The application of in vitro data in the derivation of the Acceptable Daily Intake of food additives

Food and Chemical Toxicology 37 (1999) 1175±1197

www.elsevier.com/locate/foodchemtox

Review

The Application of In Vitro Data in the Derivation of the Acceptable Daily Intake of Food Additives K. WALTON1*, R. WALKER2, J. J. M. van de SANDT3, J. V. CASTELL4, A. G. A. A. KNAPP5, G. KOZIANOWSKI6, M. ROBERFROID7 and B. SCHILTER8 1 Clinical Pharmacology Group, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK, 2School of Biological Sciences, University of Surrey, Guildford GU2 5XH, UK, 3 TNO Nutrition and Food Research Institute, PO Box 360, 3700 AJ Zeist, The Netherlands, 4 University Hospital ±La Fe-, Avda de Campanar 21, 46009 Valencia, Spain, 5National Institute of Public Health and the Environment (RJVM), PO Box 1, 3720 BA Bilthoven, The Netherlands, 6 SuÈdzucker AG, Wormer Str. 11, 67283, Obrigheim, Germany, 7Catholic University of Louvain, Ecole de Pharmacie, Tour Van Helmont, Avenue E. Mounier 73, 1200 Brussels, Belgium and 8Nestec Ltd, Case Postale 44, 100 Lausanne 26, Switzerland

(Accepted 1 June 1999) SummaryÐThe acceptable daily intake (ADI) for food additives is commonly derived from the NOAEL (no-observed-adverse-e€ect level) in long-term animal in vivo studies. To derive an ADI a safety or uncertainty factor (commonly 100) is applied to the NOAEL in the most sensitive test species. The 100-fold safety factor is considered to be the product of both species and inter-individual di€erences in toxicokinetics and toxicodynamics. Although in vitro data have previously been considered during the risk assessment of food additives, they have generally had no direct in¯uence on the calculation of ADI values. In this review 18 food additives are evaluated for the availability of in vitro toxicity data which might be used for the derivation of a speci®c data-derived uncertainty factor. For the majority of the food additives reviewed, additional in vitro tests have been conducted which supplement and support the short- and long-term in vivo toxicity studies. However, it was recognized that these in vitro studies could not be used in isolation to derive an ADI; only when sucient in vivo mechanistic data are available can such information be used in a regulatory context. Additional short-term studies are proposed for the food additives which, if conducted, would provide data that could then be used for the calculation of data-derived uncertainty factors. # 2000 Published by Elsevier Science Ltd. All rights reserved Keywords: acceptable daily intake; food additives; in vitro; metabolism; toxicokinetics; toxicodynamics and uncertainty factor. Abbreviations: ADI = acceptable daily intake; BHT = butylated hydroxytoluene; LOAEL = lowestobserved-adverse-e€ect level; NOAEL = no-observed-adverse-e€ect level; PBQ = 2-phenyl-1,4benzoquinone; PHQ = 2-phenyl-1,4-hydroquinone; THI = 2-acetyl-4(5)-tetrahydroxybutylimidazole; TSH = thyroid stimulating hormone.

Introduction Over the last 20 years there has been a clear tendency for an increased use of in vitro methods in toxicology as supplements to animal tests (Acosta, 1985). Although such studies have previously been considered during the hazard characterization of

*Corresponding author.

many compounds they generally have had no direct in¯uence on the calculation of acceptable daily intake (ADI) values. However, more commonly, the toxicological risk to humans from exposure to an individual chemical is evaluated using animal data from long-term or, less commonly, acute in vivo toxicity studies. As such, in vivo toxicity tests are internationally recognized and guidelines for their conduct have been published. These tests have two major objectives:

0278-6915/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00107-6

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1. to identify the major toxic e€ects of the substances in question by identifying and examining potential target tissues; 2. depending on the nature of toxicity, the level of intake which does not result in any adverse e€ects (NOAEL) is de®ned. Traditionally, the assessment of adverse health e€ects to humans from the consumption of food additives has been based predominantly on in vivo animal toxicity studies, using the species of animal which is most sensitive. In general, the no-observedadverse-e€ect level (NOAEL) obtained from a range of doses in the most sensitive test species (often determined from chronic or subchronic feeding studies) is then used to derive an ADI for a speci®c chemical. However, in default of any comparative human data, it is assumed that the disposition of the toxicant in the body, the dose which then reaches the target tissue, the identity of the proximate toxicant, and the target cell response to the toxicant, are events which are similar in humans and the animal species most sensitive to the compound. To account for any uncertainties in these assumptions with respect to toxicokinetics and toxicodynamics, and to allow for possible mechanistic di€erences in any of these individual events, a safety (or uncertainty) factor of 100 has been traditionally applied to the NOAEL in the most sensitive animal (Lehman and Fitzhugh, 1954). However, the use of a single uncertainty factor did not originally allow for compound-derived toxicokinetic or toxicodynamic data to contribute to the derivation of the ADI in any area of uncertainty. With this concept in mind, Renwick (1991) evaluated the 100-fold safety factor by considering two di€erent toxicological aspects: toxicokinetics (the relationship between the external dose of a compound and the internal dose) and toxicodynamics (the relationship between the internal dose of a compound and the adverse e€ect). As such, the 100-fold uncertainty factor was subdivided into two 10-fold factors, for interspecies and interindividual di€erences, respectively. Renwick (1993) suggested modi®cation of this scheme by further subdividing each 10-fold safety factor into two subfactors for toxicokinetics and toxicodynamics, each of which could be replaced by speci®c compound derived-data if it was available. The International Programme on Chemical Safety (IPCS) workshop on the derivation of guidance values (WHO, 1994) proposed that the subdivision of the 10-fold factor for human variability made by Renwick (1993) should be revised, to allow equal weighting for toxicokinetics and toxicodynamics (see Fig. 1). With this regulatory framework in place it is conceivable that compound-derived data may be used, where appropriate, to provide speci®c data-derived uncertainty factors for one or more areas of uncertainty, based on the similarities or di€erences between toxicokinetics and toxico-

Fig. 1. Subdivision of the 100-fold safety factor (WHO, 1994).

dynamics in the most sensitive test species (used in the determination of the ADI) and man. Although, most commonly for the derivation of the ADI, a default safety factor of 100 has been applied to the NOAEL, this number may vary depending on the nature of the toxic e€ect and the availability of relevant toxicity data. The most ecient approach for the risk assessment of a foreign compound is to use human data, thereby eliminating the necessity for interspecies extrapolations. Although for some food additives the NOAEL is based on studies in human subjects (e.g. erythrosine, canthaxanthin and stannous chloride), for ethical reasons such studies can only be performed after having obtained sound toxicological knowledge of the test compound. However, for many food additives and contaminants, the information available on the mechanism of toxicity is limited and human studies are therefore not very practical. Although the 100-fold uncertainty factor approach has generally proved successful in protecting consumers, the safety evaluation of food additives may be advanced further by incorporating the knowledge gained from the rapidly developing sciences that support toxicology, such as biochemistry, molecular and cellular biology and pharmacokinetics. In vitro studies may prove useful in bridging the gap between a test species and the human situation, thereby providing a more scienti®c basis for the use of a speci®c data-derived safety factor. The aim of this review is to evaluate the potential usefulness of short-term in vivo and in vitro toxicodynamic and toxicokinetic data for setting the ADI for food additives. This exercise was undertaken at the request and with the support of the International Life Sciences Institute (ISLI-Europe, Brussels, Belgium). As a ®rst step, monographs of 65 food additives prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) were screened for the NOAEL, target organ(s), critical e€ects at the lowest-observed-adverse-e€ect level (LOAEL), toxicokinetic data and reported in vitro data. From this list, 18 compounds were selected for which in vitro data were limited or absent in the JECFA monographs but could have been useful in setting the ADI (e.g. by reducing a large safety factor). For these compounds, the scienti®c literature was then searched for additional in vitro and short-term in vivo studies which were relevant to the pivotal study used in the determination of the ADI value. In vitro mutagenicity data

In vitro data in the derivation of the ADI

which were not relevant to the critical study used to derive the ADI were omitted from this review. Method Literature searches were undertaken using MEDLINE (1966±1998), BIDS-EMBASE (1980± 1998) and TOXLINE (1966±1998), employing search terms aimed primarily at identifying shortterm in vivo and in vitro studies related to the pivotal study employed in the determination of the ADI for the following compounds: curcumin, erythrosine, Red 2G, Brown HT, aluminium, gallates (propyl, octyl, dodecyl), butylated hydroxytoluene, aspartame, cyclamate, nitrate, nitrite, tartrazine, Caramel Colour III, Brilliant Blue FCF, diphenyl, o-phenylphenol, thiabendazole and ferrocyanide. For each food additive listed, the monographs prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) were also screened for in vitro data. Results Food additives with in vitro data related to the pivotal study used to calculate the ADI Cyclamate (cyclohexylamine) Cyclamate is a non-nutritive sweetener, used commonly in drinks, diet foods and table-top sweetener formulations. It was last evaluated by JECFA in 1982, when it was allocated an ADI of 0±11 mg/ kg body weight, based on a NOAEL of 100 mg/kg body weight/day in the rat (a 90-day oral study) for the metabolite cyclohexylamine. This metabolite has been found to be produced by bacterial action in the intestine, an event with varies widely between animals and man (Bickel et al., 1974; Drasar et al., 1972; Leahy et al., 1967; Renwick and Williams, 1972). The number of individuals able to convert cyclamate to cyclohexylamine and the level at which this conversion occurs has been factored into the ADI determination using averages from some studies. At the LOAEL (200 mg/kg body weight/ day) the rat showed reduced body weight; testicular e€ects were observed in the form of tubular alterations and impaired spermatogenesis. Above the LOAEL (300 mg/kg body weight/day) it was shown that the metabolite, cyclohexylamine, induced adverse e€ects in the testes (atrophy, soft consistency, livid, glassy) (WHO, 1982). The key adverse e€ect induced by cyclohexylamine, at the LOAEL, was testicular atrophy, and species di€erences in the sensitivity to testicular toxicity in laboratory animals have been described. The rat is the most sensitive species (Gaunt et al., 1976; Mason and Thompson, 1977), whereas in the mouse cyclohexylamine does not induce testicular lesions (Hardy et al., 1976). A metabolic reason for

