Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect.

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1'-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1'-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1'-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 microM did not inhibit 1'-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1'-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1'-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

Predictive models for carcinogenicity and mutagenicity: frameworks, state-of-the-art, and perspectives.

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include Vitotox, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-throughput assays combined with innovative data-mining and in silico methods. Various initiatives in this regard have begun, including CAESAR, OSIRIS, CHEMOMENTUM, CHEMPREDICT, OpenTox, EPAA, and ToxCast. In silico methods can be used for priority setting, mechanistic studies, and to estimate potency. Ultimately, such efforts should lead to improvements in application of in silico methods for predicting carcinogenicity to assist industry and regulators and to enhance protection of public health.

Induction of Nrf2-mediated cellular defenses and alteration of phase I activities as mechanisms of chemoprotective effects of coffee in the liver.

Coffee consumption has been associated with a significant decrease in the risk of developing chronic diseases such as Parkinson disease, diabetes type-2 and several types of cancers (e.g. colon, liver). In the present study, a coffee-dependent induction of enzymes involved in xenobiotic detoxification processes was observed in rat liver and primary hepatocytes. In addition, coffee was found to induce the mRNA and protein expression of enzymes involved in cellular antioxidant defenses. These inductions were correlated with the activation of the Nrf2 transcription factor as shown using an ARE-reporter luciferase assay. The induction of detoxifying enzymes GSTs and AKR is compatible with a protection against both genotoxicity and cytotoxicity of aflatoxin B1 (AFB1). This hypothesis was confirmed in in vitro and ex vivo test systems, where coffee reduced both AFB1-DNA and protein adducts. Interestingly, coffee was also found to inhibit cytochrome CYP1A1/2, indicating that other mechanisms different from a stimulation of detoxification may also play a significant role in the chemoprotective effects of coffee. Further investigations in either human liver cell line and primary hepatocytes indicated that the chemoprotective effects of coffee against AFB1 genotoxicity are likely to be of relevance for humans. These data strongly suggest that coffee may protect against the adverse effects of AFB1. In addition, the coffee-mediated stimulation of the Nrf2-ARE pathway resulting in increased endogenous defense mechanisms against electrophilic but also oxidative insults further support that coffee may be associated with a protection against various types of chemical stresses.

A toxicogenomics approach to identify new plausible epigenetic mechanisms of ochratoxin a carcinogenicity in rat.

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

Oxidative damage and stress response from ochratoxin a exposure in rats.

Ochratoxin A (OTA) is a mycotoxin found in some cereal and grain products. It is a potent renal carcinogen in male rats, although its mode of carcinogenic action is not known. Oxidative stress may play a role in OTA-induced toxicity and carcinogenicity. In this study, we measured several chemical and biological markers that are associated with oxidative stress response to determine if this process is involved in OTA-mediated toxicity in rats. Treatment of male rats with OTA (up to 2 mg/ 24 h exposure) did not increase the formation of biomarkers of oxidative damage such as the lipid peroxidation marker malondialdehyde in rat plasma, kidney, and liver, or the DNA damage marker 8-oxo-7,8-dihydro-2' deoxyguanosine in kidney DNA. However, OTA treatment (1 mg/kg) did result in a 22% decrease in alpha-tocopherol plasma levels and a 5-fold increase in the expression of the oxidative stress responsive protein haem oxygenase-1, specifically in the kidney. The selective alteration of these latter two markers indicates that OTA does evoke oxidative stress, which may contribute at least in part to OTA renal toxicity and carcinogenicity in rats during long-term exposure.

Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures.

