IPCS INCHEM Home


    WHO/Food Add./24.65
    FAO Nutrition Meetings
    Report Series No. 38A




    SPECIFICATIONS FOR IDENTITY AND 
    PURITY AND TOXICOLOGICAL EVALUATION 
    OF SOME ANTIMICROBIALS AND 
    ANTIOXIDANTS





    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met 8-17
    December 1964a





                   

    a Eighth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1965, 309; FAO
    Nutrition Meetings Report Series 1965, 38.


    BUTYLATED HYDROXYTOLUENE

    CHEMICAL NAMES      2:6-di-tert.-butyl-p-cresol; 
                        4-methyl-2:6-ditertiary-butylphenol

    SYNONYM             BHT

    EMPIRICAL FORMULA   C15H24O

    STRUCTURAL FORMULA

    MOLECULAR STRUCTURE 1

    MOLECULAR WEIGHT    220.36

    DEFINITION          Butylated hydroxytoluene should contain not less
                        than 99.0% of C15H24O.

    DESCRIPTION         A white, crystalline, odourless solid.  It is
                        insoluble in water but soluble in fats; 1 g is
                        soluble in 4 ml of ethanol.

    USE                 As an antioxidant for fats and oils or in
                        packaging material for fat-containing foods.  Its
                        activity is enhanced in combination with other
                        antioxidants and synergists.

    Biological Data

    Biochemical aspects

    BHT is readily absorbed.  Some deposition in adipose tissue bee been
    described following high dosage in the rat, and this may cause
    increased stability of the extracted perineal fat.1  The metabolism
    of BHT is complicated by the presence of the butyl groups on each side
    of the hydroxyl.  Preliminary modification is necessary before
    conjugation can occur.  This takes the form of either oxidation of one
    of the butyl groups to a 2-hydroxy-2:2-dimethyl-2-ethyl group, or
    oxidation of the methyl group to a carboxy acid.  The former yields
    glucuronide conjugates, while the latter becomes conjugated with
    glucuronic acid or glycine.  Some of the oxidized material is also
    excreted unconjugated.2,3

    Rabbits were given single or repeated doses of BHT in the range
    400-800 mg per kg body-weight.  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.4

    Rats were given doses of 100 g of BHT labelled with 3H
    intraperitoneally and the urinary output of radioactivity was measured
    for 4 consecutive days.  Four days after the injection 34.5% of the
    injected radioactivity was recovered in urine.5  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 4 days, in close
    agreement with the previous result using tritiated BHT.6

    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 ppm in males or 1.5 ppm in females.  In the body fat the level
    fluctuated round 30 ppm in males and 45 ppm in females.  Fat from rats
    returned to normal 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
    done of 14C-labelled BHT (12 mg/kg body-weight).  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.7

    Acute toxicity

                                                                                     

    Animal        Route       LD50         Approximate lethal dose     Reference
                             (mg/kg        (mg/kg body-weight)
                           body-weight)
                                                                                 

    Rat           oral       1700-1970              -                      8
    Cat           oral           -               940-2100                  8
    Rabbit        oral           -              2100-3200                  8
    Guinea-pig    oral           -                10 700                   8
                                                                                 
    
    Short-term studies

    Mouse.  BHT was given to pregnant mice in daily doses of 750 mg per
    kg body-weight for 18 days.  Another group received the same dose for
    a total of 50 to 64 days including 18 days of pregnancy.  No foetal
    abnormalities were observed.9

    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.10

    Rat.  Feeding experiments were carried out on 45 pairs of weanling
    male rats for 5 to 8 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 5 weeks.
    Female rate fed for 8 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.11

    0.3% BHT in the diet of pregnant rats that had been kept for 5 weeks
    on a diet deficient in vitamin E produced no toxic symptoms.  1.55%
    caused drastic loss of weight and foetal death.12

    BHT fed to rats in groups of 12 for a period of 7 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 litter's.13

    Groups of 6 weanling rats (3 male and 3 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 6 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 per cent. esterified liver cholesterol, total liver lipid
    or concentration of total polyunsaturated fatty acids in the liver.14

    BHT administered to rats at 250 mg per kg body-weight for 68 to 82
    days caused reduction in rate of increase in weight and fatty
    infiltration of the liver.15

    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 5 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.8

    (Work in progress) Rats were given single doses of 100 mg BHT per kg
    body-weight daily for 7 weeks before mating and then throughout
    pregnancy or were autopsied on the 20th day of pregnancy.  No evidence
    of foetal abnormality were found in any of these animals but
    abnormalities did occur in the progeny of positive control groups
    treated with vitamin A.9

    (Work in progress) 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 O.3%.  Only one male rat died in both control
    groups.16

    (Work In progress) 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.17

    Rabbit.  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 body-weight (about 2%
    in the diet).  No such effects were observed at lower dosage
    levels.18

    Dog.  A mild to moderately severe degree of diarrhoea was induced in
    a group of 4 dogs fed doses of 1.4-4.7 g/kg body-weight every 2-4 days
    over a period of 4 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 body-weight 5 days a week for a period of 12
    months.8

    Fowl.  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.19

    Long-term studies

    Rat.  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 .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 1% BHT was followed in both male and female rats by a
    sub-normal 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.8

    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 2 years. The
    effects on weight gain have already been described.13

    Comment on experimental studies reported

    Experimental studies have been carried out in several species.  There
    were, however, some important discrepancies between the results
    obtained by earlier and by some more recent observers.  Short-term and
    long-term studies in rats and metabolic studies will be discussed
    under evaluation.

