First draft prepared by
    Ms Elizabeth Vavasour
    Toxicological Evaluation Division, Bureau of Chemical Safety
    Food Directorate, Health Protection Branch, Health Canada
    Ottawa, Ontario, Canada


    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Effects on enzymes and other biochemical parameters

    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Reproductive toxicity studies
         Special studies on teratogenicity
         Special studies on genotoxicity
         Special studies on hepatotoxicity
         Special studies on nephrotoxicity
         Special studies oil pulmonary toxicity
         Special studies on haemorrhagic effects
         Special studies on effects on the thyroid
         Special studies on effects on the immmune system
         Special studies on potentiation or inhibition of cancer
         Special studies on other effect
         Observations in human


         Butylated hydroxytoluene (BHT) was previously evaluated by the
    Committee at its sixth, eighth, ninth, seventeenth, twentieth,
    twenty-fourth, twenty-seventh, thirtieth, and thirty-seventh meetings
    (Annex 1, references 6, 8, 11, 32, 41, 53, 62, 73, and 94). At the
    thirty-seventh meeting, the temporary ADI of 0 - 0.125 mg/kg bw,
    established at the thirtieth meeting, was extended pending the results
    of a study designed to elucidate the role of hepatic changes in the
    development of hepatic carcinomas observed in Wistar rats following
     in utero and lifetime exposure to BHT.

         The results of the above study were reviewed at the present
    meeting. In addition, new data relating to the previously-noted
    effects of BHT on the lung, liver, kidney, clotting mechanisms and
    promotion/inhibition of carcinogenesis, new long-term and reproductive
    toxicity studies, genotoxicity assays and human observations were

         The following consolidated monograph is a compilation of studies
    from the previous monographs and monograph addenda and those reviewed
    for the first time at the present meeting.


    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion  Mice

         A single oral dose of 14C-BHT given to male and female mice
    resulted in rapid absorption and distribution of 14C to the tissues.
    Excretion of 14C was mainly in the faeces (41-65%) and urine
    (26-50%), with lesser amounts in expired air (6-9%). The half-life for
    a single dose in the major tissues studied (stomach, intestines,
    liver, and kidney) was 9-11 h. When daily doses were given for 10
    days, the half-life for 14C in the tissues examined was 5-15 days.
    Metabolism was characterized by oxidation at one or both of the
     tert-butyl groups, followed by formation of the glucuronide
    conjugate, and excretion in the urine, or by excretion of the free
    acid in the faeces. More than 43 metabolites were present in the urine
    and faeces of mice (Matsuo  et al., 1984).  Rats

         When 14C-BHT was administered to rats, 80-90% of the 14C was
    excreted in the urine and faeces within 96 h, and less than 0.3% of
    the 14C was in expired air. Most of the 14C-BHT was excreted as
    the free acid in faeces with lesser amounts in the urine. More than 43
    metabolites were present in the urine and faeces of rats
    (Matsuo  et al., 1984).

         Male F344 rats were fed BHA/BHT mixtures at levels of 0/0,
    0.5/0.05, 1.0/0.1, or 2.0/0.2% in the diet and the levels of the
    compounds were determined in adipose tissue after 1, 2, or 4 months.
    The BHT levels found in adipose tissue were 1.4, 2.9, or 7.8 mg/kg,
    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. Considering the rat adipose tissue
    data, the mean intake of BHT by humans and the corresponding level of
    BHT found in adipose tissue from 6 humans (0.12 mg/kg), 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).

         Groups of male and female rats were maintained on diets
    containing 0 or 0.5% BHT for a period of 35 days, and then for a
    period on diets free of BHT. During the period on the test diets,
    groups of rats were killed at 5-day intervals and fat and liver
    removed for BHT analysis. During the period on BHT-free diets, rats
    were killed at 2-day intervals to measure loss of BHT from the fat and
    liver. There was no clear evidence of progressive accumulation of BHT

    in fat during the period of administration of the test compound. BHT
    levels in the fat reached a maximum level (55 mg/kg in males, 65 mg/kg
    in females) within 10 days of exposure to BHT. Thereafter there was
    considerable fluctuation in the observed levels. The levels of BHT in
    liver were very low, the maximum BHT levels being about 5 mg/kg in
    males, and 1.5 mg/kg in females. The biological half-life of BHT in
    fat and liver was estimated to be 7 to 10 days (Gage, 1964).

         Groups of two rats (one male, one female) were given 1 to 5 oral
    doses of 44 mg/kg bw BHT on alternate days and rats in each group were
    killed 24 h after the final dose. The range of the total dose
    accounted for was 92 to 104% in males and 93 to 99% in females. There
    was an indication of sex difference in the route of excretion, females
    excreting 19-43% of the radioactivity in urine and males only 3-15%.
    Eight days after administration of 5 doses, 92% of the radioactivity
    had been excreted by males and 97% by females. Subcutaneous
    administration of graded doses of BHT to female rats revealed
    substantial faecal excretion but the rate of excretion decreased with
    increasing dose. There was no evidence of accumulation of 14C-BHT in
    the body under the conditions of repeated oral dosage (Tye  et al.,

         Rats were given single oral doses (1-100 mg/rat) of 14C-BHT and
    approximately 80 to 90% of the dose was recovered within four days in
    the urine and faeces. Of the total radioactivity, 40% and 25% appeared
    in the urine of females and males, respectively. After 4 days,
    approximately 3.8% of the dose was retained, mainly in the alimentary
    tract. A substantial portion of the radioactivity was found in the
    bile collected from two rats (one male, one female) over a period of
    40 h. The relatively slow excretion of BHT is probably attributable to
    enterohepatic circulation rather than to tissue retention (Daniel &
    Gage, 1965).

         The liver and body fat of rats fed a diet containing 0.5% BHT for
    35 days were analyzed. The concentration of BHT in the liver never
    rose above 5 mg/kg in males and 1.5 mg/kg in females. In the body fat,
    the level fluctuated around 30 mg/kg in males and 45 mg/kg in females.
    Fat from rats returned to BHT-free diet showed a progressive fall in
    the concentration of BHT, the half-life being about 7 to 10 days.

         The daily excretion of radioactivity in urine and faeces was
    studied in rats given an oral dose of 14C-labelled BHT (12 mg/kg
    bw). Excretion became negligible by the sixth day after administration
    when about 70% of the injected dose had been recovered. Less than 1%
    was excreted as carbon dioxide in the expired air. About 50% of the
    radioactivity was excreted in the bile during the 24-hour period
    following the oral dose (Daniel & Gage, 1965).

         The BHT content of fat and liver of rats given diets containing
    0.5 or 1% BHT for periods up to 35 and 50 days, respectively, were
    analyzed. With 0.5% BHT in the diet, a level of approximately 30 mg/kg
    in the fat was reached in males and 45 mg/kg in females, with
    approximately 1-3 mg/kg in the liver. With 1% BHT, the level in the
    fat was 50 mg/kg in males and 30 mg/kg in females. On cessation of
    treatment, the level of BHT in fat fell with a half-life of 7 to 10
    days (Daniel & Gage, 1965).

         The level of BHT in the fat reached a plateau at approximately
    100 mg/kg after 3 to 4 days when daily doses of 500 mg/kg bw were
    given by intubation. A daily dose of 200 mg/kg bw for one week
    produced a level of about 50 mg/kg (Gilbert & Golberg, 1965).

         Rats were given doses of 100 g of 3H-labelled BHT
    intraperitoneally and the urinary output of radioactivity was measured
    for 4 consecutive days. After 4 days of dosing, 34.5% of the injected
    radioactivity was recovered in urine (Ladomery  et al., 1963). The
    same dose of BHT (100/g) labelled with 14C was given to rats, and
    34% of the radioactivity was excreted in the urine in the first four
    days, in close agreement with the previous result using tritiated BHT
    (Ladomery  et al., 1967a).

         After a single parenteral dose (100 g) of 14C-BHT, rats
    excreted 3235% of the radioactivity in the urine, and 35-37% in the
    faeces, in a 4-day period. The intestinal contents together with the
    gut wall contained most of the remaining activity. Biliary excretion
    was rapid, and the radioactive material in bile was readily absorbed
    from the gut, suggesting a rapid enterohepatic circulation
    (Ladomery  et al., 1967b).

         White male Wistar rats (290-350 g) were administered
    [14C]-labelled BHT, or its alcohol [BHT-CH2OH] or its aldehyde
    [BHT-CHO] or acid [BHT-COOH] derivative by i.v. or i.p. injection. The
    overall excretion of BHT and its related compounds excreted in urine
    and faeces was studied for a 5-day period, and biliary excretion
    monitored for 120426 h after i.p. injection. For the low doses of the
    compounds tested (100 g), there were no significant differences in
    the total recovery of 14C during the 5 days urinary and faecal
    excretion and 120-126 h biliary excretion. However, there were
    differences in ratio of urinary to faecal excretion of 14C. The
    major metabolite present in early bile after i.p. injection of the
    labelled compounds was BHT-COOH or its ester glucuronide. Late bile
    after acid hydrolysis showed BHT-COOH to be the major 14C component
    (Holder  et al., 1970a).

         Temporal concentration changes in BHT and BHT-quinone methide
    were studied following a single oral dose of 800 mg BHT/kg bw in rats,
    which were monitored in plasma, the GI tract, and some adipose tissues
    over a 48-hour period using GLC. Groups of 4 or 5 male Sprague-Dawley
    rats were sacrificed at 0.5, 1, 3, 6, 12, 18, 24, 30, 36 or 48 h after
    administration of BHT. The amount of BHT in the gastric contents
    stayed constant for 12 h, then declined rapidly to 24 h, followed by a
    gradual decline to 48 h. BHT was not detected in the plasma at any of
    the time points, while BHT-quinone methide showed a rapid rise between
    12 and 18 h after administration, followed by a gradual decline to
    48 h. BHT was detected in epididymal, subcutaneous, perirenal and
    brown dorsal adipose tissue soon after administration. A peak
    concentration was noted in epididymal and subcutaneous fat at 18 h,
    but did not show this trend in perirenal or brown dorsal fat.

         In a second experiment, in which groups of 5 or 6 rats were
    sacrificed 4-7 or 24-27 h after administration of BHT, a single oral
    dose of 800 mg/kg bw resulted in a significant increase in the weight
    of stomach contents in both fasted and non-fasted rats at 4-7 h. By
    24-27 h after administration, there was no difference in the weight of
    stomach contents between non-fasted treated animals and controls. This
    study suggested that high single doses of BHT caused a delay in
    gastric emptying which was reflected in the delay in plasma levels of

         In another experiment, 800 mg/kg bw BHT was instilled
    intraduodenally to 5 anaesthetized rats and 30 minutes later the
    concentrations of BHT and its metabolites were measured in blood from
    the portal vein and descending aorta, and in liver and epididymal
    adipose tissue. BHT and/or BHT radical was detected in all but the
    descending aorta, while BHT-quinone methide was not present in any of
    the samples collected at 30 minutes after administration (Takahashi,
    1990).  Chickens

         One-day old chicks were given 14C-BHT at a level of 200 mg/kg
    in the feed for 10 weeks. At broiler age, edible portions had residues
    amounting to 1-3 mg/kg of BHT and metabolites. Similar diets given to
    laying hens produced residues in eggs of 2 mg/kg after 7 days, the
    level thereafter remaining constant (Frawley  et al., 1965a).

         When feed containing 500 or 100 mg/kg BHT was given to laying
    hens, residues of 20 and <5 mg/kg were found in the fat fraction of
    eggs, respectively. In the broiler chicken, over a period of 21 weeks,
    the residues in body fat were 55 mg/kg on the 500 mg/kg diet and
    < 5 mg/kg on the 100 mg/kg diet (Van Stratum & Vos, 1965).  Humans

         Four human male subjects were administered a single dose of
    approximately 40 mg 14C-labelled BHT. About 75% of the administered
    radioactivity was excreted in the urine. About 50% of the dose
    appeared in the urine in the first 24 h, followed by a slower phase
    which probably represented the release of the compounds or their
    metabolites stored in tissues. In humans, the bulk of the
    radioactivity was excreted as the ether insoluble glucuronide of a
    metabolite in which the ring methyl group and one  tert-butyl methyl
    group were oxidized to carboxyl groups, and a methyl group on the
    other  tert-butyl group was also oxidized, probably to an aldehyde
    group. BHT-acid, free and conjugated, was a minor component of the
    urine and the mercapturic acid was virtually absent. The rapidity of
    the first phase of the urinary excretion in humans suggested that
    there was no considerable enterohepatic circulation as had been
    observed in the rat (Daniel  et al., 1967).

         Reported values of BHT in body fat were 0.23  0.15 mg/kg
    (11 individuals, residents of the United Kingdom) and 1.30 
    0.82 mg/kg (12 individuals, residents of the United States of America)
    (Collings & Sharratt, 1970).

         Based on reported BHT levels in human fat in Japan, United
    Kingdom and USA and the calculated dietary intakes of BHT, a
    bioconcentration factor in humans (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).

         The disposition of single oral doses of BHT was compared in
    humans and rats. A single oral dose of 0.5 mg/kg bw 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 bw
    of BHA and one week later 0.25 mg/kg bw of BHT. After another week,
    0.25 mg/kg bw of BHT plus 0.25 mg/kg bw 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, or 200 mg/kg bw of BHT.
    In rats, peak plasma concentrations of BHT (0.2, 0.3, and 2.3 g/ml)
    were seen after 2.6 h. Simultaneous administration of BHA produced
    significantly lower plasma concentrations between 0.5 and 3 h. Large
    variations were seen in humans in plasma levels of BHT. The mean peak
    plasma level was 0.09/g/ml, reached after 1.5 h. The plasma
    concentrations were not influenced by simultaneous administration of
    BHA. In the rat, 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 humans, 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 humans than in rats
    (Verhagen  et al., 1989).

    2.1.2  Biotransformation  Mice

         The oxidative metabolism of BHT by liver microsomes from 3 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 oxidation 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).

         Co-administration of BHA (200 mg/kg bw) with a subtoxic dose of
    BHT (200 mg/kg bw) enhanced the lung toxicity of BHT in male mice. BHA
    co-administration significantly increased the radioactivity covalently
    bound to lung macromolecules at 4-8 h after [14C]-BHT. The
    pretreatment also reduced the rate of  in vitro metabolism of BHT in
    mouse liver supernatant. The co-administration of BHA and BHT caused a
    decrease in metabolism of BHT in the liver with the result that the
    lung was exposed to a larger amount of BHT (Yamamoto  et al., 1988).  Rats

         Examination of the biliary metabolites from i.v. and i.p. doses
    of small amounts of 14C-BHT, showed the presence of four principal
    metabolites. Some 34 to 53% of the 14C-label in the bile was
    identified as 3-5-di- t-butyl-4-hydroxy-benzoic acid, which was
    probably present as the ester glucuronide. The other metabolites
    present were 3,5-di- t-butyl-4-hydroxybenzaldehyde, 3-5-di- t-butyl-
    4-hydroxybenzyl alcohol, and 1,2-bis (3,5-di- t-butyl-4-hydroxyphenyl)
    ethane (Ladomery  et al., 1967b).

         Rats were given a single dose of 14C-BHT and urine and
    bile collected for periods ranging from 48 to 96 h. Faeces were
    also collected during this same period. About 19% to 59% of the
    radioactivity appeared in the urine during this period, and 26% to 36%
    in the bile. The major metabolites in the urine were 3,5-di- tert-
    butyl-4-hydroxybenzoic acid, both free (9% of the dose), as well as
    glucuronide (15%) and S-(3,5-di- tert-butyl-4-hydroxybenzyl)-
    N-acetylcysteine. The ester glucuronide and mercaptic acid were also
    the major metabolites in rat bile. Free 3,5-di- tert-butyl-4-
    hydroxybenzoic acid was the major metabolite in faeces (Daniel
     et al., 1968).

         Male Wistar rats were dosed intraperitoneally with 200 mg/kg bw
    BHT-acid or 2,6-di- tert-butyl phenol (DBP). Faeces and urine were
    collected for 5 days after administration of BHT-acid and for 3 days
    after administration of DBP. Bile was collected from rats treated with
    BHT-acid for 24 h after administration. Following administration of
    BHT-acid, the metabolites DBP, 2,6-di- tert-butyl- p-benzoquinone
    (BBQ), 2,6-di- tert-butylhydroquinone (BHQ) glucuronide and BHT-acid
    glucuronide were identified in the faeces and bile. This suggested
    that BHT-acid, considered as a main metabolic end-product of BHT,
    was metabolized to the quinone and hydroquinone following its
    decarboxylation to form the di-substituted phenol, DBP. An alternative
    route for formation of the quinone, BBQ, was a homolysis reaction of
    the O-O bond of the hydroperoxide which is catalyzed by rat liver
    cytochrome P-450 (Yamamoto  et al., 1991).  Rabbits

         Rabbits were given single or repeated doses of BHT in the range
    of 400-800 mg/kg bw. About 16% of the dose was excreted as ester
    glucuronide and 19% as ether glucuronide. Unconjugated phenol (8%),
    ethereal sulfate (8%) and a glycine conjugate (2%) were also excreted.
    Excretion of all detectable metabolites was essentially complete 3 to
    4 days after administration of the compound and about 54% of the dose
    was accounted for as identified metabolites (Dacre, 1961).

         The metabolism of butylated hydroxytoluene (BHT) orally
    administered to rabbits in single doses of 500 mg/kg bw was studied.
    The metabolites 2,6-di- tert-butyl-4-hydroxymethylphenol
    (BHT-alcohol), 3,5-di- tert-butyl-4-hydroxy-benzoic acid (BHT-acid)
    and 4,4'-ethylene- bis-(2,6-di-tert-butylphenol) were identified. The
    urinary metabolites of BHT comprised 37.5% as glucuronides, 16.7%
    as ethereal sulfates and 6.8% as free phenols; unchanged BHT was
    present only in the faeces (Akagi & Aoki, 1962a); 3,5-di- tert-butyl-
    4-hydroxybenzaldehyde (BHT-aldehyde) was also isolated from rabbit
    urine (Aoki, 1962). The main metabolic pathway was confirmed by
    administering BHT-alcohol to rabbits and isolating BHT-aldehyde,
    BHT-acid, the ethylene- bis derivative and unchanged BHT-alcohol in
    the urine (Akagi & Aoki, 1962b).  Ruminants

         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).  Humans

         A group of 8 men each received 100 mg of BHT on two occasions
    separated by a 4-day interval. Urine was collected for 24 h after BHT
    administration. The metabolites were identified as BHT-COOH and
    benzoyl-glycine. In another study in which two adults were given 1.0 g
    of BHT, BHT-COOH and its ester glucuronide were the only major
    metabolites identified in urine (Holder  et al., 1970b).  Combined species

         The metabolism of BHT was studied with liver and lung microsomes
    from rats and mice. Two main metabolic processes occurred,
    hydroxylation of alkyl substituents and oxidation of the aromatic 
    electron system. The former led 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 occurred at the
    benzylic methyl position, and the resulting diol was oxidized further
    to the hydroxybenzaldehyde derivative. Oxidation of the  system led
    to BHT-quinol (2,6-di- t-butyl-4-hydroxy-4-methyl-2,5-cyclo-
    hexadienone), BHT-quinone (2,6-di- t-butyl-4-benzoquinone), and
    BHT-quinone methide (2,6-di- t-butyl-4-methylene-2,5-cyclohexa-
    dienone) probably via the hydroperoxide (BHTOOH). Derivatives of the
    quinol and quinone with a hydroxylated  t-butyl group were also
    formed. Quantitative data demonstrated that BHT-CH2OH was the
    principal metabolite in rat liver and lung microsomes. The mouse
    produced 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  in vitro peroxidase-catalyzed covalent binding of BHT to
    microsomal protein and the formation of BHT-quinone 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 humans, were also able to support this
    interaction of BHA and BHT using either hydrogen peroxide or
    arachidonic acid as the substrate (Annex 1, reference 95).

         Phenobarbital (PB) pretreatment of Sprague-Dawley rats and A/J
    mice had little or no effect on respective quinone methide formation
    from BHT or BHT-BuOH in pulmonary microsome preparations, but resulted
    in a 6-37 fold induction of this activity in hepatic microsomes from
    these species. PB administration had little effect on the two-step
    oxidation of BHT to QM-OH in pulmonary microsomes of the mouse, while
    in hepatic microsomes, PB pre-treatment resulted in a greater than
    100-fold increase in this activity. This was found to be mainly due to
    a greater than 100-fold increase in the initial  tert-butyl
    hydroxylation step in mouse liver microsomes. The enhancement was
    somewhat higher than for 7-pentoxyresorufin O-dealkylase activity,
    demonstrating that  tert-butyl hydroxylation could serve as a
    specific marker for the enzyme. The results also showed that pulmonary
    microsomes from mice, but not rats, had a relatively high constitutive
    P-450 activity for  tert-butyl hydroxylation of BHT, supporting
    the proposal that this metabolite was involved in BHT-induced
    pneumotoxicity. Two cytochrome P-450 inhibitors, SKF 525-A and
    metapyrone, inhibited the conversions of BHT to QM and QM-OH to a
    similar extent in PB-treated mouse liver microsomes; the terpenoid
    alcohol cedrol was found to selectively inhibit BHT conversion to
    QM-OH. This compound has been found previously to inhibit
    pneumotoxicity when administered to mice prior to treatment with BHT.
    In untreated mouse liver microsomes, BHT hydroxylation to BHT-MeOH
    showed the greatest activity, with oxidation to QM second and
    hydroxylation to BHT-BuOH last (Bolton & Thompson, 1991).

    2.1.3  Effects on enzymes and other biochemical parameters  Mice

         Mice (BALB/c strain) were maintained on a diet containing 7.5 g
    BHT/kg of feed. After 3 weeks on the test diet, there was an enhanced
    activity in plasma esterases which persisted throughout the
    experimental period of 20 weeks. Following electrophoretic separation
    of the esterases, the increased enzyme activity was shown to be
    located in two specific bands (Tyndall  et al., 1975).

         Dietary administration of BHT to male Swiss Webster mice resulted
    in a marked increase in hepatic microsomal epoxide hydrolase and
    glutathione-S-transferase (Hammock & Ota, 1983).  Rats

         Feeding experiments were carried out on 45 pairs of weanling male
    rats for 5 to 8 weeks with diets containing 0, 10 or 20% lard
    supplements to which 0.01, 1 or 5 g BHT/kg had been added. At
    0.01 g/kg, no changes were observed in any of the serum constituents
    studied, while at 5 g/kg an increase in the serum cholesterol level
    was seen within 5 weeks. Female rats fed for 8 months a diet
    containing a 10% lard supplement with 1 g BHT/kg showed increased
    serum cholesterol levels, but no other significant changes. Diets
    containing 5 g BHT/kg in 10% and 20% lard supplements fed to female
    rats for the same period increased serum cholesterol, phospholipid and
    mucoprotein levels (Day  et al., 1959).

         In further work with rats, it was found that increased output of
    urinary ascorbic acid accompanied liver enlargement induced by BHA or
    BHT in onset, degree and duration, being rapid but transient with BHA,
    and slower in onset but more prolonged with BHT (Gaunt  et al.,
    1965a). The simultaneous stimulation of processing enzyme activity,
    increase in urinary ascorbic acid output, and increase in relative
    liver weight brought about by BHT was unaffected by 14 days of dietary
    restriction, and all these changes except liver weight were reversible
    during 14 days' recovery on normal diet (Gaunt  et al., 1965b).

         Rats given BHT by daily intubation showed increased activity of
    some liver microsomal enzymes. Stimulation of enzyme activity
    correlated with an increase in relative liver weight. The threshold
    dose for these changes in enzyme activity in female rats was below
    25-75 mg BHT/kg bw/day. The storage of BHT in fat appeared to be
    influenced by the activity of the processing enzymes. In rats given
    500 mg/kg bw/day, the level of BHT in fat attained values of 230 mg/kg
    in females and 162 mg/kg in males by the second day, by which time the
    relative liver weight and processing enzyme activities had become
    elevated. Thereafter, liver weight and enzyme activity continued to
    rise but the BHT content of fat fell to a plateau of about 100 mg/kg
    in both sexes (Gilbert & Golberg, 1965).

         Groups of 12 SPF Carworth rats equally divided by sex were
    administered BHT dissolved in arachis oil daily for one week, at dose
    levels of 50, 100, 200 or 500 mg/kg bw. A group of 8 rats served as
    control. The animals were killed 24 h after the final dose, and
    histological and biochemical studies (glucose-6-phosphatase: and
    glucose-6-phosphate dehydrogenase) made on the livers of all animals.
    A histochemical assessment of the livers of test animals was also
    carried out. BHT caused an increase in liver weight in males at dose
    levels of 100 mg/kg bw and greater, and in females at 200 mg/kg bw and
    greater. BHT caused a decrease in glucose-6-phosphatase activity in
    females at dose levels greater than 100 mg/kg bw, and an increase in
    glucose-6-phosphate dehydrogenase in both males and females at the
    highest dose tested. In another study, rats were dosed according to

    the above schedule and then maintained for 14 to 28 days following the
    final dosing. By day 28, no biochemical changes were observed, and
    relative liver weights returned to normal by day 14 (Feuer  et al.,

         Rats fed diets containing BHT at levels of 100 to 5000 mg/kg for
    12 days showed liver enlargement, as well as increased activity of
    liver microsomal biphenyl-4-hydroxylase, at all levels except the
    lowest level of 100 mg/kg. Enzyme activity was not significantly
    altered at 5000 mg/kg BHT fed for one day (Creaven  et al., 1966).

         Rats (male and female Carworth Farm SPF) were given an oral dose
    of BHT equivalent to 500 mg/kg bw. Dosing was from 1 to 5 days, and
    rats varying in size from 100-400 g body weight were used. Microsomal
    preparations from the livers of treated rats were assayed for BHT
    oxidase, an enzyme that metabolizes BHT to the BHT alcohol
    (2,6-di- tert-butyl-4-methylphenol to 2,6-di- tert-butyl-4-hydroxy
    methylphenol). Treatment of female rats with BHT (500 mg/kg bw/day for
    5 days) caused a six fold increase in the activity of the enzyme/gram
    of liver and a 35% increase in relative liver weight, both being
    prevented by actinomycin D. The induction was more pronounced in males
    than in females, and the induction of the enzyme, low in rats in the
    100 g body-weight range, reached a maximum in rats in the 200 g
    body-weight range, and fell in larger animals (300-400 g range)
    (Gilbert & Golberg, 1967).

         Groups of female Alderly Park SPF rats were maintained on diets
    containing 0%, 0.01%, 0.1%, 1% or 5% BHT for periods up to 28 days,
    and then on diets free of BHT for 56 days. Animals were killed in
    groups of four, 2 being used for enzyme assay (aminopyrene
    demethylase) and 2 for electron microscopy. The increase in enzyme
    activity was directly related to the dietary level of BHT. No
    detectable increase was observed at the lowest level over the 28-day
    feeding period. Following withdrawal of BHT from the diet, the enzyme
    level returned to normal in all test animals. The degree of
    endoplasmic reticulum proliferation was proportional to the amount of
    BHT in the diet and the duration of feeding at the 5% and 1% level. At
    the 0.1% level, there was a transient rise in smooth endoplasmic
    reticulum. No proliferation was observed at the 0.01% level. Following
    removal of BHT from the diet, there was a rapid disappearance of the
    proliferated smooth endoplasmic reticulum.

