N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is conducted according to OECD TG 471 and GLP standards.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Principles of method if other than guideline:

- Aliquots of dose formulations were diluted in 1% (w/v) CMC.
- The radio assay of the spleen was performed in single-fold due to the small weight of this organ.
- At the time of dosing, the animals were 7 weeks old.
- The organs/tissues excised were only rinsed with saline and blotted dry when they contained large amounts of blood. This section was erroneously described in the study plan.
These deviations are considered not to have affected the validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

EC Number:

407-430-1

EC Name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Cas Number:

3741-80-8

Molecular formula:

C18H17N3S4

IUPAC Name:

bis(1,3-benzothiazol-2-ylsulfanyl)(tert-butyl)amine

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): TBSI; N-(1,1-dimethylethyl)bis(2 benzothiazolesulfen)amide; TBSI [phenyl-14C(U)]
- Physical state: packaged in toluene solution (30.14 mg/mL)
- Analytical purity: 99.7% (TBSI)
- Impurities (identity and concentrations): peak 1 & 2 (0.09 & 0.05%, respectively)
- Purity test date: February 4, 2015
- Lot/batch No.: 370-120-0067-A-20150129-DRE
- Expiration date of the lot/batch: to be completed 11 February 2020
- Radiochemical purity (if radiolabelling): 99.8%
- Specific activity (if radiolabelling): 6.7 mCi/mmol
- Locations of the label (if radiolabelling): TBSI [phenyl-14C(U)]
- Expiration date of radiochemical substance (if radiolabelling): stable
- Stability under test conditions: stable
- Storage condition of test material: −20 ºC

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Wistar WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, the Netherlands
- Age at dosing: 7 weeks
- Weight at dosing: Weight variation of the animals did not exceed ± 20% of the average weight per gender.
- Housing: During acclimatization, the animals were housed in Macrolon cages type 4 with wood shavings (Lignocel) as bedding and Enviro-Dri and a piece of Aspen wood as cage enrichment, with a maximum of 5 rats per cage. At least fifteen hours prior to dosing, all the rats were individually housed in open Plexiglas metabolism cages (Techniplast™). Just after dosing, two of the four rats per gender were individually housed in open Plexiglas metabolism cages (Techniplast™). The other two of the four rats per gender were individually placed in a whole glass metabolism cages for collection of expired air at 24-hr intervals for 48 hours; thereafter, they were transferred to open Plexiglas metabolism cages until the end of the study. After dosing, reserve animals were returned to the Macrolon cages type 4.
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Ad libitum access to a commercial rat diet diet (Rat & Mouse no.3 breeding diet)
- Water (e.g. ad libitum): The drinking water (tap-water) was given in polypropylene bottles which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC) was supplied by N.V. Vitens.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C; On one occasion, the temperature was higher than 24 °C (maximum temperature recorded: 26.3 °C).
- Humidity (%): 45% - 65%. The relative humidity did not exceed 65% other than during room cleaning
- Air changes (per hr): 9-11 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 2015 To: 4 March 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

solution of 1% (v/w) CMC

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered orally to the animals. The amount dosed of active ingredient were based on the body weight determined just prior dosing. All animals received an oral administration (1000 mg/kg) with a dose volume of maximal 20 mL/kg body weight.
The dose formulation was prepared by pipetting 770 µL [14C]TBSI (ca. 12.2 MBq), dissolved in toluene, to a glass vial. The solvent was evaporated under N2 until dryness, after which ca. 50 µL ethanol absolute and ca. 10 mL 1% CMC were added. The solution was mixed and sonificated for ca. 5 min. Next, 2.2363 g non labelled TBSI was added and an amount of 1% (w/v) CMC was added until reaching the final weight of 45.0331 g 1% (w/v) CMC to obtain a final TBSI concentration of 50 mg/mL.. The solution was mixed and stored protected from light at room temperature until dose administration.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Verification of the amount of radioactivity to be applied was performed before application. The concentration and homogeneity of [14C]TBSI in the dose preparation was checked by taking aliquots just before and directly after dosing of each gender. For homogeneity, a coefficient of variation lower than 15% was considered sufficient. The radiochemical purity in the dose preparation was checked by HPLC using radiochemical detection just before application to the rats.

