The agar diffusion test evaluates the cytotoxic potential of leachable substances released from a test item following diffusion through an agarose layer.

This test simulates the penetration of leachable substances from the test item into patient tissue through a barrier, such as skin. The method is applicable to substances that can diffuse through the agar layer without reacting with it.

As the agarose layer is stained with neutral red, the cytotoxic potential of the test material is determined by evaluating the decolourisation area and assessing cell lysis within the cell culture.

Assessment of cytotoxicity by microscopic evaluation after indirect cell contact

• A sub-confluent cell monolayer is covered with an agarose layer and stained with neutral red. 
• The test item or an extract of the test item is applied to the agarose layer in indirect contact with the cells. 
• Following an incubation period of 24 hours, cytotoxicity is evaluated microscopically. The decolourisation index and lysis index are determined qualitatively (see table).

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