Behavioral toxicology in risk assessment: problems and research needs

Behavioral methods are being used with increasing frequency in toxicology to assess the deleterious effects of chemicals to which we are exposed. The impetus for the use of behavioral techniques in risk assessment resulted from the presumption that they were more sensitive than other tests in detecting toxicity. A more logical reason for the use of behavioral tests is the fact that behavior is the functional indicator of the net sensory, motor, and integrative processes occurring in the central and peripheral nervous system. Thus, the functional capacity of the nervous system cannot be determined independent of behavioral analysis. Some of the problems confronting behavioral toxicology are (1) the translation of human subjective complaints into behavioral tests in animals; (2) determining subtle effects on the nervous system in the face of the well-known functional reserve and adaptability of the system; (3) dealing with the variety of statistical problems resulting from the use of multiple tests, multiple measurements using the same test and the (relatively) large variability inherent in some behavioral phenomena; and (4) selecting the proper tests. Three critical research needs in behavioral toxicology as they relate to risk assessment are (1) development and validation of methods, (2) determining subpopulations at greatest risk, and (3) developing a strategy for determining interactions between two or more agents.     

Workshop overview: arsenic research and risk assessment

The chronic exposure of humans through consumption of high levels of inorganic arsenic (iAs)-contaminated drinking water is associated with skin lesions, peripheral vascular disease, hypertension, and cancers. Additionally, humans are exposed to organic arsenicals when used as pesticides and herbicides (e.g., monomethylarsonic acid, dimethylarsinic acid (DMA(V)) also known as cacodylic acid). Extensive research has been conducted to characterize the adverse health effects that result from exposure to iAs and its metabolites to describe the biological pathway(s) that lead to adverse health effects. To further this effort, on May 31, 2006, the United States Environmental Protection Agency (USEPA) sponsored a meeting entitled "Workshop on Arsenic Research and Risk Assessment". The invited participants from government agencies, academia, independent research organizations and consultants were asked to present their current research. The overall focus of these research efforts has been to determine the potential human health risks due to environmental exposures to arsenicals. Pursuant in these efforts is the elucidation of a mode of action for arsenicals. This paper provides a brief overview of the workshop goals, regulatory context for arsenical research, mode of action (MOA) analysis in human health risk assessment, and the application of MOA analysis for iAs and DMA(V). Subsequent papers within this issue will present the research discussed at the workshop, ensuing discussions, and conclusions of the workshop.     

Behavioral toxicology and risk assessment

In order to establish safe exposure levels to toxic chemicals, risk assessment guidelines have been developed. These guidelines evaluate epidemiologic and animal research data on a particular chemical, as well as dose-response relationships, animal to human extrapolation and assessment of exposure levels of populations. Using the guidelines, risk characterization is established in order to determine a strategy for reducing undesirable risk to human populations. Using both human neonatal lead exposure data and results from rodent and primate studies, this review examines the possibility that behavioral measurements are sufficient to provide adequate risk assessment guidelines for lead intoxication of the developing organism. The overall trend in these data during the past 10 years has been to show that exposures to inorganic lead at levels previously considered safe have long-lasting significant alterations in behavioral measures, suggesting that central nervous system function has been altered irreversibly. The conclusion is drawn that behavioral toxicology can provide sensitive, quantitative and reliable data for risk assessment and that in the future these methodologies could be used to set exposure guidelines for other neurotoxic chemicals.     

Embryo-fetal developmental and reproductive toxicology of vinyl chloride in rats.

