mn in vivo and data interpretation

Mutation Research 455 (2000) 155–166

In vivo rodent micronucleus assay:protocol, conduct and data interpretation

Gopala Krishnaa,∗, Makoto Hayashiba Department of Worldwide Preclinical Safety, Parke-Davis Pharmaceutical Research,

Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USAb Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

Abstract

In vivo rodent micronucleus assay has been widely used to detect genotoxicity. Evaluation of micronucleus induction isthe primary in vivo test in a battery of genotoxicity tests and is recommended by the regulatory agencies around the globeto be conducted as part of product safety assessment. The assay, when performed appropriately, detects both clastogenicityand aneugenicity. Methods for performing micronucleus evaluation have evolved since its initial description in the 1970s.In recent years, the focus has been directed toward improving micronucleus detection with high efficiency by proposingdata-based recommendations to the standard initial protocol design. Such improvements include, e.g., the use of appropriateharvest time(s), inclusion of one or both sexes, number of doses tested, limit dose, integrating micronucleus assessment into theroutine toxicology studies, use of fluorescent staining, automation of micronucleus detection and assessment of micronuclei inmultiple tissues. This protocol paper describes: the mechanism of micronucleus formation, a generalized protocol for manualdetection, enumeration of micronuclei, and data interpretation in light of published information thus far, on the regulatoryaspects of this assay. Certain recent protocol issues that are practical in nature are equally valid in relation to standard manualmethod and provide robust database, which are also included for consideration. It is expected that such improvements of theprotocol will continue to drive the utility of this assay in the product safety assessment. © 2000 Elsevier Science B.V. Allrights reserved.

Keywords:Micronucleus assay; Rodents; Mice; Rats; Protocol; Automation; Recent methods; Integration with toxicology studies

1. Introduction

“The present generation is only a caretaker of thehuman genome of future generations” — a statementmade by Malling and Valcovic and quoted by Brusick[1] describes precisely the ultimate objective of ge-netic toxicologists. Genetic toxicology is the study ofadverse effects on the process of heredity. Studies ofgenetic toxicology have given rise to a number of test-ing procedures, both in vitro and in vivo, designed to

∗ Corresponding author. Tel.:+1-734-622-7985.E-mail address:[email protected] (G. Krishna).

assess the effects of chemicals on genetic mechanismsand the consequent risk to organisms, including hu-mans. Of equal importance are studies of the mecha-nisms by which adverse genetic effects are mediatedand epidemiological studies of the frequency of ge-netic effects relative to chemical exposure. Thus far,it is clear that information on three levels of mutation,e.g., gene, chromosomal, and cellular apparatus neces-sary for chromosome segregation, is necessary to pro-vide broad coverage of the mutagenic and presumablycarcinogenic potential of a chemical or radiation. Inthis regard, micronucleus assay has been widely usedto measure genotoxicity, both in vitro and in vivo. The

0027-5107/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S0027-5107(00)00117-2

156 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166

description of in vivo micronucleus assay is the pri-mary focus of this paper. The in vivo test is especiallyrelevant to assessing genotoxicity hazard in that it al-lows consideration of factors of in vivo metabolism,pharmacokinetics, and DNA-repair processes and isalso useful in further investigation of a mutagenic ef-fect detected by an in vitro genotoxicity test.

Evaluation of micronucleus frequency in vivo is theprimary test in a battery of genotoxicity tests and isrecommended by the regulatory agencies around theglobe to be conducted as part of product safety assess-ment. The assay, when performed appropriately, de-tects both clastogenicity (chromosome breakage) andaneugenicity (chromosome lagging due to dysfunc-tion of mitotic apparatus). Micronuclei, also hemato-logically known as Howell–Jolly bodies, are generallysmooth, round remnants of nuclear chromatin seen inerythrocytes and are not to be confused for Heinz orHeinz Ehrlich bodies resulting from oxidative injuryto and precipitation of hemoglobin. In vivo, the pro-cess of erythropoiesis (production of erythrocytes) hasbeen exploited in the micronucleus test.

