2.2.2. Unpublished Pre-clinical Studies

NAmSA Laboratories, 9 Morgan St., Irvine, California 92618, worked with us to perform biocompatibility studies pursuant to the FDA Tripartite and ISO 10993 guidelines which recommend a certain combination of tests for a device that will be permanently implanted in the body. Three types of tests were carried out: in vitro, short-term in vivo, and long-term in vivo tests. Because devices are so small (~0.14g), a particularly large number of devices are required for these tests. Since it would be exceptionally expensive to develop a large stock of functionally active devices, the assembly procedure for the BIONs™ was modified. Instead of attaching electrodes at either end of the glass capsule, the capsules were sealed using the same laser-welding techniques that are used for functioning devices, but without attaching the electrodes. The capsules and the free electrodes were placed together into the extraction medium in proportions equivalent to those used to produce functional devices. Because initial trials of the BION™ were conducted in Canada, this modification was approved by the Canadian government with whom we consulted prior to testing (Appendix 3).

Cytotoxicity tests and a battery of genotoxicity tests were conducted. The in vitro tests involved taking extracts of the microstimulator components and subjecting cells to these extracts. Current standards recommend assays of genotoxicity tests covering three different levels of effects: gene mutations, chromosomal aberrations, and DNA effects. Thus, the following three tests were performed: the Salmonella Typhimurium (Ames) mutagenicity test, the chromosomal aberration test, and the sister chromatid exchange test. Short-term in vivo tests involved injecting device extracts into animals and observing them for adverse effects to evaluate short-term intracutaneous reactivity, systemic toxicity, and sensitization. Long-term in vivo tests conducted in collaboration with NAmSA involved implantations of test devices into muscle in order to assess foreign body reactions and chronic toxicity of the devices. A summary of the test results follows. A copy of the final report of chronic toxicity trials including the histopathological analyses from NAmSA is found in Appendix 4.

6.4.In Vitro Tests

6.4.1. Salmonella Typhimurium Reverse Mutation Test

Test Description

This assay was conducted to evaluate whether a saline extract of the BION microstimulators would cause a mutagenic effect on any of five histidine-dependent mutant tester strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without the presence of mammalian microsome activation (rat liver S-9 fraction). The plate incorporation assay of Ames et al. (1975) was used after extraction in normal saline. Sample mutagenicity was evaluated by determining the dose-response curve of any presumptive positive response.

Test Results

The saline test article extract was found to be non-inhibitory to growth of tester strains TA98, TA100, TA1535, TA1537, and TA1538. Separate tubes containing 2 ml of molten top agar supplemented with histidine-biotin solution were inoculated with 0.1 ml of culture for each of five tester strains, and 0.1 ml of the saline extract. A 0.5 ml aliquot of S9 homogenate simulating metabolic activation was added when necessary. The mixture was poured across triplicate Minimal E plates. Parallel testing was also conducted with a negative control, and four positive controls. The mean number revertants of the triplicate test plates were compared to the mean number of revertants of the triplicate negative control plates for each of the five tester strains employed. The values (means) obtained for the positive controls were used as points of reference.

Under the conditions of the assay, the saline extract from the BION microstimulator was not considered to be mutagenic to Salmonella typhimurium tester strains. The negative and positive controls performed as expected.