Safety Assessment of Butylated Hydroxyanisole and Butylated Hydroxytoluene as Antioxidant Food Additives

Food and Chemical Toxicology 37 (1999) 1027±1038

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Safety Assessment of Butylated Hydroxyanisole and Butylated Hydroxytoluene as Antioxidant Food Additives G. M. WILLIAMS1*, M. J. IATROPOULOS1 and J. WHYSNER2 Department of Pathology, New York Medical College, American Health Foundation Valhalla, New York, 10595, USA

1

AbstractÐButylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used antioxidant food additives. They have been extensively studied for potential toxicities. This review details experimental studies of genotoxicity and carcinogenicity which bear on cancer hazard assessment of exposure to humans. We conclude that BHA and BHT pose no cancer hazard and, to the contrary, may be anticarcinogenic at current levels of food additive use. # 1999 Elsevier Science Ltd. All rights reserved Keywords: phenolic antioxidants; butylated hydroxyanisole; butylated hydroxytoluene; anticarcinogenicity. Abbreviations: AFB1 = a¯atoxin B1; BHA = butylated hydroxyanisole; BHT = butylated hydroxytoluene; DBN = N,N-dibutylnitrosamine; MNNG = N-methyl-N-nitro-N-nitrosoguanidine; NMU = N-methyl-N-nitrosourea; NOEL = no-observed-e€ect level.

Introduction Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been widely used for many years as antioxidants to preserve and stabilize the freshness, nutritive value, ¯avour and colour of foods and animal feed products (JECFA, 1996). BHT can also improve the stability of pharmaceuticals, fat-soluble vitamins and cosmetics (FDA, 1981). The service life of rubber, elastomers and plastics is increased by the addition of BHT (Sherwin-Williams, 1982), and from such use BHT may be present as an indirect food additive. Approximately 40 countries reportedly permit the use of BHT as a direct or indirect food additive (ILSI, 1984). The US Food and Drug Administration (FDA) currently permits BHA and BHT as food additives. Food-grade BHA, referred to as 2(3)-tert-butyl-4-hydroxyanisole, is generally a mixture of greater than 85% 3-tert-butyl-4-hydro*Corresponding author.

xyanisole (3-BHA) and 15% or less 2-tert-butyl-4hydroxyanisole (2-BHA), while food-grade BHT, which is 3,5-di-tert-butyl-4-hydroxytoluene, is not less than 99% (w/w) pure. A variety of experimental studies have been reported on BHA and BHT. The International Agency for Research on Cancer (IARC) has evaluated BHA and found sucient evidence for carcinogenicity in experimental animals, but no data for humans (IARC, 1986a). The evaluation of BHT concluded that there was limited evidence for carcinogenicity in experimental animals, and also no data for humans (IARC, 1986b). In this review, the data on genotoxicity and carcinogenicity of BHA and BHT, including reports which appeared subsequent to the IARC evaluations, will be put it into the perspective of information on the mode of actions of these chemicals in a€ecting neoplasia. The data on dose±response of carcinogenicity and related mechanisms of BHA and BHT are then assessed with respect to the current use of these agents.

0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00085-X

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G. M. Williams et al. Table 1. Genotoxicity studies of butylated hydroxyanisole (BHA) Results In vitro

Endpoint Test system

In vivo

Without activation

With activation

±1 ± ±

NA2 NA NA

NA NA NA

Hirose et al., 1987b Saito et al., 1989 Ito et al., 1991

Strand breaks and repair Rat/F344 hepatocytes Rat/F344 forestomach, epithelium ss fX-l74 DNA

± ± NA

NA NA ±

NA NA ND3

Williams et al., 1989, 1990a Morimoto et al., 1991 Schilderman et al., 1993

Mutagenicity assays Reverse mutation Salmonella tvphimurium TA98, 100, 1535, 1537, 1538 Salmonella typhimurium TA98, 100, 1535, 1537, 1538 Salmonella typhimurium TA98, 100 Salmonella typhimurium TA97, 100, 102, 104 Salmonella typhimurium TA97, 98, 100, 102 Salmonella typhimurium TA98, 100, 1535, 1537, 1538

NA NA NA NA NA NA

± ± ± ± ± ±

± ± ± ± ± ±

Joner, 1977 Bonin and Baker, 1980 Kawachi et al., 1980 Hageman et al., 1988 Matsuoka et al., 1990 Williams et al., 1990a

Gene mutation Staphylococcus aureus Rat ARL/HGPRT Hamster/Chinese CHO/HGPRT Hamster/Chinese V79/HGPRT

