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

Dissociation constant

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dissociation constant

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 830.7370 (Dissociation constants in water)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Remarks:

Bundesinstitut für Risikobewertung, Berlin, Germany / Food and Consumer Product Safety Authority, Den Haag, The Netherlands / National GLP Compliance Monitoring Authority, New Delhi, India

Dissociating properties:

yes

pKa:

4.65

Temp.:

20 °C

The concentration of sodium hydroxide solution used in the titration was found to be 0.0096 M.

 

The results of the dissociation constants of the test item in water are given in the tables reported below. The mean dissociation constants of the test item in water for replications R1, R2 and R3 were determined to be 4.61 ± 0.53, 4.69 ± 0.59 and 4.66 ± 0.60, respectively, with an overall mean of 4.65 ± 0.04.

The maximum and minimum temperature during the experimental period was 20.2 and 19.8 °C, respectively.

 

 

 

Dissociation constant of the test item (replication R1)

[L] volume of base (titrant) added (mL)	[P] total volume after adding L (mL)	pH	[X] moles of Base   (K * L) / 1000	[B] undissociated test item   (N - X) * 1000 / P	[BH+] dissociated test item   (X * 1000) / P	log ([B] / [BH+])	pKa
2.5	202.5	4.27	0.00002400	0.0003438	0.0001185	0.4626	3.807
3.0	203.0	4.33	0.00002880	0.0003193	0.0001419	0.3522	3.978
3.5	203.5	4.47	0.00003360	0.0002949	0.0001651	0.2519	4.218
4.0	204.0	4.54	0.00003840	0.0002707	0.0001882	0.1579	4.382
4.5	204.5	4.62	0.00004320	0.0002466	0.0002112	0.0673	4.553
5.0	205.0	4.66	0.00004800	0.0002225	0.0002341	-0.0221	4.682
5.5	205.5	4.72	0.00005280	0.0001986	0.0002569	-0.1118	4.832
6.0	206.0	4.80	0.00005760	0.0001749	0.0002796	-0.2037	5.004
6.5	206.5	4.89	0.00006240	0.0001515	0.0003022	-0.3007	5.191
7.0	207.0	5.04	0.00006720	0.0001276	0.0003246	-0.4055	5.446
Mean ± S.D.							4.61 ± 0.53

K = Concentration of base used in titration = 0.0096M

 

Weight of test item taken: 0.0250 g

Volume made up: 250 mL

Aliquot taken for titration (A): 200 mL

Molecular weight of test item: 213.623

 

Concentration of test item (C) = (0.0250 * 1000) / (213.623 x 250 (mL)) = 0.0004681

 

Total No. of moles of test item in 200 mL solution taken for titration (N) = 0.0004681 x 200 / 1000 = 0.00009362

 

Max. and Min. Temp. during the period: 20.2 and 20.0 °C, respectively

 

 

Dissociation constant of the test item (replication R2)

[L] volume of base (titrant) added (mL)	[P] total volume after adding L (mL)	pH	[X] moles of Base   (K * L) / 1000	[B] undissociated test item   (N - X) * 1000 / P	[BH+] dissociated test item   (X * 1000) / P	log ([B] / [BH+])	pKa
2.5	202.5	4.25	0.0002400	0.0003476	0.0001185	0.4614	3.783
3.0	203.0	4.35	0.0002880	0.0003231	0.0001419	0.3574	3.993
3.5	203.5	4.50	0.0003360	0.0002987	0.0001651	0.2575	4.243
4.0	204.0	4.58	0.0003840	0.0002744	0.0001882	0.1638	4.416
4.5	204.5	4.68	0.0004320	0.0002503	0.0002112	0.0738	4.606
5.0	205.0	4.76	0.0004800	0.0002262	0.0002341	-0.0149	4.775
5.5	205.5	4.85	0.0005280	0.0002023	0.0002569	0.1038	4.954
6.0	206.0	4.96	0.0005760	0.0001785	0.0002796	-0.1949	5.155
6.5	206.5	5.06	0.0006240	0.0001549	0.0003022	-0.2902	5.350
7.0	207.0	5.19	0.0006720	0.0001313	0.0003246	-0.3931	5.583
Mean ± S.D.							4.69 ± 0.59

 

K = Concentration of base used in titration = 0.0096M

 

Weight of test item taken: 0.0252 g

Volume made up: 250 mL

Aliquot taken for titration (A): 200 mL

Molecular weight of test item: 213.623

 

Concentration of test item (C) = (0.0252 * 1000) / (213.623 x 250 (mL)) = 0.0004719

 

Total No. of moles of test item in 200 mL solution taken for titration (N) = 0.0004719 x 200 / 1000 = 0.00009438

 

