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Butylated hydroxyanisole (BHA; tert-butyl-4-hydroxyanisole)
and 
Butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-p-cresol)

in 
Food Antioxidants: Technological, Toxicological, and Health Perspectives
Edited by DL Madhavi, SS Deshpande, and DK Salunkhe / Dekker 1996 

Butylated hydroxyanisole (BHA; tert-butyl-4-hydroxyanisole)

Butylated hydroxyanisole (BHA; tert-butyl-4-hydroxyanisole) is perhaps the most extensively used antioxidant in the food industry. BHA is used in fats and oils, fat-containing foods, confectioneries, essential oils, food-coating materials, and waxes. BHA is a mixture of two isomers, 2-tert-butyl-4-hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA), with the commercial compound containing 90% of the 3-isomer (47). In a recent reevaluation, JECFA allocated an ADI of 0-0.5 mg/kg bw (48).

Table 5.5 Average Terminal Body Weight and Absolute and Relative Kidney Weights of Rats Fed Nordihydroguaiaretic Acid at 0-1 % of the Diet for 74 Weeksa

                                     Kidney weight      .
Dietary   Number  Terminal body  Absolute    Relative
level    of rats    weight (g)    (g)      (g/100 g bw)
Males
0           8         395         2.82        0.71
0.5         8         335**       2.75        0.82**
1.0         7         322**       2.65        0.82**
Females
0           6         270         2.31        0.86
0.5         9         238**       2.26        0.95*
1.0         9         230**       2.36        1.03*

aValues marked with asterisks differ significantly from those of 
  controls: *P = 0.07; **P < 0.01. 
 Source: Ref. 40.

TOXICOLOGICAL STUDIES

Absorption, Metabolism, and Excretion. The absorption and metabolism of BHA has been studied in rats, rabbits, dogs, monkeys, and humans. BHA was rapidly absorbed from the gastrointestinal tract in rats (49), rabbits (50), dogs, and humans (51), rapidly metabolized, and completely excreted. No evidence of tissue accumulation of BHA was observed in rats or dogs (49,52,53). The major metabolites of BHA were the glucuronides, ether sulfates, and free phenols (Fig. 5.5). The metabolites were excreted in the urine, and unchanged BHA was eliminated in the feces. The proportions of the different metabolites varied in different species and also for different dosage levels. In rabbits dosed orally with 1 g of BHA, 46% of glucuronides, 9% ether sulfates, and 6% free phenols were observed in the urine (50). In rats at lower doses, the metabolism was similar to that of rabbits. Urinary excretion was 86% by 24 h and 91 % in 4 days (54). In dogs, nearly 60% of a 350 mg/kg dose was excreted unchanged in the feces. The remaining was excreted as ether sulfate, tert-butyl hydroquinone (TBHQ), an unidentified phenol, and some glucuronides. In humans, 22-72% of an oral dose of BHA at levels of 0.5-0.7 mg/kg bw was recovered as glucuronides in 24 h, and less than 1 % free BHA and very little ether sulfates were observed (51). El-Rashidy and Niazi (55) reported substantial quantities of TBHQ glucuronide or sulfate as a metabolite of the 3-isomer. Later studies confirmed the formation of TBHQ in rats (56,57). Tissue retention of BHA was greater in humans than in rats (58). Much lower doses of BHA were required to produce a given plasma level in humans than in rats (56).

Fig. 5.5 Major metabolites of butylated hydroxyanisole.

Acute Toxicity. The acute oral LD50 was 2200-5000 mg/kb bw in rats and 2000 mg/kg bw in mice (3,6).

Short-Term Studies. Short-term studies have been conducted in a number of species including rats, rabbits, and dogs. Rats administered 500-600 mg/kg bw for a period of 10 weeks showed decreased growth rate and reduced activity of the enzymes catalase, peroxidase, and cholinesterase (59). In rabbits, large doses of BHA (1 g/day) administered by stomach tube for 56 days caused a 10-fold increase in sodium excretion and a 20% increase in potassium excretion in the urine (60). No adverse effects were observed in dogs fed 0.3, 30, or 100 mg/kg bw BHA for 1 year (52).

