The fact that butylated hydroxytoluene (BHT) effectively promotes tumor-induced tumors is widely recognized. In tumors that are 7 weeks old, butylated hydroxytoluene (facial; 400 mg/kg; weekly) formulation boosts the efficacy of rasH2 tumorigenesis. Sensibility [3]

Absorption, Distribution and Excretion
This study compared bile metabolism in male Wistar rats following intraperitoneal or intravenous administration of BHT, BHT-COOH, BHT-OH, and BHT-aldehyde. The major metabolites of all four test compounds in the enterohepatic circulation were BHT-COOH and its ester glucuronide. … Total bile excretion was lower after intravenous or intraperitoneal administration of BHT or BHT-aldehyde. … Both rats and subjects received a single dose of BHT. Male Wistar rats (n=2–10) were treated with 20–200 mg/kg BHT. Subjects (7 non-smoking males) were treated with 0.5 mg/kg BHT. In rats, pharmacokinetic parameters increased in a dose-dependent manner. Approximately 10% of the high-dose BHT was excreted in feces as unmetabolized form after administration, primarily on day 1. Urinary excretion of BHT-COOH was slightly higher than 1%, decreasing from day 1 to day 4. In humans, the mean plasma concentration-time curve was lower than in rats. Unmetabolized BHT was not detected in feces, and the excretion of BHT-COOH in urine ranged from 0% to 5.5%.
After a single injection of [(14)C]BHT in males, at least two-thirds of the radioactive material was excreted in urine, most of which appeared within 24 hours after administration. Since individual tests showed that fecal excretion was about half that in urine, the remaining radioactive material was likely also excreted through this route. Most of the radioactive material appeared on the first day after administration, and thereafter it gradually decreased and continued to be excreted in small amounts for a considerable period of time. ……
BHT (10 mg/L) was dissolved in a 1:1 mixture of polyethylene glycol 400 and physiological saline. The solution was infused into rabbits at a constant rate of 2 mL/min for <2 minutes, with a total dose of 10 mg/kg. Blood samples were collected at 0, 0.085, 0.173, 0.33, 0.50, 0.75, 1, 1.15, 2.0, 3.0, 4.0, 6.0, and 12 hours, followed by two days of collection twice daily for two consecutive days, and then one day daily for three consecutive days. High-sensitivity and high-specificity gas chromatography was used to analyze the concentration of BHT in the blood samples. The half-life of the rapid distribution phase of BHT was approximately 1 hour, while the half-life of the slow distribution phase exceeded 11 days. These data indicate that BHT accumulates rapidly in the body but is slowly eliminated. After multiple doses, BHT tends to accumulate in tissues, and daily exposure may result in BHT accumulation exceeding 16-fold. For more complete data on the absorption, distribution, and excretion of 2,6-di-tert-butyl-p-cresol (7 compounds in total), please visit the HSDB record page. Metabolism/Metabolites The metabolism of BHT has been extensively studied in rabbits, rats, mice, and humans. The major metabolic pathways of BHT in all species involve the oxidation of methyl and tert-butyl substituents (or one or both). These two mechanisms are not mutually exclusive. Methyl oxidation is catalyzed by the microsomal enzyme BHT oxidase, and several derivatives have been identified in rat liver, including quinone methylates, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, and 4-hydroxy-4-methyl-2,6-di-tert-butylcyclohexadienone. In rats and rabbits, the oxidation of the para-methyl substituent is the major metabolic pathway, with BHT acids accounting for approximately 30% of the administered dose; while in male and female mice and humans, approximately 30-40% of the dose is excreted as metabolites involving the oxidation of one or both tert-butyl groups. BHT is primarily excreted in urine, while in rodents, 50-80% of BHT is excreted in feces. This is thought to be due to differences in the molecular weight threshold for bile excretion among different species. This study compared bile metabolism in male Wistar rats following intraperitoneal or intravenous administration of BHT, BHT-COOH, BHT-OH, and BHT-aldehyde. For all four tested compounds, the major metabolites in the enterohepatic circulation were BHT-COOH and its ester glucuronide. …Intravenous administration of BHT or BHT-aldehyde resulted in lower total bile excretion than intraperitoneal administration. …This study also compared BHT metabolism in mice and rats. In male and female DDY/Slc mice, after a single oral administration (20 or 500 mg/kg body weight) of BHT labeled with p-methyl 14C, the 14C was primarily distributed in the stomach, intestines, liver, and kidneys, and then excreted in urine, feces, and exhaled gases. Within 7 days post-treatment, 41-65%, 26-50%, and 69% of the (14)C dose were excreted via feces, urine, and exhaled gases, respectively, with an overall recovery rate of 96-98%. Seven days after treatment, (14)C levels in tissues of 21 male and 22 female mice were less than 1 μg BHT equivalent/g tissue (ppm) in mice given a 20 mg/kg dose and less than 11 ppm in mice given a 500 mg/kg dose. When male mice were orally administered [(14)C]BHT at a dose of 20 mg/kg/day for 10 consecutive days, (14)C was rapidly excreted without accumulation in any tissues. Thin-layer chromatography and high-performance liquid chromatography analysis revealed the presence of more than 43 metabolites in the urine and feces of both animals, all of which were identified to determine the metabolic pathways of BHT in mice and rats. In mice, the major metabolic reaction of [(14)C]BHT is the oxidation of the p-methyl and tert-butyl groups on the benzene ring. The product of tert-butyl oxidation reacts with the adjacent phenolic hydroxyl group, undergoing a certain degree of cyclization to form a hemiacetal or lactone. The carboxyl derivative of p-methyl oxidation binds to glucuronic acid. When male Sprague-Dawley rats were given a single oral dose of 20 or 500 mg/kg of [(14)C]BHT, similar metabolites as in mice were detected. However, the major biotransformation was the oxidation of the methyl group, while the oxidation of the tert-butyl group accounted for only a small proportion in mice. The major metabolites of 2,6-di-tert-butyl-p-cresol (BC) in mouse Clara cells of the bronchioles are 6-tert-butyl-2-(hydroxy-tert-butyl)-p-cresol (BC-butOH; 4.4 ± 1.1 pmol/10⁻⁶ cells/min) and 2,6-di-tert-butyl-p-hydroxymethylphenol (BC-OH; 1.0 ± 0.2 pmol/10⁻⁶ cells/min). This metabolic pattern is almost identical to that obtained from whole lung microsomes. BC-butOH is more likely to produce quinone methylates than BC (0.52 ± 0.14 pmol/10⁻⁶ cells/min vs. 0.41 ± 0.06 pmol/10⁻⁶ cells/min). The maximum concentration of the intermediate BC-butOH is very low relative to BC; the amount of quinone methylates produced is similar. In addition, two glutathione conjugates were found, generated by attack from BC-quinone methylate and BC-butOH-quinone methylate, respectively. Incubation time was 15 minutes (Clara cells) or 10 minutes (microsomes). Metabolites isolated from the livers of male rats orally administered BHT were identified as 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone. Microsomal monooxygenase system-mediated oxidative metabolism (phase I reaction) is the main pathway for BHT degradation. Oxidation of tert-butyl is most common in humans. Gallate and 2-tert-butylhydroquinone are primarily metabolized via non-oxidative pathways (methylation or conjugation with sulfate and glucuronic acid) (A15352). In particular, BHT is frequently metabolized to quinone methyl ethers (QMs), which are thought to be associated with tumorigenesis. 2,6-di-tert-butyl-4-methylenecyclohexadienone (BHT-QM) is an example of a quinone methyl ether. Quinone methyl ethers are strongly electrophilic and readily form adducts with proteins.

