In vivo transgenic mutation assays

Transgenic rodent gene mutation models provide quick and statistically reli able assays for mutations in the DNA From any tissue. For regulatory applic ations, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or Fewer repeated t reatments are inadequate to conclude that a compound is negative but more t han 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times mi ght be appropriate for highly proliferative tissues. For phage-based assays , 5 to 10 animals per group should be analyzed, assuming a spontaneous muta nt frequency (MF) of similar to 3 x 10(-5) mutants/locus and 125,000-300,00 0 plaque or colony forming units (PFU or CFU) per tissue. Data should be ge nerated For two dose groups but three should be treated, at the maximum tol erated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent po sitive control animals are only necessary during validation, but positive c ontrol DNA must be included in each plating. Tissues should be processed an d analyzed in a block design and the total number of PFUs or CFUs and the M F for each tissue and animal reported. Sequencing data would not normally b e required but might provide useful additional information in specific circ umstances. Statistical tests used should consider the animal as the experim ental unit. Nonparametric statistical tests are recommended. A positive res ult is a statistically significant dose-response and/or statistically signi ficant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the contr ol. (C) 2000 Wiley-Liss, Inc.