LD50 testing involves two species and two routes of administration. How many species and routes are specified?

• A. Two species and two routes
• B. One species and one route
• C. Two species and one route
• D. Three species and two routes

Answer: (A)

In LD50 testing, the goal is to estimate the dose that would kill 50% of exposed animals, and historically this was done across more than one species and more than one route of administration to account for differences in physiology and how a substance is absorbed. The standard design uses two species—typically a rodent and a non-rodent—and two routes of administration, such as oral and dermal. This combination provides a more robust hazard assessment than relying on a single species or a single exposure route. Therefore, the specified configuration is two species and two routes. Modern toxicology has moved toward alternatives to LD50, but understanding this setup helps explain why the numbers add up that way.