BUTYLATED HYDROXYTOLUENE (BHT)
Explanation
This substance has been evaluated for acceptable daily intake for
man (ADI) by the Joint FAO/WHO Expert Committee on Food Additives in
1961, 1964, 1965, 1973 and 1976 (see Annex I, Refs. 6, 8, 11, 32 and
40). Toxicological monographs were issued in 1961, 1964, 1965, 1973
and 1976 (see Annex I, Refs. 6, 9, 13, 33 and 41).
Since the previous evaluations, additional data have become
available and are summarized and discussed in the following monograph
addendum.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Effects on enzymes and other biochemical parameters
Groups of 16-24 Swiss male mice (25-30 g) received a single i.p.
injection of BHT in corn oil (62.5, 215, or 500 mg/kg bw) or corn oil
only (Tocopherol stripped 0.5 ml). Three days later the mice were
sacrificed. After BHT, wet lung weights were increased to 120% of
control, as were dry lung weights. There were significant increases in
DNA content and level of non-protein sulfhydryl (133-156% of control).
Superoxide dismutase and other oxidative enzyme levels were increased.
The authors concluded that BHT apparently increased inflammatory and
reparative-proliferative processes of the lung (Omaye et al., 1977).
BHT (500 mg/kg/day) was administered by gavage to groups of young
Wistar male and female rats (60-80 g) for seven days and the animals
were housed in metabolism cages. Control animals received corn oil
vehicle only. They were then sacrificed and liver enzymes (aniline 4
hydroxylase, biphenyl-4-hydroxylase, ethyl morphine N-demethylase, and
4-methyl umbelliferone glucoronyl transferase) were assayed and the
cytochrome P-450-CO interaction spectrum evaluated. Urinalysis, using
gas chromatography to assay for D-glucaric acid, D-glucuronic acid,
1-gulonic acid, xylitol and L-ascorbic acid was conducted.
Administration of BHT enhanced all the parameters measured with the
exception of the hepatic microsomal protein content. BHT was a more
potent inducer of xenobiotic metabolism in female rats (Lake et al.,
1976).
Groups of NMRI mice (25-35 g) and Wistar rats (160-320 g)
received BHT as a single dose of 500 mg/kg dissolved in soya bean oil,
either i.p. or by gavage. Four days later, radiolabelled C14
thymidine was given. After 90 minutes, the animals were sacrificed,
lungs removed and DNA levels were measured. In mice, DNA synthesis was
equally increased in males and females by oral or i.p. administration.
Although lung weight was increased, the concentration of DNA was not
affected. In rats, no effect was seen in males and only a slight
increase in females (Larsen & Tarding, 1977).
TOXICOLOGICAL STUDIES
Special studies
Potentiation or inhibition of carcinogenesis
The tumorigenic potency of a single i.p. injection of 1000 mg/kg
of urethan to male Swiss-Webster mice was significantly increased if
followed by repeated weekly injections with 250 mg/kg BHT. The number
of animals used per group ranged between nine and 22 and the animals
were treated from nine to 13 weeks. Only tumours on the lung surface
itself were counted. About 90% of the animals treated with urethan
alone developed lung tumours. There was a significant increase in the
number of tumours per mouse after 11 or more weeks of treatment with
BHT. Animals treated with BHT alone did not develop lung tumours. A/J
strain mice were also given the same treatment with 10 weekly
injections of BHT. The number of lung tumours per animal significantly
increased in those receiving BHT in addition to urethan in comparison
with those receiving urethan alone. With both strains of mice,
repeated injection of BHT without prior urethan treatment did not
result in an increased number of animals with lung tumours or
tumours/mouse as compared to controls dosed with corn oil. With both
mouse strains, there were fewer lung tumours in the animals given BHT
as compared to the corn oil controls. In contrast to the above
results, injection of animals with BHT for 0-7 days before urethan
injection did not increase the number of animals with tumours or
number of tumours/animal (Witschi & Cote, 1976).
A group of 93 rats (22 days old) received 0.5% BHT or control
diets for 407 days following 18 days of administration of 2-acetyl-
aminofluorene (0.02%). No control group received BHT only without
prior AAF feeding. Prolonged feeding of BHT diet after 2-AAF produced
a significant enhancement of hepatic carcinogenesis as shown by
increased number of rats with liver tumours (Peraino et al., 1977).
Mutagenicity
BHT was found not to be mutagenic using Saccharomyces
cerevisiae, D4 and Salmonella typhimurium, TA-1535, TA-1537 and
TA-1538, with and without the addition of mammalian metabolic
activation (Brusick, 1975).
