Genetic Toxicology

When DNA is exposed to particular test articles, mutations and other damage can occur, leading to cancer and/or teratogenic effects. The severity of these effects necessitates examining whether new or existing test articles intended for human use have any effect on DNA. No single test can detect all relevant genotoxic endpoints, so regulatory agencies recommend a battery of in vitro and in vivo tests for genotoxicity.

Founded in 2009, WuXi AppTec’s state-of-the-art laboratory serves worldwide customers with genotoxicity. The laboratory passed the GLP inspections by FDA, OECD and NMPA. GLP and non-GLP studies are conducted in compliance with ICH, OECD and NMPA guidelines.

A standard genetic toxicology screening battery includes screening bacterial reverse mutation assays (Ames), and screening in vitro micronucleus assays. WuXi AppTec offers (Q)SAR mutagenicity prediction for drug discovery and impurities as well.

Bacterial reverse mutation assays (commonly known as Ames) are genetic toxicology assays that identify substances that induce gene mutations. WuXi AppTec offers two formats for Ames assays:

Agar format

• Micro-Ames assay, 24-well plate
• Mini-Ames assay, 6-well plate
• Ames assay, petri dish

Liquid format

• Ames II
• Ames microplate format (MPF)

Liquid format Ames II (A); Micro-Ames in 24-well plate (B); Mini-Ames in 6-well plate (C); Standard Ames in Petri Dish (D).

The in vitro micronucleus assay identifies substances that induce clastogenicity or aneugenicity. Clastogenicty is a structural chromosomal aberration through breaks in DNA. Aneugenicity is a numerical chromosomal aberration through interactions with non-DNA cellular targets. WuXi AppTec deploys the following methods:

• CHO Microwell MNT, manual method
• MicroFlow, flow cytometer method

The International Conference on Harmonization (ICH) M7 guidelines for assessing and controlling DNA-reactive (mutagenic) impurities in pharmaceuticals allow for the consideration of in silico predictions in place of in vitro studies. This represents a significant advance in the acceptance of Quantitative Structure-active Relationship (Q)SAR models.

The QSAR mutagenicity prediction is an in silico tool for predicting the mutagenicity of compounds, impurities, and metabolites. Mutagenicity is the potential for a test article to cause genetic mutations. For impurity evaluation and drug discovery, WuXi AppTec uses:

• An expert rule-based model, Leadscope Model Applier
• A statistical model, Leadscope Model Applier

IND-enabling testing routinely includes a series of toxicology studies, including genetic toxicology. A standard GLP battery for genetic toxicology includes an Ames assay, in vitro mammalian cell assay, in vivo genotox assay, and mechanism exploration and follow-up assays.

Ames (bacterial reverse mutation assays) identifies substances that induce gene mutations. WuXi AppTec’s GLP Ames methods include:

• Plate incorporation
• Pre-incubation
• Treat and wash to avoid feeding effect
• Enhanced Ames for N-nitrosamines

Mammalian cell assays evaluate whether a chemical substance has the potential to cause genetic damage to mammalian cells. These assays include:

In vitro chromosomal aberration
In vitro micronucleus (TK6 or CHO)
Mouse lymphoma assay (MLA)

Images from in vitro cytogenetic assays. (A) Chromosomal damage observed in metaphase spreads using CHO-WBL cells; (B) Examples of binucleated cells with micronuclei (MN-BN) in the microwell micronucleus assay with CYB; (C) Fluorescent microscope images of micronucleated cells in the micronucleus assay without cytochrome B (CYB); (D) Micrograph of FISH analysis with pancentromeric probes applied to the micronucleus assay.

In vivo genotoxicity assays evaluate the potential of substances to cause genetic damage.

• In vivo micronucleus (manually or FCM based method, standalone or integrated)
• In vivo Comet, option 2

WuXi AppTec offers a series of additional genetic toxicology assays that complement the standard battery of GLP in vitro and in vivo assays, providing additional insights into how test articles might impact human DNA.

• In vivo chromosomal aberration
• In vivo Comet (standalone or integrated)
• In vivo Pig-a
• Fluorescence in situ hybridization (FISH)

Images from in vivo genetic toxicology assays. (A) Automated analysis of the formation of micronuclei in reticulocytes analyzed by flow cytometry; (B) Fluorescent microscope images of micronucleated PCEs; (C) Images of Comet analyzed by Cyto Study Manager System; (D) Chromosomal damage observed in metaphase spreads

WuXi AppTec leverages an automated reporting system to draft reports and has a dedicated study director group for report checking and peer-reviewing. Our team is committed to provide high quality study data and deliver the reports timely in order to meet your timeline.