First draft prepared by
    Ms Elizabeth Vavasour
    Toxicological Evaluation Division, Bureau of Chemical Safety
    Food Directorate, Health Protection Branch, Health Canada
    Ottawa, Ontario, Canada

    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Effects on enzymes and other biochemical parameters
    Toxicological studies
         Long-term toxicity studies
         Special studies on lung toxicity
         Special studies on carcinogenesis
         Special studies on tumour promotion
         Special studies on teratogenicity
         Special studies on genotoxicity
         Special studies on the renal pelvic epithelium
         Special studies on potentiation or inhibition of
         Observations in humans


          Tert-Butylhydroquinone (TBHQ) was previously evaluated by the
    Committee at the nineteenth, twenty-first, thirtieth and
    thirty-seventh meetings (Annex 1, references 38, 44, 73, 74 and 94).
    At the thirty-seventh meeting, the previously established temporary
    ADI of 0-0.2 per kg of body weight was extended, pending receipt of
    the results of ongoing long-term toxicity studies in rodents. This ADI
    was derived from a NOEL of 1500 mg/kg of feed (equivalent to 37.5/kg
    bw/day) in a 117-week feeding study in dogs on the basis of
    haematological abnormalities observed at the next highest dose level
    of 5000 mg/kg of feed (Annex 1, reference 39). In addition, the
    Committee requested clarification of the results of genotoxicity
    assays available at that time, which indicated that TBHQ was
    clastogenic in both  in vitro and  in vivo assays, but was
    apparently devoid of activity in bacterial mutagenicity assays. At its
    present meeting, the Committee reviewed the results of all available
    genotoxicity assays with TBHQ, in addition to some new studies on its
    metabolism and disposition, its effects on the renal pelvic
    epithelium, and its role in the promotion/inhibition of cancer. The
    final results of long-term studies in mice and rats were not available
    for review.

         This monograph addendum summarizes the information on TBHQ that
    has become available since the previous evaluation, including the
    information evaluated by the thirty-seventh meeting.


    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Oral doses of 4 ml of 0.01%, 0.1% or 1.0% butylated
    hydroxyanisole (BHA) were administered to male F344 rats. After
    3 h, the concentrations of  tert-butylquinone (TBQ) (the oxidation
    product of TBHQ) detected by HPLC in the forestomach mucosa were
    0.0045, 0.045, and 0.0552 µg TBQ/animal, compared to 1.8, 18.8 and
    216.3 µg BHA/animal, respectively (Morimoto  et al., 1991).

         TBHQ was not detected in homogenates of forestomach mucosa from
    male F344 rats which had received 4 ml of 0.01% - 2.0% [14C]BHA.
    Forestomach homogenates were therefore treated with sodium dodecyl
    sulfate in order to reduce TBQ to TBHQ, in which form it could be more
    easily measured. The TBHQ content thus generated in the forestomach
    homogenates was proportional to the dose of BHA. The ratios of the
    total tissue content of TBHQ to the total amount of covalent binding
    of 14C in forestomach were 0.010.03%, at p.o. BHA doses of 0.1-2.0%.
    The authors concluded that the covalent binding level was an important
    indicator of reactive metabolites of BHA (Morimoto  et al., 1992).

    2.1.2  Biotransformation

         Following the administration i.p. of 400 mg/kg bw BHA or
    200 mg/kg bw TBHQ to male Wistar rats, two previously undocumented
    metabolites, 3- tert-butyl-5-methylthiohydroquinone (TBHQ-5-SMe) and
    3- tert-butyl-6-methyl-thiohydroquinone (TBHQ-6-SMe), were detected
    in the urine using GC-MS. The authors suggested that these metabolites
    resulted from the metabolic conversion of glutathione conjugates of a
    quinone or semiquinone form of TBHQ. In rat liver microsomal
    preparations, the formation of two GSH conjugates at the 5- and 6-
    positions of TBHQ in the presence of an NADPH-generating system,
    molecular oxygen and GSH was confirmed. It appeared that glutathione
    S-transferase was not required for the reaction. Also, while
    inhibitors of cytochrome P-450 markedly reduced formation of TBHQ-GSH
    conjugates, indicating its role in the activation of TBHQ to TBQ,
    autooxidation was also shown to play a partial role in this reaction
    (Tajima  et al., 1991).

