N-cyclohexylbenzothiazole-2-sulfenamide

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-08-30 - 2022-12-XX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken at the beginning of the study and at different time points over the course of the tests at different pH and temperatures. For determination of the first test point 100 µL of the freshly prepared hydrolysis test solution were immediately transferred into a sample vial and diluted with 900 µL acetonitrile.
Aliquots of the test solution were directly filled into brown glass sample vials to obtain individual samples for each test point. Preparation was carried out under nitrogen as flushing gas to avoid oxygen.
The vials were closed and stored at 12 °C in a temperature-controlled water bath. The temperature was visually checked during the experiment using a digital precision thermometer.
At each subsequent test point one test sample was removed from the water bath. An aliquot of the hydrolysis test solution was transferred into a new sample vial and diluted with acetonitrile as described above.

Buffers:

Buffer pH 9: Na2B4O7/HCl

Details on test conditions:

TEST SYSTEM

- Type, material and volume of test flasks, other equipment used: 100 mL measuring flasks
- Sterility: According to OECD 111 Guideline sterility test was conducted at the end of > 90 % hydrolysis for each hydrolysis test solution. No microbes (colonies) were found. Therefore, biotic degradation can be excluded.
- Measures to exclude oxygen: Preparation of the test samples was carried out under nitrogen as flushing gas to avoid oxygen.

TEST MEDIUM

A stock solution of the test item was prepared by weighing of 10.1 mg of the test item in a 100 mL measuring flask. The flask was then filled up to the mark with acetonitrile, which when adjusted for purity of the test item, resulted in a concentration of 99.384 mg/L.
10 mL of the test item stock solution was diluted to 100 mL with acetonitrile to obtain a diluted stock solution with a test item concentration of 9.9384 mg/L.
1 mL of the diluted stock solution was mixed and diluted to 100 mL with either buffer solution (for pH 9) or demineralized water (for pH 4 and 7). For the latter the pH was adjusted with formic acid and/or sodium hydroxide.

- Kind and purity of water: Millipore water, Milli-Q-H2O ELGA
- Identity and concentration of co-solvent: Acetonitril (1% vol.)

Duration:

119.8 h

pH:

4

Temp.:

12 °C

Initial conc. measured:

82 µg/L

Duration:

142.2 h

pH:

7

Temp.:

12 °C

Initial conc. measured:

88 µg/L

Remarks:

1st determination

Duration:

144.2 h

pH:

7

Temp.:

12 °C

Initial conc. measured:

90 µg/L

Remarks:

2nd determination

Duration:

215.5 h

pH:

9

Temp.:

12 °C

Initial conc. measured:

84 µg/L

Duration:

44.8 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

84 µg/L

Duration:

48.3 h

pH:

7

Temp.:

30 °C

Initial conc. measured:

85 µg/L

Number of replicates:

For pH 7 at 12 °C the test was conducted twice.

Positive controls:

no

Negative controls:

no

Preliminary study:

No preliminary study was conducted, since it was already known from available data (e.g. Monsanto 1984) that the substance will undergo hydrolysis. Therefore, the main test at different pH values and temperatures was conducted directly.

Transformation products:

yes

Remarks:

The study was conducted with the aim to describe the hydrolysis kinetics of the substance as well as to clarify the degradation products of N-cyclohexylbenzothiazole-2-sulphenamide, which are cyclohexylamine, benzothiazole and 2-mercaptobenzothiazole.

