1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Dissociation constant

Description of key information

Due to the water solubility of the substance, the titration method could not show any significant difference in absorption spectra at extreme pH to allow determination by the spectrophotometric method. Therefore the pKa value could not be quantitatively determined. An LC-MS was completed to determine whether ZMB2 remained as a single molecule or whether it separated into separate zinc and thiomidazole ions. The qualitative analysis of the results indicate that ZMB2 does remain as a single molecule, at least to some degree (Harlan Laboratories, 2013).

Additional information

When considering the results of the investigations carried out to support the read-across from 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) to 2H-Benzimidazole-2-thione, 1,3-dihydro-4(or 5)-methyl-, zinc salt (2:1) (ZMB2) (LANXESS, 2018), the results of the dissociation constant study conducted with this substance must be reconsidered in light of new information.  The overall conclusion in LANXESS (2018) is that ZMB2 exhibits its salt characteristic in aqueous solution, i.e., dissolving in water to MB2 and zinc ions in a matter of seconds.  For full details of the investigations conducted relating to dissociation constant, refer to Section 13.2 of IUCLID.

Dissociation Constant Study (GLP, Harlan Laboratories, 2013)

According to the key study no determination of the dissociation constants in water was possible according to OECD 112, due to low solubility of the test item in aqueous media and the lack of significant differences in the ultraviolet/visible spectrum of the test item at different pHs.

In this study, the only indication suggesting that ZMB2 “is present in solution and during chromatography as the entire molecule rather than splitting into zinc and ligand ions” is discussed in the non-GLP section of the study report.  The study then identifies that UV/VIS absorption does not provide definitive evidence in itself whether or not the test item remains as a single molecule or separates into zinc and ligand, and further investigations applying MS techniques were undertaken.  

First, “eluent fractions and appropriate blanks consisting of mobile phase were analysed using inductively coupled plasma – mass spectrometry (ICP-MS) to determine whether any zinc was present in the peak observed in the HPLC analysis. Unfortunately these analyses proved inconclusive due to poor detection limits and comparatively large zinc signals in the blank solutions”.  This finding is supported by the mass spectrometry results also in LANXESS (2018), where also cross-contamination (carryover) of zinc took place in the MS trace acquired for the MB2 sample, i.e. considerable MS signals suggesting the presence of ZMB2 were detected although a solution with MB2 was analyzed.

Then a “further assessment was performed using HPLC – mass spectrometry”. The HPLC-MS system used with both an UV absorption detector and a mass spectrometry detector which is suggested to have detected ZMB2 as the entire molecule is not further described in the report  and no information on the eluent system is given.  The MS trace acquired in this non-GLP part of this Harlan study is very weak in that not even the isotopic intensity pattern for a zinc compound is sufficiently recognizable (here: [ZMB2 + H]+ expected to result in the isotopic pattern with m/z = 391, 393, 395), while the peak for m/z= 165 equaling [MB2 + H]+ is overwhelmingly strong.

It is also highly noteworthy that the HPLC-MS data recorded in this study are very similar to the ones measured on ZMB2 and MB2, described in LANXESS (2018), where these signals were shown not to originate from undissociated ZMB2.  As discussed above, MS results are not a reliable indicator of whether ZMB2 is intact or dissociated in the test item.

No further analysis is available in any of the physicochemical reports used for the REACH dossier for ZMB2 submitted in 2013 which would support the assumption that ZMB2 is present in solution and during chromatography in undissociated form rather than dissociating into zinc and ligand ions.

The overall mechanistic conclusion for this endpoint is therefore that ZMB2 exhibits its salt characteristic in aqueous solution, i.e., dissociating into water to MB2 and zinc ions in a matter of seconds.  The MB2 anion, as a moderate base, reacts with water to raise the pH, the resulting increased OH- concentration reacts with the free zinc ions to a considerable extent to form insoluble Zn(OH)2, which precipitates; and as a result, observed zinc in aqueous solution is significantly below what is expected from mass balance.