2. Process of erythropoiesis

The process of erythropoiesis and the mechanism ofmicronucleus formation in vivo are shown in Fig. 1.

In the adult rodent, both bone marrow and spleenare hemopoietic organs, in which stem cells form thebasis of erythropoiesis with proliferation and matu-ration stages. During proliferation, the cells continueto divide at which time a given test agent admin-istered may act and cause chromosome damage,such as breaks and exchanges, and may also act onmacromolecules related to the function of chromatiddisjunction, e.g., tubulin causing spindle dysfunction,depending on the mechanism of action. These anoma-lies (a fragment or a whole chromosome) may lagbehind in the cell during division and may not becomeintegrated into daughter nuclei, rather may eventuallyform micronuclei, which can be seen in the cytoplasm.During maturation, when an erythroblast develops intoa polychromatic erythrocyte (PCE, young erythrocytestill contains RNA, is basophilic and stains light blueor blue gray with Giemsa), the main nucleus is ex-truded; any micronucleus that has been formed mayremain behind in the otherwise enucleated cytoplasm.

Visualization of micronuclei is facilitated in thesecells because they lack a main nucleus. An increasein the frequency of micronucleated PCE (MNPCE) intest agent-treated animals is an indication of inducedchromosome damage. The PCEs, with time, loseRNA and contain primarily hemoglobin and becomenormochromatic erythrocytes (NCEs, mature erythro-cytes [red blood cells], somewhat smaller than PCE,acidophilic and stain light orange or orange-pink withGiemsa). These two types of erythrocytes, which staindifferentially, can be seen in bone marrow, spleen,and blood compartments. Examples of micronucle-ated cells stained with Wright’s Giemsa method anda column fractionation method that eliminates allnucleated cells are shown in Figs. 2 and 3.

In the micronucleus assay, the PCE-to-NCE ratiobetween test agent-treated animals and vehicle-controlanimals provides a cytotoxicity index. During laterstages of maturation, and on a needed basis, thesecells move into the peripheral blood compartment.A kinetochore-labeling procedure [2] can distin-guish the mechanism of either a clastogen-induceddamage (primarily chromosome breakage and ab-sence of kinetochore(s) in the micronucleus) oran aneugen-induced spindle dysfunction (primarilylagging chromosome(s) and the presence of kine-tochore(s) in the micronucleus). Examples of suchdifferentiation and aneuploidy evaluation are shownin Fig. 4. However, other methods, e.g., DNA-specificcentromere labeling [3], can also be used to evaluateaneuploidy.

The micronucleus assay [4] is devised primarily forevaluating the ability of test agents to induce structuraland/or numerical chromosomal damage. Both kinds ofdamage are associated with the appearance and/or pro-gression of tumors, and with adverse reproductive anddevelopmental outcomes. In most testing situations,the test agent is administered acutely (generally once)to mice or rats, and the frequency of MNPCE deter-mined in slides prepared from bone marrow harvested24 and 48 h after treatment. In some experimental sit-uations, peripheral blood can be sampled instead ofbone-marrow and mature erythrocytes (NCE) scoredin addition to or instead of PCE for the presence of mi-cronuclei. This assay has several important advantagesover the analysis of bone-marrow metaphase analy-sis. For example, it is technically simple, the endpointscored is more objective and amenable for automa-

G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166 157

Fig. 1. (a) The process of erythropoiesis in vivo; (b) the mechanism of micronucleus formation in the polychromatic erythrocytes (PCEs)and normochromatic erythrocytes (NCEs). Also, classification of kinetochore-positive (K+) and kinetochore-negative (K−) erythrocytes.N, nucleus; PEB, proerythroblast; MN, micronucleus.