No.:

#1

No.:

#2

No.:

#3

Details on hydrolysis and appearance of transformation product(s):

- Formation and decline of each transformation product during test:
After total hydrolysis of the parent compound the degradation products have been identified and quantified.

pH:

7

Temp.:

30 °C

Hydrolysis rate constant:

0 s-1

DT50:

13.3 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0 s-1

DT50:

13.4 h

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

12 °C

Hydrolysis rate constant:

0 s-1

DT50:

76.7 h

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

12 °C

Hydrolysis rate constant:

0 s-1

DT50:

56.4 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 2nd determination

pH:

7

Temp.:

12 °C

Hydrolysis rate constant:

0 s-1

DT50:

48.1 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: 1st determination

pH:

4

Temp.:

12 °C

Hydrolysis rate constant:

0 s-1

DT50:

26.1 h

Type:

(pseudo-)first order (= half-life)

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
For all pH values the transformation products were determined after full hydrolysis of the test item. Quantification was therefore compared to the theoretically expected concentrations if CBS is degraded completely. Based on the applied initial test item concentration, the molar concentrations of the parent substance CBS in the test solution and of the hydrolysis products in the final test solution were calculated. According to the constitution of CBS and the stoichiometry one mole of CBS will hydrolyze to one mole of either Benzothiazole (BT) or 2-Mercaptobenzothiazole (MBT) or a combination of both with corresponding shares of one mole. Therefore, the sum of the molar concentrations of BT and MBT indicates the quality of degradation, i.e. if other hydrolysis products apart from the three main products are formed and allows for deriving the ratio of those two products.

At pH4 and 12 °C: Cyclohexylamine, benzothiazole (BT) and 2-mercaptobenzothiazole (MBT) were determined. The amine was quantified in a higher concentration than theory (126%). The thiazoles were found with 28% (BT) and 18% (MBT) of the theory. The sum of the thiazoles (47%) indicates that unknown degradation products might have formed.

At pH9 and 12 °C: Cyclohexylamine, benzothiazole (BT) and 2-mercaptobenzothiazole (MBT) were determined. The amine was quantified in good agreement with the theory (102%). The thiazoles were found with 53% (BT) and 12% (MBT) of the theory. The sum of the thiazoles (65%) indicates that unknown degradation products might have formed.

At pH7:
and 12 °C: Cyclohexylamine, benzothiazole (BT) and 2-mercaptobenzothiazole (MBT) were determined in both replicates. The amine was quantified in good agreement with the theory (103% - 110%). The thiazoles were found with 38% - 41% (BT) and 45% - 49% (MBT) of the theory. The sum of the thiazoles (84% - 90%) indicates that besides these major transformation products unknown degradation products might have formed.

and 20 °C: Cyclohexylamine, benzothiazole (BT) and 2-mercaptobenzothiazole (MBT) were determined. The amine was quantified in good agreement with the theory (112%). The thiazoles were found with 42% (BT) and 52% (MBT) of the theory. The sum of the thiazoles (94%) indicates that besides these major transformation products only a small quantity of unknown degradation products might have formed.

and 30 °C: Cyclohexylamine, benzothiazole (BT) and 2-mercaptobenzothiazole (MBT) were determined. The amine was quantified in good agreement with the theory (104%). The thiazoles were found with 37% (BT) and 37% (MBT) of the theory. The sum of the thiazoles (74%) indicates that unknown degradation products might have formed.

- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:
on the - the and -th day of incubation, respectively. At the end of the study period, the corresponding concentrations were - and -- % of the applied amount, respectively.

OTHER TRANSFORMATION PRODUCTS
Maximum concentrations in % of the applied amount
- at pH4: Based on the sum of the major transformation products a maximum of 53% of other unknown transformation products can be expected.
- at pH7: Based on the sum of the major transformation products in the different replicates and with different temperatures a maximum between 26% and 6% of other unknown transformation products can be expected.
- at pH9: Based on the sum of the major transformation products a maximum of 35% of other unknown transformation products can be expected.

