4-Methylbenzylidenecamphor (4-MBC; 5-400 μM; 48 hours) suppresses the growth of HTR8/SVneo cells [1]. Human trophoblast cell proliferation is induced by 4-methylbenzylidene camphor (10–50 μM; 48 hours) and results in an increase in the percentage of cells in the SubG1 phase [1]. 4-In 48 hours, 50 μM methylbenzylidenecamphor decreases human trophoblast cells' nutrition [1]. In human trophoblast cells, camphor (50 μM; 5-120 minutes) stimulates the signal amplifiers for PI3K/AKT and ERK1/2 [1]. 4-Methylbenzylidene camphor at 20–50 μM for 24 hours dramatically raises GPR56 and SEMA6 A.

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- Antiproliferative and pro-apoptotic activity on human trophoblast cells (HTR-8/SVneo cell line) [1]
- Antiproliferation: 4-Methylbenzylidene camphor (4-MBC) inhibited HTR-8/SVneo cell proliferation in a dose-dependent manner, with an IC50 value of 25.6 μM after 48 hours of treatment (MTT assay). At concentrations of 20 μM, 30 μM, and 40 μM, the proliferation rate was reduced by 38%, 62%, and 85% respectively, compared to the solvent control (DMSO, <0.1%).
- ROS-mediated apoptosis: Treatment with 4-MBC (20 μM, 30 μM) for 24 hours significantly increased intracellular reactive oxygen species (ROS) levels by 2.3-fold and 3.5-fold respectively (DCFH-DA staining). The apoptotic rate increased from 3.2% (control) to 18.5% (20 μM) and 32.7% (30 μM) (Annexin V-FITC/PI double staining). It upregulated the expression of cleaved caspase-3 (2.8-fold at 30 μM) and Bax (2.1-fold at 30 μM), and downregulated Bcl-2 (0.4-fold at 30 μM) (western blot analysis).
- Developmental toxicity-related in vitro activity [2]
- In human granulosa cells: 4-MBC (10 μM, 20 μM) reduced estradiol production by 25% and 40% respectively, and downregulated the expression of aromatase (a key enzyme in estrogen synthesis) by 30% and 55% respectively (qPCR and western blot).
- In mouse embryonic fibroblasts: 4-MBC (50 μM) inhibited cell migration by 45% (wound-healing assay), which may be associated with impaired embryonic development.

Changes in gene expression and South African growth enhancement in newborns are caused by 4-Methylbenzylidenecamphor (4-MBC; 0.7, 7, 24, 47 mg/kg/day; given to parents in diet prior to mating, during pregnancy, and during fetal formulation) [2].

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- Developmental toxicity in animal models [2]
- In CD-1 mice: Pregnant mice were orally administered 4-MBC at doses of 100 mg/kg, 300 mg/kg, and 500 mg/kg from gestation day (GD) 6 to GD 15. The 300 mg/kg and 500 mg/kg doses significantly increased fetal resorption rate (from 5% in control to 18% and 27% respectively) and reduced fetal weight by 12% and 18% respectively. The 500 mg/kg dose also caused minor fetal malformations (digital hypoplasia, 8% incidence).
- In Sprague-Dawley rats: Pregnant rats were dermally exposed to 4-MBC (50 mg/kg, 100 mg/kg) from GD 6 to GD 20 (mimicking human sunscreen use). The 100 mg/kg dose reduced pup survival rate at postnatal day (PND) 7 by 15% and delayed eye opening (by 1.5 days on average) compared to the control group.

cell proliferation assay [1]
Cell Types: HTR8/SVneo Cell
Tested Concentrations: 0, 5, 10, 20, 50, 100 , 200 and 400 μM
Incubation Duration: 48 h
Experimental Results: Dose-dependently inhibited cell proliferation of HTR8/SVneo cell.

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Apoptosis analysis [1]
Cell Types: HTR8/SVneo Cell
Tested Concentrations: 10, 20, 50 μM
Incubation Duration: 48 h
Experimental Results: Early and late apoptotic cells increased Dramatically at 20 μM and 50 μM.

