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This document is developed in accordance with the rules given in GB/T 1.1-2020 Directives for standardization — Part 1: Rules for the structure and drafting of standardizing documents. This document is part 11 of GB/T 16886 under the general title of "Biological evaluation of medical devices". The following parts of GB/T 16886 have been issued: ——Part 1: Evaluation and testing within a risk management process; ——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 skin sensitization; ——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; ——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 of materials; ——Part 20: Principles and methods for immunotoxicology testing of medical devices. This part replaces GB/T 16886.11-2011 Biological evaluation of medical devices — Part 11: Tests for systemic toxicity. The following main changes have been made with respect to GB/T 16886.11-2011: ——The size of groups in tests for chronic toxicity has been modified (see Table 1 hereof and Table 1 of 2011 Edition). This document, by means of translation, is identical to ISO 10993-11:2017 Biological evaluation of medical devices — Part 11: Tests for systemic toxicity. The Chinese document consistent and corresponding with the normative international document in this Documentation is as follows: ——GB/T 16886.1-2011 Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk management process (ISO 10993-1:2009, IDT); ——GB/T 16886.2-2011 Biological evaluation of medical devices — Part 2: Animal welfare requirements (ISO 10993-2:2006, IDT). Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights. This document was proposed by the National Medical Products Administration of People’s Republic of China. This document is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248). The previous editions of this document and documents replaced by this document are as follows: ——It was first issued as GB/T 16886.11-1997 in 1997, first revised in 2011; ——This edition is the second revision. 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 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 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 end points are addressed in GB/T 16886.3, GB/T 16886.6, GB/T 16886.10 and GB/T 16886.20. 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 input data such as existing toxicological data, data from chemical characterization studies and/or other biological tests (including in vitro tests and less invasive in vivo tests) for the refinement of study design, dose selection, and/or selection of pathological end points to cover in the evaluation of a study. 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 (including histopathological) and clinical chemistry methods are important so as to obtain data which have sufficient scientific validity. 