Biocompatibility Testing

1. Basic Concepts

Biocompatibility: As defined in ISO 10993-1, the ability of a medical device material to perform with an appropriate host response in a specific application – i.e. the device performs its intended function without causing unacceptable local or systemic adverse effects in the recipient.

Biological evaluation: a systematic approach to assessing the biological risks that may arise when a medical device contacts the human body, ensuring product safety.

Core purposes:

  • Assess the safety of materials in contact with the human body

  • Identify potential biological risks (toxicity, sensitization, carcinogenicity, etc.)

  • Provide the biological basis for product registration

  • Meet global regulatory requirements

Important change: modern biological evaluation has moved from “run every test” to an integrated approach of “risk assessment + chemical characterization + necessary testing”.

2. Key Regulations and Standards

ISO 10993 series (international)

Standard Content
ISO 10993-1:2018 Evaluation and testing framework (overarching, core)
ISO 10993-2 Animal welfare requirements
ISO 10993-3:2014 Tests for genotoxicity, carcinogenicity and reproductive toxicity
ISO 10993-4:2017 Selection of tests for interactions with blood
ISO 10993-5:2009 Tests for in vitro cytotoxicity
ISO 10993-6:2016 Tests for local effects after implantation
ISO 10993-7:2008+A1:2019 Ethylene oxide sterilization residuals
ISO 10993-9:2019 Framework for identification and quantification of degradation products
ISO 10993-10:2021 Tests for skin sensitization (irritation moved to Part 23)
ISO 10993-11:2017 Tests for systemic toxicity
ISO 10993-12:2021 Sample preparation and reference materials
ISO 10993-13:2010 Degradation products from polymeric devices
ISO 10993-14:2001 Degradation products from ceramics
ISO 10993-15:2019 Degradation products from metals and alloys
ISO 10993-16:2017 Toxicokinetic study design for degradation products and leachables
ISO 10993-17:2023 Toxicological risk assessment of medical device constituents
ISO 10993-18:2020+A1:2022 Chemical characterization
ISO 10993-22:2017 Nanomaterials
ISO 10993-23:2021 Tests for irritation (split from Part 10)

FDA

Document Content
FDA Guidance (2020) “Use of International Standard ISO 10993-1, Biological evaluation of medical devices”
FDA Draft Guidance (2021) Chemical analysis for biocompatibility assessment

China

Standard Content
GB/T 16886 series Identical adoption of the ISO 10993 series
Guidelines for Biological Evaluation and Review of Medical Devices NMPA technical guidance

European Union

  • MDR (EU) 2017/745 emphasizes life-cycle biological evaluation

3. Key Terms

Term Meaning
Nature of contact Surface device / externally communicating device / implant device
Duration of contact Limited (≤24 h), prolonged (>24 h to 30 d), long-term (>30 d)
Contact site Skin, mucosa, breached surfaces, blood, bone/tissue, circulatory system, etc.
Biological endpoints Specific evaluation items such as cytotoxicity, sensitization, irritation and systemic toxicity
Biological evaluation plan (BEP) Biological Evaluation Plan
Biological evaluation report (BER) Biological Evaluation Report
Chemical characterization Identifying material composition and released substances
Toxicological risk assessment (TRA) Toxicological Risk Assessment

4. Device Categorization (ISO 10993-1)

Biological evaluation endpoints are determined by the device’s nature of contact + duration of contact.

By nature of contact

Category Subcategory Examples
Surface devices Intact skin Stethoscopes, ECG electrodes
Mucosal membranes Contact lenses, endoscopes
Breached/compromised surfaces Wound dressings, burn dressings
Externally communicating devices Blood path, indirect Infusion sets, blood collection sets
Tissue/bone/dentin Dental fillings, bone cement
Circulating blood Hemodialyzers, intravascular catheters
Implant devices Tissue/bone Bone screws, artificial joints, sutures
Blood Pacemakers, artificial heart valves

By duration of contact

Duration Range
A (limited) ≤24 hours
B (prolonged) >24 hours to 30 days
C (long-term) >30 days

5. ISO 10993-1 Evaluation Matrix (2018 edition)

The table below is a simplified biological evaluation endpoint matrix; in practice, the tests are determined by consulting this table:

Surface devices

Contact site Contact duration Cytotoxicity Sensitization Irritation Acute systemic toxicity Subacute/subchronic toxicity Genotoxicity Implantation Chronic toxicity Carcinogenicity Reproductive toxicity Degradation Hemocompatibility
Intact skin A/B/C ● ● ●
Mucosa A ● ● ●
Mucosa B/C ● ● ● ○ ○ ○
Breached surface A/B/C ● ● ● ○ ○ ○

