Mutation Research 455 (2000) 155–166

In vivo rodent micronucleus assay:

protocol, conduct and data interpretation
Gopala Krishna a,∗ , Makoto Hayashi b
a Department of Worldwide Preclinical Safety, Parke-Davis Pharmaceutical Research,
Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
b 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 is
the primary in vivo test in a battery of genotoxicity tests and is recommended by the regulatory agencies around the globe
to be conducted as part of product safety assessment. The assay, when performed appropriately, detects both clastogenicity
and 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 proposing
data-based recommendations to the standard initial protocol design. Such improvements include, e.g., the use of appropriate
harvest time(s), inclusion of one or both sexes, number of doses tested, limit dose, integrating micronucleus assessment into the
routine toxicology studies, use of fluorescent staining, automation of micronucleus detection and assessment of micronuclei in
multiple tissues. This protocol paper describes: the mechanism of micronucleus formation, a generalized protocol for manual
detection, enumeration of micronuclei, and data interpretation in light of published information thus far, on the regulatory
aspects of this assay. Certain recent protocol issues that are practical in nature are equally valid in relation to standard manual
method and provide robust database, which are also included for consideration. It is expected that such improvements of the
protocol will continue to drive the utility of this assay in the product safety assessment. © 2000 Elsevier Science B.V. All
rights reserved.
Keywords: Micronucleus assay; Rodents; Mice; Rats; Protocol; Automation; Recent methods; Integration with toxicology studies

1. Introduction assess the effects of chemicals on genetic mechanisms

“The present generation is only a caretaker of the
human genome of future generations” — a statement
made by Malling and Valcovic and quoted by Brusick
[1] describes precisely the ultimate objective of ge-
netic toxicologists. Genetic toxicology is the study of
adverse effects on the process of heredity. Studies of
genetic 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: gopala.krishna@wl.com (G. Krishna).
and the consequent risk to organisms, including hu-
mans. Of equal importance are studies of the mecha-
nisms by which adverse genetic effects are mediated
and 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 presumably
carcinogenic potential of a chemical or radiation. In
this regard, micronucleus assay has been widely used
to measure genotoxicity, both in vitro and in vivo. The

0027-5107/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 7 - 5 1 0 7 ( 0 0 ) 0 0 1 1 7 - 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 especially
relevant to assessing genotoxicity hazard in that it al-
lows consideration of factors of in vivo metabolism,
pharmacokinetics, and DNA-repair processes and is
also useful in further investigation of a mutagenic ef-
fect detected by an in vitro genotoxicity test.
Evaluation of micronucleus frequency in vivo is the
primary test in a battery of genotoxicity tests and is
recommended by the regulatory agencies around the
globe to be conducted as part of product safety assess-
ment. The assay, when performed appropriately, de-
tects both clastogenicity (chromosome breakage) and
aneugenicity (chromosome lagging due to dysfunc-
tion of mitotic apparatus). Micronuclei, also hemato-
logically known as Howell–Jolly bodies, are generally
smooth, round remnants of nuclear chromatin seen in
erythrocytes and are not to be confused for Heinz or
Heinz Ehrlich bodies resulting from oxidative injury
to and precipitation of hemoglobin. In vivo, the pro-
cess of erythropoiesis (production of erythrocytes) has
been exploited in the micronucleus test.
2. Process of erythropoiesis
The process of erythropoiesis and the mechanism of
micronucleus formation in vivo are shown in Fig. 1.
In the adult rodent, both bone marrow and spleen
are hemopoietic organs, in which stem cells form the
basis of erythropoiesis with proliferation and matu-
ration stages. During proliferation, the cells continue
to 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 on
macromolecules related to the function of chromatid
disjunction, e.g., tubulin causing spindle dysfunction,
depending on the mechanism of action. These anoma-
lies (a fragment or a whole chromosome) may lag
behind in the cell during division and may not become
integrated into daughter nuclei, rather may eventually
form micronuclei, which can be seen in the cytoplasm.
During maturation, when an erythroblast develops into
a polychromatic erythrocyte (PCE, young erythrocyte
still contains RNA, is basophilic and stains light blue
or blue gray with Giemsa), the main nucleus is ex-
truded; any micronucleus that has been formed may
remain behind in the otherwise enucleated cytoplasm.
Visualization of micronuclei is facilitated in these
cells because they lack a main nucleus. An increase
in the frequency of micronucleated PCE (MNPCE) in
test agent-treated animals is an indication of induced
chromosome damage. The PCEs, with time, lose
RNA and contain primarily hemoglobin and become
normochromatic erythrocytes (NCEs, mature erythro-
cytes [red blood cells], somewhat smaller than PCE,
acidophilic and stain light orange or orange-pink with
Giemsa). These two types of erythrocytes, which stain
differentially, can be seen in bone marrow, spleen,
and blood compartments. Examples of micronucle-
ated cells stained with Wright’s Giemsa method and
a column fractionation method that eliminates all
nucleated cells are shown in Figs. 2 and 3.
In the micronucleus assay, the PCE-to-NCE ratio
between test agent-treated animals and vehicle-control
animals provides a cytotoxicity index. During later
stages of maturation, and on a needed basis, these
cells move into the peripheral blood compartment.
A kinetochore-labeling procedure [2] can distin-
guish the mechanism of either a clastogen-induced
damage (primarily chromosome breakage and ab-
sence of kinetochore(s) in the micronucleus) or
an aneugen-induced spindle dysfunction (primarily
lagging chromosome(s) and the presence of kine-
tochore(s) in the micronucleus). Examples of such
differentiation and aneuploidy evaluation are shown
in Fig. 4. However, other methods, e.g., DNA-specific
centromere labeling [3], can also be used to evaluate
aneuploidy.
The micronucleus assay [4] is devised primarily for
evaluating the ability of test agents to induce structural
and/or numerical chromosomal damage. Both kinds of
damage are associated with the appearance and/or pro-
gression of tumors, and with adverse reproductive and
developmental 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 harvested
24 and 48 h after treatment. In some experimental sit-
uations, peripheral blood can be sampled instead of
bone-marrow and mature erythrocytes (NCE) scored
in addition to or instead of PCE for the presence of mi-
cronuclei. This assay has several important advantages
over the analysis of bone-marrow metaphase analy-
sis. For example, it is technically simple, the endpoint
scored 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.
158 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166

