Modified bacterial mutation test procedures for evaluation of peptides and amino acid-containing material

Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.