    Evaluation

    Level causing no significant toxicological effect in the rat

    One extensive series of studies8 indicates that the level of BHT that
    would cause no significant deleterious effect in the rat would be 0.8%
    in a diet containing 1% lard.  However, in two short-term studies11,13
    it has been reported that 0.1% BHT in a 20% lard diet resulted in
    increase of various lipid components of the blood and a significant
    reduction in weight gain.  More recently a similar trend of results
    was obtained but the effects only became definite at the 0.3%
    level.14 In another study 0.1% BHT in a 20% fat diet was found to
    have no effect on the growth rate of weanling rats of either sex and
    there was no effect on the blood cholesterol concentration at the 0.1%
    or the 0.3% levels.16  In long-term studies with rats receiving 0.5%
    BHT in a diet containing 10% lard no significant difference from the
    controls was observed.13  The addition of 10% or 20% of lard to the

    diet was found to enhance the deleterious action of BHT in some
    studies.  With a 20% lard supplement, fat provides about 33% of the
    calorie intake and human diets frequently contain this level of fat. 
    The effect of such levels of dietary fat on BHT toxicity is therefore
    relevant to its use as a food additive.  Several investigators have
    shown that BHT will cause an increase in liver weight relative to
    body-weight.  However, even in rats fed at 1% BHT in the diet for 10
    weeks, there was recovery in the liver to body-weight ratio and
    morphological appearance of the liver cells within a few weeks of
    restoration of the animals to a normal diet.17

    The chemical structure of BHT suggests the possibility of some delay
    in metabolism.  The results of studies on the excretion of labelled
    BHT5,6,7 are difficult to compare because of the different dose levels
    and routes of administration used and therefore the importance of
    possible delayed metabolism is difficult to assess.

    A possible teratogenic action of BHT was suggested by the occurrence
    of anophthalmia in some litters of mothers fed the compound.13
    Extensive reproduction studies now completed or in progress make this
    unlikely.9,10,16

    For the 0.1% level to be accepted as that causing no significant
    toxicological effect in the rat it would be necessary to disregard
    both the effect on weight gain and also the effect on blood lipids
    reported by some investigators.

    It would be inadvisable to dismiss these observations pending the
    evaluation of results of work now in progress.  The next lower dose
    studied was 0.01% (= 100 ppm) in the diet in a group of 26 rats, which
    is equivalent to 5 mg/kg body-weight per day.

    Estimate of acceptable daily intake for man

                                       mg/kg body-weight

    Conditional acceptance                  0-0.5

    Further Work Considered Desirable

    1. Further long-term studies, with particular reference to the effect
    of BHT on lipid metabolism and the relationship between the dietary
    fat load and toxicity.

    2. Metabolic studies in human subjects.

    References

    1. Johnson, A. R., O'Halloran, M. W. & Hewgill, F. R. (1956) J. Amer.
    Oil Chem. Soc., 35, 496

    2. Dacre, J. C., Denz, F. A. & Kennedy. T. H. (1956) Biochem. J., 64,
    777

    3. Dacre, J. C. (1960) J. N.Z. Inst. Chem. , 24, 161

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

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

    6. Ladomery. L. F., Ryan, A. J. & Wright, S. E. (1963) J. Pharm.
    Pharmacol., 15, 771

    7. Gage. J. C. (1964) Unpublished report, Imperial Chemical Industries
    Limited

    8. Deichmann, W. B., Clemmer, J. J., Rakoczy, R. & Bianchine, J.
    (1955) A.M.A. Arch. industr. Hlth, 11, 93

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

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

    11.  Day, A. J., Johnson, A. R., O'Halloran, M. W. & Schwartz, C. J.
    (1959) Aust. J. exp. Biol. med. Sci., 37, 295

    12.  Ames, S.R., Ludwig, H. I., Swanson, W. J. & Harris, P. L. (1956)
    Proc. Soc. exp. Biol. (N.Y.), 93, 39

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

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

    15.  Karpliuk, I. A. (1959) Vop. Pitan., 18, 24

    16.  Industrial Bio-Test Laboratories (1964) Progress report to
    Hercules Powder Company

    17.  Imperial Chemical Industries Limited (1964) Unpublished report
    No. IHR/158

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

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


    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (ICSC)
       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)
       Butylated hydroxytoluene (WHO Food Additives Series 35)
       BUTYLATED HYDROXYTOLUENE (JECFA Evaluation)