Evaluation of the Threshold of Toxicological Concern (TTC)--challenges and approaches.

Thresholds of Toxicological Concern (TTCs) have been used in the risk assessment of chemicals to which humans are exposed at very low levels. TTC values were developed using data from rodent cancer bioassays and from oral chronic and sub-chronic toxicity studies for non-cancer effects. The workshop assessed the adequacy and fitness for purpose of the TTC approach and the potential for future modifications of critical aspects. The current TTC value for cancer was considered adequate and fit for purpose because it is derived by linear extrapolation from the lowest TD(50) for each compound in the largest available rodent carcinogenicity database. The database on non-cancer endpoints was considered adequate and fit for purpose because the chemical domain, the distributions of NOAELs and the calculated TTC values are comparable across different databases. Application of the TTC approach gives conclusions compatible with the risk assessment approaches currently used by international advisory committees. The workshop recognised the desirability of developing better tools to assess the comparability of the chemical domain covered in different toxicological databases, and the need to develop an internationally acceptable framework and databases for updating the aspects critical to application of the TTC approach.

Risk assessment of micronutrients.

Risk assessment of micronutrients has to take into account two different intake-response relationships; the risk of deficiency, which decreases with increase in intake, and the risk of toxicity, which increases with increase in intake. The available databases on micronutrients tend to focus on benefits at low intakes, and there are usually few reliable data on hazard identification and characterisation at high intakes. Application of the usual default uncertainty factors for species differences, human variability and database inadequacy could result in "recommended" upper intake levels that would cause deficiency. There have been a number of comprehensive reviews that have used low, and largely arbitrary, uncertainty factors to establish tolerable upper intake levels for vitamins and minerals. A recent FAO/WHO Workshop developed a structured approach to the application of a single composite uncertainty factor. Risk-benefit approaches have been developed recently that balance the risk of toxicity against the risk of deficiency, and offer the potential for more scientifically based methods.

Risk assessment of dietary exposures to compounds that are genotoxic and carcinogenic--an overview.

The process of risk assessment of dietary exposures to genotoxic carcinogens is summarised. Exposures to six genotoxic carcinogens in food (acrylamide, aflatoxin B1, benzo(a)pyrene, dimethylnitrosamine, ethyl carbamate, PhIP) have been used to illustrate the process. The margin of exposure (MOE) approach is seen as a useful method to be used in the risk characterisation step of assessing exposures to genotoxic carcinogens. This approach combines information on animal potency and human exposure, and can be used to indicate levels of concern and also the ranking between various exposures to such agents. Both the T25 and the BMDL10 methods may be used as a reference point. Should a specific MOE value be developed as a cut-off between levels of concern and levels of low concern, the value using T25 data is proposed to be 2.5-times higher than using BMDL10 data. Linear low-dose extrapolation using either T25 or BMDL10, may also be applied. However, it should be understood that this approach should not be interpreted as giving a precise estimate of human risk. For exposures to mutagens in food lacking carcinogenicity data, it is proposed to apply the MOE approach to the lowest effective dose (LED) for in vivo genotoxicity.

The use of a sweetener substitution method to predict dietary exposures for the intense sweetener rebaudioside A.

There are more published dietary exposure data for intense sweeteners than for any other group of food additives. Data are available for countries with different patterns of sweetener approvals and also for population groups with high potential intakes, such as children and diabetic subjects. These data provide a secure basis for predicting the potential intakes of a novel intense sweetener by adjustment of the reported intakes of different sweeteners in mg/kg body weight by their relative sweetness intensities. This approach allows the possibility that a novel sweetener attains the same pattern and extent of use as the existing sweeteners. The intakes by high consumers of other sweeteners allows for possible brand loyalty to the novel sweetener. Using this method, the estimated dietary exposures for rebaudioside A in average and high consumers are predicted to be 1.3 and 3.4mg/kg body weight per day for the general population, 2.1 and 5.0mg/kg body weight per day for children and 3.4 and 4.5mg/kg body weight per day for children with diabetes. The temporary ADI defined by the JECFA for steviol glycosides [JECFA, 2005. Steviol glycosides. In: 63rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization (WHO), Geneva, Switzerland, WHO Technical Report Series 928, pp. 34-39] was set at 0-2mg/kg body weight (expressed as steviol equivalents); after correction for the difference in molecular weights, these estimated intakes of rebaudioside A are equivalent to daily steviol intakes of less than 2mg/kg. In consequence, this analysis shows that the intakes of rebaudioside A would not exceed the JECFA temporary ADI set for steviol glycosides.