Externally communicating devices

Contact site Contact duration Cytotoxicity Sensitization Irritation Acute systemic toxicity Subacute/subchronic toxicity Genotoxicity Implantation Chronic toxicity Carcinogenicity Reproductive toxicity Degradation Hemocompatibility
Indirect blood path A ● ● ● ● ●
Indirect blood path B/C ● ● ● ● ● ● ○ ○ ●
Tissue/bone B/C ● ● ● ● ● ● ● ○ ○ ○
Circulating blood A ● ● ● ● ○ ●
Circulating blood B/C ● ● ● ● ● ● ● ● ● ●

Implant devices

Contact site Contact duration Cytotoxicity Sensitization Irritation Acute systemic toxicity Subacute/subchronic toxicity Genotoxicity Implantation Chronic toxicity Carcinogenicity Reproductive toxicity Degradation Hemocompatibility
Tissue/bone A ● ● ● ● ●
Tissue/bone B/C ● ● ● ● ● ● ● ● ● ○
Blood A ● ● ● ● ○ ● ●
Blood B/C ● ● ● ● ● ● ● ● ● ●

● = usually needs to be evaluated; ○ = evaluate case by case
Consult the full ISO 10993-1 matrix for actual projects

6. Key Biological Endpoints in Detail

1. Cytotoxicity – ISO 10993-5

Purpose: assess the toxicity of materials to cells in vitro. Common methods:

  • MTT assay: measures cellular metabolic activity (most common)

  • XTT and MTS assays: similar to MTT

  • Neutral red uptake

  • Colony formation assay

Test approaches:

  • Extract method: cells treated with an extract

  • Direct contact method: material in direct contact with cells

  • Indirect contact method: agar overlay

Acceptance criteria:

  • Cell viability ≥70%: non-cytotoxic

  • 50–70%: mild

  • 30–50%: moderate

2. Sensitization – ISO 10993-10

Purpose: assess whether the material causes delayed-type hypersensitivity. Common methods:

  • Guinea pig maximization test (GPMT): the classic method

  • Buehler test: closed patch

  • Murine local lymph node assay (LLNA): better for animal welfare, preferred

Evaluation: sensitization rate, skin reaction scores

3. Irritation and intracutaneous reactivity – ISO 10993-23

Purpose: assess irritation of the skin, mucosa, eyes, etc. Common methods:

  • Rabbit intracutaneous reactivity test: the classic method

  • Skin irritation test

  • Eye irritation test

  • Oral/vaginal/rectal mucosal irritation tests

  • Reconstructed human epidermis (RhE) model: an in vitro alternative (increasingly accepted)

4. Acute systemic toxicity – ISO 10993-11

Purpose: toxic effects within 24–72 hours after a single exposure. Method: extracts administered to mice/rats intravenously, intraperitoneally, orally or subcutaneously. Observations: clinical signs, body weight, mortality

5. Subacute/subchronic/chronic toxicity – ISO 10993-11

Type Contact duration
Subacute 14–28 days
Subchronic 90 days
Chronic ≥6 months

Observations: body weight, hematology, clinical chemistry, urinalysis, histopathology

6. Genotoxicity – ISO 10993-3

Purpose: assess DNA damage and gene mutations. Common battery (three tests):

  • Ames test (bacterial reverse mutation): gene mutations

  • Chromosomal aberration test or micronucleus test: chromosomal damage

  • Mouse lymphoma assay or in vivo micronucleus test: in vivo confirmation

7. Implantation – ISO 10993-6

Purpose: assess local tissue reactions after implantation. Sites: subcutaneous, muscle, bone. Time points: 1, 4, 12, 26, 52 weeks, etc. Evaluation: inflammation, fibrous encapsulation, necrosis, vascularization, tissue reaction scores

8. Hemocompatibility – ISO 10993-4

Purpose: assess reactions when the material contacts blood. Evaluation categories:

  • Hemolysis (routinely required)

  • Coagulation (PT, APTT, TT)

  • Platelets (count, activation, aggregation)

  • Hematology (blood cell counts)

  • Complement system (C3a, C5a, SC5b-9)

Note: a hemolysis test is required for almost all blood-contacting devices.

9. Carcinogenicity – ISO 10993-3

Purpose: assess whether long-term exposure causes cancer. Duration: ≥2 years (lifetime). Alternative: may be waived if the material’s chemical composition is well defined and toxicological data are sufficient

10. Reproductive and developmental toxicity – ISO 10993-3

Purpose: assess effects on the reproductive system, embryonic development and offspring. Applies to: devices that may contact reproductive organs, pregnant women or newborns

11. Degradation products – ISO 10993-9/13/14/15

Purpose: assess the products of material degradation and their toxicity. Categories:

  • Polymers (Part 13): hydrolysis and oxidation products

  • Ceramics (Part 14): ion release

  • Metals (Part 15): metal ion release

  • Bioabsorbable materials: special attention

12. Chemical characterization – ISO 10993-18

See our Chemical Characterization page – the core input to modern biological evaluation.

