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Safety aspects of food preservatives a
D. V. Parke & D. F. V. Lewis
a
a
Division of Molecular Toxicology, School of Biological Sciences , University of Surrey , Guildford, Surrey, GU2 5XH, UK Published online: 10 Jan 2009.
To cite this article: D. V. Parke & D. F. V. Lewis (1992) Safety aspects of food preservatives, Food Additives & Contaminants, 9:5, 561-577, DOI: 10.1080/02652039209374110 To link to this article: http://dx.doi.org/10.1080/02652039209374110
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FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 5, 5 6 1 - 5 7 7
Safety aspects of food preservatives D. V. PARKE and D. F. V. LEWIS
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Division of Molecular Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK The use of food preservatives, such as benzoic acid, nitrites, and sulphites, as antimicrobials, and butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid and tocopherols, as antioxidants, has probably changed food production patterns and eating habits more than has the use of any other class of food additive. These food preservative chemicals confer substantial benefits on man, not only by the preservation and increased palatability of food, but also by affording protection against the pathological effects of reactive oxygen species (ROS) which are associated with cancer, cardiovascular disease and aging. Nevertheless, although most preservatives are now considered to be without potential adverse effects and are classified as GRAS, there have been problems concerning the safety of some of these chemicals, including the possibility of allergies from benzoic acid and sulphites, the formation of carcinogenic nitrosamines from nitrites, and the possible rodent carcinogenicity of BHA and BHT. The mechanisms of this toxicity at high dosage, the roles of the cytochromes P450, and the generation and scavenging of ROS in the toxicity of these chemicals, are reviewed and discussed. Keywords: food preservatives, antimicrobials, antioxidants, safety evaluation, antioxidant synergists, COMPACT
Introduction
The preservation of food by drying, smoking, pickling and salting, and the making of fruit conserves with sugar, were among the earliest examples of food processing, first introduced centuries ago. But whereas meat, fish, fruits and vegetables could be preserved by these means, which protected against microbial decomposition, fats of all kinds could not similarly be protected against rancidity, and unpleasant flavours were masked by the liberal use of peppers, onion, garlic, ginger, and other highly flavoured spices. The introduction of benzoic acid and other acids, nitrates and nitrites, and sulphites, as antimicrobials, and of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and other phenols, together with ascorbic acid and tocopherols as antioxidants (see table 1), has had a greater effect on the transformation of food production patterns and eating habits, than has any other class of food additive. Gone are the fears of eating meat that was not cooked fresh lest one developed food poisoning or botulism, and problems associated with the rancidity of fat-containing foods, from butter and meat to ice-cream and biscuits, appear to have gone forever. The mechanism of antimicrobial preservatives is uncertain and diverse. Propionic acid and sorbic acid, benzoic acid and />-hydroxybenzoic acid, all probably act by inhibiting microbial growth by inhibiting bacterial enzymes and by effecting a slight decrease of pH, whereas nitrites and sulphites form complexes with food components which are toxic to microorganisms, but not to mammalia. 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
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D. V. Parke Table 1. Classes of food preservatives. Antimicrobials Propionic acid, Na and K salts" Sorbic acid, Na, K and Ca saltsa Benzoic acid, Na and K saltsa p-Hydroxybenzoate Me, Et, Pr, Bu esters2 Nitrate, nitrite, Na and K salts Sulphites and sulphur dioxide"
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Antioxidants (radical scavengers) Ascorbic acid, Na salt and palmitate" Isoascorbic acid, Na salt Tocopherolsa BHA (butylated hydroxyanisole)a BHT (butylated hydroxytoluene)" Gallates (propyl, octyl, dodecyl)a ter/.-Butylhydroquinone (TBHQ) Antioxidant synergists (metal chelators) EDTA (ethylenediamine tetraacetic acid, and Ca and Na salts)" Citric acid (isopropyl and stearyl esters)" Phosphoric acid" Tartaric acid" a
GRAS substances.
The mechanism of action of antioxidants, such as ascorbic acid, tocopherol, BHT and BHA, is far better known, since all of these are oxygen radical scavengers, and it is now appreciated that oxygen radical-mediated lipid peroxidation is the mechanism by which fats and fat-containing foods undergo spoilage. This process of lipid peroxidation is readily catalysed by minute traces of iron and other transition metals contained naturally in foodstuffs, and a third group of preservatives considered by some as antioxidants, and by others as antioxidant synergists, are ethylenediamine tetraacetic acid (EDTA), citric acid, and other metal chelators, which inhibit the autoxidative action of iron and other trace metals (see table 1). These antimicrobial and antioxidant properties of food preservatives confer substantial benefits on man, not only by way of preservation and increased palatability of his food, but also by affording protection against the general background exposure to oxygen radicals that results in aging and degenerative disease, such as cancer and cardiovascular problems. It was first demonstrated more than 20 years ago that antioxidants such as a-tocopherol and selenium decreased the occurrence of cancer in mice induced by polycyclic aromatic hydrocarbons (Shamberger 1970, Shamberger et al. 1973). Wattenberg (1972) similarly reported that the synthetic antioxidants BHA, BHT and ethoxyquin were highly effective in preventing mammary and forestomach cancer in rodents caused by benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. BHA also decreased diethylnitrosamine, 4-nitroquinoline oxide, and urethane carcinogenicity, and the antitumorigenic effects of synthetic antioxidants against 2-acetamidofluorene and nitrosamines has been confirmed by other workers (Calabrese 1981). More recently, BHT has been shown to have an antiatherogenic effect in cholesterol-fed rabbits (Bjorkhem et al.