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. Biological evaluation of medical devices is proposed to consist of 21 parts as follows: ——Part 1: Evaluation and testing within a risk management process. It is developed to protect human being from potential biological risk when using medical devices, describes the biological evaluation of medical devices in the process of a risk management, and is regarded as an integral part of the overall evaluation and development process of medical devices. ——Part 2: Animal welfare requirements. It is developed to make full use of scientific and reasonable non-animal tests to ensure that animal tests used to evaluate the biological properties of materials used in medical devices conform to recognized ethical and scientific principles. ——Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity. It is developed to provide evaluation guidelines and methods for medical devices that have been confirmed with genotoxicity, carcinogenicity and reproductive toxicity. ——Part 4: Selection of tests for interactions with blood. It is developed to provide general requirements for the evaluation of interactions between medical devices and blood. ——Part 5: Tests for in vitro cytotoxicity. It is developed to provide test methods for evaluating the in vitro cytotoxicity of medical devices. ——Part 6: Tests for local effects after implantation. It is developed to provide test methods for evaluating the local effects after implantation of biomaterials used in medical devices. ——Part 7: Ethylene oxide sterilization residuals. It is developed to provide test procedures for allowable limits for ethylene oxide (EO) on individual medical device with EO sterilization and residual ethylene chlorohydrin (ECH), EO, and ECH, and provide test methods for determining whether medical devices may be released. ——Part 9: Framework for identification and quantification of potential degradation products. It is developed to provide basic principles for systematic evaluation of potential and observed biodegradation of medical devices and the design and implementation of biodegradation research. ——Part 10: Tests for irritation and skin sensitization. It is developed to provide evaluation steps for potential irritation and skin sensitization of medical devices and their constituent materials. ——Part 11: Tests for systemic toxicity. It is developed to provide a guide to test procedures for evaluating potential adverse systemic reactions caused by medical device materials. ——Part 12: Sample preparation and reference materials. It is developed to provide a guide to selection of sample preparation and reference materials for biological evaluation of medical devices. ——Part 13: Identification and quantification of degradation products from polymeric medical devices. It is developed to provide general requirements for qualitative and quantitative test design of degradation products from finished polymeric medical devices for clinical use in simulated environment. ——Part 14: Identification and quantification of degradation products from ceramics. It is developed to provide a method for obtaining solutions for quantification of degradation products from ceramics. ——Part 15: Identification and quantification of degradation products from metals and alloys. It is developed to provide general requirements for qualitative and quantitative test design of degradation products from metal medical devices or corresponding material samples for clinical purpose. ——Part 16: Toxicokinetic study design for degradation products and leachables; It is developed to provide principles for the toxicokinetic study design and