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    BUTYLATED HYDROXYTOLUENE (BHT)

    EXPLANATION

         This substance was evaluated for acceptable daily intake for man
    by the Joint FAO/WHO Expert Committee on Food Additives at its sixth,
    eighth, ninth, seventeenth, twentieth, twenty-fourth, and twenty-seven
    meetings (Annex I, references 6, 8, 11, 32, 41, 53, and 62).
    Toxicological monographs or monographs addends were published after
    each of these meetings (Annex I, references 6, 9, 12, 33, 42, 54, and
    63).

         Since the previous evaluations, additional data, including the
    results of studies requested by the twenty-seventh Committee (Annex 1,
    reference 62), have become available and are summarized and discussed
    in the following monograph addendum.

    BIOLOGICAL DATA

    Biochemical aspects

    Metabolism

         A single oral dose of 14C-BHT given to male and female mice
    resulted in rapid absorption and distribution of 14C to the tissues.
    Excretion of 14C was mainly in the faeces (41-65%) and urine
    (26-50%), with lesser amounts in expired air (6-9%). The half-life for
    a single dose in the major tissues studied (stomach, intestines,
    liver, and kidney) was 9-11 hrs. When daily doses were given for 10
    days, the half-life for 14C in the tissues examined was 5-15 days.
    Metabolism was characterized by oxidation at one or both of the
    tert-butyl groups, followed by formation of the glucuronide
    conjugate, and excretion in the urine, or by excretion of the free
    acid in the faeces. When 14C-BHT was administered to rats, 80-90% of
    the 14C was excreted in the urine and faeces within 96 hrs, and less
    than 0.3% of the 14C was in expired air. Most of the 14C-BHT was
    excreted as the free acid in faeces with lesser amounts in the urine.
    More than 43 metabolites were present in the urine and faeces of mice
    and rats (Matuso, 1984).

         Rats dosed with 14C-aflatoxin B1 and fed BHT (0.5% in the
    diet) showed an enhanced excretion of water-soluble metabolites of
    14C-aflatoxin B1 in the urine and faeces. In addition, BHT
    pre-treatment was shown to decrease the amount of 14C bound to
    hepatic nuclear DNA (Fukayama & Hsieh, 1985).

    Effect on enzymes

         Dietary BHT (300-6000 ppm) caused a dose-dependent increase in
    gamma-glutamyl transpeptidase in normal F-344 male rats. However,
    cytosolic glutathione S-transferase was only enhanced at dietary
    concentrations of 3000 or 6000 ppm BHT (Furukawa et al., 1984).

         Dietary administration of BHT to male Swiss Webster mice resulted
    in a marked increase in hepatic microsomal epoxide hydrolase and
    glutathione-S-transferase (Hammock & Ota, 1983).

    Toxicological studies

    Special studies on carcinogenicity

    Mice

         Groups of male and female C3H mice (ranging from 17-39
    mice/group), which were 6-10 weeks old at the beginning of the
    experiment, were maintained for 10 months on a semi-synthetic diet
    containing 0.05 or 0.5% BHT. Control groups were maintained on

    BHT-free semi-synthetic diet or commercial lab chow. At the end of the
    test period, the liver and lungs were excised and inspected grossly
    for proliferative lesions. Of the proliferative lesions considered to
    be clearly identifiable as tumours, approximately 50% were examined
    microscopically. Mice maintained on diets containing BHT had lower
    body weights than controls. Male mice fed BHT showed an increase in
    liver tumours, compared to controls. Histologically, the tumours were
    identified as hepatocellular adenomas. No increase was observed in
    female mice. The reported incidence of liver tumours in male C3H
    mice was 10/26 (38%), 15/26 (58%), 2/37 (5%) and 7/38 (18%) in the
    0.5% BHT, 0.05% BHT, control BHT-free, and control lab chow groups,
    respectively. In a study in which C3H mice were maintained on diets
    containing 0.5% BHT for one month followed by lab chow for 10 months,
    or control diet (BHT-free) for one month followed by lab chow for 10
    months, the incidences of liver tumours in the two groups of male mice
    were 3/35 (9%) and 5/29 (17%), respectively. Dietary BHT did not
    result in an increased incidence of lung tumours in either male or
    female C3H mice. In another study in which male BALB/c mice were
    maintained for one year on a BHT-free diet, or diets containing 0.05%
    BHT or 0.5% BHT, the incidences of liver tumours were 4/30 (131), 6/43
    (14%), and 2/28 (7%) for the respective groups
    (Lindenschmidt et al., 1986).

