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Butylated Hydroxytoluene Synthesis Essay

Butylated hydroxytoluene (BHT) is an effective, widely used, low cost antioxidant. A host of studies examining the potential of BHT to cause point mutations have been published. They include in vitro studies on various bacterial species and strains and on various types of mammalian cell lines as well as in vivo studies on Drosophila melanogaster, silk worms and also the mouse specific locus test (involving long-term exposure). Together these studies convincingly show the absence of a potential for BHT to cause point mutations.

A great number of studies on many cell types and species have also been carried out to examine the potential of BHT to cause chromosome aberrations. In vitro studies have been published using plant cells and the WI-38, CHL, CHO, and V79 mammalian cell lines. In vivo studies have been carried out on somatic and/or germ cells of Drosophila melanogaster, rats and mice. Nearly all studies, especially those using validated test systems, indicate that BHT lacks clastogenic potential.

In vitro studies on bacterial, yeast and various mammalian cell lines including DON, CHO, CHL cells and primary hepatocytes demonstrate the absence of interactions with or damage to DNA.

Taking all the existing data into account, the weight of evidence suggests that BHT does not represent a relevant mutagenic/genotoxic risk to man.

BUTYLATED HYDROXYTOLUENE (BHT) Explanation This substance has been evaluated for acceptable daily intake for man (ADI) by the Joint FAO/WHO Expert Committee on Food Additives in 1961, 1964, 1965, 1973 and 1976 (see Annex I, Refs. 6, 8, 11, 32 and 40). Toxicological monographs were issued in 1961, 1964, 1965, 1973 and 1976 (see Annex I, Refs. 6, 9, 13, 33 and 41). Since the previous evaluations, additional data have become available and are summarized and discussed in the following monograph addendum. BIOLOGICAL DATABIOCHEMICAL ASPECTS Effects on enzymes and other biochemical parameters Groups of 16-24 Swiss male mice (25-30 g) received a single i.p. injection of BHT in corn oil (62.5, 215, or 500 mg/kg bw) or corn oil only (Tocopherol stripped 0.5 ml). Three days later the mice were sacrificed. After BHT, wet lung weights were increased to 120% of control, as were dry lung weights. There were significant increases in DNA content and level of non-protein sulfhydryl (133-156% of control). Superoxide dismutase and other oxidative enzyme levels were increased. The authors concluded that BHT apparently increased inflammatory and reparative-proliferative processes of the lung (Omaye et al., 1977). BHT (500 mg/kg/day) was administered by gavage to groups of young Wistar male and female rats (60-80 g) for seven days and the animals were housed in metabolism cages. Control animals received corn oil vehicle only. They were then sacrificed and liver enzymes (aniline 4 hydroxylase, biphenyl-4-hydroxylase, ethyl morphine N-demethylase, and 4-methyl umbelliferone glucoronyl transferase) were assayed and the cytochrome P-450-CO interaction spectrum evaluated. Urinalysis, using gas chromatography to assay for D-glucaric acid, D-glucuronic acid, 1-gulonic acid, xylitol and L-ascorbic acid was conducted. Administration of BHT enhanced all the parameters measured with the exception of the hepatic microsomal protein content. BHT was a more potent inducer of xenobiotic metabolism in female rats (Lake et al., 1976). Groups of NMRI mice (25-35 g) and Wistar rats (160-320 g) received BHT as a single dose of 500 mg/kg dissolved in soya bean oil, either i.p. or by gavage. Four days later, radiolabelled C14 thymidine was given. After 90 minutes, the animals were sacrificed, lungs removed and DNA levels were measured. In mice, DNA synthesis was equally increased in males and females by oral or i.p. administration. Although lung weight was increased, the concentration of DNA was not affected. In rats, no effect was seen in males and only a slight increase in females (Larsen & Tarding, 1977). TOXICOLOGICAL STUDIES Special studies Potentiation or inhibition of carcinogenesis The tumorigenic potency of a single i.p. injection of 1000 mg/kg of urethan to male Swiss-Webster mice was significantly increased if followed by repeated weekly injections with 250 mg/kg BHT. The number of animals used per group ranged between nine and 22 and the animals were treated from nine to 13 weeks. Only tumours on the lung surface itself were counted. About 90% of the animals treated with urethan alone developed lung tumours. There was a significant increase in the number of tumours per mouse after 11 or more weeks of treatment with BHT. Animals treated with BHT alone did not develop lung tumours. A/J strain mice were also given the same treatment with 10 weekly injections of BHT. The number of lung tumours per animal significantly increased in those receiving BHT in addition to urethan in comparison with