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    BUTYLATED HYDROXYANISOLE (BHA)

    Explanation

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

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monograph has been expanded and is reproduced
    in its entirety below.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         Butylated hydroxyanisole (BHA) was absorbed from the
    gastrointestinal tract, and there was some evidence that the feeding
    of amounts 100-500 times the levels generally permitted in fats for
    human consumption (in the United States of America 200 mg/kg fat)
    caused deposition in depot fat, the stability of which was thereby
    increased (Johnson et al., 1958). However, there was no evidence of
    cumulation in other tissues (Astill et al., 1960; Bunnel et al., 1955;
    Hodge et al., 1964).

         In the rabbit, BHA was conjugated mainly with glucuronic acid or
    sulfuric acid (Dacre et al., 1956); a small amount of unchanged BHA
    was excreted in the urine.

         In rats the 2-tert-butyl isomer was chiefly excreted as
    glucuronide, while the 3-tert-butyl isomer was excreted mainly as
    ethereal sulfate (Astill et al., 1960). Thus, these animals
    effectively detoxicated BHA. The changes described occurred in the
    liver. No evidence has been found to suggest that BHA produces any
    adverse biochemical or metabolic effect in the animal body (Dacre,
    1960).

         Dogs excreted 60% of a 350 mg/kg dose unchanged in the faeces
    within three days. The remainder was excreted in the urine mainly as
    sulfate conjugates of BHA, tert-butyl-hydroquinone and an
    unidentified phenol. Only 5.5% of the dose was excreted in urine as
    the glucuronide (Astill et al., 1962). Rats were injected
    intraperitoneally with a single dose of tritium-labelled BHA.
    Approximately 90% of the radioactivity was recovered in the urine
    within four days (Golder et al., 1962).

         Pigs fed 0.1% BHA in the diet for four months, and pullets fed
    0.1% BHA in the diet for eight weeks, showed no accumulation in
    muscle, liver, kidney or the reserve fat (Francois & Pihet, 1960).

    Effects on enzymes and other biochemical parameters

         Rats administered 500 mg/kg bw (seven daily doses) of BHA showed
    no change in liver glucose-6-phosphatase activity. BHA at dose levels
    of 100 mg/kg or more (seven daily doses) caused increase in liver
    weight of male rats, but in females only at doses greater than
    200 mg/kg bw (seven daily doses). Liver weight was comparable to
    control within 14 days of withdrawal of BHA from the diet. No fatty
    changes were observed (Feuer et al., 1965a). Rats administered BHA
    (500 mg/kg day) for two days showed no increased activity of
    microsomal processing enzymes (aminopyrine demethylase) hexabarbitine
    oxidase and nitroanisol demethylase (Gilbert & Goldberg, 1965). In
    another study, rats fed 0.1, 0.25 or 0.5% BHA in the diet for 12 days
    showed no increased liver weight, but there was an increase in liver
    biphenyl-4-hydroxylase activity in the 0.5% group (Creaven et al.,
    1966).

         Groups each of eight rats (SPF Carworth strain) equally divided
    by sex were administered by intubation daily for one week BHA
    dissolved in arachis oil, at a dose level equivalent to 0, 50, 100,
    200 or 500 mg/kg body weight. BHA had no effect on the growth of the
    animals. Twenty-four hours after administration of the final dose the
    animals were sacrificed and liver preparations assayed for glucose-6-
    phosphatase, glucose-6-phosphatase dehydrogenase, hexabarbitone
    oxidase, nitro-anisol demethylase and aminopyrine demethylase
    activities. BHA had no effect on these enzyme activities.
    Histochemical studies showed that BHA caused no fatty changes in
    liver (Feuer et al., 1965b).