         It has previously been demonstrated that the incidence of
    spontaneously-occurring hepatic tumours in C3H mice is modified by
    sex, population density, level of dietary protein, and caloric intake.
    Historical data are not available for a 10-month study. The incidence
    of hepatic tumours in a 12-month study in this strain ranged from
    6-13% for females, and 41-68% for males. Thus, the reported incidence
    of hepatocellular tumours was not significantly different from other
    controls of similar age in studies with the same inbred strain
    (Peraino et al., 1973).

    Rats

         Groups of 60, 40, 40, and 60 Wistar rats of each sex
    (F0 generation) were fed a semi-synthetic diet containing 0, 25,
    100, or 500 mg BHT/kg b.w., respectively. The F0 rats were mated
    after 13 weeks of dosing. The F1 groups consisted of 100, 80, 80,
    and 100 F1 rats, respectively, of each sex from the offspring from
    each group. Because of an adverse effect on the kidney in the parents,
    the concentration of BHT in the highest-dose group was lowered to
    250 mg/kg b.w. in the F1 generation. The study was terminated when
    rats in the F1 generation were 144 weeks of age.

         Parameters studied were food consumption (weekly), body weight,
    appearance, and mortality. Autopsy and complete histopathological
    examinations were performed on all animals dying during the study, or
    sacrificed in extremus or at termination.

         The average body weights of the F1 pups at birth in the middle-
    and high-dose groups were slightly lower than those in the control
    group. Body weights of all dosed animals from weaning through the
    entire experiment were lower than those of control animals. In the
    low-, mid-, and high-dose groups, the reductions in body weight were
    7%, 11%, and 21% for males and 5%, 10%, and 16% for females. Food
    intake was comparable for all groups. Clinical appearance and
    behaviour were reported to be normal for all animals. The high-dose
    males voided a slightly reddish urine. Hematological parameters were
    reported to be unchanged by BHT treatment, but no data were given.
    Serum triglycerides were reduced in both sexes and cholesterol was
    somewhat elevated in females only.

         All animals consuming BHT had a dose-related increase in
    survival. In both sexes differences (p < 0.001) in longevity were
    seen. Histological studies indicated an increase in hepatocellular
    carcinomas in male rats (not females) and an increase in
    hepatocellular adenomas in both male and female rats. Most liver
    tumours were found during terminal sacrifice at 141-144 weeks. One
    hepatocellular carcinoma was found in a control male at 117 weeks and
    one in a high-dose male at 132 weeks. The remainder of the carcinomas
    occurred at terminal sacrifice. The first adenoma was noted in a
    high-dose male at 115 weeks. Tables 1 and 2 summarize the data on
    mortality and the appearance of adenomas and carcinomas of the liver.

         Data on mortality and tumour incidence in different groups were
    analysed using the procedure of Peto et al. (1980). The dose-related
    increases in the number of hepatocellular adenomas were statistically
    significant (at p < 0.05) in male F1 rats, when all groups were
    tested for heterogenicity or analysis of trend. The increase in
    hepatocellular adenomas and carcinomas in treated female F1 rats was
    statistically significant only for adenomas (at p < 0.05) in the
    analysis for trend.

         Reports on the spontaneous incidence of hepatocellular neoplasms
    in Wistar rats from the laboratory performing this study, as well as
    other European laboratories, indicate that it is usually less than 3%
    (Solleveld et al., 1984; Deerberg et al., 1980; Olsen et al.,
    1985). The median life span for animals in these studies ranged from
    28 months to 36 months for males and 28 months to 33 months for
    females.

