Absorption, Distribution and Excretion
Systematic exposure to 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopentanoγ-2-benzopyran (HHCB) was determined in humans under simulated exposure conditions. HHCB labeled with ring (14)C was dissolved in an alcohol solution at concentrations close to those typically found in cologne products and applied to three male volunteers without occlusion. All material was removed from the skin surface after 6 hours. Blood, fecal, and urine samples were collected over 5 days. Concentrations of both substances in blood and plasma remained below the limits of detection throughout. The total absorbed dose of HHCB was approximately 0.1% based on excretion (primarily via urine). However, 19.5% of the HHCB was recovered from the dressing during the 5-day experiment, indicating the formation of a “reservoir” in the skin, but the material in the reservoir was lost through desquamation and/or retrograde absorption and could not be absorbed systemically. An average of 22% of the HHCB evaporated under exposure conditions. …The systemic exposure of rats to 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopentano-γ-2-benzopyran (HHCB) was determined under closed conditions. Cyclo(14)C-labeled HHCB was dissolved in an alcohol solution and applied to the skin of rats at a dose of 4.5 mg/kg, followed by a 6-hour closed-loop treatment. Urine, fecal, and air samples were collected over a period of 120 hours and analyzed for radioactivity. Paired rats were periodically sacrificed for tissue and organ analysis. The total uptake of HHCB was approximately 14%. A significant amount of HHCB diffused into the skin, most of which was further absorbed, but a considerable portion was lost from the dressing via retrograde diffusion and/or desquamation. This study investigated the bioaccumulation behavior and acute toxicity of galaxolide 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopentano[γ]-2-benzopyran (HHCB) in two benthic organisms. The bioaccumulation factor (BCF) of HHCB in chironomus riparius was significantly lower than the predicted value for unconverted organic matter. The BCF of HHCB increased after co-exposure of chironomus to the cytochrome P450 inhibitor piperonyl butyl ether. Therefore, the low BCF value was a result of rapid biotransformation of HHCB in midge larvae. Bioaccumulation kinetics indicated that HHCB induced its own cytochrome P450-mediated metabolism. The acute toxicity of HHCB to midge larvae was reduced compared to the predicted baseline toxicity. The bioaccumulation of HHCB in the worm (Lumbriculus variegatus) was consistent with predictions based on the octanol-water partition coefficient of the chemical. Acute toxicity was similar to the predicted baseline toxicity values. Synthetic musk compounds are used as additives in many consumer products, including perfumes, deodorants, and detergents. Earlier studies reported the presence of synthetic musk in environmental and wildlife samples collected in the United States. This study analyzed human milk samples collected from Massachusetts and determined the concentrations of synthetic musk, including thymol (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene), muscone (4-tert-butyl-2,6-dimethyl-3,5-dinitroacetophenone), HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopentano[γ]-2-benzopyran), AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene), and the oxidation product of HHCB, HHCB lactone. Furthermore, we estimated the daily intake of synthetic musk by infants based on the rate of breast milk consumption. Synthetic musk was detected in most of the analyzed samples, with concentrations ranging from <2 to 150 ng/g for xylene musk, <2 to 238 ng/g for ketone musk, <5 to 917 ng/g for HHCB, <5 to 144 ng/g for AHTN, and <10 to 88.0 ng/g (by weight of lipids) for HHCB. The concentration of HHCB in breast milk samples was higher than that of other synthetic musk. The average concentration of HHCB (220 ng/g, by weight of lipids) was five times higher than that in breast milk samples collected in Germany and Denmark 10 years prior. Maternal age was not correlated with the concentrations of xylene musk, ketone musk, HHCB, or AHTN. Although the number of breastfed children was declining, the concentrations of xylene musk, musketone, hexachlorophenol (HHCB), and hydroxyethyl toluene (AHTN) were positively correlated with the concentrations of these substances. Based on the average daily intake of breast milk, the estimated daily intake of musk xylene by infants is 297 ± 229 ng, musketone is 780 ± 805 ng, hexachlorophenol (HHCB) is 1830 ± 1170 ng, hydroxyethyl toluene (AHTN) is 565 ± 614 ng, and hexachlorophenol (HHCB) lactone is 649 ± 598 ng. The amount of synthetic musk ingested by US infants is lower than the estimated intake of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). Based on the residue patterns and accumulation characteristics, it can be concluded that the exposure characteristics of synthetic musk differ from those of persistent organic pollutants, and its primary exposure route is likely dermal absorption or inhalation. For more complete data on absorption, distribution, and excretion of 11 types of musk, please visit the HSDB record page.

