We are very pleased to present the results from our first round of ISO 10993 testing for our latest commercial alloy, LM105. ISO 10993 is a set of standards used for evaluating the biocompatibility of a medical device prior to clinical studies. Since several biomedical device companies have shown interest in Liquidmetal alloys, we thought it would be beneficial to get a jumpstart on pre-screening the alloy for its potential use in biomedical applications. Of course, each biomedical device must undergo its own ISO certifications to account for its specific processing methods, but this set of tests serves to give potential customers confidence that LM105, our beryllium-free commercially available Zr-based amorphous metal alloy, is highly promising as a biomedical device material.
In this initial round of biocompatibility tests, we commissioned a third-party laboratory to investigate the following categories:
- Systemic Toxicity
Both in vivo and in vitro tests were conducted and reported by American Preclinical Services in Minneapolis, MN in accordance with Good Laboratory Practice (GLP). All tests were performed on as-molded parts, which were 45 x 45 x 2.1 mm3 plates of our commercial alloy, LM105, molded on our ENGEL Liquidmetal® e-motion injection molding machine. From these plates, appropriate specimen sizes were cut to obtain the appropriate volumes and surface areas required for each part of the test.
- G.P. Maximization: This tests for the potential to cause an allergic response. Extracts are inducted intradermally and topically and test specimens observed after 24 and 48 hours. A challenge phase follows with a negative control extract injection and observation of test specimens.
Result: Score of 0 for polar and non-polar extracts. Since both scores are <1.0, the material is a non-sensitizer.
- Intracutaneous Reactivity Test: This tests for the potential of any leachables to cause intracutaneous irritation (within the skin). Polar and non-polar extracts are injected on test specimens intracutaneously and observed after 24, 48, and 72 hours. A score of <1.0 for erythema (redness or rash) and edema (swelling from fluids) formation would result in a Pass for this test.
Result: Score of 0.2 for polar extract and 0.3 for non-polar extract. Since both scores are <1.0, the material is a non-irritant.
- Systemic Toxicity:
- Acute Systemic Toxicity Test: This tests for the potential to be acute systemic toxic. Extracts are injected and test specimens observed after 4, 24, 48, and 72 hours. Behavioral and clinical abnormalities, body weight changes, and effects on mortality are noted. Any nickel leaching, if present, would be revealed in this test through abnormalities of test specimens.
Result: There were no abnormalities or changes in body weight. The samples met the requirements of the test and there was no evidence of acute systemic toxicity from the extracts.
- Hemocompatibility: These tests determine whether the material causes hemolysis, the rupturing of red blood cells that leads to the release of hemoglobin into surrounding fluids.
- Hemolysis Test (ASTM F756): This test quantifies hemolysis due to the extract of the article or its direct interaction with blood cells by measuring hemoglobin concentration. The material is considered non-hemolytic if the blank corrected % hemolysis is <2.0% above the negative reference control article.
Result: The blank corrected % hemolysis of the test article was 0.5% when compared to the negative reference control for the Direct Method and 0.0% for the Extract Method. The material is considered non-hemolytic via both methods.
- Complement Activiation, C3a Assay and SC5b-9 Assay: This tests for the ability to trigger complement activation – C3a for anaphylotoxin (release of histamine), SC5b-9 for cell lysis (tissue breakdown). The test article is exposed to NHS and then the extract is deposited in triplicate wells of C3a and SC5b-9 pre-coated plates. The plates are analyzed by a microplate reader at 450 nm.
Result: The activation of the test article relative to the positive control was calculated to be 0.38% for the C3a method and 0% for the SC5b-9 method. The material is considered a non-complement-activator.
- Partial Thromboplastin Time (PTT): This tests for the ability to cause clot formation (activation of the intrinsic coagulation pathway). The test articles were exposed to plasma, then RBC and calcium chloride were added to the extract, and lastly the samples were analyzed by measurement of clotting time. The thrombogenicity of the test article is designated by the table below.
Result: Average clotting times were 97.0 and 138.4 seconds, which was 32.3% and 46.1% of the negative plasma control, for the initial and re-test investigations. The test article is considered a Moderate Activator.
Comment: Though the result of the test shows the material to be a Moderate Activator, it does not necessarily deem it non-hemocompatible. Current biomedical devices on the market can also show this same result, so to investigate this further, we plan to perform additional PTT tests on common biomedical device materials. Please stay tuned for these results.
- Prothrombin Time Assay (PT): This tests for the ability to cause clot formation (activation of extrinsic pathway). The test articles were exposed to plasma, Thromboplastin Reagent was added to the extract, and the samples were analyzed by measurement of clotting time. The increase in Prothrombin Time compared to the negative reference control determines the % of negative plasma control.
Result: The average clotting time was 13.4 seconds, which is 96.4% of the negative plasma control. Because the Prothrombin Time is < 2 times that of the negative plasma control, the test article is considered a negative result (Pass).
Overall Result: Based on the results of this series of tests, the test article could be considered hemocompatible.
- MEM Elution, L-929 Fibroblast Cells: This test determines the cytotoxicity response to the test article; it tests for the ability to cause cell lysis or inhibit cell growth. Test articles were extracted in EMEM 5%FBS, the extract was added to L-929 cell monolayer in triplicate, and the cells were microscopically evaluated at 24, 48, and 72 hours after inoculation. The cells are given a grade based on the table below.
Result: The cells were given a grade of 0, meaning the test article is considered non-cytotoxic.
The test results are summarized in this summary table. Full descriptions of test details can be found in the final test reports, which can be provided upon request.
These results suggest that LM105 in the as-molded condition is a potential candidate for 3 types of biomedical devices:
- surface contact
- blood contact*
* The tests performed thus far only partially fulfill the requirements for an external communicating device as listed in Table A.1 of ISO10993-1. The material passed all tests for limited contact duration (<24 hours) and passed some tests for prolonged contact duration (>24 hours to 30 days). Therefore, at this point in the evaluation, the material is not precluded from being used in an external communicating device.
† The tests performed thus far only fulfill one part of the requirements for the implantation category as listed in Table A.1 of ISO10993-1. The material passed all tests for tissue/bone contact with limited contact duration of < 24 hours. Therefore, at this point in the evaluation, the material is not precluded from being used in an implant device, but a number of tests would still be required before consideration.
Table A.1 of ISO10993-1 lists the evaluation tests required for considering the device in the three types of body contact for three contact durations. Because each device has its own specific processing method and must be re-evaluated and tested for biocompatibility, the set of tests we had performed for as-molded LM105 is a pre-screening and is not intended to be a comprehensive study of the alloy as a biomedical device. The purpose is to give potential customers confidence that a device produced with the alloy has a very strong likelihood of also passing the same set of tests. Please refer to the list of completed and passed tests below from Table A.1 of ISO10993-1 for as-molded LM105.
CONCLUSION: Liquidmetal’s as-molded LM105 commercial alloy produced on the ENGEL Liquidmetal® e-motion injection molding machine is non-sensitizing, non-irritating, non-systemic-toxic, hemocompatible, and non-cytotoxic, making it a promising biocompatible material for biomedical device applications.