ISO/DIS 10993-11:2025(en)

ISO/TC 194/WG 7

Secretariat: DIN

Date: 2025-01-30

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

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

© ISO 2025

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Contents Page

Foreword 5

Introduction 6

1 Scope 1

2 Normative references 1

3 Terms and definitions 1

4 General considerations 3

4.1 General 3

4.2 Selection of animal model 4

4.3 Animal status 4

4.4 Animal care and husbandry 4

4.5 Size and number of groups 4

4.5.1 Size of groups 4

4.5.2 Number of groups 5

4.5.3 Treatment controls 5

4.6 Route of exposure 5

4.7 Sample preparation 6

4.8 Dosing 6

4.8.1 Test sample administration 6

4.8.2 Dosage 6

4.8.3 Dosage frequency 7

4.9 Body weight and food/water consumption 8

4.10 Clinical observations 8

4.11 Clinical pathology 8

4.12 Anatomic pathology 8

4.13 Study designs 9

4.14 Quality of investigation 9

5 Acute systemic toxicity 9

5.1 General 9

5.2 Study design 10

5.2.1 Preparations 10

5.2.2 Experimental animals 10

5.2.3 Test conditions 10

5.2.4 Body weights 11

5.2.5 Clinical observations 11

5.2.6 Pathology 11

5.3 Evaluation criteria 12

5.3.1 General 12

5.3.2 Evaluation of results 12

5.4 Final report 12

6 Repeated exposure systemic toxicity (subacute, subchronic and chronic systemic toxicity) 14

6.1 General 14

6.2 Study design 14

6.2.1 Preparations 14

6.2.2 Experimental animals 15

6.2.3 Test conditions 15

6.2.4 Body weights 15

6.2.5 Clinical observations 15

6.2.6 Pathology 16

6.3 Evaluation criteria 17

6.3.1 General 17

6.3.2 Evaluation of results 17

6.4 Final report 17

Annex A (informative) Routes of administration 19

A.1 General 19

A.2 Dermal 19

A.3 Implantation 19

A.4 Inhalation 19

A.5 Intradermal 19

A.6 Intramuscular 19

A.7 Intraperitoneal 20

A.8 Intravenous 20

A.9 Oral 20

A.10 Subcutaneous 20

A.11 Intraneural 21

Annex B (informative) Dose volumes 22

B.1 General 22

B.2 Dosage volume references 22

Annex C (informative) Common clinical signs and observations 23

Annex D (informative) Suggested haematology, clinical chemistry and urinalysis measurements 25

D.1 Haematology 25

D.2 Clinical chemistry 25

D.3 Urinalysis (timed collection, e.g., 16 h to 24 h) 26

Annex E (informative) Suggested organ list for histopathological evaluation 27

Annex F (informative) Organ list for limited histopathology for medical devices subjected to systemic toxicity testing 29

F.1 General 29

F.2 Procedure 29

Annex G (informative) Information on material-mediated pyrogens 31

G.1 General 31

G.2 Endotoxin-mediated pyrogenicity 31

G.3 Material-mediated pyrogenicity 31

Annex H (informative) Subacute and Subchronic Toxicity in Rats — Dual routes of parenteral administration 33

H.1 General 33

H.2 Procedure 34

H.3 Dosage volume and frequency justification 34

H.3.1 Intravenous 34

H.3.2 Intraperitoneal 34

Annex ZA (informative) Relationship between this European Standard the General Safety and Performance Requirements of Regulation (EU) 2017/745 aimed to be covered 35

Bibliography 38

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 and/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:

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

b) added rabbits to Table 1 for group sizes;

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

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

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

f) 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.

