2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2002 to 12 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

Used buffer of pH 5 instead of pH 4. Not considered to have affected study validity.

Qualifier:

according to guideline

Guideline:

other: Canada PMRA DACO Number 8.2.3.2

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.5%

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

Sampling Intervals and Collection of Buffers, CO₂
Duplicate samples of each buffer at 20 °C were analysed at 3, 5, 10, 17, and 31 days after treatment (DAT) and samples at 50 °C were analysed at 3 and 5 DAT. Zero-time samples, used for the 20 and 50 °C data, were analysed immediately following dosing. Sterility and sample pH were measured at each time point.

Buffers:

Each buffer was prepared at a concentration of 0.01 M in Millipore-grade water. The buffered solutions were sterilised by autoclave.
A pH 5 buffer was prepared by adding 1 L of Millipore water to sodium acetate trihydrate. Concentrated HCl was added to adjust the pH to 5.
A pH 7 buffer was made by adding concentrated HCl to tris(hydroxymethyl) aminomethane solution.
A pH 9 buffer was made by adding concentrated HCI to sodium tetraborate solution.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks: The hydrolysis of the test material was conducted in 20-mL amber vials sealed with Teflon-lined screw caps.
- Sterilisation method: All glassware and solutions were sterilised by autoclave prior to their use in determining the hydrolytic degradation of the test material. Samples were prepared in a laminar flow hood.
- Sterility check samples: A trypticase soy broth was prepared and sterilised according to package instructions to determine the sterility of the kinetics samples. Immediately upon removal from the incubator, an aliquot (approximately 500 µL) of each sample was spiked into a labelled tube containing soy broth. The sterility check samples were then placed in a 25 °C incubator for at least 3 days. The sterility of the sample was confirmed if, after 3 days, the soy broth continued to be a clear amber solution. Sterile check samples (- controls) remained clear throughout the study period. A non-sterile check of soy broth that had been deliberately contaminated was maintained with the sterility checks. The non-sterile check was cloudy.
- Lighting: Samples were incubated in a darkened incubator
- Details of traps for volatile, if any: Samples were sealed and closed to atmosphere. No trapping of volatile organics or CO₂ was conducted.

Duration:

31 d

pH:

5

Temp.:

20

Initial conc. measured:

400 µg/L

Duration:

31 d

pH:

7

Temp.:

20

Initial conc. measured:

400 µg/L

Duration:

31 d

pH:

9

Temp.:

20

Initial conc. measured:

400 µg/L

Duration:

5 d

pH:

5

Temp.:

50

Initial conc. measured:

400 µg/L

Duration:

5 d

pH:

7

Temp.:

50

Initial conc. measured:

400 µg/L

Duration:

5 d

pH:

9

Temp.:

50

Initial conc. measured:

400 µg/L

Number of replicates:

Duplicate samples per time point were prepared, plus several extras, for a total of 25 vials per buffer. Buffer (5 mL) containing 14C-radiolabelled test material at a nominal concentration of 0.4 µg/g was pipetted into each labelled vial. Twenty samples for each pH were placed in an incubator set at 20 °C and five samples at each pH were placed in an incubator set at 50 °C.

Positive controls:

no

Negative controls:

no

Statistical methods:

Statistical analyses, including means and standard deviations, were calculated using Microsoft Excel.

Transformation products:

not measured

Key result

pH:

5

Temp.:

20 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 31 days

Key result

pH:

7

Temp.:

20 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 31 days

Key result

pH:

9

Temp.:

20 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 31 days

Key result

pH:

5

Temp.:

50 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 5 days

Key result

pH:

7

Temp.:

50 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 5 days

Key result

pH:

9

Temp.:

50 °C

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

for 5 days

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes. Sample pH was unchanged (± 0.1 pH unit). Throughout the study all samples were sterile.

Verification of Chromatographic Procedures

HPLC column recoveries were determined by directly counting an aliquot of each sample analysed by HPLC and comparing that value to total activity eluted from the HPLC column. HPLC recoveries averaged 101.3 ± 4.8 % (range 92.7 - 114.9 %).

Material Balance

At 20 °C, material balance values averaged 99.3 ± 0.8 %, 99.3 ± 1.2 % and 96.9 ± 1.2 % AR for the pH 5, 7 and 9 test systems, respectively. Material balance ranged from 98.3 to 100.6 % AR, 96.6 to 100.7 % AR and 95.0 to 98.9 % AR, respectively, for pH 5, 7 and 9.