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this species di€erence has been suggested since rats can convert up to 20% of a dose of cyclohexylamine to various aminocyclohexanols, whereas mice and man form less than 1% aminocyclohexanols, with most of the cyclohexylamine excreted unchanged (Roberts and Renwick, 1985). The metabolism of cyclamate shows wide inter-individual di€erences within animals and man. Metabolic studies showed that over 70% of the human population were unable to metabolize cyclamate (<0.1% of the dose), 3±4% metabolized >20% of the daily dose and 1% of humans converted >60% of the daily dose. In addition there are wide (two to three) day-to-day and very wide (up to 20-fold) week-toweek intra-individual variations in cyclohexylamine formation during chronic cyclamate intake (Davis et al., 1969; Litch®eld and Swan, 1971; Renwick and Williams, 1972; Wills et al., 1981). Although inter-species di€erences in cyclohexylamine metabolism do exist, the di€erence in testicular sensitivity between the rat and mouse is thought to be a consequence of di€erences in blood/testis concentrations of cyclohexylamine. Roberts and Renwick (1989) compared the toxicokinetics of chronic dietary administration of cyclohexylamine in the rat and mouse. The uptake and elimination of cyclohexylamine was more rapid in the mouse than in the rat. During steady-state continuous treatment, cyclohexylamine concentrations in the plasma and testis showed diurnal variation in the rat, which was not detected in the mouse. In addition, the steady-state plasma clearance in rats was approximately one-half of that observed in the mouse. Within the testis, the Sertoli cell has been suggested as the target for toxicity (Creasy et al., 1990). An in vitro test system has been reported for investigating the testicular e€ects induced by cyclohexylamine. Creasy et al. (1990) studied the e€ects of cyclohexylamine and 4-aminocyclohexanol in mixed cell cultures of Sertoli- and germ-cells derived from young (28-day-old) rats. These cell cultures were incubated with 0.1, 1.0, 3.0 or 10 mM cyclohexylamine or 4-aminocyclohexanol for 1, 2 or 3 days. Cyclohexylamine induced histological changes in these cells in vitro similar to those generated in vivo. The lowest e€ective concentration (1.0 mM) is equivalent to 99 mg/ml (approx. 20 times that in rat plasma and 2.5-times that found in the rat testis following a toxic dose). The studies reported for cyclohexylamine show that, when detailed mechanistic information is available on the nature of the in vivo toxicity, in vitro data can then be used to determine the sensitivity of testicular cells to this metabolite. However, this in vitro data merely con®rms the mechanism of toxicity, which in the absence of comparative human data on the relative levels of cyclohexylamine in blood and testes, and on the sensitivity of

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the target cells, could not be used by itself to derive a quantitative ADI. Caramel Colour III Caramel colour III is a food colouring used in beers and a variety of foods. It was last evaluated by JECFA in 1987, when it was allocated an ADI of 200 mg/kg body weight, based on a NOAEL of 20 g/kg body weight/day from a 90-day oral toxicity study in the rat, to which a safety factor of 100 was applied. At the NOAEL, the rat showed a reduction in weight gain with a concomitant reduction in food and water consumption. At the LOAEL [1% (w/w) in the diet, short-term rat study], a reduction in lymphocyte number and an increased neutrophil count was observed. Above the LOAEL [16% (w/w) in the diet, short-term rat study], the spleen showed a reduction in weight and the caecum and kidney increased in weight (WHO, 1987). The short-term in vivo studies on Caramel Colour III support the observations made at the LOAEL. When the rat was fed Caramel Colour III [1, 4 and 8% (w/w) in the diet] for 14 days a reduction in the lymphocyte count was observed which was correlated with reduced levels of pyridoxine (Sinkeldam, 1981, 1982). The active toxic ingredient (in terms of lymphocyte depression) of Caramel Colour III, 2acetyl-4(5)-tetrahydroxybutylimidazole (THI), was investigated in the rat following oral administration for 7 days in drinking water (2, 5 or 20 mg/litre). THI produced a marked a depression in lymphocyte number and it was suggested that this was the active component in Caramel Colour III (Sinkeldam, 1982). This was investigated further by Houben et al. (1992b), who studied the e€ects on circulating white blood cells in the rat dosed with THI (5.72 mg/litre) in drinking water for 4 wk. A reduction in cell numbers in the spleen, the popliteal lymph nodes as well as the blood was observed. In the blood, spleen and popliteal lymph nodes, CD4+ lymphocytes were reduced more than CD8+ cells. All the e€ects observed were either less pronounced or absent when the water was supplemented with 11±12 ppm pyridoxine (Houben, 1992b). The short-term in vivo studies described above are supported by mechanistic in vitro studies. Caramel colour III inhibits the activity of pyridoxal phosphatase (a key enzyme involved in pyridoxine metabolism) in rat liver homogenate. This observation is supported by the work of Spector and Huntoon (1982), who indicated that Caramel Colour III contains a heat-stable compound that can inhibit the pyridoxal kinase activity in rabbit brain slices in vitro. Comparative in vitro data for THI on the equivalent human enzyme has not been reported, although an in vivo human study has been conducted on Caramel Colour III. Elderly male volunteers with a marginal de®cit in vitamin B6 were

given Caramel Colour III at 200 mg/kg body weight/day (the current ADI level) for 7 days. Two samples were tested, a commercial sample containing 23 ppm THI or a research sample containing 143 ppm THI. The results of this study showed that all of the haematological parameters examined were una€ected by both samples of Caramel Colour III (Houben et al., 1992). Data describing either the pharmacokinetics or metabolism of THI in the rat or human were not available. Moreover, the only available human study conducted on Caramel Colour III containing THI was limited; two di€erent batches of caramel were used, one containing low and the other containing high levels of THI and these were administered as single doses to elderly male volunteers. The results of this human study showed that, unlike in the rodent, was no induction of lymphopaenia. The short-term in vivo studies described have been used to determine the nature of the toxic component in Caramel Colour III, and the in vitro studies have aided in elucidating the mechanism by which this component induces lymphopaenia. However, the variation in the dynamics of this response or its relevance to humans have not been reported in the literature. As an e€ect on pyridoxal kinase has been shown to occur in rat and rabbit tissue homogenates, comparative human/rodent inhibition studies on this enzyme may reveal toxicodynamic species di€erences and assist in de®ning data-derived safety factors for THI. Red 2G Red 2G is a water-soluble azo dye used as a food colouring. It was last evaluated by JECFA in 1981, when it was allocated an ADI of 0±0.1 mg/kg body weight, based on a NOAEL of 26±43 mg/kg body weight/day (mouse) and 8 mg/kg body weight/day (rat), obtained from long-term feeding studies. At the LOAEL (130±215 mg/kg body weight/day in the mouse and 32 mg/kg body weight/day in the rat), two target tissues were identi®ed; the spleen and the kidneys. The spleen showed enlargement with an increased deposition of iron. In the mouse, accelerated erythropoiesis was observed and the rat showed necrosis of elastica. Above the LOAEL in the rat [0.5% (w/w) in the diet] adverse e€ects were observed in the spleen, liver and bone marrow. Heinz body formation in the erythrocytes was also observed at the LOAEL in both species of rodents (WHO, 1981). In vitro studies on the metabolism of Red 2G were used to clarify the rat gut micro¯ora was capable of metabolizing this compound to 2-amino8-acetamido-1-naphtho-3,6-disulfonic acid and aniline (Jenkins et al., 1966a). These in vitro studies were supported by short-term in vivo work in the same species which demonstrated that following a single oral dose of 250 mg/kg body weight, the metabolites of Red 2G excreted in the urine

In vitro data in the derivation of the ADI

were p-aminophenol (free and conjugated) and aniline (Walker, 1971). Heinz bodies have been induced in rat erythrocytes following the oral administration of Red 2G for 75 days (1±1.5 g/kg body weight) (Rofe, 1957). Aniline, the primary rodent metabolite of Red 2G, has been used extensively in industry and its toxicological properties have been studied in detail, with the induction of Heinz bodies being the primary toxic e€ect. In addition, the proximate toxin responsible for the toxicity of aniline has been elucidated. As early as 1913, Heubner proposed that paminophenol was the active metabolite responsible for Heinz body induction by aniline but later work by Greenberg and Lester (1947) found that methaemoglobin was not induced after a single oral dose of 0.5 g p-aminophenol hydrochloride to humans. Lipschitz (1920) assumed that phenylhydroxylamine (a metabolite of aniline, formed via N'-hydroxylation) was the toxic metabolite of aniline, and von Issekutz (1939) showed that this compound was a potent inducer of methaemoglobinaemia. Later studies found that after the dietary administration of aniline or phenylhydroxylamine to rats [both at a concentration of 0.1% (w/w) in the diet for 13 days] Heinz bodies were induced in the erythrocytes (Jenkins, 1966b). A comparison of the no-e€ect dose of aniline in humans and rats showed that Heinz bodies were not detected in the red blood cells of humans following a single oral dose of up to 65 mg aniline. However, a signi®cant increase in methaemoglobin levels was observed 1, 2, and 3 hr after administration of between 25 and 65 mg aniline. At the lower doses studied (5 and 15 mg), there was no signi®cant increase in methaemoglobin; the noobserved-e€ect level in humans was therefore 15 mg (00.25 mg/kg body weight). The rat appeared to be less susceptible to methaemoglobinaemia, with a no-e€ect level of 20 mg/kg body weight of aniline (Jenkins et al., 1972). The susceptibility to methaemoglobin formation by phenylhydroxylamine has been studied in vitro using isolated human and rat red blood cells with phenylhydroxylamine concentrations ranging from 0.0005 to 0.5 ng/ml. Phenylhydroxylamine induced the formation of more methaemoglobin in the blood of the rat than in that of the human at all concentrations tested in this study, with the levels of methaemoglobin being approximately twofold greater in the rat blood (Jenkins et al., 1972). Although man is more susceptible than the rat to aniline in vivo, the methaemoglobin levels of rat blood exposed to phenylhydroxylamine in vitro exceeded that of human blood. In terms of the relevance to the pivotal study used to determine the ADI for Red 2G, the data described here only clarify that aniline is generated from Red 2G in rodents in vivo and that this occurs in the gastrointestinal tract. Once formed, it is

