BUTYLATED HYDROXYTOLUENE (BHT)
First draft prepared by Dr J.C. Larsen,
Institute of Toxicology, National Food Agency of Denmark
1. EXPLANATION
Butylated hydroxytoluene (BHT) was evaluated for acceptable
daily intake for man (ADI) by the Joint FAO/WHO Expert Committee on
Food Additives at its sixth, eight, ninth, seventeenth, twentieth,
twenty-first, twenty-fourth, twenty-seventh, and thirtieth meetings
(Annex 1, references 6, 8, 11, 32, 41, 44, 53, 62, and 73).
Toxicological monographs or monograph addenda were published after
these meetings (Annex 1, references 6, 9, 12, 33, 42, 54, 63, and
74). At its thirtieth meeting the committee established a temporary
ADI of 0-0.125 mg/kg of body weight on the basis of a no-effect
level of 25 mg/kg body weight/day in a one-generation reproduction
study in rats. The committee requested further studies or
information to elucidate the hepatocarcinogenicity of BHT in rats
after in utero exposure and studies on the mechanism of the
haemorrhagic effect of BHT in susceptible species.
Since the previous evaluation, additional data have become
available and are summarized and discussed in the following
monograph addendum.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Male F344 rats were fed BHA/BHT mixtures at levels of 0/0,
0.5/0.05, 1.0/0.1, and 2.0/0.2% in the diet and the levels of the
compounds were determined in adipose tissue after 1, 2, and 4
months. The BHT levels found in adipose tissue were 1.4, 2.9, and
7.8 ppm, respectively, in the dosed animals. On an equivalent dose
basis, BHT accumulated to ten times the level of BHA. However,
neither showed any progressive accumulation with time. Adipose
tissue from 6 humans contained 0.12 ppm BHT. Considering the mean
intake of BHT by humans, and the rat adipose tissue data, previous
observations that accumulation of BHT in the adipose tissue on a
dose/body weight basis is greater in humans than in rats were
confirmed (Conacher et al., 1986).
2.1.2 Biotransformation
The metabolism of BHT was studied with liver and lung
microsomes from rats and mice. Two main metabolic processes occur,
hydroxylation of alkyl substituents and oxidation of the aromatic pi
electron system. The former leads to the 4-hydroxymethyl product
(BHT-CH2OH) and a primary alcohol resulting from hydroxylation of a
t-butyl group (BHT-tBuOH). Additional metabolites were produced by
oxidation of BHT-CH2OH to the corresponding benzaldehyde and
benzoic acid derivatives. Hydroxylation of BHT-tBuOH occurs at the
benzylic methyl position, and the resulting diol is oxidized further
to the hydroxybenzaldehyde derivative. Oxidation of the pi system
leads to BHT-quinol (2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-
cyclohexadienone), BHT-quinone (2,6-di-t-butyl-4-benzoquinone), and
BHT-quinonemethide (2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone)
probably via the hydroperoxide (BHTOOH). Derivatives of the quinol
and quinone with a hydroxylated t-butyl group were also formed.
Quantitative data demonstrate that BHT-CH2OH is the principal
metabolite in rat liver and lung microsomes. The mouse produces
large amounts of both BHT-CH2OH and BHT-tBuOH in these tissues.
The metabolite profile was similar in rat liver and lung. Mouse
lung, however, produced more quinone relative to other metabolites
than mouse liver (Thompson et al., 1987).
The oxidative metabolism of BHT by liver microsomes from three
inbred mouse strains, NGP/N, A/J and MA/MyJ was compared. The
strain order shown is the order of increasing susceptibility of
these mice to BHT lung tumour promotion which correlates with their
increasing ability to produce BHT-BuOH, by hydroxylation of BHT at
one of the tert-butyl groups. Four weekly i.p. injections of BHT
selectively induced the BHT oxidization pathway leading to formation
of BHT-BuOH (Thompson et al., 1989).
The metabolism of BHTOOH was examined to assess the role of
reactive intermediates in mediating tumour promotion in mouse skin.
Incubation of BHTOOH with either isolated neonatal mouse
keratinocytes or a cell-free haematin system resulted in the
generation of the BHT-phenoxyl radical. Only one non-radical
metabolite of BHTOOH-BHT-quinol was detected in keratinocytes while
incubation of BHTOOH with haematin produced several metabolites:
oxacyclopentenone, BHT-quinone, BHT, BHT-stilbene quinone, and BHT-
quinone methide. In contrast to the action of BHTOOH, topical
application of epidermal doses of BHT-quinol, BHT-quinone, BHT-
stilbene quinone, as well as BHT itself, to mouse skin did not
induce epidermal ornithine decarboxylase activity (Taffe et al.,
1989).
When [14C]BHT was activated in vitro by the prostaglandin H
synthase system in microsomes from ram seminal vesicles or by
horseradish peroxidase, significant covalent binding to protein
could be detected. BHT-quinone methide was detected at only minor
concentrations, therefore an intermediate free radical was suggested
as an active metabolite. Addition of BHA to the medium greatly
increased the formation of BHT-quinone methide and covalent binding
to proteins (Thompson et al., 1986).
