Chlorhexidine Gluconate demonstrated cytotoxic effects on Chinese hamster ovary (CHO) cells in a dose-dependent and time-dependent manner (P < 0.05). Cell viability was assessed by MTT assay. At concentrations lower than 10⁻⁴% (w/v), cell viability was negatively correlated with treatment time. Treatment with 10⁻⁴% CHX for over 1 hour showed significant reduction in cellular viability, suggesting maximum effect at this concentration and time point [2].
Flow cytometry analysis using Annexin V-FITC and propidium iodide (PI) staining revealed that Chlorhexidine Gluconate induced two modes of cell death. After 1 hour treatment, at concentrations of 5×10⁻⁵%, 10⁻⁴%, and 5×10⁻⁴%, apoptosis rates were 1.62%, 5.51%, and 5.47%, respectively, while necrosis rates were 2.38%, 8.62%, and 22.50%, respectively. As CHX concentration increased, the mode of cell death shifted from apoptosis to necrosis [2].
Chlorhexidine Gluconate induced superoxide anion generation in CHO cells in a dose-dependent manner (P < 0.05). The levels of superoxide anion induced by CHX were 0.96 nmol (control), 1.35 nmol (10⁻⁵%), 2.03 nmol (5×10⁻⁵%), 1.95 nmol (10⁻⁴%), and 1.54 nmol (5×10⁻⁴%) [2].
The addition of superoxide dismutase (SOD) decreased Chlorhexidine Gluconate-induced cytotoxicity (P < 0.05). CHX at 10⁻⁴% caused approximately 50% cell death over 1 hour. Pretreatment with 10 units, 100 units, and 1000 units SOD enhanced cell viability to 64%, 88%, and 92%, respectively, suggesting that superoxide anion generation plays a role in CHX-induced cell death [2].

Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection and cultured in Eagle's minimum essential medium (MEM) with 10% fetal calf serum, 100 units/mL penicillin, and 100 mg/L streptomycin at 37°C in a humidified atmosphere of 5% CO₂ and 95% air [2].
For cytotoxicity assays, cells were seeded in 96-well plates (2×10⁴ cells/well) and allowed to attach overnight. Cells were then treated with various concentrations of Chlorhexidine Gluconate (0%, 10⁻⁵%, 5×10⁻⁵%, 10⁻⁴%, and 5×10⁻⁴% w/v) for 1, 2, or 4 hours. After treatment, 50 μL MTT dye was added to each well and incubated for 4 hours. The formazan crystals were dissolved in dimethyl sulfoxide, and optical density was measured at 550 nm using a spectrophotometer. Cell viability was expressed as a percentage relative to untreated controls [2].
For apoptosis and necrosis detection, cells were treated with various concentrations of Chlorhexidine Gluconate (10⁻⁵%, 5×10⁻⁵%, 10⁻⁴%, and 5×10⁻⁴%) for 1 hour. Cells were collected by trypsinization, washed with ice-cold PBS, and incubated with Annexin V-FITC and propidium iodide (PI) in binding buffer for 30 minutes at room temperature in the dark. Samples were analyzed by flow cytometry, and percentages of viable cells (Annexin V⁻/PI⁻), early apoptotic cells (Annexin V⁺/PI⁻), late apoptotic cells (Annexin V⁺/PI⁺), and necrotic cells (Annexin V⁻/PI⁺) were determined [2].
For superoxide anion measurement, CHO cells (1×10⁵ cells) were suspended in a cuvette with 40 μM ferricytochrome c in a final volume of 1.5 mL. A reference cuvette also received 17.5 U/mL superoxide dismutase. Various concentrations of Chlorhexidine Gluconate were added to both cuvettes, and absorbance changes were monitored continuously for 5 minutes at 550 nm. Superoxide anion amount was calculated using the formula: superoxide anion (nmol) = 71.55 × absorbance [2].
For SOD protection experiments, cells were pretreated with 10, 100, or 1000 units of superoxide dismutase for 15 minutes before adding the IC₅₀ concentration of Chlorhexidine Gluconate (10⁻⁴%) and coincubated for 1 hour. Cytotoxicity was then assessed by MTT assay [2].