         In a second study, groups of rats were fed diets containing 1%
    BHT for 10 days, and then for a second period of 10 days after an
    interval of 20 days on a normal diet. The animals were killed in
    groups of five at 10, 30, 40, 42 or 47 days. Livers were removed for
    aminopyrene demethylase assay and electron microscopy. Enzyme activity
    did not differ significantly following both 10-day periods of
    administration of BHT. Electron microscopy showed similar smooth
    endoplasmic reticulum response during both these periods (Botham
     et al., 1970).

         Microsomal preparations from livers of rats, dosed daily with
    450 mg/kg bw BHT for up to 7 days, showed an increased capacity to
    incorporate labelled amino acids, when compared to preparations from
    controls. BHT also stimulated the  in vivo incorporation of amino
    acids, mainly into the proteins of the endoplasmic reticulum
    (Nievel, 1969).

         A group of 23 female SPF rats (Wistar strain) was administered
    500 mg/kg BHT dissolved in rape-seed oil, for 11 days. The control
    group was administered rape-seed oil alone. Groups of 7 rats were
    killed following administration of the final dosing. The remaining
    rats were maintained without further exposure to BHT, and killed on
    days 28 or 63 of the study. Livers of the rats were examined for
    weight, DNA content and number of cell nuclei. Treatment with BHT
    resulted in enlargement of the liver, with a concomitant increase in
    its DNA content, and in the number and ploidy of its nuclei. The liver
    mass returned to normal within two weeks. However, the DNA content of
    the liver of BHT-treated animals remained elevated up to the time of
    termination of this study, and there was no reduction in the total
    number of nuclei or the degree of ploidy (Hermann  et al., 1971).

         Two groups of 10 male and 10 female rats (Alderly Park, SPF
    Wistar strain) were dosed by stomach tube with 200 mg/kg bw/day of BHT
    dissolved in maize oil for 7 days. Four rats/sex dosed with an
    equivalent amount of maize oil served as controls. Urinary ascorbic
    acid excretion was measured in urine samples collected following 5
    days on the test compound. The animals were killed 24 h after the
    final dose and the livers removed for biochemical assays (aminopyrine
    demethylase-AMPM, hexobarbitone oxidase-HO, cytochrome P-450, and
    glucose-6-phosphatase), and electron microscopy. Another group of
    treated rats was maintained for a 7-day recovery period, and a similar
    battery of liver studies was carried out. Administration of BHT
    resulted in an increase of urinary excretion of ascorbic acid which
    remained constant throughout the treatment period. Following cessation
    of BHT treatment there was a gradual return towards control values.
    There were significant sex differences in some of the biochemical
    responses to BHT, with the exception of the glucose-6-phosphatase
    activity. Female rats showed a marked increase in APDM and HO
    activity, which was not observed in male rats. Cytochrome P-450 levels
    were increased in both males and females. The biochemical parameters,
    with the exception of APDM activity in female rats, returned to normal
    following the 7-day recovery period. Electron microscopy showed
    significant proliferation of the smooth endoplasmic reticulum of the
    hepatic cells. No other morphological changes were detected
    (Burrows  et al., 1972).

         BHT (500 mg/kg bw/day) was administered by gavage to groups of
    young Wistar male and female rats for 7 days and the animals were
    housed in metabolism cages. Control animals received corn oil vehicle
    only. They were then sacrificed and liver enzymes (aniline-

    4-hydroxylase, biphenyl-4-hydroxylase, ethyl morphine N-demethylase,
    and 4-methyl umbelliferone glucuronyl transferase) were assayed and the
    cytochrome P-450-CO interaction spectrum evaluated. Urinalysis using
    GC was conducted to assay for D-glucaric acid, D-glucuronic acid,
    1-gulonic acid, xylitol and L-ascorbic acid. Administration of BHT
    enhanced all the parameters measured with the exception of the hepatic
    microsomal protein content. BHT was a more potent inducer of
    xenobiotic metabolism in female rats (Lake  et al., 1976).

         BHT in the diet of Sprague-Dawley rats resulted in a marked
    decrease in the NADPH-cytochrome P450 reductase activity of isolated
    liver microsomal preparations. This effect was not observed when BHT
    was added  in vitro to liver microsomes (Rikans  et al., 1981).

         Dietary BHT was also shown to affect the carboxylation process in
    the conversion of rat liver microsomal protein to prothrombin
    (Takahashi & Hiraga, 1981a).

         Rats fed a diet containing 0.4% BHT showed an increase in GSH-S
    transferase activity in the liver, but not in lungs and kidneys.
    GSH-reductase levels were increased in liver and lungs
    (Partridge  et al., 1982).

         BHT at 300-6000 mg/kg in the diet caused a dose-dependent
    increase in gamma-glutamyl transpeptidase in normal F344 male rats.
    However, cytosolic glutathione S-transferase was only enhanced at
    dietary concentrations of 3000 or 6000 mg/kg (Furukawa  et al.,

         Groups of 4 male F344 rats were pretreated with buthionine
    sulfoximine, a glutathione-depleting agent (900 mg/kg bw), and after
    1 h given intraperitoneal injections of BHT (100, 250, 400, or
    500 mg/kg bw). A dose-related elevation of serum GOT and GPT
    activities was observed. BHT or buthionine sulfoximine alone had no
    effect. In contrast, pretreatment with cysteine (100-200 mg/kg bw)
    inhibited the elevation of serum enzyme activities at a toxic dose of
    BHT (1000 mg/kg bw) (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 without
    BHT resulted in a marked loss of total hepatic nuclear envelope P-450
    (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).

         Administration of 0.5% BHT in the diet of male Wistar rats for 2
    weeks increased UDP-glucuronosyl transferase activity in liver
    microsomes for several substrates to 236-269% of controls. The amounts
    of UDP-glucuronosyl transferase protein and associated mRNA in liver
    microsomes were also increased, paralleling the increases in enzyme
    activity. In addition to induction of hepatic activity, BHT treatment
    resulted in increased activity in microsomes from the kidney and small
    intestine (Kashfi  et al., 1994).  Rabbits

         Acute effects on electrolyte excretion, similar to those
    described for large doses of BHA were also obtained following
    administration of BHT at doses of 500-700 mg/kg bw. No such effects
    were observed at lower dosage levels (Denz & Llaurado, 1957).  Monkeys

         Groups of 2-4 juvenile rhesus monkeys  (Macaca mulatta) were fed
    BHT dissolved in corn oil at dose levels of 0, 50, or 500 mg/kg bw for
    4 weeks. Blood samples were taken prior to treatment and then at
    weekly intervals from the control and test animals in the high-dose
    group, and from test animals in the low-dose group at the end of the
    4-week period, for determination of total plasma cholesterol, lipid
    phosphorus and triglyceride. Liver biopsies were taken from the test
    animals in the high group at two weeks. At the end of the test period
    all animals were fasted for 24 h and sacrificed, and liver and blood
    samples obtained. Liver samples were analyzed for succinic
    dehydrogenase and susceptibility to peroxidation. Extracted liver
    lipids were analyzed for total cholesterol, lipid phosphorus and
    triglycerides. Total cholesterol levels in plasma and liver were
    significantly lowered. Lipid phosphorus levels in the plasma were
    increased at the high-dose level, as were cholesterol:lipid phosphorus
    ratios in the plasma and liver. The susceptibility of liver lipids to
    oxidation was reduced in the high-dose group (Branen  et al., 1973).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with BHT are summarized in
    Table 1.

         Acute oral, intraperitoneal (mice) and eye irritation (rabbits)
    and skin irritation (rats) were measured for 7 breakdown products of
    BHT. All compounds tested were less toxic than the parent compound
    (Conning  et al., 1969).

        Table 1.  Acute toxicity studies with BHT

    Animal       Route      LD50           Approximate     Reference
                            (mg/kg bw)     lethal dose
                                           (mg/kg bw)

    Rat          oral       > 1700-1970    -               Deichmann et al., 1955

    Cat          oral       -              940-2100        Deichmann et al., 1955

    Rabbit       oral       -              2100-3200       Deichmann et al., 1955

    Guinea-pig   oral       -              10 700          Deichmann et al., 1955

    Rat          oral       2450                           Karplyuk, 1959

    Mouse        oral       2000                           Karplyuk, 1959
             As shown in Table 2, the LD50 (i.p.) for BHT showed
    considerable differences for strains of inbred and non-inbred male
    mice. In all cases death occurred 4 to 6 days after administration of
    BHT, and was accompanied by massive edema and haemorrhage in the lung
    (Kawano  et al., 1981).

    Table 2.  Variation in LD50 with strains of mice (Kawano et al., 1981)

    Strain                                LD50 (mg/kg bw)

    DBA/2N (inbred)                              138
    BALB/cNnN (inbred)                          1739
    C57BL/6N (inbred)                            917
    ICR-JCL (non-inbred)                        1243

    2.2.2  Short-term toxicity studies  Mice

         In order to estimate the MTD for BHT in mice, groups of 5/sex
    B6C3F1 mice, 6-week old, were fed diets containing 0, 3100, 6200,
    12 500, 25 000 or 50 000 mg BHT/kg of feed for 7 weeks. Each animal was
    weighed twice weekly. Gross necropsy was performed on all animals. One
    female mouse in the 25 000 mg/kg group, and 1 male and 4 female mice
    in the 50 000 mg/kg group died before the end of the study.

    Body-weight decrements, mostly dose-related, were noted in all
    treatment groups compared with controls. Histopathologic examination
    of male mice receiving 25 000 mg/kg revealed a very small amount of
    centrilobular cytoplasmic vacuolization of hepatocytes which was not
    observed in females receiving 12 500 mg/kg (high-dose females were not
    examined) (NCI, 1979).

         A similar study was conducted to establish the dose levels for a
    subsequent carcinogenicity study. Groups of B6C3F1 mice (10/sex)
    received BHT in the diet at concentrations of 0.25%, 0.5%, 1%, 2%, or
    4% for 10 weeks. Twenty mice/sex were used for the control group. Both
    male and female mice receiving the highest dose of BHT experienced a
    retardation of body-weight gain which exceeded 10% of control values.
    In addition, histopathological examination of mice in the 4% group,
    revealed marked starvation atrophy of the spleen, heart and kidneys.
    None of these changes were noted in mice at the next lower dose level
    (2% diet) or any of the other groups. The MTD was considered to be 2%
    diet (Inai  et al., 1988).

         Groups of male and female C3H mice (17-39 mice/group), 6-10
    weeks old, were maintained for 10 months on a semi-synthetic diet
    containing 0.05 or 0.5% BHT. Control groups were maintained on
    BHT-free semi-synthetic diet or commercial lab chow. At the end of the
    test period, the liver and lungs were excised and inspected grossly
    for proliferative lesions. Of the proliferative lesions considered to
    be clearly identifiable as tumours, approximately 50% were examined
    microscopically. Mice maintained on diets containing BHT had lower
    body weights than controls. Male mice fed BHT showed an increase in
    liver tumours, compared to controls. Histologically, the tumours were
    identified as hepatocellular adenomas. No increase was observed in
    female mice. The reported incidence of liver tumours in male C3H
    mice was 38% (10/26) in the 0.5% BHT group, 58% (15/26) in the 0.05%
    BHT group, 5% (2/37) in the control semi-synthetic diet group, and 18%
    (7/38) in the control lab chow group.

         In a study in which C3H mice were maintained on diets
    containing 0.5% BHT for one month followed by lab chow for 10 months,
    or control diet (BHT-free) for one month followed by lab chow for 10
    months, the incidence of liver tumours in the two groups of male mice
    were 9% (3/35) and 17% (5/29), respectively. Dietary BHT did not
    result in an increased incidence of lung tumours in either male or
    female mice.

         In another study in which male BALB/c mice were maintained for
    one year on a BHT-free diet, or diets containing 0.05% BHT or 0.5%
    BHT, the incidence of liver tumours were 13% (4/30), 14% (6/43), and
    7% (2/28) for the respective groups (Lindenschmidt  et al., 1986).

         It has previously been demonstrated that the incidence of
    spontaneously-occurring hepatic tumours in C3H mice is modified by
    sex, population density, level of dietary protein, and caloric intake.
    Historical dam were not available for a 10-month study. The incidence
    of hepatic tumours in a 12-month study in this strain ranged from
    6-13% for females, and 41-68% for males (Peraino  et al., 1973).
    Thus, the reported incidence of hepatocellular tumours was not
    significantly different from other controls of similar age in studies
    with the same inbred strain.  Rats

         Feeding experiments conducted for 20 or 90 days indicated that
    rats did not find food containing respectively 0.5 or 1% BHT
    palatable. However, the animals ingested food so treated more readily
    if these concentrations were attained gradually. Paired feeding
    experiments with groups of 5 or 10 rats for 25 days demonstrated that
    diets containing 0.8 and 1% BHT reduced the daily intake of food below
    control values. A level of 1% in the diet retarded weight gain
    (Deichmann, 1955).

         BHT (0.3%) in the diet of pregnant rats that had been kept for 5
    weeks on a diet deficient in vitamin E produced no toxic symptoms,
    while 1.6% caused drastic loss of weight and fetal death (Ames
     et al., 1956).

         BHT was fed to rats (12/group) for 7 weeks at a level of 0.1% BHT
    in diets containing 20% or 10% lard supplement. With the 20% lard
    supplement diet, significant reduction of the initial growth rate and
    mature weight of male rats was observed. No effect was noted in female
    or male rats receiving the 10% lard supplement diet. A paired feeding
    experiment showed that this inhibition of growth was a direct toxic
    effect of BHT and could not be explained by a reduction in the
    palatability of the diet. At this level BHT produced a significant
    increase in the weight of the liver, both absolute and relative to
    body weight. Rats under increased stress showed significant loss of
    hair from the top of the head. The toxic effect of BHT was greater if
    the fat load in the diet was increased. Anophthalmia occurred in 10%
    of the litters (Brown  et al., 1959).

         BHT administered to rats at 250 mg/kg bw/day for 68 to 82 days
    caused fatty infiltration of the liver and reduction in body-weight
    gain (Karplyuk, 1959).

         Groups of 6 weanling rats (3/sex) were fed diets containing a 20%
    lard supplement and BHT at levels of 0, 0.1, 0.2, 0.3, 0.4 or 0.5% for
    6 weeks. BHT reduced the growth rate, especially in males, the effect
    becoming significant at 0.3% BHT. It also increased the absolute liver
    weight and the ratio of liver weight to body weight in both sexes, the
    latter effect becoming significant at 0.2% BHT. BHT increased the

    ratio of left adrenal weight to body weight in male rats but had no
    consistent effect in females. There were no histological changes in
    the adrenal attributable to treatment. All dietary levels of BHT
    increased the serum cholesterol and the concentration of cholesterol
    was directly proportional to the BHT level. There was also a
    significant increase in the concentration of adrenal cholesterol. BHT
    produced no significant changes in the concentration of total or
    percentage esterified liver cholesterol, total liver lipid or
    concentration of total polyunsaturated fatty acids in the liver
    (Johnson & Hewgill, 1961).

         Rats fed diets supplemented with 20% lard, and containing 0, 0.2,
    0.3, 0.4 or 0.5% (dry weight) BHT for 6 weeks, showed an increase in
    serum cholesterol that was directly related to the level of dietary
    BHT. BHT increased the relative weight of the male adrenal and also
    caused a significantly greater decrease in growth rate of male as
    compared to the female. Increased liver weight in test animals was
    paralleled by increased absolute lipid content of the liver (Johnson &
    Hewgill, 1961).

         In another study, rats were maintained on diets containing 0.5%
    dietary BHT in the presence or absence of a 20% lard supplement. BHT
    increased the basic metabolic rate, the concentration of body
    cholesterol and the rate of synthesis of body and liver cholesterol,
    and reduced the total fatty acid content of the body, irrespective of
    the presence or absence of dietary lard. In the animals fed BHT
    without lard, BHT increased the rate of synthesis and turnover of body
    and liver fatty acids and reduced the growth rate. These effect
    occurred to a greater extent in animals fed BHT with lard (Johnson &
    Holdsworth, 1968).

         Groups of 8 young rats were fed diets containing 19.9% casein and
    0, 0.02 or 0.2% BHT for 8 weeks. The experiment was repeated with
    16.6% casein in the diet of further groups for 4 weeks and again with
    9.6% casein (and no added choline) for 7 weeks. In all three instances
    BHT caused stimulation of growth and improved protein efficiency. The
    nitrogen content of the liver was, however, greatly reduced in
    BHT-treated animals, except when the level of BHT was reduced to
    0.02%. Recovery of hepatic protein after fasting (details not given)
    was also impaired in rats on 0.2% BHT. Liver lipid content was
    increased at 0.2%, but not at 0.02% BHT. A dietary level of 0.2% BHT
    also increased the adrenal weight and ascorbic acid content, although
    if recalculated on the basis of weight of gland, there was no
    significant difference. The increase in adrenal ascorbic acid was
    interpreted as indicating a stress imposed on the organism by BHT
    (Sporn & Schbesch, 1961).

         Groups of 20 male and 20 female rats fed 1% BHT in the diet for
    10 weeks showed recovery both in liver to body weight ratios and in
    morphological appearance of the liver cells within a few weeks after
    restoring the animals to a normal diet (Goater  et al., 1964).

         Groups of 48 weanling rats (24/sex) were given diets containing 0
    or 0.1% BHT for periods of up to 16 weeks. Measurements of growth
    rate, food consumption, weight and micropathological examination of
    organs at autopsy revealed no difference between treated and control
    groups. However, increase in relative liver weight and in the weight
    of the adrenals was produced without histopathological evidence of
    damage. Biochemical measurements and histochemical assessments of
    liver glucose phosphatase and glucose 6-phosphate dehydrogenase
    activities revealed no difference from the control group
    (Gaunt  et al., 1965a).

         Groups of rats (16/sex) were fed diets containing 20% fat and BHT
    levels of 0, 0, 0.03, 0.1 or 0.3% BHT for 10 weeks. No definite effect
    on body weight was observed at any level in females and there was only
    a slight depression in males at the 0.3% level. There was no
    significant effect on blood cholesterol level in either sex after
    feeding BHT at any of the levels. Four males at the 0.3% and two at
    the 0.1% level died during the experiment. Two deaths occurred among
    females at 0.3%. Only one male rat died in both control groups
    (Frawley  et al., 1965b).

         In order to determine the MTD for BHT in rats, groups of F344
    rats (5/sex) were given diets containing 0, 6200, 12 500, 25 000 or
    50 000 mg BHT/kg diet for 7 weeks. Body weights were determined twice
    weekly. Gross necropsy was performed on all animals in the study. All
    of the male and female rats in the 50 000 mg/kg group died before the
    end of the study. With the exception of one male in the 12 500 mg/kg
    group, all of the animals in the other treatment groups and control
    groups survived to the end of the study. Body weights at week 7 of the
    study showed a dose-related decrement, with animals in the 25000 mg/kg
    group weighing only 38% to 44% of control values. At dietary levels of
    12 500 mg/kg, there was a slight increase in haematopoiesis in both
    sexes (NCI, 1979).  Dogs

         Mild to moderately severe diarrhoea was induced in a group of
    4 dogs fed BHT at doses of 1.4-4.7 g/kg bw every 2 to 4 days for
    4 weeks. Postmortem examination revealed no significant gross
    pathological changes. No signs of intoxication and no gross or
    histopathological changes were observed in dogs fed doses of
    0.17-0.94 g/kg bw, 5 days/week for a 12-month period (Deichmann
     et al., 1955).  Monkeys

         Groups of 3 infant or juvenile monkeys  (Macaca mulatta)
    received BHT at doses of 0, 50 or 500 mg/kg bw/day for 4 weeks. Blood
    analysis (complete cell count, serum sodium and potassium, bilirubin,
    cholesterol and GOT) was carried out weekly, as was a complete

    urinalysis. Liver biopsies were taken from the juvenile monkey at 2
    weeks, following a 24-h fast. At the end of the test period, all
    animals were fasted 24 h and sacrificed. Tissues from all major organs
    were prepared for light and electron microscopy. Liver tissue was 
    also analyzed for protein, RNA and cytochrome P-450. Microsomal
    preparations prepared from the livers were used to measure
    nitroanisole demethylase and glucose-6-phosphatase activity. Urine and
    blood values of test and control animals were similar. Histological
    evaluation of all organs other than the liver from either infant or
    juvenile monkeys did not indicate any compound-related changes. Test
    animals receiving BHT showed hepatocytomegaly and enlargement of
    hepatic cell nuclei. The hepatocytes of treated animals showed
    moderate proliferation of the endoplasmic reticulum. Lipid droplets
    were also prominent in cytoplasm of these hepatic cells. There was
    fragmentation of the nucleolus in 15% of the hepatic cells in the test
    animals in the high-dose group. DNA, RNA and cytochrome P-450 levels
    in the liver of test and control animals were similar. BHT-treated
    juveniles showed an increase in nitroanisole demethylase activity
    which increased with time. The enzyme activity was unaffected in
    infant monkeys. Glucose-6-phosphatase activity declined in juvenile
    monkeys but was unchanged in infant monkeys (Allen & Engblom, 1972).

    2.2.3  Long-term toxicity/carcinogenicity studies  Mice

         Groups of 60 FAF male mice were maintained on semi-synthetic
    diets containing 0, 0.25 or 0.5% BHT. The mean life-span of the test
    animals was significantly greater than controls, being 17.0  5.0 and
    20.9  4.7 months respectively for the 0.25% and 0.5% BHT, as compared
    to 14.5  4.6 months for controls (Harman, 1968).

         A group of 18, 8-week old male BALB/C mice fed BHT at a level of
    0.75% for a period of 12 months, developed marked hyperplasia of the
    hepatic bile ducts with an associated subacute cholangitis (Clapp 
     et al., 1973).

         Eleven mice (BALB/C strain) were maintained on a diet containing
    0.75% BHT for a period of 16 months. The incidence of lung tumours in
    the test group was 63.6%, compared with 24% in controls (Clapp
     et al., 1974). However, a repeat of this study using a larger group
    of test animals, showed that BHT had no effect on the incidence of
    lung tumours in either sex (Clapp  et al., 1975).

         Groups of 48 mice (CFI strain) equally divided by sex were
    maintained on diets containing 1000 mg/kg BHT. At week 4, one group
    was then fed a diet containing 2500 mg/kg BHT, and at week 8, another
    group was fed a diet containing 5000 mg/kg BHT. The animals were

    maintained on these diets until 100 weeks of age. There was no
    statistically significant reduction in survival of animals on the BHT
    diet, although survival was poorer in males at the high-dose level
    during the last quarter of the study. Animals dying or sacrificed
    during the course of the study showed greater centrilobular cytomegaly
    and karyomegaly than controls. Bile duct hyperplasia was only observed
    in 3/141 test animals. There was no significant difference in the
    incidence of malignant tumours in the high-dose group and control.
    However, there was an increased incidence of lung neoplasia in treated
    mice (75%, 74%, 53% and 47% in the 5000, 2500, 1000 mg/kg and control
    groups, respectively). There were no morphological features to
    distinguish the lung tumours in treated mice from those in controls.
    There was also an apparent increase in benign ovarian tumours in
    BHT-treated female mice, since none were observed in control animals
    (Brooks  et al., 1976).

         BHT was administered in the diet at levels of 0, 3000, or
    6000 mg/kg to groups of 20 (control) or 50 (treated) male and female
    B6C3F1 mice for 107108 weeks. The mice were observed twice daily for
    signs of ill-health. Physical examinations were performed each month
    and body weights were recorded at least once a month. Gross and
    microscopic examination of 28 major organs, tissues and gross lesions
    including the liver, thyroid and forestomach, was performed on all
    animals at the end of the study and all animals dying on test where
    possible. Peripheral blood smears were made for all animals where
    possible. The body weights of treated male and female mice were lower
    than the control mice throughout the study [numerical data not
    provided]. The magnitude of the body-weight decrements was
    dose-related. Administration of BHT in the diet resulted in similar or
    improved survival in the treated groups compared with controls. At
    termination, survival in male mice was 60%, 86% and 92% and in females
    85%, 82% and 90%, for the control, low-and high-dose groups,
    respectively. There was a marked dose-related increase in the
    incidence of hepatocytomegaly and non-neoplastic lesions of the liver
    (peliosis, hepatocellular degeneration/necrosis and cytoplasmic
    vacuolation) in males but not in females. The incidence of
    hepatocellular adenoma or carcinoma was not significantly increased in
    either treated male or female mice, although there was a small
    increase in the incidence of combined adenomas and carcinomas in the
    treated females (1/20, 4/46 and 5/49 for the control, low- and
    high-dose females, respectively) which was not statistically
    significant. The historical control incidence for hepatocellular
    neoplasms was not provided. The incidence of alveolar/bronchiolar
    carcinomas or adenomas in female mice was significantly higher than
    controls (5%) in the low dose (35%), but not the high dose (14%). The
    historical control incidence was 4.7% for alveolar/bronchiolar
    adenomas or carcinomas. Chronic ingestion of BHT in the diet was

    related to a significant reduction in the incidence of sarcomas of
    multiple organs in female mice. Four adenomas of the eye/lacrimal
    gland were observed in high-dose males (8%) and in 2 low-dose females
    but not in corresponding controls. The historical incidence of this
    tumour in male mice was 1.2%. Since the lacrimal gland was only
    examined microscopically in animals with grossly apparent lesions, the
    report states that the lacrimal gland tumours could not be clearly
    related to BHT administration (NCI, 1979).

         Groups of B6C3F1 mice (100/sex), were fed diets containing 0,
    200, 1000 or 5000 mg/kg BHT for 96 weeks, followed by a basal diet for
    8 weeks. At the end of the test period the surviving animals were
    killed. A complete autopsy was carried out, and the principal organs
    and tissues were examined microscopically. Mice that died during the
    course of the study were also autopsied. In addition, terminal blood
    samples were collected for haematological examination and serum
    clinical biochemistry. Urine samples were also examined. During the
    course of the study, food consumption was similar for test and control
    groups. Body weights of females in the 1000 and 5000 mg/kg groups were
    lower than controls, as was the body weight of males in the 5000 mg/kg
    group. There were minor changes in the absolute weight of some organs
    in the high-dose groups (salivary glands, heart and kidney). In males,
    the serum GOT and GPT levels in the 5000 mg/kg group were higher than
    controls. No other compound-related effects were observed in the
    haematological, serum and urine analysis. Neoplastic lesions were
    reported in both test and control animals. The tumours that occurred
    with greatest frequency were adenomas of the lungs, hyperplastic
    nodules and hepatocellular carcinomas of the liver and malignant
    lymphomas. However, there was no statistically significant difference
    between the BHT-treated and control groups for the incidence of any
    type of tumour (Shirai  et al., 1982).

