From simple toxicological models to prediction of toxic effects in time

The ability to predict the effects of toxicants in organisms with reasonable accuracy depends to a great extent on the toxico-kinetic models used to describe such effects. Toxic effects of organic chemicals and heavy metals have been described adequately using a hyperbolic model that considers the concentration of the toxicant and the time of exposure only. Such a model relies on the median time to effect (ET₅₀) of a chemical to estimate effects at any exposure time, but cannot make predictions for concentrations other than those tested experimentally. A complementary log-to-log model can calculate all ET₅₀ values for a toxicant, thus enabling the hyperbolic model to predict any level of effect for any combination of concentrations and times of exposure. The parameter values used in both models are obtained from experimental bioassays where the time-to-effect of a toxicant is recorded regularly in addition to standard acute or chronic toxicity data. These models will facilitate the risk assessment of chemicals by (1) predicting effects under any combination of time and concentrations, and (2) reducing to a minimum the experimental efforts required to obtain comprehensive ecotoxicity data.     

In vitro models for assessing the potential cardiovascular disease risk associated with cigarette smoking

Atherosclerotic cardiovascular disease is a prevalent human disorder and a significant cause of human morbidity and mortality. A number of risk factors may predispose an individual to developing atherosclerosis, and of these factors, cigarette smoking is strongly associated with the development of cardiovascular disease. Current thinking suggests that exposure to toxicants found in cigarette smoke may be responsible for this elevated disease likelihood, and this gives rise to the idea that reductions in the levels of some smoke toxicants may reduce the harm associated with cigarette smoking. To assess the disease risk of individuals who smoke cigarettes with altered toxicant levels, a weight-of-evidence approach is required examining both exposure and disease-related endpoints. A key element of such an assessment framework are data derived from the use of in vitro models of cardiovascular disease, which when considered alongside other forms of data (e.g. from clinical studies) may support evidence of potential reduced risk. Importantly, such models may also be used to provide mechanistic insight into the effects of smoking and of smoke toxicant exposure in cardiovascular disease development. In this review the use of in vitro models of cardiovascular disease and one of the contributory factors, oxidative stress, is discussed in the context of assessing the risk potential of both conventional and modified cigarettes. Practical issues concerning the use of these models for cardiovascular disease understanding and risk assessment are highlighted and areas of development necessary to enhance the power and predictive capacity of in vitro disease models in risk assessment are discussed.     

Time-to-event analyses of ecotoxicity data

Intensity and duration of exposure dictate the effect of a toxicant. Consequently, any assessment of ecological risk that does not include a sound understanding of both concentration and duration effects is compromised. This being the case, it is surprising that the predominant approach in ecotoxicology (concentration-effect modeling) inefficiently includes exposure duration. Ecological risk assessment can be enhanced with time-to-event models that can easily include concentration, exposure duration, and other important covariates. Time-to-event methods are described and linkage made to relevant ecological techniques, i.e. life table analyses and genetic selection models.     

Simplified models to analyse time- and dose-dependent responses of populations to toxicants

The basis of ecotoxicology lies currently in the dose–response of organisms to toxicants, as typically described by probit and logistic models. While recognising its merits, standard endpoints ignore the process of toxicity with time, and consequently our ability to predict direct toxic effects in environmental risk assessments is seriously curtailed. Although the response of toxicants with time has been studied before, its application in ecotoxicology remains underutilised. One reason is that no convincing mechanism has been proposed to explain the hyperbolic curves of such responses, whereas a variety of models have been used to describe them. The explanation of both time- and dose-dependent responses is found ultimately in the natural variability of receptor sites among individuals of populations exposed to a toxicant inhibitor with time. The process can be explained by the kinetics of inhibition, and is appropriately described by a simple mathematical expression like the Michaelis–Menten equation, though other asymptotic models (e.g. logistic model) can also be used. The advantage of the hyperbolic model is that median effect values (e.g. LC₅₀ for dose- and ET₅₀ for time-dependent responses) enable calculation of toxicity effects at any concentration level and/or time of exposure, thus making it especially attractive for risk assessment.     

