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 46 Big Blue male rats, with delivery of a target of 44 rats to Charles River Laboratories (CRL) facility, Ashland Ohio, USA. 2. Transfer of HPLC analytical method for dose formulation analysis (DFA) and method transfer summary performed by CRL. 3. Method validation, homogeneity, solubility and stability work to enable GLP dose formulation analysis during Big Blue® TGRA main study, performed by CRL. 4. 7-day dose-range finding study (DRF), wild type Fischer F344 male and female rats, to determine limit dose of 1000 mg / kg body weight /day or MTD, performed by CRL. 5. Big Blue® TGRA rat main study, male only, using vehicle, 3 dose groups and concurrent positive control conducted at CRL. Each group will contain at least 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, liver and bone marrow removed from all animals, flash frozen and stored prior to shipment to Gentronix. 6. Pig-A in vivo mutation analysis will be integrated into the Big Blue® TGRA study, with all study animals screened for elevated Pig-A locus mutation in the pre-study acclimatisation phase, and then a blood sample taken from all animals on study on day 31 as a terminal bleed to minimise animal handling procedures and impact onto the Big Blue® TGRA study. Pig-A analysis will be performed by CRL, unless Sheffield advises of an alternative third party analysis partner, consistent with GLP study principles. 7. Dose formulation analysis during conduct of the main study, performed by CRL. 8. Analysis of liver and duodenum (bone marrow will not be analysed initially) 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 and performed by Gentronix. 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. 9. GLP reporting of results.