TOXICOLOGICAL EVALUATION OF CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 10
The evaluations contained in this document were prepared by the
Joint FAO/WHO Expert Committee on Food Additives*
Rome, 21-29 April 1976
Food and Agriculture Organization of the United Nations
World Health Organization
*Twentieth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, 1976, WHO Technical Report Series No. 599, FAO Food
and Nutrition Series No. 1.
BUTYLATED HYDROXYTOLUENE
Explanation
Butylated hydroxytoluene has been evaluated for acceptable daily
intake for man by the Joint FAO/WHO Expert Committee on Food Additives
in 1961, 1964, 1965 and 1973 (see Annex 1, Refs No. 6, p. 45; No. 9,
p. 13; No. 13, p. 28; and No. 33, p. 156).
Since the previous evaluations, additional data have become
available and are summarized and discussed in the following monograph.
The previously published monographs have been expanded and are
reproduced in their entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Excretion and distribution
Groups of male and female rats were maintained on diets
containing 0 or 0.5% BHT for a period of 35 days, and then for a
period on diets free of BHT. During the period on the test diets
groups of rats were killed at 5 day intervals and fat and liver
removed for BHT analysis. Following removal of the diet rats were
killed at 2 day intervals to measure loss of BHT from the fat and
liver. There was no clear evidence of progressive accumulation of BHT
in fat during the period of administration of the test compound. BHT
levels in the fat reached a maximum level (55 ppm in males, 65 ppm in
females) within 10 days of exposure to BHT. Thereafter there was
considerable fluctuation in the observed levels. The levels of BHT in
liver were very low, the maximum BHT levels being ca 5.0 ppm in males
and 1.5 ppm in females. The biological half life of BHT in fat and
liver was estimated to be 7 to 10 days (Gage, 1964).
Groups of two rats (one male, one female) were given one to five
oral doses of 44 mg/kg bw BHT on alternate days and each group killed
24 hours after the final dose. The range of the total dose accounted
for was 92 to 103.5% in males and 92.6 to 98.6% in females. There was
an indication of sex difference in the route of excretion, females
excreting 19-43% of the radioactivity in urine and males only 3-15%.
Eight days after administration of five doses 92% of the radioactivity
had been excreted by males and 97% by females. Subcutaneous
administration of graded doses of BHT to female rats revealed
substantial faecal excretion but the rate of excretion decreased with
increasing dose. There was no evidence of accumulation of BHT-14C in
the body under the conditions of repeated oral dosage (Tye et al.,
1965).
The fate in the body of 14C-labelled BHT has been elucidated.
The relatively slow excretion of BHT is probably attributable to
enterohepatic circulation rather than to tissue retention. Rats were
given single oral doses (1-100 mg/rat) of BHT-14C and approximately
80 to 90% of the dose was recovered in four days in the urine and
faeces. Of the total radioactivity, 40% appeared in the urine of
females and 25% in males. After four days approximately 3.8% of the
dose was retained mainly in the alimentary tract. A substantial
portion of the radioactivity was found in the bile collected from two
rats (one male, one female) over a period of 40 hours (Daniel & Gage,
1965).
The liver and body fat of rats fed a diet containing 0.5% BHT for
35 days were analysed. The concentration of BHT in the liver never
rose above 5 ppm (0.0005%) in males or 1.5 ppm (0.00015%) in females.
In the body fat the level fluctuated round 30 ppm (0.003%) in males
and 45 ppm (0.0045%) in females. Fat from rats returned to normal diet
showed a progressive fall in the concentration of BHT the half-life
being about seven to 10 days. The daily excretion of radioactivity
in urine and faeces was studied in rats given an oral dose of
14C-labelled BHT (12 mg/kg bw). Excretion became negligible by the
sixth day after administration when about 70% of the injected dose had
been recovered. Less than 1% was excreted as carbon dioxide in the
expired air. About 50% of the radioactivity was excreted in the bile
during the 24-hour period following the oral dose (Daniel & Gage,
1965).
In further work with rats it was found that increased output of
urinary ascorbic acid paralleled liver enlargement induced by BHA or
BHT in onset, degree and duration, being rapid but transient with BHA
and slower in onset but more prolonged with BHT (Gaunt et al., 1965a).
The parallelism between stimulation of processing enzyme activity,
increase in urinary ascorbic acid output, and increase in relative
liver weight brought about by BHT was unaffected by 14 days of dietary
restriction, and all these changes except liver weight were reversible
during 14 days' recovery on normal diet (Gaunt et al., 1965b).
The BHT content of fat and liver of rats given diets containing
0.5 and 1.0% BHT for periods up to 35 and 50 days respectively: with
0.5% BHT in the diet, a level of approximately 30 ppm (0.003%) in the
fat was reached in males and 45 ppm (0.0045%) in females, with
approximately 1-3 ppm (0.0001-0.0003%) in the liver, while with 1.0%
BHT the level in the fat was 50 ppm (0.005%) in males and 30 ppm
(0.003%) in females. On cessation of treatment, the level of BHT in
fat fell with a half-life of seven to 10 days (Daniel & Gage, 1965).
The level of BHT in the fat reached a plateau at approximately 100 ppm
(0.01%) after three to four days when daily doses of 500 mg/kg bw were
given by intubation; 200 mg/kg bw per day for one week produced a
level of about 50 ppm (0.005%) (Gilbert & Golberg, 1965).
When feed containing 500 ppm (0.05%) BHT was given to laying
hens, 20 ppm (0.002%) was found in the fat fraction of eggs; 100 ppm
(0.01%) in the feed resulted in residues of less than 5 ppm (0.0005%).
