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    Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents



    WHO FOOD ADDITIVES SERIES NO. 5







    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization
    Geneva
    1974

              

    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.

    BUTYLATED HYDROXYANISOLE

    Explanation

         Butylated hydroxyanisole was evaluated for acceptable daily
    intake by the Joint FAO/WHO Expert Committee on Food Additives (see
    Annex 1, Ref. No. 6) in 1961.

         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

         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; Bunnell 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).

    TOXICOLOGICAL STUDIES

    Special studies on the effect on liver enzymes

         Rats administered 500 mg/kg bw (seven daily doses) of BHA, showed
    no change in liver glucose 6 phosphatase activity (Feuer et al.,
    1965). 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., 1965). Rats
    administered BHA (500 mg/kg/day) for two days showed no increased
    activity of microsomal processing enzymes (amino pyrrine demethylase)
    hexabarbitine oxidase and nitro anisole demethylase (Gilbert &
    Golberg, 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).

    Special studies on the effect on lipid metabolism

         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).

    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 one to 20 of pregnancy or
    single administration on day nine, 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 to 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).

    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
    to 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 microinvasion 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 the action of bradykinin

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

    Acute toxicity
                                                                        

                               LD50
    Animal         Route       (mg/kg bw)          References
                                                                        

    Mouse          oral        2 000               Bunnell et al., 1955;
                                                   Lehman, et al., 1951

    Rat            oral        2 200 to 5 000      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 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 3.3-7.0 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 ten-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).

    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 offsprings at the termination of the study
    did not reveal any compound-related effect (Karplyuk, 1962).

    Dog

         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 3/4 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).

    OBSERVATIONS IN MAN

         Human volunteers were dosed with 0.5-0.7 mg/kg bw BHA. 22 to 77%
    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 or hydroxylation products were 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). 60 to 70% of
    the radioactivity was excreted in the urine within two days, and by
    day 11 post-dosing, 80 to 86.5% of the radioactivity was recovered in
    the urine (Daniel et al., 1967).

    Comments:

         The available metabolic data in man indicate that absorbed BHA is
    rapidly excreted in the urine and is not likely to accumulate in the
    body. Since the increase in liver weight observed in rat feeding
    studies is only a transient effect, and since no other effects have
    been observed this should not be considered as an adverse effect. The
    significance of the effect of BHA on lipid metabolism of rats is not
    known, however there are ample long-term feeding studies showing that
    rats can be maintained on diets containing up to 0.5% BHA without any
    adverse effects. Since the metabolism of BHA in the dog is apparently
    different from that in man, the results of studies in the dog are of
    limited value for evaluation.

    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 1976.

         Studies on the effect on reproduction of mixtures of BHA, BHT and
    propyl gallate, and of BHA alone.

              

    *    As BNA, BHT or the sum of both.

    **   Temporary.

    REFERENCES

    Astill, B. D. et al. (1962) J. Ag. & Food Chem., 10, 315

    Astill, B. D., Fassett, D. W. & Roudabush, R. L. (1960) Biochem. J.,
         75, 543

    Brown, W. D., Johnson, A. R. & O'Halloran, M. W. (1959) Aust. J. exp.
         Biol. med. Sci., 37, 533

    Bunnell, R. H. et al. (1955) Poultry Sci., 34, 1068

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

    Creaven, P. J., Davies, W. H. & Williams, R. T. (1966) J. Pharm.
         Pharmacol., 18, 485

    Dacre, J. C. (1960) N. Z. J. Inst. Chem., 24, 161

    Dacre, J. C., Denz, F. A. & Kennedy, T. H. (1956) Biochem. J., 64, 777

    Daniel, J. W. et al. (1967) Fd. Cosmet. Toxicol., 5, 475

    Denz, F. A. & Llaurado, J. G. (1957) Brit. J. exp. Path., 38, 515

    Feuer, G., Golberg, L. & LePelley, J. R. (1965) Fd. Cosmet. Toxicol.,
         3, 235

    Francois, A. C. & Pihet, A. (1960) Ann. ind. natl. recherche agron.
         Ser., D9;195

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

    Golder, W. S., Ryan, A. J. & Wright, S. E. (1962) J. Pharm.
         Pharmacol., 14, 268

    Graham, W. D. & Grice, H. C. (1955) J. Pharm. (Lond.), 7, 126

    Graham. W. D.. Teed, H. & Grice, H. C. (1954) J. Pharm. (Lond.), 6,
         534

    Hodge, H. C. et al. (1966) Tox. Appl. Pharm., 9, 583

    Hodge, H. C. et al. (1964) Tox. Appl. Pharm., 6, 512

    Johnson, A. R. & Hewgill, E. R. (1961) Aust. J. exp. Biol. med. Sci.,
         39, 353

    Johnson, A. R., O'Halloran, M. W. & Hewgill, F. R. (1958) J. Amer. oil
         Chem. Soc., 35, 496

    Karplyuk, I. A. (1962) Tr. 2-01 (Vtoroi) Nauchn. Konf. po Vopr. Probl.
         Zhira v Pitanii, Leningrad, 1962:318

    Lehman, A. J. et al. (1951) Advanc. Food Res., 3, 197

    Posati, L. P. & Pallansch, M. J. (1970) Science, 168, 121

    Riley, P. A. & Seal, P. (1968) Nature, 220, 922

    Telford, I. R., Woodruff, C. S. & Linford, R. H. (1962) Am. J. Anal.,
         10, 29

    Wilder, O. H. M., Ostby, P. C. & Gregory, B. A. (1960) J. Ag. & Food
         Chem., 8, 504

    Wilder, O. H. M. & Kraybill, H. R. (1948) Summary of toxicity studies
         on butylated hydroxyanisole, American Meat Institute
         Foundation, University of Chicago


    See Also:
       Toxicological Abbreviations
       Butylated hydroxyanisole (WHO Food Additives Series 10)
       Butylated hydroxyanisole (WHO Food Additives Series 21)
       Butylated hydroxyanisole (WHO Food Additives Series 24)
       BUTYLATED HYDROXYANISOLE (JECFA Evaluation)