Significant fragments were m/z 237 (M+l), m/z 191 (237 - NO2), m/z 175 (237 - NH2NO2), and m/z 147 (237 - NH2NO2, CO).
% Recovery:
>= 94.36 - <= 99.15
pH:
7
Temp.:
25 °C
Duration:
30 d
% Recovery:
>= 89.11
pH:
9
Temp.:
25 °C
Duration:
30 d
Remarks on result:
other: Up to 102.44% recovery measured
% Recovery:
>= 96.36
pH:
7
Temp.:
40 °C
Duration:
30 d
Remarks on result:
other: Up to 114.49% recovery measured
% Recovery:
>= 94.7
pH:
9
Temp.:
40 °C
Duration:
4 d
Remarks on result:
other: Up to 100.19% recovery measured
% Recovery:
>= 75.97
pH:
7
Temp.:
60 °C
Duration:
20 d
Remarks on result:
other: Up to 140.45% recovery measured
% Recovery:
>= 99.3
pH:
9
Temp.:
60 °C
Duration:
5.5 h
Remarks on result:
other: Up to 102.92% recovery measured
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
1 253.3 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated from experimental data by means of the Arrhenius equation.
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
15.6 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated from experimental data by means of the Arrhenius equation.
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.001 d-1
DT50:
643 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.082 d-1
DT50:
8.4 d
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
40 °C
Hydrolysis rate constant:
0.005 d-1
DT50:
151 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
40 °C
Hydrolysis rate constant:
0 h-1
DT50:
28.19 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
60 °C
Hydrolysis rate constant:
0.039 d-1
DT50:
18 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
60 °C
Hydrolysis rate constant:
0.003 h-1
DT50:
4.36 h
Type:
(pseudo-)first order (= half-life)
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: The temperature variance was <± 1°c with the few exceptions. The deviations during the 25°C incubations are bracketed by the readings before and after the incidence occurred to show the short duration of the event. These deviations from the prescribed temperature were small enough and of such short duration that they did not effect the integrity of the study. There were several temperature deviations in the pH 7 at 25 °C experiment lasting approximately 20 minutes. The lowest temperature recorded was 23.6° C.This short term variation in temperature did not compromise the study. During the pH 7 at 60 °C experiment, there was a temperature spike up to 66.7°C lasting less than 20 minutes. No other deviation occurred.

MAJOR TRANSFORMATION PRODUCTS
- pH 7 at 25 °C: Parent accounted for 93.68% of total dose by the end of the 30 day period based on TLC. Individual degradates accounted for <3% of the total dose. Maximum observed concentrations for the most prominent degradates were; unidentified product 1 (1.41%, postdose samples), M1 (1.36%, Day 21, replicate 2) and M2 (2.62%, Day 30 replicate 2).
- pH 9 at 25 °C: Parent accounted for an average of 8.45% of the total dose by the last timepoint on Day 30. The maximum% of total dose for unidentified product 2, M1, and M2 were observed in the Day 30 replicate 1 sample, and were 2.23%, 59.75% and 27.98% respectively.
- pH 7 at 40 °C: The range of the average percent of the total dose recovered as parent was 96.65% on Day 0 to 83.89% by Day 30. The maximum% of total dose for M1 was observed in Day 30, replicate 1 sample and was 6.41%. The maximum% of total dose for M2 was observed in Day 30, replicate 2 sample and was 5.86%.
- pH 9 at 40 °C: The parent compound accounted for 9.15% by the end of the incubation. M1 and M2 were the most prominent degradates. After 96 hours the maximum% of total dose values for M1 reached 57.42%, and for M2 reached 27.56%. All other individual degradates accounted for <3% of total dose.
- pH 7 at 60 °C: Percent of total dose represented by parent ranged from approximately 96% on Day 0 to approximately 46% by Day 20. M1 and M2 were the most prominent degradates. By Day 20 the maximum% of total dose values for M1 and M2 were 48.11% and 1.45% respectively.
- pH 9 at 60 °C: The% total dose represented by parent.was 41.46% by the end of the incubation. Unidentified product 2, M1 and M2 were the most prominent degradates. The maximum% of total dose values for unidentified product 2, M1 and M2 were 1.29%, 40.19%, and 14.36% respectively. All other individual degradates accounted for <1% of total dose.

VOLATILIZATION
- Total volatile organics: The total volatile organics in the preliminary incubations at pH 1, pH 5, pH 7 and pH 9 accounted for 0.01%, 0.03%, 0.43% and 0.07% of the total dose by the end of the test.