         BHT was administered in the diet at concentrations of 0, 1% or 2%
    to groups of B6C3F1 mice (50/sex) for 104 weeks. After the treatment
    period, all the surviving mice were given basal diet for an additional
    16 weeks. Treated animals underwent a 16-week recovery period prior to
    pathological examination. Mean body weights of the treated male and
    female mice were lower than those of controls. The body-weight
    decrements were dose-related in both sexes and were more marked in
    female mice. Treatment with BHT was found to improve survival in a
    dose-related manner in both males and females. At the end of treatment
    (104 weeks), the percent survival for male/female mice was 40%/58%
    (control), 64%/81% (1% diet) and 74%/89% (2% diet). In male mice
    administered BHT, there was a statistically significant increase in
    the incidence of either a hepatocellular adenoma (19%, 38% and 53% in
    control, low- and high-dose groups, respectively) or a focus of
    hepatocellular alteration, showing a clear dose-response relationship.
    No increases in the number of female mice with hepatocellular adenomas
    or foci of altered hepatocytes were noted. The incidence of male mice

    with other tumours and the incidence of female mice with any
    tumour were not significantly increased as a consequence of BHT
    administration. There was a dose-related tendency to lower incidence
    of lymphoma and leukemia in both males and females (Inai  et al.,
    1988).  Rats

         Groups of rats (15/sex) given diets containing 1% lard and
    0.2, 0.5 or 0.8% BHT for 24 months showed no specific signs of
    intoxication, and micropathological studies were negative. For the
    group given a diet containing 0.5% BHT, the BHT was dissolved in lard
    and then heated for 30 minutes at 150C before incorporation in the
    diet. There were no effects on weight gain or blood constituents and
    micropathological studies of the main organs were negative. The
    feeding of 0.8% BHT was followed in both male and female rats by a
    subnormal weight gain and by an increase in the weight of the brain
    and liver and some other organs in relation to body weight.
    Micropathological studies were negative in this group also. BHT had no
    specific effect on the number of erythrocytes and leucocytes, or on
    the concentration of haemoglobin in the peripheral blood. A number of
    rats of both sexes died during this experiment, but the fatalities
    were not treatment-related. Micropathological studies supported this
    observation. At 0.5%, BHT had no effect on the rat reproductive cycle,
    the histology of the spleen, kidney, liver and skin, or on the weight
    of the heart, spleen or kidney. There was no significant increase in
    mortality of rats fed a diet containing 0.1% BHT and 10% hydrogenated
    coconut oil for a period of two years (Deichmann  et al., 1955).

         Groups of JCL strain rats (20/sex/group), reared under a barrier
    system and 4 weeks of age at the start of the study, received BHT at
    0, 0.005, 0.062 or 0.32% in the diet. Of each group of 40, 15 received
    compound for a "lifetime", 10 for 24 months, and 5 each for 3, 6 or 12
    months. At the interim and final kills, liver, kidney, heart, spleen,
    thyroid and caecum weight were determined as were haematology, serum
    biochemistry, urinalysis, and histological investigation of the
    tissues. At 24 months, heart, liver, kidney, spleen, pituitary,
    thyroid, adrenal, testes, prostate and brain were weighed,
    haematological and biochemical measurements conducted, and
    histopathology done. There was an increase in liver weight, serum
    cholesterol, serum K+ and histological changes in liver and kidney
    at the 0.32% dietary level. There was no change in quantity of food
    intake, body-weight gain, mortality during feeding or mean life span
    and no finding suspicious of tumour induction. There was no indication
    of a dose-related trend in tumour prevalence in either 24-month or
    "lifetime" groups. The tumours found were said to be typical of those
    described in aged rats. It is to be noted that the number of surviving

    rats was small and that tumour data included both lifetime groups and
    animals dead or sacrificed moribund during the 6, 12 and 24-month
    feeding. The available data do not list the number of each of the
    individual tissues examined, although the number of rats is listed.
    The data show a tendency for a decreased number of tumours per rat at
    higher BHT levels (Hiraga, 1978).

         Groups of Fischer 344 rats (50/sex) received 3000 mg/kg or
    6000 mg/ kg BHT in the diet for 105 weeks. The compound was mixed with
    autoclaved lab meal containing 4% fat. A control group of 20
    animals/sex received lab meal only. The animals were observed twice
    daily for signs of ill-health. Physical examinations were performed
    each month and body weights were recorded at least once a month. Gross
    and microscopic examination of 28 major organs, tissues and gross
    lesions including the liver, kidney, thyroid and forestomach, were
    performed on all animals at the end of the study and all animals dying
    on test where possible. Peripheral blood smears were made for all
    animals where possible. There was a dose-related decrease in body
    weights of treated male and female rats throughout the study. There
    was no significant effect of BHT on mortality and no difference in the
    incidence of various neoplasms between treated and control groups. The
    incidence of adenomas of the pituitary was significantly reduced in
    female rats with BHT administration. The incidence of focal alveolar
    histiocytosis was elevated in treated male and female rats in a
    dose-related manner compared with controls. The effect was more
    pronounced in females than in males (NCI, 1979).

         Groups of 7-week old Wistar rats (57/sex/group) were maintained
    on diets containing 0.25 or 1% of BHT for 104 weeks. Control groups
    consisted of 36 rats/sex. At the end of the test period, the surviving
    animals were killed and a complete autopsy was carried out and the
    weights of liver, spleen and kidneys were taken. The principal organs
    and tissues were examined microscopically. Terminal blood samples were
    collected for haematological examination and serum clinical
    biochemistry. Survival in test groups was between 40% and 68%. A
    significant increase in the mortality of the high-dose males was noted
    after week 96 of the study. Food intake was similar for test and
    control animals, but body-weight gain was significantly reduced in
    high-dose males up to week 60 and in high-dose females for most of the
    study. Increases in the mean absolute and relative liver weights were
    observed in all treated animals, and decreases in the absolute and
    relative spleen weights were observed in the treated females.
    Dose-related changes in serum triglyceride (reduction) and GGT
    (increase) in treated males and in total blood cholesterol (increase)
    in treated females were noted. No significant morphological changes
    were observed in the liver which were attributable to BHT treatment. A
    variety of tumours were noted on histopathological examination at the
    end of the study, with no dose-related response in either type of
    tumour or total number as compared to controls. The incidence of
    hyperplastic nodules in the liver and of pancreatic carcinomas in

    female rats and of pituitary adenomas and adeno-carcinomas in both
    sexes of test animals was higher than in controls. However, with the
    exception of the incidence of pituitary adenomas in the low-dose
    females, these differences were not significantly different from
    controls. Since this effect was not dose-related, it was concluded
    that BHT, under the conditions of this test. was not carcinogenic
    (Shibata  et al., 1979; Hirose  et al., 1981).

         Groups of 60, 40, 40, or 60 Wistar rats of each sex (F0
    generation) were fed BHT in the diet at doses of 0, 25, 100, or
    500 mg/kg bw/day, respectively. The F0 rats were mated after 13
    weeks of dosing. The F1 groups consisted of 100, 80, 80, and 100
    F1 rats, respectively, of each sex from the offspring from each
    group. Because of an adverse effect on the kidney in the parents, the
    concentration of BHT in the highest dose group was lowered to
    250 mg/kg bw/day in the F1 generation. The study was terminated when
    rats in the F1 generation were 144 weeks of age. Parameters studied
    were food consumption (weekly), body weight, appearance, and
    mortality. Autopsy and complete histopathological examinations were
    performed on all animals dying during the study, or sacrificed
     in extremis or at termination.

         All animals consuming BHT experienced a dose-related increase in
    survival. In both sexes differences (p <0.001) in longevity were
    seen. The average body weights of the F1 pups at birth in the mid-
    and high-dose groups were slightly lower than those in the control
    group. Body weights of all dosed animals from weaning through the
    entire experiment were lower than those of control animals. In the
    low-, mid-, and high-dose groups, the reductions in body weight were
    for males/females 7%/5%, 11%/10%, and 21%/16%. Food intake was
    comparable for all groups. Clinical appearance and behaviour were
    reported to be normal for all animals. The high-dose males voided a
    slightly reddish urine. Haematological parameters were reported to be
    unchanged by BHT treatment, but no data were given. Serum triglycerides
    were reduced in both sexes and cholesterol was somewhat elevated in
    females only.

         Histological studies indicated an increase in hepatocellular
    carcinomas in male rats and an increase in hepatocellular adenomas in
    both male and female rats. Most liver tumours were found during
    terminal sacrifice at 141-144 weeks. One hepatocellular carcinoma was
    found in a control male at 117 weeks and one in a high-dose male at
    132 weeks. The remainder of the carcinomas occurred at terminal
    sacrifice. The first adenoma was noted in a high-dose male at 115
    weeks. Tables 3 and 4 summarize the data on mortality and the
    appearance of adenomas and carcinomas of the liver. Data on mortality
    and tumour incidence in different groups were analyzed using the
    procedure of Peto  et al. (1980). The dose-related increases in the
    number of hepatocellular adenomas were statistically significant

    (at p < 0.05) in male F1 rats, when all groups were tested for
    heterogenicity or analysis of trend. The increase in hepatocellular
    adenomas and carcinomas in treated female F1 rats was statistically
    significant only for adenomas (at p <0.05) in the analysis for trend.
    Reports on the spontaneous incidence of hepatocellular neoplasms in
    Wistar rats from the laboratory performing this study, as well as
    other European laboratories, indicated that it was usually less than
    3% (Solleveld  et al., 1984; Deerberg  et al., 1980; Olsen  et al.,
    1984). The median life-span for animals in these studies ranged from
    28-36 months for males and 28-33 months for females. Other sites
    reported to have a slight but not statistically significant increase
    in neoplastic lesions were as follows: thyroid, pancreas, ovary,
    uterus, thymus, reticuloendothelium system, and mammary gland.

         Non-neoplastic lesions occurred incidentally and showed no
    relationship to BHT treatment, with the exception of lesions of the
    liver, which showed a dose-related increased incidence of bile duct
    proliferation and cysts in males, and focal cellular enlargement in
    females. At the highest dose (250 mg/kg bw/day), there was no adverse
    effect on the kidney (Olsen  et al., 1986).

         A long-term study was initiated in order to investigate the role
    of hepatic changes in the development of hepatocellular carcinomas in
    rats following  in utero/lifetime exposure to BHT. The dosing regimen
    of the study and strain of rat used were similar to those in the
    two-generation study conducted by Olsen  et al. (1986). Groups of
    6 male and 48 female Wistar rats, aged 13 weeks and 9 weeks,
    respectively, were fed BHT in the diet at doses of 0, 25, 100 or
    500 mg/kg bw/day for 3 weeks prior to mating (13 weeks in Olsen
    study). The rats were then mated on a 1:8 ratio for up to 21 days.
    When pregnancy had been established by abdominal palpation, dams were
    removed to individual cages. On day 20 of gestation, 5 pregnant rats
    were sacrificed for assessment of body and liver weights and liver
    histopathology. The pups were delivered by Caesarian section and
    retained for assessment of a number of parameters. The remaining
    females (20 dams in the control and high-dose groups; 24 dams in the
    low- and mid-dose groups) were allowed to deliver normally. On day 6
     post-partum, litters were either culled or augmented to comprise 8
    pups, at the same time maximizing the number of robust males in each
    litter. At weaning (3 weeks), 4 pups from each of 5 litters per group
    were selected randomly, maximizing the numbers of males, for
    assessment of a number of parameters. The male pups from the remaining
    litters (approximately 60/group) were selected to continue in the
    study and were placed in one of 4 groups corresponding to the diets
    fed to their parents, with the exception that the high dose was
    reduced to 250 mg/kg bw/day as in the Olsen study. Interim kills were
    conducted at 1, 6, 11, or 16 months. The study was terminated 22
    months after the F1 male rats were placed on test diets. All animals
    were observed daily throughout the study for clinical or behavioural
    signs of toxicity. F0 females were palpated from pregnancy to
    weaning and F1 males were palpated from 15 months to termination.

    Body weights were recorded approximately every 2 weeks and food
    consumption monitored every few days. Animals dying during the study
    or at scheduled sacrifices were subjected to gross necropsy. At each
    of the scheduled sacrifices the following parameters were measured in
    all groups of F1 fetuses and weanlings and F1 male rats: body and
    liver weights; hepatic enzyme activities for glucose-6-phosphatase,
    epoxide hydrolase, glutathione-S-transferase, ethoxyresorufin
    O-deethylase and pentoxyresorufin O-depentylase; hepatic content of
    total cytochrome P-450, total glutathione, total microsomal and
    cytosolic protein; and histopathological changes in the liver. In
    addition, immunochemical staining of liver sections for cytochrome
    P-450 1A and 2B and epoxide hydrolase was performed in the control and
    high-dose groups at all scheduled sacrifices. Cellular proliferation
    in the liver was measured in 5 animals per dose group at 4 weeks and
    all subsequent sacrifices using pulse labelling techniques with
    bromodeoxyuridine (only control and high-dose results were reported).
    At the 11, 16 and 22-month sacrifices, a number of parameters in
    addition to those listed above were monitored: histopathological
    changes in the adrenal, kidney and thyroid; assessment of distribution
    of hepatic glucose-6-phosphatase and gamma-glutamyl transferase by
    histochemical staining; and serum thyroxine concentration (presented
    for 16 and 22 months only).

         In the first 5 weeks of BHT administration, a reduction in
    body-weight gain was noted in the high-dose males. This trend started
    prior to BHT administration and continued after BHT administration was
    initiated. Body-weight gain in all other treatment groups was similar
    to that in controls. No treatment-related effects on the health of the
    animals were noted. During pregnancy and lactation, there was no
    difference in food consumption between treated and control female
    rats, and body weights of the dams were similar at weaning. At the
    sacrifice on day 20 of gestation, both absolute and relative liver
    weights of the dams were increased in a dose-related manner,
    statistically significant at the high dose. The body weights of the
    females, both including and excluding their litters, were similar in
    all groups. Histopathological examination of the liver revealed mild
    enlargement of centrilobular hepatocytes and eosinophilia in 4/5
    high-dose (500 mg/kg bw/day) animals, and 1/5 low-dose (25 mg/kg
    bw/day) animals, consistent with induction of mixed function oxidase
    activity. A decrease in the mitotic index of hepatocytes from dams
    receiving 100 and 500 mg/kg bw/day was noted; the significance of this
    result was unclear.

        Table 3.  Mortality (and combined adenomas and carcinomas of the liver) in F1 rats (Olsen et al., 1986)

    BHT         Effective
    (mg/kg      number                          Number of deaths during weeksa
    bw/day)     of rats                                                                                                             
                             0-90     91-104    105-113    114-118    119-126    127-132    133-140    141-144     Total

    0           100          20       10        13         (1)/8      11         10         (1)/12     16          (2)/100
    25          80           8        11        6          3          (1)/13     11         8          20          (1)/80
    100         90           8        12        3          2          10         7          (2)/11     (4)/27      (6)/80
    250         99           7        7         6          (1)/4      (2)/8      (2)/13     (1)/10     (20)/44     (26)/99

    0           100          16       15        18         (2)/8      11         7          8          17          (2)/100
    25          79           10       9         4          6          13         (1)/10     (1)/8      (1)/19      (3)/79
    100         80           5        17        5          5          (1)/7      (1)/9      (1)/11     (3)/21      (6)/80
    250         99           9        5         11         12         8          5          (2)/10     (12)/39     (14)/99

    a    Figures in parenthesis are combined adenomas and carcinomas occurring during that period.
        Table 4.  Incidence of hepatocellular nodular hyperplasia, adenomas,
              and carcinomas (Olsen et al., 1986)

    BHT              Effective No.   Nodular
    (mg/kg bw/day)   of rats         hyperplasia    Adenomas   Carcinomas

    0                100             2              1          1
    25               80              0              1          0
    100              90              2              5          1
    250              99              2              18a        8b

    0                100             2              2          0
    25               79              0              3          0
    100              80              4              6          0
    250              99              5              12c        2d

    a    Over-all test for heterogeneity, p< 0.001, chi-square
         18.17, 3 df.
         Test lot trend, p< 0.001, chi-square = 17.97, 1 df.
    b    Over-all test for heterogeneity, p < 0,05. chi-square
         11.12, 3 df.
         Test for trend, p< 0.01, chi-square = 9.40. 1 df.
    c    Over-all test for heterogeneity, not significant, chi-square =
         5.20, 3 df.
         Test for trend, p< 0.05, chi-square = 4.99, 1 df
    d    Over-all test for heterogeneity, not significant, chi-square =
         2.87, 3 df.
         Test for trend, not significant, chi-square = 2.59, 1 df.

         Reproductive parameters were as follows: mating index was in the
    range 54-65%; gestation index, 93-96%; viability index (birth to
    day 6), 93-96%. No effect of treatment was evident in these
    parameters. The absolute numbers of resorptions/dam were also similar
    in treated and control groups. There was a slight decrease in the
    numbers of pups/litter in the low- and high-dose groups, but a
    dose-related trend was not evident. Body weights of the pups from the
    high-dose group were significantly lower than controls at birth (10%),
    and at days 6 (12%) and 21 (21%) of lactation. Mortality of the pups
    remaining after culling, to day 21 of lactation was 2%, 8%, 12% and
    15%, in order of ascending dose. Since the culling of pups at day 6
    was non-random, these data could not be used for an unbiased
    evaluation of reproductive function in BHT-treated animals.

         Body weights of the F1 males which continued in the study were
    lower in the high-dose group, compared with controls, throughout the
    22-month treatment period. During the first year of the study, the
    difference was in the range of 10-20%. Lower body weights which
    differed from controls by about 5% were also noted in the mid-dose
    animals during the first half of the treatment period. No adverse
    reactions to treatment or effects on food consumption were noted.
    Subjective assessment of the coats of rats indicated that there was
    less age-related deterioration in the 100 and 250 mg/kg bw/day groups
    than in controls. At the scheduled sacrifices, consistent dose-related
    increases in relative, but not absolute liver weights were observed,
    which were statistically significant at the high dose.

         A dose-related incidence of enlargement and eosinophilia of the
    centrilobular hepatocytes was observed consistently at the scheduled
    sacrifices, starting at 6 months. This was indicative of proliferation
    of the SER, consistent with an induction of mixed function oxidases.
    Immunohistochemical staining of liver sections from control and
    high-dose rats revealed a marked increase in hepatocellular content
    and distribution of cytochrome P-450 2B with BHT treatment which
    persisted throughout the study. No such alteration was seen in the
    staining patterns for cytochrome P-450 1A or epoxide hydrolase.
    Histochemical staining revealed a marked induction of gamma-glutamyl
    trans-peptidase activity in the periportal hepatocytes of nearly all
    of the high-dose rats, starting at 11 months of treatment. This effect
    was noted to a lesser extent in the mid-dose group. The distribution
    of glucose-6-phosphatase activity in the treated rats was normal.
    There was no evidence of treatment-related bile duct hyperplasia or
    inflammatory cell infiltrate in the portal tract. Some rats with
    altered hepatocellular foci (AHF) were noted in treated groups at 11
    months and in all groups at 16 and 22 months. There was no treatment
    relationship in the incidence of specific types of foci. At 22 months,
    there was a higher incidence of eosinophilic and basophilic foci in
    the high-dose group. Histochemical staining of liver sections revealed
    a small number of high-dose animals with glucose-6-phosphatase-
    deficient AHF which was statistically significant. No treatment-
    related increase was noted in the incidence of AHF staining positive
    for GGT At 22 months, there was also a significant increase in the
    number of rats with hepatic nodules in the high-dose group (6/19
    animals compared with none in the other groups).

         No increases in the rate of hepatocellular proliferation were
    detected as a result of BHT administration at any point in the study
    commencing from 4 weeks post-weaning. It is of interest to note that
    Clayson  et al. (1993) observed an increase in hepatocellular
    proliferation between 2 and 4 days after initiation of treatment of
    male Wistar rats with 0.5% dietary BHT. Such a transient increase
    would be difficult to detect with the widely spaced sampling times
    used in this study.

         A number of hepatic enzyme activities and other parameters
    related to xenobiotic metabolism were altered as a result of BHT
    treatment. Total cytochrome P-450 content was increased by 30-60% in
    the high-dose animals starting at 21 days of age. Dose-related
    increases were noted in epoxide hydrolase, glutathione-S-transferase
    and pentoxyresorufin O-depentylase (PROD) activities, starting at 21
    days of age, which were statistically significant in the mid- and
    high-dose groups. The increases in PROD activity were very large,
    10-25 fold in the mid-dose, and 20-80 fold in the high-dose groups.
    Modest dose-related increases in ethoxyresomfin O-deethylase
    activities were noted which were not statistically different from
    controls. No effects on hepatic glutathione levels or glucose
    6-phosphatase activity were noted throughout the study.

         Histopathological examination of the kidneys revealed a reduction
    in severity of chronic progressive nephropathy which affected the rats
    in all groups from 11 months. No effects on the adrenal were noted,
    although in a nearly identical, but invalidated study conducted in the
    same laboratory (Robens Institute, 1989) cytomegaly of cells of the
    zona fasciculata was observed in the mid- and high-dose groups at
    weaning and at 4 weeks post-weaning, but not at subsequent time
    points. In the present study, histopathology of the adrenal was
    conducted starting at 11 months post-weaning. Evidence of thyroid
    hyper-activity, characterized by reduction of follicular size, absence
    or reduction of colloid, irregularities in the follicular outline,
    hyperaemia and increase in the number of follicular cells was noted
    starting at 11 months in both the mid-dose group (mild changes
    affecting 75-82% of the rats) and the high-dose group (marked changes
    affecting 100% of the rats). This effect was probably secondary to the
    effects of BHT on the liver. Serum thyroxine levels in treated rats
    did not differ from controls.

         The demonstrated effects on hepatic enzyme induction and
    consequent thyroid hyperactivity in the mid- and high-dose groups
    together with the tumour data from the Olsen study suggested a NOEL of
    25 mg/kg bw/day (Price, 1994).

         BHT was added to the diet of male F344 rats for periods of up to
    110 weeks. In the first experiment, a group of 36 male F344 rats
    received basal NIH-07 diet only, while groups of 21 rats received
    diets containing 300, 1000, 3000 or 6000 mg/kg BHT. Four animals were
    randomly selected from each group for measurement of altered
    hepatocellular foci at 12, 36, 48 or 76 weeks.

         The remaining animals were sacrificed at 76 weeks. In a second
    experiment, groups of 27 male F344 rats were fed basal diet, or diets
    containing 12 000 mg/kg BHT or 12 000 mg/kg BHA for 110 weeks, at
    which time all surviving animals were sacrificed. In both experiments,
    body weights were recorded every four weeks. Gross necropsy was
    performed on all animals. The livers were weighed and submitted for
    histopathology along with tumours and gross lesions from other organs.

         All the rats in experiment 1 survived the entire treatment period
    (76 weeks). In experiment 2, the survival of rats in control and
    treated groups started to decline after 84 weeks, but no
    treatment-related trend was evident. At the end of the treatment
    periods, rats fed 3000, 6000 or 12000 mg/kg BHT had significantly
    lower body weights compared with respective controls. The body-weight
    decrement for these three groups was approximately 10%. Rats fed
    6000 mg/kg BHT for 76 weeks had significantly increased absolute and
    relative liver weights compared with controls. In rats fed 12000 mg/kg
    BHT for 110 weeks, the absolute liver weights were significantly
    decreased and relative liver weights were comparable to controls. The
    density of iron storage deficient hepatocellular foci in the one
    affected rat per group was slightly but not significantly increased in
    BHT-treated animals at 48 and 76 weeks; the incidence and size of foci
    were slightly decreased at 110 weeks in rats receiving 12000 mg/kg
    BHT. No hepatocellular carcinomas were detected in any of the groups.
    Hepatocellular adenomas were detected in all groups, including
    controls, with no treatment-related trend in incidence. There was also
    no treatment-related effect of BHT on the incidence of grossly
    observable tumours in specific organs (Williams  et al., 1990a).

    2.2.4  Reproductive toxicity studies  Mice

         Diets containing 0.1 or 0.5% BHT together with two dietary levels
    of lard (10 or 20%) were given to mice. The 05% level of BHT produced
    slight but significant reduction in mean pup weight and total litter
    weight at 12 days of age. The 0.1% level of BHT had no such effect.
    Out of 7754 mice born throughout the reproductive life span of the
    mothers, none showed anophthalmia, although 12 out of the 144 mothers
    were selected from an established anophthalmic strain (Johnson, 1965).

         The chronic ingestion of 05% BHT by pregnant mice and their
    offspring resulted in a variety of behavioural changes. Compared to
    controls, BHA-treated pregnant mice showed increased exploration,
    decreased sleeping, decreased self-grooming, slower learning, and a
    decreased orientation reflex. BHT-treated offspring showed decreased
    sleeping, increased social and isolation-induced aggression, and a
    severe deficit in learning (Stokes & Scudder, 1974).

         A 3-generation study was performed in mice for the evaluation of
    reproductive, developmental, and behavioural effects. Groups of 10
    Crj:CD-1 mice/sex received BHT in the diet at concentrations of 0,
    0.015%, 0.045%, 0.135%, or 0.405% (equivalent to 0, 20, 70, 200 or
    610 mg/kg bw/day) starting at 5 weeks of age. At 9 weeks of age, the
    mice were mated on a 1:1 basis for a period of 5 days. The pups
    resulting from these matings were weaned at 4 weeks of age and 10
    mice/sex/group were randomly selected to continue in the study. Males
    and females were mated at 9 weeks as in the previous generation.

    Reproductive parameters measured for each of the F1 and F2 groups
    were: number of litters and pups, litter size and weight, sex ratio,
    pup weights on lactation days 0, 4, 7, 14, and 21, and survival to day
    21. Neurobehavioural parameters measured at various times during
    the lactation period were: surface righting, negative geotaxis,
    cliff avoidance, swimming behaviour and olfactory orientation.
    Administration of BHT in the diet to mice adversely affected only
    one of the measured reproductive parameters. Body-weight gain was
    consistently reduced from day 7 to day 21 of lactation in the F1
    high-dose pups, but no body weight differences were noted in the F2
    pups compared with controls. In the F2 males, lower scores were
    assigned to the treated groups for the 180 turn in the open field
    trial but this effect was not apparent in the F2 female pups.
    Consequently, no toxicologically significant effects of treatment were
    apparent with BHT at the dietary concentrations tested in this study
    (Tanaka  et al., 1993).  Rats

         Weanling rats (16/sex) were fed a diet containing 20% lard and 0,
    300, 1000 or 3000 mg/kg BHT and mated at 100 days of age (79 days on
    test). Ten days after weaning of the first litter, the animals were
    again mated to produce a second litter. The offspring (16 females and
    8 males) were mated at 100 days of age. Numerous function and clinical
    tests including serum cholesterols and lipids were performed on the
    parents and the first filial generation up to 28 weeks and gross and
    microscopical examination at 42 weeks. At the 3000 mg/kg dietary
    level, 10-20% reduction in growth rate of parents and offspring was
    observed. A 20% elevation of serum cholesterol levels was observed
    after 28 weeks, but no cholesterol elevation after 10 weeks. A 10-20%
    increase in relative liver weight was also observed upon killing after
    42 weeks on diet. All other observations at 3000 mg/kg and all
    observations at 1000 mg/kg and 300 mg/kg were comparable with control.
    All criteria of reproduction were normal. No teratogenic effects were
    detected (Frawley  et al., 1965b).

         Similar results to those obtained with the parental and first
    filial generations were also obtained with the second filial
    generation. Examination of two litters obtained from the latter at 100
    days of age revealed no effects except a reduction of mean body weight
    at the 3000 mg/kg level. The offspring were examined for litter size,
    mean body weight, occurrence of stillbirth, survival rate and gross
    and microscopic pathology (Kennedy  et al., 1966).

         Breeding pairs of Sprague-Dawley rats (200-220 g) received Purina
    chow supplemented with 0.125%, 0.25% or 0.5% BHT  ad libitum
    beginning from the week before mating and continuing in females
    through lactation and weaning of the pups. Growth rates and survival
    were adversely affected. Pre-weaning pups born of mothers at the

    highest dose level weighed significantly less than controls at ages 7,
    14 and 21 days. The total number of pups dying on study, born of dams
    receiving 0.25 or 0.5% BHT, was significantly higher than controls.
    Behavioural tests were conducted, consisting of righting reflex,
    pivoting, cliff avoidance, startle response, swimming, open field,
    running, wheel activity, roto-rod, active avoidance, position
    discrimination and passive avoidance. For pre-weaning testing, no
    differences were noted at the low- or mid-dose groups. At the
    high-dose level, there was significant increase in surface righting
    time, delayed forelimb swimming development and a trend to less
    activity in open field tests. In post-weaning tests, males in the
    0.25% BHT group showed an effect on passive avoidance, with more
    partial re-entries into compartments where shocked. For all other
    tests, there were no statistical differences, suggesting that BHT had
    no effect on basic motor coordination, active avoidance acquisition,
    or extinction performance (Brunner  et al., 1978).

