Absorption, Distribution and Excretion
Following a single oral administration of 2 g/kg 2-tert-butyl-4-methoxyphenol, the concentrations of 2-tert-butyl-4-methoxyphenol and 2,2'-dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl (DI-BHA) in rat plasma and intestine appeared at different times (0.15–24 hours). Peak concentrations in all analyzed tissues were observed within 1 hour of administration. The level of 2-tert-butyl-4-methoxyphenol in the intestine was approximately 10 times higher than that of DI-BHA. In plasma, its concentration was 100 to 15 times higher than in vivo. The rat intestine is capable of converting 2-tert-butyl-4-methoxyphenol to dibutylhydroxyanisole (di-BHA) in vivo and is likely the primary site of this conversion. BHA is absorbed from the digestive tract via passive diffusion. Three groups of beagle dogs were administered BHA at dose levels of 0, 0.3, 3, 30, and 100 mg/kg body weight for one year. All animals survived; no pathological damage was observed, nor was any BHA storage found… In male and female Sprague-Dawley rats, BHA was rapidly absorbed and metabolized after oral administration. In male volunteers, after oral administration of 50 mg BHA, 27-77% of the BHA was excreted in the urine as glucuronide and urinary metabolites. …Urinary excretion of BHA peaked within 17 hours and was completely eliminated within 48 hours. …In human volunteers, after a single oral administration of 14C-labeled BHA (approximately 0.5 mg/kg body weight), 60-70% of the radioactive material was excreted in the urine within 2 days, and by day 11, 80-86.5% of the radioactive material was excreted. …Four male volunteers received oral administration of 30 mg BHA, and after 10 days…

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Metabolism / Metabolites
Following oral administration of 2 g/kg 2-tert-butyl-4-methoxyphenol to rats, the metabolite 2,2'-dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl was detected in plasma and tissues within 0.15–24 hours post-administration. DI-BHA (2,2'-dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl) is a product of the reaction of 2-tert-butyl-4-methoxyphenol with commercially available horseradish peroxidase or partially purified rat intestinal peroxidase and hydrogen peroxide. Cyclic compounds (e.g., butylated hydroxyanisole, DI-BHA) contain a hydroxyl group on their ring and are competitive inhibitors of guaiacol, hydrogen peroxide, and peroxidase in systems containing guaiacol, hydrogen peroxide, and peroxidase, but are non-competitive inhibitors of catalase. In rats, the oral dose (0.4 g/kg) was primarily excreted in the urine as glucuronide conjugates (72% of the dose), with smaller amounts excreted as ethyl sulfate (14%) and unmetabolized BHA (5%). Similar metabolic patterns were observed in rabbits and humans… In dogs, only a small amount of BHA glucuronide (5.5%) was excreted in the urine, with the majority of the dose excreted in the feces as unmetabolized BHA. Canine excretion of BHA was primarily in the form of ether sulfate (23% of the dose) and formed hydroxylated and demethylated metabolites, which were not detected in human urine. In male and female Sprague-Dawley rats, oral BHA was rapidly absorbed and metabolized. The main metabolites are 4-O-conjugates: O-sulfate and O-glucuronide… (The working group did not obtain data on dermal absorption.) Following a single oral dose of 1000 mg BHA in New Zealand white rabbits, 46% of the dose was excreted in the urine as glucuronide, 9% as ether sulfate, and 6% as free phenol. The excretion of glucuronide was inversely proportional to the dose: after a 500 mg dose, 60% was excreted as glucuronide; after a 250 mg dose, 84% was excreted as glucuronide. After repeated administration (three or four times), the amount of BHA recovered as glucuronide was less than that recovered after a single dose…