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1991). However, it has also been reported that BHT may promote the tumorigenic effects of 2-acetamidofluorene (Peraino et al. 1977) and urethane (Witschi et al. 1977), although only at doses 103 to 104 times the level of the daily consumption of BHT as a food preservative. The mechanisms of protection against chemical toxicity/carcinogenesis are thought to involve oxygen radical scavenging and increased detoxication, and the mechanisms of promotion at high dosage may involve induction of cytochromes P450with possible futile cycling and oxygen radical generation. Nevertheless, despite the obvious major health advantages to the consumer from the use of food preservatives, which is in marked contrast to the questionable benefits of food colourants and flavours, these additives have to be evaluated for safety by standard toxicology programmes of short-term and long-term studies in rodents and other experimental animals, and observations in man. In the course of these studies, benzoic and sorbic acids have been associated with various allergic responses in man, as have sulphites; and nitrates and nitrites have been associated with methaemoglobinaemia, and also with the formation of nitrosamines and consequent potential tumorigenesis (see table 2). Similarly, BHT has been claimed to be a mouse liver carcinogen, BHA a rat stomach carcinogen and the alkyl gallates also have been associated with potential toxicity (see table 2). However, all of these manifestations of toxicity are seen only at high dosage, and it has been considered that they have relatively little significance for safety in man, so that the obvious benefits of food preservatives far outweigh any risk of potential toxicity, and most preservatives now in use are classified as GRAS (generally regarded as safe). Nevertheless, some preservatives, used 40 or more years ago, have been considered to lack appropriate safety as judged by animal toxicological studies, and consequently have been withdrawn from use, or their use limited to certain foodstuffs. For example, diethylpyrocarbonate, once used as a preservative in wine, fruit juice, and soft drinks, was found to have a potential to form urethane in the presence of ammonia, amino acids and proteins, and as urethane is a carcinogen, albeit a weak one even at high dosage, the use of diethylpyrocarbonate as a food additive was revoked by the WHO in 1974. Similarly, nordihydroguaiaretic acid, a food antioxidant, was found to be metabolized to the corresponding orf/joquinone, a likely cause of the faecal haemorrhage and ulceration, and mesenteric cysts, seen
Table 2. Suspected toxic effects of food preservatives. Preservative
Toxic effects
Antimicrobials
Benzoic acid Sorbic acid Sulphites Nitrites, nitrates
Allergies Contact allergy Allergies Carcinogenesis (nitrosamines) Methaemaglobinaemia
Antioxidants
BHT BHA
Mouse liver carcinogen Rat stomach carcinogen Allergy, foetal and neonatal toxicity
Alkyl gallates
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in long-term rat studies at high dosage of this chemical. The alkyl gallates, used as antioxidants in margarine, oils, meats and sausage, were found to give rise to dermatitis in bakers and others handling this material, and to buccal sensitization in some consumers; it was considered that a major cause of this sensitization was the consumption of large amounts of beer and other beverages treated with this preservative, so the use of alkyl gallates as antioxidants for beer and beverages was discontinued. In contrast to this, despite a major public outcry some 20 years ago concerning the potential carcinogenicity of nitrosamines formed by the treatment of meats and bacon with nitrites, the benefit of this class of preservative in preventing botulism, was considered to be of far greater importance to human health than the very low probability of nitrite-induced malignancy.
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Antimicrobials
The antibacterial, fungistatic activity of the antimicrobials, is probably mostly due to the inhibition of microbial enzymes, such as the dehydrogenases. Propionic acid Administered to rats at 4% of the diet, propionic acid produced tumours in rat forestomach, but at 0-4% of the diet no tumours were formed. Propionic acid exhibits no mutagenic activity, and administered to rats, mice or hamsters at 4% diet for seven days resulted in damage and cellular proliferation in the forestomach epithelium of all three species; however, at 0*4% diet no epithelial damage or proliferation occurs. It was concluded that the rat forestomach tumours probably resulted from repeated low level damage and repair caused by the high dosage of propionic acid (Harrison et al. 1991). Propionic acid is considered as GRAS and is widely used as a preservative in beverages, cheese, bakery products, jams, desserts and meat products. Sorbic acid (trans, trans-2,4-hexadienoic acid) Occurs naturally in the berries of Mountain Ash, and is widely used, also as its sodium and potassium salts, as a preservative in cheeses and flour confectionery. It manifests its fungistatic activity through the inhibition of alcohol dehydrogenase and other microbial dehydrogenase systems. Sorbic acid is rapidly metabolized by /3-oxidation to carbon dioxide, and consequently shows no overt toxicity or carcinogenicity. Benzoic acid Used as an antimicrobial in beverages, and as the calcium and sodium salts in margarine, benzoic acid is rapidly conjugated with glycine and glucuronic acid and then excreted, with no discernible toxicity, although the cat is more sensitive than other species probably because of impaired conjugation. Benzoic acid has been associated with occasional hypersensitivity reactions in humans, which have been known to include severe anaphylaxis (Michils et al. 1991). p-Hydroxybenzoic acid Used mainly as its ethyl, methyl, and propyl esters />-hydroxybenzoic acid has shown no overt toxicity or carcinogenicity in long-term animal studies; the butyl ester is associated with hypersensitivity in humans and may exacerbate dermatitis, but the lower alkyl esters do not similarly cause hypersensitivity. The ethyl, methyl,
