EN ISO 10993-11:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Biologische Beurteilung von Medizinprodukten - Teil 11: Prüfungen auf systemische Toxizität (ISO 10993-11:2006)Évaluation biologique des dispositifs médicaux - Partie 11 : Essais de toxicité systémique (ISO 10993-11:2006)Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (ISO 10993-11:2006)11.100.20Biological evaluation of medical devicesICS:Ta slovenski standard je istoveten z:EN ISO 10993-11:2009SIST EN ISO 10993-11:2009en01-julij-2009SIST EN ISO 10993-11:2009SLOVENSKI
STANDARDSIST EN ISO 10993-11:20061DGRPHãþD

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN ISO 10993-11April 2009ICS 11.100.20Supersedes EN ISO 10993-11:2006
English VersionBiological evaluation of medical devices - Part 11: Tests forsystemic toxicity (ISO 10993-11:2006)Évaluation biologique des dispositifs médicaux - Partie 11:Essais de toxicité systémique (ISO 10993-11:2006)Biologische Beurteilung von Medizinprodukten - Teil 11:Prüfungen auf systemische Toxizität (ISO 10993-11:2006)This European Standard was approved by CEN on 12 April 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 10993-11:2009: ESIST EN ISO 10993-11:2009

Relationship between this
European
Standard and the Essential Requirements
of EU Directive 93/42/EEC on Medical Devices . Error! Bookmark not defined.Annex ZB (informative)
Relationship between this
European
Standard and the Essential Requirements
of EU Directive 90/385/EEC on Active Implantable Medical Devices .5 SIST EN ISO 10993-11:2009

Relationship between this
European
Standard and the Essential Requirements
of EU Directive 93/42/EEC on Medical Devices
This European Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association to provide a means of conforming to Essential Requirements of the New Approach Directive 93/42/EEC on medical devices. Once this standard is cited in the Official Journal of the European Communities under that Directive and has been implemented as a national standard in at least one Member State, compliance with the clauses of this standard given in table ZA confers, within the limits of the scope of this standard, a presumption of conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations. Table ZA — Correspondence between this European Standard and Directive 93/42/EEC on medical devices Clause(s)/sub-clause(s) of this EN Essential Requirements (ERs) of Directive 93/42/EEC Qualifying remarks/Notes 4, 5, 6
Annex I: 7.1, 7.2, 7.5
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.
Relationship between this
European
Standard and the Essential Requirements
of EU Directive 90/385/EEC on Active Implantable Medical Devices This European Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association to provide a means of conforming to Essential Requirements of the New Approach Directive 90/385/EEC on active implantable medical devices. Once this standard is cited in the Official Journal of the European Communities under that Directive and has been implemented as a national standard in at least one Member State, compliance with the clauses of this standard given in table ZB confers, within the limits of the scope of this standard, a presumption of conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.
Table ZB — Correspondence between this European Standard and Directive 90/385/EEC on active implantable medical devices Clause(s)/sub-clause(s) of this EN Essential Requirements (ERs) of Directive 90/385/EEC Qualifying remarks/Notes 4, 5, 6 Annex I : 9
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.
