In Vivo Single Cell Gel Electrophoresis Comet Assay

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

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.

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.

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.