         Groups each of two to three Rhesus monkeys (Macaca mulatta)
    one-month-old infant or sexually-immature juvenile, of both sexes,
    were administered BHA at a dose level of 500 mg/kg body weight for
    four weeks. Another group of juveniles received 50 mg BHA/kg bw for
    the same period. Controls received equivalent amounts of corn oil.
    Urine and blood analyses were normal with the exception of serum
    cholesterol which was elevated at the high dose at week 3 of the
    study, but which returned to normal at week 4. The livers of all test
    animals were enlarged. No other organ showed any pathological change.
    Ultrastructurally, infant and juvenile monkeys treated at the high
    dose level showed pronounced proliferation of the hepatic smooth
    endoplasmic reticulum. Infants and juveniles treated with BHA
    (500 mg/kg) had lower levels of liver lipids than corn oil controls.
    Nitro-anisol demethylase activity was increased and glucose-6-
    phosphatase activity was decreased in BHA-treated juveniles but
    unaffected in infant monkeys (Allen & Engbom, 1972). No changes were
    observed in DNA, RNA and cytochrome P450 levels.

         Additional biochemical tests were carried out on the liver and
    plasma of these monkeys. BHA-treated animals showed higher plasma
    triglyceride levels than controls. There was a significant decrease in
    the level of liver cholesterol, as well as a lowering of the
    cholesterol/lipid-phosphorus ratio of the liver, in the test animals.
    Liver succinic dehydrogenase was also lowered in BHT-treated animals
    (Branen et al., 1973).

         Hepatic microsomal preparations were made from female mice (A/HcJ
    strain) fed a diet of 0 and 0.5% BHA for 14 days. The aryl hydrocarbon
    hydroxylase activity (AHH) of the preparations was similar. However,
    microsomal preparations from the BHA-fed mice showed greater
    sensitivity to in vitro inhibition of AHH activity by alpha-naphtha-
    fluorine, and continued increased amounts of cytochrome P450 per unit
    weight than preparations from control mice (Speier & Wattenberg,
    1975).

         Rats were maintained on a diet supplemented with 20% lard, and
    containing 0, 0.1, 0.2, 0.3, 0.4, 0.5% BHA, for a period of six weeks.
    BHA caused an increase in the total serum cholesterol at the 0.1%
    level, but no further elevations occurred at higher doses. There was a
    relatively greater increase in the amount of serum-free cholesterol
    than of ester-cholesterol. BHA produced enlarged adrenals in males at
    all levels, but no histological changes were observed. Increased liver
    weight at the higher dietary BHA levels was accompanied by an increase
    in the absolute lipid content of the liver. However, BHA had no effect
    on the concentration in liver of total and esterified cholesterol, or
    the composition of the polyunsaturated fatty acids (Johnson & Hewgill,
    1961).

         Groups of 10 female CD-1 mice were fed diets containing 0 or
    0.75% BHA for 10 days then sacrificed. The relative liver weight of
    the animals fed BHA was increased to 9.1% of the body weight as
    compared to a value of 5.7% in controls. The activities of a number of
    microsomal enzymes and glucose-6-phosphate dehydrogenase and
    UDP-glucose dehydrogenase was increased (Cha & Bueding, 1971).

         The activity of the microsomal mono-oxygenase system was
    inhibited when BHA was added to a microsomal preparation obtained from
    the livers of adult male Sprague-Dawley rats (Yang et al., 1974).
    Similarly, BHA at a concentration of 0.2 mM inhibited the lipid
    peroxidation activity of a microsomal preparation from male Wistar
    rats (Uainio, 1974).

         Male Sprague-Dawley rats (initial weight about 150 g) were fed a
    diet containing 0.5% BHA for 14 days. A diet free of BHA was then
    given for 24 hours and the animals were fasted for 12 hours prior to
    sacrifice. Control animals received similar treatment except that they
    were maintained on a diet containing no BHA. Compared to controls
    there was a marked increase (about 1,6-fold) in epoxide hydrase
    activity in the liver microsomes from animals receiving 0.5% BHA; and
    upon gel electrophoresis there was almost a twofold increase in the

    amount of protein staining in the epoxide hydrase band. Epoxide
    hydrase induction was greater in parallel experiments conducted with
    diets containing 0.5% BHT or ethoxyquin (Kahl & Wulff, 1979).

         A diet containing 0.75% BHA was fed to female CD-1 mice for a
    period of 3-12 days. As compared to controls epoxide hydrase activity
    from liver microsomers was ninefold higher after 12 days. When animals
    were given BHA for 12 days and then put on a normal diet epoxide
    hydrase activity fell off rapidly, falling to normal values in about
    15 days. A comparison of male Sprague-Dawley rats and female CD-1 mice
    fed diets containing 0.17% BHA for 10 days showed that the treated
    mice had an increased epoxide hydrase activity of about 11-fold as
    compared to about a 5.5-fold increase in the treated rats (Cha et al.,
    1978).

