I would like to know if it's 100% safe to remove a drive letter from secondary, bootable disk. Does it have any impact on the drive, or is it just a configuration setting used by the running Windows?
It is used only by Windows. The effect of removing the drive letter is noticed only in the Windows installation where you made this setting. The partition for which the drive letter was removed is just fine. I do this all the time and I have no issues.
Your operating system's boot entries are not dependent on the drive letters assigned in an operating system or another. It uses the volume names of each partition, information which is available at boot, independent of the configuration you make in each operating system.