Abstract

BHA has been subjected to extensive toxicological study. Since there are emerging concerns about
the possible influence of BHA on environmental and human health, this project focuses on the
genotoxicity and carcinogenicity of BHA and potential risks from exposure. The genotoxicity of
BHA has been studied in vitro and in vivo “which did not prove BHA to be mutagenic” (JECFA,
1987). BHA toxicity is low in rodents with an LD50 in mouse and rat > 2000 mg/kg bw/day. The
International Agency for Research on Cancer (IARC) categorized BHA as possibly carcinogenic to
humans due to insufficient evidence in humans but sufficient evidence in animals. In the US
National Toxicology Program Report on Carcinogens (NTP, 2005), BHA is predicted to be a
carcinogen. However, based on the human population statistics, the usual low intake levels of BHA
shows no significant association with an increased risk of cancer. Some experts recommend
banning BHA, using instead vitamin E or packing foods under nitrogen.

Background
Butylated hydroxyanisole (BHA) consists of a mixture of two isomeric compounds, 2-tert-butyl-4-
hydroxyanisole and 15% or less of 3-tert-butyl-4-hydroxyanisole. At room temperature, BHA is a
white or slightly yellow, waxy solid with a faint characteristic odor (IARC 1986).

Commonly used in food, pharmaceuticals, cosmetics, and animal feed as an antioxidant and has also
been found in water. Although deemed as a safe preservative, studies have found contradictory effects
of BHA such as its role as an antioxidant and pro-oxidant, anti-carcinogen and carcinogen, and as an
environmental endocrine disruptor (Nie et al.). Most studies have found various health risk outcomes
from exposure to BHA. Health risks associated with oral ingestion of BHA include protection of animals
against numerous xenobiotics by preventing carcinogenesis in numerous tissues such as the liver, lung,
and breast (Vandghanooni et al.). In contrast, BHA has also been observed to act as a tumor initiator or
tumor promoter and, at high doses, induces the formation of papillomas and carcinomas
(Vandghanooni et al.).

Mechanism of Action
BHA is oxidized to its metabolite, tert-butylhydroquinone (tBHQ), which is then further
oxidized to 2-tert-butyl(1,4)paraquinone (TBQ), a semiquinone anion radical and reactive
oxygen species. Many studies have documented the cytotoxic effects of BHA exposure; however,
the mechanism by which BHA induces cytotoxic effects is still relatively unclear. Some studies
have suggested that the cytotoxic nature of BHA is dependent on the formation of its reactive
intermediates while other studies have suggested that the cytotoxicity of BHA is mediated by the
release of enzymatic constituents from mitochondria (Yu et al.).

Cu-mediated oxidation of tBHQ to its reactive oxygen species and other constituents relies heavily upon the
addition of Cu(II). Without Cu(II), the absence of a Cu(II)/Cu(I) redox cycle results in the absence of BHA-
induced carcinogenesis.
In contrast, studies conducted on rat hepatocytes have found that BHA may directly interact with mitochondria
to induce apoptosis by initiating the release of caspase and cytochrome c (Yu et al.). The release of the enzymes
trigger a series of events which induces cytotoxic effects in rat hepatocytes. A diagram of the proposed
mechanism for BHA-induced cytotoxicity in rat hepatocytes is shown below.

Scavenging the reactive intermediates by antioxidants such as

NAC or ascorbic acid did not affect BHA-induced apoptosis,
suggesting that BHA alone may induce apoptosis. Also, the extent
of the effects seem to be dose-dependent. BHA exposure at the
highest dose of 1 mM BHA seems to cause rapid damage to cell
membrane as well as significant DNA fragmentation (Yu et al.).
At doses of 250 uM of BHA, rat hepatocytes were showing slight
reductions in cell viability. In addition, enzymatic activities of
caspase and cytochrome c significantly increased at doses of 500
uM of BHA. The increase in the enzymatic activities seems to
play a role in mediating the induction of apoptosis.
In contrast, studies have shown the protective effects of BHA and
another phenolic compound, Quercetin, against cellular injury
induced by oxidative stress, when exposed to each antioxidant.
There is evidence that indicates BHA is more protective than
Quercetin against ROS generation, lipid peroxidation and lipid
losses in oxidized erythrocytes. The protection against oxidative
damage provided by both antioxidants decreased while membrane cholesterol depleted. The LC50 values were
almost two-fold in the presence of quercetin as opposed to BHA in control.
If no antioxidant was used, the exposure of erythrocyte groups to BHA led to significant losses of phospholipids
and cholesterol. So antioxidants have a potent inhibition effect on cholesterol losses. Both antioxidants
perform significant role in protecting phospholipids against losses. Cholesterol depletion in the erythrocyte
membrane reduces the protective effects of antioxidants against lipid peroxidation and ROS production. This
will minimize the efficiency of antioxidants against oxidative stress in cholesterol-depleted erythrocytes.
(Sánchez-Gallego et al.).

