These treatments were selected

These treatments were selected MK-2206 datasheet for power calculations. The genotoxicity of two different 3R4F PMs were measured in each assay. Power calculations were performed on the slopes of the dose responses, pooled data and each concentration separately, to estimate the number of replicates per concentration that would detect a 30% increase or decrease in the response, with 80% power, at p < 0.05. The results are summarised in Table

1. The levels of replication typically used in these assays (e.g. 3 in the Ames test, 4 in MLA and 2 in IVMNT), could resolve a 30% difference in PM genotoxicity, in terms of slope. Replication levels of 5 (Ames test TA98), 4 (Ames test TA 100), 10 (Ames test TA1537), 6 (MLA) and 3 (IVMNT) would be required for similar resolution, in terms of pooled data or individual doses. Two 3R4F PMs were tested, to confirm the resolving power of these replication levels. These were from the same PM stock solution, but one sample was diluted to 70% (v/v), to simulate a 30% difference between PMs. The two PM samples were compared in each assay. Replication levels were as described in Table 1 for comparisons at common doses, except for IVMNT where 4 replicate cultures per dose were used, because 3 replicates might not have been powerful enough to detect differences if we had to revert to t-tests at each common

dose level. The results are shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Fig. 6. Linearity was identified in all dose responses ( Table 2a and Table 2b). Differences between the PM samples were learn more statistically significant in all three assays. This confirmed that replication levels of

5 (Ames test TA98), 4 (Ames test TA 100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) can resolve 30% differences in PM genotoxicity. The resolving power was based on estimates of intra-experiment variability. It is consistent with the differences in PM genotoxicity observed by others (Combes et al., 2012, McAdam et al., 2011, Oldham et al., 2012 and Roemer et al., 1998). 3R4F was genotoxic in the Ames test, MLA and IVMNT. This is consistent with published observations (Baker et al., 2004, Clive et al., 1997, Cobb et al., 1989, DeMarini, 2004, DeMarini et al., 2008, Guo et al., 2011, Kier et al., PRKACG 1974, McAdam et al., 2011, Mitchell et al., 1981, Richter et al., 2010, Rickert et al., 2007, Rickert et al., 2011, Roemer et al., 2002, Roemer et al., 2004 and Sato et al., 1977). Guidelines for testing genotoxicity with the Ames test, MLA and IVMNT (ICH, 1995, OECD, 1997a, OECD, 1997b and OECD, 2010) emphasize the assays’ biological responses rather than giving advice on appropriate statistical techniques. The OECD states that “biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating test results. Statistical significance should not be the only determining factor for a positive response” (OECD, 1997a).

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