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.

[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.

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.)