         A concentration of 6.70 m BHA was reported to cause a 50%
    inhibition of the synthesis of prostaglandin E1 by microsomes
    isolated from bull seminal vesicles, 3.08 g BHA caused a 50%
    inhibition in prostaglandin E2 synthesis. Higher concentrations of
    BHT than BHA were required to effect a 50% inhibition of synthesis of
    the prostaglandins (Boehme & Branen, 1977).

         Prefeeding of A/HeJ mice with diets containing (0.5%) BHA for two
    weeks was reported to cause a change in the metabolism of Benzo(a)
    pyrene (BP) by microsomal extracts. Less of the 4-5 epoxide metabolite
    of BP and more phenol formation was found in the BHA-treated animals
    (Luke et al., 1977).

         Single i.p. injections of 62.5, 215 or 500 mg/kg of BHA were
    given to adult male Swiss Webster mice. The animals were sacrificed
    three days later. No increase in lung DNA or sulfhydryl content or
    lung enzyme activity of super oxide dismutase, GSH peroxidase, GSH
    reductase and G6PDH was found in animals dosed with BHA. Slight lung
    edema may have occurred since some increase in lung weight was noted
    as compared to controls. Significant increase in lung DNA and
    sulfhydryl content and lung enzyme activities were noted in mice
    similarly treated with BHT (Omage et al., 1977).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

         Groups each of 100 mice (equally divided by sex) were given
    single s.c. injections (10 mg/mouse) of BHA in trioctanoin and
    observed for up to 575 days. Another group was given weekly skin
    applications of 0.1 mg or 10 mg of BHA in acetone for a period of
    309-459 days. Microscopic examination of the skin from the test mice
    showed no evidence of tumours (Hodge et al., 1966).

         BHA in lanolin applied to the ears of guinea-pigs once daily for
    periods of two to six weeks resulted in a micro-invasion of basal cell
    pseudopods with destruction of the superficial connective tissue and
    fragmentation of the collagen. BHA alone did not cause these changes
    (Riley & Seal, 1968).

    Special studies on mutagenicity

    (a)  Cytogenetics

         BHA was found not to induce chromosomal aberrations when tested
    in cultured Chinese hamster cells (Ishidate & Odashima, 1977). No
    effect on the occurrence of chromosomal aberrations or sister
    chromatid exchanges was found in a Chinese hamster cell line exposed
    to BHA (Abe & Sasaki, 1977).

         Butylated hydroxyanisole (BHA) was investigated at concentrations
    of 2.0, 20.0 and 200 g/ml in vitro employing WI-38 human embryonic
    lung cells for anaphase abnormalities. It was also investigated
    in vivo by the cytogenetic analysis of metaphase cells from rat bone
    marrow at dosages of 15, 150 and 1500 mg/kg. BHA did not produce any
    significant increases in abnormalities above the control values in
    either assay (Fabrizio, 1974).

    (b)  Sex-linked mutations in Drosophila

         BHA was found not to induce sex-linked lethal mutations when
    tested in Drosophila. A range of doses was tested with the compound
    being administered in a sucrose solution on which the animals were fed
    (Miyagi & Goodheart, 1976).

    (c)  Microbial mutagenicity

         A microbial mutagenicity study carried out using Salmonella
    strains TA-1535, TA-1537, TA-1538, TA-98 and TA-100 indicated BHA was
    not mutagenic when tested at concentrations of 10, 100 or 1000
    micrograms per plate. The compound was tested with and without S9
    extracts (Joner, 1978).

         Liver cytosols prepared from BHA-fed female CD-1 mice or male
    Sprague-Dawley rats reduced the mutagenicity of urinary benzo(a)
    pyrene metabolites as tested in S. typhimurium (Benson et al.,
    1978).

         CD-1 mice were treated with 0.75% BHA in the diet for 10 days and
    then given 100 mg/kg benzo(a) pyrine. The mutagenicity of the urine of
    mice pre-treated with BHA was much lower than that of animals not
    treated with BHA (Batzinger et al., 1978).

