2003 Progress Report: Mechanistic Evaluation of the Toxicity of Chemical Mixtures

EPA Grant Number: R829358
Title: Mechanistic Evaluation of the Toxicity of Chemical Mixtures
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Hahn, Intaek
Project Period: September 24, 2001 through September 23, 2006
Project Period Covered by this Report: September 24, 2002 through September 23, 2003
Project Amount: $465,281
RFA: Complex Chemical Mixtures (2000) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Hazardous Waste/Remediation , Land and Waste Management , Safer Chemicals


Evaluating the toxicity of complex chemical mixtures is one of the major challenges facing modern toxicology. The virtually infinite number of chemical combinations that constitute environmentally relevant mixtures renders standard approaches for the toxicity characterization of chemicals irrelevant. The objective of this research project is to test the hypothesis that the toxicity of complex chemical mixtures can be satisfactorily estimated by understanding the mechanism of toxicity of the individual constituents and utilizing algorithms that define interactions based upon these mechanisms. These experiments are being performed with an invertebrate model, Daphnia magna, because both acute and life-cycle toxicity evaluations can be performed with this species with reasonable cost, space, and time consideration.

Progress Summary:

Aim 1: Define the Concentration-Response Curves for the Model Chemicals That Will be Used in the Mixtures Assessment

Status: 100% Complete . A test set of seven chemicals was devised that would allow the evaluation of all four major categories of chemical interaction (concentration addition, independent joint action, antagonism, synergy). These chemicals and their proposed interactions, based upon known mechanisms of action, are described in Figure 1. Chemicals having the same mechanism of action (2- and 4-chlorophenol or malathion and parathion) are predicted to conform to a model of concentration addition and will occupy the same mechanistic cassettes in the mixtures algorithm. The chlorophenols will share a cassette for narcosis and the organophosphates will share a cassette for cholinesterase inhibition. Piperonyl butoxide and pentachlorophenol will occupy their own cassettes. The joint toxicity of all cassettes will be predicted using a model for independent joint action. Interactions (synergy and antagonism) were predicted as depicted in Figure 1 and have been confirmed experimentally. The chlorophenols were predicted to synergize with other cassettes because the membrane disruptive properties of these compounds would facilitate uptake of the other compounds. Piperonyl butoxide was predicted and confirmed to antagonize the toxicity of the organophosphates by inhibiting the metabolic activation of these compounds.

Concentration-response curves for these compounds and the modulating effects of the synergists/antagonists on the concentration-response relationships have been determined. Modulating effects were quantified by establishing coefficients of interaction (K values) that describe the degree to which a defined concentration of modulator shifts the concentration-response curve of the targeted chemical.

Figure 1. Chemicals Selected for the Test Mixture That Would Allow Evaluation of Concentration Addition, Independent Joint Action, Synergy, and Antagonism. Sites of proposed interactions are depicted in this figure.

Aim 2: Develop and Experimentally Validate Algorithms That Define the Acute Toxicity of Binary Combinations of the Chemicals

Status: 85% Complete. Algorithms that define combined toxicity of chemicals within a cassette (concentration additivity) and among cassettes (independent joint action) have been developed, and binary combinations have been experimentally validated. K values (as described above) currently are being used to describe the modulating effect of one chemical upon another. These expanded models currently are undergoing experimental validation.

Aim 3: Predict the Acute Toxicity of Complex Chemical Mixtures Using the Developed Algorithms in Combination

Status: 0% Complete. This aim is scheduled to be completed during the time remaining on this research project.

Aim 4: Expand the Approach to Include Multiple Sublethal Endpoints of Toxicity and Environmentally Chemical Mixtures

Status: 30% complete. The basic modeling methodologies developed in aims 1-3 are being applied to sublethal endpoints and environmentally relevant mixtures. These results will greatly expand the utility of the modeling approach and will provide important information regarding the toxicity of known chemical mixtures.

The eight chemicals selected for this phase of the research project were shown to be ubiquitous in surface waters of the United States (U.S. Geological Survey survey, Kolpin, et al., 2002, Environmental Science and Technology 36:1202). These chemicals are 4-nonylphenol, bisphenol A, chorpyrifos, diazinon, caffeine, DEET, carbaryl, and flouranthene. Most sensitive endpoints of toxicity were identified for each chemical and concentration-response relationships for these endpoints, over the life cycle of the organisms, were determined. Most sensitive endpoints were: reduced growth (4-nonylphenol, bisphenol A), lethality (chlorpyrifos, diazinon), developmental abnormalities among offspring (caffeine, DEET), and reduced fecundity (carbaryl, flouranthene). Concentration-response curves currently are undergoing analyses for shape (linear, sigmoidal, two-segmented, etc.), slope, and calculation of threshold concentrations. These data will be used to devise mixtures of the chemicals with which toxicity will be modeled based upon methods developed under the previous aims. Predictions will be tested experimentally.