implementation related to medical devices. ——Part 17: Establishment of allowable limits for leachable substances. It is developed to provide a method for establishment of allowable limits for leachable substances for medical devices. ——Part 18: Chemical characterization of materials. It is developed to provide a framework for the qualitative and quantitative (if necessary) identification of biological hazards and the estimation and control of biological risks in material components. ——Part 19: Physico-chemical, morphological and topographical characterization of materials. It is developed to identify and evaluate various parameters and test methods for physical properties of final medical device materials, such as the physicochemical, morphological and topographical (PMT). ——Part 20: Principles and methods for immunotoxicology testing of medical devices. It is developed to provide an immunotoxicology review of potential immunotoxicity of medical devices and a guide for testing immunotoxicity of different types of medical devices. ——Part 22: Guidance on nanomaterials. It is developed to provide guidance for the biological evaluation of medical devices containing, producing or consisting of nanomaterials. ——Part 23: Tests for irritation. It is developed to provide evaluation steps for potential irritation of medical devices and their constituent materials. 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. This document is applicable to the study of systemic toxicity of medical devices or materials. 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: Evaluation and testing within a risk management process ISO 10993-2 Biological evaluation of medical devices — Part 2: Animal welfare requirements 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. 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 within 72 h after single, multiple or continuous exposures of a test sample for 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 for rodents and non-rodents, respectively. 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 Before a decision to perform a systemic toxicity test is made, ISO 10993-1 shall be taken into account. The decision to perform a test shall be justified on the basis of an assessment of the risk of systemic toxicity. 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. 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 alternatives should be identified and implemented to satisfy the provisions of ISO 10993-2. For in vivo acute toxicity testing, in vitro cytotoxicity data are useful in estimating starting doses. 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 rodent (mouse or rat) is preferred with the option of the rabbit (lagomorph) in the case of dermal and implantation studies. 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 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. Table 1 Recommended minimum group sizes Study type Rodent Non-rodent Acutea 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 30 (15 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 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 toxic 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 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. 