In high doses (500 mg/kg bw per day), BHA induced an increase in the relative liver weight in rats and mice. In rats, the changes follow a complex course that depends on the mode of administration. When rats were given BHA by stomach tube, the relative liver weight increase followed a bimodal time course, with maxima on days 2 and 10 and a highly significant increase on day 7. On dietary administration, liver enlargement was not apparent until day 5, and a single maximum was observed by day 11 (61,62). A preliminary ultrastructural study by Allen and Engblom (63) did not reveal any nucleolar abnormalities in the liver. BHA has been reported to induce a number of hepatic enzymes in rats and mice such as epoxide hydrolase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, and biphenyl-4-hydroxylase (Table 5.6) (61,62,64-66). In dogs, BHA at levels of 1 and 1.3% induced liver enlargement, proliferation of the smooth endoplasmic reticulum, the formation of hepatic myelinoid bodies, and an increase in hepatic enzyme activity (67). Given in doses of 500 mg/kg bw to young rhesus monkeys for 28 days, BHA induced liver hypertrophy and the proliferation of the smooth endoplasmic reticulum. Some differences were observed between monkeys and rats. In monkeys, the activity of microsomal glucose-6-phosphatase was decreased and the nitroanisole demethylase activity was increased, whereas in rats no changes were observed at similar dose levels (63,65).

Long-Term and Carcinogenicity Studies. In earlier long-term studies, BHA was found to be without any toxic effects in rats after 22 months (59,68,69) and in dogs after 15 months (53). However, in later studies, Ito et al. (70-72) reported that in F344 rats, administration of BHA at a 2% level resulted in a high incidence of papilloma in almost 100% of the treated animals and squamous cell carcinoma of the forestomach in about 30% of the treated animals (Table 5.7). At lower dose levels of 0.5%, no neoplastic lesions were observed, but forestomach hyperplasia was observed. Most of the changes were close to the limiting ridge between the forestomach and the glandular stomach. Ito et al. (72) observed that in addition to 3-BHA, two metabolites p-tert-butylphenol and 2-tert-butyl-4-methylphenol, also induced papillomas in the forestomach. Verhagen et al. (73) observed that in rats not only the forestomach but also the glandular stomach, small intestine, colorectal tissues, and possibly esophageal tissues were susceptible to the proliferative effects of BHA. Hamsters were found to be more susceptible to BHA than rats (74). In hamsters fed 1 or 2% BHA for 24 and 104 weeks, forestomach papillomas were observed in almost all treated animals and carcinomas in 7-10% in the 104-week group. A lower incidence of lesions was observed in mice fed 0.5 and 1 % BHA (75).

Table 5.6 Effects of Butylated Hydroxyanisole Treatment on Hepatic Microsomal and Cytoplasmic Enzyme Activities of Mice'

Enzymes                              Control group   BHA-treated group
Number of animals                          30              12
Microsomal enzymes
  NADPH-cytochrome c reductase        0.712 ± 0.028    1.31 ± 0.07
  (µmol cytochrome c reduced
   min-1 mg-1)
  NADH-cytochrome c reductase          4.03 ± 0.24     8.22 ± 0.35
  (µmol cytochrome c reduced
   min-1 mg-1)
Cytochrome P-450 (nmol/mg)             1.53 ± 0.14      1.12± 0.10
Cytochrome b5 (nmol/mg)               0.656 ± 0.061     1.51± 0.07
Glucose-6-phosphatase                 0.509 ± 0.029   0.413 ± 0.066
  (µmol P; min-1 mg-1)
Aminopyrine demethylase                18.7 ± 1.46     13.9 ± 0.84
  (nmol formaldehyde min-1 mg-1)
Aniline hydroxylase                    2.25 ± 0.16     6.08 ± 0.59
  (nmol p-aminophenol min-1 mg-1)
Benzo[a]pyrene hydroxylase             2.07 ± 0.16     1.46 ± 0.19
  (nmol 3-hydroxybenzota]pyrene
   min-1 mg-1)
UDP-glucuronyltransferase              25.2 ± 3.15    117.3 ± 8.3
  (nmol UDP-p-aminophenol
   min-1 mg-1)
Cytoplasmic enzymes
Glucose-6-phosphate dehydrogenase      23.3 ± 5.1      88.2 ± 5
  (nmol NADPH min-1 mg-1
UDP-glucose dehydrogenase              9.79 ± 1.24     59.8 ± 10.4
  (nmol NADH min-1 mg-1 )

aBHA was fed at 0.75% (w/w) in the diet for 10 days. 
Source: Ref. 64.

Table 5.7 Proliferative and Neoplastic Lesions of the Forestomach Epithelium in F344 Rats Given Diet Containing Butylated Hydroxyanisole

 BHA     Effective    No. of rats with changes in forestomachb (%)
in diet  no. of                                 Squamous-cell
 (%)     ratsa        Hyperplasia    Papilloma    carcinoma
  0       50           0 (0)           0 (0)        0 (0)
  0.125   50           1 (2)           0 (0)        0 (0)
  0.25    50           7 (14) *        0 (0)        0 (0)
  0.5     50          16 (32) **       0 (0)        0 (0)
  1       50          44 (88) **      10 (20)*      0 (0)
  2       50          50 (100)**      50 (100)**   11 (22)**

aNumber surviving at least to week 50. 
bAsterisked values differ significantly from the control value: *P < 0.01; **P < 0.001. 
Source: Ref. 72.