Aggregating fetal liver cell cultures were tested for their ability to metabolize xenobiotics using ethoxycoumarin-O-deethylase (ECOD), as marker of phase I metabolism, and glutathione S-transferase (GST), as marker for phase II reactions. Significant basal activities, stable over 14 days in culture were measured for both ECOD and GST activities. The prototype cytochrome P450 inducers, 3-methylcholanthrene (3-MC) and phenobarbital (PB), increased ECOD and GST activities reaching an optimum 7 days after culturing, followed by a decline in activity. This decline was partially prevented by 1% dimethyl sulfoxide (DMSO) added chronically to the culture medium. DMSO was also found to induce ECOD activity and to a lesser extent GST activity. Furthermore, it potentiated in a dose-dependent manner the induction of ECOD by PB. The food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolically transformed through a number of pathways in vivo. It was therefore used to examine the metabolic capacity in fetal and adult liver cell aggregates. Metabolism of MeIQx was mainly through N2-conjugation, resulting in formation of the N2-glucuronide and sulfamate conjugates for non-induced fetal liver cells. These metabolites were also found in large amounts in non-induced adult liver cells. Low levels of cytochrome P450-mediated ring-hydroxylated metabolites were detected in both non-induced fetal and adult liver cells. After induction with arochlor (PCB) or 3-MC, the major pathway was ring-hydroxylation (cytochrome P450 dependent), followed by conjugation to beta-glucuronic or sulfuric acid. The presence of the glucuronide conjugate of N-hydroxy-MeIQx, a mutagenic metabolite, suggested an induction of P450 CYP1A2. The metabolism of MeIQx by liver cell aggregates is very similar to that observed in vivo and suggests that aggregating liver cell cultures are a useful model for in vitro metabolic studies in toxicology.

The naturally occurring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture.

The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model. As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages. The results showed that FB1 did not cause cell loss and it had no effects on neurons. However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28). The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells. However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG. The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days. In summary, FB1 selectively affected glial cells. In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides.

Aggregating brain cell cultures of fetal rat telencephalon can be grown in a chemically defined medium for extended periods of time. After a phase of intense mitotic activity, these three-dimensional cell cultures undergo extensive morphological differentiation, including synaptogenesis and myelination. To study the developmental toxicity of organophosphorus compounds (OP), aggregating brain cell cultures were treated with parathion. Protein content and cell type-specific enzyme activities were not affected up to a concentration of 10(5) M. Gliosis, characterized by an increased staining for glial fibrillary acidic protein (GFAP), was observed in immature and in differentiated cells. In contrast, uridine incorporation and myelin basic protein (MBP) immunoreactivity revealed strong differences in sensitivity between these two developmental stages. These results are in agreement with the view that in vivo the development-dependent toxicity is not only due to changes in hepatic detoxification, but also to age-related modifications in the susceptibility of the different populations of brain cells. Furthermore, they underline the usefulness of histotypic culture systems with a high developmental potential, such as aggregating brain cell cultures, and stress the importance of applying a large range of criteria for testing the developmental toxicity of potential neurotoxicants.

Anticonvulsant drug toxicity in rat brain cell aggregate cultures.

As an extension of a previous validation study, the concentration-dependent effects of a series of anticonvulsant drugs were examined in aggregating cell cultures of foetal rat telencephalon. Cultures were treated either at an early (day 5 to day 14) or at an advanced (day 20 to day 28) developmental stage, and assayed for changes in the activities of the cell type-specific enzymes choline acetyltransferase (ChAT), acetylcholinesterase (AChE), glutamic acid decarboxylase (GAD), glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). Five drugs (carbamazepine, diazepam, phenobarbital, phenytoin and valproate), currently used in the treatment of epileptic patients, were tested together with losigamone, a recently developed anticonvulsant. The results show distinct, concentration-dependent patterns of biochemical changes for the different drugs. Phenytoin, carbamazepine, losigamone and diazepam greatly reduced GAD, ChAT and AChE activities, indicating a relatively high neuron-specific toxic potential. Diazepam produced a more general pattern of toxicity and, in contrast to the anticonvulsants, showed higher toxicity in less-differentiated cultures. Phenobarbital and valproate slightly but significantly increased the activities of several enzymes. The patterns of concentration-dependent effects observed in this three-dimensional cell culture system are in good agreement with the presumed neurotoxic and/or teratogenic potential of these drugs.