4.7 Sample preparation Guidance on sample preparation and stability 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. 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. When medical devices and/or test samples in the form of nanomaterials are to be evaluated sample filtrations shall not be performed. (See ISO/TR 10993-22). 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 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. 4.8.3 Dosage frequency 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. 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 end points, as defined by national or international animal welfare guidelines, 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. 4.12 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 Chronica Body weight change + + + Clinical observations + + + Clinical pathology b a, b + Gross pathology b + + Organ weights b + + Histopathology b a, b + + Data should be provided. a Chronic systemic toxicity testing is generally a time extension of subacute/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 bodily fluids and organ/tissue analyses are included in Annex D, Annex E and Annex F. 4.13 Test designs Study designs are listed in subsequent clauses of this document. Expert consultation for study design is recommended. Note: See Annex G for the information on material-mediated pyrogens. 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 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. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the European Centre for the Validation of Alternative Methods (ECVAM) have validated the in vitro cytotoxicity test as an alternative to acute oral toxicity testing. Humane end points, as defined by national or international animal welfare guidelines, should be used in order to avoid unnecessary suffering. 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 specified 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 specified 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 specified 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. 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. 5.2.4 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 specified 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 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. Suggested parameters are listed in Annex D. 5.2.6.2 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. 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. 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. 3) If any animals treated with the sample show only slight signs of biological reactivity, and not more than one animal shows gross symptoms of biological reactivity or dies, repeat the testing using groups of 10 animals. 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. 