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assumed that aniline is then absorbed and N'-hydroxylated enzymically to generate phenylhydroxylamine, the proximate toxin. However, the extent to which Red 2G is reduced to aniline in the gastrointestinal tract of humans is unknown, although azo reduction is extensive with the closely related colour Orange G. Assuming that Red 2G undergoes reduction, the quantity of aniline that is absorbed from the gastrointestinal tract and subsequently oxidized to phenylhydroxylamine remains to be investigated. As such, the greater sensitivity of man to aniline in vivo could be due to di€erences in the absorption of aniline, variation in its metabolism to phenylhydroxylamine or a consequence of di€erences in the activities of enzymes that promote reduction of methaemoglobin in the erythrocyte, or a combination of these events, as the results of Jenkins et al. (1972) suggest. Red 2G represents a compound where the proximate toxin (phenylhydroxylamine) and the target tissue (erythrocytes) are amenable to inter-species and inter-individual toxicodynamic comparisons using in vitro tests. Alternatively, since the toxicity of aniline in humans (methaemoglobin induction) is similar to that observed in the rat and a human NOAEL has been established, the ADI for Red 2G could be calculated from the human aniline data (Jenkins et al., 1972), assuming (conservatively) 100% conversion of Red 2G to aniline. This is analogous to the cyclamate/cyclohexylamine derivation of an ADI. Butylated hydroxytoluene Butylated hydroxytoluene (BHT) is an antioxidant commonly used in foodstu€s. It was last evaluated by JECFA in 1995 when it was allocated an ADI of 0±0.3 mg/kg body weight, based on a NOAEL of 25 mg/kg body weight/day from a 2year feeding study in the rat, to which a 100-fold safety factor was applied. At the LOAEL (100 mg/ kg body weight/day) enzyme induction was observed in the liver and reproductive e€ects in the form of a reduced body weight gain of pups during lactation also were observed. Above the LOAEL (250±2300 mg/kg body weight/day) toxic e€ects were reported to occur in the blood (reduced clotting), liver (hepatocellular necrosis, in¯ammation) and kidney (tubular lesions) (WHO, 1996). In vivo toxicity studies in the rat have associated BHT with damage in the lungs (Hawkins, 1979) and haemorrhagic death (Takahashi and Hiraga, 1981). There is also limited evidence for the carcinogenicity of BHT per se in laboratory animals. Olsen et al. (1986) reported an increase in hepatocellular necrosis in rats exposed to BHT in utero and until up to 144 wk of age. Moreover, Inai et al. (1988) observed an increased incidence of liver tumours among male (but not female) mice which had been fed a diet containing 1% or 2.0% (w/w) BHT.

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For the derivation of the ADI it was concluded that the ®rst detectable e€ect from BHT exposure was enzyme induction in the liver, for which a wellde®ned threshold was demonstrated (100 mg/kg body weight/day). An BHT-mediated e€ect on hepatic enzymes is supported by the results of several anti-mutagenicity studies. BHT altered the genotoxic properties of compounds which required bioactivation to exert their mutagenic e€ects; it enhanced the mutagenicity of a¯atoxin B1 (Shelef and Chin, 1980), 3,3'-dichlorobenzidine (Ghosal and Iba, 1992), yet inhibited the mutagenicity of 2AAF (Chipman and Davies, 1988; Chipman et al., 1987; Richer et al., 1989) and 3,2'-dimethyl-4-aminobiphenyl (Reddy et al., 1983) in the Ames mutagenicity test. The pharmacokinetics and metabolism of BHT have been investigated in vivo. BHT is absorbed rapidly following ingestion, with the maximum tissue concentrations being achieved after 4 hr in the rat and mouse. There is no evidence of tissue accumulation on repeated dosage but excretion is slow (half-life, 7±10 days), which is thought to be due to some enterohepatic recirculation occurring in these species. In man, the absorption of BHT is also rapid and 50% of the dose is excreted in the urine in 24 hr. The remainder is excreted more slowly, suggesting that biliary excretion with an enterohepatic circulation might also occur in humans. In the rat and mouse the main metabolite of BHT is BHT acid, both free and conjugated, whereas in man this is a minor metabolite. The main metabolite excreted from humans is a benzofuran, as a conjugate with glucuronic acid or mercapturic acid (Conning and Phillips, 1986). The in vitro anti-genotoxicity studies in this case support the view that BHT can interact with the hepatic enzymes involved in xenobiotic metabolism and promutagen activation and therefore indicate that the hepatic toxicity induced by BHT is a consequence of enzymic induction. Moreover, in vivo pharmacokinetic studies have been conducted with BHT, showing that both inter-species and inter-individual variation in the pharmacokinetics of the parent compound does exist. The in vitro studies presented here have been used to support the mechanistic basis of the toxicity observed at the LOAEL but alone could not contribute to the quantitative determination of an ADI. However additional in vitro studies on enzyme induction could provide an indication as to the probability of this event also occurring in humans. Curcumin Turmeric is used both as a colouring and ¯avouring agent in foods. It is found naturally in the ground powder of the root of Curcuma longa which contains between 2.5 and 6% (w/w) yellow pigments (the curcuminoids), of which curcumin predominates. Turmeric oleoresin (a more concen-

trated source of curcumin) is also used as both a colouring and ¯avouring agent. Curcumin was allocated a temporary ADI of 0±1.0 mg/kg body weight by JECFA in 1995, based on a NOAEL level of 220 mg/kg body weight/day from a 2-year feeding study in the mouse, to which a safety factor of 200 was applied. At the LOAEL (440 mg/kg body weight/day) enlargement of the liver was observed; above this dose (2000 mg/kg body weight/day) ulcers and hyperplasia were detected in the gastrointestinal tract. The ADI for curcumin was based on liver enlargement (WHO, 1995). Curcumin was further reviewed by JECFA in 1998, during which the temporary ADI of 0±1.0 mg/kg body weight was extended. It is probable that hepatic enzyme induction is responsible for the hepatocellular changes associated with repeated doses of curcumin (at the LOAEL dose and above). In support of this conclusion, curcumin has been shown to inhibit hepatic cytochrome P450 enzymes and phase II xenobiotic metabolizing enzymes in vitro. Studies employing a rat liver subcellular fraction, showed that curcumin (1±100 mM) can inhibit the catalytic activities of cytochromes P450 1A1/1A2, 2B1/2B2 and 2E1, glutathione S-transferase (Oetari et al., 1996) and human phenol sulfotransferase (Eaton et al., 1996). In addition, several studies have also found that curcumin can reduce the mutagenicity elicited by compounds which are bioactivated by the hepatic cytochrome P450 isoenzymes; curcumin inhibited the enzymic activation dependent mutagenicity of benzo[a]pyrene, dimethylbenzo[a]anthracene (Deshpande and Maru, 1995; Nagabhushan et al., 1987), 2-acetamido¯uorene (Anto et al., 1996; Soudamini et al., 1995) and a¯atoxin B1 (Soni et al., 1997) in the Ames mutagenicity test using a rat hepatic subcellular activation system. Similarly, in vivo rat studies have shown that curcumin can prevent the induction of forestomach tumours induced by benzo[a]pyrene, and dimethylbenz[a]anthracene-induced skin tumours in mice, an e€ect which correlated with reduced cytochrome P450 activity (Azuine et al., 1992) and enhanced glutathione S-transferase activity in the liver (the activities in the target tissues were not examined) (Azuine et al., 1992; Susan and Rao, 1992). Using radioactive curcumin, it was demonstrated that in the rat, approximately 90% of an oral dose (2.5±1000 mg/kg body weight) is excreted in the faeces within 48 hr (Holder et al., 1978; Ravindranath and Chandrasekhara, 1982), with only 35% of the dose excreted unchanged. It was concluded that 65% of the dose was absorbed and was extensively excreted in bile (Ravindranath and Chandrasekhara, 1982). The biliary metabolites of curcumin have been identi®ed as the glucuronides of tetrahydrocurcumin and hexahydrocurcumin from the bile of rats dosed intravenously with 50 mg body weight curcumin/kg (Holder et al.,

In vitro data in the derivation of the ADI

1978). These in vivo metabolism and excretion studies suggest that the majority of an oral dose of curcumin is reduced by the intestinal micro¯ora prior to absorption. However, neither the metabolism nor the excretion of curcumin have been determined in the mouse (which was the most sensitive species, and therefore used in the derivation of the ADI) and comparative pharmacokinetic studies in humans and this species were not available in the scienti®c literature. The supplementary in vitro studies support the view that curcumin and/or its metabolites have an inhibitory e€ect on the enzymes involved in xenobiotic metabolism in the rat. Although the in vitro studies were carried out with hepatic subcellular fractions from the rat, the in vitro data does supplement and support the mechanism by which liver enlargement is induced in vivo at the LOAEL in the mouse. However, these supplementary short-term studies do not provide additional information which would either modify the uncertainty factors used for deriving the ADI nor could they be used in isolation to derive an ADI for curcumin. Gallates (propyl, octyl and dodecyl) Propyl gallate is an antioxidant used in fats, oils and waxes. It was allocated an ADI of 1.4 mg/kg body weight by JECFA in 1993, based on a NOAEL of 135 mg/kg body weight, from a 90-day oral toxicity study in rats, to which a 100-fold safety factor was applied. At the LOAEL in this species (526.5 mg/kg body weight) three target tissues were identi®ed. In the blood a reduction in both haemoglobin levels and erythrocyte counts was observed. The spleen showed extramedullary haemopoiesis. In addition, an increase in microsomal and phase II enzymes was detected in the liver (WHO, 1993a). In vitro studies have shown that propyl gallate can inhibit ethoxycoumarin deethylase and benzo[a]pyrene hydroxylase activity in rat liver subcellular fractions (Depner et al., 1982, Rahimtula et al., 1979; Yang and Strickhart, 1974). Several in vitro studies have demonstrated an interaction of propyl gallate with the hepatic cytochrome P450 family of enzymes. The microsomal activation of benzo[a]pyrene (Calle and Sullivan, 1982), and of N-methyl-N'nitroso-N-nitrosoguanidine and N-acetoxyacetylamino¯uorene to genotoxic species was reduced by propyl gallate (Rosin and Stich, 1980), while that of a¯atoxin B1 was enhanced (Calle and Sullivan, 1982). In vivo short-term studies support an e€ect on hepatic enzymes involved in the metabolic activation and/or detoxi®cation of mutagens/carcinogens. Propyl gallate failed to elicit an e€ect on the development of colon tumours in rats dosed with 1,2-dimethylhydrazine (Shirasi et al., 1985) but reduced the development of dimethylbenz[a]anthracene-induced tumours in rats pretreated with propyl gallate (Hirose et al., 1988; Raj and Katz, 1984).

