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Endpoint:
basic toxicokinetics, other
Remarks:
EU risk assessment
Type of information:
other: EU risk assessment
Adequacy of study:
supporting study
Reliability:
other: EU risk assessment
Rationale for reliability incl. deficiencies:
other: EU risk assessment report
Principles of method if other than guideline:
EU risk assessment report
GLP compliance:
no
Type:
other:
Results:
summary of toxicokinetic data

As cited in the EU risk assessment draft report (2007), two publications on the fate of 14C-radiolabelled CBS in rats were obviously based on the same experiment (Adachi 1989, Fukuoka 1995). In these experiments rats were given single oral doses of 250 mg/kg 14C-CBS. The amounts of radioactivity recorded from urine and from faeces were dependent on the position of the radioactivity label in the parent compound. Following administration of CBS radiolabel in the cyclohexyl moiety 14C-radioactivity was recovered to 89 % of the dose within three days. The extent of radioactivity was 65.4% in urine and 24.2% in faces. As the biliary excretion amounted to 5% it might be concluded that at least 70% of the radioactivity was observed from the gastro-intestinal tract. When the substance was 14C-labelded in the C2-position of the thiobenzothiazole, 92.3 % of the radioactivity was recovered within three days and similar amounts were found in urine (46.9%) as in faces (45%). In the urine, 2-mercaptobenzothiazol and cyclohexylamine were identified as metabolites of CBS. The results indicate intensive metabolism of CBS. As hydrolysis to 2-mercaptothiazol and cyclohexylamine will occur in the gastrointestinal tract, pre-systemic metabolism may play a role in the fate of CBS with different kinetic fate oft the metabolic breakdown products thus explaining the different recovery rate in urine and in faces with different positions of the 14C label.

The results after oral administration to rats indicate that CBS is readily absorbed and that intensive metabolism of CBS takes place. As hydrolysis to 2-mercaptobenzothiazol and cyclohexylamine may occur in the gastrointestinal tract, pre-systemic metabolism may play a role in the fate of CBS with different kinetic fate of the metabolic breakdown products. As cited in EU risk assessment draft report (2007), absorption of 100 % for the oral route is proposed to be taken for the risk characterisation and the inhalation absorption is assumed to be 100 % (default). No data are available for the dermal route. Therefore, a default value for dermal absorption should be applied. Based on the physico-chemical properties of CBS (molecular weight: 264.4 g/mol; log Pow 3.47; water solubility: 0.32 mg/l) a default value of 100 % would be derivated. However, this default value does not reflect the toxicity data (low toxicity via the dermal route). Therefore, an extent of absorption of 10 % will be assumed for dermal risk characterisation purposes.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report, which meets scientific principles
Objective of study:
toxicokinetics
Principles of method if other than guideline:
Kinetic study of acidic hydrolysis of CBS at pH 1 at 35°C
GLP compliance:
no
Species:
other: in vitro
Type:
other: Acidic hydrolysis of CBS
Results:
CBS is metabolised to MBTS in a two-step reaction
Type:
other: Acidic hydrolysis of CBS
Results:
acid hydrolysis of CBS at pH 1 is nearly constant 9% per day for the first 6 to 7 days, rapid drop down to 2% per day for the following time

Within the first 6.5 days (=156 h) the rate of hydrolysis is nearly linear with about 9.0% over day based on the CBS amount at t = 0h and than drops to about 2.0% for the following 9.5 days (= 228 h). Parallel to this pH increase per day within the first 6.5 days is about 0.4 and than suddenly drops to about 0 for the following days.