In the broiler chicken, over a period of 21 weeks, the residues in
body fat were 55 ppm (0.0055%) on the 500 ppm (0.05%) diet and less
than 5 ppm (0.0005%) on the 100 ppm (0.01%) diet (Van Stratum & Vos,
1965).
One-day-old chicks were given 14C-BHT at a level of 200 ppm
(0.02%) in the food for 10 weeks. At broiler age, edible portions had
residues amounting to 1-3 ppm (0.0001-0.0003%) of BHT and metabolites.
Similar diets given to laying hens produced residues in eggs of 2 ppm
(0.0002%) after seven days, the level thereafter remaining constant
(Frawley et al., 1965a).
Rats were given doses of 100 µg of BHT labelled with 3H
intraperitoneally and the urinary output of radioactivity was measured
for four consecutive days. Four days after the injection 34.5% of the
injected radioactivity was recovered in urine (Ladomery et al., 1963).
The same dose of BHT (100 µg) labelled with 14C was given to
rats and 34% of the radioactivity was excreted in the urine in the
first four days, in close agreement with the previous result using
tritiated BHT (Ladomery et al., 1967a).
White male Wistar rats (290-350 g) were administered [14C]
labelled BHT, or its alcohol [BHT-CH2OH], or its aldehyde [BHT-CHO]
or acid [BHT-COOH] derivative by i.v. or i.p. injection. The overall
excretion of BHT and its related compounds excreted in urine and
faeces was studied for a five day period, and biliary excretion
followed for 120-126 hours after i.p. injection. For the low doses of
the compounds tested (100 µg) there were no significant differences in
the total recovery of 14C during the 5 days urinary and faecal
excretion and 120-126 hours biliary excretion. However, there were
differences in ratio of urinary to faecal excretion of 14C. The major
metabolite present in early bile after i.p. injection of the labelled
compounds was BHT-COOH or its ester glucuronide. Late bile after acid
hydrolysis showed BHT-COOH to be the major 14C component (Holder et
al., 1970a).
Metabolism
Rabbits were given single or repeated doses of BHT in the range
400-800 mg/kg bw. About 16% of the dose was excreted as ester
glucuronide and 19% as ether glucuronide. Unconjugated phenol (8%)
ethoreal sulfate (8%) and a glycine conjugate (2%) were also excreted.
Excretion of all detectable metabolites was essentially complete three
to four days after administration of the compound and about 54% of the
dose was accounted for as identified metabolites (Dacre, 1961).
The metabolism of butylated hydroxytoluene (BHT) administered to
rabbits orally in single doses of 500 mg/kg bw was studied. The
metabolites 2,6-di-tertbutyl-4-hydroxymethylphenol (BHT-alc),
3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) and 4,4'-ethylene-
bis-(2,6-di-tert-butylphenol) were identified. The urinary metabolites
of BHT comprised 37.5% as glucuronides, 16.7% as ethereal sulfates and
6.8% as free phenols; unchanged BHT was present only in the faeces
(Akagi & Aoki, 1962a); 3,5-di-tert-butyl-4-hydroxybenzaldehyde
(BHT-ald) was also isolated from rabbit urine (Aoki, 1962). The main
metabolic pathway was confirmed by administering BHT-alc to rabbits
and isolating BHT-ald, BHT-acid, the ethylene-bis derivative and
unchanged BHT-alc in the urine (Akagi & Aoki, 1962b).
After a single parental dose (100 µg) of -14C BHT, rats excreted
32-35% of the radioactivity in the urine, and 33-37% in the faeces, in
a four day period. The intestinal contents together with the gut wall
contained most of the remaining activity. Biliary excretion was rapid,
and the radioactive material in bile was readily absorbed from the
gut, suggesting a rapid enterohepatic circulation (Ladomery et al.,
1967a). Examination of the biliary metabolites from i.v. and i.p.
doses of small amounts of 14C-BHT, showed the presence of four
principal metabolites. 34 to 53% of the 14C-labelled in the bile was
identified as 3-5-di-t-butyl-4-hydroxy-benzoic acid, which was
probably present as the ester glucuronide. The other metabolites
present were 3,5-di-t-butyl-4-hydroxybenzaldehyde, 3-5-di-t-butyl-4-
hydroxybenzyl alcohol, and 1,2-bis (3,5-di-t-butyl-4-hydroxyphenyl)
ethane (Ladomery et al., 1967b).
Rats were dosed with a single dose of 14C-BHT and urine and bile
collected for periods ranging from 48 to 96 hours. Faeces were also
collected during this same period. 19 to 58.5% of the radioactivity
appeared in the urine during this period, and 23.7 to 36% in the
bile. The major metabolites in the urine were 3,5-di-tert-butyl-4-
hydroxybenzoic acid, both free (9% of the dose), as well as
glucuronide (15%) and S-(3,5-di-tert-butyl-4-hydroxybenzyl)-
N-acetylcysteine. The ester glucuronide and mercaptic acid were
also the major metabolites in rat bile. Free 3,5-di-tert-butyl-
4-hydroxybenzoic acid was the major metabolite in faeces (Daniel et
al., 1968).
A group of 8 men each received 100 mg of BHT on two occasions
with a 4 day interval. Urine was collected for 24 hours after BHT
administration. The metabolites were identified as BHT-COOH and
benzoylglycine. In another study in which two adults were given 1.0 g
of BHT, the BHT-COOH and its ester glucuronide were the only major
metabolites identified in urine (Holder et al., 1970a).