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 mg/kg bw 14C-dose with a dose volume of 20 mL/kg bw
Specific activity of the active ingredient was 5.02 kBq/mg.

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Blood (tail blood, ca. 200µL; at sacrifice also from abdominal aorta)
Urine and faeces
Cage wash
Organs and tissues: blood/plasma, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary, epididymis, residual carcass, GI tract and contents
Expired air
- Time and frequency of sampling:
Blood at 30 min, 1h, 2h, 4h, 8h, 24h, 48h, 96h and 168h post-dosing
Urine and faeces at 24h-intervals up to 168h post last dose
Cage wash at 48h in the whole glass metabolism cages and at 168h in the open Plexiglas metabolism cages
Organs and tissues: blood/plasma, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary and epididymis, residual carcass, GI tract and contents at sacrifice
Expired air (CO2 and volatiles): 2 of the 4 rats were placed in a whole glass metabolism for collection of expired air at 24h-intervals for 48h. After 48 hours, the animals were transferred to an open Plexiglas metabolism cage.
- Other: Kinetic analysis of radioactivity in blood was performed using WinNonlin® applying a non-compartmental model analysis. The following pharmacokinetic parameters were calculated: Cmax, Tmax, the terminal elimination half-life (t1/2), the volume of distribution (Vd), clearance (Cl), and the area under the concentration-time curve (AUC), among others.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: Urine and faeces at 24h-intervals up to 168h post last dose
- From how many animals: (samples pooled or not) 4M/4F, samples pooled per gender and time point
- Method type(s) for identification: HPLC-RA-UV and LC-MS/MS
- Limits of detection and quantification: Limits of quantification for radioactivity in samples were calculated using the background + 3 x standard deviation of the counting error of the background as limit of quantification.
LQ sample (µgPCE/g) = 3xSDblank(DPM) / weight sample (g)/ Specific activity(kBq/mg)/60
- Other: Metabolite characterization in pooled urine/faeces was performed to cover >90% of the excreted dose.

Metabolite identification by LC-MS/MS
Unknown metabolites M1, M2 and M3 were identified using LC-MS/MS. The HPLC-PDA-HRMS system was optimized using a standard solution of 2-Mercaptobenzothiazole (MBT). After optimization, the relevant study sample containing the unknown metabolites (i.e. pooled urine of male Wistar rats) was analyzed with the HPLC-PDA-HRMS-RA system.
The relevant unknown metabolites were selected based on elution profile and retention time obtained with the on-line RA detector and subsequently identified using high resolution accurate-mass electrospray ionization in both the positive and negative ionization mode. From these data the elemental composition of the metabolites could be determined and a proposal was made for the chemical structure of the unknown metabolites. The unknown metabolites M1, M2 and M3 were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer or dibenzothiazyl disulphide (BTDS). The identity of MBT could be confirmed.

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): Not applicable

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Residual radioactivity 168 h after dosing in all the tissues and organs analyzed (blood, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary and epididymis) of male and female rats, receiving a dose of 1000 mg/kg TBSI was below 0.01% TRR.
The residual radioactivity in all the tissues and organs analysed (blood, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary gland and epididymis) of male and female rats, receiving a dose of 1000 mg/kg TBSI was below 0.005% of the administered dose, indicating low absorption and/or fast depletion in organs and tissues.

Details on distribution in tissues:

The Cmax values of 9.36 µg/mL and 7.78 µg/mL in males and in females, respectively, were found in blood in both genders at 8.0 h (Tmax) after the dose with [14C]TBSI. Radioactivity levels in blood declined slowly (terminal half-life of 166 and 188 h for males and female, respectively). This is probably caused by the fact that most of the radioactivity measured in the whole blood is bound to the red blood cells (the ratio whole blood/plasma at sacrifice is about 50), which is similar as has been observed for the metabolite MBT [ El Dareer et al., 1989]). For males, the calculated AUC168h was 617 h*µg/ml and it was 730 h*µg/ml for females.

