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    BUTYLATED HYDROXYANISOLE (BHA)

    EXPLANATION

         This substance was evaluated for acceptable daily intake for man
    by the Joint FAO/WHO Expert Committee on Food Additives at its sixth,
    ninth, seventeenth, twentieth, twenty-fourth, twenty-sixth and
    twenty-seventh meetings (Annex 1, references 6, 11, 32, 41, 53, 59,
    and 62). Toxicological monographs or monographs addenda were published
    after the sixth, seventeenth, twentieth, twenty-fourth, and
    twenty-seventh meetings (Annex 1, references 6, 33, 42, 54 and 63).

         At the twenty-seventh meeting of the Committee (Annex 1,
    reference 62), the temporary ADI of 0-0.5 mg/kg b.w. (group ADI with
    BHT and TBHQ) was maintained pending adequate studies to determine if
    the induction of hyperplasia, papilloma, and carcinoma by BHA in the
    forestomach of the rat and hamster are relevant for evaluating the
    safety of BHA for man. It was specifically requested that studies be
    submitted to show whether or not the hyperplasia is induced in the
    stomach of species that do not have a forestomach, such as the dog,
    pig, and monkey, as well as studies to determine the mechanisms
    involved in the effect of BHA on the forestomach. A multigeneration
    reproduction study was also requested.

         Since the previous evaluation additional data, including the
    results of some of the studies requested by the twenty-seventh
    Committee, have become available and are summarized and discussed in
    the following monograph addendum.

    BIOLOGICAL DATA

    Biochemical aspects

    Effects on enzymes and other biochemical parameters

         Mice fed diets containing 0.75% BHA showed no change in hepatic
    cytochrome P-450 levels, aminopyrine demethylase activity, or
    benzo(a)pyrene monooxidase activity. However, there were significant
    increases in other microsomal mixed-function oxidases (NADPH
    cytochrome reductase and aniline hydroxylase) and other enzyme
    activities (glutathione reductase and hepatic cytosol glutathione
    S-transferase). There was also a marked increase in the level of
    nonprotein thiol levels in the liver, lung, kidneys, and small
    intestine (Cha et al., 1983).

         Liver microsomal preparations from rats fed BHA did not show an
    increased rate of formation of reactive products from benzo(a)pyrene
    that bind to calf thymus DNA, as has been observed with microsomal
    preparations from rats fed other antioxidants (Dock et al., 1982a;
    Kahl & Kahl, 1983).

         Addition of BHA to the diet of mice altered the components of the
    monooxygenase system in liver nuclei and inhibited binding of
    benzo(a)pyrene metabolites to nuclear macromolecules on incubation
    with liver nuclei from the BHA-fed mice. However, there was no
    inhibition of binding of benzo(a)pyrene metabolites to macromolecules
    when benzo(a)pyrene with added DNA was incubated with liver microsomes
    from BHA-fed mice (Hennig et al., 1983).

         Dietary BHA (7.5 g/kg b.w. of diets of mice for 14 days) resulted
    in a 12-fold increase in the concentration of the predominant hepatic
    glutathione S-transferase. Its rate of synthesis and the mRNA for this
    protein were also increased (Pearson et al., 1983).

         In a study comparing the effects of dietary BHA on liver
    microsomal enzymes and benzo(a)pyrene metabolism in male and female
    mice, both sexes appeared to respond similarly to BHA feeding
    (Dock et al., 1982b).

         Lung microsomal preparations from female mice fed diets
    containing 0.5% BHA showed decreased benzo(a)pyrene metabolism and
    altered ratios of the different metabolites (Sydor et al., 1984).

         Dietary or topically-applied BHA caused an inhibition in
    epidermal ornithine decarboxylase activity induced by
    12-1-tetradec-anoylphorbol-13-acetate in mice (Kozumbo et al.,
    1983).

         BHA suppresses murine in vitro immune responses by inhibiting
    guanylate cyclase function. The suppression was reversed by addition
    of either exogenous dibutyryl cGMP or Ca+2 to the cultures
    (Wess & Archer, 1982).