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. Foreword i Introduction iii 1 Scope 2 Normative references 3 Terms and definitions 4 General considerations 4.1 General 4.2 Selection of animal species 4.3 Animal status 4.4 Animal care and husbandry 4.5 Size and number of groups 4.5.1 Size of groups 4.5.2 Number of groups 4.5.3 Treatment controls 4.6 Route of exposure 4.7 Sample preparation 4.8 Dosing 4.8.1 Test sample administration 4.8.2 Dosage volumes 4.8.3 Dosage frequency 4.9 Body weight and food/water consumption 4.10 Clinical observations 4.11 Clinical pathology 4.12 Anatomic pathology 4.13 Test designs 4.14 Quality of investigation 5 Acute systemic toxicity 5.1 General 5.2 Study design 5.2.1 Preparations 5.2.2 Experimental animals 5.2.3 Test conditions 5.2.4 Body weights 5.2.5 Clinical observations 5.2.6 Pathology 5.3 Evaluation criteria 5.3.1 General 5.3.2 Evaluation of results 5.4 Final report 6 Repeated exposure systemic toxicity (subacute, subchronic and chronic systemic toxicity) 6.1 General 6.2 Study design 6.2.1 Preparations 6.2.2 Experimental animals 6.2.3 Test conditions 6.2.4 Body weights 6.2.5 Clinical observations 6.2.6 Pathology 6.3 Evaluation criteria 6.3.1 General 6.3.2 Evaluation of results 6.4 Final report Annex A (Informative) Exposure routes Annex B (Informative) Dosage volumes Annex C (Informative) Common clinical signs and observations Annex D (Informative) Suggested haematology, clinical chemistry and urinalysis measurements Annex E (Informative) Suggested organ list for histopathological evaluation Annex F (Informative) Organ list for limited histopathology for medical devices subjected to systemic toxicity testing Annex G (Informative) Information on material-mediated pyrogens Annex H (Informative) Subchronic rat — Dual routes of parenteral administration Bibliography 医疗器械生物学评价 第11部分：全身毒性试验 1 范围 本文件规定了评价医疗器械材料导致潜在不良全身反应时的要求，给出了试验步骤指南。 本文件适用于医疗器械或材料的全身毒性研究。 2 规范性引用文件 下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件，仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本(包括所有的修改单)适用于本文件。 ISO 10993-1 医疗器械生物学评价 第1部分：风险管理过程中的评价与试验(Biological evalu-ation of medical devices—Part 1：Evaluation and testing within a risk management process) ISO 10993-2 医疗器械生物学评价 第2部分：动物福利要求(Biological evaluation of medical devices—Part 2：Animal welfare requirements) 3 术语和定义 ISO 10993-1界定的以及下列术语和定义适用于本文件。 3.1 剂量 dose／dosage 每单位体重或表面积给予的试验样品的量(如质量、体积)。 3.2 剂量效应 dose-effect 个体或群体研究对象中，剂量与特定生物学反应程度之间的关系。 3.3 剂量反应 dose-response 剂量与接触相关作用范围之间的关系。 注：剂量反应关系有两种类型，第一种是个体对剂量范围的反应，第二种是群体研究对象剂量范围反应的分布。 3.4 可沥滤物 leachable substance 水或其他与器械使用相关的液体作用于器械或材料后迁移出的化学物。 注：可沥滤物包括添加剂、灭菌残留物、加工残留物、降解产物、溶剂、增塑剂、润滑剂、催化剂、稳定剂、抗氧化剂、染色剂、填料及单体。 3.5 限度试验 limit test 采用一种适宜的试验样品剂量进行的单剂量组试验，以判定是否存在毒性危害。 3.6 全身毒性 systemic toxicity 非限定于机体与器械接触部位不良作用的毒性。 注：全身毒性是指毒性物质在进入机体至远端部位的吸收与分布中产生的有害作用。 3.7 急性全身毒性 acute systemic toxicity 在24 h内一次、多次或持续接触试验样品后在72 h内任何时间发生的不良作用。 3.8 亚急性全身毒性 subacute systemic toxicity 在24 h～28 d内多次或持续接触试验样品后发生的不良作用。 注：由于该术语在语义上不确切，在该限定时间周期内发生的不良作用也可称为短期重复性接触全身毒性研究。国际规范性导则大多选择14 d～28 d的时间周期，并考虑合理的方法。亚急性静脉研究一般规定接触时间＞24 h，但＜14 d。 3.9 亚慢性全身毒性 subchronic systemic toxicity 反复或持续接触试验样品后在动物寿命期的某一阶段发生的不良作用。 注：啮齿动物亚慢性毒性研究一般为90 d，其他种属动物在不超过其寿命期的10%的阶段内。亚慢性静脉研究一般规定啮齿类和非啮齿类动物接触时间分别为14 d～28 d。 3.10 慢性全身毒性 chronic systemic toxicity 在动物的主要寿命期内反复或持续接触试验样品后发生的不良作用。 注：慢性毒性研究一般6～12个月。 3.11 试验样品 test sample 用于生物学、化学试验或评价的材料、器械、器械的一部分、组件、浸提液或与之相关的部件。 4 基本考虑 4.1 总则 决定进行全身毒性试验之前，应考虑按照ISO 10993-1。应基于对全身毒性的风险评定对进行试验的决定进行确认。应按照ISO 10993-1中给出的接触方式和接触时间来考虑选择器械的适宜试验。 应在最终产品、最终产品和／或材料中的代表性组件上进行试验。试验样品应体现器械的正常制造和加工状态，若必需偏离，应记录在试验报告中并说明理由。出于识别危害的目的，可能需要加大对试验样品的接触。 在设计试验时要考虑的一些因素包括试验样品的物理和化学性能，如pH、稳定性、黏度、渗透压、缓冲能力、溶解度和无菌性。 当考虑动物试验时，宜分析并采用所有合理并可行的替代、减少及优化的替代方法，以符合ISO 10993-2的规定。对于体内急性毒性试验，体外细胞毒性数据可用于估计起始剂量。 