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 1980 (see Annex I, Refs. 6, 8, 11, 32, 40
and 54). Toxicological monographs were issued in 1961, 1964, 1965,
1973 and 1976 and 1980 (see Annex I, Refs. 6, 9, 13, 33, 41 and 55).
Since the previous evaluations, additional data have become
available and are summarized and discussed in the following monograph.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Effects on enzymes and other biochemical parameters
BHT in the diet of Sprague-Dawley rats resulted in a marked
decrease in the NADPH-cytochrome P-450 reductase activity of isolated
liver microsomal preparations. This effect was not observed when BHT
was added in vitro to liver microsomes (Rikans et al., 1981). Rats
fed 0.4% BHT in their diet showed an increase in GSH-S transferase
activity in the liver, but not in lungs and kidneys. GSH-reductase
levels were increased in liver and lungs (Partridge et al., 1982).
Dietary BHT was also shown to effect the carboxylation process in the
conversion of rat liver microsomal protein to prothrombin (Takahashi &
Hiraga, 1981).
Addition of cyclic GMP (cGMP added as the dibutyl or 8-bromo
form) to BHT suppressed Mishell-Dulton cultures, effected a reversal
of the BHT suppression of antibody production (Wess & Archer, 1982).
The observed increase in tumour-specific antigen activity in the
colon chromatin of rats treated with 1,2-dimethylhydrazine was
abolished by simultaneous treatment with BHT (Gabrelak et al., 1981).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
Mouse
Groups each of 100 (B6C3F1) mice, equally divided by sex were fed
diets containing 0, 200, 100 or 5000 ppm (0, 0.02, 0.1 or 0.5% of BHT
for 96 weeks, followed by a basal diet for six weeks. The diets were
made by mixing the BHT with CE-2 (CLEA Japan, Inc., Tokyo) diet at the
appropriate concentration and pelleting. At the end of the test period
the surviving animals were killed. A complete autopsy was carried out,
and the principal organs and tissues were examined microscopically.
Mice that died during the course of the study were also autopsied. In
addition terminal blood samples were collected for haematological
examination, and serum clinical biochemistry. Urine samples were also
examined. During the course of the study, food consumption was similar
for test and control groups. Body weights of females in the 1000 and
5000 ppm (0.1 and 0.5% groups were lower than controls, as was the
body weight of males in the 5000 ppm (0.5%) group. There were minor
changes in the absolute weight of some organs in the high dose groups
(salivary glands, heart and kidney). In males the serum GOT and GPT
levels in the 5000 ppm (0.5%) group were higher than controls. No
other compound related effects were observed in the haematological,
serum and urine analysis. Neoplastic lesions were reported in both
test and control animals. The tumours that occurred with greatest
frequency were adenomas of the lungs, hyperplastic nodules and
hepatocellular carcinomas of the liver and malignant lymphomas.
However, there was no statistically significant difference between the
BHT treated and control groups for the incidence of any type of tumour
(Shirai et al., 1982).
Rat
Groups of 57 Wistar rats (seven weeks old) of each sex were
maintained on diets containing 2 500 or 10 000 ppm (0.25 or 1% of BHT
for 104 weeks. Control groups consisted of 36 rats of each sex. At the
end of the test period the surviving animals were killed and a
complete autopsy was carried out. The principal organs and tissues
were examined microscopically. Terminal blood samples were collected
for haematological examination and serum clinical biochemistry. Food
intake was similar for test and control animals, but body weight gain
was reduced in both male and female rats in the high dose groups.
Increased relative liver weight was observed in all test animals, and
decreased spleen weight in the females. Total blood cholesterol was
increased in all test animals and increased red blood cell counts were
observed in females. The overall incidence of tumours was slightly but
not significantly higher in BHT treated rats than in controls. The
incidence of hyperplastic nodules and of pancreatic carcinomas in
female rats and of pituitary adenomas and adenocarcinomas in test
animals was higher than those in controls. However, with the exception
of the incidence of pituitary adenomas in the low dose females, these
differences were not significantly different from controls. Since this
effect was not dose related, it was concluded that BHT, under the
conditions of this test, was not carcinogenic (Hirose, 1980).
