Improving the safety pharmacology standard: CNS behavioral investigations using anticholinergics as a comparator against the standard Irwin test

The ICH S7A Safety Pharmacology guidelines require a novel test article to be assessed for central nervous system (CNS) effects as a part of the Core Battery. Although follow-up behavioral studies are advised, these are rarely seen as part of the GLP safety pharmacology package of work, with an Irwin screen alone conducted to address this requirement. The objective of this investigation was to provide examples using the anticholinergic drugs scopolamine and atropine, where the standard behavioral assessment (Irwin test) is limited in terms of identifying certain undesirable behavioral effects of a novel drug and in some instances, which may mistakenly lead to a conclusion of test subject animals as ‘normal’. A series of behavioral tests were conducted following intraperitoneal administration of 10 mg/kg atropine or 0.25, 1 or 4 mg/kg scopolamine to male C57BL/6J mice or male Han® Wistar rats. Memory assessments were conducted using the Y-Maze and novel object recognition (NOR) tests; the elevated plus maze (EPM), and light/dark box tests for anxiety; and nest building was assessed as an activity of daily living which is influenced by the general well-being of the animals. A modified Irwin was also conducted. Scopolamine and atropine produced statistically significant deficits on memory retrieval within the NOR or Y-Maze; anxiogenic effects within the EPM and light/dark box; and inferior nest building quality, possibly associated with the marked anxiogenic effect of these drugs resulting in lack of desire/motivation to build a nest. None of these effects were quantifiable within the Irwin test. The endpoints investigated are by no mean exhaustive but serve to provide examples of relatively simple tests which lend themselves to be incorporated as additional endpoints within repeat-dose toxicology studies or within stand-alone safety pharmacology studies, and in doing so, add value to the overall characterization of the novel drug. The actual endpoints for consideration should be determined by the on- and/or off target effects of the drug, e.g., for a serotonin activator, the inclusion of a head twitch response test would be appropriate to rule out or support 5-HT_2A activation.