A Method for Quantitative Assessment of Reproductive Risks to the Human Male

There is a need for quantitative assessment of reproductiverisks following human exposure to toxic agents. Current methodsonly estimate exposure levels that are unlikely to produce appreciable risk; they do not address the magnitude of risk. Thequantitative reproductive risk estimation (QRRE) approach presentedhere enables calculation of the increased incidence of infertilityin a human population resulting from exposure of males to atoxic agent. The four steps involve (A) obtaining a dose-responsecurve in experimental animals for the effect of a toxicant ona measure of reproductive function; (B) applying an interspeciesextrapolation factor (IEF) derived for a similar toxicant torelate doses in animals and humans that produce equivalent reproductivetoxicity; (C) using human exposure and uptake levels, alongwith the IEF and the dose-response curve, to calculate impairmentof the measure in humans; and (D) computing the increase inhuman infertility caused by that impairment. The QRRE methodis used to compute reproductive risks from dibromochloropropaneexposure.     

Research approaches to address uncertainties in the risk assessment of arsenic in drinking water

Inorganic arsenic (iAs), an environmental drinking water contaminant, is a human toxicant and carcinogen. The public health community has developed recommendations and regulations that limit human exposure to iAs in drinking water. Although there is a vast amount of information available to regulators on the exposure, disposition and the health-related effects of iAs, there is still critical information about the toxicology of this metalloid that is needed. This necessary information includes identification of the chemical species of arsenic that is (are) the active toxicant(s), the mode(s) of action for its various toxicities and information on potentially susceptible populations. Because of these unknown factors, the risk assessment of iAs still incorporates default assumptions, leading to uncertainties in the overall assessment. The characteristics of a scientifically defensible risk assessment for iAs are that it must: (1) quantitatively link exposure and target tissue dose of active metabolites to key events in the mode of action for major health effects and (2) identify sources of variation in susceptibility to arsenic-induced health effects and quantitatively evaluate their impact wherever possible. Integration of research to address these goals will better protect the health of iAs-exposed populations.     

A physiological model to predict xenobiotic concentration in fish

A physiological model was developed to estimate fish body toxicant load based on information regarding the chemical exposure regime, fish body weight, lipid content and oxygen uptake. The general model was tested in which an oxygen database (OXYREF) was used to predict fish toxicant body burden. Based on the quantitative analysis, it was shown that the model was reliable and accurate in estimating fish body burden of a number of non-metabolized aquatic toxicants. This modified model possesses some functional reality which enables more realistic predictions, making it useful in the practice of aquatic environmental risk assessment.     

Workshop on risk assessment in reproductive and developmental toxicology: addressing the assumptions and identifying the research needs

The risk assessment process is an imprecise procedure aimed at determining a toxicant exposure level with an acceptable risk to the human population. The lack of precision is due to the uncertainties in the assumptions that must be made due to the lack of specific scientific information or knowledge of how to use certain types of data. Unfortunately, every necessary piece of information cannot be obtained for every chemical requiring a risk assessment. In order to better identify and understand some of the assumptions that are made in the risk assessment of reproductive and developmental toxicants, a workshop was organized to specifically define the assumptions underlying the risk assessments for seven specific toxicants (dibromochloropropane, dioxin, glycol ethers, heptachlor, lead, tetrahydrocannabinol, and vitamin A) and to determine the potential research which would reduce the uncertainty associated with making those assumptions. The major assumptions discussed centered around the topics of heterogeneous populations, thresholds, safety factors, exposure assessment, quantitative structure-activity relationships, and mechanisms. This report is the summary of the workshop discussions.     