BHT was fed at the 5% level in a sex-linked recessive lethal test
in Drosophila. About 0.27% lethals were found in the BHT treated
flies (out of 8897), 0.29% lethals were found in the control and 5.69%
lethals were found in the positive control dosed with 100 ppm
ethylmethane sulfonate. It was concluded that BHT was not mutagenic
(Brusick, 1975).
The effect of BHT on excision repair synthesis was tested in
cultured human lymphocytes irradiated with u.v. light. Irradiated
cells cultured in the presence of BHT had decreased excision repair
based on lower incorporation of tritiated thymidine into DNA with
increasing concentration of BHT in the culture medium. In this study,
BHT also inhibited semi-conservative DNA synthesis (Daugherty et al.,
1978).
A study was conducted to confirm a reported radiosensitizing
effect of BHT on Drosophila sperm, using three radiation doses (1.2,
2.4 and 3.6 KRDS). The mutagenic activity of BHT was also tested in
the sex-linked recessive lethal system. Ten-day-old males of
D. melangaster received 0.2 µl of saline in ethanol or 0.001% BHT in
the solvent. They were exposed to gamma rays from a 137CS source at
4.1 rad/sec. They were mated to four females and two successive one
day broods were tested to obtain sensitivity of mature and slightly
immature sperm, after discarding xo males and non-disjunction females
remaining. F1 flies were pooled and F2 females obtained after
individual brother/sister mating. The absence of bar-eyed males
indicates the presence of sex-linked recessive lethals. There was no
shift in the sex ratio of the F1 generation. Without irradiation, BHT
seems to enhance the incidence of xo males. BHT almost doubled the
incidence of sex-linked lethals over saline-injected controls, but
this was not statistically significant. In comparison with gamma rays,
BHT had no influence on the sex ratio of F1 flies, and seemed to have
a negligible effect on the incidence of radiation loss of xB
chromosome. At all doses BHT exhibited a pronounced radiosensitization
of sex-linked recessive lethals (Prasad & Kamra, 1974).
Reproduction and behavioural studies
Breeding pairs of Sprague-Dawley rats (200-220 g) received Purina
chow supplemented with 0.5, 0.25 or 0.125% BHT ad libitum beginning
from the week before mating and continuing in females through
lactation and weaning of the pups. Growth rates and mortality were
adversely affected. Pre-weaning pups born of mothers at the highest
dose level (0.5%) of BHT weighed significantly less than controls at
ages seven, 14 and 21 days. The total number of pups dying on study,
born of dams receiving 0.5 or 0.2% BHT, was significantly higher than
controls. Behavioural tests were conducted consisting of righting
reflex, pivoting cliff avoidance, startle response, swimming, open
field, running, wheel activity, roto-rod, active avoidance, position
discrimination and passive avoidance. For pre-weaning testing, no
differences were noted at the low or mid dose groups. At the high dose
level, there was significant increase in surface righting time,
delayed forelimb swimming development and a trend to less activity in
open field tests. In post-weaning tests, males in 0.25% BHT groups
showed an effect on passive avoidance, with more partial re-entries
into compartments where shocked. For all other tests, there were no
statistical differences, suggesting that BHT had no effect on basic
motor coordination, active avoidance acquisition, or extinction
performance (Brunner et al., 1979).
Effect on lung
The pathological effect of BHT on the mouse lung was studied.
Sixty male Swiss mice were given i.p. injections of 400 mg/kg BHT
dissolved in corn oil. Six of the experimental animals and six of the
controls were sacrificed daily for nine days. Two hours before
sacrifice, each animal received 2 µCi/g of tritiated thymidine. No
animals died during the study and none showed signs of respiratory
distress. Two days after dosing, cellular lesions were noticed in the
type I alveolar epithelium. Abnormal giant type II cells were observed
in mitosis and many had an accumulation of tritiated thymidine.
Labelled endothelial cells were seen after day 6 in small vessels and
capillaries, and there was an increase in fibroblastic cells in the
interstitium and capillaries. There was an increase in thymidine-
labelled pulmonary cells from days 2 through 5 after which labelling
dropped off and approached control levels by day 9. Levels of lung
thymidine kinase activity rose sharply on days 1-4 after dosing and
then dropped off rapidly (Adamson et al., 1977).
Following acute exposure to BHT, the initial sequence of events
involves infiltration of type I (squamous) epithelial cells followed
by multifocal necrosis and destruction of the blood barrier. A
detailed discussion of the sequence of tissue changes and repair
mechanisms is given. It is stated that the susceptibility of the
squamous epithelium to injury is similar to that seen after oxygen
exposure, radiation exposure, and treatment with blood-borne
bleomycin, but the recovery pattern is quite different. BHT is thought
to cause cell lysis and death as a result of interaction with the cell
membrane (Anonymous, 1978).