In order to determine which isomer of BHA was carcinogenic or whether the isomers had a synergistic action, feeding studies were conducted with the pure isomers and crude BHA in hamsters for 1-4 weeks. Severe adverse effects were observed with crude BHA and the 3-isomer. The 2-isomer had no effect (76). In rats given 1 g/kg bw of the two isomers, 2-BHA was also active in the induction of forestomach papillomas (77). The forestomach hyperplasia was found to be reversible, but the time taken for recovery depended on the duration and level of treatment. In rats fed 0.1-2% BHA for 13 weeks, on cessation of treatment the forestomach reverted to normal after 9 weeks. In rats fed 2% BHA for 1, 2, or 4 weeks followed by a 4-week recovery period, the mild hyperplasia and epithelial changes observed in the 1-week group almost completely disappeared. The more severe changes observed in the 2- and 4-week groups regressed partially during the recovery period (77).

Because of the possible relevance of these observations to humans, studies were conducted in other species including monkeys and pigs, which, like humans, do not have a forestomach. In female cyanomalgus monkeys, BHA (125 or 500 mg/kg bw) given by gavage for 12 weeks failed to induce any histopathological changes in the stomach and esophagus. However, a 40% increase in the mitotic index was observed at the lower end of the esophagus (Table 5.8) (78). In dogs fed BHA at levels of 0.25, 0.5, 1, and 1.3% for 6 months, no histopathological changes were noticed in the stomach, esophagus, or duodenum. Liver weights were increased without any related histopathological changes (67,79). Administration of BHA at levels of 50, 200, and 400 mg/kg bw per day to pregnant pigs from mating to day 110 of the gestation period resulted in proliferation and parakeratotic changes in the esophageal epithelium in a few pigs in the two groups with the higher dose levels. But papillomas were not observed, and no changes were found in the glandular stomach (80). In Japanese house musk shrews (Sancus murinus), which have no forestomach, BHA was fed at levels of 0.5, 1, and 2% for 80 weeks. All the animals in the 2% group died of hemorrhage in the gastrointestinal tract. Adenomatous hyperplasia in the lungs was observed at the 0.5 and 1 % levels at a significantly higher rate (81).

The mechanism by which 3-BHA induces carcinomas in the forestomach is not clear. Studies by De Stafney et al. (82) suggest that two factors may be of importance. One of these entails thiol depletion. The second is an attack by the reactive metabolites of 3-BHA or secondary products produced by these metabolites on cellular constituents. Studies by Williams (83) also indicated that BHA has an effect on membrane systems, blocking the exchange between the hepatocytes and the epithelial cells. The data strongly suggest that BHA is an epigenetic carcinogen that produces forestomach neoplasia through a promoting effect.

Butylated hyroxyanisole has a promoting or inhibitory effect on the carcinogenic effects of a number of chemical carcinogens. BHA enhanced forestomach carcinogenesis initiated by either N-methyl-N'-nitro-N-nitrosoguanidine or N-methyinitrosourea (MNU) in rats. BHA had a promoting effect on the urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4hydroxybutyl)nitrosamine and thyroid carcinogenesis initiated by MNU in rats. It had an inhibitory effect on the liver carcinogenesis initiated by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine and on mammary carcinogenesis initiated by 7,12dimethylbenz[a]anthracene (72).

Table 5.8 Mitotic Index of the Esophageal Epithelium from Cyanomalgus Monkeys given 0-250 mg BHA/kg per Day by Gavage on 5 days per week for 12 weeks

BHA treatment       No. of    No. of cells    No. of cells    Mitotic cells
(mg kg -1 day-1)    monkeys     counted        in mitosis     (%o of total)
0 (control)            8       4860 ± 223       43 + 7          0.87 ± 0.11
125                    7       4921 ± 154       38 ± 6          0.77 ± 0.11
250                    7       5485 ± 468       91 ± 10         1.66 ± 0.17*

*P < 0.05.
Source: Ref. 78.

Reproduction. Butylated hydroxyanisole has not been reported to have any adverse effects on reproduction data or in teratogenicity studies in mice, rats, hamsters, rabbits, pigs, and rhesus monkeys (80,84-88). It has been reported to induce some behavioral abnormalities in mice. Weanling mice exposed to BHA via their mothers during pregnancy and lactation (0.5% level) and then directly for up to 3 weeks showed a significant increase in exploratory activity, decreases in sleeping and in self-grooming, slower learning, and a decrease in the orientation reflex (89). In another study, Stokes et al. (90) observed a decrease in serotonin levels and cholinesterase activity and changed noradrenaline levels in the brain of newborn mice exposed to BHA in utero, and it is postulated that these changes may have an effect on the behavioral modifications observed.