    Toxicological studies

    Special studies on the effect of BHA on the stomach

    Rats

         Groups of 50 male F344 rats from Charles River Japan, Inc., 6
    weeks of age, were maintained on powdered diets containing 0, 0.125,
    0.25, 0.5, 1, or 2% BHA. After 104 weeks the surviving animals were
    killed. These animals plus all other animals that died during the
    study were autopsied. The oesophagus and stomach from all animals were
    examined histologically.

         No treatment-related clinical signs were observed during the
    course of the study. Reduction in weight gain in BHA-treated animals
    was dose-related. Average food intake of test and control animals was
    comparable. BHA had no effect on survival. Histologically, the
    epithelial lesions which were observed in the forestomach were
    classified into three categories, hyperplasia, papilloma, and squamous
    cell carcinoma. No metastases were reported. The incidences of
    proliferative and neoplastic lesions in the forestomach were
    dose-related, as summarized in the following table (Ito et al.,
    1986):

                                                                                                  
                                                Number (%) of rats with changes
                                                      in the forestomach
                                                                                               
                       Effective
                       number                                          Squamous cell
    % BHA in diet      of ratsa        Hyperplasia      Papilloma      carcinoma
                                                                                              

    0                  50              0(0)             0(0)           0(0)
    0.125              50              1(2)             0(0)           0(0)
    0.25               50              7(14)b           0(0)           0(0)
    0.5                50              16(32)c          0(0)           0(0)
    1.0                50              44(88)c          10(20)b        0(0)
    2.0                50              50(100)c         50(100)c       11(22)c
                                                                                              

    a    Rats surviving to > 50 weeks.
    b    Significantly different from control group at p < 0.01.
    c    Significantly different from control group at p < 0.001.
    
    Special studies on mutagenicity

         BHA was shown to cause genetic alteration in S. aureus strain
    W46. The variant, obtained following incubation in S. aureus W46, is
    characterized on the basis of its hemolytic properties, its production
    of staphylokinase, and its sensivity to bacitracin (Degre & Saheb,
    1982).

         BHA showed no evidence of genotoxicity in V79 Chinese hamster
    lung cells at concentrations up to 0.3 mM with and without
    hepatocyte-mediated activation (Rogers et al., 1985).

         BHA was examined in 4 in vitro systems for genotoxicity:

    (1)  Salmonella/microsome mutation test at doses between
    0.01-10 mg/plate using 5 tester strains with and without S-9
    fractions; (2) hepatocyte primary culture/DNA repair, using 10 doses
    ranging from 10-5 to 1 mg/ml; (3) adult rat liver epithelial
    cell/hypoxanthine guanine phosphoribosyl transferase mutagenesis assay
    (rat liver line 18) using 6 doses ranging from 0.05 to 0.1 mg/ml; and
    (4) Chinese hamster ovary/sister chromatid exchange (SCE) assay, at 4
    dose levels ranging from 1 to 10-3 mg/ml. BHA was without mutagenic
    activity in any of the systems (Williams et al., 1984).

         Pre-treatment of E. coli H/r30R strain with BHA had no
    anti-mutagenic effect on UV-induced mutagenesis (Kawazoe & Kato,
    1982).

         Addition of 25-500 g of BHA/plate was shown to inhibit
    3,2'-dimethyl-4-aminobiphenyl-induced mutagenicity in Salmonella
    strains TA98 and TA100 (Reddy et al., 1983).

         BHA caused a dose-dependent reduction in revertant yield in
    benzidine-induced mutagenesis in Salmonella strain TA98 activated
    with hamster liver S-9 preparation (Josephy et al., 1985).

         BHA substantially increased aflatoxin-B 1-induced mutagenesis in
    Salmonella strains TA98 and TA100 (Shelef & Chin, 1980).

         The addition of BHA to beef patties prior to broiling resulted in
    significantly-reduced formation of mutagens detectable by
    S. typhimurium TA98 (Wang et al., 1982).

    Special studies on potentiation or inhibition of carcinogenicity

    Colon

         The effect of dietary BHA on methylazoxymethanol acetate-induced
    carcinogenesis was studied in female mice fed NIH-07 open formula diet
    or AIN-76A semi-purified diet. Lung tumour incidence was lower in mice
    fed the NIH-07 diet than in mice fed the AIN-76A diet whereas colon
    tumour incidence and tumour multiplicy were higher in animals fed the
    NIH-07 diet than animals fed the AIN-76 diet. BHA inhibited
    methylazoxymethanol-induced colon cancer and lung tumours in a
    dose-related fashion. Treated mice fed AIN-76A diet plus BHA developed
    fewer colon adenocarcinomas than did mice fed the NIH-07 diet plus BHA
    (Reddy et al., 1983a; Reddy & Maeura, 1984).

