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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2014 to October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
Aliquots were taken for analysis without any further treatment or dilution.

Sample preparation was performed either in darkness or under a non-actinic light source.
Buffers:
Buffer solution

pH 4
Citric acid 0.06 mol dm-3
Sodium chloride 0.04 mol dm-3
Sodium hydroxide 0.07 mol dm-3

pH 7
Disodium hydrogen orthophosphate (anhydrous) 0.03 mol dm-3
Potassium dihydrogen orthophosphate 0.02 mol dm-3
Sodium chloride 0.02 mol dm-3

pH9
Disodium tetraborate 0.01 mol dm-3
Sodium chloride 0.02 mol dm-3

These solutions were subjected to boiling, ultrasonication, vacuum filtration through 0.2 µm filters, purging with nitrogen and storage under nitrogen headspaces to minimize dissolved oxygen content before use.
Duration:
120 h
pH:
4
Temp.:
50
Duration:
288 h
pH:
4
Temp.:
50
Duration:
120 h
pH:
7
Temp.:
50
Duration:
64 h
pH:
7
Temp.:
40
Duration:
43 h
pH:
9
Temp.:
25
Number of replicates:
For the preliminary tests, the sample solutions were split into duplicate vessels for each sampling interval.

For Tier 2 tests, duplicate aliquots of sample solutions were taken in HPLC vials.
Preliminary study:
The results were inconclusive in that no loss in test item response was detected. Rather, an apparent increase in parent test item concentration was observed over the time period of the test. Therefore, the preliminary test was repeated (pH 4, Preliminary Test 2) incorporating a surrogate standard material (coumarin) to allow normalization of test item responses to a common standard and thus ensure accurate and valid comparison between inter-day analyses.

The results of the repeat test determination remained inconclusive in that again no loss in test item response was detected but an apparent and variable increase in test item concentration was observed over the time period of the test. The laboratory procedure was therefore adapted for further testing. For pH 4, Preliminary Test 3, the initial saturation concentration loading rate was decreased significantly to reduce the suspected initial extraction of soluble impurities into the sample solution. However, the confounding peak at approximately 2.6 minutes was later attributed to a transformation product of the parent test item.

The results indicated that the parent test item concentration in the sample solutions remained relatively constant over the 288 hour incubation period, indicating the solutions may have reached a state of equilibrium with the transformation product.

The results confirmed instability with respect to transformation, as greater than 10% reduction in the initial test item concentration was determined during the 120 hour incubation period at 50 deg. C. Tier 2 testing was therefore initiated at pH 7.
Test performance:
The results confirmed instability with respect to transformation, as more than 10% reduction in the initial test item concentration was determined during the 120 hour incubation period at 50 deg. C. Tier 2 testing was therefore initiated at pH 7.
Transformation products:
not specified
pH:
4
Temp.:
50 °C
Remarks on result:
other: Preliminary Test 1at 50 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 99.3 %.
Remarks:
No significant further transformation occurred.
pH:
4
Temp.:
50 °C
Remarks on result:
other: Preliminary Test 2 at 50 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 99.2 %.
Remarks:
No significant further transformation occurred.
pH:
4
Temp.:
50 °C
Remarks on result:
other: Preliminary Test 3 at 50 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 99.0 %.
Remarks:
No significant further transformation occurred.
pH:
7
Temp.:
50 °C
Remarks on result:
other: Preliminary Test at 50 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 75.4 %.
Remarks:
Continued concentration decrease, approximating second order kinetics.
pH:
7
Temp.:
38 °C
Remarks on result:
other: Tier 2 Test at 38 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 67.6 %.
Remarks:
Continued concentration decrease, approximating second order kinetics.
pH:
9
Temp.:
25 °C
Remarks on result:
other: Tier 2 Test at 25 °C. Approximate Degree of Transformation Observed at the Initial Timepoint Analysis: 95.4 %.
Remarks:
Continued concentration decrease, approximating second order kinetics.
Details on results:
The results indicate that for pH 4, approximately 99% of the test item rapidly transformed prior to the initial analysis, during the sample preparation procedure at ambient laboratory temperature. The remaining 1% parent test item remained approximately stable in an equilibrium state during incubation at 50 deg. C, for all three preliminary tests performed.

At pH 7, again a majority of the test item (in the range 68 to 75%) rapidly transformed prior to the initial analysis, during the sample preparation procedure at ambient laboratory temperature. The remaining parent test item then continued to transform during incubation, approximating second order kinetics.

