Antimicrobial agent

2-(Thiocyanomethylthio)benzothiazole (TCMTB) is a biocide used in the leather, pulp and paper, and water-treatment industries. TCMTB may enter aquatic ecosystems during its manufacture and use. TCMTB is environmentally unstable; therefore, it is important to evaluate the toxicity of the more persistent degradation products. This study compared the toxicity of TCMTB with its degradation products 2-mercaptobenzothiazole (2-MBT), 2-(methylthio)benzothiazole (MTBT), benzothiazole (BT), and 2-hydroxybenzothiazole (HOBT). Toxicity was determined using Ceriodaphnia dubia 48-hour acute and 7-day chronic test protocols. TCMTB was the most toxic compound evaluated in both the acute and chronic tests with EC50s of 15.3 and 9.64 microg/L, respectively. 2-MBT, the first degradation product, was the second most toxic compound with acute and chronic EC50s of 4.19 and 1.25 mg/L, respectively. The toxicity of MTBT and HOBT were similar with acute EC50s of 12.7 and 15.1 mg/L and chronic EC50s of 6.36 and 8.31 mg/L, respectively. The least toxic compound was BT with acute and chronic EC50s of 24.6 and 54.9 mg/L, respectively. TCMTB was orders of magnitude more toxic than its degradation products. Toxicity data on these benzothiazole degradation products is important because of concerns regarding their release, degradation, persistence, and non-target organism effects in aquatic ecosystems[1].

A method for biological monitoring of urinary 2-(thiocyanomethylthio)benzothiazole (TCMTB), a wood preservative and an industrial chemical, was developed. Three different doses of TCMTB in olive oil were given to male rats by gavage for 3 weeks. Urine was collected daily and the metabolites were analysed as thioethers by derivatization with pentafluorobenzyl-bromide by gas chromatography-mass spectrometry. The parent chemical was not detected in urine samples, but two metabolites of TCMTB were identified. 2-Mercaptobenzothiazole (2-MBT) was the main metabolite, and its excretion varied according to the dose. The second metabolite was 2-(mercaptomethylthio)benzothiazole. The amount of 2-MBT excreted in rat urine was 66 +/- 12% (SD), 51 +/- 20% and 44 +/- 9% for TCMTB doses of 15, 75 and 150 mg/kg, respectively. Two doses, 75 and 150 mg/kg, caused diuresis in rats during the 1 week of dosing. During the 3-week TCMTB treatment, rat liver microsomal CYP enzyme profile was not significantly changed. Urine samples of sawmill workers exposed to TCMTB were collected after their work shifts for exposure assessment. TCMTB could not be detected in the urine samples of exposed workers. Most concentrations of 2-MBT were below the limit of the detection, 0.12 mumol/l, the concentrations were 0.12-0.15 mumol/l only in few cases. The determination of 2-MBT in urine, when a sample is taken immediately after a work shift, is a suitable indicator of workers' exposure to TCMTB[2].

Experiments were performed using the cladoceran C. dubia. Neonates (<24 hours old) were obtained from laboratory cultures maintained at 25°C ± 1°C in moderately hard water and reared according to guidelines recommended by the U.S. Environmental Protection Agency (USEPA 1993, 1994). The cultures were fed a combination of the green alga Selenastrum capricornutum and YCT (a combination of yeast, Cerophyll, and trout chow) as recommended by the USEPA. The algae were cultured in Woods Hole algal growth media to the desired cell density (USEPA 1994). Buckman Laboratories International Inc. (Memphis, TN) provided TCMTB (99.4% pure; lot 946-13). 2-MBT (98% pure; lot 05426DN) and BT (96% pure, lot 04030TY) were obtained from Aldrich Chemical Company (St. Louis, MO). MTBT (98% pure; lot A010106901) and HOBT (99% pure; lot A008333701) were purchased from Acros Chemical Company (Pittsburgh, PA). Because of the low water solubility of these compounds, stocks were prepared by dissolving the compounds in dimethylformamide (DMF) (99.95% pure; lot 37H3649) (Sigma Chemical Company, St. Louis, MO). The stocks were stored in foil-covered glass beakers and kept at 4°C. Test concentrations were prepared from these stocks using moderately hard dilution water. Dilutions were prepared fresh daily to limit possible degradation. Five nominal concentrations (Tables 1 and 2) were selected based on preliminary experiments for each chemical. Two controls—a control plus DMF and a dilution water control—were also prepared. The DMF control was formulated by adding an amount of DMF equal to the greatest test concentration to the dilution water. The largest concentration of DMF used in preparing test concentrations was 2 ml/L[1].

