Validated Non-animal Alternatives
The Eurpoean Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) "endorsed three in vitro methods for embryotoxicity testing as scientifically validated" (ESAC Statements, May 1, 2002):
- Embryonic stem cell test (EST) for embryotoxicity
- Micromass embryotoxicity assay
- Whole rat embryo embryotoxicity assay
The ESAC recommended these in vitro methods as ready for regulatory acceptance but acknowledged they cannot replace the animal tests. However, when used as part of a testing strategy, they could contribute to reducing animal use.
Experts have concluded that a total replacement method for reproductive and developmental toxicity will require the development and validation of a number of in vitro assays that model the essential in vivo toxicological processes. These assays will then need to be assembled into a testing scheme that can be validated for the overall hazard assessment.
The EST uses two cell lines and three endpoints to predict embryotoxic chemicals (INVITTOX protocol). The cell lines are mouse embryonic stem cells (ES) and mouse 3T3 fibroblast cells. The assay endpoints are inhibition of differentiation of the ES cells, inhibition of ES and 3T3 cell viability, and inhibition of ES and 3T3 cell proliferation.
The micromass test evaluates effects of a test substance on the differentiation and growth of micromass cultures of rat limb bud (INVITTOX protocol). The limb bud cultures are a model that represents various developmental processes in cartilage histogenesis such as cell proliferation and differentiation, as well as cell-cell communication.
The whole rat embryo culture uses isolated and cultured early post-implantation rat embryos to study embryotoxic effects of chemicals (INVITTOX protocol). The morphology of 48-hour cultured embryos (controls and chemical exposed) are compared for delays in the development of certain organ systems or their malformation.
The micromass test and whole rat embryo culture entail the killing of animals for the tissue used in the assays, whereas the EST (which uses cell lines) does not.
Additional information on in vitro methods being developed for reproductive and developmental toxicity testing can be found in Toxicity Endpoints & Tests: Reproductive & Developmental Toxicity.
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