At 50 °C material balance averaged 98.9 ± 1.0 %, 98.8 ± 1.5 % and 96.3 ± 1.2 % AR at pH 5, 7 and 9, respectively. Material balance ranged from 97.6 to 100 % AR, 96.9 to 100.6 % AR and 95 to 98.3 % AR, respectively, for pH 5, 7 and 9.

Distribution and Compositions of Residues

At study termination, 100 % of the applied radioactivity was test material at all tested pH values and temperatures.

Identification and Characterisation of Transformation Products

No degradates were observed.

Kinetic Analysis of Data

Degradation of the test material was not observed after 31 days at 20 °C, nor in 5 days at 50 °C in sterile buffers at pH 5, 7 and 9.

Degradation Pathway

The test material was hydrolytically stable at the pH and temperatures tested.

Validity criteria fulfilled:

yes

Conclusions:

The test material was found to be hydrolytically stable at pH 5, 7, and 9 at 20 ° C for 31 days and at 50 °C for 5 days.

Executive summary:

Hydrolysis of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Guideline Subdivision N161-1, EU Method C.7 and Canada PMRA DACO Number 8.2.3.2, SETAC Section 9.

Hydrolysis of radiolabelled test material at 0.4 mg a.i./L was studied in the dark at 20 °C in sterile aqueous buffered solutions at pH 5 (acetate), pH 7 (tris hydroxymethyl aminomethane), and pH 9 (borate) for 31 days and at 50 °C for 5 days.

Samples incubated at 20 °C were analysed at 0, 3, 5, 10, 17, and 31 days and samples incubated at 50 °C were analysed at 0, 3, and 5 days. The test material residues were analysed by LSC and reverse phase HPLC.

Material balance was 99.3 ± 0.8 %, 99.3 ± 1.2 % and 96.9 ± 1.2 % of the applied radiocarbon at pH 5, pH 7 and pH 9, respectively, for the samples incubated at 20 °C. Material balance was 98.9 ± 1.0 %, 98.8 ± 1.5 % and 96.3 ± 1.2 % of the applied radiocarbon at pH 5, pH 7 and pH 9, respectively, for the samples incubated at 50 °C. At test termination, the concentration of test material remained 100 % AR in pH 5, 7, and 9 at 20 and 50 °C.

Therefore, under the conditions of the study, the test material was found to be hydrolytically stable at pH 5, 7, and 9 at 20 and 50 °C.

Description of key information

The test material was hydrolytically stable at pH 5, 7, and 9 at 20 and 50 °C, EPA Guideline Subdivision N 161-1, EU Method C.7 and Canada PMRA DACO Number 8.2.3.2, SETAC Section 9, Cook (2003)

Additional information

Hydrolysis of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA Guideline Subdivision N161-1, EU Method C.7 and Canada PMRA DACO Number 8.2.3.2, SETAC Section 9. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Hydrolysis of radiolabelled test material at 0.4 mg a.i./L was studied in the dark at 20 °C in sterile aqueous buffered solutions at pH 5 (acetate), pH 7 (tris hydroxymethyl aminomethane), and pH 9 (borate) for 31 days and at 50 °C for 5 days.

Samples incubated at 20 °C were analysed at 0, 3, 5, 10, 17, and 31 days and samples incubated at 50 °C were analysed at 0, 3, and 5 days. The test material residues were analysed by LSC and reverse phase HPLC.

Material balance was 99.3 ± 0.8 %, 99.3 ± 1.2 % and 96.9 ± 1.2 % of the applied radiocarbon at pH 5, pH 7 and pH 9, respectively, for the samples incubated at 20 °C. Material balance was 98.9 ± 1.0 %, 98.8 ± 1.5 % and 96.3 ± 1.2 % of the applied radiocarbon at pH 5, pH 7 and pH 9, respectively, for the samples incubated at 50 °C. At test termination, the concentration of test material remained 100 % AR in pH 5, 7, and 9 at 20 and 50 °C.

Therefore, under the conditions of the study, the test material was found to be hydrolytically stable at pH 5, 7, and 9 at 20 and 50 °C.