Toxicity Summary
Identification and Uses: 3-tert-butyl-4-hydroxyanisole is a component of butylated hydroxyanisole, a commonly used food preservative. This substance is an oxidation inhibitor and has been approved for use in foods where butylated hydroxytoluene is restricted. This chemical is used as a food preservative for human consumption. Human Exposure and Toxicity: There are currently no human studies on 3-tert-butyl-4-hydroxyanisole. Animal Studies: The mutagenicity of 3-tert-butyl-4-hydroxyanisole and its metabolites was determined using reverse mutagenesis assays with Salmonella Typhimurium strains and in vitro chromosomal aberration assays with Chinese hamster fibroblast cell lines. The results showed that this chemical did not exhibit any mutagenic activity. 3-tert-butyl-4-hydroxyanisole induces chromosomal aberrations only in the presence of S9 mixtures.
Identification and Uses: Butylated hydroxyanisole (BHA) is a white or slightly yellow waxy solid. The main uses of BHA are as an antioxidant and preservative in food, food packaging, animal feed, cosmetics, and rubber and petroleum products. BHA is particularly effective at protecting the flavor and color of essential oils and is considered the most potent of all food-grade antioxidants. BHA effectively controls the oxidation of short-chain fatty acids, such as those found in coconut oil and palm kernel oil, which are commonly used in cereal and confectionery products. Human Exposure and Toxicity: BHA can cause allergic contact dermatitis. Although BHA itself is not irritating, it may still cause skin reactions due to its relatively weak sensitizing properties. BHA can reduce DNA damage and micronucleus formation in peripheral lymphocytes exposed to other genotoxic substances. Animal Studies: Dietary BHA intake led to benign and malignant tumors (papilloma and squamous cell carcinoma) in the forestomach of both male and female rats, male mice, and hamsters, with increased lifespan compared to the control group. Rats tolerated dietary BHA up to 1200 ppm for 21 months. No histopathological or carcinogenic effects associated with BHA intake were found, nor were any effects on reproduction observed. The teratogenic effects of BHA were assessed in rats and mice at dose levels up to 1000 mg/kg and 500 mg/kg, respectively. No exposure-related abnormalities were observed. In another study, the teratogenic effects of BHA in rabbits were assessed at dose levels ranging from 50 to 400 mg/kg. No abnormalities were reported. Feeding BHA to juvenile rainbow trout (hermaphroditic marbled rainbow trout) led to hepatocellular carcinoma in adult fish. At doses of 5 to 500 mg/kg, BHA caused neurological changes in mice by increasing serotonin utilization in the central nervous system. Ecotoxicity studies showed that feeding BHA resulted in altered enzyme activity of the mixed-function oxidase system in rainbow trout, changes in ethyl isocyanate binding, and decreased cytochrome P-450 levels in liver microsomes.