Fig. 2. A photomicrograph of rat whole bone-marrow smear showing nucleated as well as enucleated cells. The enucleated cells (PCEsand NCEs) also contain micronuclei.

tion, it is less time consuming, the assay can detectboth clastogens and aneugens, and it can be easily in-tegrated into general toxicology studies. Although thechromosomal aberration assay does allow for an as-sessment of the types of structural damage; however,

Fig. 3. A photomicrograph of column-fractionated rat bone marrow and cytospun slide preparation showing only enucleated cells and 2PCEs containing micronuclei.

such information is rarely used for safety assessmentof test chemical along with the frequency of cells withchromosome aberrations.

A number of major methodological issues for thein vivo rodent micronucleus assay were agreed upon


Fig. 4. Photomicrographs of cells stained with antikinetochore antibody, fluoresceinated antibodies, and propidium iodide. (A) A metaphasecell showing centromere specific staining of CREST serum (V79 Chinese hamster lung cell chromosomes, for example). (B) A microscopicfield showing cellulose-column fractionated cytospun cyclophosphamide-treated (clastogen) mouse bone-marrow PCE and NCE differen-tiation; PCEs with kinetochore-negative micronuclei (arrows). (C) A microscopic field showing vincristine-treated (aneugen) spleen PCEswith kinetochore-positive micronuclei (arrows).

at the International Workshop on Genotoxicity TestProcedures (IWGTP), in Melbourne during 1993 [5].This document provided guidance in formulating theInternational Conferences on Harmonization of Tech-

nical Requirements for Registration of Pharmaceuti-cals for Human Use (ICH) [6] and also the revision ofOECD guidelines for testing of chemicals (NO. 474,Mammalian Erythrocyte Micronucleus Test) [7]. As


a follow-up and complementary to these, at the sec-ond IWGTP held in Washington, DC, March 25–26,1999, the expert working group discussed a number ofissues, including integration of the repeated-dose mi-cronucleus assay into general toxicology studies [8],the use of automated scoring techniques, micronu-cleus evaluation in tissues other than bone marrow orperipheral blood, and methods for differentiating mi-cronucleus derived from acentric chromosome frag-ments and from centromeric chromosomes [9]. Thereader of this protocol paper is strongly advised to re-fer to these prior references for additional backgrounddetail and special circumstances in conducting and re-porting micronucleus assay.

3. Acute micronucleus assay protocol

An example of a good laboratory practice (GLP)laboratory protocol using rodent bone marrow, withsingle dose and two harvest times is briefly describedhere. Any variation of this protocol could be used,depending on the objective, need, or level of interestby the researcher.

3.1. Study no. and title: study 123: rodentmicronucleus assay of test agent(s)

3.1.1. Assay principleRodents are treated with the test agent by appropri-

ate route, bone marrow extracted at appropriate timesafter treatment, smear slides are prepared either withwhole bone marrow or cellulose column-fractionatedcell suspension, stained, coded, and analyzed for thetoxicity (PCE to NCE ratio) and micronucleated cellfrequency.

3.1.2. PurposeThe purpose of the micronucleus assay is to identify

test substance(s) that cause micronuclei formation asa result of lagging of chromosome fragments (clasto-genicity) or whole chromosomes (aneugenicity), gen-erally in rodent bone-marrow erythropoietic cells. Ifthe test agent is positive in the routine test, then, spe-cific centromeric antibodies or DNA probes may beused to determine the mechanism of micronucleus for-mation (e.g., clastogenic or aneugenic), if desired.

3.1.3. Test animal selection, identification, andhousing

Test animals: [mice or rats; Age: - - -; Weight: - - -Strain: - - -; Source: - - -]. Rationale for selection ofa test animal: [e.g., rats/mice are used as an animalmodel to detect micronucleus induction because sub-stantial historical information is available and thismodel has been utilized in other studies to obtaintoxicology information regarding the test agent understudy]. Generally, commonly used laboratory strainsof young healthy sexually matured animals, wherebone marrow is expected to be actively dividing, areutilized and are acclimated to the laboratory envi-ronment for a minimum 5 days and examined priorto initiation of the study to ensure that they appearhealthy. Animals are assigned to study groups usinga randomization method and necessary information islisted for animal identification purposes using, e.g.,cage cards and/or electronic implanted transponderchips. Animals are housed individually or in treat-ment groups of same sex in appropriate cages andappropriate laboratory-specific animal husbandryprocedures are followed, e.g., appropriate room tem-perature, humidity, light cycle, laboratory rodent diet,and unlimited supply of water.