Mutagenicity. Butylated hydroxyanisole was not mutagenic in a number of test systems. It was nonmutagenic in five tester strains in the Ames Salmonella/microsome test at concentrations of up to 10 mg/mL (83). In the hepatocyte primary culture/DNA repair test, BHA was negative (91). In mammalian cell mutagenesis assay using adult rat liver epithelial cells (92) and in V79 Chinese hamster lung cells (93), BHA was negative. BHA did not induce sister chromatid exchanges in Chinese hamster ovary cells (83). In tests for chromosomal aberrations, BHA was negative in Chinese hamster lung cells and in Chinese hamster DON cells (94,95). In vivo, BHA was negative in rat bone marrow cells and in the rat dominant lethal assay (96).


Butylated Hydroxytoluene

Butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-p-cresol) is one of the antioxidants used extensively in the food industry. It is used in low-fat foods, fish products, packaging materials, paraffin, and mineral oils. BHT is also widely used in combination with other antioxidants such as BHA, propyl gallate, and citric acid for the stabilization of oils and high-fat foods. ADI values for BHT have changed over the years because of its toxicological effects in different species. The latest temporary value allocated by JECFA is 0-0.125 mg/kg bw (1).

TOXICOLOGICAL STUDIES

Absorption, Metabolism, and Excretion. The absorption, metabolism, and excretion of BHT have been studied in rats, rabbits, dogs, monkeys, and humans. In general, the oxidative metabolism of BHT was mediated by the microsomal monooxygenase system. In rats, rabbits, dogs, and monkeys, oxidation of the p-methyl group predominated, whereas in humans the tert-butyl groups were oxidized. Oxidation of both p-methyl and tert-butyl groups was observed in mice.

The metabolism of BHT is more complicated and slower than that of BHA. The relatively slow excretion of BHT has been attributed to the enterohepatic circulation (97-99).

In rats given 0.5 and 1 % BHT for 5 weeks, the concentration of BHT increased rapidly in liver and body fat. Approximately 30 ppm was observed in the body fat in males and 45 ppm in females and 1-3 ppm in the liver. On cessation of treatment the concentration in the tissue decreased with a half-life of 7-10 days. In rats given single oral doses of 14C-labeled BHT (1100 mg/rat), nearly 80-90% was recovered in 4 days, with up to 40% in the urine. Approximately 3.8% was retained in the alimentary tract (98,100-103). The major urinary metabolites observed were BHT-acid (3,5-di-tert-butyl-4-hydroxybenzoic acid) (both free and as ester glucuronide) and BHT-mercapturic acid (di-tert-butylhydroxybenzyl acetyl cysteine) in addition to many other compounds including BHT alcohol (Ionox-100 or 3,5-di-tert-butyl-4hydroxybenzyl alcohol), BHT aldehyde (3,5-di-tert-butyl-4-hydroxybenzaldehyde), and BHT dimer. Free BHT acid was the major metabolite in feces. About 10% of the dose was excreted unchanged (101,102, 104,105). Tye et al. (102) observed distinct sex differences in the mode of excretion. Female rats excreted about 40-60% of a single oral dose in feces and about 2040% in the urine. Males excreted about 70-95% in the feces and 5-9% in the urine. Females showed more tissue retention, especially in the gonads. Significant biliary excretion of BHT and metabolites has also been observed. Four major metabolites have been identified: BHT acid, BHT alcohol, BHT aldehyde, and BHT quinone methide (2,6-di-tert-butyl-4-methylene-2,5cyclohexadienone) (Fig. 5.6) (98,100,106).

The half-life of a single oral dose of BHT in mice was found to be 9-11 h in major tissues such as the stomach, intestine, liver, and kidney. The half-life was 5-10 days when daily doses were given for 10 days. The major metabolite in the urine was the glucuronide conjugate of the acid and free acid in the feces. Excretion was mainly in the feces (41-65%) and urine (26-50%). The formation of BHT quinone methide has been observed in vitro in liver microsomes and in vivo in mouse liver (101).