         Benzylthiol derivatives synthesized from TBQ had higher first
    reduction potentials than the parent compound. The authors concluded
    that TBQ maintained its potential for the generation of active oxygen
    species even after its addition to cellular thiols (Morimoto  et al.,

         The  tert-butyl semiquinone radical was shown to be formed from
    TBHQ in aerobic rat liver microsomes in the presence of NADPH. A
    concentration of 500 µM TBHQ and 5µM TBQ produced similar reductions
    in SOD-inhibitable cytochrome c which was used as an indication of
    excess superoxide anion radical production. The authors concluded that
    autooxidation of the semiquinone formed from the quinone was
    responsible for superoxide formation and that the hydroquinone entered
    the redox cycle via autooxidation. TBQ, but not TBHQ, induced toxic
    injury to rat hepatocyte plasma membrane as indicated by LDH release
    into the culture medium. The authors speculated that semiquinone-
    dependent superoxide formation was responsible for the toxic action
    (Bergman  et al., 1992).

         Incubation of TBHQ with horseradish peroxidase and hydrogen
    peroxide resulted in its rapid oxidation to TBQ. TBQ-epoxide was also
    produced at concentrations of 2.5 mM and higher of hydrogen peroxide.
    The presence of horseradish peroxidase was not a requirement for the
    production of TBQ-epoxide from TBQ (Tajima  et al., 1992).

         Three glutathione conjugates were generated by the incubation of
    TBHQ with glutathione; two of these were monoconjugates at the 5 or 6
    positions ( tert-butyl group at position 2) and one was a 5,6
    diconjugate. The redox potentials for the conjugates were twice those
    for the unconjugated hydroquinone. The monoconjugates showed an
    approximately 10-fold increase in redox cycling activity (oxygen
    consumption in the presence of a reducing agent) compared with TBHQ,
    whereas the diconjugate showed a 2-fold increase compared with TBHQ
    None of the major glutathione S-transferase isoenzymes were required
    for the formation of glutathione conjugates from TBHQ (van Ommen
     et al., 1992).

         Incubation of TBHQ in phosphate-buffered saline resulted in the
    generation of the semiquinone radical through autooxidation,
    accompanied by the formation of superoxide anion, hydroxyl radical and
    hydrogen peroxide as detected by electron spin resonance (ESR)
    spectroscopy. The addition of prostaglandin H synthase resulted in a
    substantial increase of semiquinone production with concomitant
    production of reactive species. Under the conditions of the assay,
    lipoxygenase had no effect on the formation of the semiquinone. The
    presence of either prostaglandin H synthase or lipoxygenase was found
    to substantially accelerate the metabolism of TBHQ to TBQ compared
    with the rates of autooxidation. In an  in vivo study, male Wistar
    rats were fed diets containing 1.5% BHA for 14 days, with concurrent
    administration of prostaglandin H synthase inhibitors acetylsalicylic
    acid (0.2%) or indomethacin (0.002%) in the drinking-water. Both
    agents produced a significant decrease in the amount of TBQ excreted
    into the urine compared with controls receiving drinking-water only,
    while the urinary excretion of BHA and its metabolites, TBHQ and TBQ,

    was not different between groups (46.9%, 45.4% and 43.5% of the
    ingested dose during urine collection in the control, indomethacin and
    acetylsalicylic acid groups, respectively). The results suggested an
     in vivo role for prostaglandin H synthase in the metabolism of TBHQ
    to TBQ (Schilderman  et al., 1993a).