         In vitro Salmonella TA-1530 and G46, together with
    Saccharomyces D-3, were employed. A 10% concentration was tested.
    BHA was non-mutagenic for Salmonella TA-1530 and G-46. Tests with

    Saccharomyces D-3 demonstrated a biologically significant increase
    in the frequency of recombinants. This result could not be repeated
    upon subsequent testing and was, therefore, thought to be spurious
    (Fabrizio, 1974).

         In vivo BHA was tested at 15, 150 and 1500 mg/kg in ICR Swiss
    mice employing as indicator organisms Salmonella G-46 and TA-1530
    and Saccharomyces D-3. BHA was non-mutagenic for Salmonella but
    demonstrated a biologically significant increase in the frequency of
    recombinants. Inasmuch as the host-mediated assay is no longer
    recommended for routine use, this suggested mutagenic effect was
    investigated using more sensitive (in terms of detection) procedures.
    In this study BHA was investigated employing Salmonella typhimurium
    strains TA-1535, TA-1537 and TA-1538, and Saccharomyces D-4 with and
    without metabolic activations, in plate and suspension tests. The
    percent concentrations (w/v) employed were 0.00375, 0.0075 and 0.0150
    for Salmonella, and 0.0625, 0.1250 and 0.02500 for Saccharomyces.
    Under the conditions of this investigation BHA was non-mutagenic
    (Fabrizio, 1974).

    Dominant lethal test

         Sprague-Dawley C-D strain male rats were used. Dosages of 15, 150
    and 1500 mg/kg were employed.

         Acute study - a single dose was administered with subsequent
    mating for each of eight weeks. BHA produced random statistical
    increases in dominant lethality. These were discounted due to the
    unusually low negative control value (Fabrizio, 1974).

         Subacute - five daily doses were administered (5  15, 5  150
    and 5  1500 mg/kg) and males subsequently mated for each of seven
    weeks; BHA produced a statistically significant increase in pre-
    implantation loss in weeks 6 and 7. This effect occurring alone is not
    demonstrative of mutagenicity (Fabrizio, 1974).

    Special studies on teratogenicity

         Groups of rats or mice of various strains were given BHA in
    accordance with one of three different regimes, viz, daily
    administration for seven weeks, before pairing continuing until day 18
    of pregnancy or daily administration on days 1-20 of pregnancy or
    single administration on day 9, 11 or 13 of pregnancy. Dosage ranged
    from 250 to 1000 mg/kg bw. No teratogenic effects were observed at
    dose levels as high as 300-500 mg/kg bw administered for as long as
    seven weeks, although under these conditions the mortality was 25%. At
    a higher dose level (750 mg/kg) mortality was 75% (Clegg, 1965).
    Pregnant rats receiving a total dose of 0.5 g of BHA in the diet
    showed less resorptions than rats on control diets (Telford et al.,
    1962).

         Groups of pregnant New Zealand white rabbits were dosed with 0,
    50, 200 or 400 mg/kg bw of BHA from day 7 to day 18 of pregnancy.
    There were 13-15 pregnant dams per group. The BHA was administered by
    stomach tube using a propyleneglycol vehicle. The foetuses were
    removed on day 28. No differences between doses and control groups
    were seen with respect to body weight, incidence of soft tissue or
    skeletal abnormalities, number of foetuses born dead or alive, and
    number of corpora lutea and implantations (Hansen & Meyer, 1978).

    Special studies on reproduction and behaviour

         Diets containing 0, 0.125, 0.25 or 0.5% BHA were fed to adult
    male and female Sprague-Dawley rats for two weeks prior to and during
    mating. Female rats were continued on the diets through gestation and
    weaning of their offspring. The pups were then fed the diet until the
    end of the experiment. Selected pups were sacrificed at 21 days for
    brain neuronal counts, and at 90 days for measurement of eye and
    regional brain weights and histological examination of the somatomotor
    cortex. No effect of treatment was noted on reproductive performance.
    However, cumulative offspring mortality was significantly increased in
    the 0.25% and 0.5% groups as compared to controls. The cumulative
    mortality in the controls was 4% compared to 9% and 14% in the 0.25
    and 0.5% groups, respectively. There was a significant reduction in
    pre-weaning body weights in the pups in the high dose group which
    persisted into the post-weaning period at 42 days, although animals in
    the 0.25% group weighed more than controls. There was no significant
    effect of treatment on body weight at 90 days. No effect of treatment
    was noted on surface righting, pivoting, cliff avoidance, negative
    geotaxis, swimming development, pre- and post-weaning open field,
    activity wheels, roto-rod, active avoidance, appetitive position
    discrimination or passive avoidance or vaginal patency.