Details on excretion:

The radioactivity was predominantly excreted with the faeces (the radioactivity in faeces was a factor 10 (female) to 12 (males) times higher than in the urine). The major part of the radioactivity was excreted within the first 24 hours in both genders.
The data clearly indicates that, 24 hours after dosing, most of the compound was excreted, approximately 85% in faeces and approximately 7% in urine, in both genders.
The extraction efficiency the samples of urine and faeces analyzed were above 98%. The amount of metabolites that could be identified and characterized in urine and faeces was 93.7% and 90.4% of the applied dose for males and females, respectively. The metabolic fate of TBSI could be investigated and the majority of radioactivity could be structurally identified and characterized. In urine, the major compound identified was the metabolite MBT ( 3% of the dose). In faeces, most of the radioactivity was excreted as parent compound TBSI. The identity of metabolite M1, M2 and M3 in urine (all present <3% administered dose) was elucidated further in this study, after optimization of the test method. The unknown metabolites M1, M2 and M3 were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer or dibenzothiazyl disulphide (BTDS).

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#1

Toxicokinetic parameters:

other: Terminal half life: male: 166h; female: 188 h

Toxicokinetic parameters 2

Test no.:

#2

Toxicokinetic parameters:

other: AUC168h: male: 167 h*µg/mL; female: 730 h*µg/mL

Toxicokinetic parameters 3

Test no.:

#3

Toxicokinetic parameters:

Cmax: male: 9.36 µg/mL; female 7.78 µg/mL

Toxicokinetic parameters 4

Test no.:

#4

Toxicokinetic parameters:

Tmax: male: 8.0 h; female: 8.0 h

Toxicokinetic parameters 5

Test no.:

#5

Toxicokinetic parameters:

other: Clearance: male: 808 g/h/kg; female: 604 g/h/kg

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

For identification, the final extracts were profiled on HPLC-RA-UV and retention times of metabolites found, compared to retention time of reference compounds MBT, TBBS, and TBSI. In urine, four metabolites were found: the most abundant was MBT followed by M1, M2 and M3, which remained unknown after HPLC-RA-UV (all present as <3% dosed amount). In faeces, most of the radioactivity was excreted as parent compound TBSI (89.0% in males and 86.9% in females of total excreted). In addition, small amounts of MBT (in both genders) and M2 and M3 (in females) were also detected (all present as <5% dosed amount).

Urine:
Various metabolites were detected by HPLC-RA-UV in pooled urine collected at 0-24h from males and from females: they eluted at 11.6-11.7 min, 13.9-14.0 min, 15.3-15.4 min and 24.4-24.5 min. In both genders the major peak detected was the peak eluting at 15.3-15.4 min (MBT, 3.14% and 3.46% of the dose in males and in females, respectively), followed by the peak eluting at 13.9-14.0 min (M2, 1.88% and 2.98% of the dose in males and in females, respectively), 11.6-11.7 min (M1, ca. 1% in both genders) and 24.4-24.5 min (M3, ca. 0.1% in both genders). The injected radioactivity was completely recovered from the HPLC column (98.9% for males and 99.5% for females).

Faeces:
In faeces from both genders collected at 0-24 h and 24-48 h after oral dosing, the largest peak observed corresponded to the parent compound TBSI eluting at 26.6-26.7 min (88.98% and 85.94% of the dosed amount of radioactivity in males and females, respectively). In addition, MBT (15.3-15.4 min) was also found in both genders (1.56% and 0.96% in males and in females, respectively). In females, the presence of M2 (0.03%) and M3 (0.74%) could also be detected.
The recovery from the injected radioactivity from the HPLC column ranged from 94.1-100.7%.

The extraction efficiency the samples of urine and faeces analyzed were above 98%. The amount of metabolites that could be identified and characterized in urine and faeces was 93.7% and 90.4% of the applied dose for males and females, respectively. The metabolic fate of TBSI could be investigated and the majority of radioactivity could be structurally identified and characterized. In urine, the major compound identified was the metabolite MBT ( 3% of the dose). In faeces, most of the radioactivity was excreted as parent compound TBSI. The identity of metabolite M1, M2 and M3 (all present <3% administered dose) was elucidated further in this study, after optimization of the test method .

