BUTYLATED HYDROXYTOLUENE (BHT)
EXPLANATION
This substance was evaluated for acceptable daily intake for man
by the Joint FAO/WHO Expert Committee on Food Additives at its sixth,
eighth, ninth, seventeenth, twentieth, twenty-fourth, and twenty-seven
meetings (Annex I, references 6, 8, 11, 32, 41, 53, and 62).
Toxicological monographs or monographs addends were published after
each of these meetings (Annex I, references 6, 9, 12, 33, 42, 54, and
63).
Since the previous evaluations, additional data, including the
results of studies requested by the twenty-seventh Committee (Annex 1,
reference 62), have become available and are summarized and discussed
in the following monograph addendum.
BIOLOGICAL DATA
Biochemical aspects
Metabolism
A single oral dose of 14C-BHT given to male and female mice
resulted in rapid absorption and distribution of 14C to the tissues.
Excretion of 14C was mainly in the faeces (41-65%) and urine
(26-50%), with lesser amounts in expired air (6-9%). The half-life for
a single dose in the major tissues studied (stomach, intestines,
liver, and kidney) was 9-11 hrs. When daily doses were given for 10
days, the half-life for 14C in the tissues examined was 5-15 days.
Metabolism was characterized by oxidation at one or both of the
tert-butyl groups, followed by formation of the glucuronide
conjugate, and excretion in the urine, or by excretion of the free
acid in the faeces. When 14C-BHT was administered to rats, 80-90% of
the 14C was excreted in the urine and faeces within 96 hrs, and less
than 0.3% of the 14C was in expired air. Most of the 14C-BHT was
excreted as the free acid in faeces with lesser amounts in the urine.
More than 43 metabolites were present in the urine and faeces of mice
and rats (Matuso, 1984).
Rats dosed with 14C-aflatoxin B1 and fed BHT (0.5% in the
diet) showed an enhanced excretion of water-soluble metabolites of
14C-aflatoxin B1 in the urine and faeces. In addition, BHT
pre-treatment was shown to decrease the amount of 14C bound to
hepatic nuclear DNA (Fukayama & Hsieh, 1985).
Effect on enzymes
Dietary BHT (300-6000 ppm) caused a dose-dependent increase in
gamma-glutamyl transpeptidase in normal F-344 male rats. However,
cytosolic glutathione S-transferase was only enhanced at dietary
concentrations of 3000 or 6000 ppm BHT (Furukawa et al., 1984).
Dietary administration of BHT to male Swiss Webster mice resulted
in a marked increase in hepatic microsomal epoxide hydrolase and
glutathione-S-transferase (Hammock & Ota, 1983).
Toxicological studies
Special studies on carcinogenicity
Mice
Groups of male and female C3H mice (ranging from 17-39
mice/group), which were 6-10 weeks old at the beginning of the
experiment, were maintained for 10 months on a semi-synthetic diet
containing 0.05 or 0.5% BHT. Control groups were maintained on
BHT-free semi-synthetic diet or commercial lab chow. At the end of the
test period, the liver and lungs were excised and inspected grossly
for proliferative lesions. Of the proliferative lesions considered to
be clearly identifiable as tumours, approximately 50% were examined
microscopically. Mice maintained on diets containing BHT had lower
body weights than controls. Male mice fed BHT showed an increase in
liver tumours, compared to controls. Histologically, the tumours were
identified as hepatocellular adenomas. No increase was observed in
female mice. The reported incidence of liver tumours in male C3H
mice was 10/26 (38%), 15/26 (58%), 2/37 (5%) and 7/38 (18%) in the
0.5% BHT, 0.05% BHT, control BHT-free, and control lab chow groups,
respectively. In a study in which C3H mice were maintained on diets
containing 0.5% BHT for one month followed by lab chow for 10 months,
or control diet (BHT-free) for one month followed by lab chow for 10
months, the incidences of liver tumours in the two groups of male mice
were 3/35 (9%) and 5/29 (17%), respectively. Dietary BHT did not
result in an increased incidence of lung tumours in either male or
female C3H mice. In another study in which male BALB/c mice were
maintained for one year on a BHT-free diet, or diets containing 0.05%
BHT or 0.5% BHT, the incidences of liver tumours were 4/30 (131), 6/43
(14%), and 2/28 (7%) for the respective groups
(Lindenschmidt et al., 1986).
It has previously been demonstrated that the incidence of
spontaneously-occurring hepatic tumours in C3H mice is modified by
sex, population density, level of dietary protein, and caloric intake.
Historical data are not available for a 10-month study. The incidence
of hepatic tumours in a 12-month study in this strain ranged from
6-13% for females, and 41-68% for males. Thus, the reported incidence
of hepatocellular tumours was not significantly different from other
controls of similar age in studies with the same inbred strain
(Peraino et al., 1973).
