Inclusion of a recovery period after pulse treatment in the primary human lymphocyte micronucleus assay covers potential cell cycle delays without loss of sensitivity

According to Organisation for Economic Co-operation and Development (OECD) test guideline no. (TG) 487 for the in vitro mammalian cell micronucleus test (MNT), short (pulse) treatment of primary human lymphocytes should be followed immediately by a sampling period of 1.5 - 2.0 cell cycle lengths in the presence of cytochalasin B. However, TG 487 allows extending the sampling period "if it is known or suspected that the test chemical affects the cell cycling time". It is often unknown in advance whether a test substance induces cell cycle delays, and therefore whether an extension of the sampling period is warranted. Here, we investigate whether extending the sampling time after pulse treatment - by including a recovery period prior to cytochalasin B treatment - affects the sensitivity of the in vitro MNT. Primary human lymphocytes were exposed to eleven mutagens and assessed using two protocols ("recovery" and "no recovery") in parallel from the same treatment culture. Including a recovery period after pulse treatment generally allowed testing of higher concentrations without excessive cytotoxicity. In addition, cultures subjected to a recovery period showed generally higher micronucleus rates at concentrations with acceptable levels of cytotoxicity, compared to cultures not having undergone a recovery period. To compare the sensitivity of both protocols at non-cytotoxic concentrations, benchmark concentration analysis was performed. Here, the "recovery method" proved to be at least as sensitive as the "no recovery method". The reproducibility of these findings was demonstrated in an interlaboratory ring trial using a subset of up to five mutagens. In conclusion, inclusion of a recovery period after pulse treatment per se covers potential substance-induced cell cycle delays without any loss in sensitivity towards the induced mutagenic effect. The "recovery" protocol can be used for assessing the mutagenic potential of test substances affecting and not affecting the cell cycle.