Co-administration of BHA (200 mg/kg body weight) with a
subtoxic dose (200 mg/kg body weight) of BHT enhanced the lung
toxicity of BHT in male ddy mice. BHA co-administration
significantly increased the radioactivity covalently bound to lung
macromolecules at 4-8 hr after [14C]BHT. The pretreatment also
reduced the rate of in vitro metabolism of BHT in mouse liver
supernatant. The authors suggest the co-administration of BHA and
BHT results in a decrease in metabolism of BHT in the liver with the
result that the lung is exposed to a larger amount of BHT (Yamamoto
et al., 1988).
The in vitro peroxidase-catalysed covalent binding of BHT to
microsomal protein and the formation of BHT-quinine methide was
enhanced by addition of BHA. Several other phenolic compounds
commonly used in food also enhanced the metabolic activation of BHT.
Microsomes from lung, bladder, kidney medulla and small intestine of
various animal species, including man, were also able to support
this interaction of BHA and BHT using either hydrogen peroxide or
arachidonic acid as the substrate.
Subcutaneous injections of BHA significantly enhanced the
lung/body weight ratio of mice given intraperitoneal injections of
subthreshold doses of BHT (Thompson et al., 1986).
2.1.3 Effects on enzymes and other biochemical parameters
Groups of 4 male F344 rats were pretreated with buthionine
sulfoximine (900 mg/kg bodyweight) and after one hour given
intraperitoneal injections of BHT (100, 250, 400, or 500 mg/kg body
weight). A dose related elevation of serum GOT (glutamate-oxalate-
transaminase) and GPT (glutamate-pyruvate-transaminase) activities
was observed. BHT or buthionine sulfoximine alone had no effect.
The elevation of serum enzyme activities was accompanied by a marked
depletion of the hepatic glutathione (GSH) concentration. In
contrast, pretreatment with cysteine (100-200 mg/kg body weight)
inhibited the elevation of serum enzyme activities at a toxic dose
of BHT (1000 mg/kg body weight) (Nakagawa, 1987).
Supplementation of AAF-containing diets with 0.3% BHT, which
affords protection against AAF hepatocarcinogenesis in high-fat fed
Sprague-Dawley rats, protected and/or induced total hepatic nuclear
envelope cytochrome P-450 content. Short-term feeding with AAF and
without BHT results in a marked loss of total hepatic nuclear
envelope P-450, but induction of P-450c and d (Carubelli & McCay,
1987). Immunological studies showed that BHT enhanced the AAF
dependent induction of P-450c, but not P-450d. BHT by itself had no
effect on these nuclear envelope enzymes (Friedman et al., 1989).
2.2 Toxicological studies
2.2.2 Short-term studies
See 2.2.7-2.2.11: Special Studies.
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mouse
BHT was orally administered at concentrations of 1% and 2% of
the diet to B6C3F1 mice for 104 consecutive weeks. Treated animals
underwent a 16-week recovery period prior to pathological
examination. In male mice administered BHT, the incidence of mice
with either a hepatocellular adenoma or a focus of cellular
alteration in the liver was increased showing a clear dose-response
relationship. The incidences of male mice with other tumours and
the incidences of female mice with any tumour were not significantly
increased as a consequence of BHT administration (Inai et al.,
1988).
2.2.3.2 Rat
A long-term study has been initiated to investigate the
development and role in chronic toxicity of hepatic changes in rats
fed BHT over two generations, i.e., involving in utero exposure.
The study aims to mimic the two generation study by Olsen et al.,
(1986) where an increase in liver tumours was seen in males of the
F1 generation after BHT. Only the male offspring is examined in
the study, and while Olsen et al., (1986) used a semi-synthetic
diet, this study uses a conventional standard breeding diet.
Results have been obtained from a dose ranging experiment and are
summarized under 2.2.4 Reproduction Studies. Interim results from
the main experiment (up to 7 months of the F1 generation) were
available. A review will await the final report of the study.
2.2.4 Reproduction Studies
A dose ranging experiment was initiated to determine the
maximum dietary dose of BHT tolerated by female rats exposed prior
to and through pregnancy, and by pups similarly exposed in utero
and until weaning. Groups of 3 male and 16 female Wistar rats were
administered BHT in the diet corresponding to 0, 500, 750, and 1000
mg/kg body weight/day for 3 weeks before mating. At least 8 females
per group were dosed during the pregnancy, and until weaning (21
days after the delivery). After mating, the males and the remaining
females were autopsied. No effect of treatment was seen on blood
clotting times in these animals. Food consumption of treated
females was considerably higher than controls from the fourth week
of the study onwards. No significant effect was seen on body weight
although a dose related trend to reduction was apparent. No effects
were seen on general health except for fur discoloration in treated
animals.
Successful mating occurred less frequently in rats pretreated
with 1000 mg/kg body weight/day of BHT than in the other groups. No
major differences were observed between the groups of pregnant
females. The weight gain in rats treated with the two highest doses
appeared to be inhibited in the last week of the pregnancy. There
was no significant difference between litter number or litter weight
between pups born of control rats and pups born from treated
animals, although a dose-related trend towards reduction in litter
size was seen. No evidence of teratogenic effects of BHT was
provided.
