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    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 1980 (see Annex I, Refs. 6, 8, 11, 32, 40
    and 54). Toxicological monographs were issued in 1961, 1964, 1965,
    1973 and 1976 and 1980 (see Annex I, Refs. 6, 9, 13, 33, 41 and 55).

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

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Effects on enzymes and other biochemical parameters

         BHT in the diet of Sprague-Dawley rats resulted in a marked
    decrease in the NADPH-cytochrome P-450 reductase activity of isolated
    liver microsomal preparations. This effect was not observed when BHT
    was added in vitro to liver microsomes (Rikans et al., 1981). Rats
    fed 0.4% BHT in their diet showed an increase in GSH-S transferase
    activity in the liver, but not in lungs and kidneys. GSH-reductase
    levels were increased in liver and lungs (Partridge et al., 1982).
    Dietary BHT was also shown to effect the carboxylation process in the
    conversion of rat liver microsomal protein to prothrombin (Takahashi &
    Hiraga, 1981).

         Addition of cyclic GMP (cGMP added as the dibutyl or 8-bromo
    form) to BHT suppressed Mishell-Dulton cultures, effected a reversal
    of the BHT suppression of antibody production (Wess & Archer, 1982).

         The observed increase in tumour-specific antigen activity in the
    colon chromatin of rats treated with 1,2-dimethylhydrazine was
    abolished by simultaneous treatment with BHT (Gabrelak et al., 1981).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

    Mouse

         Groups each of 100 (B6C3F1) mice, equally divided by sex were fed
    diets containing 0, 200, 100 or 5000 ppm (0, 0.02, 0.1 or 0.5% of BHT
    for 96 weeks, followed by a basal diet for six weeks. The diets were
    made by mixing the BHT with CE-2 (CLEA Japan, Inc., Tokyo) diet at the
    appropriate concentration and pelleting. At the end of the test period

    the surviving animals were killed. A complete autopsy was carried out,
    and the principal organs and tissues were examined microscopically.
    Mice that died during the course of the study were also autopsied. In
    addition terminal blood samples were collected for haematological
    examination, and serum clinical biochemistry. Urine samples were also
    examined. During the course of the study, food consumption was similar
    for test and control groups. Body weights of females in the 1000 and
    5000 ppm (0.1 and 0.5% groups were lower than controls, as was the
    body weight of males in the 5000 ppm (0.5%) group. There were minor
    changes in the absolute weight of some organs in the high dose groups
    (salivary glands, heart and kidney). In males the serum GOT and GPT
    levels in the 5000 ppm (0.5%) group were higher than controls. No
    other compound related effects were observed in the haematological,
    serum and urine analysis. Neoplastic lesions were reported in both
    test and control animals. The tumours that occurred with greatest
    frequency were adenomas of the lungs, hyperplastic nodules and
    hepatocellular carcinomas of the liver and malignant lymphomas.
    However, there was no statistically significant difference between the
    BHT treated and control groups for the incidence of any type of tumour
    (Shirai et al., 1982).

    Rat

         Groups of 57 Wistar rats (seven weeks old) of each sex were
    maintained on diets containing 2 500 or 10 000 ppm (0.25 or 1% of BHT
    for 104 weeks. Control groups consisted of 36 rats of each sex. At the
    end of the test period the surviving animals were killed and a
    complete autopsy was carried out. The principal organs and tissues
    were examined microscopically. Terminal blood samples were collected
    for haematological examination and serum clinical biochemistry. Food
    intake was similar for test and control animals, but body weight gain
    was reduced in both male and female rats in the high dose groups.
    Increased relative liver weight was observed in all test animals, and
    decreased spleen weight in the females. Total blood cholesterol was
    increased in all test animals and increased red blood cell counts were
    observed in females. The overall incidence of tumours was slightly but
    not significantly higher in BHT treated rats than in controls. The
    incidence of hyperplastic nodules and of pancreatic carcinomas in
    female rats and of pituitary adenomas and adenocarcinomas in test
    animals was higher than those in controls. However, with the exception
    of the incidence of pituitary adenomas in the low dose females, these
    differences were not significantly different from controls. Since this
    effect was not dose related, it was concluded that BHT, under the
    conditions of this test, was not carcinogenic (Hirose, 1980).