Max. and Min. Temp. during the period: 20.1 and 20.0 °C, respectively

 

 

Dissociation constant of the test item (replication R2)

[L] volume of base (titrant) added (mL)	[P] total volume after adding L (mL)	pH	[X] moles of Base   (K * L) / 1000	[B] undissociated test item   (N - X) * 1000 / P	[BH+] dissociated test item   (X * 1000) / P	log ([B] / [BH+])	pKa
2.5	202.5	4.23	0.00002400	0.0003493	0.0001185	0.4695	3.761
3.0	203.0	4.33	0.00002880	0.0003248	0.0001419	0.3596	3.970
3.5	203.5	4.45	0.00003360	0.0003004	0.0001651	0.2600	4.190
4.0	204.0	4.56	0.00003840	0.0002762	0.0001882	0.1666	4.393
4.5	204.5	4.64	0.00004320	0.0002520	0.0002112	0.0767	4.563
5.0	205.0	4.70	0.00004800	0.0002280	0.0002341	-0. 0115	4.712
5.5	205.5	4.83	0.00005280	0.0002041	0.0002569	-0.0999	4.930
6.0	206.0	4.95	0.00005760	0.0001803	0.0002796	-0.1905	5.141
6.5	206.5	5.04	0.00005240	0.0001566	0.0002538	-0.2855	5.326
7.0	207.0	5.21	0.00006720	0.0001330	0.0003246	-0.3875	5.598
Mean ± S.D.							4.66 ± 0.60

 

K = Concentration of base used in titration = 0.0096M

 

Weight of test item taken: 0.0253 g

Volume made up: 250 mL

Aliquot taken for titration (A): 200 mL

Molecular weight of test item: 213.623

 

Concentration of test item (C) = (0.0253 * 1000) / (213.623 x 250 (mL)) = 0.0004737

 

Total No. of moles of test item in 200 mL solution taken for titration (N) = 0.0004737 x 200 / 1000 = 0.00009474

 

Max. and Min. Temp. during the period: 20.0 and 19.8 °C, respectively

 

 

Dissociation constant of the test item (mean)

Replication	Mean pKa
R1	4.61
R2	4.69
R3	4.66
Mean ± S.D.	4.65 ± 0.04

 

Conclusions:

The dissociation constant (pKa) of the substance at ca. 20 °C was determined to be 4.65 ± 0.04 (OPPTS 830.7370)

Reference 2

Endpoint:

dissociation constant

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-09-16 to 2009-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 112 (Dissociation Constants in Water)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bundesinstitut für Risikobewertung, Berlin, Germany

Dissociating properties:

yes

pKa:

4.63

Temp.:

20 °C

Verification of Spectrophotometer Performance

On all occasions of use, the instrument passed all internal calibration checks.

 

Preliminary test

The absorbance of the test item was measured by adding an aliquot of an aqueous solution of test item to buffers solutions with pH from <1.96 to pH >12.1. Results shown significant difference in the absorbance value measured in the pH range between 1.96 to 12.1. The time gap between adding an aliquot of an aqueous solution of test item and capture of the UV spectrum was 2 to 7 minutes. From the preliminary test result it was observed that the dissociation constant of the test item was to be 4.63 ± 0.17.

The test was used to determine the pH range and optimize the wavelength at which the dissociation constant of the test substance was determined.

 

Definitive Test

The absorbance of the test item was measured by adding an aliquot of an aqueous solution of the test item to buffers solutions with pH from <2.02 to pH >11.85. The results are presented in Table 1.

Results shown significant difference in the absorbance value of the test item measured in the pH range between 2.02 to 11.85. The time gap between adding an aliquot of an aqueous solution of the test item and capture of the UV spectrum was 2 to 7 minutes. The time measurements are presented in Table 2. Thus, it is concluded, the compound is undergoing dissociation as a function of change in pH. From the test result it was observed that the dissociation constant of the test item is 4.63 ± 0.14.

 