Vinyl chloride (VC) exposure is primarily via inhalation in the workplace. The primary target organ of VC toxicity is the liver and occupational exposure to VC leads to hepatic angiosarcoma. However, based on epidemiological studies, researchers have been unable to ascertain the effect of occupational VC exposure on embryo-fetal development or reproductive function. A limited number of animal studies available in the literature have examined the effect of VC on embryo-fetal development, however, there are no published studies on the effect of VC exposure on reproductive capability. The current study was designed to assess the potential maternal and/or embryo-fetal developmental and 2-generation reproductive toxicity of inhaled VC in CD(R) Sprague-Dawley rats at exposure levels of 0, 10, 100, and 1100 ppm. In the embryo-fetal/developmental toxicity study, the female rats were exposed to VC daily from gestation day (GD) 6 through 19. In the reproductive toxicity study, the F(0) generation male and female rats were exposed to VC for a 10-week premating and 3-week mating periods. The F(0) generation male rats were exposed to VC until terminal euthanasia. The F(0) generation female rats were exposed from GD 0 through GD 20 and lactation day (LD) 4 through LD 25. Our results indicate that up to 1100 ppm VC exposure did not adversely affect embryo-fetal developmental or reproductive capability over 2 generations in rats. The primary target organ of VC, the liver, was affected as evidenced by an increase in liver weight and/or histologically identified cellular alterations, such as centrilobular hypertrophy at 100 and 1000 ppm. Based on the results of these studies, the no observed adverse effect level (NOAEL) for embryo-fetal/development is 1100 ppm, and the NOAEL for reproduction is 1100 ppm. The results from the current studies, which are a more comprehensive embryo-fetal/developmental and reproduction study, may be incorporated into future risk assessments of occupational exposure to VC where concerns regarding the effects of VC exposure remain.     

The challenge of reproductive and developmental toxicology under REACH.

The European Union's REACH regulation has explicit requirements for reproductive and developmental toxicity data on all substances manufactured in or imported into the European Union at > or = 10 metric tons/year. Meeting the data requirements with whole-animal testing could result in the use of almost 22 million vertebrate animals for the registration of existing chemicals and cost up to several hundred thousand dollars per registered substance. The requirement for financial and animal resources can be reduced by the use of in vitro testing, quantitative structure-activity relationship models, and grouping of related substances. Although REACH strongly encourages these methods of avoiding vertebrate animal testing, it does not appear that in vitro testing or quantitative structure-activity relationship analysis will be able to replace whole-animal reproductive and developmental toxicity testing. Grouping of related compounds offers the possibility, perhaps in conjunction with in vitro testing and structure-activity analysis, of reducing vertebrate animal testing provided there is sufficient information on the related compounds and sufficient reason to believe that the related compounds will have similar toxicological properties. The designation of a substance as a reproductive or developmental toxicant follows criteria that do not consider the dose level of the substance at which reproductive or developmental effects occur, as long as excessive generalized toxicity does not occur. This method of labeling substances without consideration of effective dose level does not provide information on the actual risk of the chemical. Designation of a substance as a reproductive or developmental toxicant may have important implications under REACH and can be expected to result in the need to obtain authorization for marketing of the substance in the European Union.     

Eological toxicology and ecologlcal risk assessment

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Integrating epidemiology and toxicology in neurotoxicity risk assessment

Neurotoxicity risk assessments depend on the best available scientific information, including data from animal toxicity studies, human experimental studies and human epidemiology studies. There are several factors to consider when evaluating the comparability of data from studies. Regarding the epidemiology literature, issues include choice of study design, use of appropriate controls, methods of exposure assessment, subjective or objective evaluation of neurological status, and assessment and statistical control of potential confounding factors, including co-exposure to other agents. Animal experiments must be evaluated regarding factors such as dose level and duration, procedures used to assess neurological or behavioural status, and appropriateness of inference from the animal model to human neurotoxicity. Major factors that may explain apparent differences between animal and human studies include: animal neurological status may be evaluated with different procedures than those used in humans; animal studies may involve shorter exposure durations and higher dose levels; and most animal studies evaluate a single substance whereas humans typically are exposed to multiple agents. The comparability of measured outcomes in animals and humans may be improved by considering functional domains rather than individual test measures. The application of predictive models, weight of evidence considerations and meta-analysis can help evaluate the consistency of outcomes across studies. An appropriate blend of scientific information from toxicology and epidemiology studies is necessary to evaluate potential human risks of exposure to neurotoxic substances.     