         Groups of 46 rats, 6-week old (Wistar outbred, SPF) were fed
    diets containing 0, or 0.5 to 0.9% BHT so that the dietary intake of
    BHT was equivalent to 500 mg/kg bw/day during the course of the study.
    At week 19, the F0 generation was mated. Twenty-four hours after
    birth of F1 rats, the size of the litters was reduced to 8, and
    half of the litters were cross-fostered. The body weight of parents
    and offspring and the developmental events of offspring were
    monitored during the course of the study, as well as the reproductive
    performance of the F0 rats. Auditory and visual function and
    locomotive coordination tests were carried out on tile F1
    generation. The F1 animals were autopsied at day 25 of age, and a
    histological examination made of the brains. Body weights and weight
    gain of test animals were reduced when compared to controls, and this
    persisted during gestation. The duration of pregnancy, average body
    weight, and litter size were similar for test and control animals. The
    average body weight and weight gain of the F1 offspring were
    significantly reduced in pups nursed by dosed mothers. Pups exposed
     in utero to BHT also showed a relatively slower development than
    controls when fostered with non-dosed mothers. Pups exposed to BHT
     in utero and/or mothers milk showed alterations in the behavioural
    patterns examined as well as higher incidence in average number of
    dead cells in the brain (Meyer & Hansen, 1980).

         Detailed comments were submitted by the Chemical Manufacturers
    Association (CMA) (1983) on studies of the effect of BHT on
    reproduction and teratogenicity. The major comments were concerned
    with the studies of Brunner  et al. (1978) and Vorhees  et al.
    (1981) previously reviewed by JECFA in 1980 (Annex 1, reference 54) as
    well as the study by Meyer & Hansen (1980).

         In the case of the Brunner  et al. and Vorhees  et al. studies,
    it was concluded that the study showed normal pup survival and
    development in pups raised by dams on diets containing 0.125% BHT.
    Normal post-weaning development was observed in pups raised by dams
    on diets containing 0.25% BHT, although increased post-weaning
    mortality occurred in pups raised by dams on the 0.25% and 0.5% diet;
    developmental delays occurred in pups in the 0.5% group. In the case
    of the Meyer and Hansen (1980) study, developmental delays were seen
    in rats raised by dams on diets containing 0.5% BHT. At the 0.25% and
    0.5% level, the effect may be due either to toxic effects of BHT on
    the dam, or direct toxicity during lactation. A number of questions
    were also raised about the design of the Brunner and Vorhees study.
    These are: (i) the pup selection, in which all litters of fewer than 8
    live pups were discarded; and (ii) the excess mortality was reported
    in terms of pup count rather than affected litters. The data from this
    study have been audited by the U.S. FDA (1983). It was concluded that
    the raw data support the authors' observations of increased mortality
    in the mid-dose and high-dose BHT offspring. However, excess mortality
    occurred in a limited number of litters. For example, in the 0.5%
    group, of the 60 deaths reported in 19 litters, 49 of the deaths
    occurred in 5 litters; in the 0.25% group, of the 42 deaths, 21
    occurred in 2 litters, and at the 0.125% level, of the 12 deaths, 11
    occurred in 1 litter. It was also noted that in the high dose-group,
    there was an increased number of litters with 8 pups or less, and no
    litters larger than 12 pups. In the other dose groups, the litter size
    was comparable to controls.

         In the case of the Meyer & Hansen (1980) study, the CMA comments
    noted that the level of BHT used in the study caused toxicity in the
    dams, which appeared to affect the pups directly or indirectly.
    Reports of teratogenicity studies and/or 1-generation reproductive
    toxicity studies in several strains of mice and rats as well as a
    3-generation reproductive toxicity study in rats were also submitted
    in the comments to support a "no effect" level of 0.1% BHT in the

         Groups of 60, 40, 40, or 60 Wistar rats of each sex, 7 weeks of
    age, were fed a semi-synthetic diet containing BHT so that the dietary
    intake was equivalent to 0, 25, 100, or 500 mg/kg bw/day, respectively.
    After 13 weeks on the test diet the rats were mated.

         Food consumption was similar in all groups. Male and female rats
    in the high-dose group showed a significant decrease in body weight
    which persisted throughout the study. Gestation rate was similar for
    all test groups. The litter size and number of males per litter were
    significantly lower in the 500 mg/kg bw/day group than in the
    controls. Viability was similar in test groups and in the control

    group during the lactation period. The average birth weight of the
    pups in the 500 and 100 mg/kg bw/day groups was slightly lower than in
    the controls. During the lactation period, BHT caused a significantly
    lower dose-related body-weight gain (5%, 7%, and 41% lower body weight
    for the 25, 100, and 500 mg/kg bw/day groups, respectively, as
    compared to controls) (Olsen  et al., 1986).

         A study 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, or 1000 mg/kg bw/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 bw/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 or 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 8 pups if possible. At weaning,
    the dams treated with 1000 mg/kg bw/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 8, the average pup weight was generally
    considerably greater. This implies that the reduced weight gains in
    litters of normal size were associated with poor milk production
    rather than BHT toxicity. Pups from two litters from each dose group
    were maintained on control diet for 4 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 Institute, 1989).  Chickens

         When BHT was fed at a level of 0.125% for 34 weeks to a group of
    10 pullets, no differences in fertility, hatchability of eggs or
    health of chicks in comparison with a similar control group were
    found. The eggs of the antioxidant-treated birds contained more
    carotenoids and vitamin A than those of the controls (Shellenberger
     et al., 1957).  Monkeys

         A group of 6 adult female rhesus monkeys were maintained on a
    test diet containing a mixture of BHT and BHA that provided an intake
    equivalent to 50 mg BHT/kg bw/day and 50 mg BHA/kg bw/day. Another
    group of 6 adult female rhesus monkeys were used as controls. The
    monkeys were fed the diet for one year prior to breeding and then for
    an additional year, including a 165-day gestation period. Haematologic
    studies including haemoglobin, haematocrit, total and differential
    WBC, cholesterol, Na+, K+, total protein, serum GPT, and GOT, were
    carried out at monthly intervals. Body weights were taken at monthly
    intervals. Records of menstrual cycles were maintained through the
    test period.

         After one year the females were bred to rhesus males not
    receiving test diets. During pregnancy complete blood counts were done
    on days 40, 80, 120 and 160 of gestation and on days 30 and 60
    post-partum. A total of 5 infants were born to the experiment monkeys
    and 6 to the control monkeys. Haematological evaluations were made on
    infants of the test and control monkeys at days 1, 5, 15, 30 and 60,
    and observations of the infants were continued through two years of
    age. Two experimental and 2 control infants, 3 months of age, were
    removed from their mothers for 1 month of psychological home cage

         No clinical abnormalities were observed in parent or offspring
    during the period of study. The gestation of test animals was free of
    complications and normal infants were delivered. Adult females
    continued to have normal infants. Infants born during the exposure
    period remained healthy, with the exception of one infant that died
    from unrelated causes. Home cage observations at the third month of
    life did not reveal any behavioural abnormalities (Allen, 1976).

    2.2.5  Special studies on teratogenicity

         In a study on the embryotoxicity of BHT, 3 dosing schedules were
    employed: single doses (1000 mg/kg bw) on a specific day of gestation,
    repeated daily doses (750 mg/kg bw) from the time of mating throughout
    pregnancy, and daily doses (250-500 mg/kg bw for mice and 500 and
    700 mg/kg bw for rats) during a 7- to 10-week period before mating,

    continuing through mating and gestation up to the time the animals
    were killed. No significant embryotoxic effects were observed on
    examination of the skeletal and soft tissues of the fully developed
    fetuses as well as by other criteria. Reproduction and postnatal
    development were also unaffected (Clegg, 1965).

    2.2.6  Special studies on genotoxicity

         The results of genotoxicity studies with BHT are summarized in
    Table 5.

         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.,

         An extensive database of genotoxicity studies for BHT, including
    those documented above, were reviewed in a paper by Bombard  et al.
    (1992). The authors concluded that the majority of evidence indicated
    a lack of potential for BHT to induce point mutations, chromosomal
    aberrations, or to interact with or damage DNA, and that BHT does not
    represent a genotoxic risk to humans.

         The ability of BHT and its metabolites to induce cleavage of
    supercoiled plasmid DNA was studied in pUC18 by agarose gel
    electrophoresis. Dose-related cleavage of DNA was noted with
    BHT-quinone in the range 106-10-3M, and to a lesser extent using
    higher doses of BHT-aldehyde and BHT-peroxyquinone. BHT, BHT- quinone
    methide and other BHT metabolites had no effect on the plasmid DNA.
    Free radical scavengers were effective against the effects of
    BHT-quinone and -peroxyquinone, but not against BHT-aldehyde.
    Formation of superoxide radical as measured by a reduction in
    cytochrome c, was noted only with BHT-quinone (Nagai  et al., 1993).

         The addition of BHT (5 to 20 g/plate) caused a two-fold increase
    in the mutagenic potency of aflatoxin B1 using  Salmonella
     typhimurium strains TA98 and TA100, with and without activation
    (Shelef & Chin, 1980).

         The addition of 50-250 g of BHT/plate inhibited 3,2'-dimethyl-
    4-aminobiphenyl-induced mutagenicity in  Salmonella typhimurium 
    strains TA98 and TA100 in the presence of rat liver S-9 fraction
    (Reddy  et al., 1983a).

        Table 5.  Results of genotoxicity assays with BHT

    Test System              Test Object              Concentration of       Results        Reference

    Point Mutation

    Ames test1               S. typhimurium           0.015-0.6%             Negative       Brusick 1975
                             TA1535, TA1537

    Ames test1               S. typhimurium                                  Negative       Hageman et al.
                             TA97, TA102                                                    1988
                             TA104, TA100

    Ames test1               S. typhimurium           100-10 000             Negative       Williams et al.
                             TA98, TA100              g/plate                              1990b
                             TA1535, TA1537

    Ames test1               S. typhimurium           100-1000 g/plate      Negative       Yoshida 1990
                             TA98, TA100

    Ames test1               S. typhimurium           10 g/plate            Negative       Detringer et al.
                             TA 98                                                          1993

    Host-mediated assay      ICR Swiss mouse/         30-1400 mg/kg -        Negative       SRI 1972
                             S. typhimurium           acute
                             G46, TA1530              30-500 mg/kg -

    Mammalian cell           rat liver epithelial     60-90 g/ml            Negative       Williams et al.
    gene mutation            cell (line 18),                                                1990b
                             HGPRT locus

    Table 5 (cont'd)

    Test System              Test Object              Concentration of       Results        Reference

    Sex-linked recessive     Drosophila               2.0  10-6 g          Negative2      Prasad &
    lethal                   melanogaster                                                   Kamra 1974

    Sex-linked               Drosophila               5% diet                Negative       Brusick 1975
    recessive lethal         melanogaster

    Clastogenic effects and chromosomal aberrations

    Chromosomal              human WI-38              2.5-250 g/ml          Positive2      SRI 1972
    aberration assay         (embryonic lung

    Sex chromosome loss      Drosophila               2.0  10-6             Negative2      Prasad &
                             melanogaster                                                   Kamra 1974

    Micronucleus assay       rat bone marrow          30, 90, 1400           Negative       SRI 1972
                                                      mg/kg (acute and

    Dominant lethal assay    Sprague-Dawley rat       30, 900, 1400          Negative       Brusick 1975
                                                      mg/kg bw (acute)
                                                      30, 250, 500           Positive
                                                      mg/kg bw/day

    Dominant lethal assay    male Sprague-Dawley      50, 150, 500           Positive2      Sheu et al.
                             rats                     mg/kg bw/day                          1986
                                                      1% diet                Negative
                             male mice

    Table 5 (cont'd)

    Test System              Test Object              Concentration of       Results        Reference

    Heritable                male mice                1% diet                Negative       Sheu et al.
    translocation assay                                                                     1986

    DNA interactions

    Mitotic recombination    Saccharomyces            0.6-2.4%               Negative       Brusick 1975
                             cerevisiae D4

    Mitotic recombination    Saccharomyces            30, 900, 1400          Negative       SRI 1972
    - Host-mediated          cerevisiae D3/ICR        mg/kg bw (acute
                             Swiss mouse              30, 250, 500           Negative
                                                      mg/kg bw/day

    Sister chromatid         CHO cells                1 - 1000 g/ml         Negative       Williams et al.
    exchange1                                                                               1984

    DNA excision repair      UV-irradiated human      ?                      Positive       Daugherty
    synthesis                lymphocytes                                                    1978

    DNA repair test          hepatocyte primary       0.01 - 10 g/ml        Negative       Williams et al.
                             culture                                                        1990b

    1    Both with and without rat liver S9 metabolic activation.
    2    A discussion of this result is contained in Bombard et al. (1992).
         The addition of 100-250/g of BHT/plate was shown to inhibit
    3,2'-dimethyl-4-aminobiphenyl-induced mutagenicity in  Salmonella
     typhimurium strains TA98 and TA100 Mutagenicity was further
    inhibited by use of S-9 preparations from rats fed dietary BHT (0.6%)
    as compared to S-9 preparation from rats fed BHT-free diets
    (Reddy  et al., 1983b).

         BHT was a moderately effective inhibitor of benzidine-induced
    mutagenicity in  Salmonella typhimurium strain TA98, activated with a
    hamster liver S-9 fraction (Josephy  et al., 1985).

         BHT (0.11-11 /M) protected against DNA damage induced in rat
    hepatocytes by AAF or N-hydroxy AAF as shown by a marked reduction of
    unscheduled DNA synthesis. BHT also inhibited AAF-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 AAF or N-hydroxy AAF (Chipman &
    Davies, 1988).

    2.2.7  Special studies on hepatotoxicity  Mice

         Groups of male ddY mice treated perorally with BHT (200-800 mg/kg
    bw) in combination with an inhibitor of GSH synthesis, buthionine
    sulfoximine (BSO, 1 h before and 2 h after BHT, 4 mmol/kg bw/dose,
    i.p.) developed hepatotoxicity characterized by an increase in SGPT
    activity and centrilobular necrosis of hepatocytes. The hepatotoxic
    response was both time- and dose-dependent. BHT (up to 800 mg/kg bw)
    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 BSO while
    inducers of drug metabolism such as phenobarbital tended to increase
    hepatic injury. The results suggested that BHT was activated by a
    cytochrome-P-450-dependent metabolic reaction and that the hepatotoxic
    effect was caused by inadequate rates of detoxification of the
    reactive metabolite in mice depleted of hepatic GSH by BSO
    administration. Based on studies with structural BHT analogues, the
    authors suggested that a BHT-quinone methide may play a role in the
    hepatotoxicity in mice (Mizutani  et al., 1987).  Rats

         Female (albino Wistar) rats, initial body weight 120-130 g, were
    maintained on a diet containing 0 or 0.4% BHT, for 80 weeks. After one
    week on the test diet, significant increases were observed in liver
    weight, microsomal protein, cytochrome P-450, cytochrome b5,
    NADPH-cytochrome c reductase, biphenyl-4-hydroxylase and ethyl
    morphine N-demethylase but not aniline-4-hydroxylase. Total liver

    protein, succinic dehydrogenase and glucose-6-phosphatase were
    slightly decreased. There was little change in this pattern during the
    period of the study. Rats removed from the BHT test diet at the end of
    the test period and maintained on BHT free diet for 18 days, showed a
    return to normal for many liver parameters except for cytochrome b5,
    cytochrome c reductase and ethylmorphine N-demethylase. Histological
    changes at the end of 80 weeks feeding consisted of centrilobular cell
    enlargement, which was reversible following 18 days on a BHT-free
    diet. The only ultrastructural change was a prolileration of smooth
    endoplasmic reticulum (Gray  et al., 1972).

         Groups of female rats (80-100 g) received 0.4% BHT with corn oil
    mixed in ground lab chow and were sacrificed at intervals of 1, 8, 16,
    32, or 80 weeks, and compared with controls. Samples of liver were
    taken for biochemical, histochemical, and morphological studies. To
    examine for reversibility of hepatic changes, control diet was
    administered for 18 days to a group of 4 rats following 80 weeks of
    BHT administration. After one week on BHT, there was a marked liver
    enlargement with relative liver weight increased up to 35% and with an
    increase in drug metabolizing activities and NADP cytochrome c
    reductase activity. After 18 days of removal from the 80-week
    treatment, there was only a slight increase in liver weight. The
    effect was therefore reversible. Histologically, after BHT treatment
    the liver was characterized by enlarged centrilobular hepatocytes,
    with a heterogenous appearance of this zone. Ultrastructurally, there
    was a proliferation of smooth endoplasmic reticulum. The authors noted
    that although the evidence of liver injury was equivocal, there were
    two features that were also seen with many hepatotoxins and
    hepatocarcinogens: depression of glucose-6-phosphatase activity and
    cell enlargement. However, there were no lysosomal changes
    characteristic of cytologic injury, and effects were reversible
    (Crampton  et al., 1977).

         Groups of 8 male Wistar rats were given diets containing 0, 0.1,
    0.25, 0.5, or 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 labelling index or mitotic index.
    In a second experiment, groups of 8 rats were treated with 0.5%
    dietary BHT for 2, 4, 8, 10, or 14 days. This treatment led to a
    time-limited increase in liver cell [3H]thymidine-labelling index
    that subsided to control values within 8 days. This increase in
    [3H]thymidine labelling in the liver was accompanied by an
    unexpectedly large increase in the mitotic index (Briggs  et al.,

         Groups of female Sprague-Dawley rats were given 700 mg BHT/kg bw
    and selected hepatic biochemical effects were determined after 4 and
    21 h. 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 bw. 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 savage at doses of
    0, 25, 250 or 500 mg/kg bw/day for 7 days (5 rats/group), or 28 days
    (10/group) and also at daily doses of 1000 or 1250 mg BHT/kg bw/day
    (5 rats/group) for up to 4 days (sublethal doses). The sublethal doses
    induced centrilobular necrosis within 48 h, whereas administration of
    250 or 500 mg/kg bw/day 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 mild cell damage was also obtained at
    250 mg/kg bw/day, while there was no evidence that BHT caused liver
    damage at 25 mg/kg bw/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).

         The acute effects of a single oral administration of 500 mg/kg bw
    BHT to rats were investigated in combination with phenobarbitone (PB),
    a microsomal enzyme inducing agent, and buthionine sulfoximine (BSO),
    a glutathione-depleting agent. Groups of 10 male Sprague-Dawley rats
    received BHT in corn oil by gavage, BHT with 3 days prior
    administration of 80 mg/kg bw/day PB in saline i.p., BHT with 1 h
    prior administration of 900 mg/kg bw BSO in saline i.p., or corn
    oil/saline alone. Thirty-six hours after administration of BHT, the
    animals were sacrificed and blood was collected for assay of serum
    enzyme activities (ALT, AST, alkaline phosphatase, lactate
    dehydrogenase), albumin, APTT and clotting factors II, VII, X and
    IX. Samples of liver (from each of 4 lobes), lung and kidney were
    processed for histopathological examination which included
    immunochemical staining of liver lot visualization of cytochrome P-450
    1A and 2B distribution. Liver homogenates were also assayed for
    reduced glutathione concentration, microsomal cytochrome P-450,
    ethoxyresorufin- O-deethylase (EROD) activity, ethoxycoumarin- O-
    deethylase (ECOD) activity, epoxide hydrolase activity and malondi-

         The results showed that a single oral dose of 500 mg/kg bw BHT
    was below the threshold for acute hepatotoxicity. BHT administration
    had no effect on any of the serum parameters, with the exception of a
    slight reduction in the levels of clotting factor IX. There was no
    evidence that BHT induced hepato-cellular necrosis, and hepatic
    malondialdehyde concentration, an indicator of lipid peroxidation, was
    reduced from controls. A single dose of BHT was also associated with
    an increase in mitotic activity of hepatocytes in 8/10 animals,
    increased hepatic activity of ECOD without affecting microsomal
    protein content or EROD activity, and a marked increase in hepatic
    epoxide hydrolase activity. The last was postulated to be related to
    BHT-induced inhibition of phylloquinone epoxide reductase activity.
    There was no effect on hepatic GSH levels, no clear treatment-related
    change in immunochemical staining of hepatocytes for the cytochrome
    P-450 1A or 2B isoenzyme families and no histopathological changes in
    the lung or kidney. Prior administration of PB or BSO resulted in
    unequivocal liver damage (hepatocyte degeneration or coagulative
    necrosis), mostly in the centrilobular areas, in about half the rats,
    without affecting serum parameters which are commonly used indices for
    tissue damage (ALT, AST, lactate dehydrogenase). Since neither of
    these treatments duplicated the periportal cell damage observed with
    repeated administration of BHT, the results could not be used as a
    model for investigation of alterations in enzyme profiles induced by
    repeated administration of BHT (Powell & Connolly 1991).

    2.2.8  Special studies on nephrotoxicity

         A single large dose of BHT (1000 mg/kg bw) in male F344 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
    bw, 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
    semi purified 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).

         Groups of 10 male ddY mice received 0, 1.35%, 1.75%, 2.28%,
    2.96%, 3.85% or 5.00% BHT in a purified diet, (equal to 0, 1570, 1980,
    2630, 3370, 4980 or 5470 mg/kg bw/day, respectively) for 30 days.
    Terminal body weights in all groups of treated mice were lower than
    those of controls, the difference ranging from 15% at the lowest dose
    to 30% at the highest dose, statistically significant at the highest
    three doses. Absolute and relative kidney weights exhibited
    dose-related decreases and increases, respectively, which were related
    to reduced body-weight gain. Results from gross pathology of the
    kidney showed 7/10 of the high-dose animals with "misshapen kidney"
    compared with none in any of the other treated or control groups.
    Histopathology of the kidney revealed a dramatic dose-related increase
    in the incidence and severity of toxic nephrosis (0, 2, 3, 6, 8, 10,
    and 10 out of 10 mice/group) as indicated by a number of tubular
    lesions (distal and proximal tubular degeneration, distal tubular
    necrosis, distal tubular regeneration, tubular dilatation and cysts).
    No pathological changes were noted in the liver of these same mice
    which could be related to treatment. The ED50 for toxic nephrosis in
    the tidy male mouse following 30 days of administration of BHT in the
    diet was calculated to be 2300 mg/kg bw/day (Takahashi, 1992).

    2.2.9  Special studies on pulmonary toxicity

         Young male Swiss Webster mice were injected i.p. with BHT at dose
    levels ranging from 63 to 500 mg/kg bw BHT. The animals were killed 1,
    3 or 5 days after BHT administration. Histopathological changes were
    well-developed 3 days after administration of 500 mg/kg bw, and
    consisted of a proliferation of many alveolar cells, formation of
    giant cells and macrophage proliferation. These changes were
    accompanied by an increase in lung weight and total amounts of DNA and
    RNA. The changes were dose dependent, the smaller effective dose being
    250 mg/kg bw (Saheb & Witschi, 1975).

         Sixty male Swiss mice were given i.p. injections of 400 mg/kg bw
    BHT dissolved in corn oil. Six experimental animals and 6 controls
    were sacrificed daily for 9 days. Two hours before sacrifice, each
    animal received 2 Ci/g of tritiated thymidine. No animals died during
    the study and none showed signs of respiratory distress. Two days
    after dosing, cellular lesions were noticed in the type I alveolar
    epithelium. Abnormal giant type II cells were observed in mitosis and
    many had an accumulation of tritiated thymidine. Labelled endothelial
    cells were seen after day 6 in small vessels and capillaries, and
    there was an increase in fibroblastic cells in the interstitium and
    capillaries. There was an increase in thymidine-labelled pulmonary
    cells from days 2 through 5, after which labelling dropped off and
    approached control levels by day 9. Levels of lung thymidine kinase
    activity rose sharply on days 1-4 after dosing and then dropped off
    rapidly (Adamson  et al., 1977).

         Groups of NMRI mice (25-35 g) and Wistar rats (160-320 g)
    received BHT as a single dose of 500 mg/kg bw dissolved in soya bean
    oil, either i.p. or by gavage. Four days later, radiolabelled 14C
    thymidine was given. After 90 minutes, the animals were sacrificed,
    lungs removed and DNA levels were measured. In mice, DNA synthesis was
    equally increased in males and females by oral or i.p. administration.
    Although lung weight was increased, the concentration of DNA was not
    affected. No effect was seen in male rats and only a slight increase
    in females (Larsen & Tarding, 1978).

         Groups of 16-24 Swiss male mice (25-30 g) received a single i.p.
    injection of BHT in corn oil (63, 215, or 500 mg/kg bw) or corn oil
    only (Tocopherol stripped 0.5 ml). Three days later the mice were
    sacrificed. After BHT treatment, wet lung weights were increased to
    120% of control, as were dry lung weights. There were significant
    increases in DNA content and level of non-protein sulfhydryl (133-156%
    of control). Superoxide dismutase and other oxidative enzyme levels
    were increased. The authors concluded that BHT apparently increased
    inflammatory and reparative-proliferative processes of the lung
    (Omaye  et al., 1977).

         Following acute exposure to BHT, the initial sequence of events
    involved infiltration of type I (squamous) epithelial cells followed
    by multifocal necrosis and destruction of the blood barrier. A
    detailed discussion of the sequence of tissue changes and repair
    mechanisms was given. It was stated that the susceptibility of the
    squamous epithelium to injury was similar to that seen after oxygen
    exposure, radiation exposure, and treatment with blood-borne
    bleomycin, but the recovery pattern was quite different. BHT was
    thought to cause cell lysis and death as a result of interaction with
    the cell membrane (BIBRA, 1977).

         The increase in lung weight and increase in thymidine
    incorporation into lung DNA observed in mice following BHT injection
    was inhibited by treatment with cedar terpenes. No increase in lung
    weight was observed in animals treated with BHT alone if they were
    less than 3-week old. This may result from the inability of infant
    mice to metabolize BHT (Malkinson, 1979).

         In a study of lung toxicity of BHT analogues in mice, it was
    established that the structural feature essential for toxic activity
    is a phenolic ring structure having a methyl group at the 4-position
    and  ortho-alkyl group(s) which can result in a moderate hindering
    effect of the hydroxyl group (Mizutani  et al., 1982).

         In another study, the toxic potency of BHT in mice was decreased
    by deuteration of the 4-methyl group, suggesting that lung damage
    following administration of BHT was caused by the metabolite
    2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone (Mizutani  et al.,

         Male mice given a single dose of BHT showed ultrastructural
    changes of the lung, which were characterized by selective destruction
    of type I epithelial cells, which were replaced by type II cuboidal
    cells. These changes were accompanied by a marked decrease in the
    number of peroxisomes, as well as catalase activity (Hirai  et al.,

         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).

         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 bw (s.c.). When injected 30 minutes prior to
    sub-threshold doses of BHT (0-250 mg/kg bw, J.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, 1988a).

         In experiments with mouse lung slices, BHA enhanced the covalent
    binding of BHT to protein. Subcutaneous administration of either BHA
    (250 mg/kg bw) or diethyl maleate (DEM, 1 ml/kg bw) 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 bw) 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, 1988b).

         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
    close 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 bw 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 I cell death) and regenerative
    response (type II cell hyperplasia and differentiation) were 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 (Maikinson
     et al., 1989).

         The synthetic corticosteroid methylprednisolone (MP; 30 mg/kg bw,
    s.c. given twice daily for 3 days) partially protected male C57BL/6N
    mice from the pulmonary toxicity of BHT when administered 0, 24 or
    48 h after BHT treatment (Okine  et al., 1986).

