IPCS INCHEM Home

    BUTYLATED HYDROXYTOLUENE (BHT)

    Explanation

         This substance has been evaluated for acceptable daily intake for
    man (ADI) by the Joint FAO/WHO Expert Committee on Food Additives in
    1961, 1964, 1965, 1973 and 1976 (see Annex I, Refs. 6, 8, 11, 32 and
    40). Toxicological monographs were issued in 1961, 1964, 1965, 1973
    and 1976 (see Annex I, Refs. 6, 9, 13, 33 and 41).

         Since the previous evaluations, additional data have become
    available and are summarized and discussed in the following monograph
    addendum.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Effects on enzymes and other biochemical parameters

         Groups of 16-24 Swiss male mice (25-30 g) received a single i.p.
    injection of BHT in corn oil (62.5, 215, or 500 mg/kg bw) or corn oil
    only (Tocopherol stripped 0.5 ml). Three days later the mice were
    sacrificed. After BHT, wet lung weights were increased to 120% of
    control, as were dry lung weights. There were significant increases in
    DNA content and level of non-protein sulfhydryl (133-156% of control).
    Superoxide dismutase and other oxidative enzyme levels were increased.
    The authors concluded that BHT apparently increased inflammatory and
    reparative-proliferative processes of the lung (Omaye et al., 1977).

         BHT (500 mg/kg/day) was administered by gavage to groups of young
    Wistar male and female rats (60-80 g) for seven days and the animals
    were housed in metabolism cages. Control animals received corn oil
    vehicle only. They were then sacrificed and liver enzymes (aniline 4
    hydroxylase, biphenyl-4-hydroxylase, ethyl morphine N-demethylase, and
    4-methyl umbelliferone glucoronyl transferase) were assayed and the
    cytochrome P-450-CO interaction spectrum evaluated. Urinalysis, using
    gas chromatography to assay for D-glucaric acid, D-glucuronic acid,
    1-gulonic acid, xylitol and L-ascorbic acid was conducted.
    Administration of BHT enhanced all the parameters measured with the
    exception of the hepatic microsomal protein content. BHT was a more
    potent inducer of xenobiotic metabolism in female rats (Lake et al.,
    1976).

         Groups of NMRI mice (25-35 g) and Wistar rats (160-320 g)
    received BHT as a single dose of 500 mg/kg dissolved in soya bean oil,
    either i.p. or by gavage. Four days later, radiolabelled C14
    thymidine was given. After 90 minutes, the animals were sacrificed,
    lungs removed and DNA levels were measured. In mice, DNA synthesis was
    equally increased in males and females by oral or i.p. administration.
    Although lung weight was increased, the concentration of DNA was not
    affected. In rats, no effect was seen in males and only a slight
    increase in females (Larsen & Tarding, 1977).

    TOXICOLOGICAL STUDIES

    Special studies

    Potentiation or inhibition of carcinogenesis

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

         A group of 93 rats (22 days old) received 0.5% BHT or control
    diets for 407 days following 18 days of administration of 2-acetyl-
    aminofluorene (0.02%). No control group received BHT only without
    prior AAF feeding. Prolonged feeding of BHT diet after 2-AAF produced
    a significant enhancement of hepatic carcinogenesis as shown by
    increased number of rats with liver tumours (Peraino et al., 1977).

    Mutagenicity

         BHT was found not to be mutagenic using Saccharomyces
    cerevisiae, D4 and Salmonella typhimurium, TA-1535, TA-1537 and
    TA-1538, with and without the addition of mammalian metabolic
    activation (Brusick, 1975).

         BHT was fed at the 5% level in a sex-linked recessive lethal test
    in Drosophila. About 0.27% lethals were found in the BHT treated
    flies (out of 8897), 0.29% lethals were found in the control and 5.69%
    lethals were found in the positive control dosed with 100 ppm
    ethylmethane sulfonate. It was concluded that BHT was not mutagenic
    (Brusick, 1975).

