Cost-effective and sensitive, zebrafish embryo toxicity testing for medical device applications is an effective alternative to USP <87> cytotoxicity testing.
Biocompatibility testing is a necessary part of any medical device validation process. It is governed by FDA’s ISO 10993 guidance document, a seemingly rigid statement that provides a general matrix dictating that some products require additional testing.1 However, the ISO 10993 guidance document does allow testing laboratories to use new and innovative biocompatibility testing methods. One such method, the zebrafish embryo toxicity (ZET) test, employs the embryos of the zebrafish (Danio rerio) to assess the toxicity of medical device polymers, drug-device combination products, and other medical device leachates.
The zebrafish is a well-characterized and extensively studied model organism. While the zebrafish biocompatibility test itself is different from past tests, it is merely a new application of previously existing tests that have a successful track record in the areas of drug testing, cardiac and neurological studies, and genetics. In addition, zebrafish embryos are already used for testing purposes in the chemical and environmental fields to determine LC50, a standard measure of the toxicity of the surrounding medium that will kill half of the sample population of a specific test animal in a specified period through exposure via inhalation. Investigated and validated by the Organization for Economic Cooperation and Development (OECD) and outside laboratories, this testing method parallels the new technique used to evaluate medical device extracts.2
Given its potential and robustness, ZET is poised to become a large and integral part of biocompatibility testing. However, in order to succeed, it must be accepted by FDA into the modified matrix of acceptable tests and be supported by data from rigorous validation testing.
ZET and Its Place in ISO 10993
According to FDA regulations, all medical devices must be tested for biocompatibility. They must also meet the following criteria:
The device materials should not, either directly or through the release of their material constituents: (i) produce adverse local or systemic effects; (ii) be carcinogenic; or, (iii) produce adverse reproductive and developmental effects.3
To ensure these criteria are met, each medical device, depending on its type, must go through a series of biocompatibility tests to ensure that it is safe for use. The process of determining which tests are required is found in ISO 10993 and outlined in a flow chart appearing in attachment C.1,3 However, because all medical devices are unique, they must be assessed on a case-by-case basis. For this reason, ISO 10993 is a modified matrix and not a rigid guide, allowing room for the use of alternative or additional tests when applicable. Hence, there is also room in the standard to use the zebrafish embryo toxicity test to determine a medical device’s biocompatibility, particularly as a preliminary screening method. ZET can fill in the gaps, helping to improve medical device testing by reducing risks, costs, and small-mammal testing.
Another test that uses zebrafish embryos is the OECD’s fish embryo toxicity test (FET), the final draft of which became available in July 2013.2 Both the fish embryo test, which exposes fish embryos to various chemical compounds to determine LC50 and toxicity, and the zebrafish embryo toxicity test rely on parallel methodologies. However, while the former analyzes known chemical compounds used in a variety of applications, the latter is specifically geared toward analyzing medical device polymer extracts. The validation of zebrafish embryo toxicity testing using the ISO 10993 guideline further supports the uses of ZET for biocompatibility screening of polymer extracts.
ZET Versus Other Methods
Figure 1. A normal zebrafish larva is shown at 72 hours old.
The most commonly used cytotoxicity test today is USP <87>. Considered the gold standard for biocompatibility testing, USP <87> relies on a cytotoxicity assay that uses a mouse fibroblast cell line.4 Although mammalian in origin, the mouse fibroblast cell line has the disadvantage of containing an oncogenic virus. Because this virus renders the cell line immortal, the line is not as natural or perhaps as sensitive as cells in a normal state. In addition, because the medium used to grow this cell line contains fetal bovine serum, it can introduce more viruses.
From a cost perspective alone, ZET is advantageous because it is inexpensive to maintain zebrafish, which are not nearly as delicate as eukaryotic cell cultures. Zebrafish can be cultured in minimally conditioned water, and the adults can live in a wide range of temperatures and pHs. In addition, as zebrafish embryos mature, lab personnel can observe the development of all types of tissues. For example, major organ precursors are observable within 36 hours. Moreover, Zebrafish typically hatch and become free-swimming larva within 72 hours, as detailed in Figure 1 (p. 44).
Between zebrafish and humans, there is a roughly 80% conservation of gene sequences.5 This conservation, in addition to the zebrafish’s lower culturing costs compared to those associated with mice, makes it a model organism for testing purposes. By using zebrafish embryos, laboratories can perform tests on whole vertebrate organisms at a relatively low cost.
|Figure 2. Shown is a comparison of a normal zebrafish larva (left) and an abnormal larva (right).|
Because zebrafish embryos are transparent after fertilization, technicians can easily observe them using a stereomicroscope. Thus, the ZET technique enables lab personnel to visually identify abnormalities resulting from contact with medical device polymer extracts and grade them with greater detail than can be achieved using USP <87> cytotoxicity testing. The cytotoxicity assay has no real endpoint; grading is performed on a scale of 0 to 4, whereby grades 1 to 3 are decided subjectively based on varying amounts of dead or unhealthy cells.