The major metabolites observed in rabbits were BHT alcohol, BHT acid, and BHT dimer. Excretion of all metabolites was essentially complete in 3-4 days (107). Urinary metabolites constituted 37.5% glucuronides, 16.7% etheral sulfates, and 6.8% free phenols. Unchanged BHT was observed only in the feces (108,109). Significant biliary excretion of BHT and metabolites has also been reported (110). In dogs, the metabolism was similar to that of rats, and significant biliary excretion was observed (111). In monkeys, the major metabolite was the ester glucuronide of BHT acid, and the rate of excretion was similar to that of humans (112). Limited studies in humans (single oral doses of approximately 0.5 mg/kg bw) have indicated that the major metabolite is in the form of an ether-insoluble glucuronide identified as 5carboxy-7-(1-carboxy-l-methyl ethyl)-3,3-dimethyl-2-hydroxy-2,3-dihydro-benzofuran (99). Daniel et al. (58) studied the excretion of single oral doses of [14C]BHT (40 mg/kg bw) in humans.

Fig. 5.6 Major metabolites of butylated hydroxytoluene.

Approximately 50% was excreted in the urine in the first 24 h followed by slower excretion for the next 10 days. Tissue retention was found to be greater in humans than in rats. Studies by Wiebe et al. (99) suggest that biliary excretion may be an important route for the elimination of BHT and also that enterohepatic circulation occurs in humans. Verhagen et al. (104) reported differences in the metabolism in rats and humans, especially in terms of plasma kinetics and plasma concentrations, and concluded that the differences were too wide to allow a hazard estimation for BHT consumption by humans on the basis of its metabolism.

Acute Toxicity. The acute oral LD50 in mg/kg bw in rats was 1700-1970; in rabbits, 21003200; in guinea pigs, 10,700; in cats, 940-2100 (113), and in mice, 2000 (114).

Short-Term Studies. In rats, BHT at the level of 0.3 or 0.5% caused an increase in the level of serum cholesterol and phospholipids within 5 weeks. Brown et al. (69) observed reduced growth rates and increases in liver weight in rats fed BHT at 0.5 % in the diet. But at lower dose levels (0.1 %) no adverse effects were observed (115). In rabbits at the 2% dose level, BHT caused an acute effect on electrolyte excretion similar to that of BHA, whereas lower levels were without any adverse effect (60). No symptoms of intoxication or histopathological changes were observed in dogs fed 0.17-0.94 mg BHT/kg bw 5 days a week for 12 months (113).

In high doses, BHT had a toxic effect on liver, lung, and kidney and also on the blood coagulation mechanism. Early studies in rats and mice revealed that BHT at 500 mg/kg per day induced liver enlargement in 2 days and stimulated microsomal drug-metabolizing enzyme activity. The effects were found to be reversible (116). Creaven et al. (65) observed that in rats, BHT at levels of 0.01-0.5% for 12 days resulted in increased liver weights and induction of liver microsomal biphenyl-4-hydroxylase activity. At levels of 500 mg/kg bw for 14 days, a reduction in the activity of glucose-6-phosphatase was observed, indicative of early liver damage (115,117). In rats, administration of BHT by gavage at levels of 25, 250, or 500 mg/kg bw for 21 days resulted in a dose-related hepatomegaly, and at the highest dose a progressive periportal hepatocyte necrosis (Table 5.9) (118). The periportal lesions were associated with a proliferation of the bile ducts, persistent fibrosis, and infammatory cell reactions. At sublethal dose levels of 1000 and 1250 mg BHT/kg bw for up to 4 days, centrilobular necrosis was observed within 48 h. At a lower dose level (25 mg), no adverse effects were observed. In mice, BHT at levels of 0.75% for 12 months resulted in bile duct hyperplasia (119). The liver hypertrophy was accompanied by a proliferation of the smooth endoplasmic reticulum, an increase in the cytochrome P-450 level, and induction of a number of enzymes, including glutathione-S-transferase, glutathione reductase, thymidine kinase, nitroanisole demethylase, epoxide hydrolase, and aminopyrene demethylase. The changes were reversible after the cessation of the treatment (62,118,120, 121). At 500 mg/kg bw for 14 days, BHT caused slight hepatomegaly, moderate proliferation of the smooth endoplasmic reticulum, a reduction in glucose-6-phosphatase, and an increase in nitroanisole demethylase activity in monkeys. At the lower dose of 50 mg/kg bw, no adverse effects were observed (63).

