Germ cell/heritable mutations are important regulatory endpoints for international agencies interested in protecting the health of future generations. However, germ cell mutation analysis has been hampered by a lack of efficient tools. The motivation for developing this AOP was to provide context for new assays in this field, identify research gaps and facilitate the development of new methods. In this AOP, a compound capable of alkylating DNA is delivered to the testes causing germ cell mutations and subsequent mutations in the offspring of the exposed parents. Although there are some gaps surrounding some mechanistic aspects of this AOP, the overarching AOP is widely accepted and applies broadly to any species that produces sperm.