Rats
Groups of 60, 40, 40, and 60 Wistar rats of each sex
(F0 generation) were fed a semi-synthetic diet containing 0, 25,
100, or 500 mg BHT/kg b.w., respectively. The F0 rats were mated
after 13 weeks of dosing. The F1 groups consisted of 100, 80, 80,
and 100 F1 rats, respectively, of each sex from the offspring from
each group. Because of an adverse effect on the kidney in the parents,
the concentration of BHT in the highest-dose group was lowered to
250 mg/kg b.w. in the F1 generation. The study was terminated when
rats in the F1 generation were 144 weeks of age.
Parameters studied were food consumption (weekly), body weight,
appearance, and mortality. Autopsy and complete histopathological
examinations were performed on all animals dying during the study, or
sacrificed in extremus or at termination.
The average body weights of the F1 pups at birth in the middle-
and high-dose groups were slightly lower than those in the control
group. Body weights of all dosed animals from weaning through the
entire experiment were lower than those of control animals. In the
low-, mid-, and high-dose groups, the reductions in body weight were
7%, 11%, and 21% for males and 5%, 10%, and 16% for females. Food
intake was comparable for all groups. Clinical appearance and
behaviour were reported to be normal for all animals. The high-dose
males voided a slightly reddish urine. Hematological parameters were
reported to be unchanged by BHT treatment, but no data were given.
Serum triglycerides were reduced in both sexes and cholesterol was
somewhat elevated in females only.
All animals consuming BHT had a dose-related increase in
survival. In both sexes differences (p < 0.001) in longevity were
seen. Histological studies indicated an increase in hepatocellular
carcinomas in male rats (not females) and an increase in
hepatocellular adenomas in both male and female rats. Most liver
tumours were found during terminal sacrifice at 141-144 weeks. One
hepatocellular carcinoma was found in a control male at 117 weeks and
one in a high-dose male at 132 weeks. The remainder of the carcinomas
occurred at terminal sacrifice. The first adenoma was noted in a
high-dose male at 115 weeks. Tables 1 and 2 summarize the data on
mortality and the appearance of adenomas and carcinomas of the liver.
Data on mortality and tumour incidence in different groups were
analysed using the procedure of Peto et al. (1980). The dose-related
increases in the number of hepatocellular adenomas were statistically
significant (at p < 0.05) in male F1 rats, when all groups were
tested for heterogenicity or analysis of trend. The increase in
hepatocellular adenomas and carcinomas in treated female F1 rats was
statistically significant only for adenomas (at p < 0.05) in the
analysis for trend.
Reports on the spontaneous incidence of hepatocellular neoplasms
in Wistar rats from the laboratory performing this study, as well as
other European laboratories, indicate that it is usually less than 3%
(Solleveld et al., 1984; Deerberg et al., 1980; Olsen et al.,
1985). The median life span for animals in these studies ranged from
28 months to 36 months for males and 28 months to 33 months for
females.
Other sites reported to have a slight but not statistically-
significant increase in neoplastic lesions were as follows: thyroid,
pancreas, ovary, uterus, thymus, reticuloendothelium system, and
mammary gland.
Non-neoplastic lesions occurred incidentally and showed no
relationship to BHT treatment, with the exception of lesions of the
liver, which showed a dose-related increased incidence of bile duct
proliferation and cysts in males, and focal cellular enlargement in
females.
At the highest level fed (250 mg/kg b.w.), there was no adverse
effect on the kidney (Olsen et al., 1986).
Special Studies on haemorrhagic effects
Groups of 10 male Sprague-Dawley CLEA rats were fed diets
containing 0.58, 0.69, 0.82, 1.00, 1.20, or 1.44% BHT for 40 days. A
dose-related effect on mortality was observed, with 21/50 rats given
0.69% or more BHT dying during the period from 9 to 37 days.
Spontaneous massive haemorrhages were observed in these animals. The
prothrombin index of survivors was decreased, which was dependent on
the BHT dose. At the lowest level fed the decrease was approximately
651 (Takahashi & Hiraga, 1978a).
The LD50 (i.p.) for BHT showed considerable differences among
strains of inbred and non-inbred male mice:
Strain LD50 (mg/kg b.w.)
DBA/2N (inbred) 138
BALB/cNnN (inbred) 1739
C57BL/6N (inbred) 917
ICR-JCL (non-inbred) 1243
In all cases, death occurred 4 to 6 days after administration of
BHT, and was accompanied by massive oedema and haemorrhage in the lung
(Kawano et al., 1981).
In another study, 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 were fed diets containing BHT (1.2% of the diet for rats and
mice; 1% of the diet for quail; 170 or 700 mg/kg b.w. for rabbits; and
173, 400, or 760 mg/kg b.w. 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 strains showed no obvious haemorrhaging. No haemorrhagic
effects were noted in rabits or dogs (Takahashi et al., 1980).
Male albino rats (CRL COB CD(SD) BR) given 3 consecutive daily
doses of 380, 760, or 1520 mg BHT/kg b.w./day showed no evidence of
haemorrhage. However, BHT produced a dose-dependent increase in
prothrombin time, with no effect on prothrombin time seen in the
380 mg/kg b.w. animals (Krasavage, 1984).