Reference numberISO 10993-11:2006(E)© ISO 2006
INTERNATIONAL STANDARD ISO10993-11Second edition2006-08-15Biological evaluation of medical devices — Part 11: Tests for systemic toxicity Évaluation biologique des dispositifs médicaux — Partie 11: Essais de toxicité systémique
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ISO 10993-11:2006(E) © ISO 2006 – All rights reserved iiiContents Page Foreword.iv Introduction.vi 1 Scope.1 2 Normative references.1 3 Terms and definitions.1 4 General considerations.2 4.1 General.2 4.2 Selection of animal species.3 4.3 Animal status.3 4.4 Animal care and husbandry.3 4.5 Size and number of groups.3 4.6 Route of exposure.4 4.7 Sample preparation.4 4.8 Dosing.5 4.9 Body weight and food/water consumption.6 4.10 Clinical observations.6 4.11 Clinical pathology.6 4.12 Anatomic pathology.7 4.13 Study designs.7 4.14 Quality of investigation.7 5 Acute systemic toxicity.7 5.1 General.7 5.2 Study design.8 5.3 Evaluation criteria.9 5.4 Final report.10 6 Repeated exposure systemic toxicity (subacute, subchronic and chronic systemic toxicity).11 6.1 General.11 6.2 Study design.12 6.3 Evaluation criteria.14 6.4 Final report.15 Annex A (informative)
Routes of administration.16 Annex B (informative)
Dosage volumes.18 Annex C (informative)
Common clinical signs and observations.19 Annex D (informative)
Suggested haematology, clinical chemistry and urinalysis measurements.20 Annex E (informative)
Suggested organ list for histopathological evaluation.22 Annex F (informative)
Information on material-mediated pyrogens.24 Bibliography.26
ISO 10993-11:2006(E) iv © ISO 2006 – All rights reserved 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. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. 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. ISO 10993-11 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices. This second edition cancels and replaces the first edition (ISO 10993-11:1993) which has been technically revised. ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices: ⎯ Part 1: Evaluation and testing ⎯ Part 2: Animal welfare requirements ⎯ Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity ⎯ Part 4: Selection of tests for interactions with blood ⎯ Part 5: Tests for in vitro cytotoxicity ⎯ Part 6: Tests for local effects after implantation ⎯ Part 7: Ethylene oxide sterilization residuals ⎯ Part 9: Framework for identification and quantification of potential degradation products ⎯ Part 10: Tests for irritation and delayed-type hypersensitivity ⎯ Part 11: Tests for systemic toxicity ⎯ Part 12: Sample preparation and reference materials ⎯ Part 13: Identification and quantification of degradation products from polymeric medical devices ⎯ Part 14: Identification and quantification of degradation products from ceramics ⎯ Part 15: Identification and quantification of degradation products from metals and alloys SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) © ISO 2006 – All rights reserved v⎯ Part 16: Toxicokinetic study design for degradation products and leachables ⎯ Part 17: Establishment of allowable limits for leachable substances ⎯ Part 18: Chemical characterization of materials ⎯ Part 19: Physico-chemical, morphological and topographical characterization ⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) vi © ISO 2006 – All rights reserved 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 leachates from the device or its materials to parts of the body with which they are not in direct contact. This part of ISO 10993 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 part of ISO 10993 is not overly prescriptive. Whilst it addresses specific methodological aspects to be considered in the design of systemic toxicity tests, proper study design must be uniquely tailored to the nature of the device’s materials and its intended clinical application. Other elements of this part of ISO 10993 are prescriptive in nature, including those aspects that address compliance with good laboratory practices and elements for inclusion in reporting. While some systemic 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 endpoints are addressed in ISO 10993-3, ISO 10993-6, ISO 10993-10 and ISO/TS 10993-20. Pyrogenicity (see Annex F) represents an additional systemic effect which has historically been included in this part of ISO 10993. However, efforts are being taken to address pyrogenicity in a dedicated, stand-alone standard. Finally, toxicology is an imperfect science. 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.
