Division 1.3.3.1 Experimental schedule

The experimental schedule of the assay is as follows:

• Day 1:

  • Individually identify and record the weight of each animal. Open application of 25µl of the appropriate dilution of the test substance, the vehicle alone, or the positive control (as appropriate), to the dorsum of each ear.

• Days 2 and 3:

  • Repeat the application procedure carried out on day 1.

• Days 4 and 5:

  • No treatment.

• Day 6:

  • Record the weight of each animal. Inject 250µl of phosphate-buffered saline (PBS) containing 20 µCi (7.4e + 8 Bq) of ³H-methyl thymidine into all test and control mice via the tail vein. Alternatively inject 250 µL PBS containing 2 µCi (7.4e + 7 Bq) of ¹²⁵I-iododeoxyuridine and 10^-5 M fluorodeoxyuridine into all mice via the tail vein.

  • Five hours later, the animals are killed. The draining auricular lymph nodes from each ear are excised and pooled in PBS for each experimental group (pooled treatment group approach); alternatively pairs of lymph nodes from individual animals may be excised and pooled in PBS for each animal (individual animal approach). Details and diagrams of the node identification and dissection can be found in Annex I of reference 10.