Table 1. Mortality (and combined adenomas and carcinomas of the Liver) in F1 rats
BHT Effective
(mg/kg number of Number of deaths during weeksa
b.w.) rats
0-90 91-104 105-113 114-118 119-126 127-132 133-140 141-144 Total
MALES
0 100 20 10 13 (1)/8 11 10 (1)/12 16 (2)/100
25 80 8 11 6 3 (1)/13 11 8 20 (1)/80
100 80 8 12 3 2 10 7 (2)/11 (4)/27 (6)/80
250 99 7 7 6 (1)/4 (2)/8 (2)/13 (1)/10 (20)/44 (26)/99
FEMALES
0 100 16 15 18 (2)/8 11 7 8 17 (2)/100
25 79 10 9 4 6 13 (1)/10 (1)/8 (1)/19 (3)/79
100 80 5 17 5 5 (1)/7 41)/9 (1)/11 (3)/21 (6)/80
250 99 9 5 11 12 8 5 (2)/10 (12)/39 (14)/99
a Figures in parenthesis are combined adenomas and carcinomas occurring during that period.
Table 2. Incidences of hepatocellular nodular hyperplasia, adenomas,
and carcinomas
BHT Effective number Nodular
(mg/kg b.w.) of rats hyperplasia Adenoma Carcinoma
MALES
0 100 2 1 1
25 80 0 1 0
100 80 2 5 1
250 99 2 18a 8b
FEMALES
0 100 2 2 0
25 79 0 3 0
100 80 4 6 0
250 99 5 12c 2d
a Over-all test for heterogeneity, p< 0.001,
chi-square = 18.17, 3 df.
Test for trend, p< 0.001, chi-square = 17.97, 1 df.
b Over-all teat for heterogeneity, p< 0.05,
chi-square = 11.12, 3 df.
Test for trend, p< 0.01, chi-square = 9.40, 1 df.
c Over-all test for heterogeneity, not significant,
chi-square = 5.20, 3 df.
Test for trend, p< 0.05, chi-square = 4.99, 1 df.
d Over-all test for heterogeneity, not significant,
chi-square = 2.87, 3 df.
Test for trend, set significant, chi-square = 2.59, 1 df.
Male Sprague-Dawley CLEA rats were maintained on diets containing
levels of 85, 170, 330, 650, 1300, 2500, or 5000 ppm BHT for 1 to 4
weeks. A significant decrease in the prothrombin index was observed at
week 1 in all groups fed BHT at levels of 170 ppm or more. However,
when the rats were maintained on the test diets for 4 weeks, a
significant decrease in the prothrombin index was observed only in the
5000 ppm group. This was the only group which showed an increase in
relative liver weights compared to those of the control group. In
another study, haemorrhagic death, haemorrhage, and a decrease in
prothrombin index in male Sprague-Dawley rats caused by 1.2% BHT were
prevented by the simultaneous addition of 0.68 mole phylloquinone/kg
b.w./day Phylloquinone oxide also prevented hypoprothrombinemia due
to BHT (Takshashi & Hiraga, 1979).
Male Sprague-Dawley rats 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 were
observed. Plasma prothrombin factors were decreased, but fibronolytic
activity was unchanged (Takahashi & Hiraga, 1981).
Male rats receiving 0.25% dietary BHT for 2 weeks showed
decreased concentrations of vitamin K in the liver and increased
faecal excretion of vitamin K (Suzuki et al., 1984).
Dietary BHT at a level of 1.2% was shown to effect platelet
morphology (distribution width), and to cause changes in the fatty
acid composition of the platelet lipids (Takahashi & Hiraga, 1984).
Special studies on mutagenicity
BHT was tested in four in vitro systems for genotoxicity: (1)
Salmonella/microsome mutation test (4 dose levels between
0.01-10 mg/plate) using 5 tester strains, with and without S-9
activation; (2) hepatocyte primary culture/DNA repair test (10 dose
levels ranging from 10-5 to 1 mg/ml); (3) adult rat liver epithelial
cell/hypoxanthine guanine phosphoribosyl transferase mutagenesis using
rat liver line 18 (6 doses ranging from 0.05 to 0.1 mg/ml); and (4)
Chinese hamster ovary/sister chromatid exchange (SCE) assay (4 doses
ranging from 10-5 to 1 mg/ml). BHT was negative in all tests
(Williams et al., 1984).
Special studies on potentiation or inhibition of carcinogenicity
G.I. Tract
Male F344 rats were treated with a single dose of
N-methyl-N'-nitro-N-nitrosoguanidine, and then maintained on diets
containing no BHT, 1.0% BHT, 5% NaCl, or 5% NaCl + 1.0% BHT for 51
weeks. The incidences of squamous cell carcinomas of the forestomach
in the respective groups were 11% in the control group, 15.8% in the
BHT group, 3.0% in the NaCl group, and 52.9% in the BHT + NaCl group
(Shirai et al., 1984).