Absorption, Distribution and Excretion
When applied topically, chlorhexidine undergoes virtually no systemic absorption. Oral chlorhexidine, such as that used in dental mouthwash, is absorbed very poorly in the gastrointestinal tract—the peak plasma concentration (Cmax) after oral administration of 300 mg chlorhexidine is 0.206 μg/g, with a time to peak concentration (Tmax) of approximately 30 minutes after ingestion. No chlorhexidine levels were detected in plasma or urine after implantation of four Periodips in 18 adult patients. Chlorhexidine gluconate is almost entirely excreted in feces, with less than 1% of the ingested dose excreted in urine. One study investigated 34 newborns who underwent standard bathing with Hibiscrub to determine whether it was absorbed percutaneously. Low concentrations of chlorhexidine were detected in blood samples from 10 infants collected via heel prick, and also in 5 of the 24 infants who received intravenous blood samples. /Chlorhexidine Gluconate/
The transdermal absorption of the antibacterial agent chlorhexidine (carbon-14 labeled) was studied in rats. Within 5 days, the absorption rate of topically applied chlorhexidine was less than 5%. The absorbed radioactive material was primarily excreted in feces.
The transdermal absorption of chlorhexidine gluconate (chlorhexidine disodium; Hibitane) through the skin (with or without stratum corneum) of hairless rats was studied. In tests conducted on intact skin, drug storage in the skin tissue was more important than diffusion after 48 hours; the opposite was observed for skin with the stratum corneum removed. When the skin was peeled, the absorption increased approximately 100-fold, and the amount stored in the skin increased approximately 10-fold. The difference in chlorhexidine diffusion observed between intact and peeled skin was related to the physicochemical properties of chlorhexidine. /Chlorhexidine Gluconate/
This study aimed to evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in dairy cows. …The study was conducted in two phases. Three cows were studied in each phase. All cows received intramammary infusion of chlorhexidine suspension after two milkings 24 hours apart. Foremilk samples (100 mL) were collected from the udders of both the treated and untreated (control) cows. Chlorhexidine was extracted from the raw milk, and residual concentrations were quantitatively analyzed using high-performance liquid chromatography (HPLC). Foremilk samples were analyzed on days 2, 5, and 8 in the first phase; and on days 0, 3, 7, 14, 21, 28, 35, and 42 in the second phase. No quantitative transfer of chlorhexidine to milk was detected in the untreated mammary gland area in either phase. During the 42-day sampling period in the second phase, measurable chlorhexidine residues were detected in the milk from the treated mammary gland area of two cows. The mean elimination half-life of chlorhexidine in milk was estimated to be 11.5 days.
Metabolism/Metabolites
Due to the extremely low absorption rate of chlorhexidine in the gastrointestinal tract, significant metabolic transformation is unlikely.

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Biological Half-Life
This study aimed to evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in dairy cows. …The study was conducted in two phases. Three cows were studied in each phase. All cows received chlorhexidine suspension infusion after two milkings 24 hours apart, with the suspension injected into the mammary gland area affected by mastitis. Foremilk samples (100 mL) were collected from the treated and untreated (control) areas of each cow. Chlorhexidine was extracted from the raw milk, and residual concentrations were quantitatively analyzed using high-performance liquid chromatography. In the first phase, foremilk samples were analyzed on days 2, 5, and 8; in the second phase, samples were analyzed on day 0 and on days 3, 7, 14, 21, 28, 35, and 42. In both phases, no quantitative transfer of chlorhexidine to milk was detected in the untreated mammary gland area. During the 42-day sampling period in the second phase, measurable chlorhexidine residues were detected in the milk from the treated mammary gland areas of two cows. The estimated mean elimination half-life of chlorhexidine in breast milk is 11.5 days.