Litter sizes were standardized to eight pups if possible. At
weaning the dams treated with 1000 mg/kg body weight/day of BHT had
lower body weights and very little body fat was observed at autopsy.
Pups from the dams treated with the lowest BHT dose were markedly
stunted in their growth, but appeared healthy. Pups from dams
treated with the two highest doses were severely stunted, showed
poor fur condition, and were less active. It was noted that in BHT
treated animals, where the litter size was less than eight, the
average pup weight was generally considerably greater. This implies
that the reduced weight gains in litters of normal size was
associated with poor milk production rather than BHT toxicity. Pups
from two litters from each dose group were maintained on control
diet for four weeks after weaning. Pups born to dams receiving BHT-
containing diets remained of lower body weight than control pups.
Pups from the two highest dose groups continued to show poor
condition. Treatment with BHT caused a marked increase in liver
weight in all dams. The liver weights were almost 10% of the body
weights, the maximum degree of enlargement possible in rats. The
relative liver weights of pups from BHT treated dams were not
different from controls (Robens, 1990).
2.2.5 Special studies on embryotoxicity
See 2.2.3.2
2.2.6 Special studies on genotoxicity
BHT was reassessed for mutagenic activity using the Salmonella
tester strains TA97, TA102 and TA104, and TA100. BHT did not show
any mutagenic activity, either with or without metabolic activation.
Combinations of BHA and BHT, tested to detect possible synergistic
effects, did not exert mutagenic activity (Hageman et al., 1988).
BHT (0.11-11 µM) protected against DNA damage induced in rat
hepatocytes by 2-acetylaminofluorene (2AAF) or N-hydroxy 2AAF as
shown by a marked reduction of unscheduled DNA synthesis. BHT also
inhibited 2AAF-induced DNA damage in human hepatocytes. In
addition, rats pre-treated with 0.5% BHT in the diet for 10 days
provided hepatocytes which exhibited less unscheduled DNA synthesis
than did hepatocytes from control rats when these cells were exposed
to either 2AAF or N-hydroxy 2AAF (Chipman & Davies, 1988).
BHT was fed to groups of 20 male Sprague-Dawley rats (50, 150,
and 500 mg/kg body weight/day) and 11 male mice (101xC3H)F1 at a
dietary level of 1% for 10 and 8 weeks, respectively, and then
tested for dominant lethal effects. The mice were also tested for
induced heritable translocation. In the rats a dominant lethal
effect of questionable significance was recorded. Results of the
mouse dominant lethal and heritable translocation study indicated no
adverse effects of BHT (Sheu et al., 1986).
At a concentration as low as 10 µg/ml (optimal 50-100 µg/ml)
BHT exerted a strong inhibitory effect on cell-to-cell dye transfer
(lucifer yellow transfer) in cultures of SV-40-transformed
Djungarian hamster fibroblasts. The effect was reversible. BHT
shared this effect with a series of well known tumour promoters
(Budunova et al., 1989).
2.2.7 Special studies on liver toxicity
2.2.7.1 Mouse
Groups of male ddy mice treated perorally with BHT (200-800
mg/kg body weight) in combination with an inhibitor of glutathione
(GSH) synthesis, buthionine sulfoximine (BOS; 1 hr before and 2 hr
after BHT, 4 mmol/kg body weight per dose, i.p.) developed
hepatotoxicity characterized by an increase in serum glutamic
pyruvic transaminase (GPT) activity and centrilobular necrosis of
hepatocytes. The hepatotoxic response was both time- and dose-
dependent. BHT (up to 800 mg/kg) alone produced no evidence of
liver injury. Drug metabolism inhibitors such as SKF-525A,
piperonyl butoxide, and carbon disulfide prevented the hepatotoxic
effect of BHT given in combination with BOS while inducers of drug
metabolism such as phenobarbital tended to increase hepatic injury.
The results suggest that BHT is activated by a cytochrome-P-450-
dependent metabolic reaction and that the hepatotoxic effect is
caused by inadequate rates of detoxification of the reactive
metabolite in mice depleted of hepatic GSH by BOS administration.
Based on studies with structural BHT analogs the authors suggested
that a BHT-quinone methide may play a role in the hepatotoxicity in
mice (Mizutani et al., 1987).
2.2.7.2 Rat
Groups of 8 male Wistar rats were given diets containing 0,
0.1, 0.25, 0.5, and 0.75% BHT for 30 days. BHT did not induce
cellular proliferation in the liver, urinary bladder or thyroid
after 30 days as measured by the [3H]thymidine labeling index or
mitotic index. In a second experiment groups of 8 rats were treated
with 0.5% dietary BHT for 2, 4, 8, 10, and 14 days. This treatment
led to a time-limited increase in liver cell [3H]thymidine labeling
index that subsided to control values within 8 days. This increase
in [3H]thymidine labeling in the liver was accompanied by an
unexpectedly large increase in the mitotic index (Briggs et al.,
1989).