Effects on enzymes and other biochemical parameters
Rats given BHT by daily intubation showed increased activity of
some liver microsomal enzymes. Stimulation of enzyme activity
correlated with an increase in relative liver weight, the threshold
dose for these changes in enzyme activity in female rats being below
25-75 mg BHT/kg bw/day. The storage of BHT in fat appeared to be
influenced by the activity of the processing enzymes. In rats given
500 mg/kg bw daily the level of BHT in fat attained values of 230 ppm
(0.023%) in females and 162 ppm (0.0162%) in males by the second day,
by which time the relative liver weight and processing enzyme
activities had become elevated. Thereafter, liver weight and enzyme
activity continued to rise but the BHT content of fat fell to a
plateau of about 100 ppm (0.01%) in both sexes (Gilbert & Golberg,
1965).
Groups each of 12 SPF Carworth rats equally divided by sex were
administered BHT dissolved in Arachis oil daily for one week, at a
dose level equivalent to 50, 100, 200 or 500 mg/kg bw. A group of 8
rats served as control. The animals were killed 24 hours after the
final dose, and histological and biochemical studies (glucose-6-
phosphatase and glucose-6-phosphate dehydrogenase) made on the livers
of all animals. A histochemical assessment of the livers of test
animals was also carried out. BHT caused an increase in liver weight
in males at dose levels of 100 mg/kg bw and greater, and in females at
200 mg/kg bw and greater. BHT caused a decrease in glucose-6-
phosphatase activity in females at dose levels greater than 100 mg/kg
bw, and an increase in glucose-6-phosphate dehydrogenase in both males
and females at the highest dose tested. In another study in which rats
were dosed according to the above schedule and then maintained for 14
to 28 days, following the final dosing. By day 28 no biochemical
changes were observed, in contrast to the return to normal by day 14
of relative liver weights (Feuer et al., 1965).
Rats (male and female Carworth Farm SPF) were dosed orally with
BHT at a level equivalent to 500 mg/kg bw. Dosing was from 1 to 5
days, and rats varying in size from 100-400 g bw were used. Microsomal
preparations from the livers of treated rats were assayed for BHT
oxidase, an enzyme that metabolizes BHT to the BHT alcohol (2,6-di
tert-butyl-4-methylphenol to 2,6-di tert butyl-4-hydroxy
methylphenol). Treatment of female rats with BHT (500 mg/kg daily for
5 days) caused a sixfold increase in the activity of the enzyme/gram
of liver and a 35% increase in relative liver weight, both being
prevented by actimomycin D. The induction was more pronounced in males
than in females, and the induction of the enzyme low in rats in the
100 g body weight range, reached a maximum in rats in the 200 g body
weight range, and fell in larger animals (300-400 g range) (Gilbert &
Golberg, 1967).
Groups of rats (female, Alderly Park SPF strain) were maintained
on diets containing 5%, 1%, 0.1%, 0.01% and 0% BHT for periods up to
28 days, and then on diets free of BHT for 56 days. Animals were
killed in groups of four, two being used for enzyme assay (aminopyrene
demethylase) and 2 for electron microscopy. The increase in enzyme
activity was directly related to the dietary level of BHT. No
detectable increase was observed at the lowest level (0.01%) over the
28 day feeding period. Following withdrawal of BHT from the diet, the
enzyme level returned to normal in all test animals. The degree of
endoplasmic reticulum proliferation was proportional to the amount of
BHT in the diet and the duration of feeding, at the 5% and 1% level.
At the 0.1% level there was a transient rise in smooth endoplasmic
reticulum. No proliferation was observed at the 0.01% level. Following
removal of BHT from the diet there was a rapid disappearance of the
proliferated smooth endoplasmic reticulum. In a second study groups of
rats were fed diets containing 1% BHT for ten days, and then for a
second period of ten days after an interval of 20 days on a normal
diet. The animals were killed in groups of five at 10, 30, 40, 42 and
47 days. Livers were removed for aminopyrene demethylase assay and
electron microscopy. Enzyme activity did not differ significantly
following both the ten day periods of administration of BHT. Electron
microscopy showed similar smooth endoplasmic reticulum response during
both these periods (Botham et al., 1969).
A group of 23 female SPF rats (Wistar strain), body weight of
about 145 g, was administered 500 mg/kg BHT dissolved in rape-seed
oil, for eleven days, starting on day 3 of the study. A control group
of rats was administered rape-seed oil alone. Groups of 7 rats were
killed following administration of the final dosing. The remaining
rats were maintained without further exposure to BHT, and killed on
day 28 and 63 of the study. Livers of the rats were examined for
weight, DNA content and number of cell nuclei. BHT was shown to result
in enlargement of the liver, with a concomitant increase in its DNA
content, and in the number and ploidy of its nuclei. The liver mass
returned to normal within two weeks. However, the DNA content of the
liver of BHT treated animals remained elevated up to the time of
termination of this study, and there was no reduction in the total
number of nuclei or the degree of ploidy (Schulte Hermann et al.,
1971).
Two groups, one male and one female rat (Alderly Park, SPF Wistar
strain) of ten test animals were dosed daily by stomach tube with
200 mg/kg bw of BHT dissolved in maize oil for seven days. Four male
and four female rats dosed with an equivalent amount of maize oil were
used as controls. Urinary ascorbic acid excretion was measured in
urine, in samples collected following 5 days on the test compound. The
animals were killed 24 hours after the final dose and the livers
removed for biochemical assays (aminopyrine demethylase-AMPM,
hexobarbitone oxidase-HO, cytochrome P450, and glucose-6-phosphatase),
and electron microscopy. Another group of treated rats was maintained
for a 7 day recovery period, and a similar battery of liver studies
carried out. Administration of BHT resulted in an increase of urinary
excretion of ascorbic acid which remained constant throughout the
treatment period. Following cessation of BHT treatment there was a
gradual return towards control values. There were significant sex
differences in some of the biochemical responses to BHT, with the
exception of the glucose-6-phosphatase activity. Female rats showed a
marked increase in APDM and HO activity, which was not observed in
male rats. Cytochrome P450 levels were increased in both males and
females. The biochemical parameters with the exception of APDM
activity in female rats, returned to normal following the 7 day
recovery period. Electron microscopy showed significant proliferation
of the smooth endoplasmic reticulum of the hepatic cells. No other
morphological changes were detected (Burrows et al., 1972).