In vitro dosimetry analyses for acrolein exposure in normal human lung epithelial cells and human lung cancer cells

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. In this study, we used acrolein as a model toxicant and defined the concentration-response relationships of the key adverse responses in normal human bronchial epithelial (NHBE) cells and human mucoepidermoid pulmonary carcinoma (NCI-H292) cells. Direct trace analysis of intracellular free acrolein is extremely challenging, if not impossible. Therefore, we developed a new method for indirectly estimating the intracellular uptake of acrolein. A 10-min treatment was employed to capture the rapid occurrence of the key alkylation reactions of acrolein. Responses, including protein carbonylation, GSH depletion, and GSH-acrolein (GSH-ACR) adduct formation, were all linearly correlated with acrolein uptake in both cell types. Compared to the NCI-H292 mucoepidermoid carcinoma cells, NHBE cells were more sensitive to acrolein exposure. Furthermore, results from the time-course studies demonstrated that depletion and conjugation of GSH were the primary adverse events and directly associated with the cytotoxicity induced by acrolein. In summary, these data suggest that cell susceptibility to acrolein exposure is closely associated with acrolein uptake and formation of GSH-ACR adducts. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.     

Use of epidemiology and clinical toxicology to determine human risk in regulating polychlorinated biphenyls in the food supply

Polychlorinated biphenyls (PCBs) became a national problem in 1971 when several accidental contaminations of foods were reported. Extensive efforts were successfully undertaken by FDA to reduce the residues of PCBs in food. However, the PCB levels in several species of freshwater fish have raised concern about PCB residues from environmental contamination. This concern prompted a reassessment of the human risk involved in consumption of such fish. The best evidence that a chemical may produce adverse health effects in humans is provided by adequate epidemiologic data confirmed or supplemented by data from valid animal tests. Traditionally, where the regulatory agencies have used results of animal toxicology experiments to evaluate hazard and predict hypothetical safety for humans, “safety factors” such as 1 to 10 or 1 to 100 have been used. The size of the safety factor and the potential exposure to a chemical are established by properly informed scientific judgment. More recent efforts have involved use of a combination of human and animal data and a variety of mathematical models to determine risk. The human epidemiology data and the animal toxicity data of PCB exposure are reviewed, as well as risk assessment in general. Specific examples of risk assessment are presented in which animal data are extrapolated to humans, based on several levels of human exposure to PCBs in fish.     

A review of the Buehler guinea pig skin sensitization test and its use in a risk assessment process for human skin sensitization

The Buehler test is a valuable procedure for screening the sensitization potential of chemicals prior to human exposure. Our experience of over 20 years has shown it to be effective in detecting strong, moderate, and most weak sensitizers. The topical exposure inherent in the Buehler test allows it to be utilized to investigate dose responses, cross reactivity between structurally related chemicals, and the sensitization potential of contaminants in raw material mixtures. For safety assessment purposes, Buehler test results provide an initial indication of the sensitization potential of the material in question under relevant, but exaggerated, exposure conditions. These results can be compared to results on benchmark chemicals to assess sensitization risk for subsequent human exposure. Optimizing the sensitivity of the Buehler test requires adherence to the published methodology and proper interpretation of the challenge and rechallenge data obtained. Adjuvant-type test methods are generally considered to be more sensitive than topical methods. However, when done properly, topical test procedures such as the Buehler test or the open epicutaneous test can accurately detect most chemicals with any realistic potential for sensitizing humans by the topical route. Moreover, from a risk assessment perspective, these topical tests avoid the problems of overestimating the weak sensitization potential of many topically applied materials or underestimating the sensitization potential of very strong sensitizers; both are potential concerns with invasive adjuvant-type test methods. The Buehler test or other topical test methods are particularly valuable for comparative sensitization risk assessment since human sensitization data on benchmark materials are all derived from topical exposure. The risk assessment is developed by comparing the guinea pig data on the new material versus relevant benchmark chemicals or formulations and also by evaluating the existing human sensitization data on the benchmark material. These data are then utilized to predict human sensitization risk from topical exposure to the new material. Confirmation of human safety can be derived from human repeat insult patch testing (HRIPT) and other clinical tests such as the product use test and the diagnostic patch test. Utilized in this manner, the Buehler test is an integral component of an overall skin sensitization safety assessment program for a new chemical or product formulation.     

