ISO/DIS 10993-11.2:2026(en)

ISO/TC 194/WG 7

Secretariat: DIN

Biological evaluation of medical devices — Part 11: Tests for systemic toxicity

Évaluation biologique des dispositifs médicaux — Partie 11: Essais de toxicité systémique

Date: 2026-04-28

© ISO 2026

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Contents Page

Foreword v

Introduction vii

4.1 General 3

4.2 Selection of animal model 4

4.3 Animal status 5

4.4 Animal care and husbandry 5

4.5 Size and number of groups 5

4.5.1 Size of groups 5

4.5.2 Number of groups 6

4.5.3 Treatment controls 6

4.6 Route of administration 6

4.7 Sample preparation 6

4.8 Dosing 7

4.8.1 Test sample administration considerations 7

4.8.2 Dose 7

4.8.3 Dosing frequency 8

4.9 Body weight and food/water consumption 8

4.10 Clinical observations 9

4.11 Clinical pathology 9

4.12 Anatomic pathology 9

4.13 Study designs 10

4.14 Quality of investigation 10

5.1 General 10

5.2 Study design 11

5.2.1 Pre-study activities 11

5.2.2 Experimental animals 11

5.2.3 Test conditions 11

5.2.4 Body weights 12

5.2.5 Clinical observations 12

5.2.6 Pathology 12

5.3 Evaluation criteria 13

5.3.1 General 13

5.3.2 Evaluation of results 13

5.4 Final report 14

6.1 General 15

6.2 Study design 16

6.2.1 Pre-study activities 16

6.2.2 Experimental animals 16

6.2.3 Test conditions 16

6.2.4 Body weights 17

6.2.5 Clinical observations 17

6.2.6 Pathology 17

6.3 Evaluation criteria 18

6.3.1 General 18

6.3.2 Evaluation of results 18

6.4 Final report 19

Annex A (informative) Routes of administration 21

Annex B (informative) Dose volumes 24

Annex C (informative) Common clinical signs and observations 25

Annex D (informative) Suggested haematology, clinical chemistry and urinalysis measurements 27

Annex E (informative) Suggested organ list for histopathological evaluation 29

Annex F (informative) Organ list for limited histopathology for medical devices subjected to systemic toxicity testing 31

Annex G (informative) Information on pyrogens 33

Annex H (informative) Repeated Dose (14 d or 28 d) Toxicity Study in Rats — Dual routes of parenteral administration 38

Annex ZA (informative) Relationship between this European Standard the General Safety and Performance Requirements of Regulation (EU) 2017/745 aimed to be covered 40

Bibliography 43

Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction or on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 194 Biological and clinical evaluation of medical devices, in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 206, Biocompatibility of medical and dental materials and devices, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

This fourth edition cancels and replaces the third edition (ISO 10993-11:2017), which has been technically revised with the following changes:

— emphasized risk assessment based on available data as a first step;

— added rabbits to Table 1 for group sizes;

— provided guidance on exaggeration of the human dose for toxicity studies;

— provided additional examples of clinical signs and observations in Annex C;

— revision of Annex G;

— provided clarification on study duration for studies described in Annex H;

— the Bibliography was updated.

A list of all parts in the ISO 10993 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www.iso.org/members.html.

Introduction

Systemic toxicity is a potential adverse effect of the use of medical devices. Generalized effects, as well as organ and organ system effects can result from absorption, distribution and metabolism of constituents from the device or its materials to parts of the body with which they are not in direct contact. This document addresses the evaluation of generalized systemic toxicity, not specific target organ or organ system toxicity, even though these effects may result from the systemic absorption and distribution of toxicants.

Given the broad range of medical devices, and their materials and intended uses, this document is not overly prescriptive. While it addresses specific methodological aspects to be considered in the design of systemic toxicity tests, proper study design should be uniquely tailored to the nature of the device’s materials and its intended clinical application.

Other elements of this document are prescriptive in nature, including those aspects that address conformity with good laboratory practices and elements for inclusion in reporting.

While some toxicity tests (e.g. long-term implantation or dermal toxicity studies) can be designed to study systemic effects as well as local, carcinogenic or reproductive effects, this document focuses only on those aspects of such studies, which are intended to address systemic effects. Studies which are intended to address other biological effects are addressed in ISO 10993-3, ISO 10993-4, ISO 10993‑5, ISO 10993-6, ISO 10993-10, ISO 10993-23 and ISO/TS 10993-20.

Prior to conducting a systemic toxicity study, all reasonably available data and scientifically sound methods in the planning and refinement of the systemic toxicity study design should be reviewed. This includes the suitability of use of existing toxicological data, chemistry data or other biological test data (including from in vitro tests and less invasive in vivo tests) for the refinement of study design (dose selection, or selection of pathological endpoints). For the long-term exposure systemic toxicity study in particular, the use of scientifically sound study design, the use of pilot studies and statistical study design and the use of unbiased, quantitative endpoints or methods in the pathological assessment (including clinical pathology, gross pathology and histopathology) are important so as to obtain data which have sufficient scientific validity.

The outcome of any single test should not be the sole basis for making a determination of whether a device is safe for its intended use.

Biological evaluation of medical devices — Part 11: Tests for systemic toxicity

This document specifies requirements and gives guidance on procedures to be followed in the evaluation of the potential for medical device materials or final products to cause adverse systemic reactions.

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10993‑1, Biological evaluation of medical devices — Part 1: Requirements and general principles for the evaluation of biological safety within a risk management process

ISO 10993‑2, Biological evaluation of medical devices — Part 2: Animal welfare requirements

ISO 10993‑6, Biological evaluation of medical devices — Part 6: Tests for local effects after implantation

ISO 10993‑12:2021, Biological evaluation of medical devices — Part 12: Sample preparation and reference materials

For the purposes of this document, the terms and definitions given in ISO 10993-1 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp

— IEC Electropedia: available at https://www.electropedia.org/

3.1

biological harm

injury to humans from one or more adverse biological effects associated with a medical device or material

[SOURCE: ISO 10993-1:2025, 3.6]

3.2

constituent

chemical that is present in or on the finished medical device or its materials of construction

Note 1 to entry: Constituents can be intentionally or unintentionally added chemicals or compounds, such as: additives (e.g. plasticizers, lubricants, stabilizers, anti-oxidants, colouring agents, fillers), manufacturing process residues (e.g. monomers, catalysts, solvents, sterilant and cleaning agents), degradation products, reaction products, or impurities or contaminants.

[SOURCE: ISO 10993-1:2025, 3.15]

3.3

dose

amount of test sample administered (e.g. mass, volume) per unit of body weight or surface area

3.4

dosage

refers to a specific amount of drug administered at a specific frequency (and over a certain duration

3.5

dose-response effect

relationship of dosage to the spectrum of effects related to the exposure either in an individual or in a population of individuals to a range of doses

3.6

leachable

substance that is released from a medical device or material during its clinical use

[SOURCE: ISO 10993-1:2025, 3.24]

EXAMPLE Additives, sterilant residues, process residues, degradation products, solvents, plasticizers, lubricants, catalysts, stabilizers, anti-oxidants, colouring agents, fillers and monomers.

Note 1 to entry: Leachable substances related to the use of gas pathway devices can be evaluated according to the ISO 18562-4.

3.7

limit test

use of a single group treated at a suitably high dosage of test sample in order to delineate the presence or absence of a toxic hazard

Note 1 to entry: If the group is not toxic at this high dose, further testing at higher dosages is generally not necessary.

3.8

Long-term exposure

medical device that has a total exposure period of more than 30 d

Note 1 to entry: More information can be found in ISO 10993-1:2025, 6.4.2 c).

3.9

systemic toxicity

harm that occurs in an organ or system other than at the contact site

Note 1 to entry: Systemic toxicity can occur after a one-time exposure (i.e. acutely) or after repeated or ongoing exposure (e.g. subacute or subchronic or chronic) to a harmful dose of a constituent released from a single medical device or from use of multiple medical devices.

Note 2 to entry: The contact site is the specific location at which the medical device interfaces or interacts with the tissue.

3.10

acute systemic toxicity

adverse effects occurring within 72 h following a single or repeated administration of a test sample for a period of up to 24 h

3.11

subacute systemic toxicity

adverse effects occurring after repeated exposure or continuous exposure of a test sample for a period of up to 28 d

Note 1 to entry: Exposure by implantation or topical application can be viewed as continuous exposure, Subacute repeated intravenous and intraperitoneal studies are generally defined as exposure durations of ≤14 to 28 d for rodents

3.12

subchronic systemic toxicity

adverse effects occurring after long-term or continuous exposure of a test sample for a period of 90 d

3.13

chronic systemic toxicity

adverse effects occurring after long-term or continuous exposure of a test sample for a major part of the life span

Note 1 to entry: Chronic toxicity studies usually have a duration of at least six months in rodents and rabbits^[35] or nine months in large animal species^[36].

