OECD 231: The Amphibian Metamorphosis Assay

The Amphibian Metamorphosis Assay (OECD 231) is designed to detect endocrine active substances acting on the thyroid system. Amphibian metamorphosis provides a well-studied, thyroid-relevant process which responds to substances active within the hypothalamic-pituitary-thyroid (HTP) axis.

This test system is of particular importance for the assessment of endocrine disrupting properties of chemicals (EFSA/ECHA 2018), as this is the case for the Larval Growth and Development Test (LAGDA, OECD 241).

Test organisms

The clawed frog, Xenopus laevis is routinely cultured in laboratories worldwide and is easily obtainable through commercial suppliers. Reproduction can be induced throughout the year using human chorionic gonadotropin injections and tadpoles are easily available.

Course of the test

Xenopus laevis tadpoles (stage 51) are exposed to a minimum of three different concentrations of a test chemical and a water control for 21 days in a flow through system. There are four replicates of each test treatment. Larval density at test initiation is 20 tadpoles per test tank for all treatment groups.

At the beginning of the study the tadpoles are in the developmental stage 51, which is approximately two weeks post hatch. At this stage, also known as premetamorphosis, the tadpole thyroid is not yet functional.

On study day 7, which is approximately when the thyroid gland begins functioning, five individual tadpoles are removed and the developmental stage is assessed. The remaining tadpoles are maintained in the test system for another 14 days. On day 21, when the tadpoles have fully functional thyroid glands and are capable of secreting thyroid hormones, the study is terminated and data are collected.


  • Hind limb length
  • Snout to vent length
  • Developmental stage
  • Wet weight
  • Thyroid histology
  • Daily observations of mortality.

Guidelines and literature

  • OECD Guideline for Testing of Chemicals, No. 231: The Amphibian Metamorphosis Assay.