Groups each of 2-4 juvenile rhesus monkeys (Macaca mulatta)
were dosed daily with BHT dissolved in corn oil at a dose level
equivalent to 0, 50, or 500 mg/kg bw. Treatment was for 4 weeks. Blood
samples were taken prior to treatment and then at weekly intervals
from the control and test animals in the high level group, and from
test animals in the low level group at the end of the four week
period, for determination of total plasma cholesterol, lipid
phosphorus and triglyceride. Liver biopsies were taken from the test
animals in the high group at two weeks. At the end of the test period
all animals were fasted for 24 hours and sacrificed, and liver and
blood samples obtained. Liver samples were analysed for succinic
dehydrogenase and susceptability to peroxidation. Extracted liver
lipids were analysed for total cholesterol, lipid phosphorus and
triglycerides. Total cholesterol levels in plasma and liver were
significantly lowered. Lipid phosphorus levels in the plasma were
increased at the high dose level, and cholesterol:lipid phosphorus
ratios in the plasma and liver. The susceptibility of liver lipids to
oxidation was reduced in the high dose group (Branen et al., 1973).
Mice (BALB/c strain) were maintained on a diet containing 0.75%
BHT. After 3 weeks on the test diet there was an enhanced activity in
plasma esterases, which persisted throughout the experimental period
of 20 weeks. Following electrophoretic separation of the esterases,
the increased enzyme activity was shown to be located in two specific
bands. (Tyndall et al., 1975).
Rats fed stock diets supplemented with 20% lard, and containing
0.2, 0.3, 0.4 or 0.5% (dry weight) BHT for six weeks, showed an
increase in serum cholesterol that was directly related to the level
of dietary BHT. BHT increased the relative weight of the male adrenal
and also caused a significantly greater decrease in growth rate of
male as compared to the female. Increased liver weight in test animals
was paralleled by increased absolute lipid content of the liver
(Johnson & Hewgill, 1961). In another study, rats maintained on diets
containing 0.5% dietary BHT in the presence or absence of a 20% lard
supplement, irrespective of the presence or absence of dietary lard,
BHT increased the basic metabolic rate, the concentration of body
cholesterol and the rate of synthesis of body and liver cholesterol,
and reduced the total fatty acid content of the body. In the animals
fed BHT without lard, BHT increased the rate of synthesis and turnover
of body and liver fatty acids and reduced the growth rate. In the
animals fed BHT with lard, BHT reduced the rate of synthesis of body
and liver fatty acids and reduced the growth rate to a greater extent
than in animals without dietary lard (Johnson & Holdsworth, 1968).
Microsomal preparations from livers of rats, dosed daily with
BHT for up to seven days at a level of 450 mg/kg bw showed an
increased capacity to incorporate labelled amino acids, when compared
to preparations from controls. BHT also stimulated the in vivo
incorporation of amino acids, mainly into the proteins of the
endoplasmic reticulum (Nievel, 1969). Rats fed diets containing BHT at
levels of 0.01 to 0.5% for 12 days, showed liver enlargement, as well
as increased activity of liver microsomal biphenyl-4-hydroxylase, at
all levels except the lowest level of 0.01%. Enzyme activity was not
significantly altered by 0.5% BHT fed for one day (Creaven et al.,
1966).
Groups each of 60 FAF, male mice were maintained on semi-
synthetic diets containing 0, 0.25 or 0.5% BHT. The mean life-span of
the test animals was significantly greater than controls, being
17.0 ± 5.0 and 20.9 ± 4.7 months respectively for the 0.25% or 0.5%
BHT, as compared to 14.5 ± 4.6 months for controls (Harman, 1968).
Groups each of 20 male and 20 female Charles River rats were
maintained on test diets (males 24 weeks, females 36 weeks) containing
BHT and/or carcinogen. (N-2-fluorenylacetamide or N-hydroxy-N-2-
fluorenylacetamide) in the molar ratio of 30:1, equivalent to 6600 ppm
(0.66%) BHT, then continued on control diets for another 12 weeks. The
N-2-fluorenylacetamide alone resulted in hepatomas in 70% of the male
rats, mammary adenocarcinoma in 20% of the females. With N-hydroxy-N-
2-fluorenylacetamide 60% of the males had hepatomas and 70% of
the females had mammary adenocarcinoma. BHT reduced the
incidence of hepatomas in males to 20% when the carcinogen was
N-2-fluorenylacetamide, and to 15% (hepatomas in males), when
N-hydroxyl-N-2-fluorenylacetamide was the test compound. Similar
results were obtained with Fischer strain rats. Liver and oesophageal-
tumour production with diethylnitrosamines (55 ppm (0.0055%)) in
drinking-water for 24 weeks was not affected by BHT (Ulland et al.,
1973).
Young adult BALB/c mice of both sexes maintained on diets
containing 0.75% BHT, for one month, and then irradiated with 525-750
R of X-ray. Radiation Protection was observed at all doses below that
which produced 100% lethality (Clapp & Satterfield, 1975a).