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Biological half-life
BHT (10 mg/L) was dissolved in a 1:1 mixture of polyethylene glycol 400 and physiological saline. This solution was infused into rabbits at a constant rate of 2 mL/min over a period of <2 minutes, with a total dose of 10 mg/kg. The rapid treatment phase half-life of BHT was approximately 1 hour, while the slow treatment phase half-life exceeded 11 days.

Toxicity Summary
Identification and Uses: Butylated hydroxytoluene (BHT) is a white, odorless crystalline solid. It is used as an antioxidant in oils and fats or as a packaging material for fatty foods. Human Exposure and Toxicity: Potential symptoms of overexposure include eye and skin irritation. Animal Studies: High doses of BHT in both male and female rats resulted in elevated serum cholesterol. Weaned rats supplemented with lard and fed BHT showed reduced growth rates, particularly in males. BHT also increased the absolute weight of the liver and the liver weight-to-body weight ratio in both male and female rats. BHT increased the left adrenal gland weight-to-body weight ratio in male rats, but had no consistent effect on female rats. Feeding rats with BHT for 68–82 days resulted in reduced weight gain rate and decreased hepatic steatosis. Adding BHT at concentrations of 3000 or 6000 ppm to the diets of male and female rats and mice for 105 weeks in rats and 107 or 108 weeks in mice did not result in tumors in either male or female rats or mice. In teratogenicity tests, BHT caused anophthalmia in rat offspring, but not in mice. Pregnant mice were fed BHT for 18 days, and another group was fed BHT for 50 to 64 days (including day 18 of gestation). No fetal malformations were observed. In a study using 144 mice, no cases of blindness were observed in 1162 litters (7765 offspring) born throughout the mother's reproductive period. The mutagenicity of BHT was tested in five Salmonella Typhimurium strains (TA1535, TA1537, TA97, TA98, and TA100) using a Salmonella/microsome pre-incubation assay with or without metabolic activation. Results showed that BHT was negative in these assays, and the highest ineffective dose tested in any Salmonella Typhimurium strain was 10 mg/plate. Ecotoxicity studies: Salmon were fed different concentrations of BHT for a 12-week feeding period, followed by a 2-week cleanup period. The results showed that BHT selectively modulated toxicological responses in the biotransformation pathway of exogenous substances during the feeding period. BHT is metabolized into quinone methylates (QMs), which are one of the factors promoting tumor formation in many animal models. 2,6-Di-tert-butyl-4-methylenecyclohexane-2,5-dienone (BHT-QM) is one such QM. Quinone compounds (QMs) are strongly electrophilic and readily form adducts with proteins. Some QMs target redox proteins, such as glutathione S-transferase P1 (GST-P1), peroxidase 6 (Prx6), copper-zinc superoxide dismutase (SOD1), carbonyl reductase, and selenium-binding protein 1, which have direct or indirect antioxidant functions (A15087, A15355). Modification of these proteins leads to decreased cellular protection against electrophilic agents and oxidants. Alkylation may also interfere with the regulation of pressure kinases by GSTP1, thereby affecting phosphorylation and cell growth. BHT can also bind to retinoic acid receptors, leading to alterations in cell development.

