Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents


    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization


    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.



         Butylated hydroxytoluene has been evaluated for acceptable daily
    intake by the Joint FAO/WHO Expert Committee on Food Additives (see
    Annex 1, Refs No. 6, No. 8, and No. 13) in 1961, 1964 and 1965.

         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

         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).

         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 tho bile collected from two
    rats (one male, one female) over a period of 40 hours (Daniel & Gage,

         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 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).

         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).

         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).

         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).

    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,


         After a single parental dose (100 µg) of -14C BHT, rats excreted
    32-35% of the radioactivity in the urine, and 35-37% 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 25.7 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).


    Special studies on liver metabolism

         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.,

    Special studies on the protective effect in vivo

         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.,

    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 a 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 (Frawley, 1967).

    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).

    Acute toxicity
                         LD50          lethal dose
    Animal      Route    (mg/kg bw)    (mg/kg bw)     Reference
    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, 1964).


         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 percent 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).

         Rats were given single doses of 100 mg BHT/kg bw daily for seven
    weeks before mating and then throughout pregnancy or were autopsied on
    the 20th day of pregnancy. No evidence of fetal abnormality was found
    in any of these animals but abnormalities did occur in the progeny of
    positive control groups treated with vitamin A (British Industrial
    Biological Research Association, 1964).

         A three-generation reproduction study was started by another
    group in May 1964. They also fed groups of 16 male and 16 female rats
    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.,

         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 (ICI, 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).


         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).


         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).

    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
    these 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 (Deichmann et al., 1955).

         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.


         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 (Daniel et al., 1967). 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 tertbutyl 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, 1970).


         Metabolic studies indicate that the prolonger enterohepatic
    circulation of BHT metabolites observed in the rat, does not operate
    in man. This is directly related to the observed differences in
    metabolism between rat and man. No extensive tissue accumulation would
    be expected, because of the rapid urinary excretion of BHT metabolites
    in man and is supported by the data on BHT residues in human fat.

         The effect of BHT on lipid metabolism in the rat is difficult to
    interpret. Long-term studies in the rat are adequate but are of
    limited significance for extrapolation to man. BHT has been shown to
    increase the longevity of mice. BHT is a weak inducer of hepatic
    microsomal enzyme systems. Reproduction studies in mice and rats have
    failed to confirm earlier observations of anophthalmia.


    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 1976.

         Studies on the effect on reproduction of mixtures of BHT, BHA and
    propyl gallate.


    Akagi, M. & Aoki, I (1962a) Chem. Pharm. Bull. (Tokyo), 10, 161

    Akagi, M. & Aoki, I (1962b) Chem. Pharm. Bull. (Tokyo), 10, 200

    Ames, S. R. et al. (1956) Proc. Soc. exp. Biol. (N.Y.), 93, 39

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

    British Industrial Biological Research Association (Unpublished report
         submitted to WHO in 1964)

    Brown, W. D., Johnson, A. R. & O'Halloran, M. W. (1959) Aust. J. exp.
         Biol. med. Sci., 37, 533

    Clegg, D. J. (1965) Fd. Cosmet. Toxicol., 3, 387

    Collings, A. J. & Sharratt, M. (1970) Fd. Cosmet. Toxicol., 8, 409

    Creaven, P. J., Davies, W. H. & Williams, R. T. (1966) J. Pharm.
         Pharmacol., 18, 485


    *    As BHA, BHT or the sum of both.

    **   Temporary.

    Dacre, J. C. (1961) Biochem. J., 78, 758

    Daniel, J. W. & Gage, J. C. (1965) Fd. Cosmet. Toxicol., 3, 485

    Daniel., J. W., Gage, J. C. & Jones, D. I. (1968) Biochem. J., 106,

    Daniel, J. W. et al. (1967) Fd. Cosmet. Toœicol., 5, 475

    Day, A. J. et al. (1959) Aust. J. exp. Biol. med. Sci., 37, 295

    Deichmann, W. B. et al. (1955) A.M.A. Arch. industr. Hlth., 11, 93

    Denz., F. A. & Llaurado, J. G. (1957) Brit. J. exp. Path., 38, 515

    Frawley, J. P. (1967) Unpublished report submitted to WHO

    Frawley, J. P., Kay, J. M. & Calandra, J. C. (1965a) Fd. Cosmet.
         Toxicol., 3, 471

    Frawley, J. P. et al. (1965b) Fd. Cosmet. Toxicol., 3, 377

    Gaunt, I. F. et al. (1965a) Ed. Cosmet. Toxicol., 3, 433

    Gaunt, I. F., Gilbert, D. & Martin, D. (1965b) Fd. Cosmet. Toxicol.,
         3, 445

    Gilbert, D. & Golberg, I. (1965) Fd. Cosmet. Toœicol., 3, 417

    Golder, W. S., Ryan, A. J. & Wright, S. E. (1962) J. Pharm.
         Pharmacol., 14, 268

    Harman, D. (1968) J. Geront., 23, 476

    I. C. I. (1964) Unpublished report No. IHR/158

    Johnson, A. R. (Unpublished report submitted to WHO in 1964)

    Johnson, A. R. (1965) Fd. Cosmet. Toxicol., 3, 371

    Johnson, A. R. & Holdsworth, E. S. (1968) J. Nutr. & Diet., 5, 147

    Johnson, A. R. & Hewgill, F. R..(1961) Aust. J. exp. Biol. med. Sci.,
         39, 353

    Karplyuk, I. A. (1959) Vop. Pitan., 18, 24

    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) J. Pharm.
         Pharmacol., 19, 383

    Ladomery, L. G., Ryan, A. J. & Wright, S. E. (1967b) J. Pharm.
         Pharmacol., 19, 388

    Nievel, J. G. (1969) Fd. Cosmet. Toxicol., 7, 621

    Shollenberger, T. E., Parrish, D. B. & Sanford, P. E. (1957) Poultry
         Sci., 36, 1313

    Sporn, A. & Schöbesch, O. (1961) Igiena (Bucharest), 9, 113

    van Stratum, D. G. C. & Vos, H. J. (1965) Fd. Cosmet. Toxicol.,
         3, 475

    Tye, R., Engel., J. D. & Rapion, I. (1965) Fd. Cosmet. Toxicol.,
         3, 475

    Ulland, B. M. et al. (1973) Fd. Cosmet. Toxicol., 11, 199

    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 10)
       Butylated hydroxytoluene (WHO Food Additives Series 21)
       Butylated hydroxytoluene (WHO Food Additives Series 35)