NA NA NA NA

+4 ±

ND ND ND

Degre and Saheb, 1982 Williams et al., 1990a Tan et al., 1982 Rogers et al., 1985

(+)5 ±

NA NA

NA NA

Prasad and Kanira, 1974 Miyagi and Goodheart, 1976

Chromosomal aberrations Hamster/Chinese Don Hamster/Chinese CHL Hamster/Chinese CHO Hamster/Chinese CHL Hamster/Chinese CHO

NA NA NA NA NA

± ± ± ± ±

ND ND + + +

Abe and Sasaki, 1977 Ishidate and Odashima, 1977 Phillips et al., 1989 Matsuoka et al., 1990 Murli and Brusick, 1992

SCEs Hamster/Chinese Don Hamster/Chinese V79 Hamster/Chinese CHO

NA NA NA

+ ± ±

ND ± ±

Abe and Sasaki, 1977 Rogers et al., 1985 Williams et al., 1990a

Reference

DNA interaction DNA adduct formation Rat/F344 forestomach Rat/F344 forestomach Rat/F344 forestomach, stomach

Sex-linked, recessive lethal Drosophila melanogaster Drosophila melanogaster Cytogenicity assays

1

± indicates negative test result. 2NA = not applicable. 3ND = indicates test was not done. 4+ indicates a positive test result. 5(+) indicates a weak positive test result.

BHA genotoxicity studies BHA has not shown DNA reactivity in assays for DNA adduct detection, and nearly all genotoxicity studies are negative (Table 1). Most importantly, investigations of BHA and its metabolites have not demonstrated DNA±adduct formation, as measured by the very sensitive [32P]postlabelling assay (Saito et al., 1989). In rats, DNA binding in the forestomach, which is the target tissue for BHA (see below), glandular stomach, kidney or liver was detected using radiolabelled BHA (Hirose et al., 1987a,b). Other tests for DNA damage such as the unscheduled DNA synthesis assay using rat hepatocytes were negative. Bacterial systems have shown a lack of mutagenicity, and cellular assays have not indicated cytogenetic e€ects, except for both positive and negative tests for chromosomal aberrations and sister chromatid exchanges (SCE) (Abe and

Sasaki, 1977; Rogers et al., 1985; Williams et al., 1990a). The causes of positive results in these tests are not necessarily due to DNA binding (Bradley et al., 1979; Williams, 1989). For example, chromosomal aberrations may be secondary to oxidative DNA damage, which has been found to be produced by BHA in human lymphocytes in vitro (Schilderman et al., 1995a), and S-9 has been shown to produce hydrogen peroxide in one of the reported positive tests (Phillips et al., 1989). Consequently, the results of DNA-reactivity and genotoxicity testing, including the mixed positive and negative results for chromosomal aberrations and SCE, do not implicate DNA reactivity as a mechanism for BHA-induced carcinogenicity. BHA is metabolized to tert-butylhydroquinone (TBHQ) and tert-butylquinone (TBQ) in the liver. Formation of these metabolites was not detected in

Phenolic antioxidant safety

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Table 2. Results from oral (diet) chronic bioassays with butylated hydroxyanisole (BHA)

Species/strain

Sex and no. per group

Mouse B6C3F1

M150

Rat F344

M150

Rat F344

M50

Rat F344

M51, F51

Exposure (ppm)

Duration (months)

10000 5000 Control 20000 10000 Control 20000 10000 5000 2500 1250 Control 10200b

24a 24a 24a 24a 24a 24a 24 24 24 24 24 24 24

c

Rat F344

M27

Syrian golden hamster

M150

Syrian golden hamster,

M10

Japanese house musk shrew,d

M30 F30

2500 Control 12000 Control 20000 10000 Control 20000 10000 Control 20000 10000 5000 Control

24 24 25 25 24a 24a 24a 5 1/2 5 1/2 5 1/2 2e 18 18 18

Results (percent incidence of forestomach neoplasms or neoplasms of other sites as indicated) *14 papillomas; 5 squarnous cell carcinomas *14 papillomas; 3 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas *92 papillomas; *14 squamous cell carcinomas *76 papillomas; 0 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas *100 papillomas; *22 squamous cell carcinomas 20 papillomas; 0 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas *100 M *96F papillomas; *35 M 29F squamous cell carcinomas 2 M 2F papillomas; 0 M 0F squamous cell carcinomas 0 M 0F papillomas; 0 M 0F squamous cell carcinornas *33 papillomas; 22 hepatocellular adenomas 0 papillomas; 36 hepatocellular adenomas *95 papillomas; * 10 squamous cell carcinornas *98 papillomas; *7 squamous cell carcinomas 0 papillomas; 0 squamous cell carcinomas * 100 papillomas * 100 papillomas 0 papillomas 6 M 0F lung adenoma 4 M 4F lung adenoma 0 M 0F lung adenoma