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Interactions
In a benzo[a]pyrene-induced forestomach tumor assay in ICR/HA mice, the addition of 3-tert-butyl-4-hydroxyanisole reduced the number and incidence of tumors. Mice fed a diet containing 0.75% 2(3)-tert-butyl-4-hydroxyanisole (BHA) for 8 days showed a 50% reduction in the maximum induction of ornithine decarboxylase (ODC) after treatment with TPA (12-O-tetradecanoylphorbol-13-acetate). Topical application of BHA (55 μmol) 30 minutes before TPA (17 nmol) treatment inhibited 80% of promoter-induced ODC activity. Administration of BHA 16 hours before or 2 hours after promoter treatment had no effect. The inhibitory effect was dose-dependent, with a dose of 6 μmol inhibiting 50% of ODC-induced activity. Structure-activity relationship studies indicated that the hydroxyl and tert-butyl substituents were key determinants of inhibitory activity.
Rats were fed a diet containing or without ciprofibrate (10 mg/kg body weight) and 2(3)-tert-butyl-4-hydroxyanisole (0.5% wt/wt) for 60 weeks. 2(3)-tert-butyl-4-hydroxyanisole significantly reduced the incidence and number of hepatocellular carcinomas larger than 5 mm. The data suggest that the inhibitory effect of BHA on ciprofibrate-induced liver tumorigenesis may be attributed to its H₂O₂ and free radical scavenging properties, as it does not inhibit the proliferation of peroxisomes and the induction of H₂O₂ peroxisome generation in the livers of rats fed ciprofibrate.
In mice and rats, the LD50 of BHA was determined by intraperitoneal or oral administration, with dimethyl sulfoxide or olive oil as the solvent. When using dimethyl sulfoxide, the LD50 of intraperitoneal BHA was about two orders of magnitude lower than the oral LD50. However, this difference was not significant when BHA was dissolved in olive oil. /BHA/
BHA and BHT both exhibit chemopreventive effects when administered concurrently with various carcinogens affecting different target organs. Antioxidants enhance the scavenging capacity of detoxification enzymes against carcinogens, and many antioxidants also scavenge free radicals. The structures of these substances suggest they are unlikely to be electrophilic, and genotoxicity tests were all negative. Both BHA and BHT have been shown to inhibit intercellular communication in vitro. /BHA/
Drinking water disinfection byproducts (DBPs) are generated during the chemical disinfection of water and may pose a threat to public health. Two main types of DBPs are present in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed after chlorine disinfection, where chlorine reacts with iodides and bromides in the water. Previous studies have shown that HAAs possess cytotoxic, genotoxic, mutagenic, teratogenic, and carcinogenic properties. This study aims to determine the effects of haloacetic acids (HAAs) on human somatic and germ cells, and whether oxidative stress is involved in their genotoxic effects. This study examined peripheral blood lymphocytes and somatic and germ cells, including sperm. The effects of three HAA compounds—iodoacetic acid (IAA), bromoacetic acid (BAA), and chloroacetic acid (CAA)—were investigated. After determining appropriate concentrations, the scavenging effects of the antioxidant butylated hydroxyanisole (BHA) and catalase on oxygen free radicals were studied under alkaline conditions (pH > 13) using single-cell gel electrophoresis (comet assay) and micronucleus assays. The comet assay results showed that both BHA and catalase reduced DNA damage in all cell types compared to HAAs alone. In the micronucleus assay, micronuclei (MNis) were found in peripheral blood lymphocytes exposed to all three HAAs. Catalase and BHA generally reduced MNi induction, indicating that oxygen free radicals played a role in both assays. These observations raise public health concerns because human somatic and germ cells exhibit similar genotoxic responses. Butylated hydroxyanisole (BHA) and propylparaben are commonly used phenolic preservatives in food, pharmaceuticals, and personal care products. Due to increasing concerns about their potential environmental and human health impacts, both chemicals have undergone extensive toxicological studies. However, the cytotoxicity and underlying mechanisms of co-exposure to these compounds have not been explored. This study analyzed a range of relevant cytotoxic endpoints, including cell viability and proliferation, oxidative stress, DNA damage, and changes in gene expression, to assess whether the antioxidant BHA could prevent the pro-oxidative effects induced by propylparaben in Vero cells. We demonstrated that the binary mixture of the two chemicals produced a stronger cytotoxic effect than exposure to each compound alone. Concomitant treatment of cells with BHA and propylparaben led to G0/G1 phase cell cycle arrest due to enhanced oxidative stress and DNA double-strand breaks. DNA microarray analysis revealed that the interaction between transforming growth factor β (TGFβ) and ataxia-telangiectasia mutant kinase (ATM) pathways regulates the response of Vero cells to test compounds in a binary mixture. Our results indicate that butylated hydroxyanisole (BHA) enhances the pro-oxidative activity of propylparaben in cultured mammalian cells and provides useful information for assessing its safety. This study evaluated the anti-androgenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG) individually and in combination (binary mixtures) 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 was monitored by measuring luminescence intensity. In assessing anti-androgenic 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 the anti-androgenic activity of PG. Neither the compounds nor the binary combinations showed androgenic activity in either individual or mixed assays. BuPB, BHA, and BHT all exhibited weak anti-androgenic activity in the presence of DHT, as confirmed in the evaluation of 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 reaction-additive models) in predicting the anti-androgenic 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 activity of the compounds in individual assays, it was impossible to estimate the effects of mixtures containing PG. This study aimed to investigate the protective effect of butylated hydroxyanisole (BHA), a commonly used phenolic antioxidant in food, against ferric triacetate (Fe-NTA)-induced nephrotoxicity. This study used 4-6 week old male albino Wistar rats weighing 125-150 g. Animals were treated with BHA (1 and 2 mg/rat/day) followed by a single dose of Fe-NTA (9 mg/kg body weight). Compared to the saline control group, Fe-NTA treatment increased ornithine decarboxylase (ODC) activity in the kidneys by 5.3-fold, [(3)H]-thymidine incorporation into DNA by 2.5-fold, while glutathione (GSH) levels and antioxidant enzyme activity decreased by 2-2.5-fold. These changes were significantly reversed in animals pretreated with BHA. Compared to the Fe-NTA treatment group, ODC activity and DNA synthesis decreased by 2.12-fold and 1.15-fold, respectively, in the higher dose (2 mg/rat/day) BHA treatment group. Pre-administration of BHA before Fe-NTA treatment increased GSH levels and antioxidant enzyme activity in the kidneys by 1.5-2-fold. The results showed that BHA could inhibit Fe-NTA-induced nephrotoxicity in male Wistar rats. For more complete data on interactions of butylated hydroxyanisoles (32 in total), please visit the HSDB record page.

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Non-Human Toxicity Values
Oral LD50 in rats: 4000 mg/kg; Oral LD50 in rabbits: 2100 mg/kg; Oral LD50 in mice: 2000 mg/kg body weight; Oral LD50 in rats: 2200 mg/kg body weight; Intraperitoneal LD50 in male rats: 881 mg/kg body weight.