Use of threshold and mode of action in risk assessment

Under current guidelines, exposure guidelines for toxicants are determined by following one of two different tracks depending on whether the toxicant’s mode of action (MOA) is believed to involve an exposure threshold. Although not denying the existence of thresholds, this paper points out problems with how the threshold concept and MOA is used in risk assessment. Thresholds are frequently described using imprecise terms that imply some unspecified increase in risk, which robs them of any meaning (any reasonable dose response will satisfy such a definition) and tacitly implies a value judgment about how large a risk is acceptable. MOA is generally used only to inform a threshold’s existence and not its value. Often MOA is used only to conclude that the adverse effect requires an upstream cellular or biochemical response for which a threshold is simply assumed. Data to inform MOA often come from animals, which complicates evaluation of the role of human variation in genetic and environmental conditions, and the possible interaction of the toxicant with processes already producing background toxicity in humans. In response to these and other problems with the current two-track approach, this paper proposes a modified point of departure/safety factor approach to setting exposure guidelines for all toxicants. MOA and the severity of the toxic effect would be addressed using safety factors calculated from guidelines established by consensus and based on scientific judgment. The method normally would not involve quantifying low-dose risk, and would not require a threshold determination, although MOA information regarding the likelihood of a threshold could be used in setting safety factors.     

Cellular and biochemical indices of bronchoalveolar lavage for detection of lung injury following insult by airborne toxicants

Cellular and biochemical profiles of bronchoalveolar lavage (BAL) material after inhalation or intratracheal exposure to various airborne toxicants clearly reflect that BAL has the potential of being a useful tool for the rapid screening of lung injury. The cellular and biochemical responses not only predict inflammation, extent of tissue damage and toxic nature of the substances, but could also help in understanding the molecular mechanisms of pathogenicity. Depending upon the changes of BAL in animals acutely exposed to a pulmonary toxicant, future in-depth studies along with complete histopathological evaluations could be made. Also, the assessment of macromolecules of pharmacological importance in the lavage, especially the secretory products of alveolar macrophages and other lung cell types, could be very useful in predicting the toxic potential of various airborne substances and could also serve as important indicators of developing chronic lung diseases and, therefore, necessitate further studies.     

Factors affecting the toxicity of dioxin-like toxicants: a molecular approach to risk assessment of dioxins.

The numerous toxic responses of dioxin-like compounds are mediated by the intracellular Ah (aryl hydrocarbon) receptor. It has been suggested that the regulation of dioxins and similar substances could be placed on a molecular foundation by considering the proportion of Ah-receptor sites occupied by toxicant molecules. The present work has shown that the following formation not yet available would be needed in order to develop this approach: correlation between dioxin exposure and human tissue levels; accurate determination of the association constants for human Ah-receptor with toxicant, and for human receptor-ligand complex with DNA; and knowledge of the intracellular concentrations of both receptor binding sites and DNA binding sites. Furthermore, since not all dioxin-like substances behave identically, this information would need to be gathered for a wide variety of substances.     

A multi-route model of nicotine–cotinine pharmacokinetics,pharmacodynamics and brain nicotinic acetylcholine receptor binding in humans

The pharmacokinetics of nicotine, the pharmacologically active alkaloid in tobacco responsible for addiction, are well characterized in humans. We developed a physiologically based pharmacokinetic/pharmacodynamic model of nicotine pharmacokinetics, brain dosimetry and brain nicotinic acetylcholine receptor (nAChRs) occupancy. A Bayesian framework was applied to optimize model parameters against multiple human data sets. The resulting model was consistent with both calibration and test data sets, but in general underestimated variability. A pharmacodynamic model relating nicotine levels to increases in heart rate as a proxy for the pharmacological effects of nicotine accurately described the nicotine related changes in heart rate and the development and decay of tolerance to nicotine. The PBPK model was utilized to quantitatively capture the combined impact of variation in physiological and metabolic parameters, nicotine availability and smoking compensation on the change in number of cigarettes smoked and toxicant exposure in a population of 10,000 people presented with a reduced toxicant (50%), reduced nicotine (50%) cigarette Across the population, toxicant exposure is reduced in some but not all smokers. Reductions are not in proportion to reductions in toxicant yields, largely due to partial compensation in response to reduced nicotine yields. This framework can be used as a key element of a dosimetry-driven risk assessment strategy for cigarette smoke constituents.