OECD 488 transgenic rodent mutation assay (TGRA) using the Big Blue® rat

The conduct of an OECD 488 transgenic rodent mutation assay (TGRA) using the Big Blue® rat using cII mutant selection for the University of Sheffield. Studies will be conducted to GLP, and are based on the following preliminary study design: 1. Breeding of target cohort of 40 Big Blue male rats, with delivery to Charles River Laboratories (CRL) facility, Ashland Ohio, USA. 2. Transfer of HPLC analytical method for dose formulation analysis and method transfer summary. 3. Method transfer, validation, homogeneity, solubility and stability work to enable GLP dose formulation analysis during Big Blue® TGRA main study. 4. 7-day dose-range finding study, wild type Fischer F344 male and female rats, to determine limit dose of 1000 mg / kg body weight /day or MTD. 5. Big Blue® TGRA rat main study, male only, using vehicle, 3 dose groups and concurrent positive control. Each group will contain 6 male rats, conducted in accordance with OECD 488; daily test item administration by oral gavage for 28 days (positive control will be dosed days 1, 3, 10, 17 & 24 only); termination and schedule necropsy on day 31 with duodenum and liver removed from all animals, flash frozen and stored prior to shipment to Gentronix Limited. 6. Dose formulation analysis during conduct of the main study. 7. Analysis of liver and duodenum from 5 animals per dose group in the in vitro phase of the Big Blue® TGRA study as per OECD 488 will be conducted at Gentronix Limited. DNA will be extracted, packaged into lambda bacteriophage used to infect E. coli strain G1250 to determine phage packaging efficiency and cII mutation detection after plating on agar. Phage packaging titres of at least 125,000 per animal will be determined, to facilitate estimation of mutation frequency. 8. GLP reporting of results.