Experimental designs and risk assessment in combination toxicology: Panel discussion

Advancing our knowledge on the toxicology of combined exposures to chemicals and implementation of this knowledge in guidelines for health risk assessment of such combined exposures are necessities dictated by the simple fact that humans are continuously exposed to a multitude of chemicals. A prerequisite for successful research and fruitful discussions on the toxicology of combined exposures (mixtures of chemicals) is the use of defined terminology implemented by an authoritative international body such as, for example, the International Union of Pure and Applied Chemistry (IUPAC) Toxicology Committee. The extreme complexity of mixture toxicology calls for new research methodologies to study interactive effects, taking into account limited resources. Of these methodologies, statistical designs and mathematical modelling of toxicokinetics and toxicodynamics seem to be most promising. Emphasis should be placed on low-dose modelling and experimental validation. The scientifically sound so-called bottom-up approach should be supplemented with more pragmatic approaches, focusing on selection of the most hazardous chemicals in a mixture and careful consideration of the mode of action and possible interactive effects of these chemicals. Pragmatic approaches may be of particular importance to study and evaluate complex mixtures; after identification of the ‘top ten’ (most risky) chemicals in the mixture they can be examined and evaluated as a defined (simple) chemical mixture. In setting exposure limits for individual chemicals, the use of an additional safety factor to compensate for potential increased risk due to simultaneous exposure to other chemicals, has no clear scientific justification. The use of such an additional factor is a political rather than a scientific choice.     

Biomarkers in toxicology and risk assessment: informing critical dose-response relationships

Tremendous advances have been made in the study of biomarkers related to carcinogenesis during the past 20 years. This perspective will briefly review improvements in methodology and instrumentation that have increased our abilities to measure the formation, repair, and consequences of DNA adducts. These biomarkers of exposure, along with surrogates such as protein adducts, have greatly improved our understanding of species differences in metabolism and effects of chemical stability and DNA repair on tissue differences in molecular dose. During this same time frame, improvements in assays for biomarkers of effect have provided better data and an improved understanding of the dose responses for both gene and chromosomal mutations. A framework analysis approach was used to examine the mode of action of genotoxic chemicals and the default assumption that cancer can be expected to be linear at very low doses. This analysis showed that biomarkers of exposure are usually linear at low doses, with the exception being when identical adducts are formed endogenously. Whereas biomarkers of exposure extrapolate down to zero, biomarkers of effect can only be interpolated back to the spontaneous or background number of mutations. The likely explanation for this major difference is that at high exposures, the biology that results in mutagenesis is driven by DNA damage resulting from the chemical exposure. In contrast, at very low exposures, the biology that results in mutagenesis is driven by endogenous DNA damage. The shapes of the dose-response curves for biomarkers of exposure and effect can be very different, with biomarkers of effect better informing quantitative estimates of risk for cancer, a disease that results from multiple mutations. It is also clear, however, that low dose data on mutagenesis are needed for many more chemicals.     

Asbestos: toxicology and risk assessment for the general population in The Netherlands

Within the scope of the preparation of Integrated Criteria Documents for priority compounds in The Netherlands, the possible health effects of oral and inhalatory exposure to asbestos for the general population were evaluated. It was concluded from the results of experiments in animals that exposure to asbestos by the oral route is not carcinogenic and is not expected to present a health risk to the general population. Inhaled asbestos, however, is distinctly carcinogenic to man, giving rise to lung tumours, and mesotheliomas of the pleura and peritoneum. Chrysotile asbestos appears to be less potent in inducing mesotheliomas than the amphiboles, but all types of asbestos appear to have a similar potency for inducing lung cancer. The risk of mesothelioma is not expected to be influenced by smoking, whereas the risk of lung cancer is expected to be ten times higher in smokers than in non-smokers exposed to the same asbestos concentrations. Risk-assessment models for the inhalatory route, for the general population, are based on linear non-threshold extrapolation of occupational exposure to much lower environmental concentrations. These models give only a rough approximation of the risk of environmental exposure to asbestos. In accordance with the Air Quality Guidelines of the World Health Organization (World Health Organization, 1987), it was estimated that an extra risk of lung cancer of one in 10(6) (in the general population, with 30% smokers) may be presented by lifetime exposure to asbestos fibres longer than 5 microns, measured by electron microscopy, at concentrations of 100-1000/m3. It was further estimated that an extra risk of mesothelioma of one in 10(6) may be presented by lifetime exposure to 10-100 amphibole fibres/m3 or to a range of 100-10000 chrysotile fibres/m3 (fibres longer than 5 microns). From the current asbestos concentrations, the risk of mesothelioma for the general population in The Netherlands appears to be negligible; the extra risk of lung cancer is expected to be higher than 1 in 10(6) near asbestos sources, whereas it appears to be negligible in background areas and in most large cities and industrial areas. However, it must be borne in mind that the validity of the risk figures given is difficult to judge.     

