Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Dissociation constant

Link to relevant study record(s)

Reference

Endpoint:

dissociation constant

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jun 2012 to 05 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 112 (Dissociation Constants in Water)

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Dissociating properties:

no

Results preliminary screening

A consistent spectral profile over the wavelength range of approximately 320 nm to 225 nm was observed for the neutral and basic samples, supportive of a common unionised species. For the basic condition, the increase in absorbance below 225 nm was attributed to the reduced optical transparency of the sodium hydroxide present in the sample matrix as the wavelength decreased.

However, under acidic conditions, a significant shift in the spectrum profile was observed, indicating possible ionisation of the test item. This change in absorbance profile was confirmed through the analysis of a replicate acidic sample (pH 1.89).

Experimental procedures therefore continued with the investigation of the acidic pH region in order to quantify the dissociation constant and to assess its relevance to solubility and partitioning characteristics of the test item in the environment.

 

Results Investigative Work Employing a 1 % v/v Methanol co-Solvent

Initial investigative work in an attempt to quantify the apparent acidic dissociation constant was performed within the constraints of the method guideline, in that the sample concentration was limited to less than half the water solubility of the test item and the organic co-solvent content was restricted to 1 % v/v.

The spectra were problematic for a number of reasons. Absorbance was limited (even when using a 5 cm pathlength), spectral profiles were inconsistent, and most critically, no obvious pH dependent trend. was observed. Further to this, the concentration of hydrochloric acid required to obtain estimated pH’s required to achieve a completely ionised form of the test item were not optically transparent over the region of interest, such that no absorbance data was available at either a nominal pH value of 1 or a nominal pH value of 0.

As it was evident that conclusive data could not be obtained within the constraints of the method guideline, testing was therefore continued employing a higher sample solution concentration and an increased methanol co-solvent content to compensate for this. As the dissociation constant of a substance may be influenced by the identity and percentage of cosolvent present, the next stage of testing aimed to investigate the influence of the co-solvent content on the spectral profile.

 

Results Investigative Work Assessing the Influence of the Proportion of the Methanol

co-Solvent

A systematic decrease in absorbance at 254 nm was seen with a decrease in the proportion of methanol present in the sample solution; however this may be partially attributable to a slight drift in pH, with values of 1.15, 1.53 and 1.61 being recorded for the samples. As the cosolvent content was identified as having a potential influence on the degree of ionisation and/or spectral profile of the test item, testing was continued employing a uniform 20% v/v methanol co-solvent content, the influence of which is assessed within the discussion.

 

Results Definitive Test employing a 20% v/v Methanol co-Solvent

Data for the sample solution pH and absorbance at 250 nm for each of the definitive test sample solutions is presented in Table 1.

 

Discussion

Preliminary testing indicated the potential presence of a dissociation constant for the test item under acidic conditions. However, due to the limited solubility of the test item in water and the method guideline constraint of working at a maximum concentration of half the saturation concentration in water, resulting spectra from samples generated within the constraints of the method guideline were concluded to be too variable and thus unreliable for the purpose of quantifying this dissociation constant.

The definitive test subsequently required an adaptation of the procedure, increasing both the nominal sample solution concentration to give significant absorbance and also an increase in the proportion of organic co-solvent to establish and maintain dissolution of the test item.

Assessing the definitive test data generated graphically, a significant decrease in the absorbance of the test item at 250 nm was observed below approximately pH 2.0. However the data did not cover a pH range which allowed evaluation of the point of inflection (which would approximate to the dissociation constant or pKa value) or the absorbance properties of the completely ionised form of the test item.

It can however be confidently concluded that the pKa value for this weakly basic dissociation constant was below 1.0. Due to the relationship between pKa, pH and the degree of ionization, the shift from 95% of one species to 95% of the second species occurs over a pH range of approximately 2.6 pH units. Therefore it was estimated that the dissociation constant was approximately 0.7 (i.e. 1.3 units below pH 2.0, where a noticeable shift in relative absorbance was first observed).

Finally the influence of the increased proportion of methanol in the definitive test sample solutions must be addressed. The proportion of methanol in the sample solutions had previously been demonstrated to have an influence on the spectral profile of the test item and therefore possibly the degree of ionisation. This originates from a differing solvating ability and/or dielectric constant for methanol compared to that of water.