3.14

test sample

medical device, component or material (or a representative sample thereof, manufactured and processed by equivalent methods), or an extract or portion thereof that is subjected to biological evaluation testing

[SOURCE: ISO 10993-12:2021, 3.14]

Before a decision to perform a systemic toxicity test is made, a biological evaluation as described in ISO 10993-1 shall be conducted. To evaluate potential systemic toxicity risks of medical devices, consideration should first be given to the availability and applicability of chemical characterization and toxicological risk assessment data before pursuing systemic toxicity testing using an animal model. When there is not sufficient data to estimate the risk of systemic toxicity using either relevant existing information or chemical characterization according to ISO 10993-18 followed by a toxicological risk assessment according to ISO 10993-17, then in vivo systemic toxicity studies should be considered. For example, when the outcome of chemical characterization and toxicological risk assessment is inconclusive to assess tolerable risk and worst-case exposure dose of extractable constituent is not well understood, then an in vivo systemic toxicity test can be considered.

NOTE 1 Some devices can contain such low concentrations of extractable or leachable constituents that it is unlikely to result in an observed adverse effect in a systemic toxicity test (see ISO 10993-18 and ISO 10993-17). Chemical analysis of test sample extracts can provide information on whether in vivo systemic toxicity testing is potentially useful to the overall biological evaluation.

EXAMPLE 1 The analytical results from a water extract can provide a reasonable estimate of the composition and concentration of device constituents in the saline extract used for dosing an in vivo study, if:

— extraction conditions are similar and

— identification and quantitation are adequate.

EXAMPLE 2 The analytical results from all extracts in an exhaustive extraction study can provide a reasonable estimate of the potential systemic exposure from a systemic toxicity study performed using implantation as the route of exposure.

If available, such information shall be considered when designing a systemic toxicity study. Where constituent concentrations correspond to a dose to the animal are less than approximately 0,015 mg/kg/day to 0,15 mg/kg/day, in vivo effects are unlikely to be observed. Particularly, chemical characterization according to ISO 10993-18 should inform whether the study will be useful for the overall biological evaluation^[4].

NOTE 2 A reasonable estimate of very low levels of extractables is that which would equate to a dose to the animal of approximately 0,015 to 0,15 mg/kg/day. The high end of the range is based on the 5th percentile of the oral NOAELs used to calculate the lowest Cramer Class TTC value.^[5] The low end of the range accounts for a potential 10-fold difference in toxicity between oral NOAELs used to derive the TTC and parenteral routes used in most medical device extract testing.

EXAMPLE 3 A long-term medical device with a surface area of 300 cm² was exhaustively extracted according to ISO 10993-18. The water extract of the device contained a single extractable that was identified, and the total quantity was reported to be 10 μg. For a systemic toxicity test using a saline extract, the 300-cm² device would be extracted at 6 cm²/ml with a total extraction volume of 50 ml. Assuming the extraction efficiency of the extractable from the device remains the same between water and saline and the same extraction conditions are used, the resulting saline extract would contain the extractable at a concentration of 0,2 μg/ml. At a dose volume of 50 ml/kg IP, the dose to the animal would be 10 μg/kg. If given daily, the dose would be 10 μg/kg/day (0,01 mg/kg/day), which is less than 0,015 mg/kg/day. This calculation indicates that a systemic toxicity test using a saline extract would be unlikely to result in adverse effects and would not be useful for the overall biological evaluation of the device.

Testing shall be performed on the final product or representative component samples, or materials of the final product. Test samples shall reflect the conditions under which the device is normally manufactured and processed. If modifications to the manufacturing and processing conditions are necessary, these should be documented by the manufacturer, together with their justification and captured in the biological evaluation plan. For hazard identification purposes, it can be necessary to exaggerate exposure to the test samples. It could also be necessary to determine the dose for implantation-based systemic toxicity studies, including, but not limited to calculation of dose based on animal weight and worst-case clinical use, and accounting for a safety factor.

Physical and chemical properties of the test sample including, for example, pH, stability, viscosity, osmolality, buffering capacity, solubility and sterility, are some factors to consider when designing the study.

When animal tests are considered, all reasonably and practically available replacement, reduction and refinement (3Rs) alternatives should be identified and implemented to satisfy the provisions of ISO 10993-2. Animal studies should be tailored to address the specific type of systemic toxicity for which data or information are lacking. For instance, if sufficient data exists for chronic toxicity but not for acute toxicity, in vivo studies should be limited to acute toxicity designs.

There is no absolute criterion for selecting a particular animal species for systemic toxicity testing of medical devices. However, the species used shall be scientifically justified and in line with the provisions of ISO 10993-2. For acute oral, intravenous, dermal and inhalation studies of medical devices, rodents (mouse or rat) are preferred. Rabbits, which are lagomorphs, are an option in dermal studies and preferred in the case of implantation studies where a larger model is needed due to the size of the implant. Other non-rodent species can be considered for testing, recognizing that a number of factors might dictate the number or choice of species for study.

It is preferred that a single animal species and strain are used when a series of systemic toxicity studies of different durations are performed, e.g. subacute, subchronic and chronic systemic toxicity. This minimizes the variability between species and strains and facilitates an evaluation based primarily on study duration. Should multiple species or strains be used, justification for their selection shall be documented.

Generally, healthy purpose-bred young adult animals, as determined by qualified veterinary personnel, and, of known species, strain, substrain, age, sex, source and with defined microbiological and pathogen health status should be used. At the commencement of the study, the weight variation of animals used within a sex should not exceed ±20 % of the mean weight. When females are used, they should be nulliparous and non-pregnant. Animal selection shall be justified.

Care and handling of animals shall be in accordance with the animal care guidelines of the country in which the test facility is located. Animals shall be acclimatized to the laboratory conditions prior to treatment and the period of time documented. Control of environmental conditions and proper animal care techniques are essential to animal well-being, minimization of stress-related physiological responses and the quality of the results. Dietary constituents and bedding materials that are known to produce or influence toxicity should be properly characterized and their potential to influence test results taken into account.

The temperature and the relative humidity in the experimental animal rooms should be appropriate for the species, e.g. (22 ± 3) °C and relative humidity of 30 % to 70 %, for rodents. Typically, the artificial lighting sequence should be 12 h light, 12 h dark.

For feeding, standardized commercial laboratory diets may be used with an unlimited supply of drinking water. Animals should be caged in-groups by sex or individually, as appropriate; for group housing not more than five animals shall be housed per cage.

The study should use the least number of animals to detect meaningful differences in biological responses and provide meaningful interpretation of the data (see ISO 10993-2). Recommended minimum group sizes, with all routes of test sample administration considered, are given in Table 1.

Table 1 — Recommended minimum group sizes^a

When the testing is designed to address both systemic toxicity and implantation endpoints, group sizes shall meet both ISO 10993-11 and ISO 10993-6 requirements. If both sets of requirements can’t be met, justification shall be provided and documented.

One dose group treated at a suitable dosage of test sample in a single species could delineate the presence or absence of a hazard (i.e. limit test). However, other multi-dose or dose response studies require multiple groups to delineate the toxic response.

The number of treatment groups may be increased when attempting to characterize a dose response using exaggerated doses. The following examples for exaggerating the dose should be considered:

— multiples of the human dose based on device mass or number/per patient body weight;

— multiples of the surface area of human exposure per patient body weight;

— multiples of the duration of exposure;

— multiples of the amounts of extractable fraction or individual chemicals from the device per patient body weight;

— multiple administrations within a 24-h period.

Other methods to exaggerate the dose may be acceptable. The method used shall be justified.

Depending on the objective of the study, the nature of the test sample and the route of exposure, negative control, vehicle control, or sham-treated controls, shall be incorporated into all systemic toxicity studies. These controls shall mimic the test sample preparation and treatment procedure.

Medical devices or their leachable constituent s may gain access to the body by multiple routes of exposure. The test route of administration for a systemic toxicity test should be chosen based on the ability to exaggerate the systemic dose balanced with clinical relevance. Sometimes parenteral dosing with device extracts can exaggerate the dose more readily than implantation. However, there is limitation in repeated administration of extracts into animals due to their inherent stress and physiological tolerance. Therefore, extract studies (via intravenous and intraperitoneal administration) are typically limited to systemic toxicity up to 28 d, see Annex H. The route of administration shall be justified. Examples of routes of administration can be found in Annex A.

The test and control samples and their preparation (such as pH, stability, homogeneity, osmolality, and sterility, as appropriate) shall be described and justified. All samples and vehicles for parenteral delivery should be prepared aseptically. Further guidance on sample preparation is given in ISO 10993-12.

Procedures should be designed to avoid physiological changes or animal welfare problems not directly related to the toxicity of the test material. Current proper handling and restraint techniques that minimize aversion and anxiety levels in animals shall be practiced (for example, picking up mice through tunnel or cupped hand instead of by the tail). If a single daily dose of a sufficient volume or concentration is not possible, the dose may be given in smaller fractions over a period not exceeding 24 h.

Test samples shall be delivered at a physiologically acceptable temperature. Aseptic techniques shall be used when samples are given parenterally. In general, test samples are used at or near room temperature (e.g. 25 °C) or body temperature (e.g. 37 °C), with the temperature documented and justified.

Vehicles administered by a parenteral route should be physiologically compatible. When necessary, if gravimetric sedimentation is ineffective, extract centrifugation or filtration to remove particulates can be used. These post-extraction manipulations shall be documented, and justified. In addition, alternate administration routes (e.g. intraperitoneal injections) can be considered and shall be justified. When medical devices or test samples in the form of nanomaterials are to be evaluated, special considerations may be necessary for the sample preparation (e.g. the use of nano-object dispersions instead of extracts).

NOTE For more information, see ISO/TR 10993-22:2017, Clause 6.