         Dietary BHA given after the post-initiation phase of colon tumour
    development in male F344 rats treated with 1,2-dimethylhydrazine did
    not modify tumour development (Shirai et al., 1985).

         In a study in mice, BHA decreased the incidence and multiplicity
    of 1,2-dimethylhydrazine-induced colon cancer (Jones et al., 1984).

    Forestomach and bladder

         Rats were treated with methylnitrosourea (MNU) for 4 weeks and
    then maintained on a diet containing 0 or 2% BHA for 32 weeks. BHA in
    the diet resulted in a significant increase in the number per rat of
    papillomas and papillary and nodular hyperplasia of the urinary
    bladder and in the number of cancers per rat. There was also a
    significant increase in the incidence of cancer and papillomas in the
    forestomach of the MNU-induced rats (Imaida et al., 1984).

    Gastric tumours

         BHA and BHA plus NaCl exerted a promoting activity on
    N-methyl-N'-nitro-N-nitrosoguanidine-induced forestomach
    carcinogenesis in rats. The promotion was more marked in the BHA plus
    NaCl group, suggesting a synergistic effect on tumour promotion
    (Shirai et al., 1984).

    Liver

         BHA fed to rats for 5 weeks after administration of
    N-ethyl-N-hydroxyethylnitrosamine resulted in an inhibition of the
    induction of preneoplastic and neoplastic lesions (Ito et al.,
    1983b).

         Sequential administration of BHA resulted in a dose-dependent
    inhibition of hepatic tumourigenesis in rats treated with
    diethylnitrosamine (as measured by gamma-glutamyltranspeptidase and
    the placental form of glutathione S-transferase, as markers of altered
    focal populations of liver cells) (Thamavit et al., 1985).

         Rats were fed diets containing aprofibrate alone or in
    combination with BHA. There was a significant reduction in the number
    of hepatic tumours greater than 5 mm, but not in the overall number of
    tumours. Peroxisome proliferation was seen in the liver cells of
    non-tumorous portions of the liver in rats fed aprofibrate with or
    without BHT (Rao et al., 1984).

    Liver and bladder

         Rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine for 4
    weeks, and then administered 2% BHA in the diet for 32 weeks, showed a
    significant increase in the incidence of cancer and papillomas, in the
    average number of papillomas, and in papillary or nodular hyperplasia
    of the urinary bladder. In another study, dietary BHA significantly
    inhibited the induction of gamma-glutamyl transpeptidase-positive foci
    in the livers of rats treated with diethylnitrosamine (Imaida et al.,
    1983).

    Lung

         Dietary administration of BHA to mice treated with hydrazine
    sulfate had no effect on induction of lung tumours. However, BHA
    inhibited the formation of lung tumours in mice receiving isoniazide
    (Maru & Bhide, 1982).

         BHA did not significantly increase the multiplicity of lung
    tumours induced in mice treated with urethane, 3-methylcholanthrene,
    or dimethylnitrosamine (Witschi et al., 1981).

         In another study, dietary BHA did not enhance the development of
    lung tumours in A/J mice treated with urethane, benzo(a)pyrene, or
    dimethylnitrosamine. Pre-feeding mice with diets containing BHA prior
    to treatment with urethane or benzo(a)pyrene did not affect tumour
    incidence, but caused a marked decrease in tumour multiplicity.
    Neither 3-BHA nor 2-BHA stimulated the proliferation of mouse lung
    cells (Witschi & Doherty, 1954).

    Mammary tumours

         The incidence of mammary tumours induced in rats by
    7,12-dimethylbenz(a)anthracene (DMBA) was shown to be increased when
    the level of polyunsaturated fat in the diet was increased. BHA
    supplementation of the diet was not effective in inhibiting the
    DMBA-induced tumors. However, in another study, dietary BHA fed up to
    1 week prior to, and even 1 week after, administration of DMBA was
    effective in reducing the incidence of rat mammary tumours (King &
    McCay, 1983; McCormick et al., 1984).

