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 µm BHA was reported to cause a 50%
inhibition of the synthesis of prostaglandin E1 by microsomes
isolated from bull seminal vesicles, 3.08 µg 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 µg/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 µg/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)