Validation of whole chick embryo cultures, whole rat embryo cultures and aggregating embryonic brain cell cultures using six pairs of coded compounds.

A comparative study was performed to assess the effects of six pairs of coded compounds using cultures of whole chick and rat embryos as well as aggregating brain cell cultures. Developed originally for basic studies in developmental biology, these three culture systems have been adapted for the screening of chemicals in the field of prenatal toxicology. Chick and rat embryos were cultured for 2 days during the early stages of organogenesis. Aggregating cell cultures were prepared from early foetal rat telecephalon and grown for 14 days in a chemically defined medium. Concentration-response relationships were established by treating whole embryos in vitro for 2 days, and aggregating brain cell cultures for 9 days. After decoding the compounds, the results showed that, in the three test systems, specific effects were induced at comparable concentration levels. Similar compound-related malformations could be observed in both chick and rat whole embryo cultures. In aggregating brain cell cultures, neuron- and glia-specific effects could be distinguished. Based on the results obtained in the three in vitro systems, the following concentration ranges were determined for the teratogenic/toxic potencies of the test compounds (in mol/litre): <10(-6): retinoids (Ro 13-6307, Ro 1-5488), 6-aminonicotinamide, ketoconazole; 10(-6)-10(-3): 4-hydroxypyridine, sulfadiazine, sulfanilamide, caffeine, theophylline, metronidazole, methoxyacetic acid; >10(-3): methoxyethanol. In general, the three in vitro test systems were found to provide concordant and complementary data on the toxicity and teratogenicity of a given compound. These data were also comparable with those available from in vivo studies. It is therefore concluded that such a test battery could contribute significantly to risk assessment and to the reduction of in vivo experimentation in reproductive toxicology.

Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells.

The coffee-specific diterpenes cafestol and kahweol (C + K) have been reported to be anticarcinogenic in several animal models. Proposed mechanisms involve a co-ordinated modulation of several enzymes responsible for carcinogen detoxification, thus preventing reactive agents interacting with critical target sites. To address the human relevance of the chemoprotective effects of C + K against aflatoxin B(1) (AFB1) genotoxicity observed in rat liver, and to compare the mechanisms of protection involved in both species, animal and human hepatic in vitro test systems were applied. In rat primary hepatocytes, C + K reduced the expression of cytochrome P450 CYP 2C11 and CYP 3A2, the key enzymes responsible for AFB1 activation to the genotoxic metabolite aflatoxin B1-8,9 epoxide (AFBO). In addition, these diterpenes induced significantly GST Yc2, the most efficient rat GST subunit involved in AFBO detoxification. These effects of C + K resulted in a marked dose-dependent inhibition of AFB1-DNA binding in this rat in vitro culture system. Their relevance in humans was addressed using liver epithelial cell lines (THLE) stably transfected to express AFB1 metabolising cytochrome P450s. In these cells, C + K also produced a significant inhibition of AFB1-DNA adducts formation linked with an induction of the human glutathione S-transferase GST-mu. Altogether, these results suggest that C + K may have chemoprotective activity against AFB1 genotoxicity in both rats and humans.

Cafestol and kahweol, two coffee specific diterpenes with anticarcinogenic activity.

