The agar overlay assay or agar diffusion test is an in vitro cytotoxicity assay designed to show the presence of toxic material eluted from a test sample as it affects L929 cells cultured in the presence of the test article.

Test articles are placed onto the agar surface overlaid onto a L929 cell monolayer preventing direct damage while allowing diffusion of leachable chemicals from the sample. After treatment, the cells are exposed to neutral red dye to differentiate between the viable and stressed or lysed cells.  The degree of irritancy is determined microscopically by measuring the zone of reactivity. IIVS offers the United States Pharmacopoeia (USP) and International Organization for Standardization 10993 compliant Agar Diffusion Test. The characteristics of the particular medical device, polymer, container, etc. determine the dosing method. Users of the assay include consumer products and cosmetic companies with products defined as medical devices.

Grading System for Microscopic Examination

Grade Reactivity Description of Reactivity Zone
  0  None No detectable zone around or under specimen
  1  Slight Some malformed or degenerated cells under specimen
  2  Mild Zone limited to area under specimen**
  3  Moderate Zone extends 0.5 to 1.0 cm beyond specimen
  4  Severe Zone extends greater than 1.0 cm beyond specimen but does not involve entire dish

Assays of Interest