ES/RP 531 Fundamentals of Environmental Toxicology Fall 2005 ESRP531 Lecture 19 Mixtures.doc Page 1 of 9 October 31, 2005 Lecture 19: Chemical Mixtures & Interactions (Dose-Response Assessment 2) I. Living in a Chemical Soup A. Although the vast majority of risk assessments are conducted on chemicals one at a time, exposure is to a plethora of chemicals, naturally occurring and synthetic. B. No one seems to flinch at the prospects of simultaneous or sequential multiple chemical exposures when we eat food. 1. However, food is full of bioactive secondary plant metabolites that have as great a probability as synthetic chemicals of testing positive in rodent assays for carcinogenicity or for interactions with the endocrine system that could be interpreted as adverse. 2. For example, here is a list of selected compounds that have been found in Camembert cheese and are associated with its complex flavor (Sable & Cottenceau 1999, J. Agric. Food Chem. 47:4825. a. Acetic acid; propionic acid; butanoic acid; oleic acid; methanol; ethanol; octanol; acetone; ethyl acetate; diethyl phthalate; hydrogen sulfide; methyl mercaptan; phenol; cresol. C. An examination of pesticide residues in food by the USDA Pesticide Data Program, shows that two or more different types of pesticide residues occur together in about 25% of analyzed foods. (Figure 1). Figure 1. Proportion of food samples analyzed by the USDA in its Pesticide Data Program (PDP) that have one or more pesticide residues detected. Note that the trend is for less detection of any pesticide residues and lower frequency of detection of multiple residues. Reports can be downloaded from URL: http://www.ams.usda.gov/science/pdp/ D. The USGS NAWQA (National Water Quality Assessment) program reports multiple detections of some pesticides in the same water sample.ES/RP 531 Fundamentals of Environmental Toxicology Fall 2005 ESRP531 Lecture 19 Mixtures.doc Page 2 of 9 1. The most frequent simultaneous occurrence is atrazine and some other herbicide. More infrequent are insecticide detections, but in Oregon, along the Willamette Basin tributaries, a number of insecticides were found simultaneously in water samples. (Figure 2) 2. All available reports for pesticides and other water quality parameters measured in the NAWQA program can be viewed and downloaded at URL: http://www.ams.usda.gov/science/pdp/ Figure 2. Detection of multiple pesticide residues in water samples collected from Zollner Creek in the Willamette Basin watershed of Oregon (Larson et al. 1999) E. Under the Food Quality Protection Act, which is a 1996 amendment to the overarching federal pesticide regulatory law, FIFRA (Federal Insecticide Fungicide and Rodenticide Act), EPA was mandated by Congress to cumulate exposure for risk assessment when multiple residues of compounds with identical mechanisms of toxicity (i.e., identical pharmacodynamics) were present in food and/or water. 1. Thus regulatory science policy, at least for pesticide regulations, must take into account multiple occurrences of residues, although they must have the same mechanism of causing toxicity. a. The OP insecticides were the first group of insecticides subjected to this “mixture” exposure analysis because they all have the same basic mechanism of toxicity through inhibition of brain acetylcholinesterase.ES/RP 531 Fundamentals of Environmental Toxicology Fall 2005 ESRP531 Lecture 19 Mixtures.doc Page 3 of 9 2. Historically, the FDA as far back as 1957 mandated that companies examine possible synergistic effects of OP insecticides for purposes of tolerance establishment. (Discussed in Hayes 1991) a. It had been noted in studies of this time that some OP insecticides, when simultaneously administered to rodents, could substantially increase the expected toxicity. II. Classification of Potential Interactions Between Chemicals A. Independent (or neutral) Effects 1. Substances exert their own toxicity independently of one another; 2. The toxicity of one substance does not affect the toxicity of a second substance. B. Additive Effects (Two types) (Koneman and Pieters 1996) 1. Dose (Concentration) Addition: Compounds having similar mechanisms of toxicity cause a response that is simply the sum of the effects produced by the individual compounds alone. a. Example: 1X Dose Compd. A + 1X Dose Compd. B = 2X Effect 2. Response Addition (also called Independent Action): Chemicals can act on completely different physiological systems, or on the same physiological system but they are functionally independent. a. Response additivity will occur only when the individual compounds exceed their own thresholds of tolerance. b. If the individual compounds do not exert an effect on their own, response additivity is unlikely to occur. C. Antagonistic Effects 1. One compound interferes with the expression of toxicity of another compound resulting in a combined effect that is lower than expected from one compound alone. 2. Example: 1X Dose Compd. A + 1X Dose Compd. B = 0.5X Effect D. Potentiation (or Synergism) 1. Two compounds given simultaneously or close in time cause an effect that is greater than the sum of either alone. 2. One of the compounds may not cause a reaction at all, but in combination with another biologically active compound toxic effects are greatly magnified. 3. Example: 1X Dose Compd. A + 1X Dose Compd. B = 10X Effect E. Dose Response Relationship (Figure 3) 1. The relationship between the dose and response for any two chemicals can be visualized by examining the isobole graph in Figure 3.ES/RP 531 Fundamentals of Environmental Toxicology Fall 2005 ESRP531 Lecture 19 Mixtures.doc Page 4 of 9 Figure 3. Lines of equal effect (isoboles) for different doses of chemicals in mixtures. 2. For example, in a synergistic interaction, a 2.5X dose of chemical A mixed with a 5X dose of chemical B causes a toxic effect of equal magnitude to a 5X A plus 5X B dose. III. Testing for Combined Interactions of Chemical Mixtures A. Risk assessment as practiced today normally analyzes compounds in isolation. 1. The reason is not due to lack of desire; the lack of testing is due as much to practicality and the myriad of possible interactions. (Table 1) Table 1. Cost of testing multiple chemicals, assuming the single test of one compound is $1000. Cost of Entire Series ($ Millions) Cost of Individual Test Number of Chemicals