KEYNOTE LECTURES-KL1 New development in risk assessment of genotoxic carcinogens in foods

The no-observed-effect level (NOEL) in a study of carcinogenicity for compounds that are both genotoxic and carcinogenic represents the limit of detection in that bioassay, rather than an estimate of a possible threshold. Therefore, for those genotoxic and carcinogenic contaminants (e.g. acrylamides, PAHs, etc.) in foods it is not possible to develop health-based guidance values (e.g. ADI or PTWI) using the traditional NOEL and safety/uncertainty factors.     

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.     

Risk assessment of peak exposure to genotoxic carcinogens: a pragmatic approach

Short-term exposures to relatively high concentrations or doses are a regular cause of concern. Since carcinogenicity is often of great personal and social relevance the question arises whether short-term exposure (1-10 days) to a carcinogenic substance may contribute to tumour development and, if so, whether this contribution to the cancer risk can be quantified. The present object was to explore the possibility of a pragmatic estimation of the cancer risk of peak exposure to a genotoxic carcinogen relative to the cancer risk of the same cumulative dose of this carcinogen distributed over lifetime. A report published by the Health Council of The Netherlands served as point of departure. Published data strongly suggests that short-term or single exposure can indeed give rise to tumour formation in animal experiments. The application of a dose-rate correction factor (DRCF), defined as a factor by which the tumour incidence caused by a specific dose of a chemical carcinogen at low-dose rates is multiplied to derive the tumour incidence at high-dose rates, appears to be a feasible approach. Theoretical models calculated maximum values for the DRCF of up to seven for a young child acutely exposed to an initiator or first-stage carcinogen. A maximum value of 8.3 was calculated from animal experiments. A decision tree is presented which allows the pragmatic assessment of the carcinogenic risk following short-term exposure to genotoxic carcinogens. It is recommended to validate this decision tree with model-substances.     

Contribution of toxicology towards risk assessment of carcinogens

In the last decade many tests have been designed to detect possible carcinogenicity of compounds. Presently, many more or less simple and convenient systems are available to detect mutations, effects on chromosomes, DNA binding and damage and malignant transformation. These systems, which have been extensively refined during the last years, often show reasonably good relevance to carcinogenicity. Although inconsistencies in the patterns of response do indicate that their role as predictive indicators of carcinogenicity remains still uncertain, the use of such short-term tests in carcinogen risk assessment does seem feasible.Factors other than these tests should also be taken into consideration, since other characteristics like chemical structure, biotransformation, toxicokinetics, qualitative and quantitative physiological and/or morphological effects, species, strains, organ specificity, dose-response relation and information on studies in man, if available, are of importance too.In conjunction with the results of adequately performed carcinogenicity tests in mammals, one may attempt to classify carcinogens. Current knowledge does not permit a rigid classification, but may warrant a subclassification into carcinogens acting via a genetic or a non-genetic mechanism. It is emphasized that on theoretical and practical grounds a different extrapolation system should be used for the different types of carcinogens in risk assessment procedures. Evaluations on individual compounds should be made to decide whether such genotoxic or non-genotoxic compounds should be permitted in the human environment.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Clinical risk assessment of GM foods

The main concerns about adverse effects of genetically modified (GM) foods on health are the transfer of antibiotic resistance, toxicity and allergenicity. There are two issues from an allergic standpoint. First, the transfer of a known allergen may occur from a crop into a non-allergenic target crop. The second scenario is the creation of a neo-allergen where de novo sensitisation occurs in the population. The first scenario occurred in 1996 when the 2S albumen protein from Brazil nut was transferred into soy bean (N. Engl. J. Med. 334 (1996) 688). 2S albumen was found to be a major Brazil nut allergen and the newly expressed protein in transgenic soy retained its allergenicity. Patients allergic to Brazil nuts and not to soy bean now showed an IgE mediated response towards GM soy bean. We argue that it is possible to prevent such occurrences by doing IgE-binding studies and taking into account physico-chemical characteristics of proteins and referring to known allergen databases. The second possible scenario of de novo sensitisation does not easily lend itself to risk assessment. We compare GM technology to traditional plant breeding and food processing methods. There is no evidence that the technology used for the production of GM foods poses an allergic threat per se compared to other methodologies widely accepted in the food industry. We need to proceed cautiously in the future, assessing individual GM foods on the basis of their individual merits and risks prior to introducing them into the market.     