3.1.4. Test agent and controlsTest agent-specific number or name designation,

chemical name, therapeutic and/or chemical class (ifany), source, active moiety, stability and homogeneityin vehicle may be included. Special storage and han-dling conditions, assays for confirmation of potencyfollowing completion of treatment. Information on ve-hicle (solvent control, generally nontoxic at the dosevolume used and not known to produce chemical re-action with the test agent, e.g., water, or methylcellu-lose aqueous solution) and positive control [an agentthat is known to induce micronuclei, used to veri-fy performance of the assay, e.g., cyclophosphamidei.p.−20 mg/kg for rats, 40 mg/kg for mice; mitomycinC i.p. −0.5 mg/kg rats and mice].

3.1.5. Route of administration and samplepreparation

Justification for route of administration (e.g., oral,intraperitoneal, or intravenous) is generally included:this route has been intended for human exposure,and/or was used in a single-dose rat/mouse acute


toxicity study of (test agent). Test agent dose calcula-tions is based on active moiety, and given on a mg/kgbody weight basis at a dose volume 10 to 20 ml/kg.The positive control, cyclophosphamide, e.g., is dis-solved in distilled water at 2 mg/ml and administeredat 20 mg/kg to rats or 40 mg/kg to mice by a singlei.p. injection of 10 ml/kg.

3.1.6. Test-agent dose selection criteriaRegulatory guidelines recommend that the high

dose selected for the rodent micronucleus assay shouldproduce some toxicity, be at maximum tolerated dose(MTD), or be administered at 2000 mg/kg. Generally,the MTD is the highest dose that can be adminis-tered without inducing lethality or excessive toxicityduring the study causing moribund euthanasia. It hasalso been recommended that the intermediate dose beone-half of the high dose and the low dose be one-halfof the intermediate dose. Information on acute toxi-cology data is included, if available or a dose-rangefinding study matching schedule of treatment and/oreuthanasia times used in the micronucleus assay maybe conducted to select appropriate doses.

3.1.7. Treatment groups, number and sex of animalsFor a standard study, based on a regulatory recom-

mendation regarding the in vivo micronucleus assay,a single dosing regimen and two euthanasia times (24and 48 h) were selected for this study. If the test agentdoes cause differential toxicity in males and females,then use of both sexes is necessary, otherwise theuse of an appropriate sex, usually male, is sufficient.In the case where sex-related difference in toxicityis apparent, groups of five males and five femalesscheduled for the 24- and 48-h euthanasia will beadministered vehicle control. Generally, three doselevels of test agent are used and each treatment groupconsists of five animals per sex per euthanasia time.If unexpected lethality is anticipated at the high dose,a few extra animals may be included at this group toreplace, if necessary. Also, additional animals maybe included at each group, if exposure (plasma drugconcentration) data are needed as part of the micronu-cleus assay. Positive control animals are includedonly at the 24-h euthanasia time. Five animals of eachsex per group will be euthanized in a sequence at 24and 48 h after treatment and bone marrow harvestedaccording to the following schedule:

Table of treatment groups

Treatment Dose(mg/kg)

Group number

Sex Sex

Vehicle control 0 M 1 (10)a F 6 (10)Test agent Low M 2 (10) F 7 (10)Test agent Mid M 3 (10) F 8 (10)Test agent High M 4 (10) F 9 (10)Positive controlb 20/40 M 5 (5) F 10 (5)

aValues in parentheses are the numbers of animalsdosed per group;bPositive control, e.g., cyclophos-phamide — 20 mg/kg for rats and 40 mg/kg for mice.

