You just got the news. Your cytotoxicity (AKA cytotox) test was positive. What are you do do with this information? Is it time to find a new job? On a side note, in the world of biocompatibility, the word positive is a bad thing, and negative is a good thing because the test is looking for signs of cytotoxicity, irritation or sensitization. It is similar as being tested for a disease, infection or drugs. So, if proper grammar were used, it would be said that your product tested positive for cytotoxicity. Those immersed in the world of biocompatibility speak will just say a “cytotox is positive” and a newbie may be happy about that news. In any case, the test device failed the test.
First, and most important, thing to do is take a deep breath. The cytotoxicity test is a very sensitive test and is the most likely test to cause you anguish. The biocompatibility evaluation of a product is not intended to be taken lightly, or just some regulatory hurdle you must jump. It is a step-wise evaluation that increases in rigor as the nature of the device’s contact with the patient increases.
Just like the 5 stages of grief; denial, anger, bargaining, depression and acceptance. There are 5 stages of coping with any failed biocompatibility test. These are the stages that are typically gone thru coping with failure, but are not necessarily the correct order to proceed.
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First, and most important, thing to do is take a deep breath. The cytotoxicity test is a very sensitive test and is the most likely test to cause you anguish. The biocompatibility evaluation of a product is not intended to be taken lightly, or just some regulatory hurdle you must jump. It is a step-wise evaluation that increases in rigor as the nature of the device’s contact with the patient increases.
Just like the 5 stages of grief; denial, anger, bargaining, depression and acceptance. There are 5 stages of coping with any failed biocompatibility test. These are the stages that are typically gone thru coping with failure, but are not necessarily the correct order to proceed.
Continue reading below
The first stage is “Blame the Lab”:
After all, a lot can go wrong in the conduct of a cytotox test. The pH of the extract could affect the outcome as could the temperature or the concentration of Carbon Dioxide in the incubator chambers. Ask the lab whether or not a failure investigation was conducted. Are they confident that this was not a technical error on their part. Did both the positive and negative controls behave as usual? If all that checks out, you need to consider the reality that the positive is a true positive and the investigation should shift focus to your product. Unfortunately, many jump to the second stage.
The second stage is to “Blame the Test”:
This test method is not appropriate for my product. You steered me in the wrong direction. Aren’t there other tests and why didn’t we do those other tests. The test device has to be evaluated according to the ISO 10993 series of standards they specify what endpoints need to be assessed. The 10993 series also specifies what test methods are appropriate for specific kinds of devices. There are three categories of surface patient contact: intact skin, breached or compromised tissue and mucosal contact and the differences in their testing requirements reflect that. For instance, the agar diffusion test (direct application test) is primarily designed to evaluate intact skin contacting devices, but extract based tests like the neutral red uptake (NRU) and MTT are designed to evaluate devices that are internalized in some way or are mucosal contact. It should be understood that changing the test doesn’t necessarily mean that your regulatory body (FDA etc.) will accept the chosen method.
The third stage is to “Tweak the technical parameters”:
Maybe if we used less material in the extract, or extracted for a shorter period of time or at a lower temperature we can get a pass. This is similar to blaming the test but at least shows a hint of problem solving. This approach usually does not solve the problem but is an upgrade from the initial reactions.
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After all, a lot can go wrong in the conduct of a cytotox test. The pH of the extract could affect the outcome as could the temperature or the concentration of Carbon Dioxide in the incubator chambers. Ask the lab whether or not a failure investigation was conducted. Are they confident that this was not a technical error on their part. Did both the positive and negative controls behave as usual? If all that checks out, you need to consider the reality that the positive is a true positive and the investigation should shift focus to your product. Unfortunately, many jump to the second stage.
