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    FAO Nutrition Meetings
    Report Series No. 40A,B,C
    WHO/Food Add./67.29




    TOXICOLOGICAL EVALUATION OF SOME
    ANTIMICROBIALS, ANTIOXIDANTS, EMULSIFIERS,
    STABILIZERS, FLOUR-TREATMENT AGENTS, ACIDS AND BASES





    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met at Rome,
    13-20 December, 19651 Geneva, 11-18 October, 19662




                   

    1 Ninth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, 1966 No. 40; 
    Wld Hlth Org. techn. Rep. Ser., 1966, 339

    2 Tenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, 1967, in press; 


    Food and Agriculture Organization of the United Nations
    World Health Organization
    1967


    BUTYLATED HYDROXYTOLUENE

    Synonyms                      BHT

    Chemical Name                 2,6-ditertiarybutyl-p-cresol;
                                  4-,methyl-2,6-ditertiarybutylphenol

    Empirical formula             C15H24O

    Structural formula

    MOLECULAR STRUCTURE 8

    Molecular weight              220.36

    Definition                    Butylated hydroxytoluene contains not
                                  less than 99 per cent. of C15H24O.

    Description                   Butylated hydroxytoluene is a white,
                                  crystalline or flaked solid, having a
                                  characteristic faint aromatic odour.

    Use                           As an antioxidant.

    Biological Data

         The document entitled "Specifications for Identity and Purity and
    Toxicological Evaluation of some Antimicrobials and Antioxidants"
    (FAO/WHO, 1965) contains a monograph on BHT summarizing the data
    available at the end of 1964. Since then a substantial amount of new
    information has become available for consideration. This and other
    data are summarized in the present monograph.

    Biochemical aspects

         Excretion and tissue distribution

         The metabolism of butylated hydroxytoluene (BHT) administered to
    rabbits orally in single doses of 500 mg/kg body-weight was studied. 
    The metabolites 2,6-di-tertbutyle-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 per cent. as glucoronides,
    16.7 per cent. as ethereal sulfates and 6.8 per cent. 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 derative 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
    80-90 per cent. of the dose was recovered in 4 days in the urine and
    faeces. Of the total radioactivity, 40 per cent. appeared in the urine
    of females and 25 per cent. in males. After 4 days approximately 3.8
    per cent. 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 (1 male, 1 female) over a period of 40 hours
    (Daniel & Gage, 1965).

         Groups of two rats (1 male, 1 female) were given 1-5 oral doses
    of 44 mg/kg body-weight BHT on alternate days and each group killed 24
    hours after the final dose. The range of the total dose accounted for
    was 92-103.5 per cent. in males and 92.6-98.6 per cent. in females.
    There was an indication of sex difference in the route of excretion,
    females excreting 19-43 per cent. of the radioactivity in urine and
    males only 3-15 per cent. Eight days after administration of 5 doses
    92 per cent. of the radioactivity had been excreted by males and 97
    per cent. 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 at al., 1965).

         The BHT content of fat and liver of rats given diets containing
    0.5 and 1.0 per cent. BHT for periods up to 35 and 50 days
    respectively: with 0.5 per cent. BHT in the diet, a level of
    approximately 30 ppm in the fat was reached in males and 45 ppm in
    females, with approximately 1-3 ppm in the liver, while with 1.0 per
    cent. BHT the level in the fat was 50 ppm in males and 30 ppm in
    females. On cessation of treatment, the level of BHT in fat fell with
    a half-life of 7-10 days (Daniel & Gage, 1965). The level of BHT in

    the fat reached a plateau at approximately 100 ppm after 3-4 days when
    daily doses of 500 mg/kg body-weight were given by intubation; 200
    mg/kg body-weight/day for 1 week produced a level of about 50 ppm
    (Gilbert & Golberg, 1965).

         When feed containing 500 ppm BHT was given to laying hens, 20 ppm
    was found in the fat fraction of eggs; 100 ppm in the feed resulted in
    residues of less than 5 ppm. In the broiler chicken, over a period of
    21 weeks, the residues in body fat were 55 ppm on the 500 ppm diet and
    less than 5 ppm on the 100 ppm diet (Van Stratum & Vos, 1965).

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

         Stimulation of liver microsomal processing enzymes

         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 body-weight/day. The storage of BHT in fat appeared to
    be influenced by the activity of the processing enzymes. In rats given
    500 mg/kg body-weight daily the level of BHT in fat attained values of
    230 ppm in females and 162 ppm 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 activities
    continued to rise but the BHT content of fat fell to a plateau of
    about 100 ppm in both sexes (Gilbert & Golberg, 1965).

         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).

    Special studies

         Diets containing 0.1 or 0.5 per cent. BHT together with two
    dietary levels of lard (10 and 20 per cent.) were given to mice. The
    0.5 per cent. level of BHT produced slight but significant reduction
    in mean pup weight and total litter weight at 22 days of age. The 0.1
    per cent. 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).

