An empirical approach to sufficient similarity: combining exposure data and mixtures toxicology data.

When assessing risks posed by environmental chemical mixtures, whole mixture approaches are preferred to component approaches. When toxicological data on whole mixtures as they occur in the environment are not available, Environmental Protection Agency guidance states that toxicity data from a mixture considered "sufficiently similar" to the environmental mixture can serve as a surrogate. We propose a novel method to examine whether mixtures are sufficiently similar, when exposure data and mixture toxicity study data from at least one representative mixture are available. We define sufficient similarity using equivalence testing methodology comparing the distance between benchmark dose estimates for mixtures in both data-rich and data-poor cases. We construct a "similar mixtures risk indicator"(SMRI) (analogous to the hazard index) on sufficiently similar mixtures linking exposure data with mixtures toxicology data. The methods are illustrated using pyrethroid mixtures occurrence data collected in child care centers (CCC) and dose-response data examining acute neurobehavioral effects of pyrethroid mixtures in rats. Our method shows that the mixtures from 90% of the CCCs were sufficiently similar to the dose-response study mixture. Using exposure estimates for a hypothetical child, the 95th percentile of the (weighted) SMRI for these sufficiently similar mixtures was 0.20 (i.e., where SMRI <1, less concern; >1, more concern).

Metabolite analyses of grain from maize hybrids grown in the United States under drought and watered conditions during the 2002 field season.

Understanding natural variation in the composition of conventional crop germplasms is critical in establishing a baseline for comparison of biotechnology-derived crops. This is particularly relevant to such traits as tolerance to drought stress. Thus, there is both a need to understand the contribution of stress conditions to natural variation in plant nutritional components and to determine whether levels of small molecule metabolites such as osmoprotectants and stress metabolites are also affected. As a first step in developing such information for maize, seven conventional hybrids were grown under different moisture regimens and the impact of moisture on composition was assessed. The regimens included well-watered conditions, water restriction during the vegetative phase, and water restriction during grain fill. Compositional analyses of the harvested grain included assessments of the levels of proximates (moisture, protein, oil, starch) and small molecule metabolites such as fatty acids, free amino acids, organic acids, sugars, total glycerol, glycine betaine, and abscisic acid. Ranges for these analytes were determined across all moisture regimens, and the effect of the different water regimens on these analytes was also evaluated. The number and type of grain analytes that showed statistically significant differences in levels between different water regimens differed quite markedly by maize hybrid. However, the magnitude of mean differences between well-watered and water-restricted samples was typically small, and statistically significant differences for any given analyte were typically observed in only one to three of the seven maize hybrids. Only two analytes, free glutamine and free proline, showed a significant drought-induced difference in at least four maize hybrids.

Impact of genetics and environment on nutritional and metabolite components of maize grain.

The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnology-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnology-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly associated with a newly introduced trait. Therefore, steps have been initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against two different testers. These were grown at three different, but geographically similar, locations in the United States. In addition to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, organic acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant organic acid was citric acid, with only minor amounts of other organic acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and organic acids appeared to be markedly higher than that observed for the OECD analytes.

Testing for additivity at select mixture groups of interest based on statistical equivalence testing methods.

Several assumptions, defined and undefined, are used in the toxicity assessment of chemical mixtures. In scientific practice mixture components in the low-dose region, particularly subthreshold doses, are often assumed to behave additively (i.e., zero interaction) based on heuristic arguments. This assumption has important implications in the practice of risk assessment, but has not been experimentally tested. We have developed methodology to test for additivity in the sense of Berenbaum (Advances in Cancer Research, 1981), based on the statistical equivalence testing literature where the null hypothesis of interaction is rejected for the alternative hypothesis of additivity when data support the claim. The implication of this approach is that conclusions of additivity are made with a false positive rate controlled by the experimenter. The claim of additivity is based on prespecified additivity margins, which are chosen using expert biological judgment such that small deviations from additivity, which are not considered to be biologically important, are not statistically significant. This approach is in contrast to the usual hypothesis-testing framework that assumes additivity in the null hypothesis and rejects when there is significant evidence of interaction. In this scenario, failure to reject may be due to lack of statistical power making the claim of additivity problematic. The proposed method is illustrated in a mixture of five organophosphorus pesticides that were experimentally evaluated alone and at relevant mixing ratios. Motor activity was assessed in adult male rats following acute exposure. Four low-dose mixture groups were evaluated. Evidence of additivity is found in three of the four low-dose mixture groups. The proposed method tests for additivity of the whole mixture and does not take into account subset interactions (e.g., synergistic, antagonistic) that may have occurred and cancelled each other out.

Thermoregulatory response to an organophosphate and carbamate insecticide mixture: testing the assumption of dose-additivity.

Most toxicity data are based on studies using single compounds. This study assessed if there is an interaction between mixtures of the anticholinesterase insecticides chlorpyrifos (CHP) and carbaryl (CAR) using hypothermia and cholinesterase (ChE) inhibition as toxicological endpoints. Core temperature (T(c)) was continuously monitored by radiotelemetry in adult Long-Evans rats administered CHP at doses ranging from 0 to 50mg/kg and CAR doses of 0-150 mg/kg. The temperature index (TI), an integration of the change in T(c) over a 12h period, was quantified. Effects of mixtures of CHP and CAR in 2:1 and 1:1 ratios on the TI were examined and the data analyzed using a statistical model designed to assess significant departures from additivity for chemical mixtures. CHP and CAR elicited a marked hypothermia and dose-related decrease in the TI. The TI response to a 2:1 ratio of CHP:CAR was significantly less than that predicted by additivity. The TI response to a 1:1 ratio of CHP and CAR was not significantly different from the predicted additivity. Plasma and brain ChE activity were measured 4h after dosing with CHP, CAR, and mixtures in separate groups of rats. There was a dose-additive interaction for the inhibition of brain ChE for the 2:1 ratio, but an antagonistic effect for the 1:1 ratio. The 2:1 and 1:1 mixtures had an antagonistic interaction on plasma ChE. Overall, the departures from additivity for the physiological (i.e., temperature) and biochemical (i.e., ChE inhibition) endpoints for the 2:1 and 1:1 mixtures studies did not coincide as expected. An interaction between CHP and CAR appears to depend on the ratio of compounds in the mixture as well as the biological endpoint.