Prolonged restraint of animals in long-term exposure systemic toxicity studies should be scientifically justified and performed in a manner that is as humane as possible. Animals shall have adequate room for thoracic and abdominal expansion during breathing, and comfortable surface for resting the head and body. The nature and the duration of restraint should be the minimum required to meet the scientific objectives and should not of themselves compromise the welfare of the test animals. Deviations shall be justified.

Further guidelines on prolonged restraint can be found in the Guide for the Care and Use of Laboratory Animals.^[22] When restraint is required animals should be acclimatized to the restraint device prior to test sample administration. Minimal effective restraint of test animals is a key factor to be considered for prolonged infusion.

Guidance on dose volume is summarized in Annex B. Multiple dose volume groups and use of dose volumes greater than those given in Annex B shall be justified.

Large dose volumes administered by the oral route should be avoided because they have been shown to overload the stomach capacity and pass immediately into the small bowel. Large volumes may also reflux into the oesophagus.

Intramuscular administration is also volume-limited, depending on size of the animal and the muscular site. Species-specific intramuscular administration volumes are addressed in Annex B.

Bolus intravenous injection volumes are usually given over a period of time dependent on the species. The rate of injection is an important factor and a maximum of 2 ml/min is suggested for rodents.

Slow or timed injection, or intravenous infusion, may be required for large volume administration. Regardless of the calculated rate, the rate of fluid administration shall be stopped or decreased if the animal demonstrates a marked change in clinical condition.

Slow intravenous injection rates may be necessary for test samples limited by solubility or irritancy.

Continuous infusion may be used if clinically indicated. The volume and rate of administration will depend on the substance being given and take into account standard fluid therapy practice.

For subcutaneous administration of test sample, refer to Annex B. The rate and extent of absorption depends on the test sample formulation.

For implantable devices, often the most clinically relevant route of exposure is by implantation. Where practical, the route of administration should mimic the clinical route or tissues of exposure. When possible, the implanted sample should represent an exaggeration of the human clinical dose on a mg/kg body weight basis. In some cases, a surface area (cm²/kg body weight) or volume (ml/kg body weight) basis of exaggeration may be used based on what is most appropriate for the subject device and its indication(s). Other bases of providing an exaggerated dose may be used with justification. A suggested exaggeration is 10 to 100 times the proportional human dose unless not technically achievable. Ideally, the highest exaggeration factor is to be considered when feasible, e.g. 100X. Additionally, this exaggeration should consider the worst case human population for use, e.g. – adult, children, or infants, as appropriate. When the 10X minimum exaggerated dose is impractical, the dose <10X should be justified. Depending on the nature and size of the device an exaggerated dose, it may not be feasible to implant in the clinical location of use or tissues of exposure. Implantation in alternative tissue(s) may be considered with justification. For example, while relevant route of exposure is preferred, subcutaneous implant sites have the advantage of being able to accommodate proportionately large doses, allowing for exaggeration of the typical clinical dose. This could be a consideration when the most clinically relevant route of exposure is not possible, and an alternative exposure route needs to be selected. For devices that are in contact with internal tissues or reside in the vascular system (e.g. hemodialysis filter), intravenous or intraperitoneal administration of extracts may be an appropriate means to provide exposure (see Annex H). For devices in which the patient is exposed to leachables via inhalation, see the ISO 18562-4 for additional guidance.

For studies where the route of administration is via implantation, the amount/volume of a final finished device, portion thereof, or material implanted should be compatible with the test system and not be excessively large. If a large device or a device with multiple components is being implanted, the entire device shall be implanted in the same animal so that systemic toxicity of the entire device can be assessed. If it is not technically feasible to implant the entire device, representative portions of the entire device should be implanted in each test animal. If the test sample has sharp edges/corners this could potentially result in skin perforations in a subcutaneous implant study. In cases where toxic effect may be expected, multiple dose levels may be advisable rather than a single limit type dose.

The dosing frequency should be based on clinical relevancy. Exaggerated dose volumes shall be clearly specified and justified. Single or repeated dosing or extract delivery should consider technique refinements that can enhance animal comfort and to prevent compromising test results due to undue stress,^[13] including, but not limited to, the use of the smallest needle, plastic orogastric needle^[14,15] or catheter size possible to minimize trauma.

In acute systemic toxicity studies, the animals should be exposed to the test sample in a single dose or with multiple fractions of the dose given within a 24 h period.

In repeated exposure studies with extracts, the animals should be dosed with the test sample daily, seven days each week for the duration of the test (see Annex H for further discussion of dual route exposure of extracts). Other dosage regimens may be acceptable but shall be justified.

Body weight change and changes in food and water consumption may be attributed to the effects of a test sample. Consequently, individual weights of the animals shall be determined shortly before the test sample is administered (e.g. usually within 24 h for single or acute dosing, and no more than 7 d for long-term exposure studies), at regular intervals throughout the study and at study termination. If dose volume/amount of extracts or solutions for each animal is calculated by body weight, the most recent body weight should be utilized.

Body weight measurements shall be conducted by using a balance that is properly calibrated and maintained, and, has proper accuracy and readability. For weight measurement of mice, balance that has at least 0,1 g readability and calibrated to 0,1 g accuracy shall be used.

Animals shall be properly restrained when conducting the weight measurement so that the balance reading can be stabilized before recording the animal weights.

Measurements of food and water consumption, as appropriate, can be considered for longer-term (e.g., subacute, subchronic or chronic) exposure studies.

Clinical observations should be performed by trained individuals to ensure consistent reporting. The frequency and duration of observation should be determined by the nature and severity of the clinical signs, toxic reactions, rate of onset and recovery period. Increased frequency of observation may be necessary in the early phase of a study, especially acute studies. The time at which signs of toxicity appear and disappear, their duration and the time of death are important, especially if there is a tendency for adverse clinical signs or deaths to be delayed. Humane endpoints, as defined by national or international animal welfare guidelines, shall be preferable to death or moribundity since humane endpoints minimize pain and distress^[25],^[31]. General clinical observations shall consider the peak period of anticipated effects after dosing.

Observations shall be recorded systematically and contemporaneously as they are made. Records shall be maintained for each animal.

Cage-side observations for viability or overt clinical signs shall be recorded at least once each day using common laboratory descriptors of clinical effects (see Table C.1 and C.2). For some clinical observations of pain, these observations can be captured quantitatively using the Grimace Scale^[27][28].

A more extensive screening for adverse clinical signs may be considered on at least a weekly basis for longer-term (longer than 6 months) long-term exposure studies.

Haematology and clinical chemistry analyses are performed to investigate toxic effects in tissues, organs and other systems. When indicated, these analyses should be performed on blood samples obtained from study animals at least just prior to, or as a part of, the procedure for scheduled animal termination. Fasting of animals prior to blood sampling may be necessary in some cases. When scientifically indicated, urinalysis can be performed during the study or during the last week of a long-term long-term exposure study using timed (e.g. 16 h to 24 h) urine volume collection.

Suggested haematology, clinical chemistry and urinalysis parameters for evaluation are listed in Annex D.

When clinically indicated, gross pathological evaluations should be considered for acute systemic toxicity studies. Gross findings and organ weight data should be reviewed in conjunction with histology slides, when available.

All animals in subacute or long-term systemic toxicity studies shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Selected organs for weighing should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible to avoid drying.

Annex E suggests the tissues that should be weighed and preserved in an appropriate fixation medium for histopathological examination.

A summary of minimum observations for each type of study is given in Table 2. See 5.2.6.2 and 6.2.6.2 for more information.

Table 2 — Summary of observations

Study designs are listed in subsequent clauses of this document. Expert consultation for study design is recommended.

Good laboratory practices deal with the organization, process and conditions under which laboratory studies are planned, performed, monitored, recorded, reported and archived. These practices are intended to ensure the quality, integrity and validity of the test data. They also support the global harmonization effort by facilitating the memoranda of understanding between trading nations. For example, the OECD (Organisation for Economic Co-operation and Development) Mutual Acceptance of Data (MAD) framework allows for test study data generated in any member country in accordance with OECD Test Guidelines and principles of good laboratory practice^[34] to be accepted in other member countries. Systemic toxicity studies shall be conducted following such principles.

Acute systemic toxicity provides general information on biological harms likely to arise from an acute exposure by the intended clinical route. An acute toxicity study can be an initial step in establishing a dosage regimen in subacute/subchronic and other studies and may provide information on the mode of toxic action of a constituent by the intended clinical exposure route. Subsequent to test sample administration in acute systemic toxicity testing, observations are made of effects (e.g. adverse clinical signs, body weight change, gross pathological findings) and deaths. Animals showing severe and enduring signs of distress and pain need to be euthanized immediately. Grimace Scale^[27][28] along with other observations can be used collectively to determine if an animal should be euthanized. Corrosive or irritating materials known to cause marked pain or distress should be reported as such and need not be tested. Humane endpoints, as defined by national or international animal welfare guidelines, shall be preferable to death or moribundity since humane endpoints minimize pain and distress.

NOTE The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the European Centre for the Validation of Alternative Methods (ECVAM) have developed the concept of in vitro cytotoxicity tests that can be considered for use in a weight-of-evidence approach to determine starting doses in acute oral toxicity studies. This is presented in OECD Series on Testing and Assessment No. 129^[10].

