Benzo[a]pyrene deposited on a glass fiber filter reacts rapidly in the dark or light with ambient levels of ozone to yield a mixture of products that display strong direct mutagenicity in the Ames assay. The major stable contributor to this activity has been identified as benzo[a]pyrene-4,5-oxide, a DNA-binding metabolite in biological systems, known to be a strong direct mutagen with Salmonella typhimurium strain TA98.

Factors determining the precision and variability of the Ames Salmonella test for mutagenicity were investigated. The most important source of variability in the agar-overlay method was nonuniformity in the soft-agar layer thickness. Solution of this problem resulted from application of an agar-leveling table described in this paper. Several other procedural elements also contribute to improved precision, including temperature uniformity during incubation, incubation interval, consistency of plate agar volume, completeness of mixing the soft-agar overlay, peculiarities in the interaction of mutagens and mammalian liver microsomal extract (S9), and methods of storage and controls for tester strains. When these and other effects were well-controlled, variability of the test results was reduced from 200 or 300% to only +/- 10% or less. The significance of the factors affecting precision are discussed and an improved experimental protocol is presented.

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