Hybrid (C31F1) male mice, 10 to 12 weeks of age were maintained
on diets containing 0 or 0.75% BHT, for a period of 30 days, and then
injected i.p. with alkylating materials. There was a marked reduction
in the 30 day mortality in mice fed BHT. Males were protected against
ethyl methanesulphonate, n-propyl or isopropyl methanesulphonate,
ethyl dibromide, diethylnitrosamine and cyclophosphamide, but not
against methyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguaridine
or dipropylnitrosamine (Cumming & Walton, 1973).
TOXICOLOGICAL STUDIES
Special studies on embryotoxicity
In a study on the embryotoxicity of BHT three dosing schedules
were employed: single doses (1000 mg/kg bw) on a specific day of
gestation, repeated daily doses (750 mg/kg bw) from the time of
mating throughout pregnancy and daily doses (250-500 mg/kg bw for mice
and 500 and 700 mg/kg bw for rats) during a seven to 10 week period
before mating, continuing through mating and gestation up to the time
the animals were killed. No significant embryotoxic effects were
observed on examination of the skeletal and soft tissues of the fully
developed fetuses as well as by other criteria. Reproduction and
postnatal development were also unaffected (Clegg, 1965).
Diets containing 0.1 or 0.5% BHT together with two dietary levels
of lard (10 and 20%) were given to mice. The 0.5% level of BHT
produced slight but significant reduction in mean pup weight and total
litter weight at 12 days of age. The 0.1% level of BHT had no such
effect. Out of 7754 mice born, none showed anophthalmia, although 12
out of the 144 mothers were selected from an established anophthalmic
strain (Johnson, 1965).
Special studies on mutagenicity
(a) Cytogenetics
BHT was investigated at concentrations of 2.5, 25 and 250 µg/ml
in vitro, employing WI-38 human embryonic lung cells for anaphase
abnormalities. BHT produced a biologically significant increase in the
number of abnormal anaphase figures. These effects are thought to be
cytogenetic in nature but not mutagenic. The distinction between the
terms is that inhibition of cell division can be a cytogenetic effect,
but not mutagenic in the sense that this effect is not transmissible
through cell division to progency cells. Additionally 70% of the
substances tested by this procedure yielded significant biological
effects; therefore there is the possibility of generating false
positive effects (S.R.I., 1972).
BHT was also investigated in vivo by the cytogenetic analysis
of rat bone marrow cells. Dosages of 30, 900 and 1400 mg/kg were
employed. Acute and subacute regimens were done. No evidence of
significant cytogenetic damage was found (S.R.I., 1972).
(b) Host-mediated assay
In vitro-Salmonella TA-1530 and C-46, together with
Saccharomyces D-3 were employed. A 50% concentration was
tested. No mutagenicity was seen (S.R.I., 1972).
In vivo-BHT was tested at levels of 30.0, 900.0 and 1400 mg/kg
in the acute tests and at 30.0, 250.0 and 500.0 mg/kg in the subacute
tests in ICR Swiss mice employing as indicator organisms Salmonella
G-46 and TA-1530 and Saccharomyces D-3. No mutagenicity was seen.
BHT has also been investigated by more sensitive (in terms of
detection) test methods which are a part of the tier one screen. In
this study BHT was investigated using Salmonella typhimurium strains
TA-1535, 1537 and 1538 and Saccharomyces D-4 with and without
metabolic activation in plate and suspension tests. The percentage
concentrations (w/v) employed were 0.15, 0.3, and 0.6 for bacteria and
0.6, 1.2 and 2.4 for yeasts. Under the conditions of this
investigation BHT was non-mutagenic (Brusick, 1975).
(c) Dominant lethal study
BHT was investigated in Sprague Dawley rats at acute dosages of
30, 900 and 1400 mg/kg and at subacute dosages of 30×5, 250×5 and
500×5 mg/kg.
Significant postimplantation losses were produced in weeks three,
four and six by the subacute regimen (Brusick, 1975).
Special studies on reproduction
Weanling rats (16 of each sex) were fed a diet containing 20%
lard and 0, 300, 1000 or 3000 ppm (0, 0.03, 0.1 or 0.3%) BHT and mated
at 100 days of age (79 days on test). Ten days after weaning of the
first litter the animals were again mated to produce a second litter.
The offspring (16 females and eight males) were mated at 100 days of
age. Numerous function and clinical tests including serum cholesterols
and lipids were performed on the parents and the first filial
generation up to 28 weeks and gross and microscopical examination at
42 weeks. At the 3000 ppm (0.3%) dietary level 10-20% reduction in
growth rate of parents and offspring was observed. A 20% elevation of
serum cholesterol levels was observed after 28 weeks, but no
cholesterol elevation after 10 weeks. A 10-20% increase in relative
liver weight was also observed upon killing after 42 weeks on diet.
All other observations at 3000 ppm (0.3%) and all observations at
1000 ppm (0.1%) and 300 ppm (0.03%) were comparable with control. All
criteria of reproduction were normal. No teratogenic effects were
detected (Frawley et al., 1965b). Similar results to those obtained
with the parental and first filial generations were also obtained with
the second filial generation. Examination of two litters obtained from
the latter at 100 days of age revealed no effects except a reduction
of mean body weight at the 3000 ppm (0.3%) level. The offspring were
examined for: litter size, mean body weight, occurrence of stillbirth,
survival rate and gross and microscopic pathology (Kennedy et al.,
1966).
Other special studies
Acute oral, intraperitoneal (mice) and eye irritation (rabbits)
and skin irritation (rats) were measured for 7 breakdown products of
BHT. All compounds tested were less toxic than the parent compound
(Conning et al., 1969).
The chronic ingestion of 0.5% of the butylated hydroxytoluene
(BHT) by pregnant mice and their offspring resulted in a variety of
behavioural changes. Compared to controls, BHA-treated offspring
showed increased exploration, decreased sleeping, decreased self-
grooming, slower learning, and a decreased orientation reflex.