The risks of risk assessment in foods.

This report is the outcome of a workshop organized by the International Life Sciences Institute-European Branch (ILSI Europe), on the "Risks of Risk Assessment in Foods" held on 18 February 1998 in Brussels, Belgium. The meeting discussed Risk Assessment as the principal means by which the European Union evaluates the potential harm arising from the use of existing and new products. The experiences of the parties involved have often shown that the concepts underlying risk assessment are complex and not always fully understood. There is an urgent need to familiarize industry, policy makers and the scientific community with developments in the basic principles and terminology of risk assessment. Therefore, the workshop aimed to review key areas in risk assessment and to provide an open forum for learning and discussion between all interested parties.     

Human risk assessment of carcinogens from an administrative perspective

For evaluation of the carcinogenic potency of an environmental chemical or a mixture of chemicals, epidemiological or workplace studies, animal studies, and in vitro tests are the three major strategies used. Asbestos fibers and 1,2-dichloroethane are chosen as examples. They illustrate that the strategies may enable the implementation of measures by health authorities.     

Chronic bioassays: relevance to quantitative risk assessment of carcinogens

Conventional carcinogenic potency estimates for chemicals have been compared across rodent species. The high correlations previously demonstrated between maximum likelihood estimates (MLE) for chemicals identified as positive carcinogens in both rats and mice (r = 0.83, n = 83) are shown to occur also for chemicals that were negative in both species (r = 0.85, n = 51), for chemicals causing tumors in rats but not mice (r = 0.55, n = 15), and for those causing tumors in mice but not rats (r = 0.68, n = 25). Corresponding upper-bound estimates of carcinogenic potency are also highly correlated across rodent species. The correlations arise from (i) the strong interspecies correlation between maximum doses tested in chronic bioassays, (ii) the small group sizes utilized, and (iii) the narrow range of doses typically tested. These factors constrain conventional ML and upper-bound potency estimates to lie very close to the inverse maximum doses tested, irrespective of the bioassay's qualitative outcomes. The potency estimates are thus artifacts of experimental design and would seem to provide little information on the actual human cancer risks from chemical exposure. Risk assessment models can reveal true potency differences across species, but they must incorporate relevant mechanistic information before quantitative risk extrapolation from rodents to humans is scientifically defensible.     

Identification of genotoxic and epigenetic carcinogens in liver culture systems

Liver culture systems are available for identifying the DNA reactivity of carcinogens and a cell membrane effect, which appears to be associated with neoplasm-promoting ability. Using these and other approaches, carcinogens can be categorized as genotoxic or epigenetic. This distinction has implications for risk assessment.     

Quantitative risk assessment of foods containing peanut advisory labeling

Foods with advisory labeling (i.e. “may contain”) continue to be prevalent and the warning may be increasingly ignored by allergic consumers. We sought to determine the residual levels of peanut in various packaged foods bearing advisory labeling, compare similar data from 2005 and 2009, and determine any potential risk for peanut-allergic consumers. Of food products bearing advisory statements regarding peanut or products that had peanut listed as a minor ingredient, 8.6% and 37.5% contained detectable levels of peanut (>2.5 ppm whole peanut), respectively. Peanut-allergic individuals should be advised to avoid such products regardless of the wording of the advisory statement. Peanut was detected at similar rates and levels in products tested in both 2005 and 2009. Advisory labeled nutrition bars contained the highest levels of peanut and an additional market survey of 399 products was conducted. Probabilistic risk assessment showed the risk of a reaction to peanut-allergic consumers from advisory labeled nutrition bars was significant but brand-dependent. Peanut advisory labeling may be overused on some nutrition bars but prudently used on others. The probabilistic approach could provide the food industry with a quantitative method to assist with determining when advisory labeling is most appropriate.     