    Pancreas

         Rats maintained on diets containing 0.45% BHA were each given an
    i.p. injection of 30 mg azaserine once a week for 3 weeks. Evaluation
    of the pancreas 4 months after treatment showed dietary BHA resulted
    in a 42% reduction in the number of acidophilic foci per pancreas, but
    it was without effect on focal size (Roebuck et al., 1984).

    Perinatal effects

         Perinatal administration of BHA to pregnant mice treated with a
    single dose of 7,12-dimethylbenz(a)anthracene resulted in substantial
    reduction in tumour incidence in the F1, F1 and F2 generations
    (Rao, 1982).

    Special studies on reversibility of the effect of BHA on the rat
    forestomach

         BHA (food grade) was mixed dry into powdered diet, incorporated
    into the diet in corn oil, or incorporated into a pelleted diet at 0
    or 2% and fed to male F344 rats for 9 or 27 days. The increase in the
    3H-thymidine labelling index (a standardized measure of
    methyl-3H-thymidine labelling of squamous cells of the stomach
    epithelium) in the forestomach region adjacent to the fundic
    epithelium at 9 and 27 days were comparable for BHA in the ground and
    pelleted diet, while larger increases were observed at 9 days than at
    27 days for BHA in corn oil. Stomach lining thickness increases due to
    BHA were markedly higher at 27 days than at 9 days. When BHA was mixed
    dry into powdered diet at dose levels of 0, 0.1, 0.25, 0.5, 1, or 2%
    and fed to rats for 9 days the no-effect level for the proliferative
    effect, as measured by the 3H-thymidine labelling index, was 0.25%
    (Nera et al., 1984).

         Male F344 rats were fed diets containing 0, 0.1, 0.25, 0.5, or 2%
    BHA, which was mixed dry into the powdered diet, for 13 weeks. Rats
    fed 2% BHA showed lower food consumption and weight gain than animals
    in the other groups and in the control group. At the end of the test
    period, proliferative lesions were observed in the forestomach of the

    2% group, but not in the other test groups. Elevation of the
    3H-thymidine labelling index showed a dose-related response, and a
    no-effect level of 0.25% BHA in the diet. Only the 2% group showed
    histological changes of the forestomach. Removal of BHA from the diet
    after 13 weeks, and maintenance on the control diet, resulted in a
    rapid decline in the labelling index, and after 1 week was comparable
    with the controls in all test groups. After 9 weeks on the normal
    diet, the mucosae in the 2% treatment group had reverted almost to
    normal (Iverson et al., 1985).

         Groups of male and female Wistar Han/BGA rats were fed diets
    containing 2% BHA for periods of 1, 2, or 4 weeks. Control groups were
    pair-fed BHA-free diets. After one week epithelial damage, mild
    hyperplasia, and hyperkeratosis of the forestomach mucosa were
    reported. The hyperplasia and hyperkeratosis increased in severity at
    weeks 2 and 4, but the other epithelial changes had decreased by that
    time. The hyperplastic changes occurred in the region of the limiting
    ridge. After a 4-week recovery period on a BHA-free diet, the
    epithelial changes and mild hyperplasia induced in animals maintained
    on the test diet for one week had completely disappeared, and only a
    minimal increase in cellularity and basophilic staining of the
    limiting ridge was noted. The more severe hyperplastic changes
    observed in the groups maintained 2 and 4 weeks on the test diets
    regressed only partially during a 4-week recovery period. In another
    study, male rats were dosed daily by gavage with a solution of BHA in
    arachis oil (5 ml/kg b.w.) of a dose equivalent to 1 g BHA/kg b.w./day
    for periods of 1, 2, 4, 8, 16, and 32 days. Forestomach changes
    occurred mainly in the area remote from the limiting ridge. After 1
    dose mild inflammation, minor epithelial defects, and increased
    mitotic activity were observed in the forestomach. Mild hyperplasia
    and hyperkeratosis, with a marked increase in mitotic activity, were
    apparent after 2 days. Epithelial hyperplasia became more marked with
    4 and 8 daily intubations, but mitotic activity that was markedly
    increased after 4 doses was less pronounced after 8 doses. After 16 or
    32 daily intubations the hyperplastic lesions in the forestomach
    appeared to have regressed (Altmann et al., 1985).

