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Environmental fate & pathways

Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
The test item was dissolved to the maximum reachable solubility (saturated solution) at neutral pH-value
Principles of method if other than guideline:
As the test item is only soluble in an unquantifiable amount a modified test design was carried out as follows:
The test item was dissolved to the maximum reachable solubility (saturated solution) at a preferably neutral pH-value. After filtration of undissolved particles 1H-NMR spectra were recorded. The filtered solution was then acidified with deuterated acetic acid to decrease the pH to approx. 3. The test solution was then repeatedly measured by 1H-NMR after 3 and 29 minutes. The tests were carried out at room temperature (20 – 25 °C).
After measurement the chemical shifts from MBT and ZMBT at neutral and acidic pH values were analyzed. Due to the low solubility of ZMBT a quantification via the mass content of ZMBT was not possible. Therefore the analysis was performed in a semi-quantitative manner.

The pH determination was done with pH indicator Strips (1-14). The determination with a glass electrode was not possible due to the small amount of test solution.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- test item was dissolved to the maximum reachable solubility (saturated solution)
- filtration of undissolved particles
- 1H-NMR spectrum recorded after sample preparation.
- acidification to pH 3 and immediate record of 1H-NMR spectrum, approx. 9 minutes after first measurement
- two more measurements after 3 and 29 minutes
- record of 1H-NMR spectrum of the reference substance of the assumed hydrolysis product 2-Marcaptobenzothiazole immediately after sample preparation
- acidification to pH 3 and immediate record of 1H-NMR spectrum

Buffers:
The measurements were performed in NMR grade D2O. For acidification of the stock solution deuterated acetic acid was employed.
Details on test conditions:
1H-NMR spectroscopy:
The test item ZMBT was weighed to the nearest 0.01 mg (167.45 mg) and dissolved in 11 mL of D2O. A vein of 3-(Trimethylsilyl)-propionic acid-d4-Na salt was added as a standard of the chemical shift.
The solution was shaken for approx. 5 minutes. The insoluble parts were filtered off and a clear solution was obtained. The pH value of this solution was determined using pH-stripes.
The pH value was 7-8. 1 mL of this solution was abstracted and a 1H-NMR spectrum was recorded.
The rest of the stock solution was set to pH 3 using deuterated acetic acid. Immediately another 1H-NMR measurement was carried out. The time difference between both measurements (neutral pH and pH 3) was 9 minutes.
Two more measurements were carried out after 3 and 29 minutes.

In addition a reference substance of the assumed hydrolysis product 2-Mercaptobenzothiazole was dissolved and measured in analogy to ZMBT.
111.69 mg of the reference substance MBT were weighed to 5 mL D2O. A trace of 3-(Trimethylsilyl)-propionic acid-d4-Na salt was added as a reference.
The solution was shaken for approx. 5 minutes. The insoluble parts were filtered off and a clear solution was obtained.
The pH value of this solution was determined using pH-stripes. The pH value was approx. 6. 1 mL of this solution was abstracted and a 1H-NMR spectrum was recorded.
The rest of the stock solution was set to pH 3 using deuterated acetic acid. Immediately another 1H-NMR spectrum was recorded.
Duration:
38 min
pH:
3
Temp.:
25 °C
Remarks:
as the test substance did not fully dissolve and precipitate a subsequent filtration step to remove undissolves particles was necessary, no initial concentration was determinable
Positive controls:
no
Negative controls:
no
Preliminary study:
As the test item is expected to be hydrolytically unstable at the relevant pH value a preliminary test (Tier 1) according to OECD TG 111 is not required.
Transformation products:
yes
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
ZMBT undergoes rapid hydrolysis. The 1H-NMR spectrum recorded immediately after acidification shows no trace of residual ZMBT but the fully formed MBT. The spectra of the hydrolyzed ZMBT and the reference substance MBT are identical at pH = 3.