         There was a delay in the 0.5% and 0.25% groups in auditory
    startle development. No effect of treatment was seen on eye or
    regional brain weights or brain histology. The authors noted that less
    toxicity was observed with BHA than was observed in a similar design
    where BHT was fed (Vorhees et al., 1979).

         Groups of mated pairs of Swiss Webster mice (Mus musculus) were
    maintained on diets containing 0, or 0.5% BHA. The litters obtained
    from the mated pairs were mated at 21 days and then maintained on a
    diet similar to that of their mother. At six weeks of age the mice
    were subjected to behavioural tests. The BHA-treated offspring showed
    increased exploration, decreased sleeping, decreased self-grooming,
    slower learning and a decreased orientation complex than did the
    control group (Stokes & Scudder, 1974).

    Special studies on immunology

         A number of reports have been published recently concerning the
    possible effect of BHA on immune function. For example, it was
    reported that BHA suppressed the in vitro immune response of spleen
    cells at concentrations that were not cytotoxic (Archer et al., 1977).
    A total of 112 consecutive patients were patch-tested with BHA and BHT
    for contact dermatitis. The substances were tested at concentrations
    of 2% in petrolatum. Two patients had positive reactions to both, one
    to BHA only, and one to BHT only. Two of the possible responding
    patients who were being treated for eczema remained symptom-free on a
    diet free of antioxidants but redeveloped eczema upon challenge with 5
    or 10 mg of BHA daily given orally. Eighty-three consecutive patients
    were tested with 5% BHA and 5% BHT in alcohol; all tests were negative
    (Roed-Petersen & Hjorth, 1976).

         Seven patients suffering from asthma or rhinitis of unknown
    etiology were challenged with 300-450 mg of BHA given orally, except
    one patient who received 850 mg. None of the patients suffered an
    exacerbation of their symptoms immediately following challenge,
    although a few patients noted drowsiness from 10 to 45 minutes after
    dosing (Cloninger & Novey, 1974).

    Special studies on carcinogenicity

         BHA was tested for its ability to induce lung tumours when given
    to groups of 15 male and 15 female strain A mice in series of 24 i.p.
    injections given three times weekly for eight weeks. The mice were
    killed and examined for the presence of lung tumours 24 weeks after
    the initial injection. Two dose levels were used amounting to a total
    dose of 1200 or 6000 mg/kg over the course of 24 injections. No
    increase in the incidence of lung tumours was noted at either dose
    (Stover et al., 1973).

         In a study carried out using groups of 30 CD-1 female mice, aged
    6-8 weeks, BHA did not promote skin tumours when applied to the skin
    of animals previously initiated with 7,12-dimethyl-benzanthracene
    (DMBA). One milligram of BHA dissolved in acetone was applied to the
    shaved back of the mice twice-weekly for 30 weeks. Treatment began one
    week after initiation with topical application with DMBA.

         A study carried out using groups of 10 Sprague-Dawley rats fed
    BHA in the diet for three weeks at a level corresponding to an intake
    of 4.2 mmol/kg bw indicated that BHA did not produce haemorrhagic
    death or alteration in the animal's prothrombin index. Haemorrhagic
    death and a significant drop in the prothrombin time was produced in
    animals fed similar levels of BHT (Takahshi & Hiraga, 1978).

         Concentrations of 0.01 to 0.0001% BHA caused a significant
    decrease in the frequency and amplitude of atrial contractions from
    isolated preparations of rabbit atria. A significant depression of
    metacholine-induced ileal contractions was also reported in the
    presence of 0.05 to 0.001% BHA (Gad et al., 1978).