Microbial hydrolysis of steviol glycosides.

A review of the role of gut microbiota in the metabolism of the steviol glycosides, stevioside and rebaudioside A, indicates that they are not absorbed intact but undergo hydrolysis by the intestinal microflora to steviol. Steviol is not metabolized by the intestinal flora and is absorbed from the intestine. The rate of hydrolysis for stevioside is greater than for rebaudioside A. Recent studies using mass spectrometry have shown that steviol-16,17-epoxide is not a microbial metabolite of steviol glycosides. Bacteroides species are primarily responsible for hydrolysis via their beta-glucosidase activity. Fecal incubation studies with both human and animal mixed flora provide similar results, and this indicates that the rat is an appropriate model for studies on steviol glycosides. Given the similarity in the microbial metabolism of stevioside and rebaudioside A with the formation of steviol as the single hydrolysis product that is absorbed from the intestinal tract, the toxicological data on stevioside are relevant to the risk assessment of rebaudioside A.

The Threshold of Toxicological Concern (TTC) in risk assessment.

The Threshold of Toxicological Concern (TTC) is a level of human intake or exposure that is considered to be of negligible risk, despite the absence of chemical-specific toxicity data. The TTC approach is a form of risk characterisation in which uncertainties arising from the use of data on other compounds are balanced against the low level of exposure. The approach was initially developed by the FDA for packaging migrants, and used a single threshold value of 1.5 microg/day (called the threshold of regulation). Subsequent analyses of chronic toxicity data resulted in the development of TTC values for three structural classes with different potentials for toxicity (1,800, 540 and 90 microg/day). These TTC values have been incorporated into the procedure that is used internationally for the evaluation of flavouring substances. Further developments included additional TTC values for certain structural alerts for genotoxicity (0.15 microg/day), and for the presence of an organophosphate group (18 microg/day). All of these TTC values were incorporated into an extended decision tree for chemicals, such as contaminants, which might be present in human foods. The TTC approach has been shown to have potential applications to risk assessments of cosmetic ingredients, household products and impurities in therapeutic drugs.

Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats.

The toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol were examined in rats for comparative purposes to determine whether toxicological studies conducted previously with stevioside would be applicable to the structurally-related glycoside, rebaudioside A. Single, oral doses of the radiolabelled compounds were extensively and rapidly absorbed with plasma concentration-time profiles following similar patterns for stevioside and rebaudioside A. Elimination of radioactivity from plasma was essentially complete within 72h. All plasma samples had similar metabolite profiles; the predominant radioactive component in all samples was steviol, with lower amounts of steviol glucuronide(s) and low levels of one or two other metabolites. Rebaudioside A, stevioside, and steviol were metabolized and excreted rapidly, with the majority of the radioactivity eliminated in the feces within 48h. Urinary excretion accounted for less than 2% of the administered dose for all compounds in both intact and bile duct-cannulated rats, and the majority of the absorbed dose was excreted via the bile. After administration of the compounds to intact and bile duct-cannulated rats, radioactivity in the feces was present primarily as steviol. The predominant radioactive compound detected in the bile of all cannulated rats was steviol glucuronide(s), indicating de-conjugation in the lower intestine. Overall, the data on toxicokinetics and metabolism indicate that rebaudioside A and stevioside are handled in an almost identical manner. These studies support the use of toxicological safety studies conducted with stevioside for the safety assessment of rebaudioside A.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route. The physicochemical characteristics of the chemical and the pattern of cosmetic use would affect the long-term average internal dose that is compared with the relevant TTC value. Analysis has shown that the oral TTC values are valid for topical exposures and that the relationship between the external topical dose and the internal dose can be taken into account by conservative default adjustment factors. The TTC approach relates to systemic effects, and use of the proposed procedure would not provide an assessment of any local effects at the site of application. Overall the TTC approach provides a useful additional tool for the safety evaluation of cosmetic ingredients and impurities of known chemical structure in the absence of chemical-specific toxicology data.

Absorption, tissue distribution, metabolism and elimination of taurine given orally to rats.

