WORLD HEALTH ORGANIZATION
Toxicological evaluation of some food colours, thickening
agents, and certain other substancse
WHO FOOD ADDITIVES SERIES NO. 8
The evaluations contained in this publication were prepared
by the Joint FAO/WHO Expert Committee on Food Additives which
met in Geneva, 14-23 April 19751
World Health Organization, Geneva 1975
1 Nineteenth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Wld Hlth Org. techn. Rep. Ser., 1975, No. 576;
FAO Nutrition Meetings Report Series, 1975, No. 55.
The monographs contained in the present volume are
also issued by the Food and Agriculture Organization
of the United Nations, Rome, as
FAO Nutrition Meetings Report Series, No. 55A
ISBN 92 4 166008 2
(C) FAO and WHO 1975
TERTIARY BUTYL HYDROQUINONE (TBHQ)
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Metabolic studies in the rat
Male and female rats, body weight 250 g, were given a single dose
of TBHQ, dissolved in corn oil (10% w/w) by intubation at dose levels
equivalent to 100, 200, 300, or 400 mg/kg. At the 400 mg/kg level
there was a rapid onset of ataxia, followed by recovery in two or
three hours. Urine samples were collected daily for three days before
dosing and then for six days after dosing. At all dose levels
excretion appeared to be complete in three or four days. About 66% of
the dose was excreted as the o-sulfate conjugate and less than 10% as
the glucuronide. At the 100 mg/kg level, urinary excretion accounted
for almost all the dose. At higher levels about 33% could not be
accounted for in the urine, nor be detected in the faeces. Excretion
of the free TBHQ at the 100 mg/kg level was about 12% but this level
decreased at the higher dose levels (2% at 400 mg/kg). No other major
metabolites were detected (Astill et al., 1968).
Urine samples were collected from two animals, from the 0, 0.16%,
and 0.5% dietary TBHQ groups of a long-term feeding study at months
12 and 20. Sera samples were collected from groups of five rats at
months six, 12, 20 and at autopsy. Samples of perirenal, omental and
subcutaneous fat were removed at autopsy, pooled by sex and dose. At
12 months, males at both levels excreted about equal amounts of the
conjugates in the urine (o-sulfate and o-glucuronide). About two-
thirds of the excretory products in females was the o-sulfate form and
the remainder the o-glucuronide. At 20 months in both male and female,
most of the conjugate excreted was in the o-sulfate form with little
evidence for glucuronide excretion. Only negligible amounts of TBHQ
were detected in serum or fat (Astill et al., 1968).
Portions of the fat of control animals and animals that had been
maintained on 0.5% TBHQ were examined for stability by the active
oxygen method (oxidative stability) and also for TBHQ content. There
were no apparent differences in the oxidative stability of fats from
treated and control animals, nor did polarographic and colorimetric
methods of analysis (sensitive to 5 ppm) indicate the presence of TBHQ
in the fat of test animals (Astill, 1968).
In a single dose study, rats received a dose of C14-labelled
TBHQ equivalent to 15, 48, 92, 383, 380, or 400 mg/kg. Urine and
faeces were collected daily as was expired CO2. At the end of the
test period the animals were sacrificed and blood, brain, kidneys,
liver, GI tract and perirenal, omental and subcutaneous fat removed
for assay. Seventy-eight to 88 per cent. of the administered
radioactivity was recovered in the urine, the bulk of this being
excreted within the first 24 hours (55-82.7% of the administered
dose). Of the recovered radioactivity, 70-76% was in the form of the
o-sulfate conjugate: 1-2% as the o-glucuronide. Faecal excretion was
2-6%. Only traces of radioactivity were detected in the tissues at the
92 mg/kg level; no values were given for the higher levels (Astill et
al., 1967a).
