Benzisothiazolone targets microbial cells through its isothiazolone ring structure, which reacts with cellular thiols (cysteine residues) in enzymes and proteins, disrupting essential metabolic processes. The compound's fused benzene and isothiazolone ring structure gives it strong biocidal properties. BIT inhibits the growth of bacteria and fungi by interfering with cellular respiration and energy metabolism. Some BIT derivatives have been evaluated for antiproliferative activity against leukemia and solid tumor cell lines, showing cytotoxicity at micromolar concentrations.

In vitro, Benzisothiazolone demonstrates growth inhibitory activity against Escherichia coli ATCC 8739 and yeast NCYC 1354. The compound is effective against a broad spectrum of bacteria, fungi, and algae. BIT derivatives have been evaluated for their antiproliferative activity against leukemia and solid tumor cell lines, with cytotoxicity observed at micromolar concentrations. The presence of the heterocyclic ring, methyl group, and phenyl ring is crucial for optimal antimicrobial and antiproliferative activity. The compound's activity is concentration-dependent.

In vivo, Benzisothiazolone is not used as a therapeutic agent but is applied in industrial and consumer products as a preservative and biocide. Its in vivo effects have been studied in the context of environmental toxicology and occupational exposure. BIT has been frequently detected in aquatic environments, raising concerns over its potential endocrine-disrupting effects and toxicity to aquatic organisms. The compound is not administered to animals for therapeutic purposes.

Non-cell-based assays for Benzisothiazolone involve antimicrobial susceptibility testing. Minimum inhibitory concentration (MIC) is determined against bacterial and fungal strains using broth microdilution or agar dilution methods. The compound's chemical properties are characterized by NMR, MS, and HPLC. Its stability in various formulations and its degradation products are studied. Binding studies with target enzymes or proteins may be performed to elucidate the mechanism of action. Cytotoxicity assays using cancer cell lines may also be conducted.

Cellular assays for Benzisothiazolone are performed using bacterial and fungal strains for antimicrobial testing. Broth microdilution assays are used to determine MIC values. Time-kill assays are used to evaluate bactericidal or fungicidal activity. For antiproliferative activity, cancer cell lines (leukemia, solid tumors) are treated with BIT derivatives at various concentrations, and cell viability is measured by MTT or CellTiter-Glo assays. Apoptosis is assessed by Annexin V/PI staining. The compound's effects on cellular metabolism and signaling pathways are studied.

Benzisothiazolone is not used in animal experiments as a therapeutic agent. It is a biocide used in industrial applications. In environmental toxicology studies, BIT is tested on aquatic organisms (fish, algae, daphnia) to assess ecotoxicity. Endocrine-disrupting effects are evaluated in animal models. Occupational exposure studies assess the compound's toxicity to workers handling BIT-containing products. However, the compound is not administered to animals for pharmacological evaluation.

Absorption, Distribution and Excretion
A skin penetration study was conducted in rats, where 10 mg/kg of 1,2-benzisothiazolin-3-one was applied to the skin for 4, 8, 24, 48, or 72 hours. After 72 hours, the skin penetration reached a maximum of 40.6%.

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Pharmacokinetic properties are not applicable to Benzisothiazolone as it is not a therapeutic agent. The compound is used as an industrial biocide and preservative. Its chemical stability, solubility, and compatibility with various formulations are characterized for industrial applications. Environmental persistence and degradation are studied for regulatory purposes. No PK data are available as the compound is not a drug.