Epidemiological studies have found an inverse association between coffee consumption and the risk of certain types of cancers such as colorectal cancers. Animal data support such a chemopreventive effect of coffee. Substantial research has been devoted to the identification of coffee components that may be responsible for these beneficial effects. In animal models and cell culture systems, the coffee diterpenes cafestol and kahweol (C+K) were shown to produce a broad range of biochemical effects resulting in a reduction of the genotoxicity of several carcinogens including 7,12-dimethylbenz[a]anthracene (DMBA), aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Different mechanisms appear to be involved in these chemoprotective effects: an induction of conjugating enzymes (e.g. glutathione S-transferases, glucuronosyl S-transferases), an increased expression of proteins involved in cellular antioxidant defense (e.g. gamma-glutamyl cysteine synthetase and heme oxygenase-1) and an inhibition of the expression and/or activity of cytochromes P450 involved in carcinogen activation (e.g. CYP2C11, CYP3A2). In animal models, the C+K-mediated induction of conjugating and antioxidant enzymes has been observed in hepatic, intestinal and kidney tissues. In the small intestine, these inductions were shown to be mediated by Nrf2-dependent transcriptional activation. In vitro investigations obtained in cell cultures of human origin indicate that the effects and mechanisms observed in animal test systems with C+K are likely to be of relevance for humans. In human liver epithelial cell lines transfected to express AFB(1)-activating P450s, C+K treatment resulted in a reduction of AFB(1)-DNA binding. This protection was correlated with an induction of GST-mu, an enzyme known to be involved in AFB(1) detoxification. In addition, C+K was found to inhibit P450 2B6, one of the human enzymes responsible for AFB(1) activation. Altogether, the data on the biological effects of C+K provide a plausible hypothesis to explain some of the anticarcinogenic effects of coffee observed in human epidemiological studies and in animal experiments.

Threshold of toxicological concern for chemical substances present in the diet: a practical tool for assessing the need for toxicity testing.

The de minimis concept acknowledges a human exposure threshold value for chemicals below which there is no significant risk to human health. It is the underlying principle for the US Food and Drug Administration (FDA) regulation on substances used in food-contact articles. Further to this, the principle of Threshold of Toxicological Concern (TTC) has been developed and is now used by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in their evaluations. Establishing an accepted TTC would benefit consumers, industry and regulators, since it would preclude extensive toxicity evaluations when human intakes are below such threshold, and direct considerable time and cost resources towards testing substances with the highest potential risk to human health. It was questioned, however, whether specific endpoints that may potentially give rise to low-dose effects would be covered by such threshold. In this review, the possibility of defining a TTC for chemical substances present in the diet was examined for general toxicity endpoints (including carcinogenicity), as well as for specific endpoints, namely neurotoxicity and developmental neurotoxicity, immunotoxicity and developmental toxicity. For each of these endpoints, a database of specific no-observed-effect levels (NOELs) was compiled by screening oral toxicity studies. The substances recorded in each specific database were selected on the basis of their demonstrated adverse effects. For the neurotoxicity and developmental neurotoxicity databases, it was intended to cover all classes of compounds reported to have either a demonstrated neurotoxic or developmentally neurotoxic effect, or at least, on a biochemical or pharmacological basis were considered to have a potential for displaying such effects. For the immunotoxicity endpoint, it was ensured that only immunotoxicants were included in the database by selecting most of the substances from the Luster et al. database, provided that they satisfied the criteria for immunotoxicity defined by Luster. For the developmental toxicity database, substances were selected from the Munro et al. database that contained the lowest NOELs retrieved from the literature for more than 600 compounds. After screening these, substances showing any effect which could point to developmental toxicity as broadly defined by the US were recorded in the database. Additionally, endocrine toxicity and allergenicity were addressed as two separate cases, using different approaches and methodology. The distributions of NOELs for the neurotoxicity, developmental neurotoxicity and developmental toxicity endpoints were compared with the distribution of NOELs for non-specific carcinogenic endpoints. As the immunotoxicity database was too limited to draw such a distribution of immune NOELs, the immunotoxicity endpoint was evaluated by comparing immune NOELs (or LOELs-lowest-observed-effect levels-when NOELs were not available) with non-immune NOELs (or LOELs), in order to compare the sensitivity of this endpoint with non-specific endpoints. A different methodology was adopted for the evaluation of the endocrine toxicity endpoint since data currently available do not permit the establishment of a clear causal link between endocrine active chemicals and adverse effects in humans. Therefore, this endpoint was analysed by estimating the human exposure to oestrogenic environmental chemicals and evaluating their potential impact on human health, based on their contribution to the overall exposure, and their estrogenic potency relative to endogenous hormones. The allergenicity endpoint was not analysed as such. It was addressed in a separate section because this issue is not relevant to the overall population but rather to subsets of susceptible individuals, and allergic risks are usually controlled by other means (i.e. labelling) than the Threshold of Toxicological Concern approach. (ABSTRACT TRUNCATED)

Structure-based thresholds of toxicological concern (TTC): guidance for application to substances present at low levels in the diet.

