Follow-Up Genetic
Toxicology Assays

Uncertain Genotoxicity Results? Learn How to Assess Their Significance.

Studies have shown that 29% of marketed drugs contained positive findings in one of the four categories of standard battery assays (bacterial mutation, in vitro mammalian cell gene mutation, in vitro and in vivo cytogenetics). For these reasons, a range of approaches are available to assess the relevance of a positive genotoxicity result. While there are techniques such as QSAR and FISH/CREST to provide additional support for a positive result, follow-up assays can be conducted to further define the biological relevance of the result.

In Vivo Single Cell Gel Electrophoresis Comet Assay

commet-assay The comet assay, also called single cell gel electrophoresis, is a rapid and sensitive technique for accurately analyzing DNA damage (genotoxicity) in individual cells resulting from exposure to environmental and industrial chemicals, consumer products, agrochemicals, and pharmaceuticals. The comet assay is a common follow up test to a positive in vitro assay (such as the chromosome aberration or in vitro micronucleus assay). The technique is applicable to any tissue from which a single cell suspension can be prepared, but it is most commonly used to examine organs involved in metabolism of substances or that are the first site of contact during chemical exposure.

The comet assay can be used to detect multiple types of DNA damage, including single and double stranded breaks, alkali labile sites, oxidative base damage, and DNA cross-linking. It can also be used to monitor effects on DNA repair. Thus, it can be useful for exploring a chemical’s mechanism of action.

Comet Assay Options:

Species: Mouse and Rat
Qualified tissues (species and method of collection dependent) include: peripheral blood, bone marrow, brain, colon, duodenum, jejunum, kidney, liver, lung, nasal cavity, skin, spleen, stomach

At Inotiv, we offer Comet assays in multiple formats including:

In vivo rodent Comet assays (different dosing regimens are available) in fresh or frozen tissues
In vivo Comet assay in combination with in vivo Micronucleus and/or Pig-a assays
Comet assay slide evaluation (slides prepared at another lab and shipped to Inotiv for scoring)
Shipped sample evaluation (tissues flash frozen at another lab and shipped to Inotiv for processing and scoring)
Mobile Comet — we bring the assay to your lab to include as part of an ongoing toxicology study!

Quality of Storing Tissue and Slide Samples for a Comet Assay

OECD 489

Comet Assay FAQs:

Which species is preferred for the Comet assay?

The most commonly used species are rats and mice. Species selection is often based on test material availability, existing exposure information, and previous experience with the test substance in repeat dose toxicity studies.

What is the difference between fresh and frozen tissues?

Fresh tissue methodology involves processing tissue(s) to cell suspensions and preparation of agarose slides on the day of collection. The advantage of using fresh tissues is that they are captured in their most pristine state.

Frozen tissue methodology involves processing tissues(s) to cell suspensions or small cubes on the day of harvest and flash freezing to store for later use. The disadvantage is that frozen cell suspensions can have higher baseline levels of damage as compared to fresh tissue. Properly prepared flash frozen cubes of tissue tend to have baseline levels of damage comparable to fresh tissue but require more backend effort to process samples to slides.

What are the most common tissues collected for Comet?

The most common tissue collected is liver because it is the primary site of xenobiotic metabolism and is often exposed to both parent substance(s) and metabolite(s). The most common tissues collected to evaluate responses at the site of first contact for orally administered substances are the gastrointestinal tract tissues, stomach, and duodenum. Jejunum and colon may also be evaluated. For inhalation studies, lung and nasal cavity are most often evaluated.

How do you know which tissue to take for Comet analysis?

It depends. The tissue(s) to collect for Comet analysis are generally dependent upon the results of 1) in vitro testing, 2) in vivo toxicity testing, and/or 3) target tissue for the test material. Tissues selected on the basis of in vitro testing reflect whether the observed positive response required metabolic activation. Tissues may also be selected for follow-up assessments based on pathological findings on in vivo toxicity studies (e.g., 7-, 14-, or 28-day repeat dose toxicity). Tissues are also selected based on known target specificities of the test material.

In Vivo Pig-a Gene Mutation Assay

The Pig-a gene mutation assay is a validated technique for measuring mutations caused by genotoxic effects of exposure to drugs or chemicals. Gene mutation assays, like the Pig-a assay, measure the induction of mutations at a specific gene location as opposed to measuring overall DNA damage.

Clear advantages of the Pig-a Assay include:

Easily integrates into standard repeat dose toxicity studies
Reduces animal use
Use of standard laboratory rodents alleviates the need for expensive transgenic rodent mutation assays
Fast, quantifiable results with flow cytometry

The entire Pig-a assay can be performed at Inotiv, or blood samples collected at another lab can be sent to Inotiv for analysis. The assay can be performed alone or in conjunction with other endpoints (Micronucleus Assay, Comet Assay, etc.)

OECD Guideline 470

Pig-a FAQs:

When is Pig-a required to be run?

The Pig-a assay is used as a follow-up to a positive Ames or in vitro gene mutation test. It can be used when metabolic activation was not required for the in vitro positive response if the test material is bioavailable to the bone marrow compartment.

Which species is preferred for Pig-a?

The Pig-a assay can be performed in rats or mice.

Is Pig-a run in both sexes or one?

Typically, one sex is sufficient. When data demonstrate relevant differences for the test chemical between males and females (e.g., differences in systemic toxicity, metabolism, and bioavailability), evaluation of both sexes is recommended.

How long does it take to run a Pig-a assay?

Typically, a 7-day (or longer) dose range finder test is first performed to select doses for the definitive study which is conducted for 28 days.

Integrated Genetic Toxicology Endpoints and Repeat Dose Studies

Genetic toxicology endpoints may be integrated into rodent repeat dose toxicology studies. Endpoints can be evaluated by flow cytometry (when available) or manual scoring (microscopy).

Endpoints include: Micronucleus, Comet, and Pig-a gene mutation

View our posters on integrating in vivo micronucleus in a repeat dose toxicology study here.

Integrating Comet Assay in a Repeat Dose Study

Integrating Genetox Endpoints into a Subchronic Toxicity Study

Duplex Sequencing

The assays typically used to follow up on a positive Ames or other in vitro mutagenicity assay are currently the Pig-a gene mutation and transgenic rodent (TGR) mutation assays. Duplex sequencing is an error-corrected next generation sequencing technology that can supplement and potentially replace these assays for the detection of in vivo mutagenicity.

Inotiv utilizes the TwinStrand Duplex Sequencing™ technology, which is a tag-based ultra-accurate method that reveals true and ultra-rare mutations with a >10,000-fold increase in accuracy over standard next-generation sequencing (NGS). The technology can be applied to analysis of rat, mouse, and human tissues and cells.

Advantages of Duplex Sequencing:

Mutagenicity can be assessed in any tissue type
Mutagenicity can be assessed in any rat or mouse strain to match previous toxicity studies
Can be incorporated into ongoing general toxicology studies in support of the 3 R’s
Avoid high animal costs and long lead times associated with use of specialized transgenic models.
Can be applied to in vitro mutagenicity testing
Mutation spectra signatures can help elucidate mechanism of damage
Demonstrated excellent interlaboratory reproducibility (both in vitro and in vivo)

Duplex Sequencing technology.

Follow-Up Genetic
Toxicology Assays