Groups of female Sprague-Dawley rats were given 700 mg BHT/kg
body weight and selected hepatic biochemical effects were determined
after 4 and 21 hours. Ornithine decarboxylase (ODC) activity and
cytochrome P-450 content were increased 190 and 30% respectively.
No effect was seen on hepatic glutathione content or serum alanine
aminotransferase activity. Indication of hepatic DNA damage was
obtained as measured by an increased alkaline DNA elution. No
effects on these parameters could be detected when the BHT dose was
140 mg/kg body weight. It was concluded that BHT in high doses may
have a DNA damaging effect (Kitchin & Brown, 1987).
BHT was administered to male Wistar rats by gavage at doses of
0, 25, 250 or 500 mg/kg body weight/day for 7 days (5 animals per
group), or 28 days (10 rats per group) and also at daily doses of
1000 and 1250 mg BHT/kg body weight (5 animals per group) for up to
4 days (sublethal doses). The sublethal doses induced centrilobular
necrosis within 48 hr, whereas administration of the lower doses of
BHT for 7 or 28 days caused dose-related hepatomegaly and at the
highest dose level induced progressive periportal hepatocyte
necrosis. The periportal lesions were associated with proliferation
of bile ducts, persistent fibrous and inflammatory cell reactions,
hepatocyte hyperplasia and hepatocellular and nuclear hypertrophy.
Evidence of cell damage was also obtained after 250 mg/kg body
weight/day, while there was no evidence that BHT causes liver damage
at a dose level of 25 mg/kg body weight/day. Biochemical changes
consisted of dose-related induction of epoxide hydrolase, dose-
related changes in the ratio of cytochrome P-450 isoenzymes and
depression of glucose-6-phosphatase. Measurement of BHT
demonstrated a dose-related accumulation in fat but not in the liver
(Powell et al., 1986).
2.2.8 Special studies on haemorrhagic effects
Groups of 4-5 male Sprague-Dawley rats (5-6 weeks old) were fed
a diet containing 1.2% butylated hydroxytoluene (BHT) for 1-7 days,
and blood coagulation factors II(prothrombin), VII, VIII, IX and X,
and platelet aggregation were measured. The average intake of BHT
was about 1000 mg/kg body weight/day. The plasma concentrations of
factors II, VII, IX and X were significantly reduced in a time-
dependent fashion when BHT was administered for 2-7 days and
haemorrhages in epididymis were found in rats given BHT for 4-7
days. On the contrary, thrombin-induced and calcium-required
aggregation of washed platelets was unchanged throughout the
experiment. These results suggest that factors II, VII, IX and X
rapidly decrease immediately after the administration of BHT, but
hypoaggregability of platelets may be a secondary defect caused by
bleeding (Takahashi, 1986).
Groups of 4-10 male Sprague-Dawley rats (5-6 weeks old) were
given single oral doses of 800 mg BHT/kg body weight, and 0.5-72
hours later plasma concentrations of blood coagulation factors II
(prothrombin), VII, IX and X and hepatic levels of BHT and BHT-
quinone methide were determined. Levels of the coagulation factors
were reduced 36-60 hours after BHT treatment, but by 72 hours some
recovery had occurred. Hepatic levels of BHT reached maxima at 3 (a
major peak) and 24 hours after BHT dosing and BHT-quinone methide
reached maxima at 6 and 24 hours (a major peak). When BHT was given
in doses of 200, 400 and 800 mg/kg body weight, factors II, VII and
X decreased after 48 hours only in rats given the highest dosage,
but factor IX was more susceptible to BHT and showed a dose-
dependent decrease. Neither pretreatment with phenobarbital for 3
days nor the feeding of 1% cysteine in the diet throughout the
experiment prevented the decrease in vitamin-K-dependent factors by
800 mg BHT/kg. In contrast, pretreatment with cobaltous chloride or
SKF 525A partially prevented the decrease in the blood coagulation
factors. The results indicate that the anticoagulant effect may
require the metabolic activation of BHT (Takahashi, 1987).
The diets used in the above mentioned studies, and in previous
studies from the same laboratory (Annex 1, references 54, 63, and
74) contain no added vitamin K, and the animals apparently were
depleted of stored vitamin K and were marginally vitamin K deficient
(Faber, 1990).
BHT was less efficient than synthetic retinoids in elevating
the prothrombin times and causing haemorrhagic deaths in male
Sprague-Dawley rats maintained on a diet devoid of vitamin K
(McCarthy et al., 1989).
2.2.9 Special studies on potentiation or inhibition of cancer
2.2.9.1 Bladder
Groups of 20 male (six week old) F344 rats were pretreated with
0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water
for 2 weeks and thereafter given diets containing 0, 0.25, 0.5, or
1% BHT. On day 22 of the experiment the lower section of the left
ureter of each rat was ligated. Animals were killed at week 24 of
the experiment. BHT increased dose-dependently the incidence and
number of preneoplastic lesions, papillary or nodular hyperplasia of
the urinary bladder. The incidence of bladder lesions was increased
particularly at 1% BHT (Fukushima et al., 1987).