         The activity of a metabolite of BHT hydroxylated on one
     tert-butyl group, (BHT-BuOH) in inducing pneumotoxicity was
    investigated in the mouse on the basis that pneumotoxicity had been
    observed following administration of BHT in all inbred strains of mice
    tested, but not in rats, and BHT-BuOH was a major product of mouse
    liver and lung microsomes and formed only in traces in rat microsomes.
    Lung damage was assessed by determining lung/body weight ratios,
    Ca2+-dependent protease (calpain) activity and by histopathological
    examinations of the lungs. The liver, heart and kidneys were
    investigated for histopathological changes resulting from i.p.
    injection of BHT-BuOH. BHT-BuOH induced nearly a doubling of lung/body
    weight ratios 4 days after an i.p. injection of 50 mg/kg bw. By
    comparison, doses of 200 mg/kg bw BHT or greater were required to
    produce consistent increases in this parameter. Other BHT metabolites,
    DBQ, BHT-MeOH, BHT-OOH and BHT-OH, had no effect at i.p. doses of
    200 mg/kg bw. Pulmonary calpain activity was significantly decreased
    in mice which received 50 mg/kg bw BHT-BuOH, reaching a maximal loss
    at 3-4 days after administration. This effect was similar in time
    course and extent to that induced by 400 mg/kg bw BHT. A dose of
    10 mg/kg bw BHT-BuOH also resulted in a significant decrease in
    calpain activity which was less marked than the higher dose. Alveolar
    deterioration and compensatory Type II cell hyperplasia, inflammatory
    response and bronchiolar cell hyperplasia were observed in response to
    i.p. doses of 50 mg/kg bw BHT-BuOH. Less extensive effects were noted
    with doses of 10 mg/kg bw and these effects were considered comparable
    to those induced with doses of 400 mg/kg bw BHT. On the basis of the
    qualitative similarity in the effects of BHT and BHT-BuOH on the
    mouse lung, the higher potency of the metabolite, and the species
    correlation of formation of BHT-BuOH and pneumotoxicity, the authors
    concluded that BHT-BuOH was an intermediate in the biotransformation
    of BHT to a toxic metabolite in the mouse (Malkinson  et al., 1989).

         A quinone methide metabolite of BHT which is formed subsequent to
    hydroxylation of the  tert-butyl side group (QM-OH) was investigated
    as the metabolite responsible for pulmonary toxicity in mice. QM-OH
    was more strongly electrophilic than BHT-quinone methide as indicated
    by a reaction time with GSH which was 6 times faster. Liver and lung
    microsomes from both rats and mice produced quinone methide from BHT,
    but only microsomes from the mouse produced QM-OH readily from BHT.
    Microsomes from rat liver produced traces of QM-OH and lung microsomes
    produced none. These results were used to reconcile previous results
    linking pulmonary toxicity in the mouse to quinone methide formation
    with species specificity of this effect. The authors postulated that
    the organ specificity of BHT toxicity in mice was due to lower
    concentrations of GSH in the lung compared with the liver for
    inactivation of toxic metabolites (Bolton  et al., 1990).

         The time course for repair of BHT-induced lung injury was
    investigated in four strains of mice with i.p. LD50s ranging from
    350-1700 mg/kg bw. The four strains of mice tested developed similar
    levels of injury at equivalent doses and no correlation could be made
    between lung injury and lethality (Kehrer & DiGiovanni, 1990).

         A number of compounds which modify the activity of specific
    cytochrome P-450 isoenzymes were used to identify the isoenzymes
    involved in bioactivation of some compounds, including BHT, to
    pulmonary toxins. Pretreatment of mice with O,O,S-trimethylphosphoro-
    dithioate, bromophos,  p-xylene, -naphthoflavone or pyrazole all
    produced a reduction of pneumotoxicity in MF1 outbred mice induced by
    a single i.p. dose of 400 mg/ kg bw BHT as indicated by a lowering of
    lung/body weight ratios measured 3 days after administration of BHT.
    The first three agents greatly reduced lethality of BHT in mice as
    indicated by an increase in LD50 values. The prevention of lung
    toxicity by these agents was proportional to the reduction in
    lethality of BHT. These three agents also markedly inhibited
    pentoxyresomfin (PROD) activity, an action attributed to CYP 2B1 in
    rat lung microsomes. -Naphthofiavone exerted a less marked effect on
    these LD50 values and PROD activity, and pyrazole-induced PROD
    activity. The authors concluded that since the three agents which
    prevented the pneumotoxicity of BHT in mice also inhibited PROD
    activity in rat lung microsomes, CYP 2B1 was the most likely candidate
    for the bioactivation of BHT in mouse lung. However, since BHT is not
    toxic to the rat lung, results obtained with rat lung microsomes would
    not necessarily be relevant to the mouse. In addition, the authors did
    not mention that pretreatment of mice with pyrazole, while reducing
    the lung/body weight ratio increase induced by BHT, also induced PROD
    activity in rat lung microsomes (Verschoyle  et al., 1993).

         The ability of isolated Clara (non-ciliated bronchiolar
    epithelial) cells from mouse lung to metabolize BHT to the putative
    toxic quinone methide QM-OH was investigated as well as comparison of
    the toxic effects of BHT and BHT-BuOH on these cells. These cells
    contain most of the monooxygenase activity of the lung, mainly as CYP
    2B1/2B2. Analysis of quantitative metabolite data revealed that
    hydroxylation of BHT occurred 5 times more readily at a  tert-butyl
    group, producing BHT-BuOH, than at the 4-methyl position to produce
    BHT-MeOH in mouse Clara cells. The data also suggested that QM-OH was
    more readily produced from BHT-BuOH than was BHT-QM from BHT. BHT-BuOH
    more effectively reduced the viability of Clara cells in culture than
    did BHT. Concentrations of 5 and 10 M BHT-BuOH reduced viability in a
    comparable manner to 75 and 100 M BHT. Inhibition of cytochrome P-450
    with SKF 525-A reduced damage to Clara cells induced by both BHT and
    BHT-BuOH. The authors concluded that BHT-BuOH is an intermediate in
    P-450-catalyzed oxidation of BHT to a cytotoxic species which they
    propose is QM-OH (Bolton  et al., 1993).

         Groups of 20 male Swiss albino mice received BHT in olive oil or
    olive oil alone by a single i.p. injection at doses of 0, 200, 400 or
    800 mg/kg bw. Five animals from each group were sacrificed at 24 h,
    48 h or 7 days after exposure. Lavage fluid was collected from the
    lungs and assayed for total protein content and lactate dehydrogenase
    (LDH) activity. Cells in the sediment were counted. Histopathological
    examination of the lungs was performed. A time- and dose-dependent
    increase in the number of cells in the bronchoalveolar lavage fluid
    and in the total protein content and LDH activity was noted at 48 h
    and 7 days. The severity of histopathological lesions, described as
    congestion of capillaries and small blood vessels, and increased
    cellularity and diffuse thickening of alveolar septa, was also
    increased in a time- and dose-dependent manner (Waseem & Kaw, 1994).

    2.2.10  Special studies on haemorrhagic effects  Mice

         See Combined species, section  Rats

         Groups of male Sprague-Dawley rats (6 weeks of age) received BHT
    in a semi-synthetic diet at concentrations ranging from 0.6% to 1.4%
    or control diet only. Deaths occurred within 40 days, at levels of
    0.7% or greater. Spontaneous massive bleeding to the pleural and
    peritoneal cavities, or as external haemorrhage, was observed in all
    dead or dying animals. The prothrombin index was decreased as the
    daily dose of BHT was increased. Mild diarrhoea was noted after 4
    days. Rough hair coat, and redness of urine was noted. Death was due
    to haemorrhage and was classified by the authors as a secondary type
    of toxicity, probably due to a decrease in prothrombin concentration.
    According to the authors, the effect seemed to depend on strain of
    rats and dietary concentration (Takahashi & Hiraga, 1978a).

         Groups of 10 male Sprague-Dawley CLEA rats were fed diets
    containing 0.6, 0.7, 0.8, 1.0, 1.2, or 1.4% BHT for 40 days. A
    dose-related effect on mortality (21 death/50 rats) was observed with
    rats given 0.7% or more BHT during the period from 9 to 37 days.
    Spontaneous massive haemorrhages were observed in these animals. The
    prothrombin index of survivors was decreased, which was dependent on
    the BHT dose. At the lowest level, the decrease was approximately 65%
    (Takahashi & Hiraga, 1978b).

         Male Sprague-Dawley CLEA rats were maintained on diets containing
    levels of 85, 170, 330, 650, 1300, 2500, or 5000 mg/kg BHT for 1 to 4
    weeks. A significant decrease in the prothrombin index was observed at
    week 1 in all groups fed BHT at levels of 170 mg/kg or higher.
    However, when the rats were maintained on the test diets for 4 weeks,

    a significant decrease in the prothrombin index was observed only in
    the 5000 mg/kg group. This was the only group which showed an increase
    in relative liver weights compared to those of the control group. In
    another study, haemorrhagic death, haemorrhage, and a decrease in
    prothrombin index in male Sprague-Dawley rats caused by 1.2% BHT were
    prevented by the simultaneous addition of 0.68 mole phylloquinone/kg
    bw/day. Phylloquinone oxide also prevented hypoprothrombinemia due to
    BHT (Takahashi & Hiraga, 1979).

         Male Sprague-Dawley rats were fed diets containing 0 or 1.2% BHT
    for one week. BHT-treated rats showed haemorrhages in most organs.
    There was a significantly increased leakage of Evans Blue into the
    epididymis. In addition, inhibition of ADP-induced platelet
    aggregation and decreased platelet factor 3 availability were
    observed. Plasma prothrombin factors were decreased, but fibrinolytic
    activity was unchanged (Takahashi & Hiraga, 1981b).

         Male albino rats (CRL COB CD(SD) BR) given 3 consecutive daily
    doses of 380, 760, or 1520 mg BHT/kg bw/day showed no evidence of
    haemorrhage. However, BHT produced a dose-dependent increase in
    prothrombin time, with no effect on prothrombin time seen in the
    380 mg/kg bw/day group (Krasavage, 1984).

         Male rats receiving 0.25% dietary BHT for 2 weeks showed
    decreased concentrations of vitamin K in the liver and increased
    faecal excretion of vitamin K (Suzuki  et al., 1983).

         Dietary BHT at a level of 1.2% was shown to affect platelet
    morphology (distribution width), and to cause changes in the fatty
    acid composition of the platelet lipids (Takahashi & Hiraga, 1984).

         Groups of 4-5 male Sprague-Dawley rats (5-6 weeks old) were fed a
    diet containing 1.2% BHT for 1-7 days, and blood coagulation factors
    II(prothrombin), VII, VIII, IX and X, and platelet aggregation were
    measured. The average dose of BHT was about 1000 mg/kg bw/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 effect 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 bw and 0.5-72 h 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 h

    after BHT treatment, but by 72 h some recovery had occurred. Hepatic
    levels of BHT reached maxima at 3 h (a major peak) and 24 h after BHT
    dosing and BHT-quinone methide reached maxima at 6 and 24 h (a major
    peak). When BHT was given in doses of 200, 400 or 800 mg/kg bw,
    factors II, VII and X decreased after 48 h only in rats given the
    highest dose, 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 at
    800 mg/kg bw. 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 contained no added vitamin K, and the
    animals apparently 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).

         The effects of BHT on platelet aggregation were examined.
     In vitro experiments indicated that BHT, at concentrations greater
    than about 10-3M, inhibited both ADP- and collagen-induced platelet
    aggregations, but not those induced by arachidonic acid. BHT-quinone
    methide also inhibited ADP- and collagen-induced aggregations to a
    lesser extent. In another experiment, male Sprague-Dawley rats were
    fed a diet containing 1.2% BHT (650-740 mg/kg bw/day) for 4 or 7 days.
    The treated animals showed marked decreases in body-weight gain
    compared with controls. Haemorrhage was detected in epididymal adipose
    tissues of all animals receiving BHT in the diet. Platelet aggregation
    capacity induced by ADP or collagen in platelet-rich plasma collected
    from these rats was considered to be normal. Although aggregation
    induced by 3.9 mM arachidonic acid was markedly inhibited in platelets
    from the rats fed 1.2% BHT for 7 days, platelet aggregation induced by
    the optimal concentration of arachidonate (2.0 mM) was normal. These
    results suggested a difference in plasma or platelet properties
    between the BHT-treated and control rats. The authors concluded that
    BHT-induced haemorrhage in rats was not due to a direct effect of BHT
    on platelet aggregation. The differences between  in vitro and
     in vivo results were attributed to the low plasma concentrations of
    BHT or BHT-quinone methide which were present  in vivo (Takahashi,

         This study reported on the effect of BHT on vitamin K-dependent
    clotting factors in rats receiving vitamin K-sufficient and vitamin
    K-supplemented diets. Groups of 5-6 male Wistar rats received diets
    with BHT added to give a nominal intake of: (1) 0, 3000 mg/kg bw/day
    or 3000 mg/kg bw/day plus 250 mg vitamin K3/kg of feed for 7, 14 or
    21 days; (2) the same regimen for 7 or 14 days with a 250 mg/kg
    Vitamin K control group added; (3) 0, 12.5, 125 or 600 mg/kg bw/day or
    600 mg/kg bw/day plus 3 mg/kg vitamin K3 for 28 days. The basic diet
    (SDS Ltd., Witham, Essex, UK) was found to contain a minimum of
    3 mg/kg vitamin K3 which was considered more than adequate to supply
    the recommended intake of this vitamin. In the first experiment,
    prothrombin time (PT) was measured as well as specific vitamin
    K-dependent factor deficiencies. The results showed that 3000 mg/kg
    bw/day BHT had no effect on PT, but specifically decreased the levels
    of vitamin K-dependent clotting factors II, VII, X and IX in rats
    receiving a diet containing adequate vitamin K. In experiments 2 and
    3, a Thrombotest optimized for rodents was used in addition to
    different assay methods for PT and APTT. Serum fibrinogen levels were
    measured in experiment 2 only. Thrombotest time, PT and APTT were
    significantly increased from controls within 7 days at a close of
    3000 mg/kg bw/day. Vitamin K3 supplementation at 250 mg/kg in the
    diet prevented the BHT-induced increases in Thrombotest time and APTT
    and reduced the effects of BHT on PT. BHT administration had no effect
    on serum fibrinogen concentrations. Thrombotest time, but not PT or
    APTT, was significantly increased from controls after administration
    of 600 mg BHT/kg bw/day for 28 days. The effect was prevented by
    concurrent dietary supplementation with 3 mg vitamin K3/kg of feed.
    This study confirmed an antagonistic effect of BHT on vitamin K in
    rats, which resulted in a reduction in blood-clotting factors even
    when the diets contained adequate vitamin K. The authors pointed out
    that this was a high-dose phenomenon with a threshold and a steep
    dose-response curve (Cottrell  et al., 1994).  Combined species

         A number of strains of rats (Sprague-Dawley, Wistar, Donryu and
    Fischer), mice OCR, ddY, DBA/c, C3H/HBe, BALB/CaAn and C57BL/6), New
    Zealand White-Sat rabbits, beagle dogs, and Japanese quail were fed
    diets containing BHT (1.2% in the diet for rats and mice; 1% for
    quail; 170 or 700 mg/kg bw/day for rabbits; and 173,400, or 760 mg/kg
    bw/day for dogs) for a period of 14-17 days. Haemorrbagic deaths
    occurred among male rats of all strains and female rats of the Fischer
    strain. Female rats of the Donryu and Sprague-Dawley strains showed no
    obvious haemorrhaging. No haemorrhagic effects were noted in quails,
    rabbits or dogs (Takahashi  et al., 1980).

         Administration of 0.5%, 1.0% or 2.0% BHT in a purified diet
    (equal to 660, 1390 or 2860 mg/kg bw/day) for 21 days resulted in a
    dose-related increase in the mortality due to massive haemorrhage of
    the lungs in male ddY mice housed in wire-bottomed cages. The
    surviving animals exhibited a dose-related increase in both absolute
    and relative lung and liver weights at termination. Haemorrhagic
    deaths and increased absolute lung and liver weights were not observed
    in ddY male mice fed diets containing 1.35% 5.0% BHT for 30 days and
    housed in cages with soft-wood chip bedding, or in Hartley guinea-pigs
    fed 0.125% - 2.0% BHT in the diet for 14-17 days. The prothrombin
    times were significantly increased in all groups of ddY mice receiving
    BHT in the diet at levels of 1.0% or higher (with the exception of the
    2.0% wire-caged group). The effect was of a similar magnitude
    (approximately 25%) in treated groups housed on soft-wood bedding,
    regardless of the dose. Results for kaolin-activated partial
    thromboplastin time were similar. Effects on coagulation in
    guinea-pigs were equivocal. The authors suggested that the coagulation
    defect in mice in the absence of a dose-response relationship might be
    due to minor damage to hepatobiliary function and/or fatty liver and
    the haemorrhages in the lungs to injury to that organ. The haemorrhage
    and coagulation defect would consequently not have the same cause as
    that observed in rats. BHT was detected in the livers of mice and
    guinea-pigs in dose-related concentrations (0.2-4/g/g tissue).
    BHT-quinone methide was not detected in these species although it has
    been detected in the livers of rats (7-40 g/g tissue) in a study by
    Takahashi  et al. (1980) (Takahashi, 1992).

    2.2.11  Special studies on effects on the thyroid

         Male MOL/WIST SPF rats, outbred strain (approximately 200 g) were
    used for the study. BHT was added to a semi-synthetic diet in which
    the iodine content was controlled at about 12/g/100 g (nutritional
    requirement for the rat is 15 g/100 g). In one study, rats were fed
    0, 500 or 5000 mg BHT/kg of feed for 8, 26 or 90 days, and the uptake
    of 125I by the thyroid was determined. The presence of BHT in the
    diet resulted in a marked increase in the uptake of 125I at all time
    periods studied. When rats were fed BHT in diets containing varying
    amounts of iodine (12, 150 or 300/g/100 g) for 30 days, there was a
    significant increase in thyroid weight in BHT-treated animals when
    compared to controls. BHT in the diet of rats increased liver and
    thyroid weights at 5000 mg/kg of the diet, but only thyroid weight at
    500 mg/kg. BHT did not change levels of T3 and T4 in the blood.
    The biological half-life of thyroxine was increased after 13 days on a
    BHT diet but returned to normal after 75 days. Electron microscopy of
    the thyroid glands of rats exposed to 5000 mg/kg BHT for 28 days
    showed an increase in the number of follicle cells (Sondergaard &
    Olsen, 1982).

    2.2.12  Special studies on effects on the immune system

         In vitro, BHT (50 /g/culture) suppressed the plaque-forming cell
    response of mouse spleen cell cultures as measured by the method of
    Mishell & Dulton (Archer  et al., 1978).

         Addition of cyclic GMP (cGMP added as the dibutyl or 8-bromo
    form) to BHT suppressed Mishell-Dulton cultures and effected a
    reversal of the BHT suppression of antibody production (Wess & Archer,

    2.2.13  Special studies on potentiation or inhibition of cancer

         Male Strain A mice were injected i.p. with 500 mg/kg bw urethan,
    then one week later received repeated injections (1/week for 8 weeks)
    of either 300 mg/kg bw BHT, or 500 mg/kg bw BHA, or 1000 mg/kg bw
    Vitamin E all dissolved in corn oil. At the termination of the study,
    only BHT was shown to produce a significant increase in tumour yield.
    Although the number of tumours produced by BHA treatment was greater
    than usual, it was not statistically significant. A/J mice treated
    with 3-methylcholanthrene or dimethylnitrosamine, followed by
    treatment with BHT (i.p.), resulted in an increase in tumour yield
    (Witschi  et al., 1981).

         Groups of Charles River rats (20/sex) were fed diets (males 24
    weeks, females 36 weeks) containing 6600 mg BHT/kg of feed and/or
    carcinogen (N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenyl-
    acetamide) in the molar ratio of 30:1, then continued on control diets
    for another 12 weeks. The N-2-fluorenylacetamide alone resulted in
    hepatomas in 70% of the male rats, and mammary adenocarcinoma in 20%
    of the females. With N-hydroxy-N-2-fluorenylacetamide, 60% of the
    males had hepatomas and 70% of the females had mammary adenocarcinoma.
    BHT reduced the incidence of hepatomas in males to 20% when the
    carcinogen was N-2-fluorenylacetamide, and to 15% when N-hydroxyl-
    N-2-fluorenylacetamide was the test compound. Similar results were
    obtained with Fischer strain rats. Liver and oesophageal tumour
    production with diethylnitrosamines at 55 mg/litre in drinking-water
    for 24 weeks was not affected by BHT (Ulland  et al., 1973).

         Groups of 20 male F344 rats were given a single intragastric
    administration of 100 mg/kg bw MNNG or 750 mg/kg bw EHEN, 2 s.c.
    injections of 0.5 mg/kg bw MBN or 4 s.c. injections of 40 mg/kg bw
    DMH. At the same time, the rats received 0.1% DBN for 4 weeks,
    followed by 0.1% DHPN for 2 weeks in drinking-water for a total
    carcinogen exposure period of 6 weeks. Three days after completion of
    these treatments, the rats received 0 or 0.7% BHT in the diet for 36
    weeks. Control groups of 10 or 11 animals received 0.7% BHT alone or
    basal diet alone. Final body weights of both BHT-treated groups were
    significantly lower than those of respective controls (by 7% compared

    with carcinogen-treated control and by 13% compared with basal diet
    control) and this was reflected in higher relative kidney weights.
    Relative liver weights were increased by about 60-75%. Dietary BHT
    following carcinogen treatment eliminated the appearance of colon
    carcinomas and reduced the incidence and multiplicity of some
    preneoplastic and neoplastic lesions of the kidney. BHT administration
    increased the incidence of hyperplasia, adenomas and carcinomas of the
    thyroid gland and had no effect on tongue, oesophagus, forestomach,
    glandular stomach, duodenum, small intestine, liver, lung or urinary
    bladder (Hirose  et al., 1993).  Bladder

         Male F344 rats were treated with 0.01 or 0.05% N-butyl-N
    (4-hydroxy-butyl) nitrosamine (BBN) in drinking-water for 4 weeks, then
    fed diets containing 0 or 1% BHT for 32 weeks. BHT in the diet was
    associated with a significant increase in the incidence of cancer and
    papilloma of the bladder of rats treated with 005%, but not 0.01% BBN
    (Imaida  et al., 1983).

         Rats were administered 200 mg/kg N-2-fluorenylacetamide (FAA) in
    the diet alone or with 6000 mg/kg BHT for 25 weeks. No bladder
    neoplasms resulted from feeding FAA alone, but the combination of FAA
    and BHT resulted in 17/41 papillomas and 3/41 carcinomas in the
    bladder (Williams  et al., 1983).

         Four dietary levels of BHT (300, 1000, 3000, or 6000 mg/kg)
    were simultaneously fed with 200 mg/kg FAA for 25 weeks. FAA
    feeding alone produced no neoplasms, but when combined with BHT at
    3000 or 6000 mg/kg, the incidence of bladder tumours were 18% and
    44%, respectively. The incidence of bladder tumours in the 300 and
    1000 mg/kg BHT groups was low and not significantly different from the
    incidence with FAA alone (see also effects on liver) (Maeura &
    Williams, 1984).

         Male F344 rats were given injections of methylnitrosourea (MNU)
    twice a week for 4 weeks, and then a basal diet containing 1% BHT for
    32 weeks. BHT significantly increased the incidence of papilloma and
    papillary or nodular hyperplasia of the urinary bladder, and the
    incidence of adenoma (but not adenocarcinoma) of the thyroid (Imaida
     et al., 1984).

         Groups of 20 male F344 rats (6-week old) 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., 1987a).

         Groups of 20 male F344 rats (6-week old) 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 incidence in liver
    lesions. 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).

         Groups of 20 male F344 rats (6-week old) 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 mg/kg vitamin K. A decrease in body weight was observed.
    DNA synthesis in the urinary bladder epithelium was increased after 4
    weeks while no morphological changes were seen after 8 weeks using
    light microscopy. 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.,

         A study was performed to investigate early proliferation-related
    responses of the renal pelvic epithelium in response to bladder tumour
    promoters. Groups of 10 male F344 rats received 0 or 1% BHT. At week
    4, the DNA-labelling index of the renal pelvic epithelial cells was
    determined from 1000 cells in 5 rats/group. At week 8, kidney sections
    were prepared for SEM examination. Body weights of the treated animals
    were significantly lower than for controls, 18% at 4 weeks and 25% at
    8 weeks (Shibata  et al., 1989). The mean DNA labelling index in the
    renal pelvic epithelium after 4 weeks treatment was slightly higher
    than in controls, but without statistical significance. No cell
    surface alterations were observed by SEM after 8 weeks of treatment
    (Shibata  et al., 1991).

         Groups of 20 F344 rats, 4 or 54 weeks of age, were injected s.c.
    with 50 mg/kg bw of 3,2'-dimethyl-4-aminobiophenyl (DMAB), a
    multi-organ carcinogen, once a week for 10 weeks. At the same time,
    the animals received 1% BHT in the diet for 11 weeks. The study was
    terminated 55 weeks after initiation of treatment. Combined treatment
    with DMAB and BHT resulted in the development of urinary bladder
    papillomas and carcinomas in more than 95% of both young and old rats.

    The induction of liver foci and pancreatic acinar cell foci by DMAB
    was inhibited by concurrent treatment with BHT. Increased formation of
    DMAB-DNA adducts, detected by immunohistochemical staining was
    demonstrated in urinary bladder epithelial cells from the BHT-treated
    rats. BHT treatment suppressed the formation of these adducts in the
    liver and had no effect on formation of adducts in the colon, pancreas
    or prostate. The authors suggested that these effects of BHT were
    related to its ability to induce a number of drug-metabolizing
    enzymes, thus altering the quantity of active metabolites in a
    particular tissue (Shirai  et al., 1991).

         Simultaneous feeding for 76 weeks of 6000 mg/kg BHT with 50 mg/kg
    AAF, a minimally effective carcinogenic dose of this compound in F344
    rats, resulted in an increase in the incidence and multiplicity of
    urinary bladder neoplasms, including carcinomas. Feeding of both 3000
    and 6000 mg/kg BHT in conjunction with AAF resulted in an increase in
    the incidence of nodular hyperplasia of the urinary bladder, and a
    positive trend in the incidence of this lesion was noted down to
    dietary levels of 300 mg/kg BHT. In the same study, dietary
    administration of 100 - 6000 mg/kg BHT resulted in a dose-related
    decrease in induction of altered hepatic foci (iron-storage deficient
    and GGT-positive) and a decrease in the multiplicity of AAF-induced
    adenomas and carcinomas of the liver and the incidence of carcinomas
    (Williams  et al., 1991).  GI tract


         Groups of male BALB/c mice treated intrarectally with
    methyl-nitrosurea, and then maintained on diets containing BHT, showed
    a marked increase in the incidence and multiplicity of GI tract
    tumours when compared to treated mice maintained on BHT-free diets. In
    another study, BALB/c mice were injected with dimethylhydrazine (6
    weekly injections) and then maintained on control (BHT-free) diets or
    on diets containing 0.05% or 0.5% BHT. The colon tumour incidence were
    10%, 0%, and 32%, in the respective groups (Lindenschmidt  et al.,


         The observed increase in tumour-specific antigen activity in the
    colon chromatin of rats treated with 1,2-dimethylhydrazine was
    eliminated by simultaneous treatment with BHT (Gabryelak  et al.,

         Male F344 rats were treated with a single dose of N-methyl-
    N'-nitro-N-nitrosoguanidine, and then maintained on diets containing
    no BHT, 1.0% BHT, 5% NaCl, or 5% NaCl + 1.0% BHT for 51 weeks. The
    incidence of squamous cell carcinomas of the forestomach were 11%,
    16%, 3%, and 53% in the respective groups (Shirai  et al., 1984).