BHA was also examined for its capacity to modulate the cytotoxic effects of ∆9-tetrahydrocannabinol (THC).
THC was not cytotoxic at low concentrations, but induced cell necrosis at higher levels. BHA, at concentrations
of 10–200 _M, induced partial cell toxicity but considerably boosted the necrotic death resulting from
concurrent exposure to THC. In the presence of BHA at 200 _M, the LC50 for THC decreased. Neither of
inhibitor compounds (diphenyleneiodonium or dicumarol ), protected cells from the effects of combined THC
and BHA, but rather enhanced necrotic cell death. Both THC and BHA reduced ATP levels in A549 cells, and
showed toxic effects on mitochondrial electron transport. The combination of BHA and THC was synergistic in
this respect, reducing ATP levels. Exposure to marijuana smoke in conjunction with BHA, may promote
deleterious health effects in the lung. (Theodore A. Sarafian et al.)
Effects of BHA in Aquatic Organisms
The use of BHA is very ubiquitous, so industrial wastewater and surface water containing BHA may effortlessly
enter into the environment and disrupt the atmospheric, terrestrial, and aquatic ecosystems. BHA is known to
have low soil mobility, volatilizes slowly from water, and has a high bioconcentration factor—these properties
raise a concern that, over time, BHA may potentially bioaccumulate in the environment and in the tissues of
organisms in the environment.
Currently, very few literature has been published on the metabolic breakdown of xenobiotics in exposed algae—
less than 1% of ecotoxicological data is available for pharmaceuticals (Sanderson et al.). The available studies,
thus far, have assessed the behavioral response of phase I and phase II enzymes in algae as well as analyzing
the potential ecotoxicological effects of BHA bioaccumulation (Nie et al.; Jos et al.)
One of the few existing literature includes a study involving a battery with six different types of organisms and
eighteen endpoints over a range of exposure times. Below is a table which summarizes the different organisms
and respective endpoints of the study by Jos et al.

The study by Jos et al. found that exposure to BHA had various EC50 values ranging from 1.2 uM to more than
500 um for 5, 15, and 60 minutes. The results of the study suggest that the most sensitive to the least sensitive
organisms is as follows: V. fischeri >D. magna > C. vulgaris > Vero mammalian cell line > A. cepa (Jos et al.).
V. fischeri seem to be the most sensitive to BHA and had immediately showed signs of bioluminescent decline
after 15 minutes of BHA exposure. As for the rest of the organisms, most began showing apparent signs of BHA
toxicity after 48 hours of BHA exposure.
The toxic effects of norfloxacin (NOR) and butylated hydroxyanisole (BHA) on the microalga Scene desmus
obliquus were assessed in terms of growth rate. Scenedesmus obliquus is an ideal model aquatic organism.
Growth of S. obliquus was decreased under high concentrations of BHA and NOR over the 96 h exposure
period. Growth inhibition was 82.4% at 60.0 mg/l for NOR and 60.6% at 16.0 mg/l for BHA after 96 h. These
types of assays, revealing toxic effects of NOR and BHA to phototrophs, could be employed to assess the
potential risks of these xenobiotics to aquatic ecological systems. Increasing BHA concentration increased the
inhibition of S. obliquus growth, displaying an obvious dose-response relationship (Nie et al.).
BHA was classified by the IARC as a possible carcinogen to humans according to the evidences in experimental
animals. BHA and propylparaben (PPB) both are phenolic preservatives which have been reported to have
possible impacts on environmental and human health. Though, the cytotoxicity of co-exposure to these
compounds have not been discovered. This study, shows whether the BHA could stop the pro-oxidant effects of
propylparaben in Vero cells. According to the results, concurrent exposure to both BHA and PPB induce
greater cytotoxic effects and shows synergistic characteristics of their co-exposure. Binary mixture of these two
chemicals caused G0/G1 cell cycle arrest as a result of oxidative stress and DNA double strand breaks. The
evidences indicate that BHA stimulates the pro-oxidant effects of propylparaben (Lee et al.).
Animal Health Effects of BHA
Routes of human exposure to BHA are ingestion, inhalation, and dermal contact. The estimated average daily
intake of BHA in the human diet was 4.3 mg (IARC, 1986). Workers are also potentially at risk of exposure to
BHA in industries such as food production, and animal feed production, cosmetic manufacturers, and
petroleum workers (Report on Carcinogens, 13th edition). Currently, there has only been one epidemiological
study which contained adequate data to evaluate the relationship between human cancer and BHA exposure.
The population-based nested case-control study of stomach cancer in men and woman within the Netherlands
Cohort Study of dietary intake found no increase in risk at ordinary levels of dietary intake of BHA (Botterweck
et al., 2000).
However, oral exposure to BHA have been observed to induce benign and malignant forestomach tumors in
F334 rats, and the effects seem to be dose-related. There have also been a higher incidence of hyperplasia
development in the forestomach in both male and female rats (Nobuyuki et al.). Male and female rats were
orally fed a low or high dose of BHA, 98 and 414 mg/kg/day and 108 and 474 mg/kg/day, respectively, for a
total of 112 weeks. Male and female rats treated with a higher dose of BHA had a significantly higher incident of
developing papilloma and squamous cell carcinoma.

