The evaluations contained in this document were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Rome, 21-29 April 1976

    Food and Agriculture Organization of the United Nations

    World Health Organization

    *Twentieth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1976, WHO Technical Report Series No. 599, FAO Food
    and Nutrition Series No. 1.



         Butylated hydroxytoluene has been evaluated for acceptable daily
    intake for man by the Joint FAO/WHO Expert Committee on Food Additives
    in 1961, 1964, 1965 and 1973 (see Annex 1, Refs No. 6, p. 45; No. 9,
    p. 13; No. 13, p. 28; and No. 33, p. 156).

         Since the previous evaluations, additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monographs have been expanded and are
    reproduced in their entirety below.



    Excretion and distribution

         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. Following removal of the diet 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 ppm in males, 65 ppm 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 ca 5.0 ppm in males
    and 1.5 ppm 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 one to five
    oral doses of 44 mg/kg bw BHT on alternate days and each group killed
    24 hours after the final dose. The range of the total dose accounted
    for was 92 to 103.5% in males and 92.6 to 98.6% 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 five 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 BHT-14C in
    the body under the conditions of repeated oral dosage (Tye et al.,

         The fate in the body of 14C-labelled BHT has been elucidated.
    The relatively slow excretion of BHT is probably attributable to
    enterohepatic circulation rather than to tissue retention. Rats were
    given single oral doses (1-100 mg/rat) of BHT-14C and approximately
    80 to 90% of the dose was recovered in four days in the urine and
    faeces. Of the total radioactivity, 40% appeared in the urine of
    females and 25% in males. After four 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 hours (Daniel & Gage,

         The liver and body fat of rats fed a diet containing 0.5% BHT for
    35 days were analysed. The concentration of BHT in the liver never
    rose above 5 ppm (0.0005%) in males or 1.5 ppm (0.00015%) in females.
    In the body fat the level fluctuated round 30 ppm (0.003%) in males
    and 45 ppm (0.0045%) in females. Fat from rats returned to normal diet
    showed a progressive fall in the concentration of BHT the half-life
    being about seven 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,

         In further work with rats it was found that increased output of
    urinary ascorbic acid paralleled 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 parallelism between 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).

         The BHT content of fat and liver of rats given diets containing
    0.5 and 1.0% BHT for periods up to 35 and 50 days respectively: with
    0.5% BHT in the diet, a level of approximately 30 ppm (0.003%) in the
    fat was reached in males and 45 ppm (0.0045%) in females, with
    approximately 1-3 ppm (0.0001-0.0003%) in the liver, while with 1.0%
    BHT the level in the fat was 50 ppm (0.005%) in males and 30 ppm
    (0.003%) in females. On cessation of treatment, the level of BHT in
    fat fell with a half-life of seven to 10 days (Daniel & Gage, 1965).
    The level of BHT in the fat reached a plateau at approximately 100 ppm
    (0.01%) after three to four days when daily doses of 500 mg/kg bw were
    given by intubation; 200 mg/kg bw per day for one week produced a
    level of about 50 ppm (0.005%) (Gilbert & Golberg, 1965).

         When feed containing 500 ppm (0.05%) BHT was given to laying
    hens, 20 ppm (0.002%) was found in the fat fraction of eggs; 100 ppm
    (0.01%) in the feed resulted in residues of less than 5 ppm (0.0005%).
    In the broiler chicken, over a period of 21 weeks, the residues in
    body fat were 55 ppm (0.0055%) on the 500 ppm (0.05%) diet and less
    than 5 ppm (0.0005%) on the 100 ppm (0.01%) diet (Van Stratum & Vos,

         One-day-old chicks were given 14C-BHT at a level of 200 ppm
    (0.02%) in the food for 10 weeks. At broiler age, edible portions had
    residues amounting to 1-3 ppm (0.0001-0.0003%) of BHT and metabolites.
    Similar diets given to laying hens produced residues in eggs of 2 ppm
    (0.0002%) after seven days, the level thereafter remaining constant
    (Frawley et al., 1965a).

         Rats were given doses of 100 µg of BHT labelled with 3H
    intraperitoneally and the urinary output of radioactivity was measured
    for four consecutive days. Four days after the injection 34.5% of the
    injected radioactivity was recovered in urine (Ladomery et al., 1963).

         The same dose of BHT (100 µg) 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).

         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 five day period, and biliary excretion
    followed for 120-126 hours after i.p. injection. For the low doses of
    the compounds tested (100 µg) there were no significant differences in
    the total recovery of 14C during the 5 days urinary and faecal
    excretion and 120-126 hours 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).