Groups of 20 male (six week old) F344 rats were pretreated with
0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water
for 4 weeks and thereafter maintained on diets containing 0, 0.4%
BHA + 0.4% BHT + 0.4% TBHQ, or 0.8% BHT. The study was terminated
after 36 weeks. An increase in urinary crystals and incidence and
density of papillary or nodular hyperplasia of urinary bladder
epithelium was observed in all groups fed BHT containing diets. The
incidence of papillomas and carcinomas of the bladder was not
increased and no proliferative changes were seen in renal pelvis.
Hepatocyte hypertrophy was induced in the group administered 0.8%
BHT (Hagiwara et al., 1989).
Ten male F344 rats (6 week old) were given a diet containing 1%
BHT with 7 ppm vitamin K. A decrease in body weight was observed.
DNA synthesis in the urinary bladder epithelium was increased after
4 weeks (5 rats) while no morphological changes were seen after 8
weeks (5 rats) using light microscophy. Using electron microscopy,
morphologic surface alterations such as formation of pleomorphic or
short, uniform microvilli and ropy or leafy microridges were seen
(Shibata et al., 1989).
Groups of 20 male (six week old) F344 rats were given 0.05%
N,N-dibutylnitrosamine in their drinking water for 16 weeks, and
simultaneously administered 0 or 0.7% BHT in the diet. The
simultaneous administration of BHT led to increased incidences in
liver lesions (hyperplastic nodules 16/16 (18/20); hepatocellular
carcinomas 16/16 (8/20); metastasis in the lungs 8/16 (0/20)). The
incidence of transitional cell carcinomas or papillary or nodular
hyperplasia of the urinary bladder and papillomas or carcinomas of
oesophagus was not altered. A decrease in hyperplastic nodules in
the forestomach was observed (Imaida et al., 1988).
2.2.9.2 Mammary gland
A dose related inhibition of 7,12-dimethylbenz[a]anthracene
(DMBA) induced mammary tumorigenesis in female Sprague-Dawley rats
was seen after long-term exposure to dietary BHT. BHT was given
from 2 weeks before carcinogen administration to termination at 210
days. In animals fed the cereal-based NIH-07 diet and receiving a
low dose (5 mg/rat) of DMBA, there was a significant overall
inhibitory trend in tumour incidence observed among those receiving
300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was
approximately 50% at the highest concentration of BHT (6,000 ppm).
The inhibitory effect of BHT on mammary tumour incidence was less
pronounced when BHT was administered to rats initiated with a high
carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20%
at the highest concentration of BHT (6,000 ppm). Similar results
were obtained when BHT was fed in the casein-based AIN-76A diet.
The inhibition seen in this study was less pronounced than that seen
in an earlier study using short-term exposure to BHT (-2 weeks/+2
weeks) (Cohen et al., 1986).
Retinyl acetate (RA) and BHT had additive effects in inhibiting
mammary carcinogenesis in female Sprague-Dawley rats. Chronic
exposure to RA plus BHT induced a high incidence of hepatic fibrosis
and bile duct hyperplasia; these changes were not observed in
controls and were seen in low incidence in animals exposed to RA
only or BHT only (McCormick et al., 1986).
2.2.9.3 Skin
BHT had no tumour initiating activity when tested in a two-
stage mouse skin carcinogenesis model using 12-O-tetradecanoyl
phorbol-13-acetate (TPA) as a promoter. BHT was applied twice
weekly for 5 weeks at a total dose of 100 mg (Sato et al., 1987).
The hydroperoxide metabolite of BHT, BHTOOH (2,6-di-tert-butyl-
4-hydroperoxyl-2,5-cyclohexadienone), was an effective inducer of
epidermal ODC activity in SENCAR mice. Maximal induction of ODC
activity was observed 12 hours after a single application of BHTOOH.
Papilloma and carcinoma formation was observed when BHTOOH was
applied twice weekly for 50 weeks to mice previously initiated with
DMBA. Doses of 2, 8, and 20 µmol BHTOOH gave maximal papilloma
responses. Progression of papillomas to carcinomas occurred after
60 weeks. The data suggest that BHTOOH, unlike BHT, is an effective
tumour promoter in mouse skin. No papillomas or carcinomas were
observed in uninitiated mice treated with BHTOOH only (Taffe &
Kensler, 1988).
2.2.9.4 Gastro-intestinal tract
Seven week old male Wistar rats (20/group) were given in the
drinking water (100 mg/1) for 8 weeks, and were also fed a diet
supplemented with 10% sodium chloride. Thereafter, they were
maintained on a diet containing 1% BHT for 32 weeks. A carcinogen
control group was fed the basal diet without BHT supplementation.
The experiment was terminated 40 weeks after the beginning of
administration of MNNG. BHT did not increase the incidence of
tumours in the glandular stomach or in the forestomach (Takahashi
et al., 1986).
