No animal testing to prove biocompatibility

In order to evaluate the biocompatibility of standard materials, medical device manufacturers can, in most cases, avoid animal testing.

In recent years, the Johner Institute had always succeeded in showing alternatives to animal testing, even when authorities and notified bodies demanded them.

Learn here how you can contribute to animal welfare, save money, and bring your medical devices to market faster.

1. About animal testing

a) Verification for which animal tests are suitable

Manufacturers must verify the safety, effectiveness, and efficacy of medical devices. Animal testing is one way to provide this evidence:

Property to be verified	Example
Biological safety, biocompatibility	Animals are placed in contact with a material/extract of the medical device to demonstrate that it has no biological reactions.
Effectiveness	Newly developed orthopedic or cardiac implants are inserted into animals to evaluate their effectiveness.
Efficacy	Animals with liver failure are connected to a liver dialysis machine to demonstrate that it adequately removes liver-derived substances and increases animal survival.

b) Proof of biocompatibility

Manufacturers must prove that no substances are dissolved out of the material or from the surface (e.g., residues from production/cleaning) in quantities that are toxicologically relevant, i.e., have hazardous health effects.

Different adverse health effects may occur depending on the type and duration of use of the medical device. These effects are called clinical endpoints. A distinction can be made between local endpoints, i.e., effects related to the site of application, and systemic endpoints, which affect the whole organism.

For most endpoints, animal testing can be used for evaluation (see Tab. 2). However, depending on the application and materials used, biocompatibility does not have to be evaluated using animal tests. There are suitable ways to address the endpoints without animal testing.

Endpoints for biological evaluation	Systemic endpoint?	Is the TTC concept* intended for this?	Can the endpoint be evaluated with animal testing?	Does the endpoint need to be evaluatedwith animal testing?
Physical and/or chemical information (material properties)	No	No	No	No
Cytotoxicity (cell damage)	No	No	No	No
Skin sensitization (e.g., allergic reaction)	No	No	Yes	No
Irritation of the skin or tissue (e.g., redness and itching).	No	No	Yes	No
Systemic toxicity: acute, subacute, subchronic and chronic toxicity (e.g., organ damage, death).	Yes	Yes	Yes	No
Material-related pyrogenicity (generation of fever)	Yes	No	Yes	No
Genotoxicity (damage to DNA)	Yes	Yes	Yes	No
Carcinogenicity (causing cancer)	Yes	Yes	Yes	No
Reproductive toxicity (impairment of the ability to reproduce)	Yes	Yes	Yes	No
Implantation effects (e.g., rejection of implants)	No (local effects) Yes (systemic effects)	Yes (systemic effects)	Yes (joint consideration possible)	Usually
Blood compatibility (e.g., thrombosis)	No	No, but in vitro tests	Yes	No (except implants)

Animal testing procedure

To prove biocompatibility, the animals (e.g., their skin, eyes, and blood) are brought into direct contact with the material to be tested or an extract thereof. This requires several animals (usually at least five per endpoint, dose, and route of ingestion).

For necessary concentration determinations, animals are subjected to preliminary tests. For the study itself, additional animals are needed for the control groups to show the quality of the test system.

The test animals are mainly guinea pigs, rabbits, rats, or mice. At the end of the study, these are killed painlessly in order to be able to carry out further investigations for the evaluation of the endpoint.

Effort and costs of animal testing

Animal tests typically take several weeks. Sometimes they delay approval by several months. This is especially the case when

• the manufacturer has many endpoints to determine (usually only one endpoint and route of ingestion can be studied per investigation),
• the experimental design has yet to be developed and validated,
• the outputs suggest that biocompatibility is not given, and
• in the case of inconclusive results and product-independent influences.

The costs depend heavily on the type and number of endpoints. They almost always amount to five-digit sums. For example, the determination of a single endpoint (e.g., a sensitization test) usually takes 14 to 16 weeks. The costs for this are approximately between EUR 5,000 and 10,000.

2. Regulatory framework

There is no legal or normative obligation to conduct animal tests.

The standards on biocompatibility (ISO 10993 series) require “only” an adequate biocompatibility evaluation of the final medical device.

