6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic acid

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2007 - 10 Mar 2008

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:

2004

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority, Den Haag, The Netherlands; National GLP Compliance Monitoring Authority, Department of Science & Technology, New Dlhi, India

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: immediately after the test substance was placed into the test vessels at Day 0 and when sampled on Day 1, 3 and 5

Buffers:

The tests were performed in solutions buffered at pH levels of 4, 7, and 9 (acetate, phosphate, and borate buffers): appropriate volume of each reagent was added to a volumetric flask, diluted to volume with Milli-Q water, where necessary and mixed. After
preparation, the buffer solutions were filter sterilized by passing through 0.2-µm filters under vacuum.

- pH: 4
- Type and final molarity of buffer: Acetate, 0.01 M
- Composition of buffer: 82 mL 0.01M acetic acid solution, 18 mL 0.01 M sodium acetate (final volume: 100 mL), sonicated for about 5 minutes and the pH of the prepared solutions were recorded using a pre-calibrated pH meter

- pH: 7
- Type and final molarity of buffer: Phosphate, 0.01 M
- Composition of buffer: 19.5 mL 0.02 M sodium phosphate manobasic solution, 30.5 mL 0.02 M, Milli-Q-water (final volume: 100 mL), sonicated for about 5 minutes and the pH of the prepared solutions were recorded using a pre-calibrated pH meter

- pH: 9
- Type and final molarity of buffer: Borate, 0.01 M
- Composition of buffer: 2 mL 0.5 M boric acid solution, Milli-Q-water (final volume: 100 mL), sonicated for about 5 minutes and the pH of the prepared solutions were recorded using a pre-calibrated pH meter

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: glass hydrolysis vessels (5 mL capacity) with tightly sealed Teflon-lined caps
- Sterilisation method: autoclaving
- Lighting: dark

TEST MEDIUM
- Volume used/treatment: 4.5 mL
- Kind and purity of water: Milli-Q Water
- Preparation of test medium: 600 µL aliquots of the 14C-DPX-MAT28 stock solution added into three 50 mL volumetric flasks, evaporation of the solvent using a gentle stream of nitrogen gas, residues were reconstituted and flasks were brought to volume with buffer solutions

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Dissolved oxygen: de-oxygenated by bubbling for approximately 5 minutes with nitrogen gas before adding the test item

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

2.03 mg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

2.03 mg/L

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

2.03 mg/L

Number of replicates:

8 (2 replicates were removed from the water bath at each interval for each pH)

Positive controls:

no

Negative controls:

no

Preliminary study:

DT50 at pH 4, 7, 9 and 50°C: no calculation, less than 10% degradation after 5 days

Transformation products:

no

% Recovery:

96.3

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

99.2

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

89.3

pH:

9

Temp.:

50 °C

Duration:

5 d

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

pH:

9

Temp.:

25 °C

DT50:

> 1 yr

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: no

SUPPLEMENTARY EXPERIMENT (Testing of Adsorption to Apparatus):

RESULTS: No significant loss of radioactivity due to adsorption to apparatus was observed

Table 1: Recovery of the test item in pH 4.0 buffer solution incubated at 50 ± 0.5°C expressed as percent of applied radioactivity

COMPOUND	REPLICATE	SAMPLING INTERVAL(DAYS)
		0	1	3	5
DPX-MAT28	R1	99.2	98.0	98.6	96.1
	R2	100.2	98.2	98.9	96.4
	Mean	99.7	98.1	98.8	96.3
Others	R1	0.3	0.2	0.2	0.2
	R2	0.3	0.2	0.2	0.2
	Mean	0.3	0.2	0.2	0.2
Total	R1	99.5	98.2	98.8	96.3
	R2	100.5	98.4	99.1	96.6
	Mean	100.0	98.3	99.0	96.5

Table 2: Recovery of the test item in pH 7.0 buffer solution incubated at 50±0.5°C expressed as percent of applied radioactivity

COMPOUND	REPLICATE	SAMPLING INTERVAL(DAYS)
		0	1	3	5
DPX-MAT28	R1	100.2	96.4	99.8	98.7
	R2	101.1	97.1	98.1	99.7
	Mean	100.6	96.8	99.0	99.2
Others	R1	0.2	0.1	0.2	0.3
	R2	0.2	0.2	0.2	0.3
	Mean	0.2	0.2	0.2	0.3
Total	R1	100.4	96.5	100.0	99.0
	R2	101.3	97.3	98.3	100.0
	Mean	100.8	96.9	99.2	99.5

Table 3: Recovery of the test item in pH 9.0 buffer solution incubated at 50±0.5°C expressed as percent of applied radioactivity

COMPOUND	REPLICATE	SAMPLING INTERVAL(DAYS)
		0	1	3	5
DPX-MAT28	R1	100.2	96.1	99.0	89.0*
	R2	100.2	99.2	98.4	89.6*
	Mean	100.2	97.7	98.7	89.3*
Others	R1	0.2	0.1	0.1	0.4
	R2	0.1	0.1	0.2	0.2
	Mean	0.2	0.1	0.2	0.3
Total	R1	100.4	96.2	99.1	89.4*
	R2	100.3	99.3	98.6	89.8*
	Mean	100.4	97.8	98.9	89.6*

*    Althoughmassbalancewasslightlybelow90%,nocomponentotherthan14C-DPX-MAT28 was observed in the radiochemical chromatogram of pH 9 buffersolution.

Description of key information

DT50 > 1 year at 25°C, pH 4, 7, 9 (OECD 111)

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

Additional information

The hydrolytic stability of the test item was investigated in a GLP study conducted according to OECD 111. Sterile test item solutions were prepared in 0.01 M acetate buffer (pH 4), 0.01 M phosphate buffer (pH 7) and 0.01 M borate buffer (pH 9) at a nominal test concentration of 2.0 µg/mL. The solutions were incubated at 50°C for 5 days. At selected time intervals, samples were analyzed directly by Liquid Scintillation
Counting (LSC), to determine the quantity of radioactivity present in each sample. Radioactivity was quantitatively recovered from each test solution. The mass balances were in a range between 90.8 and 100.5%. Test solutions were subjected to chromatographic analysis (HPLC) to investigate the nature of any hydrolysis products formed. The hydrolysis of the test item at 50 ± 0.5°C after 5 days of incubation was < 10% in pH 4, 7 and 9 buffer solutions. The test item is therefore considered to be stable (t1/2 at 25°C > 1 year) at pH 4, 7 and 9 and no further tests were performed.

Based on the results of this study, hydrolysis is not expected to be a relevant route of degradation of the test item in the environment.