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Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Chlorhexidine has been used prenatally for topical application to the vagina, abdomen, or perineum to prevent infection. There are currently no reports of toxicity in breastfed infants, and in these cases, chlorhexidine is significantly less toxic than povidone-iodine. One study suggests that topical application of chlorhexidine to the breast before and after breastfeeding does not appear to have adverse effects on breastfed infants. Mothers using chlorhexidine mouthwash during lactation are unlikely to have adverse effects on their infants.
◉ Effects on Breastfed Infants
A group of researchers in Belgium reviewed the results of thyroid-stimulating hormone (TSH) levels on day 5 postpartum in 4,745 newborns delivered at their hospital over two years. Infants were divided into two groups: one group whose mothers experienced iodine overload during delivery due to topical application of 10% povidone-iodine solution (n = 3086), and another group whose mothers did not experience iodine overload (n = 1659). There are two methods of applying povidone-iodine to mothers: one is a single application to 900 square centimeters of skin during epidural anesthesia, and the other is three applications to the entire abdominal wall during cesarean section. Compared with infants without iodine overload (0.1%), breastfed infants whose mothers experienced iodine overload had a higher risk of elevated thyroid-stimulating hormone (TSH) levels and the need for follow-up testing (3.2% in the cesarean section group and 2.7% in the epidural anesthesia group). Bottle-fed infants were significantly less affected than breastfed infants. After the institution replaced povidone-iodine with 0.5% chlorhexidine (dissolved in 70% isopropanol) for disinfection for 6 months, no abnormal elevations in thyroid function tests were found in 1178 infants born at the institution, and the recall rate of breastfed infants was also reduced. A Spanish study observed mothers who, starting in the final stage of labor, applied 10% povidone-iodine (n=21) or chlorhexidine (n=13) topically to the perineum and continued to apply it to the episiotomy site daily postpartum. The results showed no difference in thyroid-stimulating hormone (TSH), thyroxine, or free thyroxine levels in breastfed infants 5 to 7 days postpartum. Some studies in Africa have attempted to reduce the incidence of mother-to-child transmission (MTCT) of HIV by using chlorhexidine vaginally before delivery. One study included 4078 women who had their vaginal walls wiped with swabs soaked in 0.25% chlorhexidine solution every 4 hours from admission to delivery. Another study included 309 women who had their vaginas doucheed with 120 ml of 0.2% or 0.4% chlorhexidine solution every 3 hours from admission to delivery. The average number of douches was 2.1 (range 1 to 11). For women whose membranes had ruptured for more than 4 hours, wiping with 0.25% chlorhexidine swabs reduced the rate of MTCT, but this effect was not observed in other women. Vaginal douching showed a trend toward reducing the rate of MTCT, but the effect was not statistically significant; the 0.4% group was more effective than the 0.2% group. Almost all infants in these studies were breastfed. No adverse events were reported in the infants, but follow-up focused primarily on infant mortality and HIV infection status, rather than the effects of chlorhexidine itself.
◉ Effects on Breastfeeding and Milk
A randomized study compared the effects of a 0.2% chlorhexidine alcohol solution versus a distilled water spray on the breasts of 200 breastfeeding mothers. Mothers sprayed their breasts with the solution before and after each breastfeeding session. Mothers and infants were assessed at discharge and weekly thereafter. Compared to the placebo group, the chlorhexidine group had a lower incidence of discomfort and nipple damage, especially at the initial assessment. Although the skin flora on the breasts of treated mothers was reduced, there was no difference in the incidence of mastitis between the treatment and placebo groups. No significant side effects were observed in breastfed infants, and there was no difference in the incidence of thrush between the two treatment methods. A systematic review concluded that, based on current evidence, this practice is unreasonable.
Protein Binding

Chlorhexidine is known to bind to albumin in serum and saliva, but the extent of this binding is unclear.

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Chlorhexidine Gluconate exhibits significant cytotoxicity in a dose-dependent and time-dependent manner. At concentrations lower than 10⁻⁴%, cell viability decreased with longer exposure times. The IC₅₀ (50% inhibitory concentration) for a 1-hour exposure was approximately 10⁻⁴% w/v. At higher concentrations, the mode of cell death shifts from apoptosis to necrosis, with necrosis reaching 22.50% at 5×10⁻⁴% after 1 hour [2].
Chlorhexidine Gluconate induces superoxide anion generation, and the addition of the free radical scavenger superoxide dismutase significantly reduces CHX-induced cytotoxicity, indicating that oxidative stress contributes to its toxic effects [2].

[1]. Peritoneal Lavage Using Chlorhexidine Gluconate at the End of Colon Surgery Reduces Postoperative Intra-Abdominal Infection in Mice. J Surg Res. 2015 May 1;195(1):121-7.

[2]. Assessment of the cytotoxicity of chlorhexidine by employing an in vitro mammalian test system. Journal of Dental Sciences. Volume 9, Issue 2, June 2014, Pages 130-135.

[3]. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli. PLoS One. 2012;7(5):e36659.

Chlorhexidine gluconate is an organochlorine compound and a D-gluconic acid adduct. It is an antibacterial agent with functions similar to chlorhexidine. Chlorhexidine is a broad-spectrum antibacterial biguanide compound used as a topical disinfectant and in dentistry to treat inflammatory dental diseases caused by microorganisms. It is one of the most commonly used skin and mucous membrane disinfectants. The molecule itself is a cationic biguanide, consisting of two 4-chlorobenzene rings and two biguanide groups linked by a central hexamethylene chain. Topical chlorhexidine used for disinfection, as well as dental mouthwash, are active against a variety of pathogens, including bacteria, yeasts, and viruses. Chlorhexidine was developed by Imperial Chemical Industries in the early 1950s and introduced to the United States in the 1970s. Due to numerous reports of chemical and thermal burns caused by the use of 0.5% chlorhexidine gluconate tincture, the U.S. Food and Drug Administration (FDA) revoked the product's marketing authorization. Other formulations of chlorhexidine remain available for use. Chlorhexidine is a biguanide compound used as a disinfectant with topical antibacterial activity. Chlorhexidine carries a positive charge and reacts with the negatively charged cell surface of microorganisms, thereby disrupting the cell membrane integrity. Subsequently, chlorhexidine penetrates the cell, causing leakage of intracellular components and ultimately leading to cell death. Gram-positive bacteria, which carry a predominantly negative charge, are more sensitive to chlorhexidine. Chlorhexidine gluconate is the gluconate salt form of chlorhexidine, a biguanide compound used as a disinfectant with local antibacterial activity. Chlorhexidine gluconate carries a positive charge and reacts with the negatively charged cell surface of microorganisms, thereby disrupting the cell membrane integrity. Subsequently, chlorhexidine gluconate penetrates the cell, causing leakage of intracellular components and ultimately leading to cell death. Gram-positive bacteria, which carry a predominantly negative charge, are more sensitive to this drug. See also: chlorhexidine gluconate; isopropanol (ingredient); alcohol; chlorhexidine gluconate (ingredient); benzalkonium chloride; chlorhexidine gluconate (ingredient)... See more...