However, if a test agent is relatively nontoxic, es-pecially at≥2000 mg/kg and genotoxicity would notbe expected based on data from structurally relatedagents, a full study using three dose levels may notbe considered necessary and a limit dose test at 2000mg/kg may be sufficient.

3.1.8. Test procedures: animal observations,euthanasia and bone-marrow processing

Animals are observed for clinical signs of toxicity atvarious intervals after treatment, and at 24 and 48 h orat the discretion of study scientist. Bone-marrow cellswill be harvested, cells processed and/or slides pre-pared for evaluation according to standard procedures.Briefly, animals will be euthanized by CO2 asphyxi-ation and one usable femur or tibia excised, skin andmuscle tissue trimmed, and both ends of the bone tipssevered with bone snips, marrow flushed gently fromthe channel into a tube with fetal bovine serum (FBS,approximately 3 ml/femur). Cells are processed formanual (standard smear or cellulose column fraction-ation, Appendix A [10,11] or flow cytometry methodof evaluation of micronuclei according to proceduresdescribed in the literature [9,12–14].

3.1.9. Data collection, analysis, and evaluation andinterpretation

In the micronucleus assay, the proportion of MN-PCE constitute the primary endpoint and the pro-portion of PCEs is the supportive endpoint to assesscytotoxicity, which helps demonstrate a target cell ex-posure with the test chemical. Generally, in the manual


method, the slides are coded to not to reveal the treat-ment groups by the scorer and 2000 PCE/animal areevaluated for the presence of micronuclei. The unit ofanalysis is PCE and not the number of micronuclei perPCE, as a PCE may contain more than one micronu-cleus. For bone-marrow toxicity, at least 200 total ery-throcytes are evaluated per animal for the proportionof PCE in bone marrow and 1000 in peripheral blood.In the automated method (e.g., flow cytometry), thenumber of cells evaluated could be much higher andup to 100,000 total erythrocytes and the proportionsof PCE, NCE, MNPCE and MNNCE may be quan-tified depending on the purpose of the study. Indivi-dual animal data are listed in a tabular form along withmeans, standard deviations and statistical significance,if any.

Numerical data are analyzed using appropriate sta-tistical tests, e.g., MNPCE data are analyzed with aone-sided test for an increase and PCE data may bealso be analyzed with a one-sided test for a decrease.If there is no evidence for a difference in responsebetween sexes, the data from both sexes may be com-bined for statistical analysis.

While evaluating data, both statistical and biologi-cal criteria are considered. One criterion for a positiveresult would be a statistically significant dose-relatedincrease in MNPCE frequency at any time pointwith at least 1 value significantly exceeding the his-torical vehicle control range. Equivocal results maybe clarified by further testing using, preferably, themodified experimental conditions. Positive resultsindicate that the test agent induces micronuclei un-der the experimental conditions, which have beenthe result of chromosome damage and/or damageto the mitotic apparatus. Negative results mean thatthe test agent does not produce micronuclei underthe experimental conditions. The data may be put inperspective by taking test agent’s pharmacokineticand metabolism pathways and systemic toxicity intoconsideration. If a positive micronucleus response forthe test agent is obtained, additional studies can beconducted to identify the mechanistic origin of themicronuclei. Generally, micronuclei that arise fromclastogenic damage lack a kinetochore, and thoseassociated with numerical chromosomal damage con-tain a kinetochore. The presence of a kinetochore canbe identified using commercially available antibodies[2,3].

3.1.10. Test report procedureAt the conclusion of the study, the results of the

study are reported in a report, with a study number anda report number, giving the experimental design, pro-tocol, raw data, evaluation of results, and a conclusion.Archival and retrieval information is also included inthe report. An example of information typically in-cluded in a research report is shown in Appendix B.