A correlation of the CBS loss with the formation of MBTS shows that at the beginning the rate of hydrolysis is remarkably higher than the rate of MBTS formation which easily be explained with the low MBT concentration in this period. When there is enough MBT present, which at pH<5 is a precipitated solid like CBS and MBTS, the MBTS formation rate increases rapidly to slow down again after 8 to 9 days (= 216 h) to nearly the same rate than that of the CBS hydrolysis. The trend of the sums of the MBT moieties of CBS and MBTS shows that within analytical accuracy all hydrolyzed CBS is metabolized to MBTS in two steps; in the beginning free mercaptobenzothiazole (MBT) and cyclohexylamine appeared. The free MBT then reacted directly with the starting substance CBS leading to dimeric MBT (mercaptobenzothiazyl disulfide, MBTS) and cyclohexylamine. The interim formed MBT was not measured as it is dissolved during work-up of the sample.

Executive summary:

Within the first 6.5 days (=156 h) the rate of hydrolysis is nearly linear with about 9.0% over day based on the CBS amount at t = 0h and than drops to about 2.0% for the following 9.5 days (= 228 h). Parallel to this pH increase per day within the first 6.5 days is about 0.4 and than suddenly drops to about 0 for the following days.

A correlation of the CBS loss with the formation of MBTS shows that at the beginning the rate of hydrolysis is remarkably higher than the rate of MBTS formation which easily be explained with the low MBT concentration in this period. When there is enough MBT present, which at pH<5 is a precipitated solid like CBS and MBTS, the MBTS formation rate increases rapidly to slow down again after 8 to 9 days (= 216 h) to nearly the same rate than that of the CBS hydrolysis. The trend of the sums of the MBT moieties of CBS and MBTS shows that within analytical accuracy all hydrolyzed CBS is metabolized to MBTS in two steps; in the beginning free mercaptobenzothiazole (MBT) and cyclohexylamine appeared. The free MBT then reacted directly with the starting substance CBS leading to dimeric MBT (mercaptobenzothiazyl disulfide, MBTS) and cyclohexylamine. The interim formed MBT was not measured as it is dissolved during work-up of the sample.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: data are generally regarded as sufficient, publication written in japanese with an abstract and tables in english
Objective of study:
metabolism
Principles of method if other than guideline:
Metabolism study in rats.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C-2 CBS
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Dose / conc.:
250 other: mg/kg
No. of animals per sex per dose:
no data
Type:
excretion
Results:
ca 65% and 24 % of the dose were excreted into urine and feces, respectively for 3 days after administration of labeled CBS (cyclohexyl 14C)
Type:
metabolism
Results:
cyclohexylamine and 2-mercaptobenzothiazole identified as urinary metabolites
Details on excretion:
ca 65% and 24 % of the dose were excreted into urine and feces, respectively

About 65% and 24 % of the dose were excreted into urine and feces, respectively, for 3 days after administration of labeled CBS (cyclohexyl-14C). Biliary excretion amounted to about 5% of the dose for 5 days. While about 92 % of the dose was recovered in urine and feces at a ratio of 1: 1 within 3 days when 14C-2CBS was given. Cyclohexylamine and 2 -mercaptobenzothiazole were identified as urinary metabolites.

Executive summary:

In an oral study with rats about 65% and 24 % of the dose were excreted into urine and feces, respectively, for 3 days after administration of labeled CBS. Biliary excretion amounted to about 5% of the dose for 5 days. While about 92 % of the dose was recovered in urine and feces at a ratio of 1: 1 within 3 days when 14C-2CBS was given. Cyclohexylamine and 2 -mercaptobenzothiazole were identified as urinary metabolites (Adachi 1989).

Endpoint:
basic toxicokinetics, other
Remarks:
in vivo and in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable documented publication, which meets basic scientific principles
Objective of study:
toxicokinetics
Principles of method if other than guideline:
Metabolic fates of N-oxydiethylene-2-benzothiazyl sulfenamide and N-cyclohexyl-2-benzothiazyl sulfenamide in rats were studied using tracer technique. Additionally CBS was incubated in an artificial gastric juice and degradation products determined.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Weanling male wistar rats (body weight, 200 - 230 g and about 300 g, Nippon, Bio-supp. Center, Tokyo).
Sex:
male
Route of administration:
oral: gavage
Vehicle:
DMSO
Dose / conc.:
250 other: mg/kg
No. of animals per sex per dose:
5 rats.
Type:
excretion
Results:
92.3 % of CBS recovered within 3 d: urine 46.9 %, faces 45 %, total 92%
Details on excretion:
14C-labelded in the C2-position of the thiobenzothiazole, 92.3 % of the radioactivity recovered within 3 d: urine 46.9 %, faces 45 %, total 92%

In vivo: metabolic change of CBS in the stomach: after 2 h: conversion into BTDS detected.