Reference Masui et al., 1986 Masui et al., 1986 Ito et al., 1986

Ito et al., 1982, 1983a Williams et al., 1990b Masui et al., 1986 Ito et al., 1983b Amo et al., 1990

*Indicates statistically di€erent from controls. aAnimals were sacri®ced sequentially at 8-wk intervals from wk 8 to wk 104. bThe BHA concentration in the diet decreased from 20,000 ppm to 10,200 ppm during the processing of the pellets. cThe BHA concentration in the diet decreased from 5000 ppm to 2500 ppm during the processing of the pellets. dThe Japanese house musk shrew has no forestomach. eAll animals in this group died within 8 wk after commencement of treatment due to bleeding in the gastrointestinal tract.

the rat forestomach epithelium by Hirose et al. (1987a), although a small amount of TBQ was found by Morimoto et al. (1991). Forestomach DNA damage, as measured by single-strand breaks, was detected for TBQ, but not BHA or TBHQ (Morimoto et al., 1991); however, no DNA adducts for TBQ have been detected in rats by [32P]postlabelling (Saito et al., 1989). Thus, there is no evidence that BHA or its metabolites can react with DNA in the forestomach, which is the target organ of interest. BHA carcinogenicity studies The results of bioassays for BHA are summarized in Table 2. The ®rst reported carcinogenicity bioassay was performed by Ito et al. (1982, 1983a), using initial concentrations of 5000 ppm and 20,000 ppm BHA in the diet of 6-wk-old male and female F344 rats, later reduced to 2500 and 10,200 ppm for 2 yr. Food intake was measured, and the resulting ®nal daily doses were 98 and 414 mg/kg/day for males and 108 and 474 mg/kg/day for females. The incidences of proliferative lesions of the forestomach in the high-dose groups were 100% for hyperplasia in males and 98% in females and for papillomas, 100% in males and 96.1% in females. The incidences of forestomach squamous cell carcinoma were 34.6% in the males and 29.4% in the females. In the low-dose groups, there were no carcinomas,

only 2% papillomas in males and females and 26.0% and 19.6% hyperplasia in males and females, respectively. No neoplastic changes were seen in the forestomachs of controls. There were no BHA-related changes in occurrence of any other tumour types. Ito et al. (1986) further studied the dose±response of BHA in the induction of forestomach neoplasia in 6-wk-old male F344 rats. The doses used were 0, 1250, 2500, 5000, 10,000 and 20,000 ppm BHA in the diet for 104 wk. Hyperplasia, papillomas and squamous cell carcinomas exhibited dose-related increases, with no-observed-e€ect levels (NOELs) for BHA of 1250 ppm for hyperplasia, 5000 ppm for papillomas and 10,000 ppm for carcinomas. Ito et al. (1983b) and Masui et al. (1986) reported that 6-wk-old F344 rats and Syrian golden hamsters developed hyperplasia, papillomas and carcinomas in the forestomach when fed 20,000 ppm BHA in the diet for up to 104 wk. A dose of 10,000 ppm caused similar lesions in hamsters, but in rats, only hyperplasia and papillomas occurred. B6C3F1 mice fed 10,000 ppm BHA, which was the highest dose, had hyperplasia, papillomas and carcinomas, but the incidence of carcinomas did not achieve statistical signi®cance (Masui et al. 1986). Controls showed some hyperplasia, but no tumours. No changes were observed in the glandular portion of the stomach in any animal species. The e€ects of 12,000 ppm BHA in the diet for up to 110 wk in

1030

G. M. Williams et al.

male F344 rats, beginning at 6 or 11 wk of age, were reported by Williams (1986) and Williams et al. (1990b). By 20 months in animals that had died, only one papilloma was detected, resulting in a 4% incidence (Williams, 1986). By the end of the study when survival was 63%, a substantial incidence of papillomas occurred (Williams et al., 1990b). No signi®cant increase in any tumours of other organs, including the glandular stomach, was found, and in particular, there were no visible lesions of the oesophagus. Amo et al. (1990) have reported a study of Japanese house musk shrews, which have no forestomach. BHA was given to both males and females for 80 wk at doses of 5000, 10,000 and 20,000 ppm BHA in the diet. All animals of the high dose group died in the ®rst 8 wk from gastrointestinal haemorrhage. The low- and mid-dose groups of both sexes showed a 50±67% incidence of adenomatous hyperplasia of the lung compared with 0± 3% of controls. In addition, one low- and two middose animals had lung adenomas. There were no other signi®cant ®ndings of tumours compared with controls in any of the tissues examined. Park et al. (1990) reported hepatocarcinoma in the ®sh Rivulus ocellatus marmoratus. Neither of these studies has been con®rmed.