         In a study on the embryotoxicity of BHT three dosing schedules
    were employed: single doses (1000 mg/kg body-weight) on a specific day
    of gestation, repeated daily doses (250-500 mg/kg body-weight) from
    the time of mating throughout pregnancy and daily doses (250-500 mg/kg
    body-weight for mice and 500 and 700 mg/kg body-weight for rats)
    during a 7-10 week period before mating, continuing throughout mating
    and gestation up to the time the animals were killed. No significant
    embryotoxic effects were observed on examination of the skeletal and
    soft tissue of the fully developed foetuses as well as by other
    criteria.  Reproduction and post-natal development were also
    unaffected (Clegg, 1965)

    Acute toxicity

                                                                       

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

    Rat          oral         2 450               Karplyuk, 1960
    Mouse        oral         2 000               Karplyuk, 1960
                                                                       

    Short-term studies

         Rat. BHT (2000 ppm) incorporated in a diet containing 19.9 per
    cent. casein was administered to a group of 8 weeks; a further group
    of 8 rats served as controls. The experiment was repeated with 16.6
    per cent. casein in the diet of further groups for 4 weeks and again
    with 9.6 per cent. casein (and no added choline) for 7 weeks. In all 3
    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. Recovery of hepatic protein after fasting (details not given)
    was also impared in rats on 2000 ppm BHT. Liver lipid content was
    increased with 2000 ppm but not with 200 ppm BHT. A dietary level of
    2000 ppm 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 & Sch÷besch, 1961).

         Groups of 48 weanling rats (24 of each sex) were given diets
    containing 1000 ppm 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

    measurement and histochemical assessments of liver glucose
    -phosphatase and glucose 6-phosphate dehydrogenase activities revealed
    no difference from the control group (Gaunt at al., 1965a).

    Multigeneration study

         Weanling rats (16 of each sex) were fed a diet containing 20 per
    cent. lard and 0, 300, 1000 or 3000 ppm BHT and mated at 100 days of
    age (79 days on test). Ten days after weaning of the first litter the
    animals were again mated to produce a second litter. The offspring (16
    females and 8 males) ware 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 dietary level a 10-20 per cent. reduction in growth rate of
    parents and offspring was observed. A 20 per cent. elevation of serum
    cholesterol levels was observed after 28 weeks, but no cholesterol
    elevation after 10 weeks. A 10-20 per cent. increase in relative liver
    weight was also observed upon killing after 42 weeks on diet. All
    other observations at 3000 ppm and all observations at 1000 ppm and
    300 ppm were comparable with control. All criteria of reproduction
    were normal. No teratogenic effects ware 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 level. The offspring were examined for: litter size,
    mean body-weight, occurrence of still-birth, survival rate and gross
    and microscopic pathology (Frawley, 1967).

    Comments

         After single and repeated oral doses BHT is eliminated almost
    completely in four days in the urine and faeces. The relatively slower
    rate of excretion than that shown by BHA could be due to enterohepatic
    circulation. Results of the analysis of tissues of rats suggest that
    unchanged BHT does not tend to accumulate in the body.

         The browning, thinning and loss of hair reported in one strain of
    rat has not been reproduced in other strains. Reproduction studies
    carried out on several strains of rats and mice fail to reveal
    anophthalmia in the offspring reported earlier in a different strain
    of rats.

         Diet containing 20 per cent. lard and 1000 ppm BHT has no effect
    on serum cholesterol and lipid levels, relative liver and other organ
    weights, liver lipids and growth rate.

         Metabolic studies in man are desirable.

    Evaluation

    Level causing no toxicological effect

         Rat.  1000 ppm in the diet, equivalent to 50 mg/kg
    body-weight/day.

    Estimate of acceptable daily intake for man

                                      mg/kg body-weight1

        Unconditional acceptance            0-0.5

        Conditional acceptance              0.5-2

    REFERENCES

    Akagi, M. & Aoki, I. (1962a) Chem, Pharm. Bull. (Tokyo), 10, 101

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

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

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

    Daniel, J. W. & Gage, J. C. (1965) Fd Cosmet.  Toxicol., 3, 405

    FAO/WHO (1965) FAO Nutrition Meetings Report Series, No. 38;
    WHO/Food Add./24.65

    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., Kohn, F.E. Kay, J. H. & Calandra, J. C. (1965b) 
    Fd Cosmet. Toxicol., 3, 377

    Gaunt. I. F., Feuer, G., Fairweather, F. A. & Gilbert, D. (1965a) 
    Fd Cosmet.Toxicol., 3, 433

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

    Gilbert, D. & Golberg, L. (1965) Fd Cosmet. Toxicol., 3, 417


                   

    1 As sum of BHA and BHT.

    Johnson, A. R. (1965) Fd Cosmet. Toxicol., 3, 417

    Sporn, A. & Sch÷besch, 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

    


    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (ICSC)
       Butylated hydroxytoluene (FAO Nutrition Meetings Report Series 38a)
       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)