those receiving urethan alone. With both strains of mice, repeated injection of BHT without prior urethan treatment did not result in an increased number of animals with lung tumours or tumours/mouse as compared to controls dosed with corn oil. With both mouse strains, there were fewer lung tumours in the animals given BHT as compared to the corn oil controls. In contrast to the above results, injection of animals with BHT for 0-7 days before urethan injection did not increase the number of animals with tumours or number of tumours/animal (Witschi & Cote, 1976). A group of 93 rats (22 days old) received 0.5% BHT or control diets for 407 days following 18 days of administration of 2-acetyl- aminofluorene (0.02%). No control group received BHT only without prior AAF feeding. Prolonged feeding of BHT diet after 2-AAF produced a significant enhancement of hepatic carcinogenesis as shown by increased number of rats with liver tumours (Peraino et al., 1977). Mutagenicity BHT was found not to be mutagenic using Saccharomycescerevisiae, D4 and Salmonella typhimurium, TA-1535, TA-1537 and TA-1538, with and without the addition of mammalian metabolic activation (Brusick, 1975). BHT was fed at the 5% level in a sex-linked recessive lethal test in Drosophila. About 0.27% lethals were found in the BHT treated flies (out of 8897), 0.29% lethals were found in the control and 5.69% lethals were found in the positive control dosed with 100 ppm ethylmethane sulfonate. It was concluded that BHT was not mutagenic (Brusick, 1975). The effect of BHT on excision repair synthesis was tested in cultured human lymphocytes irradiated with u.v. light. Irradiated cells cultured in the presence of BHT had decreased excision repair based on lower incorporation of tritiated thymidine into DNA with increasing concentration of BHT in the culture medium. In this study, BHT also inhibited semi-conservative DNA synthesis (Daugherty et al., 1978). A study was conducted to confirm a reported radiosensitizing effect of BHT on Drosophila sperm, using three radiation doses (1.2, 2.4 and 3.6 KRDS). The mutagenic activity of BHT was also tested in the sex-linked recessive lethal system. Ten-day-old males of D. melangaster received 0.2 µl of saline in ethanol or 0.001% BHT in the solvent. They were exposed to gamma rays from a 137CS source at 4.1 rad/sec. They were mated to four females and two successive one day broods were tested to obtain sensitivity of mature and slightly immature sperm, after discarding xo males and non-disjunction females remaining. F1 flies were pooled and F2 females obtained after individual brother/sister mating. The absence of bar-eyed males indicates the presence of sex-linked recessive lethals. There was no shift in the sex ratio of the F1 generation. Without irradiation, BHT seems to enhance the incidence of xo males. BHT almost doubled the incidence of sex-linked lethals over saline-injected controls, but this was not statistically significant. In comparison with gamma rays, BHT had no influence on the sex ratio of F1 flies, and seemed to have a negligible effect on the incidence of radiation loss of xB chromosome. At all doses BHT exhibited a pronounced radiosensitization of sex-linked recessive lethals (Prasad & Kamra, 1974). Reproduction and behavioural studies Breeding pairs of Sprague-Dawley rats (200-220 g) received Purina chow supplemented with 0.5, 0.25 or 0.125% BHT ad libitum beginning from the week before mating and continuing in females through lactation and weaning of the pups. Growth rates and mortality were adversely affected. Pre-weaning pups born of mothers at the highest dose level (0.5%) of BHT weighed significantly less than controls at ages seven, 14 and 21 days. The total number of pups dying on study, born of dams receiving 0.5 or 0.2% BHT, was significantly higher than controls. Behavioural tests were conducted consisting of righting reflex, pivoting cliff avoidance, startle response, swimming, open field, running, wheel activity, roto-rod, active avoidance, position discrimination and passive avoidance. For pre-weaning testing, no differences were noted at the low or mid dose groups. At the high dose level, there was significant increase in surface righting time, delayed forelimb swimming development and a trend to less activity in open field tests. In post-weaning tests, males in 0.25% BHT groups showed an effect on passive avoidance, with more partial re-entries into compartments where shocked. For all other tests, there were no statistical differences, suggesting that BHT had no effect on basic motor coordination, active avoidance acquisition, or extinction performance (Brunner et al., 1979). Effect on lung The pathological effect of BHT on the mouse lung was studied. Sixty male Swiss mice were given i.p. injections of 400 mg/kg BHT dissolved in corn oil. Six of the experimental animals and six of the controls were sacrificed daily for nine days. Two hours before