Because of 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 has to 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 compliance 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 toxicological end points are addressed in ISO 10993-3, 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 and/or other biological test data (including from in vitro tests and less invasive in vivo tests) for the refinement of study design (dose selection, and/or selection of pathological end points). For the repeated 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 end points/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 — 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 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 10993‑11, Biological evaluation of medical devices - Part 11: Tests for systemic toxicity

ISO 10993‑12, 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 terminological databases for use in standardization at the following addresses:

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

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

3.1

dose

dosage

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

3.2

dose-effect

relationship between the dosage and the magnitude of a defined biological effect either in an individual or in a population sample

3.3

dose-response

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

3.4

leachable substance

chemical released from a device or material by the action of solvents related to the use of the device

Note 1 to entry: Examples of leachable substances are additives, sterilant residues, process residues, degradation products, solvents, plasticizers, lubricants, catalysts, stabilizers, anti-oxidants, colouring agents, fillers and monomers.

Note 2 to entry: Leachable substances related to the use of gas pathway devices can be evaluated according to the ISO 18562 series.

3.5

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; if not toxic at this high dose, further testing at higher dosages is generally not necessary

3.6

systemic toxicity

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

3.7

acute systemic toxicity

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

3.8

subacute systemic toxicity

adverse effects occurring after single or repeated exposure of a test sample between 24 h and 28 d

Note 1 to entry: Since this term is semantically incorrect, the adverse effects occurring within the specified time period may also be described as a short-term repeated exposure systemic toxicity study. The selection of time intervals between 14 d and 28 d is consistent with most international regulatory guidelines and considered a reasonable approach. Subacute repeated intravenous and intraperitoneal studies are generally defined as exposure durations of ≤14 d for rodents.

3.9

subchronic systemic toxicity

adverse effects occurring after repeated administration or continuous exposure of a test sample for a period of up to 10 % of the lifespan of the species

Note 1 to entry: Subchronic toxicity studies are usually 90 d in rodents or rabbits but not exceeding 10 % of the lifespan of other species. Subchronic repeated dose intravenous and intraperitoneal studies are generally defined as treatment durations of 28 d for rodents.

3.10

chronic systemic toxicity

adverse effects occurring after the repeated or continuous administration 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 6 months in rodents or exceeding 10 % of the lifespan of other species.

3.11

test sample

material, device, device portion, component, chemical, extract or portion thereof subjected to biological or chemical testing or evaluation

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 toxicological risks of medical devices, first, consideration of applicability of chemical characterization and toxicological risk assessment should be given before pursuing systemic toxicity testing using an animal model. Only when there is not sufficient data to assess the risk of systemic toxicity using the chemical characterization and toxicological risk assessment or when this cannot be adequately performed due to the nature of the device, the in vivo toxicity studies should be considered.

Some devices contain such low concentrations of extractable constituents that adverse effects are unlikely to be observed in a systemic toxicity test. 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 comparable 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 are less than approximately 0,015 – 0,15 mg/kg/day, in vivo effects are unlikely to be observed. Particularly, chemical information should inform whether the study will be useful for the overall biological evaluation^[11].

Testing shall be performed on the final product and/or representative component samples of the final product and/or materials. 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, or deviations to the protocol occurred, they shall be recorded in the test report, together with their justification. For hazard identification purposes, it could 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 per the intended 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.

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. Rabbit (lagomorph) is an option in dermal studies and preferred in the case of implantation studies where a large model is needed due to the size of the implant. Other non-rodent species may also need to 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. acute, subacute, subchronic and/or chronic systemic toxicity. This minimizes the variability between species and strains and facilitates an evaluation related solely to study duration. Should multiple species or strains be used, justification for their selection shall be documented.

Generally, healthy purpose-bred young adult animals of known origin and with defined microbiological 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 precision of the systemic toxicity test is dependent to a large extent on the number of animals used per dose level. The degree of precision needed and, in turn, the number of animals per dose group needed depends on the study design.

Group sizes should logically increase with the duration of treatment, such that at the end of the study sufficient animals in every group are available to help meet the objectives of the study. Group sizes shall meet both ISO 10993-11 and ISO 10993-6 requirements on the group size when the testing is designed to address both implantation and systemic toxicity endpoints, otherwise, additional justification shall be provided and documented. 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

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/body weight;

— multiples of the clinical surface area of exposure;

— multiples of the duration of exposure;

— multiples of the extractable fraction or the individual chemicals;

— 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 article and the route of exposure, negative, vehicle and/or sham-treated controls should be incorporated into all systemic toxicity studies. These controls shall mimic the test sample preparation and treatment procedure.

