Rodent estrous cycle pattern: Harmonizing the cycle evaluation and interpretation

The estrous cycle is a sensitive endpoint from an endocrine disruptor perspective and is included in rodent reproductive toxicity studies. In this paper, the methods for estrous cycle timing and different approaches followed by testing laboratories for evaluating the days of the estrous cycle were reviewed. No major differences are identified for counting 4-day estrous cycle. However, when extended episodes of estrus (E) stages occur, the cycle counting differs between testing laboratories potentially resulting in misinterpretation and inaccurate outcome. Appearance of extended episodes of two to three E in an estrous cycle need explanation. Therefore, the following are proposed, 1) follow OECD guidance document for normal 4/5 day cycles, 2) recommends an update of the OECD document as in the current one it is unclear for a 5-day cycle ending with Proestrus(P) followed by episode(s) of estrus(E), 3) two episodes of E after P in a 5-day cycle and three consecutive episodes of E should be considered abnormal, and 4) stages prior to first E of the first cycle or after last E (last P of a 5-day) of last cycle should not be assumed a complete cycle. Additionally, call for collaboration is made to harmonize the cycle counting approach.