Not reported in the provided literature [1]

For CMIT/MIT mixture: mitochondria (electron transport chain complexes, oxidative phosphorylation); induces mitochondrial fragmentation via increased Drp1 Ser616 phosphorylation and decreased Mfn-1 expression [2]

MIT (2, 4, 8 μg/mL) induced dose-dependent cytotoxicity in BEAS-2B human bronchial epithelial cells after 24 h exposure, with cell viability reduced to 81.0±3.8%, 67.9±5.1%, and 35.1±5.2% of control, respectively [1]

MIT induced apoptotic cell death: early apoptosis proportions were 4.5±0.5%, 4.1±0.4%, 7.3±0.2%, and 9.0±0.4% at 0, 2, 4, and 8 μg/mL, respectively; late apoptosis proportions were 6.2±1.2%, 6.6±0.7%, 18.3±1.2%, and 39.4±0.7%, respectively. Apoptotic bodies were observed under phase-contrast microscopy [1]

MIT did not cause cell cycle blockade [1]

MIT impaired organelle structure and function: TEM images showed abnormal double membrane formation within the nucleus, altered mitochondrial structure, and nucleolus aggregation at 8 μg/mL [1]

MIT increased mitochondrial volume to 119.3±2.0% and mitochondrial membrane potential to 147.4±3.5% of control at 8 μg/mL, while ATP production decreased dose-dependently (almost completely inhibited at 8 μg/mL). ER volume decreased to 80.7±8.5%, 69.8±18.8%, and 43.6±3.7% of control at 2, 4, and 8 μg/mL, respectively. LDH release was not significantly changed [1]

MIT increased intracellular ROS to 123.8±1.7% of control at 8 μg/mL, decreased NO level (control 17.3±1.3 μM, 8 μg/mL 14.9±1.4 μM), and elevated catalase activity (control 6.7±0.1, 2 μg/mL 7.9±0.8, 4 μg/mL 8.0±1.1, 8 μg/mL 10.4±0.8 nmol/μg) [1]

Microarray analysis (8 μg/mL MIT for 24 h) identified 232 down-regulated genes (e.g., histone cluster-related genes, microRNA1206, metallothionein1F) and 1197 up-regulated genes (e.g., MMP-1, MMP-3, MMP-10, FBJ osteosarcoma viral oncogene homolog B, keratin associated protein 4-12, small nucleolar RNAs) [1]

MIT increased secretion of inflammatory mediators: IL-1β (24.4±0.8 pg/mL control vs 150.6±12.4 pg/mL at 8 μg/mL), IL-6 (201.0±52.3 vs 682.8±65.4 pg/mL), IL-10, IFN-γ, and IL-8. MMP-1 and MMP-3 levels were also elevated (MMP-1: 92.1±2.7 vs 294.3±52.1 pg/mL; MMP-3: 3.4±0.5 vs 62.7±38.6 pg/mL) [1]

MIT enhanced expression of Apaf-1, fibrillarin (with aggregation), p-p53, cytochrome C, and sodium potassium ATPase proteins. LC3-I was enhanced but LC3-II was not notable [1]

For CMIT/MIT mixture (3:1) in bEND.3 murine brain endothelial cells: at 1 h treatment, MTT reduction decreased significantly before LDH leakage. At 2.5 μg/mL, LDH leakage increased after 3 h. CMIT/MIT (1 and 2.5 μg/mL for 1 h) significantly increased mitochondrial ROS, decreased mitochondrial membrane potential (JC-1), and decreased mitochondrial mass (NAO staining). Oxygen consumption rate (OCR) was inhibited within 20 min of exposure, and impairment persisted after washout. CMIT/MIT decreased Mfn-1 expression and increased Drp1 Ser616 phosphorylation, leading to mitochondrial fragmentation. Mitophagy was activated (increased LC3BII/LC3BI ratio, colocalization of mitochondria with autophagosomes and lysosomes). COX IV protein level decreased at 2.5 μg/mL [2]

In hCMEC/D3 human brain endothelial cells: CMIT/MIT (1 and 2.5 μg/mL for 1 h) increased mitochondrial ROS, decreased MMP, decreased mitochondrial mass (at 2.5 μg/mL), and inhibited OCR (basal respiration, ATP production, reserved capacity, maximal respiration) [2]