Healthy young animals are acclimatized to the laboratory conditions for at least 5 d prior to the test. Shorter durations shall be justified. Animals are then randomized and assigned to the treatment groups.

Selection of species

Typically, a rodent species (rat, mouse) will be used. Signalment characteristics of the model (species, strain/stock, age, sex, weight, etc.) are as specified in 4.2 and 4.3. If non-rodent species are used their use shall be scientifically justified.

NOTE Regarding the age for rodents, OECD 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, clause 7 suggest “Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old.”

Number and sex

The number and type of treatment groups, animals per group, and sex are as specified in 4.5.

Housing and feeding conditions

The temperature and the relative humidity in the experimental animal rooms should be appropriate for the species, e.g. (22 ± 3) °C and relative humidity of 30 % to 70 %, for mice. Typically, the artificial lighting sequence should be 12 h light, 12 h dark.

For feeding, standardized commercial laboratory diets may be used with an unlimited supply of drinking water. Animals should be caged in-groups by sex or individually, as appropriate; for group housing not more than five animals shall be housed per cage.

Dose levels

Dose levels shall be as specified in 4.8.

Animals in the control group should be handled in an identical manner to the test group subjects but should be dosed with the same volume per body weight dosing vehicle used for dosing the treated animals.

Dosing and observation procedures

The animals receive a single dose of the test sample or, when necessary, multiple doses within a single 24 h period. Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.

Daily observation (physical appearance and behaviour) and regular physical examination of the animals are necessary to assess their health and well-being. Any animal showing marked clinical signs shall be promptly reported to the veterinary staff for evaluation, diagnosis and possible treatment. At the end of the study, all surviving animals are euthanized. Any moribund animals shall be removed, euthanized and evaluated post-mortem to investigate the cause of their illness. Methods used for euthanasia should be in accordance with national or international animal welfare guidelines.

The observation schedules and humane endpoints applied should preclude the possibility of animals being found dead as a direct consequence of test sample toxicity.

Body weight measurements should be made just prior to dosing, daily for the first three days after dosing, weekly after the first dose if indicated by study duration, and at the end of the study.

The observation period for an acute systemic toxicity study shall be at least 3 d, or longer when deemed appropriate. Specifics of frequency and observation type are specified in 4.10 and Annex C. In all cases, observations shall be made daily within the observation period. Cage-side observations should include, but not be limited to, changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern, using the descriptors provided in Annex C. Weak animals shall be isolated, and moribund animals shall be euthanized. For animals that are found dead or euthanized early, necropsy including organ examination (histopathology)^[8] shall be done.

Clinical pathology

Clinical pathology evaluations shall be considered when there is an indication, such as for device materials with expected or observed toxicity (from a prior study), or for new device materials where there is no previous experience. When clinical pathology evaluations are performed, the following examinations shall be considered.

a) Haematology, as specified in Annex D, should be considered for investigation at the end of the test period.

b) Clinical biochemical determination on blood, as listed in Annex D, should be considered at the end of the test period. Test areas which are considered appropriate to acute exposure studies are liver and kidney function. Additional clinical biochemistry may be utilized where necessary to extend the observation of the observed effects.

Urinalysis (see Annex D) is not necessary on a routine basis but only when there is an indication based on expected or observed toxicity.

Gross pathology

Gross pathological evaluations shall be considered when there is an indication, such as for device materials with expected or observed toxicity (from a prior study), or for new device materials where there is no previous experience. This should include an examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. When appropriate, consideration should also be given to recording the weight of the brain, liver, kidneys, adrenals and testes or ovaries, which should be weighed wet as soon as possible after dissection to avoid drying and subsequent falsely low values.

Histopathology

Full histopathology is not typically carried out on organs and tissues from animals in the acute systemic toxicity study, unless indicated specifically by unique gross necropsy findings. When animals are found dead or are euthanized prior to the end of the study, it can be useful to conduct organ histopathology to determine a cause of death or morbidity.

Depending on the test design utilized, the following evaluation criteria apply:

a) For pharmacopoeia-type testing:

1) If, during the observation period of an acute systemic toxicity test, none of the animals treated with the test sample shows a substantially greater biological reactivity than animals treated with the vehicle control, the sample meets the requirements of this test.

2) Using five animals, if two or more animals die, or have marked symptoms of toxicity or abnormal behaviour such as convulsions or prostration occurs in two or more animals, or if a final (end of study) body weight loss greater than 10 % occurs in three or more animals, the sample does not meet the requirements of the test. Any transitory body weight loss should be critically evaluated along with other clinical observations in the assessment of systemic toxicity. Any significant body weight losses, i.e. >10 %, shall be discussed in the final report. A transitory body weight loss suggesting a transitory toxicity is not sufficient for performing a repeat test and the sample still meets the requirements of this test.

3) If any animals treated with the sample show only slight signs of toxicity, and not more than one animal shows moderate or marked signs of toxicity or dies, repeat the testing using groups of 10 animals (see Table C.1).

4) On the repeat test, if all 10 animals treated with the sample show no substantially greater reaction than the vehicle control animals during the observation period, the sample meets the requirements of this test.

5) If any animal dies, or other adverse events are noted, these shall be reported and a root cause investigation shall be conducted, including necropsy (gross pathology) and histopathological evaluation, as appropriate.

b) For non-pharmacopoeia acute systemic toxicity tests:

The option exists to perform evaluations using more extensive methods including clinical and anatomic pathology, which may eliminate the need for a repeat test. Acute exposure may include a re-evaluation if there are equivocal differences from concurrent controls. Differences should be explained, and the study extended to include an additional five animals, if applicable.

The findings of an acute systemic toxicity study should be evaluated in conjunction with the findings of preceding studies, if available, and considered in terms of the toxic effects and the gross necropsy findings, if observed. The evaluation shall include the relationship between the dose of the test substance and the presence or absence and the incidence and severity of abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, effects on mortality and any other general or specific effects.

The following information, where applicable, shall be contained in the final test report for the acute systemic toxicity study:

a) Details of the testing laboratory and study sponsor, and rationale for selection of the study design.

b) Test sample:

1) physical nature, purity, and physiochemical properties, as appropriate;

2) other identification data.

c) Extraction solvent or vehicle (if appropriate): justification for choice of extraction solvent or vehicle if other than those listed in ISO 10993-12.

d) Test animals:

1) species/strain used;

2) number, age and sex of animals;

3) source including microbiological status (e.g. barrier raised, conventional), housing conditions (temperature, humidity, bedding, lighting, diet, acclimatization period, etc.);

4) weights at study initiation.

e) Test conditions:

1) rationale for dose selection;

2) details of test sample formulation/preparation; achieved concentrations; stability and homogeneity, if appropriate;

3) details of the administration of the test sample;

4) conversion from test sample concentration (ppm) to the actual dose (mg/kg BW), if applicable;

5) details of food, water and bedding quality.

f) Results:

1) data may be summarized in tabular form, showing for each control and test group the number of animals at the start of the test, the number of animals showing adverse clinical signs, and the number of animals displaying body weight changes;

2) all body weight measurements and body weight change;

3) food and water consumption, if applicable;

4) toxic response data by sex and dose level, if applicable;

5) clinical observations, and the nature, severity and duration of all adverse clinical observations (whether reversible or not);

6) neurological and behavioural assessments, if applicable;

7) haematological tests utilized and results with relevant control data, if applicable;

8) clinical biochemistry tests utilized and results with relevant control data, if applicable;

9) urinalysis tests utilized and results with relevant control data, if applicable;

10) necropsy findings, terminal organ weight data, if applicable;

11) detailed description of all histopathological findings, and narrative that include discussion on the frequency and severity of the findings in test and control animals, if applicable;

12) statistical evaluation of results, if used, and a discussion of their biological significance.

g) Discussion of results:

h) Conclusions;

i) Quality assurance statement.

An acute systemic toxicity study will provide information on the effects of acute exposure to a test substance. Extrapolation of the results of the study to humans is valid to a limited degree but it can provide useful information on permissible exposure.

While acute toxicity deals with the adverse effects of single doses (or limited exposure), a more common form of human exposure to many medical devices is in the form of repeated or continuous exposures. Effects from repeated or continuous exposure may potentially occur due to accumulation of chemicals in tissues or by other mechanisms. Longer duration testing (subacute, subchronic, chronic) can identify these potential effects. For a long-term device, it may not be necessary to conduct all three exposure durations when justified by considering all existing data; e.g. acute systemic toxicity data, preclinical safety study covering both short and long-term implant durations and chemical characterization data may be used to justify omission of subacute/subchronic/chronic systemic toxicity studies. In another example, if data from a subacute and subchronic toxicity study via implantation on a long-term implant device is acceptable, it may not be necessary to conduct a chronic toxicity study. The approach taken should be justified and documented.

Long-term exposure systemic toxicity tests provide information on biological harms likely to arise from a prolonged to long-term exposure by the intended clinical route. It can also provide information on the mode of toxic action of a constituent by the intended clinical exposure route.

Long-term exposure systemic toxicity studies will provide detailed information on toxic effects, target organs, reversibility or other effects and may serve as the basis for safety estimation. Results of these studies provide important information that is reflected in the extent of the guidance of clinical and anatomic pathology investigations.

Long-term exposure studies do not generally provide a retest criterion. Rather, group sizes are designed to accommodate a statistical assessment of the recorded observations (see Table 1).