Three biodisposition studies with taurine were performed in male and female adult rats at dosages of 30 and 300 mg/kg. A single oral dose of (14)C-taurine was rapidly absorbed, distributed to tissues and excreted unchanged in urine. Elimination of radioactivity from intracellular pools was slow. Pre-treatment of animals for 14 days with unlabelled taurine did not significantly affect the fate of (14)C-taurine. At the higher dose there was more extensive excretion combined with a lower percentage of the dose in the carcass, indicating the possibility of saturation of the tubular reabsorption mechanism for taurine. Daily administration of unlabelled taurine for 14 days did not result in an increase in total taurine in the brain. The data indicate that exogenous taurine rapidly equilibrates with endogenous body pools and that any excess is rapidly eliminated by the kidneys.

First European conference on aspartame: putting safety and benefits into perspective. Synopsis of presentations and conclusions.

A Conference was held in Paris in 2006 to review the safety and benefits arising from the replacement of sucrose with the intense sweetener aspartame. The intakes of aspartame are only about 10% of the acceptable daily intake, even by high consumers, so that the safety margin is about 3 orders of magnitude. The safety of aspartame was confirmed in the EFSA Opinion of a recent controversial rodent cancer bioassay. There is increasing evidence that even modest reductions in the intake of calories can reduce the risk factors associated with a number of diseases, such as diabetes and cardiovascular disease. A key issue addressed at the conference was whether the replacement of sucrose with aspartame could result in a prolonged decrease in calorie intake that was of similar magnitude to that necessary to produce a health benefit. A recent meta-analysis of published data showed that an adequate, prolonged weight reduction could be achieved with aspartame. It was recognised that risk assessment alone gave an unbalanced impression to regulators and consumers, and that in the future quantitative risk-benefit analyses should be able to provide more comprehensive advice.

Evaluation of certain food additives and contaminants.

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to recommending acceptable daily intakes (ADIs) and to preparing specifications for identity and purity. The Committee also evaluated the risk posed by two food contaminants, with the aim of advising on risk management options for the purpose of public health protection. The first part of the report contains a general discussion of the principles governing the toxicological evaluation and assessment of intake of food additives (in particular flavouring agents) and contaminants. A summary follows of the Committee's evaluations of technical, toxicological and intake data for certain food additives (acidified sodium chlorite, asparaginase from Aspergillus oryzae expressed in Aspergillus oryzae, carrageenan and processed Eucheuma seaweed, cyclotetraglucose and cyclotetraglucose syrup, isoamylase from Pseudomonas amyloderamosa, magnesium sulfate, phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae, sodium iron(III) ethylenediaminetetraacetic acid (EDTA) and steviol glycosides); eight groups of related flavouring agents (linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters; aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances; simple aliphatic and aromatic sulfides and thiols; aliphatic acyclic dials, trials and related substances; aliphatic acetals; sulfur-containing heterocyclic compounds; aliphatic and aromatic amines and amides; and aliphatic alicyclic linear alpha, beta -unsaturated di- and trienals and related alcohols, acids and esters); and two food contaminants (aflatoxin and ochratoxin A). Specifications for the following food additives were revised: maltol and ethyl maltol, nisin preparation, pectins, polyvinyl alcohol, and sucrose esters of fatty acids. Specifications for the following flavouring agents were revised: maltol and ethyl maltol, maltyl isobutyrate, 3-acetyl-2,5-dimethylfuran and 2,4,5-trimethyl-delta-oxazoline (Nos 1482, 1506 and 1559), and monomenthyl glutarate (No. 1414), as well as the method of assay for the sodium salts of certain flavouring agents. Annexed to the report are tables summarizing the Committee's recommendations for intakes and toxicological evaluations of the food additives and contaminants considered.

Risk assessment of substances that are both genotoxic and carcinogenic report of an International Conference organized by EFSA and WHO with support of ILSI Europe.