The threshold of toxicological concern (TTC) is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept that there are levels of exposure that do not cause adverse effects is inherent in setting acceptable daily intakes (ADIs) for chemicals with known toxicological profiles. The TTC principle extends this concept by proposing that a de minimis value can be identified for many chemicals, in the absence of a full toxicity database, based on their chemical structures and the known toxicity of chemicals which share similar structural characteristics. The establishment and application of widely accepted TTC values would benefit consumers, industry and regulators. By avoiding unnecessary toxicity testing and safety evaluations when human intakes are below such a threshold, application of the TTC approach would focus limited resources of time, cost, animal use and expertise on the testing and evaluation of substances with the greatest potential to pose risks to human health and thereby contribute to a reduction in the use of animals. An Expert Group of the European branch of the International Life Sciences Institute-ILSI Europe-has examined the TTC principle for its wider applicability in food safety evaluation. The Expert Group examined metabolism and accumulation, structural alerts, endocrine disrupting chemicals and specific endpoints, such as neurotoxicity, teratogenicity, developmental toxicity, allergenicity and immunotoxicity, and determined whether such properties or endpoints had to be taken into consideration specifically in a step-wise approach. The Expert Group concluded that the TTC principle can be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate. The use of a decision tree to apply the TTC principle is proposed, and this paper describes the step-wise process in detail. Proteins, heavy metals and polyhalogenated-dibenzodioxins and related compounds were excluded from this approach. When assessing a chemical, a review of prior knowledge and context of use should always precede the use of the TTC decision tree. The initial step is the identification and evaluation of possible genotoxic and/or high potency carcinogens. Following this step, non-genotoxic substances are evaluated in a sequence of steps related to the concerns that would be associated with increasing intakes. For organophosphates a TTC of 18microg per person per day (0.3 microg/kg bw/day) is proposed, and when the compound is not an OP, the TTC values for the Cramer structural classes III, II and I, with their respective TTC levels (e.g. 1800, 540 and 90 microg per person per day; or 30, 9 and 1.5 microg/kg bw /day), would be applied sequentially. All other endpoints or properties were shown to have a distribution of no observed effect levels (NOELs) similar to the distribution of NOELs for general toxicity endpoints in Cramer classes I, II and III. The document was discussed with a wider audience during a workshop held in March 2003 (see list of workshop participants).

Guidance for the safety assessment of botanicals and botanical preparations for use in food and food supplements.

There is a growing interest by both consumers and industry for the development of food products with 'functional' properties, or health benefits. These products may take the form of dietary supplements or of foods. The health benefits are given by particular ingredients, and in many cases these are derived from botanicals. The variety of plants providing these functions is large, ranging from staple food sources such as cereals, fruits and vegetables, to herbals as used in traditional medicine. The food or ingredient conferring health properties may consist of the plants themselves, extracts thereof, or more purified components. The scientific literature is abundant with articles not only on the beneficial properties, but also on possible adverse health effects of plants and their components. The present report discusses the data required to determine the safe use of these types of ingredients, and provides advice on the development of risk assessment strategies consistent with due diligence under existing food regulations. Product specifications, composition and characterisation of standardised and authentic materials, documented history of use and comparison to existing products (taking into account the effect of industrial processing), description of the intended use and consequent exposure are highlighted as key background information on which to base a risk evaluation. The extent of experimental investigation required, such as in vitro, animal, and/or human studies, depends on the adequacy of this information. A decision tree is presented as an aid to determine the extent of data requirements based on product comparison. The ultimate safety in use depends on the establishment of an adequate safety margin between expected exposure and identified potential hazards. Health hazards may arise from inherent toxicities or contaminants of the plant materials, including the mechanism of the intended beneficial effect. A lower safety margin may therefore be expected than for food ingredients or additives where no physiological effects are intended. In rare cases, post launch monitoring programmes may be envisaged to confirm expected exposures and adequacy of the safety margin. This guidance document was elaborated by an expert group of the Natural Toxin Task Force of the European Branch of the International Life Sciences Institute--ILSI Europe and discussed with a wider audience of scientists at a workshop held on 13-15 May 2002 in Marseille, France.