Medical devices or their leachable substances may gain access to the body by multiple routes of exposure. The test route of exposure shall be the most clinically relevant to the use of the device, where possible. If an alternative route of exposure is necessary, it shall be justified. Examples of routes of administration can be found in Annex A.

Route of exposure should be chosen based on consideration of 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. If dosing with device extracts is chosen, the ability of the extraction method to extract the constituents of interest should be considered.

Chemical characterization performed on the device can provide information on the potential systemic exposure in a toxicity study. For example, the analytical results from a water extract from an extractables study can provide a reasonable estimate of the composition and concentration of device constituents in the saline extract used for dosing if extraction conditions are comparable. Similarly, the analytical results from all extracts in an extractables study can provide a reasonable estimate of the potential systemic exposure from an implantation study. Such information shall be considered when designing a systemic toxicity study including whether the study will be useful for the overall biological evaluation of the device in accordance with ISO 10993-1.

The test and control samples and their preparation (such as pH, stability, homogeneity, osmolality, and/or sterility) shall be described and justified. 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. 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, sample filtration to remove particulates should be used, documented, and justified. In addition, alternate administration routes (e.g. intraperitoneal injections) can be considered and shall be justified. When medical devices and/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.

Prolonged restraint of animals in repeated exposure systemic toxicity studies should be scientifically justified and performed in a manner that is as humane as possible. 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^[19]. 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^[21].

Guidance on dosage volume is summarized in Annex B. When multiple dosage groups are used, variability in the test volume may be minimized by adjusting the concentration to ensure a constant volume at all doses. Use of dosage 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 approximately 1 min. 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 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 or volume basis of exaggeration may be used. Other bases of providing an exaggerated dose may be used and if justified. A suggested exaggeration is 10-100 times the proportional human dose unless not technically achievable. 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 and/or tissues of exposure. Implantation in alternative tissue(s) may be considered. For example, subcutaneous implant sites have the advantage of being able to accommodate proportionately large doses, allowing for exaggeration of the typical clinical dose. The implant location(s) and dose implanted shall be justified. For devices that are externally communicating or reside in the vascular system (e.g., hemodialysis filter), intravenous and/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 series 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 the test article has sharp edges/corners this could potentially result in skin perforations in a subcutaneous implant study. In general, the amount implanted, when possible, should represent a 10X to 100X exaggeration of the human dose (i.e., per kg body weight) to provide for an adequate safety assessment. The amount implanted may be expressed as mass of material (g), surface area (cm²), or volume (mL), with justification based on what is most appropriate for the subject device and its indication(s). Exaggeration of 100 times the human dose should be ideally reached unless justified. In cases where toxic effect may be expected, multiple dose levels may be advisable rather than a single limit type dose.

Systemic toxicity studies performed using implantation can be utilized to also address local effects (see ISO 10993-6). When addressing local effects, the study shall satisfy the requirements in both ISO 10993-11 and ISO 10993-6.

The dosage frequency should be based on clinical relevancy. Exaggerated procedures shall be clearly specified and justified.

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

Body weight change and changes in food and water consumption may be attributed to the effects of a test article. 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 repeated exposure studies), at regular intervals throughout the study and at study termination. If dosage volume/amount of each animal is calculated by body weight, the most recent body weight should be utilized.

Measurements of food and water consumption, as appropriate, can be considered for longer-term repeated 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.^[35],^[41] General clinical observations shall consider the peak period of anticipated effects after dosing.

Observations shall be recorded systematically 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 Annex C). For some clinical observations of pain, these observations can be captured quantitatively using the Grimace Scale^[35][36].

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

Haematology and clinical chemistry analyses are performed to investigate toxic effects in tissues, organs and other systems. When indicated, these analyses shall be performed on blood samples obtained from repeated exposure 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 last week of a long-term repeated exposure study using timed (e.g., 16 h to 24 h) urine volume collection. When deemed necessary, additional urinalysis can also be included to provide additional information.