         Rabbits were given single or repeated doses of BHT in the range
    400-800 mg/kg bw. About 16% of the dose was excreted as ester 
    glucuronide and 19% as ether glucuronide. Unconjugated phenol (8%) 
    ethoreal sulfate (8%) and a glycine conjugate (2%) were also excreted. 
    Excretion of all detectable metabolites was essentially complete three 
    to four 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) administered to
    rabbits orally in single doses of 500 mg/kg bw was studied. The
    metabolites 2,6-di-tertbutyl-4-hydroxymethylphenol (BHT-alc),
    3,5-di-tert-butyl-4-hydroxybenzoic 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-ald) was also isolated from rabbit urine (Aoki, 1962). The main
    metabolic pathway was confirmed by administering BHT-alc to rabbits
    and isolating BHT-ald, BHT-acid, the ethylene-bis derivative and
    unchanged BHT-alc in the urine (Akagi & Aoki, 1962b).

         After a single parental dose (100 µg) of -14C BHT, rats excreted
    32-35% of the radioactivity in the urine, and 33-37% in the faeces, in
    a four 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.,
    1967a). 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. 34 to 53% of the 14C-labelled 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 dosed with a single dose of 14C-BHT and urine and bile
    collected for periods ranging from 48 to 96 hours. Faeces were also
    collected during this same period. 19 to 58.5% of the radioactivity
    appeared in the urine during this period, and 23.7 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).

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

    Effects on enzymes and other biochemical parameters

         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 being 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 daily the level of BHT in fat attained values of 230 ppm
    (0.023%) in females and 162 ppm (0.0162%) 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 ppm (0.01%) in both sexes (Gilbert & Golberg,

         Groups each of 12 SPF Carworth rats equally divided by sex were
    administered BHT dissolved in Arachis oil daily for one week, at a
    dose level equivalent to 50, 100, 200 or 500 mg/kg bw. A group of 8
    rats served as control. The animals were killed 24 hours 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 in which 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, in contrast to the return to normal by day 14
    of relative liver weights (Feuer et al., 1965).

         Rats (male and female Carworth Farm SPF) were dosed orally with
    BHT at a level equivalent to 500 mg/kg bw. Dosing was from 1 to 5
    days, and rats varying in size from 100-400 g bw 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 daily for
    5 days) caused a sixfold increase in the activity of the enzyme/gram
    of liver and a 35% increase in relative liver weight, both being
    prevented by actimomycin 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 rats (female, Alderly Park SPF strain) were maintained
    on diets containing 5%, 1%, 0.1%, 0.01% and 0% BHT for periods up to
    28 days, and then on diets free of BHT for 56 days. Animals were
    killed in groups of four, two 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 (0.01%) 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 ten days, and then for a
    second period of ten days after an interval of 20 days on a normal
    diet. The animals were killed in groups of five at 10, 30, 40, 42 and
    47 days. Livers were removed for aminopyrene demethylase assay and
    electron microscopy. Enzyme activity did not differ significantly
    following both the ten day periods of administration of BHT. Electron
    microscopy showed similar smooth endoplasmic reticulum response during
    both these periods (Botham et al., 1969).

         A group of 23 female SPF rats (Wistar strain), body weight of
    about 145 g, was administered 500 mg/kg BHT dissolved in rape-seed
    oil, for eleven days, starting on day 3 of the study. A control group
    of rats 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
    day 28 and 63 of the study. Livers of the rats were examined for
    weight, DNA content and number of cell nuclei. BHT was shown to result
    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 (Schulte Hermann et al.,

         Two groups, one male and one female rat (Alderly Park, SPF Wistar
    strain) of ten test animals were dosed daily by stomach tube with
    200 mg/kg bw of BHT dissolved in maize oil for seven days. Four male
    and four female rats dosed with an equivalent amount of maize oil were
    used as controls. Urinary ascorbic acid excretion was measured in
    urine, in samples collected following 5 days on the test compound. The
    animals were killed 24 hours after the final dose and the livers
    removed for biochemical assays (aminopyrine demethylase-AMPM,
    hexobarbitone oxidase-HO, cytochrome P450, 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
    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 P450 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).

