Temporal analyses of germ cell mutations using the MutaMouse model support the recommended design in OECD test guideline 488

Abstract

The Organisation for Economic Co-operation and Development test guideline (TG) 488 uses transgenic rodent models to assess in vivo mutagenesis. TG 488 recommends 28 consecutive days of exposure with sampling of germ cells from seminiferous tubules 28 days post-exposure (i.e., 28 + 28d). We analyzed mutant frequencies (MF) in male germ cells up to 70 days post-exposure to determine whether designs other than 28 + 28d are necessary for assessing germ cell mutagenicity. Adult MutaMouse males received various doses of benzo(a)pyrene (BaP), N -ethyl- N -nitrosourea (ENU), isopropyl methanesulfonate (iPMS), or procarbazine (PRC) alongside vehicle controls for 28 days orally. Germ cells were collected from seminiferous tubules at + 3d, + 28d, + 42d, or + 70d post-exposure and MF quantified using the lacZ assay. Significant increases in lacZ MF were observed for all four chemicals at 28 + 28d. No further increases occurred at later sampling times. There was no significant effect with BaP at 28 + 3d, and a significantly stronger response with ENU and BaP at 28 + 28d compared to 28 + 3d. PRC produced the strongest response at 28 + 3d, while there was no impact of different sampling times for iPMS. Both these chemicals significantly reduced testis weight at 28 + 3d and 28 + 28d. Finally, benchmark dose modeling generated overlapping confidence intervals among the four sampling times for ENU, iPMS, and PRC. However, for BaP, the confidence interval was significantly greater at 28 + 3d than at the other sampling times. These results support the use of the 28 + 28d design as the recommended experimental design for germ cells in TG 488 and that later sampling times are not necessary.

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