In recent years, ethical concerns over the use of live animals in testing have been increasing.

2023 is the Chinese Year of the Rabbit, making it the perfect time to move irritation validations away from traditional rabbit irritation testing to non-animal alternatives.

The use of rabbits for irritation testing dates back to the early 20th century. For many years, it has been a widely accepted method for evaluating the irritation potential of various substances, including medical devices. This type of testing involves applying a test substance (such as a medical device extract) under the skin of a rabbit and then observing the resulting reaction. The severity of the reaction is then used to determine the irritation potential of the test substance.

A comparison of histology between the RhE tissues (in vitro) and a normal human epidermis (in vivo).

In the past, rabbit irritation testing has been considered the de facto method for reliably predicting the irritation potential of substances that might come into contact with patients.

However, in recent years, ethical concerns over the use of live animals in testing have been increasing, particularly for cases not directly related to advancing human health. These concerns have motivated a push to find alternative methods for evaluating the irritation potential of medical devices.

In 2021, a new ISO Standard (ISO 10993-23:2021, Biological Evaluation of Medical Devices — Part 23: Tests for Irritation), emphasized this shift to in vitro testing, such as the reconstructed human epidermis (RhE) method.

RhE is a laboratory-grown tissue model that mimics the structure and function of human skin. RhE is typically created using human keratinocyte cells, which are cultured in a laboratory setting to produce a three-dimensional tissue model that can be used for a variety of purposes, including irritation testing.

In addition to the ethical benefits, RhE is considered to be a more reliable predictor of irritation potential than animal models due to its closer resemblance to human skin. This can be especially important for medical devices that will be used on human skin, as the response of animal skin can differ. Other advantages of this in vitro test include the potential for decreased turn time and better testing control.

Table 1. Results summary.

Animal tests require the ordering of animals and then acclimation to the animal facility; animal testing has built-in delays. With the RhE test, tissues are on hand when samples arrive and testing can start immediately. The tissues also have less variability between test specimens making repeatability of test results more consistent over the inherent variability of animals (see Table 1 and Figure 1).

Fig. 1 - With the RhE test, the tissues have less variability between test specimens, making repeatability of test results more consistent over the inherent variability of animals.

Despite these benefits, the use of RhE for irritation testing is not without its challenges. The first is the need for standardized methods for producing and testing RhE. This is an area of active research, and there are currently several different approaches used for creation and testing.

The second challenge is the cost of producing RhE, which can be relatively high compared to other methods. Fortunately, reliability and cost have made significant progress in the last few years, resulting in the ISO standard and routine production of these tissues.

MTT test results. A spectrophotometer is used to measure the amount of purple color in each well. The more purple detected, the more viable the cells. The more viability, the less irritation.

The last obstacle remaining is global regulatory acceptance of the method. As of today, most of the world’s regulatory bodies have publicly announced the acceptance of the RhE method in place of the rabbit, with the notable exception of U.S. FDA.

In 2023, the U.S. regulatory tides might be turning. Recently FDA has hinted that they are making progress to officially accept the RhE irritation test along with its animal test counterpart early this year. If that becomes a reality, the use of RhE for irritation testing is likely to continue to grow in popularity as a more ethical, reliable, and faster alternative to traditional methods.

It is difficult to predict exactly when the FDA will accept RhE irritation testing for medical devices. Even though they have indicated progress toward acceptance of the in vitro method, FDA has not provided a specific timeline.

The process for evaluating and performing the test for submissions would require a discussion with FDA, consensus, and approval for the testing method. The discussion with the FDA is an important part, as there may be lab-to-lab variability in test procedures and therefore the FDA should sign off on test protocol before starting testing.

The evaluation of the in vitro irritation test: an MTT cytotoxicity assay is run on the tissues to evaluate viability of the cell; viability is an indicator of non-irritation.

That being said, FDA has expressed interest in the use of RhE for irritation testing and has provided guidance on the use of RhE for this purpose. In particular, FDA has stated that RhE can be used as an alternative to animal testing for the evaluation of the irritation potential of medical devices, provided that the RhE method is validated and performs adequately. This means that if a manufacturer wants to explore performing the RhE test method for FDA submissions, it should present the validated test method to the FDA in a pre-sub, as FDA currently does not have an official acceptance position.

Overall, the transition from rabbit-based irritation testing to RhE testing represents a significant advancement in the field of medical device testing. While the use of RhE for irritation testing is still an emerging field, there are ongoing efforts to establish standardized methods for producing and testing RhE. As more data becomes available and the use of RhE becomes more widely accepted, it is possible that FDA may begin to accept RhE testing as a viable alternative to traditional animal-based methods.

As of today, FDA does not have an official position on the use and acceptance of the RhE test in place of the rabbit irritation test, but will consider the results of the method if appropriate validation and evidence can be provided to demonstrate equivalency with the animal model.

Much progress has been made over the past years to demonstrate the validity of the RhE test method to replace the rabbit irritation model. We are nearing the time where universal acceptance of this test is a real possibility, and this year might indeed by the year of the rabbit.

This article was written by Thor Rollins, Sr. Director of Toxicology and E&L Consulting, Nelson Laboratories, Salt Lake City, UT. For more information, visit