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

tion, it is less time consuming, the assay can detect
both clastogens and aneugens, and it can be easily in-
tegrated into general toxicology studies. Although the
chromosomal aberration assay does allow for an as-
sessment of the types of structural damage; however,
such information is rarely used for safety assessment
of test chemical along with the frequency of cells with
chromosome aberrations.
A number of major methodological issues for the
in vivo rodent micronucleus assay were agreed upon

Fig. 3. A photomicrograph of column-fractionated rat bone marrow and cytospun slide preparation showing only enucleated cells and 2
PCEs containing micronuclei.
 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166 159

Fig. 4. Photomicrographs of cells stained with antikinetochore antibody, fluoresceinated antibodies, and propidium iodide. (A) A metaphase
cell showing centromere specific staining of CREST serum (V79 Chinese hamster lung cell chromosomes, for example). (B) A microscopic
field 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 PCEs
with kinetochore-positive micronuclei (arrows).

at the International Workshop on Genotoxicity Test nical Requirements for Registration of Pharmaceuti-
Procedures (IWGTP), in Melbourne during 1993 [5]. cals for Human Use (ICH) [6] and also the revision of
This document provided guidance in formulating the OECD guidelines for testing of chemicals (NO. 474,
International Conferences on Harmonization of Tech- Mammalian Erythrocyte Micronucleus Test) [7]. As
160 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166
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 of
issues, 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 or
peripheral blood, and methods for differentiating mi-
cronucleus derived from acentric chromosome frag-
ments and from centromeric chromosomes [9]. The
reader of this protocol paper is strongly advised to re-
fer to these prior references for additional background
detail 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, with
single dose and two harvest times is briefly described
here. Any variation of this protocol could be used,
depending on the objective, need, or level of interest
by the researcher.
3.1. Study no. and title: study 123: rodent
micronucleus assay of test agent(s)
3.1.1. Assay principle
Rodents are treated with the test agent by appropri-
ate route, bone marrow extracted at appropriate times
after treatment, smear slides are prepared either with
whole bone marrow or cellulose column-fractionated
cell suspension, stained, coded, and analyzed for the
toxicity (PCE to NCE ratio) and micronucleated cell
frequency.
3.1.2. Purpose
The purpose of the micronucleus assay is to identify
test substance(s) that cause micronuclei formation as
a result of lagging of chromosome fragments (clasto-
genicity) or whole chromosomes (aneugenicity), gen-
erally in rodent bone-marrow erythropoietic cells. If
the test agent is positive in the routine test, then, spe-
cific centromeric antibodies or DNA probes may be
used to determine the mechanism of micronucleus for-
mation (e.g., clastogenic or aneugenic), if desired.
3.1.3. Test animal selection, identification, and
housing
Test animals: [mice or rats; Age: - - -; Weight: - - -
Strain: - - -; Source: - - -]. Rationale for selection of
a test animal: [e.g., rats/mice are used as an animal
model to detect micronucleus induction because sub-
stantial historical information is available and this
model has been utilized in other studies to obtain
toxicology information regarding the test agent under
study]. Generally, commonly used laboratory strains
of young healthy sexually matured animals, where
bone marrow is expected to be actively dividing, are
utilized and are acclimated to the laboratory envi-
ronment for a minimum 5 days and examined prior
to initiation of the study to ensure that they appear
healthy. Animals are assigned to study groups using
a randomization method and necessary information is
listed for animal identification purposes using, e.g.,
cage cards and/or electronic implanted transponder
chips. Animals are housed individually or in treat-
ment groups of same sex in appropriate cages and
appropriate laboratory-specific animal husbandry
procedures 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 controls
Test agent-specific number or name designation,
chemical name, therapeutic and/or chemical class (if
any), source, active moiety, stability and homogeneity
in vehicle may be included. Special storage and han-
dling conditions, assays for confirmation of potency
following completion of treatment. Information on ve-
hicle (solvent control, generally nontoxic at the dose
volume 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 agent
that is known to induce micronuclei, used to veri-
fy performance of the assay, e.g., cyclophosphamide
i.p. −20 mg/kg for rats, 40 mg/kg for mice; mitomycin
C i.p. −0.5 mg/kg rats and mice].
3.1.5. Route of administration and sample
preparation
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
 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166 161