         When rats were given 0.5% BHT in the diet for 36 weeks following
    4 injections (1 per week) of 1,2-dimethylhydrazine, BHT did not affect
    the number of rats with colon tumours, but the number of tumours per
    rat occurring in the distal colon was significantly decreased (Shirai
     et al., 1985).

         Wistar rats fed 1.0% BHT in the diet during treatment with
    N-methyl-N'-nitro-N-nitrosoguanidine (administered in drinking-water
    at a concentration of 1.0 mg/ml) for 25 weeks, and then maintained on
    the test diet for another 14 weeks, showed a significant reduction in
    the incidence of gastric cancer, when compared to rats receiving
    BHT-free diets (82% versus 37%) (Tatsuta  et al., 1983).

         Seven-week old male Wistar rats (20/group) were given
    N-methyl-N'-nitro-N-nitrosoguanidine in the drinking-water
    (100 mg/litre) 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 0.5 g/litre N,N-dibutyl-
    nitrosamine in drinking-water for 4 weeks and then treated with a
    basal diet containing 1% BHT with 7 mg/kg 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. BHT induced an increased incidence of papillary or
    nodular hyperplasia and papilloma in the bladder, while no
    statistically significant increase was seen in liver lesions
    (Fukushima  et al., 1987b).

         Groups of 5 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 diets containing 0, 0.5% or
    1.0% BHT for 5 or 6 months immediately following initiation with 2 or
    4 s.c. injections of DMH (40 mg/kg bw), a significantly higher
    incidence of colon tumours (5-month 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 bw given orally) produced stomach and colon tumours;
    0.5% BHT in the diet did not affect 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 weeks 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 labelling index was observed at any time during the experiment
    (Hirose  et al., 1986).  Liver

         Groups of 93 rats (22-day old) received 0 or 0.5% BHT diets for
    407 days following 18 days of administration of AAF (0.02%). Prolonged
    feeding of BHT diet after AAF produced a significant increase of liver
    tumours (Peraino  et al., 1977).

         Rats were administered 200 mg/kg AAF in the diet, alone or with
    6000 mg/kg BHT for 25 weeks. AAF alone induced a 100% incidence of
    liver neoplasms. Simultaneous administration of BHT resulted in a
    decreased frequency of benign neoplasms, neoplastic nodules and
    malignant neoplasms, and hepatocellular carcinomas (Williams  et al.,

         BHT at concentrations of 300, 1000, 3000, or 6000 mg/kg was fed
    simultaneously with 200 mg/kg AAF for 6, 12, 18, or 25 weeks. BHT
    produced a reduction in the incidence of tumours in a dose-dependent
    manner (100% incidence in the absence of BHT to 56% at 6000 mg/kg BHT)
    (see also effects on the bladder) (Maeura  et al., 1984).

         Rats were fed 200 mg/kg AAF for 8 weeks, then diets containing
    BHT at levels of 300, 1000, 3000, or 6000 mg/kg for up to 22 weeks.
    The area of altered hepatocellular foci, identified by iron exclusion
    and gamma-glutamyl transferase (GGT) activity, that was induced by
    feeding the AAF, showed increased development at the highest level of

    BHT (the number of foci, the area occupied by GGT-positive
    preneoplastic and neoplastic lesions, and the neoplasm incidence were
    increased). These parameters were unaffected at the lower BHT levels
    (Maeura & Williams, 19841.

         Rats were given a single i.p. injection of 200 mg/kg bw of
    diethylnitrosamine, and then maintained on a diet containing 1% BHT
    for 6 weeks. At week 3 the rats were subjected to partial hepatectomy.
    The number of gamma-glutamyl transpeptidase positive foci in the liver
    of BHT-fed rats was significantly decreased when compared to controls
    (Imaida  et al., 1983).

         BHT was compared to phenobarbital (PB) and DDT 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 bw). Two weeks
    later, selection was carried out by feeding AAF for 2 weeks and giving
    a necrogenic dose of carbon tetrachloride after 1 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,
    or 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 male Wistar rats when given
    after initiation (Prat  et al., 1986).

         Initiation of liver carcinogenesis with a single dose of
    diethylnitrosamine (DEN), and selection with 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. When the diet
    was supplemented with PB (after I-S), the increase in diploid nuclei
    started earlier. At the time carcinomas appeared (22 weeks PB
    treatment) a decrease in the frequency of 2N nuclei was found.
    BHT-treated animals which develop no carcinoma within the considered
    time span showed a clear increased amount of 2N nuclei in the
    precancerous lesions only after 14 weeks treatment (Haesen  et al.,

         Dietary administration of 1% BHT for 26 weeks was commenced
    during or immediately after 2 weekly i.p. injections of azaserine
    (30 mg/kg bw) 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, no
    effect was seen in the liver, while the frequency of preneoplastic
    lesions in the pancreas was significantly reduced (Thornton  et al.,

         A study was conducted to establish whether the modulating effect
    of dietary fats and BHT on AAF-induced hepatocarcinogenesis was
    related to the levels of cytochrome P-450 in the nuclear envelope
    (NE). Treatment of weanling rats with 0.3% dietary BHT for 2 weeks was
    followed by up to 16 weeks of treatment with 0.05% AAF in the diet.
    Prior treatment with BHT protected against the AAF-induced reduction
    in the NE cytochrome P-450 content of liver cells for 9 weeks in rats
    fed a diet with a high saturated fat content, compared with 3 weeks in
    rats fed a diet with a high polyunsaturated fat content. The magnitude
    of this effect of BHT on cytochrome P-450 content was apparently
    correlated with its tumour reduction effect in rats fed AAF in
    high-fat diets. The results from rats fed low-fat diets, in which BHT
    apparently does not exert a significant effect on AAF-induced
    carcinogenesis, were different from those in rats fed the high-dose
    diets. In rats fed low-fat diets, AAF did not reduce cytochrome P-450
    content until 9 weeks of feeding, at which point prior treatment with
    BHT resulted in a partial reversal of this effect. In the first 3
    weeks when AAF-treated rats had cytochrome P-450 content similar to
    control animals, prior treatment with BHT resulted in its induction in
    the AAF-treated animals (Carubelli & McCay 1989).

         BHT at concentrations of 1-14 mM in the presence of a rat liver
    microsomal preparation reduced the binding of AAF to calf thymus DNA
    to 20% of control values. When N-hydroxylated 2-AAF, a cytochrome
    P-450 activated oxidation product of AAF was used, BHT (0.1-8.0 mM)
    reduced its binding to DNA to only 80% of control levels. In rat
    hepatocyte cultures, BHT concentrations of 0.01-0.10 mM resulted in a
    reduction of AAF binding to DNA to about 85% of control levels. These
    results showed that mechanisms in addition to those demonstrated
     in vivo (i.e. induction of detoxifying enzymes) may be involved in
    the anticarcinogenic effects of BHT (Richer  et al., 1989).

         Male B6C3F1 mice were injected i.p. with 100 or 200 mol/kg bw
    of diethylnitrosamine (DEN) once a week for 10 weeks. After a recovery
    interval of 4 weeks, the mice were fed diets containing 5000 mg/kg BHT
    or 500 mg/kg phenobarbital (positive control) for 24 weeks. At the end
    of this time, DEN alone induced a dose-related incidence of altered
    hepatic foci and hepatocellular adenomas. Treatment with BHT following
    DEN administration had no effect on the incidence or multiplicity of
    these lesions, whereas phenobarbital administration potentiated the
    effects of DEN 2 to 3 fold (Tokumo  et al., 1991).

         Dietary administration of 50 mg/kg AAF was found to induce a 100%
    incidence of liver neoplasms after 76 weeks. Concurrent administration
    of BHT at levels of 100 - 6000 mg/kg of feed inhibited the induction
    of altered hepatic foci and reduced the multiplicity of hepatocellular
    adenomas and carcinomas and the incidence of carcinomas (Williams
     et al., 1991).

         Concurrent administration of BHT in the diet at 5, 25 or
    125 mg/kg of feed for 42 weeks with gavage administration of aflatoxin
    B1 (5 g/kg bw, 3 times a week) for the final 40 weeks resulted in a
    reduction in the density of hepatic foci staining positive for the
    placental form of glutathione S-transferase at the highest dose of BHT
    (Iatropoulos  et al., 1994).  Lung

         The tumorigenic potency of a single i.p. injection of 1000 mg/kg
    bw of urethane to male Swiss-Webster mice was significantly increased
    if followed by repeated weekly injections with 250 mg/kg bw BHT. The
    number of animals/group ranged from 9 to 22 and the animals were
    treated for 9 to 13 weeks. Only tumours on the lung surface itself
    were counted. About 90% of the animals treated with urethane alone
    developed lung tumours. There was a significant increase in the number
    of tumours/mouse after 11 or more weeks of treatment with BHT. Animals
    treated with BHT alone did not develop lung tumours. A/J strain mice
    were also given the same treatment with 10 weekly injections of BHT
    The number of lung tumours/mouse significantly increased in those
    receiving BHT in addition to urethane in comparison with those
    receiving urethane alone. With both strains of mice, repeated
    injection of BHT without prior urethane treatment did not result in an
    increased number of animals with lung tumours or tumours/mouse as
    compared to controls dosed with corn oil. With both mouse strains,
    there were fewer lung tumours in the animals given BHT as compared to
    the corn oil controls. In contrast to the above results, injection of
    animals with BHT for 0-7 days before urethane injection did not
    increase the number of animals with tumours or number of tumours/mouse
    (Witschi & Cote. 1976).

         Groups of Swiss mice were given 50, 250, or 1000 mg/kg bw
    urethane or 0.9% NaCl. After 7 days, half the urethane-treated
    animals and half the controls received 300 mg/kg bw BHT i.p., the
    remaining animals receiving corn oil alone. The animals received 13
    injections/week. The number of tumours/lung found 14-24 weeks after
    the initial urethane doses was significantly increased in the
    BHT-treated animals.

         In another study, when the interval between injection of the
    urethane and the first treatment with BHT was delayed for 6 weeks, BHT
    treatment produced more tumours. When the number of BHT injections
    commencing 1 week after urethane treatment was reduced from 13 to 4,
    the same significant increase in tumour yield was observed as in the

    13-dose study. However, 1 or 2 doses of BHT had no significant effect.
    When the mice were pretreated with 13 injections of BHT, and then
    treated with urethane 1 week later, there was no enhancement of tumour
    yield. Simultaneous administration of BHT and urethane resulted in
    fewer tumours compared to animals treated with urethane alone. When
    mouse strains (C57BL, C3H and BALB/C) which have a low naturally
    occurring incidence of lung adenoma were treated with urethane and
    then with multiple injections of BHT, the BHT treatment did not
    significantly increase tumour incidence or average numbers of lung
    tumours (Witschi & Lock, 1979).

         Male A/J mice were injected i.p. with a single dose of urethane
    and then fed 0.75% of either BHT, BHA, or ethoxyquin in the diet, once
    a week or continuously for 8 weeks. Lung tumour yield was scored 4
    months after the urethane treatment. Dietary BHT, but not BHA or
    ethoxyquin, under both test conditions, enhanced lung tumour

         Mice were fed diets containing BHA or BHT for 2 weeks prior to
    urethane treatment, and then maintained on conventional laboratory
    diets for 4 months. The BHT diet had no effect on tumour yield, but
    the BHA treatment significantly decreased the average number of
    tumours (Witschi, 1981).

         A/J mice were given a single dose of BHT i.p. (400 mg/kg bw),
    sufficient to cause acute lung damage and produce cell proliferation
    in the lung for 6 to 7 days. Urethane was administered continuously by
    implanted mini pumps during this period. Continuous presence of
    urethane during the period of cell division did not result in an
    enhanced number of the tumours. When urethane-injected mice were dosed
    i.p. with SKF525A (2-diethylaminoethyl-2-,2-di-phenylvalerate
    hydrochloride) and BHT (SKF inhibits lung cell division normally seen
    following BHT administration), or BHT alone, both treatment gave a
    very significant increase in lung tumour yield compared to
    urethane-treated controls. Repeated pulmonary cell division brought
    about by other treatment e.g., 95-100% oxygen, were also shown not to
    enhance tumour development (Witschi & Kehrer, 1982).

         BHT was shown to enhance the lung tumour incidence in mice
    treated with doses of urethane greater than 50 mg/kg bw. At lower
    doses of urethane (subcarcinogenic doses) BHT did not enhance tumour
    development. In another study, it was shown that following treatment
    of mice with urethane, a two-week exposure to 0.75% BHT in the diet
    was sufficient to enhance tumour development, and that 0.1% BHT was an
    effective enhancer when fed for 8 weeks.

         BHT, administered within 24 h post-treatment and fed for 8
    weeks, enhanced tumour development in mice treated once with
    3-methylcholanthrene, benzo(a)pyrene, or N-nitrosodimethylamine.

         When mice were injected weekly with BHT, there was a rapid
    increase in cell proliferation, and in both the cumulative labelling
    index (incorporation of 3H-thymidine) and the number of labelled
    type II cells. These effects were smaller after each injection, and by
    the fifth injection, no increase was observed (Witschi & Morse, 1985).

         A single i.p. injection of BHT (200 mg/kg bw) 6 h before a single
    urethane injection (1000 mg/kg bw) 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 injections/week) following urethane,
    while multiplicities increased several-fold with such treatment in
    adult mice of this strain (Malkinson & Thaete, 1986).

         A/J mice given 1000 mg/kg bw urethane followed by 400 mg/kg bw
    BHT by injection, developed 40% more lung tumours than mice treated
    with urethane alone. In mice treated with 3-methylcholanthrene,
    repeated injections of BHT (300 mg/kg bw) 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 3 of its metabolites was
    compared in the inbred mouse strain MA/MyJ. MA/MyJ mice were given a
    single injection of urethane (50 mg/kg bw) followed by 6 weekly i.p.
    injections of 50 or 200 mg/kg bw BHT, BHT-BuOH, 2,6-di- tert-butyl-
    4-hydroxymethyl phenol (BHT-MeOH) or 2,6-di- tert-butyl-1,4-benzo-
    quinone (DBQ). 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).

         The susceptibility of different strains of mouse to the lung
    tumour-promoting effects of BHT was correlated with ability of hepatic
    microsomal preparations from each strain to produce a metabolite of
    BHT, BHT-BuOH, which is hydroxylated on one  tert-butyl group. No
    correlation existed between tumour promotion and microsomal production
    of BHT-MeOH (hydroxylated on the methyl group) or DBQ (the quinone
    metabolite). In the MA/MyJ strain, which was found to be the most

    sensitive to promotion of urethane-induced lung tumours by BHT, 6
    weekly i.p. injections of 50 mg/kg bw of the BHT metabolite, BHT-BuOH,
    resulted in a similar promotional effect to 200 mg/kg bw BHT, while
    200 mg/kg bw of the BHT metabolites, BHT-MeOH or DBQ, had no
    promotional effect. The authors cited other evidence which implicated
    the  tert-butyl hydroxylation pathway in lung-tumour promotion by
    BHT: preferential  in vitro formation of this metabolite relative to
    other metabolic products of BHT was correlated with species (rat
    versus mouse) and strain susceptibility to lung tumour promotion by
    BHT; the repeat-dose administration regimen of BHT associated with
    lung tumour formation was also associated with induction of
    hydroxylation on the  tert-butyl group; and this pathway is a major
    route of metabolism in the mouse lung (Thompson  et al., 1989).

         Evidence was cited to show that the genes which regulate
    sensitivity to the lung tumour-promoting effects of BHT are distinct
    from the  pas (pulmonary adenoma susceptibility) genes which
    predispose some inbred strains of mice to the development of lung
    tumours. It was suggested that strain differences in response to the
    effects of BHT are mediated through genes which regulate the ability
    to metabolize BHT along specific pathways (Malkinson, 1991).  Mammary gland

         Groups of female Sprague-Dawley rats were treated with
    7,12-dimethylbenz[a]anthracene (DMBA) or nitrosomethylurea (NMU), and
    then fed diets containing 0 or 0.3% BHT for 30 weeks. Rats treated
    with DMBA and maintained on the control diet developed 100% tumour
    incidence (mammary gland) by week 27, whereas those maintained on the
    BHT supplemented diet had an incidence of 54% by the end of the study.
    Dietary BHT had no effect on the incidence of tumours induced by NMU
    treatment (King  et al., 1981).

         Female rats were fed diets containing 0, 0.25, or 0.5% BHT. The
    test diets were administered either (a) 2 weeks before until 1 week
    after DMBA administration or (b) 1 week after DMBA administration to
    the end of the study (30 weeks). The DMBA was administered as a single
    dose of 8 mg. BHT was an effective inhibitor of mammary carcinogenesis
    when administered during either of these time frames (20% inhibition
    by regime (a) and 50% by regime (b)) (McCormick  et al., 1984).

         Dietary BHT was shown to decrease the incidence of mammary
    tumours induced in female Sprague-Dawley rats by DMBA but had no
    effect on animals treated with MNU (King  et al., 1981).

         The inhibitory effect of BHT was strongly influenced by the dose
    of initiating carcinogen and the type of diet in which BHT was fed.
    Administration of BHT in the AIN-76A diet, showed a markedly different
    effect from BHT in the NIH-07 diet. In the AIN-76A diet, 6000 mg/kg
    BHT had no effect on the incidence of mammary tumours induced by 15 mg
    DMBA, whereas a similar level of BHT in the NIH-07 diet resulted in a
    40% inhibition of tumour development (Cohen  et al., 1984).

         A dose-related inhibition of DMBA-induced mammary tumorigenesis
    in female Sprague-Dawley rats was seen after long-term exposure to
    dietary BHT. BHT was given from 14 days 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, 1000, 3000, or 6000 mg BHT/kg of
    feed. Maximal inhibition was approximately 50% at the highest
    concentration of BHT. 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. 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 (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).

         The effect of dietary administration of BHT on the formation of
    DNA adducts in the mammary gland by DMBA was investigated in female
    Sprague-Dawley rats using 32P post-labelling techniques. Diets
    containing 0.4% or 0.8% BHT were fed to 39-day old rats for 2 weeks
    followed by oral administration of 32 mg/kg bw DMBA. BHT treatment
    resulted in a 42% or 36% reduction in the formation of all types of
    DMBA-derived DNA adducts 22 h later compared with DMBA-treated
    controls. Dietary BHT selectively inhibited the formation of adducts
    derived from the  anti diastereomer of DMBA as opposed to the  syn
    diastereomer. Dietary concentrations of BHT within the range used in
    this study have been reported to significantly inhibit the initiation
    of DMBA-induced carcinogenesis (Singletary & Nelshoppen, 1991).

         In this study, the effects of BHT and its metabolites BHT-MeOH
    and DBQ (BHT-quinone) on DMBA-induced rat mammary carcinogenesis and
    the  in vivo formation of rat mammary DMBA-DNA adducts were tested.
    The selection of these metabolites was based on the observation that
    they were the major metabolites detected following incubation of BHT

    with rat liver microsomes and that no metabolites were detected
    following inhibition with rat mammary microsomes. Each of the
    compounds were administered 2 weeks before and 1 week after oral
    administration of 33 mg/kg bw DMBA. The i.p. administration of
    200 mg/kg bw BHT and DBQ (but not BHT-MeOH) resulted in 39% and 25%
    inhibition of mammary tumour formation, respectively. There was a good
    quantitative correlation between inhibition of mammary tumorigenesis
    by BHT and DBQ and the inhibition of DMBA-DNA adduct formation. Doses
    of 100 and 200 mg/kg bw BHT inhibited DMBA-DNA binding to a similar
    degree. BHT and DBQ differed in their selectivity of inhibition of
    specific adducts. A decrease in the formation of the  anti-dihydro-
    diolepoxide adduct of DMBA to deoxyguanosine was most closely
    correlated with the abilities of BHT and DBQ to inhibit mammary
    tumorigenesis (Singletary  et al., 1992).  Pancreas

         Male LEW inbred rats were given an injection of 30 mg azaserine
    once a week for 3 weeks, and then maintained on diets containing
    0 or 0.45% BHT for 4 months. BHT treatment reduced the number of
    acidophilic foci per pancreas by 32%, but was without effect on focal
    size. BHT had no effect on the occurrence of basophilic foci (Roebuck
     et al., 1984).  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, 1987a).

         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 h 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).

    2.2.14  Special studies on other effects

         Young adult BALB/c mice of both sexes were maintained on diets
    containing 0.75% BHT for 1 month, and then irradiated with 525-750 R
    of X-ray. Radiation protection was observed at all doses below that
    which produced 100% lethality (Clapp & Satterfield, 1975).

         Hybrid (C31F1 male mice, 10-12 weeks of age, were maintained
    on diets containing 0 or 0.75% BHT, for a period of 30 days, and then
    injected i.p. with alkylating materials. There was a marked reduction
    in the 30-day mortality in mice fed BHT Males were protected against
    ethyl methane-sulphonate, n-propyl or isopropyl methanesulphonate,
    ethyl dibromide, diethylnitrosamine and cyclophosphamide, but not
    against methyl methane-sulphonate, N-methyl-N'-nitro-N-nitroso-
    guaridine or dipropylnitrosamine (Cumming & Walton, 1973).

         Rats dosed with 14C-aflatoxin B1 and fed BHT (0.5% in the
    diet) showed an enhanced excretion of water-soluble metabolites of
    14C-aflatoxin B1 in the urine and faeces. In addition, BHT
    pretreatment was shown to decrease the amount of 14C bound to
    hepatic nuclear DNA (Fukayama & Hsieh, 1985).

    2.3  Observations in humans

         Double-blind, placebo-controlled challenge tests with a 1:1
    mixture of BHT and BHA (50 mg) were carried out in 44 cases of chronic
    urticaria, 91 cases of atopic dermatitis, and 123 cases of contact
    dermatitis. No positive reactions were seen (Hannuksela & Lahti,

         Support bandages containing BHT were found to induce allergic
    contact dermatitis in two leg ulcer patients. The lesions showed
    improvement when use of the bandages in question was discontinued.
    Both patients gave a positive response to skin patch testing with BHT
    (Dissanayake & Powell, 1989).

         A study was conducted to evaluate the sensitizing risk of BHT
    based on patch testing of 1336 eczema patients and estimates of
    exposure derived from a data base on chemical products. During a
    2-year period in which the patients were tested, BHT produced no
    positive reactions. It was concluded that these results, together with
    the frequent use of BHT in industrial settings and consumer products,
    suggested that concentrations of BHT encountered in normal use would
    not induce allergic contact dermatitis (Flyvholm & Menn, 1990).

         Two patients with chronic idiopathic urticaria were subjected to
    double-blind, placebo-controlled, oral challenges with a series of
    food additives. During testing, BHT and BHA were identified as
    causative agents. Avoidance of foods containing BHT and BHA resulted
    in long-term reduction in severity and frequency of urticarial
    episodes (Goodman  et al., 1990).

         Using platelets from healthy volunteers, the basis for the
    inhibitory effects of BHT on thrombin-induced platelet activation was
    investigated. Concentrations of 20-300 M BHT in incubation mixtures
    of platelets resulted in a dose-dependent activation of protein kinase
    C. The authors indicated that this action desensitizes platelets
    against subsequent phospholipase C activation associated with platelet
    activation by the physiological agonists thrombin and collagen
    (Ruzzene  et al., 1991).

         BHT concentrations of 100 g/ml were cytotoxic to human
    peripheral lymphocytes in culture. Concentrations up to 60 g/ml had
    no effect on tritiated thymidine uptake in phytohaemagglutinin-
    stimulated lymphocytes, although a dose-related synergistic inhibition
    with cortisol or prednisolone was noted. The mixed lymphocyte reaction
    (MLR) was suppressed by concentrations of 50 g/ml BHT. There was a
    linear relationship between the stimulation ratio for lymphocytes from
    each pair of subjects and the extent of inhibition of the MLR by
    50 g/ml BHT. The concentrations of BHT used in lymphocyte cultures in
    this study were not considered by the authors to be relevant to plasma
    concentrations achieved with dietary exposures (Klein & Bruser, 1992).


         The effects of long-term BHT administration have been adequately
    documented in a number of rodent studies, in only one of these
    studies (Olsen  et al., 1986) conducted in the Wistar rat, was a
    hepatocarcinogenic effect evident. This study differed from those
    conducted previously in that the rats were exposed to BHT  in utero,
    during the lactation period, and for a further 40 weeks after the
    standard 2-year exposure period. The dose levels employed were 25, 100
    or 500 mg/kg bw/day. It was necessary to reduce the highest dose from
    500 mg/kg bw/day in the reproduction segment to 250 mg/kg bw/day in the
    long-term feeding portion of the study. A statistically significant
    increase in the survival-adjusted incidence of hepatocellular
    neoplasms was observed in both male and female rats at the highest
    dose tested. The majority of these tumours were not malignant;
    however, the incidence of hepatocarcinomas was significantly higher in
    male rats in the high-dose group than in untreated males. The tumours
    were detected very late in the study, in most cases when the animals
    were killed following 141-144 weeks of treatment. The NOEL was
    25 mg/kg bw/day, based on effects on litter size, sex ratio, and pup
    body-weight gain during the lactation period in the reproduction
    segment of the study.

         The Committee was aware that the above study had been reviewed by
    the International Agency for Research on Cancer (IARC, 1986), and
    concluded that it was difficult to draw conclusions about the observed
    incidence of liver lesions in the treated groups because of the large
    differences in survival between treatment and control groups. The
    carcinogenicity of BHT to humans could not be evaluated.

         The protocol employed in the new study (Price, 1994), the purpose
    of which was to investigate the hepatic changes in male Wistar rats
    that occur following  in utero and lifetime exposure to BHT for up to
    22 months, was almost identical to that of the Olsen  et al., 1986
    study. In the new study, hepatomegaly was observed in the F0 dams
    receiving the highest dose (500 mg/kg bw/day), while toxicologically
    significant liver enlargement was not observed in any F1 dose group
    up to 250 mg/kg bw/day, the highest dose tested. The body weights of
    the pups from the highest dose group were significantly lower than
    those of control pups throughout the lactation period, and mortality
    was increased in the treated pups in a dose-related manner between
    days 6 and 21 of lactation. In the F1 males, BHT administration
    resulted in a persistent, marked induction of cytochrome P-450 2B and
    gamma-glutamyl transpeptidase activity in the centrilobular and
    periportal hepatocytes in the highest dose group throughout the study,
    commencing at a very early stage (21 days of age); gamma-glutamyl
    transpeptidase activity was also increased, but to a lesser extent, at
    the middle dose (cytochrome P-450 2B activity was not determined).
    Total cytochrome P450 content and epoxide hydrolase, ethoxyresorufin

     O-deethylase, and glutathione  S-transferase activities were
    also consistently elevated in a dose-related manner in the mid- and
    highest dose groups. In a separate study, uridine diphosphate
    (UDP)-glucuronosyl transferase activity was induced in the liver of
    male Wistar rats receiving BHT at a dose level of 5 g/kg (equivalent
    to 500 mg/kg bw/day). Consistent enlargement of the centrilobular
    hepatocytes was evident starting at 6 months in rats receiving the
    highest dose of 250 mg/kg bw/day, indicative of proliferation of the
    smooth endoplasmic reticulum consistent with the induction of
    mixed-function oxidases. However, histopathological examination failed
    to reveal any signs of hepatocellular necrosis in this group. In
    addition, no evidence of hepatoxicity as indicated by a decrease in
    glucose 6-phosphatase activity and intracellular glutathione content
    in the treated groups, was seen. There was no indication of sustained
    hepatocellular proliferation during the study. A small, but
    significant, number of altered hepatic foci deficient in glucose-6-
    phosphatase were found in the highest dose group (250 mg/kg bw/day) at
    22 months. In this same group, lesions described as hepatic nodules
    were detected in 6/19 animals as compared with none in the lower-dose
    groups and controls. Evidence of thyroid enlargement with follicular
    hyperplasia, in the absence of elevated serum thyroxine levels, was
    noted in the groups receiving 100 and 250 mg/kg bw/day. There was
    evidence in the mid- and highest dose groups of an early, transient
    effect on the adrenal cortex before sexual maturation of the F1
    males, which took the form of cytomegaly of the cells of the zona

         BHT has been shown to induce hepatocellular necrosis and
    proliferation in male Wistar rats at doses higher than those used in
    either of these long-term studies and which exceeded the maximum
    tolerated dose. Sublethal oral doses of 1000 or 1250 mg/kg bw/day for
    4 days induced hepatocellular necrosis in the centrliobular region
    within 48 hours. When a lower dose of BHT (500 mg/kg bw/day) was
    administered for periods of 1 to 4 weeks, allowing for enzyme
    induction to occur, bile duct proliferation, hepatocellular
    hyperplasia, and persistent fibrous and inflammatory cell reactions
    were observed in the periportal region. The shift in the localization
    of the damage from one area of the liver to another suggested the
    involvement of inducible hepatic drug-metabolizing enzymes in the
    production of reactive metabolites.