In another experiment, rats (body weight 200-250 g) were
maintained on a daily diet which allowed an intake of 5.7 mg/kg
(0.029% level), of TBHQ-C14 daily for 17 days. Urine and faeces were
collected throughout the experiment. At the end of the test period the
rats were starved overnight before sacrifice, and brain, liver,
kidney, and fat samples collected. Tissue levels were as follows
(mg TBHQ/g wet tissue): liver, 0.06-0.34; kidney, 0.09-0.38; brain,
0.06-0.56; fat, 0.06-0.37) (Astill et al., 1967a).
Pregnant albino S-D rats 380-440 g body weight age 48 weeks were
selected from the third litter of second generation females in a
reproduction study and had received 0.5% of TBHQ in the diet since
weaning. Animals were given one day before term an oral dose of
TBHQ-C14 (40 mg/kg) as a 10% solution in corn oil. Urine and faeces
were collected up to time of sacrifice (7.6-16.7 hours after dosing).
Foetuses were removed by Caesarean section. The uterus, amniotic
fluid, GI tract, liver, brain, kidneys, and fat specimens were
collected for radioassay. About 74% of the dose was excreted in the
urine in the 16.7 hour period. Only 10% of the dose was detected in
the GI tract at 7.4 hours after dosing, and 8.5% at 17.6 hours. The
level of radioactivity in foetuses was 0.2% of the dose at 7.6 hours
and 0.02% at 16.7 hours. Similar small proportions of the dose were
present in the uterus and amniotic fluid and other tissues examined.
Based on these results, extrapolation to possible known exposures
suggest that at the highest possible intake (0.1 mg/kg/day), the human
foetus would be exposed to the order of 1% of the daily intake in the
form of unchanged TBHQ and probably higher levels of the conjugate
(Astill & Walton, 1968).
Metabolic studies in the dog
Male beagle dogs about 11 kg weight fed Purina Chow and TBHQ
as a single 100 mg/kg oral dose, via ground meat capsule. Urine was
collected three days before dosing and six days after dosing.
Excretion was essentially complete within 48 hours. The major urinary
excretory products were the o-sulfate, and o-glucuronide conjugates
and a small amount of unchanged TBHQ. Total recoveries ranged from
77-98%. About two-thirds of this was as the o-sulfate and one-third as
the o-glucuronide (Astill et al., 1967b).
In another study, 26 male and female dogs were used. The dogs
were maintained on diets containing TBHQ dissolved in corn oil at
levels equivalent to 0, 0.05, 0.1, and 0.5%. Urine and serum samples
were collected on day nine and one day before commencement of feeding
TBHQ, and at months three, six, 12, 13 and 24 of the test period.
Serum was collected 23 hours after feeding. At autopsy, performed on
one dog of each sex at each dose level at 12 months, and the remaining
at 24 months, samples of perirenal, omental and subcutaneous fat were
removed. Chromatographic studies of the urine indicated excretion of
both the o-sulfate and o-glucuronide conjugates, at all dose levels.
In the case of males the o-sulfate/glucuronide ratios were 2/1,
whereas in females the bulk of the conjugate was in the form of the
o-sulfate. Only insignificant quantities of TBHQ were detected in the
fat (in fat, maximum in males was 7 ppm, and in females 17 ppm, in
most cases value was 0), and serum (0-0.7 ppm) (Astill et al., 1967).
Portions of the fat from test animals and animals that had been
maintained on the highest level of TBHQ (0.5%) for two years were
examined for stability by the active oxygen method. There was no
apparent difference in the oxidative stability of fats from treated or
control animals (Anonymous, 1968a). In another study TBHQ residues
were assayed in fat, brain, liver and kidney of dog and rats from the
long-term feeding studies. Storage appeared to be negligible (Astill &
Jones, 1969).
Metabolic studies in human subjects
Human subjects (male) received TBHQ under the following
conditions:
(1) gelatin capsule containing 150 mg TBHQ;
(2) a mixture of TBHQ (2%) in corn oil and graham cracker crumbs,
equivalent to a dose of 125 mg TBHQ;
(3) 100 mg dissolved in cottonseed oil contained in a gelatin
capsule;
(4) 20 g of mixture containing TBHQ, 2% cottonseed oil and 2%
confectioners sugar in graham cracker crumbs. Doses of TBHQ
ranged from 20 to 70 mg.