    Potentiation or inhibition of carcinogenesis

         Groups of Swiss mice were given 1000, 250, or 50 mg/kg urethan or
    0.9% NaCl. Seven days later, half the urethan treated animals and half
    the controls received 300 mg/kg BHT i.p. the remaining animals

    receiving corn oil alone. Thirteen weekly injections were given. The
    number of tumours/lung found 14-24 weeks after the initial urethan
    doses was significantly increased in the BHT treated animals. In
    another study, when the interval between injection of the urethan and
    the first treatment with BHT was delayed for six weeks, BHT treatment
    produced more tumours. When the number of BHT injections commencing
    one week after urethan treatment was reduced from 13 to four, the same
    significant increase in rumour yield was observed as in the 13-dose
    study. However, one or two doses of BHT had no significant effect.
    When the mice were pretreated with 13 injections of BHT, and then
    treated with urethan one week later, there was no enhancement of
    tumour yield. Simultaneous administration of BHT and urethan, resulted
    in fewer rumours compared to animals treated with urethan alone. When
    mouse strains (C57BL, C3H and BALB/C) which have a low naturally
    occurring incidence of lung adenoma were treated with urethan and then
    with multiple injections of BHT, the BHT treatment did not
    significantly increase rumour incidence or average numbers of rumours
    per lung (Witschi & Lock, 1979).

         Male Strain A mice were injected i.p. with 500 mg/kg urethan,
    then one week later received repeated injections (one a week for eight
    weeks) of either 300 mg/kg BHT, or BHA, 500 mg/kg, or Vitamin E,
    1000 mg/kg, all dissolved in corn oil. At the termination of the
    study, only BHT was shown to produce a significant increase in rumour
    yield. Although the number of rumours produced by BHA treatment was
    greater than usual, it was not statistically significant. A/J mice
    treated with 3-methylcholanthrene or dimethylnitrosamine, followed by
    treatment with BHT (i.p.), resulted in an increase in rumour yield
    (Witschi et al., 1981). In another study, male A/J mice were injected
    i.p. with a single dose of urethan and then fed either 0.75% BHT, or
    BHA or ethoxyquin in the diet, once a week, or continuously for eight
    weeks. Lung tumours yield was scored four months after the urethan
    treatment. Dietary BHT, but not BHA or ethoxyquin, under either
    conditions of the test, enhanced lung rumour formation. Mice were
    prefed with diets containing either BHA or BHT for two weeks prior to
    urethan treatment, and then maintained on conventional laboratory
    diets for four months. The BHT diet had no effect on tumour yield, but
    the BHA treatment significantly decreased the average number of
    tumours (Witschi, 1981).

         In another study A/J mice were given a single dose of BHT i.p.
    (400 mg/kg), sufficient to cause acute lung damage and produce cell
    proliferation in the lung for six to seven days. Urethan was
    administered continuously by implanted minipumps during this period.
    Continuous presence of urethan during the period of cell division did
    not result in an enhanced number of the rumours. When urethan injected
    mice were dosed i.p. with SKF525A (2-diethylaminoethyl-2-,
    2-di-phenylvalerate hydrochloride) and BHT (SKF inhibits lung cell
    division normally seen following BHT administration), or BHT alone,
    both treatments gave a very significant increase in lung tumour yield
    compared to urethan treated controls (Witschi & Kehrer, 1982).

    Repeated pulmonary cell division brought about by other treatments,
    e.g., 95-100% oxygen, were also shown not to enhance tumour
    development (Witschi & Kehrer, 1982).

         Groups of female Sprague-Dawley rats were treated with either
    7-12-dimethylbenz[a]anthracene (DMBA) or nitrosomethylurea (NMU) and
    then fed diets containing 0 or 0.3% added BHT for 30 weeks. Rats
    treated with DMBA and maintained on the control diet developed 100%
    tumour incidence (mammary gland) by week 27, whereas, those maintained
    on the BHT supplemented diet had an incidence of 54% by the end of the
    study. Dietary BHT had no effect on the incidence of rumours induced
    by NMU treatment (King, McCay & Kosanke, 1981).