7. Biological Evaluation Process (ISO 10993-1)

1. Gather device information (materials, processes, sterilization, clinical use)
 ↓
2. Risk management (ISO 14971)
 ↓
3. Chemical characterization (ISO 10993-18) + literature review
 ↓
4. Biological evaluation plan (BEP)
 ↓
5. Gap analysis
 Are existing data sufficient?
 ↓
 ┌────┴────┐
 ↓ ↓
Sufficient data Insufficient data
 ↓ ↓
 │ Perform necessary biological tests
 ↓ ↓
 └────┬────┘
 ↓
6. Toxicological risk assessment (ISO 10993-17)
 ↓
7. Biological evaluation report (BER)
 ↓
8. Ongoing evaluation (life cycle)

8. The Modern Approach to Biological Evaluation

Traditional vs. modern approach

Dimension Traditional approach Modern approach (2018 edition onwards)
Starting point Run every test in the matrix Based on risk assessment
Chemical information Secondary Core input
Animal testing Extensive Minimized (3R principles)
Use of literature Limited Make full use of existing data
Depth of evaluation Test-result driven Risk driven

Reducing animal testing (3R principles)

  • Replacement: in vitro tests, in silico models

  • Reduction: optimized design, fewer animals

  • Refinement: improved animal welfare

9. Sample Preparation (ISO 10993-12)

Extraction conditions

Contact duration Recommended extraction conditions
≤24 hours 37 ±1 °C, 72 h or 50 ±2 °C, 72 h
24h~30d 37 ±1 °C, 72 h or 70 ±2 °C, 24 h
>30d 37 ±1 °C, 72 h or 50 ±2 °C, 72 h
Exaggerated 121 °C, 1 hour (heat-resistant materials only)

Extraction vehicles

  • Polar: saline, culture medium, water for injection

  • Non-polar: vegetable oil, polyethylene glycol

  • Where necessary: ethanol/water, DMSO

Extraction ratio (surface area/volume)

Material thickness/shape Ratio
Thickness <0.5 mm 6 cm²/mL
Thickness 0.5–1 mm 3 cm²/mL
Thickness >1 mm 3 cm²/mL or 6 cm²/mL
Irregular/granular 0.2 g/mL

10. Typical Evaluation Endpoints for Different Devices

Device type Typical endpoints
Adhesive bandages/dressings Cytotoxicity, sensitization, irritation
Contact lenses Cytotoxicity, sensitization, eye irritation
Infusion sets Cytotoxicity, sensitization, irritation, acute systemic toxicity, hemolysis
Intravenous catheters (short-term) Cytotoxicity, sensitization, irritation, acute systemic toxicity, hemolysis, chemical characterization
Coronary stents Full set (except a few special items)
Artificial joints Cytotoxicity, sensitization, irritation, systemic toxicity, genotoxicity, implantation, degradation, chemical characterization
Bone cement Cytotoxicity, sensitization, irritation, acute systemic toxicity, implantation, degradation
Surgical instruments (reusable) Cytotoxicity, sensitization, irritation
Dental implants Cytotoxicity, sensitization, irritation, implantation, genotoxicity, degradation

11. Factors Affecting Biological Evaluation

1. Material factors

  • Chemical composition, additives

  • Surface condition (coatings, roughness)

  • Physical form (sheet, granules, fibers)

2. Process factors

  • Residual processing aids (mold release agents, lubricants)

  • Cleaning processes

  • Sterilization method (EO, irradiation, moist heat)

  • Sterilization residues (EO, ECH)

3. Clinical factors

  • Contact site

  • Contact time and frequency

  • Patient population (children, pregnant women, immunocompromised)

  • Cumulative exposure

4. Packaging factors

  • Leachables from packaging materials

  • Effect of storage conditions on materials

12. Common Challenges and Pitfalls

1. Misunderstanding the matrix

  • Misjudging the nature or duration of contact, leading to missing or excessive endpoints

2. Improper sample preparation

  • Not reflecting actual processing (after sterilization, cleaning)

  • Unscientific extraction conditions

3. Insufficient chemical characterization

  • TOC alone cannot replace full chemical characterization

  • Incorrect AET calculation

4. Weak literature review

  • Not making full use of data on marketed equivalent devices

  • Insufficient chemical equivalence arguments

5. Misinterpreting test results

  • Giving up as soon as mild cytotoxicity is found

  • Not considering the clinical context as a whole

6. Lack of change management

  • Material, process or supplier changes not re-evaluated

7. Regulatory differences

  • The FDA and NMPA have different requirements for some tests

  • The EU MDR is stricter on post-market surveillance

13. Regulatory Considerations

FDA

  • Accepts ISO 10993-1 as the main basis

  • Emphasizes chemical characterization + toxicological assessment to reduce animal testing