The ISO 10993 series even requires in several places that material characterization and in vitro tests should be carried out first and that animal tests may only be used if the data obtained are not sufficient.

Description of medical device chemical constituents and consideration of material characterization including chemical characterization (see ISO 10993-18) shall precede any biological testing (see Figure 1). Chemical characterization with an appropriate toxicological threshold can be used to determine if further testing is needed (see Annex B, ISO 10993-17 and ISO 10993-18).

EN ISO 10993-1:2020, 4.3

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 (see ISO 10993-2). 

EN ISO 10993-1:2020, 4.6

Suppose the material characterization does not reveal any indications of released substances with toxicologically relevant potential. In that case, the waiver of additional testing is not only justified but also mandatory.

The FDA calls for a “3Rs approach” with regard to animal testing: “reduce, refine and replace”. In this context, the FDA has include Appendix G, “Biocompatibility of Certain Devices in Contact with Intact Skin,” in the currently revised version of the guidance “Use of International Standard ISO 10993-1, “Biological evaluation of medical devices – Part 1: Evaluation and testing within a risk management process” (Sept.2023). It identifies product materials with a low risk of contact with intact skin surfaces that have been used safely in medical devices for years and describes a procedure to avoid animal testing. Requirements for the quality system and other controls are listed to immediately recognise and avoid biocompatibility problems with medical devices that come into contact with intact skin.

”FDA believes that these materials pose a very low biocompatibility risk because they have a long history of safe use in legally marketed medical devices that contact intact skin. The policy outlined in this Attachment describes a least burdensome approach for these devices that recommends specific material information to be included in a premarket submission in lieu of biocompatibility testing. This approach also supports the principles of the “3Rs,” to replace, reduce, and/or refine animal use in testing when feasible”

Guidance document of the FDA on the application of ISO 10993-1:2023

In addition, the FDA’s guidance document generally calls for a procedure to avoid or reduce animal testing when assessing the biocompatibility of medical devices made from standard materials.

“Some devices are made of materials that have been well characterized both chemically and physically in the published literature and/or have a long history of safe use in legally marketed medical devices. It may not be necessary to conduct testing for all or a portion of the biocompatibility endpoints suggested in the FDA matrix of this guidance.“

Guidance document of the FDA on the application of ISO 10993-1:2023

“For FDA submissions, biocompatibility information for the device in its final finished form, either developed through the risk management process or from biocompatibility testing (using both in vitro and in vivo models), and/or adequate chemical, physical, morphological, and topographical characterization in conjunction with supplementary biocompatibility information that adequately address the biocompatibility risks of the device should be provided.” (FDA)

Guidance document of the FDA on the application of ISO 10993-1:2023

In chemical characterization, the so-called TTC concept is then applied to ensure the biocompatibility of medical devices and, thus, patient safety.

3. The TTC concept: Threshold of Toxicological Concern

a) Objective of the TTC concept

The TTC concept was developed to qualitatively assess the risk from substances present in small quantities [EFSA].

The objective of the concept is to establish an exposure level for all chemicals, whether or not there are chemical-specific toxicity data, below which there is no appreciable risk to human health.

b) Determination of the TTC

The TTC is thus a threshold for toxicological concern. It is typically established for chemicals of unknown toxicity, below which the likelihood of adverse human health effects is negligible due to chemical structure and low dose.

This concept is based on the assumption that a daily exposure of 1.5 µg of an unknown organic compound is associated with an acceptable lifetime cancer risk of less than 1:1,000,000.

Accordingly, this implies that exposures shorter than a lifetime are possible with a proportionally higher daily intake. Hence, the TTC for the use of fewer than ten years is 10 µg/day, and for short-term use of less than one month is 120 µg/day.

c) Use of the TTC

Based on the TTC determined, an analytical evaluation threshold (AET = Analytical Evaluation Threshold) is calculated, on which the required limit of quantification of the chemical analysis depends. The AET value ensures that the analyses are performed with sufficient sensitivity. In addition to the TTC, other parameters such as the number of products used, the extraction volume, patient groups, the uncertainty factor, and the application (number of products used per patient and day) are included in the calculation of the AET.