    2.1.3  Effects on enzymes and other biochemical parameters

         Groups of 50 rainbow trouts were fed 0 or 5.6 mmol/kg of diet of
    TBHQ (0.1%), BHT, BHA, or ethoxyquin for 6 weeks. The treated trouts
    had reduced liver weight/body weight ratios. Compared to controls,
    TBHQ-treatment led to a decrease in hepatic microsomal protein and
    cytochrome P-450 contents, and nitroanisole-O-demethylase activity. In
    contrast, the hepatic activities of benzo-[a]-pyrene hydroxylase,
    epoxide hydratase, ethoxycoumarin-O-deethylase, and NADPH-cytochrome
    c-reductase were elevated following TBHQ-treatment (Eisele  et al.,

         TBHQ was found to be an inducer of quinone reductase in cell
    mutants and mouse strains which lack the Ah-receptor. In these target
    tissues, agents that induce both phase I and phase II enzymes (such as
    polycyclic aromatics) were ineffective (Prochaska, 1987).

         TBHQ was found to stimulate the production of superoxide,
    hydrogen peroxide, and hydroxyl radicals in microsomes from rat liver
    and forestomach. The oxidation product of TBHQ,  tert-butylquinone,
    exceeded TBHQ in its capacity to induce oxygen radical formation
    (Kahl  et al., 1989).

         TBHQ (30µM) was found to induce quinone reductase activity in
    cultures of bone marrow stromal cells from C57BL/6 and DBA/2 mice
    (Twerdok & Trush, 1990).

         Hepatocytes were isolated from male F344 rats and incubated at
    37°C at a concentration of 106 cells/ml with 0.5mM TBHQ. Incubation
    with TBHQ resulted in 100% cell death between 1 and 2 h following its
    addition to the medium. A drop in intracellular GSH to undetectable
    levels was observed in the first hour, prior to the increase in cell
    death. Decreases in ATP and reduced protein thiol concentrations were
    observed concurrently with the increase in cell death. Although
    superoxide anion radicals were generated by autooxidation of TBHQ,
    intracellular malondialdehyde was not increased during the incubation
    period (Nakagawa & Moldéus, 1992).

    2.2  Toxicological studies

    2.2.1  Long-term toxicity studies  Dogs

         Groups of 8 pure bred beagle dogs (4/sex), approximately 6 to 8
    months of age were maintained on a commercial diet to which 6%
    cottonseed oil was added. TBHQ was added to the test diets at level of
    0, 500, 1500 or 5000 mg/kg of feed. Diets were available  ad lib for
    one hour, 6 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 the 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.

         Haematological studies consisted of haemoglobin concentration,
    haematology, RBC, WBC and differentials. Biochemical studies consisted
    of serum BUN, glucose, LDM, SAP, 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 1, 2, 3, 4, 7, 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 were normal at all times and physical examinations did not
    reveal any treatment-related problems. Growth and food consumption
    were 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 concentration and haematocrits of some animals. At week
    99, and a subsequent test period there was a slightly 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 (Eastman Chemical Products. 1968).

    2.2.2  Special studies on lung toxicity  Mice

         TBHQ was tested for its potential to produce lung damage in mice
    similar to that seen following administration of BHT. Groups of 10
    mice were given single intraperitoneal injections of 63, 125, 250, or
    500 mg/kg bw TBHQ, or 300, 625, or 1230 mg/kg bw BHT in corn oil.
    After 5 days, all animals were necropsied and the lungs were weighed
    and examined histomorphologically. TBHQ led to mortality at doses of
    125 mg/kg bw (4/10), 250 mg/kg bw (9/10), and 500 mg/kg bw (10/10).
    Two of the mice which received 1230 mg/kg bw BHT died before the end
    of the observation period. Body weights as well as absolute and
    relative organ weights were comparable in all groups. While BHT
    produced hyperplasia of pulmonary pneumocytes, TBHQ did not lead to
    any treatment-related lung lesions (Krasavage & O'Donoghue, 1984).

