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    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 M) 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 g/ml (optimal 50-100 g/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 mol 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 (Prat  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)