N-cyclohexylbenzothiazole-2-sulfenamide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

There is no direct data available regarding the chronic effect of CBS on fish. As the primary metabolite from CBS, MBT has a long-term study available for fish and hence a read-across approach is used to support the toxicity discussion regarding CBS. However the effective concentration reported in this study is higher than the one reported for the long-term effect on Daphnia, and hence this value is not used in risk assessment for the PNEC estimation.

For hydrolysable substances used in aquatic ecotox tests, REACH Guidance Document R7b (2017), p. 86 states: :
“Where degradation is rapid (e.g. half-life < 1 hour), the available test data will frequently define the hazard of the degradation products since it will be these that have been tested. These data may be used to classify the parent substance in the normal way.
Where degradation is slower (e.g. half-life > 3 days), it may be possible to test the parent substance and thus generate hazard data in the normal manner using a suitable renewal regime. The subsequent degradation may then be considered in determining whether an acute or chronic hazard class should apply.
Where degradation rates fall between these two, testing of either parent and/or degradates should be considered on a case-by-case basis. “

CBS hydrolyses with a DT50 of 13.4 h thus falling in the category where either parent or degradation product could be tested. In the following considerations are discussed which kind of study is recommended:

1. In the case of the sulphenamide category, a study with the degradation product benzothiazole-2-thiol (MBT) is already available. This study (FELS test) has been performed in accordance similar to an accepted international guideline (OECD 210, FELS test) and has been evaluated as Klimisch 2.

2. There are acute and chronic ecotox studies with MBT and CBS available. The results are presented in the table below (effect values are based on measured concentrations):

CBS (mg/L) MBT (mg/L)
Daphnia 48h-EC 50 0.79 0.71
Daphnia 21d-NOEC 0.058 0.08
Fish 96h LC 50 2.1 0.73
Fish 14d-NOEC 0.14 n/a
Fish 89d NOEC use MBT data 0.048
Algae 72h-EC 50 0.15 0.5
72-h-NOEC 0.0084 0.066
* 14d-EC50

Whereas for algae, CBS appears to be more toxic, the effect values for CBS and MBT in acute daphnia and fish are comparable. This can also be seen in the chronic tests in daphnia. These results suggest that CBS and its degradation product MBT have a similar toxicological profile and no relevant differences in a chronic test for CBS would be expected.

3. MBT was identified as the hydrolysis product in studies for the category members TBBS and DCBS. In a study for CBS (Monsanto 1984) however, Benzothiazole (BT) was designated as the hydrolysis product. Therefore a new comprehensive hydrolysis study (Currenta 2022) was conducted, where the degradation products were identified as cyclohexylamine, MBT and BT.

As a conclusion the existing chronic fish test for MBT is sufficient to explain the toxicity of CBS.

Qualifier:

according to guideline

Guideline:

other: TSCA Test Standard No. 797.1600, comparable with OECD TG 210

Deviations:

not specified

Principles of method if other than guideline:

Federal Register 50: TSCA Test Standard No. 797.1600. This test is comparable with the OECD-Guideline 210.

GLP compliance:

yes

Specific details on test material used for the study:

The test material, 2-mercaptobenzothiazole, (lot N8F-228, CAS #149-3~) was received from Monsanto Company, St. Louis, Missouri, on 27 January 1989. The test material was a yellow-green powder and was identified by the study sponsor to be 96.2% active ingredient (A.I.). Upon receipt at SU, the test material was stored in the dark under refrigerated conditions (approximately 4 °C). Determination of the dissociation constant of 2-mercaptobenzothiazole was performed at SLI. The mean ± SD pKs is reported to be 7.03 ± 0.04 at a temperature of 20 ± 1 °C. Test concentrations are expressed in this report as micrograms of 2-mercaptobenzothiazole (as active ingredient) per liter of test solutions (J.Ig A.I./L).

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

Clear yellow-colorp.d diluter stock solutions of 21.4 mg A.l./mL (nominal) were prepared weekly for the definitive exposure by diluting 2-mercaptobenzothiazole with acetone (e.g., 2.14 grams as active ingredient with 100 mL acetone, CAS #67-64-1). Weekly analysis of these stocks resulted in measured concentrations which averaged 101% of nominal.
In addition, a stock stability study was performed at SU during the initial phase of testing to
establish the stability of the test material in acetone. A stock solution, at a nominal concentration of 21.4 mg A.I./L, was prepared on 23 May 1989 and analyzed over a period of seven weeks. During the sampling period, this stock solution was stored under the same conditions as the diluter stock (e.g., ambient temperature, laboratory light etc.). Weekly analysis of the toxicant stock solution demonstrated that measurements were consistent over a seven week period and ranged from 94 - 105% of nominal. Stability of the stock solution was therefore established for a period of at least seven weeks.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Unfertilised rainbow trout eggs and sperm were individually packaged and shipped under refrigeration from Mount Lassen Farm, a certified disease-free trout hatchery located in Red Bluffs, California.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