Validity criteria fulfilled:

yes

Conclusions:

In a guideline study according to OECD TG 111 the hydrolytic behavior of N-cyclohexylbenzothiazole-2-sulphenamide was investigated in a Tier 2 study. At three pH values (pH 4, 7 and 9) and different temperatures (12 °C, 20 °C and 30 °C) the hydrolysis rate constant and half-life as well as major transformation products were determined. At pH 7 and 20 °C the hydrolysis rate constant was found to be 1.44e-6 s-1 and the half life was t1/2 = 13.4 h. The major transformation products were cyclohexylamine (112% of theory for full degradation of the parent compound), benzothiazole (BT, 42% of theory) and 2-mercaptobenzothiazole (MBT, 52% of theory). According to the constitution of CBS and the stoichiometry one mole of CBS will hydrolyze to one mole of either Benzothiazole (BT) or 2-Mercaptobenzothiazole (MBT) or a combination of both with corresponding shares of one mole. Therefore the sum of BT and MBT (94% of theory) indicate that only minor quantities of unknown transformation products occure at pH7 and 20 °C.

Executive summary:

In a guideline study according to OECD TG 111 the hydrolytic behavior of N-cyclohexylbenzothiazole-2-sulphenamide (CBS) was investigated in a Tier 2 study. At three pH values (pH 4, 7 and 9) and different temperatures (12 °C, 20 °C and 30 °C) the hydrolysis rate constant and half-life as well as major transformation products were determined. In accordance with the guideline testing was continued until the concentration of the parent compound was found to be <10%. When this threshold was reached, determination of the major expected transformation products was conducted. Available information from other studies (Monsanto 1984) and related substances indicated that the parent substance might degrade to cyclohexylamine, 2-mercaptobenzothiazole (MBT) and possibly benzothiazole (BT). Those major transformation products could be identified and quantified in all pH and temperature combinations. Specifically for cyclohexylamine good agreement with theoretical quantities in case of full degradation of the parent compound was observed in all tests. This indicates that CBS indeed completely hydrolysed in the determined timeframes. According to the constitution of CBS and the stoichiometry one mole of CBS was expected to hydrolyze to one mole of either Benzothiazole (BT) or 2-Mercaptobenzothiazole (MBT) or a combination of both with corresponding shares of one mole. At pH 7 these two substances were found between 37% and 42% (BT) and 37% and 52% (MBT) of the theoretical quantities, resulting in summarized quantities between 74% (at 30 °C) and 94% (at 20 °C). This indicates that only small quantities of other degradation products might form besides the major transormation products at pH 7. At pH 4 and 9 (both at 12 °C) the sum of BT and MBT lies between 47% (28% BT and 18% MBT) and 65% (53% BT and 12% MBT). At those pH values a larger quantity of unknown transformation products seem possible. Specifically MBT is susceptible to further transformation (e.g. oxidation to MBTS or photodegradation to BT and BTon), while stable at pH 7 (see disseminated dossier for MBT).

 

Hydrolysis temperature	Half-life time of CBS t(1/2) in hours			Hydrolysis rate constant k in s-1
	pH 4	pH 7	pH 9	pH 4	pH 7	pH 9
12 °C	26.1 h	52.3 h	76.7 h	7.37 ∙ 10-6 s-1	3.71 ∙ 10-6 s-1	2.51 ∙ 10-6 s-1
20 °C	--	13.4 h	--	--	1.44 ∙ 10-5 s-1	--
30 °C	--	13.3 h	--	--	1.45 ∙ 10-5 s-1	--

 

 

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

Only hydrolysis at pH 7.0 was measured and at ambient temperature

Principles of method if other than guideline:

Analytical Bio-Chemistry Laboratories (ABC) Protocol #M-8305
Determination of the hydrolysis of CBS in pH 7.0 buffered deionized water with light excluded at 25°C and identification of hydrolysis products by GC/MS.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Monsanto Polymer Products Company, lot no. NC06-304
- Purity, including information on contaminants, isomers, etc.: 96%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at ambient temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Primary stock solutions of 1.00 mg/ml in nanograde acetone were produced
- Preliminary purification step (if any): no preliminary purification


OTHER SPECIFICS
- The pH was adjusted to 7.00 ± 0.05 using 0.1 M NaOH/0.1 M KH2PO4.

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: 0.2, 4.4, 8.4 12.1, 16.0 and 25 hours

Buffers:

pH 7.00±0.05 with 0.1 M NaOH/0.1 M KH2PO4
pH was measured with a Corning pH meter using a one-point calibration method.