Subjects one, two and three drank milk immediately after
ingesting test material, subject four ate doughnuts and drank coffee.
Urine was collected from subjects 24 hours before dosing and
during the 72-hour period after dosing. Blood was collected by
venipuncture at three or five and 24 hour post-dosing. Clinical
observations were made immediately before ingestion and three to six
hours after and consisted of blood pressure, pulse response, condition
of pharynx, conjunctivae and pupils and neurologic effects.
Haematologic studies consisted of haemoglobin, cell volume, WBC,
differentials, reticulocytes, platelets and total protein. Urinalysis
consisted of SpGr, albumin, reducing sugars, ketone bodies, occult
blood, pH and sediment. Levels of TBHQ in serum and metabolites of
TBHQ in urine were also determined.
There was no evidence of any systemic effect following ingestion
of TBHQ. No significant changes were observed in haematological
studies or urinalysis. Examination of urine indicated that TBHQ was
excreted as the o-sulfate and o-glucuronide conjugates (ratio
approximately 3:1). These were mainly recovered during the first
24 hours. No free TBHQ was detected at any time. The manner of
ingestion had a marked effect on the proportion of the dose recovered
from urine. TBHQ administered by methods one and three resulted in
only 22-40% of the dose being recovered in the urine, whereas method
two resulted in 90-100% recovery. In all cases, the same metabolic
products were present in urine. High recoveries of TBHQ metabolites in
urine were accompanied by a serum level of TBHQ 3.1-3.7 mg/100 ml at
three hours for two, compared to 0.4-1.2 mg/100 ml at three hours (one
and three). At 24 hours these levels had fallen to 1.5 mg/100 ml for
two and 0.2-1.2 mg/100 ml for one and three (Astill et al., 1967c).
TOXICOLOGICAL STUDIES
Special studies on reproduction
TBHQ was fed in the diet to groups of 15 male and 15 female S-D
rats for three successive generations at levels of 0 and 0.5%. Pairs
of rats were mated to produce two litters per generation, with the
next generation selected from weanlings of the second titter. Data
recorded in each were as follows: number of inseminations, number of
pregnancies, gestation period, average litter size, mortality of young
from birth to weaning, weaning to one week after weaning, one week and
two weeks after weaning to sacrifice. The average body weight per pup
at weaning, one week after weaning, and two weeks after weaning were
also recorded. Tissues were collected from all breeders. Animals that
were not used as breeders were sacrificed at seven weeks and those
selected as possible breeders but not used, at 14 weeks. All of the
pups in the b generation were sacrificed and autopsied at
approximately seven weeks of age. All animals were examined for gross
pathology, and micropathology was studied on at least four animals of
each litter. Organs examined included: trachea, lung, heart, tongue,
oesophagus, stomach, small and large intestines, liver, kidneys,
urinary bladder, pituitary, adrenal, pancreas, thyroid, parathyroid,
gonads, uterus, spleen, bone marrow, cerebrum, cerebellum, and eye. To
terminate study F3b litters were delivered by uterotomy on the
nineteenth day of gestation. Foetuses were examined for gross
abnormalities. One-third were stained with alizarin red for skeletal
defects, while the other two-thirds, after fixation, were sectioned
freehand and examined for abnormalities. The percentage of
inseminations and pregnancies, gestation and average litter size
appeared normal for both matings of each generation. In the F1a and
F2a test groups there were more deaths than in control in birth to
weaning period. The effect was not observed in the subsequent F1b and
F2b generations. The parent rats (Fo generation) of the test group
ate less and showed lower weight gain than their respective controls.