Because of the variable durations for long-term exposure studies, test samples shall be prepared as required, to ensure their stability.

Healthy young adult animals are acclimatized to the laboratory conditions for at least 5 d prior to the test. Animals are then randomized and assigned to the treatment groups.

Selection of species

Typically, the rodent (rat, mouse) or rabbits are used. Characteristics of the model (species, strain/stock, age, sex, weight etc.) are specified in 4.2 and 4.3. When other species are used their selection shall be scientifically justified.

Number and sex

The number and type of treatment groups, animals per group, and sex are as specified in 4.5. When scientifically justified, consideration should be given to the use of satellite animals treated with the high dose level along with satellite controls for a predetermined period beyond the terminal euthanasia. This group, with its controls, may be used to examine treatment effects including reversibility, persistence or delayed toxic effects.

Housing and feeding conditions

The temperature and the relative humidity in the experimental animal rooms should be appropriate for the species, e.g. (22 ± 3) °C and 30 % to 70 % RH, for rats. Typically, the artificial lighting sequence should be 12 h light, 12 h dark.

For feeding, standardized commercial laboratory diets may be used with an unlimited supply of drinking water. Animals may be caged in groups by sex or individually with justification, as appropriate; for group housing not more than five animals should be housed per cage.

Dose levels

The dose to use for toxicity tests of medical devices shall be defined in relation to the results of risk assessment, balancing the clinical exposure dose with the use of safety factors, as applicable. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects.

Unlike classical chemical studies of long-term exposure systemic toxicity, long-term exposure studies with medical devices often do not result in a dose-response effect, thus a toxic effect at the dose level investigated is not mandatory.

Dosing procedures

Dosing regimen should be based on clinical use of the medical device. Ideally, animals should be dosed 7 d/week for the duration of the study. For longer term repeated exposure studies, dosing on 5 d/week basis is acceptable but should be documented and justified. Consideration to injection site and animal welfare should be given when defining an appropriate dosing frequency, e.g. daily intraperitoneal injections can cause animal distress.

Body weight measurements should be made just prior to dosing, weekly after the first dose if indicated by study duration, and at the end of the study.

The observation period for a long-term systemic toxicity study shall be appropriate for the duration of the study. Specifics of frequency and observation type are specified in 4.10 and Annex C. In all cases, cage-side observations shall be made daily within the observation period. Weak animals should be isolated, and moribund animals shall be euthanized. For animals that are found dead or euthanized early, necropsy including organ examination (histopathology) shall be done^[11]. Cage-side observations should include, but not be limited to, changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern, using the descriptors provided in Annex C.

Typically, when relevant, ophthalmologic examinations, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If changes in the eyes are detected, all animals should be examined.

Clinical pathology

The following examinations should be made:

a) Haematology, as specified in Annex D, should be investigated at the end of the test period. Depending on the length of the study, more frequent sampling should be considered.

b) Clinical biochemical determination on blood should be carried out at the end of the test period. Depending on the length of the study, more frequent sampling should be considered. Test areas that are considered appropriate to all long-term exposure studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests may be influenced by observations on the mode of action of the test substance or chemical characterization data. Suggested determinations are listed in Annex D. Additional clinical biochemistry may be utilized where necessary to extend the observation of the observed effects.

Additional clinical pathology evaluation testing intervals should be selected based on probable understanding of when changes of toxicological significance may be anticipated to occur during the study, whenever possible.

Urinalysis (see Annex D) is not necessary on a routine basis but only when there is an indication based on expected or observed renal toxicity or renal devices.

Historical data for normal values are useful for establishing baseline levels and for comparison with concurrent study controls. If historical baseline data are deemed inadequate, consideration should be given to the collection of this information for animals of the same age, sex, strain and source, preferably within the same laboratory.

Gross pathology

All animals should be subjected to full gross necropsy, which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The adrenals, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testes or ovaries, thymus, and uterus should be weighed wet as soon as possible after dissection to avoid drying and subsequent falsely low values. The organs and tissues listed in Annex E should be preserved in a suitable fixation medium for possible future histopathological examination.

Histopathology

a) Full histopathology should be carried out on organs and tissues from animals in the control and the test group, or the high dose group, if multiple dosage groups are used.

b) All gross lesions should be examined.

c) The lungs of animals in the low and intermediate dose groups, if used, should be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals. Consideration should also be given to histopathological examination of the liver and kidneys in these groups. Further histopathological examination may not be required routinely on the animals in these groups but shall always be carried out in organs which showed evidence of lesions in the high dose group.

d) When a satellite group is used, histopathology may be performed on tissues and organs identified as showing effects in the treated groups.

e) In general, for chronic studies, sentinel animals should be used for monitoring the occurrence of infectious agents. Serology or histology of sentinel groups may be performed as indicated. Microbiological monitoring methods that do not use sentinel animals are available. In non-animal methods, samples collected non-invasively from animals, such as faeces, oral swabs, and body surface swabs, can be used.

f) During selection of organs for histopathology, consideration should be given to chemical characterization of device materials. For example, if the device is coated with drugs/pharmaceutical agents, then target organs for those chemicals should be studied in treated animals for any adverse effects. For devices that are intended to be placed into or onto a specific organ during clinical use (e.g. devices intended to be placed into the cranial cavity or onto the skin), the histopathological analysis shall include that target organ.

Data may be summarized in tabular form, showing, for each test group, the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion. Statistical evaluations should be performed but biological relevance should be considered. Generally accepted statistical methods should be used and selected during the design phase of the study.

The findings of a long-term exposure study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation shall include the relationship between the dose of the test substance and the observed effects. Observed effects including behavioural and clinical abnormalities, gross lesions, microscopic changes, effects on mortality and any other effects should be evaluated for their biological significance. Evaluation of observed effects should also consider their relevance to humans.

The information given in 5.4 a) through e) shall be contained in the final report for the long-term exposure systemic toxicity study. In addition, the following information shall be provided:

— number of animals in each control and test group at the start and the end of the study;

— number of animals euthanized before the scheduled termination or found dead, if applicable;

— number of animals replaced during the study and when they were replaced, if applicable;

— all body weight measurements including initial, interim and terminal body weights;

— food and water consumption, if applicable;

— toxic response data by sex and dose level, if applicable;

— clinical observations, including a description of the nature, severity and duration of all adverse clinical observations (whether reversible or not)

— neurological and behavioural assessments, if applicable;

— haematological tests utilized and results with relevant control data;

— clinical biochemistry tests utilized and results with relevant control data;

— urinalysis tests utilized and results with relevant control data, if applicable;

— necropsy findings;

— terminal organ weights;

— histopathological findings and narrative that include discussion on the frequency and severity of the findings in test and control treated animals;

— a statistical evaluation of results, where used, and a discussion of their biological significance.

A long-term systemic toxicity study will provide information on the effects of long-term exposure to a test substance. Extrapolation of the results of the study to humans is valid to a limited degree but it can provide useful information on permissible human exposure.

The concept of material mediated pyrogenicity (MMP) is discussed in Annex G. Additional guidance on MMP is provided ISO/TR 21582. Methods for performing the rabbit pyrogen test (RPT) can be found in the pharmacopeia (e.g., United States Pharmacopoeia). Should other methods for detecting non-endotoxin pyrogenicity be developed, and become validated, these will be considered for replacement of the RPT.

Endotoxin-mediated and non-endotoxin microbial-mediated pyrogenicity assays and risk assessments (see Figure G.1) must be conducted before proceeding with a Rabbit Pyrogenicity Test (RPT). This approach helps ensure that RPT is both necessary and appropriate for the specific test article. To ensure the quality and validity of RPT data while prioritizing animal welfare, standard operating procedures and protocols must incorporate refinements that minimize pain, stress, and distress to the rabbits.

Rabbits shall be maintained in optimal health through observations conducted by qualified veterinary staff. Housing and environment should meet or exceed local specific, recognized national and international standards for construction, size (area), height, along with providing social interaction and enrichment. Refer to ISO 10993-2 for Animal welfare requirements for additional animal requirements.

Re-use of rabbits for RPT is permissible and recognized in various pharmacopeial standards. However, laboratories should have procedures established to limit the number of times an animal may be reused balancing reduction of animal use and cumulative distress to the individual animal. To minimize stress during testing, rabbits shall be gradually acclimated to prolonged restraint. Continuous monitoring for distress is essential. A suggested seven-day conditioning protocol might progress as follows: 15 min (day 1), 30 min (day 2), 1 h (day 3), 1,5 h (day 4), 2 h (day 5), 2,5 h (day 6), and 3 h (day 7). Rabbits that do not tolerate restrainer acclimation should not be used for pyrogen testing.

Restraint devices should be designed to allow rabbits of different weights and body condition scores to maintain a normal sitting position, prevent injuries, and provide adequate space for normal thoracic and abdominal expansion as well as slight movements for comfort adjustments. A clean soft, lubricated, flexible rectal thermometer probe shall be used to minimize discomfort and prevent tissue trauma during temperature measurements.

The test article solution shall be physiologically compatible for intravenous administration and should be given at a rate of no faster than 2 ml/kg/min to minimize adverse reactions. For soluble devices or unknown solutions, pH and osmolality should be measured and adjusted to a physiological range. For solutions with apparent viscosities higher than blood, adjustments may also need to be made to be compatible with an intravenous bolus injection.