INTERNATIONAL STANDARD ISO 10993-11:2006(E) © ISO 2006 – All rights reserved 1Biological evaluation of medical devices — Part 11: Tests for systemic toxicity 1 Scope This part of ISO 10993 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 referenced documents are indispensable for the application of this document. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements 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. 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 NOTE There are two types of dose-response relationships. The first type is the response of an individual to a range of doses. The second type is the distribution of responses of a population of individuals to a range of doses. 3.4 leachable substance chemical removed from a device or material by the action of water or other liquids related to the use of the device NOTE Examples of leachable substances are additives, sterilant residues, process residues, degradation products, solvents, plasticizers, lubricants, catalysts, stabilizers, anti-oxidants, colouring agents, fillers and monomers. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) 2 © ISO 2006 – All rights reserved 3.5 limit test use of a single group treated at a suitable dosage of test sample in order to delineate the presence or absence of a toxic hazard 3.6 systemic toxicity toxicity that is not limited to adverse effects at the site of contact between the body and the device NOTE Systemic toxicity requires absorption and distribution of a toxicant from its entry point to a distant site at which deleterious effects are produced. 3.7 acute systemic toxicity adverse effects occurring at any time after single, multiple or continuous exposures of a test sample within 24 h 3.8 subacute systemic toxicity adverse effects occurring after multiple or continuous exposure between 24 h and 28 d NOTE 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 intravenous studies are generally defined as treatment durations of > 24 h but < 14 d. 3.9 subchronic systemic toxicity adverse effects occurring after the repeated or continuous administration of a test sample for a part of the lifespan NOTE Subchronic toxicity studies are usually 90 d in rodents but not exceeding 10 % of the lifespan of other species. Subchronic intravenous studies are generally defined as treatment durations of 14 d to 28 d. 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 Chronic toxicity studies usually have a duration of 6 months to 12 months. 3.11 test sample material, device, device portion, component, extract or portion thereof that is subjected to biological or chemical testing or evaluation 4 General considerations 4.1 General Selection of the appropriate test(s) for a device shall be in accordance with ISO 10993-1, giving due consideration to mode and duration of contact. 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 deviations are necessary, they shall be recorded in the test report, together with their justification. For hazard identification purposes, it may be necessary to exaggerate exposure to the test samples. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) © ISO 2006 – All rights reserved 3Physical 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, to satisfy the provisions of ISO 10993-2, all reasonably and practically available replacement, reduction and refinement alternatives should be identified and implemented. For in vivo acute toxicity testing, in vitro cytotoxicity data are useful in estimating starting doses [9]. 4.2 Selection of animal species 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 the mouse or rat is preferred with the option of the rabbit in the case of dermal and implantation studies. 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 is used when a series of systemic toxicity studies of different durations are performed, e.g. acute, subacute, subchronic and/or chronic systemic toxicity. This controls 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. 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 conform to accepted animal husbandry guidelines. 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 necessary for meaningful 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 purpose of the study. Group sizes should logically increase with the duration of treatment, such that at the end of the study enough animals in every group are available for thorough biological evaluation. However, the minimum number of animals should be used consistent with obtaining meaningful results (see ISO 10993-2). Recommended minimum group sizes, all routes considered, are given in Table 1. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) 4 © ISO 2006 – All rights reserved Table 1 — Recommended minimum group sizes Study type Rodent Non-rodent Acute a 5 3 Subacute 10 (5 per sex) a 6 (3 per sex) a Subchronic 20 (10 per sex) a 8 (4 per sex) a Chronic 40 (20 per sex) b, c c a Testing in a single sex is acceptable. When a device is intended for use in only one sex, testing should be done in that sex. b The recommendation 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 group size is recommended. The number of animals tested should be based on the minimum required to provide meaningful data. Enough animals must 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 toxic hazard (i.e., limit test). However, other multi-dose or dose response studies require multiple groups to delineate the toxic response. Group numbers may increase when attempting to exaggerate the dose. The following examples for exaggerating dose should be considered: ⎯ multiples of the clinical surface area 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. 4.7 Sample preparation Guidance on sample preparation and stability is given in ISO 10993-12. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) © ISO 2006 – All rights reserved 54.8 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. In general, room or body temperature is a common practice. Deviations shall be justified. Vehicles administered by a parenteral route should be physiologically compatible. When necessary, sample filtration to remove particulates should be used and documented. Restraint of animals in repeated exposure systemic toxicity studies should generally be limited to between 4 h and 6 h per day. 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. When restraint is required animals should be acclimatized to the restraint device prior to test sample administration. 4.8.2 Dosage volumes 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 short period of approximately 1 min. The rate of injection is an important factor and it is suggested that, for rodents, the rate shall not exceed 2 ml/min. 