The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals
- DOI
- Language of the publication
- English
- Date
- 2021-01-28
- Type
- Article
- Author(s)
- Marchetti, Francesco
- Zhou, Gu
- LeBlanc, Danielle
- White, Paul A.
- Williams, Andrew
- Yauk, Carole L.
- Douglas, George R.
- Publisher
- Springer
Abstract
The Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.
Plain language summary
Health Canada supports the development of test guidelines for the Organisation for the Economic Co-operation and Development (OECD). These guidelines are routinely used for assessing the safety of chemicals before they come on the market. One of these guidelines (TG 488) uses genetically-modified mouse models, called transgenic rodents (TGR), to detect mutations (changes in the sequence of the DNA) in any tissue in the body. TG 488 was recently updated to improve the analysis of mutations in germ cells (ie, sperm) by recommending an experimental design of 28 days of exposure followed by tissue sampling 28 days later (ie, 28+28d). The same design is recommended for somatic tissues, such as the liver, that have low rates of cell division. However, it is unclear what is the impact of this design for somatic tissues with active cell divisions, such as the bone marrow. In this study, Health Canada scientists evaluated the persistence of mutations in the bone marrow of TGR mice after exposure to four chemicals known to induce mutations. The analysis of bone marrow of TGR mice up to 70 days after the end of the exposure showed that the number of mutations is relatively stable over the time period investigated. These results strongly suggest that the 28+28d design is an appropriate design for the comprehensive evaluation of the mutagenicity of environmental chemicals in somatic cells and germ cells to aid in their risk assessment and guide subsequent regulatory decisions. These results provide critical information for optimizing OECD TG 488 to greatly reduce the number of animals required for regulatory testing.
Subject
- Health,
- Health and safety