3.1.11. Standard operating procedures, GLPcompliance, laboratory safety, and study schedule

The methods used in the study are described in de-tail in the laboratory-specific Manual of Operations. Astatement as to whether the study will be conducted incompliance with the OECD or any other global regu-latory body’s GLP regulations for nonclinical labora-tory studies, is included. Exceptions to this may bestated, as needed in exploratory studies. A statementas to the potential toxicity of positive control (e.g.,cyclophosphamide), is included and appropriate pre-cautions taken to minimize exposure, as well as stepsto be taken if exposure takes place, by accident, areincluded. Information regarding tentative start date,completion date, and final report date are includedprior to protocol approval by the study director andthe facility management.

4. Repeat-dose integrated micronucleus study

In the industrial toxicology laboratories, micronu-cleus assay can be integrated into routine toxicologi-cal studies [8]. This approach utilizes: (i) the generalprinciples of toxicology that govern the overall toxi-city profile of a test substance; (ii) factors, such asthe dose and/or route of administration, metabolism,principles of toxicokinetics, and saturation of de-fense mechanisms, are considered in evaluatinggenotoxicity; (iii) the concept of administering mul-tiple tolerable doses achieving steady-state plasmadrug concentrations that are more relevant in humanrisk assessment compared to high acute doses; and(iv) this approach would minimize the amount of testcompound, number of animals, and other resourcesthat are generally utilized in the conduct of standardacute micronucleus assay.

While integrating micronucleus assessment intogeneral toxicology repeat-dose studies, e.g., 2-to-4-


Table 1Rodent micronucleus assay: historical control database (1987–1998): Parke-Davis Pharmaceutical Laboratory

PCEa/100 TEb MNPCEc/1000 PCE

Males Females M+Fd Males Females M+F

MiceVehicle controlNo. of mice 243 245 430 230 215 430No. of assays 47 45 44 47 45 44Mean assay range 42.0–66.9 48.1–75.6 46.0–69.7 0.4–3.8 0.6–3.6 0.9–3.1

Positive control (CPe - 40 mg/kg)No. of mice 130 130 230 115 115 220No. of assays 24 24 23 24 24 23Mean assay range 42.9–67.6 39.6–69.3 41.3–66.8 7.7–42.7 8.0–44.7 8.8–42.1

RatsVehicle controlNo. of rats 185 180 360 185 180 360No. of assays 37 36 36 37 36 36Mean assay range 40.1–63.3 34.0–65.0 37.1–59.6 1.1–6.4 0.8–4.9 1.3–5.3

Positive control (CP - 20 mg/kg)No. of rats 135 120 240 135 120 240No. of assays 24 24 24 24 24 24Mean assay range 25.5–54.9 25.3–50.8 27.8–52.7 9.0–43.8 5.8–25.1 10.4–33.8

aPolychromatic erythrocytes.bTotal erythrocytes.cMicronucleated polychromatic erythrocytes.dCombined sexes, where available.eCyclophosphamide. Data based on CD1 mice and Wistar rats of age 5 to 8 weeks at study initiation.

week studies and possibly 13-week studies, the fol-lowing approach may be followed. Generally, a femuror tibia from five animals/sex/group used for thetoxicological assessment is utilized for micronucleusassay. Positive control animals may be included tomatch with the euthanasia schedule of study animalsand given a single 20-mg/kg i.p. dose of cyclophos-phamide dissolved in sterile water at a concentrationof 2 mg/ml and administered i.p. at a dose volume of10 ml/kg for rats. Body weights of the positive con-trol animals are determined prior to dosing and anyclinical signs noted at termination may be recorded.The positive control animals are treated and bonemarrow collected approximately 24 h after treatment.Inclusion of both vehicle and positive controls is in-tended to minimize any staining variations in slideevaluation and/or to adjust automated measurementsettings, if appropriate. The data are collected similarto acute micronucleus assay. In light of minimizinganimal use in research and still obtain required data

from a study, the routine use of positive control inevery micronucleus assay has been questioned by thescientific community, especially in laboratories whichhave demonstrated assay reproducibility and conductstudies under GLP regulations. Based on a reviewof available data, such as shown in Table 1, on thereproducibility of positive control response, the mi-cronucleus assay Expert Panel recently recommendedthat the use of positive control may not be necessaryin every study [9].