Studies on BHA mode of action Based on genotoxicity studies, BHA clearly does not operate through a chemical DNA-reactive mechanism. An expert panel proposed that the cancer-producing e€ects of BHA in the forestomach might be due to TBHQ formation resulting in oxidative damage to DNA (FASEB, 1994). Subsequently, TBHQ, was tested for carcinogenicity and found to be inactive (NTP, 1997). Nevertheless, the oxidative DNA damage hypothesis needs further investigation. Several reports described promoting e€ects of BHA in the forestomach when given after initiating carcinogens. Forestomach neoplasia in female F344 rats initiated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was enhanced by the subsequent administration of 5000 ppm BHA in the diet for 51 wk, compared with basal diet controls (Shirai et al., 1984). No increase in neoplastic changes was observed in the glandular stomach. Forestomach papillomas and squamous cell carcinomas were also promoted by 10,000 ppm BHA given for up to 32 wk to N-methyl-N-nitrosourea (NMU)-initiated F344 rats (Imaida et al., 1984; Tsuda et al., 1984). Takahashi et al. (1986) found a promoting e€ect in forestomach cancer in male Wistar rats given 10,000 and 20,000 ppm BHA for 32 wk after initiation with MNNG. Fukushima et al. (1987) found that feeding of 20,000 ppm BHA for 32 wk enhanced tumours of the forestomach, but not of the oesopha-

gus, in male F344 rats initiated with N,N-dibutylnitrosamine (DBN). A study of tumour promotion following initiation with several DNA-reactive initiating agents found that 4000 ppm, but not 800 ppm BHA, produced a non-statistically signi®cant increased incidence of hyperplasia and papillomas of the forestomach (Hirose et al., 1997). These reports are supported by the observation that BHA inhibits intercellular molecular transfer (Williams et al., 1990a), a property of neoplasm-promoting agents (Budunova and Williams, 1994; Trosko et al., 1990; Williams, 1981; Yamasaki, 1996). BHA speci®cally promoted MNNG-initiated squamous cell tumours in the forestomach in a lifetime study, but did not a€ect the MNNG-initiated tumours of the glandular stomach or produce adverse e€ects elsewhere at doses up to 12,000 ppm in the diet for up to 110 wk (Whysner et al., 1994; Williams, 1986). This study did not ®nd tumours of the oral cavity, oesophagus, duodenum, intestine or colon attributable to BHA. Accordingly, the evidence supports the conclusion that BHA acts as a tumour promoter only in a part of the rodent stomach, the forestomach, that does not exist in humans. Several dose levels were included in this study ranging from 60 to 12,000 ppm. A NOEL of 3000 ppm was found in this experiment for tumour promotion, which was comparable to NOELs found for other e€ects of BHA in the male F344 rat associated with tumour formation. Studies by Altman et al. (1985) revealed that epithelial damage, mild hyperplasia, in¯ammation, and an increase in mitotic activity are the initial changes produced by BHA. Later changes include hyperplasia, papilloma formation, and ®nally cancer. The hyperplastic e€ect of BHA is very pronounced in the forestomach and signi®cantly enhanced cell proliferation rates persist as long as BHA is given in the diet. Studies have demonstrated BHA-induced gap junction e€ects (Williams, 1986; Williams et al., 1990a), which are characteristic of many promoters (Budunova and Williams, 1994; Trosko et al., 1990; Williams, 1981; Yamasaki, 1996) and are reversible. The mechanism underlying BHA-induced cytotoxicity is not understood. Although it has been suggested that oxidative damage, including DNA damage, may be responsible (Iverson, 1995), Ito et al. (1991) reported that no 8-oxodeoxyguanosine or evidence of lipid peroxidation were found in the forestomach following exposure to 20,000 ppm BHA for 2 wk. Schilderman et al. (1995b) demonstrated increased levels of 8-oxodeoxyguanosine in the glandular stomach, which is not a target tissue for BHA-induced carcinogenicity or tumour promotion (Whysner et al., 1994), but did not obtain sucient DNA from the forestomach for measurement. Accordingly, further studies are required to explore the possible prooxidant e€ects of BHA, which is a property of certain antioxidants at high levels (Aruoma, 1994).

