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    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 375F followed by four
    hours at 375F). 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
    375F 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
         technical report No. 495-F submitted to the World Health
         Organization by Eastman Chemical Products, Inc.

    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
         Organization by Eastman Chemical Products, Inc.

    Astill, B. D., Blakeley, R. V. & Cantor, E. E. (1967a) The metabolic
         fate of TBHQ in rats and dogs and of TBHQ-14C in rats.
         Unpublished report of the Biochemical Laboratory, Eastman Kodak,
         submitted to the World Health Organization by Eastman Chemical
         Products, Inc.

    Astill, B. D. Cantor, E. E. & McEwan, D. B. (1967b) Long-term feedings
         of TBHQ to rats and dogs: urinary conjugate excretions, serum
         TBHQ levels and autopsied fat analyses. Unpublished report from
         the Biochemistry Laboratory, Eastman Kodak, submitted to the
         World Health Organization by Eastman Chemical Products, Inc.

    Astill, B. D., Cantor, E. E., Ely, T. S., Jones, W. H. & Uskavitch,
         R. J. (1967c) The oral ingestion of t-butylhydro-quinone (TBHQ)
         by humans; clinical observations and metabolic fate. Unpublished
         report from the Laboratory of Industrial Medicine, Eastman Kodak,
         submitted to the World Health Organization by Eastman Chemical
         Products, Inc.

    Astill, B. D. & Walton, D. A. (1968) Distribution of radioactivity in
         pregnant rats receiving 14C-labeled TBHQ. Unpublished report
         from the Biochemistry Laboratory, Eastman Kodak, submitted to the
         World Health Organization by Eastman Chemical Products, Inc.

    Astill, B. D., Blakeley, R. V., Cantor, E. E., Tischer, K. S., Walton,
         D. A., McEwan, D. B., Jones, W. H. & Ely, T. S. (1968)
         Biochemical studies on Tert-Butylhydroquinone (TBHQ). A Summary.
         Unpublished report from the Laboratory of Industrial Medicine,
         Eastman Kodak, submitted to the World Health Organization by
         Eastman Chemical Products, Inc.

    Astill, B. D. & Jones, B. E. (1969) Long-term feedings of TBHQ to rats
         and dogs: levels of TBHQ in tissues and organs of autopsied
         animals. Unpublished report from the Biochemistry Laboratory,
         Eastman Kodak, submitted to the World Health Organization by
         Eastman Chemical Products, Inc.

    Fassett, D. W., Terhaar, C. J. & Astill, B. D. (1968) Summary of the
         safety evaluation of monotertiary butyl hydroquinone. Unpublished
         report from the Laboratory of Industrial Medicine, Eastman Kodak,
         submitted to the World Health Organization by Eastman Chemical
         Products, Inc.

    Krasavage, W. J. & Terhaar, C. J. (1970) The reproductive performance
         of rats fed monotertiary butyl hydroquinone: a single generation
         study. Unpublished report from the Toxicology Laboratory, Eastman
         Kodak, submitted to the World Health Organization by Eastman
         Chemical Products, Inc.

    Krasavage, W. J. (1973) A study of the teratogenic potential of
         monotertiary butyl hydroquinone in rats. Unpublished report from
         the Health and Safety Laboratory, Eastman Kodak, submitted to the
         World Health Organization by Eastman Chemical Products, Inc.

    Terhaar, C. J. & Krasavage, W. J. (1968a) The reproductive performance
         of rats fed monotertiary butyl hydroquinone. Unpublished report
         from the Toxicology Laboratory, Eastman Kodak, submitted to the
         World Health Organization by Eastman Chemical Products, Inc.

    Terhaar, C. J. & Krasavage, W. J. (1968b) Monotertiary butyl
         hydroquinone (TBHQ) in heated and unheated cottonseed oil. A
         six-month dietary feeding study in rats. Unpublished report from
         the Toxicology Laboratory, Eastman Kodak, submitted to the World
         Health Organization by Eastman Chemical Products, Inc.

    Terhaar, C. J., Vis, E. A. & Kesel, H. J. (1968a) Acute oral toxicity
         of monotertiary butyl hydroquinone (TBHQ). Unpublished report
         from the Laboratory of Industrial Medicine, Eastman Kodak,
         submitted to the World Health Organization by Eastman Chemical
         Products, Inc.

    Terhaar, C. J., Krasavage, W. J., Wolf, G. L. & Leonard, W. J. (1968b)
         Study of the tolerance of rats to monotertiary butyl hydroquinone
         in the diet. Final report at 20 months. Unpublished report from
         the Toxicology Laboratory, Eastman Kodak, submitted to the World
         Health Organization by Eastman Chemical Products, Inc.

    Tischer, K. S. & Walton, D. A. (1968) Dietary feeding of TBHQ and
         related compounds to rats and dogs: the response of liver
         processing enzymes and liver glucose-6-phosphatase activity.
         Unpublished report from the Biochemistry Laboratory, Eastman
         Kodak, submitted to the World Health Organization by Eastman
         Chemical Products, Inc.

    Wolf, G. L. & Fassett, D. W. (1968a) Electron microscope study of
         livers of rats fed monotertiary butyl hydroquinone. Unpublished
         report from the Laboratory of Industrial Medicine, Eastman Kodak,
         submitted to the World Health Organization by Eastman Chemical
         Products, Inc.

    Wolf, G. L. & Fassett, D. W. (1968b) Electron microscope study of
         livers of dogs fed monotertiary butyl hydroquinone for two years.
         Unpublished report from the Laboratory of Industrial Medicine,
         Eastman Kodak, submitted to the World Health Organization by
         Eastman Chemical Products, Inc.


    See Also:
       Toxicological Abbreviations