When rats were maintained on dietary 0.5% BHT for 36 weeks
following 4 injections (1 per week) of 1,2-dimethylhydrazine, the
presence of dietary BHT did not effect the number of rats with colon
tumours, but the number of tumours per rat occurring in the distal
colon was significantly decreased (Shirai et al., 1985).
Wistar rats fed 1.0% BHT in the diet during treatment with
N-methyl-N'-nitro-N-nitrosoguanidine (administered in drinking water
at a concentration of 1.0 mg/ml) for 25 weeks, and then maintained on
the test diet for another 14 weeks, showed a significant reduction in
the incidence of gastric cancer, when compared to rats receiving
BHT-free diets (82% versus 36.8%) (Tatsuta et al., 1983).
Groups of male BALB/c mice treated intrarectally with
methylnitrosurea, and then maintained on diets containing BHT, showed
a marked increase in the incidence and multiplicity of G.I. tract
tumours when compared to treated mice maintained on BHT-free diets. In
another study, BALB/c mice were injected with dimethylhydrazine
(6 weekly injections) and then maintained on control (BHT-free) diets
or on diets containing 0.05% BHT or 0.5% BHT. The colon tumour
incidences were 3/30 (10%), 0/43, and 9/28 (32%), in the respective
groups (Lindenschmidt et al., 1986).
Bladder
Male F344 rats were treated with 0.01 or 0.05% N-butyl-N-
(4-hydroxybutyl) nitrosamine (BBN) in drinking water for 4
weeks, and then fed diets containing 0 or 1% BHT for 32 weeks. BHT in
the diet was associated with a significant increase in the incidence
of cancer and papilloma of the bladder of rats treated with 0.05% (but
not 0.01%) BBN (Imaida et al., 1983).
Rats were administered 200 ppm N-2-fluorenylacetamide (FAA) in
the diet alone or with 6000 ppm BHT for 25 weeks. No bladder neoplasms
resulted from feeding FAA alone, but the combination of FAA and BHT
resulted in 17/41 papillomas and 3/41 carcinomas in the bladder
(Williams et al., 1983).
Four dietary levels of BHT (300, 1000, 3000, or 6000 ppm) were
simultaneously fed with 200 ppm FAA for 25 weeks. FAA feeding alone
produced no neoplasms, but when combined with BHT at 3000 or 6000 ppm,
the incidences of bladder tumours were 18% and 44%, respectively. The
incidence of bladder tumours in the 300 and 1000 ppm BHT groups was
low and not significantly different from the incidence with FAA alone
(see also effects on liver) (Maeura et al., 1984)
Male F344 rats were given injections of methylnitrosourea (MNU)
twice a week for 4 weeks, and then a basal diet containing 11 BHT for
32 weeks. Dietary BHT significantly increased the incidences per group
and numbers per rat of papilloma and papillary or nodular hyplasia of
the urinary bladder. The incidence of adenoma (but not adenocarcinoma)
of the thyroid was also increased by treatment with HNU + BHT
(Imaida et al., 1984).
Liver
Rats were administered 200 ppm FAA in the diet, alone or with
6000 ppm BHT for 25 weeks. FAA alone induced a 1001 incidence of liver
neoplasms. Simultaneous administration of BHT resulted in a decreased
frequency of benign neoplasms, neoplastic nodules and malignant and
hepatocellular carcinomas (Williams et al., 1983).
Four dietary levels of BHT (300, 1000, 3000, or 6000 ppm) were
fed simultaneously with 200 ppm FAA for 6, 12, 15, or 25 weeks. BHT
produced a reduction in the incidence of tumours in a dose-dependent
manner (100% incidence in the absence of BHT to 56% at 6000 ppm BHT)
(see also effects on the bladder) (Maeura et al., 1984).
Rats were fed 200 ppm FAA for 8 weeks, then diets containing BHT
at levels of 300, 1000, 3000, or 6000 ppm for up to 22 weeks. The area
of hepatocellular altered foci, identified by iron exclusion and
gamma-glutamyl transferase (GGT) activity, that was induced by feeding
the FAA, showed increased development at the highest dietary level of
BHT (the number of foci, the area occupied by GGT-positive
preneoplastic and neoplastic lesions, and the neoplasm incidence were
increased). These parameters were unaffected at the lower BHT levels
(Maeura & Williams, 1954).
Rats were given a single i.p, injection of 200 mg/kg b.w. of
diethylnitrosamine, and then maintained on diets containing 1% BHT for
6 weeks. At week 3 the rats were subjected to partial hepatectomy. The
number of gamma-glutamyl transpeptidase positive loci in the liver of
BHT-fed rats was significantly decreased when compared to controls
(Imaida et al., 1983).