Co-exposure of bEND.3 cells to CMIT/MIT with oxygen-glucose deprivation (OGD) or methylglyoxal (MG) potentiated cell damage: increased LDH release, decreased MTT reduction, increased Drp1 phosphorylation, decreased TEER. Co-exposure with MG decreased glyoxalase-2 expression and reduced tight junction proteins ZO-1, claudin-5, and occludin [2]

In male ICR mice (6-week-old, 4 mice/group), a single intratracheal instillation of MIT (0, 25, 50, 100 μg/mouse) was administered. Blood was collected at day 1 after instillation. Hematological analysis showed that the proportion of neutrophils in whole blood significantly increased in mice exposed to 2 μg MIT compared to control (preliminary test) [1]

For CMIT/MIT mixture in male SD rats (7-8 weeks old, 230-300 g): intravenous administration via caudal vein of saline or CMIT/MIT (0.05 and 0.15 mg/kg body weight) at a volume of 0.4 mL. Three hours after injection, brain tissue was isolated. Brain endothelial cells were isolated by density gradient centrifugation. CMIT/MIT (0.15 mg/kg) significantly decreased mitochondrial membrane potential and mitochondrial mass, increased mitochondrial ROS production, and decreased tight junction proteins ZO-1 (0.472-fold), claudin-5 (0.259-fold), and occludin (0.558-fold) compared to vehicle control [2]

Measurement of catalase activity: cell pellets were homogenized in cold assay buffer and centrifuged at 10,000g for 15 min at 4°C. Supernatants were reacted with 1 mM hydrogen peroxide at 25°C for 30 min, then stop solution added. Absorbance measured at 540 nm, and values calculated using a standard curve [1]

PAD activity not applicable (no enzyme target reported for MIT). For CMIT/MIT: mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored using an extracellular flux analyzer. Cells were seeded on XFp plates. Assay medium contained 5.5 mM glucose, 2 mM L-glutamine, 1 mM sodium pyruvate for bEND.3; 25 mM glucose, 2 mM L-glutamine, 1 mM sodium pyruvate for hCMEC/D3. CMIT/MIT, oligomycin, FCCP, and rotenone/antimycin A were serially injected. Parameters (basal respiration, ATP production, maximal respiration, reserved capacity) were calculated from OCR profiles [2]

Cell viability (MTT assay): BEAS-2B cells (1×10⁴ cells/mL) seeded in 96-well plates, stabilized overnight, then incubated with MIT (0,2,4,8 μg/mL) for 24 h. Medium removed, FBS-free medium with MTT added, incubated 4 h at 37°C. Formazan dissolved in DMSO, absorbance at 540 nm [1]

Cell cycle and apoptosis: BEAS-2B cells (2×10⁵ cells/mL) in 6-well plates, exposed to MIT (0,2,4,8 μg/mL) for 24 h. For cell cycle, cells fixed in 70% ethanol, treated with RNase and PI. For apoptosis, annexin V and PI staining. Analyzed by flow cytometry [1]

Oxidative stress: After MIT exposure, cells incubated with carboxy-DCF-DA for 30 min, fluorescence measured by flow cytometry. NO level: supernatant reacted with NO detection solution, absorbance at 540 nm [1]

Organelle function: Mitochondrial mass and membrane potential assessed with MitoTracker Green FM and Red CMXRos; ER volume with ER Tracker Green; flow cytometry. ATP production measured with luminescent ATP assay kit [1]

Immunofluorescence: BEAS-2B cells on glass slides fixed with paraformaldehyde and methanol, blocked with BSA, incubated with primary antibodies (sodium potassium ATPase, fibrillarin, Apaf-1, cytochrome C, p-p53), then fluorescent secondary antibodies, mounted with DAPI, visualized by confocal microscopy [1]

ELISA: Cell supernatants collected after 24 h MIT exposure, cytokines (IL-1β, TNF-α, IFN-γ, IL-6, IL-10), chemokines (IL-8, CXCL1), and MMPs (MMP-1, MMP-3) quantified using ELISA kits, absorbance at 450 nm [1]