         Other sites reported to have a slight but not statistically-
    significant increase in neoplastic lesions were as follows: thyroid,
    pancreas, ovary, uterus, thymus, reticuloendothelium system, and
    mammary gland.

         Non-neoplastic lesions occurred incidentally and showed no
    relationship to BHT treatment, with the exception of lesions of the
    liver, which showed a dose-related increased incidence of bile duct
    proliferation and cysts in males, and focal cellular enlargement in
    females.

         At the highest level fed (250 mg/kg b.w.), there was no adverse
    effect on the kidney (Olsen et al., 1986).

    Special Studies on haemorrhagic effects

         Groups of 10 male Sprague-Dawley CLEA rats were fed diets
    containing 0.58, 0.69, 0.82, 1.00, 1.20, or 1.44% BHT for 40 days. A
    dose-related effect on mortality was observed, with 21/50 rats given
    0.69% or more BHT dying during the period from 9 to 37 days.
    Spontaneous massive haemorrhages were observed in these animals. The
    prothrombin index of survivors was decreased, which was dependent on
    the BHT dose. At the lowest level fed the decrease was approximately
    651 (Takahashi & Hiraga, 1978a).

         The LD50 (i.p.) for BHT showed considerable differences among
    strains of inbred and non-inbred male mice:

         Strain                   LD50 (mg/kg b.w.)

         DBA/2N (inbred)              138
         BALB/cNnN (inbred)          1739
         C57BL/6N (inbred)            917
         ICR-JCL (non-inbred)        1243

         In all cases, death occurred 4 to 6 days after administration of
    BHT, and was accompanied by massive oedema and haemorrhage in the lung
    (Kawano et al., 1981).

         In another study, a number of strains of rats (Sprague-Dawley,
    Wistar, Donryu and Fischer), mice (ICR, ddY, DBA/c, C3H/He, BALB/CaAn
    and C57BL/6), New Zealand white-sat rabbits, beagle dogs, and Japanese
    quail were fed diets containing BHT (1.2% of the diet for rats and
    mice; 1% of the diet for quail; 170 or 700 mg/kg b.w. for rabbits; and
    173, 400, or 760 mg/kg b.w. for dogs) for a period of 14-17 days.
    Haemorrhagic deaths occurred among male rats of all strains and female
    rats of the Fischer strain. Female rats of the Donryu and Sprague-
    Dawley strains showed no obvious haemorrhaging. No haemorrhagic
    effects were noted in rabits or dogs (Takahashi et al., 1980).

         Male albino rats (CRL COB CD(SD) BR) given 3 consecutive daily
    doses of 380, 760, or 1520 mg BHT/kg b.w./day showed no evidence of
    haemorrhage. However, BHT produced a dose-dependent increase in
    prothrombin time, with no effect on prothrombin time seen in the
    380 mg/kg b.w. animals (Krasavage, 1984).


        Table 1.  Mortality (and combined adenomas and carcinomas of the Liver) in F1 rats
                                                                                                                                              

    BHT         Effective
    (mg/kg      number of                                     Number of deaths during weeksa
    b.w.)       rats                                                                                                                          

                             0-90       91-104     105-113    114-118    119-126    127-132    133-140    141-144    Total
                                                                                                                                              

    MALES
    0           100          20         10         13         (1)/8      11         10         (1)/12     16         (2)/100
    25          80           8          11         6          3          (1)/13     11         8          20         (1)/80
    100         80           8          12         3          2          10         7          (2)/11     (4)/27     (6)/80
    250         99           7          7          6          (1)/4      (2)/8      (2)/13     (1)/10     (20)/44    (26)/99

    FEMALES
    0           100          16         15         18         (2)/8      11         7          8          17         (2)/100
    25          79           10         9          4          6          13         (1)/10     (1)/8      (1)/19     (3)/79
    100         80           5          17         5          5          (1)/7      41)/9      (1)/11     (3)/21     (6)/80
    250         99           9          5          11         12         8          5          (2)/10     (12)/39    (14)/99
                                                                                                                                              

    a    Figures in parenthesis are combined adenomas and carcinomas occurring during that period.
    