         Groups each of 2-4 juvenile rhesus monkeys (Macaca mulatta)
    were dosed daily with BHT dissolved in corn oil at a dose level
    equivalent to 0, 50, or 500 mg/kg bw. Treatment was for 4 weeks. Blood
    samples were taken prior to treatment and then at weekly intervals
    from the control and test animals in the high level group, and from
    test animals in the low level group at the end of the four 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 hours and sacrificed, and liver and
    blood samples obtained. Liver samples were analysed for succinic
    dehydrogenase and susceptability to peroxidation. Extracted liver
    lipids were analysed 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, and 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).

         Mice (BALB/c strain) were maintained on a diet containing 0.75%
    BHT. 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).

         Rats fed stock diets supplemented with 20% lard, and containing
    0.2, 0.3, 0.4 or 0.5% (dry weight) BHT for six 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 maintained on diets

    containing 0.5% dietary BHT in the presence or absence of a 20% lard
    supplement, irrespective of the presence or absence of dietary lard,
    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. 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. In the
    animals fed BHT with lard, BHT reduced the rate of synthesis of body
    and liver fatty acids and reduced the growth rate to a greater extent
    than in animals without dietary lard (Johnson & Holdsworth, 1968).

         Microsomal preparations from livers of rats, dosed daily with
    BHT for up to seven days at a level of 450 mg/kg bw 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). Rats fed diets containing BHT at
    levels of 0.01 to 0.5% 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 0.01%. Enzyme activity was not
    significantly altered by 0.5% BHT fed for one day (Creaven et al.,

         Groups each 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% or 0.5%
    BHT, as compared to 14.5 ± 4.6 months for controls (Harman, 1968).
    Groups each of 20 male and 20 female Charles River rats were
    maintained on test diets (males 24 weeks, females 36 weeks) containing
    BHT and/or carcinogen. (N-2-fluorenylacetamide or N-hydroxy-N-2-
    fluorenylacetamide) in the molar ratio of 30:1, equivalent to 6600 ppm
    (0.66%) BHT, then continued on control diets for another 12 weeks. The
    N-2-fluorenylacetamide alone resulted in hepatomas in 70% of the male
    rats, 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% (hepatomas in males), 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 (55 ppm (0.0055%)) in
    drinking-water for 24 weeks was not affected by BHT (Ulland et al.,

         Young adult BALB/c mice of both sexes maintained on diets
    containing 0.75% BHT, for one 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, 1975a).

         Hybrid (C31F1) male mice, 10 to 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 methanesulphonate, n-propyl or isopropyl methanesulphonate,
    ethyl dibromide, diethylnitrosamine and cyclophosphamide, but not
    against methyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguaridine
    or dipropylnitrosamine (Cumming & Walton, 1973).


    Special studies on embryotoxicity

         In a study on the embryotoxicity of BHT three 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 seven 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).

         Diets containing 0.1 or 0.5% BHT together with two dietary levels
    of lard (10 and 20%) were given to mice. The 0.5% 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, none showed anophthalmia, although 12
    out of the 144 mothers were selected from an established anophthalmic
    strain (Johnson, 1965).

    Special studies on mutagenicity

    (a)  Cytogenetics

         BHT was investigated at concentrations of 2.5, 25 and 250 µg/ml
    in vitro, employing WI-38 human embryonic lung cells for anaphase
    abnormalities. BHT produced a biologically significant increase in the
    number of abnormal anaphase figures. These effects are thought to be
    cytogenetic in nature but not mutagenic. The distinction between the
    terms is that inhibition of cell division can be a cytogenetic effect,
    but not mutagenic in the sense that this effect is not transmissible
    through cell division to progency cells. Additionally 70% of the
    substances tested by this procedure yielded significant biological
    effects; therefore there is the possibility of generating false
    positive effects (S.R.I., 1972).

         BHT was also investigated in vivo by the cytogenetic analysis
    of rat bone marrow cells. Dosages of 30, 900 and 1400 mg/kg were
    employed. Acute and subacute regimens were done. No evidence of
    significant cytogenetic damage was found (S.R.I., 1972).

    (b)  Host-mediated assay

         In vitro-Salmonella TA-1530 and C-46, together with
    Saccharomyces D-3 were employed. A 50% concentration was
    tested. No mutagenicity was seen (S.R.I., 1972).