         Incubation of 100 ppm (0.55 mM) BHA with primary cultures of
    beating myocardial and endothelioid cells caused a marked depression
    of the beating frequency and a release of LDH from the cells into the
    culture medium without inducing changes in the morphology of the
    cells. Culture of the cells for one hour with 1000 ppm of BHA resulted
    in marked cell death within one hour.

         Incubation of red blood cells isolated from man, guinea-pigs and
    rats with 1 mM BHA resulted in haemolysis of 12, 70 and 35% of the
    cells, respectively. Incubation of mitochondrial-lysosomal suspensions
    of rat liver with BHA resulted in leakage of protein, acid phosphatase
    and glutamic acid dehydrogenase from the mitochondrial-lysosomal
    membrane (Cumming & Walton, 1973).

         BHA at concentrations as low as 8  10-10 mol/l can inhibit the
    guinea-pig's smooth muscle contraction caused by bradykinin (Posati &
    Pallanich, 1970).

         Using an in situ method of perfusion for rat intestine, BHA at
    a level of 2 mg/ml has been shown to reduce the absorption of glucose
    and methionine, but not butyric acid (Fritsch et al., 1975a). BHA at
    levels of 400 g/ml caused an inhibition of the metabolism (as
    measured by gas evolution) of culture of bacteria isolated from the
    faecal flora of rats (Fritsch et al., 1975b).

    Acute toxicity

                                                                        

                                LD50
    Animal      Route        (mg/kg bw)            References
                                                                        

    Mouse       Oral         2000              Bunnell et al., 1955;
                                               Lehman et al., 1951

    Rat         Oral         2200-5000         Bunnell et al., 1955;
                                               Lehman et al., 1951
                                                                        

    Short-term studies

    Rat

         No effect on potassium excretion, as described below for the
    rabbit, was observed in a short-term feeding study in the rat (Dacre,
    1960).

         Groups of seven recently-weaned rats were fed for six months on
    rations containing 0, 0.5, 1, 2 and 3% of BHA. The rats at the 3%
    level did not eat enough to gain weight and were put on to the 2%
    diet for a time, then returned to 3%. Even at the 2% level, food
    consumption was not optimal. Histopathological examination revealed no
    pathological condition attributable to BHA (Wilder & Kraybill, 1948).

         Combinations of BHA with other food additives, such as chlorine
    dioxide, sodium propionate, propyl gallate, or polyoxyethylene-8-
    stearate, at 50 times the normal levels of use in bread, had no
    deleterious effects when they were fed in bread to groups of 26 rats
    for a period of 32 weeks. The treated bread formed 75% of the animals'
    diet. The daily dosage levels of BHA were from 3.3 to 7.0 mg/kg bw
    (Graham et al., 1954; Graham & Grice, 1955).

         Rats were maintained on test diets containing 0, or the
    equivalent of 500-600 mg/kg bw BHA (1/5 of the LD50), for a period
    of 10 weeks. The test animals showed decreased growth rate, and
    reduced activity of the blood enzymes, catalase, peroxidase and
    cholinesterase. Chemical analysis of livers of test animals showed a
    decrease in the amount of phospholipid as compared to controls, but
    there was no lipid accumulation. Histological examination of the
    tissues and organs did not show any compound-related effects
    (Karplyuk, 1962).

    Rabbit

         In rabbits, a dose of 1 g given daily for five to six days by
    stomach tube caused a 10-fold increase in sodium excretion and a 20%
    increase in potassium excretion in the urine. Extracellular fluid
    volume fell, and this prevented any marked change in the plasma sodium
    level. The serum potassium fell after five days' treatment and
    potassium was being replaced by sodium in muscle cells. In heart
    muscle the changes occurred later than in skeletal muscle and were
    less marked. The antioxidant may have a direct effect on the kidney;
    the adrenal cortex showed changes in the zona glomerulosa and there
    was increased excretion of aldosterone in the urine, associated with
    the sodium and potassium loss (Denz & Llaurado, 1957).

    Dog

         When BHA was fed to dogs at dose levels of 0, 0.3, 30 and
    100 mg/kg bw for one year, no ill-effects were observed. Renal
    function, haematology and histopathology of the main tissues were
    normal. Organ weights were within normal limits and there was no
    demonstrable storage of BHA. The urine did not contain a demonstrable
    increase of reducing substances, even when 100 mg/kg bw of BHA was
    fed. Groups of three dogs were used at each dose level for these
    experiments (Hodge et al., 1964).

