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OECD Guideline: Test No. 489, In Vivo Mammalian Alkaline Comet Assay

(text taken directly from the OECD website)
OECD Test Guidelines are a collection of the most relevant internationally agreed test methods used by government, industry and independent laboratories to determine the safety of chemicals and chemical preparations, including pesticides and industrial chemicals. They are covered by the OECD system of Mutual Acceptance of Data.

On 26 September 2014, the OECD Council adopted three new, five updated and one corrected OECD Test Guidelines for the testing of chemicals, one of these was 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.

Publication Date : 26 Sep 2014
Pages : 25
ISBN : 9789264224179 (PDF)
DOI : 10.1787/9789264224179-en