Toxicity Summary
Based on the results of a quantitative risk assessment (QRA) or similar method, the panel concluded that the ingredient mixture MCI/MI is safe in cosmetics when the formulation is non-allergenic; however, its concentration should not exceed 7.5 ppm in rinse-off products and 15 ppm in rinse-off products.
Safe for use in cosmetics under specific conditions
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 devices. 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 necessary to consult federal, state, and local authorities for currently 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 containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, which appear to be a common cause of contact dermatitis in Europe. Animal Studies: A mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMI/MIT) was dissolved in corn oil and 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, respectively. At the highest CMIT/MIT exposure dose, 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 mild histological changes in the liver. The CMI/MIT mixture induced point mutations in bacteria lacking the rat liver metabolic system (TA 100 strain) 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 needs to be increased 10-fold. No mutagenic activity was observed in the metabolic system or in *Salmonella typhimurium*. No positive results were obtained in the Drosophila sex-linked recessive lethal assay, in vivo cytogenetics assay in mice, non-programmed DNA synthesis assay in cultured rat hepatocytes, and in vitro cell transformation assay. In immunotoxicity studies, protein-bound CMI induced a proliferative response in ear lymph node cells, while MI, with weaker protein binding, neither stimulated a proliferative response nor induced lymph node enlargement at similar concentrations to CMI.

---

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, leading to apoptosis and necrosis. In this study, flow cytometry analysis showed that CMI/MI induces early disruption of calcium homeostasis, resulting in 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, as well as the loss of ΔΨm and S-glutathioneyl groups induced by necrotic doses of CMI/MI, but did not inhibit CMI/MI-induced apoptosis, mitochondrial calcium uptake, and mitochondrial hyperpolarization. This suggests that increased cytoplasmic calcium concentration is not a direct cause of apoptosis, but rather that the interaction between the ER and mitochondria may be a key factor in inducing apoptosis. GSH-OEt pretreatment increases intracellular GSH levels, decreases S-glutathioneization levels, and reduces cytoplasmic and mitochondrial calcium ion concentrations, thereby inhibiting apoptosis and the transition from necrosis to apoptosis. Therefore, changes in GSH consumption and protein S-glutathioneization levels may play a crucial role in the increase in calcium ion concentration. Increased mitochondrial calcium ion concentration may be a cause of apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathioneization of specific proteins may serve as a molecular link between calcium ions and redox signaling.

[1]. In situ chemical behaviour of methylisothiazolinone (MI) and methylchloroisothiazolinone (MCI) in reconstructed human epidermis: a new approach to the cross-reactivity issue. Contact Dermatitis. 2016 Mar;74(3):159-67.

5-Chloro-2-methyl-4-isothiazolin-3-one is a 1,2-thiazole compound with the structure 4-isothiazolin-3-one, 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 has dual effects against bacteria, exogenous substances, and environmental pollutants. It is an organochlorine compound belonging to the 1,2-thiazole class of compounds. Its function is similar to that of methylisothiazolinone. The expert panel concluded, based on the results of a quantitative risk assessment (QRA) or similar method, that MCI/MI mixtures are safe in cosmetics when formulated as non-allergenic; however, in no-rinse products, the concentration should not exceed 7.5 ppm, and in rinse-off products, the concentration should not exceed 15 ppm. Methylchloroisothiazolinone (MCI) is an isothiazolinone compound commonly used as a preservative with antibacterial and antifungal properties. It is found in many commercially available cosmetics, lotions, and makeup removers. It is also a known skin sensitizer and allergen; some side effects include peeling or scaling of the skin, rash, redness or itching, and moderate to severe swelling around the eyes. The American Contact Dermatology Association named methylchloroisothiazolinone its 2013 Contact Allergen of the Year. Sensitivity to methylchloroisothiazolinone can be identified through clinical patch testing. Methylchloroisothiazolinone is a standardized chemical allergen. The physiological effects of methylchloroisothiazolinone are achieved through increased histamine release and cell-mediated immunity.

---

Drug Indication
Methylchloroisothiazolinone has been approved for use in allergic skin patch testing, which is indicated for the auxiliary diagnosis of allergic contact dermatitis (ACD) in individuals aged 6 years and older.

C4H4CLNOS

149.60

148.97

26172-55-4

5-Chloro-2-methylisothiazol-3(2H)-one-d3;1329611-34-8

33344

Liquid

1.5±0.1 g/cm3

200.2±50.0 °C at 760 mmHg

54-55 °C

74.9±30.1 °C

0.3±0.4 mmHg at 25°C

1.628

0.24

0

2

0

8

156

0

ClC1=C([H])C(N(C([H])([H])[H])S1)=O

DHNRXBZYEKSXIM-UHFFFAOYSA-N

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

5-chloro-2-methyl-1,2-thiazol-3-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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