Phenolic antioxidant safety

Anticarcinogenicity studies of BHA Numerous studies have shown that BHA inhibits carcinogenic e€ects of other chemicals when given at high concentrations of 3000 ppm or greater, either before or during carcinogen administration. Some of the earlier studies have been reviewed by Wattenberg (1980) and include BHA inhibition of neoplasia in the lung, forestomach, skin, large intestine, breast and lymphatic system induced by a variety of DNA-reactive carcinogens known to require metabolic activation. Subsequent to that review, several additional studies have shown inhibition of carcinogenesis by BHA (Williams, 1993b; Williams et al., 1986). Among the studies which examined neoplasia (as opposed to preneoplasia), in approximately half of the studies, BHA treatment clearly decreased the incidence of chemical-induced neoplasms (Chung et al., 1986; McCormick et al., 1984; Reddy et al., 1983; Wattenberg, 1972; Wattenberg and Sparnins, 1979; Williams et al., 1986). In the remaining studies, only tumour multiplicity was clearly reduced by BHA. The anticarcinogenic e€ects can also be seen at doses much lower than 3000 ppm. Williams et al. (1986) have shown that BHA administered to rats at 1000 ppm starting 1 wk before a¯atoxin B1 (AFB1) administration and continuing 1 wk after cessation, decreased liver neoplasia. In a subsequent study, BHA at 125 ppm inhibited the initiation of hepatocarcinogenesis by AFB1, in rats studied over 42 wk (Williams and Iatropoulos, 1996). Thus, the e€ective chemoprotective concentrations of BHA extend below 1000 ppm to 125 ppm. There was suggestive evidence in the experiment by Whysner et al. (1994) that BHA at doses below the NOEL of 3000 ppm were protective and showed antipromoting activity. Also, at 800 ppm in the diet, BHA in the study of Hirose et al. (1997) appeared to be protective against carcinoma of the large intestine produced by the initiating agents. Presumably, in that study the exposure level for the large intestine would have been substantially lower than the administered level or that in the forestomach. These studies suggest that low doses of BHA inhibit carcinogenesis, when given prior to and during exposures to DNA reactive carcinogens. Anticarcinogenic activity at low concentrations has been suggested to be due to free radical trapping activity (Williams and Iatropoulos, 1997).

BHT genotoxicity studies Genotoxicity studies are summarized in Table 3. BHT did not cause DNA damage in Bacillus subtilis (Kinae et al., 1981) or mutation in Salmonella typhimurium (Ben-Hur et al., 1981; Brusick, 1993; McKee and Tometsko, 1979; Shelef and Chin, 1980; Williams et al., 1990a). It did not induce

1031

chromosomal aberrations in plants (Alekperov et al., 1975) or mutation and chromosomal aberrations in Drosophila melanogaster (Prasad and Kamra, 1974). In one study, it was reported to be mutagenic to cultured Chinese hamster V79 cells in the presence of an exogenous metabolic system (Paschin and Bahitova, 1984). Binding of BHT to the DNA of liver of rats exposed in vivo has been reported (Nakagawa et al., 1980), but no adduct was identi®ed. Moreover, BHT was negative for DNA repair in isolated hepatocytes (Williams et al., 1990a). BHT did not induce micronuclei in bone marrow or dominant lethal mutations in mice (Bruce and Heddle, 1979; Epstein et al., 1977). In studies in our laboratory, BHT also exhibited no evidence of mutagenicity in the Salmonella/microsome mutagenesis assay or the adult rat liver epithelial cell/hypoxanthine±guanine phosphoribosyl transferase mutagenicity assay (Williams et al., 1990a). The weight of evidence, therefore, supports the conclusion that BHT is not genotoxic.

