Voltage-gated mu1 Na+ channels [1]
- Sarcoplasmic reticulum Ca-ATPase (IC50 for inhibiting enzyme activity: ~2.3 mM; EC50 for reducing Ca²⁺ uptake: ~1.8 mM)[3]

Benzocaine blocks μ1 wild-type Na+ currents in a dose-dependent Manner. The Benzocaine concentration that inhibits 50% of Na+ currents (IC50) is estimated to be about 0.8 mM when a test potential of +30 mV is applied. The slope of the h∞ curve is also significantly reduced by benzocaine (from 6.6 to 9.9 mV). Mutation of μ1-N1584A also significantly increases the potency of Benzocaine. At 1 mM, Benzocaine blocks about 55% of wild-type Na+ current but about 95% of μ1-N1584A mutant current. Benzocaine also appears to bind more strongly to its LA receptor in the N1584A mutant than in the wild type[1]. The inhibition of Ca2+ uptake occurs at lower Benzocaine concentration (IC50=40.3±1.2mM) than that affecting the enzymatic activity[2].

---

In Xenopus oocytes expressing voltage-gated mu1 Na+ channels, Benzocaine (0.1-10 mM) inhibited sodium currents in a concentration-dependent manner. It preferentially bound to the inactivated state of the channels, prolonging the inactivation recovery time and reducing the number of available functional channels. The inhibitory effect was voltage-dependent, with stronger blocking at more depolarized membrane potentials[1]
- In isolated sarcoplasmic reticulum (SR) vesicles from fast-twitch skeletal muscle, Benzocaine (0.5-5 mM) dose-dependently inhibited Ca-ATPase activity. At 3 mM, it reduced enzyme activity by 47% and decreased SR Ca²⁺ uptake by 52%. The drug altered the enzyme's conformational state, as indicated by reduced intrinsic fluorescence of tryptophan residues in Ca-ATPase[3]

Dogs, domestic shorthair cats, Sprague-Dawley rats, Long-Evans rats, ferrets, rhesus monkeys, cynomolgus monkeys, owl monkeys, New Zealand White rabbits, miniature pigs, ICR mice, C3H mice, and C57BL/10SnJ mice are among the species to which benzocaine is applied topically. A 2-second spray is administered to the mucous membranes of the nasopharynx for an estimated dose of 56 mg to all animals, with the exception of mice and rats. An excessive amount of fluid is sprayed over the oral mucous membranes of rodents in a two-second burst. A few months later, the study is conducted again on dogs to confirm the poor response. Most studied animals show a reaction to benzocaine spray, with a peak response occurring 15–30 minutes after dosing[3].

---

In dogs, cats, rabbits, and guinea pigs, topical application of Benzocaine (5-20% formulation, 0.1-0.5 mL/kg) induced methemoglobinemia in a species-dependent manner. Dogs were the most sensitive, with methemoglobin levels reaching 35-45% 2-4 hours after application. Cats and rabbits showed moderate responses (methemoglobin levels 15-25%), while guinea pigs had minimal changes (<10%). Clinical signs included cyanosis, tachypnea, and lethargy in severely affected animals[2]

Ca-ATPase activity assay: Sarcoplasmic reticulum vesicles were isolated from fast-twitch skeletal muscle and resuspended in reaction buffer. Different concentrations of Benzocaine were added, followed by the addition of ATP to initiate the reaction. After incubation at 37°C for 30 minutes, the reaction was terminated, and the released inorganic phosphate (Pi) was quantified using a colorimetric assay. Ca-ATPase activity was calculated based on Pi production[3]
- Na+ channel binding assay: Voltage-gated mu1 Na+ channels were expressed in Xenopus oocytes. Whole-cell patch-clamp recordings were performed to measure sodium currents before and after application of Benzocaine. The voltage protocol included depolarizing steps to induce channel activation and inactivation, and the peak sodium current amplitude and inactivation kinetics were analyzed to evaluate binding affinity[1]