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The e€ect of propyl gallate on hepatic drug metabolizing enzymes was studied in vivo in male rats following dietary exposure (1% in the diet) for 14 wk. Cytochrome P450 levels were not enhanced, however epoxide hydratase activity was increased (Depner et al., 1982). The in vitro metabolism of propyl gallate has been studied only to limited extent. Using rat hepatocytes the major metabolites of propyl gallate were identi®ed as gallic acid, 4-O-methylgallic acid, dipropyl-4,4',5,5',6,6'-hexahydroxydiphenate and ellagic acid (Nakagawa et al., 1995). It would appear probable that hydrolysis by hepatic carboxylesterases is the primary step in the in vivo and in vitro metabolism of propyl gallate. In support of this, the parent compound was shown to be the most toxic species in vitro since hepatotoxicity was enhanced following the addition of a carboxylesterase inhibitor to the incubate (Nakagawa et al., 1995). The pivotal e€ect of propyl gallate on the hepatic drug metabolizing enzymes (an e€ect observed at the LOAEL) is supported by the in vitro studies, in which the modulation of genotoxic carcinogens that require bioactivation was reported. However, the proximate compound responsible for this e€ect has not yet been elucidated, although the in vitro studies of Nakagawa et al. (1995) have suggested that it may be the parent compound. The pharmacokinetic pro®les of propyl gallate in either the human or the rat have not been reported in the scienti®c literature to date. Octyl gallate is a preservative used in fats, oils and waxes. It was last evaluated by JECFA in 1993, when it was noted that a slight hypochromic anaemia occurred at 100 mg/kg body weight/ day in a feeding study in rats in which the substance was administered for 2 generations. A temporary ADI of 0±0.1 mg/kg body weight was allocated based on a NOAEL of 17.5 mg/kg body weight/day, in a reproduction study with rats to which a safety factor of 200 was applied. Very few short-term in vivo and in vitro studies that were relevant to the study used for the determination of the ADI were reported. In vivo octyl gallate failed to either enhance or inhibit the activity of several drug metabolising enzymes (Depner et al., 1982). Additional studies showed that octyl gallate was not absorbed or metabolized to any extent in rats following a single oral administration (15 mg/kg body weight). Dodecyl gallate is a preservative used in fats, oils and waxes. It was last evaluated by JECFA in 1993 and allocated a temporary ADI of 0±0.05 mg/kg body weight, based on a rodent study for 150 days, to which a 200-fold safety factor was applied. At the NOAEL of 10 mg/kg body weight/day, a reduction of spleen weight was observed along with pathological changes in the liver, kidney and spleen (150-day gavage study with rats). Dodecyl gallate showed no e€ect on drug metabolizing enzymes,

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either in vitro or in vivo (Depner et al., 1982). Neither of these studies addressed the mechanism by which pathological changes were induced in the liver, kidney or spleen and show no connection to the primary study used in the derivation of the ADI for dodecyl gallate. The gallates (propyl, octyl and dodecyl) originally had a group ADI, based on the assumption that esterase hydrolysis prior to absorption would result in the formation of gallic acid from all three of the esters. However, the group ADI was withdrawn by JECFA in 1993 due to di€erences in toxicological potency observed in long-term animal studies and a lack of supporting metabolic studies. The gallates represent a group of compounds where simple in vitro metabolic studies may provide an explanation for the toxicological di€erences within this group. Erythrosine Erythrosine (FD&C Red No. 3) is an iodine-containing colour used in foods, drugs and cosmetics. It was allocated an ADI of 0±0.1 mg/kg body weight, by JECFA in 1991, calculated from a human study (males only) during which erythrosine was ingested for 14 days (20, 60 or 200 mg/person/ day). The NOAEL in this human study was 1 mg/ kg body weight, to which a safety factor of 10 was applied. The data for the LOAEL and above this level was obtained from a 2-year feeding study in rats. At the LOAEL (3.3 mg/kg body weight/day), erythrosine induced changes in the thyroid hormones, and above this level (3029 mg/kg body weight/day) adenomas and carcinomas were observed in this organ (WHO, 1991). The carcinogenic e€ect shown by erythrosine in the rat (at doses above the LOAEL) is commonly perceived to be a secondary event, where tumour formation is due to an e€ect on the feedback mechanism involved in the secretion of thyroid stimulating hormone (TSH). It is believed that in the rat, erythrosine inhibits the peripheral conversion of thyroxine (T4) to tri-iodotyrosine (T3) resulting in an increased production, and release, of the thyrotropic hormone, TSH, from the pituitary gland. The released TSH can then stimulate cell proliferation in the thyroid, resulting in hyperplasia, and in the case of erythrosine (above the LOAEL), the subsequent formation of tumours. That erythrosine is carcinogenic via a non-genotoxic mode of action is supported by the negative results of in vitro genotoxicity studies; erythrosine failed to elicit mutagenic activity in the Ames mutagenicity test (in the absence or presence of metabolic activation) (Auletta et al., 1977; Bonin and Baker, 1980; Brown et al., 1978; Haveland-Smith et al., 1981; Lakdawalla and Netrawali, 1988a; Tarjan et al., 1986), nor did it induce gene mutations in eukaryotic cells (Ishidate et al., 1984; Lakdawalla and

Newtrawali, 1988a,b; Matula and Downie, 1984; Rogers et al., 1988; Sankaranarayanan et al., 1979). The short-term human in vivo study, which was used in the derivation of the ADI for erythrosine, concerned the e€ects of erythrosine (20, 60 or 200 mg/person/day) on thyroid function and serum urinary iodide concentrations in 30 men for 14 days (10 subjects per dose). In humans, as in the rat, levels of TSH were found to increase; however, in the human study there was no concomitant increase in serum T4 levels. The increase in TSH in humans was considered to be an e€ect of increased iodide concentrations in the serum rather than an e€ect of erythrosine on secretion of the thyroid hormones or peripheral metabolism of T3 and T4 (Gardner et al., 1987). However, a later study suggested that inorganic iodine per se was not the causative agent responsible for increasing the levels of TSH in the human study by Gardner et al. (1987). Paul et al. (1988) determined the e€ect of small amounts of iodine in the diet. Humans were fed 250, 500 or 1500 mg iodine for 14 days. The doses employed were selected to correspond to the amounts of iodine which might be bioavailable from the doses of erythrosine used by Gardner et al. (1987). At levels of 1500 mg iodine/day a small but signi®cant decrease in serum T4 and T3 concentrations were observed, along with a small compensatory increase in serum TSH concentration and in the TSH response to TRH. However, all of these values remained within the normal range, and no changes occurred following daily administration of the lower doses (250 or 500 mg/day) of iodine. Although the mode of action by which erythrosine induces an increase in TSH in the study of Gardner et al. (1987) remains to be elucidated, the mechanism by which this occurs in the rat has been described. Dietary administration of erythrosine for 3 wk in this species caused an increase in the pituitary TSH response to TRH by preventing conversion of T4 to T3 in the liver (Jennings et al., 1990). This was supported by an investigation on the metabolism of [125I]T4 and [125I]T3 by liver homogenates from rats dosed orally with erythrosine (2.5±250 mg/kg body weight), which demonstrated that erythrosine can inhibit the 5'-monodeiodination of T4 to T3 (Ruiz and Ingbar, 1982). The published data on erythrosine suggests that the underlying e€ect by which erythrosine induces toxicity (enhancing the production of TSH) and subsequent carcinogenicity in the rat may also apply to humans. Even though the human study of Gardner et al. (1987) failed to show an e€ect on levels of serum T4, an increase in TSH was observed. In this situation in vitro studies utilizing rat and human whole liver homogenate might aid in clarifying whether the inhibitory e€ect of erythrosine on 5-monodeiodinase is a species-speci®c phenomenon or whether the increased TSH arises from a direct e€ect of erythrosine on the pituitary.

In vitro data in the derivation of the ADI

An erythrosine-induced increase in TSH production does occur in humans and this raises the question of to what extent does thyroid stimulation represent an aetiological factor in the formation of thyroid tumours in humans? In man, although thyroid goitres are common in certain populations, progression to thyroid adenomas appears to be a rare event. In addition, it has been suggested that since the rat lacks thyroxine-binding globulin, it is not a good model for studying the e€ects of thyrotoxic agents in humans. In humans circulating T4 is bound to a speci®c, high-anity protein (thyroxinebinding globulin) and because of this high anity, T4 has a circulating half-life in man of about 7 days. In rodents, this protein does not exist and 75% of thyroxine is bound to albumin. This has a low anity for T4, and consequently the half-life of T4 in the rat is only about 12 hr. This results in a much higher turnover of thyroid hormones and the histomorphology of the thyroid appears to be hypertrophic and hyperplastic, even in healthy untreated animals. The tumours observed in the rat may therefore be a consequence of the enhanced sensitivity of this species to thyrotoxic agents. For the derivation of the ADI the most sensitive index of e€ects on the thyroid for the evaluation of erythrosine is changes in serum TSH, an e€ect which was observed in humans dosed orally with this compound (Gardner et al., 1987). The human data allowed a 10-fold safety factor to be applied to the NOAEL obtained by Gardner et al. (1987). The negative in vitro genotoxicity data reported in this review supports the hypothesis that the thyroid tumours observed in rodents above the LOAEL are the result of a non-genotoxic mechanism of action, but do not assist per se a quantitative evaluation of the ADI for erythrosine. For the further modi®cation of the ADI for erythrosine, studies describing the extent of human variability in erythrosine toxicokinetics and toxicodynamics would be needed, neither of which have been suciently studied to date. Nitrite Nitrite was last evaluated by JECFA in 1996 when it was allocated an ADI of 0±0.06 mg/kg body weight based on a NOAEL of 5.4 mg/kg body weight (90-day feeding study) and 6.7 mg/kg body weight/day (2-year feeding study) in the rat, to which a 100-fold safety factor was applied (WHO, 1996). At the LOAEL (16.3 mg/kg body weight/day in the rat) hypertrophy in the zona glomerulosa of the adrenal gland was observed. Above the LOAEL (67 mg/kg body weight/day in the mouse), the target organs for toxicity were the blood, heart and the lung. In the blood, dose-related methaemoglobinaemia was observed. In addition, heart tissue showed dilation of the coronary arteries and the lung was in®ltrated with lymphocytes.