Table 5.9 Summary of Hepatic Histopathology in Rats Treated with Butylated Hydroxytoluene for 7 or 28 Days

                               No. of rats with lesiona
                               after treatment with BHT
                              in doses (mg kg -1 day') of:
Observation                           25   250  500     
After 7 days
Periportal region
  Hepatocyte necrosis                  0    0    2
  Fibrosis                             0    0    3
  Hepatocyte hypertrophy               0    0    3
  Hepatocyte hyperplasia               0    0    4
  Glycogen accumulation                0    4    4
After 28 days
Periportal region
  Hepatocyte necrosis                  0    0    6
  Fibrosis                             0    0    5
  Bile-duct cell proliferation         0    0    4
  Hepatocyte hypertrophy               0    0    2
  Hepatocyte hyperplasia               0    0    3
  Pigment-laden macrophages            0    0    3
  Glycogen depletion                   0    0    7
  Glycogen accumulation                0    8    0
  Midzonal glycogen accumulation       0    0    5

a Out of a total of five per group treated for 7 days and 10 per group treated for 28 days. 
  Source: Ref. 118.

In a more recent study, Takahashi (122) reported that BHT in very high doses (1.35-5% for 30 days) caused a dose-related toxic nephrosis with tubular lesions in mice. The lesions appeared as irregular patches or wedge-shaped proximal tubules, necrosis, and cyst formation. Renal toxicity has also been reported in rats (123,124).

Butylated hydroxytoluene was reported to cause extensive internal and external hemorrhages in rats due to a disruption of the blood coagulation mechanism, resulting in increased mortality (125,126). The minimum effective dose was found to be 7.5 mg/kg bw per day. The disruption observed in the blood coagulation was due to hypoprothrombinemia brought about by inhibition of phylloquinone epoxide reductase activity in the liver by BHT quinone methide, one of the reactive metabolites of BHT (127). Administration of vitamin K prevented the BHT-induced hemorrhage. Takahashi and Hirage (128) suggested that BHT may inhibit absorption of vitamin K in the intestines or uptake by the liver. An increased fecal excretion of vitamin K was observed in rats receiving 0.25% BHT for 2 weeks. BHT was also reported to affect platelet morphology, fatty acid composition of the platelets, and vascular permeability, which may play a role in the hemorrhagic effect (129).

In mice, Takahashi (122) observed that BHT at levels of 0.5, 1, or 2% for 21 days caused massive hemorrhages in the lungs and blood pooling in various organs but only a slight reduction in blood coagulating activity. It was suggested that the hemorrhages might be due to a severe lung injury and not to a coagulation defect as observed in rats. BHT did not cause significant hemorrhaging in guinea pigs at dietary levels of 0-2%. The prothrombin index was slightly reduced at the 1 % level. BHT quinone methide was not detected in guinea pigs, whereas 7-40 mg/g liver was detected in rats. In rabbits, dogs, and Japanese quail fed BHT for 14-17 days at levels of 170 or 700 mg/kg bw, 173, 400, or 760 mg/kg bw, and 1 %, respectively, no hemorrhages were observed (Table 5.10) (122,126).

A number of studies have shown that BHT causes acute pulmonary toxicity in mice at levels of 400-500 mg/kg bw. The effects include hypertrophy, hyperplasia, and a general thickening of the alveolar walls of the lungs. A substantial proliferation of the pulmonary cells accompanied by a dose-dependent increase in total DNA, RNA, and lipids in the lungs was also observed within 3-5 days of a single intraperitoneal (IP) injection of BHT (130-132). The effect was generally reversible in 6-10 days of cessation of the treatment. However, exposure to a second stress such as hyperbaric OZ after administration of BHT impeded the repair process, resulting in pulmonary fibrosis (133). Some of the morphological and cytodynamic events include perivascular edema and cellular infiltration in type I epithelial cells followed by multifocal necrosis, destruction of the air-blood barrier, and fibrin exudation by day 2 after a single IP injection of 400 mg/kg bw BHT (134). Ultrastructural studies indicated that the type I cells were damaged by day 1 and cell destruction was complete within 2-3 days. Elongation of the type II cells with large nuclei and abundant cytoplasm was evident in 2-7 days (135). It has been postulated that BHT causes cell lysis and death as a result of interaction with the cell membrane (136). However, the mechanism of BHT toxicity still remains unclear.

