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Frog Embryo Teratogenesis Assay: Xenopus, or FETAX, is a test used to determine the developmental toxicity of chemicals in aquatic toxicology. Developmental toxicity results when a chemical affects any process of an organism’s development usually focusing on concentrations lower than that which cause harm to the parent. This is because developmental stages of organisms are often more sensitive to chemical exposures in comparison to adult stages indicating FETAX as a quick and sensitive toxicity test of exposure to contaminants. A chemical capable of causing developmental malformations is called a teratogen. The organism most commonly used for FETAX is Xenopus laevis, the African clawed frog. Originally, X. laevis was used for a relatively simple and low-cost pregnancy test, discovered by Lancelot Hogben. Further testing revealed that the embryos of amphibians were sensitive indicators of water quality. Using this information, Greenhouse. made the initial developments for FETAX as a quick test for teratogenesis. One current use for FETAX is detecting potential human developmental health hazards. However, the test has not been generally accepted as a regulatory tool. It is also currently used for understanding complex mixtures and single chemical exposures in the environment[4]. The test is run by exposing the X. laevis embryos to the toxicant of concern for 96 hours, while replenishing the toxicant solution every 24 hours.

Mechanism

A chemical, or toxicant, exhibiting teratogenic effects on X. laevis embryos during the test will interrupt one or more stages of embryogenesis. These stages include cell division, cell interaction, cell migration, cell differentiation, and selective cell death. The interruption of these stages can present itself as a physical malformation at one or more levels of biological organization, ranging from abnormal development of specific tissues, to abnormal limb development all of which can sometimes result in the death of the organism. These outcomes, or endpoints, are important indicators of significant ecological effects as organisms experiencing teratogenesis are more likely to be preyed on or die of starvation, removing them from the population.

Regulation

The use of FETAX is not accepted as a regulatory test for developmental toxicity in the United States of America under USEPA, FIFRA, CAA, TSCA or FFDCA, all of which require mammalian test organisms. On the other hand, FHSA will accept any available evidence while OECD does not designate the specific use of mammalian test organisms. However, neither FHSA or OECD specify FETAX as an accepted test.

A review was requested by USEPA, asking the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to organize a workshop to evaluate the possible use of FETAX in regulatory applications. A public meeting was held during May 16 - 18 in 2000 with a panel of over 50 experts contributing to the discourse. The panel concluded that FETAX was not “sufficiently validated or optimized to be used for regulatory applications". They concluded that FETAX databases which relate animal and human developmental toxicity needed increased accuracy and reliability before they can be accepted as indicators of human health.

Procedure

The FETAX test uses embryos of the South African clawed frog X. Laevis. Healthy adult breeders are chosen and placed into a breeding tank to breed overnight. To induce breeding, X. laevis will receive an injection of human chorionic gonadotropin (HCG) into the dorsal lymph sac[13]. After eggs have been laid and fertilized, they are removed from the breeding tank. Once fertilization has occurred, the eggs have begun development and are now considered to be embryos. After deposition in the breeding tank, the embryos are covered in a viscous, sticky jelly coating. The embryos are de-jellied with an amino acid solution immediately after being laid[13]. Acceptable embryos for the test must be between the midblastula (Stage 8) and early gastrula (Stage 11) stages of development at the initiation of the assay. Acceptable embryos are placed into petri dishes where the de-jellying solution is then removed via pipette, and a soft water solution containing the test material or chemical is immediately added to the dish. The temperature of the solution should be maintained at 24°C for the entirety of the testing period. The test material solution is removed and replaced with a new solution of equal concentration at every 24-hour increment throughout the testing period. If the temperature is held constant, 90% of the embryo controls should reach Stage 46 of development at the 96-hour test termination time. This development must be reached in order for the test to be acceptable.

At the end of the 96-hour exposure, the embryos are examined for developmental malformations which are recorded in regards to the severity and type of malformation. The Atlas of Abnormalities may be used as a guide in the characterization and determination of various malformations that may exist. The development of hindlimb buds are a telling and easily identified marker of embryo development. Results obtained from the assay are able to generate concentration-response data in terms of the mortality, malformation, and growth of the organism. Test materials are assigned a value based on the severity and amount of malformations which occurred to the embryos. This value ranks the materials in the teratogenic index (TI), where their toxicity to X. laevis is easily compared.

History

Prior to its use as the test organism for FETAX, X. laevis was used as a relatively quick and simple pregnancy test for women as early as the 1930’s. The use of X. laevis as the test organism for FETAX was initially pioneered by Greenhouse as they recognized the advantages compared to other organisms. There was not, however, a consensus on the proper procedure for conducting the test. Major developments were made by Dumont et al.[7], including the formal naming of the test, identifying more defined endpoints, fully developing the test protocol, and the proposed concept of the teratogenic index (TI). A final test protocol was developed by the American Society for Testing and Materials (ASTM) in 1998 and was reapproved in 2004. An Atlas of Abnormalities was written In order to assist in the identification of malformations visible in the assay and was created to accompany the ASTM procedure.