The European Food Safety Authority (EFSA) and the World Health Organization (WHO), with the support of the International Life Sciences Institute, European Branch (ILSI Europe), organized an international conference on 16-18 November 2005 to discuss how regulatory and advisory bodies evaluate the potential risks of the presence in food of substances that are both genotoxic and carcinogenic. The objectives of the conference were to discuss the possible approaches for risk assessment of such substances, how the approaches may be interpreted and whether they meet the needs of risk managers. ALARA (as low as reasonably achievable) provides advice based solely on hazard identification and does not take into account either potency or human exposure. The use of quantitative low-dose extrapolation of dose-response data from an animal bioassay raises numerous scientific uncertainties related to the selection of mathematical models and extrapolation down to levels of human exposure. There was consensus that the margin of exposure (MOE) was the preferred approach because it is based on the available animal dose-response data, without extrapolation, and on human exposures. The MOE can be used for prioritisation of risk management actions but the conference recognised that it is difficult to interpret it in terms of health risk.

Approaches to the risk assessment of genotoxic carcinogens in food: a critical appraisal.

The present paper examines the particular difficulties presented by low levels of food-borne DNA-reactive genotoxic carcinogens, some of which may be difficult to eliminate completely from the diet, and proposes a structured approach for the evaluation of such compounds. While the ALARA approach is widely applicable to all substances in food that are both carcinogenic and genotoxic, it does not take carcinogenic potency into account and, therefore, does not permit prioritisation based on potential risk or concern. In the absence of carcinogenicity dose-response data, an assessment based on comparison with an appropriate threshold of toxicological concern may be possible. When carcinogenicity data from animal bioassays are available, a useful analysis is achieved by the calculation of margins of exposure (MOEs), which can be used to compare animal potency data with human exposure scenarios. Two reference points on the dose-response relationship that can be used for MOE calculation were examined; the T25 value, which is derived from linear extrapolation, and the BMDL10, which is derived from mathematical modelling of the dose-response data. The above approaches were applied to selected food-borne genotoxic carcinogens. The proposed approach is applicable to all substances in food that are DNA-reactive genotoxic carcinogens and enables the formulation of appropriate semi-quantitative advice to risk managers.

Toxicology of micronutrients: adverse effects and uncertainty.

The establishment of safe upper intake levels for micronutrients must consider the intake-response relations for both deficiency and toxicity. Limited data are available on the toxicities of most micronutrients, and few studies that meet the criteria considered essential for the risk assessment of other chemicals in food, such as pesticides and food additives, have been performed. In some cases, the application of large uncertainty factors, which are used to establish the amount of a chemical that would be safe for daily intake throughout life, could result in nutritionally inadequate intakes of micronutrients. As a consequence, lower than normal uncertainty factors have been applied to determine safe or tolerable intakes of many micronutrients. There is no clear scientific rationale, on the basis of the metabolism and elimination of micronutrients or the nature of the adverse effects reported for high intakes, for the use of reduced uncertainty factors for micronutrient toxicity. A review of recent evaluations of selected vitamins and minerals shows little consistency in the application of uncertainty factors by different advisory groups, such as the Institute of Medicine in the United States and the Scientific Committee on Foods in the European Union. It is apparent that, in some cases, the uncertainty factor applied was selected largely to give a result that is compatible with nutritional requirements; therefore, the uncertainty factor represented part of risk management rather than hazard characterization. The usual risk assessment procedures for chemicals in food should be revised for micronutrients, so that the risks associated with intakes that are too low and too high are considered equally as part of a risk-benefit analysis.

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

Health-based guidance values, such as the ADI, use chemical-specific data to determine the highest intake that would be without significant adverse health effects. A threshold of toxicological concern (TTC) is a level of intake predicted to be without adverse effects based on the toxicity of structurally related compounds. The main advantage of the use of TTCs is that the risk of low exposures can be evaluated without the need for chemical-specific animal toxicity data. TTCs have been used for many years for screening the safety of packaging migrants by the FDA in the USA, and of flavoring substances, by the JECFA. A recent reassessment of the use of TTCs, organized by ILSI Europe, has developed a decision tree which allows a systematic approach to the evaluation of low levels of diverse chemicals in food. The decision tree incorporates a series of increasing TTC values into a step-wise approach. Potentially genotoxic carcinogens are considered first, based on the presence of known structural alerts. Aflatoxin-like, azoxy- and nitroso-compounds are removed from consideration because they are the most potent, and a practical TTC could not be established. Other compounds with structural alerts for genotoxicity are allocated a TTC of 0.15 microg/person per day. Compounds without structural alerts for genotoxicity are evaluated based on chemical structure and intake using a series of TTC values derived by the application of a 100-fold uncertainty factor to the 5th percentile of the distribution of NOAELs from chronic studies on compounds sharing similar structural characteristics.