Duration:

25

pH:

7

Temp.:

25 °C

Initial conc. measured:

0 µg/L

Duration:

16

pH:

7

Temp.:

25 °C

Initial conc. measured:

8.8 µg/L

Duration:

12.1 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

16 µg/L

Duration:

8.4 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

15 µg/L

Duration:

4.4 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

0 µg/L

Remarks:

This measurement was disregarded. Following measurements show linear degradation.

Duration:

0.2 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

26 µg/L

Transformation products:

yes

No.:

#1

Details on hydrolysis and appearance of transformation product(s):

Benzothiazole was identified as sole hydrolysis product. Although Cyclohexylamine was expected as a potential degradation product as a result of stoichiometric hydrolysis, it was not identified by either gas liquid chromatography or GC/MS techniques.

Key result

pH:

7

Temp.:

25 °C

DT50:

12.5 h

The apparent hydrolysis rate is 1µg/hr. The test substance hydrolyzed completely over 24.9 hours.

Conclusions:

An apparent hydrolysis rate of 1µg/hr and a t1/2 of 12.5 hours were determined for CBS in pH 7.0 buffered deionized water with light excluded at 25 °C (Monsanto, 1984). In the same condition, a complete hydrolysis of CBS in 24.9 hours was also observed with the sole hydrolysis product as benzothiazole identified (Monsanto, 1984). However, the expected degradation products as a result of stoichiometric hydrolysis (Cyclohexylamine) was not identified.

Executive summary:

The substance N-cyclohexylbenzothiazole-2-sulphenamide was examined according OECD TG 111. An apparent hydrolysis rate of 1µg/hr and a t1/2 of 12.5 hours were determined for CBS in pH 7.0 buffered deionized water with light excluded at 25 °C (Monsanto, 1984). In the same condition, a complete hydrolysis of CBS in 24.9 hours was also observed with the sole hydrolysis product as benzothiazole identified (Monsanto, 1984). However, the expected degradation products as a result of stoichiometric hydrolysis (Cyclohexylamine) was not identified.

Description of key information

In a guideline study according to OECD TG 111 the hydrolytic behavior of N-cyclohexylbenzothiazole-2-sulphenamide was investigated in a Tier 2 study. At three pH values (pH 4, 7 and 9) and different temperatures (12 °C, 20 °C and 30 °C) the hydrolysis rate constant and half-life as well as major transformation products were determined. At pH 7 and 20 °C the hydrolysis rate constant was found to be 1.44e-6 s-1 and the half life was t1/2 = 13.4 h. The major transformation products were cyclohexylamine (112% of theory for full degradation of the parent compound), benzothiazole (BT, 42% of theory) and 2-mercaptobenzothiazole (MBT, 52% of theory). According to the constitution of CBS and the stoichiometry one mole of CBS will hydrolyze to one mole of either Benzothiazole (BT) or 2-Mercaptobenzothiazole (MBT) or a combination of both with corresponding shares of one mole. Therefore the sum of BT and MBT (94% of theory) indicate that only minor quantities of unknown transformation products occure at pH7 and 20 °C.

 

In a supporting study an apparent hydrolysis rate of 1µg/hr and a t1/2 of 12.5 hours were determined for CBS in pH 7.0 buffered deionized water with light excluded at 25 °C (Monsanto, 1984). In the same condition, a complete hydrolysis of CBS in 24.9 hours was also observed with the sole hydrolysis product identified as benzothiazole (Monsanto, 1984). However, the expected degradation product as a result of stoichiometric hydrolysis (i.e. Cyclohexylamine) was not found.

 

Key value for chemical safety assessment

Half-life for hydrolysis:

13.4 h

at the temperature of:

20 °C

Additional information

Under oxidative environment of 0.5 M phosphate buffer at pH 6.5, trace amount of MBTS (CAS: 120-78-5) and MBT (CAS: 149-30-4) were also detected (Hansson & Agrup, 1993).