Genotoxicity testing is an essential tool for drug developers, as it can ensure patient safety in new and established drugs. Genotoxic events can cause irreversible damage and particularly severe health events, which makes it imperative these tests are conducted accurately and efficiently.
The mammalian erythrocyte micronucleus assay is a key type of genotoxicity testing but traditionally relies on labor and time-intensive data collection methods. The flow cytometry method offers significant advantages, but while it’s been performed extensively in rats in preclinical studies, only a handful of tests have been carried out with mice.
WuXi AppTec researchers conducted extensive research to validate the use of flow cytometry as an analysis method for mouse peripheral blood micronucleus assay.
The importance of micronucleus assays
A micronucleus is a small extra nucleus that forms in a cell when a chromosome or fragment of a chromosome isn’t incorporated into a daughter nuclei during division. For drug developers, micronuclei act as warning flags, as they usually indicate genomic damage has occurred. A micronucleus assay detects micronuclei, assessing the potential genotoxicity of a drug.
The mammalian erythrocyte micronucleus assay involves collecting bone marrow and manually counting up micronucleus frequency to assess the potential for genotoxic damage. However, this can take a lot of time, slowing the testing process and making it more expensive. Fortunately, flow cytometry offers a more efficient method.
How flow cytometry works
Flow cytometry is an automated technique that analyzes the physical and chemical properties of cells and particles as they pass through a laser. This technique offers three major advantages over manual scoring:
Speed: Because flow cytometry is automated, it can analyze thousands of cells in a second and produce results much faster than manual methods. This also reduces the labor required to carry out testing such as the mammalian erythrocyte micronucleus assay.
Accuracy: Flow cytometry can produce more accurate and consistent results by reducing human error. It uses laser technology to detect cell components and produces standardized results.
Robustness: The data produced from flow cytometry is more robust and detailed than standard testing methods. It provides more information and allows for more exhaustive analysis. Testing using this method is also extremely sensitive and can detect very rare events.
The flow cytometry method was adopted and recommended by the ICH-S2(R1) guideline and OECD Testing Guideline 474.
Objectives of the study
WuXi AppTec’s research aimed to validate the mouse peripheral blood micronucleus assay using flow cytometry as the analysis method. To do this, three daily doses were given, including a negative control (0 mg/kg/day), hydroquinone (20, 40 and 80 mg/kg/day), and a single dose of the positive control, cyclophosphamide monohydrate, at 75 mg/kg.
Samples were collected 20-24 hours after the last dose and were processed using a Litron commercial analysis kit manual. Our researchers measured the percentage of reticulocytes (%RET) and micronucleus frequency (%MN-RET) and assessed the results for precision, carryover effect, and stability across different days.
What the data tells us
The flow-cytometric-based peripheral blood micronucleus assay in mice was found to be valid. The micronucleus frequency in the negative control group showed micronucleus levels that were comparable with literature and expected.
The research found that hydroquinone, a known genotoxic compound, caused a significant dose-related increase in micronucleus frequency for both male and female mice. The positive control also produced statistically significant increases in micronucleus formation.
Blood samples stored in methanol or a long-term storage solution remained stable for at least 30 days. Researchers noted no obvious carryover between samples, and both the intra-day and inter-day assay variation were within acceptable limits.
The results show that all validation requirements were met. As a result, WuXi AppTec has successfully validated and is able to perform flow cytometry-based peripheral blood micronucleus assay in mice under Good Laboratory Practice (GLP) conditions.
A final word
Advanced, automated testing tools like flow cytometry play a crucial role in ensuring patient safety from genotoxic risks. With accurate, robust, and efficiently gathered data, drug developers can identify potential issues early in their process, ensuring safer compounds move on to clinical trials. These results enhance the role of flow cytometry in micronucleus assays further, validating their use in mice. With this testing, WuXi AppTec has again proven its ability to provide the latest testing techniques while adhering to the highest GLP standards. By collaborating with lab partners that use the most cutting-edge technology and testing methods, developers can save time and money and minimize the potential for risks. Ultimately, this benefits the patient as treatments are brought to market quicker and safer.