Butylated hydroxyanisole is a white, beige, or slightly yellow waxy solid with an aromatic odor and a slightly bitter, burning taste. (NTP, 1992)
3-tert-butyl-4-hydroxyanisole is an aromatic ether, a derivative of 4-methoxyphenol, in which a hydrogen atom at the ortho position of the phenolic hydroxyl group is replaced by a tert-butyl group. It has antioxidant properties and is also a byproduct of human xenobiotic metabolism. It belongs to the phenolic class of compounds and is an aromatic ether.
2-tert-butyl-4-methoxyphenol has been reported to exist in sage (Salvia officinalis), Murraya paniculata, and Dillenia indica, but relevant data are unavailable.
Butylated hydroxyanisole is a white waxy solid mixture of 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole, with a faint aromatic odor. Butylated hydroxyanisole (BHA) is a synthetic antioxidant widely used in food, cosmetics, and pharmaceuticals, primarily for preserving oils and fats. BHA is considered potentially carcinogenic to humans. (NCI05)
A mixture of 2- and 3-tert-butyl-4-methoxyphenol is used as an antioxidant in food, cosmetics, and pharmaceuticals.

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Mechanism of Action
Administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA) to rodents can protect various target tissues from tumorigenesis induced by a variety of chemical carcinogens. BHA can reduce the levels of benzo[a]pyrene and mutagenic metabolites produced by various therapeutic drugs in vivo; increase the activity of hepatic microsomal epoxide hydratase and cytoplasmic glutathione S-transferase; alter the activity of other hepatic enzymes and affect the levels of certain hepatic catalytic components; and increase the concentration of non-protein thiols in the liver and other tissues. This study investigated the effects of butyl hydroxyanisole (BHA) on the activity of aryl hydrocarbon hydroxylase (AHH) in the liver, lungs, and skin of rats and mice to explore the possible mechanism of its anticancer effect. The AHH inducers 3-methylcholanthrene, phenobarbital, and 2,3,7,8-tetrachlorodibenzo-p-dioxin were used in the experiment. Observations revealed that 2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, and a commercial mixture of BHA (85% 3-BHA and 15% 2-BHA) exhibited roughly the same inhibitory efficacy against microsomal AHH, although the 2-isomer appeared to have a slightly stronger inhibitory effect. Data suggest that the inhibitory effect observed in commercial BHA is due to the combined action of the two isomers and is dependent on animal species, tissue type, and inducer treatment. Both BHA and BHT have chemopreventive effects when co-administered with various carcinogens affecting different target organs. Antioxidants can increase the activity of carcinogen-detoxifying enzymes, and many antioxidants also act as free radical scavengers. The structures of these substances suggest they are unlikely to be electrophilic, and genotoxicity tests were all negative. Both BHA and BHT have been shown to inhibit intercellular communication in cultured cells. /BHA/
Estrogen metabolism-mediated oxidative stress is considered to play an important role in estrogen-induced breast cancer development. We have previously demonstrated that the antioxidants vitamin C (Vit C) and butylated hydroxyanisole (BHA) can inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and suppress breast cancer development in female August Copenhagen Irish (ACI) rats. This study aimed to elucidate the mechanism by which these antioxidants prevent DNA damage during breast cancer development. Female ACI rats were treated with E2, Vit C, Vit C+E2, and BHA, respectively. Over a period of 240 days, the mRNA and protein levels of the DNA repair enzyme 8-oxoguanine DNA glycosidase (OGG1) and the transcription factor NRF2 in rat mammary tissue and breast tumor tissue were quantitatively analyzed and compared with rats treated with antioxidants alone or with antioxidants in combination with E2. The results showed that OGG1 expression was suppressed in mammary tissue and breast tumors of rats treated with E2. NRF2 expression was also significantly suppressed in E2-treated mammary tissue and breast tumors. Treatment with vitamin C or BHA prevented E2-mediated reductions in OGG1 and NRF2 levels in breast tissue. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated OGG1 induction is achieved by increasing the direct binding of NRF2 to the OGG1 promoter region. Studies using silent RNA confirmed the role of OGG1 in inhibiting oxidative DNA damage. Our study suggests that the antioxidants vitamin C and BHA provide protection against oxidative DNA damage and E2-induced breast cancer, at least in part, through NRF2-mediated OGG1 induction.

C11H16O2

180.25

180.115

121-00-6

8456

Off-white to light yellow solid powder

1.0±0.1 g/cm3

269.1±28.0 °C at 760 mmHg

58-64 °C

105.6±8.9 °C

0.0±0.6 mmHg at 25°C

1.508

3

1

2

2

13

160

0

O([H])C1C([H])=C([H])C(=C([H])C=1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

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.)