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 the histology slides.

All animals in repeated exposure 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.

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^[42] 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 might 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 substance 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^[24][25] 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 end points, as defined by national or international animal welfare guidelines, should be used in order to avoid unnecessary suffering.

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 test 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^[17].

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. Characteristics of the model (age, weight, etc.) are as specified in 4.2 and 4.3. If non-rodent species are used their use 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.

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 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 of dose vehicle used for dosing the treated animals.

Procedure

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 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 end points applied should preclude the possibility of animals being found dead as a direct consequence of test sample toxicity.

Body weight measurements should be made immediately before 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)^[18] 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, 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.

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 significantly 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 if 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 %, should 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 to moderate signs of biological reactivity, and not more than one animal shows gross (marked) symptoms of biological reactivity or dies, repeat the testing using groups of 10 animals (see Annex C, Table C.1).

4) On the repeat test, if all 10 animals treated with the sample show no scientifically meaningful biological reactivity above the vehicle control animals during the observation period, the sample meets the requirements of this test.

5) If any animal dies, and/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):

1) 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, 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) body weight/body weight change;

3) food and water consumption, if applicable;

4) toxic response data by sex and dose level, including signs of toxicity;

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

6) neurobehavioural 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) terminal body weight and organ weight data, if applicable;

11) necropsy findings, if applicable;

12) detailed description of all histopathological findings, if applicable;

13) 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 considering all existing data, e.g. if data from a subacute and chronic toxicity/implant study on a long-term device is acceptable, it may not be necessary to conduct a subchronic toxicity study.

Repeated 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 might also provide information on the mode of toxic action of a substance by the intended clinical exposure route.

Repeated 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.

Repeated 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 repeated 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) will be used. Characteristics of the model (age, weight, etc.) are specified in 4.2 and 4.3. When non-rodent 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. For subchronic studies the satellite animals shall be maintained for not less than 28 d.

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 repeated exposure systemic toxicity, repeated 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.

Procedure

Animals should be dosed ideally 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 immediately before dosing, weekly after the first dose if indicated by study duration, and at the end of the study.

The observation period for a repeated dose 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 shall be isolated, and moribund animals shall be euthanized. For animals that are found dead or euthanized early, comprehensive necropsy shall be done. 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 repeated 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. 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 toxicity.

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, 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.

f) During selection of organs for histopathology, due 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, etc.), 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 repeated 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 shall be contained in the final report for the repeated exposure systemic toxicity study. In addition, the following information shall be provided:

— initial, interim, and terminal body weights as applicable;

— terminal organ weights;

— haematological tests utilized and results with relevant control data;

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

— histopathological findings;

— 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 repeated 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.

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 shall be determined based on the clinical relevancy and the ability to exaggerate the dose. The selected route of administration shall be justified.

• 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 leaching 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.

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 rodents are obligate nose breathers), dose extrapolation (breathing volume and rates), exposure scenarios (whole-body, nose-only), and confounding doses from additional routes of exposure (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 need to 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 NSAIDs, 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. 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 the stomach or intestinal tract.

• 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 infusion of aqueous extracts of test article to 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 stress of the animals being repeated dosed 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.

• 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. 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.

• 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 articles, clinical procedure, etc. shall be clearly specified and justified.

1. 
   (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. These dose volumes should not be taken as a recommendation in this document, but investigators should apply upper limits with regard to factors such as body weight/surface area, rate of administration, number and frequency of administrations, physical-chemical and biological properties of the test sample, and animal model. 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 (ml/kg) for test sample administration

• 1. Dosage volume references

See References [20], [21], [22], [23], [24], [25], [26], [27], [28].

1. 
   (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^[9])

See References [34] [35] [36], [37], [38], [39].