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and propyl esters are completely and rapidly hydrolysed to the free acid and then conjugated with glycine, and with glucuronic acid to form both ether and ester glucuronides. The acyl glucuronides of aromatic carboxylic acids are metastable and undergo deglucuronidation with the simultaneous aroylation of endogenous proteins, which may lead to neoantigen formation and consequent allergies (Parke et al. 1991). Nitrites and nitrates (sodium and potassium salts) These are antimicrobial preservatives used in the curing of meats to fix colour, develop flavour and to inhibit contamination by Clostridium botulinum, and also in the production of bacon, and the preservation of fish and poultry. Concern for the safety of nitrites and nitrates has been the consequence of acute toxic effects resulting from oxidation of haemoglobin to methaemoglobin to which infants are particularly susceptible (WHO 1978), and the formation of the potentiallycarcinogenic nitrosamines and nitrosamides, in food and in the digestive tract, after food consumption (Daniel 1985). Nitrates are also present in drinking water and vegetables, and these natural sources are considered to produce 5 to 100 times more nitrite in vivo then does the ingestion of preserved meats and bacon. An FDA estimate of the annual cancer risk from the consumption of cured meat was given as 1 in 106, which has to be offset against the greater risk of botulism in the absence of nitrate preservatives. Furthermore, the potential to form nitrosamines from nitrite may be decreased by addition of ascorbic acid, which inhibits the nitrosation of amines and amides by converting nitrite to nitrous oxide, but does not impair the protection afforded by nitrite against Clostridium botulinum in food (Walters 1985). Ascorbic acid, a powerful antioxidant, is naturally secreted into the stomach and is highly protective against nitrosation and gastric cancer (Sobala et al. 1989); sorbic acid, like ascorbic acid, also reacts rapidly with nitrite and inhibits the formation of nitrosamines from nitrite and secondary amines. Naturally-occurring phenolics (jo-hydroxybenzoic acid, propyl gallate) and synthetics (BHA and BHT) present in food can trap nitrite thereby also inhibiting nitrosamine formation, but paradoxically may also enhance nitrosation by forming active nitrosating species (Stich and Rosin 1982). Dimethylnitrosamine and diethylnitrosamine, and other hepatocarcinogenic nitrosamines induce oxidative stress and hepatic lipid peroxidation in rat, whereas the oesophageal carcinogen Af-nitrosomethylbenzylamine, which is not hepatocarcinogenic, does not give rise to hepatic lipid peroxidation (Ahotupa et al. 1987). The levels of nitrosamine-induced lipid peroxidation are seen to parallel their hepatocarcinogenicity; and oxygen radical production results in the formation of 8hydroxydeoxyguanosine in DNA, which is considered to be the probable cause of mutations and malignancy (Bartsch et al. 1989). In addition to enzymic demethylation, the denitrosation of dimethylnitrosamine has also been shown to be a major metabolic pathway in rat (Streeter et al. 1990), and cytochrome P450 IIE1 of rat liver microsomes catalyses both the dealkylation and denitrosation of dimethylnitrosamine and diethylnitrosamine, the metabolic activation of these carcinogens, and the production of oxygen radicals (Yoo et al. 1990). This is a good indication that dialkylnitrosamine carcinogenicity is mediated, at least in part, by oxygen radicals, and would therefore be a much greater problem in small rodents than in man due to the high susceptibility of rats and mice to oxygen radical toxicity (Parke and Ioannides 1990a);
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Sulphites Sulphur dioxide, as sodium sulphite or metabisulphite, is widely used as a preservative in wine, fruit juices, dried fruit and vegetables. Sulphites are potent inhibitors of lactate dehydrogenase and other bacterial dehydrogenases, and this is thought to be the basis of the antimicrobial action of sulphite. Sulphites are highly reactive and interact in many ways with food constituents (Walker 1985a). They cleave protein disulphide bonds yielding a thiol and an 5-sulphonic acid; they form complexes with aldehydes, ketones and sugars, and interact with nucleic acids and vitamins. They are strongly bound by plasma proteins in the form of S-sulphonates which are gradually cleared from the blood into the urine as sulphates. Small amounts of sulphite are formed naturally in intermediary metabolism, in the catabolism of cysteine to pyruvate plus sulphur dioxide, and sulphite is readily metabolized to sulphate by sulphite oxidase and thus shows no overt toxicity in experimental animal studies (Daniel 1985). However, at high dietary concentrations, sulphites cause ulceration of the forestomach, and necrosis and inflammation of the glandular stomach of rats (Walker, 1985b). Til et al. (1972) showed that at 4% of the diet, sulphite produced haemorrhagic erosions, 6% of diet caused inflammation, and 8% resulted in necrosis, in the stomach of rats. Sulphites have a potential to convert the base cytosine (present in DNA and RNA) into uracil (present only in RNA) and hence may cause point mutations, but this effect has been seen in cells and tissue cultures only at very high concentrations of >1000ppm (0-1%), and the relevance to human health is questionable. Sulphur dioxide is a co-carcinogen for benzo(a)pyrene in the respiratory tract of rodents, by enhancing the mutagenicity of the diol-epoxide reactive intermediate, partly by depletion of glutathione (Reed et al. 1990). Mutagenicity studies in mammals in vivo were negative, as were rodent carcinogenicity studies, and three-generation studies in rats, with dietary thiamine supplements, showed a no-effect level of 0-125% sodium metabisulphite (equivalent to 70 mg SO2/kg body wt/day) (WHO 1987). The treatment of food with sulphites destroys its thiamine content, but sulphite administered to humans at 400 mg per day for 25 days, in wine and other beverages, did not adversely affect the thiamine status of these individuals or result in any adverse effect. However, a single dose of 4 g of sodium sulphite given to humans produces toxic symptoms of gut irritation, vomiting and diarrhoea, in the majority of cases. The widespread practice, particularly in the United States, of using sulphite sprays to enliven salad bars in restaurants has given rise to public aversion to the use of sulphites in freshening foods, and has been blamed for a widespread syndrome of so-called 'sulphite allergy'. Biphenyl This is used as a fungistat on citrus fruit; it produces polyuria when fed to rats, and inhibits growth, possibly by depletion of glutathione. It is extensively metabolized to 4-hydroxybiphenyl, other hydroxylated products and to mercapturic acids (Parke 1968). Dehydroacetic acid (DHA, 3-acetyl-6-methyl-l,2-pyran-2,4 (3H)-dione) DHA has been used as a fungicide and bactericide in food wrappings. Its use is now discontinued because of its impairment of kidney function, and its binding to tissue proteins through the reactive carbonyl group in the side-chain. It is
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extensively metabolized in rat and rabbit to hydroxy-DHA, triacetic lactone, triacetic lactone 3-carboxylic acid, and various imino analogues (Parke 1968).