    2.2.3  Special studies on carcinogenesis  Rats

         In a study designed to investigate the hyperplastic activity of
    BHA and related phenols on rat forestomach, groups of 5-10 male and
    female rats were fed powdered diets containing BHA, BHT, TBHQ, or one
    of 8 structural analogs for 90 days. TBHQ added to the diet at a
    concentration of 2% led to brownish discolorations and mild
    hyperplasia of basal cells. The local basal cell hyperplasia did not
    tend to differentiate. The hyperplastic activity of TBHQ was however
    considerably lower than that of BHA (Altmann  et al., 1986).  Hamsters

         Syrian golden hamsters (16 males/group) were fed a powdered basal
    diet containing 0.5% TBHQ (purity > 98%) for 20 weeks. This dose is
    approximately one quarter of the LD50. The control group received a
    basal diet only while 12 other groups received diets containing one of
    12 other phenolic compounds in concentrations corresponding to one
    quarter of their respective LD50. At the end of the experiment,
    animals were killed and organs were fixed. Five sections each were cut
    from the anterior and posterior walls of the forestomach, 2 from the
    glandular stomach, and 4 from the urinary bladder. Tissues were
    processed for histopathology and auto-radiography. Analysis of the
    labelling index was made on 4000 cells of urinary bladder epithelium,
    3000 cells of pyloric gland epithelium (1000 cells each of fundic
    side, middle portion, and pyloric side) and 2000 basal cells of
    the forestomach epithelium. Unlike some of the other phenolic
    compounds, TBHQ did not induce hyperplasia or tumorous lesions of
    the forestomach, the glandular stomach or the urinary bladder.
    Furthermore, it did not increase the labelling index in the tissues
    investigated (Hirose  et al., 1986).

    2.2.4  Special studies on tumour promotion  Rats

         The promoting activity of TBHQ in urinary bladder carcinogenesis
    initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in male
    F344/Dulrj rats was examined and compared with the effect of
    alpha-tocopherol (TP) or propyl gallate (PG). Rats (6-week old) were
    treated with 0.05% BBN in drinking-water for 4 weeks. Groups of 20
    rats received thereafter control diet or diet containing 2.0% TBHQ,
    1.0% PG, or 0.4, 0.75, or 1.5% TP. After 36 weeks, the urinary
    bladders of all animals were examined histologically. The incidence of
    papillary or nodular hyperplasia was significantly higher in BBN
    +TBHQ-treated rats as compared to rats initiated with BBN but
    receiving control diet. However, there was no significant differences
    for papillomas or cancer. This indicated a weak promoting activity of
    TBHQ in BBN-initiated urinary bladder carcinogenesis. TP and PG were
    inactive in this respect (Tamano  et al., 1987).

         The effects of TBHQ and 7 other antioxidants on
    7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland,
    ear duct, and forestomach carcinogenesis were examined in female
    Sprague-Dawley rats. Groups of 20 rats were given a single dose of
    25 mg/kg bw of DMBA in 0.5 ml of sesame oil by stomach tubing at 50
    days of age. Starting one week later, rats were given a basal diet
    containing 0.8% of TBHQ or one of the other antioxidants for 51 weeks.
    Controls received basal diet only. Groups of 15 rats served as
    carcinogen-free controls and received the different diets without

    prior treatment with DMBA. Groups receiving antioxidants had reduced
    body weights at the end of the experiment. Histological examinations
    revealed a reduced rate of mammary tumour development in TBHQ-treated,
    and in DMBA-initiated rats as compared to DMBA-treated controls. The
    incidence of ear duct and forestomach tumours was not affected by TBHQ
    treatment (Hirose  et al., 1988).

         The modifying activities of BHA, BHT, and TBHQ and of paired
    combinations of these phenolic antioxidants on urinary bladder
    carcinogenesis in male F344 rats pretreated with BBN were
    investigated. Groups of 20 animals (6-weeks old) were given 0.05% BBN
    in their drinking-water for 4 weeks, followed by BHA, BHT, or TBHQ
    alone (0.8% each) or in pairs (0.4% each) in their diet for 32 weeks.
    Controls received no further treatment after BBN administration. A
    decrease in body-weight gain was observed in all antioxidant-treated
    groups. The incidence of preneoplastic papillary or nodular
    hyperplasia (PN hyperplasia) was slightly but significantly higher in
    the group treated with BHA+TBHQ after BBN than in controls receiving
    BBN only. The densities of PN hyperplasia were also significantly
    increased in all treated groups. However, no synergistic enhancing
    effects were observed. No differences were seen with respect to the
    incidence and densities of papillomas or carcinomas. Thus BHA, BHT,
    and TBHQ all exerted enhancing effects in BBN-induced urinary bladder
    carcinogenesis in rats, but no synergism regarding this promotion
    occurred (Hagiwara  et al., 1989).

