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 B(1), 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.     

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.     

Reproductive toxicants have a threshold of adversity

This paper surveys the scientific basis for the current threshold approach for reproductive hazard and risk assessment. In some regulatory areas it was recently suggested to consider reproductive toxicants under the stringent linear extrapolation risk assessment paradigm that was developed for genotoxic carcinogens. First, the current risk assessment paradigm for genotoxic carcinogens is addressed, followed by an overview of reproductive toxicology and its threshold dose approach for hazard and risk assessment, the testing procedures for assessing the reproductive toxicity of chemicals, and the derivation of conclusions on their risk assessment and Classification, Labelling and Packaging (CLP). Relevant details of testing methodologies are discussed, such as exposure time windows, parameters determined, and the coverage of the entire reproductive cycle. In addition, the dose-response relationship is considered, illustrated with several examples. It is concluded that the current risk assessment methodology for genotoxic carcinogens is a debatable worst-case scenario and that for risk assessment of reproductive toxicants the threshold dose approach remains valid.     

Reproductive toxicants have a threshold of adversity

This paper surveys the scientific basis for the current threshold approach for reproductive hazard and risk assessment. In some regulatory areas it was recently suggested to consider reproductive toxicants under the stringent linear extrapolation risk assessment paradigm that was developed for genotoxic carcinogens. First, the current risk assessment paradigm for genotoxic carcinogens is addressed, followed by an overview of reproductive toxicology and its threshold dose approach for hazard and risk assessment, the testing procedures for assessing the reproductive toxicity of chemicals, and the derivation of conclusions on their risk assessment and Classification, Labelling and Packaging (CLP). Relevant details of testing methodologies are discussed, such as exposure time windows, parameters determined, and the coverage of the entire reproductive cycle. In addition, the dose-response relationship is considered, illustrated with several examples. It is concluded that the current risk assessment methodology for genotoxic carcinogens is a debatable worst-case scenario and that for risk assessment of reproductive toxicants the threshold dose approach remains valid.     

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.     

Assessment of safety/risk of chemicals: inception and evolution of the ADI and dose-response modeling procedures.

This article reviews the procedures for the assessment of safety/risk of chemicals to human health. Because the nature and severity of toxicity and the extent of the database vary from chemical to chemical, the assessment is done on a case by case basis. Essentially 5 steps are involved in the assessment: (a) identification of hazards based on appropriate human and animal data; (b) determination of the dose-response relationship of the adverse effects of the chemical; (c) extrapolation of the dose-response data from test subjects to human populations; (d) estimation of the exposure; and (e) assessment of the safety/risk of the chemical under a specified exposure. Emphasis in this article, however, is placed on the extrapolation of the dose-response data to the human situation. The extrapolation is done by the identification of a no-observed-adverse-effect level (NOAEL) and the application of a safety factor, thereby arriving at an acceptable daily intake (ADI). The safety factor is selected on the basis of, inter alia, the severity of the adverse effect and the adequacy of the database. On the other hand, with genotoxic carcinogens, mathematical modeling is used for extrapolation. This is because the effects of genotoxic carcinogens are generally believed to have no threshold. The ADI approach, which involves the identification of a NOAEL, is therefore not applicable. A number of mathematical models have been developed to assess, from the dose-response data, either the risks that may be associated with a specified dose, or the 'virtually safe dose' at a specified risk level. The evolution, application and shortcomings of these procedures and the potential improvements in the ADI approach and in the dose-response characterization based on these mathematical models are also discussed.     

Human carcinogenic risk evaluation, part II: contributions of the EUROTOX specialty section for carcinogenesis.

There is growing recognition that carcinogenic risk extrapolation to low doses (and standard setting) should consider the mode of action of a given chemical. So far, there is agreement on distinguishing between genotoxic and nongenotoxic chemicals; yet, further differentiations seem appropriate. For genotoxic carcinogens, case studies of chemicals point to many possibilities for assessing carcinogenic risk. There are numerous, apparently genotoxic carcinogens where practical thresholds are a matter of discussion. For instance, positive data of chromosomal effects only, in the absence of mutagenicity, may support the characterization of a compound that produces carcinogenic effects only at high, toxic doses. There is a wide consensus that for non-DNA-reactive genotoxicants, such as aneugens, thresholds should be defined. Specific mechanisms of clastogenicity have been repeatedly addressed as also having thresholds, such as topoisomerase II poisons, or mechanisms based on reactive oxygen. These and other arguments together lead to the distinction of four groups of carcinogens, which have been introduced (C. Streffer et al., 2004, Springer-Verlag). There are nonthreshold genotoxic carcinogens (for low-dose risk assessment, the linear nonthreshold [LNT] model appears appropriate); genotoxic carcinogens, for which the existence of a threshold cannot be sufficiently supported (in these cases the LNT model is used as a default assumption, based on the scientific uncertainty and backed by the precautionary principle); genotoxic carcinogens for which a practical threshold is supported; and nongenotoxic carcinogens and non-DNA-reactive carcinogens (for these compounds a true [perfect] threshold is associated with a clearly founded no-observed-adverse-effect level).     

Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency

The assessment of the potency of a skin sensitizing chemical is a key starting point for its subsequent risk assessment/management. The Local Lymph Node Assay can provide information on the relative skin sensitizing potency of contact allergens by interpolation from the dose response curve the concentration of a chemical required to elicit a threshold positive response (EC3 value). However, interpolation requires that the dose response curve have at least one stimulation index (SI) value above and one SI value below the threshold value of 3. For instances where all test concentrations result in SI values above 3, there was a need to develop a method that would permit estimation of EC3 values. This has been achieved by log-linear extrapolation using the two lowest test concentrations from the dose response curve. Before applying this approach, it is important that data quality is assessed. The dose response must include concentrations on the linear portion of the curve and, ideally, the SI induced by the lowest dose should approach 3. Judicious use of this approach for extrapolating EC3 values can provide information on a likely potency classification for use in risk assessment and may avoid the need for repeat animal testing.     

T25: A Simplified Carcinogenic Potency Index: Description of the System and Study of Correlations between Carcinogenic Potency and Species/Site Specificity and Mutagenicity

Abstract: A simplified carcinogenic potency index, the T25, is proposed as a practical method for the inclusion of potency considerations in carcinogen classification systems. The T25 is the chronic daily dose in mg per kg bodyweight which will give 25% of the animals tumours at a specific tissue site, after correction for spontaneous incidence, within the standard life span of that species. Calculated T25 values of a set of 113 US National Cancer Institute/National Toxicology Program (NC/NTP) carcinogens showed excellent correlation (correlation coefficient 0.96, P<0.0001) with the carcinogenic potency index TD50 of Peto et al. (1984). The mean of T25 values for 51 transspecies, multiple common site NCI/NTP carcinogens were 10-fold lower than those for 62 NCI/NTP single species, single site carcinogens. For these 113 carcinogens, the mean T25 values were approximately 3-fold lower for agents that were also mutagenic in Salmonella compared to the non-mutagenic agents.     

A proposed framework for assessing risk from less-than-lifetime exposures to carcinogens

Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.     

A proposed framework for assessing risk from less-than-lifetime exposures to carcinogens

Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.     

Mutagenic impurities in pharmaceuticals: A critique of the derivation of the cancer TTC (Threshold of Toxicological Concern) and recommendations for structural-class-based limits

The cancer TTC (Threshold of Toxicological Concern) concept is currently employed as an aid to risk assessment of potentially mutagenic impurities (PMIs) in food, cosmetics and other sectors. Within the pharmaceutical industry the use of one default cancer TTC limit of 1.5 μg/day for PMIs is being increasingly questioned. Its derivation, originally in the context of foodstuffs, can be broken down into five key elements: dataset composition; determination of carcinogenicity/mutagenicity status and carcinogenic potency (based on TD₅₀s) of compounds in the dataset; linear extrapolation of carcinogenic potencies; evaluation of the more potent compounds in each structural category, and presence of representative structural alerts amongst the more potent compounds. A detailed evaluation reveals that the derivation process is distorted by the use of the lowest statistically significant TD₅₀s (which can produce a false-carcinogen phenomenon) and by employing linear extrapolation for non-mutagenic carcinogens. By correcting for these two factors, it is concluded that only around 50% of conventional structural-alert categories were adequately addressed and that limits higher than the default value appear to be justified in many cases. Using similar criteria for PMIs in pharmaceuticals, four distinct potency categories of conventional structural alerts can be distinguished, ranging from alerts with questionable validity to those with high potency, which are considered to provide a range of flexible and pragmatic limits for such impurities.     