Unknown metabolites M1, M2 and M3 were identified using LC-MS/MS. The HPLC-PDA-HRMS system was optimized using a standard solution of 2-Mercaptobenzothiazole (MBT). After optimization, the relevant study sample containing the unknown metabolites (i.e. pooled urine of male Wistar rats) was analyzed with the HPLC-PDA-HRMS-RA system.
The relevant unknown metabolites were selected based on elution profile and retention time obtained with the on-line RA detector and subsequently identified using high resolution accurate-mass electrospray ionization in both the positive and negative ionization mode. From these data the elemental composition of the metabolites could be determined and a proposal was made for the chemical structure of the unknown metabolites. The unknown metabolites M1, M2 and M3 were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer or dibenzothiazyl disulphide (BTDS). The identity of MBT could be confirmed.

Any other information on results incl. tables

The homogeneity and purity of [¹⁴C]TBSI in the dose formulation was well within specifications.

 

Table 1. Recovered radioactivity (expressed as % of the dose)

 

	Nominal dose: 1000 mg.kg^-1
Time group	Males	Females
Urine                         (0-24 h)	6.87 ± 3.52	7.78 ±  5.54
(24-48 h)	0.28 ±  0.18	0.67±  0.34
(48-72 h)	0.03 ±  0.02	0.04 ±  0.03
Total                       (0-168 h)	7.21 ±  3.67	8.53 ± 5.72
Faeces                       (0-24 h)	86.54 ± 6.83	83.95 ±  9.60
(24-48 h)	4.03 ±  4.33	3.73 ±  3.11
(48-72 h)	0.01 ±  0.01	0.04 ±  0.05
Total                       (0-168 h)	90.59 ± 3.97	87.72 ± 7.24
Cage wash	0.13 ±  0.14	0.10 ±  0.10
Total Excreted	97.93 ±  0.70	96.37 ±  1.46
Expired air
Volatiles	<0.01 ± <0.01	0.01 ± <0.01
CO2	0.01± <0.01	0.01 ± <0.01
Organ/Tissue residues
Organs	<0.01 ± <0.01	<0.01± <0.01
Excised^1	0.01 ± <0.01	0.01 ±  0.01
GI-tract and contents	<0.01 ± <0.01	<0.01 ± <0.01
Carcass	0.01 ±  0.01	0.01 ± <0.01
Total retained	0.03± 0.01	0.03 ±  0.01
Total recovery	97.96 ±0.69	96.41 ± 1.44

  Excised = blood, bone, muscle and fat.

 

Table 2. Distribution of radioactivity in organs and tissues 168 h after dosing of [¹⁴C]TBSI

 

	Males		Females
	µg PCE/g	% TRR	µg PCE/g	% TRR
Blood at sacrifice	2.327	0.0080	2.799	0.0102
Plasma	0.045	-	0.056	-
Liver	0.421	0.0020	0.264	0.0014
Kidney	0.574	0.0004	0.501	0.0004
Lung	0.383	0.0002	0.452	0.0003
Heart	0.202	0.0001	0.333	0.0001
Testes	< LQ	<0.0001	--	--
Ovaries	--	--	<LQ	<0.0001
Brain	0.050	<0.0001	0.103	0.0001
Spleen	0.343	0.0001	0.643	0.0001
Epididymus	0.152	<0.0001	--	--
Adrenals	<LQ	<0.0001	<LQ	<0.0001
Pituitary gland	<LQ	<0.0001	<LQ	<0.0001
Bone marrow	<LQ	<0.0001	<LQ	<0.0001
Bone	0.053	<0.0001	0.097	<0.0001
Muscle	0.047	0.0001	0.057	0.0001
Fat	0.191	0.0001	0.208	0.0002
Residual carcass	0.169	0.0135	0.112	< 0.0124

Note: For tissues the %TRR was calculated for the amount of tissue excised from the body and not the total amount in the body.