4.2 动物种属选择 对于医疗器械全身毒性试验具体动物种属的选择没有绝对的要求。但是，试验所用动物种属应经过科学论证并符合ISO 10993-2的规定。对于医疗器械急性经口、静脉、皮肤和吸入研究，首选啮齿动物(小鼠或大鼠)，皮肤和植入研究选择家兔。认识到许多因素可能会影响研究物种的数量或选择，也可能考虑用其他非啮齿动物进行测试。 在进行不同时间周期的全身毒性系列研究时，如急性、亚急性、亚慢性、和／或慢性全身毒性研究，首选采用同一种动物种属和动物品系，这样可控制动物种属和品系间的变异性，以利于对研究周期进行完全相关性评价。如使用多种动物种属或品系，对其选择理由应形成文件。 4.3 动物状况 一般情况下，宜使用已知来源并符合规定微生物健康状况的健康、性成熟的饲育动物。在研究的开始阶段，同一性别的动物体重差异应不超过平均值的±20%。如使用雌性动物，宜未育并无孕对动物选择应进行论证。 4.4 动物饲养与管理 动物饲养与管理应符合有关动物管理规定。在处置之前以及规定的试验周期内，应使动物适应实验室条件。为了获得有效的试验结果，环境条件的控制以及适当的动物饲养技术是必要的。宜对已知产生或影响毒性的饲料成分和垫料进行适当鉴别，并考虑对试验结果的潜在影响。 4.5 剂量组大小和数目 4.5.1 剂量组大小 全身毒性试验的精确度在很大程度上取决于每一剂量水平所采用的动物数量。试验需要达到的精确程度，或者说每剂量组需要达到的动物数量要根据研究的目的来确定。 剂量组宜根据试验周期而增加动物数量，这样在试验终结时每组能有足够的动物进行有效的生物学评价。但是，宜采用最少的动物来获取有效结果(见ISO 10993-2)。推荐的各接触途径的最少动物数量见表1。 表1 推荐的剂量组最少动物数量 试验类别 啮齿动物 非啮齿动物 急性a 5只 3只 亚急性 10只(每种性别各5只)a 6只(每种性别各3只)a 亚慢性 20只(每种性别各10只)a 8只(每种性别各4只)a 慢性 30只(每种性别各15只)b，c c a 可采用单一性别动物进行试验。如预期器械仅用于一种性别时，试验宜在该性别动物体上进行。 b 啮齿类单剂量组试验可参考该推荐数量，额外增加的剂量组可减少至每种性别各10只。 c 建议与统计学专家商讨非啮齿类慢性试验剂量组的动物数量，实验动物的数量宜根据能提供有效数据所需的最少动物数来确定，试验终结时应有足够的动物以保证能对结果进行适当的统计学评价。 4.5.2 剂量组数目 在单一种属动物中进行适宜剂量的单剂量组试验组可以用来判断是否存在毒性危害(即限度试验)，但是在其他多剂量试验或剂量反应试验中需要采用多个剂量组来对毒性反应进行描绘。 如准备采用加大剂量表征剂量反应时，可增加剂量组。加大剂量宜考虑下列参数： ——临床接触表面积的倍数； ——接触时间的倍数； ——浸提组分或具体化学物的倍数； ——24h内多次接触。 也可采用其他加大剂量方法，所用方法应进行论证。 4.5.3 对照设置 根据试验目的，宜结合试验样品状态、接触途径、阴性对照、介质对照、和／或假处理对照(sham-treated controls)来进行全身毒性试验。这些对照应模拟试验样品制备和处理步骤。 4.6 接触途径 医疗器械或其可沥滤物可通过多种接触途径进入人体，试验接触途径应尽可能与器械的应用具有临床相关性，如必需采用其他途径应进行论证。接触途径示例见附录A。 4.7 样品制备 ISO 10993-12给出了样品制备和稳定性导则。 4.8 剂量 4.8.1 试验样品接触 宜设计试验样品接触程序，以避免与试验材料毒性无直接关系的生理变化或动物福利问题。如单次单日剂量达不到足够的体积或浓度，可采取不超过24 h分次小剂量给予的方式。 试验样品应在生理可接受温度下给予，一般常规采用室温或体温，如偏离应进行论证。 胃肠外接触途径宜是生理相容性溶液，必要时可对样品进行过滤除去微粒并形成文件。当评价纳米材料形式的医疗器械和／或试验样品时，不应进行过滤(见ISO／TR 10993-22)。 重复接触全身毒性试验中，动物的活动限制每天一般在4 h～6 h。动物活动限制的程度和时间宜最小，要求符合科学目的，不宜仅考虑实验动物福利的要求。如偏离应进行论证。 如必需限制动物活动，在接触试验样品之前宜使动物与固定器械相适应。 4.8.2 剂量体积 剂量体积导则见附录B。当采用多剂量组时，可通过调整浓度使各剂量组的试验体积差异降至最小，确保各剂量组剂量体积恒定。采用大于附录B给出的剂量体积时需进行论证。 宜避免经口途径给予大剂量体积，因为超出胃容量会快速进入小肠，也可能会逆流返回食管。 肌肉注射也有体积限制，要根据动物大小和肌肉部位来确定。各种属动物肌肉注射剂量见附录B。 单次快速静脉注射体积一般在约1 min左右的时间内注射完毕。注射速度是一个重要因素，推荐对啮齿动物大鼠注射速度应不超过2 mL／min。 注射较大剂量体积时可缓慢或分次注射，也可静脉输液。动物如果出现明显的临床症状，则应不再按照预定的速率，应停止给液或减慢给液速率。 试验样品受溶解度或刺激方面的限制时可采用缓慢静脉注射速度。 如有临床方面的指征可采取连续输液方式，根据所给物质以及标准液体治疗规范来确定给液体积和速度，2 h内一次给液量小于循环血量的10%可作为一种参考。对于持续长时间输液来说，最低限度有效地限制动物活动也是要考虑的一个重要因素。 皮下给予试验样品见附录B，吸收速率和程度与试验样品的组成有关。 4.8.3 剂量频率 宜根据临床相关性确定剂量频率。加严过程应详细描述并进行论证。 在急性全身毒性试验中，动物宜在24 h内一次或分次给予试验样品。 在重复接触试验中，动物宜每日给予试验样品，试验期间每周7 d给予。也可采用其他给予方式，但应进行论证。 4.9 体重和饲料／水消耗 试验样品可能造成实验动物体重的变化和饲料及水消耗的改变，因此在给予试验样品前的短时间内(如一次接触或急性试验一般在24 h内，重复接触试验在不超过7 d内)应称量每只动物的体重，并在整个试验期间定期以及试验结束时测量动物体重。如要根据动物体重确定给予剂量，则应采用最新测量的体重。 必要时，较长期重复接触研究应考虑测定饲料和水的消耗量。 4.10 临床观察 临床观察宜由受过培训的人员来进行，以保证报告的一致性。观察频率和间隔时间宜根据毒性反应的性质和严重程度、反应速度和恢复周期来确定。在试验的早期阶段尤其在急性试验中，可能需要增加观察次数。毒性症状出现和消失的时间、持续时间和动物死亡时间都是很重要的，特别是出现延迟性不良临床症状或死亡的迹象。为避免动物遭受不必要的痛苦，宜对动物实施国家或国际动物福利导则中规定的人道终点。接触试验样品后基本临床观察应考虑预期作用的高峰期。 应系统记录观察到的现象，并保留每只动物的观察记录。 每天应至少一次采用实验室通用临床反应术语记录观察到的动物存活情况和明显的临床反应(见附录C)。 长期重复接触研究每天应至少两次观察记录动物的发病率和死亡率。更长周期重复接触研究可考虑至少每周一次观察记录更大范围内的不良临床症状。 4.11 临床病理学 采用血液学和临床生化分析来研究组织、器官和其他系统的毒性反应。如有指征，应至少在预定的动物处死之前、或在动物处死的过程中采集重复接触实验动物的血样来进行此类分析。有些情况下采血前可能需要动物禁食。如出于科学研究的需要，长期重复接触试验的最后一周可进行尿液检验，采用定时(如16 h～24 h)尿量采集方法。 建议用于评价的血液学、临床生化和尿液分析参数见附录D。 4.12 解剖病理学 如有临床指征，急性全身毒性试验宜考虑大体病理学评价。 重复接触试验的全部动物应进行完整详细的大体尸检，包括检查体表、体表孔口、头部、胸(腹)腔及内脏等。