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. Toœicol., 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