In vitro irritation

Audrey Turley, senior biocompatibility expert at Nelson Labs, explains the value for medical device manufacturers to understand ISO 10993-23 and how to navigate the transition from in vivo irritation testing to in vitro testing. 

Every medical device on the market must be evaluated for its potential to cause irritation, from a knee brace to an artificial cardiac pacemaker, regardless of whether it has a short-term or long-term exposure to the user/patient. This is a requirement set forth in International Organization for Standardization (ISO) 10993-1:2018: Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk management process. This standard requests manufacturers to evaluate the safety of a medical device to ensure it is manufactured using materials and processes that will not cause an adverse response (that is, for example, irritation, allergy, fever, inadvertent weight loss) when used in a clinical setting. 

Currently, the requirements for assessing irritation for medical devices are covered in two different ISO 10993-series standards: ISO 10993-10:2010 and the newly released ISO 10993-23:2021 Tests for irritation. This situation prompts the questions: Which standard should be referenced in internal documentation and used in laboratory testing? and Which standard is accepted by the regulatory authorities? The ultimate questions, however, are Which standard guidance or testing approach should the device manufacturers use now? and How will this be handled for regulatory submissions in the meantime? 

Why assess irritation?

First, it is important to understand why irritation is assessed and what has driven a change in the standard. Irritation is defined in ISO 10993-23 as, “a localized, non-specific inflammatory response to single, repeated, or continuous application of a substance/material”. The standard also specifies that, “It is a reversible reaction and is mainly characterized by local erythema (redness) and oedema (swelling) of the skin.” Many people have experienced an irritation response, typically after using a particular detergent, with itching and possibly a rash. Then, once the detergent was no longer used, the rash disappeared. A similar reaction can also be seen with medical devices such as bandages.

Although irritation is not typically a life-threatening response on its own, if left unattended over a long period of time, it can lead to more problematic health issues. To predict an irritation response to medical devices, the most commonly performed irritation test is (and has been) the intracutaneous reactivity test. This test is performed by injecting extracts of a medical device into the subcutaneous tissue of rabbits and observing the local tissue response for redness and swelling over several days. During the test, a final score from 0–4 is given for each injection site based on the noted reactivity level (that is, redness or swelling), providing a conclusion on the irritation potential of the device. 

The need for an alternative

Considering that that majority of medical devices available on the market are tested for irritation, and that ISO 10993-10 only outlines several in vivo testing approaches, it was urgent to find an alternative in vitro approach, in line with the principle of the 3Rs (replace, reduce, refine) of ISO 10993-2:2006: Animal welfare requirements. 

The international community involved with the ISO standards has, therefore, been working for the last several years to demonstrate that an adapted OECD method used to assess irritation for chemicals and cosmetics (OECD 439) in vitro can accurately predict irritation responses for medical devices. This involved several global collaborations to develop the protocol adjustments needed to assess medical devices, which resulted in a massive round robin study involving 18 labs in nine countries. The results demonstrated that the in vitro method accurately identified the irritation potential of several materials commonly used in the manufacturing of medical devices, and that the method would result in reproducible data among the different labs.¹

A separate standard: ISO 10993-23

It is this global success that has led to a change in the ISO irritation standard, which is now covered in its own separate standard, ISO 10993-23, apart from skin sensitisation (which remains in ISO 10993-10). The in vitroirritation method uses a reconstructed human epidermis (RhE) model with normal human-derived epidermal keratinocytes which have been cultured to form a multilayered, differentiated model of the human epidermis. The devices are extracted in the same manner as in the in vivo assay. After the extraction period, the extract is applied directly to the RhE tissues and then incubated for 18–24 hours. 

The in vitro method uses MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) to determine cell viability. This is a commonly used dye with a reactivity that is based on cellular reduction of MTT from yellow to purple by viable cells. This colour change is then quantified on a spectrophotometer and compared to the negative control. It is important to note that although MTT is more commonly associated with cytotoxicity assays, it is not the dye that makes the assay, but rather the complexity of the test system to which it is applied. Thus, while we are examining cell viability, it is of complex tissue rather than a typical cytotoxicity experiment, which consists of a monolayer of cells. 

With ISO 10993-23, the in vitro method is now identified as the preferred test method for initial consideration. Yet, the most common question asked now is, “Which regulatory agencies/countries accept the in vitro method?” The general input from around the globe is that the in vitro method is preferred and will be accepted widely, even while the standard is being translated for official international inclusion and acceptance. Japan was ahead of the mark and has included the in vitro test in their guidance document already. The US FDA has yet to officially recognise the new standard, but there have been direct discussions with FDA to facilitate a speedy acceptance of this method. 

Technically, it is true that irritation testing is covered in two standards, ISO 10993-10:2010: Tests for irritation and skin sensitization and ISO 10993-23:2021 Tests for irritation, yet the most updated and current standard, ISO 10993-23 is considered state of the art and includes the information applicable to assess irritation from ISO 10993-10 (meaning all of the in vivo testing options from ISO 10993-10 are also included in ISO 10993-23). It is, therefore, appropriate to comply with ISO 10993-23. Besides, it is anticipated that ISO 10993-10 will soon be updated (Summer 2021) to remove all references to the irritation endpoint.

Making the transition

There are a couple of points to consider when navigating this transition to ISO 10993-23. First, the new standard indicates a preference for the in vitro test. This is also in line with ISO 10993-1:2018, which quotes from ISO 10993-2 Animal welfare requirements: “In vitro test methods, which are appropriately validated, reasonably and practically available, reliable and reproducible, shall be considered for use in preference to in vivo tests.” As such, device manufacturers should consider transitioning their biocompatibility test plans to include the in vitro method, which may include a discussion with the regulatory body that is being used, to ensure that the method will be accepted for the subject device(s). Second, test labs will transition to the ISO 10993-23 standard according to their own internal procedures, so you will want to verify that the standard referenced in the test protocols matches your internal documentation. 

This move from in vivo to in vitro irritation testing in the medical device industry is no small feat; experts around the world have collaborated for almost a decade to bring this about. It is valuable for medical device manufacturers to understand the new test method, the current and anticipated state of the two relevant standards (ISO 10993-10 and ISO 10993-23), and how to navigate the transition from in vivo irritation testing to in vitro testing.

A double win

The ultimate goal in assessing irritation is to protect patient safety, which has been proven by showing comparability between the two main testing options. It is also worth noting that as a side goal, this approach is targeted to preserving animal lives. To help understand the impact on animal lives used in medical device testing, in 2018 the US FDA had 1,608 510(k) submissions just in the first quarter. For each device, let’s assume that irritation testing was probably performed, which consumes three animals. With that in mind, moving to the in vitro option would effectively save 4,824 animals in the first quarter or almost 20,000 in one year, which is an underestimate as it only considers the US submissions, and one type of device submission.

Reference

De Jong, W.H. et al., “Roundrobin study to evaluate the reconstructed human epidermis (RhE) model as an in vitro skin irritation test for detection of irritant activity in medical device extracts,” Toxicology in Vitro 50, 429–449 (August 2018).