Appendix A. Micronucleus assay

A.1. Preparation of cells and/or slides

A.1.1. Standard smear method with Wright’s GiemsaThe bone marrow with serum in tubes is centrifuged

at approximately 150×g for 5 min, the supernatant re-moved, the pellet resuspended using a few drops of ad-ditional serum, if necessary. A drop of cell suspension


is placed onto the slide and using a pusher slide a smearis prepared. Generally at least two slides are preparedper animal. Slides are air dried, fixed in methanol,and stained with 5% to 10% Wright’s Giemsa withHaemastainer. All slides are cover-glassed using Per-mount and coded for evaluation.

A.1.2. Cellulose column fractionation methodSlides may also be prepared according to the cel-

lulose column fractionation and cytocentrifugation[10,11]. The most common method of preparingslides is to use whole bone marrow, which containsvarious types of blood cells and their precursors. Acellulose column fractionation procedure has beenrecommended to remove all nucleated cells as wellas artifact-producing cell debris from bone marrowto facilitate scoring of micronuclei [10,11]. Withthis technique, one can overcome the problem ofmicronucleus-imitating mast cell granules, espe-cially, in rat bone marrow. The column-fractionatedcells may be used for making standard smears, forcytocentrifugation, or for flow cytometric analysisdepending on the objective of the experiment. If acolumn method is used, each laboratory should assurethat micronucleated cell frequencies are obtained,which are comparable with those obtained using di-rect scoring. For this purpose, samples should beused that contain elevated micronucleus frequenciesinduced by a known clastogen and also an aneugen[9].

A.1.3. Preparation of cellulose column [10,11]One part of microcrystalline cellulose and one part

of a-cellulose fiber are weighed, mixed by shaking ina securely capped bottle for approximately 3 min. A20-mm disc of microscope-cleaning tissue is placedat the bottom of a 20 ml plastic syringe and 1.0 to1.1 g of cellulose mixture is added to the column,packed by tapping the syringe on a hard surface (ap-proximately 20 sharp taps from a height of about1 cm), followed by slightly pressing down the cel-lulose mixture with a modified syringe plunger un-til the 3 ml mark of the syringe is reached. Bonemarrow is isolated in a routine fashion using 3 mlFBS per two femurs in mouse and 3 ml FBS perone femur in rat. Bone marrow is aspirated and dis-charged about 20 times using Pasteur pipette to breakup clumps. Using a pipette, cell suspension is care-

fully, dropped into the center of the column so that itis absorbed outwards to the plastic edge of the cellu-lose column.

Following this, 20 ml Hanks’ balanced salt solu-tion (HBSS, without phenol red) is carefully added tothe column surface and approximately 20 ml eluted(takes 20 to 30 min). The eluate generally contains ery-throcytes. The eluate is centrifuged at approximately800×g for 10 pellet resuspended in a few drops of FBSfor smears, or resuspend the pellet in 200ml of sup-plemented MEM culture media for cytospun smears.For manual micronucleus scoring, slides are preparedusing a cytocentrifuge and labeled sufficiently to iden-tify the treatment group and experiment.

Slides are stained for micronucleus analysisin a routine fashion with Wright’s Giemsa tech-nique using a Haemastain automatic staining ma-chine, coverslipped, blind coded, and used for datacollection.