Mammary glands
Female rats were fed diets containing 0, 0.25, or 0.5% BHT. The
test diets were administered either (a) 2 weeks before until 1 week
after 7,12-dimethylbenz(a)anthracene (DMBA) administration or (b) 1
week after DMBA administration to the end of the study (30 weeks). The
DMBA was administered as a single dose of 8 mg. BHT was an effective
inhibitor of mammary carcinogenesis when administered during either of
these time frames (20% by regime (a) and 50% by regime (b))
(McCormick et al., 1984).
In another study, dietary BHT was shown to decrease the incidence
of mammary tumours induced in female Sprague-Dawley rats by DMBA, but
it was without effect on animals treated with MNU (King et al.,
1981).
The inhibitory effects of BHT were strongly influenced by the
dose of initiating carcinogen and the type of diet in which BHT was
fed. Administration of BHT in the AIN-76A diet showed a markedly
different effect from BHT in the NIH-07 diet. In the AIN-76A diet
6000 ppm BHT had no effect on the incidence of mammary tumours induced
by 15 mg DMBA, whereas a similar level of BHT in the NIB-07 diet
resulted in a 40% inhibition of tumour development (Cohen et al.,
1984).
Pancreas
Male LEW inbred rats were given an injection of 30 mg azaserine
once a week for 3 weeks, and then maintained on diets containing 0 or
0.45% BHT for 4 months. BHT treatment reduced the number of
acidophilic foci per pancreas by 32%, but was without effect on focal
size. BHT had no effect on the occurrence of basophilic foci
(Roebuck et al., 1984).
Special studies on potentiation or inhibition of mutagenesis
The addition of 50-250 µg of BHT/plate inhibited 3,2'-dimethyl-
4-aminobiphenyl-induced mutagenicity in Salmonella typhimurium
strains TA98 and TA100 in the presence of rat liver S-9 fraction (Reddy
et al., 1983a).
BHT was a moderately effective inhibitor of benzidine-induced
mutagenicity in Salmonella typhimurium strain TA98, activated with a
hamster liver S-9 fraction (Josephy et al., 1985).
The addition of 100-250 µg of BHT/plate was shown to inhibit
3,2'-dimethyl-4-aminobiphenyl-induced mutagenicity in Salmonella
typhimurium strains TA98 and TA100. Mutagenicity was further
inhibited by use of S-9 preparations from rats fed dietary BHT (0.6%)
as compared to S-9 preparation from rats fed BHT-free diets
(Reddy et al., 1983b).
The addition of BHT (5 to 20 µg/plate) caused a two-fold increase
in the mutagenic potency of aflatoxin B1 using Salmonella
typhimurium strains TA98 and TA100, with and without activation
(Shelef & Chin, 1980).
Special studies on pulmonary toxicity
In a study of lung toxicity in mice of BHT analogues, it was
established that the structural feature essential for toxic activity
is a phenolic ring structure having (i) a methyl group at the
4-position and (ii) ortho-alkyl group(s) which can result in a
moderate hindering effect of the hydroxyl group (Mizutani et al.,
1982).
In another study, the toxic potency of BHT in mice was decreased
by deuteration of the 4-methyl group, suggesting that lung damage
following administration of BHT is caused by the metabolite
2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (Mizutani
et al., 1983).
Male mice given a single dose of BHT showed ultrastructural
changes of the lung, which were characterized by selective destruction
of type I epithelial cells, which were replaced by type II cuboidal
cells. These changes were accompanied by a marked decrease in the
number of peroxisomes, as well as catalase activity (Hirai et al.,
1983).
BHT was shown to enhance the lung tumour incidence in mice
treated with doses of urethan greater than 50 mg/kg. At lower doses of
urethan (subcarcinogenic doses) BHT did not enhance tumour
development. In another study, it was shown that following treatment
of mice with urethan, a two-week exposure to dietary 0.75% BHT was
sufficient to enhance tumour development, and that 0.1% BHT was an
effective enhancer when fed for 8 weeks. BHT, administered within 24
hours post-treatment and fed for 8 weeks, enhanced tumour development
in mice treated once with 3-methylcholanthrene, benzo(a)pyrene, or
N-nitrosodimethylamine. When mice were injected weekly with BHT, there
was a rapid increase in cell proliferation, and in both the cumulative
labelling index (incorporation of 3H-thymidine) and the number of
labelled type II cells. These effects were smaller after each
injection, and by the fifth injection, no increase was observed
(Witschi & Morse, 1985).
Special study on reproduction
Rats
Groups of 60, 40, 40, and 60 Wistar rats of each sex, 17 weeks of
age, were fed a semi-synthetic diet containing BHT so that the dietary
intake was equivalent to 0, 25, 100, or 500 mg/kg b.w./day,
respectively. After 13 weeks on the test diet the rats were mated.
Food consumption was similar in all groups. Male and female rats
in the high-dose group showed a significant decrease in body weight
which persisted throughout the study. Gestation rate was similar for
all test groups. The litter size and number of males per litter were
significantly lower in the 500 mg/kg b.w. group than in the controls.