toxicity study of (test agent). Test agent dose calcula- Table of treatment groups
tions is based on active moiety, and given on a mg/kg
body weight basis at a dose volume 10 to 20 ml/kg. Treatment Dose Group number
The positive control, cyclophosphamide, e.g., is dis- (mg/kg)
solved in distilled water at 2 mg/ml and administered Sex Sex
at 20 mg/kg to rats or 40 mg/kg to mice by a single Vehicle control 0 M 1 (10)a F 6 (10)
i.p. injection of 10 ml/kg. Test agent Low M 2 (10) F 7 (10)
Test agent Mid M 3 (10) F 8 (10)
3.1.6. Test-agent dose selection criteria
Test agent High M 4 (10) F 9 (10)
Regulatory guidelines recommend that the high
Positive controlb 20/40 M 5 (5) F 10 (5)
dose selected for the rodent micronucleus assay should
produce some toxicity, be at maximum tolerated dose
a Values
in parentheses are the numbers of animals
(MTD), or be administered at 2000 mg/kg. Generally,
the MTD is the highest dose that can be adminis- dosed per group; b Positive control, e.g., cyclophos-
tered without inducing lethality or excessive toxicity phamide — 20 mg/kg for rats and 40 mg/kg for mice.
during the study causing moribund euthanasia. It has
also been recommended that the intermediate dose be However, if a test agent is relatively nontoxic, es-
one-half of the high dose and the low dose be one-half pecially at ≥2000 mg/kg and genotoxicity would not
of the intermediate dose. Information on acute toxi- be expected based on data from structurally related
cology data is included, if available or a dose-range agents, a full study using three dose levels may not
finding study matching schedule of treatment and/or be considered necessary and a limit dose test at 2000
euthanasia times used in the micronucleus assay may mg/kg may be sufficient.
be conducted to select appropriate doses.
3.1.8. Test procedures: animal observations,
3.1.7. Treatment groups, number and sex of animals euthanasia and bone-marrow processing
For a standard study, based on a regulatory recom- Animals are observed for clinical signs of toxicity at
mendation regarding the in vivo micronucleus assay, various intervals after treatment, and at 24 and 48 h or
a single dosing regimen and two euthanasia times (24 at the discretion of study scientist. Bone-marrow cells
and 48 h) were selected for this study. If the test agent will be harvested, cells processed and/or slides pre-
does cause differential toxicity in males and females, pared for evaluation according to standard procedures.
then use of both sexes is necessary, otherwise the Briefly, animals will be euthanized by CO2 asphyxi-
use of an appropriate sex, usually male, is sufficient. ation and one usable femur or tibia excised, skin and
In the case where sex-related difference in toxicity muscle tissue trimmed, and both ends of the bone tips
is apparent, groups of five males and five females severed with bone snips, marrow flushed gently from
scheduled for the 24- and 48-h euthanasia will be the channel into a tube with fetal bovine serum (FBS,
administered vehicle control. Generally, three dose approximately 3 ml/femur). Cells are processed for
levels of test agent are used and each treatment group manual (standard smear or cellulose column fraction-
consists of five animals per sex per euthanasia time. ation, Appendix A [10,11] or flow cytometry method
If unexpected lethality is anticipated at the high dose, of evaluation of micronuclei according to procedures
a few extra animals may be included at this group to described in the literature [9,12–14].
replace, if necessary. Also, additional animals may
be included at each group, if exposure (plasma drug 3.1.9. Data collection, analysis, and evaluation and
concentration) data are needed as part of the micronu- interpretation
cleus assay. Positive control animals are included In the micronucleus assay, the proportion of MN-
only at the 24-h euthanasia time. Five animals of each PCE constitute the primary endpoint and the pro-
sex per group will be euthanized in a sequence at 24 portion of PCEs is the supportive endpoint to assess
and 48 h after treatment and bone marrow harvested cytotoxicity, which helps demonstrate a target cell ex-
according to the following schedule: posure with the test chemical. Generally, in the manual