1183

The majority of the in vivo and in vitro studies currently reviewed have examined the formation of nitrosoamines from sodium nitrite and secondary and tertiary amines. However, epidemiological studies of the human population have failed to show any convincing association between nitrite intake and cancer, the inconsistency of the ®ndings from the studies of stomach cancer thus failing to establish a causative link. Animal feeding studies on the whole support the view that nitrite itself is noncarcinogenic (Maekawa et al., 1982), and although some studies have shown a signi®cant increase in tumour incidences in nitrite-exposed rats, there is no consistent pattern in either the type of tumour seen or the gender a€ected. In addition, in two long-term rodent studies nitrite was associated with a reduced incidence of malignancies (monocytic leukaemias) (Lijinsky et al., 1983; Maekawa et al., 1982), observations which do appear to con¯ict with the in vitro nitrosation studies. The e€ects induced by nitrite on the adrenal zona glomerulosa, which were pivotal in determining the ADI, may be a secondary consequence of vasodilation. The vascular dilating e€ects of nitrite are well known and used clinically in the treatment of angina, and have been extensively studied in vitro using standard pharmacological techniques. It terms of pharmacokinetics, nitrite is probably absorbed from the stomach. It is then rapidly oxidized to nitrate in the blood and in vitro studies have indicated that this results from a coupled oxidation with haemoglobin, leading to methaemoglobin formation. As a consequence, oxygen transport to the tissues is impaired. Comparative studies have also clari®ed why the neonate is more sensitive to nitrite-induced methaemoglobinaemia (a combination of foetal type haemoglobin and lower methaemoglobin reductase levels in the neonate). There seems to be further potential for in vitro studies in elucidating the e€ects of nitrite on the adrenal and its relationship with vasodilation, which could assist in re-evaluating the ADI for nitrite. Tartrazine Tartrazine (FD&C Yellow No. 5) is a monoazo pyrazolone dye, used mainly to colour sweets, soft drinks, hand lotions and other food, drugs and cosmetics. It was last evaluated by JECFA in 1964, when it was allocated an ADI of 0±7.5 mg/kg body weight, based on a NOAEL of 750 mg/kg body weight/day from a 2-year feeding study in the rat, to which a safety factor of 100 was applied. At the LOAEL of 1000 mg/kg body weight/day, a signi®cant number of animals su€ered from laxation. Above the LOAEL (2500 mg/kg body weight) the target organ was the kidney where the disposition of ``gritty material'' (presumably calcinosis) was found to occur in the renal pelvis (WHO, 1964a).

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A number of metabolism studies have been conducted on tartrazine. An early comparative in vivo metabolic study showed that when tartrazine was administered intraperitoneally to the rat or rabbit, only the parent compound was excreted. However, following the oral administration of tartrazine to the same species (and man) complete metabolism was observed, with the major metabolite identi®ed as sulfanilic acid. As sulfanilic acid was not detected after the intraperitoneal administration of tartrazine to the rat or rabbit, it was suggested that the intestinal micro¯ora are responsible for its metabolism following oral exposure (Jones et al., 1964). This in vivo observation was supported by later in vitro studies which showed that tartrazine was metabolized by at least one species of intestinal bacteria (the Proteus species) and the metabolites were con®rmed as sulfanilic acid and 1-p-sulfophenyl-3-carboxy-4-amino-5-pyrazolone (Allan and Roxon, 1974; Chung et al., 1978; Pradhan and Majumdar, 1986; Roxon et al., 1966). Although these comparative in vivo and in vitro metabolic studies are scienti®cally interesting, they do not assist in explaining the pivotal study which was used to determine the ADI for tartrazine, nor could they be used in isolation to derive an ADI. The chemical nature of the substance responsible for the in vivo e€ects of tartrazine has not been elucidated. However, since the toxicity observed at the LOAEL in the rat was a presystemic, gastrointestinal e€ect it is postulated that either tartrazine or its hydrolysis products (sulfanilic acid and 1-p-sulfophenyl-3-carboxy-4-amino-5-pyrazolone) are responsible for the laxation observed in the pivotal study used for the derivation of the ADI. It is plausible that the adverse e€ect of tartrazine on the gastrointestinal tract could be induced by a number of mechanisms, such as induction of smooth muscle motility, enhanced release of substances which stimulate muscle motility, stimulation of epithelial cells lining the gut lumen, stimulation of enteric nerves in the gut, or a tartrazine-induced osmotic e€ect. The possible pharmacological e€ect of tartrazine on the gastrointestinal tract has been examined in a single in vitro study. Tartrazine was shown to stimulate smooth muscle motility in guinea pig small intestine, via binding to the acetylcholine receptor (Hutchinson et al., 1992). It may be that tartrazine can stimulate acetylcholine receptors in human gastrointestinal smooth muscle. This could be readily tested in vitro using human colonic strips which are responsive to acetylcholine. If tartrazine was able to stimulate acetylcholine receptors in the intestinal epithelial cells then this may result in an increase in ¯uid and chloride secretions into the bowel lumen and subsequent diarrhoea, all of which could be examined using human mucosal sheets in Ussing chambers or in human colonic epithelial cell lines responsive to acetylcholine. Clearly, a simple study investigating the ability of tartrazine

(and its primary metabolites) to induce a similar response, employing human in vitro systems, may aid in determining the relevance to humans of the rodent gastrointestinal response observed in the pivotal study used in the determination of the ADI. Food additives with in vitro data unrelated to the pivotal study used to calculate the ADI Aspartame Aspartame is an arti®cial low-calorie sweetener added to a number of foods and beverages. It was last evaluated by JECFA in 1981, and allocated an ADI of 0±40 mg/kg body weight based on a NOAEL from a 2-year feeding study in the rat (4 g/ kg body weight/ day), to which a safety factor of 100 was applied. In other studies in this species, at 4 g/kg body weight/day, there was a reduced body weight gain and decreased food consumption with e€ects in numerous tissues, including, kidneys (increased weight, nephrocalcinosis), thyroid (decreased weight), seminal vesicle (atrophy), pancreas (®brosis, mild atrophy, nodular hyperplasia), stomach (gastritis, ulceration), liver (increased weight, hyperplastic nodules, nodular hyperplasia in cortex), adrenal glands (reduced weight in weanlings). At the LOAEL (8 g/kg body weight/day) reduced survival was observed (due to body weight reduction, reduced food consumption, thyroid, seminal vesicle). In addition, the kidneys showed a signi®cant weight increase and a high level of red and white blood cells was observed in the urine (WHO, 1981). Aspartame is a dipeptide, consisting of aspartic acid and phenylalanine, with the carboxyl group of the latter methylated. Aspartame is metabolized initially by hydrolysis of the methyl ester by intestinal esterases followed by hydrolysis of the dipeptide within the intestinal mucosa (Heizer and Laster, 1969). Therefore the three metabolites of aspartame are methanol, aspartic acid and phenylalanine. The extensive presystemic metabolism results in little or no parent compound reaching the general circulation. However, it is possible that the potential toxicity of aspartame would result from an increase in the blood levels of metabolites. Above the LOAEL, aspartame showed toxic e€ects in several target tissues. Although natural constituents of the human diet, the metabolites of aspartame are not without toxic e€ects at high doses. Aspartic acid has been shown to produce lesions of the central nervous system in mice when the dose was at least 1000 mg/kg body weight. However, negligible changes were observed in levels of aspartate in blood in humans given high doses of aspartame (20 mg/kg body weight) (Burns et al., 1990). It has been proposed that phenylalanine may interfere with the brain transport of neurotransmitter precursors and alter the synthesis of the mono-

In vitro data in the derivation of the ADI

amine neurotransmitters (norepinephrine, dopamine and serotonin). However, most in vivo studies have shown that oral doses of aspartame have no e€ect on catecholamines or indolamines in the brain in rodents (Reilly et al., 1989). Methanol, another metabolic product of aspartame, is produced in quantities that do not exceed that formed during the consumption of many natural foods (Davoli et al., 1986). The neurotoxic properties of aspartame have been examined in several in vitro studies. A positive e€ect on brain nerve cell cultures was observed, as measured by a reduction in calcium in¯ux and an increase in lactate dehydrogenase leakage, indicative of aspartame-induced cytotoxicity (Sonnewald et al., 1995). However, aspartame had no e€ect on rat hippocampal slices (Fountain et al., 1988). The limited in vitro studies reviewed on aspartame showed little relevance to the e€ects seen in the pivotal in vivo study used for the determination of the ADI, particularly since aspartame is largely hydrolysed prior to absorption. Nitrate Nitrate was last evaluated by JECFA in 1996, when it was allocated an ADI of 0±3.7 mg/kg body weight (as the nitrate ion) based on a NOAEL of 370 mg/kg body weight/day from a 2-year feeding study in the rat, to which a safety factor of 100 was applied. At the LOAEL in rats (1850 mg/kg body weight/day), reduced weight gain was observed and in pigs (730 mg/kg body weight/day) thyroid function was inhibited. Above the LOAEL (3700 mg/kg body weight/day) in rats inanition was observed (WHO, 1996). Once ingested, nitrate has been shown to be rapidly absorbed from the rat upper intestine. Once absorbed, nitrate is selectively distributed to the salivary glands and actively secreted in saliva in humans and most animals but not in the rat (Fritsch et al., 1985). Following salivary secretion, nitrate is reduced in the oral cavity; this reduction has been studied in vitro and shown to be largely dependent on the oral micro¯ora. The in vitro data describing the toxicity of sodium nitrate available in the literature is limited. In vitro studies indicated a lack of mutagenicity (Konetzka, 1974), which were supported by the results of in vivo genotoxicity studies (Luca et al., 1985) and negative carcinogenicity studies (Maekawa et al., 1982). However, toxic e€ects have been reported in vivo in the kidney, spleen and liver of rodents, but these were later shown to be reversible (Rasheva et al., 1990). In addition, minor doserelated changes were observed to the function and histology of the thyroid in rats in short-term studies at doses up to 4000 mg/litre in drinking water (Gatseva et al., 1992; Horing et al., 1986; Se€ner, 1983). Moreover, it has been suggested that thyroid hypertrophy may be observed in humans when

1185

nitrate exceeded 50 mg/litre in drinking water (van Maanen et al., 1994). Studies on the mechanism responsible for this e€ect were not available, although short-term in vivo studies have suggested that it is due to an e€ect on thyroid hormones (Jahreis et al., 1986). The rat, a species which is generally considered to be more sensitive to thyrotoxic agents, did not develop thyroid e€ects in long-term studies (Maekawa et al., 1982) and the thyrotropic e€ects have been discounted during the derivation of an ADI for sodium nitrate. In species other than the rat, nitrate has been shown to share common secretory pathways with iodide (and thiocycanate), and the e€ects on the thyroid may be due to competition with pathways of iodine metabolism/translocation. These interspecies biochemical di€erences have not been clari®ed in a way that might modify the derivation of the ADI. Diphenyl Diphenyl is a broad spectrum antimicrobial, which is employed as a fungicide in the post-harvest treatment of fruits and vegetables. It was last evaluated by JECFA in 1964, when it was allocated an ADI of 0±0.05 mg/kg body weight, based on a NOAEL of 50 mg/kg body weight/day from a 2year feeding study in the rat, to which a safety factor of 100 was applied. At the LOAEL (250 mg/kg body weight/day) a reduction in body weight, reduced food consumption and non-speci®c e€ects on the kidney were reported. Above the LOAEL (500 mg/kg body weight/day) the same e€ects as those observed at the LOAEL were noted, along with decreased haemoglobin values (WHO, 1964b). The in vitro metabolism of diphenyl has been investigated in some detail, mainly because of its early extensive use as a ``P450`` substrate. Species di€erences in the metabolism of diphenyl have been shown in vitro, using hepatic homogenates. Rabbit liver homogenate was shown to hydroxylate diphenyl to both the o- and p-phenylphenol metabolites (Mitoma et al., 1956). Rat liver subcellular fractions were also capable of hydroxylating diphenyl to pphenylphenol. However, the levels of o-phenylphenol generated by tissue from this species were too small to measure. Hepatic subcellular fractions from at least one of the several mouse strains tested showed measurable amounts of o-phenylphenol (Creaven et al., 1965). Later studies, employing HPLC showed that both o-phenylphenol and p-phenylphenol were generated from diphenyl when either rat or human liver microsomes were employed as activation systems in vitro. The formation of the p-phenylphenol was 35 times greater in the rat than in human microsomes, whereas the generation of o-phenylphenol was similar in both of the species studied (Benford and Bridges, 1980; Parkinson and Safe, 1982). In isolated rat hepatocytes, diphenyl was hydroxylated to