    Table 2.  Incidences of hepatocellular nodular hyperplasia, adenomas,
              and carcinomas
                                                                        

    BHT            Effective number   Nodular
    (mg/kg b.w.)   of rats            hyperplasia   Adenoma  Carcinoma
                                                                        

    MALES
    0               100                2              1          1
    25              80                 0              1          0
    100             80                 2              5          1
    250             99                 2              18a        8b

    FEMALES
    0               100                2              2          0
    25              79                 0              3          0
    100             80                 4              6          0
    250             99                 5              12c        2d
                                                                        

    a    Over-all test for heterogeneity, p< 0.001,
         chi-square = 18.17, 3 df.
         Test for trend, p< 0.001, chi-square = 17.97, 1 df.
    b    Over-all teat for heterogeneity, p< 0.05,
         chi-square = 11.12, 3 df.
         Test for trend, p< 0.01, chi-square = 9.40, 1 df.
    c    Over-all test for heterogeneity, not significant,
         chi-square = 5.20, 3 df.
         Test for trend, p< 0.05, chi-square = 4.99, 1 df.
    d    Over-all test for heterogeneity, not significant,
         chi-square = 2.87, 3 df.
         Test for trend, set significant, chi-square = 2.59, 1 df.

         Male Sprague-Dawley CLEA rats were maintained on diets containing
    levels of 85, 170, 330, 650, 1300, 2500, or 5000 ppm BHT for 1 to 4
    weeks. A significant decrease in the prothrombin index was observed at
    week 1 in all groups fed BHT at levels of 170 ppm or more. However,
    when the rats were maintained on the test diets for 4 weeks, a
    significant decrease in the prothrombin index was observed only in the
    5000 ppm group. This was the only group which showed an increase in
    relative liver weights compared to those of the control group. In
    another study, haemorrhagic death, haemorrhage, and a decrease in
    prothrombin index in male Sprague-Dawley rats caused by 1.2% BHT were
    prevented by the simultaneous addition of 0.68 mole phylloquinone/kg
    b.w./day  Phylloquinone oxide also prevented hypoprothrombinemia due
    to BHT (Takshashi & Hiraga, 1979).

         Male Sprague-Dawley rats were fed diets containing 0 or 1.2% BHT
    for one week. BHT-treated rats showed haemorrhages in most organs.
    There was a significantly-increased leakage of Evans Blue into the
    epididymis. In addition, inhibition of ADP-induced platelet
    aggregation and decreased platelet factor 3 availability were
    observed. Plasma prothrombin factors were decreased, but fibronolytic
    activity was unchanged (Takahashi & Hiraga, 1981).

         Male rats receiving 0.25% dietary BHT for 2 weeks showed
    decreased concentrations of vitamin K in the liver and increased
    faecal excretion of vitamin K (Suzuki et al., 1984).

         Dietary BHT at a level of 1.2% was shown to effect platelet
    morphology (distribution width), and to cause changes in the fatty
    acid composition of the platelet lipids (Takahashi & Hiraga, 1984).

    Special studies on mutagenicity

         BHT was tested in four in vitro systems for genotoxicity: (1)
    Salmonella/microsome mutation test (4 dose levels between
    0.01-10 mg/plate) using 5 tester strains, with and without S-9
    activation; (2) hepatocyte primary culture/DNA repair test (10 dose
    levels ranging from 10-5 to 1 mg/ml); (3) adult rat liver epithelial
    cell/hypoxanthine guanine phosphoribosyl transferase mutagenesis using
    rat liver line 18 (6 doses ranging from 0.05 to 0.1 mg/ml); and (4)
    Chinese hamster ovary/sister chromatid exchange (SCE) assay (4 doses
    ranging from 10-5 to 1 mg/ml). BHT was negative in all tests
    (Williams et al., 1984).

