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Abstract
It is common practice to conduct laying hen pilot studies when developing pesticides in order to determine potential metabolites. The laying hen assay has been the industry standard, but there is motivation for a more economical, less time consuming assay to determine metabolite production. Three experiments were conducted to determine the similarity between metabolites produced by a Single Comb White Leghorn laying hen (DuPont AMR, 1993) with those produced by a viable egg assay when administered with diuron, pyrithiobac sodium, and nicotine, a nonpolar herbicide, a polar pesticide, and a xenobiotic compound, respectively. Fertile Single Comb White Leghorn eggs were incubated at 99°F and 50% humidity. A dosing solution of 14C N,-(3,4 dichlorophenyl)-N, N-dimethylurea (Diuron) was solubilized in polyethylene glycol 400 at a concentration of 6.4 mg/ml and a specific activity of 6.04 μCi/mg. The dosing solutions for 14C phenyl label and 14C pyrimidine label pyrithiobac sodium were solubilized in water at concentrations of 6.34 and 6.44 mg/ml and specific activities of 6.63 and 6.04 μCi/mg, respectively. The dosing solution for 14C nicotine was solubilized in water at a concentration of 15.12 mg/ml and a specific activity of 0.13 μCi/mg. The incubated eggs were checked for viability at d 6. Viable eggs were allotted to replicate groups (10eggs/treatment) to minimize the differences in egg weight and randomly assigned to the treatments utilizing two sites of injection (total volume dosed: 200ul/egg). Viable eggs were harvested on d 15 and fractioned into allantoic fluid, yolk, embryo, shell, and remaining fluid (pooled by treatment). Samples were frozen until analysis. Each sample was extracted, and the final supernatant was analyzed using reverse phase HPLC analysis, column and gradient in accordance with the procedure utilized in the laying hen studies. Samples were analyzed for confirmation with electrospray ionization LC/MS, using the same LC method employed in the HPLC analysis. Liquid Scintillation Counting analysis and pellet combustions were conducted to determine total radioactive recovery.
The total radioactive recovery was in accordance with Good Laboratory Practice (GLP) guidelines for all three experiments, 70–120%. The site of injection did not influence the distribution of radioactivity among the fractions nor the metabolites produced in each fraction for both pesticide experiments. The metabolites identified and confirmed in the diuron and pyrithiobac sodium experiments were analogous to those of the DuPont laying hen metabolism studies. Additional metabolites were identified and confirmed in the pyrithiobac sodium study, which were analogous to metabolites found in the DuPont rat metabolism study. Metabolites identified and confirmed in the nicotine study were analogous to major metabolites present in humans. This study suggests that the viable egg assay is a reliable, economical, potential surrogate for the pilot laying hen metabolism assay. It also suggests that the viable egg assay has the potential to be utilized in studying human nicotine metabolism.
