In Vivo Comet Assay
When determining if a compound is genotoxic, the comet assay (also known as the single-cell gel electrophoresis [SCGE] assay) helps to evaluate DNA strand breaks in individual cells. A single-cell suspension from the selected tissue(s) is embedded in low melting point agarose, lysed, and electrophoresed under alkaline (pH > 13) conditions. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often looks like a celestial comet.
In Vitro 3D Comet Assay
The 3D Skin Comet assay uses reconstructed, full thickness (FT) skin models as an in vitro alternative to animal testing. These models are made with primary keratinocytes and fibroblasts, forming a highly differentiated and complete skin model comprised of the epidermis and dermis. Although the skin tissues can be topically exposed to a variety of compounds, the DNA damage levels can be measured separately in both skin layers.
This in vitro model is constructed from p53 human-competent cells and comprises an in vivo-like human skin tissue including epidermal and dermal layers.
When to Perform Comet Assay Techniques
- IND-enabling studies
- As part of the ICH S2 (R1) standard battery (Option 2)
- As a follow up to a positive in vitro mammalian cell clastogenic response
- REACH requirements
- As a follow up to a positive result in an Annex VII or VIII genotoxicity test
- 3D Skin Comet assay
- Cosmetics testing
- Investigation of first site-of-contact (skin) of potential genotoxicants in toxicological safety assessment
We offer testing for liver, blood, bone marrow, stomach, duodenum, kidney, lung, nasal epithelium, eyeball, and a reconstructed 3D skin tissue model. Other tissues can be validated upon request.
Frequently Asked Questions (FAQs) About Comet Assay Studies
I got a positive response in my in vitro mammalian cells test; what can I do?
Assuming negative results in the bacterial reverse mutation assay (Ames assay), if there is no sufficient weight of evidence or mechanistic information to rule out relevant genotoxic potential, two in vivo tests are usually required, with two different tissues and demonstrated target organ exposure. Generally, a micronucleus test and a comet assay analysis will be recommended. Negative results in two appropriate in vivo endpoints are considered sufficient to demonstrate the absence of significant genotoxic risk.
Can I include the comet assay in my toxicology repeat dose study?
Like many of the in vivo genotoxicity tests, and in the spirit of the 3Rs, the comet assay can be integrated into a repeat-dose toxicity study designed for other purposes, or the endpoint can be combined with other genotoxicity endpoints such as the in vivo mammalian erythrocyte micronucleus assay, provided the dose levels and dosing regimen are appropriate. However, because the DNA damage that is measured usually does not persist, longer exposure does not result in increased sensitivity. Therefore, it is necessary to collect the tissues for comet assay analysis within 2-6 hours following the last administration, which can sometimes be challenging.
In addition, the selected tissue to be evaluated should be based on the intended route of human exposure, metabolism and distribution, potential for site-of-contact effects, structural alerts, other genotoxicity or toxicity data, and the purpose of the study. Because it is the primary site of metabolism, the liver is almost always included for evaluation, provided systemic exposure is demonstrated. However, the stomach or duodenum could be evaluated when the test article is administered by oral gavage since they are considered site-of-contact tissues with highest exposure.
How does the in vitro skin model correlate to human skin?
Phenion® FT Skin Models are full-thickness, reconstructed 3D skin models comprised by the two main layers of human skin – epidermis and dermis. These models retain full epidermal differentiation as observed in human skin, including stratum basal, spinosum, granulosum, lucidum, and corneum. This 3D skin comet assay may serve as great in vitro alternative to animal testing.