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p-phenylphenol, which was then extensively conjugated with either sulfate or glucuronic acid (Wiebkin et al., 1978). In vivo studies in the rat showed that the major metabolites of diphenyl were p-phenylphenol and p,p'-dihydroxybiphenyl (Meyer and Scheline, 1979). A relationship between the toxicity of this compound and the e€ect of the location of the hydroxyl group has been studied in vitro using hepatocytes from rats. This study showed that p-phenylphenol exhibited more potent cytotoxicity than the parent compound (Nakagawa et al., 1995). The relevance of this study to the metabolites generated from diphenyl remain to be established. At least one of these (o-phenylphenol) has also been shown to induce changes in the kidney; however, whether ophenylphenol represents the toxic metabolite of diphenyl remains to be established. Despite these comparative metabolic studies in vitro, the relevance of these to the study which was used to derive the ADI remains to be elucidated. At the LOAEL, the major toxic e€ects observed in the rat were a reduction in body weight and nonspeci®c e€ects on the kidney. The nature of the proximate toxin responsible for the in vivo e€ects of diphenyl has not yet been elucidated (see o-phenylphenol, below) and comparative human/rodent pharmacokinetic studies were not available in the scienti®c literature. o-Phenylphenol o-Phenylphenol (OPP) and its sodium salt are used as a post-harvest treatment for fruits and vegetables, to prevent microbial decay. It was last evaluated by JECFA in 1964 and allocated an ADI of 0.2 mg/kg body weight, based on a NOAEL of 100 mg/kg body weight/day, from a long-term feeding study in rats, to which a safety factor of 50 was applied. At the LOAEL (1000 mg/kg body weight/ day), the target organs were the kidney (tubular dilation) and body weight (reduced weight gain). No studies above the LOAEL were reported in the evaluation (WHO, 1964b). A number of studies have been conducted since OPP was last evaluated by JECFA. OPP has been shown to induce urinary bladder tumours in the male Fischer rat, following chronic administration at high dose (1±4% in the diet) (Hiraga and Fujii, 1984; Wahle et al., 1997). However, bladder tumours have not been seen in any other species tested chronically with OPP (Allan, 1994; Quast et al., 1997). It has been postulated that the formation of bladder tumours in the rat is caused by 2-phenyl-1,4benzoquinone (PBQ), an oxidative metabolite generated enzymically from OPP (Morimoto et al., 1989; Nakao et al., 1983). Oxidation of OPP to 2phenyl-1,4-hydroquinone (PHQ) was found to occur at high dose levels in the rat (500±1400 mg/kg body weight), possibly due to saturation of the

major conjugative pathways of metabolism (Nakao et al., 1983; Reitz et al., 1983). PHQ was present in the urine as an acid-labile conjugate (Reitz et al., 1983). Formation of PHQ was also found to be gender dependent, with higher levels being present in the male than the female rat (Morimoto et al., 1989; Nakao et al., 1983). The majority of an oral dose of OPP administered to the rat is eliminated in the urine primarily as the glucuronide and sulfate conjugates of the parent compound (Ernst, 1965; Reitz et al., 1983). A comparative metabolic study of OPP in the rat, mouse and human showed that OPP was well absorbed in all species and eliminated in the urine. Sulfation was shown to be the primary metabolic pathway at low doses in all three species, accounting for 57%, 82% and 69% of the urinary radioactivity in the male mouse (15 mg/kg body weight, oral administration), male rat (28 mg/kg body weight, oral) and male volunteers (0.006 mg/kg body weight, dermal administration), respectively. OPP glucuronide was also present in the urine from all species, representing 29%, 7% and 4% of the total urinary metabolites in the low dose groups of mouse, rat and human volunteers, respectively. Conjugates of PHQ in these single-dose studies accounted for 12%, 5% and 15% of the dose in the mouse, rat and human respectively. Little or no free OPP was found in any of the species studied, and no free PHQ or PBQ was found in the mouse, rat or human. A high degree of similarity was found in the routes of metabolism of this compound in these species. A saturable conjugation of OPP with sulfate has been found in both the rat and mouse. This sulfate conjugate was also found to be the major metabolite of OPP in man (Bartels et al., 1998). The dose-dependent increase in the hydroxylated metabolite PHQ has been correlated to the dosedependent formation of urinary bladder tumours in the male rat at high doses (Morimoto et al., 1989; Nakao et al., 1983). They proposed that this metabolite in its oxidized form of PBQ, may be the ultimate carcinogenic metabolite of OPP. The amount of free PHQ found as urinary metabolite represents less than 3% of the dose administered via the diet in chronic studies (Morimoto et al., 1989). The dose-dependent increase in levels of unconjugated PHQ greater than 0.5% in the diet have been seen by several groups (Reitz et al., 1983). A similar increase was seen in the mouse (Bartels et al., 1998). However, since no urinary bladder e€ects have been seen following chronic administration of OPP to the mouse, the dose-dependent increase in total PHQ does not in itself explain the di€erences in species-selective urinary bladder carcinogenicity of OPP. Clearly, additional factors such as localized deconjugation of PHQ-G or PHQ-S followed by oxidation to PBQ may be responsible. The pH-

In vitro data in the derivation of the ADI

dependent auto-oxidation of urinary PHQ to PBQ may also play a role (Kwok and Eastmond, 1997). The majority of the short-term in vitro tests show OPP to be non-genotoxic (Reitz et al., 1983), although some suggest that this compound will damage DNA (Suzuki et al., 1985; Tayama and Natagawa, 1994; Ushiyama et al., 1992). The metabolism of OPP has been studied in vitro. Using rat liver subcellular fraction the major metabolites detected were PHQ and PBQ. PHQ was generated by a P450 monoxygenase and this was further oxidized non-enzymically to PBQ via a phenylhydroquinone-semiquinone intermediate. PHQ has been shown to increase the formation of 8-hydroxy2'deoxyguanine, demonstrating free radical damage by reactive oxygen species (Nakagawa et al., 1995). This was supported by further studies using the microsomal fraction from rat liver (Reitz et al., 1983, 1984; Roy et al., 1990). OPP was metabolized to OPP-glucuronide, PHQ-glucuronide, PHQ-glutathione and PHQ hepatocytes from rats (Nakagawa et al., 1992). Since the last evaluation of this compound by JECFA, several studies have reported an increase in tumours in the bladder of the male Fischer rat. As a consequence of this, OPP has undergone extensive testing in a range of in vitro systems. The relevance of these results to the study that was used for the derivation of the ADI remains to be elucidated. It might be predicted that the metabolite (PHQ), which has been proposed as the proximate compound responsible for bladder tumours in the male rat, is also involved in the kidney lesions observed at the LOAEL, although the relevance of this is currently not known. The limited metabolic studies in the mouse, rat and human showed that there was little di€erence in the nature of the urinary metabolites between these species. However, comparative pharmacokinetic studies on either OPP or its metabolites have not been reported in the literature. Moreover, the relevance of the metabolic similarities and di€erences between species is questionable in relation to the pathogenesis, which shows some similarities with sodium saccharin and sodium ascorbate, speci®cally in the male Fischer rat. Aluminium Aluminium was last evaluated by JECFA in 1988, when it was allocated a provisional tolerable weekly intake of 7 mg/kg body weight, based on a NOAEL of 110 mg/kg body weight/day, from a 189-day feeding study in dogs. No doses were reported above the NOAEL level in this species (WHO, 1988). It has been suggested that aluminium may contribute to the pathogenesis of several neurological disorders that may have an environmental component in their aetiology such as Alzheimer's disease, dialysis encephalopathy, endemic amyotropic lateral sclerosis and parkinsonism-dementia.

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However, the majority of the pharmacokinetic studies show that, due to the formation of insoluble aluminium phosphate in the gastrointestinal tract, only a minor amount of an orally administered aluminium salt is absorbed into the systemic circulation. For example, in the rat after a single oral dose of 11 mg AlCl3, between 0.06 and 0.2% was absorbed and, following dietary exposure, humans have been shown to absorb between 0.1 and 0.8% (Greger and Baier, 1983); although a single study reported aluminium absorption between 2 and 24% after the oral administration of Al(OH)3 ranging from 1 and 3 g/day (Gorsky et al., 1979). However, other studies using Al(OH)3 show that aluminium is absorbed at levels ranging from 0.001 to 0.007% (Haram and Werberg, 1987; Werberg and Berstad, 1986). Following absorption, aluminium binds to several plasma proteins, the most important of these being transferrin (Trapp, 1983). The extent of aluminium binding to protein is reported to range from 0 to 98% and is believed to be in¯uenced by the serum level of aluminium (Gidden et al., 1980). As the majority of ingested aluminium is not absorbed, a large proportion of an oral dose is excreted in the faeces (Ondreicka et al., 1966). Following absorption of a minor amount of the dose into the blood stream, urinary excretion is then the major route of elimination, although some evidence does suggest that systemic aluminium can be eliminated in the bile to some extent (Anderson et al., 1979; Kovalchik et al., 1978). The quantitative role played by the kidneys in the excretion of aluminium is controversial. The wide range of clearance values for aluminium is explained by the fact that renal elimination depends on aluminium plasma concentration, so that the proportion of the dose of aluminium cleared by the kidney in a given time decreases with increasing plasma aluminium concentration (Hohr et al., 1989; Pai and Melethil, 1989). Although di€erent mechanisms for renal elimination have been suggested, it is likely that reduced renal aluminium clearance at high plasma levels is due to increased plasma protein binding (Wilhelm et al., 1986). Normal renal excretion of humans is less than 20 mg/day, although values up to 3800 mg/day have been reported in patients receiving aluminium for therapeutic purposes (Klein et al., 1982). The association of aluminium with neurological disorders was suggested by the observation that the risk of developing dementia was high among individuals receiving kidney dialysis treatment. The initial diagnosis of dialysis encephalopathy was con®rmed in 150 patients treated in 65 dialysis centres in Europe in 1976 and 1977. The prevalence was approximately 500 per 100,000 cases (Wing et al., 1980). However, owing to its poor intestinal absorption, the intake of aluminium orally has not been associated with aluminium-induced encephalopathy. Dialysis patients with clinical symptoms of