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Metabolism / Metabolites
This study investigated the metabolism and mechanism of action of thymol (HHCB) in European sea bass (Dicentrarchus labrax) via a single intraperitoneal injection of 50 mg/kg body weight. Additionally, a group of fish was injected with 50 mg/kg ketoconazole (KCZ), a bactericide known to interfere with multiple cytochrome P450 isoenzymes. The results showed that HHCB was efficiently metabolized in European sea bass and acted as a weak inhibitor of oxyandrogen synthesis in the gonads of male fish. HHCB and its hydroxylated metabolites were detected in bile. The bactericide ketoconazole was a strong inhibitor of Cyp11β and Cyp3a catalytic activity. This study contributes to a better understanding of the effects of synthetic thymol on fish and suggests that measuring HHCB and its hydroxylated metabolites in bile could serve as an indicator for assessing environmental exposure levels in wild fish. This study also compared the interactions of emerging pollutants with exogenous and endogenous metabolic systems in deep-sea fish. The drugs diclofenac, fluoxetine, and gemfibrozil belong to different drug classes with different mechanisms of action; the personal care products triclosan, galesulide, and nonylphenol represent antibacterial agents, nitromustine, and surfactants, respectively. The fish species compared represent the mid-to-lower slope zones of deep-sea habitats. The study included adult four fish species: black sea bream (Trachyrynchus scabrus), moro sea bream (Mora moro), broadhead catfish (Cataetix laticeps), and long-snout catfish (Alepocehalus rostratus). This study investigated the activity of several cytochrome P450 isoenzymes (CYPs) in the hepatic metabolic system, including 7-ethoxycarbamoline-O-deethylase (EROD), benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxyase (BFCOD), and 7-ethoxycoumarin-O-deethylase (ECOD). The results showed differences in baseline activity and sensitivity to chemicals across different species, chemicals, and metabolic pathways. Among all species, *T. scabrous* was most sensitive to the interaction of chemicals with both exogenous and endogenous metabolic systems (EROD and BFCOD), particularly when diclofenac interfered with BFCOD activity (IC50 = 15.7 ± 2.2 μM). Furthermore, both *T. scabrous* and *A. rostratus* exhibited high basal ECOD activity, and in vitro diclofenac exposure significantly affected the ECOD activity of *T. scabrous* (IC50 = 6.86 ± 1.4 μM). These results highlight the susceptibility of marine fish to emerging pollutants and suggest that *T. scabrous* (medium slope) and *A. rostratus* (low slope) serve as indicator species, with ECOD activity as a sensitive biomarker for such exposures.