         The effect of BHT on excision repair synthesis was tested in
    cultured human lymphocytes irradiated with u.v. light. Irradiated
    cells cultured in the presence of BHT had decreased excision repair
    based on lower incorporation of tritiated thymidine into DNA with
    increasing concentration of BHT in the culture medium. In this study,
    BHT also inhibited semi-conservative DNA synthesis (Daugherty et al.,
    1978).

         A study was conducted to confirm a reported radiosensitizing
    effect of BHT on Drosophila sperm, using three radiation doses (1.2,
    2.4 and 3.6 KRDS). The mutagenic activity of BHT was also tested in
    the sex-linked recessive lethal system. Ten-day-old males of
    D. melangaster received 0.2 l of saline in ethanol or 0.001% BHT in
    the solvent. They were exposed to gamma rays from a 137CS source at
    4.1 rad/sec. They were mated to four females and two successive one
    day broods were tested to obtain sensitivity of mature and slightly
    immature sperm, after discarding xo males and non-disjunction females
    remaining. F1 flies were pooled and F2 females obtained after
    individual brother/sister mating. The absence of bar-eyed males
    indicates the presence of sex-linked recessive lethals. There was no
    shift in the sex ratio of the F1 generation. Without irradiation, BHT
    seems to enhance the incidence of xo males. BHT almost doubled the
    incidence of sex-linked lethals over saline-injected controls, but
    this was not statistically significant. In comparison with gamma rays,
    BHT had no influence on the sex ratio of F1 flies, and seemed to have
    a negligible effect on the incidence of radiation loss of xB
    chromosome. At all doses BHT exhibited a pronounced radiosensitization
    of sex-linked recessive lethals (Prasad & Kamra, 1974).

    Reproduction and behavioural studies

         Breeding pairs of Sprague-Dawley rats (200-220 g) received Purina
    chow supplemented with 0.5, 0.25 or 0.125% BHT ad libitum beginning
    from the week before mating and continuing in females through
    lactation and weaning of the pups. Growth rates and mortality were
    adversely affected. Pre-weaning pups born of mothers at the highest
    dose level (0.5%) of BHT weighed significantly less than controls at
    ages seven, 14 and 21 days. The total number of pups dying on study,
    born of dams receiving 0.5 or 0.2% BHT, was significantly higher than
    controls. Behavioural tests were conducted consisting of righting
    reflex, pivoting cliff avoidance, startle response, swimming, open
    field, running, wheel activity, roto-rod, active avoidance, position
    discrimination and passive avoidance. For pre-weaning testing, no
    differences were noted at the low or mid dose groups. At the high dose
    level, there was significant increase in surface righting time,
    delayed forelimb swimming development and a trend to less activity in
    open field tests. In post-weaning tests, males in 0.25% BHT groups
    showed an effect on passive avoidance, with more partial re-entries
    into compartments where shocked. For all other tests, there were no
    statistical differences, suggesting that BHT had no effect on basic
    motor coordination, active avoidance acquisition, or extinction
    performance (Brunner et al., 1979).

    Effect on lung

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

         Following acute exposure to BHT, the initial sequence of events
    involves infiltration of type I (squamous) epithelial cells followed
    by multifocal necrosis and destruction of the blood barrier. A
    detailed discussion of the sequence of tissue changes and repair
    mechanisms is given. It is stated that the susceptibility of the
    squamous epithelium to injury is similar to that seen after oxygen
    exposure, radiation exposure, and treatment with blood-borne
    bleomycin, but the recovery pattern is quite different. BHT is thought
    to cause cell lysis and death as a result of interaction with the cell
    membrane (Anonymous, 1978).

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

    Immunological studies

         In vitro BHT (50 g/culture) suppressed the plaque-forming cell
    response of mouse spleen cell cultures as measured by the method of
    Mishell & Dutton (Archer et al., 1977).