Toxicity Summary
Identification and Uses: 1,2-Benzisothiazolin-3-one (BIT) is a grayish-white to pale yellow solid. It is registered as an insecticide in the United States, but its approved insecticide uses may change periodically, so it is essential to consult federal, state, and local authorities for currently approved uses. BIT is used as an antimicrobial agent in cosmetics; as a slime-killing agent in the manufacture of disposable powder-free PVC gloves; and widely in industry as a preservative for aqueous solutions such as pastes, paints, and cutting oils. It is also used in disinfectants and other sterilizing products in private and public health areas, as an in-can preservative, a film preservative, a preservative for fibers, leather, rubber, and polymers, a masonry preservative, a preservative for liquid cooling and processing systems, a preservative for metalworking fluids, and as a preservative solution and specimen preparation solution. Human Exposure and Toxicity: Skin contact with adequate doses and durations of BIT can cause sensitization and allergic contact dermatitis in susceptible individuals. There have been reports of a 26-year-old male working in a chemical plant (producing detergents) developing occupational asthma and rhinitis after inhaling BIT. Animal studies show that BIT has severe eye irritation. Subchronic oral toxicity studies in rats have shown systemic effects after repeated oral administration, including weight loss, increased incidence of forestomach hyperplasia, and nonglandular gastric lesions. In dogs, BIT produces these effects at lower doses, including changes in blood chemistry (decreased plasma albumin, total protein, and alanine aminotransferase) and an increase in absolute liver weight. In developmental toxicity studies in rats, maternal effects were observed, including decreased weight gain, reduced food intake, and clinical toxicity symptoms (audible breath sounds, staining of hair in the anal and genital areas, and dry brown material around the nose), as well as increased mortality. Developmental effects manifested as increased skeletal abnormalities (increased ossification of the skull, and unossified sternum), but no external or visceral abnormalities were observed.

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Non-human toxicity values
Oral LD50 in rats: 1020 mg/kg
Oral LD50 in mice: 1150 mg/kg

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The toxicity of Benzisothiazolone has been evaluated in the context of its use as an industrial biocide. BIT can cause skin irritation and sensitization in humans, and occupational exposure is a concern. The compound is toxic to aquatic organisms and has been detected in aquatic environments, raising concerns over its potential endocrine-disrupting effects. Comprehensive toxicological studies including genotoxicity, reproductive toxicity, and organ toxicity have been conducted for regulatory purposes. BIT is not intended for human consumption or therapeutic use.

[1]. Collier PJ, et al. Growth inhibitory and biocidal activity of some isothiazolone biocides. J Appl Bacteriol. 1990 Oct;69(4):569-77.

Benzo[d]isothiazol-3-one is an organic heterobicyclic compound whose structure is based on a fused 1,2-thiazole and benzene bicyclic skeleton, with the sulfur atom located in an adjacent position on the fused ring. It can be used as a disinfectant, platelet aggregation inhibitor, environmental pollutant, exogenous substance, drug allergen, and sensitizer. It is an organic nitrogen heterocyclic compound and an organic heterobicyclic compound. It is an industrial bactericide. 1,2-Benzisothiazol-3(2H)-one is found in can sealing adhesives. 1,2-Benzisothiazol-3(2H)-one belongs to the benzothiazolium class of compounds. These organic compounds are formed by the fusion of a benzene ring and a thiazolium ring (a five-membered ring consisting of four carbon atoms, one nitrogen atom, and one sulfur atom).

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Benzisothiazolone (CAS# 2634-33-5) is an isothiazolinone fungicide and antimicrobial biocide with the molecular formula C₇H₅NOS and a molecular weight of 151.19. It is also known as 1,2-benzisothiazol-3(2H)-one and BIT. The compound exhibits broad-spectrum antimicrobial activity against bacteria, fungi, and algae and is widely used in industrial and consumer products including paints, adhesives, metalworking fluids, and household products. BIT has growth inhibitory activity against E. coli ATCC 8739 and yeast NCYC 1354. The compound is not approved as a therapeutic agent and is used exclusively for industrial and research applications.

C7H5NOS

151.19

151.009

2634-33-5

Benzisothiazolinone-13C5;1329616-16-1

17520

Off-white to yellowish solid
White to off-white fine, crystalline powder
Technical product (commercial) can be in the form of a solid paste that is off-white to brown in color

1.4±0.1 g/cm3

204.5±23.0 °C at 760 mmHg

156.6 °C ; 157 - 158 °C

77.5±22.6 °C

0.2±0.4 mmHg at 25°C

1.745

1.78

1

2

0

10

160

0

S1C2=C([H])C([H])=C([H])C([H])=C2C(N1[H])=O

DMSMPAJRVJJAGA-UHFFFAOYSA-N

InChI=1S/C7H5NOS/c9-7-5-3-1-2-4-6(5)10-8-7/h1-4H,(H,8,9)

1,2-benzothiazol-3-one

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.)