Viability was similar in test groups and in the control group during
the lactation period. The average birth weight of the pups in the
500 mg/kg b.w./day and 100 mg/kg b.w./day groups was slightly lower
than in the controls. During the lactation period, dietary BHT caused
a significant dose-related lower body-weight gain (5%, 7%, and 41%
lower body weight for the 25, 100, and 500 mg/kg b.w./day groups,
respectively, as compared to controls) (Olsen et al., 1986).
Comments
The haemorrhagic effects of massive doses of BHT seed in certain
strains of mice and rats, but not in dogs, may be related to its
ability to interfere with vitamin K metabolism. However, in a
susceptible strain of rat, the decrease in prothrombin index was shown
to be transient. The effect can be reversed by feeding a vitamin K
analogue. This suggests that the effect may be due to the presence of
antivitamin K activity possibly associated with diets marginal in
vitamin K. Additional studies will be required to elucidate the
mechanism of the haemorrhagic effect.
Additional studies have been carried out on the ability of BHT to
modify the carcinogenicity of chemical agents. Enhancement or
reduction of the incidence of tumours varies with the chemical
administered, the time of addition of BHT to the diet, concentration
of BHT in the diet, and also the organ site effected. For example, in
the case of rats fed FAA concurrently with BHT, there was a
significant increase in bladder tumours even when rats were exposed to
subcarcinogenic levels of dietary FAA. However, under these conditions
there was a reduction in the incidence of liver tumours. In both
instances the effects were dose related, and the effects were only
observed at high dietary levels of BHT. Dietary BHT also modified the
carcinogenicity of chemicals causing tumours of the G.I. tract,
mammary gland, and pancreas in experimental animals. The inhibitory
effects of BHT may also be influenced by the type of diet in which it
is fed, since in one study BHT was an effective inhibitor of
DMBA-induced mammary tumours in rats when a NIH-07 diet was used, but
was without effect when the rats were fed an AIN-76A diet. In another
study on mice, conventional laboratory chow containing BHT reportedly
produced a higher incidence of spontaneous-occurring tumours than did
a BHT-free semi-synthetic diet.
BHT was not mutagenic in a number of in vitro tests. However,
BHT inhibited 3,2'-dimethyl-4-aminobiphenyl- and benzidine-induced
mutagenicity in the Salmonella test.
In a recent two-generation lifetime study in Wistar rats, BHT
caused a dose-related increased number of hepatocellular adenomas and
carcinomas. Most of the hepatocellular tumours were detected when the
rats were more than two years old. Previously-reported single-
generation carcinogenicity studies in Fischer 344 and Wistar
rats with BHT were negative (Annex 1, references 51 and 63). The
significance of the hepatocarcinogenicity of BHT in the recent studies
to the toxicological evaluation raises a number of questions. These
relate to the differences in design of the recent rat study and those
previously reported to be negative, such as in utero exposure,
duration of the study, and low body weights of the test animals. An
elucidation of the reasons for the in utero effect is a priority.
In a study with male and female C3H mice fed semi-synthetic
diets containing BHT for 10 months there was a significant increase in
the incidence of liver tumours in male but not female animals under
the conditions of the study. This effect was not dose related, the
lower dose showing a higher incidence of these tumours than the higher
dose (58% versus 38%. In addition, the tumour incidence was not
significantly different from other controls of similar age in studies
with the same inbred strain and the reported increase of
hepatocellular tumours (adenomas) appeared to be strain specific,
since an increased incidence of liver tumours was not observed in male
BALB/c mice fed diets containing 0.51 BHT for 12 months. In the
previously-reported carcinogenicity studies in B6D3F1 mice, BHT was
not carcinogenic under the conditions of the test (Annex 1, references
54 and 63).
In a one-generation reproduction study in rats, BHT had no effect
on gestation rate, but showed a dose-related response in litter size,
number of males per litter, and body-weight gain during the lactation
period. The Committee based its evaluation on the no-effect level in
this study.
EVALUATION
Level causing no toxicological effect
Rat: 25 mg/kg b.w./day (based on one-generation reproduction
study)
Estimate of temporary acceptable daily intake for man
0 - 0.125 mg/kg b.w.
Further work or information
Required by 1990
1. Elucidation of in utero exposure on hepatocarcinogenicity of
BHT in the rat.
2. Studies on the mechanism of the haemorrhagic effect of BHT in
susceptible species.
REFERENCES
Cohen, L.A., Polansky, M., Furuya, K., Reddt, M., Berke, B. &
Weisburger, J.H. (1984). Inhibition of chemically induced
mammary carcinogenesis in rats by short-term exposure to BHT:
Interrelationships among BHT concentration, carcinogen dose
and diet. J. Natl. Cancer Inst., 72, 165-173.
Deerberg, F., Rapp, K.G., Pittermann, W. & Rehm, S. (1980). Zum
tumorspektrum der Han: Wis-Ratte. Z. Versuchstierk,
22, 267-280.
Fukayama M.Y. & Hsieh, D.P.H. (1985). Effects of butylated
hydroxytoluene pretreatment on the excretion, tissue distribution
and DNA binding of (14C)aflatoxin B1 in the rat.
