Alfred Grafe

A European collaborative study of the Ames assay: I. Results and general interpretation

Results are presented of a collaborative study between 19 European laboratories on the variability of the Ames test. Examples are shown of various methods that are generally used to evaluate an Ames test without reference to a specific statistical model: the number of revertants per plate, mutation factors (increase over the spontaneous value) and determination of the doubling concentration. Considerable variations between test results occurred, between laboratories as well as within laboratories. Partly this was due to different interpretations of the guidelines given, as these allowed some flexibility. The results were also influenced by other factors, some of which are perhaps not yet generally recognized. Apart from the level and quality of the S9 preparations, the most important factor might be the number and physiological condition of the cells plated. When the results from all experiments were considered together, 60-80% of the test results were found within the half- to two-fold range of the overall median. This might be considered satisfactory for a study not using rigidly standardized test conditions. From the experience with the present study, several recommendations are given for the design and performance of future collaborative studies.

Small numbers in mutagenicity tests.

Experimental control material for statistical analysis of the results of the micronuclei test in the mouse (NMRI strain) and the Chinese hamster and for the host-mediated assay in the mouse (NMRI strain) using auxotrophic bacterial strains are presented. The binomial distribution of the micronuclei makes it possible to analyse the sample size according to the formula of Cochran and Cox (1957).For the host-mediated assay, the experimental principles are given which make it possible to evaluate the results obtained even with a weakly mutagenic, unknown substance.Critical points in comparative tests are not only the methodological questions, but also pharmacokinetic problems of the substance being tested which can only be clarified in the species used for the mutagenicity test. If this is ignored then even experimentally based findings can only be recorded as speculations.ZusammenfassungDas experimentelle Kontrollmaterial zur statistischen Auswertung der Ergebnisse des Mikrokerntests an der Maus (Stamm NMRI) und dem Chinesischen Hamster sowie des Host-mediated assay an der Maus (Stamm NMRI) bei Verwendung auxotropher Bakterienstämme werden vorgelegt. Die binomiale Verteilung der Mikrokerne ermöglicht ihre Auswertung nach der Formel von Cochran und Cox (1957).Für den Host-mediated assay werden die experimentellen Grundlagen genannt, die eine Auswertbarkeit erzielter Ergebnisse auch mit schwach mutagenen unbekannten Substanzen ermöglichen.Kritische Punkte in Vergleichstesten sind nicht nur methodische Fragen sondern pharmakokinetische Probleme der Prüfsubstanz, die nur in der im Mutagenitätstest verwendeten Spezies aufgeklärt werden können. Werden diese Probleme ignoriert, dann sind auch experimentell begründete Feststellungen nur als Vermutungen zu bewerten.

Testing the mutagenic potency of chemical substances in a linear host-mediated assay I. Experimental microbiological basis

By the use of the mutagenic substance hydrazine sulphate it is shown that the currently used single determination method for determining point mutations in host-mediated assays with calculation of mutation frequencies can lead to erroneous results. The microbiological basis for a linear-method is presented in which the population growth of the auxotrophic and substance-induced mutants used int he test can be described mathematically during their logarithmic growth phase with regression lines.