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Interactions
In DPPH scavenging assays in nonpolar aprotic solvents such as toluene, BHT can regenerate tocopherol radicals into α-tocopherol; however, in polar protic solvents such as methanol, no regeneration was observed due to the rapid electron transfer reaction of tocopherol radicals to reactive DPPH radicals. …In the presence of small amounts of alcohol, the synergistic effect is enhanced; due to the nucleophilic addition of short-chain alcohols to the oxidation products of BHT, BHT can regenerate twice the amount of α-tocopherol, generating a new phenolic co-antioxidant.
Both natural retinyl acetate (RA) and the phenolic antioxidant butylated hydroxytoluene (BHT) can effectively inhibit the occurrence of breast cancer in rats. This study aims to determine whether the combined use of retinoic acid (RA) and benzo[a]anthracene (BHT) can enhance the inhibitory effect on breast cancer. Unmated female Sprague-Dawley rats aged 50 days (time 0) were administered a single intragastric gavage of 16 mg of 7,12-dimethylbenzo[a]anthracene dissolved in 1 mL of sesame oil. Thirty rats in each group treated with the carcinogen were fed Wayne Lab Chow diets supplemented (per kg of feed) with 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT, according to the following regimens: -2 to +1 weeks; +1 week to the end of the experiment; -2 weeks to the end of the experiment; or no drug was added. The results showed that the RA plus BHT combination regimen administered from -2 weeks to the end of the experiment was more effective in preventing breast cancer than RA or BHT alone; the interaction between RA and BHT had an additive effect. Similarly, the chemopreventive activity of the RA plus BHT regimen administered from -2 weeks to the end of the experiment was higher than that of the RA plus BHT regimen administered from -2 weeks to +1 week or +1 week to the end of the experiment. Long-term exposure to RA combined with BHT led to a high incidence of liver fibrosis and bile duct hyperplasia; these changes were not observed in the control group, but were less frequent in animals exposed to RA alone or BHT alone. These data suggest that the use of a “combined chemoprevention” protocol can enhance anticancer activity; however, interactions between chemopreventive compounds may not only inhibit carcinogenicity but also induce toxicity. The antiandrogenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG), individually and in combination (binary mixtures), were evaluated using the MDA-kb2 cell line. Exposure of these cells to androgen receptor (AR) agonists induced the expression of a reporter gene (encoding luciferase), and the activity of the reporter protein could be monitored by measuring luminescence intensity. In assessing antiandrogenic effects, individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a potent AR agonist (1000 pM 5α-dihydrotestosterone; DHT). Cell viability was assessed using a resazurite-based assay. This is the first report in the literature of (anti)androgenic activity for PG. No androgenic activity was observed in any of the compounds or binary combinations, whether tested individually or in mixtures. BuPB, BHA, and BHT exhibited weak antiandrogenic activity in the presence of DHT, which was also confirmed when evaluating binary mixtures (BuPB+BHA, BuPB+BHT, and BHA+BHT). In addition to in vitro testing of the binary combinations, the accuracy of two mathematical models (dose-additive and response-additive models) in predicting the antiandrogenic effects of the selected binary mixtures was evaluated. The dose-additive model ensured a good correlation between experimental and predicted data. However, due to the lack of effect of the compounds in individual tests, it was impossible to estimate the effect of mixtures containing PG. This study aimed to compare the effects of L-arginine (L-arg) and the food antioxidant butylated hydroxytoluene (BHT) on hepatic oxidative stress induced by Escherichia coli endotoxin (LPS). Ninety Wistar albino rats were randomly assigned to three groups. Before intraperitoneal injection of 5 mg/kg LPS, rats were pretreated with one of the following for 3 days: saline, L-arg (NO donor, 100 mg/kg), or BHT (250 mg/kg/day). Plasma nitrite and nitrate levels, circulating liver enzymes, glutathione levels, superoxide dismutase (SOD) and glutathione peroxidase (GPO) activities were measured at 2, 4, and 6 hours. Compared with L-arginine-pretreated rats, BHT-pretreated animals showed the most significant liver injury at all time points. Although BHT enhanced SOD activity after LPS administration, it simultaneously decreased glutathione levels compared to the L-arginine group. ...
For more complete data on interactions with 2,6-di-tert-butyl-p-cresol (15 items in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 890 mg/kg
Oral LD50 in mice: 650 mg/kg
Intraperitoneal LD50 in mice: 138 mg/kg
Intravenous LD50 in mice: 180 mg/kg
For more complete non-human toxicity data for 2,6-di-tert-butyl-p-cresol (out of 11), please visit the HSDB record page.