162 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166
method, the slides are coded to not to reveal the treat-
ment groups by the scorer and 2000 PCE/animal are
evaluated for the presence of micronuclei. The unit of
analysis is PCE and not the number of micronuclei per
PCE, 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 proportion
of PCE in bone marrow and 1000 in peripheral blood.
In the automated method (e.g., flow cytometry), the
number of cells evaluated could be much higher and
up to 100,000 total erythrocytes and the proportions
of 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 with
means, standard deviations and statistical significance,
if any.
Numerical data are analyzed using appropriate sta-
tistical tests, e.g., MNPCE data are analyzed with a
one-sided test for an increase and PCE data may be
also be analyzed with a one-sided test for a decrease.
If there is no evidence for a difference in response
between 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 positive
result would be a statistically significant dose-related
increase in MNPCE frequency at any time point
with at least 1 value significantly exceeding the his-
torical vehicle control range. Equivocal results may
be clarified by further testing using, preferably, the
modified experimental conditions. Positive results
indicate that the test agent induces micronuclei un-
der the experimental conditions, which have been
the result of chromosome damage and/or damage
to the mitotic apparatus. Negative results mean that
the test agent does not produce micronuclei under
the experimental conditions. The data may be put in
perspective by taking test agent’s pharmacokinetic
and metabolism pathways and systemic toxicity into
consideration. If a positive micronucleus response for
the test agent is obtained, additional studies can be
conducted to identify the mechanistic origin of the
micronuclei. Generally, micronuclei that arise from
clastogenic damage lack a kinetochore, and those
associated with numerical chromosomal damage con-
tain a kinetochore. The presence of a kinetochore can
be identified using commercially available antibodies
[2,3].
3.1.10. Test report procedure
At the conclusion of the study, the results of the
study are reported in a report, with a study number and
a report number, giving the experimental design, pro-
tocol, raw data, evaluation of results, and a conclusion.
Archival and retrieval information is also included in
the report. An example of information typically in-
cluded in a research report is shown in Appendix B.
3.1.11. Standard operating procedures, GLP
compliance, laboratory safety, and study schedule
The methods used in the study are described in de-
tail in the laboratory-specific Manual of Operations. A
statement as to whether the study will be conducted in
compliance with the OECD or any other global regu-
latory body’s GLP regulations for nonclinical labora-
tory studies, is included. Exceptions to this may be
stated, as needed in exploratory studies. A statement
as to the potential toxicity of positive control (e.g.,
cyclophosphamide), is included and appropriate pre-
cautions taken to minimize exposure, as well as steps
to be taken if exposure takes place, by accident, are
included. Information regarding tentative start date,
completion date, and final report date are included
prior to protocol approval by the study director and
the 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 general
principles of toxicology that govern the overall toxi-
city profile of a test substance; (ii) factors, such as
the dose and/or route of administration, metabolism,
principles of toxicokinetics, and saturation of de-
fense mechanisms, are considered in evaluating
genotoxicity; (iii) the concept of administering mul-
tiple tolerable doses achieving steady-state plasma
drug concentrations that are more relevant in human
risk assessment compared to high acute doses; and
(iv) this approach would minimize the amount of test
compound, number of animals, and other resources
that are generally utilized in the conduct of standard
acute micronucleus assay.
While integrating micronucleus assessment into
general toxicology repeat-dose studies, e.g., 2-to-4-
 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166 163