Characteristic expression profiles induced by genotoxic carcinogens in rat liver.

When applied in toxicological studies, the recently developed gene expression profiling techniques using microarrays, which brought forth the new field of toxicogenomics, facilitate the interpretation of a toxic compound's mechanism of action. In this study, we investigated whether genotoxic carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate a common set of genes in a short-term in vivo study and, if so, whether these deregulated genes represent defined biological pathways. Rats were dosed with the four genotoxic hepatocarcinogens dimethylnitrosamine (4 mg/kg/day), 2-nitrofluorene (44 mg/kg/day), aflatoxin B1 (0.24 mg/kg/day), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 20 mg/kg/day). After treatment for up to 14 days, the expression profiles of the livers were analyzed on Affymetrix RG_U34A microarrays. Among the significantly upregulated genes were a set of target genes of the tumor suppressor protein p53, indicating a DNA damage response. Such a response was expected and, therefore, confirmed the validity of our approach. In addition, the gene expression changes suggest a specific detoxification response, the activation of proliferative and survival signaling pathways, and some cell structural changes. These responses were strong throughout the 14 day time course for 2-nitrofluorene and aflatoxin B1; in the case of dimethylnitrosamine and NNK, the effects were weakly detectable at day 1 and then increased with time. For dimethylnitrosamine and aflatoxin B1, which caused observable inflammation in vivo, we found a corresponding upregulation of inflammatory genes at the same time points. Thus, by the toxicogenomic analysis of short-term in vivo studies, we identified genes and pathways commonly deregulated by genotoxic carcinogens, which may be indicative for the early events in tumorigenesis and, thus, predictive of later tumor development.     

Risk assessment of carcinogens in food

Approaches for the risk assessment of carcinogens in food have evolved as scientific knowledge has advanced. Early methods allowed little more than hazard identification and an indication of carcinogenic potency. Evaluation of the modes of action of carcinogens and their broad division into genotoxic and epigenetic (non-genotoxic, non-DNA reactive) carcinogens have played an increasing role in determining the approach followed and provide possibilities for more detailed risk characterisation, including provision of quantitative estimates of risk. Reliance on experimental animal data for the majority of risk assessments and the fact that human exposures to dietary carcinogens are often orders of magnitude below doses used in experimental studies has provided a fertile ground for discussion and diverging views on the most appropriate way to offer risk assessment advice. Approaches used by national and international bodies differ, with some offering numerical estimates of potential risks to human health, while others express considerable reservations about the validity of quantitative approaches requiring extrapolation of dose-response data below the observed range and instead offer qualitative advice. Recognising that qualitative advice alone does not provide risk managers with information on which to prioritise the need for risk management actions, a “margin of exposure” approach for substances that are both genotoxic and carcinogenic has been developed, which is now being used by the World Health Organization and the European Food Safety Authority. This review describes the evolution of risk assessment advice on carcinogens and discusses examples of ways in which carcinogens in food have been assessed in Europe.     

A note on the role of background tumor incidence in risk assessment for carcinogens

The extrapolation of animal bioassay data to humans for the purpose of estimating risk from food additives, drugs, pesticides, etc. is still the primary approach to carcinogenic risk assessment. When the extrapolation is based on tumor data with high background incidence (e.g., hepatocellular tumors in rodents), its direct relevance to the human situation is sometimes questioned. This paper suggests a method for adjusting the acceptable excess risk level for humans according to the background incidence of the tumor type used for extrapolating from animals to humans. Application of the suggested "fixed relative risk" extrapolation method in setting acceptable low-risk exposure levels for humans is illustrated for methylene chloride.     