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Tetrachloroisophthalonitrile (BRA VO; 2,4,5, 6-tetrachloro-3-cyano-benzonitrile) TCPN (BRAVO) is a bactericide, nematocide and fungicide, used at one time as a fungicide in the preservation of fruit and vegetables, but discontinued when an IARC carcinogenesis assay showed clear evidence of potential carcinogenesis in the rat. Smoke flavourings (liquid smoke) These are produced by fractionation of wood smoke condensate and purification to remove toxic products such as polycyclic aromatic hydrocarbons. The major components are phenolic compounds (0-1 to 16%, as 2,6dimethoxyphenol), carbonyls (2 to 17% as heptaldehyde) and carboxylic acids, with a benzo(a)pyrene content of < 10 jtg/kg. The material is used as a flavouring agent and in the preservation of bacon, poultry, cheese and sausage (JECFA 1988). The polycyclic hydrocarbon content is determined by chromatographic procedures, which would not necessarily detect or quantify other potentially toxic/carcinogenic constituents such as dioxins and polychlorobiphenyls. In view of the known highly potent carcinogenic constituents of smoke it would seem prudent to use a more reliable and comprehensive analytical procedure to monitor the safety of this product, such as the Enzyme Induction Analysis for Chemical Toxicity (ENACT), which is based on the observed induction of cytochrome P4501 (CYP1) and other carcinogen-activating enzymes (Parke, 1990, Parke and Ioannides 1990b). Antioxidants These substances are added to fats to prevent rancidity, and include ascorbic acid, the tocopherols, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and the alkyl gallates. Ascorbic acid and isoascorbic acid (erythorbic acid) These are antioxidant, oxygen radical scavengers; ascorbate reduces the peroxyl radicals that propagate lipid peroxidation, and reduces the oxidized form of the lipid antioxidant, vitamin E thereby maintaining the antioxidant potential of the latter. However, in the presence of metals, ascorbic acid has pro-oxidant activity, but this is eliminated by EDTA (Laudicina and Marnett 1990). These compounds are considered to be natural nutrients and, as they show no overt toxicity in long-term rodent studies, are widely used as antioxidants in foods and drink (WHO 1972, 1991). Ascorbic acid is also used as the palmitate and stearate in margarine, bread and breakfast foods. Tocopherols These forms of vitamin E, the major natural antioxidants in vegetable oils, are widely used as antioxidant preservatives in bacon, fats and poultry, and are considered as GRAS. a-Tocopherol has been shown to be non-mutagenic and noncarcinogenic in animal studies, but there is evidence that an excessive intake of a-tocopherol could cause haemorrhage. Clinical studies indicate that, generally, intakes of 85% of 3-tert.-butyl-4-hydroxyanisole and 0-25). The data for the food preservatives (indicated by x) are shown in table 5. Data for other chemicals (indicated by •) are published elsewhere (Lewis et al. 1989a,b). Abbreviations are: DEN, diethylnitrosamine; DMN, dimethylnitrosamine; TBHQ, terf.-butylhydroquinone; TCDD, 2,3,7,8-tetrachlorodibenzo-/>-dioxin; TCPN, tetrachloroisophthalonitrile.
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promoting oxygen radical formation and oxidative stress. In contrast, P450 IIB is mostly concerned in the detoxication of chemicals. The COMPACT data and the predicted cytochrome P450 specificity for a number of preservatives are given in table 5, and the COMPACT ratios are plotted in figure 2. From the figure and table it may be seen that tetrachloroisophthalonitrile, once a food fungicide, has the highest COMPACT ratio (0-56) and has COMPACT data characteristics of a P450 I substrate; it is significant that this chemical has been shown to be carcinogenic in rodents. Gallic acid (0·48) and biphenyl (0-47) similarly, have high COMPACT ratios; neither is carcinogenic, but biphenyl exhibits toxicity, probably through its depletion of tissue glutathione, and gallic acid causes hypersensitivity reactions, similar to other P450 I substrates of low planarity (e.g. benoxaprofen and tienilic acid) (Parke et al. 1991). The nitrosamines, like TBHQ and dehydroacetic acid, all have the COMPACT characteristics of P450 IIE1 substrates and consequently will be associated with oxygen radical generation at high dosage, and exhibit toxicity/carcinogenicity in rodents that probably has little significance for man. Acknowledgements
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chelation and diuresis on iron-nitrilotriacetate-induced lipid peroxidation in rats and mice. Xenobiotica, 20, 879-886. REED, G. Α., RYAN, M. J., and ADAMS, K. S., 1990, Sulfite enhancement of diolepoxide mutagenicity: the role of altered glutathione metabolism. Carcinogenesis, 11, 1635-1639. SHAMBERGER, R. J., 1970, Relationship of selenium to cancer. I. Inhibitory effect of selenium on carcinogenesis. Journal of the National Cancer Institute, 44, 931-936. SHAMBERGER, R. J., BAUGHMAN, F. F., CALCHERT, S. L., WILLIS, C. E., and HOFFMAN, G. E.,
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Carcinogen-induced chromosomal breakage decreased by antioxidants. Proceedings of the National Academy of Sciences, USA, 70, 1401-1403. SKAARE, J. U., and NAFSTAD, I., 1979, The distribution of l4C-ethoxyquine in rat. Acta Pharmacologica et Toxicologica (Copenhagen), 44, 303-307. SOBALA, G. M., SCHORAH, C. J., SANDERSON, M., DIXON, M. F., TOMPKINS, D. S., GODWIN, P., and
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C. T., and KEEFER, L. K., 1990, Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat. Cancer Research, 50, 1144-1150. TAKAHASHI, O., and HIRAGA, K., 1978, Dose response study of the hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats. Toxicology and Applied Pharmacology, 43 399-406.