BHT-treated offspring showed decreased sleeping, increased social and
isolation-induced agression, and a severe deficit in learning (Stokes
& Scudder, 1974).
Young male Swiss Webster mice were injected i.p. with BHT at dose
levels ranging from 62.5 to 500 mg/kg bw BHT. The animals were killed
on days 1, 3 and 5 after BHT administration. Histopathological changes
were well developed 3 days after administration of 500 mg/kg bw, and
consisted of a proliferation of many alveolar cells, formation of
giant cells and macrophage proliferation. These changes were
accompanied by an increase in lung weight and total amounts of DNA and
RNA. The changes were dose dependent, the smaller effective dose being
250 mg/kg bw (Saheb & Witschi, 1975).
Acute toxicity
LD50 Approximate
Animal Route (mg/kg bw) lethal dose Reference
(mg/kg bw)
Rat oral 1 700-1 970 - Deichmann et al.,
1955
Cat oral - 940-2 100 Deichmann et al.,
1955
Rabbit oral - 2 100-3 200 Deichmann et al.,
1955
Guinea-pig oral - 10 700 Deichmann et al.,
1955
Rat oral 2 450 Karplyuk, 1959
Mouse oral 2 000 Karplyuk, 1959
Short-term studies
Mouse
BHT was given to pregnant mice in daily doses of 750 mg/kg bw for
18 days. Another group received the same dose for a total of 50 to 64
days including 18 days of pregnancy. No fetal abnormalities were
observed (Clegg, 1965).
In a statistically planned experiment using 144 female mice no
blindness was observed in any of the 1162 litters representing 7765
babies born throughout the reproductive life span of the mothers
(Johnson, 1965).
Rat
Feeding experiments were carried out on 45 pairs of weanling male
rats for five to eight weeks with diets containing 0, 10 and 20% lard
supplements to which 0.001, 0.1 or 0.5% BHT had been added. 0.001%
caused no changes in any of the serum constituents studied. 0.5%
produced increase in the serum cholesterol level within five weeks.
Female rats fed for eight months on a diet containing a 10% lard
supplement with 0.1% BHT showed increased serum cholesterol levels,
but no other significant changes. 0.5% BHT in 10% and 20% lard
supplements fed to female rats for the same period increased serum
cholesterol, phospholipid and mucoprotein levels (Day et al., 1959).
0.3% BHT in the diet of pregnant rats that had been kept for five
weeks on a diet deficient in vitamin E produced no toxic symptoms.
1.55% caused drastic loss of weight and fetal death (Ames et al.,
1956).
BHT fed to rats in groups of 12 for a period of seven weeks at a
dietary level of 0.1% in conjunction with a 20% lard supplement
significantly reduced the initial growth rate and mature weight of
male rats. No effect was noted in female or male rats with a 10% lard
supplement. A paired feeding experiment showed that this inhibition of
growth was a direct toxic effect of BHT and could not be explained by
a reduction in the palatability of the diet. At this level BHT
produced a significant increase in the weight of the liver, both
absolute and relative to body weight. Rats under increased stress
showed significant loss of hair from the top of the head. The toxic
effect of BHT was greater if the fat load in the diet was increased.
Anophthalmia occurred in 10% of the litters (Brown et al., 1959).
Groups of six weanling rats (three male and three female) were
fed BHT at dietary levels of 0, 0.1, 0.2, 0.3, 0.4 and 0.5% in
conjunction with a 20% lard supplement for six weeks. BHT reduced the
growth rate, especially in the males, the effect appearing to become
significant at the 0.3% level. It also increased the absolute liver
weight and the ratio of liver weight to body weight in both sexes, the
latter effect appearing to become significant at the 0.2% level. BHT
increased the ratio of left adrenal weight to body weight in male rats
but had no consistent effect in females. There were no histological
changes attributable to the treatment in the adrenal. All dietary
levels of BHT increased the serum cholesterol and the concentration of
the cholesterol was directly proportional to the BHT level. There
was also a significant increase in the concentration of adrenal
cholesterol. BHT produced no significant changes in the concentration
of total or percentage esterified liver cholesterol, total liver lipid
or concentration of total polyunsaturated fatty acids in the liver
(Johnson & Hewgill, 1961).
BHT administered to rats at 250 mg/kg bw for 68 to 82 days caused
reduction in rate of increase in weight and fatty infiltration of the
liver (Karplyuk, 1959).
Feeding experiments conducted for 20 and 90 days respectively
indicated that rats do not find food containing 0.5 or 1% BHT
palatable. However, the animals ingest foods so treated more readily
if these concentrations are attained gradually. Paired feeding
experiments with groups of five or 10 rats for 25 days demonstrated
that diets containing 0.8 and 1% BHT will reduce the daily intake of
food below control values. A level of 1% in the diet retarded weight
gain (Deichmann, 1955).
BHT (2000 ppm (0.2%)) incorporated in a diet containing 19.9%
casein was administered to a group of eight young rats for eight
weeks; a further group of eight rats served as controls. The
experiment was repeated with 16.6% casein in the diet of further
groups for four weeks and again with 9.6% casein (and no added
choline) for seven weeks. In all three instances BHT caused
stimulation of growth and improved protein efficiency. The N content
of the liver was, however, greatly reduced in BHT-treated animals,
except when the level of BHT was reduced to 200 ppm (0.02%). Recovery
of hepatic protein after fasting (details not given) was also impaired
in rats on 2000 ppm (0.2%) BHT. Liver lipid content was increased with
2000 ppm (0.2%) but not with 200 ppm (0.02%) BHT. A dietary level of
2000 ppm (0.2%) BHT also increased the adrenal weight and ascorbic
acid content, although if recalculated on the basis of weight of
gland, there was no significant difference. The increase in adrenal
ascorbic acid is interpreted as indicating a stress imposed on the
organism by BHT (Sporn & Schöbesch, 1961).