Potentiation or inhibition of carcinogenesis
Groups of Swiss mice were given 1000, 250, or 50 mg/kg urethan or
0.9% NaCl. Seven days later, half the urethan treated animals and half
the controls received 300 mg/kg BHT i.p. the remaining animals
receiving corn oil alone. Thirteen weekly injections were given. The
number of tumours/lung found 14-24 weeks after the initial urethan
doses was significantly increased in the BHT treated animals. In
another study, when the interval between injection of the urethan and
the first treatment with BHT was delayed for six weeks, BHT treatment
produced more tumours. When the number of BHT injections commencing
one week after urethan treatment was reduced from 13 to four, the same
significant increase in rumour yield was observed as in the 13-dose
study. However, one or two doses of BHT had no significant effect.
When the mice were pretreated with 13 injections of BHT, and then
treated with urethan one week later, there was no enhancement of
tumour yield. Simultaneous administration of BHT and urethan, resulted
in fewer rumours compared to animals treated with urethan alone. When
mouse strains (C57BL, C3H and BALB/C) which have a low naturally
occurring incidence of lung adenoma were treated with urethan and then
with multiple injections of BHT, the BHT treatment did not
significantly increase rumour incidence or average numbers of rumours
per lung (Witschi & Lock, 1979).
Male Strain A mice were injected i.p. with 500 mg/kg urethan,
then one week later received repeated injections (one a week for eight
weeks) of either 300 mg/kg BHT, or BHA, 500 mg/kg, or Vitamin E,
1000 mg/kg, all dissolved in corn oil. At the termination of the
study, only BHT was shown to produce a significant increase in rumour
yield. Although the number of rumours produced by BHA treatment was
greater than usual, it was not statistically significant. A/J mice
treated with 3-methylcholanthrene or dimethylnitrosamine, followed by
treatment with BHT (i.p.), resulted in an increase in rumour yield
(Witschi et al., 1981). In another study, male A/J mice were injected
i.p. with a single dose of urethan and then fed either 0.75% BHT, or
BHA or ethoxyquin in the diet, once a week, or continuously for eight
weeks. Lung tumours yield was scored four months after the urethan
treatment. Dietary BHT, but not BHA or ethoxyquin, under either
conditions of the test, enhanced lung rumour formation. Mice were
prefed with diets containing either BHA or BHT for two weeks prior to
urethan treatment, and then maintained on conventional laboratory
diets for four months. The BHT diet had no effect on tumour yield, but
the BHA treatment significantly decreased the average number of
tumours (Witschi, 1981).
In another study A/J mice were given a single dose of BHT i.p.
(400 mg/kg), sufficient to cause acute lung damage and produce cell
proliferation in the lung for six to seven days. Urethan was
administered continuously by implanted minipumps during this period.
Continuous presence of urethan during the period of cell division did
not result in an enhanced number of the rumours. When urethan injected
mice were dosed i.p. with SKF525A (2-diethylaminoethyl-2-,
2-di-phenylvalerate hydrochloride) and BHT (SKF inhibits lung cell
division normally seen following BHT administration), or BHT alone,
both treatments gave a very significant increase in lung tumour yield
compared to urethan treated controls (Witschi & Kehrer, 1982).
Repeated pulmonary cell division brought about by other treatments,
e.g., 95-100% oxygen, were also shown not to enhance tumour
development (Witschi & Kehrer, 1982).
Groups of female Sprague-Dawley rats were treated with either
7-12-dimethylbenz[a]anthracene (DMBA) or nitrosomethylurea (NMU) and
then fed diets containing 0 or 0.3% added BHT for 30 weeks. Rats
treated with DMBA and maintained on the control diet developed 100%
tumour incidence (mammary gland) by week 27, whereas, those maintained
on the BHT supplemented diet had an incidence of 54% by the end of the
study. Dietary BHT had no effect on the incidence of rumours induced
by NMU treatment (King, McCay & Kosanke, 1981).
Reproduction and behavioural studies
Groups each of 46 rats, six weeks old (Wistar outbred, SPF) were
fed diets containing 0, or 0.5 to 0.9% BHT so that the dietary intake
of BHT was equivalent to 500 mg/kg during the course of the study. At
week 19, the F0 generation was mated. Twenty-four hours after birth
of F1 rats, the size of the litters was reduced to eight, and half of
the litters wore cross-fostered. Body weight of parents and offspring
and developmental events of offspring were monitored during the course
of the study, as well as the reproductive performance of the F0 rats.
Auditory and visual function and locomotive coordination tests were
carried out on the F1 generation. The F1 animals were autopsied at
day 25 of age, and a histological examination made of the brains. Body
weights and weight gain of test animals were reduced when compared to
controls, and this persisted during gestation. The duration of
pregnancy, average body weight, and litter size were similar for test
and control animals. The average body weight and weight gain of the
F1 offsprings was significantly reduced in pups nursed by dosed
mothers. Pups exposed in utero to BHT also showed a relatively
slower development than controls when fostered with non-dosed mothers.