    Reproduction and behavioural studies

         Groups each of 46 rats, six weeks old (Wistar outbred, SPF) were
    fed diets containing 0, or 0.5 to 0.9% BHT so that the dietary intake
    of BHT was equivalent to 500 mg/kg during the course of the study. At
    week 19, the F0 generation was mated. Twenty-four hours after birth
    of F1 rats, the size of the litters was reduced to eight, and half of
    the litters wore cross-fostered. Body weight of parents and offspring
    and developmental events of offspring were monitored during the course
    of the study, as well as the reproductive performance of the F0 rats.
    Auditory and visual function and locomotive coordination tests were
    carried out on the F1 generation. The F1 animals were autopsied at
    day 25 of age, and a histological examination made of the brains. Body
    weights and weight gain of test animals were reduced when compared to
    controls, and this persisted during gestation. The duration of
    pregnancy, average body weight, and litter size were similar for test
    and control animals. The average body weight and weight gain of the
    F1 offsprings was significantly reduced in pups nursed by dosed
    mothers. Pups exposed in utero to BHT also showed a relatively
    slower development than controls when fostered with non-dosed mothers.
    Pups exposed to BHT in utero and/or mothers milk showed alterations
    in the behavioural patterns examined as well as higher incidence in
    average number of dead cells in the brain (Meyer & Hansen, 1980).

         Detailed comments were submitted by the Chemical Manufacturers
    Association (CMA) (1983) on studies of the effect of BHT on
    reproduction and teratogenicity. The major comments were concerned
    with the studies of Brunner et al. (1978) and Vorhees et al. (1981)
    previously reviewed by JECFA in 1980 as well as the study by Meyer &
    Hansen (1980).

         In the case of the Brunner et al. and Vorhees et al. study, it
    was concluded that the study showed normal pup survival and
    development in pups raised by rat dams on diets containing 0.125% BHT.
    Normal post-weaning development was observed in pups raised by rat
    dams on diets containing 0.25% BHT, although increased post-weaning
    mortality occurred in pups raised by dams on the 0.25% and 0.5% diet;

    developmental delays occurred in pups in the 0.5% group. In the case
    of the Meyer and Hansen (1980) study, developmental delays were seen
    in rats raised by rat dams on diets containing 0.5% BHT. At the 0.25%
    and 0.5% level, the effect may be due either to toxic effects of BHT
    on the rat dam, or direct toxicity during lactation. A number of
    questions were also raised about the design of the Brunner or Vorhees
    study. These are: (1) the pup selection, in which all litters of fewer
    than eight live pups were discarded; (2) the excess mortality was
    reported in terms of pup count rather than affected litters. The data
    from this study have been audited by the United States FDA (1983). It
    was concluded that the raw data support the authors observations of
    increased mortality in the mid-dose and high-dose BHT offspring.
    However, excess mortality occurred in a limited number of litters;
    e.g., in the 0.5% group, of the 60 deaths reported in 19 litters, 49
    of the deaths occurred in five litters, and in the 0.25% group of the
    42 deaths, 21 occurred in two litters, and at the 0.125% level of the
    12 deaths, 11 occurred in one litter. It was also noted that in the
    high dose group; that there was an increased number of litters with
    eight pups or less, and no litters larger than 12 pups. In the other
    dose groups, the litter size was comparable to controls.

         In the case of the Meyer & Hansen (1980) study, the CMA comments
    note that the level of BHT used in the study caused toxicity in the
    dams, which appears directly or indirectly to affect the pups. Reports
    of teratogenicity studies and/or one generation reproduction studies
    in several strains of mice and rats as well as a three generation
    reproduction study in rats were also submitted in the comments to
    support a "no effect" level of 0.1% BHT in the diet.

    Special studies on the effect of BHT on the thyroid

         Male MOL/WIST SPF rats, outbred strain (approximately 200 g) were
    used for the study. BHT was added to a semi-synthetic diet in which
    the iodine content was controlled at about 12 g/100 g (nutritional
    requirement for the rat is 15 g/100 g). In one study, rats were fed
    0, 500 or 5000 ppm (0, 0.05 or 0.5%) BHT in the diet for eight, 26 and
    90 days, and the uptake of 125I by the thyroid determined. The
    presence of BHT in the diet resulted in a marked increase in the
    uptake of 125I at all time periods studied. When rats were fed BHT in
    diets containing varying amounts of iodine (12, 150 or 300 g/100 g)
    for 30 days there was a significant increase in thyroid weight in BHT
    treated animals when compared to controls. BHT in the diet of rats
    increased liver and thyroid weights at 5000 ppm (0.5%) of the diet,
    but only thyroid weight at 500 ppm (0.05%). BHT did not change levels
    of T3 and T4 in the blood. The biological half life of thyroxine was
    increased after 13 days on a BHT diet but returned to normal after 75
    days. Electron microscopy of the thyroid glands of rats exposed to
    dietary BHT (5000 ppm (0.5%)) for 28 days showed an increase in the
    number of follicle cells (Sondergaard & Olsen, 1982).

    Special studies on haemorrhagic toxicosis

         The LD50 (i.p.) for BHT showed considerable differences for
    strains of inbred and non-inbred male mice.