Groups of 21 male F344 rats were given 0.05% N,N-
dibutylnitrosamine in their drinking water for 4 weeks and then
treated with a basal diet containing 1% BHT with 7 ppm vitamin K for
32 weeks. BHT enhanced oesophageal carcinogenesis (papillomas:
16/21 versus 3/21; carcinomas 9/21 versus 0/21) but did not enhance
forestomach carcinogenesis. In the bladder BHT induced an increased
incidence of papillary or nodular hyperplasia and papilloma, while
no statistically significant increase was seen in liver lesions
(Fukushima et al., 1987).
Groups of five male F344 rats were given diets containing 0 or
0.7% BHT for 4 weeks. Histological examination of the forestomach
showed that BHT did not induce hyperplasia in the forestomach
epithelium (Hirose et al., 1987).
When male Fischer 344 rats were fed a diet containing 0.5% or
1.0% BHT for 5 and 6 months immediately following initiation with
two or four injections of DMH, 40 mg/kg sc, a significantly higher
incidence of colon tumours (5 months study) and a significantly
increased incidence of small intestinal tumours (duodenum, jejunum,
and ileum) were seen in the BHT-treated animals than in the animals
fed a BHT-free control diet. Administration of N-nitroso-N-
methylurea (NMU; 90 mg/kg given orally) produced stomach and colon
tumours; 0.5% BHT in the diet did not modulate tumour incidence. It
was concluded that dietary BHT may enhance development of
gastrointestinal tumours produced by DMH, but not by NMU, provided
exposure to BHT occurs after exposure to the carcinogen
(Lindenschmidt et al., 1987).
Male Syrian golden hamsters were given a diet containing 1%
BHT. Induction of hyperplasia and neoplastic lesions of the
forestomach were examined histopathologically and
autoradiographically at week 1, 2, 3, 4, and 16. Mild hyperplasia
occurred slightly more often in hamsters fed the BHT diet than in
the control group. BHT induced no severe hyperplasia or
papillomatous lesions. No significant increase in the labeling
index was observed at any time during the experiment (Hirose et
al., 1986).
2.2.9.5 Liver
BHT was compared to phenobarbital (PB) and with respect to its
effect on liver carcinogenesis in male Wistar rats using an
initiation-selection-promotion protocol. The rats were initiated
with a single dose of diethylnitrosamine (DEN; 200 mg/kg body
weight). Two weeks later selection was carried out by feeding 2-AA+
for two weeks and giving a necrogenic dose of carbon tetrachloride
after one week. After another week the rats were maintained on a
diet with the promoters, or BHT at a level of 0.5%. Groups of 8-10
animals were examined after 3, 6, 14, and 22 weeks on the diet.
BHT, as PB and DDT, had strongly increased the frequency of GGT-
positive lesions in the liver at week 14, but in contrast to PB and
DDT, BHT did not enhance the development of hepatocellular
carcinomas at week 22. It was suggested that BHT was not a promoter
of liver carcinomas in the male Wistar rat when given after
initiation (Préat et al., 1986).
Initiation of liver carcinogenesis with a single dose of
diethylnitrosamine (DEN), and selection with 2-acetylaminofluorene
(2-AAF) combined with a proliferative stimulus (CCl4
administration), was followed by a treatment with PB or BHT (0.5% in
the diet) for periods up to 22 weeks. Control animals received no
treatment after the initiation and selection procedure. An increase
in the amount of 2N nuclei was found in the putative preneoplastic
lesions of animals that received initiation and selection (I-S) and
3 weeks basal diet (BD). When the diet was supplemented with PB
(after I-S), the increase in diploid nuclei started earlier. At the
time carcinomas arise (22 weeks PB treatment) a decrease in the
frequency of 2N nuclei was found. BHT-treated animals which develop
no carcinoma within the considered timespan showed a clear increased
amount of 2N nuclei in the precancerous lesions only after 14 weeks
treatment (Haesen et al., 1988).
Dietary administration of 1% BHT for 26 weeks was commenced
during or immediately after two weekly i.p. injections of azaserine
(30 mg/kg body weight) to male Wistar rats. Administration of BHT
after azaserine enhanced the frequency of GST-A positive focal
pancreatic acinar lesions while GST-P positive hepatocellular
lesions were significantly reduced. When BHT was given together
with azaserine BHT no effect was seen in the liver while the
frequency of preneoplastic lesions in the pancreas was significantly
reduced (Thornton et al., 1989).
2.2.9.6 Lungs
A single i.p. injection of BHT (200 mg/kg body weight) 6 hours
before a single urethane injection (1000 mg/kg body weight) had
varying effects on lung tumorigenesis in mice of different strains
and ages. Strains exhibiting both high (A/J, SWR/J) and low
(BALB/cByJ, 129/J, C57BL/6J) susceptibility to urethane
tumorigenesis were tested. BHT treatment decreased tumour
multiplicity by an average of 32% in adult A/J mice but acted as a
cocarcinogen by increasing tumour number 48% in adult SWR/J mice,
240% in adult C57BL/6J mice, 655% in adult 129/J mice, and 38% in
14-day-old A/J mice. The numbers of both alveolar type 2 cell-
derived and bronchiolar Clara cell-derived lung adenomas were
similarly affected by these BHT treatments. BHT pre-treatment had
no effect on adenoma multiplicity in either young or adult BALB/cByJ
mice. Multiplicity in young BALB cByJ mice was also unaffected by
chronic BHT administration (6 weekly injections) following urethane,
while multiplicities increased several-fold with such treatment in
adult mice of this strain (Malkinson & Thaete, 1986).