         The effects of TBHQ on urine composition, bladder epithelial
    morphology, and DNA synthesis was studied in comparison with other
    antioxidants and bladder tumour promoters. Groups of 10 male Fischer
    344 rats, 5-week old, were given powdered basal diet containing 2%
    TBHQ or one of 11 other compounds, or basal diet only (controls); two
    further groups received two other tumour promoters in their
    drinking-water. Five rats in each group were killed after 4 weeks for
    estimation of DNA synthesis levels and histopathological examination
    by light microscopy. The remaining rats were killed at week 8 for
    light and scanning electron microscopic examination of the urinary
    bladder. During week 4, fresh urine specimens were obtained from rats
    in each group and analyzed for pH as well as electrolyte content. TBHQ
    brought about an elevation of DNA synthesis in the urothelium and
    produced morphological surface alterations such as the formation of
    pleomorphic or short, uniform microvilli and ropey or leafy
    microridges. The ability to induce proliferation and cell surface
    alterations was common to all bladder tumour promoters investigated.
    TBHQ also caused an increase in urinary pH, and a decrease in
    potassium and phosphate contents as well as in osmolality (Shibata
     et al., 1989).

    2.2.5  Special studies on teratogenicity  Rats

         Groups of 20 female Sprague-Dawley rats were fed basal diet
    containing 0, 0.125, 0.25, or 0.50% TBHQ from days 6 to 16 of
    gestation. During the mating period and on all other days of
    gestation, all treatment groups received control diet only. The
    experiment was terminated on day 20 of gestation. Total doses of 970,
    1880, or 3600 mg/kg bw/day TBHQ had no effect on the mean body-weight
    gain or feed consumption of the dams. The average number of corpora
    lutea, implantation sites, viable fetuses, resorptions, fetal body
    weights, and mortality did not differ between the control and
    treatment groups. A significant number of skeletal variations
    (rudimentary ribs) were seen in all groups, but the incidence of these
    variations was two times greater in the control group than in any
    treatment group. It was concluded that TBHQ was not teratogenic in
    rats at all doses employed (Krasavage, 1977).

    2.2.6  Special studies on genotoxicity

         The results of genotoxicity studies with TBHQ are summarized in
    Table 1.

         An alkaline elution assay was conducted with DNA extracted from
    forestomach epithelium of male F344 rats which had received an oral
    dose of 220 mg/kg bw BHA, 0.22 mg/kg bw TBHQ, 0.22 mg/kg bw TBQ, or
    0.0022 mg/kg bw TBQ and sacrificed 3 hours later. The BHA or TBHQ
    treatments caused no detectable DNA damage, but treatment with TBQ (an
    oxidation product of TBHQ) induced dose-related DNA damage. The
    elution rate at a dose of TBQ of 0.22 mg/kg bw was significantly
    higher than for BHA or controls and was similar to that induced by
    15 mg/kg bw MNNG in rat forestomach epithelial cells. The linearity of
    the elution pattern suggested that DNA damage was not due to cell
    necrosis at a dose of 0.22 mg/kg bw TBQ. TBQ appeared to be cytotoxic
    to the forestomach epithelial cells of rats at doses of 220 mg/kg bw
    (Morimoto  et al., 1991).

         The potential of BHA, TBHQ and TBQ to induce oxidative DNA damage
    were studied by measuring biological inactivation of single-stranded
    bacteriophage phiX-174 DNA and the formation of 7-hydro-8-oxo-2'-
    deoxyguanosine (8-oxodG) from dG by these compounds,  in vitro, in
    the absence and presence of peroxidases. TBHQ, but not BHA or TBQ,
    appeared to be a strong inducer of DNA damage as indicated by a strong
    inactivation of phage DNA and a potent induction of 8-oxodG formation.
    This damage was shown to be due to the formation of superoxide anion,
    hydrogen peroxide and hydroxyl radicals. The lack of activity of the
    quinone metabolite was attributed by the authors to a lack of
    cytochrome P-450 reductase  in vitro (Schilderman  et al., 1993b).