The second stage is to “Blame the Test”:
This test method is not appropriate for my product. You steered me in the wrong direction. Aren’t there other tests and why didn’t we do those other tests. The test device has to be evaluated according to the ISO 10993 series of standards they specify what endpoints need to be assessed. The 10993 series also specifies what test methods are appropriate for specific kinds of devices. There are three categories of surface patient contact: intact skin, breached or compromised tissue and mucosal contact and the differences in their testing requirements reflect that. For instance, the agar diffusion test (direct application test) is primarily designed to evaluate intact skin contacting devices, but extract based tests like the neutral red uptake (NRU) and MTT are designed to evaluate devices that are internalized in some way or are mucosal contact. It should be understood that changing the test doesn’t necessarily mean that your regulatory body (FDA etc.) will accept the chosen method.
The third stage is to “Tweak the technical parameters”:
Maybe if we used less material in the extract, or extracted for a shorter period of time or at a lower temperature we can get a pass. This is similar to blaming the test but at least shows a hint of problem solving. This approach usually does not solve the problem but is an upgrade from the initial reactions.
Continue reading below
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The fourth stage is “Exhaustion”:
You have gone around and around with everybody, from your boss, to the engineers, to your vendors, and the testing lab. But you are still in the same place. Holding the bag with this report saying that your product is cytotoxic.
The fifth stage is “Addressing the Issue”:
This is where we are going to get down to the brass tacks. The initial four stages are understandable but they could be much more effective if they were all part of the fifth stage. If the exact same points were addressed in a methodical failure investigation instead of an emotional fight or flight response everybody could get to addressing the issue that much faster. The cytotoxicity evaluation serves as a front line defense for you financially, the patient’s safety, and animal welfare. The cytotox test is the “screening test”. A failure is not the end of the world. To follow the rationale behind a comprehensive biocompatibility testing plan, the cytotoxicity endpoint is to be addressed first before any other tests. This allows for the opportunity to get the results and then to decide how to proceed.
Why did it fail? Is this completely unexpected? Is it a catheter impregnated with an antimicrobial or is the product an inert polypropylene implant. The implications of a positive result on these two different products are an example of the extremes. The antimicrobial catheter failing could be expected. Is it the end of the world? Absolutely not. The polypropylene implant failing is a complete and utter surprise. Is this the end of the world? It very much could be.
Continue reading below
You have gone around and around with everybody, from your boss, to the engineers, to your vendors, and the testing lab. But you are still in the same place. Holding the bag with this report saying that your product is cytotoxic.
The fifth stage is “Addressing the Issue”:
This is where we are going to get down to the brass tacks. The initial four stages are understandable but they could be much more effective if they were all part of the fifth stage. If the exact same points were addressed in a methodical failure investigation instead of an emotional fight or flight response everybody could get to addressing the issue that much faster. The cytotoxicity evaluation serves as a front line defense for you financially, the patient’s safety, and animal welfare. The cytotox test is the “screening test”. A failure is not the end of the world. To follow the rationale behind a comprehensive biocompatibility testing plan, the cytotoxicity endpoint is to be addressed first before any other tests. This allows for the opportunity to get the results and then to decide how to proceed.
Why did it fail? Is this completely unexpected? Is it a catheter impregnated with an antimicrobial or is the product an inert polypropylene implant. The implications of a positive result on these two different products are an example of the extremes. The antimicrobial catheter failing could be expected. Is it the end of the world? Absolutely not. The polypropylene implant failing is a complete and utter surprise. Is this the end of the world? It very much could be.
Continue reading below
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What is the next step? In both cases you want to assess how bad the failure actually was. This assessment can be done internally or you can contract an external expert. These experts can help you identify if a certain component, or manufacturing process may have been a leading contributor to the failure. There are a number of materials which are known to be cytotoxic and should both be considered as a part of the original device design as well as during the process of understanding and justifying the failed test. Review the linked document here: Mitigating Risk in Biocompatibility for a list of materials known to fail the test, as well as other factors to consider when performing general biocompatibility testing.