In vitro: incubation of CBS in artificial gastric juice: 5 min incubation: 47 % CBS, 9 % MBT, 23% BTDS; 10 min incubation: 45 % CBS, 31 % BTDS; 30 min incubation: CBS (trace), 63 % BTDS, 5 h incubation: 63 % BTDS

In vivo:

Oral administration CBS 250 mg/kg (5 rats):

Excretion %: 46.9 +/- 7.1 (urine), 45.4 +/- 15 (feaces), 92.3 +/- 16.6 (total)

Executive summary:

Metabolic fates of N-oxydiethylene-2-benzothiazyl sulfenamide and N-cyclohexyl-2-benzothiazyl sulfenamide in rats were studied using tracer technique. These compounds given orally to rats were excreted rapidly in the urine and feces. Five urinary metabolites, 2-mercaptobenzothiazoIe (MBT), its three conjugates, mercapturate, glucuronide and sulfate, and 2,2 -dibenzothiazyl disulfide (BTDS) were confirmed.

Furthermore, BTDS was found as a fecal metabolite. The sulfenamides were partly transformed in the stomach to BTDS, which was predominantly excreted into the feces. In the liver, the sulfenamides were mainly transformed to MBT and its conjugates. The 5-glucuronide and 5-sulfate conjugates were predominantly excreted into the bile.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable documented publication, which meets basic scientific principles
Principles of method if other than guideline:
Analytic
GLP compliance:
no
Type:
metabolism
Results:
CBS can undergo hydrolysis to 2 -mercaptobenzothiazole (MBT) and MBTS (2,2'-benzothiazyl disulfide) at pH 6.5
Metabolites identified:
yes
Details on metabolites:
2 -mercaptobenzothiazole (MBT) and MBTS (2,2'-benzothiazyl disulfide).

Detection: CBS can undergo hydrolysis to 2-mercaptobenzothiazole (MBT) and MBTS (2,2'-benzothiazyl disulfide).

CBS showed instability in buffer solution at pH 6.5. After 2 h, 2/3 of the original CBS amount were recorded, but only small amount was converted into MBTS and even less 2-MBT was found. When 1 µmol CBS and 1 µmol 2-MBT were mixed together in buffer solution at pH 6.5 and analysed after 15 min, 0.5 µmol of 2-MBT was found but only 0.3 µmol of CBS. MBTS was found in an amount of 0.6 µmol. In the presence of excess glutathione, CBS laregly disappeared within 10 min, and most of it was present as 2-MBT. After 1 h reaction time, all CBS had disappeared and was found as 2-MBT. Faster formation of 2-MBT from CBS took place when cysteine was used as sulhydryl compound instead of glutathione.

Executive summary:

CBS can undergo hydrolysis to 2 -mercaptobenzothiazole (MBT) and MBTS (2,2'-benzothiazyl disulfide).