对选择进行称重的器官，应采用适当方法去除粘附组织，并尽快称量其湿重以防止干燥。 宜称量的组织示例见附录E，将其置于适当的固定液中保护以进行组织病理学检查。 各类型试验至少应观察的项目见表2。 表2 观察项目汇总 观察项目 急性 亚急性／亚慢性 慢性a 体重变化 ＋ ＋ ＋ 临床观察 ＋ ＋ ＋ 临床病理学 b a，b ＋ 大体病理学 b ＋ ＋ 器官称重 b ＋ ＋ 组织病理学 b a，b ＋ ＋宜提供的资料。 a 慢性全身毒性试验一般是亚急性／亚慢性试验在时间上的再延长，根据人体接触周期来确定，记录和报告的很多参数是相同的。慢性全身毒性试验可增加剂量组，包括卫星剂量组，可观察其中的一些项目或全部项目。 b 当有临床指征或预期不进行更长期接触试验时，宜考虑这些项目。建议的体液和器官／组织分析项目见附录D、附录E和附录F。 4.13 试验设计 本文件在后面的章条将列出试验设计，推荐由专家协商来进行试验设计。 注：材料介导的热原信息见附录G。 4.14 试验质量 良好实验室质量管理规范涉及组织、过程和条件，在该规范条件下计划、实施、监控、记录并报告实验室试验。该规范可保证试验数据的质量和有效性，符合全球一体化要求，为贸易双方签署谅解备忘录提供便利。进行全身毒性试验应遵循此类原则。 5 急性全身毒性 5.1 总则 急性全身毒性提供根据预期临床途径进行的急性接触所产生的健康危害方面的基本信息。急性毒性试验可作为亚急性／亚慢性和其他试验确定剂量接触方式的初试步骤，并且可提供物质预期临床接触途径毒性作用模式方面的信息。本章中将涉及急性全身毒性试验中试验样品的给予方式、反应观察项目(如不良临床症状、体重变化、大体病理学发现)以及死亡。对出现严重及持续不适和痛苦的动物应立即实施安乐死，已知可导致显著疼痛或不适的腐蚀性或刺激性的材料宜报告，无需再进行试验。 美国替代方法论证协调委员会(ICCVAM)和欧洲替代方法验证中心(ECVAM)已经认可体外细胞毒性试验作为急性经口毒性试验的替代方法。为避免动物遭受不必要的痛苦，宜对动物实施国家或国际动物福利导则中规定的人道终点。 5.2 研究设计 5.2.1 准备 在试验前至少5 d使健康初成年动物适应实验室环境，如不到5 d则应进行论证。将动物随机分组。 5.2.2 实验动物 5.2.2.1 种属选择 典型试验是采用啮齿类动物(大鼠、小鼠)，4.2和4.3中规定了动物模型的要求(年龄、体重等)。如采用非啮齿类动物应对其使用进行科学论证。 5.2.2.2 数量与性别 4.5中规定了剂量组数目和类别、每组动物数和动物性别。 5.2.2.3 设施与饲养条件 实验室动物房间的温度和相对湿度宜适合于动物种属，如小鼠需(22±3)℃、相对湿度30%～70%条件。典型的人工照明宜设置为12 h开启、12 h关闭。 饲养方面，可采用标准商业实验动物饲料，无限制性饮用水供应。适当时，动物宜按性别群养或单独饲养，群养时每一笼具应不多于5只动物。 5.2.3 试验条件 5.2.3.1 剂量水平 剂量水平应按4.8的要求。 对照组动物除了不接触试验样品，其他处置方式应与试验组动物相同。 5.2.3.2 步骤 在24 h内，动物接受试验样品的一次剂量或必要时接受多次剂量。记录观察到的毒性症状，包括开始的时间、程度和持续时间。 动物的定期观察是很必要的，可保证动物不会由于自残、尸体组织自溶或动物错放造成损耗。试验终结时对全部存活动物实施人道方式处死，观察到濒死动物时应取出实施人道方式处死。采用的安乐死方法应与国家或国际动物福利导则一致。 观察中和实施人道终点时宜排除死亡动物由于试验样品毒性直接作用的可能性。 5.2.4 体重 在给予样品前即时测量动物体重，接触后前三天每天一次，如试验周期需要，则在首次接触后每周一次和试验终结时测量体重。 5.2.5 临床观察 急性全身毒性试验观察周期应至少3 d，必要时可延长。4.10中规定了具体观察次数和观察项目(见附录C)。应进行适当次数的观察并采取相应的措施，使实验动物的损失降至最低，如对发现死亡的动物进行尸检或冷冻，并隔离或处死病弱或濒死的动物。笼边观察宜包括(但不仅限于此)皮肤与被毛、眼与黏膜的改变，以及呼吸、循环、自主和中枢神经系统、躯体运动神经活动性和行为模式等状况。附录C给出了描述语。 5.2.6 病理学 5.2.6.1 临床病理学 有临床指征时，如器械材料预期有毒性或预试验观察到毒性，或无以往经验的新器械材料应考虑进行临床病理学评价。当进行临床病理学评价时，应进行下列检查： a) 试验终结时考虑测定附录D中给出的血液学检查项目。 b) 试验终结时考虑测定附录D中给出的临床生化血液方面的项目。急性接触试验适宜的测定项目包括肝和肾功能，必要时可进行其他临床生化检验，对观察到的作用进行扩展研究。 尿液检验(见附录D)不作为常规检验项目，仅在预期或观察到这方面的毒性反应的情况下才考虑进行。附录D给出了建议的测定项目。 5.2.6.2 大体病理学 有临床指征时，如器械材料预期有毒性或预试验观察到毒性，或无以往经验的新器械材料应考虑进行大体病理学评价，包括检查体表、体表孔口、头部、胸(腹)腔及内脏等。必要时宜考虑记录脑、肝、肾、肾上腺和睾丸的重量，在取出后尽快称量其湿重，以防止干燥以及由此造成的重量减轻。 5.2.6.3 组织病理学 急性全身毒性研究一般不进行动物器官和组织完整的病理学检查，除非有大体病理学明确指征的情况。 5.3 评价标准 5.3.1 总则 在现有试验设计的基础上，采用下列评价标准： a) 药典方法试验 1) 在急性全身毒性试验观察期间，如接触试验样品的动物生物学反应不大于介质对照组动物，则试验样品符合试验要求。 2) 采用5只动物，如2只或2只以上出现死亡、或2只或2只以上出现抽搐或俯卧、或3只或3只以上出现体重下降超过10%，则试验样品不符合试验要求。在全身毒性评价中，任何短暂的体重下降宜连同其他临床观察一起进行严格的评价。 3) 如试验组动物仅显示轻微生物学反应，而且不多于1只动物出现一般生物学反应症状或死亡，应采用10只动物为试验组重复进行试验。 4) 重复试验时，如全部10只接触试验样品的动物在观察阶段显示没有大于介质对照组动物的科学意义上的生物学反应，则试验样品符合试验要求。 b) 非药典方法急性全身毒性试验 可采用更大范围的方法进行评价，包括临床和解剖病理学检查，这样可以排除重复试验的必要。急性接触试验如出现与平行对照组不确定的差异可进行再评价，宜解释差异性，适宜时再用5只动物扩展试验。 5.3.2 结果评价 宜结合以前已有的研究信息对急性全身毒性研究中的发现进行评价，并分析观察到的毒性作用及大体尸检发现。评价应包括试验物质剂量与发病率和异常症状严重性之间的关系，如行动和临床异常症状、大体损伤、体重变化、致死作用以及其他一般性或特异性作用。 5.4 最终报告 适用时，急性全身毒性试验最终研究报告应包括下列信息： a) 实验室及委托方具体信息及试验设计选择的说明。 b) 试验样品： 1) 适宜时，物理性质、纯度和生理化学性能； 2) 其他表征数据。 c) 浸提溶剂或介质(适宜时)： 1) 如选择ISO 10993-12给出之外的浸提溶剂或介质，进行论证。 d) 实验动物： 1) 所用动物种属、品系； 2) 动物数量、年龄和性别； 3) 来源，包括微生物学状况(如屏障饲养、普通饲养)，动物设施条件(温度、湿度、垫料、光照、饲料等)； 4) 研究开始时的体重。 e) 试验条件： 1) 剂量选择说明； 2) 适宜时，试验样品配方／制备的详细说明；达到的浓度；稳定性和同质性； 3) 试验样品接触的详细说明； 4) 适用时，试验样品浓度(mg／mL)转换至实际剂量(mg／kg体重)； 5) 饲料、水和垫料质量的详细说明。 