1. Isn't that exposure too long?
In a non synchronized cell culture some cells are present in each of the phases of the cell cycle.
Let us assume that substance X arrests cells in G₂. Cells that are in S-phase at the beginning of the exposure to substance X will arrest fast  because they are close to the point of arrest, whereas cells that are in M-phase at the beginning of the exposure will require an exposure period that equals the full duration of the cell cycle to reach the point of arrest. (A cell cycle of mammalian fibroblasts lasts about 24 hours.)
If the exposure of the culture to substance X is not  long enough, only part of the cells will be arrested, and this will obscure the effect of substance X on cell cycle progression.