CORRELATION COEFFICIENTS
- pH 7 at 25 °C: -0.9582
- pH 9 at 25 °C: -0.9999
- pH 7 at 40 °C: -0.9973
- pH 9 at 40 °C: -0.9993
- pH 7 at 60 °C: -0.9711
- pH 9 at 60 °C: -0.9825

Table: Preliminary incubations, 60 °C. Percentage of parent compound based on dpm recovered from TLC and HPLC

Sample

TLC pH 1 %

HPLC pH 1 %

TLC pH 5 %

HPLC pH 5 %

TLC pH 7 %

HPLC pH 7 %

DAY 0 R1

97.12

97.54

93.22

98.80

97.24

97.70

DAY 0 R2

96.76

98.86

96.12

98.47

97.25

97.55

POSTDOSE

97.06

98.00

95.51

98.32

97.09

99.49

DAY 0 AVERAGE

96.94

98.20

94.67

98.64

97.25

97.63

DAY 1 R1

96.63

98.10

96.52

99.68

90.74

93.89

DAY 2 R1

95.11

98.39

96.82

98.69

86.64

88.51

DAY 3 R1

94.75

95.32

96.42

98.91

81.03

85.58

DAY 4 R1

93.41

96.13

96.27

99.48

77.08

78.68

DAY 5 R1

91.77

94.86

96.76

98.82

72.65

75.79

 

Sample

TLC pH 9 %

HPLC pH 9 %

0 HOUR R1

96.78

100.00

0 HOUR R2

96.28

100.00

POSTDOSE

95.60

96.94

0 HOUR AVERAGE

96.53

100.00

2 HOUR

71.88

75.37

3 HOUR

56.01

59.22

4 HOUR

43.54

44.49

5 HOUR

31.96

33.82

24 HOUR

0.67

2.26

  

Table: HPLC and TLC results from the final incubation at pH 7, 25 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

Total % Incubation Recovery

% HPLC Recovery

% TLC Recovery

DAY 0 R1*

NA

738844

NA

98.92

93.35

DAY 0 R2*

NA

737467

NA

102.18

91.38

FILTERED**

NA

772622

NA

NA

NA

POSTDOSE

NA

763111

NA

98.70

93.83

DAY 2 R1

767867

733222

95.49

98.88

92.69

DAY 2 R2

767867

728489

94.87

99.30

94.52

DAY 7 R1

767867

749889

97.66

97.40

90.42

DAY 7 R2

767867

735889

95.S4

100.51

93.59

DAY 9 R1

767867

761311

99.15

96.16

92.53

DAY 9 R2

767867

728089

94.82

99.92

94.20

DAY 14 R1

767867

731711

95.29

98.89

91.96

DAY 14 R2

767867

724578

94.36

99.55

94.20

DAY 16 R1

767867

732822

95.44

98.99

93.46

DAY 16 R2

767867

739444

96.30

97.36

97.21

DAY 21 R1

767867

746822

97.26

101.62

93.84

DAY 21 R2

767867

748311

97.45

99.25

92.72

DAY 23 R1

767867

746800

97.26

100.38

95.64

DAY 23 R2

767867

744578

96.97

97.60

95.59

DAY 28 R1

767867

733822

95.57

96.30

93.00

DAY 28 R2

767867

730889

95.16

97.02

94.69

DAY 30 R1

767867

747556

97.35

96.71

93.14

DAY 30 R2

767867

745956

97.15

96.23

93.13

NA = Not Applicable

NOTE:Repeat radioassay was performed on the Postdose prior to TLC and HPLC.

* Day 1, R1&R2 = Subsamples removed before sterility filtration.

** Filtered (Predose) and Postdose used for average dpm removed after sterility filtration.

 

Table: HPLC and TLC results from the final incubation at pH 9, 25 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