Quantitative factors in chemical carcinogenesis: variation in carcinogenic potency

The quantitative assessment of toxicological data on the carcinogenic potential of chemicals requires consideration of a number of factors, including mathematical models of the mechanism of carcinogenic action and pharmacokinetic models for the metabolic activation of the parent compound to its reactive metabolite. In this article, the use of such models in estimating carcinogenic potency and in predicting risks at low levels of exposure is discussed, along with other factors involved in the evaluation of carcinogen bioassay data. The Carcinogenic Potency Database (CPDB) established by Gold et al. (1984, Environ. Health Perspect. 58, 9-322) is used to illustrate the application of quantitative approaches to carcinogenic risk assessment and to examine the variation in the potency of chemical carcinogens. Based on an analysis of 585 experiments selected from the CPDB, the risk-specific (10(-6) doses (RSDs) obtained by linear extrapolation from the TD50 were generally within a factor of 5-10 of those derived from the linearized multistage model. The RSDs obtained by linear extrapolation from the TD50 are roughly log-normally distributed with a median of about 20-90 ng/kg/day, depending on the subset of the CPDB considered. This distribution has been used by Rulis (1986, in Food Protection Technology (C. W. Felix, Ed.), pp. 29-37, Lewis, Chelsea, MI) to explore the concept of a threshold of regulation for chemical carcinogens present in the environment at low levels.     

K6/ODC transgenic mice as a sensitive model for carcinogen identification

Ornithine decarboxylase (ODC), an important enzyme in the polyamine biosynthetic pathway, is aberrantly regulated in many epithelial tumors of rodents and humans. In murine skin, it has been shown that ODC overexpression provides a sufficient condition for tumor promotion. Therefore, we hypothesized that K6/ODC transgenic mice in which ODC overexpression was targeted to hair follicle keratinocytes might provide a sensitive model for identifying genotoxic carcinogens. Ten known carcinogens or noncarcinogens have been tested in the model so far and results are highly concordant with 2-year rodent bioassays (100% concordant). More importantly, each of two chemicals tested that is recognized as a human carcinogen was identified as a carcinogen in K6/ODC transgenic mice. In addition, 7, 12-dimethylbenz(a)anthracene (DMBA) dose response studies indicated that even at a very low dose, 2 nmol, a high percentage of mice (50%) had already developed tumors 8 weeks after treatment. We conclude that the K6/ODC transgenic mouse model is very sensitive to topical application of genotoxic carcinogens and could therefore be a useful mouse model for carcinogen identification and chemical risk assessment.     

Early Indicators of Response in Biologically Based Risk Assessment for Nongenotoxic Carcinogens

The proposed existence of dose-response thresholds for nongenotoxic carcinogens has led to a major controversy in the risk extrapolation process. To resolve this debate, there has been a significant investment in mechanism-based risk assessment research. The ability to utilize this mechanistic research for risk assessment procedures is still limited and may not warrant the expense. Alternatively, an approach can be used to identify dose-response thresholds through the utilization of sensitive indicators of biological response. This approach does not rely upon a mechanistic framework for the development of pathology, is solely dependent on already existing technology, and takes into account the possibility of background levels of pathway activation. For this approach, sensitive biochemical responses need to be identified and linked to the introduction of the toxicant through dose response, by time of response, and, when possible, through a proposed biochemical mechanism. The weakness of this approach is that more sensitive unidentified responses may exist requiring that a safety factor of 10 be used to define a NOEL. For dioxin-like compounds, using a surrogate marker of response CYP1A1 induction, this approach yields an estimate of the acceptable daily intake of 5-50 fg/kg/day. This limit is remarkably similar to the results of the original EPA linear extrapolation (6 fg/kg/day). A similar approach can be used for other nongenotoxic carcinogens and the analysis can be completed within 1 year.     

Risk-Assessment Implications of Mechanistic Model��S Prediction of Low-Dose Nonlinearity of Liver Tumor Risk for Mice Fed Fumonisin B1

A two-stage, clonal-expansion model of liver tumor risk in mice was developed by Kodell et al. (Food Addit Contam 18:237–253, 2001) based on the hypothesis that fumonisin B₁, a naturally occurring mycotoxin in corn, is not genotoxic, but rather causes cancer through the disruption of sphingolipid metabolism. This disruption is assumed to cause an increase in apoptosis, in response to which cells proliferate to compensate for reduced tissue mass. The resulting differential increase in the number of pre-neoplastic cells at risk of mutation during cell division is assumed to lead to an increase in the incidence of tumors. Two-year liver tumor incidences predicted by the model using data on organ weight, cell proliferation, and sphingolipid metabolism provided a reasonable match to the actual 2-year observed incidences in a study conducted at the National Center for Toxicological Research. The predictions indicated no risk at low doses (even a possible hormetic effect) and high risk at high doses in females, as well as a complete absence of a dose response (or perhaps, a hormetic effect) in males. This paper provides a commentary on the risk-assessment implications of the modeling results, pointing out that the model’s low-dose predictions provide scientific support and justification for the U.S. Food and Drug Administration’s low-ppm guidance levels in corn products. These guidance levels are significantly higher than would be obtained using linear extrapolation, the method most often used for genotoxic carcinogens and other carcinogens for which low-dose linearity cannot be ruled out.