Is "cooling then freezing" a humane way to kill amphibians and reptiles

What is the most humane way to kill amphibians and small reptiles that are used in research? Historically, such animals were often killed by cooling followed by freezing, but this method was outlawed by ethics committees because of concerns that ice-crystals may form in peripheral tissues while the animal is still conscious, putatively causing intense pain. This argument relies on assumptions about the capacity of such animals to feel pain, the thermal thresholds for tissue freezing, the temperature-dependence of nerve-impulse transmission and brain activity, and the magnitude of thermal differentials within the bodies of rapidly-cooling animals. A review of published studies casts doubt on those assumptions, and our laboratory experiments on cane toads (Rhinella marina) show that brain activity declines smoothly during freezing, with no indication of pain perception. Thus, cooling followed by freezing can offer a humane method of killing cane toads, and may be widely applicable to other ectotherms (especially, small species that are rarely active at low body temperatures). More generally, many animal-ethics regulations have little empirical basis, and research on this topic is urgently required in order to reduce animal suffering.