How Genotoxicity Testing Guides Safer Drug Development

Given the potential risks associated with genotoxic substances, it is essential to conduct genotoxicity testing. This testing is a standard part of the drug development process. Genotoxicity studies can be done during the early stages of drug development, when the structure of the molecule can be modified and the manufacturing process can be changed if necessary.

Standard Genotoxicity Tests Used in Drug Development

A wide range of tests are used to test the genotoxicity of drugs, including specific assays for different types of genotoxic effects. These tests are often performed in a tiered approach, with initial screens using in vitro assays. The most common in vitro assays for genotoxicity are:

1. In Vitro Genotoxicity Assays

Ames Test (Bacterial Reverse Mutation Assay)

The Ames test is the most widely used initial screen for genotoxicity. This assay uses specific strains of Salmonella typhimurium and Escherichia coli that are unable to grow on selective media due to mutations that inactivate genes necessary for the synthesis of essential amino acids. The assay evaluates the ability of a drug candidate to produce reverse mutations which allow bacteria to grow on specific selective media. The Ames test is very sensitive to point mutations and frameshift mutations, but does not detect chromosomal damage. To simulate mammalian metabolic activation, the assay is performed in the presence and absence of a liver microsomal fraction, also known as an S9 mix, that can convert pro-genotoxins to their active form.

Mammalian Cell Gene Mutation Test

The in vitro mammalian cell gene mutation test detects mutation induction in cultured mammalian cells, such as Chinese hamster ovary (CHO), Chinese hamster lung (CHL), or mouse lymphoma L5178Y cells. Mutation at defined loci, such as hypoxanthine-guanine phosphoribosyltransferase (HPRT) or thymidine kinase (TK), are detected based on resistance to selective agents. This test has greater relevance to human biology and can detect both point and frameshift mutations compared with bacterial assays. Metabolic activation is also assessed using an S9 mix, as with the Ames test.

Chromosomal Aberration Assay

Structural damage to mammalian cell chromosomes including deletions and translocations emerges through the detection capabilities of the in vitro chromosomal aberration assay. After exposure to the test compound, cells are arrested in metaphase and are analyzed microscopically for chromosomal abnormalities. This assay is especially useful for identifying agents that cause DNA double-strand breaks or interfere with DNA repair processes. The assay is performed with and without metabolic activation.

2. In Vivo Genotoxicity Assays

In Vivo Micronucleus Test

The in vivo micronucleus test is a standard technique for assessing chromosomal breakage or loss in rodent bone marrow or peripheral blood cells. The animals receive the test compound and cells are harvested at set intervals for micronuclei detection. Micronuclei are formed from chromosomes that are not properly segregated or are fragmented during cell division. The assay is relatively quick and can detect both structural and numerical chromosome abnormalities. The use of peripheral blood sampling also allows for repeat measurements and long-term monitoring.

Comet Assay (Single-Cell Gel Electrophoresis)

The in vivo comet assay is a sensitive method for detecting DNA strand breaks in individual cells from various tissues, such as liver, kidney, and blood. Cells are lysed and subjected to electrophoresis, causing damaged DNA to migrate and form a distinct comet-like tail. DNA damage levels directly affect the distance DNA migrates. The assay is well-suited for detecting low-level or tissue-specific genotoxic effects.

Transgenic Rodent Mutation Assays

The in vivo mammalian bone marrow chromosomal aberration test is similar to the in vitro chromosomal aberration test, but it is performed in rodents (usually mice or rats). After administering the drug candidate to test animals researchers collect bone marrow cells at designated times. Researchers process the cells to enable chromosome visualization and then count the chromosomal aberrations. The test evaluates whether a drug causes structural chromosomal damage in living organisms by considering absorption, distribution, metabolism and excretion.

Key Considerations in the Interpretation of Genotoxicity Results

Weight-of-Evidence Interpretation

The interpretation of genotoxicity data is based on a weight-of-evidence approach that considers multiple assays, biological relevance, and clinical context of the drug. Regulatory guidance documents like ICH S2(R1) provide criteria for a systematic evaluation of qualitative and quantitative findings, rather than relying on the outcome of a single test.

Evaluation of Individual Test Results

The results of individual genotoxicity assays are first considered for an indication of a positive response, usually a statistically significant and dose-related increase in genetic damage compared to controls. Positive findings are further evaluated for biological significance against historical control ranges and in the context of assay-specific considerations.

Consistency Across Multiple Assays

Consistency of findings across multiple genotoxicity assays, both in vitro and in vivo, is a key consideration in the overall assessment of genotoxic potential. Concordant positive results across tests strengthen the likelihood of genotoxic risk, while consistent negative results support a low-risk assessment. Discordant results between in vitro and in vivo assays, such as in vitro positives with in vivo negatives, may warrant further investigation and additional targeted studies.

Biological Relevance and Exposure Considerations

Interpretation of genotoxicity findings also considers species differences, metabolic competence, DNA repair capacity, and achieved exposure levels. Genotoxic effects observed only under non-physiological conditions or at doses far exceeding anticipated human exposure may have limited clinical relevance, particularly when appropriate safety margins are established.

Impact of Intended Clinical Use and Risk-Benefit Balance

The acceptable threshold for genotoxic risk is influenced by the intended clinical use of the drug and the therapeutic context. Drugs developed for serious or life-threatening ailments may tolerate higher levels of genotoxic risk than medications intended for self-limiting conditions or those with alternative treatments available. The acceptable risk level depends on both the targeted patient population and treatment duration.

Contribution of Genotoxic Impurities

When genotoxic effects are identified, it is important to determine whether they are related to the active pharmaceutical ingredient or to the presence of trace genotoxic impurities. The identification and control of such impurities may mitigate genotoxic risk, while genotoxicity that is an inherent property of the drug substance may lead to considerations for structural modifications or halting of development.

Limitations of Genotoxicity Test Systems

Because each genotoxicity assay exhibits unique advantages and limitations scientists cannot expect a single test to discover all types of genetic damage. A combination of assays, along with other related toxicological data such as carcinogenicity and reproductive toxicity findings, is necessary to form a complete and scientifically defensible safety profile.

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Reference

1. Mišík M, Nersesyan A, et al. Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems. Mutat Res Genet Toxicol Environ Mutagen. 2022. 881:503524.