Groups of 48 weanling rats (24 of each sex) were given diets
containing 1000 ppm (0.1%) BHT for periods of up to 16 weeks. A group
of 48 rats served as controls. Measurements of growth rate, food
consumption, weight and micropathological examination of organs at
autopsy revealed no difference from untreated rats. However, increase
in relative liver weight and in the weight of the adrenals was
produced without histopathological evidence of damage. Biochemical
measurements and histochemical assessments of liver glucose
phosphatase and glucose 6-phosphate dehydrogenase activities revealed
no difference from the control group (Gaunt et al., 1965a).
Groups of 16 male and 16 female rats were fed on levels of 0.03,
0.1 and 0.3% BHT in a diet containing 20% fat for 10 weeks. There were
two control groups each containing 16 male and 16 female animals. No
definite effect on body weight was observed at any level in the
females and there was only a slight depression in the males at the
0.3% level. There was no significant effect on blood cholesterol level
in either sex after feeding BHT at any of the levels for 10 weeks.
Four of the males at the 0.3% and two at the 0.1% level died during
the experiment. Two deaths occurred among the females at 0.3%. Only
one male rat died in both control groups (Frawley et al., 1965a).
Groups of 20 male and 20 female rats fed 1% BHT in the diet for
10 weeks showed recovery both in liver to body weight ratios and in
morphological appearance of the liver cells within a few weeks after
restoring the animals to a normal diet (Goater et al., 1964).
Rabbit
Acute effects on electrolyte excretion similar to those described
for large doses of BHA were also obtained following administration of
doses of BHT of 500-700 mg/kg bw (about 2% in the diet). No such
effects were observed at lower dosage levels (Denz & Llaurado, 1957).
Fowl
When BHT was fed at a level of 0.125% for 34 weeks to a group of
10 pullets, no differences in fertility, hatchability of eggs or
health of chicks in comparison with a similar control group were
found. The eggs of the antioxidant-treated birds contained more
carotenoids and vitamin A than those of the controls (Shollenberger et
al., 1957).
Dog
A mild to moderately severe degree of diarrhoea was induced in a
group of four dogs fed doses of 1.4-4.7 g/kg bw every two to four days
over a period of four weeks. Post-mortem examination revealed no
significant gross pathological changes. No signs of intoxication and
no gross or histopathological changes were observed in dogs fed doses
of 0.17-0.94 g/kg bw five days a week for a period of 12 months
(Deichmann et al., 1955).
Monkey
A group of six adult female rhesus monkeys were maintained on a
test diet containing a mixture of BHT and BHA that provide an intake
equivalent to 50 mg BHT and 50 mg BHA/kg bw. Another group of six
adult female rhesus monkeys were 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. Hematologic studies
including hemoglobin, 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 5 infants were born to the experiment monkeys
and 6 to the control monkeys. Hematological 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, 3 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 delivered. Adult
females continued to have normal infants. 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,
1976).
In another study, groups each of 3 infant or juvenile monkeys
(Macaca mulatta) were dosed daily with BHT at a level equivalent to
500 mg/kg bw. Another group of juvenile monkeys received 50 mg/kg bw
BHT. Treatment was for 4 weeks. Blood analysis (complete cell count,
serum sodium and potassium, bilirubin, cholesterol and glutamic
oxalactic transaminase) was carried out weekly, as was a complete
urinalysis. Liver biopsies were taken from the juvenile monkey at 2
weeks, following a 24 hour fast. At the end of the test period, all
animals were fasted 24 hours and sacrificed. Tissues from all major
organs were prepared for light and electron microscopy. Liver tissue
was also analysed for protein, RNA and cytochrome P450. Microsomal
preparations prepared from the livers were used to measure
nitroanisole demethylase and glucose-6-phosphatase activity. Urine and
blood values of test and control animals were similar. Histological
evaluation of all organs other than the liver from either infant or
juvenile did not indicate any compound related changes. Test animals
receiving BHT showed hepatocytomegaly and enlargement of hepatic cell
nuclei. Ultrastructurally, the hepatocytes of treated animals showed
moderate proliferation of the endoplasmic reticulum. Lipid droplets
were also prominent in cytoplasm of these hepatic cells. There was
fragmentation of the nucleolus in 15% of the hepatic cells in the test
animals in the high level group. DNA and RNA and cytochrome P450
levels in the liver of test and control animals were similar. BHT
treated juveniles showed an increase in nitroanisole demethylase
activity which increased with time. The enzyme activity was unaffected
in infant monkeys. Glucose-6-phosphatase activity declined in juvenile
monkeys but was unchanged in infant monkeys (Allen & Engblom, 1972).
Long-term studies
Rat
Groups of 15 male and 15 female rats given diets containing 1%
lard and 0.2, 0.5 or 0.8% BHT for 24 months showed no specific signs
of intoxication, and micropathological studies were negative. For one
group given a diet containing 0.5% BHT, the BHT was dissolved in lard
and then heated for 30 minutes at 150°C before incorporation in the
diet. There were no effects on weight gain or blood constituents and
micropathological studies of the main organs were negative. The
feeding of 1% BHT was followed in both male and female rats by a
subnormal weight gain and by an increase in the weight of the brain
and liver and some other organs in relation to body weight.