Total % Incubation Recovery

% HPLC Recovery

% TLC Recovery

DAY 0 R1*

NA

1178567

NA

94.81

98.31

DAY 0 R2*

NA

1134367

NA

99.54

99.11

FILTERED**

NA

NA

NA

NA

NA

POSTDOSE

NA

1108267

NA

103.55

103.99

4 HOUR R1

1198020

1179633

98.47

93.25

97.80

4 HOUR R2

1198020

1067500

89.11

103.76

106.71

DAY 1 R1

1198020

1227200

102.44

97.02

95.55

DAY 1 R2

1198020

1205000

100.58

98.80

98.46

DAY 2 R1

1198020

1084800

90.55

109.12

105.35

DAY 2 R2

1198020

1164300

97.19

97.89

98.57

DAY 3 R1

1198020

1116300

93.18

99.98

102.95

DAY 3 R2

1198020

1142700

95.38

97.79

101.91

DAY 7 R1

1198020

1154167

96.34

99.37

99.58

DAY 7 R2

1198020

1179967

98.49

93.53

96.06

DAY 9 R1

1198020

1177067

98.25

96.28

96.99

DAY 9 R2

1198020

1190133

99.34

95.23

97.49

DAY 14 R1

1198020

1169533

97.62

99.79

96.25

DAY 14 R2

1198020

1135533

94.78

99.09

97.50

DAY 16 R1

1198020

1182533

98.71

94.81

93.01

DAY 16 R2

1198020

1194300

99.69

95.02

94.78

DAY 21 R1

1198020

1151433

96.11

96.29

99.01

DAY 21 R2

1198020

· 1142200

95.34

93.84

90.26

DAY 23 R1

1198020

1120633

93.54

97.73

104.37

DAY 23 R2

1198020

1133267

94.59

94.81

89.80

DAY 30 R1

1198020

1110200

92.67

97.56

99.46

DAY 30 R2

1198020

1078067

89.99

99.23

101.67

NA = Not Applicable

NOTE:Repeat radioassay was performed on the Postdose prior to TLC and HPLC.

* Day 1, R1&R2 = Subsamples removed before sterility filtration.

** Filtered (Predose) and Postdose used for average dpm removed after sterility filtration.

 

Table: HPLC and TLC results from the final incubation at pH 7, 40 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

Total % Incubation Recovery

% HPLC Recovery

% TLC Recovery

DAY 0 R1*

NA

1416200

NA

90.29

96.38

DAY 0 R2*

NA

1346833

NA

97.51

97.46

FILTERED**

NA

1334820

NA

NA

NA

POSTDOSE

NA

1372900

NA

92.86

93.56

DAY 2 R1

1335174

1362500

102.05

99.60

100.75

DAY 2 R2

1335174

1317233

98.66

101.72

95.76

DAY 7 R1

1335174

1379667

103.33

94.30

93.47

DAY 7 R2

1335174

1342333

100.54

95.57

97.15

DAY 9 R1

1335174

1324433

99.20

97.26

98.75

DAY 9 R2

1335174

1342933

100.58

96.38

97.15

DAY 14 R1

1335174

1455367

109.00

97.95

99.05

DAY 14 R2

1335174

1420633

106.40

94.62

89.85

DAY 16 R1

1335174

1343200

100.60

93.00

97.35

DAY 16 R2

1335174

1353200

101.35

95.14

96.62

DAY 21 R1

1335174

1322633

99.06

96.54

98.96

DAY 21 R2

1335174

1363367

102.11

93.81

92.80

DAY 23 R1

1335174

1304933

97.74

96.30

94.45

DAY 23 R2

1335174

1304833

97.73

95.55

96.08

DAY 28 R1***

1335174

1528667

114.49

95.72

98.74

DAY 28 R2

1335174

1297167

97.15

97.81

102.56

DAY 30 R1

1335174

1295900

97.06

97.79

95.69

DAY 30 R2

1335174

1286567

96.36

98.59

96.74

NA = Not Applicable

NOTE:Repeat radioassay was performed on the Postdose prior to TLC and HPLC.

* Day 1, R1&R2 = Subsamples removed before sterility filtration.

** Filtered (Predose) and Postdose used for average dpm removed after sterility filtration.

*** Day 28 Rep 1 dpm/ml was high due to loose vial top and sample concentrated

 

Table: HPLC and TLC results from the final incubation at pH 9, 40 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

***Sample Total Vol (mL) before pH adjust.

*** Sample Total Vol (mL) after pH adjust.

Sample total dpm after pH adjust.