 

Table 3. Summary of results of HPLC profiling: results are expressed as % of the dosed amount of radioactivity

 

Sample	Retention time (min) radioactive profiles
	M1 (11.6-11.7 min)	M2 (13.9-14.0 min)	MBT (15.3-15.4 min)	TBBS (21.1 min)	M3 (24.4-24.5 min)	TBSI (26.5-26.7 min)	Rest	Total
Males
Urine 0-24h	1.13	1.88	3.14	n.d.	0.07	n.d.	0.49	6.70
Faeces 0-24h	n.d.	n.d.	1.51	n.d.	n.d.	85.03	0.00	86.54
Faeces 24-48h	n.d.	n.d.	0.05	n.d.	n.d.	3.95	0.00	4.00
Total faeces profiled	n.d.	n.d.	1.56	n.d.	n.d.	88.98	0.00	90.54
Total urine and faeces	1.13	1.88	4.70	n.d.	0.07	88.98	0.49	97.24
Females
Urine 0-24h	1.28	2.98	3.46	n.d.	0.08	n.d.	0.00	7.80
Faeces 0-24h	n.d.	n.d.	0.88	n.d.	0.74	82.33	0.00	83.95
Faeces 24-48h	n.d.	0.03	0.08	n.d.	n.d.	3.62	0.00	3.73
Total faeces profiled	n.d.	0.03	0.96	n.d.	0.74	85.94	0.00	87.68
Total urine and faeces	1.28	3.01	4.43	n.d.	0.82	85.94	0.00	95.48

n.d. not detected

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
The data clearly indicates that, 24 hours after dosing, most of the compound was excreted, approximately 85% in faeces and approximately 7% in urine, in both genders. Residual radioactivity 168 h after dosing in all the tissues and organs analysed (blood, plasma, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary and epididymis) of male and female rats, receiving a dose of 1000 mg/kg TBSI was below 0.01% TRR.

Executive summary:

Objectives

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide (TBSI) was tested to evaluate the absorption, kinetics, distribution, metabolism and routes of elimination or excretion after dosing of [14C]TBSI after one single oral dose of TBSI at 1000 mg/kg body weight in male and female Wistar rats.

 

The study was performed with the following objectives;

•        To generate data on the toxicokinetic fate of the test substance after single administration.

•        To determine the radioactivity in blood at various time points (i.e. blood kinetics)

•        To determine the rate and route of excretion of the absorbed test substance in urine and faeces

•        To determine the pattern of tissue distribution of the test substance

•        To produce metabolite profiles of urine and feces to assess whether radioactivity is associated with parent or metabolite(s) including conjugates (to evaluate the extent of metabolism and possible metabolic pathways)

 

Conduct of the study

The animals (5 male and 5 female rats, housed in groups of 5 per cage) were acclimated in the animal room in macrolon cages containing wood shavings as bedding and cage enrichment. One day before dosing, 4 male and 4 females rats were selected based on their body weights and they were housed individually in open Plexiglas metabolic cages. The day of dosing, all selected animals (n=8) received an oral administration of [¹⁴C]TBSI (1000 mg/kg, 5 MBq/kg, 20 mL/kg) and then returned to the metabolic cages. Urine and faecal samples were collected at 24-hours intervals up to 168 hours. Blood samples (tail blood, ca. 200 µL) were collected at 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h and 168 h post-dosing. In addition, expired air (CO₂and volatiles) was sampled at 24 hour intervals for 48 hours in a closed glass metabolism chamber in two male and in two female rats. After 48 hours, the animals were transferred to an open Plexiglas metabolism cage. About 168 hours after dosing, the animals were sacrificed. The animals were anesthetized with CO₂/ O₂and terminated by exsanguination from the abdominal aorta. Subsequently, the following samples were collected: blood, plasma, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary gland, epididymis, GI tract and contents and residual carcass. Radioactivity in all samples was determined by liquid scintillation counter (LSC).

Metabolite profiling was conducted in urine and faecal samples pooled per time point and gender to cover > 90% of the dose.