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Drug Indications
Chlorhexidine is an over-the-counter drug available in various dosage forms (e.g., solution, sponge, cloth, swabs) and can be used as a topical disinfectant for sterilization before surgery and/or medical procedures. Prescription-only dental preparations include mouthwashes for treating gingivitis and sustained-release "chips" that can be implanted in periodontal pockets to reduce pocket depth in adults with periodontitis, as an adjunct to scaling and root planing.
FDA Label

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Mechanism of Action
Chlorhexidine's broad-spectrum antibacterial action stems from its ability to disrupt microbial cell membranes. Positively charged chlorhexidine molecules react with negatively charged phosphate groups on the surface of microbial cells—a reaction that both disrupts cell integrity, leading to leakage of intracellular substances, and allows chlorhexidine to enter the cell, causing precipitation of cytoplasmic components, ultimately leading to cell death. The specific manner of cell death depends on the concentration of chlorhexidine—lower concentrations have an antibacterial effect, leading to leakage of intracellular substances (such as potassium and phosphorus), while higher concentrations have a bactericidal effect, leading to cytoplasmic precipitation.

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Chlorhexidine Gluconate is a synthetic cationic molecule containing two 4-chlorophenyl rings and two biguanide groups connected by a central hexamethylene chain. It is a broad-spectrum antiseptic widely used in dentistry for purposes such as root canal irrigation, mouthwash, toothpaste, gels, sprays, and as an additive to dental materials like calcium hydroxide sealers and mineral trioxide aggregate [2].
While Chlorhexidine Gluconate has broad antibacterial activity, its toxic effects are not reserved entirely for bacteria. It has been shown to be cytotoxic to various cell types including murine fibroblasts, human dermal fibroblasts, human gingival fibroblasts, human periodontal ligament cells, human alveolar bone cells, and human osteoblastic cell lines. This study demonstrates that CHX disrupts cellular redox balance, increasing free radical generation and leading to cell death through both apoptosis and necrosis [2].

C28H40CL2N10O7

699.59

896.319

18472-51-0

Chlorhexidine dihydrochloride;3697-42-5;Chlorhexidine;55-56-1;Chlorhexidine diacetate;56-95-1;Chlorhexidine acetate hydrate;206986-79-0

9552081

Colorless to light yellow liquid

1.06 g/mL at 25 °C(lit.)

699.3ºC at 760 mmHg

134ºC

376.7ºC

0mmHg at 25°C

18

16

23

60

819

8

N=C(NC(NCCCCCCNC(NC(NC1=CC=C(Cl)C=C1)=N)=N)=N)NC2=CC=C(Cl)C=C2.O[C@H]([C@@H](O)C(O)=O)[C@H](O)[C@H](O)CO.O[C@H]([C@@H](O)C(O)=O)[C@H](O)[C@H](O)CO

YZIYKJHYYHPJIB-UUPCJSQJSA-N

InChI=1S/C22H30Cl2N10.2C6H12O7/c23-15-5-9-17(10-6-15)31-21(27)33-19(25)29-13-3-1-2-4-14-30-20(26)34-22(28)32-18-11-7-16(24)8-12-18;2*7-1-2(8)3(9)4(10)5(11)6(12)13/h5-12H,1-4,13-14H2,(H5,25,27,29,31,33)(H5,26,28,30,32,34);2*2-5,7-11H,1H2,(H,12,13)/t;2*2-,3-,4+,5-/m.11/s1

(1E)-2-[6-[[amino-[(E)-[amino-(4-chloroanilino)methylidene]amino]methylidene]amino]hexyl]-1-[amino-(4-chloroanilino)methylidene]guanidine;(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoic acid

Chlorhexidine digluconate UniseptChlorhexidine Gluconate Peridex GibitanUNII-MOR84MUD8E Hexidine Hibiclens Hibident Hibisol HibitaneHibiscrub Perio Chip

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)

H2O : ~100 mg/mL (~111.39 mM)
DMSO : ≥ 38 mg/mL (~42.33 mM)

Solubility in Formulation 1: 100 mg/mL (111.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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