sacrifice, each animal received 2 µCi/g of tritiated thymidine. No animals died during the study and none showed signs of respiratory distress. Two days after dosing, cellular lesions were noticed in the type I alveolar epithelium. Abnormal giant type II cells were observed in mitosis and many had an accumulation of tritiated thymidine. Labelled endothelial cells were seen after day 6 in small vessels and capillaries, and there was an increase in fibroblastic cells in the interstitium and capillaries. There was an increase in thymidine- labelled pulmonary cells from days 2 through 5 after which labelling dropped off and approached control levels by day 9. Levels of lung thymidine kinase activity rose sharply on days 1-4 after dosing and then dropped off rapidly (Adamson et al., 1977). Following acute exposure to BHT, the initial sequence of events involves infiltration of type I (squamous) epithelial cells followed by multifocal necrosis and destruction of the blood barrier. A detailed discussion of the sequence of tissue changes and repair mechanisms is given. It is stated that the susceptibility of the squamous epithelium to injury is similar to that seen after oxygen exposure, radiation exposure, and treatment with blood-borne bleomycin, but the recovery pattern is quite different. BHT is thought to cause cell lysis and death as a result of interaction with the cell membrane (Anonymous, 1978). The increase in lung weight and increase in thymidine incorporation into lung DNA observed in mice following BHT injection was inhibited by treatment with cedar terpenes. No increase in lung weight was observed in animals treated with BHT alone if they were less than three weeks old. This may result from the inability of the infant mice to metabolize BHT (Malkinson, 1979). Immunological studiesIn vitro BHT (50 µg/culture) suppressed the plaque-forming cell response of mouse spleen cell cultures as measured by the method of Mishell & Dutton (Archer et al., 1977). Carcinogenicity Groups of 50 Fischer 344 rats/sex and 50 B6C3F1 mice/sex received 3000 ppm or 6000 ppm BHT in the diet. The compound was mixed with autoclaved lab meal containing 4% fat. A control group of 20 animals per sex received lab meal only. The rats were on study for 105 weeks and the mice for 107 or 108 weeks. There was a dose-related depression in the body weights of the rats and mice of both sexes throughout the study. In rats there was no significant effect of BHT on mortality. In male mice BHT was significantly associated with decreased mortality, while BHT feeding had no effect on mortality in female mice. There was a high dose-related incidence of hepatocytomegaly and hepatocellular degeneration and necrosis in the liver of male mice, but the incidence of hepatocellular carcinomas was reduced in a dose-related manner. The incidence of alveolar/bronchiolar carcinomas or adenomas was significantly higher than controls in the low dose, but not the high dose female mice. BHT feeding was related to a significant reduction in the incidence of sarcomas of multiple organs in female mice. Four adenomas of the eye/lacrimal gland were observed in the high dose male mice and in two low dose females but not in corresponding controls. The historical incidence of this tumour was 1.2%. Since the lacrimal gland was only examined microscopically in animals with grossly apparent lesions, the report states that the lacrimal gland tumours could not be clearly related to BHT administration. There was an increased incidence of focal alveolar histiocytosis in treated rats, especially in high dose females. The incidence of adenomas of the pituitary was significantly reduced in female rats with BHT administration. The report concluded that no neoplastic lesions occurred in rats or mice that could be clearly related to BHT administration. Under the conditions of the test, there were increased incidences of focal alveolar histiocytosis in dosed male rats which may have been related to the administration of BHT. BHT was not, however, carcinogenic for F344 rats or B6C3F1 mice of either sex. The report was evaluated by the Data Evaluation/Risk Assessment subgroups of the Clearinghouse on Environmental Carcinogens. The subgroup accepted the conclusions of the report but, because of the widespread human exposure, evidence of hepatotoxicity and a suggestion of a tumorigenic effect in the lung, the subgroup suggested that the compound be considered for retest by the chemical selection working group (Anonymous, 1979). Short-term studies Rat Groups of male Sprague-Dawley rats (six weeks of age) received BHT in a semi-synthetic diet at concentrations ranging from 0.58% to 1.44% or control diet only. Deaths occurred within 40 days, at levels of 0.69% or greater. Spontaneous massive bleeding to the pleural and peritoneal cavities, or as external haemorrhage, was observed in all dead or dying animals. The prothrombin index was decreased as the daily