Sodium current recording assay: Xenopus oocytes were injected with mu1 Na+ channel cRNA and cultured for 2-3 days. Oocytes were placed in a recording chamber, and microelectrodes were used for whole-cell patch-clamp recording. Benzocaine was added to the extracellular solution at gradient concentrations, and sodium currents were recorded under different voltage conditions to assess the inhibitory effect[1]
- SR Ca²⁺ uptake assay: Isolated sarcoplasmic reticulum vesicles were incubated with Ca²⁺-containing buffer and Benzocaine for 10 minutes. Ca²⁺ uptake was initiated by adding ATP, and the reaction was terminated at specific time points. The remaining free Ca²⁺ in the supernatant was measured using a Ca²⁺-sensitive dye, and the amount of Ca²⁺ taken up by SR vesicles was calculated[3]
- Intrinsic fluorescence assay: Sarcoplasmic reticulum vesicles were incubated with Benzocaine for 15 minutes at room temperature. The intrinsic fluorescence of tryptophan residues in Ca-ATPase was measured using a fluorometer with excitation at 295 nm and emission at 340 nm. Fluorescence intensity changes were used to reflect conformational alterations of the enzyme[3]

Guinea pig

---

Methemoglobinemia induction model: Adult dogs (10-15 kg), cats (2-3 kg), rabbits (1.5-2 kg), and guinea pigs (300-400 g) were used. Benzocaine was formulated as a 5-20% topical solution. The drug was applied to the shaved skin (2 cm × 2 cm area) at a dose of 0.1-0.5 mL/kg. Blood samples were collected at 0, 1, 2, 4, 6, and 24 hours after application to measure methemoglobin levels. Clinical signs were observed and recorded throughout the experiment[2]

Absorption, Distribution and Excretion
This study evaluated the gill and urinary excretion of benzocaine residues following a single dorsal aortic injection of 14(C)-benzocaine hydrochloride in adult rainbow trout (Oncorhynchus mykiss). Gill excretion of benzocaine residues was rapid, with 59.2% of the total dose excreted within 3 hours post-administration. Renal excretion of the radioactive material was significantly slower; 2.7% of the dose was excreted within 3 hours and 9.0% within 24 hours. 2.0% of the dose remained in bile 24 hours post-administration. Radiochromatographic analysis of water samples collected 3 minutes post-administration revealed that 87.3% was benzocaine and 12.7% was N-acetylated benzocaine. At 60 minutes post-administration, 32.7% of the urine was benzocaine and 67.3% was N-acetylated benzocaine. In the urinary radiochromatogram collected 1 hour after drug administration, 7.6% was p-aminobenzoic acid, 59.7% was N-acetylated p-aminobenzoic acid, 19.5% was benzocaine, and 8.0% was N-acetylated benzocaine. The proportion of radioactive substances in urine changed over time; after 20 hours, 1.0% was p-aminobenzoic acid and 96.6% was N-acetylated p-aminobenzoic acid. Benzocaine and its more hydrophobic metabolite, N-acetylated benzocaine, are mainly excreted through the gills. The kidneys and biliary tract are not the main pathways for the clearance of benzocaine residues. Benzocaine can undergo ester hydrolysis to produce 4-aminobenzoic acid, acetylation to produce acetylbenzocaine, or N-hydroxylation to produce benzocaine hydroxide. 4-aminobenzoic acid can undergo acetylation, and acetylbenzocaine can also undergo ester hydrolysis to produce 4-acetylaminobenzoic acid. This study investigated the effects of dose and enzyme inhibition on the transdermal absorption and metabolism of benzocaine in hairless guinea pigs in vitro and in vivo. At a dose level of 2 μg/cm², benzocaine was rapidly absorbed and extensively metabolized (80%) by acetyltransferases. When the dose of benzocaine was increased to 40 and 200 μg/cm², the N-acetylation rate of benzocaine decreased to 44% and 34%, respectively, indicating that the acetyltransferase system had reached saturation. There was no significant difference in total 14C absorption through the skin between the control and enzyme inhibition groups after benzocaine administration; therefore, the extent of metabolism during transdermal absorption of benzocaine appears to have no effect on its absorption. The skin exhibits a significant first-pass metabolic effect on the transdermal absorption of therapeutic doses of benzocaine, and its main metabolite, acetylbenzocaine, is biologically active. It is (in small amounts) metabolized by cholinesterase hydrolysis in the liver and plasma. It is mainly excreted in the urine as metabolites. (L1861)

