Passive dosing: An approach to control mutagen exposure in the Ames fluctuation test

One of the major challenges for mutagenicity assessment of environmental samples and individual compounds for example in the Ames fluctuation test (AFT) is the establishment and control of a well defined exposure concentration. Thus, a combination of passive dosing with silicone O-rings (SRs) together with an analytical confirmation of the freely dissolved concentration (FDC) is presented. FDCs are often determined with a combination of solid phase micro-extraction (SPME) with gas chromatography (GC). For compounds with poor performance in GC, a high performance liquid chromatography–mass spectrometry (HPLC–MS) analysis of bi-distilled water dosed with identically loaded SRs is suggested to avoid interference of the bacterial culture. The approach was tested for six amino-, nitro-, and keto-substituted polycyclic aromatic compounds with a log KOW range of 2.5–5.1 without metabolic activation. The method provided reliable concentration-effect relationships and freely dissolved 50% effect concentrations (DEC50) 3–33 times lower than nominal effect concentrations (NEC50) derived in parallel solvent-dosed AFT. Partition coefficients and NEC50/DEC50 ratios were well correlated with lipophilicity.

Research highlights
► Passive dosing for AFT with silicon rings for amino- and nitropyrenes.
► Combination with concentration confirmation of thermolabile compounds using LC–MS.
► Nominal effect concentrations may underestimate hazards by a factor of more than 30.
► Partition coefficients and dissolved effect concentrations estimated using log KOW.