At pH 9, approximately 95% of the test item rapidly transformed prior to the initial analysis, during the sample preparation procedure at ambient laboratory temperature. During incubation at 25 deg. C, the remaining parent test item continued to transform, approximating second order kinetics.

Although the test item rapidly transformed at environmental relevant pHs (pH 4, 7 and 9) and temperatures tested, the observed kinetics did not fit the pseudo-first order model on which the regulatory guidelines are based. For first order kinetics, a plot of the logarithm of concentration versus time equates to a linear relationship, from which the rate constant and half-life can be calculated from the slope. For second order kinetics, the plot of the logarithm of concentration versus time equates to a non-linear relationship. In the absence of a constant slope, the rate constant and half-life cannot be determined since these two values are constantly changing as the reaction progresses. Therefore, quantification of the rate of transformation was considered to be beyond the scope of the referenced testing guidelines. 

Based on structural information supplied by the Sponsor and with reference to available literature, the almost immediate decrease in parent test item concentration was attributed to the tautomerism of the thione functional groups to thiol functional groups, in aqueous solution. This transformation would present second order kinetics and an equilibrium between the two tautomer forms, consistent with the data determined experimentally within the study.

The various tautomers of both the dimer and monomer may or may not respond consistently to typical analytical techniques. Various spectroscopic techniques may preferentially detect one species over another, or they may detect the equilibrium-weighted average of the species present. In other cases, a method may not differentiate among the species at all. For example, mass spectroscopy (MS) will not differentiate between the tautomers as they have equal mass. Furthermore, the disulfide bond is expected to cleave during MS analysis, so MS of the dimer will appear as the monomeric form in a mass spectrum.

Analytical monitoring of a sample solution of test item directly fortified in pH 4 buffer solution using 1% v/v methanol co-solvent, at ambient laboratory temperature, eliminated the delay in analysis caused by the previously employed sample solution preparation procedure and thus captured kinetics data beginning at a much higher initial concentration of the parent test item. Due to the potential catalytic effect of the co solvent, however, no quantification was performed from this additional test. The results of this test however suggest that only the latter stages of transformation were observed in aqueous solutions free from co-solvent. Without a co-solvent at pH 4, transformation appeared to have reached equilibrium prior to the initial analysis and thus analytical data may represent only the final stable state. At pH 7 and pH 9, any transformation rate determined from the analytical data likely would be significantly underestimated, as the data in these tests appear to represent only the final, decelerating stages of the reaction.

Validity criteria fulfilled:
yes
Conclusions:
Although the observed transformation was not considered to originate from true hydrolysis, i.e. chemical breakdown due to reaction with water, critically it has highlighted a rapid transformation of the test item in aqueous solution.
Executive summary:

In an OECD 111 study, conducted according to GLP, transformation of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was observed atenvironmental relevant pHs (pH 4, 7 and 9), however, transformation was not considered to originate from true hydrolysis ,i.e. chemical breakdown due to reaction with water.Significant, extremely rapid transformation occurred prior to analysis.5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is expected to undergo non-hydrolytic transformations.

Two reaction pathways must be considered in any experiments involving 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione:

• Tautomerization; and

• Disulfide exchange.

Tautomerization should rapidly produce an equilibrium mixture of thione and thiol forms of the test substance upon exposure to a protic solvent (such as water). The position of the equilibrium will depend on temperature, solvent, and pH of the solution.

Disulfide exchange, involving the cleavage and reformation of the disulfide bridge, may be slower and the rate will depend on exposure to heat, light, and reducing or oxidizing species. Both the dimer and monomer present during this dynamic exchange will undergo tautomerization.

Description of key information

Key value for chemical safety assessment

Additional information

Key Study:

In an OECD 111 study, conducted according to GLP, transformation of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was observed at environmental relevant pHs (pH 4, 7 and 9), however, transformation was not considered to originate from true hydrolysis ,i.e. chemical breakdown due to reaction with water. Significant, extremely rapid transformation occurred prior to analysis.5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is expected to undergo non-hydrolytic transformations (Envigo Research Limited, 2016a).

Two reaction pathways must be considered in any experiments involving 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione:

• Tautomerization; and

• Disulfide exchange.

Tautomerization should rapidly produce an equilibrium mixture of thione and thiol forms of the test substance upon exposure to a protic solvent (such as water). The position of the equilibrium will depend on temperature, solvent, and pH of the solution.

Disulfide exchange, involving the cleavage and reformation of the disulfide bridge, may be slower and the rate will depend on exposure to heat, light, and reducing or oxidizing species. Both the dimer and monomer present during this dynamic exchange will undergo tautomerization.