Body weight of pups from treated animals at various time periods from
weaning were lower than those of the control. Deaths during the
period, weaning to five weeks were always more frequent in treated
group (deaths as percentage total number animals). Abnormalities were
reported in 22 foetuses from the F3b generation but 13 of these were
in control group. Minor skeletal changes were noted in two animals in
the test group. No compound related histological abnormalities were
observed (Terhaar & Krasavage, 1968a).
In another study groups each of 20 male and 20 female rats (S-D)
were maintained on diets containing TBHQ at concentrations of 0.015,
0.15, 0.5 and 0% along with Mazola Corn Oil at 5% (w/w) incorporated
into a basic diet of Purina Chow. Diets were fed for 66 days prior to
breeding. Rats of the same dose level were mated (1:1) to produce two
groups of first generation litters (F1a and F1b). F1a litters
were maintained on assigned diets. Data recorded included number of
inseminations and pregnancies, gestation period, litter size,
mortality of young at birth, birth to weaning, weaning to one week
post-weaning and one week post-weaning to two weeks post-weaning.
Litters of F1b generation were treated similarly to F1a litters up to
the tenth day after birth. At day 10, litters and dams within test
groups were paired, and one pair placed on control diet, while others
remained on the test diet. Also half the litters and dams of control
groups were allowed to remain on the control diet and half on high
level TBHQ diet (0.5%). At five weeks of age pups were sacrificed and
examined for gross pathology. A number of deaths of parent animals
occurred during the study (one male high dose, one control group
during first five days, and later three males, one low, one middle,
one high dose level, and one female control). None of the deaths
appeared to be compound related. Food intake of parent rats was
similar to control except at the 0.5% level, where there was a slight
decrease at the commencement of the test. Male rats in this group
showed a slightly decreased weight gain compared to control. There was
no apparent effect on gonadal function, oestrus cycle, mating
behaviour, conception rates, gestation period, parturition and
lactation during the two breedings. The average litter size, neonatal
viability and growth of the new-born pups appeared normal. Autopsy of
F1b pups failed to reveal any gross pathology. The average litter
weights of the F1a test pups were similar to controls. Pups from the
F1b control changed to the 0.5% TBHQ showed a somewhat lower body
weight than those left on control diet, for the period of study (up to
two weeks past weaning). This may be related to rejection of the diet
(Krasavage & Terhaar, 1970).
Special studies on teratogenicity
Groups each of 20 pregnant female S-D rats were fed test diets
containing 0, 0.125, 0.25 and 5% TBHQ, and 0.03 and 0.12 Apholate
(as positive control), at days 6-16 of gestation. On day 20 all
females were sacrificed, and total implantation sites evaluated for
resorptions, live foetuses and dead foetuses. The foetuses were
weighed and examined for gross anomalies and either soft or skeletal
tissue anomalies. All females on the TBHQ test diets showed weight
gains similar to control. The mean body weight of both male and female
foetuses of all treated groups were comparable to control, with the
exception of the high Apholate group. Two foetuses in the high dose
TBHQ showed soft tissue anomalies. The percentage skeletal tissue
anomalies in the TBHQ test groups was not greater than controls
(Krasavage, 1973).