BHA exposure has also been associated with acting as a weak endocrine disruptor and inducing cytotoxic
effects on the reproductive system of rats such as reproductive function, maturation, and hormonal status
before, during, and after fetus development. Exposure to BHA at 100 mg/kg or 500 mg/kg seems to decrease
the weight of sex organs such as vagina, testes, and ventral prostate while increasing the weight of liver, adrenal
and thyroid gland. Although the toxic effects seem to be weak, the decreased weight of sex organs, low steroid
and thyroid hormone levels, and minor histopathological lesions of the thyroid gland give cause to suspect
BHA as an endocrine disruptor (Jeong et al.).
A study which evaluated the estrogenic and androgenic activity of butylated hydroxyanisole (BHA), suggested
the anti-estrogenic activity of BHA which decreases the uterine weight in immature female rat while it doesn’t
have effect on the androgenic activity in castrated male rats. So many in vitro studies has also been performed
about the endocrine disruptive trait of BHA. According to .Jeong et al. (2005), high dose BHA (500 mg/kg) can
cause impaired reproductive functions such as delayed sexual maturation, and decreased vaginal weight. BHA
250 mg/kg also was shown that increased the TP-stimulated relative ventral prostate gland weight.
Antioxidants were reported to have synergistic effects on testosterone in apoptosis of prostate cancer cells in
castrated rats. High doses of BHA may induce forestomach carcinomas followed by cell injuries in rodents.
Therefore BHA could be a rodent carcinogen. According to the evaluation of WHO (1989), BHA can cause
hemorrhage, liver lesion, hyperplasia and hyperkeratosis in stomach mucosa in Rodents. (WHO, 1989; IARC,
1986; Botterweck et al., 2000). Body weight showed significant decrease while treated with BHA 250 and 500
mg/kg., but relative liver weight was increased at BHA 500 mg/kg.
Regulations
The 13th Report on Carcinogens, published on October 2, 2014, listed current regulations for BHA. According to
the Food and Drug Administration (FDA), BHA is:
• Generally recognized as safe for use in food when the total of antioxidants is not greater than 0.2% of fat
or oil content.
• May be used as a food additive for direct addition to food intended for human consumption.
• May be used in manufacturing of food packaging materials as long as the amount does not exceed
0.005%.
• May be used as an antioxidant in defoaming agents for processed foods as long as the amount does not
exceed 0.1% by weight of defoamer
In addition, the EPA has listed BHA as a contaminant on Candidate List 3. Contaminant candidates on this list:
• Currently not subjected to any proposed national primary drinking water regulation(s)
• Known or anticipated to occur in public water systems
• May require regulation under the Safe Drinking Water Act (SDWA)
• Remediation
• Currently, there is an absence of water treatment methods to remediate water sources in which BHA is
found. As such, EPA has assigned BHA on the list of chemicals for Tier 1 Screening. BHA is identified
under the Safe Drinking Water Act (SDWA) because the EPA believes this this chemical should be a
candidate, at least for screening purposes, under EDSP testing because it may occur in sources of
drinking water to which a substantial population may be exposed (EPA 2014).
• Conclusions
• Studies support the concept that BHA does not raise concern with respect to carcinogenicity and
genotoxicity. BHA exposure, at high doses, has been shown to induce forestomach carcinomas in
rodents. Since humans do not have forestomach, BHA does not pose as a hazard. BHA at lower
concentrations such as 100 ppm, exert anticarcinogenic properties. Thus, an ADI of 0.5 mg/kg bw/day
was established. Most of our studies concluded that BHA exposure had no association between rodent
forestomach hyperplasia or human health effects. Therefore, authorities have recommended continued
use of BHA (Iverson, 1995; JECFA, 1996). Based on these studies, BHA at levels found in consumer
products is not significant enough to pose a risk to human health.
Abstract
BHA has been subjected to extensive toxicological study. Since there are emerging concerns about
the possible influence of BHA on environmental and human health, this project focuses on the
genotoxicity and carcinogenicity of BHA and potential risks from exposure. The genotoxicity of
BHA has been studied in vitro and in vivo “which did not prove BHA to be mutagenic” (JECFA,
1987). BHA toxicity is low in rodents with an LD50 in mouse and rat > 2000 mg/kg bw/day. The
International Agency for Research on Cancer (IARC) categorized BHA as possibly carcinogenic to