prEN ISO 10993-11

Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (ISO/DIS 10993-11:2025)

ISO 10993-11:2017 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.

Biologische Beurteilung von Medizinprodukten - Teil 11: Prüfungen auf systemische Toxizität (ISO/DIS 10993‑11:2025)

Évaluation biologique des dispositifs médicaux - Partie 11: Essais de toxicité systémique (ISO/DIS 10993-11:2025)

ISO 10993-11:2017 spécifie des exigences et fournit des préconisations quant aux modes opératoires à suivre pour évaluer les éventuelles réactions systémiques défavorables causées par les matériaux de dispositifs médicaux.

Biološko ovrednotenje medicinskih pripomočkov - 11. del: Preskusi sistemske toksičnosti (ISO/DIS 10993-11:2025)

General Information

• Status: Not Published
• Publication Date: 25-Oct-2026
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: CEN/TC 206 - Biocompatibility of medical and dental materials and devices
• Current Stage: 4060 - Closure of enquiry - Enquiry
• Start Date: 20-Jun-2025
• Completion Date: 20-Jun-2025
• Directive: 2017/745 - Regulation (EU) 2017/745 of the European Parliament and of the Council of 5 April 2017 on medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 and repealing Council Directives 90/385/EEC and 93/42/EEC
• Mandate: M/575 - Medical devices in support of Regulation (EU) 2017/745 and in vitro diagnostic medical devices in support of Regulation (EU) 2017/746
• Mandate: M/575 AMD 2 - Medical devices in support of Regulation (EU) 2017/745 and in vitro diagnostic medicial devices in support of Regulation (EU) 2017746
• Ref Project: oSIST prEN ISO 10993-11:2025 - Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (ISO/DIS 10993-11:2025)

Relations

• Revises: EN ISO 10993-11:2018 - Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (ISO 10993-11:2017)
• Effective Date: 26-Apr-2023

Overview

The prEN ISO 10993-11:2025 standard, titled Biological evaluation of medical devices - Part 11: Tests for systemic toxicity, is an international draft standard developed by ISO/TC 194 in collaboration with CEN/TC 206. It provides detailed requirements and guidance for evaluating medical device materials to identify their potential to cause adverse systemic toxic effects following exposure. This fourth edition updates and supersedes the 2017 version, with an emphasis on risk assessment throughout the evaluation process.

This standard is key for manufacturers, regulators, and testing laboratories engaged in biocompatibility testing of medical devices. It outlines protocols for acute and repeated systemic toxicity tests, specifying animal model selection, dosing, clinical observation, pathology, and reporting criteria. By following prEN ISO 10993-11:2025, stakeholders ensure medical devices comply with global safety regulations to minimize systemic health risks to patients.

Key Topics

• Scope and Application: The standard covers systemic toxicity tests necessary for biological evaluation of medical devices' materials, focusing on identification and characterization of adverse systemic reactions.
• Animal Model Selection: Guidance on choosing appropriate species and health status of test animals to achieve reliable and reproducible results.
• Exposure Routes and Dosage: Detailed instructions on administration methods (oral, intravenous, subcutaneous, etc.), dosage levels, frequency, and sample preparation to simulate realistic patient exposure.
• Study Design and Procedures: Framework for conducting acute and repeated systemic toxicity studies, including control groups, group sizes, clinical observations, body weight monitoring, and pathology assessments.
• Evaluation Criteria: Methods for assessing and interpreting toxicological data to determine safety profiles, including clinical pathology, anatomic pathology, and toxic signs.
• Reporting Requirements: Template for comprehensive final reports that include all test conditions, observations, results, analytical procedures, and conclusions.
• Risk Assessment: Emphasis on integrating toxicological data into overarching risk assessment to support regulatory submissions and safe device design.
• Informative Annexes: Additional resources like dose volume specifications, common clinical signs, hematology and pathology guidelines, and correlation with regulatory requirements such as EU MDR.