For CMIT/MIT in bEND.3 and hCMEC/D3 cells: MTT reduction and LDH release assays as described. Oxidative stress: DCF-DA fluorescence; GSH levels measured using GSH-Glo assay (luminescence). Mitochondrial membrane potential (JC-1), mtROS (MitoSOX Red), mitochondrial mass (NAO) by flow cytometry. Western blot for Drp1, p-Drp1 (Ser616), Mfn-1, Mfn-2, LC3B, COX IV. Confocal microscopy for MitoTracker and Lysotracker colocalization. TEM for mitochondrial morphology. TEER for barrier function using transwell inserts and EVOM2 voltmeter [2]

For MIT intratracheal instillation: Six-week-old male ICR mice (4 mice/group) were anesthetized with isoflurane. A single 100 μL instillation of MIT (0, 25, 50, 100 μg/mouse) was administered via trachea. Mice were sacrificed at day 1 after instillation. Blood was obtained from abdominal aorta under anesthesia. Hematological analysis performed using a blood autoanalyzer [1]

For CMIT/MIT intravenous administration: Male SD rats (7-8 weeks old, 230-300 g) were used. CMIT/MIT solution was diluted with saline (0.9% sodium chloride). Saline or diluted CMIT/MIT (0.05 and 0.15 mg/kg body weight) solution was intravenously injected through the caudal vein at a volume of 0.4 mL. Three hours after injection, brain tissue was carefully isolated. Brain endothelial cells were isolated by homogenization, density gradient centrifugation with Ficoll and dextran, and then characterized by PECAM (CD31) and NeuN staining. Purity of ECs (~92.0% CD31 positive) confirmed by flow cytometry [2]

Toxicity Summary
Identification and Uses: 5-Chloro-2-methyl-4-isothiazolin-3-one (CMI) forms crystals. It is used as an antimicrobial preservative in cosmetics, hygiene products, paints, emulsions, cutting oils, paper coatings, and water storage and cooling systems. CMI is also used in hydraulic fracturing fluids. It is registered as a pesticide in the United States, but approved pesticide uses may change periodically, so it is essential to consult federal, state, and local authorities for current approved uses. CMI is often used in combination with 2-methyl-4-isothiazolin-3-one (MI). Human Exposure and Toxicity: Kathon CG is a cosmetic preservative with the active ingredients 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, and appears to be a common cause of contact dermatitis in Europe. Animal experiments: A mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMI/MIT) dissolved in corn oil was administered to male and female rats by gavage at doses of 0, 0.26, 0.78, 2.33, and 7.0 mg/kg body weight/day. At the highest CMIT/MIT exposure, decreased serum triglyceride levels were observed in male rats, and increased activity of phase I hepatic xenobiotic metabolic enzymes was observed in female rats, accompanied by slight histological changes in the liver. The CMI/MIT mixture induced point mutations in bacteria (TA 100 strain) lacking the rat liver metabolic system and in cultured mammalian cells. In the presence of the rat liver metabolic system, the concentration required to induce point mutations in cultured mammalian cells was 10-fold higher. No mutagenic activity was observed in the metabolic system and in Salmonella typhimurium. Negative results were obtained in Drosophila sex-linked recessive lethality assays, in vivo cytogenetic assays in mice, non-programmed DNA synthesis assays in cultured rat hepatocytes, and in vitro cell transformation assays. In immunotoxicity studies, protein-bound CMI induced a proliferative response in ear lymph node cells, while MI, with weaker protein binding, neither stimulated proliferation nor induced lymph node enlargement under similar CMI concentrations.

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Interactions
We previously reported that transient exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces oxidative stress, thereby inducing apoptosis and necrosis. In this study, flow cytometry analysis revealed that CMI/MI can induce early disturbances in calcium homeostasis, leading to increased cytoplasmic and mitochondrial calcium concentrations and depletion of the intracellular endoplasmic reticulum (ER) calcium pool. The calcium chelator BAPTA-AM alleviated necrosis and secondary necrosis induced by necrotic doses of CMI/MI, as well as the loss of ΔΨm and S-glutathioneylation, but did not inhibit CMI/MI-induced apoptosis, mitochondrial calcium uptake, and mitochondrial hyperpolarization. This suggests that elevated cytoplasmic calcium concentration is not a direct cause of apoptosis, but rather that the interaction between the endoplasmic reticulum and mitochondria may be a key factor in inducing apoptosis. GSH-OEt pretreatment increased intracellular GSH levels, decreased S-glutathioneylation levels, and reduced cytoplasmic and mitochondrial calcium ion levels, thereby protecting cells from apoptosis and necrosis and promoting apoptosis. Therefore, the degree of GSH consumption is closely related to protein S-glutathioneylation levels and may play a causal role in elevated calcium ion levels. Elevated mitochondrial calcium ion levels may be a cause of apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathioneylation of specific proteins plays a molecular linking role between calcium ions and redox signaling.

