A new test has been developed for medical device manufacturers and biomaterials researchers to screen plastics, composites, and polymers for toxicity. This ZET test utilizes zebrafish (Danio rerio). Zebrafish are extensively used in drug development studies due to their transparent embryos and fast development times. This test demonstrates similarities to mammalian models and humans. Its unique approach to fast-track medical device toxicity screens will save manufacturers time and reduce expenses.

Regulatory Background
The U.S. Food and Drug Administration (FDA) requires all medical device manufacturers and suppliers to those manufacturers to have a biocompatibility program. This program is defined as one set by the manufacturer to assess medical device toxicology. Most device manufacturers must test their products (including packaging: ref. ASTM-F2475) according to the modified ISO 10993-1 matrix. However, changes to suppliers and devices require that manufacturers repeat portions of these tests according to their internal biocompatibility program, or add new testing programs that are deemed necessary to determine the extent of new toxins found in medical devices. New toxins, such as phthalates (BPA), can be materials that, upon further scientific information or studies, have been shown to be toxic to vertebrates in vivo.

Any biocompatibility program should utilize the FDA guidance document G95-1, entitled Use of International Standard ISO-10993, “Biological Evaluation of Medical Devices Part-1: Evaluation and Testing.” This document includes an FDA- modified matrix that designates the type of testing needed for various medical devices. However, the FDA also states that the matrix is only a framework for testing selection and not a checklist of every required test. According to the FDA, these tests are not all-encompassing, and each device should be judged on its merits regarding patient use:

“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.

“Therefore, evaluation of any new device intended for human use requires data from systematic testing to ensure that the benefits provided by the final product will exceed any potential risks produced by device materials.

“When selecting the appropriate tests for biological evaluation of a medical device, one must consider the chemical characteristics of device materials and the nature, degree, frequency and duration of its exposure to the body. In general, the tests include: acute, sub-chronic and chronic toxicity; irritation to skin, eyes and mucosal surfaces; sensitization; hemocompatibility; genotoxicity; carcinogenicity; and, effects on reproduction including developmental effects. However, depending on varying characteristics and intended uses of devices as well as the nature of contact, these general tests may not be sufficient to demonstrate the safety of some specialized devices. Additional tests for specific target organ toxicity, such as neurotoxicity and immunotoxicity may be necessary for some devices.”

The international standards document ISO 10993-1 (section 5.1 b) 7) indicates its primary goal is to protect human beings, and its secondary goal to reduce animal testing:

“…that the protection of humans is the primary goal of this document, a secondary goal being to ensure animal welfare and to minimize the number and exposure of test animals.”

Zebrafish embryo toxicity testing addresses these primary and secondary goals.

Zebrafish Facts
Testing has revealed that zebrafish embryos are highly susceptible to toxins including reproductive toxins such as androgens. In our research, a suspected toxic compound BPA (bis-phenol A) was tested against the gold standard, i.e., the USP biological reactivity test <87> in vitro (cell cytotoxicity) assay. The USP cytotoxicity assay utilizes a mouse fibroblast (L929) cell line in culture. BPA in concentrations of 100 µM failed our new ZET zebrafish embryo test, while it passed the USP cytotoxicity test. BPA or phthalates in general have been suspected of having both carcinogenic and teratogenic effects on humans. ZET may be the best way to screen for teratogenic effects.

Zebrafish are tropical freshwater fish belonging to the minnow family (Cyprinidae). They have the capability to lay hun- dreds of eggs in 1 day. The fertilized eggs become transparent immediately. This characteristic makes D. rerio an excellent research and testing model. Embryos are removed from the aquarium, cleaned, and placed into sterile multi- welled plates. The embryos are examined for fertility. Polymer materials are extracted according to USP <1031> conditions using both saline (Extract A) and saline with 0.1% DMSO (Extract B) as extractants. Extracts are pipetted into multi-well plates 2 to 4 hours post-fertilization (hpf).

Zebrafish embryos at 24 hours post-fertilization (30X mag.). Positive control: 100 µM BPA — the fertilized embryos exhibit no development.Embryo development progresses very rapidly. Major organ precursors are visible within 36 hours post-fertilization. The test reveals anatomical observations of the brain, spinal cord, and heart. Within 72 hours, the embryos grow into larvae (fry) and swim around the well.

The table reveals that ZET sensitivity is greater than the USP gold standard test for BPA.

Zebrafish embryos at 24 hours post- fertilization (30X mag.). Negative controls are shown. Normal development.The testing is performed in duplicate with five embryos per well. BPA at concentration of 100 uM is used for the positive control on each sample run. Negative controls are Extract A and Extract B run in duplicate on five embryos.

The embryos are observed at 24, 48, and 72 hours post-fertilization using a stereomicroscope at 30X magnification. Observations include brain, heart, and spinal cord development. Once hatched, the fry are evaluated for typical darting activity when subjected to light stimulus. Mutagenicity testing can be performed, if requestedNormal 72 hour post-fertilization zebrafish fry at 30X mag. Normal development (negative controls)..

Microtest has developed a simple new whole-vertebrate toxicology test to screen medical device polymers/plastics and nanotechnology composites. Hundreds of material can be screened relatively inexpensively using this ZET test. The test is robust and scientifically sound. Zebrafish models are well categorized and utilized in the drug discovery community. Some cardiac drugs are screened by actually measuring cardiac electrical signals for abnormalities in this model. Neuropathic drugs are screened by researchers to determine neurological abnormalities in zebrafish.

The FDA states in their guidance documents that additional testing may be required when the device is used in a specific clinical area:

“For example, a neurological device with direct contact with brain parenchyma and cerebrospinal fluid (CSF) may require an animal implant test to evaluate its effects on the brain parenchyma, susceptibility to seizure, and effects on the func- tional mechanism of choroid plexus and arachnoid villi to secrete and absorb (CSF). The specific clinical application and the materials used in the manufacture of the new device determines which tests are appropriate.”

ZET vs. USP Cytotoxicity
The ZET test translates well with current USP cytotoxicity testing. The USP test utilizes the mouse fibroblast cell line that has an oncogene in the genome to maintain the cell line’s immortality. It should be noted that the USP test has major scientific issues, so utilizing a whole animal test that meets the requirements and the intent of the FDA guidance document makes sense.

Ongoing Studies
The ZET test translates well to the mouse embryo toxicity test that is required for medical devices used in in vitro fertilization procedures. Side-by-side correlation studies so far are equivalent. Cost and time comparisons alone have major benefits. Reduction of small-animal testing would be an immediate benefit due to the fact that the ZET test would eliminate candidate toxic polymers that otherwise would be tested in mice, rabbits, or dogs.