This Guidance Document describes methods to determine the in vitro basal cytotoxicity of test substances using NRU assays and the use of the in vitro data to determine starting doses for in vivo acute oral systemic toxicity tests. The NRU assay is performed in a dose-response format to determine the concentration that reduces NRU by 50% compared to the controls (i.e. the IC50). The IC50 value is used in a linear regression equation to estimate the oral LD50 value (dose that produces lethality in 50% of the animals tested), which is then used to determine a starting dose for acute oral systemic toxicity testing using rats for the UDP, the ATC method, or FDP. The use of the NRU test method in a weight-of-evidence approach to determine starting doses for these acute oral systemic toxicity tests might reduce the number of animals required for the tests, and for relatively toxic substances, might reduce the number of animals that die or require humane euthanasia due to severe toxicity.