         In view of the probable involvement of hepatic enzyme induction
    in the development of the hepatocellular damage associated with
    repeated doses of BHT, the Committee concluded that, in this case,
    enzyme induction was the most sensitive index of effects on the liver.
    A well-defined threshold was demonstrated at 100 mg/kg bw/day in the
    long-term study reviewed for the first time at this meeting, giving a
    NOEL of 25 mg/kg bw/day. Effects observed in the reproduction segments
    of the  in utero/lifetime exposure studies were also taken into
    account in the derivation of this NOEL. The Committee used a safety
    factor of 100 to allocate an ADI of 0-0.3 mg/kg bw for BHT.


    ADAMSON, I.Y.R., BOWDEN, D.H., COTE, M.G. & WITSHI, H. (1977).
    Lung injury induced by butylated hydroxytoluene: cytodynamic and
    biochemical studies in mice.  Lab. Invest., 36: 26-32.

    AKAGI, M. & AOKI, I. (1962a).  Chem. Pharm. Bull. (Tokyo), 10: 101.

    AKAGI, M. & AOKI, I. (1962b). Studies on food additives. VIII
    metabolism of alpha-hydroxy-2,6-di- tert-butyl-p-cresol. Isolation of
    metabolites.  Chem. Pharm. Bull. (Tokyo), 10: 200-204.

    ALLEN, J.R. (1976). Long-term antioxidant exposure effects on female
    primates.  Arch. Environ. Health, 31: 47-50.

    ALLEN, I.R. & ENGBLOM, J.F. (1972). Ultrastructural and biochemical
    changes in the liver of monkeys given butylated hydroxytoluene and
    butylated hydroxyanisole.  Food Cosmet. Toxicol., 10: 769-779.

    AMES, SR., LUDWIG, M.I, SWANSON, W.J. & HARRIS, P.L. (1956).  Proc.
     Soc. Exp. Biol. (N.Y.), 93: 39.

    AOKI, I. (1962).  Chem. Pharm. Bull. (Tokyo), 10: 105.

    ARCHER, D.L., SMITH, B.G. & BUKOVIC-WESS, J.A. (1978). Use of an
     in vitro antibody-producing system for recognizing potentially
    immunosuppressive compounds,  Int. Arch. Allergy Appl. Immunol.,
    56: 90-93.

    BIBRA. (1977). British Industrial Biological Research Association. BHT
    and lung tumours.  BIBRA information Bulletin, 17(2): 88.

    BLUMENTHAL, E.J., & MALKINSON, A.M. (1987). Changes in pulmonary
    calpain activity following treatment of mice with butylated
    hydroxytoluene.  Arch. Biochem. Biophys., 256(1): 19-28.

    BOLTON, J.L. & THOMPSON, J.A. ( 1991). Oxidation of butylated
    hydroxytoluene to toxic metabolites. Factors influencing hydroxylation
    and quinone methide formation by hepatic and pulmonary microsomes.
     Drug Metab. Dispos., 19(2): 467-472.

    BOLTON, J.L., SEVESTRE, H., IBE, B.O. & THOMPSON, J.A. (1990).
    Formation and reactivity of alternative quinone methides from buylated
    hydroxytoluene: Possible explanation for species-specific
    pneumotoxicity.  Chem. Res. Toxicol., 3: 65-70.

    A.M. (1993). Metabolic activation of butylated hydroxytoluene by mouse
    bronchiolar Clara cells.  Toxicol. Appl. Pharmacol., 123: 43-49.

    BOMHARD, E.M., BREMMER, J.N. & HERBOLD, B.A. (1992). Review of the
    mutagenicity/genotoxicity of butylated hydroxytoluene.  Mutat. Res.,
    277: 187-200.

    T.F. (1970). Effects of butylated hydroxytoluene on the enzyme
    activity and ultrastructure of rat hepatocytes.  Food Cosmet.
     Toxicol., 8: 1-8.

    BRANEN, A.L., RICHARDSON, T., GOEL, M.C. & ALLEN, J. R. (1973). Lipid
    and enzyme changes in the blood and liver of monkeys given butylated
    hydroxytoluene and butylated hydroxyanisole.  Food Cosmet. Toxicol.,
    11: 797-806.

    BRIGGS, D., LOK, E., NERA, E.A., KARPINSKI, K. & CLAYSON, D.B. (1989).
    Short-term effects of butylated hydroxytoluene on the Wistar rat
    liver, urinary bladder and thyroid gland.  Cancer Letters, 46: 31-36.

    BROOKS, T.M., HUNT, P.F., THORPE, E. & WALKER, A.I.T. (1976). Effects
    of prolonged exposure of mice to butylated hydroxytoluene. Unpublished
    report from Shell Research, Ltd., Tunstell Lab., Sittingbourne, Kent,
    U.K submitted to the World Health Organization by the authors.

    BROWN, W.D., JOHNSON, A.R. & O'HALLORAN, M.W. (1959).  Aust. J. Exp.
     Biol. Med. Sci., 37: 533.

    BRUNNER, R., VORHEES, C. & BUTCHER, K. (1978). Psychotoxicity of
    selected food additives and related compounds. Report prepared under
    FDA contract 223-75-2030.

    BRUSICK, D. (1975). Mutagenic evaluation of compound FDA 71-25:
    butylated hydroxytoluene (IONOL). Unpublished report from Litton
    Biometrics, Inc., Kensington, Md., U.S. submitted to the World Health
    Organization by the U.S. Food and Drug Administration.

    Identification of tumor promoters by their inhibitory effect on
    intercellular transfer of lucifer yellow.  Cell. Biol. Toxicol.,
    5(1): 77-89.

    (1972). Topanol(R) 354 (BHT and BHA): Comparison of biochemical and
    ultrastructural effects on rat hepatocytes. Unpublished report
    (HO/IH/P49) from ICI Special Centre, Toxicology Bureau, submitted to
    the World Health Organization by the Imperial Chemical Industries
    Ltd., Alderley Park, Cheshire, U.K.

    CARUBELLI, R. & McCAY, P.B. (1987). Dietary butylated hydroxytoluene
    protects cytochrome P-450 in hepatic nuclear membranes of rats fed
    2-acetylaminofluorene.  Nutr. Cancer, 10: 145-148.

    CARUBELLI, R. & McCAY, P.B. (1989). Hepatic nuclear envelope
    cytochrome P-450 in rats fed 2-acetylaminofluorene. Effect of dietary
    fats and butylated hydroxytoluene.  Cancer Lett., 47: 83-89.

    CHIPMAN, J.K, & DAVIES, J.E. (1988). Reduction of 2-acetylamino-
    fluorene-induced unscheduled DNA synthesis in human and rat
    hepatocytes by butylated hydroxytoluene.  Mutat. Res.,
    207(3-4): 193-198.

    CLAPP, N.K. & SATTERFIELD, L.C. (1975). Modification of radiation
    lethality by previous treatment with butylated hydroxytoluene.
     Radiation Res., 64: 388-392.

    CLAPP, N.K., TYNDALL, R.L. & CUMMING, R.B. (1973). Hyperplasia of
    hepatic bile ducts in mice following long-term administration of
    butylated hydroxytoluene.  Food Cosmet. Toxicol., 11: 847.

    CLAPP, N.K., TYNDALL, R.L. CUMMING, R.B. & OTTEN J.A. (1974). Effects
    of butylated hydroxytoluene alone or with diethylnitrosamine in mice.
     Food Cosmet. Toxicol., 12: 367-371.

    CLAPP, N.K., SATTERFIELD, L.C. & KLIMA, W.C. (1975). Modification of
    diethylnitrosamine lung tumorigenesis by concomitant treatment with
    butylated hydroxytoluene. Unpublished report from Oak Ridge National
    Lab., Oak Ridge, Tenn., USA, submitted to the World Health
    Organization by Eastman Chemical Product, Inc., Kingsport, Tenn., USA.

    CLAYSON, D.B., IVERSON, F., NERA, E.A. & LOK, E. (1993). The
    importance of cellular proliferation induced by BHA and BHT.  Toxicol.
     Industr. Health, 9(1-2): 231-242.

    CLEGG, D.J. (1965). Absence of teratogenic effect of butylated
    hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in rats and
    mice.  Food Cosmet. Toxicol., 3: 387-403.

    & WEISBURGER, J.H. (1984). Inhibition of chemically induced
    mammary carcinogenesis in rats by short-term exposure to BHT:
    Interrelationships among BHT concentration, carcinogen dose, and diet.
     J. Natl. Cancer Inst., 72: 165-174.

    J.H. (1986). Inhibition of chemically induced mammary carcinogenesis
    in rats by long-term exposure to butylated hydroxytoluene (BHT):
    interrelations among BHT concentration, carcinogen dose, and diet.
     J. Natl. Cancer Inst., 76(4): 721-730.

    COLLINGS, A.J. & SHARRATT, M. (1970). The BHT content of human adipose
    tissue.  Food Cosmet. Toxicol., 8: 409-412.

    CONACHER, H.B., IVERSON, F., LAU, P.Y., & PAGE, B.D. (1986). Levels
    of BHA and BHT in human and animal adipose tissue: interspecies
    extrapolation.  Food Chem. Toxicol., 24: 1159-1162.

    CONNING, D.M., KIRCH, D.A. & STYLES, J.A. (1969). The toxicity of BHT
    and several oxidation products  in vivo and  in vitro. Unpublished
    report (IHR/260) from the Industrial Hygiene Research Labs., Alderley
    Park, Cheshire, U.K., submitted to the World Health Organization by
    the Imperial Chemical Industries, Ltd.

    COTTRELL, S., ANDREWS, C.M., CLAYTON, D. & POWELL, C.J. (1994). The
    dose-dependent effect of BHT (butylated hydroxytoluene) on vitamin
    K-dependent blood coagulation in rats.  Food Chem. Toxicol.,
    32(7): 589-594.

    CRAMPTON, R.F., GRAY, T.J., GRASSO, P. & PARKE, D.V. (1977). Long-term
    studies on chemically induced liver enlargement in the rat. I.
    Sustained induction of microsomal enzymes with absence of liver damage
    on feeding phenobarbitone or butylated hydroxytoluene.  Toxicology,
    7: 289-306.

    CREAVEN, P.J., DAVIES, W.H. & WILLIAMS, R.T. (1966). The effect of
    butylated hydroxytoluene, butylated hydroxyanisole and octyl gallate
    upon liver weight and biphenyl 4-hydroxylate activity in the rat.
     J. Pharm Pharmacol., 18: 485.

    CUMMING, R.B. & WALTON, M.F. (1973). Modification of the acute
    toxicity of mutagenic and carcinogenic chemicals in the mouse by
    prfeeding with antioxidants.  Food Cosmet. Toxicol., 11: 547-553.

    DACRE, J.C. (1961).  Biochem. J., 78: 758.

    DANIEL, J.W. & GAGE, J.C. (1965). The absorption and excretion of
    butylated hydroxytoluene (BHT) in the rat.  Food Cosmet. Toxicol.,
    3: 405-415.

    DANIEL, J.W., GAGE, J.C., JONES, D.I. & STEVENS, M.A. (1967).
    Excretion of butylated hydroxyoluene (BHT) and butylated
    hydroxyanisole (BHA) by man.  Food Cosmet. Toxicol., 5: 475-479.

    DANIEL, J.W., GAGE, I.C. & JONES, D.I. (1968). The metabolism of
    3,5-di- tert-butyl-4-hydroxytoluene in the rat and in man.
     Biochem. J., 106: 783-790.

    DAUGHERTY, J.P., DAVIS, S. & YIELDING, K.L. (1978). Inhibition
    by butylated hydroxytoluene of excision repair synthesis and
    semiconservative DNA synthesis.  Biochem. Biophys. Res. Commun.,
    80(4): 963-969.

    DAY, A.J., JOHNSON, A.R., O'HALLORAN, M.W & SCHWARTZ, C.J. (1959).
     Aust. J. Exp. Biol. Med. Sci., 37: 295.

    DEERBERG, F., RAPP, K.G., PITTERMANN, W. & REHM, S. (1980). Zum
    tumorspektrum der Han: Wis-Ratte. [Tumour spectrum of the Han:WIST
    rat]  Z. Versuchstierk, 22: 267-280.

     A.M.A. Arch. Industr. Hlth., 11: 93.

    DENZ, F.A. & LLAURADO, I.G. (1957).  Brit. J. Exp. Path., 38: 515.

    DERTINGER, S.D., TOROUS, D.K. & TOMETSKO, A.M. (1993).  In vitro
    system for detecting non-genotoxic carcinogens.  Environ. Molecular
     Mutagen., 21: 332-338.

    DISSANAYAKE, M. & POWELL, S.M. (1989). Allergic contact dermatitis
    from BHT in leg ulcer patients.  Contact Dermatitis, 21: 195.

    FABER, W. (1990). Hemorrhagic effects of butylated hydroxytoluene
    (BHT). Unpublished report. Submitted to WHO by the BHT Special Program
    Panel, Chemical Manufacturers Association, Washington, D.C., USA.

    FEUER, G., GAUNT, I.F., GOLBERG, L. & FAIRWEATHER, F.A. (1965). Liver
    response tests. VI Application to comparative study of food
    antioxidants and hepatotoxic agents.  Food Cosmet. Toxicol.,
    3: 457-469.

    FLYVHOLM, M.-A. & MENN, T. (1990). Sensitizing risk of butylated
    hydroxytoluene based on exposure and effect data.  Contact Dermatitis,
    23: 341-345.

    FRAWLEY, J.P., KAY, J.H. & CALANDRA, J.C. (1965a). The residue of
    butylated hydroxytoluene (BHT) and metabolites in tissue and eggs of
    chickens fed diets containing radioactive BHT.  Food Cosmet. Toxicol.,
    3: 471-474.

    FRAWLEY, J.P., KOHN. F.E., KAY, J.H. & CALANDRA, J.C. (1965b).
    Progress report n multigeneration reproduction studies in rats fed
    butylated hydroxytoluene (BHT).  Food Cosmet. Toxicol., 3: 377-386.

    CARUBELLI, R. (1989). Induction of rat liver microsomal and nuclear
    cytochrome P-450 by dietary 2-acetylaminofluorene and butylated
    hydroxytoluene.  Biochem. Pharmacol., 38(18): 3075-3081.

    FUKAYAMA, M.Y. & HSIEH, D.P. (1985). Effect of butylated
    hydroxytoluene pretreatment on the excretion, tissue distribution and
    DNA binding of [14C]aflatoxin B1 in the rat.  Food Chem. Toxicol.,
    23: 567-573.

    FUKUSHIMA, S., OGISO, T., KURATA, Y., HIROSE, M. & ITO, N. (1987a).
    Dose-dependent effects of butylated hydroxyanisole, butylated
    hydroxytoluene and ethoxyquin for promotion of bladder carcinogenesis
    in N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated, unilaterally
    ureter-ligated rats.  Cancer Lett., 34: 83-90.

    ITO, N. (1987b). Different modifying response of butylated
    hydroxyanisole, butylated hydroxytoluene, and other antioxidants in
    N,N-dibutylnitrosamine esophagus and forestomach carcinogenesis of
    rats.  Cancer Res., 4., 47:2113-2116.

    Induction by butylated hydroxytoluene of rat liver y-glutamyl
    transpeptidase activity in comparison to expression in
    carcinogen-induced altered lesions.  Chem Biol. Interact.,
    48: 43-58.

    GABRYELAK, T., PUMO, D.E. & CHIU, J.F. (1981). Changes in
    tumor-specific nuclear antigen activity in carcinogen-treated colon
    by tumor promotor and carcinogen inhibitors,  Cancer Res.,
    41: 3392-3394.

    GAGE, J.C. (1964). The tissue retention of 3,5-Di-t-butyl-4-hydroxy-
    toluene (butylated hydroxytoluene, BHT). Unpublished report
    (No. IHR/157) from the Industrial Hygiene Research Labs., Alderley
    Park, Cheshire, U.K., submitted to the World Health Organization by
    the Imperial Chemical Industries, Ltd., U.K.

    GAUNT, I.F., FEUER, G., FAIRWEATHER, F.A. & GILBERT, D. (1965a). Liver
    response tests. IV. Application to short-term feeding studies with
    butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).
     Food Cosmet. Toxicol., 3: 433-443.

    GAUNT, I.F., GILBERT, D. & MARTIN, D. (1965b). Liver response tests.
    V. Effect of dietary restriction on a short-term feeding study with
    butylated hydroxytoluene (BHT).  Food Cosmet. Toxicol., 3: 445-456.

    GEYER, H., SCHEUNERT, I. & KORTE, F. (1986). Bioconcentration
    potential of organic environmental chemicals in humans.  Regul.
     Toxicol. Pharmacol., 6(4): 313-347.

    GILBERT, D. & GOLBERG, L. (1965).  Food Cosmet. Toxicol., 3: 417.

    GILBERT, D. & GOLBERG, L. (1967). BHT oxidase. A liver-microsomal
    enzyme induced by the treatment of rats with butylated hydroxytoluene.
     Food Cosmet. Toxicol., 5: 481-490.

    GOATER, T.O., KENYON, A.J. & & HURST, E.W. (1964). The subacute
    toxicity of Topanol(R) BHT. Unpublished report from the Industrial
    Hygiene Research Labs., Alderley Park, Cheshire, U.K., submitted to
    the World Health Organization by the Imperial Chemical Industries
    Ltd., U.K.

    WEBER, R.W. (1990). Chronic urticaria exacerbated by the antioxidant
    food preservatives, butylated hydroxyanisoie (BHA) and butylated
    hydroxytoluene (BHT).  J. Allergy Clin. Immunol., 86: 570-575.

    GRAY, T.J., PARKE, D.V., GRASSO, P.. CRAMPTON, RF (1972). Biochemical
    and pathological differences in hepatic response to chronic feeding of
    safrole and butylated hydroxytoluene to rats.  Biochem. J.,
    130(2): 91P.

    PRAT, V. & KIRSCH-VOLDERS, M. (1988). The influence of phenobarbital
    and butylated hydroxytoluene on the ploidy rate in rat hepato-
    carcinogenesis.  Carcinogenesis, 9(10): 1755-1761.

    HAGEMAN, G.J., VERHAGEN, H. & KLEINJANS, J.C. (1988). Butylated
    hydroxyanisole, butylated hydroxytoluene and  tert-butylhydroquinone
    are not mutagenic in the  Salmonella/microsome assay using new tester
    strains.  Mutat. Res., 208:207-211.

    Modulation of N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat
    urinary bladder carcinogenesis by post-treatment with combinations of
    three phenolic antioxidants.  J. Toxicol. Pathol., 2: 33-39.

    HAMMOCK, B.D. & OTA, K. (1983). Differential induction of cytosolic
    epoxide hydrolase, microsomal epoxide hydrolase and glutathione
    S-transferase activities.  Tox. Appl. Pharmacol., 71: 254-265.

    HANNUKSELA, M. & LAHTI, A. (1986). Peroral challenge tests with food
    additives in urticaria and atopic dermatitis,  Int. J. Dermatol.,
    25(3): 178-180.

    HARMAN, D. (1968). Free radical theory of aging: effect of free
    radical reaction inhibitors on the mortality rate of male LAF mice.
     J. Gerontol., 23: 476-482.

    HERMANN, R.S, KORANSKY, W., LEBERL, C. & NOACK, G. (1971). Hyperplasia
    and hypertrophy of rat liver induced by hexachloro-cyclohexane and
    butylhydroxytoluene. Retention of the hyperplasia during involution of
    the enlarged organ.  Virchows Arch. B. Zellpath., 9: 125-134.

    HIRAGA, K. (1978). Life-span oral toxicity study of 3,5-di- tert-
    hydroxytoluene (BHT) in rats.  Ann. Rep. Tokyo Metropolitan Research
     Lab. Public Health, 32: 83.

    HIRAI, K., YAMAUCHI, M., WITSCHI, H, & COTE, M.G. (1983).
    Disintegration of lung peroxisomes during differentiation of type II
    cells to type I cells in butylated hydroxytoluene-administered mice.
     Exper. Mol. Pathal., 39: 129-138.

    HIROSE, M., SHIBATA, M., HAGIWARA, A., IMAIDA, K & ITO, N. (1981).
    Chronic toxicity of butylated hydroxytoluene in Wistar rats.
     Food Cosmet. Toxicol., 19: 147-151.

    (1986). Histologic and autoradiographic studies on the forestomach of
    hamsters treated with 2- tert-butylated hydroxyanisole, 3- tert-
    butylated hydroxyanisole, crude butylated hydroxyanisole, or butylated
    hydroxytoluene.  J. Natl. Cancer Inst., 76(1): 143-147.

    (1987). Induction of forestomach lesions in rats by oral
    administrations of naturally occurring antioxidants for 4 weeks.
     Jpn. J. Cancer. Res., 78(4): 317-321.

    HIROSE, M., YADA, H., HAKOI, K. TAKAHASHI, S. & ITO, N. (1993).
    Modification of carcinogenesis by alpha-tocopherol, t-butylhydro-
    quinone, propyl gallate and butylated hydroxytoluene in a rat
    multi-organ carcinogenesis model.  Carcinogenesis, 14(11): 2359-2364.

    HOLDER, G.M., RYAN, A.J., WATSON, T.R. & WIEBE, L.I. (1970a). The
    biliary metabolism of butylated hydroxytoluene and its derivatives in
    the rat.  J. Pharm. Pharmacol., 22: 832-838.

    HOLDER, G.M., RYAN, A.J., WATSON, T.R. & WIEBE, L.I. (1970b). The
    metabolism of butylated hydroxytoluene in man.  J. Pharm. Pharmacol.,
    22: 375-376.

    occuring and synthetic food components, furocoumarins and ultraviolet
    radiation. Lyon, (IARC Monographs on the Evaluation of Carcinogenic
    Risks to Humans,  Vol 40, 161-206.

    IATROPOULOS, M.J., WILLIAMS, G.M., CONAWAY. C.C. (1994). Inhibition of
    the action of the food-borne carcinogen aflatoxin B1 by butylated
    hydroxyanisole and butylated hydroxytoluene in male rats. Unpublished
    report to ILSI Research Foundation, 77 pp.

    ITO, N. (1983). Promoting activities of BHA and BHT on 2-stage urinary
    bladder carcinogenesis and inhibition of gamma-glutamyl transpeptidase-
    positive foci development in the liver of rats.  Carcinogenesis,
    4: 895-899.

    (1984). Promoting activities of BHA, BHT and sodium-L-ascorbate on
    forestomach and urinary bladder carcinogenesis initiated with
    methylnitrosourea in F-344 male rats.  Gann, 75: 769-775.

    (1988). Modifying effects of concomitant treatment with butylated
    hydroxyanisole or butylated hydroxytoluene on N,N-dibutylnitrosamine-
    induced liver, forestomach and urinary bladder carcinogenesis in F344
    male rats.  Cancer Lett., 43: 167-172.

    Hepatocellular tumorigenicity of butylated hydroxytoluene administered
    orally to B6C3F1 mice.  Jpn. J. Cancer Res., 79(1): 49-58.

    JOHNSON, A.R. & HEWGILL, F.R. (1961).  Aust. J. Exp. Biol. Med. Sci.,
    39: 353.

    JOHNSON, A.R. & HOLDSWORTH, E.S (1968).  J. Nutr. & Diet., 5: 147.

    JOHNSON, A.R, (1965). A re-examination of the possible teratogenic
    effects of butylated hydroxytoluene (BHT) and its effect on the
    reproductive capacity of the mouse.  Food Cosmet. Toxicol.,
    3: 371-375.

    JOSEPHY, P.D., CARTER, M.H., & GOLDBERG, M.T. (1985). Inhibition of
    benzidine mutagenesis by nucleophiles: a study using the Ames test
    with hamster hepatic S9 activation.  Mut. Res., 143: 5-10.

    A.J. (1994). Regulation of uridine diphosphate glucuronosyltransferase
    expression by phenolic antioxidants.  Cancer Res., 54: 5856-5859.

    KARPLYUK, I.A. (1959).  Vop. Pitan., 18: 24.

    KAWANO, S., NAKAO, T & HIRAGA, K. (1981). Strain differences in
    butylated hydroxytoluene-induced deaths in male mice.  Tox. Appl.
     Pharmacol., 61: 475-479.

    KEHRER, J.P. & DIGIOVANNI, J. (1990). Comparison of lung injury
    induced in 4 strains of mice by butylated hydroxytoluene.  Taxicol.
     Lett., 52: 55-61.

    KENNEDY, G., FANCHET, O.E. & CALANDRA, J.C. (1966). Three-generation
    reproduction study in albino rats. Butylated Hydroxytoluene. Final
    Report. Unpublished report from Bio-Test Labs., Inc., Northbrook,
    Ill., USA, submitted to the World Health Organization by Hercules,
    Inc., Wilmington, Delaware, USA.

    KING, M.M., MCCAY, P.B. & KOSANKE, S.D. (1981). Comparison of the
    effects of butylated hydroxytoluene on N-nitrosomethylurea and
    7,12-dimethylbenz[a]-anthracene-induced mammary tumours,  Cancer
     Letters, 14: 219-226.

    KITCHIN, K.T. & BROWN, J.L. (1987). Biochemical effects of two
    promoters of hepatocarcinogenesis in rats  Food Chem. Toxic.,
    25(8): 603-607.

    KLEIN, A. & BRUSER, B. (1992). The effect of butylated hydroxytoluene
    with and without cortisol, on stimulated lymphocytes.  Life Sciences,
    50: 883-889.

    KRASAVAGE, W.J. (1984). The lack of effect of tertiary butylhydro-
    quinone on prothrombin time in male rats.  Drug Chem Toxicol.,
    7: 329-334.

    LADOMERY, L.G., RYAN, A.J. & WRIGHT, S.E. (1963).  J. Pharm.
     Pharmacol., 15: 771

    LADOMERY, L.G., RYAN, A.J. & WRIGHT, S.E. (1967a). The excretion of
    [14C] butylated hydroxytoluene in the rat.  J. Pharm. Pharmacol.,
    19: 383-387.

    LADOMERY, L.G., RYAN, A.J. & WRIGHT, S.E. (1967b). The biliary
    metabolite of butylated hydroxytoluene in the rat.  J. Pharm.
     Pharmacol., 19: 388-394.