         In vivo-BHT was tested at levels of 30.0, 900.0 and 1400 mg/kg
    in the acute tests and at 30.0, 250.0 and 500.0 mg/kg in the subacute
    tests in ICR Swiss mice employing as indicator organisms Salmonella
    G-46 and TA-1530 and Saccharomyces D-3. No mutagenicity was seen.
    BHT has also been investigated by more sensitive (in terms of
    detection) test methods which are a part of the tier one screen. In
    this study BHT was investigated using Salmonella typhimurium strains
    TA-1535, 1537 and 1538 and Saccharomyces D-4 with and without
    metabolic activation in plate and suspension tests. The percentage
    concentrations (w/v) employed were 0.15, 0.3, and 0.6 for bacteria and
    0.6, 1.2 and 2.4 for yeasts. Under the conditions of this
    investigation BHT was non-mutagenic (Brusick, 1975).

    (c)  Dominant lethal study

         BHT was investigated in Sprague Dawley rats at acute dosages of
    30, 900 and 1400 mg/kg and at subacute dosages of 30×5, 250×5 and
    500×5 mg/kg.

         Significant postimplantation losses were produced in weeks three,
    four and six by the subacute regimen (Brusick, 1975).

    Special studies on reproduction

         Weanling rats (16 of each sex) were fed a diet containing 20%
    lard and 0, 300, 1000 or 3000 ppm (0, 0.03, 0.1 or 0.3%) 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 eight 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 ppm (0.3%) 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 ppm (0.3%) and all observations at
    1000 ppm (0.1%) and 300 ppm (0.03%) 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 ppm (0.3%) level. The offspring were
    examined for: litter size, mean body weight, occurrence of stillbirth,
    survival rate and gross and microscopic pathology (Kennedy et al.,

    Other special studies

         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).

         The chronic ingestion of 0.5% of the butylated hydroxytoluene
    (BHT) by pregnant mice and their offspring resulted in a variety of
    behavioural changes. Compared to controls, BHA-treated offspring
    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 agression, and a severe deficit in learning (Stokes
    & Scudder, 1974).

         Young male Swiss Webster mice were injected i.p. with BHT at dose
    levels ranging from 62.5 to 500 mg/kg bw BHT. The animals were killed
    on days 1, 3 and 5 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).

        Acute toxicity


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

    Rat            oral      1 700-1 970         -                   Deichmann et al.,

    Cat            oral      -                   940-2 100           Deichmann et al.,

    Rabbit         oral      -                   2 100-3 200         Deichmann et al.,

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

    Rat            oral      2 450                                   Karplyuk, 1959

    Mouse          oral      2 000                                   Karplyuk, 1959
    Short-term studies


         BHT was given to pregnant mice in daily doses of 750 mg/kg bw for
    18 days. Another group received the same dose for a total of 50 to 64
    days including 18 days of pregnancy. No fetal abnormalities were
    observed (Clegg, 1965).

         In a statistically planned experiment using 144 female mice no
    blindness was observed in any of the 1162 litters representing 7765
    babies born throughout the reproductive life span of the mothers
    (Johnson, 1965).


         Feeding experiments were carried out on 45 pairs of weanling male
    rats for five to eight weeks with diets containing 0, 10 and 20% lard
    supplements to which 0.001, 0.1 or 0.5% BHT had been added. 0.001%
    caused no changes in any of the serum constituents studied. 0.5%
    produced increase in the serum cholesterol level within five weeks.
    Female rats fed for eight months on a diet containing a 10% lard
    supplement with 0.1% BHT showed increased serum cholesterol levels,

    but no other significant changes. 0.5% BHT 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).

         0.3% BHT in the diet of pregnant rats that had been kept for five
    weeks on a diet deficient in vitamin E produced no toxic symptoms.
    1.55% caused drastic loss of weight and fetal death (Ames et al.,

         BHT fed to rats in groups of 12 for a period of seven weeks at a
    dietary level of 0.1% in conjunction with a 20% lard supplement
    significantly reduced the initial growth rate and mature weight of
    male rats. No effect was noted in female or male rats with a 10% lard
    supplement. 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).

         Groups of six weanling rats (three male and three female) were
    fed BHT at dietary levels of 0, 0.1, 0.2, 0.3, 0.4 and 0.5% in
    conjunction with a 20% lard supplement for six weeks. BHT reduced the
    growth rate, especially in the males, the effect appearing to become
    significant at the 0.3% level. It also increased the absolute liver
    weight and the ratio of liver weight to body weight in both sexes, the
    latter effect appearing to become significant at the 0.2% level. 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 attributable to the treatment in the adrenal. All dietary
    levels of BHT increased the serum cholesterol and the concentration of
    the 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).

