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

Endpoint summary

Description of key information

A. First, predictions on the toxicokinetic behaviour of TBSI were made, based on the intrinsic properties and toxicological profile of TBSI. 
B. In second instance, dermal absorption was assessed qualitatively and quantitatively. 
C. Potential hydrolysis and metabolism of TBSI to TBBS (N-tert-butylbenzothiazole-2 -sulfenamide), MBT (2 -mercaptobenzothiazole) and TBA (tertiary butylamine) were tested. First, an in vitro dissolution study in stomach and intestinal fluid was conducted; there were no significant changes in TBSI concentration . Further in absorption, kinetics, distribution, metabolism and elimination or excretion were studied in vivo after dosing of [14C]TBSI after single oral dose of TBSI at 1000 mg/kg bw in male and female Wistar rats. The data indicated that  most of the compound was excreted, ca. 85% in faeces and ca.7% in urine, in both genders. Residual radioactivity 168 h after dosing in all the tissues and organs analysed was below 0.01% TRR. 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 <3% of the dose) were identified as respectively MBT-S-glucuronide, MBT-S-mercapturate and the MBT-dimer = dibenzothiazyl disulphide (BTDS or MTBS). In conclusion, at least 7% was absorbed and excreted as metabolites in the urine, whereas the major fraction was excreted mainly unchanged in the faeces.
D. Other studies with MBT and MBTS demonstrated that these compounds were also excreted rapidly in the urine as MBT conjugates. MBTS  was mainly found as a fecal metabolite formed in the gastro-intestinal tract. 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

7

Additional information

A. In first instance, some reasonable predictions on the toxicokinetic behaviour of TBSI can be made based on the available information on the intrinsic properties and toxicological profile of the substance.

 

A1. Relevant exposure routes:

Inhalatory:

TBSI is a solid substance with a vapour pressure of 5.3E^-06hPa. As a consequence, inhalation exposure related to the substance’s volatility will be minimal. However, as the particle size distribution ranges from ca. 1.5 to ca. 16.9 µm (i.e. respirable and thoracic size fractions), inhalation of dust particles cannot be excluded, and the inhalatory route is to be considered a relevant exposure route.

Dermal:

The dermal uptake of solid substances is mainly determined by the molecular weight, log Pow and water solubility of the substance (source: Toxicological risk assessment of chemicals – A practical guide, E. Nielsen et al., Informa Healthcare, 2010).

The molecular weight of TBSI (404 g/mol) enables dermal uptake. Dermal uptake is less favourable at higher molecular weights (>500 g/mol). The Log Pow of TBSI is ca. 6.7. According to the above noted source, the dermal uptake is optimal when the Log Pow is 2 - 3. At Log Pow > 4, the substance will be taken up into the stratum corneum, but further migration into the epidermis is slow. At Log Pow > 6, both the uptake in the stratum corneum and the migration into the epidermis are considered to be slow.

The water solubility of TBSI is ca. 0,001 mg/L. Such a low water solubility results in a slow partitioning from the stratum corneum into the epidermis. It can thus be concluded that the uptake of TBSI via the dermal exposure route will be slow.

Oral:

With a log Pow of ca. 6.7, TBSI is rather lipophilic and passive diffusion across biological membranes is therefore not optimal. Assessment of hydrolysis and metabolism is further explained under Section C.

 

A2. Absorption, Distribution, Metabolism, Excretion:

Initially a basic toxicokinetic assessment was made, however as recent data were provided, this paragraph is replaced by the sections B, C and D below.

 

B. In second instance, dermal absorption was assessed qualitatively and quantitatively.

 

B1. Assessment

The qualitative assessment was performed according to most recent ECHA Guidance on information requirements and chemical safety assessment (Chapter R.7c: Endpoint specific guidance, November 2014 Version 2.0), which confirms that most arguments are not in favour of dermal absorption. The substance is a solid, and its low water solubility prevents the substance to partition from the stratum corneum into the epidermis. In addition the high log Pow >4 limits the transfer rate between stratum corneum and epidermis. The substance does not have surfactant properties, and no irritation, sensitisation or acute dermal toxicity were detected.

