Test No. 231: Amphibian Metamorphosis Assay

This Test Guideline describes an amphibian metamorphosis assay intended to screen substances which may interfere with the normal functioning of the hypothalamo-pituitary-thyroid axis. The assay was validated with the species Xenopus laevis, which is recommended for use in the Guideline. The assay uses three test chemical concentrations and the necessary controls, including a carrier control if necessary. The assay starts with tadpoles at the development stage 51 on the Nieuwkoop and Faber scale and is extended for a duration of 21 days. Four replicate test vessels are used for each treatment level and control(s). After 7 days of exposure, a sub-set of tadpoles from each treatment level is sampled for the measurement of the length of the hind-limb. At termination of 21-day exposure period, developmental stage, snout-to-vent length and hind limb length are measured on all remaining tadpoles. A sub-set of tadpoles from each treatment level is fixed (whole-body or dissected) for histopathology of the thyroid gland.