A/J mice given urethane (1000 mg/kg) followed by four
injections of BHT (400 mg/kg body weight) developed 40% more lung
tumours than mice treated with urethane alone. In mice treated with
3-methylcholanthrene, repeated injections of BHT (300 mg/kg body
weight) increased tumour multiplicity by a much larger factor (500-
800). Pretreatment of mice with BHT reduced the number of tumours
produced by methylcholanthrene. The enhancing effect of BHT on lung
tumour development was not due to the production of diffuse alveolar
cell hyperplasia (Witschi, 1986).
Lung tumour promotion by BHT and three of its metabolites was
compared in the inbred mouse strain MA/MyJ. Six weekly i.p.
injections of 50 or 200 mg/kg body weight BHT, BHT-BuOH, or two
other metabolites, 2,6-di-tert-butyl-4-hydroxymethyl phenol (BHT-
MeOH) or 2,6-di-tert-butyl-1,4-benzoquinone (DBQ) to MA/MyJ mice
followed a single injection of urethane (50 mg/kg body weight). The
only metabolite that enhanced lung tumour formation was BHT-BuOH,
and it was effective at one-fourth the effective dose of BHT. The
study implicates BHT-BuOH formation as an important step in the
chain of events leading to promotion of lung tumours (Thompson et
al., 1989).
2.2.10 Special studies on pulmonary toxicity
The ability of BHA to modify BHT-induced changes in lung weight
was studied in male CD-1 mice. BHA alone had no effect on lung
weight up to a dose of 500 mg/kg body weight (s.c.). When injected
30 minutes prior to sub-threshold doses of BHT (0-250 mg/kg body
weight, i.p.), BHA significantly enhanced lung weight in a dose-
dependent manner. The ability of BHA to enhance BHT-induced changes
in lung weight was dependent on both the time and the route of
administration of BHA relative to BHT (Thompson & Trush, 1988).
In experiments with mouse lung slices, BHA enhanced the
covalent binding of BHT to protein. Subcutaneous administration of
either BHA (250 mg/kg body weight) or diethyl maleate (DEM, 1 ml/kg
body weight) to male CD-1 mice produced a similar enhancement of
BHT-induced lung toxicity. In contrast to DEM, the administration
of BHA (250 or 1500 mg/kg body weight) did not decrease mouse lung
glutathione levels. In vitro results suggested that BHA
facilitates the activation of BHT in the lung as a result of
increased formation of hydrogen peroxide and subsequent peroxidase-
dependent formation of BHT-quinone methide (Thompson & Trush, 1988).
BHT administration lowered cytosolic Ca++-activated neutral
protease (calpain) activity in the lungs of male and female A/J
mice. The altered proteolytic activity occurred earlier (day 1) and
at a dose lower than that which caused observable lung toxicity as
assessed by the lung weight/body weight ratio (day 4) (Blumenthal &
Malkinson, 1987).
A range of doses from 10-200 mg/kg body weight of BHT or BHT-
BuOH, a metabolite of BHT, were administered i.p. to groups of 2-3
inbred, C57BL/6J mice. BHT-BuOH had a 4- to 20-fold greater potency
than BHT in increasing the relative lung weight, decreasing lung
cytosolic Ca++-dependent protease activity, and causing pulmonary
histopathology. Nature of damage (type 1 cell death) and
regenerative response (type II cell hyperplasia and differentiation)
was identical with the two compounds. BHT-BuOH also caused damage
to liver, kidney or heart. The authors suggested that BHT-BuOH
formation may be an essential step in the conversion of BHT to the
ultimate pneumotoxin, which might be the corresponding BHT-BuOH-
quinone methide (Malkinson et al., 1989).
The synthetic corticosteroid methylprednisolone (MP; 30 mg/kg
body weight, s.c. given twice daily for 3 days) partially protected
male C57BL/6N mice from the pulmonary toxicity of BHT when
administered 0, 24 and 48 hours after BHT treatment (Okine et al.,
1986).
2.2.11 Special studies on nephrotoxicity
A single large dose of BHT (1000 mg/kg body weight) in male
Fischer 344 rats produced some renal damage, as measured by reduced
accumulation of p-aminohippuric acid in renal slices, proteinuria
and enzymuria, in addition to hepatic damage. Administration of
phenobarbital (80 mg/kg body weight, i.p., daily for 4 days) prior
to BHT treatment of male rats produced renal damage accompanied by
slight tubular necrosis and more pronounced biochemical changes.
Female rats were less susceptible to BHT-induced renal and hepatic
damage than male rats (Nakagawa & Tayama, 1988).
The nephrocalcinogenic effect of BHT was studied in groups of
10-20 female Wistar rats (5 weeks old) fed 1% BHT for 13-48 days in
semipurified diets using sodium caseinate or lactalbumin as the only
protein source. BHT induced nephropathy in female rats irrespective
of the diet used. Pronounced nephrocalcinosis was only found in
rats fed the sodium caseinate diet. Thus a connection between the
development of nephropathy and nephrocalcinosis after BHT was not
established (Meyer et al., 1989).
