RECOMMENDATIONS FOR STATISTICAL DESIGNS OF IN-VIVO MUTAGENICITY TESTSWITH REGARD TO SUBSEQUENT STATISTICAL-ANALYSIS

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherbe rg, Germany, to start a discussion of experimental design and statisti cal analysis issues for three in vivo mutagenicity test systems, the m icronucleus test in mouse bone marrow/peripheral blood, the chromosoma l aberration tests in mouse bone marrow/differentiating spermatogonia, and the mouse dominant lethal test. The discussion has now come to co nclusions which we would like to make generally known. Rather than dwe ll upon specific statistical tests which could be used for data analys is, serious consideration was given to test design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system. Concerning the statistical sensitivity parameters, a type I er ror of 0.05 tone tailed), a type II error of 0.20 and a dose related i ncrease of twice the background (negative control) frequencies were ge nerally adopted. It was recommended that sufficient observations (cell s, implants) be planned for each analysis unit (animal) so that at lea st one adverse outcome (micronucleus, aberrant cell, dead implant) wou ld likely be observed. The treated animal was the smallest unit of ana lysis allowed. On the basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000 im mature erythrocytes/animal should be scored for micronuclei from each of at least 4 animals in each comparison group in the micronucleus ass ays. A minimum of 200 cells should be scored for chromosomal aberratio ns from each of at least 5 animals in each comparison group in the abe rration assays. In the dominant lethal test, a minimum of 400 implants (40-50 pregnant females) are required per dose group for each mating period. The analysis unit for the dominant lethal test would be the tr eated male unless the background frequency of dead implants (DI) is so low that multiple males would need to be integrated to meet the minim um observation of one adverse outcome (DI) per analysis unit. A three- step strategy of data analysis was proposed for the cytogenetic assays . Use of negative historical controls was allowed in certain circumsta nces for interpretation of results from micronucleus tests and chromos omal aberration tests. (C) 1998 Elsevier Science B.V. All rights reser ved.