Test No. 489: In Vivo Mammalian Alkaline Comet Assay
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet
Assay is a method measuring DNA strand breaks in eukaryotic cells.
Each treated group is composed of a minimum of 5 animals of one sex (or of each sex
as appropriate). A positive and a vehicle control group are also used. Administration
of the treatment consists of daily doses over duration of 2 days or more, ensuring
the test chemical reaches the target tissue which can be the liver, the kidney or
other tissues if justified.
Tissues of interest are dissected and single cells/nuclei suspensions are prepared
and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove
cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to
electrophoresis at high pH. This results in structures resembling comets which by
using suitable fluorescent stain, can be observed by fluorescent microscopy. Based
on their size DNA fragments migrate away from the head to the tail, and the intensity
of the comet tail relative to the total intensity (head plus tail) reflects the amount
of DNA breakage.
Published on July 29, 2016Also available in: French
In series:OECD Guidelines for the Testing of Chemicals, Section 4: Health Effectsview more titles