Additional Projects

Experiments were conducted in an effort to define mechanisms of toxicity that would result in synergistic interactions. We hypothesized that a chemical that lowered endogenous levels of a steroid hormone would synergistically interact with a chemical that competitively inhibited the receptor of that steroid. This assumption of synergism was based upon the prediction that by lowering levels of the endogenous receptor ligand (the hormone), the receptor antagonist would be better able to compete for receptor occupancy. This prediction of synergy was modeled and experimentally tested using the ecdysone synthesis inhibitor fenarimol and the ecdysone receptor antagonist testosterone. Ecdysone-dependent embryo development was used as the indicator of toxicity. The combined toxicity of these chemicals was poorly predicted using our model for independent joint action. The toxicity of mixtures of these two chemicals, however, was accurately predicted when K values were calculated that defined the effects of fenarimol on the toxicity of testosterone. These K values were incorporated into the model.

Future Activities:

Aim 2: Experiments are underway to experimentally validate the use of K values to quantify the synergistic effects of the chlorophenol cassette on the other chemical cassettes that will be used to model the toxicity of the test mixture. These experiments should be completed by January 2004.

Aim 3: Model components developed and experimentally validated with binary combinations will be expanded to accommodate larger combinations of the test chemicals. Ultimately, the approach will be used to predict the toxicity of various combinations of the six compounds that constitute the mixture. Predictions will be experimentally validated. This aim will be completed during the Year 3 of the project.

Aim 4: Combinations of the eight chemicals (listed above) used in this aim will be formulated at environmentally relevant ratios. Toxicity (both lethal and sublethal effects) will be modeled according to independent joint action. This model will assume that no synergistic/antagonistic interactions occur among the chemicals. The model predictions will be tested experimentally. If the model accurately predicts measured toxicity, then results will be used to judge risk associated with these chemicals at the levels in which they occur in the environment. Results will be published and similar analyses will be pursued with additional environmentally relevant mixtures. If the model does not accurately predict toxicity, then sources of interaction among the chemicals will be investigated, mechanisms of interaction will be defined, K values will be developed that quantify these interactions, and the K values will be incorporated into the model. Such modifications to the model will continue until the model accurately predicts the toxicity of the mixture. The final model then will be used to judge risk associated with these chemicals at concentrations found in the environment.

Additional experiments are planned to further evaluate mechanisms by which chemical constituents of mixtures can synergize. We will investigate the consequence of exposure to chemical mixtures whose constituents target different components of the same signaling pathway. Mechanisms of synergy will be hypothesized and experimentally evaluated. Planned investigations include the potential synergistic toxicity resulting from exposure to combinations of chemicals that target different constituents of the same signaling receptor molecule.

Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other project views: All 29 publications 21 publications in selected types All 17 journal articles
Type Citation Project Document Sources
Journal Article Mu XY, LeBlanc GA. Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna. ournal of Experimental Zoology 2002;292(3):287-292. R829358 (2003)
R826120 (Final)
R826129 (2001)
R826129 (Final)
not available
Journal Article Mu XY, LeBlanc GA. Synergistic interaction of endocrine-disrupting chemicals: model development using an ecdysone receptor antagonist and a hormone synthesis inhibitor. Environmental Toxicology and Chemistry 2004;23(4):1085-1091. R829358 (2003)
R829358 (2004)
R829358 (Final)
R826129 (Final)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
  • Other: DeepDyve-PDF Article Preview
  • Journal Article Mu XY, LeBlanc GA. Developmental toxicity of testosterone in the crustacean Daphnia magna involves anti-ecdysteroidal activity. General and Comparative Endocrinology 2002;129(2):127-133. R829358 (2003)
    R826129 (Final)
    not available
    Journal Article Olmstead AW, LeBlanc GA. Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. Journal of Experimental Zoology 2002;293(7):736-739. R829358 (2003)
    R826129 (Final)
    not available
    Journal Article Olmstead AW, LeBlanc GA. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environmental Health Perspectives 2003;111(7):919-924. R829358 (2003)
    R826129 (Final)
    not available
    Journal Article Wolf CJ, Hotchkiss A, Ostby JS, LeBlanc GA, Gray LE. Effects of prenatal testosterone propionate on the sexual development of male and female rats: A dose-response study. Toxicological Sciences 2002;65(1):71-86. R829358 (2003)
    R826129 (2001)
    R826129 (Final)
    not available
    Journal Article Wolf CJ, LeBlanc GA, Gray Jr. LE. Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. Toxicological Science 2004;78(1):135-143. R829358 (2003)
    not available

    Supplemental Keywords:

    hazard assessment, narcotics, computational toxicology, toxicity, complex chemical mixtures, chemicals, chlorophenols, organophosphates, cassettes, compounds, analytical models, biodegradation, contaminated sediments, hazardous organic substances,, RFA, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, chemical mixtures, Fate & Transport, Hazardous Waste, Ecology and Ecosystems, Hazardous, complex mixtures, contaminated sediments, fate and transport, fate and transport , biodegradation, hazardous organic substances, toxicity testing, environmental transport and fate, chemical kinetics, hazardous chemicals, complex toxic chemical mixtures, mechanisitic research, analytical models

    Relevant Websites:

    http://www.tox.ncsu.edu/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2004 Progress Report
  • 2005
  • Final Report