The increase in lung weight and increase in thymidine
incorporation into lung DNA observed in mice following BHT injection
was inhibited by treatment with cedar terpenes. No increase in lung
weight was observed in animals treated with BHT alone if they were
less than three weeks old. This may result from the inability of the
infant mice to metabolize BHT (Malkinson, 1979).
Immunological studies
In vitro BHT (50 µg/culture) suppressed the plaque-forming cell
response of mouse spleen cell cultures as measured by the method of
Mishell & Dutton (Archer et al., 1977).
Carcinogenicity
Groups of 50 Fischer 344 rats/sex and 50 B6C3F1 mice/sex received
3000 ppm or 6000 ppm BHT in the diet. The compound was mixed with
autoclaved lab meal containing 4% fat. A control group of 20 animals
per sex received lab meal only. The rats were on study for 105 weeks
and the mice for 107 or 108 weeks. There was a dose-related depression
in the body weights of the rats and mice of both sexes throughout the
study. In rats there was no significant effect of BHT on mortality. In
male mice BHT was significantly associated with decreased mortality,
while BHT feeding had no effect on mortality in female mice. There was
a high dose-related incidence of hepatocytomegaly and hepatocellular
degeneration and necrosis in the liver of male mice, but the incidence
of hepatocellular carcinomas was reduced in a dose-related manner. The
incidence of alveolar/bronchiolar carcinomas or adenomas was
significantly higher than controls in the low dose, but not the high
dose female mice. BHT feeding was related to a significant reduction
in the incidence of sarcomas of multiple organs in female mice. Four
adenomas of the eye/lacrimal gland were observed in the high dose male
mice and in two low dose females but not in corresponding controls.
The historical incidence of this tumour was 1.2%. Since the lacrimal
gland was only examined microscopically in animals with grossly
apparent lesions, the report states that the lacrimal gland tumours
could not be clearly related to BHT administration. There was an
increased incidence of focal alveolar histiocytosis in treated rats,
especially in high dose females. The incidence of adenomas of the
pituitary was significantly reduced in female rats with BHT
administration. The report concluded that no neoplastic lesions
occurred in rats or mice that could be clearly related to BHT
administration. Under the conditions of the test, there were increased
incidences of focal alveolar histiocytosis in dosed male rats which
may have been related to the administration of BHT. BHT was not,
however, carcinogenic for F344 rats or B6C3F1 mice of either sex. The
report was evaluated by the Data Evaluation/Risk Assessment subgroups
of the Clearinghouse on Environmental Carcinogens. The subgroup
accepted the conclusions of the report but, because of the widespread
human exposure, evidence of hepatotoxicity and a suggestion of a
tumorigenic effect in the lung, the subgroup suggested that the
compound be considered for retest by the chemical selection working
group (Anonymous, 1979).
Short-term studies
Rat
Groups of male Sprague-Dawley rats (six weeks of age) received
BHT in a semi-synthetic diet at concentrations ranging from 0.58% to
1.44% or control diet only. Deaths occurred within 40 days, at levels
of 0.69% or greater. Spontaneous massive bleeding to the pleural and
peritoneal cavities, or as external haemorrhage, was observed in all
dead or dying animals. The prothrombin index was decreased as the
daily dose of BHT was increased. Mild diarrhoea was noted after four
days. Rough hair coat, and redness of urine was noted. Death was due
to haemorrhage and was classified by the authors as a secondary type
of toxicity, probably due to a decrease in prothrombin concentration.
According to the authors, the effect seems to depend on strain of rats
and dietary concentration (Takahashi & Hiraga, 1978).
Long-term studies
(See also under "Carcinogenicity")
Rats
Groups of female rats (80-100 g) received 0.4% BHT with corn oil
mixed in ground lab chow and were sacrificed at intervals of 1, 8, 16,
32, or 80 weeks, and compared with controls. Samples of liver were
taken for biochemical, histochemical, and morphological studies to
examine for reversibility of hepatic changes. Control diet only was
administered for 18 days to a group of four rats following 80 weeks of
BHT administration. After one week on BHT there was a marked liver
enlargement with relative liver weight increased up to 35% and with an
increase in drug metabolizing activities and NADP-cytochrome C
reductase activity. After 18 days of removal from the 80-week
treatment there was only a slight increase in liver weight. The effect
was therefore reversible. Histologically, after BHT treatment the
liver was characterized by enlarged centrilobular hepatocytes, with a
heterogenous appearance of this zone. Ultrastructurally, there was a
proliferation of smooth endoplasmic reticulum. The authors note that,
although there is no unequivocal evidence of liver injury, there were
two features that are also seen with many hepatotoxins and
hepatocarcinogens: depression of glucose-6-phosphatase activity and
cell enlargement. However, there were no lysosomal changes
characteristic of cytologic injury, and effects were reversible
(Crampton et al., 1977).