The application of in vitro data in the derivation of the acceptable daily intake of food additives.

The acceptable daily intake (ADI) for food additives is commonly derived from the NOAEL (no-observed-adverse-effect 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 differences 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 influence 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 specific 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 sufficient 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.

Application of gastrointestinal modelling to the study of the digestion and transformation of dietary glycidyl esters.

Glycidyl esters (GEs) are known to be formed during vegetable oil processing. Because of their structure, it has been hypothesised that GEs, like fatty acid esters of chloropropanols (MCPD esters), may be accepted as substrates by gut lipases to release the epoxide glycidol. If confirmed such a hypothesis would be important for risk assessment since glycidol is considered as a genotoxic carcinogen. In the present study, biotransformation was investigated using static and dynamic gastrointestinal models. During the experiments, aliquots were analysed for non-digested GEs using liquid chromatography-time-of-flight-mass spectrometry (LC-ToF-MS). In the static model, a fast hydrolysis of GEs was observed as a result of lipase action. Lipase was very efficient at pH 4.8, and totally inhibited at very low pH (1.7). In the absence of lipase, GEs were found to be relatively stable. The potential impact of food matrix was studied using milk in a dynamic model simulating human physiological conditions. The fast, pH-dependent hydrolysis of GEs was further confirmed. The possible transformation of the digestion products was then investigated using gas chromatography coupled to mass spectrometry (GC-MS), mainly the epoxide ring-opening to glycerol followed by additional reactions. In any conditions applied, neither 2- nor 3-mono-chloropropanediol (2- nor 3-MCPD) were formed, indicating that a ring-opening of the epoxide group of GEs or glycidol followed by a reaction with chloride was unlikely. A small transformation of glycidol into glycerol was observed after longer incubation time correlated with a low pH. This suggested that ring-opening and reaction with water is possible in strongly acidic conditions. Overall, it is concluded that GEs are rapidly digested by gut lipases to form glycidol. Consequently, GEs should be considered as sources of glycidol exposure. In addition, risk assessment of GEs can likely rely on hazard identification and characterisation data specific for glycidol.

Evidence for a role of oxidative stress in the carcinogenicity of ochratoxin a.

The in vitro and in vivo evidence compatible with a role for oxidative stress in OTA carcinogenicity has been collected and described. Several potential oxido-reduction mechanisms have been identified in the past. More recently, the possibility of a reduction of cellular antioxidant defense has been raised as an indirect source of oxidative stress. Consequences resulting from the production of oxidative stress are observed at different levels. First, OTA exposure has been associated with increased levels of oxidative DNA, lipid, and protein damage. Second, various biological processes known to be mobilized under oxidative stress were shown to be altered by OTA. These effects have been observed in both in vitro and in vivo test systems. In vivo, active doses were often within doses documented to induce renal tumors in rats. In conclusion, the evidence for the induction of an oxidative stress response resulting from OTA exposure can be considered strong. Because the contribution of the oxidative stress response in the development of cancers is well established, a role in OTA carcinogenicity is plausible. Altogether, the data reviewed above support the application of a threshold-based approach to establish safe level of dietary human exposure to OTA.