Toxicity Summary
Identification and Uses: Gylesyl (HHCB) is a nearly colorless, viscous liquid. It is a synthetic artificial musk fragrance used as a laundry detergent fragrance and is also an ingredient in perfumes, soaps, and cosmetics. Human Studies: In a human repeated stimulation patch test, no irritation was observed in any of the 42 subjects, even with repeated occlusive application of undiluted HHCB. High concentrations of HHCB can affect steroid production in the H295R cell line in vitro. In the in vitro human lymphocyte micronucleus assay, HHCB did not exhibit genotoxicity regardless of metabolic activation. Animal Studies: HHCB was added to the diet of rats at average daily doses of 5, 15, 50, or 150 mg HHCB/kg. After a 90-day treatment period, three males and three females from each of the high-dose and control groups underwent a four-week treatment-free period. No adverse reactions were found in clinical examination or extensive histopathological examination. No significant abnormalities were found in any of the dose groups. Developmental studies in rats have shown axial skeletal deformities at a daily dose of 500 mg/kg. Rats exposed to HHCB (35 mg/kg) for extended periods exhibited reduced birth weight in their F1 generation, upregulated CYP2B and CYP3A expression, and slightly elevated high-density lipoprotein cholesterol (HDL-C) levels in male rats. Metamorphosis studies in African clawed frogs revealed accelerated development in tadpoles on day 14, along with hypertrophy of the thyroid follicular epithelium, suggesting a potential agonist effect of HHCB. Intraperitoneal injection of HHCB at a dose of 3.64 mg/kg into rainbow trout inhibited E2-induced vitellogenin production. Using sensitive and specific reporter gene cell lines, the interactions of HHCB with estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR) were assessed. The results indicate that HHCB is an antagonist of ERβ, AR, and PR. With or without metabolic activation, HHCB showed no genotoxicity to *Salmonella typhimurium* strains TA97, TA98, TA100, and TA102. Ecotoxicity studies: In zebra mussels (*Dreissena polymorpha*), HHCB significantly increased lipid peroxidation and protein carbonyl levels. Furthermore, exposure to the highest concentrations of HHCB significantly increased DNA strand breaks, but no fixed genetic damage was observed. This study examined the mRNA expression levels of four representative protein-coding genes (HSP70, CRT, cyPA, and TCTP) in earthworms (*Eisenia fetida*) exposed to HHCB. The results suggest that the expression patterns of HSP70 and CRT genes may serve as potential early molecular biomarkers for predicting harmful HHCB exposure levels and their ecotoxicological effects. Another study on earthworms indicated that superoxide dismutase and catalase appear to be the most sensitive biomarkers of HHCB-induced oxidative stress.

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Interactions
This study investigated cadmium accumulation in wheat seedlings under single stress and combined cadmium (Cd) and haloxyfop-methyl (HHCB) stress, and assessed its phytotoxicity and oxidative stress, including the activities of chlorophyll (CHL), malondialdehyde (MDA), superoxide dismutase (SOD), and peroxidase. The results showed that cadmium accumulation in wheat seedlings increased with increasing soil cadmium concentration. Low concentrations of HHCB inhibited cadmium accumulation, while high concentrations promoted it. Chlorophyll content in wheat seedlings significantly increased under treatments of 1–50 mg/kg Cd. However, under combined cadmium and HHCB stress, chlorophyll content in wheat seedlings was significantly lower than in the control group. Furthermore, MDA content in wheat leaves and roots was significantly affected by both HHCB and cadmium, especially under combined stress. Co-contamination with HHCB significantly affected the activity of antioxidant enzymes in wheat seedlings under cadmium stress. In conclusion, HHCB may exacerbate the phytotoxicity of cadmium in wheat seedlings. Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are commonly found in natural aquatic environments. This study aimed to evaluate the toxic effects of the combined effects of PAHs and heavy metals with suspended solids stress on *Daphnia magna*. Gynostemma pentaphyllum and lead were used as typical pollutants. In experiments using Gynostemma pentaphyllum alone, the toxic effects on *Daphnia magna* were reduced after the addition of suspended solids at both 24 and 48 hours. Similar results were observed for lead toxicity on *Daphnia magna*, with a decrease in toxicity after the addition of suspended solids. Based on additive index analysis, in the combined experiments of Gynostemma pentaphyllum and lead, both showed a synergistic effect on *Daphnia magna* at both 24 and 48 hours. The addition of suspended solids significantly reduced the combined toxic effects of Gynostemma pentaphyllum and lead. The results can provide useful information for the toxicity risk assessment of surface water bodies. This study used outdoor pot experiments to investigate the biomass of wheat seedlings planted in alluvial and cinnamon soils, as well as the accumulation of HHCB and/or Cd in different parts of the wheat seedlings. The biomass order of wheat seedlings under different treatments was: HHCB treatment alone > HHCB and Cd combined treatment > Cd treatment alone. HHCB accumulation in wheat seedlings planted in alluvial soil was higher than in cinnamon soil, and the effect of Cd on HHCB accumulation in wheat seedlings differed between alluvial and cinnamon soils. In alluvial soil, the accumulation order of HHCB in different parts of wheat seedlings was: roots > stems > leaves. Cd significantly induced HHCB accumulation in wheat roots but inhibited HHCB accumulation in wheat stems and leaves, with a maximum inhibition rate of 44.07%. In cinnamon soil, the accumulation order of HHCB in different parts of wheat seedlings was: roots > leaves > stems. Cadmium had no significant effect on HHCB accumulation in wheat roots, but medium to high concentrations of cadmium significantly induced HHCB accumulation in wheat stems and leaves, with a maximum induction rate of 35.95%. Furthermore, cadmium accumulation in alluvial soil was lower than in cinnamon soil, and HHCB significantly induced cadmium accumulation in wheat seedlings in both soil types. The increase rates of cadmium accumulation in roots, stems, and leaves in alluvial soil were 30.84%, 61.82%, and 61.82%, respectively, while those in cinnamon soil were 41.53%, 184.16%, and 206.18%, respectively. This study indicates that HHCB in cinnamon soil is more likely to induce cadmium accumulation in wheat seedlings than HHCB in alluvial soil. This study investigated the single and combined toxic effects of polycyclic musk compounds, including 1,3,4,6,7,8-hexahydro-4,6,6,6,7,8,8-hexamethylcyclopentano[g]-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN), and cadmium (Cd) on wheat (Triticum aestivum) seed germination and seedling growth. The results showed that the toxicity order of HHCB to wheat seed germination and seedling growth was similar to that of AHTN, i.e., germination rate > shoot elongation > root elongation; while according to LC50 and EC50 values, the toxicity order of Cd was root elongation > shoot elongation > germination rate. This suggests that polycyclic musk and Cd have different toxicological mechanisms. Wheat root and shoot elongation may be good bioindicators of polycyclic musk and Cd pollution in soil. When root elongation was used as the toxicological endpoint, the mixture of polycyclic musk and Cd had a synergistic effect on common wheat (T. aestivum) according to the isotoxicity mixture method. Therefore, the combined toxicity of HHCB and Cd was significantly higher than that of HHCB or Cd alone, which was also confirmed by the EC50 value of the mixture (EC50 mix = 0.530 TUmix). The EC50 value of the AHTN and Cd mixture was 0.614 TUmix, indicating that the mixed toxicity was enhanced when AHTN and Cd coexisted.