Applications

prEN ISO 10993-11:2025 is essential for:

• Medical Device Development: Ensuring new materials and device components do not cause systemic toxicity, enabling safe product launches.
• Regulatory Compliance: Supporting submissions for approvals under frameworks such as the EU Medical Device Regulation (MDR) 2017/745 and FDA requirements by providing standardized safety data.
• Biocompatibility Testing Laboratories: Establishing test protocols and evaluation criteria for systemic toxicity assessments.
• Quality Assurance Programs: Integrating toxicity testing into product safety risk management and quality control.
• Clinical Risk Evaluation: Informing clinical use considerations by identifying systemic toxicological risks associated with devices.

Related Standards

• ISO 10993 Series: This part 11 complements other parts addressing biological evaluation aspects like cytotoxicity (Part 5), sensitization (Part 10), and genotoxicity (Part 3).
• ISO 10993-1: Provides general principles and strategies for the biological evaluation of medical devices.
• ISO 10993-12: Specifies requirements for sample preparation and reference materials for biological testing.
• EU MDR Regulation (EU) 2017/745: prEN ISO 10993-11 supports conformity to general safety and performance requirements related to systemic toxicity.
• ISO/IEC Directives: Framework for developing and maintaining ISO standards.

By adhering to prEN ISO 10993-11:2025, stakeholders can confidently assess systemic toxicity risks, ensuring safer medical devices and compliance with international regulatory expectations. This fosters innovation, patient safety, and global market access for medical device manufacturers.

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2025
Biološko ovrednotenje medicinskih pripomočkov - 11. del: Preskusi sistemske
toksičnosti (ISO/DIS 10993-11:2025)
Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (ISO/DIS
10993-11:2025)
Biologische Beurteilung von Medizinprodukten - Teil 11: Prüfungen auf systemische
Toxizität (ISO/DIS 10993-11:2025)
Évaluation biologique des dispositifs médicaux - Partie 11: Essais de toxicité systémique
(ISO/DIS 10993-11:2025)
Ta slovenski standard je istoveten z: prEN ISO 10993-11
ICS:
11.100.20 Biološko ovrednotenje Biological evaluation of
medicinskih pripomočkov medical devices
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 10993-11
ISO/TC 194
Biological evaluation of medical
Secretariat: DIN
devices —
Voting begins on:
Part 11: 2025-03-28
Tests for systemic toxicity
Voting terminates on:
2025-06-20
Évaluation biologique des dispositifs médicaux —
Partie 11: Essais de toxicité systémique
ICS: 11.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 10993-11:2025(en)
DRAFT
ISO/DIS 10993-11:2025(en)
International
Standard
ISO/DIS 10993-11
ISO/TC 194
Biological evaluation of medical
Secretariat: DIN
devices —
Voting begins on:
Part 11:
Tests for systemic toxicity
Voting terminates on:
Évaluation biologique des dispositifs médicaux —
Partie 11: Essais de toxicité systémique
ICS: 11.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2025
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BE CONSIDERED IN THE LIGHT OF THEIR
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
CH-1214 Vernier, Geneva
NOTIFICATION OF ANY RELEVANT PATENT
Phone: [phone]
RIGHTS OF WHICH THEY ARE AWARE AND TO
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Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 10993-11:2025(en)
ii
ISO/DIS 10993-11:2025(en)
Contents Page
Foreword .v
Introduction .vi
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 .3
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 .4
4.5.3 Treatment controls .5
4.6 Route of exposure .5
4.7 Sample preparation .5
4.8 Dosing .5
4.8.1 Test sample administration .5
4.8.2 Dosage .6
4.8.3 Dosage frequency .7
4.9 Body weight and food/water consumption .7
4.10 Clinical observations .7
4.11 Clinical pathology .8
4.12 Anatomic pathology .8
4.13 Study designs .8
4.14 Quality of investigation .8
5 Acute systemic toxicity . 9
5.1 General .9
5.2 Study design .9
5.2.1 Preparations .9
5.2.2 Experimental animals .9
5.2.3 Test conditions .10
5.2.4 Body weights . .10
5.2.5 Clinical observations .10
5.2.6 Pathology .10
5.3 Evaluation criteria .11
5.3.1 General .11
5.3.2 Evaluation of results .11
5.4 Final report . 12
6 Repeated exposure systemic toxicity (subacute, subchronic and chronic systemic
toxicity) .13
6.1 General . 13
6.2 Study design . 13
6.2.1 Preparations . 13
6.2.2 Experimental animals . 13
6.2.3 Test conditions .14
6.2.4 Body weights . .14
6.2.5 Clinical observations .14
6.2.6 Pathology . 15
6.3 Evaluation criteria .16
6.3.1 General .16
6.3.2 Evaluation of results .16
6.4 Final report .16