With respect to literature data examining the dissociation constants of a number of amines (n = 30) in water and in methanol, even on comparison between 100 % aqueous and 100 % methanol matrices, all pKa values remained within 2 units for all substances and critically, for 39 of the 40 substances, a higher pKa value was obtained in methanol than in water. For the single sample where the pKa value decreased in the presence of methanol, the deviation was only 0.10 units.

Reference: Acidity in methanol–water. Rived, Fernando; Canals, Immaculada; Bosch, Elisabeth; Rosés, Martí. Analytica Chimica Acta vol. 439 issue 2 July 25, 2001. p. 315-333.

 

Therefore the overall conclusion was that the test item does not present any environmentally relevant dissociation constant values (greater than 2 for bases and less than 12 for acids). This was since irrespective of the need to increase the proportion of methanol co-solvent in the sample preparation procedure, from literature references it can be confirmed that methanol has a tendency to promote ionisation of bases, such that the experimental value of approximately 0.7 may remain a systematic overestimation.

Such a conclusion was further supported by computer estimation of dissociation constants for the test item. Using SPARC (version 4.6, October 2011, release reference w4.6.1691- s4.6.1687), no basic dissociation constant greater than -0.2 was detected. ACD/Labs (Algorithm Version: v12.1.0.50374) gave a predicted basic dissociation constant value of 0.90 ± 0.4 units for the amide functional group.

Data generated was normalised to a test temperature of a 25 °C through the use of an automatic temperature compensation probe for all solution pH readings.

 

Conclusion

This study confirmed the absence of any dissociation constant for the test substance in the range greater than 2 and less than 12 (i.e. within a range of environmental relevance).

Due to the low water solubility of the test item and hence limited absorbance for sample concentrations within the constraints of the method guideline, it was necessary to adapt the procedure by increasing both the sample solution concentration and the proportion of methanol co-solvent content in the sample solutions to compensate for this. This was concluded not to impact on the validity of the study as it was confirmed from literature data that methanol has limited influence on the observed dissociation constant values for amines and typically promotes ionisation.

The dissociation constant of the test item in water has been estimated using these modified conditions to be approximately 0.7 at 25 °C and has been attributed to that of a very weak basic functionality, most likely the acceptance of a proton by the amide functional group.

It must be noted that the value of approximately 0.7 may still overestimate the true value of the dissociation constant.

 

Table 1. Definite test sample solutions pHs and observed absorbance at 250 nm

Nominal Sample Solution pH	Actual Sample Solution pH	Absorbance at 250 nm
4.0	4.75	0.325
3.0	3.14	0.313
2.5	2.62	0.346
2.0	2.09	0.336
1.0	1.13	0.269
0.0	0.18	0.190

 

Conclusions:

The study confirmed the absence of any dissociation constant for the test substance in the range greater than 2 and less than 12 (i.e. within a range of environmental relevance).

Executive summary:

The study was performed to determine the dissociation constant of the test item in water. The method employed was based on OECD TG 112 and followed GLP principles.

This study confirmed the absence of any dissociation constant for the test substance in the range greater than 2 and less than 12 (i.e. within a range of environmental relevance). Due to the low water solubility of the test item and hence limited absorbance for sample concentrations within the constraints of the method guideline, it was necessary to adapt the procedure by increasing both the sample solution concentration and the proportion of methanol co-solvent content in the sample solutions to compensate for this. This was concluded not to impact on the validity of the study as it was confirmed from literature data that methanol has limited influence on the observed dissociation constant values for amines and typically promotes ionisation.

The dissociation constant of the test item in water has been estimated using these modified conditions to be approximately 0.7 at 25 °C and has been attributed to that of a very weak basic functionality, most likely the acceptance of a proton by the amide functional group. It must be noted that the value of approximately 0.7 may still overestimate the true value of the dissociation constant.

Description of key information

Dissociation constant: The study confirmed the absence of any dissociation constant for the test substance in the range greater than 2 and less than 12 (i.e. within a range of environmental relevance), OECD TG 112, spectrophotometric filtration, O'Connor 2012