    Strain                         LD50 (mg/kg)
                                                  


    DBA/2N (inbred)                         138

    BALB/cNnN (inbred)                    1 739

    C57BL/6N (inbred)                       917

    ICR-JCL (non-inbred)                  1 243

    In all cases death occurred four to six days after administration of
    BHT, and was accompanied by massive oedema and haemorrhage in the lung
    (Kawano et al., 1981).

         Male rats (Sprague-Dawley) were fed diets containing 0 or 1.2%
    BHT for one week. BHT treated rats showed haemorrhages in most organs.
    There was a significantly increased leakage of Evans blue into the
    epididymis. In addition, inhibition of ADP induced platelet
    aggregation and decreased platelet factor 3 availability was observed.
    Plasma prothrombin factors were decreased, but fibronolytic activity
    was unchanged (Takahashi & Hiraga, 1981).

         In another study in which the haemorrhagic response was studied
    in a number of strains of rats (Sprague-Dawley, Wistar, Donryu and
    Fischer), mice (ICR, ddY, DBA/c, C3H/He, BALB/CaAn and C57BL/6), New
    Zealand White-Sat rabbits, beagle dogs, and Japanese quail fed diets
    containing BHT (1.2% of the diet for rats and mice), (1% of the diet
    for quail), (170 or 700 mg/kg bw for rabbits) and (173, 400 or 760
    mg/kg bw for dogs) for a period of 14-17 days. Haemorrhagic deaths
    occurred among male rats of all strains and female rats of the Fischer
    strain. Female rats of the Donryu, and Sprague-Dawley strain showed no
    obvious haemorrhaging. No haemorrhagic effects were noted in rabbits
    or dogs (Takahashi et al., 1980).

    Comments

         A recent lifetime study in mice and a 104-week study in rats
    showed that under the conditions of the test BHT was not carcinogenic.

         Additional studies are available on the role of BHT in the
    enhancement of lung tumour yield by chemical carcinogens, in
    susceptible species of mice. BHT has also been shown to be effective
    as a promoting agent in this assay system when the test animals were
    treated with either polycyclic hydrocarbons or nitrosamine. The lowest

    dose at which BHT can act as a promoter of urethan induced lung
    tumours in the mouse has not been established. No additional studies
    are available on the possible promotion of hepatic carcinogenesis
    caused by chemical carcinogens. BHT only acts as a promoter when
    administered after exposure to the chemical carcinogen, but not
    before. BHT has also been shown to inhibit the effect of some chemical
    carcinogens. The protective effect may be associated with changes in
    the metabolism of the carcinogen resulting from enzyme induction
    caused by BHT. More information is required on the conditions as well
    as the mechanisms of the inhibitory or promotional activity of BHT on
    chemical carcinogens, to assist in interpretation of these studies
    before they provide a useful basis for the toxicological evaluation.

         A recent study on the behavioural and development effects of BHT
    on rats exposed in utero during lactation showed that BHT caused a
    significant decrease in body weight gain of both offspring and parent.
    Altered behavioural patterns, as well as brain lesions were noted in
    the offspring. However, only one dose level (0.5% BHT in the diet) was
    used in this study, and this level was toxic to the dams. A detailed
    analysis of data from the study of Vorhees et al. (1981) showed that
    at both the mid and high (0.5% and 0.25%) dose levels, the excess
    mortality may reflect litter effects. The data indicate a "no effect"
    level for BHT-induced reproductive effects to be 0.1% of the diet of
    rats. A lifetime feeding study with rats, which involves a single
    generation reproduction study, is under way. The data from this study
    will provide additional information to support a "no effect" level
    from BHT in reproduction studies in the rat.

         The haemorrhagic effects of massive doses of BHT seen in certain
    species of mice but not in dogs and certain species of rats may be
    related to its ability to interfere with vitamin K metabolism.

         Rats exposed to 500 or 5000 ppm (0.05 or 0.5% of dietary BHT
    showed a significant increase in thyroid weights, as well as the
    ability of the thyroid to take up iodine. However, lifetime studies in
    rats maintained on diets containing up to 10 000 ppm (1%) BHT have not
    shown adverse effects on the thyroid.

         Previously reported studies on induction of microsomal enzymes,
    reproduction and behavioural effects provide a basis for setting a "no
    effect" level.

    EVALUATION

    Level causing no toxicological effect

         Mouse:    5000 ppm (0.5%) in the diet, equivalent to 250 mg/kg.
         Rat  :    1000 ppm (0.1%) in the diet, equivalent to 50 mg/kg.

