induced mutation by the test compound. In the CHO Cell/BrdU-VL assay, each 

 cell screened will either be auxotrophic for one or more of the nine nutrilites 

 omitted for F12D (operational definition of mutant), or it will not be 

 (operational definition of wild-type). The actual criteria employed to classify 

 mammalian cell variants as true mutants are somewhat complex, and have been 

 reviewed recently (16, 40, 46). For the purpose of this paper, the operational 

 definitions given above shall be used. 



In the standard procedure, a sample of 10 cells from each test or control 

 population is distributed among fifty 60 mm dishes, and subjected to the 

 selective process. Surviving clones are then sampled and tested for the presence 

 of auxotrophs. The total number of auxotrophs expected per 10 viable cells is 

 then estimated from the data by equation [2] , where (y) is the estimated 

 number of auxotrophs, (x) is the number of auxotrophs observed, (n) is the 

 number of replica experiments, (A) is the total number of cells surviving 

 selection, (B) is the total number of colonies picked and tested, (C) is the 

 initial number of 60 mm dishes, (D) is the final number of dishes (some may be 

 lost to contamination during the course of the experiment), and (E) is the 

 absolute plating efficiency (defined as the ratio of macroscopic colonies 

 produced to cells inoculated) as measured in low density control dishes. 

 Because mutants are randomly distributed among wild-types in mixed 

 populations, the probability that any given survivor will be a mutant should be 

 constant over all survivors. Moreover, as only a small number of mutants is 

 generally found in any given population, the distribution of mutants in such 

 populations should be Poisson. Accordingly, mutagenesis data from sets of 

 replica experiments were tested for goodness of fit to a Poisson model and 

 found to be consistent with this type of distribution (33). 



For two independent Poisson variables (X, Y), a new statistic (V) has been 

 proposed by Best (9) for testing the difference between two Poisson 

 expectations (e.g., the estimated mean number of mutants in experimental (X) 

 versus control (Y) populations). This statistic, given by equation [3] , is similar 

 in performance to the more familiar square root of the Poisson Index of 

 Dispersion (20), except in the tails of the distribution where (V) is superior. 

 Although (V) is a function of the Poisson variables (X, Y), (V) itself shows an 

 approximately normal distribution. This statistic may be pafticularly applicable 

 to mutagenesis data where the difference in variance observed between 

 experimental and control populations is large. This is the situation at the 

 present time with the CHO Cell/BrdU-VL system where mutants are rarely 

 observed in control populations. All mutagenesis data considered below were 

 scaled via equation [2] and compared to an historical control (Y) in 

 accordance with equation [3] and appropriate confidence limits. The model given by 



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