    Carcinogenicity

         Groups of 50 Fischer 344 rats/sex and 50 B6C3F1 mice/sex received
    3000 ppm or 6000 ppm BHT in the diet. The compound was mixed with
    autoclaved lab meal containing 4% fat. A control group of 20 animals
    per sex received lab meal only. The rats were on study for 105 weeks
    and the mice for 107 or 108 weeks. There was a dose-related depression
    in the body weights of the rats and mice of both sexes throughout the

    study. In rats there was no significant effect of BHT on mortality. In
    male mice BHT was significantly associated with decreased mortality,
    while BHT feeding had no effect on mortality in female mice. There was
    a high dose-related incidence of hepatocytomegaly and hepatocellular
    degeneration and necrosis in the liver of male mice, but the incidence
    of hepatocellular carcinomas was reduced in a dose-related manner. The
    incidence of alveolar/bronchiolar carcinomas or adenomas was
    significantly higher than controls in the low dose, but not the high
    dose female mice. BHT feeding was related to a significant reduction
    in the incidence of sarcomas of multiple organs in female mice. Four
    adenomas of the eye/lacrimal gland were observed in the high dose male
    mice and in two low dose females but not in corresponding controls.
    The historical incidence of this tumour was 1.2%. Since the lacrimal
    gland was only examined microscopically in animals with grossly
    apparent lesions, the report states that the lacrimal gland tumours
    could not be clearly related to BHT administration. There was an
    increased incidence of focal alveolar histiocytosis in treated rats,
    especially in high dose females. The incidence of adenomas of the
    pituitary was significantly reduced in female rats with BHT
    administration. The report concluded that no neoplastic lesions
    occurred in rats or mice that could be clearly related to BHT
    administration. Under the conditions of the test, there were increased
    incidences of focal alveolar histiocytosis in dosed male rats which
    may have been related to the administration of BHT. BHT was not,
    however, carcinogenic for F344 rats or B6C3F1 mice of either sex. The
    report was evaluated by the Data Evaluation/Risk Assessment subgroups
    of the Clearinghouse on Environmental Carcinogens. The subgroup
    accepted the conclusions of the report but, because of the widespread
    human exposure, evidence of hepatotoxicity and a suggestion of a
    tumorigenic effect in the lung, the subgroup suggested that the
    compound be considered for retest by the chemical selection working
    group (Anonymous, 1979).

    Short-term studies

    Rat

         Groups of male Sprague-Dawley rats (six weeks of age) received
    BHT in a semi-synthetic diet at concentrations ranging from 0.58% to
    1.44% or control diet only. Deaths occurred within 40 days, at levels
    of 0.69% or greater. Spontaneous massive bleeding to the pleural and
    peritoneal cavities, or as external haemorrhage, was observed in all
    dead or dying animals. The prothrombin index was decreased as the
    daily dose of BHT was increased. Mild diarrhoea was noted after four
    days. Rough hair coat, and redness of urine was noted. Death was due
    to haemorrhage and was classified by the authors as a secondary type
    of toxicity, probably due to a decrease in prothrombin concentration.
    According to the authors, the effect seems to depend on strain of rats
    and dietary concentration (Takahashi & Hiraga, 1978).

    Long-term studies

    (See also under "Carcinogenicity")

    Rats

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

         Groups of 40 JCL strain rats/sex/group, reared under a barrier
    system and four weeks of age at the start of the study, received
    either control diet or BHT at 0.005, 0.062, 0.32% in the diet. Of each
    group of 40, 15 received compound for a "lifetime", 10 for 24 months,
    and five each for three, six or 12 months. At interim and final kill,
    liver, kidney, heart, spleen, thyroid and caecum weight were
    determined as were haematology, serum biochemistry, urinalysis, and
    histological investigation of the tissues. At 24 months, heart, liver,
    kidney, spleen, pituitary, thyroid, adrenal, testes, prostate and
    brain were weighed, haematological and biochemical measurements
    conducted and histopathology done. There was an increase in liver
    weight, serum cholesterol, serum K+ and histological changes in
    liver and kidney at the 0.32% dietary level. There was no change in
    quantity of food intake, body weight gain, mortality during feeding or
    mean life span and no finding suspicious of tumour induction. There
    was no indication of a dose-related trend in tumour prevalence in
    either 24-month or "life span" groups. The tumours found were said to
    be typical of those described in aged rats. It is to be noted that the
    number of surviving rats is small and that tumour data include both
    life span groups and animals dead or sacrificed moribund during the
    six, 12 and 24 month feeding. The available data do not list the
    number of each of the individual tissues examined, although the number
    of rats is listed. The data show a tendency for a decreased number of
    tumours per rat at higher dose levels (Hiraga, 1978).