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Short- and long-term feeding studies in pigs. Food and Cosmetics Toxicology, 10, 463-473. WALKER, R., 1985a, Sulphur dioxide: A fugitive additive. Food Toxicology: Real or Imaginary Problems, edited by G. G. Gibson and R. Walker (London: Taylor & Francis), pp. 355-367. WALKER, R., 1985b, Sulphiting agents in foods: some risk/benefit considerations. Food Additives and Contaminants, 2, 5-24. WALTERS, C. J. 1985, Nitrosamines--can the hazard be controlled? Food Toxicology--Real or Imaginary Problems?, edited by G. G. Gibson and R. Walker (London: Taylor & Francis), pp. 342-354. WATTENBERG, L. W., 1972, Inhibition of carcinogenic and toxic effects of polycyclic hydrocarbons by phenolic antioxidants and ethoxyquin. Journal of the National Cancer Institute, 48, 1425-1430. WATTENBERG, L. W., 1978, Inhibition of chemical carcinogenesis. Advances in Cancer Research, 26, 197-226. WILLIAMS, G. M., MCQUEEN, C. Α., and TONG, C., 1990a, Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. I. Genetic and cellular effects. Food and Chemical Toxicology, 28, 793-798. WILLIAMS, G. M., WANG, C. X., and IATROPOULOS, M. J., 1990b, Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. II. Chronic feeding studies. Food and Chemical Toxicology, 28, 799-806. WITSCHI, H. P., MALKINSON, A. M., and THOMPSON, J. Α., 1989, Metabolism and pulmonary toxicity
of butylated hydroxytoluene (BHT). Pharmacology and Therapeutics, 42, 89-113. WITSCHI, H. P., WILLLIAMSON, D., and LOCK, S., 1977, Enhancement of urethane tumorigenesis in
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Safety aspects of food preservatives a
D. V. Parke & D. F. V. Lewis
a
a
Division of Molecular Toxicology, School of Biological Sciences , University of Surrey , Guildford, Surrey, GU2 5XH, UK Published online: 10 Jan 2009.
To cite this article: D. V. Parke & D. F. V. Lewis (1992) Safety aspects of food preservatives, Food Additives & Contaminants, 9:5, 561-577, DOI: 10.1080/02652039209374110 To link to this article: http://dx.doi.org/10.1080/02652039209374110
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FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 5, 5 6 1 - 5 7 7
Safety aspects of food preservatives D. V. PARKE and D. F. V. LEWIS
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Division of Molecular Toxicology, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK The use of food preservatives, such as benzoic acid, nitrites, and sulphites, as antimicrobials, and butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid and tocopherols, as antioxidants, has probably changed food production patterns and eating habits more than has the use of any other class of food additive. These food preservative chemicals confer substantial benefits on man, not only by the preservation and increased palatability of food, but also by affording protection against the pathological effects of reactive oxygen species (ROS) which are associated with cancer, cardiovascular disease and aging. Nevertheless, although most preservatives are now considered to be without potential adverse effects and are classified as GRAS, there have been problems concerning the safety of some of these chemicals, including the possibility of allergies from benzoic acid and sulphites, the formation of carcinogenic nitrosamines from nitrites, and the possible rodent carcinogenicity of BHA and BHT. The mechanisms of this toxicity at high dosage, the roles of the cytochromes P450, and the generation and scavenging of ROS in the toxicity of these chemicals, are reviewed and discussed. Keywords: food preservatives, antimicrobials, antioxidants, safety evaluation, antioxidant synergists, COMPACT
Introduction
The preservation of food by drying, smoking, pickling and salting, and the making of fruit conserves with sugar, were among the earliest examples of food processing, first introduced centuries ago. But whereas meat, fish, fruits and vegetables could be preserved by these means, which protected against microbial decomposition, fats of all kinds could not similarly be protected against rancidity, and unpleasant flavours were masked by the liberal use of peppers, onion, garlic, ginger, and other highly flavoured spices. The introduction of benzoic acid and other acids, nitrates and nitrites, and sulphites, as antimicrobials, and of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and other phenols, together with ascorbic acid and tocopherols as antioxidants (see table 1), has had a greater effect on the transformation of food production patterns and eating habits, than has any other class of food additive. Gone are the fears of eating meat that was not cooked fresh lest one developed food poisoning or botulism, and problems associated with the rancidity of fat-containing foods, from butter and meat to ice-cream and biscuits, appear to have gone forever. The mechanism of antimicrobial preservatives is uncertain and diverse. Propionic acid and sorbic acid, benzoic acid and />-hydroxybenzoic acid, all probably act by inhibiting microbial growth by inhibiting bacterial enzymes and by effecting a slight decrease of pH, whereas nitrites and sulphites form complexes with food components which are toxic to microorganisms, but not to mammalia. 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
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D. V. Parke Table 1. Classes of food preservatives. Antimicrobials Propionic acid, Na and K salts" Sorbic acid, Na, K and Ca saltsa Benzoic acid, Na and K saltsa p-Hydroxybenzoate Me, Et, Pr, Bu esters2 Nitrate, nitrite, Na and K salts Sulphites and sulphur dioxide"
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Antioxidants (radical scavengers) Ascorbic acid, Na salt and palmitate" Isoascorbic acid, Na salt Tocopherolsa BHA (butylated hydroxyanisole)a BHT (butylated hydroxytoluene)" Gallates (propyl, octyl, dodecyl)a ter/.-Butylhydroquinone (TBHQ) Antioxidant synergists (metal chelators) EDTA (ethylenediamine tetraacetic acid, and Ca and Na salts)" Citric acid (isopropyl and stearyl esters)" Phosphoric acid" Tartaric acid" a
GRAS substances.