Pups exposed to BHT in utero and/or mothers milk showed alterations
in the behavioural patterns examined as well as higher incidence in
average number of dead cells in the brain (Meyer & Hansen, 1980).
Detailed comments were submitted by the Chemical Manufacturers
Association (CMA) (1983) on studies of the effect of BHT on
reproduction and teratogenicity. The major comments were concerned
with the studies of Brunner et al. (1978) and Vorhees et al. (1981)
previously reviewed by JECFA in 1980 as well as the study by Meyer &
Hansen (1980).
In the case of the Brunner et al. and Vorhees et al. study, it
was concluded that the study showed normal pup survival and
development in pups raised by rat dams on diets containing 0.125% BHT.
Normal post-weaning development was observed in pups raised by rat
dams on diets containing 0.25% BHT, although increased post-weaning
mortality occurred in pups raised by dams on the 0.25% and 0.5% diet;
developmental delays occurred in pups in the 0.5% group. In the case
of the Meyer and Hansen (1980) study, developmental delays were seen
in rats raised by rat dams on diets containing 0.5% BHT. At the 0.25%
and 0.5% level, the effect may be due either to toxic effects of BHT
on the rat dam, or direct toxicity during lactation. A number of
questions were also raised about the design of the Brunner or Vorhees
study. These are: (1) the pup selection, in which all litters of fewer
than eight live pups were discarded; (2) the excess mortality was
reported in terms of pup count rather than affected litters. The data
from this study have been audited by the United States FDA (1983). It
was concluded that the raw data support the authors observations of
increased mortality in the mid-dose and high-dose BHT offspring.
However, excess mortality occurred in a limited number of litters;
e.g., in the 0.5% group, of the 60 deaths reported in 19 litters, 49
of the deaths occurred in five litters, and in the 0.25% group of the
42 deaths, 21 occurred in two litters, and at the 0.125% level of the
12 deaths, 11 occurred in one litter. It was also noted that in the
high dose group; that there was an increased number of litters with
eight pups or less, and no litters larger than 12 pups. In the other
dose groups, the litter size was comparable to controls.
In the case of the Meyer & Hansen (1980) study, the CMA comments
note that the level of BHT used in the study caused toxicity in the
dams, which appears directly or indirectly to affect the pups. Reports
of teratogenicity studies and/or one generation reproduction studies
in several strains of mice and rats as well as a three generation
reproduction study in rats were also submitted in the comments to
support a "no effect" level of 0.1% BHT in the diet.
Special studies on the effect of BHT on the thyroid
Male MOL/WIST SPF rats, outbred strain (approximately 200 g) were
used for the study. BHT was added to a semi-synthetic diet in which
the iodine content was controlled at about 12 µg/100 g (nutritional
requirement for the rat is 15 µg/100 g). In one study, rats were fed
0, 500 or 5000 ppm (0, 0.05 or 0.5%) BHT in the diet for eight, 26 and
90 days, and the uptake of 125I by the thyroid determined. The
presence of BHT in the diet resulted in a marked increase in the
uptake of 125I at all time periods studied. When rats were fed BHT in
diets containing varying amounts of iodine (12, 150 or 300 µg/100 g)
for 30 days there was a significant increase in thyroid weight in BHT
treated animals when compared to controls. BHT in the diet of rats
increased liver and thyroid weights at 5000 ppm (0.5%) of the diet,
but only thyroid weight at 500 ppm (0.05%). BHT did not change levels
of T3 and T4 in the blood. The biological half life of thyroxine was
increased after 13 days on a BHT diet but returned to normal after 75
days. Electron microscopy of the thyroid glands of rats exposed to
dietary BHT (5000 ppm (0.5%)) for 28 days showed an increase in the
number of follicle cells (Sondergaard & Olsen, 1982).
Special studies on haemorrhagic toxicosis
The LD50 (i.p.) for BHT showed considerable differences for
strains of inbred and non-inbred male mice.
Strain LD50 (mg/kg)
DBA/2N (inbred) 138
BALB/cNnN (inbred) 1 739
C57BL/6N (inbred) 917
ICR-JCL (non-inbred) 1 243
In all cases death occurred four to six days after administration of
BHT, and was accompanied by massive oedema and haemorrhage in the lung
(Kawano et al., 1981).