Micropathological studies were negative in this group also. BHT in the
concentrations had no specific effect on the number of erythrocytes
and leucocytes, or on the concentration of haemoglobin in the
peripheral blood. A number of rats of both sexes died during this
experiment, but as the fatalities were in no relation to the
concentration of BHT fed, it was believed that the cause of death was
unrelated to the feeding of this substance. Micropathological studies
support this observation. When fed at the 0.5% level in the diet, BHT
had no effect in rats on the reproductive cycle, the histology of the
spleen, kidney, liver and skin, or on the weight of the heart, spleen
or kidney. There was no significant increase in mortality of rats fed
on a diet containing 0.1% BHT and 10% hydrogenated coconut oil for a
period of two years. The effects on weight gain have already been
described (Deichmann et al., 1955).
Female (albino Wistar) rats, initial body weight 120-130 g were
maintained on a diet containing 0 or 0.4% W/W BHT, for 80 weeks. After
one week on the test diet significant increases were observed in
liver weight, microsomal protein, cytochrome P450, cytochrome b5,
NADPH-cytochrome c reductase, biphenyl-4-hydroxylase and ethyl
morphine N-demethylase but not aniline-4-hydroxylase. Total liver
protein, succinic dehydrogenase and glucose-6-phosphatase were
slightly depressed. There was little change in this pattern during the
period of the study. Rats removed from the BHT test diet at the end of
the test period and maintained on BHT free diet for 18 days, showed a
return to normal for many liver parameters. However, cytochrome b5,
cytochrome c reductase and ethylmorphine N-demethylase remained
increased. Histological changes at the end of 80 weeks feeding the
diet consisted of centrilobular cell enlargement, which was
reversible, following 18 days on a BHT free diet. The only
ultrastructural change was a proliferation of smooth endoplasmic
reticulum (Gray & Parke, 1974).
A group of 18, 8 week-old male BALB/c mice fed dietary BHT at a
level of 0.75% for a period of 12 months, developed marked hyperplasia
of the hepatic bile ducts with an associated sub-acute cholangitis
(Clapp et al., 1973). In another study eleven mice (BALB/c strain)
were maintained on a diet containing 0.75% BHT for a period of 16
months. The incidence of lung tumours in the test group was 63.6%,
compared with 24% in controls (Clapp et al., 1974). However, a repeat
of this study using a larger group of test animals, showed that BHT
had no effect on the incidence of lung tumours in either sex (Clapp et
al., 1975b).
Groups each of 48 mice (CFI strain) equally divided by sex were
maintained on diets containing 1000 ppm BHT. At week 4, one group was
then fed a diet containing 2500 ppm BHT, and then at eight weeks
another group was fed a diet containing 5000 ppm BHT. The animals were
maintained on these diets until 100 weeks of age. There was no
statistically significant reduction in survival of animals on the BHT
diet, although survival was poorer in males at the high dose level
during the last quarter of the study. Animals dying or sacrificed
during the course of the study showed greater centrilobular cytomegaly
and karyomegaly than controls. Bile duct hyperplasia was only observed
in 3/141 test animals. There was no significant difference in the
incidence of malignant tumours in the high level group and control.
However, there was an increased incidence of lung neoplasia in treated
rats (75% in 5000 ppm group, 73.9% in 2500 ppm group, 53.2% in the
1000 ppm group and 46.8% in controls). There were no morphological
features to distinguish the lung tumours in treated mice from those in
controls. There was also an apparent increase in benign ovarian
tumours in BHT treated female mice, since none were observed in
control animals (Brooks et al., 1976).
OBSERVATIONS IN MAN
Four human male subjects were administered a single dose of
approximately 40 mg 14C-labelled BHT. About 75% of the administered
radioactivity was excreted in the urine. About 50% of the dose
appeared in the urine in the first 24 hours, followed by a slower
phase which probably represents the release of the compounds or their
metabolites stored in tissues. In man, the bulk of the radioactivity
is excreted as the ether insoluble glucuronide of a metabolite in
which the ring methyl group and one tert-butyl methyl group are
oxidized to carboxyl groups, and a methyl group on the other tert-
butyl group is also oxidized, probably to an aldehyde group. BHT-acid
free and conjugated is a minor component of the urine and the
mercapturic acid is virtually absent. The rapidity of the first phase
of the urinary excretion in man suggests that there is no considerable
enterohepatic circulation as has been observed in the rat (Daniel et
al., 1967).
Reported values of BHT in the body fat were 0.23 ± 0.15 ppm
(0.000023% ± 0.000015%) (11 individuals, residents of the United
Kingdom) and 1.30 ± 0.82 ppm (0.000130 ± 0.000082) (12 individuals,
residents of the United States of America) (Collings & Sharratt,
1970).
Comments
Further elucidation of the metabolism of BHT in man as well as
the rat indicate a rapid urinary excretion of oxidation products, some
conjugated with glycine.
Swiss-Webster mice that were the offspring of parents fed 0.5%
BHT and fed the same diet were subjected to behavioural tests at 6
weeks of age. Results indicated behavioural changes, but these changes
are difficult to evaluate in the light of the fact that infant monkeys
from BHT-treated dams showed no behavioural abnormalities.
Several carcinogenicity studies have been undertaken on mice.
Balb C mice did not have an increased incidence of lung tumours
whereas CFI mice had an increased incidence of lung tumours compared
with controls. As regards other tumours no increased incidence was
seen compared with controls and long-term rat studies have been
negative. BHT has been studied in several in vitro and in vivo
systems and was found to be non-mutagenic. The negative mutagenic
studies provide additional evidence for a non-carcinogenic potential
of BHT. However, the Committee considered that an appropriate
carcinogenic study meeting modern standards would be desirable.
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
Mouse: 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 1980
Appropriate carcinogenicity study, meeting currently accepted
standards.
* As BHA, BHT or the sum of both.
** Temporary.
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