dose of BHT was increased. Mild diarrhoea was noted after four days. Rough hair coat, and redness of urine was noted. Death was due to haemorrhage and was classified by the authors as a secondary type of toxicity, probably due to a decrease in prothrombin concentration. According to the authors, the effect seems to depend on strain of rats and dietary concentration (Takahashi & Hiraga, 1978). Long-term studies (See also under "Carcinogenicity") Rats Groups of female rats (80-100 g) received 0.4% BHT with corn oil mixed in ground lab chow and were sacrificed at intervals of 1, 8, 16, 32, or 80 weeks, and compared with controls. Samples of liver were taken for biochemical, histochemical, and morphological studies to examine for reversibility of hepatic changes. Control diet only was administered for 18 days to a group of four rats following 80 weeks of BHT administration. After one week on BHT there was a marked liver enlargement with relative liver weight increased up to 35% and with an increase in drug metabolizing activities and NADP-cytochrome C reductase activity. After 18 days of removal from the 80-week treatment there was only a slight increase in liver weight. The effect was therefore reversible. Histologically, after BHT treatment the liver was characterized by enlarged centrilobular hepatocytes, with a heterogenous appearance of this zone. Ultrastructurally, there was a proliferation of smooth endoplasmic reticulum. The authors note that, although there is no unequivocal evidence of liver injury, there were two features that are also seen with many hepatotoxins and hepatocarcinogens: depression of glucose-6-phosphatase activity and cell enlargement. However, there were no lysosomal changes characteristic of cytologic injury, and effects were reversible (Crampton et al., 1977). Groups of 40 JCL strain rats/sex/group, reared under a barrier system and four weeks of age at the start of the study, received either control diet or BHT at 0.005, 0.062, 0.32% in the diet. Of each group of 40, 15 received compound for a "lifetime", 10 for 24 months, and five each for three, six or 12 months. At interim and final kill, liver, kidney, heart, spleen, thyroid and caecum weight were determined as were haematology, serum biochemistry, urinalysis, and histological investigation of the tissues. At 24 months, heart, liver, kidney, spleen, pituitary, thyroid, adrenal, testes, prostate and brain were weighed, haematological and biochemical measurements conducted and histopathology done. There was an increase in liver weight, serum cholesterol, serum K+ and histological changes in liver and kidney at the 0.32% dietary level. There was no change in quantity of food intake, body weight gain, mortality during feeding or mean life span and no finding suspicious of tumour induction. There was no indication of a dose-related trend in tumour prevalence in either 24-month or "life span" groups. The tumours found were said to be typical of those described in aged rats. It is to be noted that the number of surviving rats is small and that tumour data include both life span groups and animals dead or sacrificed moribund during the six, 12 and 24 month feeding. The available data do not list the number of each of the individual tissues examined, although the number of rats is listed. The data show a tendency for a decreased number of tumours per rat at higher dose levels (Hiraga, 1978). Six-week-old random bred Wistar rats were allocated to groups of 57 males and 57 females fed control diet and 36 males and 36 females fed BHT at either 0.25% or 1% in the diet for 104 weeks. Survival by tests groups was between 40% and 68%. A variety of tumours were noted on histopathological examination at the end of the study, with no dose-related response in either type of tumour or total number as compared to controls (Shibata et al., 1979). Comments Several new studies were available on BHT, including a behavioural study in newborn rats that had been exposed to the material in utero and during lactation. Decreased pup survival and slight behavioural effects were noted at levels above 0.1% of the diet. It was noted that behavioural effects were not seen in newborn monkeys whose mothers had been treated with the chemically related BHA. At its twentieth meeting, the Committee had noted that BHT had been reported to enhance the occurrence of lung adenomas in mice. At that meeting, it had considered that BHT was not likely to be carcinogenic but had requested additional long-term studies. Recently completed long-term studies in mice and rats have been negative and confirm the view that BHT is not carcinogenic. Two series of studies with BHT have demonstrated that it enhances the effect of certain chemical carcinogens. In one study, it was shown that mice initially receiving injections of the carcinogen urethane developed more lung adenomas if treated subsequently for some weeks with BHT. In another study, rats treated