[1]. Butylated hydroxytoluene (BHT): a review. Environ Res. 1982 Oct;29(1):1-29.

[2]. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. ell. 2017 Oct 5;171(2):273-285.

[3]. Butylhydroxytoluene (BHT) increases susceptibility of transgenic rasH2 mice to lung carcinogenesis. J Cancer Res Clin Oncol. 2001 Oct;127(10):583-90.

Therapeutic Uses
This study aimed to evaluate the efficacy of potential therapies in Rdh8(-/-)Abca4(-/-) mice (a rodent model of human age-related macular degeneration (AMD). In the recently reported AMD mouse model Rdh8(-/-)Abca4(-/-) mice, we evaluated the therapeutic effects of several antioxidants (ascorbic acid, alpha-lipoic acid, alpha-tocopherol, Mn(III)-tetra(4-benzoic acid)-porphyrin, and butylated hydroxytoluene), an immunosuppressant with anti-vascular endothelial growth factor (VEGF) activity (sirolimus, also known as rapamycin), a retinoid cycling inhibitor (retinamine), and an artificial chromophore (9-cis-retinyl acetate). The animals exhibited retinopathy due to delayed clearance of all-trans retinol caused by the loss of ATP-binding cassette transporter 4 (Abca4) and retinol dehydrogenase 8 (Rdh8) activity. Drug efficacy was assessed by retinal histology and electroretinography (ERG). All investigated drugs partially prevented atrophic changes in the retina of Rdh8(-/-)Abca4(-/-) mice, with retinamine showing the best efficacy. A significant reduction in complement deposition on Bruch's membrane was observed in sirolimus-treated mice, but the severity of retinal degeneration was similar to that in mice treated with antioxidants and 9-cis-retinyl acetate. Four months of sirolimus treatment in 6-month-old Rdh8(-/-)Abca4(-/-) mice prevented choroidal neovascularization without affecting retinal vascular endothelial growth factor (VEGF) levels. Mechanism-based retinamine therapy significantly reduced retinal degeneration in Rdh8(-/-)Abca4(-/-) mice. ...
/EXPL THER/ This study aimed to evaluate the potential ameliorative effect of butylated hydroxytoluene (BHT) on ferric nitrogen triacetic acid (Fe-NTA)-induced oxidative stress and liver injury in mice. Compared with the saline-treated control group, Fe-NTA treatment alone increased ornithine decarboxylase activity by 4.6-fold, protein carbonylation by 2.9-fold, and DNA synthesis (expressed as [(3)H]thymidine incorporation) by 3.2-fold, while antioxidants and antioxidant enzymes decreased by 1.8-2.5-fold. These changes were significantly reversed in animals pretreated with BHT (p < 0.001). Our data suggest that BHT can counteract the toxic effects of Fe-NTA and may serve as an effective chemopreventive agent.

C15H24O

220.36

220.182

128-37-0

Butylated hydroxytoluene-d21;64502-99-4;Butylated hydroxytoluene-d24;1219805-92-1;Butylated hydroxytoluene-d3;86819-59-2

31404

White to off-white solid powder

1.048

265 ºC

69-71 ºC

127 ºC

0.0±0.6 mmHg at 25°C

1.499

5.32

1

1

2

16

207

0

NLZUEZXRPGMBCV-UHFFFAOYSA-N

InChI=1S/C15H24O/c1-10-8-11(14(2,3)4)13(16)12(9-10)15(5,6)7/h8-9,16H,1-7H3

Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-

Butylated hydroxytoluene NSC-6347 NSC6347NSC 6347

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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)

DMSO : ~100 mg/mL (~453.82 mM)
H2O : ~1 mg/mL (~4.54 mM)

Solubility in Formulation 1: 2.5 mg/mL (11.35 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 (11.35 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (11.35 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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Solubility in Formulation 4: 100 mg/mL (453.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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