         Six-week-old random bred Wistar rats were allocated to groups of
    57 males and 57 females fed control diet and 36 males and 36 females
    fed BHT at either 0.25% or 1% in the diet for 104 weeks. Survival by
    tests groups was between 40% and 68%. A variety of tumours were noted
    on histopathological examination at the end of the study, with no
    dose-related response in either type of tumour or total number as
    compared to controls (Shibata et al., 1979).

    Comments

         Several new studies were available on BHT, including a
    behavioural study in newborn rats that had been exposed to the
    material in utero and during lactation. Decreased pup survival and
    slight behavioural effects were noted at levels above 0.1% of the
    diet. It was noted that behavioural effects were not seen in newborn
    monkeys whose mothers had been treated with the chemically related
    BHA.

         At its twentieth meeting, the Committee had noted that BHT had
    been reported to enhance the occurrence of lung adenomas in mice. At
    that meeting, it had considered that BHT was not likely to be
    carcinogenic but had requested additional long-term studies. Recently
    completed long-term studies in mice and rats have been negative and
    confirm the view that BHT is not carcinogenic.

         Two series of studies with BHT have demonstrated that it enhances
    the effect of certain chemical carcinogens. In one study, it was shown
    that mice initially receiving injections of the carcinogen urethane
    developed more lung adenomas if treated subsequently for some weeks
    with BHT. In another study, rats treated with low levels of the
    carcinogen N-fluorenyl acetamide and subsequently with BHT developed
    more hepatomas, more rapidly than with the carcinogen alone.

         In these studies, it is implied that BHT is a "promoting" agent.
    Possible mechanisms of action include enzyme induction and the
    production of hyperplasia and hypertrophy in both the lung and the
    liver by BHT.

         The phenomenon of "promotion" of carcinogenesis in various
    systems, including the skin and urinary bladder, as well as these
    examples, are attracting much attention in cancer research. Mechanisms
    of action, although under intense study, are not yet understood.
    In addition, since BHT has been shown to inhibit the action of
    carcinogens under other conditions, it is felt premature to use such
    information for toxicological evaluation.

         As regards effects on microsomal enzymes, reproduction and
    behaviour, a no-effect level of 0.1% equal to 50 mg/kg in the diet of
    rodents can be set.
    

    EVALUATION

    Levels causing no toxicological effect

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

    Rat:      1000 ppm (0.1%) in the diet, equivalent to 50 mg/kg bw

    Estimate of acceptable daily intake for man

    0-0.5* mg/kg bw**

    FURTHER WORK OR INFORMATION

    Required by 1981

    (1) A study to clarify the effects on rat pup survival.

    (2) Elucidation of the significance of the behavioural effects
    observed in newborn rats.

              

    *    BHT, TBHQ, or the sum of the three compounds.
    **   Temporary.
    