Other special studies
(a) Response to liver processing enzyme and liver G-6-P activity to
TBHQ
Rat
Adult male rats S-D strain were maintained on standard diets
containing the following additions: (1) none; (2) 5% heated cottonseed
oil; (3) DL-Ethionine, 2.5% level for 10 days; (4) 100 mg/kg/day
phenobarbital for five days (intraperitoneal injection), (5) 1% corn
oil + 0.05% BHA; (6) 4% corn oil + 0.2% BHA; (7) 1% corn oil + 0.05%
TBHQ; (8) 4% corn oil + 0.2% TBHQ; and (9) 5% heated cottonseed oil +
0.025% TBHQ. A liver microsomal fraction was prepared from each group
of animals and glucose-6-phosphatase (G-6-P), p-nitroanisole
demethylase (pNaD) and aniline hydroxylase (AHase) activities
determined. The expected elevation of pNaD (5X) and AHase (3X)
occurred with phenobarbital and DL-ethionine had no significant effect
on these enzymes. Phenobarbital produced a depression of G-6-P
activity (25%), TBHQ at 0.05% level produced a 25% depression in
G-6-Pase which was absent at the 0.2% level. TBHQ had no effect on
pNaD at the 0.05% level, but produced at the 0.2% level a 60%
elevation of pNaD. There was no clear effect on AHase. In contrast,
BHA produced a 30% decrease in G-6-P at both levels, a 50% increase in
pNaD at 0.05% and 700% increase of pNaD at the 0.2% level. There was
no effect on AHase. Inclusion of heated oil in the diet had no marked
effect on previous changes. In another experiment in which enzyme
activities were measured of microsomal preparation from livers of rats
fed for 180 days diets containing 0.5% TBHQ dissolved in either heated
or unheated cottonseed oil, no significant differences were observed
that could be attributed to heat treatment of oil before addition to
the diet (Tischer & Walton, 1968).
Dog
pNaD, AHase, and G-6-Pase activities of microsomal fractions from
dogs which had been maintained on diets containing 0, 0.05, 0.16, and
0.5% levels of TBHQ for two years were within range of control values
(Tischer & Walton, 1968).
Electron microscopy studies of liver and kidney tissue from
both dog and rat showed that long-term administration of TBHQ did
not significantly alter the subcellular constituents, or cause a
proliferation of the endoplasmic reticulus of liver cells (Wolf &
Fassett, 1968a and b).
(b) TBHQ in heated or unheated oil six month dietary study in rats
Eight groups, each of 30 rats (S-D) (equally divided by sex),
were fed four levels of TBHQ in unheated oil and at the same level in
heated oil (one hour to raise temperature to 375°F followed by four
hours at 375°F). The levels of TBHQ in the oils were 0, 0.02, 0.1 and
0.5%. Oils were incorporated at a 5% level into a standard diet of
ground Purina Chow. Animals were housed five/cage and water was
available at all times. Body weight and gross feed consumption was
recorded weekly for the first two months and thereafter fornightly.
General appearance and behaviour was observed during the test period.
Haemograms and urinalysis were done on the 0.5% unheated and heated
and control at one, five, and six months. Haemograms consisted of
haemoglobin, haematocrit, WBC and differentials and protein
determination. Urinalysis consisted of pH, SpGr, occult blood,
albumin, reducing sugar and microscopic examinations of the sediment.
SGOT and SAP were done on the high and control groups at three and six
months. At autopsy, liver, kidney, heart, spleen, lung, brain and
testes weights were determined. The following tissues were examined
microscopically: lung, heart, tongue, oesophagus, stomach, small and
large intestines, liver, kidney, urinary bladder, pituitary gland,
adrenal, pancreas, thyroid, gonads, spleen, bone marrow, cerebrum,
cerebellum and eye. Three deaths occurred during the test period, but
these were not compound related. Male rats on the 0.5% TBHQ/unheated
fat showed a slight depression in weight gain and those in the 0.02%
TBHQ/unheated fat a significant increase in weight gain over control.
These effects were not observed in female rats on diets containing
heated fats. Female rats in all groups showed similar weight gains to
controls. Food intake of test groups was comparable or better than
controls. Haematologic tests gave similar values for test and control
groups with the exception of the 0.5% TBHQ/unheated fat male group at
three months, where the WBC was slightly elevated. This effect was not
noted at six months. Urinalysis, SGOT and SAP values of test and
control groups were comparable and within normal limits. Organ/body
weight ratios indicated a slight increase in ratios for testes and
livers of the male rats from 0.5% TBHQ/heated oil group and liver
weight ratio of the female rats of the 0.5% and 0.2% heated oil group.