Interpretation of studies on the developmental reproductive toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring

There have been several studies on the maternal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and effects in the reproductive tract of male offspring, subsequent to risk assessments undertaken in 2001. This review compares the methodology and results to examine key methodological features, and consistency in reported outcomes. Maternal dosing at >0.8 μg TCDD/kg causes lethality and weight loss, and it is difficult to distinguish between direct and indirect effects of TCDD at these dose levels. Statistically significant effects of maternal doses of <1 μg TCDD/kg (i.e. the dose levels relevant for risk assessment) on prostate weight or epididymal sperm counts in offspring were reported in the minority of studies. The pharmacokinetics of TCDD differs considerably between acute and chronic dosing, and with dose level of TCDD. On the basis of body burden, TCDD had different potency at inducing adverse effects in the only comparison study between acute and chronic dosing. Understanding of the pharmacokinetics of TCDD and relationship to adverse effects in offspring is required. These analyses identify key features of TCDD developmental toxicity in male offspring, and identify data needs for future risk assessment.     

Issues in Australian contaminated land risk assessment toxicology

Since implementation of the National Environmental Protection (Assessment of Site Contamination) Measure (NEPM) guidelines in 1999, a number of improvements are needed to enhance the contaminated land risk assessment process. In particular, due to advances in toxicological research there is a need to update the Australian adopted health investigation levels (HILs), presented in the NEPM guidelines, and to significantly expand the number of HILs to generate a comprehensive list of soil screening criteria. Furthermore, there is a need to develop Australian benchmark doses for carcinogens and to incorporate benchmark doses more widely in Australian risk assessment practice. The NEPM is currently under revision and consequently there is an excellent opportunity to undertake such development; however, this will require appreciation of the need for improvement and significant financial investment.     

Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group II report: testing methods in developmental neurotoxicity for use in human risk assessment

The Work Group addressed the design and content of a basic screening battery for detecting or flagging developmental neurotoxicity. It was agreed that a basic screening battery should be incorporated routinely into any developmental or reproductive toxicity study conducted for risk assessment purposes, and that a "triggered" stand-alone developmental neurotoxicity study, as exemplified by the current EPA proposal, should include greater in-depth evaluation of CNS function and pathology. Time constraints did not permit the group to address the design or content of such a stand-alone study. It was acknowledged unanimously that a basic screening battery may provide more information than simply the detection of neurotoxicity; however, it should not be expected to provide detailed dose-response information nor to identify the precise mechanism of agent action. The Work Group also agreed that a basic screening battery should include evaluation of multiple CNS functions, that observed alterations may be indicative of primary or secondary effects on the nervous system, that the test methods selected may differ based on what is known about the agent, and that the protocol for study conduct can be as important as the methods employed. In light of these assumptions, the Work Group recommended schedules for monitoring offspring development using measures of: 1) physical landmarks, 2) brain weights, 3) neuropathology, 4) functional observations, 5) motor activity, 6) reactivity, and 7) learning and memory. The species tested, sample sizes, treatment parameters, etc., would be determined by the type of developmental or reproductive toxicity study into which the basic screening battery was incorporated.(ABSTRACT TRUNCATED AT 250 WORDS)