Description of key information

As cited in the EU risk assessment final report (2008), two publications on the fate of 14C-radiolabelled CBS in rats were obviously based on the same experiment (Adachi 1989, Fukuoka 1995). In these experiments rats were given single oral doses of 250 mg/kg 14C-CBS. The amounts of radioactivity recorded from urine and from faeces were dependent on the position of the radioactivity label in the parent compound. Following administration of CBS radiolabel in the cyclohexyl moiety 14C-radioactivity was recovered to 89 % of the dose within three days. The extent of radioactivity was 65.4% in urine and 24.2% in faces. As the biliary excretion amounted to 5% it might be concluded that at least 70% of the radioactivity was observed from the gastro-intestinal tract. When the substance was 14C-labelded in the C2-position of the thiobenzothiazole, 92.3 % of the radioactivity was recovered within three days and similar amounts were found in urine (46.9%) as in faces (45%). In the urine, 2-mercaptobenzothiazol and cyclohexylamine were identified as metabolites of CBS. The results indicate intensive metabolism of CBS. As hydrolysis to 2-mercaptothiazol and cyclohexylamine will occur in the gastrointestinal tract, pre-systemic metabolism may play a role in the fate of CBS with different kinetic fate oft the metabolic breakdown products thus explaining the different recovery rate in urine and in faces with different positions of the 14C label.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential

Additional information

Limited data are available on the kinetic and metabolism of CBS which are restricted to some oral studies in the rat. CBS can undergo hydrolysis to 2-mercaptobenzothiazole (MBT) and cyclohexylamine (CHA) (Adachi 1989, Hansson 1993, Lanxess 2007).

As cited in the EU risk assessment final report (2008), two publications on the fate of 14C-radiolabelled CBS in rats were obviously based on the same experiment (Adachi 1989, Fukuoka 1995). In these experiments rats were given single oral doses of 250 mg/kg 14C-CBS. The amounts of radioactivity recorded from urine and from faeces were dependent on the position of the radioactivity label in the parent compound. Following administration of CBS radiolabel in the cyclohexyl moiety 14C-radioactivity was recovered to 89 % of the dose within three days. The extent of radioactivity was 65.4% in urine and 24.2% in faces. As the biliary excretion amounted to 5% it might be concluded that at least 70% of the radioactivity was observed from the gastro-intestinal tract. When the substance was 14C-labelded in the C2-position of the thiobenzothiazole, 92.3 % of the radioactivity was recovered within three days and similar amounts were found in urine (46.9%) as in faces (45%). In the urine, 2-mercaptobenzothiazol and cyclohexylamine were identified as metabolites of CBS. The results indicate intensive metabolism of CBS. As hydrolysis to 2-mercaptothiazol and cyclohexylamine will occur in the gastrointestinal tract, pre-systemic metabolism may play a role in the fate of CBS with different kinetic fate oft the metabolic breakdown products thus explaining the different recovery rate in urine and in faces with different positions of the 14C label.

In a more recent study the acid hydrolysis of CBS at pH1 and 35°C were evaluated (Lanxess 2007). The study shows that CBS exerts a hydrolysis rate of 9% per day at pH 1 and 35°C for the first 6 to 7 days followed a drop to 2% until the end of the study on day 16. The study of the hydrolysis products done in parallel showed that in the beginning free mercaptobenzothiazole (MBT) and cyclohexylamine (CHA) appeared. The free MBT than reacted directly with the starting substance CBS leading to dimeric MBT (mercaptobenzothiazy disulfide, MBTS) and cylohexylamine.

Summary and discussion on toxicokinetics

The results after oral administration to rats indicate that CBS is readily absorbed and that intensive metabolism of CBS takes place. As hydrolysis to 2-mercaptobenzothiazol and cyclohexylamine may occur in the gastrointestinal tract, pre-systemic metabolism may play a role in the fate of CBS with different kinetic fate of the metabolic breakdown products. As cited in EU risk assessment final report (2008), absorption of 100 % for the oral route is proposed to be taken for the risk characterisation and the inhalation absorption is assumed to be 100 % (default). No data are available for the dermal route. Therefore, a default value for dermal absorption should be applied. Based on the physico-chemical properties of CBS (molecular weight: 264.4 g/mol; log Pow 3.47; water solubility: 0.32 mg/l) a default value of 100 % would be calculated. However, this default value does not reflect the toxicity data (low toxicity via the dermal route in repeated dose study). Therefore, an extent of absorption of 10 % will be assumed for dermal risk characterisation purposes.