These minor differences appeared to be related to heated versus
unheated fat rather than a compound effect. Histological studies did
not reveal any compound related effects (Terhaar & Krasavage, 1968b).
Acute toxicity
LD50 References
Animal Route (mg/kg body weight)
955 (10% in corn oil)
Rat (fed) Oral 890 ( 5% in corn oil) Terhaar et al., 1968a
756 (10% in corn oil)
Rat (fasted) Oral 802 ( 5% in corn oil) Terhaar et al., 1968a
Mice (fasted) Oral 1 260 Terhaar et al., 1968a
Guinea-pig Oral 790 Terhaar et al., 1968a
Dog Oral <400 (consistently Terhaar et al., 1968a
regurgitated)
Short-term studies
Rat
Rats were injected (intraperitoneally) with 200 mg/kg TBHQ daily
for one month without mortality but some loss of weight. At 100 mg/kg
(intraperitoneally) daily for one month, there was no loss of weight.
No histopathologic changes occurred in either case. Rats were
maintained for 22 days on a diet containing 1% TBHQ. Initial rejection
of food was followed by near normal food intake and growth curve
paralleling control group. There were no mortalities, nor was there
any gross or microscopic pathology (Fassett et al., 1968).
Long-term studies
Rat
Groups of each of 100 albino rats (A & C Farms, Altamont, N.Y.)
equally divided by sex were maintained on diets containing 0, 0.016,
0.05, 0.16 and 0.5% TBHQ for 20 months. Animals were observed for
changes in appearance and behaviour. Body weight was reported at
approximately 14-day intervals and group food intake at approximately
23-day intervals. Haematology was carried out at three, six, 12 and
20 months on 10 rats (five of each sex) from each of the 0, 0.16 and
0.5% level groups. Haematological tests consisted of haemoglobin,
PCV, WBC and differentials. Clinical chemical tests and urinalysis
were carried out on the same groups of animals at six, 12 and 20
months. The clinical chemical tests consisted of SGPT at six months,
and SGPT, SGOT and SAP at 12 and 20 months. Urinalysis consisted
of SpGr, albumin, sugar and appearance. At six and 12 months,
approximately 20 rats (10 of each sex) of each group were sacrificed
and at 20 months all surviving animals were sacrificed and autopsied.
Organ weights were determined for liver, adrenal, kidney, spleen,
heart, brain, lung and testes. Haematological studies were made on
trachea, lung, heart, oesophagus, stomach, small intestine, large
intestine, liver, kidney, urinary bladder, adrenal, pancreas, thyroid,
ovary or testes, uterus, spleen, femoral bone marrow, cerebrum,
cerebellum and eye. There were no adverse changes in appearance and
behaviour of the rats during the test period. Mortalities occurred
with equal frequency in all groups and were particularly heavy during
the 12 to 20-month period. Deaths did not appear to be compound
related. Growth rate, food intake and feed efficiency were comparable
for all groups during the experimental period. Haematologic,
biochemical tests and urinalysis of test and control groups were
similar and within normal limits. Although there were some decreases
in the absolute organ weight of spleen and brain of males of the 0.16
and 0.05 group at 20 months, these were not significantly different
from control when expressed as organ/body weight ratio. No gross or
microscopic lesions that could be attributed to the test compound were
detected (Terhaar et al., 1968b).
Dog
Four groups each of eight pure bred beagle dogs (four male, four
female), approximately six to eight months of age were used. The dogs
were maintained on a commercial diet to which 6% cottonseed oil was
added. TBHQ was added to the test diets at levels equivalent to 500,
1500 and 5000 ppm. Diets were available ad lib for one hour, six
days/week. Water was available ad lib at all times. The dogs were
housed individually.
Daily inspection was made for appearance, behaviour, survival and
physical signs. Body weight was determined weekly for first 12 weeks,
and thereafter biweekly. Food intake was determined weekly during the
first 12 weeks, and thereafter periodically. Complete physical
examinations were conducted at various times during the test period.