The mechanism of action of antioxidants, such as ascorbic acid, tocopherol, BHT and BHA, is far better known, since all of these are oxygen radical scavengers, and it is now appreciated that oxygen radical-mediated lipid peroxidation is the mechanism by which fats and fat-containing foods undergo spoilage. This process of lipid peroxidation is readily catalysed by minute traces of iron and other transition metals contained naturally in foodstuffs, and a third group of preservatives considered by some as antioxidants, and by others as antioxidant synergists, are ethylenediamine tetraacetic acid (EDTA), citric acid, and other metal chelators, which inhibit the autoxidative action of iron and other trace metals (see table 1). These antimicrobial and antioxidant properties of food preservatives confer substantial benefits on man, not only by way of preservation and increased palatability of his food, but also by affording protection against the general background exposure to oxygen radicals that results in aging and degenerative disease, such as cancer and cardiovascular problems. It was first demonstrated more than 20 years ago that antioxidants such as a-tocopherol and selenium decreased the occurrence of cancer in mice induced by polycyclic aromatic hydrocarbons (Shamberger 1970, Shamberger et al. 1973). Wattenberg (1972) similarly reported that the synthetic antioxidants BHA, BHT and ethoxyquin were highly effective in preventing mammary and forestomach cancer in rodents caused by benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. BHA also decreased diethylnitrosamine, 4-nitroquinoline oxide, and urethane carcinogenicity, and the antitumorigenic effects of synthetic antioxidants against 2-acetamidofluorene and nitrosamines has been confirmed by other workers (Calabrese 1981). More recently, BHT has been shown to have an antiatherogenic effect in cholesterol-fed rabbits (Bjorkhem et al.
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1991). However, it has also been reported that BHT may promote the tumorigenic effects of 2-acetamidofluorene (Peraino et al. 1977) and urethane (Witschi et al. 1977), although only at doses 103 to 104 times the level of the daily consumption of BHT as a food preservative. The mechanisms of protection against chemical toxicity/carcinogenesis are thought to involve oxygen radical scavenging and increased detoxication, and the mechanisms of promotion at high dosage may involve induction of cytochromes P450with possible futile cycling and oxygen radical generation. Nevertheless, despite the obvious major health advantages to the consumer from the use of food preservatives, which is in marked contrast to the questionable benefits of food colourants and flavours, these additives have to be evaluated for safety by standard toxicology programmes of short-term and long-term studies in rodents and other experimental animals, and observations in man. In the course of these studies, benzoic and sorbic acids have been associated with various allergic responses in man, as have sulphites; and nitrates and nitrites have been associated with methaemoglobinaemia, and also with the formation of nitrosamines and consequent potential tumorigenesis (see table 2). Similarly, BHT has been claimed to be a mouse liver carcinogen, BHA a rat stomach carcinogen and the alkyl gallates also have been associated with potential toxicity (see table 2). However, all of these manifestations of toxicity are seen only at high dosage, and it has been considered that they have relatively little significance for safety in man, so that the obvious benefits of food preservatives far outweigh any risk of potential toxicity, and most preservatives now in use are classified as GRAS (generally regarded as safe). Nevertheless, some preservatives, used 40 or more years ago, have been considered to lack appropriate safety as judged by animal toxicological studies, and consequently have been withdrawn from use, or their use limited to certain foodstuffs. For example, diethylpyrocarbonate, once used as a preservative in wine, fruit juice, and soft drinks, was found to have a potential to form urethane in the presence of ammonia, amino acids and proteins, and as urethane is a carcinogen, albeit a weak one even at high dosage, the use of diethylpyrocarbonate as a food additive was revoked by the WHO in 1974. Similarly, nordihydroguaiaretic acid, a food antioxidant, was found to be metabolized to the corresponding orf/joquinone, a likely cause of the faecal haemorrhage and ulceration, and mesenteric cysts, seen
Table 2. Suspected toxic effects of food preservatives. Preservative
Toxic effects
Antimicrobials
Benzoic acid Sorbic acid Sulphites Nitrites, nitrates
Allergies Contact allergy Allergies Carcinogenesis (nitrosamines) Methaemaglobinaemia
Antioxidants
BHT BHA
Mouse liver carcinogen Rat stomach carcinogen Allergy, foetal and neonatal toxicity
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in long-term rat studies at high dosage of this chemical. The alkyl gallates, used as antioxidants in margarine, oils, meats and sausage, were found to give rise to dermatitis in bakers and others handling this material, and to buccal sensitization in some consumers; it was considered that a major cause of this sensitization was the consumption of large amounts of beer and other beverages treated with this preservative, so the use of alkyl gallates as antioxidants for beer and beverages was discontinued. In contrast to this, despite a major public outcry some 20 years ago concerning the potential carcinogenicity of nitrosamines formed by the treatment of meats and bacon with nitrites, the benefit of this class of preservative in preventing botulism, was considered to be of far greater importance to human health than the very low probability of nitrite-induced malignancy.