Groups of 40 JCL strain rats/sex/group, reared under a barrier
system and four weeks of age at the start of the study, received
either control diet or BHT at 0.005, 0.062, 0.32% in the diet. Of each
group of 40, 15 received compound for a "lifetime", 10 for 24 months,
and five each for three, six or 12 months. At interim and final kill,
liver, kidney, heart, spleen, thyroid and caecum weight were
determined as were haematology, serum biochemistry, urinalysis, and
histological investigation of the tissues. At 24 months, heart, liver,
kidney, spleen, pituitary, thyroid, adrenal, testes, prostate and
brain were weighed, haematological and biochemical measurements
conducted and histopathology done. There was an increase in liver
weight, serum cholesterol, serum K+ and histological changes in
liver and kidney at the 0.32% dietary level. There was no change in
quantity of food intake, body weight gain, mortality during feeding or
mean life span and no finding suspicious of tumour induction. There
was no indication of a dose-related trend in tumour prevalence in
either 24-month or "life span" groups. The tumours found were said to
be typical of those described in aged rats. It is to be noted that the
number of surviving rats is small and that tumour data include both
life span groups and animals dead or sacrificed moribund during the
six, 12 and 24 month feeding. The available data do not list the
number of each of the individual tissues examined, although the number
of rats is listed. The data show a tendency for a decreased number of
tumours per rat at higher dose levels (Hiraga, 1978).
Six-week-old random bred Wistar rats were allocated to groups of
57 males and 57 females fed control diet and 36 males and 36 females
fed BHT at either 0.25% or 1% in the diet for 104 weeks. Survival by
tests groups was between 40% and 68%. A variety of tumours were noted
on histopathological examination at the end of the study, with no
dose-related response in either type of tumour or total number as
compared to controls (Shibata et al., 1979).
Comments
Several new studies were available on BHT, including a
behavioural study in newborn rats that had been exposed to the
material in utero and during lactation. Decreased pup survival and
slight behavioural effects were noted at levels above 0.1% of the
diet. It was noted that behavioural effects were not seen in newborn
monkeys whose mothers had been treated with the chemically related
BHA.
At its twentieth meeting, the Committee had noted that BHT had
been reported to enhance the occurrence of lung adenomas in mice. At
that meeting, it had considered that BHT was not likely to be
carcinogenic but had requested additional long-term studies. Recently
completed long-term studies in mice and rats have been negative and
confirm the view that BHT is not carcinogenic.
Two series of studies with BHT have demonstrated that it enhances
the effect of certain chemical carcinogens. In one study, it was shown
that mice initially receiving injections of the carcinogen urethane
developed more lung adenomas if treated subsequently for some weeks
with BHT. In another study, rats treated with low levels of the
carcinogen N-fluorenyl acetamide and subsequently with BHT developed
more hepatomas, more rapidly than with the carcinogen alone.
In these studies, it is implied that BHT is a "promoting" agent.
Possible mechanisms of action include enzyme induction and the
production of hyperplasia and hypertrophy in both the lung and the
liver by BHT.
The phenomenon of "promotion" of carcinogenesis in various
systems, including the skin and urinary bladder, as well as these
examples, are attracting much attention in cancer research. Mechanisms
of action, although under intense study, are not yet understood.
In addition, since BHT has been shown to inhibit the action of
carcinogens under other conditions, it is felt premature to use such
information for toxicological evaluation.
As regards effects on microsomal enzymes, reproduction and
behaviour, a no-effect level of 0.1% equal to 50 mg/kg in the diet of
rodents can be set.
EVALUATION
Levels causing no toxicological effect
Mouse: 5000 ppm (0.5%) in the diet, equivalent to 250 mg/kg bw
Rat: 1000 ppm (0.1%) in the diet, equivalent to 50 mg/kg bw
Estimate of acceptable daily intake for man
0-0.5* mg/kg bw**
FURTHER WORK OR INFORMATION
Required by 1981
(1) A study to clarify the effects on rat pup survival.
(2) Elucidation of the significance of the behavioural effects
observed in newborn rats.
* BHT, TBHQ, or the sum of the three compounds.
** Temporary.
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