Haematological and biochemical studies and urine analyses were made
twice before commencement of the feeding study, and at weeks 12, 26,
52, 78 and 104.
Haematologic studies consisted of haemoglobin conc., haematology,
RBC, WBC and differentials. Biochemical studies consisted of serum
BUN, glucose, LDM, SAP and SGPT and bilirubin at week 104 only.
Urinalysis consisted of pH, albumin, glucose, ketone bodies and occult
blood. Because some minor abnormalities were noted in the haematology,
the test period was increased to 117 weeks to permit additional
observations. At week 108, peripheral blood samples were taken. At
week 108, TBHQ was withdrawn from the diet of two dogs of the high
level group. The animals were maintained in metabolism cages and
24-hour urine samples were collected daily. Blood samples were
collected on days one, two, three, four, seven, 10 and 13 of this
period. An interim sacrifice of one male and one female of each test
group was made at one year. The remaining animals were sacrificed at
week 117. At autopsy, animals were examined for gross pathological
changes. The liver, kidneys, spleen, heart, brain, lungs, gonads,
adrenals, thyroid and pituitary of all dogs were weighed. Tissues from
the following organs of all dogs of control and high level groups were
examined microscopically: liver, spleen, gallbladder, stomach, small
and large intestines, pancreas, kidneys, urinary bladder, adrenals,
gonads and adnexa, pituitary, thymus, thyroid, salivary glands, lymph
nodes, heart, lungs, marrow, aorta, skin, muscle, spinal cord and
brain. The liver, stomach, small and large intestines and kidneys of
all dogs on low-and mid-level test diets were also examined
microscopically. In addition, specimens of liver and kidney tissues
were prepared for electron microscopy. No deaths occurred during the
test period. Behaviour and appearance was normal at all times and
physical examinations did not reveal any treatment related problems.
Growth and food consumption was similar for control and test groups
except at the beginning of the test when dogs were adjusting to the
test diet. Biochemical studies and urinalysis showed variations within
animals and groups, but none of these differences appeared to be
compound related effects. Haematologic studies showed variable effects
in the high level group. RBC were slightly lower in both male and
female dogs than their respective controls. These shifts were also
reflected in the haemoglobin conc. and haematocrits of some animals.
At week 99, and a subsequent test period there was a slight deviation
of reticulocytes in the high level groups. Peripheral blood smears
also showed more normoblasts as well as occasional increase in
erythrocyte basophilia. These effects were not observed in the lower
level groups. Organ weight and gross pathology and histopathology
failed to reveal any compound related changes. Electron microscopy of
liver and kidney showed normal cellular constituents in test animals.
There was no increase in the endoplasmic reticulum in liver cells of
treated animals (Anonymous, 1968b).
OBSERVATIONS IN MAN
See "Biochemical aspects".
Comments:
Metabolic studies in rats indicate >90% of an ingested dose of
TBHQ is absorbed. Excretion is rapid, primarily as the o-sulfate. At
higher levels of intake there is an increasing urinary excretion of
the o-glucuronide. About 10% of the urinary excretion is unchanged
TBHQ. Even after chronic administration of 0.5% TBHQ in the diet, less
than 1 ppm is stored in any tissue and the oxidative stability of the
fat of chronically fed animals does not differ from that of controls.
TBHQ is shown to pass across the placenta with about 0.2% of the
chronically administered dose remaining in the foetus.
In dog the metabolic, excretory and storage patterns were
qualitatively similar to rats, except that a greater percentage of the
o-glucuronide was excreted in the case of males, and a greater amount
was stored in fat. Storage in fat was two times greater for females
than males.
In humans dosed with TBHQ absorption by capsule resulted in
22-40% absorption, whereas absorption approached 100% when
incorporated into food. No free TBHQ was excreted in the urine.