f) 结果： 1) 可以表格形式摘要给出数据，包括试验开始时每一对照组和试验组的动物数量、出现不良临床反应的动物数量、出现体重变化的动物数量； 2) 体重／体重变化； 3) 适用时，饲料和水的消耗； 4) 性别和剂量水平的毒性反应数据，包括毒性迹象； 5) 临床观察情况、严重程度和持续时间(是否为可逆性)； 6) 适用时，神经行为性评价； 7) 适用时，所采用的血液学试验和结果以及相关对照数据； 8) 适用时，所采用的临床生化试验和结果以及相关对照数据； 9) 适用时，所采用的尿分析试验和结果以及相关对照数据； 10) 适用时，最终体重和器官重量数据； 11) 尸检发现； 12) 适用时，全部组织病理学检查的详细描述； 13) 所采用的结果统计学评价(如使用)及其生物学意义讨论。 g) 结果讨论。 h) 结论。 i) 质量保证声明。 急性全身毒性研究可提供试验物质急性接触作用方面的信息，研究结果外推至人具有一定的局限性，但能够提供允许接触方面的有效信息。 6 重复接触全身毒性(亚急性、亚慢性和慢性全身毒性) 6.1 总则 急性毒性与单剂量(或限制性接触)的不良作用有关，但许多医疗器械更常见的人体接触方式是重复或持续接触形式。重复或持续接触可能会由于化学物在组织内的积聚或其他机制产生潜在反应，长期试验(亚急性、亚慢性、慢性)可识别此类潜在反应。 重复接触全身毒性试验可提供预期临床途径持续接触产生健康危害方面的信息，还可提供物质经预期临床接触途径毒性作用模式方面的信息。 重复接触全身毒性研究可提供毒性作用、靶器官、可逆性或其他作用方面的详细信息，并且可作为安全评估的依据。这些研究结果提供临床和解剖病理学研究导则范围方面的重要信息。 重复接触研究一般不给出复测的标准，但剂量组的动物数量要适应记录结果的统计学评价要求(见表1)。 由于重复接触研究的周期不同，应按照要求制备试验样品以确保其稳定性。 注：胃肠外双途径接触进行大鼠亚慢性全身毒性的试验方法见附录H。 6.2 研究设计 6.2.1 准备 至少在试验前5 d使健康初成年动物适应实验室条件，将动物随机分为各试验组。 6.2.2 实验动物 6.2.2.1 种属选择 典型试验采用啮齿动物(大鼠、小鼠)，4.2和4.3中规定了动物模型的要求(年龄、体重等)。如采用非啮齿动物应进行科学论证。 6.2.2.2 数量与性别 4.5中规定了剂量组、每组动物的数量和类别以及动物性别。如经科学论证，宜考虑采用高剂量水平卫星实验动物和对照动物，在超过最终处死期的预定时间，该试验组以及对照组动物可用于检查包括可逆性、持续性或迟发性毒性作用在内的试验反应。亚慢性研究的卫星组动物应至少保留28 d。 6.2.2.3 设施与饲养条件 实验动物房间温度和湿度宜适合动物种属，如大鼠需(22±3)℃、相对湿度30%～70%条件。典型的人工照明宜设置为12 h开启、12 h关闭。 饲养方面，可采用标准商业实验室饲料，无限制性饮用水供应。适当时，动物可按性别群养或单独饲养，群养时每一笼具不宜多于5只动物。 6.2.3 试验条件 6.2.3.1 剂量水平 适当情况下，医疗器械毒性试验所用剂量应根据风险评价的结果、临床接触剂量与安全因素的权衡而确定。对照组动物除了不接触试验物质，其他处理方式宜与试验组动物完全一致。 重复接触全身毒性研究与经典的化学物毒性研究不同，医疗器械重复接触试验通常不会产生剂量反应效应，因此不一定要研究剂量水平产生的毒性效应。 6.2.3.2 步骤 试验周期内动物最好每周7d接触试验样品，较长期重复接触研究每周5d接触是可接受的，但宜形成文件并进行论证。 6.2.4 体重 在给予样品前即时测量动物体重，以及第一次接触后的每周(如试验周期需要)、试验终结时测量动物体重。 6.2.5 临床观察 重复剂量全身毒性研究观察期应与研究周期相适应，4.10中规定了具体观察次数和观察项目(见附录C)。无论何种情况，观察次数和采取的适宜方式应能最大限度降低实验动物的损耗，如对死亡动物进行尸检或冷冻、隔离或处死病弱或濒死动物。笼边观察宜包括但不限于皮肤与被毛、眼与黏膜的改变，以及呼吸、循环、自主和中枢神经系统、躯体运动神经活动性和行为模式等状况，附录C给出了描述语。 典型的眼科检查采用检眼镜或其他同类适宜器具，宜在接触试验物质之前和试验期间进行检查。最好检查所有动物，至少检查高剂量组和对照组动物。如检出眼部改变，则宜对全部动物进行检查。异常检查宜形成文件并进行论证。 6.2.6 病理学 6.2.6.1 临床病理学 宜进行下列检查： a) 试验终结时测定附录D中给出的血液学检查项目，根据研究周期宜考虑增加检验次数。 b) 试验终结时测定血液临床生化方面的项目，根据研究周期宜考虑增加检验次数。适合所有重复接触研究的测定项目包括电解质平衡、碳水化合物代谢，以及肝和肾功能。具体检验项目的选择还要根据试验物质作用模式方面的观察，附录D给出了建议的测定项目。必要时可进行其他临床生化检验，对观察到的作用进行扩展研究。 尿液检验(见附录D)不作为常规检验项目，仅在预期或观察到这方面的毒性反应的情况下才考虑进行。 正常值的历史数据有利于建立基础水平，并可用于与当前研究对照进行比较。如历史基础数据不充分时，可考虑从同一年龄、性别、品系和来源的动物中采集该类信息，最好在同一实验室内进行。 6.2.6.2 大体病理学 全部动物宜进行完整的大体尸检，包括检查体表、体表孔口、头部、胸(腹)腔及内脏等。肾上腺、脑、附睾、心脏、肾、肝、卵巢、脾、睾丸、胸腺和子宫在取出后宜尽快称量其湿重，以防止干燥以及由此造成的重量减轻。器官和组织(见附录E)置于适宜的介质中保存，以进行下一步组织病理学检查。 6.2.6.3 组织病理学 宜进行下列检查： a) 宜对对照组和高剂量组动物的器官和组织进行完整的组织病理学检查。 b) 宜检查所有大体损害。 c) 如设有低、中剂量组，宜在这些组中对动物肺脏进行组织病理学检查是否有感染迹象，因为这种检查便于判定动物的健康状态。还宜考虑进行肝和肾的组织病理学检查，进一步的组织病理学检查可不必常规进行，但在高剂量组显示有损害迹象的器官则应在这些组中进行组织病理学检查。 d) 如设有卫星组，可根据试验组动物出现的反应来确定需进行的组织和器官病理学检查。 e) 慢性试验一般设有哨兵动物来监控感染性因子的出现，必要时可对哨兵动物进行血清学和组织学检查。 f) 在选择进行组织病理学检查的器官时，适当考虑器械材料的化学表征。举例来说，若该材料表面有药物／药品成分涂层，则需在实验动物中针对这些化学物的靶器官进行研究，考察是否有副作用发生。 6.3 评价标准 6.3.1 总则 以表格形式总结给出数据，包括试验开始时每一试验组动物数量、出现损害迹象的动物数量、损害的类型以及出现每种损害类型动物的百分率。宜进行统计学评价，但要考虑生物学相关性。在研究设计阶段应选择和使用已广泛接受的统计学方法。 6.3.2 结果评价 宜结合以前的研究结果并考虑毒性作用、尸检与组织病理学方面的结果对重复接触研究结果进行评价。评价应包括试验物质剂量与观察到的反应之间的关系。应评价观察到的反应包括行为和临床异常症状、大体损害、显微改变、对死亡率的影响以及具有生物学意义的其他反应。评价观察到的反应还要考虑与人的相关性。 6.4 最终报告 重复接触全身毒性研究最终报告应包括5.4中给出的信息，还应给出下列信息： ——所采用的血液学试验和结果以及相关对照数据； ——所采用的临床生化试验和结果以及相关对照数据； ——组织病理学发现； ——所采用的结果统计学评价及其生物学意义讨论。 长期全身毒性研究可提供试验物质重复接触作用方面的信息，研究结果外推至人具有一定的限定性，但能够提供人体允许接触方面的有效信息。 |
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