BHT carcinogenicity studies Several reviews discuss chronic carcinogenicity bioassays of BHT in rodents (Babich, 1982; IARC, 1986b; Ito et al., 1985; Kahl, 1984; WHO, 1983, 1987). A number of chronic carcinogenicity bioassays were conducted in mice and rats by oral administration in the diet (Table 4). In one mouse study, there was no di€erence in tumour incidence among exposed and control groups. In another mouse study, 7500 ppm BHT increased the number of lung tumours (Clapp et al., 1974). When larger number of animals were used by the same investigators, this ®nding was not con®rmed (Clapp et al., 1978). In one of the two other mouse studies, alveolar/bronchiolar neoplasms were present in the mid (3000 ppm) but not in the high (6000 ppm) dose BHT group (NCI, 1979). In the other, BHT at up to 5000 ppm did not induce any tumours (Shirai et al., 1982). In one study in Wistar rats, no increase in tumour incidence was seen with BHT at 10,000 ppm (Deichmann et al., 1955). In the same rat strain, an increased incidence of pituitary adenoma in females was present at the lower (2500 ppm) but not at the higher (10,000 ppm) dose level (Hirose et al., 1981), but the incidence of this and all other tumours was not signi®cantly di€erent from that in controls. The pituitary tumour ®nding also was not con®rmed in a subsequent study (Olsen et al., 1986) in Wistar rats. In an unique study by Olsen et al. (1986), BHT was administered to Wistar rats prenatally, during nursing and for 144 wk. F0 generation rats of each sex were fed BHT at 0, 375, 1500 or 7500 ppm in the diet for 13 wk prior to mating. After mating, the F0 males and after weaning, the F0 females were removed from the study. After weaning, F1 rats were continued on

1032

G. M. Williams et al. Table 3. Genotoxicity studies of butylated hydroxytoluene Results In vitro

Endpoint Test system

In vivo

Without activation

With activation

Reference

DNA interaction DNA adduct formation DNA adducts/rat/F344 forestomach, stomach DNA damage DNA binding/calf thymus DNA C (BHT-PMS) DNA binding/calf thymus DNA B(PB/3 MC-rabbit- Cyt-P450 and PB/3MC-rabbit-Micr) DNA binding/human bronchial cells DNA binding/Sprague±Dawley rat M/liver DNA DNA binding/Bacillus subtilis rec + /ÿ Sister chromatid exchange CHO cells

±1

Strand breaks and repair Rat hepatocyte primary/rat/F344 hepatocytes culture/DNA repair/ DNA repair/human lymphocytes DNA repair/human skin ®broblasts DNA repair/Chinese hamster V79

NA

NA

3

ND

±

Fukayama and Hsieh, 1984

NA

ND

±

Belvedere et al., 1980

NA ± NA NA

± NA ± ±

ND NA ND ±

Harris et al., 1976 Goodman et al., 1976 Kinae et al., 1981 Williams et al., 1990a

NA

ND

±

Williams et al., 1990a

ND ND ND

Daugherty et al., 1978 Wei et al., 1981 Goodman et al., 1976

NA NA NA

Mutagenicity assays Reverse mutation Salmonella typhimurium/TA98,100,1537,1538 Salmonella typhimurium/TA98, 100 Salmonella typhimurium/TA 100 Salmonella typhimurium/TA97,100,102,104 Salmonella typhimurium/TA98,100,1535,1537,1538 Salmonella typhimurium/TA98, 100 Salmonella typhimurium/TA98 Gene mutation HGPRT/adult rat epithelial cells Sex-linked, recessive lethal Sex-linked gene mutation/Drosophila melanogaster Sex-linked gene mutation/Drosophila melanogaster

2

NA

NA NA NA NA NA NA NA

± ND ± ± ± ± ±

ND +4 ND ± ± ± ±

McKee and Tometsko, 1979 Shelef and Chin, 1980 Ben-Hur et al., 1981 Hageman et al., 1988 Williams et al., 1990a Yoshida, 1990 Dertinger et al., 1993

NA

±

ND

Williams et al., 1990a

5

NA NA

NA NA

Kamra, 1974 Prasad and Mazar-Barnett and MunÄoz 1980

(+) ±

NA NA

NA NA

Kamra, 1973 Mazar-Barnett and MunÄoz 1980

± ± NA + ± ±

NA NA (±)6 NA NA NA

NA NA ND NA NA NA

Prasad and Kamra, 1974 Sankaranarayanan, 1983 Shamberger et al., 1973 Sheu et al., 1986 Sheu et al., 1986 Sheu et al., 1986

(+) ±

Cytogenicity assays Chromosomal aberrations Drosophila melanogaster/translocations Drosophila melanogaster/translocation (dominant lethal) Drosophila melanogaster/chromosome loss (ring X) Drosophila melanogaster/chromosome loss (X) Human lymphocytes/aberrations (structural) SD rats germ cells), male/dominant lethal assay CD- 1 mice (germ cells), male/dominant lethal assay CD- 1 ni ice germ cells), male/heritable translocation assay 1

Indicates negative test result. 2NA = not applicable. 3ND = indicates test was not done. 5 (+) Indicates a weak positive test result.6High number of aberration in the negative control.