Male rats (Sprague-Dawley) were fed diets containing 0 or 1.2%
BHT for one week. BHT treated rats showed haemorrhages in most organs.
There was a significantly increased leakage of Evans blue into the
epididymis. In addition, inhibition of ADP induced platelet
aggregation and decreased platelet factor 3 availability was observed.
Plasma prothrombin factors were decreased, but fibronolytic activity
was unchanged (Takahashi & Hiraga, 1981).
In another study in which the haemorrhagic response was studied
in a number of strains of rats (Sprague-Dawley, Wistar, Donryu and
Fischer), mice (ICR, ddY, DBA/c, C3H/He, BALB/CaAn and C57BL/6), New
Zealand White-Sat rabbits, beagle dogs, and Japanese quail fed diets
containing BHT (1.2% of the diet for rats and mice), (1% of the diet
for quail), (170 or 700 mg/kg bw for rabbits) and (173, 400 or 760
mg/kg bw for dogs) for a period of 14-17 days. Haemorrhagic deaths
occurred among male rats of all strains and female rats of the Fischer
strain. Female rats of the Donryu, and Sprague-Dawley strain showed no
obvious haemorrhaging. No haemorrhagic effects were noted in rabbits
or dogs (Takahashi et al., 1980).
Comments
A recent lifetime study in mice and a 104-week study in rats
showed that under the conditions of the test BHT was not carcinogenic.
Additional studies are available on the role of BHT in the
enhancement of lung tumour yield by chemical carcinogens, in
susceptible species of mice. BHT has also been shown to be effective
as a promoting agent in this assay system when the test animals were
treated with either polycyclic hydrocarbons or nitrosamine. The lowest
dose at which BHT can act as a promoter of urethan induced lung
tumours in the mouse has not been established. No additional studies
are available on the possible promotion of hepatic carcinogenesis
caused by chemical carcinogens. BHT only acts as a promoter when
administered after exposure to the chemical carcinogen, but not
before. BHT has also been shown to inhibit the effect of some chemical
carcinogens. The protective effect may be associated with changes in
the metabolism of the carcinogen resulting from enzyme induction
caused by BHT. More information is required on the conditions as well
as the mechanisms of the inhibitory or promotional activity of BHT on
chemical carcinogens, to assist in interpretation of these studies
before they provide a useful basis for the toxicological evaluation.
A recent study on the behavioural and development effects of BHT
on rats exposed in utero during lactation showed that BHT caused a
significant decrease in body weight gain of both offspring and parent.
Altered behavioural patterns, as well as brain lesions were noted in
the offspring. However, only one dose level (0.5% BHT in the diet) was
used in this study, and this level was toxic to the dams. A detailed
analysis of data from the study of Vorhees et al. (1981) showed that
at both the mid and high (0.5% and 0.25%) dose levels, the excess
mortality may reflect litter effects. The data indicate a "no effect"
level for BHT-induced reproductive effects to be 0.1% of the diet of
rats. A lifetime feeding study with rats, which involves a single
generation reproduction study, is under way. The data from this study
will provide additional information to support a "no effect" level
from BHT in reproduction studies in the rat.
The haemorrhagic effects of massive doses of BHT seen in certain
species of mice but not in dogs and certain species of rats may be
related to its ability to interfere with vitamin K metabolism.
Rats exposed to 500 or 5000 ppm (0.05 or 0.5% of dietary BHT
showed a significant increase in thyroid weights, as well as the
ability of the thyroid to take up iodine. However, lifetime studies in
rats maintained on diets containing up to 10 000 ppm (1%) BHT have not
shown adverse effects on the thyroid.
Previously reported studies on induction of microsomal enzymes,
reproduction and behavioural effects provide a basis for setting a "no
effect" level.
EVALUATION
Level causing no toxicological effect
Mouse: 5000 ppm (0.5%) in the diet, equivalent to 250 mg/kg.
Rat : 1000 ppm (0.1%) in the diet, equivalent to 50 mg/kg.
Estimate of temporary acceptable daily intake for man
0-0.5* mg/kg bw.
FURTHER WORK OR INFORMATION
Required by 1986.
Submission of the lifetime feeding study known to be in progress
which includes a single generation reproduction study.
* Group ADI: As BHA, BHT, and TBHQ, singly or in combination.
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See Also:
Toxicological Abbreviations
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 15)
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 28)
Butylated hydroxytoluene (BHT) (WHO Food Additives Series 42)
Butylated Hydroxytoluene (BHT) (IARC Summary & Evaluation, Volume 40, 1986)