The protocols for the toxicity tests were performed as recommended by the USEPA. Exposures were conducted in 30-ml plastic cups containing 20 ml solution. In the 48-hour acute tests, 4 replicates containing 5 neonates each were used for each exposure concentration. Neonates <24 hours old were pipetted into the exposure chambers. The 48-hour acute tests were a static nonrenewal exposure, and the organisms were not fed during this period. Survival counts were made after 24- and 48-hour periods in accordance with standard USEPA protocols (1993). In the chronic toxicity tests, each concentration had 10 replicates (1 daphnid/container,) and the organisms were fed daily with a combination of Selenastrum capricornutum and YCT, as recommended by the USEPA (1994). The chronic tests were static renewal, where concentrations were renewed daily for a period of 7 days, which is sufficient time for 1 organism to produce 3 broods. Mortality and reproduction were recorded every 24 hours for each organism. Two reference toxicant tests were performed using sodium dodecyl sulfate and NaCl to assure that the organisms were responding appropriately to toxicant exposure. All toxicity tests were conducted at 25°C ± 1°C with a 16 hours light/8 hours dark photoperiod.[1]
Standard water-quality parameters were measured including dissolved oxygen, pH, conductivity, temperature, alkalinity, hardness, ammonia, and chlorine. Dissolved oxygen, pH, conductivity, and temperature were measured daily using probes and meters. Alkalinity and hardness were measured at the initiation and termination of each test using Hach titrant kits. Ammonia and chlorine levels were similarly determined using a Hach colorimetry kit.[1]
Measured concentrations were determined by high-pressure liquid chromatography (HPLC) analysis at George Mason University, Fairfax, VA. To limit possible degradation, samples were shipped overnight on ice in amber glass bottles. 2-MBT, MTBT, BT, and HOBT samples were prepared by using 5 ml sample water diluted with 5 ml acetonitrile. This diluted sample was spiked with 119 μg 2-phenylbenzothiazole, the internal standard. HPLC analyses were performed using 2 ml of each prepared sample. Because of the low concentration of TCMTB, 500 to 700 ml of sample were extracted using C18-bonded phase-extraction cartridges. The eluates for each sample were combined and concentrated to 1 ml using dry nitrogen gas blow-down. The sample was spiked with the internal standard and analyzed using HPLC. TCMTB extraction yielded recoveries of 86% ± 15% for 2 μg/L and 99% ± 10% for 10 μg/L TCMTB. A previous study indicated that gas chromatography is not a preferred technique because TCMTB is not stable at high temperatures (Daniels and Swan 1987). For the acute tests, 2 L of each concentration were prepared to ship two 800-ml volumes of sample as initial and final concentrations to be analyzed by HPLC. The final 800 ml was exposed to the same conditions as the acute test. This procedure allowed detection of any degradation that may have occurred during the 48-hour static nonrenewal period. The detection limit for TCMTB was 0.45 μg/L, for 2-MBT was 0.35 μg/L, for MTBT was 0.17 μg/L, for BT was 0.11 μg/L, and for HOBT was 0.13 μg/L. Similar steps were performed for the chronic tests; however, additional samples were analyzed because of the length of the test. Concentrations were taken on days 0, 4, and 7 to be analyzed. Averages were performed using the measured concentrations to determine each mean for statistical analyses. In addition, several spiked samples were analyzed to ensure quality control.[1]
For each chemical, measured concentration averages were used to calculate EC50 estimates and associated 95% confidence intervals using the probit analysis where applicable. Where the probit analysis was not applicable because of a lack of two partial mortalities, either the graphical method, the Spearman-Karber, or the trimmed Spearman-Karber method was used to determine EC50 values as recommended by the USEPA (1993, 1994). Sublethal effects were also examined by observing the number of neonates produced during the 7-day period in each concentration and the control. Statistical differences were determined using Dunnett’s Test. Differences were considered significant at p ≤ 0.05.[1]