    REFERENCES

    Adamson, I. Y. R., Bowden, D. H., Cote, M. G. & Witshi, H. Lung injury
         induced by butylated hydroxytoluene: Cytodynamic and biochemical
         studies in mice. Lab. Invest., 36: 26-32, 1977

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

    Anonymous. National Cancer Institute. Bioassay of butylated
         hydroxytoluene (BHT) for possible carcinogenicity. DHEW
         Publication No. (NIH) 79-1706, 1979

    Archer, D. L., Smith, B. G. & Bukovic-Wess. Use of in vitro antibody
         producing system for recognizing potentially immunosuppressive
         compounds. Int. Arch. Allergy, 56: 90-93, 1978

    Brunner, R., Vorhees, C. & Butcher, K. Psychotoxicity of selected food
         additives and related compounds. Report prepared under FDA
         contract 223-75-2030

    Brusick, D. Mutagenic evaluation of compound FDA 71-25 (butylated
         hydroxytoluene). Unpublished report from Litton Bionetics,
         Incorp., submitted to WHO by Eastman Chemical Products, Inc.,
         1975

    Crampton, R. F., Gray, R. J. B., Gray, P., Grasso, R. & Parke, D. V.
         Long term studies on chemically induced liver enlargement in the
         rat. 1. Sustained induction of microsomal enzymes and absence of
         liver damage on feeding phenobarbitone or butylated
         hydroxytoluene. Toxicology, 7: 289-306, 1977

    Daugherty, J. P., Davis, S. & Yielding, K. L. Inhibition by butylated
         hydroxytoluene of excision repair synthesis and semiconservative
         DNA synthesis. Biochem. biophys. Res. Commun., 80(4):
         963-969, 1978

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

    Lake, B. G., Longland, R. C., Gangolli, S. D. & Lloyd, A. G. The
         influence of some foreign compounds on hepatic xenobiotic
         metabolism and the urinary excretion of D-glucoronic acid
         metabolites in the rat. Toxicol. appl. Pharmacol., 35:
         113-122, 1976

    Larsen, J. C. & Tarding, F. Stimulation of DNA synthesis in mouse
         and rat lung following administration of butylated
         hydroxytoluene. Archives of Toxicol. Suppl., 1: 147-150,
         1978 (Proceedings of the European Soc. of Toxicology Meeting held
         in Copenhagen, 19-22 June 1977)

    Malkinson, A. M. Prevention of BHT-induced lung damage in mice by
         cedar terpene administration. Pre-print of paper accepted for
         publication in Toxicol. appl. Pharmacol., 1979

    Omaye, S. T., Reddy, A. & Cross, C. E. Effect of butylated
         hydroxytoluene and other antioxidants on mouse lung metabolism.
         J. Toxicol. Environ. Health, 3: 829-836, 1977

    Prasad, O. & Kamra, O. P. Radio sensitization of Drosophila sperm by
         commonly used food additives, butylated hydroxyanisole and
         butylated hydroxy toluene. Int. J. Radiat. Biol., 25: 67-72,
         1974

    Peraino, C., Fray, R. J. M., Staffeld, E. & Christopher, J. P.
         Enhancing effects of phenobarbitone and butylated hydroxytoluene
         on 2-acetylaminofluorene induced hepatogenesis in the rat. Food
         and Cosmetics Toxicol., 15: 93-96, 1977

    Shibata, M., Hagiwara, A., Miyata, Y., Imaida, K., Arai, M. & Ito, N.
         Experimental study on carcinogenicity of butylated hydroxy
         toluene (BHT) in rats. Translation of the Proceedings of the 38th
         Annual Meeting of the Japanese Cancer Assoc., Tokyo, September
         1979

    Takahashi, D. & Hiraga, K. Dose response study of hemorrhagic death by
         dietary butylated hydroxytoluene (BHT) in male rats. Toxicol.
         appl. Pharmacol., 43: 399-406, 1978

    Witschi, H. & Cote, M. G. Biochemical pathology of lung damage
         produced by chemicals. Fed. Proc., 35(1): 89-94, 1976
    


    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (BHT) (WHO Food Additives Series 18)
       Butylated hydroxytoluene (BHT) (WHO Food Additives Series 28)
       Butylated hydroxytoluene (BHT) (WHO Food Additives Series 42)
       Butylated Hydroxytoluene (BHT) (IARC Summary & Evaluation, Volume 40, 1986)