1、Designation: E 1263 97 (Reapproved 2003)Standard Guide forConduct of Micronucleus Assays in Mammalian BoneMarrow Erythrocytes1This standard is issued under the fixed designation E 1263; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide provides recommended guidelines for per-forming the mammalian in vivo bone marrow micronucl
3、eusassay. Under appropriate test conditions, measurement of thefrequency of newly formed micronucleated erythrocytes inbone marrow provides a convenient index of chromosomaldamage in nucleated erythrocyte precursor cells. The rationalefor the occurrence of micronuclei in conjunction with chromo-soma
4、l damage has been described previously (1).2This guidedescribes conditions under which the frequency of micronucle-ated erythrocytes in mammalian bone marrow is an appropriatemeasure of in vivo chromosomal damage, and provides guide-lines for the design and technical execution of assays employ-ing t
5、his endpoint.1.2 The following guidelines for mammalian bone marrowerythrocyte micronucleus assays have been published by orga-nizations concerned with the evaluation of genotoxicity testdata. These references should be consulted for recommenda-tions on details not covered in depth by this guide and
6、 forrequirements of specific organizations or government agencies(2-6).1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine th
7、e applica-bility of regulatory limitations prior to use.2. Summary of Guide2.1 Animals are exposed either acutely or chronically to atest substance. At predetermined times after or during expo-sure, animals are sacrificed and the bone marrow is extracted,spread on slides, and stained. The frequency
8、of micronucleatedcells among the newly-formed (RNA-containing) erythrocytesis determined, and this frequency is compared among treatmentgroups. The newly-formed erythrocytes are identified by stain-ing the residual RNA which remains in the newly-formed cellsfor about 2 days after enucleation. Cells
9、that stain uniformlypositive for RNA are referred to as polychromatic, or poly-chromatophilic, erythrocytes (PCEs). Cells that do not stainpositively for RNA are referred to as normochromatic eryth-rocytes (NCEs). An increase in the frequency of micronucle-ated PCEs relative to the vehicle control g
10、roup indicates thatthe test substance induced structural chromosomal damage orlagging chromosomes aneuploidy in the nucleated erythrocyticcells.3. Significance and Use3.1 This guide provides guidelines for the selection ofanimal species, dosage and sampling conditions, sampling andscoring methods, s
11、tatistical design, and analysis of genotoxic-ity assays in which the endpoint measured is the frequency ofmicronucleated erythrocytes in mammalian bone marrow.4. Animal Selection and Care4.1 Laboratory species that are suitable for use in this assayinclude the mouse (Mus musculus), rat (Rattus rattu
12、s), andChinese hamster (Cricetulus griseus) (1). Other species prob-ably are equally suitable. If species or strains not previouslyused are employed, it must be established that the preparationprocedure adequately visualizes RNA-containing erythrocytesand micronuclei, that potential artifacts such a
13、s aggregatedRNA and mast cell granules do not interfere with the identifi-cation of micronuclei under the conditions employed, and thatthe micronucleus frequency is responsive to known clastogensand aneuploidy-inducing agents in that species and strain.4.2 In choosing the species and strain of test
14、animal,consideration should be given both to the availability ofhistorical data on the response of that species and strain toknown genotoxins and to the availability of other toxicity dataon the same test material in the species and strain chosen.Choice of the same strain to be used in other genotox
15、icityassays of the same test material, or in long-term toxicity orcarcinogenicity bioassays, has the advantage that the micro-nucleus frequency can be directly compared with other end-points. The species for which the largest data base on knowngenotoxins is available is the mouse (1).1This guide is
16、under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Sept. 10, 2003. Published September 2003. Originallyapproved in 1988. Last previous edition appro
17、ved in 1997 as E 1263 97.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.3 Animals should be obtained from a recognized source oflaborator
18、y animals and should be acclimated to laboratoryconditions prior to use. Upon arrival, the age, sex, weight, andhealth of each animal should be documented. Only healthyanimals should be used. Animal care and housing shouldconform to prevailing guidelines for the country and institutionwhere the work
