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

In August 2021 the University of Sheffield entered into a contract with WuXi AppTec (Hong Kong) Ltd (Find A Tender Service Contract Award Notice Publication Reference: 2021/S 000-021618) for the provision of IND enabling toxicology study services for M102 (small-molecule drug candidate; oral route, once daily), including 2 animal studies.

It is now the intention of the University of Sheffield to contract the third animal study (Big Blue) to Gentronix Limited. Studies will be conducted to GLP, and are based on the following preliminary study design:

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.

Schedule - subject to CRL timelines and Study Plan

• In-life study phases commence at CRL in either December 2022 (if dose range finding study required) or January 2023 (if main phase OECD 488 study only)
• In-life study phases complete at CRL in February 2023
• Post-life in vitro phases commence at Gentronix Limited in February 2023
• Post-life in vitro phases complete at Gentronix Limited in April 2023
• Draft report - by end May 2023

Gentronix's Limited's choice of partnership with CRL was based on 4 main factors:

1. They are a significant contract research organisation, high animal welfare standards and with capabilities at their Ashland facilities to conduct all necessary aspects of the in-life Big Blue studies. These include supporting toxicological and analytical endpoints and in particular they have coverage for all expected routes of exposure/administration including via inhalation;
2. The laboratory team at Ashland has previous experience of acting as GLP main test site and Study Director for Big Blue® studies, commissioned by the previous asset owner being BioReliance Corporation. This substantially de-risks the GLP compliance elements of these studies, and was viewed favourably in our discussions with the UK MHRA prior to commencing work on this project;
3. The USA location and relative close proximity to our animal facility breeding location in NY. This enables ground transportation of animals within a timeframe that provides no compromise on animal welfare and shipments are routine from our contract breeder to these facilities;
4. In many circumstances they were and are an established service provider to the same clients Gentronix Limited receives enquiries from for Big Blue® studies. This de-risks commercial, contractual and legal discussions.

At present the CRL Ashland facility is the only site capable of conducting GLP compliant in-life subcontract work for Big Blue studies on behalf of Gentronix.