In the case of the catheter which has the intention of killing microbes, there may be some collateral damage at the patient/device interface, we can proceed cautiously through the rest of the studies. If an acute systemic toxicity test is conducted, and that study passes it indicates that a whole body biological system has the ability to buffer the effects of the antimicrobial. Next, how the intracutaneous reactivity test performs is of paramount importance. As the acute systemic injection evaluates systemic toxicity at a high dose, the intracutaneous reactivity will be injecting the device, or extract thereof, into the dermis of rabbits, and this is really the target tissue for this kind of product. If the test passes you are heading in the right direction, but if it fails here is your opportunity to adjust the concentration of your coating or the elution profile.
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In the case of the catheter which has the intention of killing microbes, there may be some collateral damage at the patient/device interface, we can proceed cautiously through the rest of the studies. If an acute systemic toxicity test is conducted, and that study passes it indicates that a whole body biological system has the ability to buffer the effects of the antimicrobial. Next, how the intracutaneous reactivity test performs is of paramount importance. As the acute systemic injection evaluates systemic toxicity at a high dose, the intracutaneous reactivity will be injecting the device, or extract thereof, into the dermis of rabbits, and this is really the target tissue for this kind of product. If the test passes you are heading in the right direction, but if it fails here is your opportunity to adjust the concentration of your coating or the elution profile.
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A failing cytotox, indicates that your genotoxicity tests are going to have some technical hurdles because they utilize cells and bacteria and therefore, a different strategy needs to be considered. In the case of the failed polypropylene implant, this is a big deal. The material is one of the safest on the market and may be embedded in some deep tissue. Put everything on the shelf. This shouldn’t fail, this can’t fail. Something is wrong in the manufacturing process or the supply chain. Sure you could proceed with acute systemic and the intracutaneous reactivity tests and see how that fares. And quite possibly pass the other tests and not even run into an issue conducting genotoxicity endpoints.
In both cases, or in any case, you will have to demonstrate that you have addressed cytotoxicity endpoints. If you submit failing data, FDA will ask you to justify moving forward with failed results. Be prepared to address the results, either with a scientific rationale or additional testing that typically is in the realm of some clinically relevant model. For instance, there have been more than a few cases where a study such as wound healing has been requested in order to show that your product performs equivalent to a legally marketed IN THE UNITED STATES (or any other geographical region of submission) product. Part of your justification should include the other biocompatibility tests that were conducted, assuming they all passed. If your strategy is to rerun your test with dilutions, or use the agar diffusion test (if your original was extract based) and you have a wound care product, implant or externally communicating device, be aware that the reviewer likely will likely see right through this and will tell you that your cytotoxicity evaluation is insufficient or incomplete and might ask for evaluations above and beyond.
Continue reading below
In both cases, or in any case, you will have to demonstrate that you have addressed cytotoxicity endpoints. If you submit failing data, FDA will ask you to justify moving forward with failed results. Be prepared to address the results, either with a scientific rationale or additional testing that typically is in the realm of some clinically relevant model. For instance, there have been more than a few cases where a study such as wound healing has been requested in order to show that your product performs equivalent to a legally marketed IN THE UNITED STATES (or any other geographical region of submission) product. Part of your justification should include the other biocompatibility tests that were conducted, assuming they all passed. If your strategy is to rerun your test with dilutions, or use the agar diffusion test (if your original was extract based) and you have a wound care product, implant or externally communicating device, be aware that the reviewer likely will likely see right through this and will tell you that your cytotoxicity evaluation is insufficient or incomplete and might ask for evaluations above and beyond.
Continue reading below
As this paper stated starting out, this testing is not just a regulatory hurdle and it will likewise end on that note. It’s not just a cytotox test that happens to be the cheapest, and shortest turnaround time, test in the biocompatibility battery. It can have clinical implications and a failure shouldn’t be taken lightly like sometimes it is. Use this opportunity wisely. Investigate, evaluate and adjust appropriately.