2.3 Observations in humans
In double-blind, placebo controlled challenge tests with a 1:1
mixture of BHT and BHA (50 mg) in 44 cases of chronic urticaria, 91
cases of atopic dermatitis, and 123 cases of contact dermatitis, no
positive reactions were seen (Hannuksela & Lahti, 1986).
The disposition of single oral doses of BHT was compared in man
and rat. A single oral dose of 0.5 mg/kg body weight of BHT was
ingested by 7 healthy male volunteers after fasting overnight.
Blood samples were taken after 0, 15, 30, 45, 60, 75, 90, 120, 150,
180 and 240 minutes. Total urine and faeces were collected for 2
days. In another experiment 5 healthy female volunteers ingested
0.25 mg/kg body weight of BHA and one week later 0.25 mg/kg body
weight of BHT and after another week 0.25 mg/kg body weight of BHT
plus 0.25 mg/kg body weight of BHA were given simultaneously. After
each dosing blood samples were taken as described above. Similar
experiments were conducted in male Wistar rats, except that the
doses used were 20, 63, and 200 mg/kg body weight of BHT. In rats
peak plasma concentrations of BHT (0.2, 0.3, and 2/3 ug/ml) were
seen after 2.6 hour. Simultaneous administration of BHA produced
significantly lower plasma concentrations between 0.5 and 3 hour.
Large variations were seen in man in plasma levels of BHT. The mean
peak plasma level was 0.09 ug/ml reached after 1.5 hour. The plasma
concentrations were not influenced by simultaneous administration of
BHA. In rat urine approximately 2% of the dose was excreted as BHT-
COOH in the urine (equal amounts of conjugated and unconjugated
compound) and 10% as BHT in the faeces in 4 days. In man 2.8% of
the dose was found in the urine as BHT-COOH (mainly conjugated) and
no BHT could be detected in the faeces. On a comparative dose basis
it seems that BHT in plasma reaches a higher level in man than in
rats (Verhagen et al., 1989).
Based on reported BHT levels in human fat in Japan, United
Kingdom and United States and the calculated dietary intakes of BHT
a bioconcentration factor in man (BCF; wet weight basis) of 0.36 was
calculated for BHT. This BCF was 45 times higher than that
calculated for the rat. In comparison, the BCF for total DDT was
calculated at 1279 (Geyer et al., 1986).
3. COMMENTS
As a long-term study in Wistar rats involving exposure in
utero to BHT had shown hepatocarcinogenicity in male rats at a high
dose level, in contrast to several previously reviewed single-
generation long-term studies in Fischer 344 and Wistar rats, the
Committee requested further investigation of the
hepatocarcinogenicity of BHT in rats after in utero exposure. The
Committee also noted that in several studies from one laboratory,
feeding of high doses of BHT caused haemorrhage in rats fed a diet
containing low amounts of vitamin K which suggested an anti-vitamin
K effect of BHT. The Committee therefore requested further studies
on the mechanism of the haemorrhagic effect of BHT.
The requirements of the Committee have been partially met. In
further studies on the haemorrhagic effect of BHT in the male
Sprague-Dawley rat, the compound caused very rapid decrease in
levels of vitamin K-dependent coagulation factors in the plasma,
while platelet aggregation did not seem to be affected initially.
The causative agent is probably a metabolite of BHT as it was
demonstrated that inhibitors of hepatic drug metabolism reduced the
effect on coagulation factors. The Committee noted that high doses
of BHT are required to cause haemorrhage in vitamin K-deficient
rats; it did not consider this effect to be critical with respect
to the safety evaluation of BHT as a food additive in the human
population.
The Committee was informed that a study had been initiated on
the development and role of hepatic changes in chronic toxicity in
male Wistar rats after exposure to BHT in utero. The Committee
reviewed results from a "range finding" study and from the main
study in which the F1 generation had been exposed to BHT in the
diet for 7 months after weaning. The study design was very similar
to that of the previous reported long-term study in which rats were
exposed to the compound in utero.
Additional studies have confirmed the non-genotoxicity of BHT,
and several short-term studies on the liver toxicity of BHT in the
rat have indicated that induction of liver necrosis requires high
doses of BHT while 25 mg/kg b.w. per day was devoid of toxic effects
on the liver. In addition the Committee noted that BHT, in contrast
to phenobarbital and DDT, was not able to enhance hepatocellular
carcinomas in the Wistar rat after initiation with
diethylnitrosamine in 22 weeks.
4. EVALUATION
The Committee extended the previously established temporary ADI
of 0-0.125 mg/kg b.w. pending the results in rats involving in
utero exposure to BHT. The Committee requested the final results
of this study for re-evaluation of BHT in 1994.
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See Also:
Toxicological Abbreviations
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 15)
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 18)
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 42)
Butylated Hydroxytoluene (BHT) (IARC Summary & Evaluation, Volume 40, 1986)