the same doses, except that the top dose was lowered to 3750 ppm because of concern for nephrotoxicity seen in F0 females at 7500 ppm. BHT at 3750 ppm, but not lower doses, increased the incidences of benign hepatocellular adenoma in both sexes and of hepatocellular carcinoma only in males. For both tumour types, the earliest occurrence of tumour was after 114 wk in both sexes. In this study, there was a 41% reduction in the body weight of F1 pups in high dose groups at weaning, and a 21% reduction in males, and a 16% reduction in females in body weight gain in high dose rats from wk 5 until the end of exposure. Thus, increases in liver neoplasia were found only at ex-

Ito et al., 1991

4

+Indicates a positive test result.

posures that exceeded the MTD and only after a duration beyond that of conventional studies. With such drastic growth rate reduction at 3750 ppm, there is likely to be under-utilization of protein and triglycerides in the liver, with gluconeogenesis and hypermetabolism leading to glutathione depletion, cell death and hepatocellular compensatory hyperplasia. Such e€ects may in part explain the hepatocellular neoplasia. Additionally, the very high mortality in the control animals compromised the statistical comparison to BHT exposure groups. Liver neoplasia was not observed in any other study in rats, including in the same Wistar strain receiving 10,000 ppm (Hirose et al., 1981) or in

M50

M100, F50 M50, F50

M51, F52

M15, F15

M50, F50

M57, F57

M100, F100

Mouse BALB/c

Mouse BALB/c Mouse B6C3F1

Mouse B6C3F1

Rat Wistar

Rat F344

Rat Wistar

Rat Wistar1

Exposure included in utero/25 months post partum;

1

Sex and no. per group

Species/strain

2

18 18 18 25 25 25 23 23 23 24 24 24 24 25 25 24 24 24 25 25 25 25

Duration (months)

Results (% incidence of neoplasms)

3F pituitary adenoma; 7F pit. carcinoma; M 6F pituitary adenoma; 3F pit. carcinomas 0F pituitary adenoma, 3F pit. carcinomas *18 M *12F hepatocellular adenoma; *8 M 2F hepatocarcinoma 5 M 6F hepatocellular adenoma, 1 M 0F hepatocarcinoma 1 M 3F hepatocellular adenoma; 0 M 0F hepatocarcinoma 1 M 2F hepatocellular adenoma; 1 M 0F hepatocarcinoma

2

No signi®cant exposure-related tumours observed

No signi®cant exposure-related tumours observed

*64 pulmonary adenomas; 12.5 reticulum cell sarcomas 24 pulmonary adenomas; 56 reticulum cell sarcomas No observed pulmonary tumours and no reticulum cell sarcomas 35 M 14 F alveolar/bronchiolar neoplasms 42 M 35 F alveolor/bronchiolar neoplasms 35 M 5 F alveolor/bronchiolar neoplasms No signi®cant exposure-related tumours observed

no exposure-related tumours in males. *Signi®cant at P < 0.05 or less.

7500 Control 7500 6000 3000 Control 5000 1000 200 10000 8000 5000 2000 6000 3000 10000 2500 Control 3750 1500 375 Control

Exposure (ppm)

Table 4. Results from oral (diet) chronic bioassays with butylated hydroxytoluene

Olsen et al., 1986

Hirose et al., 1981

NCI, 1979

Deichmann et al., 1955

Shirai et al., 1982

Clapp et al., 1978 NCI, 1979

Clapp et al., 1974

Reference

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F344 rats, in which BHT exposure at 3000 and 6000 ppm was not associated with any tumours (NCI, 1979). Thus, the ®ndings in the study by Olsen et al. (1986), which have not been con®rmed, may be attributable to study conditions, not the administration of BHT. Overall, these data do not provide convincing evidence that BHT has carcinogenic activity in either mice or rats. Interestingly, 2,2'-methylenebis (4methyl-6-tert-butylphenol), an antioxidant which is essentially two molecules of BHT and has all attributes of BHT, was also non-carcinogenic at up to 0.1% for 18 months to Wistar rats (Takagi et al., 1994). Studies on BHT mode of action BHT is not genotoxic or patently carcinogenic. Several reports indicated neoplasia promoting activity when given after an initiating carcinogen for mouse lung (Witschi et al., 1977) and colon (Lindenschmidt, 1986), and rat liver (Maeura and Williams, 1984) and urinary bladder (Imaida et al., 1984). Consistent with these observations, BHT inhibits intercellular molecular transfer (Williams et al., 1990a), a property of neoplasm-promoting agents (Budunova and Williams, 1994; Trosko et al., 1990; Williams, 1981; Yamasaki, 1996). In a study in our laboratory, however, BHT (5000 ppm) had no promoting or syncarcinogenic e€ect on diethylnitrosoamine-induced mouse liver neoplasia by 38 wk, whereas under the same conditions, phenobarbital (500 ppm) acted as an enhancer (Tokumo et al., 1991). Thus, BHT at high doses can exert tumour-promoting e€ects, apparently due to blocking of cellular communication channels, but this does not seem to be sucient for de®nite enhancement of tumour development when administered on its own. Anticarcinogenicity studies of BHT BHT has been shown to inhibit the carcinogenicity of a variety of carcinogens in di€erent tissues in mice and rats when given at high concentrations of greater than 3000 ppm (Wattenberg, 1985; Williams, 1993b; Williams and Iatropoulos, 1997). BHT inhibition of liver and mammary gland carcinogenesis in rats (Ulland et al., 1973), as well as colon carcinogenesis in rats (Weisburger et al., 1977), has been demonstrated. Additionally, in studies that examined liver neoplasia, BHT inhibited the hepatocarcinogenicity of both AFB1 and 2acetylamino¯uorene in rats (Maeura et al., 1984; Williams et al., 1986, 1991). In contrast to these high dose studies, Williams et al. (1986) have shown that BHT administered to rats at 1000 ppm, starting 1 wk before AFB1 administration and continuing 1 wk after cessation,