A further quantitative assessment was performed by means of Dermwin application, based on US EPA risk assessment for dermal absorption, resulting in following results for TBSI:

- Dermal permeability coefficient (Kp)of 0.241 cm/hr.

- Dermally Absorbed Dose (DAD) of 0.416 µg/kg/day for a life time exposure.

- However, the latter takes into account full dermal exposure (>17500 cm²in adults), whereas for chemicals mostly only default surface areas are used, e.g. hands (857 cm², or ca. 5% of total body surface). This leads to a realistic worst case exposure of 0.021 µg/kg bw/day systemic exposure to TBSI by dermal route.

However as the correlation of the DermWin method was limited (r2 = 0.66) ,a second method was applied, starting from a similar equation for but applying a correction factor for high lipophilicity (Kroes et al., 2007). By applying this correction, the method is considered to be more specific and reliable. Results were as follows:

- Corrected Permeability coefficient (Kp) = 0.097 cm/hr for TBSI.

- From this value, Maximum flux (Jmax) was calculated to be 0.00017 µg/cm²/h for TBSI.

- Maximum potential absorption (Qabs) for hands (857,5 cm²for 8 h) was calculated to be 1.17 µg/adult/day or 0.019 µg/kg bw/day (assuming 60 kg body weight).

Reference: Kroes, R. et al. Food and Chemical Toxicology 45 (2007) 2533-2562, Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

 

B2. Conclusion

Quantitative estimation of dermal absorption for TBSI was performed by two methods, of which the second method was more specific for substances with high logKow. In conclusion:       

- Dermal permeability coefficient was very low, especially when corrected for high LogKow. This confirms a slow permeation through the skin.

- Potential dermal absorption by hands was also calculated to be very low via both methods used (0.021 and 0.019 µg/kg bw/day, respectively). Both methods used realistic worst case exposure scenarios of 5% body surface either for 24 or 8 h/day and 70 or 60 kg bw for adult weight.

- Based on the results given, systemic exposure via the dermal route is confirmed to be very limited. For testing, the oral route is considered to be most appropriate.

 

C. A step-wise testing was performed for potential hydrolysis and metabolism of TBSI to TBBS (N-tert-butylbenzothiazole-2 -sulfenamide), MBT (2 -mercaptobenzothiazole) and TBA (tertiary butylamine).

 

C1. Method qualification was performed for in vitro testing of TBSI in gastric and duodenal fluid and potential hydrolysis products TBBS, MBT and TBA (Buscher, 2014):

The methods for the quantitative determination of TBSI, TBBS, MBT (LC-UV) and TBA (LC-MS) in gastric and duodenum fluid have been qualified successfully. The results are summarized as follows:

Sensitivity

For TBSI, TBBS and MBT, the LLOQ of the method was 0.1mg/mL. For TBA, the LLOQ of the method was 5 ng/mL.

Selectivity

The selectivity of the method was accepted. One outlier was observed (tert-butylamine in one of the two blank duodenum samples).

Calibration

For TBSI, TBBS and MBT, the methods were linear from approx. 0.1mg/mL to 100mg/mL. For TBA, the method was linear from 0.005 to 2mg/mL.

Accuracy and repeatability

QC samples were prepared by adding TBSI, TBBS, MBT and TBA to gastric and duodenum fluid. The accuracy/recovery was within 70 - 110% for all four compounds. The repeatability, expressed as the coefficient of variation (CV, %), was within 20% for each of the four compounds.

Carry-over

The carry-over in the system was accepted for TBSI, TBBS, MBT and TBA.

 

C2. In vitro study of TBSI in gastric and duodenum fluid, including analysis of TBSI and potential hydrolysis products TBBS, MBT and TBA (Cnubben & Buscher, 2014):

The metabolic fate of the test substance TBSI was evaluated using in vitro incubation conditions mimicking the human stomach and small intestine. Potential hydrolysis products (metabolites) are TBBS, MBT and TBA. MBT might potentially dimerise to form MBTS.