    Special studies on potentiation or inhibition of carcinogenicity

    G.I. Tract

         Male F344 rats were treated with a single dose of
    N-methyl-N'-nitro-N-nitrosoguanidine, and then maintained on diets
    containing no BHT, 1.0% BHT, 5% NaCl, or 5% NaCl + 1.0% BHT for 51
    weeks. The incidences of squamous cell carcinomas of the forestomach
    in the respective groups were 11% in the control group, 15.8% in the
    BHT group, 3.0% in the NaCl group, and 52.9% in the BHT + NaCl group
    (Shirai et al., 1984).

         When rats were maintained on dietary 0.5% BHT for 36 weeks
    following 4 injections (1 per week) of 1,2-dimethylhydrazine, the
    presence of dietary BHT did not effect the number of rats with colon
    tumours, but the number of tumours per rat occurring in the distal
    colon was significantly decreased (Shirai et al., 1985).

         Wistar rats fed 1.0% BHT in the diet during treatment with
    N-methyl-N'-nitro-N-nitrosoguanidine (administered in drinking water
    at a concentration of 1.0 mg/ml) for 25 weeks, and then maintained on
    the test diet for another 14 weeks, showed a significant reduction in
    the incidence of gastric cancer, when compared to rats receiving
    BHT-free diets (82% versus 36.8%) (Tatsuta et al., 1983).

         Groups of male BALB/c mice treated intrarectally with
    methylnitrosurea, and then maintained on diets containing BHT, showed
    a marked increase in the incidence and multiplicity of G.I. tract
    tumours when compared to treated mice maintained on BHT-free diets. In
    another study, BALB/c mice were injected with dimethylhydrazine
    (6 weekly injections) and then maintained on control (BHT-free) diets
    or on diets containing 0.05% BHT or 0.5% BHT. The colon tumour
    incidences were 3/30 (10%), 0/43, and 9/28 (32%), in the respective
    groups (Lindenschmidt et al., 1986).

    Bladder

         Male F344 rats were treated with 0.01 or 0.05% N-butyl-N-
    (4-hydroxybutyl) nitrosamine (BBN) in drinking water for 4
    weeks, and then fed diets containing 0 or 1% BHT for 32 weeks. BHT in
    the diet was associated with a significant increase in the incidence
    of cancer and papilloma of the bladder of rats treated with 0.05% (but
    not 0.01%) BBN (Imaida et al., 1983).

         Rats were administered 200 ppm N-2-fluorenylacetamide (FAA) in
    the diet alone or with 6000 ppm BHT for 25 weeks. No bladder neoplasms
    resulted from feeding FAA alone, but the combination of FAA and BHT
    resulted in 17/41 papillomas and 3/41 carcinomas in the bladder
    (Williams et al., 1983).

         Four dietary levels of BHT (300, 1000, 3000, or 6000 ppm) were
    simultaneously fed with 200 ppm FAA for 25 weeks. FAA feeding alone
    produced no neoplasms, but when combined with BHT at 3000 or 6000 ppm,
    the incidences of bladder tumours were 18% and 44%, respectively. The
    incidence of bladder tumours in the 300 and 1000 ppm BHT groups was
    low and not significantly different from the incidence with FAA alone
    (see also effects on liver) (Maeura et al., 1984) 

         Male F344 rats were given injections of methylnitrosourea (MNU)
    twice a week for 4 weeks, and then a basal diet containing 11 BHT for
    32 weeks. Dietary BHT significantly increased the incidences per group
    and numbers per rat of papilloma and papillary or nodular hyplasia of
    the urinary bladder. The incidence of adenoma (but not adenocarcinoma)
    of the thyroid was also increased by treatment with HNU + BHT
    (Imaida et al., 1984).

    Liver

         Rats were administered 200 ppm FAA in the diet, alone or with
    6000 ppm BHT for 25 weeks. FAA alone induced a 1001 incidence of liver
    neoplasms. Simultaneous administration of BHT resulted in a decreased
    frequency of benign neoplasms, neoplastic nodules and malignant and
    hepatocellular carcinomas (Williams et al., 1983).