         Groups each of four weanling dogs were fed BHA at 0, 5, 50 and
    250 mg/kg for 15 months. General health and weight gains of the dogs
    were within normal range, as were haematologic parameters. Urine from
    test dogs contained higher ratios of total to inorganic sulfate and
    glucuronates than controls. At autopsy, microscopic examination of
    tissues and organs showed that three out of four of the animals at the
    highest dose level tested had a liver cell degeneration and a diffuse
    granulocytic infiltration. The lobular structure of the livers of
    these animals was normal, and there was no excessive connective tissue
    proliferation (Wilder et al., 1960).

    Monkey

         A group of six adult female Rhesus monkeys was maintained on a
    test diet containing a mixture of BHT and BHA that provided an intake
    equivalent to 50 mg BHT and 50 mg BHA/kg bw. Another group of six
    adult female Rhesus monkeys was used as controls. The monkeys were fed
    the diet for one year prior to breeding and then for an additional
    year, including a 165-day gestation period. Haematological studies
    including haemoglobin, hematocrit, total as well as differential white
    blood cell count, cholesterol, Na+, K+, total protein, serum glutamic
    pyruvic transaminase, and serum glutamic oxylacetic transaminase, were
    carried out at monthly intervals. Body weights were taken at monthly
    intervals. Records of menstrual cycles were maintained through the
    test period.

         After one year, the females were bred to Rhesus males not
    receiving test diets. During pregnancy, complete blood counts were
    done on days 40, 80, 120 and 160 of gestation and on days 30 and 60
    post-partum. A total of five infants was born to the experimental
    monkeys and six to the control monkeys. Haematological evaluations
    were made on infants of the test and control monkeys at days 1, 5, 15,
    30 and 60, and observations of the infants were continued through two
    years of age. Two experimental and two control infants, three months
    of age, were removed from their mothers for one month of psychological
    home cage observations. No clinical abnormalities were observed in
    parent and offspring during the period of study. The gestation of test
    animals was free of complications and normal infants were produced.
    Infants born during the exposure period remained healthy, with the

    exception of one infant that died from unrelated causes. Home cage
    observations at the third month of life did not reveal any behavioural
    abnormalities (Allen, 1974, 1976).

    Long-term studies

    Rat

         Groups of 15 or more newly-weaned rats were placed on diets
    containing 0, 0.05, 0.5 and 1% BHA in lard (0, 0.003, 0.03 and 0.06%
    of the total diet) for 22 months. Weight gain was comparable in all
    groups. Reproduction was normal, and young rats kept on the same
    ration grew normally. Number, size, weight, weight gain and mortality
    of the litters were comparable for animals of all groups. After one
    year on test, the colony suffered from an infectious respiratory
    disease and many died. There was no significant difference in
    mortality among the groups. After 22 months, the remaining animals
    were killed; histopathological examination revealed no changes
    attributable to the antioxidant (Wilder & Kraybill, 1948).

         A similar series of tests was undertaken, with an additional
    group on a diet of 2% BHA in lard (0.12% of the total diet). There
    were 17 rats in each group. After 21 months, the survivors were
    killed. Histopathological examination revealed no significant
    differences compared with the control animals. The rate of gain in
    weight during the growing period was unchanged, and all rats appeared
    normal in every respect (Wilder & Kraybill, 1948).

         In another rat feeding test carried out over a period of two
    years on groups of 40 rats, there was a small reduction in the mature
    weight and an increase in relative liver weight in some cases with the
    highest level of BHA used (0.5% of the diet), but there were no
    effects on any of the following: the reproductive cycle; histology of
    the spleen, kidney, liver, or skin; ratio of weight of heart, spleen,
    or kidneys to total body weight; mortality. The toxicity of BHA was
    not affected by the dietary fat load (Brown et al., 1959).