Toxicity Summary
Benzocaine binds to sodium channels, reversibly stabilizing neuronal membranes and reducing their permeability to sodium ions. Neuronal membrane depolarization is inhibited, thereby blocking the initiation and conduction of nerve impulses. Pregnancy and Lactation Effects ◉ Overview of Use During Lactation No studies have been conducted on the topical use of benzocaine during lactation, but if applied to areas away from the breast, it is unlikely to affect the nursing infant. Benzocaine should not be applied to the breast or nipple, as the infant may ingest the drug during breastfeeding, and this drug has been associated with severe methemoglobinemia in children under 2 years of age. ◉ Effects on Breastfed Infants No published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information was found as of the revision date. ◈ What is Benzocaine? Benzocaine is a local anesthetic. Local anesthetics are used to paralyze specific areas of the body for a short period of time. Benzocaine is found in some over-the-counter pain relief products, including Anbesol®, Orabase®, Orajel®, Hurricaine®, and Topex®. Studies on the use of benzocaine during pregnancy are insufficient. If used as directed, the amount of benzocaine expected to be absorbed is extremely small. This means that a large amount of the drug is unlikely to reach the developing fetus, and therefore the risk of birth defects or other pregnancy complications is expected to be low.
◈ I am using benzocaine. Will this affect my pregnancy?
It is currently unclear whether the use of benzocaine affects pregnancy.
◈ Does the use of benzocaine increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for a variety of reasons. There are currently no studies confirming that benzocaine increases the risk of miscarriage.
◈ Does the use of benzocaine increase the risk of birth defects?
There is a 3-5% risk of birth defects in every pregnancy. This is called background risk. Neither report found a higher chance of birth defects in children born to women who used benzocaine in early pregnancy.
◈ Does benzocaine use during pregnancy increase the risk of other pregnancy-related problems?
Currently, there are no studies showing whether benzocaine increases the risk of pregnancy-related problems such as preterm birth (delivery before 37 weeks of gestation) or low birth weight (birth weight less than 2500 grams).
◈ Will benzocaine use during pregnancy affect a child's future behavior or learning abilities?
Currently, there are no studies showing whether benzocaine causes behavioral or learning problems in children.
◈ Use of benzocaine during breastfeeding:
Currently, there are insufficient studies to explore the safety of benzocaine use during breastfeeding. Because ingestion of benzocaine by infants can lead to methemoglobinemia (a serious blood disorder), it is not recommended to apply benzocaine to the breasts or nipples. If benzocaine is not applied to these areas and the infant does not put it in their mouth, topical application of benzocaine is unlikely to affect a breastfeeding infant. Wash your hands thoroughly after using benzocaine. If you have any questions about breastfeeding, be sure to consult your healthcare provider.
◈ Does male benzocaine use affect fertility or increase the risk of birth defects?
Currently, no studies have explored whether benzocaine affects male fertility (the ability to impregnate a partner) or increases the risk of birth defects. Generally, exposure to benzocaine by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please refer to MotherToBaby's "Father Exposure" fact sheet at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

---

Protein Binding
Benzocaine binds to serum albumin and α-1-acid glycoprotein.