89 d

Hardness:

CaCO3, 26 - 30 mg/l

Test temperature:

12 +/- 2°C

pH:

6.9 - 7.4

Dissolved oxygen:

7.1 - 11.2 mg/l

Conductivity:

100 - 140 µmhos/cm

Key result

Duration:

89 d

Dose descriptor:

NOEC

Effect conc.:

0.041 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

larval development

Key result

Duration:

89 d

Dose descriptor:

LOEC

Effect conc.:

0.078 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

larval development

Details on results:

MATC = Maximum Acceptable Toxicant Concentration
geometric mean = 0.057 mg/l

At the termination of the test, data obtained on embryo viability, survival at hatch, larval survival and larval growth (wet weight and total length) were statistically analysed to establish treatment level effects.

Conclusions:

The Maximum Acceptable Toxicant Concentration (MATC) in an early-life stage test on rainbow trout was determined to be 0.041 - 0.078 mg/l within 89 days. The concentration of 0.078 mg/l was reported as LOEC and 0.041 mg/l as NOEC (Chemical Manufactures Association Rubber Additives Panel, 1989).

Executive summary:

The Maximum Acceptable Toxicant Concentration (MATC) in an early-life stage test on rainbow trout was determined to be 0.041 - 0.078 mg/l within 89 days. The concentration of 0.078 mg/l was reported as LOEC and 0.041 mg/l as NOEC (Chemical Manufactures Association Rubber Additives Panel, 1989).

Description of key information

A read-across is carried out from MBT to CBS, as the primary metabolite of CBS. The Maximum Acceptable Toxicant Concentration

(MATC) in an early-life stage test on rainbow trout was determined to be 0.041 - 0.078 mg/l within 89 days for MBT. The concentration of 0.078 mg/l was reported as LOEC and 0.041 mg/l as NOEC (Chemical Manufactures Association Rubber Additives Panel, 1989).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.041 mg/L

Additional information

For hydrolysable substances used in aquatic ecotox tests, REACH Guidance Document R7b (2017), p. 86 states: :

“Where degradation is rapid (e.g. half-life < 1 hour), the available test data will frequently define the hazard of the degradation products since it will be these that have been tested. These data may be used to classify the parent substance in the normal way.

Where degradation is slower (e.g. half-life > 3 days), it may be possible to test the parent substance and thus generate hazard data in the normal manner using a suitable renewal regime. The subsequent degradation may then be considered in determining whether an acute or chronic hazard class should apply.

Where degradation rates fall between these two, testing of either parent and/or degradates should be considered on a case-by-case basis. “

CBS hydrolyses with a DT50 of 13.4 h thus falling in the category where either parent or degradation product could be tested. In the following considerations are discussed which kind of study is recommended:

1.       In the case of the sulphenamide category, a study with the degradation product benzothiazole-2-thiol (MBT) is already available. This study (FELS test) has been performed in accordance similar to an accepted international guideline (OECD 210, FELS test) and has been evaluated as Klimisch 2.

2.       There are acute and chronic ecotox studies with MBT and CBS available. The results are presented in the table below (effect values are based on measured concentrations):

 

                                  CBS (mg/L)       MBT (mg/L)

Daphnia 48h-EC 50       0.79                     0.71

Daphnia 21d-NOEC     0.058                     0.08

Fish 96h LC 50              2.1                         0.73

Fish 14d-NOEC             0.14                     n/a

Fish 89d NOEC       Read-across              0.048

Algae 72h-EC 50            0.15                     0.5

          72-hNOEC       0.0084               0.066

 

Whereas for algae, CBS appears to be more toxic, the effect values for CBS and MBT in acute daphnia and fish exhibit very comparable. This can also be seen in the chronic tests in daphnia. These results suggest that CBS and its degradation product MBT have a similar toxicological profile and no relevant differences in a chronic test for CBS would be expected.

3.       MBT was identified as the hydrolysis product in studies for the category members TBBS and DCBS. In a study for CBS (Monsanto 1984) however, Benzothiazole (BT) was designated as the hydrolysis product. Therefore a new comprehensive hydrolysis study (Currenta 2022) was conducted, where the degradation products were identified as cyclohexylamine, MBT and BT.

As a conclusion the existing chronic fish test for MBT is sufficient to explain the toxicity of CBS.