The MBT spectra at pH = 3 and pH = 6 vary, as MBT exhibits a thioamide-iminethiol-tautomerism (cf. illustration below).
Key result
pH:
3
Temp.:
25 °C
Remarks on result:
other: The test item is completely hydrolytically degraded after a period of less than nine minutes.
Remarks:
Due to the low solubility of ZMBT a quantification via the mass content of ZMBT was not possible. Therefore the analysis was performed in a semi-quantitative manner.
pH:
7
Temp.:
25 °C
Remarks on result:
other: hydrolytically stable
Details on results:
With the first measurement of the test item solution at pH 7 - 8 the presence of ZMBT in the test solution could be approved.
The test item was dissolved to the maximum reachable solubility (saturated solution) at a preferably neutral pH-value. After filtration of undissolved particles 1H-NMR spectra were recorded and the presence of the test item could be confirmed. Additionally, no hydrolysis to MBT was detected as the obtained chemical shifts of the aromatic protons differ significant from the chemical shifts of the assumed hydrolysis product MBT at a comparable pH value.
The filtered solution was then acidified with deuterated acetic acid to decrease the pH to approx. 3. The test solution was then repeatedly measured by 1H-NMR. Differences in the chemical shifts of the aromatic protons between MBT and ZMBT were noticed. Also a difference in the chemical shifts of the aromatic protons of the possible hydrolysis product MBT was observed as this compound can be present in two isomeric forms depending on the pH of the solution (see box illustration: Figure 1, Ia and Ib).

It could be shown that the chemical shifts of the 1H-NMR spectra of the acidified ZMBT solutions perfectly fit to the chemicals shifts of the acidified MBT sample where MBT is present in isomeric form Ib. As no remaining signals derived from ZMBT after acidification were observed, it was concluded that all ZMBT used in the sample is immediately hydrolyzed under acidic conditions to MBT (complete but unquantifiable after filtration) in less than 9 minutes. The measurement was repeated after 3 and 29 minutes and no changes in the chemical shifts were observed, concluding that no further dissociation of the hydrolysed ZMBT sample took place.

Sterility test:

A plate count test according to prEN ISO 7218 (2005) was conducted at the end the hydrolysis tests with the test item stock solution.

The test solution was proved to be sterile.

Validity criteria fulfilled:
not specified
Conclusions:
The test item ZMBT is hydrolytically unstable at a pH value of approx. 3. The test item is completely hydrolytically degraded after a period of less than nine minutes.
Executive summary:

Due to the properties of the test item Zinc di(benzothiazol-2-yl) disulphide (ZMBT) (low solubility, hydrolysis) a hydrolysis study according to OECD Guidelines for Testing of Chemicals, Section 1 – Physical-Chemical Properties, OECD TG 111 (2004), Council Regulation (EC) No 440/2008, Guideline Part C – Methods for the Determination of Ecotoxicity, C.7. “Abiotic Degradation: Hydrolysis as a function of pH” could not be carried out.

Therefore the test design was adjusted to fit the test items properties.

Because the test item is expected to be hydrolytically unstable at the relevant pH value a preliminary test (Tier 1) according to OECD TG 111 is not required.

With the first measurement of the test item solution at pH 7 - 8 the presence of ZMBT in the test solution could be approved.

The test item was dissolved to the maximum reachable solubility (saturated solution) at a preferably neutral pH-value. After filtration of undissolved particles 1H-NMR spectra were recorded and the presence of the test item could be confirmed. Additionally, no hydrolysis to MBT was detected as the obtained chemical shifts of the aromatic protons differ significant from the chemical shifts of the assumed hydrolysis product MBT at a comparable pH value.

The filtered solution was then acidified with deuterated acetic acid to decrease the pH to approx. 3. The test solution was then repeatedly measured by 1H-NMR. Differences in the chemical shifts of the aromatic protons between MBT and ZMBT were noticed. Also a difference in the chemical shifts of the aromatic protons of the possible hydrolysis product MBT was observed as this compound can be present in two isomeric forms depending on the pH of the solution (see illustration above: Figure 1: Isomeric forms of MBT)

It could be shown that the chemical shifts of the 1H-NMR spectra of the acidified ZMBT solutions perfectly fit to the chemicals shifts of the acidified MBT sample where MBT is present in isomeric form Ib. As no remaining signals derived from ZMBT after acidification were observed, it was concluded that all ZMBT used in the sample is immediately hydrolyzed under acidic conditions to MBT (complete but unquantifiable after filtration) in less than 9 minutes. The measurement was repeated after 3 and 29 minutes and no changes in the chemical shifts were observed, concluding that no further dissociation of the hydrolysed ZMBT sample took place.