        Table 1.  Results of genotoxicity assays on TBHQ

    Test System            Test Object             Concentration of      Results            Reference

    Point mutation

    Ames test              S. typhimurium          < 50 µg/ml            Negative1          Société Kemin
                           TA98, TA100,            (TA1535)                                 Europa 1982a
                           TA1535, TA1537,         < 15 µg/ml
                           TA1538                  (TA1537)
                                                   < 50 µg/ml
                                                   (other strains)

    Ames test              S. typhimurium          < 450 µg/plate        Negative1          Mueller &
                           TA98, TA100,            (-S9)                                    Lockhart 1983
                           TA1535, TA1537          < 2700 µg/plate
                           TA1538                  (+S9)

    Ames test              S. typhimurium          1-1000 µg/plate       Negative1          Hageman et al.,
                           TA97, TA100,                                                     1988
                           TA102, TA104

    Ames test              S. typhimurium          3-3333 µg/plate       Negative2          NTP,
                           TA97, TA98,                                                      unpublished
                           TA100, TA102                                                     results

    Ames test              S. typhimurium          0-10 µg/plate         Negative1          Matsuoka et al.,
                           TA97, TA98,             (-S9)                                    1990
                           TA100, TA102            0.5-250 µg/plate

    Table 1 cont'd

    Test System            Test Object             Concentration of      Results            Reference

    Reverse mutation       Saccharomyces           100-500 µg/ml         Negative1          Rogers et al.,
    in yeast               cerevisiae D7           (-S9)                                    1992
                                                   50-200 µg/ml

    In vitro mammalian     Mouse lymphoma          < 31.3 µg/ml          Positive1          Litton Bionetics
    point mutation assay   cells line L5178Y                                                1982a

    In vitro mammalian     CHO cells/HGPRT         < 6 µg/ml (-S9)       Negative1          Beilman &
    point mutation         locus                   < 250 µg/ml                              Barber 1985
    assay                                          (+ S9)

    In vitro mammalian     Chinese hamster         0.17-3.40 µg/ml       Negative3,4        Rogers et al.,
    point mutation         V79 cells, HGPRT                                                 1992
    assay                  locus

    Clastogenic effects and chromosomal aberrations

    In vitro chromosomal   Chinese hamster V79     < 330 µg/ml           Positive1 (-S9     Societé Kemin
    aberration             cells                                         only)              Europa 1982b

    In vitro chromosomal   Chinese hamster         15-62 µmol/l          Positive1 (-S9     Phillips et al.,
    aberration             ovary cells                                   only)              1989

    Table 1 cont'd

    Test System            Test Object             Concentration of      Results            Reference

    In vitro               Chinese hamster         5-25.2 µg/ml          Positive1          NTP,
    chromosomal            ovary cells             (S9)                  (+S9 only)         unpublished
    aberration                                     100.5-300µg/ml                           results

    In vitro               Chinese hamster         12.5-50 µg/ml         Positive1,5        Matsuoka et al.,
    chromosomal            lung fibroblast         (-S9)                 (+S9 only)         1990
    aberration             cells                   20-40 µg/ml

    In vivo chromosomal    Mouse bone marrow       < 200 mg/kg bw        Negative           Litton Bionetics
    aberration assay                                                                        1985

    In vivo chromosomal    Mouse bone marrow       200 mg/kg i.p.        Positive           Giri et al., 1984

    In vivo chromosomal    Mouse bone marrow       2 x 30 mg/kg          Positive           Giri et al., 1984
    aberration                                     bw/day, p.o.