         Four dietary levels of BHT (300, 1000, 3000, or 6000 ppm) were
    fed simultaneously with 200 ppm FAA for 6, 12, 15, or 25 weeks. BHT
    produced a reduction in the incidence of tumours in a dose-dependent
    manner (100% incidence in the absence of BHT to 56% at 6000 ppm BHT)
    (see also effects on the bladder) (Maeura et al., 1984).

         Rats were fed 200 ppm FAA for 8 weeks, then diets containing BHT
    at levels of 300, 1000, 3000, or 6000 ppm for up to 22 weeks. The area
    of hepatocellular altered foci, identified by iron exclusion and
    gamma-glutamyl transferase (GGT) activity, that was induced by feeding
    the FAA, showed increased development at the highest dietary level of
    BHT (the number of foci, the area occupied by GGT-positive
    preneoplastic and neoplastic lesions, and the neoplasm incidence were
    increased). These parameters were unaffected at the lower BHT levels
    (Maeura & Williams, 1954).

         Rats were given a single i.p, injection of 200 mg/kg b.w. of
    diethylnitrosamine, and then maintained on diets containing 1% BHT for
    6 weeks. At week 3 the rats were subjected to partial hepatectomy. The
    number of gamma-glutamyl transpeptidase positive loci in the liver of
    BHT-fed rats was significantly decreased when compared to controls
    (Imaida et al., 1983).

    Mammary glands

         Female rats were fed diets containing 0, 0.25, or 0.5% BHT. The
    test diets were administered either (a) 2 weeks before until 1 week
    after 7,12-dimethylbenz(a)anthracene (DMBA) administration or (b) 1
    week after DMBA administration to the end of the study (30 weeks). The
    DMBA was administered as a single dose of 8 mg. BHT was an effective
    inhibitor of mammary carcinogenesis when administered during either of
    these time frames (20% by regime (a) and 50% by regime (b))
    (McCormick et al., 1984).

         In another study, dietary BHT was shown to decrease the incidence
    of mammary tumours induced in female Sprague-Dawley rats by DMBA, but
    it was without effect on animals treated with MNU (King et al.,
    1981).

         The inhibitory effects of BHT were strongly influenced by the
    dose of initiating carcinogen and the type of diet in which BHT was
    fed. Administration of BHT in the AIN-76A diet showed a markedly
    different effect from BHT in the NIH-07 diet. In the AIN-76A diet
    6000 ppm BHT had no effect on the incidence of mammary tumours induced
    by 15 mg DMBA, whereas a similar level of BHT in the NIB-07 diet
    resulted in a 40% inhibition of tumour development (Cohen et al.,
    1984).

    Pancreas

         Male LEW inbred rats were given an injection of 30 mg azaserine
    once a week for 3 weeks, and then maintained on diets containing 0 or
    0.45% BHT for 4 months. BHT treatment reduced the number of
    acidophilic foci per pancreas by 32%, but was without effect on focal
    size. BHT had no effect on the occurrence of basophilic foci
    (Roebuck et al., 1984).

    Special studies on potentiation or inhibition of mutagenesis

         The addition of 50-250 g of BHT/plate inhibited 3,2'-dimethyl-
    4-aminobiphenyl-induced mutagenicity in Salmonella typhimurium
    strains TA98 and TA100 in the presence of rat liver S-9 fraction (Reddy
    et al., 1983a).

         BHT was a moderately effective inhibitor of benzidine-induced
    mutagenicity in Salmonella typhimurium strain TA98, activated with a
    hamster liver S-9 fraction (Josephy et al., 1985).

         The addition of 100-250 g of BHT/plate was shown to inhibit
    3,2'-dimethyl-4-aminobiphenyl-induced mutagenicity in Salmonella
    typhimurium strains TA98 and TA100. Mutagenicity was further
    inhibited by use of S-9 preparations from rats fed dietary BHT (0.6%)
    as compared to S-9 preparation from rats fed BHT-free diets
    (Reddy et al., 1983b).

