2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Dissociation constant

Link to relevant study record(s)

Reference

Endpoint:

dissociation constant

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-01 to 2015-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD TG 112 (1981) with deviations regarding the solubility of the test item but without any impact on the validity of data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 112 (Dissociation Constants in Water)

Deviations:

yes

Remarks:

Adaptation of OECD Method 112: increasing the sample solution concentration above that of half the water solubility of the test item and also the proportion of dimethyl sulfoxide co-solvent content in sample solutions. No impact on the validity of data.

GLP compliance:

no

Remarks:

No formal claim. Study conducted in a facility operating to GLP.

Dissociating properties:

no

No.:

#1

pKa:

6.7

Temp.:

25 °C

Results

On extrapolation of the Yasuda-Shedlovsky plot to the reciprocal of the dielectric constant of water (1/78.36 = 1.276 x 10e-2), a dissociation constant (pKa) value of 6.70 was obtained for the test item in pure water.

On titration of the basic functional group evaluation sample solutions, no dissociation constants were evident. As a further confirmation of this, duplicate sample blank solutions, with no test item present, were also titrated. This indicated that the test item sample solution titration curves were not significantly different from the sample blank titration curves; therefore supporting the absence of any basic functional groups with a dissociation constant above 2.

Spectrophotometric evaluation of aqueous solutions of the test item resulted in essentially identical spectra, both with respect to absorbance wavelength and intensity at pH 2 and pH 4.

Test temperature: 25.0 ± 0.5 °C

Discussion

On assessment of the functional groups present, two potential dissociating functionalities were identified. These were the weakly acidic donation of a proton by the cyclic amide functional group and a weakly basic acceptance of a proton by the triazole functional group. As a consequence of this, titrations of test item solutions were performed using both potassium hydroxide solution and hydrochloric acid solution.

On titration with potassium hydroxide solution, the dissociation constant of a weakly acidic functional group was detected and quantified. On titration with hydrochloric acid, no significant differences were identified between the sample solutions and that of blank solutions with the test item absent. This allowed a conclusion that there were no basic dissociating groups with a dissociation constant greater than 2, as the limit value for the titration method is the negative logarithm of the molar concentration of the test item in solution.

The solubility of the test item was insufficient to allow dissolution of the test item in water at a concentration of 0.01 M, considered optimal for the investigation of dissociation constants, and also critical to allow evaluation of the basic functional group down to a possible dissociation constant of 2. As a result of this, it was necessary to adapt the OECD Method 112 procedure by increasing the sample solution concentration above that of half the water solubility of the test item and the proportion of dimethyl sulfoxide co-solvent content in the sample solutions to compensate for this. This was concluded not to impact on the validity of the resulting data, as through the use of a series of test item solutions with differing water to dimethyl sulfoxide compositional ratios, it was possible to obtain a value for the dissociation constant in pure water by the Yasuda-Shedlovsky extrapolation method.

The nominal mass of test item taken for each sample solution generated a concentration of 0.010 M at a sample volume of 50.0 mL, i.e. half titration.

On titration of the basic functional group evaluation sample solutions, no dissociation constants were evident. As a further confirmation of this, duplicate sample blank solutions, with no test item present, were also titrated. This indicated that the test item sample solution titration curves were not significantly different from the sample blank titration curves; therefore supporting the absence of any basic functional groups with a dissociation constant above 2. As sample solution pH’s were observed to be slightly variable during titrations, possibly in the absence of any buffering capacity and working at the extreme limit of the methodology, evaluation of the test item by a spectrophotometric method was also performed as a precautionary step.

Spectrophotometric evaluation of aqueous solutions of the test item resulted in essentially identical spectra, both with respect to absorbance wavelength and intensity at pH 2 and pH 4. Based on the proposed weakly basic functional group being acceptance of a hydrogen by the major chromophore present within the structure, i.e. the triazole ring, any change in ionization state would be expected to have a significant influence on the absorbance properties of the test item in the ultraviolet region. Comparison was completed between pH 2 and pH 4 sample solutions as evaluation was limited to pH 2 as this addressed any possible ionization across the environmentally relevant pH range, and based on the weakly acidic dissociation constant quantified, pH 4 ensured a valid unionized test item form for comparison purposes.

Conclusions:

In a valid, reliable and conclusive study, the test item was evaluated for functional groups with dissociation constants within the range 2 to 12, which would allow ionization of the test item within the environmentally relevant pH range of 4 to 10. The value for the weakly acidic dissociation constant of the amide functional group in pure water was determined to be 6.70 at 25 °C, by a Yasuda-Shedlovsky extrapolation method.

Executive summary:

The study was performed according to OECD 112 to determine the dissociation constants of the test item in water. By titrating a series of test item solutions of differing water to dimethyl sulfoxide compositional ratios with potassium hydroxide solution, it was possible to obtain a value for the weakly acidic dissociation constant of the amide functional group in pure water. The dissociation constant of this functional group in pure water was determined to be 6.70 at 25 °C, by a Yasuda-Shedlovsky extrapolation method. By titrating similar solutions with hydrochloric acid solution and spectrophotometric evaluation, no basic functional groups were identified with a dissociation constant greater than 2.

Description of key information

In a valid, reliable and conclusive study according to OECD TG 112 (1981), the test substance was evaluated for functional groups with dissociation constants within the range 2 to 12, which would allow ionization of the test item within the environmentally relevant pH range of 4 to 10. The value for the weakly acidic dissociation constant of the amide functional group in pure water was determined to be 6.70 at 25 °C, by a Yasuda-Shedlovsky extrapolation method.

Key value for chemical safety assessment

pKa at 20°C:

6.7