    Mouse micronucleus     Mouse bone marrow       162, 325, or 650      Negative           Litton Bionetics
    assay                                          mg/kg bw, p.o.                           1982b

    Mouse micronucleus     Mouse bone marrow       250 mg/kg bw,         Positive           Société Kemin
    assay                                          p.o.                                     Europa 1982c

    Table 1 cont'd

    Test System            Test Object             Concentration of      Results            Reference

    Dominant lethal        Sprague-Dawley          < 565 mg/kg           Negative           Krasavage &
    assay                  rats                    bw/day, 83 days                          Farber, 1983

    DNA interactions

    Mitotic gene           S. cerevisiae D7        100-500 µg/ml         Negative1          Rogers et al.,
    conversion                                     (-S9)                                    1992
                                                   50-200 µg/ml

    Sister chromatid       Chinese hamster         0.5-16.7 µg/ml        Positive1          NTP,
    exchange               ovary cells             (-S9)                 (+S9 only)         unpublished
                                                   5-166.7 µg/ml                            results
                                                   (+ S9)

    Sister chromatid       Chinese hamster         0.17-3.4 µg/ml        Negative3,4        Rogers et al.,
    exchange               V79 cells                                                        1992

    Sister chromatid       Mouse bone marrow       0.5 - 200 mg/kg       Positive           Mukerjee et al.,
    exchange                                       bw, i.p.                                 1989

    1   Both in the absence and presence of a metabolic activation system derived from rat liver S9 fraction.
    2   Both in the absence and presence of a metabolic activation system derived from rat or hamster liver S9
    3   Cultures with or without added rat or hamster hepatocytes.
    4   The highest dose tested did not achieve a 50% reduction in cloning efficiency.
    5   The oxidative metabolites of TBHQ, TBQ and TBQ-epoxide induced chromosomal aberrations in this system
        in the presence (TBQ and TBQ-epoxide) and absence (TBQ-epoxide) of S-9 metabolic activation
         TBHQ induced strand breaks in double stranded phiX-174 relaxed
    form I DNA in the presence of micromolar concentrations of copper. The
    induced DNA strand breaks were inhibited by a Cu(I) chelator or by
    catalase, indicating that a Cu(II)/Cu(I) redox cycle and H2O2
    generation were requirements for the observed DNA damage. The authors
    concluded that DNA-associated copper in cells may have the potential
    to activate phenolic compounds, producing reactive oxygen and
    electrophilic phenolic intermediates capable of inducing a spectrum of
    DNA lesions (Li & Trush, 1994).

    2.2.7  Special studies on the renal pelvic epithelium

         This study was performed to investigate early proliferation-
    related responses of the renal pelvic epithelium in response to
    bladder tumour promoters. Groups of 10 male F344 rats received control
    diet or 2% TBHQ. At week 4, the DNA-labelling index of the renal
    pelvic epithelial cells was determined from 1000 cells in 5
    rats/group. At week 8, kidney sections were prepared for SEM
    examination. At the end of the study, body weights of the treated
    animals were statistically significantly lower than controls as
    demonstrated previously by these investigators (Shibata  et al.,
    1989; 25% at 4 weeks and 15% at 8 weeks). The mean DNA labelling index
    in the renal pelvic epithelium after 4 weeks treatment was 10-fold
    higher than in controls, but without statistical significance. Slight
    cell surface alterations were observed by SEM after 8 weeks of
    treatment in some of the treated rats (Shibata  et al., 1991).

