Egg LD50 Assay for Categorization

About This Grant

Project Summary: Correctly identifying a substance’s acute oral toxicity is crucial for labeling with the proper safety precautions needed for health and safety. The classification, handling, and labeling requirements of substances is determined by their oral toxicity category, based on their acute exposure hazard as determined by the Environmental Protection Agency (EPA) and Globally Harmonized System (GHS). Acute oral toxicity data from rat mortality studies are needed to ascertain the median lethal dose of 50% (LD50). This LD50 is a requirement needed to specify governmental agencies' labeling demands and for risk assessment for human health and environmental hazards. The determination of the LD50 (a single endpoint of lethality) meets the oral and systemic toxicity data requirements of categorization for international regulatory authorities in the areas of industrial chemicals, consumer products, and mixtures such as pesticides [1] [2] [3] [4]. Under considerable public pressure, the US government now has an interest in fostering the development of in vitro and alternative replacements for animal tests [1]. Although in vitro and alternative assays have been developed and codified for other toxicity hazards (i.e., eye and skin irritation, skin sensitization), the alternative assays developed for assessing acute oral systemic toxicity are limited and inaccurate. Additionally, the high cost of the zebrafish models, in vitro 3D constructs, and organ-on-a-chips generally dissuade chemical registrants and product stewards who only rely upon the lower expense and accuracy of LD50 assays. The Egg LD50 Assay for Categorization (ELDAC) would fill a large gap between mammalian LD50 testing and other anticipated but yet-to-be-validated and very complex alternatives. ELDAC uses an already well-characterized network of organ systems. Since the yolk and albumen are the sole nutrition sources for the embryo, test chemical administration to these food sources is a good approximation of oral administration. To demonstrate the feasibility of the ELDAC, we administered 18 chemicals (from GHS 1-5 and EPA IV oral toxicity categories) by injecting directly into the yolk or the albumen of fertilized chicken eggs. Our work demonstrates that these fertilized chicken eggs respond to different chemicals’ toxicity in a potency-responsive manner. We correctly predicted the exact GHS or EPA category for 16 of the 18 (88.9%) chemicals (and all 18 of 18 were predicted correctly within one category). Thus, the ELDAC can easily stratify lethality responses based on the oral toxicity categories as well as the rat LD50 values of these chemicals. Our research establishes the novel utility and the approach necessary for evaluating the LD50 of chemicals specifically, or for acute oral toxicity categories assignment. In summary, we conducted requisite studies for establishing the ELDAC as a feasible non-mammalian test system that is very capable of categorizing oral toxicity hazards of chemicals according to their relative toxicity.