Three different doses of TCMTB in olive oil were given to male rats by gavage for 3 weeks. Urine was collected daily and the metabolites were analysed as thioethers by derivatization with pentafluorobenzyl-bromide by gas chromatography-mass spectrometry. The parent chemical was not detected in urine samples, but two metabolites of TCMTB were identified. 2-Mercaptobenzothiazole (2-MBT) was the main metabolite, and its excretion varied according to the dose. The second metabolite was 2-(mercaptomethylthio)benzothiazole. The amount of 2-MBT excreted in rat urine was 66 +/- 12% (SD), 51 +/- 20% and 44 +/- 9% for TCMTB doses of 15, 75 and 150 mg/kg, respectively. Two doses, 75 and 150 mg/kg, caused diuresis in rats during the 1 week of dosing. During the 3-week TCMTB treatment, rat liver microsomal CYP enzyme profile was not significantly changed[2].

Metabolism / Metabolites
TCMTB has several metabolites, including 2-mercaptobenzothiazole (2-MBT) and 2-benzothiazolesulfonic acid (2-BTSA).
... Three different doses of TCMTB in olive oil were given to male rats by gavage for 3 weeks. Urine was collected daily and the metabolites were analysed as thioethers by derivatization with pentafluorobenzyl-bromide by gas chromatography-mass spectrometry. The parent chemical was not detected in urine samples, but two metabolites of TCMTB were identified. 2-Mercaptobenzothiazole (2-MBT) was the main metabolite, and its excretion varied according to the dose. The second metabolite was 2-(mercaptomethylthio)benzothiazole. The amount of 2-MBT excreted in rat urine was 66 +/- 12% (SD), 51 +/- 20% and 44 +/- 9% for TCMTB doses of 15, 75 and 150 mg/kg, respectively. Two doses, 75 and 150 mg/kg, caused diuresis in rats during the 1 week of dosing. During the 3-week TCMTB treatment, rat liver microsomal CYP enzyme profile was not significantly changed.
Organic nitriles are converted into cyanide ions through the action of cytochrome P450 enzymes in the liver. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)

Nawrocki ST, Drake KD, Watson CF, Foster GD, Maier KJ. Comparative aquatic toxicity evaluation of 2-(thiocyanomethylthio)benzothiazole and selected degradation products using Ceriodaphnia dubia. Arch Environ Contam Toxicol. 2005 Apr;48(3):344-50. Epub 2005 Feb 15. PubMed PMID: 15750776.

TCMTB is a wood preservative, marine biocide, contact fungicide, and as a preservative in paint.

C9H6N2S3

238.35234

237.969

C, 45.35; H, 2.54; N, 11.75; S, 40.35

21564-17-0

21564-17-0;

30692

Oil
Vivid orange liquid

1.5±0.1 g/cm3

405.6±47.0 °C at 760 mmHg

<-10 °C

199.1±29.3 °C

0.0±0.9 mmHg at 25°C

1.743

3.12

0

5

3

14

238

0

C1=CC=C2C(=C1)N=C(SCSC#N)S2

TUBQDCKAWGHZPF-UHFFFAOYSA-N

InChI=1S/C9H6N2S3/c10-5-12-6-13-9-11-7-3-1-2-4-8(7)14-9/h1-4H,6H2

1,3-benzothiazol-2-ylsulfanylmethyl thiocyanate

2-(Thiocyanatomethylthio)benzothiazole; 2-(Thiocyanomethylthio)benzothiazole; Benthiazole; Superdavloxan; Thiocyanic acid, Acticide WB 300, Superdavloxan; 2-((Thiocyanatomethyl)thio)benzo[d]thiazole; Alentisan;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)