Micronuclei are identified according to the criteriaestablished by Schmid [4] and are darkly stained (pur-ple) and generally round or almond shaped, althoughlightly stained, ring shaped micronuclei occasionallyoccur. Micronuclei have sharp borders and are gen-erally 5–20% the size of the PCE and may occurin either PCE or NCE. However, in a single-doseregimen only MNPCEs are counted. The unit of scor-ing is the MNPCE, not the number of micronucleiper PCE, as occasionally more than one micronu-cleus may appear per PCE. In a repeated dosingregimen, in addition to the bone-marrow analysis,micronuclei may be scored in PCEs and/or NCEsfound in the blood at the discretion of the studyscientist. Initially, the ratio of PCE to total erythro-cytes (PCE+NCE) for each animal is determinedby examining at least 200 and 1000 erythrocytes forbone-marrow and peripheral blood, respectively, peranimal and then, the number of MNPCE in 2000PCE/animal is determined and data recorded on theappropriate assay form and data are uncoded by thestudy scientist and analyzed by appropriate statisticaltests.

A.1.4. Slide staining using acridine orange [15]The acridine orange stock solution is prepared as a

0.1% aqueous solution that could be available for sev-eral weeks stored at 4◦C. Acridine orange, 0.24 mMin 1/15 M Sörensen’s phosphate buffer (pH 6.8) (two


parts of stock solution and 30 parts of the buffer), isused as a working solution. The fixed cells are stainedin this solution for 3 min at room temperature. Theslides are rinsed in the buffer three times for 1–3 mineach time. If the nuclei emit a reddish fluorescence, theslides are rinsed for another several minutes to opti-mally stain nuclei with green fluorescence. The prepa-rations are mounted with the same buffer, and sealedwith Balsam paraffin or suitable media. An alternativemore simple method can be used: one drop of 0.04mM acridine orange solution in the same Sörensen’sphosphate buffer is placed on the fixed cells and cov-ered with cover slip. The excess solution is blotted andsealed if necessary. The slide is already ready for fluo-rescent microscopy. Observations can be made withina day using fluorescent microscopy equipped with blueexcitation and 515–530 nm barrier filter. Cytoplasm ofPCE emits red fluorescence and micronuclei as wellas nucleus of nucleated cells fluoresce yellowish greenor yellow.

Appendix B. Test report

The research report should include the followinginformation.

B.1. Test agent

Source, identification data and CAS No., if known,physical nature and purity, physiochemical propertiesrelevant to the conduct of the study, stability of thetest agent, if known.

B.2. Solvent/vehicle

Justification for choice of solvent/vehicle; solubi-lity, homogeneity, and stability of the test agent in thesolvent/vehicle, if known.

B.3. Test animals

Species/strain used, number, age, and sex of ani-mals, source, housing conditions, diet, individualweight of the animals at the start of the test, includingbody weight range, mean, and standard deviation foreach group.

B.4. Test conditions

Positive and negative control data, data fromrange-finding study, if conducted, rationale for doseselection, details of test agent preparation, detailsof the administration of the test agent, rationale forroute of administration, methods for verifying thatthe test agent reached the general circulation or targettissue, if applicable, details of food and water quali-ty, detailed description of treatment and samplingschedules, methods of slide preparation, methods formeasurement of toxicity, criteria for scoring MNPCE,number of cells analyzed per animal, criteria for con-sidering studies as positive, negative, or equivocal.

B.5. Results and discussion

Clinical signs of animal toxicity, percentage ofPCE among total erythrocytes or ratio of PCE toNCE, number of MNPCE, given separately for eachanimal, mean±standard deviation of MNPCE pergroup, treatment–response relationship, where possi-ble, statistical analyses and methods applied, concur-rent and historical negative-control data, concurrentand historical positive control data, where applicable,and discussion of the results, as appropriate.

B.6. Conclusion

A one or two sentence conclusion as to whether ornot the test agent was negative, positive, or equivocalunder the conditions of the assay.

B.7. Authorized signatures and GLP compliancestatement (if necessary)

Signatures of study director, responsible staff andmanagement, are included. A GLP compliance state-ment along with quality assurance unit’s statement isalso included.

References

[1] D. Brusick, Principles of Genetic Toxicology, 2nd edn.,Plenum, New York, 1987.

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