In contrast, while the ZET method is also subjective at this time, it can provide more-descriptive detail than the cytotoxicity assay about the shape, pigmentation, organ formation, and activity of affected embryos. For example, Figure 2 (p.44) illustrates the differences between a normal and an abnormal embryo. In addition, the ZET technique is robust; using mutant zebrafish lines and monitoring their development using more sophisticated equipment than is used in USP <87> testing, it can be developed into a tertiary test for pinpointing toxic areas exactly.
Table I. Shown is a comparison of ZET versus USP cytotoxicity <87> testing.
The advantages of ZET over USP <87> are supported by data collected from tests comparing the ability of both methods to detect Bisphenol A (BPA) and other compounds. A phthalate that is suspected of being a teratogen, the ubiquitous BPA plasticizer was tested at varying concentrations and exposed to both the mouse fibroblast cell line and zebrafish embryos. As demonstrated in Table I, the zebrafish embryos were able to detect the BPA in concentrations down to 25 µM, in contrast to the USP <87> test. This and other comparisons indicate that zebrafish embryos are more sensitive than mouse fibroblast cell lines and can detect toxic compounds that other tests cannot identify. In no case did mouse fibroblast cell lines achieve higher sensitivity than the zebrafish embryos.
In addition to evaluating ZET’s ability to detect BPA toxicity, the technique was used to test uncured and cured BPA-free dental composites. Both the uncured and cured resins caused complete toxicity in all embryos within 24 hours, indicating that they were more toxic than the positive control. However, because the cured composite resin is the most common source of long-term exposure to humans, it underwent tests at dilution rates of 1:10 and 1:100. While the 1:10 dilution did not produce the same toxicity level as the undiluted resin, it caused some significant embryo abnormalities within 72 hours, including a curved spine, yellowing, and enlarged or deformed yolk sacs. In contrast, the embryos subjected to the resin at the 1:100 dilution rate appeared normal within 72 hours.
To ensure that the test compounds rather than the water conditions in the testing wells caused the toxicity, the researchers conducted a pH study. There was no indication that death or abnormalities resulted from changing the pH of the water from 5 to 8. And to ensure that the embryos were not overly sensitive to compounds, they were subjected to over-the-counter amoxicillin and acetaminophen prepared at 100-ppm concentrations. The lack of toxicity corresponds to drug-testing studies that often rely on zebrafish.
|Figure 3. Abnormal zebrafish larva are shown at 72 hours old.|
Studies were also conducted on various in vitro fertilization media. While the results indicated toxicity in some solutions, this outcome seemed to be related to the salinity of the solutions. To confirm this hypothesis, the researchers conducted a salinity study in which zebrafish embryos were tested in saline solutions ranging from 0.2 to 0.8%. A freshwater fish, the zebrafish cannot tolerate water with a saline concentration of 0.2%. As shown in Figure 3 (p. 46), such abnormalities as yellowing, a deformed spine, and clouding of the yolk sac were followed by death at or after 72 hours.
While ZET is similar in many ways to the mouse embryo assay, mouse embryos are far more delicate and difficult to work with. Thus, while mouse embryos require only an 80% survival rate to be acceptable as controls or to be considered nontoxic, zebrafish embryos must demonstrate a 90% survival rate, according to the OECD methodology.2
Most importantly, ZET testing reduces patient risk by rendering medical device testing more comprehensive. Given its track record as a model organism, the zebrafish is a robust and promising choice of test subject that has already proven itself in drug and neurological studies. And given the high conservation of gene sequences between zebrafish and humans, researchers may even succeed in the future in pinpointing various compounds’ affected toxicity areas.
Reducing animal testing is one of FDA’s stated goals in ISO 10993, and zebrafish toxicity testing can help achieve this. Since one female zebrafish can produce hundreds of eggs a day while a mouse can produce only about a dozen, the use of zebrafish embryo testing would greatly reduce the demand for small-mammal testing and help to reduce the demand for test animals overall. And because zebrafish embryo toxicity testing can be employed as a preliminary screening method, its use could rule out many compounds before they reach the animal testing phase, saving animal lives and reducing costs.
Featuring greater sensitivity than other current biocompatibility testing methods, ZET can potentially screen out more compounds before they are released to the market. While FDA has not yet validated ZET, this technique meets the medical device industry’s goals of protecting humans.
Natalie Soares is a scientist at Microtest Laboratories, where her experience includes research and development in Zebrafish toxicology testing and testing and validation studies in the specialized microbiology group. Soares received a bachelor’s degree in biotechnology from the Rochester Institute of Technology. Contact her at email@example.com.
Bob Michaels is senior technical editor at UBM Canon.