decreased liver neoplasia. Also, in a subsequent study, BHT at 125 ppm inhibited the initiation of hepatocarcinogenesis by AFB1 in rats studied over 42 wk (Williams and Iatropoulos, 1996). In another study by Williams et al. (1991), BHT at 100 ppm fed together with AAF at a low concentration of 50 ppm inhibited the induction of liver altered foci and reduced the incidence of liver carcinomas by wk 76. Thus, the e€ective chemoprotective concentrations of BHT extend below 1000 ppm to 100 ppm (Williams and Iatropoulos, 1997). Activity at such low concentrations has been suggested to be due to free radical trapping activity (Williams and Iatropoulos, 1997).

Conclusions BHA, at high doses above 3000 ppm, has been found to induce forestomach squamous cell carcinomas in rodents, but not glandular cell or other types of neoplasms in the glandular stomach. BHA is not DNA-reactive, and the epigenetic mechanism of tumour formation appears to involve tumour promotion. Experimental studies support the concept that BHA fed at above 3000 ppm in the diet causes cellular damage and proliferation in the forestomach, which are critical events underlying the promotion of cancer. Also, BHA inhibits cell±cell communication. Humans do not have a forestomach and therefore are predicted to be much less sensitive than rodents to the e€ects of BHA. Moreover, the exposures to humans are well below those producing the epigenetic e€ects in rodents, such as cell proliferation. We conclude, therefore, that BHA is a rodent carcinogen which is species-speci®c for all practical purposes, and not relevant to humans. Moreover, human exposures (<0.1 mg/kg/day) are well below the lowest e€ect level of 230 mg/kg/day for hyperplasia, which is the most sensitive e€ect in rodents associated with forestomach tumorigenesis (Whysner and Williams, 1996; Williams and Whysner, 1995). This supports the decisions of authorities that have reviewed these data to recommend continued use of BHA (Iverson, 1995; JECFA, 1996), despite the International Agency for Research on Cancer evaluation that BHA is possibly carcinogenic to humans (IARC, 1987). Furthermore, at concentrations as low as 125 ppm, which is closer to food additive levels, BHA exhibits anticarcinogenic properties. Based on the entire body of evidence and data from mechanistic studies, BHT is not genotoxic or reproducibly carcinogenic, although at high doses, 250 mg/kg/day or greater, it was associated with some uncon®rmed increases in spontaneous neoplasms and, like BHA, has some tumour-promoting activity. The overall evaluation of IARC was that BHT is not classi®able as to its carcinogenicity to humans (IARC, 1987). Based on these consider-

Phenolic antioxidant safety

ations, we support the conclusion of authorities that the use of BHT as a food additive does not pose any cancer hazard to humans (JECFA, 1996). Moreover, human exposures (<0.1 mg/kg/day) are at least 1000-fold below those associated with any possibly neoplastic e€ects in rodents. Also, like BHA, at concentrations as low as 100 ppm, BHT exerts anticarcinogenic properties. Thus, we conclude that BHA and BHT at current food additive levels represent no cancer hazard and may actually be reducing human cancer, as previously suggested (Williams, 1994), as well as providing other potential health bene®ts (see Williams et al., 1993). AcknowledgementsÐThe authors wish to thank Melissa Mohan for assistance in preparing tables and Nancy Rivera for typing the manuscript.

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