         Groups of Wistar rats were fed diets containing 0, 0.125, 0.5, or
    2% BHA in crystalline form for 90 days. At the highest level fed there
    were marked changes in the forestomach, characterized as
    hyperkeratosis and massive papillary hyperplasia with epithelial
    changes in the basal area. Less-pronounced effects were observed at
    the lower dose levels. Another group of rats was fed diets containing
    2% BHA for 90 days, and then maintained on a BHA-free diet for 4
    weeks. After the recovery period only mild hyperkeratosis and moderate
    hyperplasia of the forestomach were observed. In another study, groups
    of Wistar rats were fed diets containing 0, 0.125, 0.5, or 2% BHA

    dissolved in arachis oil for 90 days. In the highest-dose group,
    pronounced hyperplasia and in some cases papillary hyperplasia of the
    forestomach were observed. In contrast to the study in which the BHA
    in the diet was in the crystalline form, the lesions were confined to
    the limiting ridge. No changes of the forestomach were observed at the
    lower dose levels. When rats were maintained on 2% BHA for 90 days and
    then maintained on a BHA-free diet for 4 weeks or 8 weeks, only 1/10
    rats showed any effect on the forestomach (mild hyperplasia), and this
    effect persisted in 2/10 rats after an 8-week recovery period. None of
    the rats in these studies showed any changes in the oesophagus
    (Altmann, 1986).

         In another feeding study, rats were maintained on diets with 2%
    BHA dissolved in arachis oil for periods of 6, 12, or 15 months with
    or without a subsequent 2 or 7-month period of BHA-free diet.
    Histological studies were carried out on (1) the oesophagus near the
    cardiac orifice, (2) forestomach and glandular stomach at the greater
    curvature adjacent to the limiting ridge, and (3) forestomach and
    glandular stomach near the oesophageal entry. Generalized hyperplasia
    with hyperkeratosis or parakeratosis were observed, especially in the
    vicinity of the limiting ridge. After 12 months of exposure, BHA
    induced focal dysplasia of the forestomach epithelium in 3/10 rats and
    generalized dysplasia of the forestomach epithelium in 4/10 rats. The
    type and extent of lesions were similar in the different sections of
    the forestomach. After 12 months on the test diet and a recovery
    period of 2 months, lesions at the greater curvature had almost
    completely regressed, whereas lesions in the stomach near the
    oesophageal entry were still present. After a recovery period of 7
    months following a 15-month period on the test diet, there was a
    nearly complete regression of the forestomach changes that included
    extensive hyperplasia, papillomas, dysplastic changes, and invasive
    growth (which did not reach the muscularis mucosae to become malignant
    under the conditions of this study) (Altmann, 1986).

    Special studies on species differences

    Mice

         NMRI mice were dosed daily by gavage with BHA dissolved in
    arachis oil at dose levels equivalent to 0 or 1000 mg BHA/kg b.w./day
    for 28 days. When examined macroscopically, visible lesions were
    observed in the forestomach, which were similar to hyperkeratosis
    observed in rats (Altmann, 1986).

    Hamsters

         Male Syrian golden hamsters were maintained on a diet containing
    0 or 2% BHA (pelleted) or 1% BHA (powdered) for 24 weeks. Tumours of
    the forestomach were observed in all hamsters in the treated group. No
    tumours were reported in the control group or in other organs examined
    (Ito et al., 1983).

         In another study, groups of Syrian golden hamsters were
    maintained on powdered diets containing 0 BHA, 1% "crude BHA" (98%
    3-tert isomer and 2% 2-tert isomer), 3-tert BHA, or 2-tert BHA
    for 1 to 4 weeks. Hyperplasia of the forestomach was found to be more
    progressive and severe in hamsters fed 3-tert BHA or "crude BHA"
    than those fed 2-tert BHA (Ito et al., 1984).

         Groups of hamsters were fed diets containing 0 or 2% BHA for a
    period of 90 days. The forestomach changes differed from those
    observed in rats and mice, showing no hyperkeratosis when examined
    macroscopically.  When examined histologically, mild epithelial
    hyperplasia and hyperkeratosis were observed. The effects were more
    marked in the females (Altmann, 198b).