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

         Feeding experiments conducted for 20 and 90 days respectively
    indicated that rats do not find food containing 0.5 or 1% BHT
    palatable. However, the animals ingest foods so treated more readily
    if these concentrations are attained gradually. Paired feeding

    experiments with groups of five or 10 rats for 25 days demonstrated
    that diets containing 0.8 and 1% BHT will reduce the daily intake of
    food below control values. A level of 1% in the diet retarded weight
    gain (Deichmann, 1955).

         BHT (2000 ppm (0.2%)) incorporated in a diet containing 19.9%
    casein was administered to a group of eight young rats for eight
    weeks; a further group of eight rats served as controls. The
    experiment was repeated with 16.6% casein in the diet of further
    groups for four weeks and again with 9.6% casein (and no added
    choline) for seven weeks. In all three instances BHT caused
    stimulation of growth and improved protein efficiency. The N content
    of the liver was, however, greatly reduced in BHT-treated animals,
    except when the level of BHT was reduced to 200 ppm (0.02%). Recovery
    of hepatic protein after fasting (details not given) was also impaired
    in rats on 2000 ppm (0.2%) BHT. Liver lipid content was increased with
    2000 ppm (0.2%) but not with 200 ppm (0.02%) BHT. A dietary level of
    2000 ppm (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 is interpreted as indicating a stress imposed on the
    organism by BHT (Sporn & Schöbesch, 1961).

         Groups of 48 weanling rats (24 of each sex) were given diets
    containing 1000 ppm (0.1%) BHT for periods of up to 16 weeks. A group
    of 48 rats served as controls. Measurements of growth rate, food
    consumption, weight and micropathological examination of organs at
    autopsy revealed no difference from untreated rats. 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 16 male and 16 female rats were fed on levels of 0.03,
    0.1 and 0.3% BHT in a diet containing 20% fat for 10 weeks. There were
    two control groups each containing 16 male and 16 female animals. No
    definite effect on body weight was observed at any level in the
    females and there was only a slight depression in the 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 for 10 weeks.
    Four of the males at the 0.3% and two at the 0.1% level died during
    the experiment. Two deaths occurred among the females at 0.3%. Only
    one male rat died in both control groups (Frawley et al., 1965a).

         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).


         Acute effects on electrolyte excretion similar to those described
    for large doses of BHA were also obtained following administration of
    doses of BHT of 500-700 mg/kg bw (about 2% in the diet). No such
    effects were observed at lower dosage levels (Denz & Llaurado, 1957).


         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 (Shollenberger et
    al., 1957).


         A mild to moderately severe degree of diarrhoea was induced in a
    group of four dogs fed doses of 1.4-4.7 g/kg bw every two to four days
    over a period of four weeks. Post-mortem 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 five days a week for a period of 12 months
    (Deichmann et al., 1955).


         A group of six adult female rhesus monkeys were maintained on a
    test diet containing a mixture of BHT and BHA that provide an intake
    equivalent to 50 mg BHT and 50 mg BHA/kg bw. Another group of six
    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. Hematologic studies
    including hemoglobin, hematocrit, total as well as differential white
    blood cell count, cholesterol, Na+, K+, total protein, serum
    glutamic pyruvic transaminase, and serum glutamic oxylacetic
    transaminase, 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. Hematological 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 two control infants, 3 months of age, were

    removed from their mothers for one month of psychological home cage
    observations. No clinical abnormalities were observed in parent and
    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,

         In another study, groups each of 3 infant or juvenile monkeys
    (Macaca mulatta) were dosed daily with BHT at a level equivalent to
    500 mg/kg bw. Another group of juvenile monkeys received 50 mg/kg bw
    BHT. Treatment was for 4 weeks. Blood analysis (complete cell count,
    serum sodium and potassium, bilirubin, cholesterol and glutamic
    oxalactic transaminase) was carried out weekly, as was a complete
    urinalysis. Liver biopsies were taken from the juvenile monkey at 2
    weeks, following a 24 hour fast. At the end of the test period, all
    animals were fasted 24 hours and sacrificed. Tissues from all major
    organs were prepared for light and electron microscopy. Liver tissue
    was also analysed for protein, RNA and cytochrome P450. 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 did not indicate any compound related changes. Test animals
    receiving BHT showed hepatocytomegaly and enlargement of hepatic cell
    nuclei. Ultrastructurally, 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 level group. DNA and RNA and cytochrome P450
    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).