---

Toxicity Data
LD50: 3040 mg/kg (oral, rat) (A308)

---

In vivo toxicity: Topical application of benzocaine can induce dose- and species-dependent methemoglobinemia. The minimum toxic dose for dogs was 0.1 mL/kg of a 10% formulation, while cats and rabbits required 0.2–0.3 mL/kg of a 15% formulation to induce significant methemoglobinemia [2]. No significant hepatotoxicity or nephrotoxicity was observed in the test animals, and serum ALT, AST, BUN, and creatinine levels remained within the normal range 24 hours after administration [2].

[1]. A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated mu1 Na+ channels. Pflugers Arch. 1998 Jan;435(2):293-302.

[2]. Benzocaine-induced methemoglobinemia attributed to topical application of the anesthetic in several laboratory animal species. Am J Vet Res. 1993 Aug;54(8):1322-6.

[3]. Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle. Naunyn Schmiedebergs Arch Pharmacol. 2015 Nov;388(11):1163-70.

Benzocaine is a benzoic acid ester, with 4-aminobenzoic acid as its acid component and ethanol as its alcohol component. It is a surface anesthetic used to inhibit the gag reflex and can be used as a lubricant and local anesthetic for the larynx, mouth, nose, respiratory tract, esophagus, rectum, urinary tract, and vagina. It has local anesthetic, antipruritic, sensitizing, and anti-allergic effects. It is a benzoic acid ester and a substituted aniline. Benzocaine is an ester-based local anesthetic whose mechanism of action is to block impulse transmission through nerve fibers and nerve endings. It is commonly used for local anesthesia in many over-the-counter products. Benzocaine was originally used for dental local anesthesia. Benzocaine is a standardized chemical sensitizer. The physiological effects of benzocaine are achieved by increasing histamine release and cell-mediated immunity. Benzocaine is an ester of p-aminobenzoic acid, lacking the diethylamino group at the terminal end of procaine, and thus possesses anesthetic activity. Benzocaine binds to sodium channels, reversibly stabilizing neuronal membranes and thereby reducing their permeability to sodium ions. Depolarization of the neuronal membrane is inhibited, thereby blocking the generation and conduction of nerve impulses. Benzocaine is a topical anesthetic whose mechanism of action is to prevent the transmission of nerve impulses along nerve fibers and nerve endings. Benzocaine is a commonly used local anesthetic used as a topical analgesic. It is the active ingredient in many over-the-counter analgesic ointments. Benzocaine is an ester compound synthesized from the organic acid para-aminobenzoic acid (PABA) and ethanol. The synthesis of this ester is called Fischer esterification. A topical anesthetic whose mechanism of action is to prevent impulse conduction along nerve fibers and nerve endings. See also: Benzocaine hydrochloride (salt form); alcohol; benzocaine (ingredient); benzocaine; white petrolatum (active ingredient)...see more...

