thiamethoxam (ISO); 3-(2-chloro-thiazol-5-ylmethyl)-5-methyl[1,3,5]oxadiazinan-4-ylidene-N-nitroamine

Hydrolysis

Link to relevant study record(s)

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

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

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Version / remarks:

October 1982

Qualifier:

according to guideline

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. QA statement)

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

The hydrolysis of the substance was studied under GLP at different temperatures and pH conditions in accordance with OECD TG 111. The substance was stable in pH 1 and pH 5 buffers at 60 °C in a preliminary study. A definitive study in pH 7 and pH 9 buffers showed that the substance was stable in pH 7 buffer at 25 °C (half-life of 643 days), whereas it was hydrolytically relatively instable in pH 9 buffer at 25 °C (half-life of 8.4 days) and 40 °C (half-life of 28.19 hours).