Table 5.10 Hemorrhagic Effects of BHT in Various Species

                                Mean         % Total population
                              intake of        population with     Mean       Hepatic level of
Species and            Dose      BHTa            hemorrhages    prothrombin   quinone methidec
strain            Sex  of BHT  (mg kg-1 day-1) Dead Surviving    indexb (%)      (µ/g liver)    .
Rat
  Sprague-Dawley  M    0         0 (31)         0      0          101
                       1.2%    693 (50)        44     50           18***             NC
                  F    0         0 (5)          0      0          101
                       1.2%   1000 (10)         0     30           73***             NC
Wistar            M    0         0 (5)          0      0          100
                       1.2%    638 (10)        10     90           22***             38
                  F    0         0 (5)          0      0          100
                       1.2%    854 (10)         0    100           38***              7
Donryu            M    0         0 (5)          0      0          100
                       1.2%   1120 (10)        10     70           13***             41
                  F    0         0 (5)          0      0          100
                       1.2%   1000 (10)         0      0           92                27
Fischer           M    0         0 (5)          0      0          100
                       1.2%    821 (10)        30     70            5***             16
                  F    0         0 (5)          0      0          100
                       1.2%    895 (10)        20     70           18***             11
Mouse
  ddY             M    0         0 (5)          0      0          100
                       1.2%   1701 (10)         0     30           79**              ND
ICR               M    0         0 (10)         0      0          100
                       1.2%   1344 (10)         0      0           96                ND
DBA/2             M    0         0 (10)         0      0          100
                       1.2%    847 (10)         0      0          138**              NC
BALB/cAN          M    0         0 (5)          0      0          100
                       1.2%   1730 (10)         0      0           84**              NC
C31-1/He          M    0         0 (10)         0      0          100
                       1.2%   1858 (10)         0      0          115***             NC
C57BL/6           M    0         0 (5)          0      0          100
                       1.2%   1925 (10)         0      0           91*               NC
Hamster
   Syrian golden  M    0         0 (5)          0      0          101
                               380 (4)          0      0          101
                               760 (6)          0      0           87                ND d
Guinea pig
   Hartley        M    0         0 (5)          0      0          100
                               190 (5)          0      0           78
                               380 (5)          0      0           73                ND d
Japanese quail
   White egged    M    0         0 (5)          0      0          100
                       1%     1056 (5)          0      0           53*               ND

a The numbers in brackets are the numbers of animals in each group. 
b
The values marked with asterisks differ significantly from the corresponding control values: *P < 0.05; **P < 0.01; ***P < 0.001. The times of treatment for rats, hamsters, guinea pigs, and quail were 3 weeks, 1 week, 3 days, 3 days, and 17 days. respectively. 
c NC = Not calculated; ND = Not detected. 
d
Determined in separate experiments in hamsters or guinea pigs at 1.2 or I % in the diet, respectively, for 3 days. Source: Ref. 126.

Long-Term and Carcinogenicity Studies. In early studies, Deichmann et al. (113) observed no adverse effects in rats fed 0.2, 0.5, 0.8, or 1% BHT for 2 years. In a 104-week long-term feeding study in rats, Hirose et al. (137) reported that BHT at the 0.25 and 1 % levels was not carcinogenic. Treated rats of both sexes showed reduced body weight gain and increased liver weights. Only males showed increased y-glutamyl transferase levels. Tumors were observed in various organs, but their incidence was not statistically significant compared to controls (Table 5.11). In a two-generation carcinogenicity study with in utero exposure in rats, BHT was fed at levels of 25, 100, or 500 mg/kg bw per day from 7 weeks of age to the weaning of the F1 generation. The Fl generation were given 25, 100, or 250 mg/kg bw per day from weaning to 144 weeks of age. At weaning, the BHT-treated F1 rats, especially the males, had lower body weights than untreated controls. Dose-related increases in the numbers of hepatocellular adenomas and carcinomas were statistically significant in male F1 rats tested for heterogeneity or analyzed for trend. In F1 females the increases were statistically significant only for adenomas in the analysis of trend. However, all tumors were detected when the Fl rats were more than 2 years old (138). Unlike BHA, BHT had no adverse effects on the forestomach of rats or hamsters at the 1 % level (76,77).

Carcinogenicity studies have been conducted in various strains of mice. In a 2-year study in B6C3F1 mice, Shirai et al. (139) reported that BHT at the level of 0.02, 0.1, 0.5% was not carcinogenic. A reduction in body weight gain was noticed, the effect being more pronounced in males. Nonneoplastic lesions related to BHT treatment were lymphatic infiltration of the lung in females and of the urinary bladder in both sexes at the highest dose level. Tumors were observed in various organs, with a high incidence in the lung, liver, and the lymph nodes. But the incidence was not statistically significant. In another study in the same strain at higher dose levels of 1 or 2% in the diet, a significant dose-dependent increase in hepatocellular adenomas and foci of alterations in the liver were observed in males but not in females (140). Clapp et al. (141) reported an increase in the incidence of lung tumors and hepatic cysts in BALB/c mice fed 0.75% BHT for 16 months. Brooks et al. (142) reported a dose-related increase in both benign and malignant tumors in the lung in both sexes of CF1 mice and benign ovarian tumors in females. In C3H mice, which are more likely to develop spontaneous liver tumors with age, BHT fed at levels of 0.05 or 0.5% for 10 months increased the incidence of liver tumors in males, but it was not dose-related. The incidence of lung tumors was increased in males at both dietary levels but in females only at the higher dose level (143).