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. As a guide, the volume administered on a single occasion will be < 10 % of the circulating blood volume over 2 h. Minimal effective restraint of test animals is a key factor to be considered for prolonged infusion. For subcutaneous administration of test article, refer to Annex B. The rate and extent of absorption depends on the test sample formulation. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) 6 © ISO 2006 – All rights reserved 4.8.3 Dosage frequency The dosage frequency should be based on clinical relevancy. Exaggerated procedures shall be clearly described 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. 4.9 Body weight and food/water consumption 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. When dosing by body weight, the most recent body weight should be utilized. Measurements of food and water consumption, as appropriate, shall be considered for longer-term repeated exposure studies. 4.10 Clinical observations 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 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 should be used in order to avoid unnecessary suffering. 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). Morbidity and mortality observations shall be recorded at least twice daily for long-term repeated exposure studies. A more extensive screening for adverse clinical signs may be considered on at least a weekly basis for longer-term repeated exposure studies. 4.11 Clinical pathology 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. Suggested haematology, clinical chemistry and urinalysis parameters for evaluation are listed in Annex D. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) © ISO 2006 – All rights reserved 74.12 Anatomic pathology When clinically indicated, gross pathological evaluations should be considered for acute systemic toxicity studies. 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 Observation Acute Subacute Subchronic/chronic aBody weight change + + + Clinical observations + + + Clinical pathology b a, b + Gross pathology b + + Organ weights b + + Histopathology b a, b + a Chronic systemic toxicity testing is generally a time extension of subchronic testing, justified by the human exposure period. Many of the same parameters are recorded and reported. Group sizes may be increased to include satellite groups for which some, or all, of these observations may be made. b Consideration should be given to these measurements when clinically indicated or if longer exposure testing is not anticipated. Lists of suggested blood and organ/tissue analyses are included in Annex D and Annex E. 4.13 Study designs Study designs are listed in subsequent sections of this part of ISO 10993. Expert consultation for study design is recommended. 4.14 Quality of investigation Good laboratory practices deal with the organization, process and conditions under which laboratory studies are planned, performed, monitored, recorded and reported. These practices are intended to promote the quality and validity of the test data. They also support the global harmonization effort by facilitating the memoranda of understanding between trading nations. Systemic toxicity studies shall be conducted following such principles. 5 Acute systemic toxicity 5.1 General Acute systemic toxicity provides general information on health hazards 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 SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) 8 © ISO 2006 – All rights reserved 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. Corrosive or irritating materials known to cause marked pain or distress should be reported as such and need not be tested. NOTE ICCVAM and ECVAM are currently validating in vitro cytotoxicity tests as an alternative to acute toxicity testing. 5.2 Study design 5.2.1 Preparations Healthy young adult 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. 5.2.2 Experimental animals 5.2.2.1 Selection of species Typically, a rodent species (rat, mouse) will be used. Characteristics of the model (age, weight, etc.) are as described in 4.2 and 4.3. If non-rodent species are used their use shall be scientifically justified. 5.2.2.2 Number and sex The number and type of group, animals per group, and sex are as described in 4.5. 5.2.2.3 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. 5.2.3 Test conditions 5.2.3.1 Dose levels Dose levels shall be as described in 4.8. Animals in the control group should be handled in an identical manner to the test group subjects with the exception of not being dosed with the test sample. 5.2.3.2 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. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to cannibalism, autolysis of tissues or misplacement. At the end of the study all surviving animals are euthanized. Any moribund animals should be removed and euthanized when noticed to exhibit such behaviour. The observation schedules and humane endpoints applied should preclude the possibility of animals being found dead as a direct consequence of test sample toxicity. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) © ISO 2006 – All rights reserved 95.2.4 Body weights 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. 5.2.5 Clinical observations 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 described in 4.10 and Annex C. In all cases, observations shall be made at a frequency, and appropriate actions taken, to minimize the loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. 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. 5.2.6 Pathology 5.2.6.1 Clinical pathology Clinical pathology evaluations shall be considered when clinically indicated. The following examinations should be made. a) Haematology, as described 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 is not necessary on a routine basis but only when there is an indication based on expected or observed toxicity. Suggested parameters are listed in Annex D. 5.2.6.2 Gross pathology Gross pathological evaluations shall be considered when clinically indicated. 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. 5.2.6.3 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. 5.3 Evaluation criteria 5.3.1 General Depending on the test design utilized, the following evaluation criteria apply. a) For pharmacopoeia-type testing ⎯ 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. SIST EN ISO 10993-11:2009

ISO 10993-11:2006(E) 10 © ISO 2006 – All rights reserved ⎯ Using five an
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