Urinary excretion was as the o-sulfate or o-glucuronide in a ratio of
3:1. Excretion was almost complete in 24 hours and studies of
haematology, urine analysis, blood chemistry and clinical signs showed
no effects due to administration of TBHQ.
In a 20-month study in rats fed TBHQ in their diet there were no
adverse effects noted.
In a 117-week study dogs were given TBHQ in their diets.
Parameters studied were similar to those for rats and in addition,
electron microscopy of liver and kidney were performed. Aside from a
slight decrease in RBC, haemoglobin and haematocrits, more normoblasts
and occasional increases in erythrocyte basophilia, seen at the
highest dose, there were no TBHQ-related effects observed.
A three-generation reproduction study including an "a" and "b"
littering, done in rats, showed scattered incidents of increased
deaths from birth to weaning and deaths from weaning to five weeks
were more frequent in the TBHQ-fed group. No other abnormalities were
noted in the fed animals as compared to the controls.
In a cross-over study with rats fed TBHQ, pups from untreated
parents nursed with TBHQ-treated parents gained less weight than the
corresponding cross-over, suggesting that diet rejection is the major
factor rather than an organic deficiency in pups from TBHQ-fed
parents.
A teratology study in rats showed no abnormal effects.
In a study designed to demonstrate the safety of reacted TBHQ,
rats were fed TBHQ in oils that had been heated for four hours at
375°F for six months. Groups of rats were fed the same diets using
unheated oils. Except for a slight depression in weight gain in the
high level rats fed the 0.5% TBHQ, there were no deleterious compound
related effects noted.
Enzyme induction experiments were done with rat and dog.
Glucose-6-phosphatase, paranitroanisole demethylase (pNaD) and aniline
hydroxylase (AHase) activities in liver microsomes were studied. In
dog no induction effects were noted. In rat and dog heated oil had no
effect on enzyme activities. In rat induction of pNaD and AHase were
noted. There was a non-dose related elevation in G-6-P that is
probably artifact. Chronic administration did not alter the picture in
the rat. Since there was no change in enzyme induction seen in
chronically exposed as opposed to acutely exposed rats, it is
reasonable to conclude the enzyme induction activity noted for TBHQ is
freely reversible.
In the seventeenth report of the Joint FAO/WHO Expert Committee,
reference was made to recent evidence indicating possible effects on
reproduction in the rat when butylated hydroxyanisole, alone or with
propylgallate, was mixed with lard in the diet. The Committee, noting
that TBHQ was chemically similar to both BHA and BHT, concluded that
the same type of data should be forthcoming for this compound.
EVALUATION
Level causing no toxicological effect in the dog
3000 ppm in the diet equivalent to 75 mg/kg body weight.
Estimate of acceptable daily intake for man
0-0.75 mg/kg body weight.*
FURTHER WORK OR INFORMATION
Required by 1978.
Appropriate studies on reproduction using tertiary butyl
hydroquinone in mixtures with propylgallates.
* Temporary.
REFERENCES
Anonymous (1968a) Determination of the stability and tertiary butyl
hydroquinone content of rat and dog fat extracts. Unpublished
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Anonymous (1968b) Two-year chronic feeding studies with tertiary butyl
hydroquinone (TBHQ) in dogs. Unpublished report from the Food and
Drug Research Labs, Inc. submitted to the World Health
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Astill, B. D., Blakeley, R. V. & Cantor, E. E. (1967a) The metabolic
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Unpublished report of the Biochemical Laboratory, Eastman Kodak,
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Astill, B. D. Cantor, E. E. & McEwan, D. B. (1967b) Long-term feedings
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Astill, B. D., Cantor, E. E., Ely, T. S., Jones, W. H. & Uskavitch,
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Unpublished report from the Laboratory of Industrial Medicine,
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Terhaar, C. J. & Krasavage, W. J. (1968b) Monotertiary butyl
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Terhaar, C. J., Krasavage, W. J., Wolf, G. L. & Leonard, W. J. (1968b)
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See Also:
Toxicological Abbreviations