Total % Incubation Recovery

*** % HPLC Recovery

*** % TLC Recovery

TIME 0 R1*

NA

696558

5.00

5.100

3552438

NA

96.57

94.29

TIME 0 R2*

NA

718111

5.00

5.100

3662368

NA

98.42

92.83

PREDOSE

731443

722953

5.00

5.000

3614765

NA

NA

NA

POSTDOSE

731443

739'933

5.00

5.000

3699665

NA

97.27

94.36

1.5 HR

731443

691067

5.00

5.100

3524442

98.37

97.38

95.62

3 HR

731443

700800

5.00

5.000

3504000

95.81

NA

94.48

4 HR

731443

681933

4.00

4.100

2795925

95.58

98.77

97.35

5.5 HR

731443

688400

3.00

3.100

2134040

97.25

NA

95.52

7 HR

731443

694733

4.00

4.100

2848405

97.38

NA

94.99

8.5 HR

731443

670358

3.00

3.100

2078104

94.70

89.88

95.48

10 HR

731443

688089

3.00

3.100

2133078

97.21

NA

94.88

11.5 HR

731443

690378

5.00

5.100

3520928

96.27

NA

94.58

24 HR

731443

713333

5.00

5.100

3637998

99.47

94.54

92.38

28 HR

731443

694267

4.00

4.100

2846495

97.29

94.50

97.49

31 HR

731443

720800

3.00

3.050

2198440

100.19

89.12

92.28

52 HR

731443

679287

2.00

2.100

1426461

97.51

91.79

95.52

98 HR

731443

671667

2.00

2.100

1410501

98.42

94.92

95.52

 

* Time 0, R1 and R2 removed before sterility filtration

** Predose and Postdose used for average dpm was removed after sterility filtration

*** A malfunction of the metering device occurred during filling of the sample vials for incubation. Some vials did not contain exactly 5.0 ml. A measured subsample was removed from each vial for analysis at the conclusion of its incubation period.

NA = Not Applicable

NOTE: Repeat radioassay was performed on the postdose prior to TLC and HPLC.

 

Table: HPLC and TLC results from the final incubation at pH 7, 60 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

Total % Incubation Recovery

% HPLC Recovery

% TLC Recovery

TIME 0 R1*

NA

1418133

NA

96.41

98.61

TIME 0 R2*

NA

1369300

NA

100.66

· 95.33

POSTDOSE

NA

1385033

NA

95.26

98.90

DAY1 R1

1416220

1375600

97.13

97.80

97.24

DAY1 R2

1416220

1390767

98.20

94.51

94.13

DAY 3 R1

1416220

1450367

102.41

94.07

91.33

DAY 3 R2

1416220

1378467

97.33

96.98

95.68

DAY 6 R1

1416220

1374967

97.09

98.30

97.61

DAY6 R2

1416220

1306567

92.26

98.38

91.80

DAY 7 R1

1416220

1268900

89.60

102.23

97.67

DAY 7 R2

1416220

1284833

90.72

97.70

97.88

DAY 8 R1

1416220

1260500

89.00

96.38

92.05

DAY 8 R2

1416220

1285900

90.80

96.29

93.24

DAY 9 R1

1416220

1246700

88.03

96.09

96.50

DAY 9 R2

1416220

1255667

88.66

91.80

90.08

DAY 13 R1

1416220

1989100

140.45

99.71

101.49

DAY 13 R2

1416220

1212067

85.58

86.07

91.57

DAY 14 R1

1416220

1644800

116.14

97 7

94.90

DAY 14 R2

1416220

1215567

85.83

85.55

86.01

DAY 17 R1

1416220

1197400

84.55

89.45

79.14

DAY 17 R2

1416220

1075933

75.97

98.64

97.11

DAY 20 R1

1416220

1373000

96.95

95.08

92.20

DAY 20 R2

1416220

1204433

85.05

99.05

95.33

 

NA = Not Applicable

NOTE: Repeat radioassay was performed on the postdose prior to TLC and HPLC.

* Day 0, R1 and R2 = subsamples removed before sterility filtration

** Filtered (Predose) and Postdose used for average dpm removed after sterility

filtration.

Day 13 rep 1, Day 14 rep 1 and Day 17 rep 2 (data was not used for % half-life calculations}.

  

Table: HPLC and TLC results from the final incubation at pH 9, 60 °C

Sample

Average dpm/5ml**Pre/Post dose

Dpm/ml sample

*** Sample Total Vol after pH adjust.

Sample total dpm after pH adjust.

Total % Incubation Recovery

% HPLC Recovery

% TLC Recovery

TIME0R1*

NA

753822

5.12

3859569

NA

93.79

91.84

TIME0R2*

NA

746000

5.125

3823250

NA

96.94

90.75

PREDOSE

NA

744553

5.125

3815834

NA

NA

NA

POSTDOSE

NA

722911

5.125

3704919

NA

97.18

94.64

15MIN

3668660

743400

5.12

3806208

103.75

NA

90.45

30MIN

3746100

739356

5.12

3785503

101.05

NA

90.36

45MIN

3746100

739511

5.12

3786296

101.07

NA

89.73

60MIN

3746100

739533

5.12

3786409

101.08

96.95

92.29

75MIN

3746100

741022

5.12

3794033

101.28

NA

91.42

90MIN

3746100

742933

5.12

3803817

101.54

NA

94.45

105MIN

3746100

746356

5.1

3806416

101.61

NA

91.93

120MIN

3746100

748556

5.1

3817636

101.91

109.05

91.98

185MIN

3746100

756000

5.1

3855600

102.92

94.24

89.39

210MIN

3746100

747400

5.115

3822951

102.05

NA

97.29

270MIN

3746100

744622

5.1

3797572

101.37

96.17

93.52

330MIN

3746100

729400

5.1

3719940

99.3

109.59

95.41

NA = Not Applicable

Note: The Postdose was reassayed prior to HPLC and TLC analysis.