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encephalopathy have been shown to have extremely high plasma aluminium levels (up to 555.0 2 86 mg/ litre) (Pogglitsch et al., 1980). However the plasma aluminium concentration in such patients depends on the aluminium content of the dialysate ¯uid. When the dialysate ¯uid was rich in aluminium (200 mg/litre) 16 out of 23 patients showed encephalographic alterations (Vecchierini et al., 1980.) There do appear to be species di€erences in the susceptibility to the neurological e€ects of aluminium. Rats, for example, do not develop aluminium-induced encephalopathy even when the concentration of aluminium in the brain has reached six times the levels necessary to elicit encephalopathy in cats, rabbits and dogs (Boegman and Bates, 1984). Learning, memory, behavioural de®cits and electrophysiologic (seizures) and neurochemical alterations are observed in experimental animals with elevated brain aluminium concentrations resulting from exposure to high aluminium via brain infusion or injection. However, these e€ects were not reported following the oral administration of very high doses of aluminium (as sodium aluminium phosphate) in the dog (the NOAEL was 110 mg Al/kg body weight/day in the dog, the highest dose tested). As a consequence of the neurological associations with aluminium consumption the majority of the in vitro studies on this metal have concentrated on its potential involvement in the development of neurological disorders. In vitro studies showed that aluminium has e€ects on interneurone connections (Van et al., 1989), Ca2 + ATPase in rat brain synaptosomes (Julka and Gill, 1996), protein kinase C (Katsuyama et al., 1989), nerve cell viability and development in culture (Atterwill et al., 1996; Muller and Bruinink, 1994; Roll et al., 1989) and induced a conformational change in the human neuro®lament protein (Hollosi et al., 1995). The short-term in vivo studies reported here show that orally administered aluminium is absorbed only in minute quantities in experimental animals and humans. Consequently, the systemic levels following oral exposure in the normal healthy population never approach those observed in dialysis patients in whom dementia has been observed. Neurotoxicity in dialysis patients is therefore a consequence of the high levels of systemic aluminium from its intravenous administration and reduced renal clearance of aluminium. The in vitro studies reported here could be validated by comparing aluminium concentrations with those which induce neurological changes in dialysis patients. However, neurotoxicity was not the basis for the NOAEL in the ADI determination for aluminium, where the NOAEL was actually the highest dose tested. The in vitro neurotoxicity data is therefore not relevant for the consideration of the human risk associated with aluminium consumption.

Brown HT Brown HT is a water-soluble azo dye, used as a colouring for a variety of food and drink products. It was allocated an ADI of 0±1.5 mg/kg body weight, by JECFA in 1984, based on a NOAEL of 150 mg/kg body weight/day, from a 2-year feeding study in the mouse to which a safety factor of 100 was applied. At the LOAEL (750 mg/kg body weight/ day, 80-wk oral study in the mouse), four target tissues of toxicity were identi®ed (liver, ovaries, heart, blood). Increased leucocyte in®ltration was observed in the liver, and cystic ovaries were induced in the female mouse. In addition, there was a reduction in heart weight and an increase in the total leucocyte count. Above the LOAEL (3000 mg/ kg body weight/day), from a short-term study in the rat, pigment disposition was observed in the intestine, lymph and kidneys. The kidneys of both sexes showed mild dysfunction (WHO, 1984). In vitro studies with rat intestinal micro¯ora showed that Brown HT undergoes azo reduction to naphthionic acid and two unidenti®ed metabolites (Mallett et al., 1986). This is supported by shortterm in vivo studies in the rat, mouse and guinea pig (50±200 mg/kg body weight, single dose, 14Clabelled), where the major urinary metabolite in the three species was identi®ed as naphthionic acid (Phillips et al., 1987). It has been suggested that the brown coloration associated with several internal organs (liver, kidney, intestine, ovaries, uterus and lymph nodes) of the rat and mouse following prolonged exposure to high levels of Brown HT in the diet was due to absorbed colouring or metabolites (Chambers et al., 1966; Drake et al., 1978; Grant and Gaunt, 1987; Hall et al., 1966). Following administration of a single radiolabelled oral dose to the rat (250 mg/kg body weight), the major metabolite was naphthionic acid, and radioactivity was found to be associated with gastrointestinal tract, kidney and lymph nodes. However, no evidence for pathological changes in these tissues was demonstrated in this study. The short-term in vivo and in vitro studies reported in this review provide only limited support to the pivotal study which was used to derive the ADI for Brown HT. Despite the fact that naphthionic acid has been identi®ed as a major metabolite of Brown HT in the rodent, the proximate toxin responsible for the adverse e€ects at the LOAEL (and above this dose level) is as yet unknown. Comparative human/rodent pharmacokinetic studies have not been conducted on either Brown HT or its reduction product, naphthionic acid. Food additives with limited in vitro data Brilliant Blue FCF Brilliant Blue FCF (both the diammonium salt and disodium salt) is used as a commercial food colour. It has an ADI of 0±12.5 mg/kg body

In vitro data in the derivation of the ADI

weight, which was allocated in 1969 by JECFA, based on a NOAEL of 2500 mg/kg body weight from a 2-year feeding study in the rat, to which 200-fold safety factor was applied (WHO, 1969). The excretion of Brilliant Blue FCF was investigated in the rat, rabbit and dog following a single oral administration (200 mg). Less then 5% of the dye was shown to be absorbed in any of the species studied, with the majority excreted in the faeces (Hess and Fitzhugh, 1953, 1955). This early work was supported by a similar, later, study in the same species (Brown et al., 1980). In addition, neither absorption nor metabolism was detected following a single oral administration (30±10,000 mg/kg body weight) to the rat, mouse or guinea pig (Phillips et al., 1980). There were no reported e€ects above the NOAEL for Brilliant Blue FCF which was the highest level tested in the rat (2500 mg/kg body weight), and therefore a speci®c target for toxicity has not been described for this food additive. In terms of its toxicokinetics, this compound is absorbed only to a limited extent in all of the species so far studied and is excreted largely unchanged in the faeces. It could be predicted that Brilliant Blue FCF is sparsely absorbed in humans; a short-term human in vivo study would clarify this. In addition, the absence of metabolism by human gut micro¯ora could be con®rmed using in vitro techniques. In terms of the toxicodynamics neither the inter-species nor the human inter-individual default factor can be replaced with a data-derived safety factor using the available short-term in vivo studies. Thiabendazole Thiabendazole is used as a broad spectrum anthelmintic in various animal species and is also employed for the control of parasitic infection in humans. An ADI of 0.1 mg/kg body weight was allocated in 1993 by JECFA, based on a NOAEL of 10 mg/kg body weight/day from a 2-year feeding study in rats, to which a 100-fold safety factor was applied. At the LOAEL (40 mg/kg body weight/ day) and above the LOAEL (160 mg/kg body weight/day) a reduction in body weight was observed (WHO, 1993b). The most sensitive index of the toxic e€ects of thiabendazole appeared to be non-speci®c, with reduced weight gain being the principal adverse e€ect at the LOAEL. Human in vivo data describing the pharmacokinetics and metabolism of this compound are very sparse. A metabolism study conducted in four human males following a single oral dose (1.0 g, 14C-thiabendazole 25 mCi) showed that the majority (80%) of the radiolabel was excreted within 24 hr following administration, of which less than 1% represented either unchanged thiabendazole or its hydroxylated metabolite, 5hydroxythiabendazole. The majority was shown to

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be excreted as the sulfate (13%) or glucuronide (25%) conjugates of 5-hydroxythiabendazole (Tocco et al., 1966). In the rat (25 mg/kg body weight) the major urinary metabolites were identi®ed as unchanged thiabendazole (3%), 5-hydroxythiabendazole (4%), the sulfate conjugate of 5hydroxythiabendazole (39%) and the glucuronide conjugate of 5-hydroxythiabendazole (28%). The remaining metabolites were not identi®ed in either species. The usefulness of this information for modifying the ADI remains to be elucidated, especially in view of the fact that the only adverse e€ect observed at the LOAEL was a reduction in body weight, which is a non-speci®c event rather than a mechanistic consequence of a toxic insult occurring on a speci®c tissue. In addition, no in vitro studies were found which were relevant to determining the mechanism of the weight de®cit. Ferrocyanide (calcium, potassium and sodium) Ferrocyanide (its calcium, potassium and sodium salts) is used as an anticaking agent and the potassium salt is also used as a precipitant for removing surplus copper and iron from white wine. It was last evaluated by JECFA in 1974 when it was allocated an ADI of 0.025 mg/kg body weight, based on a NOAEL of 25 mg/kg body weight/day from a short-term feeding study in rats, to which a 1000fold safety factor was applied. At the LOAEL (250 mg/kg body weight/day) the target organ was the kidney, which showed increased weight and minimal tubular damage (females only). Above the LOAEL (2500 mg/kg body weight/day) a number of target tissues were identi®ed. The kidneys increased in weight and showed marked tubular damage and calci®ed deposits. The adrenal gland (males only) and pituitary gland (females only) also increased in weight. These animals also showed depressed haemocrit and haemoglobin values (WHO, 1974). It is generally considered that ferrocyanide is non-toxic, due to the great stability of the complex ion, which is only capable of separating from the CNÿ group in unusual circumstances (e.g. exposure to strong acid) . The biological fate of this compound has been studied in humans and nephrectomized dogs. In four healthy human subjects (all male), ferrocyanide clearance following intravenous administration was estimated to be 37 29.8 ml/min. One patient with renal failure showed reduced clearance (3.0 ml/min). It was concluded that following intravenous infusion, ferrocyanide is cleared predominantly by the kidney. Glomerular function was studied in 115 humans; 25% was excreted in 80 minutes and the remainder in the next 90 minutes by glomerular ®ltration. As expected, patients with renal impairment had slower rates of excretion (Forero and Kock, 1942). The half-life in normal humans was 135 minutes and in normal dogs this

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was 40±50 minutes (Kleeman and Epstein, 1956). In infants, the clearance values for sodium ferrocyanide were 4.3 and 6.0 ml/min for two subjects studied. Intravenous infusion of ferrocyanide and creatine into dogs gave an average clearance value for ferrocyanide of 0.966 2 0.41 ml/min. No pharmacokinetic studies in the rat (the species used in the determination of the ADI) have been reported. No other studies on this compound were reported in the literature. It is clear from these studies that the kidney is the target site for toxicity for this compound, yet the underlying mechanism responsible for the toxic response is unknown. Moreover, the reporting of the JECFA evaluation was not detailed enough to indicate how the ADI was derived but the large safety factor used (1000-fold) suggests that the database was probably considered rather limited and the evaluation was based on a short-term study. No recent in vitro studies were located that might assist in deriving the ADI.