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Antimicrobials
The antibacterial, fungistatic activity of the antimicrobials, is probably mostly due to the inhibition of microbial enzymes, such as the dehydrogenases. Propionic acid Administered to rats at 4% of the diet, propionic acid produced tumours in rat forestomach, but at 0-4% of the diet no tumours were formed. Propionic acid exhibits no mutagenic activity, and administered to rats, mice or hamsters at 4% diet for seven days resulted in damage and cellular proliferation in the forestomach epithelium of all three species; however, at 0*4% diet no epithelial damage or proliferation occurs. It was concluded that the rat forestomach tumours probably resulted from repeated low level damage and repair caused by the high dosage of propionic acid (Harrison et al. 1991). Propionic acid is considered as GRAS and is widely used as a preservative in beverages, cheese, bakery products, jams, desserts and meat products. Sorbic acid (trans, trans-2,4-hexadienoic acid) Occurs naturally in the berries of Mountain Ash, and is widely used, also as its sodium and potassium salts, as a preservative in cheeses and flour confectionery. It manifests its fungistatic activity through the inhibition of alcohol dehydrogenase and other microbial dehydrogenase systems. Sorbic acid is rapidly metabolized by /3-oxidation to carbon dioxide, and consequently shows no overt toxicity or carcinogenicity. Benzoic acid Used as an antimicrobial in beverages, and as the calcium and sodium salts in margarine, benzoic acid is rapidly conjugated with glycine and glucuronic acid and then excreted, with no discernible toxicity, although the cat is more sensitive than other species probably because of impaired conjugation. Benzoic acid has been associated with occasional hypersensitivity reactions in humans, which have been known to include severe anaphylaxis (Michils et al. 1991). p-Hydroxybenzoic acid Used mainly as its ethyl, methyl, and propyl esters />-hydroxybenzoic acid has shown no overt toxicity or carcinogenicity in long-term animal studies; the butyl ester is associated with hypersensitivity in humans and may exacerbate dermatitis, but the lower alkyl esters do not similarly cause hypersensitivity. The ethyl, methyl,
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and propyl esters are completely and rapidly hydrolysed to the free acid and then conjugated with glycine, and with glucuronic acid to form both ether and ester glucuronides. The acyl glucuronides of aromatic carboxylic acids are metastable and undergo deglucuronidation with the simultaneous aroylation of endogenous proteins, which may lead to neoantigen formation and consequent allergies (Parke et al. 1991). Nitrites and nitrates (sodium and potassium salts) These are antimicrobial preservatives used in the curing of meats to fix colour, develop flavour and to inhibit contamination by Clostridium botulinum, and also in the production of bacon, and the preservation of fish and poultry. Concern for the safety of nitrites and nitrates has been the consequence of acute toxic effects resulting from oxidation of haemoglobin to methaemoglobin to which infants are particularly susceptible (WHO 1978), and the formation of the potentiallycarcinogenic nitrosamines and nitrosamides, in food and in the digestive tract, after food consumption (Daniel 1985). Nitrates are also present in drinking water and vegetables, and these natural sources are considered to produce 5 to 100 times more nitrite in vivo then does the ingestion of preserved meats and bacon. An FDA estimate of the annual cancer risk from the consumption of cured meat was given as 1 in 106, which has to be offset against the greater risk of botulism in the absence of nitrate preservatives. Furthermore, the potential to form nitrosamines from nitrite may be decreased by addition of ascorbic acid, which inhibits the nitrosation of amines and amides by converting nitrite to nitrous oxide, but does not impair the protection afforded by nitrite against Clostridium botulinum in food (Walters 1985). Ascorbic acid, a powerful antioxidant, is naturally secreted into the stomach and is highly protective against nitrosation and gastric cancer (Sobala et al. 1989); sorbic acid, like ascorbic acid, also reacts rapidly with nitrite and inhibits the formation of nitrosamines from nitrite and secondary amines. Naturally-occurring phenolics (jo-hydroxybenzoic acid, propyl gallate) and synthetics (BHA and BHT) present in food can trap nitrite thereby also inhibiting nitrosamine formation, but paradoxically may also enhance nitrosation by forming active nitrosating species (Stich and Rosin 1982). Dimethylnitrosamine and diethylnitrosamine, and other hepatocarcinogenic nitrosamines induce oxidative stress and hepatic lipid peroxidation in rat, whereas the oesophageal carcinogen Af-nitrosomethylbenzylamine, which is not hepatocarcinogenic, does not give rise to hepatic lipid peroxidation (Ahotupa et al. 1987). The levels of nitrosamine-induced lipid peroxidation are seen to parallel their hepatocarcinogenicity; and oxygen radical production results in the formation of 8hydroxydeoxyguanosine in DNA, which is considered to be the probable cause of mutations and malignancy (Bartsch et al. 1989). In addition to enzymic demethylation, the denitrosation of dimethylnitrosamine has also been shown to be a major metabolic pathway in rat (Streeter et al. 1990), and cytochrome P450 IIE1 of rat liver microsomes catalyses both the dealkylation and denitrosation of dimethylnitrosamine and diethylnitrosamine, the metabolic activation of these carcinogens, and the production of oxygen radicals (Yoo et al. 1990). This is a good indication that dialkylnitrosamine carcinogenicity is mediated, at least in part, by oxygen radicals, and would therefore be a much greater problem in small rodents than in man due to the high susceptibility of rats and mice to oxygen radical toxicity (Parke and Ioannides 1990a);
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Sulphites Sulphur dioxide, as sodium sulphite or metabisulphite, is widely used as a preservative in wine, fruit juices, dried fruit and vegetables. Sulphites are potent inhibitors of lactate dehydrogenase and other bacterial dehydrogenases, and this is thought to be the basis of the antimicrobial action of sulphite. Sulphites are highly reactive and interact in many ways with food constituents (Walker 1985a). They cleave protein disulphide bonds yielding a thiol and an 5-sulphonic acid; they form complexes with aldehydes, ketones and sugars, and interact with nucleic acids and vitamins. They are strongly bound by plasma proteins in the form of S-sulphonates which are gradually cleared from the blood into the urine as sulphates. Small amounts of sulphite are formed naturally in intermediary metabolism, in the catabolism of cysteine to pyruvate plus sulphur dioxide, and sulphite is readily metabolized to sulphate by sulphite oxidase and thus shows no overt toxicity in experimental animal studies (Daniel 1985). However, at high dietary concentrations, sulphites cause ulceration of the forestomach, and necrosis and inflammation of the glandular stomach of rats (Walker, 1985b). Til et al. (1972) showed that at 4% of the diet, sulphite produced haemorrhagic erosions, 6% of diet caused inflammation, and 8% resulted in necrosis, in the stomach of rats. Sulphites have a potential to convert the base cytosine (present in DNA and RNA) into uracil (present only in RNA) and hence may cause point mutations, but this effect has been seen in cells and tissue cultures only at very high concentrations of >1000ppm (0-1%), and the relevance to human health is questionable. Sulphur dioxide is a co-carcinogen for benzo(a)pyrene in the respiratory tract of rodents, by enhancing the mutagenicity of the diol-epoxide reactive intermediate, partly by depletion of glutathione (Reed et al. 1990). Mutagenicity studies in mammals in vivo were negative, as were rodent carcinogenicity studies, and three-generation studies in rats, with dietary thiamine supplements, showed a no-effect level of 0-125% sodium metabisulphite (equivalent to 70 mg SO2/kg body wt/day) (WHO 1987). The treatment of food with sulphites destroys its thiamine content, but sulphite administered to humans at 400 mg per day for 25 days, in wine and other beverages, did not adversely affect the thiamine status of these individuals or result in any adverse effect. However, a single dose of 4 g of sodium sulphite given to humans produces toxic symptoms of gut irritation, vomiting and diarrhoea, in the majority of cases. The widespread practice, particularly in the United States, of using sulphite sprays to enliven salad bars in restaurants has given rise to public aversion to the use of sulphites in freshening foods, and has been blamed for a widespread syndrome of so-called 'sulphite allergy'. Biphenyl This is used as a fungistat on citrus fruit; it produces polyuria when fed to rats, and inhibits growth, possibly by depletion of glutathione. It is extensively metabolized to 4-hydroxybiphenyl, other hydroxylated products and to mercapturic acids (Parke 1968). Dehydroacetic acid (DHA, 3-acetyl-6-methyl-l,2-pyran-2,4 (3H)-dione) DHA has been used as a fungicide and bactericide in food wrappings. Its use is now discontinued because of its impairment of kidney function, and its binding to tissue proteins through the reactive carbonyl group in the side-chain. It is
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extensively metabolized in rat and rabbit to hydroxy-DHA, triacetic lactone, triacetic lactone 3-carboxylic acid, and various imino analogues (Parke 1968).