         The addition of BHT (5 to 20 g/plate) caused a two-fold increase
    in the mutagenic potency of aflatoxin B1 using Salmonella
    typhimurium strains TA98 and TA100, with and without activation
    (Shelef & Chin, 1980).

    Special studies on pulmonary toxicity

         In a study of lung toxicity in mice of BHT analogues, it was
    established that the structural feature essential for toxic activity
    is a phenolic ring structure having (i) a methyl group at the
    4-position and (ii) ortho-alkyl group(s) which can result in a
    moderate hindering effect of the hydroxyl group (Mizutani et al.,
    1982).

         In another study, the toxic potency of BHT in mice was decreased
    by deuteration of the 4-methyl group, suggesting that lung damage
    following administration of BHT is caused by the metabolite
    2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (Mizutani
    et al., 1983).

         Male mice given a single dose of BHT showed ultrastructural
    changes of the lung, which were characterized by selective destruction
    of type I epithelial cells, which were replaced by type II cuboidal
    cells. These changes were accompanied by a marked decrease in the
    number of peroxisomes, as well as catalase activity (Hirai et al.,
    1983).

         BHT was shown to enhance the lung tumour incidence in mice
    treated with doses of urethan greater than 50 mg/kg. At lower doses of
    urethan (subcarcinogenic doses) BHT did not enhance tumour
    development. In another study, it was shown that following treatment
    of mice with urethan, a two-week exposure to dietary 0.75% BHT was
    sufficient to enhance tumour development, and that 0.1% BHT was an
    effective enhancer when fed for 8 weeks. BHT, administered within 24
    hours post-treatment and fed for 8 weeks, enhanced tumour development
    in mice treated once with 3-methylcholanthrene, benzo(a)pyrene, or
    N-nitrosodimethylamine. When mice were injected weekly with BHT, there
    was a rapid increase in cell proliferation, and in both the cumulative
    labelling index (incorporation of 3H-thymidine) and the number of
    labelled type II cells. These effects were smaller after each
    injection, and by the fifth injection, no increase was observed
    (Witschi & Morse, 1985).

    Special study on reproduction

    Rats

         Groups of 60, 40, 40, and 60 Wistar rats of each sex, 17 weeks of
    age, were fed a semi-synthetic diet containing BHT so that the dietary
    intake was equivalent to 0, 25, 100, or 500 mg/kg b.w./day,
    respectively. After 13 weeks on the test diet the rats were mated.

         Food consumption was similar in all groups. Male and female rats
    in the high-dose group showed a significant decrease in body weight
    which persisted throughout the study. Gestation rate was similar for
    all test groups. The litter size and number of males per litter were
    significantly lower in the 500 mg/kg b.w. group than in the controls.
    Viability was similar in test groups and in the control group during
    the lactation period. The average birth weight of the pups in the
    500 mg/kg b.w./day and 100 mg/kg b.w./day groups was slightly lower
    than in the controls. During the lactation period, dietary BHT caused
    a significant dose-related lower body-weight gain (5%, 7%, and 41%
    lower body weight for the 25, 100, and 500 mg/kg b.w./day groups,
    respectively, as compared to controls) (Olsen et al., 1986).

    Comments

         The haemorrhagic effects of massive doses of BHT seed in certain
    strains of mice and rats, but not in dogs, may be related to its
    ability to interfere with vitamin K metabolism. However, in a
    susceptible strain of rat, the decrease in prothrombin index was shown
    to be transient. The effect can be reversed by feeding a vitamin K
    analogue. This suggests that the effect may be due to the presence of
    antivitamin K activity possibly associated with diets marginal in
    vitamin K. Additional studies will be required to elucidate the
    mechanism of the haemorrhagic effect.