The refinement of uncertainty/safety factors in risk assessment by the incorporation of data on toxicokinetic variability in humans.

The derivation of safe levels of exposure in humans for compounds that are assumed to cause threshold toxicity has relied on the application of a 100-fold uncertainty factor to a measure for the threshold, such as the no observed adverse effect level (NOAEL) or the benchmark dose (BMD). This 100-fold safety factor consists of the product of two 10-fold factors allowing for human variability and interspecies differences. The International Programme on Chemical Safety has suggested the subdivision of these 10-fold factors to allow for variability in toxicokinetics and toxicodynamics. This subdivision allows the replacement of the default uncertainty factors with a chemical-specific adjustment factor (CSAF) when suitable data are available. This short review describes potential options to refine safety factors used in risk assessment, with particular emphasis on pathway-related uncertainty factors associated with variability in kinetics. These pathway-related factors were derived from a database that quantified interspecies differences and human variability in phase I metabolism, phase II metabolism, and renal excretion. This approach allows metabolism and pharmacokinetic data in healthy adults and subgroups of the population to be incorporated in the risk-assessment process and constitutes an intermediate approach between simple default factors and chemical-specific adjustment factors.

Human variability in xenobiotic metabolism and pathway-related uncertainty factors for chemical risk assessment: a review.

This review provides an account of recent developments arising from a database that defined human variability in phase I metabolism (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, hydrolysis, alcohol dehydrogenase), phase II metabolism (N-acetyltransferases, glucuronidation, glycine conjugation, sulphation) and renal excretion. This database was used to derive pathway-related uncertainty factors for chemical risk assessment that allow for human variability in toxicokinetics. Probe substrates for each pathway of elimination were selected on the basis that oral absorption was >95% and that the metabolic route was the primary route of elimination of the compound (60-100% of a dose). Intravenous data were used for compounds for which absorption was variable. Human variability in kinetics was quantified for each compound from published pharmacokinetic studies (after oral and intravenous dosing) in healthy adults and other subgroups of the population using parameters relating to chronic exposure (metabolic and total clearances, area under the plasma concentration-time curve (AUC)) and acute exposure (Cmax) (data not presented here). The pathway-related uncertainty factors were calculated to cover 95%, 97.5% and 99% of the population of healthy adults and of each subgroup. Pathway-related uncertainty factors allow metabolism data to be incorporated into the derivation of health-based guidance values. They constitute an intermediate approach between the general kinetic default factors (3.16) and a chemical-specific adjustment factor. Applications of pathway-related uncertainty factors for chemical risk assessment and future refinements of the approach are discussed. A knowledge-based framework to predict human variability in kinetics for xenobiotics showing a threshold dose below which toxic effects are not observed, is proposed to move away from default assumptions.

Risk-benefit analysis of micronutrients.

Traditionally, different approaches have been used to determine the recommended dietary allowances for micronutrients, above which there is a low risk of deficiency, and safe upper levels, below which there is a negligible risk of toxicity. The advice given to risk managers has been in the form of point estimates, such as the recommended dietary allowance (RDA) and the tolerable upper level (UL). In future, the gap between the two intake-response curves may become narrower, as more sensitive indicators of deficiency and toxicity are used, and as health benefits above the recommended daily allowance are taken into account. This paper reviews the traditional approaches and proposes a novel approach to compare beneficial and adverse effects across intake levels. This model can provide advice for risk managers in a form that will allow the risk of deficiency or the risk of not experiencing the benefit to be weighed against the risk of toxicity. The model extends the approach used to estimate recommended dietary allowances to make it applicable to both beneficial and adverse effects and to extend the intake-incidence data to provide a range of estimates that can be considered by the risk manager. The data-requirements of the model are the incidence of a response at one or more levels of intake, and a suitable coefficient of variation to represent the person-to-person variations within the human population. A coefficient of variation of 10% or 15% has been used for established recommended dietary allowances and a value of 15% is proposed as default for considerations of benefit. A coefficient of variation of 45% is proposed as default for considerations of toxicity, based on analyses of human variability in the fate and effects of therapeutic drugs. Using this approach risk managers, working closely with risk assessors, will be able to define ranges of intake based on a balance between the risks of deficiency (or lack of benefit) and toxicity.