The demethylase mutants contain knockout versions of three genes controlling nicotine to nornicotine conversion (CYP82E4, CYP82E5v2 and CYP82E10). These triple mutants have many advantages; lower conversion, lower NNN, cheaper and easier identification, elimination of expensive seed screening. However, there are several potential disadvantages. As a result of the lower conversion, nicotine levels in the mutants can be too high; three triple mutant lines failed the RQT (Regional Quality Test) on nicotine in 2013. The enantiomeric ratio is different in ultra-low converter lines, with a higher ratio of the more toxic S isomer of both nornicotine and NNN; this could result in higher absolute levels of S-NNN. We have developed a method to measure absolute amounts of R and S-NNN; previously, it was only possible to measure the ratio. It is also possible that the mutations could have an adverse effect on plant growth. In this study, we compared single (e4), double (e4e5) and triple (e4e5e10) mutants with the RNAi transgenic and equivalent wild types. Nicotine was not significantly higher in the triple mutants and transgenic than in the LC equivalent, nor was the absolute amount of S-NNN, despite the higher ratio of S-NNN. There were some consistent differences in growth parameters. The triple mutants generally had longer, narrower leaves and were taller with fewer leaves. The triple mutant and transgenic were later flowering with longer internodes in one variety but not in the other. In general, we conclude that the advantages of these triple mutants outweigh the potential disadvantages; they have a marked potential for harm reduction because the S-NNN, as well as the total amount of NNN, is greatly reduced in the triple mutants.