         Additional studies have been carried out on the ability of BHT to
    modify the carcinogenicity of chemical agents. Enhancement or
    reduction of the incidence of tumours varies with the chemical
    administered, the time of addition of BHT to the diet, concentration
    of BHT in the diet, and also the organ site effected. For example, in
    the case of rats fed FAA concurrently with BHT, there was a
    significant increase in bladder tumours even when rats were exposed to
    subcarcinogenic levels of dietary FAA. However, under these conditions
    there was a reduction in the incidence of liver tumours. In both
    instances the effects were dose related, and the effects were only
    observed at high dietary levels of BHT. Dietary BHT also modified the
    carcinogenicity of chemicals causing tumours of the G.I. tract,
    mammary gland, and pancreas in experimental animals. The inhibitory
    effects of BHT may also be influenced by the type of diet in which it
    is fed, since in one study BHT was an effective inhibitor of
    DMBA-induced mammary tumours in rats when a NIH-07 diet was used, but
    was without effect when the rats were fed an AIN-76A diet. In another
    study on mice, conventional laboratory chow containing BHT reportedly
    produced a higher incidence of spontaneous-occurring tumours than did
    a BHT-free semi-synthetic diet.

         BHT was not mutagenic in a number of in vitro tests. However,
    BHT inhibited 3,2'-dimethyl-4-aminobiphenyl- and benzidine-induced
    mutagenicity in the Salmonella test.

         In a recent two-generation lifetime study in Wistar rats, BHT
    caused a dose-related increased number of hepatocellular adenomas and
    carcinomas. Most of the hepatocellular tumours were detected when the
    rats were more than two years old. Previously-reported single-
    generation carcinogenicity studies in Fischer 344 and Wistar
    rats with BHT were negative (Annex 1, references 51 and 63). The
    significance of the hepatocarcinogenicity of BHT in the recent studies
    to the toxicological evaluation raises a number of questions. These
    relate to the differences in design of the recent rat study and those
    previously reported to be negative, such as in utero exposure,
    duration of the study, and low body weights of the test animals. An
    elucidation of the reasons for the in utero effect is a priority.

         In a study with male and female C3H mice fed semi-synthetic
    diets containing BHT for 10 months there was a significant increase in
    the incidence of liver tumours in male but not female animals under
    the conditions of the study. This effect was not dose related, the
    lower dose showing a higher incidence of these tumours than the higher
    dose (58% versus 38%. In addition, the tumour incidence was not
    significantly different from other controls of similar age in studies
    with the same inbred strain and the reported increase of
    hepatocellular tumours (adenomas) appeared to be strain specific,
    since an increased incidence of liver tumours was not observed in male
    BALB/c mice fed diets containing 0.51 BHT for 12 months. In the
    previously-reported carcinogenicity studies in B6D3F1 mice, BHT was
    not carcinogenic under the conditions of the test (Annex 1, references
    54 and 63).

         In a one-generation reproduction study in rats, BHT had no effect
    on gestation rate, but showed a dose-related response in litter size,
    number of males per litter, and body-weight gain during the lactation
    period. The Committee based its evaluation on the no-effect level in
    this study.

    EVALUATION

    Level causing no toxicological effect

    Rat:      25 mg/kg b.w./day (based on one-generation reproduction
              study)

    Estimate of temporary acceptable daily intake for man

              0 - 0.125 mg/kg b.w.

    Further work or information

    Required by 1990

         1. Elucidation of in utero exposure on hepatocarcinogenicity of
    BHT in the rat.

         2. Studies on the mechanism of the haemorrhagic effect of BHT in
    susceptible species.

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    See Also:
       Toxicological Abbreviations
       Butylated hydroxytoluene (ICSC)
       Butylated hydroxytoluene (FAO Nutrition Meetings Report Series 38a)
       Butylated hydroxytoluene (FAO Nutrition Meetings Report Series 40abc)
       Butylated hydroxytoluene (WHO Food Additives Series 5)
       Butylated hydroxytoluene (WHO Food Additives Series 10)
       Butylated hydroxytoluene (WHO Food Additives Series 35)
       BUTYLATED HYDROXYTOLUENE (JECFA Evaluation)