Fd. Chem. Toxicol., 23, 567-573.
Furukawa, K., Maeura, Y., Furukawa, N.T., & Williams, G.M. (1984).
Induction by butylated hydroxytoluene of rat liver gamma-glutamyl
transpeptidase activity in comparison to expression in
carcinogen-induced altered lesions. Chem. Biol. Interact.,
48, 43-58.
Hnmmock, B.D. & Ota, K. (1983). Differential induction of cytosolic
epoxide hydrolase, microsomal epoxide hydrolase and glutathione
S-transferase activities. Tox. Appl. Pharm., 71, 254-265.
Hirai, K.I., Yamauchi, M., Witschi, H. & Cote, M.G. (1983).
Disintegration of lung peroxisomes during differentiation of type
II cells in butylated hydroxytoluene-administered mice.
Exper. Mol. Pathol., 39, 129-138.
Imaida, K., Fukushima, S., Shirai, T., Ohtani, M., Nakanishi, K. &
Ito, N. (1983). Promoting activities of BHA and BHT on 2-stage
urinary bladder carcinogenesis and inhibition of GGT-positive
foci development in the liver of rats. Carcinogenesis,
4, 895-899.
Imaida, K., Fukushima, S., Shirai, T., Masui, T., Ogiso, T. & Ito, N.
(1984). Promoting activities of BHA, BHT and sodium-L-ascorbate
on forestomach and urinary bladder carcinogenesis initiated with
methylnitrosourea in F-344 rats. Gann, 75, 769-775.
Josephy, P.D., Carter, M.H., & Goldberg, M.T. (1985). Inhibition of
benzidine mutagenesis by nucleophiles; a study using the Ames
test with hamster S-9 activation. Hut. Res., 143, 5-10.
Kawano, S., Nakao, T. & Kiraga, K. (1981). Strain differences in
butylated hydroxytoluene induced deaths in male mice.
Tox. Appl. Pharm., 61, 475-479.
King, M.M., McCay, P.B. & Kosanke, S.D. (1981). Comparison of the
effect of butylated hydroxytoluene on N-nitrosomethylurea and
7,12-dimethylbenz(a)anthracene-induced mammary tumours.
Cancer Lett., 14. 219-226.
Krasavage, W.J. (1984). The lack of effect of tertiary
butylhydroquinone on prothrombin time in male rats.
Drug Chem. Tox., 7, 329-334.
Lidenschmidt, R.C., Tryka, A.F., Goad, M.E., & Witschi, H.P. (1986),
The effect of dietary BHT on liver and colon tumour development
in mice. Toxicology., 38. 151-160.
Maeura, Y. & Williams, G.M. (1984). Enhancing effect of butylated
hydroxytoluene on the development of liver altered foci and
neoplasms induced by N-2-fluorenylacetamide in rats.
Fd. Chem. Tox., 22, 191-198.
Maeura, Y., Weisburger, J.H. & Williams, G.M. (1984). Dose-dependent
reduction of N-2-fluorenylacetamide-induced liver cancer and
enhancement of bladder cancer in rats by butylated
hydroxytoluene. Cancer Res., 44, 1604-1610.
Matsuo, M., Mihara, K., Okuno, M., Ohkawa, H., & Miyamoto, J. (1984).
Comparative metabolism of 3,5-tert-butyl-4-hydroxytoluene (BHT)
in mice and rats. Fd. Chem. Toxicol., 22, 345-354.
McCormick, D.L., Major, N., & Moon, R.C. (1984). Inhibition of
7,12-dimethylbenz(a)anthracene induced rat mammary carcinogenesis
by concomitant or postcarcinogen antioxidant exposure.
Cancer Res., 44, 2858-2863.
Mizutani, T., Ishida, I., Yamanotok, K. & Tajima, K. (1982). Pulmonary
toxicity of butylated hydroxytoluene and related alkylphenols:
Structural requirements for toxic potency in mice.
Tox. Appl. Pharm., 62, 273-281.
Mizutani, T., Yamamoto, K. & Tajima, K. (1983). Isotope effects on the
metabolism and pulmonary toxicity of butylated hydroxytoluene in
mice by deuteration of the 4-methyl group. Tox. Appl. Pharm.,
69, 283-290.
Olsen, P., Gry, I., Knudsen, I., Meyer, O., & Poulsen, E. (1985).
Investigations on nitrite treated meat. In N-nitroso compounds:
Occurence, biological effects, and relevance to human cancer.
O'Neill, I.K., von Borstel, R.C., Miller, C.T., Long, J., &
Bartsch, H. (eds.), IARC Scient. Publ. No.57, pp. 667-675.
International Agency for Research on Cancer, Lyon.
Olsen, P., Meyer, O., Bille, N., & Wurtzen, G. (1986). Carcinogenicity
study on butylated hydroxytoluene (BHT) in Wistar rats exposed in
utero. Fd. Chem. Toxicol., 24, 1-12.