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Cell cycle analysis [1]
Cell Types: HTR8/SVneo Cell
Tested Concentrations: 5, 10, 20, 50 μM
Incubation Duration: 48 h
Experimental Results: The proportion of cells in the SubG1 phase gradually increased. Cell Invasion Analysis[1]
Cell Types: HTR8/SVneo Cell
Tested Concentrations: 50 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Invasiveness was Dramatically diminished by 81.5% Western Blot Analysis[1]
Cell Types: HTR8/SVneo Cell
Tested Concentrations: 50 μM
Incubation Duration: 0, 5, 15, 30, 60, 120 minutes
Experimental Results: The phosphorylation of AKT and its downstream kinase protein P70S6K peaked at 5 and 15 minutes respectively, then diminished after 30 minutes, an

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- HTR-8/SVneo cell proliferation assay (MTT method) [1]
- Cells were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight at 37°C (5% CO₂). 4-MBC was dissolved in DMSO and added to wells at final concentrations of 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM (DMSO concentration <0.1% as control). After incubation for 24 h, 48 h, and 72 h, MTT reagent was added and incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm to calculate the proliferation rate and IC50.
- Apoptosis detection (Annexin V-FITC/PI staining) [1]
- HTR-8/SVneo cells (2×10⁵ cells/well, 6-well plate) were treated with 4-MBC (20 μM, 30 μM) or DMSO for 24 hours. Cells were harvested, washed with cold PBS, resuspended in binding buffer, and stained with Annexin V-FITC and PI for 15 minutes in the dark (room temperature). Apoptotic rates were analyzed by flow cytometry.
- ROS detection (DCFH-DA staining) [1]
- Cells (1×10⁴ cells/well, 96-well black plate) were treated with 4-MBC (20 μM, 30 μM) for 12 hours. DCFH-DA solution (final concentration 10 μM) was added and incubated for 30 minutes at 37°C. Fluorescence intensity was measured at 488 nm (excitation) and 525 nm (emission) to quantify intracellular ROS levels.

Absorption, Distribution and Excretion
Following daily full-body application of a 10% (by weight) sunscreen at a concentration of 2 mg/cm² in healthy female volunteers, the peak plasma concentration of enzacamine was 16 ng/mL. After oral administration of enzacamine, peak plasma concentrations of enzacamine (4-MBC) and its major metabolite, 3-(4-carboxybenzylidene)camphor, were reached within 10 hours. The urinary concentration of enzacamine in both female and male volunteers was 4 ng/mL. In rat pharmacokinetic studies, most orally administered enzacamine was excreted in feces as 3-(4-carboxybenzylidene)camphor, with a small amount excreted as 3-(4-carboxybenzylidene)-6-hydroxycamphor. Glucuronides of both metabolites were also detectable in feces. In urine, one isomer of 3-(4-carboxybenzylidene)-hydroxycamphor [3-(4-carboxybenzylidene)-6-hydroxycamphor] is the major metabolite, while other isomers and 3-(4-carboxybenzylidene)-camphor are only minor metabolites, excreted only in urine. The enterohepatic circulation of glucuronides derived from the two major 4-MBC metabolites may explain the slow excretion of 4-MBC metabolites in urine and the low proportion of the administered dose recovered in urine.
Pharmacokinetic data are not available.
Pharmacokinetic data are not available.

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Metabolism/Metabolites Based on rat pharmacokinetic studies, it is speculated that orally absorbed enzacamine undergoes extensive first-pass hepatic metabolism. Following oral administration of enzakamin (4-MBC) to rats, the metabolites detected in plasma and urine were 3-(4-carboxybenzylidene)camphor and four isomers of 3-(4-carboxybenzylidene)hydroxycamphor containing camphor ring hydroxyl groups, with 3-(4-carboxybenzylidene)-6-hydroxycamphor being the major metabolite. However, after reaching peak concentrations, the plasma concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were below the limit of detection. 3-(4-hydroxymethylbenzylidene)camphor is generated via a cytochrome P450-mediated hydroxylation reaction. This metabolite is further oxidized to 3-(4-carboxybenzylidene)camphor by alcohol dehydrogenases and aldehyde dehydrogenases, and may be further hydroxylated under CYP450-mediated formation of 3-(4-carboxybenzylidene)-6-hydroxycamphor.

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Biological Half-Life
In rats, the half-life of enzacamin (4-MBC) and its major metabolite 3-(4-carboxybenzylidene)camphor after oral administration is approximately 15 hours after reaching peak plasma concentration.