危险度评价最新进展

在过去的几十年里 ,人们开发了许多新的方法来改进化学品危险度评价中的剂量 反应关系的评定 :如采用定义较为完善的有效作用剂量来替代无可见有害作用水平(NOAEL) ,以及改进了的不确定 /安全系数的评定方法。本文将介绍目前国际上使用较为广泛的几种评定方法 :①采用剂量 反应关系数学模拟的方法来估计有效剂量以取代NOAEL作为安全剂量的出发起点值的剂量 反应关系的评定方法 ,包括 :“基准剂量法”和“分类回归法” ;②使用种系内和种系间毒代动力学和毒效动力学的资料来选择不确定系数的“基于数据的不确定系数法” ;③用于估计不确定系数的可信区间 ,以及计算安全剂量值的可信区间的“概率参考剂量法”。     

Recent developments in the risk assessment of potentially genotoxic impurities in pharmaceutical drug substances.

Controlling the quality of medicines is just as important as demonstrating efficacy. The International Conference on Harmonisation has published general guidance on the quality and safety assessment of impurities in pharmaceutical drug substances and drug products. More recently, the European Medicines Evaluation Agency has published a guideline focusing on limits for genotoxic impurities. This is based on a Threshold of Toxicological Concern (TTC) derived from animal carcinogenicity data using multiple worst case assumptions to estimate a daily dose (1.5 microg/day) associated with a lifetime cancer risk of 1 in 100,000, a risk level considered acceptable for genotoxic impurities in human medicines. Based on these assumptions, presentation of the TTC as a single figure infers an unwarranted level of precision and supports the adoption of a more flexible approach by regulatory authorities when evaluating new drug products; a range within fivefold of the TTC limit would seem sensible. Furthermore, the limit is based on 70 years continuous daily exposure, a scenario that is uncommon for most medicines and irrelevant to the preregistration clinical development phase. To address this latter point, a staged TTC has been developed that proposes limits based on shorter durations of treatment, e.g., up to 1 year. Based on recent history, this approach has been acceptable to some authorities but not to others, and it is imperative that steps are taken to reach a common agreement between the pharmaceutical industry and regulatory authorities globally in order that new medicines can continue to be developed and delivered to benefit patients in a safe and timely manner.     

Combinations of genotoxic tests for the evaluation of group 1 IARC carcinogens

Many of the known human carcinogens are potent genotoxins that are efficiently detected as carcinogens in human populations but certain types of compounds such as immunosuppressants, sex hormones, etc. act via non‐genotoxic mechanism. The absence of genotoxicity and the diversity of modes of action of non‐genotoxic carcinogens make predicting their carcinogenic potential extremely challenging. There is evidence that combinations of different short‐term tests provide a better and efficient prediction of human genotoxic and non‐genotoxic carcinogens. The purpose of this study is to summarize the in vivo and in vitro comet assay (CMT) results of group 1 carcinogens selected from the International Agency for Research on Cancer and to discuss the utility of the comet assay along with other genotoxic assays such as Ames, in vivo micronucleus (MN), and in vivo chromosomal aberration (CA) test. Of the 62 agents for which valid genotoxic data were available, 38 of 61 (62.3%) were Ames test positive, 42 of 60 (70%) were in vivo MN test positive and 36 of 45 (80%) were positive for the in vivo CA test. Higher sensitivity was seen in in vivo CMT (90%) and in vitro CMT (86.9%) assay. Combination of two tests has greater sensitivity than individual tests: in vivo MN + in vivo CA (88.6%); in vivo MN + in vivo CMT (92.5%); and in vivo MN + in vitro CMT (95.6%). Combinations of in vivo or in vitro CMT with other tests provided better sensitivity. In vivo CMT in combination with in vivo CA provided the highest sensitivity (96.7%).