Table 1
Rodent micronucleus assay: historical control database (1987–1998): Parke-Davis Pharmaceutical Laboratory
PCEa /100 TEb MNPCEc /1000 PCE

Males Females M+F d Males Females M+F

Mice
Vehicle control
No. of mice 243 245 430 230 215 430
No. of assays 47 45 44 47 45 44
Mean 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 130 130 230 115 115 220
No. of assays 24 24 23 24 24 23
Mean 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
Rats
Vehicle control
No. of rats 185 180 360 185 180 360
No. of assays 37 36 36 37 36 36
Mean 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 135 120 240 135 120 240
No. of assays 24 24 24 24 24 24
Mean 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
a Polychromatic erythrocytes.
b Totalerythrocytes.
c Micronucleated polychromatic erythrocytes.
d Combined sexes, where available.
e Cyclophosphamide. 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 femur
or tibia from five animals/sex/group used for the
toxicological assessment is utilized for micronucleus
assay. Positive control animals may be included to
match with the euthanasia schedule of study animals
and given a single 20-mg/kg i.p. dose of cyclophos-
phamide dissolved in sterile water at a concentration
of 2 mg/ml and administered i.p. at a dose volume of
10 ml/kg for rats. Body weights of the positive con-
trol animals are determined prior to dosing and any
clinical signs noted at termination may be recorded.
The positive control animals are treated and bone
marrow collected approximately 24 h after treatment.
Inclusion of both vehicle and positive controls is in-
tended to minimize any staining variations in slide
evaluation and/or to adjust automated measurement
settings, if appropriate. The data are collected similar
to acute micronucleus assay. In light of minimizing
animal use in research and still obtain required data
from a study, the routine use of positive control in
every micronucleus assay has been questioned by the
scientific community, especially in laboratories which
have demonstrated assay reproducibility and conduct
studies under GLP regulations. Based on a review
of available data, such as shown in Table 1, on the
reproducibility of positive control response, the mi-
cronucleus assay Expert Panel recently recommended
that the use of positive control may not be necessary
in every study [9].
Appendix A. Micronucleus assay
A.1. Preparation of cells and/or slides
A.1.1. Standard smear method with Wright’s Giemsa
The 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
164 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166
is placed onto the slide and using a pusher slide a smear
is prepared. Generally at least two slides are prepared
per animal. Slides are air dried, fixed in methanol,
and stained with 5% to 10% Wright’s Giemsa with
Haemastainer. All slides are cover-glassed using Per-
mount and coded for evaluation.
A.1.2. Cellulose column fractionation method
Slides may also be prepared according to the cel-
lulose column fractionation and cytocentrifugation
[10,11]. The most common method of preparing
slides is to use whole bone marrow, which contains
various types of blood cells and their precursors. A
cellulose column fractionation procedure has been
recommended to remove all nucleated cells as well
as artifact-producing cell debris from bone marrow
to facilitate scoring of micronuclei [10,11]. With
this technique, one can overcome the problem of
micronucleus-imitating mast cell granules, espe-
cially, in rat bone marrow. The column-fractionated
cells may be used for making standard smears, for
cytocentrifugation, or for flow cytometric analysis
depending on the objective of the experiment. If a
column method is used, each laboratory should assure
that micronucleated cell frequencies are obtained,
which are comparable with those obtained using di-
rect scoring. For this purpose, samples should be
used that contain elevated micronucleus frequencies
induced 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 ␣-cellulose fiber are weighed, mixed by shaking in
a securely capped bottle for approximately 3 min. A
20-mm disc of microscope-cleaning tissue is placed
at the bottom of a 20 ml plastic syringe and 1.0 to
1.1 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 about
1 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. Bone
marrow is isolated in a routine fashion using 3 ml
FBS per two femurs in mouse and 3 ml FBS per
one femur in rat. Bone marrow is aspirated and dis-
charged about 20 times using Pasteur pipette to break
up clumps. Using a pipette, cell suspension is care-
fully, dropped into the center of the column so that it