Possible formation of the metabolites TBBS, MBT and TBA during in vitro incubations with stomach fluid and small intestine fluid were monitored in time at a concentration of approximately 14.3 mg/mL. The rationale for this test concentration is as follows; the NOAEL for TBSI in rats is 1000 mg/kg body weight. Assuming a rat dose volume of 10 mL/kg, the dose will contain 100 mg TBSI/mL. Allometric scaling of this dose to the human situation results in an equivalent concentration of approximately 14.3 mg/mL.

Gastric fluid samples

No significant increase or decrease of the TBSI concentration was observed in the stomach fluid samples between t=0 and t=8 h. The average TBSI concentration in the stomach fluid fluctuated between 100% and 114%.

The average TBBS percentage varied from 0.045% (t=0) to 0.020% (t=8 h) relative to the TBSI concentration in the same sample. The average MBT percentage varied from 0.035% (t= 8h) to 0.084 % (t=2 h). The average TBA percentage was 0.09 – 0.11% in the first 8 hours.

Duodenum fluid samples

No significant increase or decrease of the TBSI concentration was observed in the duodenum fluid samples between t=0 and t=24 h. The average TBSI concentration in the duodenum fluid fluctuated between 95% and 112%.

The average TBBS percentage varied from 0.02% (t=8 h) to 0.13% (t=4 h) relative to the TBSI concentration in the same sample. The average MBT percentage varied from 0.06% to 0.16%. The average TBA percentage was 0.10 – 0.14% in the first 24 hours.

   

C3. ADME study of [¹⁴C]N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide (TBSI) in male and female Wistar rats (Maas et al., 2015).

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 bw(5MBq/kg bw) in male and female Wistar rats. 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 and tissues 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.

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

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.

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 (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). 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 indicate 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.

 

C4. ADME studies with 2-mercaptobenzothiazole (MBT), 2-mercaptobenzothiazole disulphide (MBTS) and other

2-benzothiazyl sulfenamides

In a first study, ADME data were available from radioactive MBT and MBTS study in rats (El Dareer et al., 1989); this was used for comparison with the TBSI ADME profile. After IV administration of MBT, in 72 h, 90.9 - 101% of the dose appeared in the urine and 3.79 - 15.1% in the faeces. At this time, a small portion of the administered radioactivity (1.52 - 1.96% of the dose) remained associated with erythrocytes. Oral dosing for 14 days with unlabeled MBT (0.510 mg/kg·d) prior to a single dose of radiolabeled MBT (0.503 mg/kg) showed that 1.20 - 1.69% of the dose remained associated with the erythrocytes, 60.8 - 101% of the radioactivity administered appeared in the urine and 3.46 - 9.99% in the faeces in 96 h. At this time, only trace amounts of radioactivity remained in tissues other than blood. Of these tissues, thyroid contained the highest concentration. In the urine, there was no detectable MBT or MBTS, but there were two metabolites, one of which was identified as a thioglucuronide derivative of MBT and the other a sulfonic acid derivative of MBT. In conclusion, there were similarities in absorption, distribution, and metabolism of MBT and MBTS in rats and in guinea pigs, indicating that MBTS was readily converted to MBT.

In a second study, metabolic fates of 2-benzothiazyl sulfenamides (Mercaptobenzothiazole (MBT), N-oxydiethylene-2-benzothiazyl sulfenamide and N-cyclohexyl- 2-benzothiazyl sulfenamide) in rats were studied using tracer techniques (Fukuoka et al., 1995). These compounds given orally to rats were excreted rapidly in the urine and feces. Five urinary metabolites, 2-mercaptobenzothiazole (MBT), its three conjugates, mercapturate, glucuronide and sulfate, and 2,2' -dibenzothiazyl disulfide (BTDS) were confirmed.Furthermore, BTDS (dimer of MBT) 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 S- glucuronide and S-sulfate conjugates were predominantly excreted into the bile.