Table 5.11 Tumor Incidence in Rats Fed BHT

                                                     No. of animals with tumors a                    .
                                                  Males                        Females               .
                     Treatment group: Control     0.25%       1%       Control    0.25%        1%
Site and type of tumor  No. of ratsb:   26         43         38         32         46         51    .
Liver
  Hyperplastic nodule                 2 (7.7)    2 (4.7)    1 (2.6)    0          3 (6.5)    3 (5.9)
Pancreas
  Carcinoma                           0          0          1 (2.6)    0          1 (2.2)    4 (7.8)
  Islet-cell adenoma                  0          1 (2.3)    2 (5.3)    0          0          0
Mammary gland
  Fibroadenoma                           —         —         —      6 (18.8)   8 (17.4)   8 (15.7)
  Adenoma                                —        —          —      1 (3.4)    1 (2.2)    1  (2.0)
Uterus
  Leiomyoma                              —         —         —      1 (3.4)    1 (2.2)    0
  Carcinoma                              —               —          —      1 (3.1)    2 (4.3)    1 (2.0)
Pituitary gland
  Adenoma                             2 (7.7)    3 (7.0)    1 (2.6)    0          6 (13.0)   3 (11.8)
  Carcinoma                           0          2 (4.7)    5 (13.2)   3 (9.4)    3 (6.5)    7 (13.7)
Adrenal gland
  Adenoma                             1 (3.8)    3 (7.0)    0          0          2 (4.3)    1 (2.0)
  Carcinoma                           0                     0          0          0          1 (2.0)
Others                                2 (7.7)    2 (4.7)    4 (10.5)   2 (6.3)    4 (8.7)    3 (11.8)
Total                                 6 (23.1)  13 (30.2)  10 (26.3)  11 (34.4)  25 (54.3)  25 (49.0)

a Percent of group given in parentheses. 
b
Animals that survived more than 69 weeks were included. 
c
Differs significantly (chi-square test) from the corresponding control value (P < 0.05). 
Source: Ref. 137.

A number of studies have been conducted on the modifying effects of BHT on chemical carcinogenesis. These effects depend on a number of factors including target organs, type of carcinogen, species and strain differences, type of diet used, and time of administration. In general, BHT inhibited the induction of neoplasms in the lung and forestomach in mice and neoplasms in the lung, liver, and forestomach in rats when given before or with the carcinogen. BHT had a promoting effect on urinary bladder, thyroid, and lung carcinogenesis (144).

Reproduction. In an earlier study, Brown et al. (69) reported that rats fed 0.1 or 0.5% BHT showed a 10% incidence of anophthalmia. These findings were not confirmed in any other laboratory. BHT had no adverse effects on reproduction data and was not teratogenic in single or multigeneration reproduction studies in rats, mice, hamsters, rabbits, and monkeys at lower doses, and the no-effect level was equivalent to 50 mg/kg bw (84,88,145-149). At higher dose levels, some of the significant effects observed in rats include a dose-related response in litter size, number of males per litter, and body weight gain during lactation, but the effects were significant only at 500 mg/kg bw per day. In rabbits given 3-320 mg/kg bw per day by gavage during embryogenesis, an increase in intrauterine deaths was observed at high doses. In mice at 500 mg/kg bw per day, prolonged time to birth of first litters and a reduction in pup numbers and pup weight were observed.

In a developmental neurobehavioral toxicity test, the offspring of rats fed 0.5% BHT before conception and throughout pregnancy and lactation showed delayed eyelid opening, surface righting development, and limb coordination in swimming in males and reduced female open-field ambulation. However, the results did not suggest any specific toxicity of BHT for the central nervous system (150). In another study weanling mice fed 0.5% BHT for 3 weeks, whose parents had been maintained at the same level during their entire mating, gestation, and preweaning period, decreased sleeping, increased social and isolation-induced aggression, and learning disabilities were observed under the experimental conditions employed (89).

Mutagenicity. Butylated hydroxytoluene was found to be negative in several strains of Salmonella typhimurium with or without metabolic activation (151,152). In in vitro tests using mammalian cells, BHT was weakly positive in the test for gene mutation in Chinese hamster V79 cells (153). BHT was positive in tests for chromosomal aberrations in human lymphocyte cultures (154) and in Chinese hamster ovary cells (155). In in vivo tests, BHT was negative in tests for chromosomal damage in bone marrow cells and liver cells of rats (156,157). In mice, BHT was negative in three dominant lethal tests, but in rats at high doses BHT was positive in two dominant lethal tests (158,159). In general, the mutagenic effects were observed only at higher levels of BHT.

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