Due to their fast development, transparency, and similar responses to chemicals as humans, zebrafish have been used extensively in drug development. But it wasn't until Microtest Laboratories began exploring the broader potential for zebrafish embryos that a fast-track toxicity test was introduced to help medical device manufacturers test implantable materials.
|Toxicity testing using zebrafish embryos is more sensitive than small-animal tests recommended by FDA.|
The 'gold standard' for toxicity testing is the USP cytotoxicity assay, which uses live mouse fibroblast cells for in vitro tests performed in petri dishes. But there remains a fair amount of ambiguity about these tests because the cells have to be graded and the tests are not transferrable to humans, according to Steven Richter, Microtest's president and scientific director.
While researching zebrafish about five years ago, however, Richter was struck by their speedy growth, observing their development from embryo to larvae in just three days. "The embryo is transparent, so you can see cell division, structure development, and heart and brain development under a microscope," Richter says. "After studying it, the question was: Why aren't we using this for medical device testing?"
An opportunity quickly presented itself. Suspected of being linked to a variety of health issues, phthalates such as bisphenol A (BPA) were gathering controversy in the news for their widespread use in such products as baby bottles and medical device components. "With the current cytotoxicity assay test, samples weren't coming up as positive," Richter explains. "There were some problems with the test that weren't catching toxins that were leaching into patients."
In light of this flaw, Microtest decided to test for phthalates using zebrafish embryos. The company employed a multiwell plate and two relatively small concentrations of BPA--100 and 500 µM. The zebrafish embryos had a definite reaction. "With BPA, the embryos just don't develop two to four hours after fertilization," Richter notes. This outcome diverged from the usual cell-division timeframe. Teratogenic or embryogenic toxicity, he adds, was even detected at a 24-µM concentration.
Based on the success of the BPA testing, Microtest has added zebrafish toxicity testing for its medical device clients. Submitted materials are cut into saline and saline with 0.1% dimethyl sulphoxide nonpolar solvent to extract leachables, which are introduced to the embryos two to four hours after fertilization. The effects are observed while the transparent embryo develops. "One of the benefits of the screening process is that it eliminates a fair amount of animal work that may or may not give you the info you need," Richter says. "This test gives you the whole animal development, from specific parts to swimming behavior, which gives you a lot of information in a week as to whether you want to go forward with a certain polymer."
While the costs and timeframe associated with zebrafish embryo testing are similar to those of the cytotoxicity assay test, hundreds of samples can be tested during the same period. "Our assay," Richter states, "also has better sensitivity and generates more scientific data than the small-animal tests currently recommended by FDA."