  • Requires complete BEP/BER submissions

  • In some cases requires chemical characterization as evidence of biocompatibility equivalence

EU MDR

  • Strict life-cycle evaluation

  • Ongoing post-market surveillance requirements

  • Emphasis on integrating risk management and clinical evaluation

China NMPA

  • Identical adoption of the ISO 10993 series (GB/T 16886)

  • Accepts comparison with equivalent devices to waive some tests

  • Stricter requirements for novel materials

  • Emphasis on traditional animal test data (changing)

International trends

  • Global adoption of the 3R principles

  • Chemical characterization + TRA becoming mainstream

  • In vitro alternatives increasingly accepted (e.g. RhE models)

14. Key Contents of a Biological Evaluation Report (BER)

  1. Device information: name, intended use, composition, clinical application

  2. Material information: formulation, suppliers, processing

  3. Contact category: nature, duration and site of contact

  4. Risk management input: identified biological risks

  5. Chemical characterization results

  6. Review of existing data: literature, equivalent devices, historical data

  7. Gap analysis: determining which tests need to be added

  8. Summary of test reports: method, results and conclusion for each test

  9. Toxicological risk assessment (TRA)

  10. Overall evaluation and conclusion: whether the risk is acceptable

  11. Life-cycle monitoring plan

15. Typical Project Timelines and Costs

Timelines for a complete biocompatibility evaluation

Endpoint Timeline
Cytotoxicity 2–4 weeks
Sensitization (GPMT/LLNA) 6–8 weeks
Irritation (intracutaneous/skin) 2–4 weeks
Acute systemic toxicity 4–6 weeks
Subacute/subchronic toxicity 4–6 months
Genotoxicity (three-test battery) 2–4 months
Implantation Depending on duration, 3–12 months
Chronic toxicity/carcinogenicity 1–2 years
Chemical characterization + TRA 2–4 months

Cost ranges

  • Basic package (cytotoxicity + sensitization + irritation): tens of thousands of RMB

  • Intermediate package (including systemic toxicity, implantation): from over a hundred thousand to several hundred thousand RMB

  • Complete package (high-risk implants): from several hundred thousand to a million RMB

  • Chemical characterization + TRA: from tens of thousands to hundreds of thousands of RMB

16. Recommendations and Best Practices

1. Plan early

  • Start planning the biological evaluation early in product development

  • Consider biocompatibility when selecting materials

2. Prioritize chemical characterization

  • Chemical data can guide the design of subsequent tests

  • May waive a large number of animal tests

3. Make full use of historical data

  • Equivalent device comparisons, literature reviews, supplier data

  • Justify the applicability of the data

4. Choosing a third-party laboratory

  • Choose GLP-qualified laboratories

  • China: CMA, CNAS accreditation

  • International: FDA GLP, OECD GLP

5. Life-cycle perspective

  • Not just a one-time evaluation at registration

  • Ongoing for changes and post-market surveillance

6. Interdisciplinary collaboration

  • Collaboration across materials, toxicology, clinical and regulatory

  • Engage toxicology experts where necessary

7. Complete documentation

  • Write the BEP/BER using structured templates

  • Traceable data and well-founded decisions

17. Emerging Trends

1. Microphysiological systems (organ-on-chip)

  • Simulating organ function in vitro, with potential to replace animal testing

2. Computational toxicology (in silico)

  • Predicting toxicity with QSAR and PBPK models

3. High-throughput screening

  • Rapid early screening of materials

4. Digital twins

  • Simulating the long-term behavior of devices in the body

5. Evaluation of nanomaterials

  • The new ISO 10993-22 specifically addresses nanomaterials

6. 3D-printed/personalized devices

  • Process diversity brings new challenges

7. Bioabsorbable materials

  • Dynamic evaluation of the degradation process

18. Relationship Between Biocompatibility and Other Tests

Material selection → chemical characterization → biocompatibility evaluation
 ↓ ↓
 Toxicological assessment Clinical evaluation
 ↓ ↓
 Risk management ←——————————┘
 ↓
 Registration submission + post-market surveillance

  • With chemical characterization: mutual inputs; chemical data guide the biological evaluation

  • With the sterile barrier: the sterilization method affects biocompatibility

  • With aging tests: material degradation after aging may affect biocompatibility

  • With clinical evaluation: biological data support clinical safety

  • With risk management: biological risk is part of the overall risk

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