         Rats were maintained on diets containing 0, and the equivalent of
    500-600 mg/kg bw BHA (1/5 of the LD50) for a period of one year.
    During the course of this study, rats were bred to produce three
    successive generations. Two generations were maintained on the test
    diet for six months. BHA had no effect on reproductive performance, as
    measured by litter size, birth weight, date of appearance of incisors,
    and opening of eyes. Autopsy and histological examination of tissues
    and organs of parents and offspring at the termination of the study
    did not reveal any compound-related effect (Karplyuk, 1962).

    OBSERVATION IN MAN

         Human volunteers were dosed with 0.5-0.7 mg/kg bw BHA. Twenty-two
    to seventy-seven per cent. was excreted in the urine as the
    glucuronide within 24 hours. Less than 1% was excreted in the urine as
    unchanged BHA, and no dealkylation of hydroxylation products was
    detected (Astill et al., 1962). In another study, human volunteers
    were administered a single dose of 14C-labelled BHA (approximately
    0.5 mg/kg bw). Sixty to seventy per cent. of the radioactivity was
    excreted in the urine after two days, and by day 11 post-dosing
    80-86.5% of the radioactivity was recovered in the urine (Daniel et
    al., 1967).

         The time course of disappearance of BHA from the plasma and
    appearance in the urine was measured in two healthy male human adults
    (body weight 55 and 60 kg) given a single dose of 100 mg of BHA in a
    gelatin capsule. Blood samples were withdrawn at 0.5, 1, 2, 4, 6 and 8
    hours following administration and urine samples were collected for
    0-8, 8-16, 16-24 and 24-36 hour intervals. Inter-subject variability
    in blood levels of BHA was large. In one subject, the peak BHA level
    of about 600 nano g/ml occurred two hours after dosing, while in the
    other subject a peak value of about 220 nano g/ml occurred 30 minutes
    after dosing. Less than 1% of dose was eliminated in the urine in 36
    hours. In both subjects, peak urinary levels occurred in the period
    between 8 and 16 hours following dosing (El-Rashidy & Niazi, 1979).

    Comments

         Several metabolic studies with orally administered BHA were
    available in rats, mice and monkeys. In the rat, at the levels tested,
    there appeared no change in the activity of the liver enzymes studied.
    In the case of mice, although there appeared to be no increase in aryl
    hydrocarbon hydroxylase, there was an increase in cytochrome P450 and
    the nature of the enzyme appears altered. With monkeys, on the other
    hand, cytochrome P450 levels appeared unaffected, whereas some enzyme
    activities were affected. In this regard, infant monkeys, as opposed
    to juveniles, did not show these changes. BHA appeared to lower liver
    cholesterol, whereas plasma triglycerides were increased.

         Female monkeys maintained on a dietary intake of 50 mg/kg bw for
    one year prior to breeding, and bred to untreated males, gave birth to
    normal offspring. BHA had no effect on any of the indices tested.
    These included haematology and clinical biochemistry as well as
    behavioural observations on the young.

         Mutagenic activity of BHA has been tested in several in vitro
    and in vivo systems. The collective assessment of these tests does
    not show BHA to be mutagenic; however, pre-treatment of animals with
    BHA may induce enzymes which alter the metabolism of some mutagenic
    compounds.

         Some new studies with BHA, notably behavioural studies in the
    newborn rat after exposure to the material in utero and through
    lactation, have become available. In contrast to the monkey, the rat
    showed slight behavioural impairment due to exposure. In addition, at
    the highest dosage there was increased pup mortality.

         The Committee extended the temporary ADI of 0-0.5 mg/kg bw
    pending the completion of a multigeneration reproduction study in the
    Sprague-Dawley rat.

         The previously stated requirement for studies on the effect on
    reproduction of mixtures of BHA, BHT and propyl gallate was considered
    to be no longer necessary.
    

    EVALUATION

    Level causing no toxicological effect

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

    Estimate of acceptable daily intake for man

    0-0.5* mg/kg bw.**

    FURTHER WORK OR INFORMATION

    Required by 1982

         Multigeneration reproduction feeding study in Sprague-Dawley rat.

              

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

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    See Also:
       Toxicological Abbreviations
       Butylated hydroxyanisole (BHA) (WHO Food Additives Series 18)
       Butylated hydroxyanisole (BHA) (WHO Food Additives Series 42)
       Butylated Hydroxyanisole (BHA) (IARC Summary & Evaluation, Volume 40, 1986)