    2.2.8  Special studies on potentiation or inhibition of cancer

         The effects of dietary TBHQ were tested in a multi-organ
    carcinogenesis model. Groups of 20 male F344 rats were given a
    single intragastric administration of 100 mg/kg bw MNNG, a single
    intragastric administration of 750 mg/kg bw EHEN, 2 s.c. injections of
    0.5 mg/kg bw MBN and 4 s.c. injections of 40 mg/kg bw DMH. At the same
    time, the rats received 0.1% DBN for 4 weeks, followed by 0.1% DHPN
    for 2 weeks in the drinking-water for a total carcinogen exposure
    period of 6 weeks. Three days after this regime, the rats received in
    the diet 1% TBHQ or control diet for 36 weeks. Control groups of 10 or
    11 animals received 1% TBHQ alone or basal diet alone. Final body
    weights of both TBHQ-treated groups were significantly lower than
    those of respective controls (19% for carcinogen-treated control and
    9% for basal diet control) and this was reflected in higher relative
    liver and kidney weights. Dietary TBHQ following carcinogen treatment
    reduced the incidence and multiplicity of colon carcinomas and
    slightly reduced the incidence and multiplicity of some preneoplastic
    and neoplastic lesions of the kidney. At the same time, this treatment
    increased the incidence of oesophageal and forestomach papillomas and
    oesophageal papillary or nodular hyperplasia compared with controls
    and had no effect on tongue, glandular stomach, duodenum, small
    intestine, liver, lung, urinary bladder or thyroid gland (Hirose
     et al., 1993).

    2.3  Observations in humans

         No new information was available.


         TBHQ was shown to be oxidatively metabolized to  tert-
    butylquinone both enzymatically and by autoxidation. The results
    of a number of studies indicated that  tert-butylquinone
    participates in redox cycling through the formation of a semiquinone
    radical, such cycling being accompanied by the production of reactive
    oxygen species. Glutathione conjugates of  tert-butylquinone were
    also shown to have the potential to participate in redox cycling
    reactions, suggesting that reactive oxygen species might be generated
    even after covalent binding of  tert-butylquinone to cellular thiols.

         The results of bacterial mutagenicity studies available to the
    Committee were uniformly negative in both the presence and the absence
    of a rat liver metabolic activation system, as observed previously. In
    mammalian cell mutagenicity assays, TBHQ was positive in one study at
    the thymidine kinase (TK) locus and negative in two studies at the
    hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus. It was
    noted that the TK locus is responsive to reactive oxygen species and
    can respond to clastogens, while the HGPRT locus is considerably less
    sensitive. Clastogenicity  in vitro was demonstrated in four
    independent assays and  in vivo in one of two independent studies.
    Micronuclei were induced in mouse bone marrow in one of two  in vivo

         TBHQ caused DNA damage  in vitro as a result of the formation of
    reactive oxygen species, but did not do so in the rat forestomach
    after administration by gavage.  tert-Butylquinone, the oxidation
    product of TBHQ previously mentioned, did cause DNA damage in this
    system at a non-cytotoxic concentration. In this context, it has been
    shown that  tert-butylquinone is produced in the rat forestomach
    following the administration of butylated hydroxyanisole (BHA), TBHQ
    being formed as an intermediate.

         In a 117-week study, dogs were given TBHQ in their diets. Aside
    from a slight decrease in RBC, haemoglobin and haematocrits, more
    normoblasts and occasional increases in erythrocyte basophilia seen at
    the highest level of 5000 mg/kg of feed, there were no TBHQ-related
    effects observed. The NOEL in this study was 1500 mg/kg of feed,
    equivalent to 37.5 mg/kg bw/day. This NOEL was the basis of the
    temporary ADI established in 1975 (Annex 1, reference 39).


         The Committee extended the temporary ADI of 0-0.2 mg/kg bw. The
    final results of the long-term studies in mice and rats that are known
    to have been completed are required for review in 1997.


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    LITTON BIONETICS (1985). Mutagenicity evaluation of EK 81-0318 (TBHQ)
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    (1990). Mutagenicity of 3- tert-butyl-4-hydroxyanisole (BHA) and
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    KITSUTAKA, H. & TAKAHASHI, A. (1991). DNA damage in forestomach
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    MUKHERJEE, A., TALUKDER, G. & SHARMA, A. (1989). Sister chromatid
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    NAKAGAWA, Y. & MOLDÉUS, P. (1992). Cytotoxic effects of
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    SOCIÉTÉ KEMIN EUROPA (1982b). Recherche d'aberrations chromosomiques
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    SOCIÉTÉ KEMIN EUROPA (1982c). Recherche de l'éventuelle potentialité
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    Identification and structure characterization of S-containing
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    See Also:
       Toxicological Abbreviations