iii
ISO/DIS 10993-11:2025(en)
Annex A (informative) Routes of administration . 17
Annex B (informative) Dose volumes .20
Annex C (informative) Common clinical signs and observations .21
Annex D (informative) Suggested haematology, clinical chemistry and urinalysis measurements .23
Annex E (informative) Suggested organ list for histopathological evaluation .25
Annex F (informative) Organ list for limited histopathology for medical devices subjected to
systemic toxicity testing .27
Annex G (informative) Information on material-mediated pyrogens .28
Annex H (informative) Subacute and Subchronic Toxicity in Rats — Dual routes of parenteral
administration .30
Annex ZA (informative) Relationship between this European Standard the General Safety and
Performance Requirements of Regulation (EU) 2017/745 aimed to be covered .32
Bibliography .34

iv
ISO/DIS 10993-11:2025(en)
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.

v
ISO/DIS 10993-11:2025(en)
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.
vi
DRAFT International Standard ISO/DIS 10993-11:2025(en)
Biological evaluation of medical devices —
Part 11:
Tests for systemic toxicity
1 Scope
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.
2 Normative references
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
3 Terms and definitions
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

ISO/DIS 10993-11:2025(en)
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

ISO/DIS 10993-11:2025(en)
4 General considerations
4.1 General
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
[11]
overall biological evaluation .
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.
4.2 Selection of animal model
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.

ISO/DIS 10993-11:2025(en)
4.3 Animal status
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.
4.4 Animal care and husbandry
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.
4.5 Size and number of groups
4.5.1 Size of groups
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.
a
Table 1 — Recommended minimum group sizes
Study type Rodent Rabbit Non-rodent
Acute 5 3 3
Subacute 10 (5 per sex) 6 (3 per sex) 6 (3 per sex)
Subchronic 20 (10 per sex) 8 (4 per sex) 8 (4 per sex)
b c
Chronic 30 (15 per sex) 12 (6 per sex)
a
Testing in a single sex is typical for acute and pharmacopeia-type testing. When a
device is intended for use in only one sex, testing should be done in that sex.
b
The recommendation for rodents refers to one dose-level group testing. Where
additional exaggerated dose groups are included the recommended group size may be
reduced to 10 per sex.
c
Expert statistical consultation for chronic study non-rodent group size is
recommended. The number of animals tested should be based on the minimum required
to provide meaningful data. Enough animals shall remain at the termination of the study
to ensure proper statistical evaluation of the results.
4.5.2 Number of groups
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.

ISO/DIS 10993-11:2025(en)
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.
4.5.3 Treatment controls
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.
4.6 Route of exposure
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.
4.7 Sample preparation
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.
4.8 Dosing
4.8.1 Test sample administration
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.

ISO/DIS 10993-11:2025(en)
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
[19]
Animals . 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
[21]
prolonged infusion .
4.8.2 Dosage
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.

ISO/DIS 10993-11:2025(en)
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
...