The TTC concept provides a valuable alternative to animal testing. In principle, this approach was developed for systemic endpoints. However, it can also be used to evaluate local endpoints, such as irritation and sensitization of standard materials to evaluate the final medical device. For example, ISO 10993-1 describes, on the one hand, that a chemical characterization with a suitable TTC concept is sufficient to determine whether further investigations are necessary. On the other hand, ISO 10993-10, for example, requires a step-by-step approach to address skin sensitization using material characterization and chemical analysis to evaluate this endpoint.

ISO 10993-10: “This part of ISO 10993 requires a step-wise approach, which shall include one or more of the following: 
a) characterization of test material, involving chemical characterization … 
b) literature review, including an evaluation of chemical … and information on the irritation and sensitization potential of any product constituent as well as structurally-related chemicals and materials; 
c) in accordance with ISO 10993-2, in vitro tests in preference… 
In vivo animal tests are appropriate when test materials cannot be characterized and risk assessments cannot be undertaken using information obtained by the means set out in a), b) and c).”

ISO 10993-10:2020, 4

Existing data from the literature on animal tests already carried out on the material itself, on the additives used as well as release products found can be used for assessment.

4. Waiver of animal testing

a) When a waiver is necessary

For standard materials, biocompatibility evaluation via material characterization, in vitro tests, and chemical characterization using the TTC concept is possible and even required.

b) When a waiver is possible

In general, a waiver of animal testing is possible for all of the above endpoints (see Tab. 2, right column), especially for those endpoints for which the TTC concept can be applied without difficulty (see Tab. 2, middle column).

c) When a waiver is not possible

Animal testing is necessary if no literature data and experience on the material or product are available and cannot be derived. This is especially the case for novel materials.

d) Benefits of not testing on animals

The strategy via in vitro tests has many advantages compared to animal testing:

• Animal welfare is not compromised.
• The tests are more sensitive.
• The tests are more reproducible.
• Multiple endpoints and intake routes (oral/dermal/inhalation) are assessable.
• The tests are (therefore) usually less expensive and less time-consuming.
• The tests allow better comparability of products.
• Outputs can be transferred to materials for root cause analysis if necessary.

5. Procedure

In order to avoid animal testing, you, as a manufacturer, should take the following steps:

1. Create a Biological Evaluation Plan (BEP) individually for the medical device. In it, you must justify your strategy for evaluating biocompatibility without animal testing and describe the procedure. In addition, the test parameters and the respective limit values must be explicitly defined for the medical device, depending on the type of exposure (e.g., via the TTC concept).

2. In case of uncertainties, the Johner Institute recommends coordinating this BEP with the authorities or notified bodies.

3. Look for a suitable laboratory specialized in the necessary tests. Preference should be given to accredited/certified laboratories with appropriate experience in the particular area. 4. Summarize the laboratory’s output in a Biological Evaluation Report (BER). The BER will also include a toxicological evaluation of these data based on the limit values established in the BEP and data from literature and experience.

6. Summary and conclusion

Unnecessary animal testing is unethical, expensive, and lengthy.

In many cases, animal testing is even unsuitable. It makes little sense to test standard materials, such as a polyethylene housing of a control element, for the umpteenth time using an animal test to determine whether an allergic reaction can be triggered.

While the evaluation of the final medical device is required, it is not fundamentally about animal testing. On the contrary:

ISO 10993-1 calls for preferring material characterization as a method and avoiding animal testing. In its published guidance documents on the biocompatibility of medical devices, the FDA also calls for animal testing to be reduced and replaced by suitable in vitro strategies.

This means that authorities and notified bodies cannot refuse a stringent and data-based line of argumentation in these cases and must refrain from demanding animal testing. In this way, you, as a manufacturer, can not only contribute to animal welfare but also save effort, costs, and time.

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Change history

• 2023-09-14: Citations and link to current FDA Guidance on the application of ISO 10993-1:2023 adapted and reference to the new Annex G of the Guidance inserted
• 2023-02-09: Creation of the blog article