Table C.2 — Common clinical signs and observations

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

— Clotting potential (PT, APTT)

— Haemoglobin concentration

— Haematocrit

— Platelet count

— Red blood cell count

— White blood cell count

— WBC differential

• 1. Clinical chemistry

— 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^[24]

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

— 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 (*) below 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. 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 material-mediated pyrogens
   1. General

Pyrogenicity is the ability of a chemical agent or other substance to produce a febrile response. Pyrogenic responses may be material-mediated, endotoxin-mediated, or mediated by other substances, such as components of gram-positive bacteria and fungi. This document is concerned with material-mediated pyrogenicity. This topic is covered in more detail in ISO/TR 21582^[7].

It is not necessary to test all new medical devices for in vivo pyrogenicity. However, materials containing substances that have previously elicited a pyrogenic response, and/or new chemical entities where the pyrogenic potential is unknown should be evaluated for material-mediated pyrogenicity. For medical devices that may be used in a combination product, testing to satisfy the product pyrogenicity should be considered. Endotoxin contamination may be a source of a pyrogenic response and should not be confused with a material-mediated pyrogenic response.

• 1. Endotoxin-mediated pyrogenicity

This form of pyrogenicity originating from biologically active endotoxin of gram-negative bacteria, which is usually a fever-inducing contamination in the manufacturing process of medical devices (e.g., use of water that has been contaminated during processing, non-sterile equipment surfaces or device components in contact with contaminated water), is evaluated by measuring the amount of endotoxin in the devices by endotoxin-specific methods such as the LAL (Limulus Amebocyte Lysate) test and an in vitro pyrogen test using human immune cells, the human cell-based pyrogen test (HCPT), both without performing a rabbit test (see Reference [10]).

• 1. Material-mediated pyrogenicity

This type of pyrogenicity originates from non-endotoxin related factors. The following is a list of substances which are known to generate a pyrogenic response, without being endotoxins:

— endogenous pyrogens (e.g., IL-1, IL-6, TNFα, INF-γ);

— prostaglandin;

— inducers (e.g., polyadenylic, polyuridylic, polybionosinic and polyribocytidylic acids);

— substances disrupting the function of thermoregulatory centres (e.g., LSD, cocaine, morphine);

— uncoupling agents of oxidative phosphorylation (e.g., 4, 6-dinitro-o-cresol, dinitrophenol, picric acid);

— N-phenyl-β-naphthylamine and aldo-α-naphthylamine (the febrile mechanism is unknown);

— bacterial exotoxins (e.g., TSST-1, SEA, Spe F, Spe C);

— neurotransmitters (e.g., noradrenaline, serotonin);

— metals such as nickel salts, in some instances.

For detection of material-mediated pyrogenicity, the rabbit pyrogen test, which has a wide range for detecting pyrogenic activity, is currently recommended. Methods for performing the rabbit pyrogen test can be found in the United States Pharmacopoeia, the European Pharmacopoeia and the Japanese Pharmacopoeia. The LAL test is not suitable for determining the pyrogenicity of these substances. The HCPT assay also known as the Monocyte Activation test (MAT), is based on cytokine release by monocytes/macrophages, which is able to detect pyrogenicity related to components of gram-negative and gram-positive bacteria and fungi. However, the MAT has not been validated as an accepted method for detecting material-mediated pyrogenicity. Should the MAT and/or other methods for detecting non-endotoxin pyrogenicity become validated, these will be considered for replacement of the rabbit test.

1. 
   (informative)
   
   Subacute and Subchronic Toxicity in Rats — Dual routes of parenteral administration
   1. General

Many devices requiring subacute/subchronic toxicity testing are implantable devices and thus the most clinically relevant route of exposure in the animal model is via implantation. However, when the device is not intended for implant, exposure of the device via dosing of extracts is an option. Concurrent parenteral administration of polar and nonpolar extracts can be an option. Under circumstances where implantation of an implant medical device is not possible and preparation of coupons of the device is impractical, concurrent infusion of extracts 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. This model may not be appropriate when there is a need to study the administration routes separately. In that case Clause 6 should be considered.

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

Table H.1 — Recommended dosing parameters - Subacute

Table H.2 — Recommended dosing parameters - Subchronic

• 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 [28]) 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 [21].

• • 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 [29] [30]). 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

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.

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