NOTE High infusion rates and excessive intravenous fluid volumes can compromise circulatory function and cause pleural effusion, pulmonary edema, and peripheral edema.

Rabbits shall be monitored during and after intravenous infusion, throughout the study period, and post-study for adverse clinical observations. Any adverse clinical observations shall be documented.

1. 
   (informative)
   
   Routes of administration
   1. General

Several routes of administration are listed in A.2 to A.11, however, other routes of administration may be more clinically relevant and should be utilized. The most relevant route of administration should be determined based on the clinical relevancy and the ability to exaggerate the dose. The selected route of administration should be justified. Only trained personnel should perform a specific route of administration.

• 1. Dermal

Tests for systemic toxicity by the dermal route may be appropriate for surface-contacting devices. Consideration should be given to limiting animal oral access to the test sample (e.g. collars or single animal housing). If justified by the intended clinical use, breached skin exposure should be considered, e.g. wound healing devices.

• 1. Implantation

Tests for systemic toxicity by implantation may be appropriate for implanted devices. The test may be appropriate for direct testing of a material by application to a general or specific area. Shape and texture of the test sample should be taken into consideration. Methods for implantation can be found in ISO 10993-6.

• 1. Inhalation

Tests for systemic toxicity by the inhalation route may be appropriate for devices with a contact environment conducive to volatile chemical vapour releasing or for an aerosol/particulate test sample with potential for inhalation. Protocol specifics for this route of administration may be found in most dedicated texts for inhalation toxicology (see ISO 18562-4).

A number of factors should be carefully considered while selecting species and exposure settings, including relevance to humans, physiological and anatomical differences in test species (rabbits and rats are obligate nose breathers), dose extrapolation (breathing volume and rates), exposure scenarios (whole-body, nose-only), and confounding doses from additional routes of exposure (e.g. oral and dermal routes).

• 1. Intradermal

Tests for systemic toxicity by the intradermal route may be appropriate for a device with an intradermal contact environment conducive to chemical leaching. Test samples are typically administered directly to the intradermal region by injection. The use of multiple treatment sites should be clearly specified and justified.

• 1. Intramuscular

Tests for systemic toxicity by the intramuscular route may be appropriate for devices with a muscle tissue contact environment conducive to chemical leaching. Test samples are typically administered directly to the muscle tissue by injection or surgical implantation. Sites should be chosen to minimize the loss of function or the possibility of pain from nerve damage caused by muscle fibre tension from the injected or implanted test sample. If the animal is presumed to be in constant pain, use of the anesthetics or analgesics should be considered. In these cases, drugs with no anti-inflammatory properties, such as acetaminophen or opioids, should be used rather than non-steroidal anti-inflammatory drugs, which may affect the results. Sites should be rotated for repeated dose studies since, for example, non-aqueous formulations may remain as a depot for >24 h. The use of multiple treatment sites should be clearly specified and justified.

• 1. Intraneural

Tests for systemic toxicity by the neural route may be appropriate for a device with a neural tissue contact, directly or indirectly. Animal species, animal numbers, selection of control and test samples, clinical procedure, should be clearly specified and justified.

• 1. Intraperitoneal

Tests for systemic toxicity by the intraperitoneal route may be appropriate for devices with a fluid-path or peritoneal cavity contact environment conducive to chemical leaching. This is also an appropriate route when the extract should not be given intravenously, such as with non-polar oil extracts and where particulates might be present. This route is preferable to filtering for an intravenous injection. Test samples are typically administered directly to the peritoneal cavity. Dose frequency calculations should consider that test sample administered by this route are absorbed primarily through the portal circulation and therefore shall pass through the liver before reaching general circulation. Care should be taken not to inject into any intra-abdominal tissues.

• 1. Intravenous

Tests for systemic toxicity by the intravenous route may be appropriate for devices (or extracts of) with a direct or indirect fluid-path or blood contact environment conducive to chemical leaching. Intravenous administration of aqueous extracts of test sample may be justifiable when simulation of clinical exposure is not practical or technically achievable (see Annex H). Test samples are typically placed in or administered directly to the vascular system. If particulates are present, delivery by the intraperitoneal route or sample filtration should be considered. For the evaluation of nanomaterials, nanomaterial dispersions themselves may be considered for intravenous administration. Recommended dosage volumes and rates of administration for intravenous studies with the most commonly used laboratory animal species are listed in Annex B.

Care should be taken to minimize the possibility of extra vascular injection of test sample. For injection taking 5 min or more, consideration should be given to the use of a butterfly needle or an intravenous cannula. Intravenous administration in rodents can be limited to 28 d due to the potential stress imposed on animals, resulting from repeated intravenous injections, over time.

• 1. Oral

Tests for systemic toxicity by the oral route may be appropriate for devices contacting the oral mucosa directly or indirectly, or for products with other enteral application. Test samples are typically administered by gavage. Except for repeated dosing, experimental animals should generally be fasted prior to test sample administration. The period of fasting may range from hours to overnight, with the shorter periods for animals with higher metabolic rates. Following the period of fasting, the animals should be weighed and then the test sample administered in a single dose based on body weight. After the test sample has been administered, food may be withheld for an additional 3 h to 4 h. Where a dose is administered in fractions over a certain period, it may be necessary to provide the animals with food and water depending on the length of the period to maintain the health of the animal.

• 1. Subcutaneous

Tests for systemic toxicity by the subcutaneous route may be appropriate for a device with a subcutaneous contact environment conducive to chemical leaching. Subcutaneous implantation is often used as a route of exposure for devices implanted into soft tissue, i.e. – subcutaneous tissue, muscle, and other soft tissues. This route can also be suitable for addressing toxicity of topically applied products used for example in wound therapy, if compatible. Test samples are typically administered directly to the subcutaneous region by injection or by implantation. The use of multiple treatment sites should be clearly specified and justified.

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   (informative)
   
   Dose volumes
   1. General

The principles of humane animal research require that all reasonable efforts be made to minimize pain and distress. The dose volumes listed in Table B.1 are intended to be informative and represent maximum volume limits reported in the literature for single dose administrations that takes into consideration the welfare of laboratory animals. These dose volumes should be taken as a recommendation in this document. Should investigators use other dose volumes beyond this recommendation, it should be explained and justified with appropriate literature references. Upper limits with regard to factors such as body weight/surface area, rate of administration, number and frequency of administrations, physico-chemical and biological properties of the test sample, and animal model should be applied. For repeated dose administrations attempts should be made to minimize the dose volume while taking into consideration these adjustment factors.

Table B.1 — Maximum single dosage volumes for test sample administration

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   (informative)
   
   Common clinical signs and observations

Two examples of clinical signs and observation are suggested as follows.

Table C.1 — Mouse clinical signs and grading scheme (from ASTM F750-20^[12])

For more information can be found in References [12] [27] [28], [30], [31], [32].

Table C.2 — Common clinical signs and observations (for animals other than mice)

1. 
   (informative)
   
   Suggested haematology, clinical chemistry and urinalysis measurements
   1. Haematology

The following parameters are routinely assessed haematology endpoints that are evaluated in systemic toxicity studies:

— Clotting potential (PT, APTT);

— Haemoglobin concentration;

— Haematocrit;

— Platelet count;

— Red blood cell count;

— White blood cell count;

— WBC differential.

• 1. Clinical chemistry

The following parameters are routinely assessed clinical chemistry endpoints that are evaluated in systemic toxicity studies:

— Albumin;

— ALP;

— ALT;

— AST;

— Calcium;

— Chloride;

— Cholesterol;

— Creatinine;

— GGT;

— Glucose;

— Inorganic phosphorus;

— Potassium;

— Sodium;

— Total bilirubin;

— Total protein;

— Triglycerides;

— Urea nitrogen;

— Additional enzymes, as scientifically appropriate;

— Measurement of IgG and IgM levels in rats may be included in future guidelines as an indicator of immunotoxicity.

• 1. Urinalysis (timed collection, e.g. 16 h to 24 h)

The following parameters are routinely assessed urinalysis endpoints that are evaluated in systemic toxicity studies:

— Appearance;

— Bilirubin;

— Glucose;

— Ketones;

— Occult Blood;

— Protein;

— Sediment;

— Specific gravity or osmolality;

— Volume;

— Other scientifically appropriate tests if test sample is suspected to cause specific organ toxicity (generally requires refrigerated sample collection).

1. 
   (informative)
   
   Suggested organ list for histopathological evaluation

In addition to the histopathological evaluation, organs/tissues marked with an asterisk (*) in this annex should be weighed, with other organs weighed if scientifically appropriate. The clinical and other findings may suggest the need to examine additional tissues. Also, any organs considered likely to be target organs based on the known properties of the test substance should be preserved. For devices that are intended to be placed into or onto a specific organ during clinical use (e.g. devices intended to be placed into the cranial cavity or onto the skin, etc.), the histopathological analysis shall include that target organ.

Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and highest dose group. Limited histopathology may be employ as outlined in Annex F. These examinations, targeted to specific organs/tissues as necessary, should be extended to animals of all other dosage groups if treatment-related changes are observed in the highest dosage group:

— Adrenals*;

— All gross lesions (including treatment sites);

— Aorta;

— Bone marrow (femur, rib or sternum);

— Brain* (representative sections including cerebrum, cerebellum and pons);

— Caecum;

— Colon;

— Duodenum;

— Epididymis*;

— Oesophagus;

— Eyes;

— Gall bladder (if present);

— Heart*;

— Ileum;

— Jejunum;

— Kidneys*;

— Liver*;

— Lungs and bronchi (preserved by inflation with fixative and then immersion);

— Lymph nodes (local to cover site of administration and distant to cover systemic effects);

— Mammary gland (female);

— Muscle (skeletal);

— Nasal turbinates (for inhalation studies);

— Nerve (sciatic or tibial) preferably in close proximity to the muscle;

— Ovaries*;

— Pancreas;

— Parathyroid;

— Pituitary;

— Prostate;

— Rectum;

— Salivary glands;

— Seminal vesicles;

— Skin;

— Spinal cord;

— Spleen*;

— Sternum;

— Stomach;

— Testes*;

— Thymus*;

— Thyroid;

— Trachea;

— Urinary bladder;

— Uterus* (including cervix and oviducts);

— Vagina;

1. 
   (informative)
   
   Organ list for limited histopathology for medical devices subjected to systemic toxicity testing
   1. General

Many medical devices employ commonly used materials differing only in the amount or type of chemical additives, processing or sterilization methods.

When a toxicological risk assessment of the device extractables/leachables determines that a biocompatibility/safety assessment for potential systemic effects is required a reduced histopathology evaluation may be considered. In this model, a limited number of potential target organs/tissues are examined using a tiered approach.

• 1. Procedure

All tissues indicated in Annex E should be collected and preserved.

Limited histopathological analysis should be completed for all Tier I organs/tissues listed in Table F.1.

If abnormal or questionable findings are observed in Tier I tissues, or in the concurrent clinical pathology (clinical chemistry and haematology), proceed to Tier II (examine the full list of organs/tissues in Annex E).

Table F.1 — Organ list for limited histopathology

1. 
   (informative)
   
   Information on pyrogens
   1. General

Any substance that causes an elevation in body temperature can be considered a pyrogen. The term “material-mediated pyrogen” has historically been used to describe any non-endotoxin substance with the ability to produce a febrile response, regardless of its origin or mechanism of action. This term is not scientifically accurate since increased body temperature can be due to several mechanisms of action and a single test is unable to detect all mechanisms of action or “material-mediated pyrogens”.

This topic is covered in more detail in ISO/TR 21582.

According to ISO/TR 21582, substances that alter the body temperature can be divided into three groups:

a) chemical agents arising from materials used to manufacturer medical devices that alter body temperature;

b) bacterial endotoxin-mediated pyrogenicity;

c) pyrogenicity mediated by microbial components other than endotoxin.

This annex is focused on chemical agents arising from materials used to manufacturer medical devices that alter body temperature. ISO/TR 21582 addresses bacterial endotoxin-mediated pyrogenicity and pyrogenicity mediated by microbial components other than endotoxin.

Per ISO 10993-1, “material mediated pyrogenicity” is rare and has been shown to occur only in the case of a small number of constituents very unlikely to be found associated with medical devices^[39].

By contrast, endotoxin-mediated pyrogenicity is much more likely. This form of pyrogenicity originating from biologically active endotoxins (Lipopolysaccharides (LPS)) of Gram-negative bacteria, which are usually a contaminate occurring during the manufacturing process of medical devices (e.g. water that has been contaminated during processing, non-sterile equipment surfaces or device components in contact with contaminated water). Endotoxins are evaluated by measuring the amount of endotoxin in the devices using a bacterial endotoxin-specific tests such as those that utilize Limulus amebocyte lysate (LAL), recombinant Factor C (rFc), or recombinant cascade reagent (rCR). In vitro pyrogen test using human immune cells, and the human cell-based pyrogen test (HCPT) can also detect endotoxin but are nonspecific and will react with other pyrogens. The requirements and guidance for testing of endotoxin-mediated pyrogens are defined by ISO 11737-3.

Although non-endotoxin microbial pyrogens, e.g., Lipoteichoic acid (LTA), Peptidoglycan, bacterial exotoxins such as TSST-1, SEA, Spe F, Spe C caused by Gram-positive bacteria, fungi, and viruses can be pyrogenic, they do so through different mechanisms and to a lesser degree than Gram-negative bacteria. This is because extremely high levels of Gram-positive bacteria are required to elicit a pyrogenic response due to Gram-positive cell wall constituents or intact bacteria, with a potency several orders of magnitude less than endotoxin.^{[51][53][63][64]} Such levels are easily notable by an increase in bioburden counts and can be monitored and controlled per the bioburden standard ISO 11737-1.

Neither endotoxin-mediated pyrogens, nor non-endotoxin microbial mediated pyrogens have historically been considered “material-mediated pyrogen” and their presence on devices often leads to a pyrogenic response depending on the sensitivity of the pyrogenicity test method. These pyrogens, their testing and control are managed outside of biological evaluation according to ISO 10993-1 and can be clearly distinguished from other substances that elevate body temperature, because the febrile reaction is originated from microbial contamination rather than as an intrinsic property of the material itself.

• 1. Historical “Material-Mediated Pyrogen” Assessment

Historically, according to ISO 10993-11:2017, Annex G, “material-mediated pyrogenicity” has referred to categories of substances, both non-endotoxin biological in origin (e.g. endogenous signalling molecules, synthetic inducers, bacterial exotoxins from select Gram-positive bacteria) and chemical in origin (e.g. legal and illegal drugs, and uncouplers of oxidative phosphorylation), that had been reported to elicit a pyrogenic response. Literature reviews have been conducted to understand the mechanisms by which these substances produce local or systemic heat.^[38][39]

• • 1. Non-Microbial Biological Pyrogens

Borton and Coleman (2018, 2025) indicate cytokine-mediated fevers are generated by substances of biological-origin either naturally occurring (e.g. endogenous signalling molecules) or synthetically produced substances that mimic endogenous human substances (e.g. synthetic double-stranded RNA molecules or “inducers” like polyadenylic acid [poly(A)], polyuridylic acid [poly(U)], polyinosinic acid [poly(I)], and polycytidylic acid [poly(C)]).^[38][39]

• • 1. Non-Biologic Pyrogens

Borton and Coleman (2025)^[39] collated data on pyrogens that they term ‘thermogens’ that do not operate through the cytokine network, specifically chemical agents that work through uncoupling of oxidative phosphorylation as previously reported by References [48], [49], [61] and others. During uncoupling of oxidative phosphorylation, the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) is disrupted leading to an increased metabolic rate and heat production^[57].

Illicit and controlled drugs such as lysergic acid diethylamide (LSD), cocaine, morphine, bupivacaine and numerous others, have been reported to be thermogenic or pyrogenic and have been extensively reviewed in several papers by Clark and colleagues.^{[40][41][42][43]} These restricted substances, including many pharmaceuticals, have demonstrated hyperthermic activity.^[42] The presence of controlled or illicit drugs within a medical device is regulated by device design and manufacturing process controls, and therefore, should not be considered a hazard within an ISO 10993-1 risk-management framework. Evaluation of illicit and controlled drugs is not necessary in the context of ISO 10993-1 biological evaluation.

In ISO 10993-11:2017, Metallics and N-Phenyl-2-naphthylamine and 1-naphthylamine were mentioned a potential pyrogens. The literature reviews indicated that metals and their salts are not pyrogenic or thermogenic.^[39] Borton and Coleman (2018, 2025)^[38][39] also found no studies implicating n-phenyl-2-naphthylamine or 1-naphthylamine as fever-inducing substances. Therefore, these substances should not be considered a hazard regarding altered body temperature within an ISO 10993-1 risk-management framework.

• 1. Application of a Risk-Based Approach

Due to the documented extreme rarity of chemical agents arising from materials used to manufacturer medical devices that alter body temperate, and the well-established chemical nature of many materials used for medical devices, it is not necessary to test all new medical devices for in vivo pyrogenicity. Consistent with ISO 10993-1:2025, 6.5.10.5, it can be sufficient to present a well-documented risk assessment demonstrating that the composition of the finished medical device and the use of controlled manufacturing processes (including processing aids and reasonable potential for low levels of manufacturing contaminants) do not present a significant biological risk of pyrogenicity.

A process for the conducting of a pyrogenic assessment is depicted in Figure G.1. The flowchart considers whether the device’s materials including processing aids and manufacturing contaminants present a significant biological risk associated with pyrogenicity or thermogenicity. ISO 10993-1 evaluates medical devices within the context of a risk-based approach, not a hazards-based approach. Based on the evidence presented by Borton and Coleman (2025)^[39] and a risk-based approach in accordance with ISO 14971 and ISO 10993-1, unintentional addition or contamination is unlikely to pose a pyrogenic response and therefore do not require assessment.