Peraino, C., Fry, M.R.J., & Staffeldt (1973). Enhancement of
spontaneous hepatic tumourigenesis in C3H mice by dietary
phenol-barbitol. J. Natl. Cancer Inst., 51, 1349-1350.
Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S.,
Peto, J., Richards, S., & Wahrendorf, J. (1980). Guidelines for
simple, sensitive significance tests for carcinogenic effects in
long-term animal experiments. In: Long-term and Short-term
Screening Assays for Carcinogens: A Critical Appraisal. IARC
Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Humans, Suppl. 2, pp. 311-426. International Agency
for Research on Cancer, Lyon.
Reddy, B.S., Hansen, D., Mathews, L., & Sharma, C. (1983a). Effect of
micronutrients, antioxidants and related compounds on the
mutagenicity of 3,2'-dimethyl-4-aminobiphenyl, a colon and breast
carcinogen. Fd. Chem. Toxicol., 21, 129-132.
Reddy, B.S., Sharma, C., & Mathews, L. (1983b). Effect of butylated
hydroxytoluene and butylated hydroxyanisole on the mutagenicity
of 3,2'-dimethyl-4-aminobiphenyl. Nutr. Cancer, 5, 153-158.
Roebuck, B.D., MacMillan, D.L., Bush, D.M. & Kensler, T.W. (1984).
Modulation of azaserine-induced pancreatic foci by phenolic
antioxidants in rats. J. Natl. Cancer Inst., 72, 1405-1409.
Shelef, L.A. & Chin, F. (1980). Effect of phenolic antioxidants on the
mutagenicity of aflatoxin B1. Appl. Environ. Microbiol.,
40, 1039-1043.
Shirai, T., Fukushima, S., Ohshima, M., Masuda, A., & Ito, N. (1984).
Effects of BHA, BHT and NaCl on grastic carcinogenesis with
N-methyl-N'-nitro-N-nitrosoguanidine in F-344 rats. J. Natl.
Cancer Inst., 72, 1189-1198.
Shirai, T., Ikawa, E., Hirose, M., Thamavit, W., & Ito, N. (1985).
Modification by five antioxidants of 1,2-dimethyl-hydrazine-
initiated colon carcinogenesis in F-344 rats. Carcinogenesis,
6, 637-639.
Solleveld, H.A., Haseman, J.K., & McConnell, E.E. (1984). Natural
history of body weight gain, survival and neoplasia in the F-344
rat. J. Natl. Cancer Inst., 72, 929-940.
Suzuki, N., Nakao, T. & Hiraga, K. (1983). Vitamin K content of liver
and faeces from vitamin K-deficient and butylated hydroxy-toluene
(BHT)-treated male rats. Tox. Appl. Pharm., 67, 152-155.
Takahashi, O. & Hiraga, K. (1978a). Dose-response study of
haemorrhagic death by dietary butylated hydroxytoluene (BHT) in
male rats. Tox. Appl. Pharm., 43, 399-406.
Takahashi, O. & Hiraga, K. (1978b). Effects of low levels of butylated
hydroxytoluene in the prothrombin index of male rats.
Fd. Cosmet. Tox., 16, 475-477.
Takahashi, O. & Hiraga, K. (1979). Preventative effects of
phylloquinone on haemorrhagic death induced by butylated
hydroxytoluene in male rats. J. Nutr., 109, 453-457.
Takahashi, O., Hayashida, S., & Hiraga, K. (1980). Species differences
in the haemorrhagic response to butylated hydroxytoluene.
Fd. Cosmet. Tox., 18, 229-235.
Takahashi, O. & Hiraga, K. (1981). Haemorrhagic toxicosis in rats
given butylated hydroxytoluene. Acta Pharmacol. Toxicol.,
49, 14-20.
Takahashi, O. & Hiraga, K. (1984). Effects of dietary butylated
hydroxytoluene on functional and biochemical properties of
platelets and plasma preceeding the occurrence of haemorrhage in
rats. Fd. Chem. Tox., 22, 97-103.
Tatsuta, M., Mikuni, T., & Taniguchi, H. (1983). Protective effect of
butylated hydroxytoluene against induction of gastric cancer by
N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.
Int. J. Cancer, 32, 253-254.
Williams G.M., Maeura, Y., & Weisburger, J.H. (1983). Simultaneous
inhibition of liver carcinogenicity and enhancement of bladder
carcinogenicity of N-2-fluorenylacetamide by butylated
hydroxytoluene. Cancer Lett., 19, 55-60.
William G.M., Shimada, T., Mcqueen, C., Tong, C., & Ved Brat, S.
(1984). Lack of genotoxicity of butylated hydroxyanisole (BHA)
and butylated hydroxytoluene (BHT). The Toxicologist, 4, 104.
Witschi, H.P. & Morse, C.C. (1985). Cell kinetics in mouse lung
following administration of carcinogens and butylated
hydroxytoluene. Tox. Appl. Pharm., 78, 464-472.