with low levels of the carcinogen N-fluorenyl acetamide and subsequently with BHT developed more hepatomas, more rapidly than with the carcinogen alone. In these studies, it is implied that BHT is a "promoting" agent. Possible mechanisms of action include enzyme induction and the production of hyperplasia and hypertrophy in both the lung and the liver by BHT. The phenomenon of "promotion" of carcinogenesis in various systems, including the skin and urinary bladder, as well as these examples, are attracting much attention in cancer research. Mechanisms of action, although under intense study, are not yet understood. In addition, since BHT has been shown to inhibit the action of carcinogens under other conditions, it is felt premature to use such information for toxicological evaluation. As regards effects on microsomal enzymes, reproduction and behaviour, a no-effect level of 0.1% equal to 50 mg/kg in the diet of rodents can be set. EVALUATION Levels causing no toxicological effect Mouse: 5000 ppm (0.5%) in the diet, equivalent to 250 mg/kg bw Rat: 1000 ppm (0.1%) in the diet, equivalent to 50 mg/kg bw Estimate of acceptable daily intake for man 0-0.5* mg/kg bw** FURTHER WORK OR INFORMATION Required by 1981 (1) A study to clarify the effects on rat pup survival. (2) Elucidation of the significance of the behavioural effects observed in newborn rats. * BHT, TBHQ, or the sum of the three compounds. ** Temporary. REFERENCES Adamson, I. Y. R., Bowden, D. H., Cote, M. G. & Witshi, H. Lung injury induced by butylated hydroxytoluene: Cytodynamic and biochemical studies in mice. Lab. Invest., 36: 26-32, 1977 Anonymous. British Industrial Biological Research Association. BHT and lung tumours. BIBRA Information Bulletin, 17(2): 88, 1977 Anonymous. National Cancer Institute. Bioassay of butylated hydroxytoluene (BHT) for possible carcinogenicity. DHEWPublication No. (NIH) 79-1706, 1979 Archer, D. L., Smith, B. G. & Bukovic-Wess. Use of in vitro antibody producing system for recognizing potentially immunosuppressive compounds. Int. Arch. Allergy, 56: 90-93, 1978 Brunner, R., Vorhees, C. & Butcher, K. Psychotoxicity of selected food additives and related compounds. Report prepared under FDA contract 223-75-2030 Brusick, D. Mutagenic evaluation of compound FDA 71-25 (butylated hydroxytoluene). Unpublished report from Litton Bionetics, Incorp., submitted to WHO by Eastman Chemical Products, Inc., 1975 Crampton, R. F., Gray, R. J. B., Gray, P., Grasso, R. & Parke, D. V. Long term studies on chemically induced liver enlargement in the rat. 1. Sustained induction of microsomal enzymes and absence of liver damage on feeding phenobarbitone or butylated hydroxytoluene. Toxicology, 7: 289-306, 1977 Daugherty, J. P., Davis, S. & Yielding, K. L. Inhibition by butylated hydroxytoluene of excision repair synthesis and semiconservative DNA synthesis. Biochem. biophys. Res. Commun., 80(4): 963-969, 1978 Hiraga, K. Life-span oral toxicity study of 3,5-di-tert-hydroxy- toluene (BHT) in rats. Ann. Rep. Tokyo Metropolitan ResearchLab. Public Health, 32: 83, 1978 Lake, B. G., Longland, R. C., Gangolli, S. D. & Lloyd, A. G. The influence of some foreign compounds on hepatic xenobiotic metabolism and the urinary excretion of D-glucoronic acid metabolites in the rat. Toxicol. appl. Pharmacol., 35: 113-122, 1976 Larsen, J. C. & Tarding, F. Stimulation of DNA synthesis in mouse and rat lung following administration of butylated hydroxytoluene. Archives of Toxicol. Suppl., 1: 147-150, 1978 (Proceedings of the European Soc. of Toxicology Meeting held in Copenhagen, 19-22 June 1977) Malkinson, A. M. Prevention of BHT-induced lung damage in mice by cedar terpene administration. Pre-print of paper accepted for publication in Toxicol. appl. Pharmacol., 1979 Omaye, S. T., Reddy, A. & Cross, C. E. Effect of butylated hydroxytoluene and other antioxidants on mouse lung metabolism. J. Toxicol. Environ. Health, 3: 829-836, 1977 Prasad, O. & Kamra, O. P. Radio sensitization of Drosophila sperm by commonly used food additives, butylated hydroxyanisole and butylated hydroxy toluene. Int. J. Radiat. Biol., 25: 67-72, 1974 Peraino, C., Fray, R. J. M., Staffeld, E. & Christopher, J. P. Enhancing effects of phenobarbitone and butylated hydroxytoluene on 2-acetylaminofluorene induced hepatogenesis in the rat. Foodand Cosmetics Toxicol., 15: 93-96, 1977 Shibata, M., Hagiwara, A., Miyata, Y., Imaida, K., Arai, M. & Ito, N. Experimental study on carcinogenicity of butylated hydroxy toluene (BHT) in rats. Translation of the Proceedings of the 38th Annual Meeting of the Japanese Cancer Assoc., Tokyo, September 1979 Takahashi, D. & Hiraga, K. Dose response study of hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats. Toxicol.appl. Pharmacol., 43: 399-406, 1978 Witschi, H. & Cote, M. G. Biochemical pathology of lung damage produced by chemicals. Fed. Proc., 35(1): 89-94, 1976

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