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.
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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.
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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.
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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.
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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