Table 1: Calculation of dissociation constant - definitive test

pH of the buffer only solution	pH of solution after addition of test item	abs @ 260 nm	mean	pKa
2.02	2.02	0.368	0.3690	N/C
		0.369		N/C
		0.370		N/C
2.65	2.65	0.361	0.3630	N/C
		0.363		N/C
		0.365		N/C
3.06	3.07	0.382	0.3777	N/C
		0.384		N/C
		0.367		N/C
3.71	3.72	0.321	0.3307	4.459
		0.342		4.902
		0.329		4.590
4.24	4.28	0.289	0.2787	4.658
		0.276		4.544
		0.271		4.503
4.59	4.63	0.250	0.2543	4.687
		0.250		4.687
		0.263		4.789
4.88	4.98	0.196	0.1860	4.602
		0.178		4.420
		0.184		4.485
5.31	5.42	0.169	0.1577	4.750
		0.151		4.451
		0.153		4.493
5.91	5.98	0.135	0.1373	4.414
		0.138		4.596
		0.139		4.644
6.48	6.56	0.132	0.1320	4.687
		0.132		4.687
		0.132		4.687
6.96	7.01	0.130	0.1303	4.656
		0.131		4.959
		0.130		4.656
7.41	7.46	0.125	0.1257	N/C
		0.127		N/C
		0.125		N/C
7.98	8.04	0.132	0.1300	N/C
		0.129		N/C
		0.129		N/C
8.41	8.44	0.124	0.1253	N/C
		0.126		N/C
		0.126		N/C
8.86	8.9	0.125	0.1253	N/C
		0.125		N/C
		0.126		N/C
9.54	9.54	0.125	0.1260	N/C
		0.126		N/C
		0.127		N/C
10.01	10.03	0.120	0.1207	N/C
		0.119		N/C
		0.123		N/C
10.54	10.58	0.113	0.1117	N/C
		0.112		N/C
		0.110		N/C
10.93	10.93	0.124	0.1223	N/C
		0.124		N/C
		0.119		N/C
11.49	11.49	0.131	0.1297	N/C
		0.130		N/C
		0.128		N/C
11.85	11.85	0.127	0.1277	N/C
		0.129		N/C
		0.127		N/C
AVERAGE				4.625
STANDARD DEVIATION				0.141
RSD%				3.055%

 

 

Table 2: Time duration between addition of test item and analysis

pH	Spiked time in hours	Analysis time in hours	Time gap in minutes
11.85	10.45	10.47	2.0
	10.45	10.49	4.0
	10.45	10.51	6.0
11.49	10.53	10.56	3.0
	10.53	10.59	6.0
	10.53	11.00	7.0
10.93	11.04	11.06	2.0
	11.04	11.08	4.0
	11.05	11.10	5.0
10.58	11.13	11.15	2.0
	11.13	11.18	5.0
	11.14	11.20	6.0
10.03	11.27	11.29	2.0
	11.27	11.31	4.0
	11.27	11.33	6.0
9.54	11.36	11.38	2.0
	11.37	11.40	3.0
	11.37	11.42	5.0
8.90	11.49	11.51	2.0
	11.50	11.53	3.0
	11.50	11.54	4.0
8.44	11.58	12.01	3.0
	11.58	12.03	5.0
	11.58	12.05	7.0
8.04	12.12	12.16	4.0
	12.12	12.17	5.0
	12.12	12.18	6.0
7.46	12.21	12.23	2.0
	12.21	12.24	3.0
	12.21	12.26	5.0
7.01	12.28	12.30	2.0
	12.31	12.34	3.0
	12.36	12.41	5.0
6.56	12.51	12.57	6.0
	12.51	12.58	7.0
	12.59	13.03	4.0
5.98	13.54	13.56	2.0
	13.58	14.01	3.0
	13.58	14.03	5.0
5.42	14.10	14.13	3.0
	14.10	14.15	5.0
	14.10	14.16	6.0
4.98	14.19	14.21	2.0
	14.19	14.22	3.0
	14.19	14.24	5.0
4.63	14.25	14.28	3.0
	14.25	14.30	5.0
	14.25	14.32	7.0
4.28	14.34	14.36	2.0
	14.34	14.38	4.0
	14.34	14.40	6.0
3.72	14.42	14.44	2.0
	14.45	14.48	3.0
	14.45	14.52	7.0
3.07	14.53	14.56	3.0
	14.53	14.58	5.0
	14.53	15.00	7.0
2.65	15.06	15.10	4.0
	15.06	15.09	3.0
	15.06	15.11	5.0
2.02	15.20	15.24	4.0
	15.20	15.25	5.0
	15.20	15.27	7.0

 

 

Conclusions

The dissociation constant of the test item is 4.63 ± 0.14 when conducting the measurements within 2 to 7 minutes after the addition of test item in to the respective buffer solutions. This dissociation constant value compares favourably with the dissociation constant for technical grade in water, which was determined to be 4.65 ± 0.04 at 20 ± 1 °C (see study M-539985-02-1).

Conclusions:

The dissociation constant (pKa) of the test item at ca. 20 °C was determined to be 4.63 ± 0.14 (OECD Guideline 112, Spectrophotometric method)

Description of key information

4.65 at 20 °C (EPA OPPTS 830.7370, titration method)

Key value for chemical safety assessment

pKa at 20°C:

4.65

Additional information

In another study performed using spectrophotometric method (OECD Guideline 112) a pKa of 4.63 at 20 °C was measured starting from a soluble concentrate of the test item.