 

Recovery of radioactivity

The total recovery of administered radioactivity was 98.0% and 96.4% in males and females, respectively.

 

Absorption, distribution and excretion

The radioactivity was predominantly excreted with the faeces (the radioactivity in faeces was a factor 10 (female) to 12 (males) times higher than in the urine). The major part of the radioactivity was excreted within the first 24 hours in both genders. The residual radioactivity in all the tissues and organs analysed (blood, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary gland and epididymis) of male and female rats, receiving a dose of 1000 mg/kg TBSI was below 0.005% of the administered dose, indicating low absorption and/or fast depletion in organs and tissues.

 

Blood kinetics

The C_maxvalues of 9.36 µg/mL and 7.78 µg/mL in males and in females, respectively, were found in blood in both genders at 8.0 h (T_max) after the dose with [¹⁴C]TBSI. Radioactivity levels in blood declined slowly (terminal half-life of 166 and 188 h for males and females, respectively). This is probably caused by the fact that most of the radioactivity measured in the whole blood is bound to the red blood cells (the ratio whole blood/plasma at sacrifice is about 50, which is similar as has been observed for the metabolite MBT [El Dareer et al., 1989]). For males, the calculated AUC_lastwas 617 h*µg/mL and it was 730 h*µg/mL for females.

 

Metabolite profiling and identification

For all the urine and faecal samples analyzed, the % recoveries in the extracts were above 98%.

For identification, the final extracts were profiled on HPLC-RA-UV and compared by retention time with reference compounds (2-mercaptobenzothiazole [MBT], N-tert-butylbenzothiazole-2-sulfenamide [TBBS] and N-(1,1-dimethylethyl)bis(2-benzothiazole-sulfen)-amide [TBSI]). In urine, four metabolites were found: the most abundant was MBT (ca 3 - 3.5% of the dose) followed by M1, M2 and M3, which remained unknown after HPLC-RA-UV (all present as <3% dosed amount). In faeces, most of the radioactivity was excreted as parent compound TBSI (89.0% in males and 86.9% in females of total excreted). In addition, small amounts of MBT (in both genders) and M2 and M3 (in females) were also detected (all present as <3% dosed amount).

 

Metabolite identification by LC-MS/MS

Unknown metabolites M1, M2 and M3 were identified using LC-MS/MS. The HPLC-PDA-HRMS system was optimized using a standard solution of 2-Mercaptobenzothiazole (MBT). After optimization, the relevant study sample containing the unknown metabolites (i.e. pooled urine of male Wistar rats) was analyzed with the HPLC-PDA-HRMS-RA system.

The relevant unknown metabolites were selected based on elution profile and retention time obtained with the on-line RA detector and subsequently identified using high resolution accurate-mass electrospray ionization in both the positive and negative ionization mode. From these data the elemental composition of the metabolites could be determined and a proposal was made for the chemical structure of the unknown metabolites. The unknown metabolites M1, M2 and M3 were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer or dibenzothiazyl disulphide (BTDS). The identity of MBT could be confirmed.

 

In Conclusion

The data clearly indicates that, 24 hours after dosing, most of the compound was excreted, approximately 85% in faeces and approximately 7% in urine, in both genders. Residual radioactivity 168 h after dosing in all the tissues and organs analysed (blood, plasma, liver, spleen, kidneys, adrenals, heart, testes/ovaries, lungs, brain, muscle, fat, bone, bone marrow, pituitary and epididymis) of male and female rats, receiving a dose of 1000 mg/kg TBSI was below 0.01% TRR.

The extraction efficiency the samples of urine and faeces analysed were above 98%. The amount of metabolites that could be identified and characterized in urine and faeces was 93.7% and 90.4% of the applied dose for males and females, respectively. The metabolic fate of TBSI could be investigated and the majority of radioactivity could be structurally identified and characterized. In urine, the major compound identified was the metabolite MBT (3% of the dose). In faeces, most of the radioactivity was excreted as parent compound TBSI. The minor metabolites M1, M2 and M3 (all present <3% administered dose) were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer or dibenzothiazyl disulphide (BTDS). TBBS was not detected in this study.