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Tetrachloroisophthalonitrile (BRA VO; 2,4,5, 6-tetrachloro-3-cyano-benzonitrile) TCPN (BRAVO) is a bactericide, nematocide and fungicide, used at one time as a fungicide in the preservation of fruit and vegetables, but discontinued when an IARC carcinogenesis assay showed clear evidence of potential carcinogenesis in the rat. Smoke flavourings (liquid smoke) These are produced by fractionation of wood smoke condensate and purification to remove toxic products such as polycyclic aromatic hydrocarbons. The major components are phenolic compounds (0-1 to 16%, as 2,6dimethoxyphenol), carbonyls (2 to 17% as heptaldehyde) and carboxylic acids, with a benzo(a)pyrene content of < 10 jtg/kg. The material is used as a flavouring agent and in the preservation of bacon, poultry, cheese and sausage (JECFA 1988). The polycyclic hydrocarbon content is determined by chromatographic procedures, which would not necessarily detect or quantify other potentially toxic/carcinogenic constituents such as dioxins and polychlorobiphenyls. In view of the known highly potent carcinogenic constituents of smoke it would seem prudent to use a more reliable and comprehensive analytical procedure to monitor the safety of this product, such as the Enzyme Induction Analysis for Chemical Toxicity (ENACT), which is based on the observed induction of cytochrome P4501 (CYP1) and other carcinogen-activating enzymes (Parke, 1990, Parke and Ioannides 1990b). Antioxidants These substances are added to fats to prevent rancidity, and include ascorbic acid, the tocopherols, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and the alkyl gallates. Ascorbic acid and isoascorbic acid (erythorbic acid) These are antioxidant, oxygen radical scavengers; ascorbate reduces the peroxyl radicals that propagate lipid peroxidation, and reduces the oxidized form of the lipid antioxidant, vitamin E thereby maintaining the antioxidant potential of the latter. However, in the presence of metals, ascorbic acid has pro-oxidant activity, but this is eliminated by EDTA (Laudicina and Marnett 1990). These compounds are considered to be natural nutrients and, as they show no overt toxicity in long-term rodent studies, are widely used as antioxidants in foods and drink (WHO 1972, 1991). Ascorbic acid is also used as the palmitate and stearate in margarine, bread and breakfast foods. Tocopherols These forms of vitamin E, the major natural antioxidants in vegetable oils, are widely used as antioxidant preservatives in bacon, fats and poultry, and are considered as GRAS. a-Tocopherol has been shown to be non-mutagenic and noncarcinogenic in animal studies, but there is evidence that an excessive intake of a-tocopherol could cause haemorrhage. Clinical studies indicate that, generally, intakes of 85% of 3-tert.-butyl-4-hydroxyanisole and 0-25). The data for the food preservatives (indicated by x) are shown in table 5. Data for other chemicals (indicated by •) are published elsewhere (Lewis et al. 1989a,b). Abbreviations are: DEN, diethylnitrosamine; DMN, dimethylnitrosamine; TBHQ, terf.-butylhydroquinone; TCDD, 2,3,7,8-tetrachlorodibenzo-/>-dioxin; TCPN, tetrachloroisophthalonitrile.
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promoting oxygen radical formation and oxidative stress. In contrast, P450 IIB is mostly concerned in the detoxication of chemicals. The COMPACT data and the predicted cytochrome P450 specificity for a number of preservatives are given in table 5, and the COMPACT ratios are plotted in figure 2. From the figure and table it may be seen that tetrachloroisophthalonitrile, once a food fungicide, has the highest COMPACT ratio (0-56) and has COMPACT data characteristics of a P450 I substrate; it is significant that this chemical has been shown to be carcinogenic in rodents. Gallic acid (0·48) and biphenyl (0-47) similarly, have high COMPACT ratios; neither is carcinogenic, but biphenyl exhibits toxicity, probably through its depletion of tissue glutathione, and gallic acid causes hypersensitivity reactions, similar to other P450 I substrates of low planarity (e.g. benoxaprofen and tienilic acid) (Parke et al. 1991). The nitrosamines, like TBHQ and dehydroacetic acid, all have the COMPACT characteristics of P450 IIE1 substrates and consequently will be associated with oxygen radical generation at high dosage, and exhibit toxicity/carcinogenicity in rodents that probably has little significance for man. Acknowledgements
We are grateful to the Humane Research Trust and to the Ministry of Agriculture, Fisheries and Food, for the financial support of our development of COMPACT. References AHOTUPA,
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