NOTE 1 Borton and Coleman (2025)^[39] reported that over 90 % of Table G.1 chemicals are poorly soluble in saline and water – the only extraction vehicles employed in the rabbit pyrogen test (RPT). Therefore, the detection of substance in Table G.1. by the RPT is highly unlikely. As a result, a large proportion of Table G.1 substances can only be detected by ISO 10993-18 chemical characterization (a priori materials assessment or analytical chemistry techniques) and an ISO 10993-17 toxicological risk assessment as evidenced by marked adverse effects associated with substances listed in Table G.1. in systemic toxicity studies by the following clinical signs: significantly decreased body weight, increased body temperature, increased respiration rate, and other related effects.^{[37][57][58][65]}

NOTE 2 Long-chain fatty acids are ubiquitous in medical device materials and manufacturing processes and were consistently identified in medical device extracts^[39]. Long-chain fatty acids, specifically C12-C18, were identified as pseudo-uncouplers capable of oxidative phosphorylation under specific biological conditions^[65][66][67]. These C12-C18 long chain fatty acids are involved in non-shivering thermogenesis, an essential biological process.^[44][65] Long-chain fatty acids are part of a normal biological response. Only when there is excessive dietary fatty acid consumption and low physical activity do long-chain fatty acids become a human health risk due to pathological conditions such as heart disease, atherosclerosis, and hypertension, and not due to thermogenesis^[45].

Figure G.1 — Flowchart – Materials Assessment

Table G.1 — List of Established Chemical Thermogens Acting via Uncouplers of Oxidative Phosphorylation

1. 
   (informative)
   
   Repeated Dose (14 d or 28 d) Toxicity Study in Rats — Dual routes of parenteral administration
   1. General

To assess systemic toxicity, for devices not intended for implant, exposure of the device via dosing of extracts is an option. Dual route administration of polar and nonpolar extracts into the same animal can be an option.

Under circumstances where implantation of an implantable medical device is not feasible and preparation of coupons of the device is impractical, dual routes administration of extracts (i.e., administration of extracts via intravenous and intraperitoneal routes into the same animal) may be considered with justification.

Clinically, when a medical device is contacting with either breached or compromised surfaces (skin or mucosal membranes) or internal tissues, exposure to polar and nonpolar leachables may be concurrent. One approach to assess toxicity is to inject both polar and nonpolar extracts into the same animal. This approach would result in the exposure of the test animals to a more clinically relevant set of extractables (from both polar and non-polar extracts). The dual exposure route also reduces the number of animals used in the study. Therefore, the dual exposure route model is preferred unless there is a need to study the administration routes separately. In that case, a justification should be provided and Clause 6 should be considered.

Recommended dosing parameters for the dual routes of parenteral administration model for 14 d and 28 d durations are as specified in the Table H.1 and Table H.2, respectively.

Table H.1 — Recommended dosing parameters — 14 d

Table H.2 — Recommended dosing parameters — 28 day

• 1. Procedure

The test animals are dosed with the polar test sample extract, and the control animals are dosed with polar vehicle, intravenously 7 d/week over the duration of the study (i.e. 14 doses or 28 doses). For nonpolar test samples the same test animals are dosed with the nonpolar test sample extract, and the control animals are dosed with the nonpolar vehicle, intraperitoneally every third day over the duration of the study (i.e. 5 or 10 doses).

• 1. Dosage volume and frequency justification
     1. Intravenous

Table B.1 describes the maximum dosage volumes for test sample administration when a single or a very limited number of intravenous treatments are required. For daily-repeated or recurrent administrations by any route the test sample volume should be reduced. LASA (see Reference [22]) recommends a maximum intravenous dosage volume of 5 ml/kg for a bolus injection in the rat carried out over a relatively short period of time (less than 1 min), and for once daily dosing on a routine basis (injection rate ≤2 ml/min). For repeated intravenous administration of medical device extracts a dose volume of 10 ml/kg is considered unlikely to cause undue stress in the animals, see Reference [16].

• • 1. Intraperitoneal

Table B.1 describes the maximum dosage volumes for test sample administration when a single or a very limited number of intraperitoneal treatments are required. Current experience indicates that 5 ml/kg of sesame oil extract given intermittently is well tolerated. Sufficient anecdotal evidence suggests that a peritoneal residual volume (PRV) of non-aqueous injectates of 5 ml/kg to 10 ml/kg may persist for up to three days. Consequently, for repeated intraperitoneal administration of nonpolar medical device extracts, when administered concurrent with an intravenous injection of 10 ml/kg, a dose volume of 5 ml/kg is considered unlikely to cause undue stress in the animal and represents an acceptable protracted exposure volume for nonpolar extracts.

Several complications by the intraperitoneal route of administration are well documented. These include bleeding at the injection site, paralytic ileus due to substance injected, laceration of abdominal organs, peritonitis, and injection into the gastrointestinal tract or bladder. In that regard, the frequency of erroneous intraperitoneal injections by skilled investigators has been reported to range from 11 % to 20 % (see References [22] and [23]). In view of this, and with consideration of the PRV and the potential for increasing intraperitoneal injectate volume with too frequent dosing, a thrice weekly administration schedule is considered unlikely to cause undue stress in the animal and represents an acceptable protracted exposure frequency for non-aqueous injectates.

General aspects of study design are covered in Clause 6.

Annex ZA
(informative)

Relationship between this European Standard the General Safety and Performance Requirements of Regulation (EU) 2017/745 aimed to be covered

NOTE Annex ZA is not included in the final ISO publication.

This European standard has been prepared under M/575 to provide one voluntary means of conforming to the General Safety and Performance Requirements of Regulation (EU) 2017/745 of 5 April 2017 concerning medical devices [OJ L 117] and to system or process requirements including those relating to quality management systems, risk management, post-market surveillance systems, clinical investigations, clinical evaluation or post-market clinical follow-up.

Once this standard is cited in the Official Journal of the European Union under that Regulation, compliance with the normative clauses of this standard given in Table ZA.1 and application of the edition of the normatively referenced standards as given in Table ZA.2 confers, within the limits of the scope of this standard, a presumption of conformity with the corresponding General Safety and Performance Requirements of that Regulation, and associated EFTA Regulations.

Where a definition in this harmonised standard differs from a definition of the same term set out in Regulation (EU) 2017/745, the differences shall be indicated in the Annex Z. For the purpose of using this standard in support of the requirements set out in Regulation (EU) 2017/745, the definitions set out in this Regulation prevail.

Where the European standard is an adoption of an International Standard, the scope of this document can differ from the scope of the European Regulation that it supports. As the scope of the applicable regulatory requirements differ from nation to nation and region to region the standard can only support European regulatory requirements to the extent of the scope of the European Regulation for medical devices ((EU) 2017/745).

NOTE 1 Where a reference from a clause of this standard to the risk management process is made, the risk management process needs to be in compliance with Regulation (EU) 2017/745. This means that risks have to be ‘reduced as far as possible’, ‘reduced to the lowest possible level’, ‘reduced as far as possible and appropriate’, ‘removed or reduced as far as possible’, ‘eliminated or reduced as far as possible’, ’removed or minimized as far as possible’, or ‘minimized’, according to the wording of the corresponding General Safety and Performance Requirement.

NOTE 2 The manufacturer’s policy for determining acceptable risk must be in compliance with General Safety and Performance Requirements 1, 2, 3, 4, 5, 8, 9, 10, 11, 14, 16, 17, 18, 19, 20, 21 and 22 of the Regulation.

NOTE 3 When a General Safety and Performance Requirement does not appear in Table ZA.1, it means that it is not addressed by this European Standard.

NOTE 4 General Safety and Performance Requirements listed in Table ZA.1 are specific to biological safety for patients only.

Table ZA.1 — Correspondence between this European Standard and Annex I
of Regulation (EU) 2017/745 [OJ L 117] and to system or process requirements including those relating to quality management systems, risk management, post-market surveillance systems, clinical investigations, clinical evaluation or post-market clinical follow-up

Table ZA.2 — Applicable Standards to confer presumption of conformity as described in this Annex ZA

The documents listed in the Column 1 of table ZA.2, in whole or in part, are normatively referenced in this document, i.e. are indispensable for its application. The achievement of the presumption of conformity is subject to the application of the edition of Standards as listed in Column 4 or, if no European Standard Edition exists, the International Standard Edition given in Column 2 of table ZA.2.

WARNING 1 — Presumption of conformity stays valid only as long as a reference to this European standard is maintained in the list published in the Official Journal of the European Union. Users of this standard should consult frequently the latest list published in the Official Journal of the European Union.

WARNING 2 — Other Union legislation may be applicable to the product(s) falling within the scope of this standard.

Bibliography

ISO 10993‑3, Biological evaluation of medical devices — Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity

ISO 10993‑4, Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood

ISO 10993‑5, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity

ISO 10993‑10, Biological evaluation of medical devices — Part 10: Tests for skin sensitization

ISO 10993‑17, Biological evaluation of medical devices — Part 17: Toxicological risk assessment of medical device constituents

ISO 10993‑18, Biological evaluation of medical devices — Part 18: Chemical characterization of medical device materials within a risk management process

ISO 10993‑23, Biological evaluation of medical devices — Part 23: Tests for irritation

ISO/TR 10993‑22:2017, Biological evaluation of medical devices — Part 22: Guidance on nanomaterials

ISO/TS 10993‑20, Biological evaluation of medical devices — Part 20: Principles and methods for immunotoxicology testing of medical devices

ISO 11737‑1, Sterilization of health care products — Microbiological methods — Part 1: Determination of a population of microorganisms on products

ISO 11737‑3, Sterilization of health care products — Microbiological methods — Part 3: Bacterial endotoxin testing

ISO 14971, Medical devices — Application of risk management to medical devices

ISO 18562‑4, Biocompatibility evaluation of breathing gas pathways in healthcare applications — Part 4: Tests for leachables in condensate

ISO/TR 21582, Pyrogenicity — Principles and methods for pyrogen testing of medical devices

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