It can be summarized that the test item ZMBT is hydrolytically stable at a pH value of 7 -8. However, ZMBT is hydrolytically unstable at a pH value of approx. 3. At this pH value ZMBT is completely hydrolytically degraded to MBT after a period of less than nine minutes.

Description of key information

Due to the low solubility of ZMBT a hydrolysis study according to OECD 111 was not possible. The study design was adjusted to fit the test items properties.

The test item was dissolved to the maximum achievable concentration and the identity of the substance was determined by 1H-NMR before acidification (pH = 7 - 8) and after (pH = 3).

Because of the low solubility and consequential sample preparation the initial concentration of the test solution was not determinable.

It can be summarized that the test item ZMBT is hydrolytically stable at a pH value of 7 -8. However, ZMBT is hydrolytically unstable at a pH value of approx. 3. At this pH value ZMBT is completely hydrolytically degraded to MBT after a period of less than nine minutes.

Key value for chemical safety assessment

Additional information

Due to the properties of the test item Zinc di(benzothiazol-2 -yl) disulphide (ZMBT) (low solubility, hydrolysis) a hydrolysis study according to OECD Guidelines for Testing of Chemicals, Section 1 – Physical-Chemical Properties, OECD TG 111 (2004), Council Regulation (EC) No 440/2008, Guideline Part C – Methods for the Determination of Ecotoxicity, C.7. “Abiotic Degradation: Hydrolysis as a function of pH” could not be carried out.

Therefore the test design was adjusted to fit the test items properties.

Because the test item is expected to be hydrolytically unstable at the relevant pH value a preliminary test (Tier 1) according to OECD TG 111 is not required.

In a first measurement of the test item solution at pH 7 - 8 the presence of ZMBT in the test solution could be proved by 1H-NMR measurements. No hydrolysis to MBT was detected.

The filtered solution was then acidified with deuterated acetic acid to decrease the pH to approx. 3 and then repeatedly measured by 1H-NMR.

The first 1H-NMR spectrum (after approx. 9 minutes) exhibited already total degradation of ZMBT to a new set of signals accountable to MBT.

It could be shown that the chemical shifts of the 1H-NMR spectra of the acidified ZMBT solutions perfectly fit to the chemicals shifts of the acidified MBT sample where MBT is present in an isomeric form.

As no remaining signals derived from ZMBT after acidification were observed, it was concluded that all ZMBT used in the sample is immediately hydrolyzed under acidic conditions to MBT (complete but unquantifiable after filtration) in less than 9 minutes. The measurement was repeated after 3 and 29 minutes and no changes in the chemical shifts were observed, concluding that no further dissociation of the hydrolysed ZMBT sample took place.

In a supporting non-GLP study (Currenta 2019) rapid degradation to MBT following a short term extraction with 3% acetic acid could be demonstrated qualitatively by HPTLC, wheras no degradation after a short term extraction with water was indicated.

In a supporting study (Currenta 2020) the hydrolytical stability of ZMBT for the purpose of the completion of genotoxicity studies (OECD 476 In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes, OECD 487 In vitro Micronucleus Assay) was investigated, where the laboratory employed an extended sample preparation, i.e. dissolution of the sample ZMBT in DMSO and consequent heating (50 °C) and sonication for complete dissolution.

It can be summarized that ZMBT is hydrolytically stable in DMSO under neutral conditions over the course of 24 h as well as during sample preparation under heating (50 °C) and sonication conditions for 90 minutes.

ZMBT is an organic complex with where the relation of Zn to Sulfur bridge is considered to be ionic.

The dissolution of MBT determines the hazard profile of ZMBT aqueous solution; and hence the environmental fate and behavior of MBT is used as read-across approach to support the risk assessment of ZMBT.

The read-across is justified as ZMBT is the mainly ionic form of MBT.