    Estimate of temporary acceptable daily intake for man

         0-0.5* mg/kg bw.

    FURTHER WORK OR INFORMATION

    Required by 1986.

         Submission of the lifetime feeding study known to be in progress
    which includes a single generation reproduction study.

              

    *    Group ADI: As BHA, BHT, and TBHQ, singly or in combination.

    REFERENCES

    Babryelak, T., Pumo, E. D. & Chiu, J. F. (1981) Changes in tumor-
         specific nuclear antigen activity in carcinogen-treated colon by
         tumor promotor and carcinogen inhibitors, Cancer Research,
         41, 3392-3394

    Hirose, M. et al. (1981) Chronic toxicity of butylated
         hydroxytoluene in Wistar rats, Fd. Cosmet. Tox., 19,
         147- 151

    Kawano, S., Nakao, T. & Kiraga, K. (1981) Strain differences in
         butylated hydroxytoluene induced deaths in male mice,
         Tox. Appl. Pharm., 61, 475-479

    King, M. M., McCay, P. B. & Kosanke, S. D. (1981) Comparison of the
         effects of butylated hydroxytoluene on N-nitrosamethylurea and
         7,12-dimethylbenz[a]anthracene-induced mammary tumours, Cancer
         Letters, 14, 219-226

    Nakagawa, Y., Hiraya, K. & Suga, T. (1980) Biological fate of BHT-
         binding of BHT to nucleic acid in vivo, Biochemical
         Pharmacology, 29, 1304-1306

    Meyer, O. & Hansen, E. (1980) Behavioral and developmental effects of
         butylated hydroxytoluene dosed to rats in utero and in the
         lactation period, Toxicology, 16, 247-258

    Partridge, C. A., Dao, D. D. & Awasthi, Y. C. (1982) Induction of
         glutathione-linked detoxification system by dietary antioxidants,
         Fed. Proc., 41, Abstract 2152

    Rikans, L. E. et al. (1981) Effects of butylated hydroxytoluene and
         acetylaminofluorene on NADPH-cytochrome P-450 reductase activity
         in rat liver microsome, Fd. Cosmet. Toxicol., 19, 89-92

    Shirai, T. et al. (1982) Lack of carcinogenicity of butylated
         hydroxytoluene on long-term administration to B6C3F1, Fd.
         Chem. Tox., 20, 861-865

    Sondergaard, D. & Olsen, P. (1982) The effect of butylated
         hydroxytoluene (BHT) on the rat thyroid, Toxicology Letters,
         10, 239-244

    Takahashi, O., Hayashida, S. & Hiraga, K. (1980) Species differences
         in the haemorrhagic response to butylated hydroxytoluene,
         Fd. Cosmet. Tox., 18, 229-235

    Takahashi, O. & Hiraga, K. (1981) Haemorrhagic toxicosis in rats given
         butylated hydroxytoluene, Acta Pharmacol. Toxicol., 49, 14-20

    Takahashi, O. & Hiraga, K. (1981) Inhibition of phylloquinone
         expoxide-dependent carboxylation of microsomal proteins from rat
         liver by 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadenone,
         Fd. Cosmet. Tox., 19, 701-706

    Wess, J. A. & Archer, D. L. (1982) Evidence from in vitro
         immunologic assays that some phenolic food additives may function
         as anti-promotors by lowering intracellular cyclic GMP levels,
         Proc. Soc. Exp. Biol. Med., 170, 427-430

    Witschi, H. P. & Lock, S. (1979) Enhancement of adenoma formation in
         mouse lung by butylated hydroxytoluene, Tox. Appl. Pharm.,
         50, 391-400

    Witschi, H. P., Hakkinen, P. J. & Kehrer, J.P. (1981) Modification of
         lung tumor development in A/J mice, Toxicology, 21, 37-45

    Witschi, H. P. (1981) Enhancement of tumor formation in mouse lung by
         dietary butylated hydroxytoluene, Toxicology, 21, 95-104

    Witschi, H. P. & Kehrer, J. P. (1982) Adenoma development in mouse
         lung following treatment with possible promoting agents, J. Am.
         Coil. Toxicology, 1, 171

    Vorhees, C. V. et al. (1981) Developmental neurobehavioral toxicity of
         butylated hydroxytoluene in rats, Food Cosmet. Tox., 19,
         153-162
    


    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (BHT) (WHO Food Additives Series 15)
       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)