    Guinea pigs

         Test animals were dosed daily by gavage with BHA dissolved in
    arachis oil at dose levels equivalent to 0 or 1000 mg BHA/kg b.w./day
    for 28 days. No gross changes were observed in the stomach. (Note: The
    guinea pig is a rodent species that does not have a forestomach)
    (Altmann, 1986).

    Dogs

         Beagle dogs were fed diets containing 0, 1.0, or 1.3% BHA for 180
    days. At autopsy no gross or microscopic lesions were found in the
    stomach that could be attributed to the presence of BHA in the diet.
    Neither hyperplasia nor cellular proliferation were observed. Electron
    microscopic examination of the oesophagus (above the stomach and at
    the cardio-oesophageal junction), of the cardia area, and of the main
    body of the stomach revealed no differences in ultrastructure between
    the tissues from test and control animals. Enzyme analysis of hepatic
    tissues showed a significant increase in mixed-function oxidases, UDP
    glucuronyltransferase, glutathione-S-transferase, and epoxide
    hydrarase activities in the BHA-treated animals (Ikeda et al., 1986).

         Groups of 3 or 4 male and female beagle dogs were maintained on
    diets containing 0, 0.25, 0.5, or 1% BHA for 6 months. A dose-related
    retardation of growth was observed. Serum biochemical analyses carried
    out at 1, 3, and 6 months showed a slight decrease in albumin content
    and increases in alkaline phosphatase and leucine aminopeptidase
    activities in the high-dose groups. At autopsy, liver weights were
    increased in the test groups, but no histological changes were
    reported. Histopathological and histometrical studies showed no
    significant mucosal alterations in the stomach (fundic and pyloric
    areas), oesophagus, or duodenum in the test animals. The mitotic index
    in the basal layer of the squamous epithelium of the distil part of
    the oesophagus was similar in test and control groups (Tobe et al.,
    1986).

    Pigs

         Pregnant pigs (Danish landacre) were fed diets containing 0,
    0.5, 1.9, or 3.7% BHA for 16 weeks. At necropsy, epithelial changes in
    the oesophageal part of the stomach were similar in test and control
    groups. No papillomas and no changes in the glandular part of the
    stomach were reported. Linear yellow-brown rough epithelium was seen
    in the whole length of the oesophagus in a few pigs in the middle- and
    high-dose groups. Microscopic examination of these tissues showed
    proliferative and parakeratotic changes of the epithelium. The changes
    in the oesophagus were not seen in the low-dose or control groups and
    have not been observed in historical controls (Wurtzen & Olsen, 1986).

    Monkeys

         Monkeys (Macaca fascicularis) were given 0, 125, or 500 mg
    BHA/kg b.w./day by gavage in corn oil 5 times/week for 20 days, after
    which time the high dose was halved. After 85 days the study was
    terminated. Histopathology of the stomach showed no treatment-related
    effects. However, there was a significant increase in the mitotic
    index of the squamous epithelial cell lining of the distal oesophagus
    in the high-dose group (Iverson et al., 1985b).

    Special studies on the effect of structurally-related substances
    on the forestomach of the rat

         Groups of 5 to 10 male and female rats were fed diets containing
    2% BHA, tert-butylhydroquinone (TBHQ), 1,4-dimethoxybenzene,
    hydroquinone, 4-methoxyphenol, 3-methxyphenol, 2-methoxyphenol,
    anisol, rho-cresol, or phenol or 1% BHT for 28 days. BHT did not
    induce forestomach lesions. TBHQ-treated animals showed mild
    hyperplasia of the forestomach mucosa with locally-increased
    hyperplasia of the basal cells. 4-Methoxyphenol caused circular deep
    ulceration parallel to the limiting ridge, with hyperplasia and mild
    hyperkeratosis in the adjoining mucosa. Neither 3- nor 2-methoxyphenol
    nor 1,4-dimethoxy-benzene had any effect on the forestomach,
    rho-cresol and phenol also failed to cause any effect on the
    forestomach epithelium (Altmann, 1986).