    LAKE, B.G., LONGLAND, R.C., GANGOLLI, S.D. & LLOYD, A.G. (1976). The
    influence of some foreign compounds on hepatic xenobiotic metabolism
    and the urinary excretion of D-glucuronic acid metabolites in the rat.
     Toxicol. Appl. Pharmacol., 35:113-122.

    LARSEN, J.C. & TARDING, F. (1978). Stimulation of DNA synthesis in
    mouse and rat lung following administration of butylated
    hydroxytoluene.  Archives of Toxicol. Suppl., 1: 147-150.

    The effects of dietary BHT on liver and colon tumor development in
    mice.  Toxicology, 38:151-160.

    LINDENSCHMIDT, R.C., TRYKA, A.F. & WITSCHI, H. (1987). Modification of
    gastrointestinal tumor development in rats by dietary butylated
    hydroxytoluene.  Fundam. Appl. Toxicol., 8(4): 474-481.

    MAEURA, Y. & WILLIAMS, G.M. (1984). Enhancing effect of butylated
    hydroxytoluene on the development of liver altered foci and neoplasms
    induced by N-2-fluorenylacetamide in rats.  Food Chem. Toxicol.,
    22: 191-198.

    MAEURA, Y., WEISBURGER, J.H. & WILLIAMS, G.M. (1984). Dose-dependent
    reduction of N-2-fluorenylacetamide-induced liver cancer and
    enhancement of bladder cancer in rats by butylated hydroxytoluene.
     Cancer Res., 44: 1604-1610.

    MALKINSON, A.M. (1979). Prevention of butylated hydroxytoluene-induced
    lung damage in mice by cedar terpene administration. Pre-print of
    paper accepted for publication in  Toxicol. Appl. Pharmacol.,
    49: 551-560.

    MALKINSON, A.M. (1991). Genetic studies on lung tumor susceptibility
    and histogenesis in mice.  Environ. Health Perspect., 93: 149-159.

    MALKINSON, A.M. & THAETE, L.G. (1986). Effects of strain and age on
    prophylaxis and co-carcinogenesis of urethane-induced mouse lung
    adenomas by butylated hydroxytoluene.  Cancer. Res., 46: 1694-1697.

    (1989). Evidence for a role of  tert-butyl hydroxylation in the
    induction of pneumotoxicity in mice by butylated hydroxytoluene.
     Toxicol. Appl. Pharmacol., 101: 196-204.

    MATSUO, M., MIHARA, K., OKUNO, M., OHKAWA, B., & MIYAMOTO, J. (1984).
    Comparative metabolism of 3,5-di- tert-butyl-4-hydroxytoluene (BHT)
    in mice and rats.  Food Chem. Toxicol., 22: 345-354.

    McCARTHY, D.J., LINDAMOOD, C., GUNDBERG, C.M. & HILL, D.L. (1989).
    Retinoid-induced hemorrhaging and bone toxicity in rats fed diets
    deficient in vitamin K.  Toxicol. Appl. Pharmacol., 97: 300-310.

    McCORMICK, D.L., MAJOR, N., & MOON, R.C. (1984). Inhibition of
    7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by
    concomitant or postcarcinogen antioxidant exposure.  Cancer Res.,
    44: 2858-2863.

    McCORMICK, D.L., MAY, C.M., THOMAS, C.F. & DETRISAC, C.J. (1986).
    Anticarcinogenic and hepatotoxic interactions between retinyl acetate
    and butylated hydroxytoluene in rats.  Cancer Res., 46: 5264-5269.

    MEYER, O. & HANSEN, E. (1980). Behavioural and developmental effects
    of butylated hydroxytoluene dosed to rats  in utero and in the
    lactation period.  Toxicology, 16: 247-258.

    MEYER, O.A., KRISTIANSEN, E. & WURTZEN, G. (1989). Effects of dietary
    protein and butylated hydroxytoluene on the kidneys of rats.
     Lab. Anim., 23: 175-179.

    MIZUTANI, T., ISHIDA, I., YAMANOTOK, K. & TAJIMA, K. (1982). Pulmonary
    toxicity of butylated hydroxytoluene and related alkylphenols:
    Structural requirements for toxic potency in mice.  Toxicol. Appl.
     Pharmacol., 62: 273-281.

    MIZUTANI, T., YAMAMOTO, K. & TAJIMA, K. (1983). Isotope effects on the
    metabolism and pulmonary toxicity of butylated hydroxytoluene in mice
    by deuteration of the 4-methyl group.  Toxicol. Appl. Pharmacol.,
    69: 283-290.

    Hepatotoxicity of butylated hydroxytoluene and its analogs in mice
    depleted of hepatic glutathione.  Toxicol. Appl. Pharmacol.,
    87: 166-176.

    NAGAI, F., USHIYAMA, K. & KANO, I. (1993). DNA cleavage by metabolites
    of butylated hydroxytoluene.  Arch. Toxicol., 67: 552-557.

    NAKAGAWA, Y. (1987). Effects of buthionine sulfoximine and cysteine on
    the hepatotoxicity of butylated hydroxytoluene in rats.  Toxicol.
     Lett., 37: 251-256.

    NAKAGAWA, Y. & TAYAMA, K. (1988). Nephrotoxicity of butylated
    hydroxytoluene in phenobarbital-pretreated male rats.  Arch. Toxicol.,
    61: 359-365.

    NCI (1979) National Cancer Institute. Bioassay of butylated
    hydroxytoluene (BHT) for possible carcinogenicity.  DHEW Report
    No. NIH 79-1706. Technical Report Series No. 150.

    NIEVEL, J.G. (1969). Effect of coumarin, BHT and phenobarbitone on
    protein synthesis in the rat liver.  Food Cosmet. Toxicol.,
    7: 621-634.

    (1986). Protection by methylprednisolone against butylated
    hydroxytoluene-induced pulmonary damage and impairment of microsomal
    monooxygenase activities in the mouse: lack of effect on fibrosis.
     Exp. Lung Res., 10: 1-22.

    OLSEN, P., GRY, J., KNUDSEN, L., MEYER, O, & POULSEN, E. (1984).
    Animal feeding study with nitrite-treated meat. In: N-nitroso
    compounds; Occurrence, biological effects, and relevance to human
    cancer. O'Neill, I.K., von Borstel, R.C., Miller, C.T., Long, J., &
    Bartsch, B. (eds.),  IARC Scient. Publ. No.57: pp. 667-675.
    International Agency for Research on Cancer, Lyon.

    OLSEN, P., MEYER, O., BILLE, N. & WURTZEN, G. (1986). Carcinogenicity
    study on butylated hydroxytoluene (BHT) in Wistar rats exposed
     in utero. Food Chem. Toxicol., 24: 112.

    OMAYE, S.T., REDDY, K.A. & CROSS, C.E. (1977). Effect of butylated
    hydroxytoluene and other antioxidants on mouse lung metabolism.
     J. Toxicol. Environ. Health, 3: 829-836.

    PARTRIDGE, C.A., DAO, D.D. & AWASTHI, Y.C. (1982). Induction of
    glutathione-linked detoxification system by dietary antioxidants,
     Fed. Proc., 41: Abstract 2152.

    PERAINO, C., FRY, R.J., & STAFFELDT, E. (1973). Enhancement of
    spontaneous hepatic tumorigenesis in C3H mice by dietary
    phenolbarbital.  J. Natl. Cancer Inst., 51: 1349-1350.

    Enhancing effects of phenobarbitone and butylated hydroxytoluene on
    2-acetylaminofluorene-induced hepatic tumorigenesis in the rat.
     Food and Cosmetics Toxicol., 15: 93-96.

    PETO, R., PIKE, M.C., DAY, N.E., GRAY, R.G., LEE, P.N., PARISH, S.,
    PETO, J., RICHARDS, S., & WAHRENDORF, J. (1980). Guidelines for
    simple, sensitive significance tests for carcinogenic effects in
    long-term animal experiments. In: Long-term and Short-term Screening
    Assays for Carcinogens: A Critical Appraisal. IARC Monographs on the
    Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 2,
    pp. 311-426. International Agency for Research on Cancer, Lyon.

    POWELL, C.J. & CONNOLLY, A.K. (1991). The site specificity and
    sensitivity of the rat liver to butylated hydroxytoluene-induced
    damage.  Toxicol. Appl. Pharmacol., 108: 67-77.

    (1986). Hepatic responses to the administration of high doses of BHT
    to the rat: their relevance to hepatocarcinogenicity.  Food. Chem.
     Toxicol., 24: 1131-1143

    PRASAD, O. & KAMRA O.P. (1974). Radiosensitization of  Drosophila
    sperm by commonly used food additives-butylated hydroxyanisole and
    butylated hydroxytoluene,  Int. J. Radiat. Biol., 25: 67-72.

    (1986). Comparative analysis of the effect of phenobarbital,
    dichlorodiphenyltrichloroethane, butylated hydroxytoluene and
    nafenopin on rat hepatocarcinogenesis.  Carcinogenesis,
    7(6): 1025-1028.

    PRICE, S.C. (1994). The role of hepatocellular injury in the chronic
    toxicity of BHT: Two generation Wistar albino rat study. Robens
    Institute, U. of Surrey, Guildford, Surrey, U.K. Study No: 1/91/Tx.
    Final Report No:  R193/TOX/0020. Vol. 1-8. Submitted to WHO by Robens

    REDDY, B.S., HANSEN, D., MATHEWS, L., & SHARMA, C. (1983a). Effect of
    micronutrients, antioxidants and related compounds on the mutagenicity
    of 3,2'-dimethyl-4-aminobiphenyl, a colon and breast carcinogen
     Food Chem. Toxicol., 21: 129-132.

    REDDY, B.S., SHARMA, C., & MATHEWS, L. (1983b). Effect of butylated
    hydroxytoluene and butylated hydroxyanisole on the mutagenicity of
    3,2'-dimethyl-4-aminobiphenyl.  Nutr. Cancer, 5: 153-158.

    RICHER, N., MARION, M. & DENIZEAU, F. (1989) Inhibition of binding of
    2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated
    hydroxyanisole in vitro.  Cancer Lett., 47: 211-216.

    RIKANS, L.E., GIBSON, D.D., McCAY, P.B., KING, M.M. (1981). Effects of
    butylated hydroxytoluene and acetylaminofluorene on NADPH-cytochrome
    P-450 reductase activity in rat liver microsome,  Food Cosmet.
     Toxicol., 19: 89-92.

    ROBENS INSTITUTE (1989). Dose ranging experiment on the role of
    hepatocellular injury in the chronic toxicity of BHT. Final report
    7/88/TX, Robens Institute of Health and Safety, University of Surrey,
    Guildford, Surrey, United Kingdom. Unpublished report. Submitted to
    WHO by European BHT Manufacturers Association (EBMA). CEFIC,
    Bruxelles, Belgium.

    ROEBUCK, B.D., MACMILLAN, D.L., BUSH, D.M. & KENSLER, T.W. (1984).
    Modulation of azaserine-induced pancreatic foci by phenolic
    antioxidants in rats.  J. Natl. Cancer Inst., 72: 1405-1409.

    DEANA, R. (1991). The antioxidant butylated hydroxytoluene stimulates
    platelet protein kinase C and inhibits subsequent protein
    phosphorylation induced by thrombin.  Biochim. Biophys. Acta,
    1094: 121-129.

    SAHEB, W. & WITSCHI, H. (1975). Lung growth in mice after a single
    dose of butylated hydroxytoluene.  Toxicol. & Appl. Pharm.,
    33: 309-319.

    TOYODA, K., HAYASHI, Y. (1987). Initiating potential of 2-(2-furyl)-
    3-(5-nitro-2- furyl)acrylamide (AF-2), butylated hydroxyanisole (BHA),
    butylated hydroxytoluene (BHT) and 3,3',4',5,7-pentahydroxyflavone
    (quercetin) in two-stage mouse skin carcinogenesis.  Cancer Lett.,
    38(1-2): 49-56.

    SHELEF, L.A. & CHIN, B. (1980) Effect of phenolic antioxidants on the
    mutagenicity of aflatoxins B1.  Appl. Environ. Microbiol.,
    40: 1039-1043.

    SHELLENBERGER, T.E., PARRISH, D.B. & SANFORD, P.E. (1957).  Poultry
     Sci., 36: 1313.

    W.M. (1986). Tests for mutagenic effects of ammoniated glycyrrhizin,
    butylated hydroxytoluene, and gum Arabic in rodent germ cells.
     Environ. Mutagen., 8(3): 357-367.

    (1979). Experimental study on carcinogenicity of butylated
    hydroxytoluene (BHT) in rats. Translation of the Proceedings of the
    38th Annual Meeting of the Japanese Cancer Assoc., Tokyo, September

    (1989). Changes in urine composition, bladder epithelial morphology,
    and DNA synthesis in male F344 rats in response to ingestion of
    bladder tumour promoters.  Toxicol. Appl. Pharmacol., 99: 37-49.

    (1991). DNA synthesis and scanning electron microscopic lesions in
    renal pelvic epithelium of rats treated with bladder cancer promoters.
     Toxicol. Lett., 55: 263-272.

    ITO, N. (1982). Lack of carcinogenicity of butylated hydroxytoluene on
    long-term administration to B6C3FI mice.  Food. Chem. Tox.,
    20: 861-865.

    SHIRAI, T., FUKUSHIMA, S., OHSHIMA, M., MASUDA, A., & ITO, N. (1984).
    Effects of BHA, BHT and NaCl on gastric carcinogenesis initiated with
    N-methyl-N'-nitro-N-nitrosoguanidine in F-344 rats.  J. Natl. Cancer
     Inst., 72: 1189-1198.

    SHIRAI, T., IKAWA, E., HIROSE. M., THAMAVIT, W., & ITO, N. (1985).
    Modification by five antioxidants of 1,2-dimethylhydrazine-initiated
    colon carcinogenesis in F-344 rats.  Carcinogenesis, 6: 637-639.

    TADA, M. & ITO, N. (1991). Selective induction of rat urinary bladder
    tumors by simultaneous administration of 3,2'-dimethyl-4-aminobiphenyl
    (DMAB) and butylated hydroxyanisole or butylated hydroxytoluene is
    associated with increased DMAB-DNA adduct formation.  Carcinogenesis,
    12(7): 1335-1339.

    SINGLETARY, K.W. & NELSHOPPEN, J.M. (1991). Selective  in vivo
    inhibition of rat mammary 7,12-dimethylbenz[alpha]anthracene - DNA
    adduct formation by dietary butylated hydroxytoluene.  Carcinogenesis,
    12(10): 1967-1969.

    (1992). Inhibition by butylated hydroxytoluene and its oxidative
    metabolites of DMBA-induced mammary tumorigenesis and of mammary
    DMBA-DNA adduct formation  in vivo in the female rat.  Food Chem.
     Toxicol., 30(6): 455-465.

    SOLLEVELD, H.A., HASEMAN, J.K., & MCCONNELL, E.E. (1984). Natural
    history of body weight gain, survival and neoplasia in the F344 rat.
     J. Natl. Cancer Inst., 72: 929-940.

    SONDERGAARD, D. & OLSEN, P (1982). The effect of butylated
    hydroxytoluene (BHT) on the rat thyroid.  Toxicology Letters,
    10: 239-244.

    SPORN, A. & SCHOBESCH, O. (1961).  Igiena (Bucharest), 9: 113.

    S.R.I. Stanford Research Institute (1972). Report 14 submitted to U.S.
    Food and Drug Administration.

    STOKES, J.D. & SCUDDER, C.L. (1974). The effect of butylated
    hydroxyanisole and butylated hydroxytoluene on behavioral development
    of mice.  Devel. Psychobiol., 7: 343-350.

    SUZUKI, H., NAKAO, T. & HIRAGA, K. (1983). Vitamin K content of liver
    and faeces from vitamin K-deficient and butylated hydroxytoluene
    (BHT)-treated male rats.  Toxicol. Appl. Pharmacol., 67: 152-155.

    TAFFE, B.G. & KENSLER, T.W. (1988). Tumor promotion by a hydroperoxide
    metabolite of butylated hydroxytoluene, 2,6-di- tert-butyl-4-
    hydroperoxy-4-methyl-2,5-cyclohexadienone, in mouse skin.  Res. Commun.
     Chem. Pathol. Pharmacol., 61(3): 291-303.

    TAFFE, B.G., ZWEIER, J.L., PANNELL, L.K. & KENSLER, T.W. (1989).
    Generation of reactive intermediates from the tumor promoter butylated
    hydroxytoluene hydroperoxide in isolated murine keratinocytes or by
    hematin.  Carcinogenesis, 10(7): 1261-1268.

    TAKAHASHI, O. (1986). Feeding of butylated hydroxytoluene to rats
    caused a rapid decrease in blood coagulation factors II (prothrombin),
    VII, IX and X.  Arch. Toxicol., 58(3): 177-181.

    TAKAHASHI, O. (1987). Decrease in blood coagulation factors II
    (prothrombin), VII, IX and X in the rat after a single oral dose of
    butylated hydroxytoluene.  Fd. Chem. Toxicol., 25(3): 219-224.

    TAKAHASHI, O.(1990). Gastric retention and delayed absorption of a
    large dose of butylated hydroxytoluene in the rat.  Xenobiotica,
    20(12): 1319-1329.

    TAKAHASHI, O. (1991). Some properties of rat platelet aggregation and
    effects of butylated hydroxytoluene, warfarin and aspirin.  Food Chem.
     Toxicol., 29(3): 173-183.

    TAKAHASHI, O. (1992). Haemorrhages due to defective blood coagulation
    do not occur in mice and guinea-pigs fed butylated hydroxytoluene, but
    nephrotoxicity is found in mice.  Food Chem. Toxicol., 30(2): 89-97.

    TAKAHASHI, O. & HIRAGA, K. (1978a). Dose-response study of hemorrhagic
    death by dietary butylated hydroxytoluene (BHT) m male rats.  Tox.
     Appl. Pharm., 43: 399-406.

    TAKAHASHI, O. & HIRAGA, K. (1978b). Effects of low levels of butylated
    hydroxytoluene on the prothrombin index of male rats.  Food Cosmet.
     Toxicol., 24: 16, 475-477.

    TAKAHASHI, O. & HIRAGA, K. (1979). Preventive effects of phylloquinone
    on hemorrhagic death induced by butylated hydroxytoluene in male rats.
     J. Nutr., 109: 453-457.

    TAKAHASHI, O. & HIRAGA, K (1981a). Inhibition of phylloquinone
    expoxide-dependent carboxylation of microsomal proteins from rat liver
    by 2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone.  Food Cosmet.
     Toxicol., 19: 701-706.

    TAKAHASHI, O. & HIRAGA, K. (1981b). Haemorrhagic toxicosis in rats
    given butylated hydroxytoluene,  Acta Pharmacol. Toxicol., 49: 14-20.

    TAKAHASHI, O. & HIRAGA, K. (1984). Effects of dietary butylated
    hydroxytoluene on functional and biochemical properties of platelets
    and plasma preceeding the occurrence of haemorrhage in rats.
     Food Chem. Toxicol., 22: 97-103.

    TAKAHASHI, O., HAYASHIDA, S., & HIRAGA, K. (1980). Species differences
    in the haemorrhagic response to butylated hydroxytoluene.
     Food Cosmet. Toxicol., 18: 229-235.

    HAYASHI, Y. (1986). Effects of four antioxidants on N-methyl-N'-nitro-
    N-nitrosoguanidine initiated gastric tumour development in rats.
     Cancer Lett., 30(2): 161-168.

    TANAKA, T., OISHI, S. & TAKAHASHI, O. (1993). Three generation
    toxicity study of butylated hydroxytoluene administered to mice.
     Toxicol. Lett., 66: 295-304.

    TATSUTA, M., MIKUNI, T., & TANIGUCHI, H. (1983). Protective effect of
    butylated hydroxytoluene against induction of gastric cancer by
    N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.  Int. J. Cancer,
    32, 253-254.

    THOMPSON, D.C. & TRUSH, M.A. (1988a). Enhancement of butylated
    hydroxytoluene-induced mouse lung damage by butylated hydroxyanisole.
     Toxicol. Appl. Pharmacol., 96(1): 115-121.

    THOMPSON, D.C. & TRUSH, M.A, (1988b). Studies on the mechanism of
    enhancement of butylated hydroxytoluene-induced mouse lung toxicity by
    butylated hydroxyanisole.  Toxicol. Appl. Pharmacol., 96(1): 122-131.

    THOMPSON, D.C., CHA, Y.N. & TRUSH, M.A (1986). The peroxidative
    activation of butylated hydroxytoluene to BHT-quinone methide and
    stilbenequinone.  Adv. Exp. Med. Biol., 197: 301-309.

    MEAD, E.W., SCHULLEK, K.M. & LAUDENSCHLAGER, W.G. (1987). Oxidative
    metabolism of butylated hydroxytoluene by hepatic and pulmonary
    microsomes from rats and mice.  Drug Metab. Dispos., 5: 833-840.

    (1989). A metabolite of butylated hydroxytoluene with potent
    tumor-promoting activity in mouse lung.  Carcinogenesis,
    10(4): 773-775.

    THORNTON, M., MOORE, M.A. & ITO, N. (1989). Modifying influence of
    dehydroepiandrosterone or butylated hydroxytoluene treatment on
    initiation and development stages of azaserine-induced acinar
    pancreatic preneoplastic lesions in the rat.  Carcinogenesis,
    10(2): 407-410.

    TOKUMO, K., IATROPOULOS, M.J. & WILLIAMS, G.M. (1991). Butylated
    hydroxytoluene lacks the activity of phenobarbital in enhancing
    diethylnitrosamine-induced mouse liver carcinogenesis.  Cancer Lett.,
    59: 193-199.

    TYE, R., ENGEL, J.D. & RAPIEN, I. (1965). Summary of toxicological
    data. Disposition of butylated hydroxytoluene (BHT) in the rat.
     Food Cosmet Toxicol., 3: 547-551.

    TYNDALL, R.L., COLYER, S. & CLAPP, N. (1975). Early alterations in
    plasma esterases with associated pathology following oral
    administration of diethylnitrosamine and butylated hydroxytoluene
    singly or in combination,  Int. J. Cancer, 16: 184-191.

    (1973). Antioxidants and carcinogenesis: butylated hydroxytoluene, but
    not diphenyl-p-phenyl-enediamine, inhibits cancer induction by
    N-2-fluorenylacetamide and by N-hydroxy-N-2-fluorenyl acctamide in
    rats  Food Cosmet. Toxicol., 11: 199-207.

    VAN STRATUM, P.G. & VOS, H.J. (1965). The transfer of dietary
    butylated hydroxytoluene (BHT) into the body and egg fat of laying
    hens.  Food Cosmet. Toxicol., 3: 475-477.

    F.TEN, HENDERSON, P.T. & KLEINJANS, J.C.S. (1989). Disposition of
    single oral doses of butylated hydroxytoluenc in man and in rat.
     Fd. Chem. Toxicol., 27(12): 765-772.

    VERSCHOYLE, R.D., WOLF, C.R. & DINSDALE, D. (1993). Cytochrome P450
    2B isoenzymes are responsible for the pulmonary bioactivation and
    toxicity of butylated hydroxytoluene, O,O,S-trimethylphosphorothioate
    and methylcyclopentadienyl manganese tricarbonyl.  J. Pharmacol. Exp.
     Ther., 266(2): 958-963.

    Developmental neurobehavioral toxicity of butylated hydroxytoluene in
    rats.  Food Cosmet. Toxicol., 19: 153-162.

    WASEEM, M. & KAW, J.L. (1994). Pulmonary effects of butylated
    hydroxytoluene in mice.  Food Addit. Contain., 11(1): 33-38.

    WESS, J.A. & ARCHER, D.L. (1982). Evidence from  in vitro murine
    immunologic assays that some phenolic food additives may function as
    antipromotors by lowering intracellular cyclic GMP levels.  Proc. Soc.
     Exp. Biol. Med., 170: 427-430.

    WILLIAMS, G.M., MAEURA, Y., & WEISBURGER, J.H. (1983). Simultaneous
    inhibition of liver carcinogenicity and enhancement of bladder
    carcinogenicity of N-2-fluorenylacetamide by butylated hydroxytoluene.
     Cancer Lett., 19: 55-60.

    (1984). Lack of genotoxicity of butylated hydroxyanisole (BHA) and
    butylated hydroxytoluene (BHT).  The Toxicologist, 4: 104.

    WILLIAMS, G.M., WANG, C.X. & IATROPOULOS, M.J. (1990a). Toxicity
    studies of butylated hydroxyanisole and butylated hydroxytoluene. II.
    Chronic feeding studies.  Food Chem. Toxicol., 28(12): 799-806.

    WILLIAMS, G.M., MCQUEEN, C.A. & TONG, C (1990b). Toxicity studies of
    butylated hydroxyanisole and butylated hydroxytoluene. I. Genetic and
    cellular effects.  Food Chem. Toxicol., 28(12): 793-798.

    & ZANG, E. (1991). Modulation by butylated hydroxytoluene of liver and
    bladder carcinogenesis induced by chronic low-level exposure to
    2-acetylaminofluorene.  Cancer Res., 51: 6224-6230.

    WITSCHI, H. P. (1981). Enhancement of tumor formation in mouse lung by
    dietary butylated hydroxytoluene.  Toxicology, 21: 95-104.

    WITSCHI, H.P. (1986). Separation of early diffuse alveolar cell
    proliferation from enhanced tumour development in mouse lung.  Cancer
     Res., 46(6): 2675-2679.

    WITSCHI, H. & COTE, M. G. (19761. Biochemical pathology of lung damage
    produced by chemicals.  Fed. Proc., 35(1): 89-94.

    WITSCHI H.P. & KEHRER, J.P. (19821. Adenoma development in mouse lung
    following treatment with possible promoting agents.  J. Am. Coll.
     Toxicology, 1: 171.

    WITSCHI, H.P. & LOCK, S. (19791. Enhancement of adenoma formation in
    mouse lung by butylated hydroxytoluene.  Tox. Appl Pharm., 50: 391-400.

    WlTSCHI H.P. & MORSE, C.C. (19851. Cell kinetics in mouse lung
    following administration of carcinogens and butylated hydroxytoluene.
     Tox. Appl. Pharm., 78: 464-472.

    WITSCHI H.P., HAKKINEN, P.J. & KEHRER, J.P. (19811. Modification of
    lung tumor development in A/J mice,  Toxicology, 21: 37-45.

    YAMAMOTO, K., TAJIMA, K, OKINO, N. & MIZUTANI, T. (19881. Enhanced
    lung toxicity of butylated hydroxytoluene in mice by coadministration
    of butylated hydroxyanisole.  Res. Commun. Chem Pathol. Pharmacol.,
    59(2): 219-231.

    YAMAMOTO, K., TAJIMA, K., TAKEMURA, M. & MIZUTANI, T. (1991). Further
    metabolism of 3,5-di- tert-butyl-4-hydroxybenzoic acid, a major
    metabolite of butylated hydroxytoluene, in rats.  Chem. Pharm. Bull.,
    39(2): 512-514.

    YOSHIDA, Y. (1990). Study on mutagenicity and antimutagenicity of BHT
    and its derivatives in a bacterial assay.  Mutation Res.,
    242: 209-217.

    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (ICSC)
       Butylated hydroxytoluene (FAO Nutrition Meetings Report Series 38a)
       Butylated hydroxytoluene (FAO Nutrition Meetings Report Series 40abc)
       Butylated hydroxytoluene (WHO Food Additives Series 5)
       Butylated hydroxytoluene (WHO Food Additives Series 10)
       Butylated hydroxytoluene (WHO Food Additives Series 21)