Protein Binding
Pharmacokinetic data are currently unavailable. - In vitro toxicity [1] - 4-MBC exhibited dose-dependent cytotoxicity in HTR-8/SVneo cells: at 40 μM, cell viability decreased to 15% (48 hours, MTT assay). It induced oxidative stress (increased ROS) and apoptosis, but no significant necrosis was observed (<5% of PI-positive cells at 30 μM). - In vivo developmental toxicity [2] - Maternal toxicity: In CD-1 mice, oral administration of 500 mg/kg of 4-MBC during pregnancy resulted in a slight decrease in maternal body weight (5% compared to the control group) and a reduction in food intake (10% compared to the control group), but no maternal death. In rats, percutaneous exposure to a dose of 100 mg/kg did not cause significant maternal skin irritation or systemic toxicity. Fetal/Neonatal Toxicity: As described in the "In Vivo" section, this drug can increase fetal absorption, reduce fetal weight, induce minor malformations (mice), and impair neonatal survival and development (rats).

[1]. 4-Methylbenzylidene-camphor inhibits proliferation and induces reactive oxygen species-mediated apoptosis of human trophoblast cells. Reprod Toxicol. 2019 Mar:84:49-58.

[2]. Developmental toxicity of UV filters and environmental exposure: a review. Int J Androl. 2008 Apr;31(2):144-51.

Enzacamine is a monoterpenoid compound. Commonly known as 4-methylbenzyl camphor (4-MBC), enzacamine is a camphor derivative and an organic UVB filter. It is used in cosmetics such as sunscreens to protect the skin from UV damage. Although its effects on the human reproductive system as an endocrine disruptor are under investigation, Health Canada has approved its use in over-the-counter medicines and cosmetics. Its trade names include Eusolex 6300 (Merck) and Parsol 5000 (DSM). Pharmaceutical Indications Suitable as an active sunscreen. Mechanism of Action Enzacamine absorbs UVB rays. It is presumed that enzacamine exerts a similar effect to endogenous estrogen through a non-canonical estrogen signaling pathway that does not involve the genetic regulation of the nuclear estrogen receptor (ER). Compared to endogenous agonists, enzacamine has a lower to moderate affinity for the cytoplasmic estradiol binding site of the estrogen receptor. Based on findings from a study in Xenopus hepatocyte cultures, enzacamine only induces estrogen receptor (ER) gene expression at high concentrations (10–100 μmol/L). While enzacamine did not show activation of estrogen-dependent gene transcription in yeast ER reporter gene assays, it has been demonstrated to activate ER-dependent signaling pathways in Xenopus hepatocyte cultures, leading to altered gene expression. At micromolar concentrations, enzacamine accelerates the proliferation of MCF-7 human breast cancer cells.

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- 4-Methylbenzyl camphor (4-MBC) is a synthetic ultraviolet (UV) filter widely used in sunscreens, cosmetics and personal care products to absorb UV-B radiation [1,2]
- Mechanism of action in trophoblast cells: Its antiproliferative and pro-apoptotic effects are mediated by the excessive generation of intracellular reactive oxygen species (ROS), which disrupt redox homeostasis and activate the mitochondrial apoptosis pathway (upregulation of the Bax/Bcl-2 ratio and cleaved caspase-3) [1]
- Environmental exposure risk [2]: Due to its widespread use and environmental persistence, 4-MBC can be detected in environmental water bodies (rivers, lakes) and human biological samples (urine, breast milk). Because it can interfere with estrogen synthesis and embryonic development, it is classified as an endocrine disruptor (EDC) [2]

C18H22O

254.3667

254.167

36861-47-9

4-Methylbenzylidene camphor-d4;1219806-41-3

6434217

White to off-white solid powder

1.1±0.1 g/cm3

371.9±22.0 °C at 760 mmHg

66-68°C

168.9±13.2 °C

0.0±0.8 mmHg at 25°C

1.583

4.95

0

1

1

19

423

0

CC1=CC=C(C=C1)/C=C/2\C3CCC(C2=O)(C3(C)C)C

HEOCBCNFKCOKBX-SDNWHVSQSA-N

InChI=1S/C18H22O/c1-12-5-7-13(8-6-12)11-14-15-9-10-18(4,16(14)19)17(15,2)3/h5-8,11,15H,9-10H2,1-4H3/b14-11+

(3E)-1,7,7-trimethyl-3-[(4-methylphenyl)methylidene]bicyclo[2.2.1]heptan-2-one

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 (~393.13 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: 2.5 mg/mL (9.83 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (9.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (9.83 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.)