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MIT: LDH release (membrane integrity marker) was not significantly changed at 2,4,8 μg/mL in BEAS-2B cells, indicating no necrotic cell death under these conditions [1]

[1]. Methylisothiazolinone induces apoptotic cell death via matrix metalloproteinase activation in human bronchial epithelial cells. Toxicol In Vitro. 2020 Feb;62:104661.

[2]. Functional and dynamic mitochondrial damage by chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture in brain endothelial cell lines and rat cerebrovascular endothelium. Toxicol Lett. 2022 Aug 1;366:45-57.

[3]. Methylisothiazolinone: dermal and respiratory immune responses in mice. Toxicol Lett. 2015 Jun 15;235(3):179-88.

[4]. Effects of chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) on Th2/Th17-related immune modulation in an atopic dermatitis mouse model. Sci Rep. 2020 Mar 5;10(1):4099.

4-Isothiazolinone (MCI) is a 1,2-thiazole compound with the structure 4-isothiazolin-3-one, consisting of a methyl group attached to the nitrogen atom and a chlorine atom attached to the C-5 position. It is a potent bactericide and preservative, and the main active ingredient in the commercial product Kathon™. It possesses antibacterial, xenobiotic, and environmentally friendly properties. It belongs to the 1,2-thiazole class of compounds and organochlorine compounds. Its function is related to methylisothiazolinone. Methylchloroisothiazolinone (MCI) is a commonly used isothiazolinone compound with antibacterial and antifungal properties, often used as a preservative. It is found in many commercially available cosmetics, lotions, and makeup removers. Furthermore, it is a known skin sensitizer and allergen. Some of its side effects include skin peeling or scaling, rash, redness or itching, and moderate to severe swelling around the eyes. The American Contact Dermatology Association named MCI the 2013 Contact Allergen of the Year. Sensitivity to methylchloroisothiazolinone can be identified through clinical patch testing. Methylchloroisothiazolinone is a standardized chemical allergen. Its physiological effects are achieved through increased histamine release and cell-mediated immunity. See also: Magnesium chloride (note moved to).

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Drug Indications
Methylchloroisothiazolinone is approved for use in allergic skin patch testing to aid in the diagnosis of allergic contact dermatitis (ACD) in individuals aged 6 years and older.

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MIT is an isothiazolinone biocide used as preservative. In Korea, MIT has been suspected to induce idiopathic pulmonary fibrosis associated with humidifier disinfectant use. This study demonstrates that MIT induces apoptotic cell death and inflammatory response via matrix metalloproteinase activation in human bronchial epithelial cells. KEGG pathway analysis suggested possible carcinogenesis following MIT exposure [1]

CMIT/MIT is a 3:1 mixture of chloromethylisothiazolinone and methylisothiazolinone, used as a biocide in consumer products and humidifier disinfectants. This study shows that CMIT/MIT impairs mitochondrial function and dynamics in brain endothelial cells, leading to barrier dysfunction. The effects are potentiated by sub-threshold pathological stimuli such as hypoxia (OGD) or glycative stress (methylglyoxal). Intravenous administration in rats induced mitochondrial damage and decreased tight junction proteins in brain endothelium [2]

C4H5NOS

115.15

115.009

2682-20-4

Methylisothiazolinone hydrochloride;26172-54-3

33344

White to off-white solid powder

1.3±0.1 g/cm3

182.8±23.0 °C at 760 mmHg

254-256 °C(lit.)

64.3±22.6 °C

0.8±0.3 mmHg at 25°C

1.589

0.05

0

2

0

8

156

0

DHNRXBZYEKSXIM-UHFFFAOYSA-N

InChI=1S/C4H4ClNOS/c1-6-4(7)2-3(5)8-6/h2H,1H3

2-methyl-3(2H)-isothiazolone

MI N-Methylisothiazolin-3-oneMethylisothiazolinone free base KB-838

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)

DMSO : ~100 mg/mL (~868.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (21.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (21.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (21.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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