19、 is conducted. General information on guide-lines for animal care and use can be obtained from theAmerican Association for Accreditation of Laboratory AnimalCare.3For any given experiment, all animals should be fromthe same source and should be approximately the same age(within one week for young ad
20、ults). In the absence of specialrequirements for a particular age and sex, young adults of bothsexes are recommended. Data from each sex should beanalyzed independently.5. Route of Administration and Choice of Vehicle5.1 The choice of exposure route depends on the objectiveof the experiment. The obj
21、ective of most micronucleus assaysis to determine if the test substance induces types of chromo-somal damage known to result in the formation of micronuclei.In this case, it is desirable to choose a route of administrationand a vehicle that maximize the dose delivered to the targettissue. For this p
22、urpose, intraperitoneal and oral routes havebeen used most commonly, although others may also beappropriate. In other cases, the objective may be to evaluatespecifically in vivo activity under conditions based upon knownexposure routes in man. In such cases, the appropriate route isthe one that prov
23、ides the best experimental model of theexpected exposure route in man.5.2 The choice of a solvent or vehicle is influenced byseveral factors, including the chemical nature and solubility ofthe test substance, its toxicity to the test organism, and theroute of exposure. In all cases care must be take
24、n to ensure thatthe vehicle selected will not produce measurable toxicity orinterfere with the normal uptake and metabolism of the testsubstance at the dose employed. In particular, the vehicleshould not alter the spontaneous micronucleus frequency. Ifpossible, it is desirable to use isotonic saline
25、 for parenteraladministration and water or isotonic saline for oral adminis-tration. For oral administration of organic substances notreadily soluble in aqueous solution, a pharmaceutical grade ofcorn or other vegetable oil may be used. Vegetable oil is lesssuitable for intraperitoneal administratio
26、n because it is poorlyabsorbed from the peritoneal cavity. Other acceptable choicesof vehicle include carboxymethylcellulose or suspension ingum arabic. Dimethylsulfoxide (DMSO) is an effective solventfor a wide range of substances and has frequently been used inexperiments with mice, although there
27、 are a few reports offoreign intermediates being produced by interaction of DMSOwith certain test substances (7) and one unconfirmed report thatDMSO increases the frequency of chromosomal aberrations inthe rat (8).6. Dose Selection6.1 The doses to be employed should be selected on thebasis of either
28、 toxicity data obtained in the same laboratory orpublished toxicity data, if available. Preliminary range-findingexperiment(s) should employ a minimum of two animals perdose group and should use the solvent and route of exposure tobe employed in the final experiment. The highest dose levelshould be
29、chosen to meet one or more of the following criteriain the experiment carried out with the full test group:6.1.1 It should cause a marked and significant increase inthe micronucleus frequency in the target cell population.6.1.2 It should produce a statistically significant suppressionof the frequenc
30、y of RNA-positive erythrocytes.6.1.3 It should cause compound-related signs of toxicity orsignificantly reduce survival.6.1.4 It should be the maximum practical dose that can beadministered. The maximum practical dose of a nontoxic testmaterial is determined by the physical bulk and solubility.Testi
31、ng at such a maximum dose level has been referred to asa “limit test” in OECD and EPA/TSCA testing guidelines. Thisdose will vary with test agent, but will generally be in the range5 to 10 g/kg for acute oral or intraperitoneal (i.p.) administra-tion (3, 5).6.2 The doses employed should include a mi
32、nimum of two,and preferably three, doses, at least one of which does notseverely reduce the frequency of RNA-positive erythrocytes(the frequency should be at least 10 to 20 % of the controlvalue) and which does not significantly reduce the survival ofthe test animals. The rationale for selecting tes
33、t doses haspreviously been discussed in the U.S. Environmental Protec-tion Agency Gene-Tox Program report on the bone marrowpolychromatic erythrocyte assay (1) and by Salamone andHeddle (9). Because the maximum cytogenetic effect is likelyto be found at doses near the maximum tolerated dose (MTD),th
34、e lower doses should be spaced at relatively small incrementsbelow the highest dose (for example, no more than12 and14 ofthe upper dose).7. Controls7.1 Vehicle or Solvent ControlA vehicle or solvent controlshall be included for each sampling condition (dose, time, sex)in each experiment. Animals are