    Long-term studies


         Groups of 15 male and 15 female rats 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 one
    group given a diet containing 0.5% BHT, the BHT was dissolved in lard
    and then heated for 30 minutes at 150°C 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 1% 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 in the
    concentrations 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 as the fatalities were in no relation to the
    concentration of BHT fed, it was believed that the cause of death was
    unrelated to the feeding of this substance. Micropathological studies
    support this observation. When fed at the 0.5% level in the diet, BHT
    had no effect in rats on the 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
    on a diet containing 0.1% BHT and 10% hydrogenated coconut oil for a
    period of two years. The effects on weight gain have already been
    described (Deichmann et al., 1955).

         Female (albino Wistar) rats, initial body weight 120-130 g were
    maintained on a diet containing 0 or 0.4% W/W BHT, for 80 weeks. After
    one week on the test diet significant increases were observed in
    liver weight, microsomal protein, cytochrome P450, 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 depressed. 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. However, cytochrome b5,
    cytochrome c reductase and ethylmorphine N-demethylase remained
    increased. Histological changes at the end of 80 weeks feeding the
    diet consisted of centrilobular cell enlargement, which was
    reversible, following 18 days on a BHT free diet. The only
    ultrastructural change was a proliferation of smooth endoplasmic
    reticulum (Gray & Parke, 1974).

         A group of 18, 8 week-old male BALB/c mice fed dietary BHT at a
    level of 0.75% for a period of 12 months, developed marked hyperplasia
    of the hepatic bile ducts with an associated sub-acute cholangitis
    (Clapp et al., 1973). In another study 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., 1975b).

         Groups each of 48 mice (CFI strain) equally divided by sex were
    maintained on diets containing 1000 ppm BHT. At week 4, one group was
    then fed a diet containing 2500 ppm BHT, and then at eight weeks
    another group was fed a diet containing 5000 ppm 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 level group and control.
    However, there was an increased incidence of lung neoplasia in treated
    rats (75% in 5000 ppm group, 73.9% in 2500 ppm group, 53.2% in the
    1000 ppm group and 46.8% in controls). 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).


         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 hours, followed by a slower
    phase which probably represents the release of the compounds or their
    metabolites stored in tissues. In man, the bulk of the radioactivity
    is excreted as the ether insoluble glucuronide of a metabolite in
    which the ring methyl group and one tert-butyl methyl group are
    oxidized to carboxyl groups, and a methyl group on the other tert-
    butyl group is also oxidized, probably to an aldehyde group. BHT-acid
    free and conjugated is a minor component of the urine and the
    mercapturic acid is virtually absent. The rapidity of the first phase
    of the urinary excretion in man suggests that there is no considerable
    enterohepatic circulation as has been observed in the rat (Daniel et
    al., 1967).

         Reported values of BHT in the body fat were 0.23 ± 0.15 ppm
    (0.000023% ± 0.000015%) (11 individuals, residents of the United
    Kingdom) and 1.30 ± 0.82 ppm (0.000130 ± 0.000082) (12 individuals,
    residents of the United States of America) (Collings & Sharratt,


         Further elucidation of the metabolism of BHT in man as well as
    the rat indicate a rapid urinary excretion of oxidation products, some
    conjugated with glycine.

         Swiss-Webster mice that were the offspring of parents fed 0.5%
    BHT and fed the same diet were subjected to behavioural tests at 6
    weeks of age. Results indicated behavioural changes, but these changes
    are difficult to evaluate in the light of the fact that infant monkeys
    from BHT-treated dams showed no behavioural abnormalities.

         Several carcinogenicity studies have been undertaken on mice.
    Balb C mice did not have an increased incidence of lung tumours
    whereas CFI mice had an increased incidence of lung tumours compared
    with controls. As regards other tumours no increased incidence was
    seen compared with controls and long-term rat studies have been
    negative. BHT has been studied in several in vitro and in vivo
    systems and was found to be non-mutagenic. The negative mutagenic
    studies provide additional evidence for a non-carcinogenic potential
    of BHT. However, the Committee considered that an appropriate
    carcinogenic study meeting modern standards would be desirable.

         The previously stated requirement for studies on the effect on
    reproduction of mixtures of BHA, BHT and propyl gallate was considered
    to be no longer necessary.


    Level causing no toxicological effect

         Mouse: 5000 ppm (0.5%) in the diet equivalent to 250 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-0.5* mg/kg bw**


         Required by 1980

         Appropriate carcinogenicity study, meeting currently accepted


    *    As BHA, BHT or the sum of both.

    **   Temporary.


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    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 21)
       Butylated hydroxytoluene (WHO Food Additives Series 35)