Discussion In vitro test systems are increasingly becoming an essential tool as part of an integrated toxicology testing strategy, and scienti®c progress in the ®elds of cellular and molecular biology now permit the utilization of in vitro test systems which can be used to study toxicological mechanisms under circumstances that are more relevant to humans. To a large degree, in vivo toxicity tests have, in the past, been used as the almost exclusive source of data for the quantitative hazard characterization of food additives and contaminants. In terms of identifying a likely human hazard, the need for data to complement the results obtained in vivo and provide greater con®dence in extrapolating toxicity data between species is well recognized. However, in vitro toxicity data is currently considered on an ad hoc basis during the safety assessment of a speci®c compound. The aim of the current study was to determine the availability of in vitro toxicity data for a range of food additives, the applicability of this in vitro data to the pivotal study used for the determination of the ADI, and to assess how results from in vitro studies might be incorporated into the current regulatory framework. Present use of in vitro data for the determination of an ADI Of the 18 food additives reviewed in this study, nine were found to have in vitro data related to the pivotal in vivo study used for the calculation of an ADI value. Of these eight compounds, the arti®cial sweetener cyclamate was found to be the compound for which the supplementary toxicity data was most relevant to the pivotal study used in the ADI determination. First, the proximate toxic metabolite of cyclamate (cyclohexylamine) has been identi®ed. In

addition, the e€ects of this metabolite at the target organ have been well described in the most sensitive test species. As a result of the extensive in vivo database, one in vitro toxicodynamic study has been conducted using rat testicular Sertoli cells at concentrations comparable to those found in the plasma and testes following a toxic dose to the same species (Creasy et al., 1990). However, the in vitro data on cyclohexylamine, although con®rming the mechanism of toxicity, in the absence of comparative human studies, could not be used to replace any of the existing default uncertainty factors for this compound. Cyclamate was the only food additive in this review for which in vitro studies have been designed to relate speci®cally to the toxicological changes which occur in vivo at the LOAEL. The current review described one food additive in which in vitro tests have been used to establish the mechanism by which toxicity occurs in vivo. Two in vitro studies were used to show that the mechanism of lymphopaenia induced by a speci®c component of Caramel Colour III [2-acetyl-4(5)-tetrahydroxybutylimidazole] was due to the inhibition of pyridoxine phosphatase, a key enzyme involved in pyridoxine metabolism. However, since the pharmacokinetics of this component of the colour has not been established in either the test species or in humans, this data in isolation could not replace the current default uncertainty factor in an ADI determination. The in vitro tests on other food additives in the current review, showed that although not speci®cally designed to aid in the safety assessment procedure, supplementary in vitro tests could be used to support a speci®c mechanism of in vivo toxicity. For example, the in vitro antigenotoxicity studies reported for butylated hydroxytoluene, curcumin and propyl gallate show an interaction with hepatic drug metabolising enzymes which correlates with the liver enlargement induced by these compounds at doses above the LOAEL. Six of the 18 food additives reported had in vitro data which was unrelated to the pivotal study used in the ADI determination. The in vitro studies conducted on aspartame, for example, were used to investigate the potential neurotoxic e€ects of this arti®cial sweetener. However, neurotoxicity was not a response observed in the pivotal study used in the ADI determination for aspartame and these in vitro studies are therefore not useful for re®ning the ADI for this compound. Similarly for the food additives diphenyl and o-phenylphenol, the metabolism of which has been studied extensively, the substance responsible for the toxic e€ects observed at the LOAEL has not yet been elucidated for either compound. The remaining three food additives (Brilliant Blue FCF, thiabendazole and ferrocyanide) were compounds for which no in vitro toxicity data was found. Brilliant Blue FCF was not toxic in the rat at the highest dose tested and kinetic

In vitro data in the derivation of the ADI

studies showed that it was absorbed from the gastrointestinal tract only to a very limited extent in all of the species studied. Although comparative rat/ human in vivo studies would con®rm inter-species similarities in the absorption of Brilliant Blue FCF, it is dicult to envisage how in vitro studies might further improve the safety assessment of this compound. Thiabendazole induced reduced body weight at the LOAEL, an e€ect which is not amenable to in vitro investigations. The toxicity data on ferrocyanide was also very limited. Although the kidney was described as the target tissue, the mechanism by which this occurs in vivo was not well described. Suggestions for safety approaches using in vitro data It is evident from the food additives discussed in this review that in the majority of cases in vitro studies have been conducted in isolation, that is, without taking into account the toxic e€ects observed in the pivotal study used in the determination of the ADI. However, for in vitro data on a food additive to be incorporated into the current regulatory framework and therefore assist in the re®nement of default uncertainty factors, these studies should be designed using information obtained from in vivo studies. Detailed prior toxicological knowledge should be available in the following areas: 1. The chemical nature of the proximate toxin (and the mechanism involved in its formation) should have been well studied. 2. The main target tissue(s) for toxicity should have been clearly de®ned in long-term in vivo studies in the most sensitive test species. 3. Peak plasma levels (or preferably tissue levels at the target site) of the proximate toxin following oral administration in the most sensitive test species, should have been studied at doses where toxicity has been observed in vivo.

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available; these would also take into account enzymic variations such as P450 polymorphisms, which will in¯uence toxicity in vivo. Although it appears unlikely that the results of such isolated metabolic studies could be used in by themselves to derive an ADI, such data would provide crucial information for the design of future short-term in vivo and in vitro toxicity studies. Toxicokinetics For uncertainty arising as a consequence of interspecies and inter-individual variability in toxicokinetics, a data-derived safety factor would have to be determined from short-term in vivo pharmacokinetic studies, where a mean parameter for the proximate toxin in the most sensitive test species (such as systemic plasma clearance or the area under the plasma concentration curve) is compared with the same parameter in man. Although pharmacokinetic parameters for the proximate toxin would be more commonly derived from short-term in vivo studies, attempts have been made to predict the plasma clearance of drugs using in vitro systems. For example, the prediction of plasma clearance has been addressed for the ergot derivative CQA 206291 (a pro-drug). The metabolism of this compound in human, dog and rat in vitro and in vivo is similar, with cytochrome P450 3A playing a major role. The pharmacokinetic prediction for hepatic slices and microsomes from the three species correlated well with the in vivo data, resulting in the same species ranking order (rat>dog>man) (Vickers et al., 1993). In addition, saturation kinetics might also be studied in vitro employing concentrations of the parent compound which are related to the plasma concentration in vivo at a toxic dose. However, these in vitro approaches have yet to be systematically incorporated into the risk assessment of food additives. Toxicodynamics

Metabolism When the proximate toxin has been shown to be a metabolite of the food additive the formation of this from the parent can be compared between human and the most sensitive test species. The in vitro metabolism of drugs and other xenobiotics using isolated hepatic systems, intended to mimic in vivo metabolism (such as hepatic subcellular fractions and isolated hepatocytes), have been used in toxicology and drug metabolism for over 30 years. In addition, a more recent development in this area is the use of precision-cut tissue slices. Precision-cut liver slices have been used to study the metabolism of a chemically diverse range of compounds such as a¯atoxin B1, benzene, ca€eine and coumarin (Ekins, 1996). Hepatic liver slices could be used for studying inter-species di€erences in the metabolism of food additives. Human tissue slices are also

For the modi®cation of default uncertainty factors arising from toxicodynamic variability, it is envisaged that in vitro studies might be used both as an initial screen to identify the putative toxin, and for studying inter-species and inter-individual di€erences in the mechanism of toxicity. This could be achieved using cellular models corresponding to the most sensitive tissue, previously identi®ed from long-term toxicity studies (and used for the derivation of the ADI), as shown with cyclohexylamine. Several in vitro systems, based on target organs, are already used to investigate speci®c organ toxicity in other areas of toxicology. For example, neurotoxicity has been addressed using organotypic neural cultures in vitro and the adverse e€ects induced by neurotoxic agents have been shown to correlate with changes induced in vivo (Spencer et al., 1986). Similar neuronal cell-derived models have been used to investigate the in vitro toxicity of aluminium and

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aspartame. However, for these food additives the neurological e€ects were not observed in pivotal the animal study and were therefore of no relevance to re®ning the ADI for these compounds. In vitro models mimicking renal toxicity have also been used in toxicology. Rodent cortical epithelial cells have been tested with nephrotoxic metals and acetaminophen. Moreover, the in vitro toxicity induced by these compounds correlated with e€ects induced in the intact organism (Smith et al., 1987). Although three of the food additives investigated in this review showed adverse e€ects on the kidney in long-term toxicity studies in vivo (diphenyl, o-phenylphenol and ferrocyanide), these have not been studied in vitro with cells from this tissue. In addition, the reticuloendothelial system has also been mimicked using isolated erythrocytes and in vitro e€ects were shown to be similar to those observed in vivo for several compounds (Stammati et al., 1981). The proximate toxin responsible for erythropoietic e€ects of Red 2G, phenylhydroxylamine, has been tested for toxicity using isolated erythrocytes and was shown to induce methaemoglobin in both the rodent and human cell (Jenkins et al., 1972). However, since this in vitro data could not be related to plasma levels of phenylhydroxylamine following oral administration of Red 2G in either species this information could not be used to determine a speci®c data-derived safety factor for Red 2G. Cultured thyroid cells have also been used as both a screening and mechanistic tool to investigate a range of compounds which show thyroid-speci®c toxicity in vivo (Brown, 1988). However, thyroid toxicity can be induced by a wide range of mechanisms. The thyrotoxic dye erythrosine, for example, inhibits peripheral conversion of T3 to T4. Therefore, for thyrotoxic agents such as erythrosine where the mechanism of toxicity is a peripheral e€ect a simple thyroid cell system would give no indication as to the potential in vivo e€ects of this compound. Testicular toxicity can also be studied in vitro. Sertoli and germ cell cultures have been used to study the adverse e€ects from phytholate esters and glycol esters, the results of which have been to correlate with the in vivo testicular e€ects of these compounds (Garside, 1988). A similar in vitro system was used to investigate the toxicity of cyclohexylamine, using concentrations relevant to toxic plasma and testicular concentrations of this compound (Creasy et al., 1990). Several of the food additives showed induction of the cytochrome P450 isoenzymes (BHT, gallates and curcumin), yet none of these has been tested with hepatic culture systems derived from rodents or humans. The induction of drug-metabolizing enzymes could also be studied in vitro using hepatic tissue slices. For example, Lake et al. (1997) have

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