35、 treated with the solvent orvehicle in the absence of the test substance. The quantity ofsolvent or vehicle administered should be equivalent to themaximum given to the animals receiving the test substance.This control helps discriminate any test-substance effect fromany that may have been induced b
36、y the solvent.7.2 Untreated ControlThe use of untreated animals isgenerally not necessary during routine testing. It is important,however, that each laboratory determine the frequency ofmicronucleated cells in animals treated with the vehicle orsolvent control relative to the spontaneous frequency i
37、n un-treated animals, so that any effect of the vehicle or solvent isknown.7.3 Positive Control SubstanceA positive control sub-stance, that is, a substance known to induce micronuclei inbone marrow, should be included with each experiment toconfirm that all features of the protocol have been carrie
38、d out3American Association for Accreditation of Laboratory Animal Care, 208ANorth Cedar Rd., New Lenox, IL 60451.E 1263 97 (2003)2correctly. The positive control agent preferably should be onethat is chemically related to the test substance and preferablyadministered by the same route as the test ar
39、ticle. In addition,the agent or dose should be chosen to produce a mild or weaklypositive result. This provides a better evaluation of the sensi-tivity of the assay than does the use of a high dose of a potentclastogen which would almost always be detected regardless ofwhether or not the sensitivity
40、 of the assay were optimal.8. Number of Animals/Sex8.1 It is desirable to have data for both sexes. For routinescreening, both sexes should be tested using a minimum of fiveanimals of each sex at each test dose. If a positive result isobtained in one sex, a test agent may be classified as activewith
41、out data from the other sex, but both sexes must be testedto verify a negative result.9. Treatment and Sampling Schedule9.1 The main requirement of the treatment/sampling sched-ule is to obtain at least one sample at or near the time of themaximum incidence of micronucleated cells among the RNA-posi
42、tive erythrocytes in bone marrow. The time of maximumincidence varies with the test agent, dose, and treatmentschedule.9.2 Treatment Schedule:9.2.1 Treatment protocols using single, double, and multipletreatments have been reported (9). Although each of thesetreatment schedules has been reported to
43、be advantageous withspecific test agents, there is insufficient evidence at present tosupport the exclusive use of a specific treatment schedule forall test substances. Accordingly, the choice of single, multiple,or continuous dosing protocols must be made by the investi-gator, based on the specific
44、 objectives of a particular study andthe available knowledge of the pharmacokinetic behavior ofthe test substance. The use of a single- or double-dosetreatment has the advantage that these protocols have beenmost often employed in studies reported to date, so a largercomparative data base will be av
45、ailable if these treatmentschedules are used.9.2.2 Although the interval between multiple treatments canaffect the response obtained, little data are available to supportthe choice of an optimum interval. Since historical data onmultiple-treatment schedules in the mouse and rat are basedprimarily on
46、 a 24-h dosing interval, it appears best to use thisinterval until definitive data supporting an alternative areavailable.9.3 Sampling Schedule:9.3.1 Following each treatment, there is a particular timeinterval during which micronucleated RNA-positive erythro-cytes, if induced, would be present. Sin
47、ce micronuclei areformed during division of the nucleated erythropoietic cells butscored in the anucleate mature erythrocyte, micronuclei cannotappear earlier after treatment than the interval between comple-tion of the final erythroblast mitosis and enucleation. In themouse, this minimum time betwe
48、en treatment and appearanceof micronuclei is about 5 h (10). For most chemicals, substan-tial increases in the micronucleus frequency have not beenfound earlier than 9 to 12 h after treatment. Since the life spanof the RNA-positive erythrocyte within the bone marrow hasbeen reported to be between 10
49、 and 30 h in the mouse and rat(for review, see (9), any micronucleated RNA-positive eryth-rocytes formed will remain in the bone marrow for at least 10to 12 h. It is therefore not necessary to sample earlier than 19to 24 h after the first treatment.9.3.2 Due to differences between test agents in the time aftertreatment at which the peak frequency of micronuclei occurs, itis important that two or more samples be taken if only one ortwo treatments are given. Available data indicate that this peakfrequency usually occurs between 24 and 48 h after treatment,but that in certain cases