         Weanling male F344 rats were maintained on diets containing one
    of the following phenols or acids: 2% 3-BHA, 4% 4-hydroxybenzoic acid,
    4% methyl-4-hydroxybenzoic acid ester, 4% ethyl-4-hydroxybenzoic acid
    ester, 4% n-propyl-4-hydroxybenzoic acid ester, 4% n-butyl-
    4-hydroxybenzoic acid ester, 2% 4-methoxyphenol, 4% propionic acid, or
    0.5% acetylsalicylic acid for periods ranging from 9 to 27 days. At
    the end of the test period studies were carried out to determine the
    effects of administration of these compounds on the methyl-3H-
    thymidine labelling index and histological appearance of the
    forestomach.

         3-BHA was as effective as food grade BHA in inducing cellular
    proliferation in the prefundic region. Methyl-4-hydroxybenzoic acid
    ester did not increase the thymidine labelling index in the
    forestomach epithelium in the prefundic area or the main body of the
    tissue. However, the thymidine labelling index in the prefundic region
    was progressively increased in going from the ethyl to the n-butyl
    ester, the 4% n-butyl ester being as active as 2% BHA in this system.
    Propionic acid was without effect after 9 days, but it caused a 2-fold
    increase in the labelling index in the main body of the forestomach
    epithelium after 27 days. Acetylsalicylic acid alone did not affect
    thymidine labelling of either the prefundic or midregion of the rat
    stomach. However, simultaneous administration of 2% BHA and 0.5%
    acetylsalicylic acid resulted in a marked reduction (43%) of the
    proliferative effect of BHA in the prefundic region and appeared to
    protect the midregion of the forestomach. Histopathological changes
    paralleled the results obtained from the thymidine labelling index
    studies (Rodrigues et al., 1986).

    Human studies

    Metabolism

         Two males were given 100 mg BHA. There was a significant
    conversion of BHA to TBHQ (0-demethylation) prior to its conjugation
    and excretion, primarily as the glucoronide with lesser amounts as the
    O-sulphate (El-Rashidy & Niazi, 1983).

    Comments

         Studies have been conducted that provide additional information
    on the proliferative changes observed in the forestomach of rats fed
    BHA, and also the effect of BHA on the stomach and oesophagus of
    species that do not have a forestomach. The data show that induction
    of hyperplasia in the forestomach of the rat is dose-dependent, and
    can be reversed when BHA is removed from the diet. In one species that
    does not have a forestomach, the dog, levels of BHA that produced
    effects in the forestomach of the rat had no effect on either the
    stomach or oesophagus. However, in monkey and pig studies there was
    some evidence that BHA produced effects in the oesophagus. In the case
    of the monkey, an increased mytotic rate was reported, and in the case
    of the pig largely macroscopically-diagnosed hyperkeratosis was
    reported.

         In both these cases the Committee believes that the studies
    should be repeated. In particular, studies should be performed in the
    monkey in which BHA is administered in the diet (only gavage data are
    presently available in the monkey).

         The results of several tests on the genotoxicity of BHA,
    involving both bacterial and mammalian cells, lend additional support
    to previous evaluation of several in vitro and in vivo systems
    which do not show BHA to be mutagenic.

         New data were not submitted to meet the requirements for a
    multigeneration reproduction study.

         The present evaluation is based upon the results of long-term
    feeding studies showing that rats can be maintained on diets
    containing up to 0.125% BHA without any significant adverse effect
    (summarized under "Special studies on the effect of BHA on the
    stomach").

    EVALUATION

    Level causing no toxicological effect

    Rat:      1250 ppm (0.125%) in the diet, equivalent to 62.5 mg/kg
              b.w./day

    Estimate of temporary acceptable daily intake for man

              0-0.3 mg/kg b.w.

    Further work or information

    Required (by 1988)

         1. Studies in pigs and monkeys to explore the potential for BHA
    to cause oesophageal hyperplasia in these species. These studies
    should be carried out with dietary BHA. The Committee recognizes the
    technical difficulties in carrying out this study in the monkey due to
    potential diet rejection, but emphasizes that it should be attempted.

         2. Mutigeneration reproduction study.

    Desirable

         Studies to determine the mechanism involved in the effects of BHA
    on the forestomach.

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    See Also:
       Toxicological Abbreviations
       Butylated hydroxyanisole (WHO Food Additives Series 5)
       Butylated hydroxyanisole (WHO Food Additives Series 10)
       Butylated hydroxyanisole (WHO Food Additives Series 24)
       BUTYLATED HYDROXYANISOLE (JECFA Evaluation)