* Day 0 = Predose before sterility filtration

** Predose used for average dpm was removed after sterility filtration.

***A measured sample volume of 5ml, acidified with a measured volume of 1M hci.

This is exact volume used for incubation recovery determination.

Validity criteria fulfilled:
yes
Remarks:
most recoveries were in the range 90-110%; any recoveries below 84% and over 110% were not used for half-life calculations.
Conclusions:
The test substance is base labile as demonstrated by the rapid degradation in the pH 9 buffer at all temperatures. It was stable in pH 1 and pH 5 buffers at 60°C and in the pH 7 buffer at 25°C. The rate of degradation was both pH and temperature-dependent in the pH 7 and pH 9 buffers as seen by the decrease in half-life as the temperature was increased and by the more rapid degradation at pH 9 versus pH 7. The hydrolytic rate for each incubation was determined using pseudo first order kinetics. There are two significant degradates under these conditions: M1 and M2.
Executive summary:

The hydrolysis potential of the radiolabelled test item was investigated in a study according to OECD TG 111 and in compliance with GLP criteria. In this study, a preliminary test was performed at 60 °C for 5 days at pH values of 1, 5, and 7, and for 24 hours at pH 9. In this pre-test, at pH values of 1, 5 percentages degradation after 5 days incubation were 8.23, 3.24 respectively, based on TLC. At pH 7 and pH 9 the percentages degradation after 5 days incubation were 27.35 and 99.33%. Therefore, the test compound was concluded to be stable under acidic conditions and further testing was performed at pH 7 and pH 9. The final test was performed at temperatures of 25, 40 and 60 °C. Based on half-lives estimated from the pre-test, samples were incubated for 30, 30 and 20 days, respectively at pH 7 and for 30 days, 4 days and 5.5 hours, respectively at pH 9. All incubations were conducted under sterile and dark conditions. The degradation of the parent compound and the accumulation and decline of its degradates over a period of time were measured using both thin layer and high performance liquid chromatography. Total [14C]-guanidine material balance was calculated for each sample and the degradation rates at the different test temperatures calculated by assuming pseudo first-order reaction kinetics. Half-lives were calculated from degradation rates. The half-lives at 25, 40 and 60 °C were determined to be 643, 151 and 18 days, respectively at pH 7 and 8.4 days, 28.19 hours and 4.36 hours, respectively at pH 9. From these values the half-life at 20 °C was estimated by means of the Arrhenius equation to be 1253.3 days at pH 7 and 15.6 days at pH 9. Two degradates were identified that accounted for greater than 10% of the applied dose. These two degradates were identified by mass spectral analysis as M1 and M2, and represented a maximum of 59.54 and 27.88% of the applied dose, respectively. Other degradates were present at levels well below 10% and therefore not further analysed.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and the following information was selected for the CSA (based on OECD TG 111, Lowery, 1997):

The test item is hydrolytically stable at pH values of 1 and 5.

DT50 at 25 °C at pH 7: 643 days

DT50 at 25 °C at pH 9: 8.4 days

Key value for chemical safety assessment

Half-life for hydrolysis:
643 d
at the temperature of:
25 °C

Additional information

Table: Overview of available data for the test item on hydrolytic stability as a function of pH.

Method

Guideline / GLP

Endpoint

Value (DT50)

Comment

Reference

Temperature

pH

Duration

25 °C

7

30 d

OECD TG 111

DT50

643 d

The substance was stable in pH 1 and pH 5 buffers at 60°C in a preliminary study. The half-life at 20 °C at pH 7 and 9 were calculated to be 1253.3 and 15.6 days, respectively.

Lowery, 1997

25 °C

9

30 d

DT50

8.4 d

40 °C

7

30 d

DT50

151 d

40 °C

9

4 d

DT50

28.19 h

60 °C

7

20 d

DT50

18 d

60 °C

9

5.5 h

DT50

4.36 h