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Non-Human Toxicity Values
Dermal LD50 in rats: 10000 mg/kg body weight [ECHA; 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindene]
Oral LD50 in rats: 4640 mg/kg body weight [ECHA; 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindene]

[1]. Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids. Toxicol In Vitro. 2018 Apr:48:342-349.

[2]. Potential of environmental concentrations of the musks galaxolide and tonalide to induce oxidative stress and genotoxicity in the marine environment. Mar Environ Res. 2020 Sep:160:105019.

Galaxolide is an organic heterocyclic tricyclic compound with the structure 1,3,4,6,7,8-hexahydrocyclopentano[g]isochromene, substituted with methyl groups at positions 4, 6, 6, 7, 8, and 8. It is a synthetic musk used as a cosmetic fragrance. It possesses fragrance properties. It is an organic heterocyclic tricyclic compound belonging to the isochromene class of compounds.
Musk fragrance; structure see first source.

C18H26O

258.40

258.198

1222-05-5

91497

Colorless to light yellow liquid

0.9±0.1 g/cm3

326.3±11.0 °C at 760 mmHg

57-58 °C ; MP: -10 to 0 °C (mixture of isomers)

146.1±15.5 °C

0.0±0.7 mmHg at 25°C

1.499

6.23

0

1

0

19

357

0

O1C([H])([H])C([H])(C([H])([H])[H])C2C(C1([H])[H])=C([H])C1=C(C=2[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])(C([H])([H])[H])C1(C([H])([H])[H])C([H])([H])[H]

ONKNPOPIGWHAQC-UHFFFAOYSA-N

InChI=1S/C18H26O/c1-11-9-19-10-13-7-15-16(8-14(11)13)18(5,6)12(2)17(15,3)4/h7-8,11-12H,9-10H2,1-6H3

4,6,6,7,8,8-hexamethyl-1,3,4,7-tetrahydrocyclopenta[g]isochromene

Musk Galact

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)

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.

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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).

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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)

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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).

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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

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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.

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