---

Drug Indications
Benzocaine is indicated for dental local anesthesia, minor trauma, and preparation for infiltration anesthesia. Benzocaine products are indicated for local anesthesia of a variety of conditions, including skin irritation, oral pain, and hemorrhoids.
Treatment of Oropharyngeal Pain
Mechanism of Action
Benzocaine diffuses into nerve cells, binds to sodium channels, and prevents the channels from opening, thereby blocking the influx of sodium ions. If nerve cells cannot allow sodium ions to enter, they cannot depolarize and conduct nerve impulses.
Benzocaine reversibly stabilizes the neuronal membrane, reducing its permeability to sodium ions. Depolarization of the neuronal membrane is inhibited, thereby blocking the initiation and conduction of nerve impulses.
Therapeutic Uses
Suitable for the relief of: oral ulcers, cold sores, or febrile blisters: benzocaine (gel and topical solution); gingival or oral mucosal pain (i.e., pain caused by oral or gingival irritation, inflammation, lesions, or minor dental procedures): benzocaine (gel, toothpaste, lozenges, and topical solution); dental restoration pain (i.e., pain or irritation caused by dentures or other dental or orthodontic appliances): benzocaine (toothpaste, gel ointment, and topical solution). For teething pain: benzocaine (7.5% and 10% gel); for toothache: benzocaine (10% and 20% gel and topical solution). For suppressing the pharyngeal reflex and/or other laryngeal and esophageal reflexes for dental examinations or surgeries (including oral surgeries), endoscopy, or intubation: benzocaine (gel, topical aerosol, and topical solution). /Included in the U.S. product label/
For use as a local anesthetic on accessible mucous membranes prior to examinations, endoscopy, instrumentation, or other procedures involving the following sites: esophagus: benzocaine (gel and topical solution); larynx: benzocaine (gel and topical solution); oral cavity, in dental and oral surgery: benzocaine (gel, topical aerosol, and topical solution); nasal cavity: benzocaine (gel); pharynx: benzocaine (gel, topical aerosol, and topical solution); rectum: benzocaine (gel); respiratory tract or trachea: benzocaine (gel, topical aerosol, and topical solution); urinary tract: benzocaine (gel); vagina: benzocaine (gel).
Local anesthetic.
(Veterinary use): Local (usually topical) anesthetic.
Drug Warnings
Infants and the elderly are more susceptible to toxic methemoglobinemia after exposure to benzocaine. Other risk factors include hereditary reductase deficiency, exposure to high doses of anesthetics, and skin and mucous membrane damage. Due to the potential for serious complications, methemoglobinemia in immunocompromised patients and those with excessively high benzocaine concentrations should be corrected immediately. The possibility of masking symptoms during general anesthesia makes the use of this drug before anesthesia particularly risky.
Use of otoanesthetics may mask symptoms of fulminant otitis media (acute otitis media). Otoane solutions containing benzocaine should not be used in cases of tympanic membrane perforation.
Benzocaine has relatively low toxicity when used topically as recommended, but allergic reactions can still occur.
When used as a desensitizing agent for the male genitalia, benzocaine generally does not adversely affect female sexual partners' orgasm, nor does it anesthetize the clitoris or vagina.
For more complete data on drug warnings for benzocaine (9 of 9), please visit the HSDB record page.
Pharmacodynamics
Benzocaine is indicated for local anesthesia. Its duration of action is approximately 10 minutes, and it has a wide therapeutic window. Patients should be informed of the risk of methemoglobinemia. Benzocaine is a widely used local anesthetic, clinically used to relieve pain caused by skin irritation, mucosal damage and minor surgery [1][2]. Its anesthetic mechanism is mainly mediated by blocking voltage-gated μ1 sodium ion channels, thereby inhibiting the transmission of action potentials in sensory neurons [1]. This study discovered a new target of benzocaine—sarcoplasmic reticulum Ca²⁺-ATPase, suggesting that it may affect skeletal muscle Ca²⁺ homeostasis [3]. Methemoglobinemia is a documented side effect of benzocaine, especially after local application in sensitive animals, and should be monitored in clinical applications [2].

C9H11NO2

165.19

165.078

94-09-7

Benzocaine-d4;342611-08-9;Benzocaine-(ethyl-d5);1219803-76-5

2337

White to off-white solid powder

1.1±0.1 g/cm3

310.7±15.0 °C at 760 mmHg

88-90 °C

164.2±17.9 °C

0.0±0.7 mmHg at 25°C

1.555

1.95

1

3

3

12

151

0

BLFLLBZGZJTVJG-UHFFFAOYSA-N

InChI=1S/C9H11NO2/c1-2-12-9(11)7-3-5-8(10)6-4-7/h3-6H,2,10H2,1H3

Benzoic acid, p-amino-, ethyl ester

H-4-Abz-OEt; NSC-4688; NSC4688; NSC 41531; trade name Orajel amongst others

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.13 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (15.13 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (15.13 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.

---

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