is 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 to
the column surface and approximately 20 ml eluted
(takes 20 to 30 min). The eluate generally contains ery-
throcytes. The eluate is centrifuged at approximately
800×g for 10 pellet resuspended in a few drops of FBS
for smears, or resuspend the pellet in 200 ␮l of sup-
plemented MEM culture media for cytospun smears.
For manual micronucleus scoring, slides are prepared
using a cytocentrifuge and labeled sufficiently to iden-
tify the treatment group and experiment.
Slides are stained for micronucleus analysis
in a routine fashion with Wright’s Giemsa tech-
nique using a Haemastain automatic staining ma-
chine, coverslipped, blind coded, and used for data
collection.
Micronuclei are identified according to the criteria
established by Schmid [4] and are darkly stained (pur-
ple) and generally round or almond shaped, although
lightly stained, ring shaped micronuclei occasionally
occur. Micronuclei have sharp borders and are gen-
erally 5–20% the size of the PCE and may occur
in either PCE or NCE. However, in a single-dose
regimen only MNPCEs are counted. The unit of scor-
ing is the MNPCE, not the number of micronuclei
per PCE, as occasionally more than one micronu-
cleus may appear per PCE. In a repeated dosing
regimen, in addition to the bone-marrow analysis,
micronuclei may be scored in PCEs and/or NCEs
found in the blood at the discretion of the study
scientist. Initially, the ratio of PCE to total erythro-
cytes (PCE+NCE) for each animal is determined
by examining at least 200 and 1000 erythrocytes for
bone-marrow and peripheral blood, respectively, per
animal and then, the number of MNPCE in 2000
PCE/animal is determined and data recorded on the
appropriate assay form and data are uncoded by the
study scientist and analyzed by appropriate statistical
tests.
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 mM
in 1/15 M Sörensen’s phosphate buffer (pH 6.8) (two
 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166
parts of stock solution and 30 parts of the buffer), is
used as a working solution. The fixed cells are stained
in this solution for 3 min at room temperature. The
slides are rinsed in the buffer three times for 1–3 min
each time. If the nuclei emit a reddish fluorescence, the
slides are rinsed for another several minutes to opti-
mally stain nuclei with green fluorescence. The prepa-
rations are mounted with the same buffer, and sealed
with Balsam paraffin or suitable media. An alternative
more simple method can be used: one drop of 0.04
mM acridine orange solution in the same Sörensen’s
phosphate buffer is placed on the fixed cells and cov-
ered with cover slip. The excess solution is blotted and
sealed if necessary. The slide is already ready for fluo-
rescent microscopy. Observations can be made within
a day using fluorescent microscopy equipped with blue
excitation and 515–530 nm barrier filter. Cytoplasm of
PCE emits red fluorescence and micronuclei as well
as nucleus of nucleated cells fluoresce yellowish green
or yellow.
Appendix B. Test report
The research report should include the following
information.
B.1. Test agent
Source, identification data and CAS No., if known,
physical nature and purity, physiochemical properties
relevant to the conduct of the study, stability of the
test agent, if known.
B.2. Solvent/vehicle
Justification for choice of solvent/vehicle; solubi-
lity, homogeneity, and stability of the test agent in the
solvent/vehicle, if known.
B.3. Test animals
Species/strain used, number, age, and sex of ani-
mals, source, housing conditions, diet, individual
weight of the animals at the start of the test, including
body weight range, mean, and standard deviation for
each group.
165
B.4. Test conditions
Positive and negative control data, data from
range-finding study, if conducted, rationale for dose
selection, details of test agent preparation, details
of the administration of the test agent, rationale for
route of administration, methods for verifying that
the test agent reached the general circulation or target
tissue, if applicable, details of food and water quali-
ty, detailed description of treatment and sampling
schedules, methods of slide preparation, methods for
measurement 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 of
PCE among total erythrocytes or ratio of PCE to
NCE, number of MNPCE, given separately for each
animal, mean±standard deviation of MNPCE per
group, treatment–response relationship, where possi-
ble, statistical analyses and methods applied, concur-
rent and historical negative-control data, concurrent
and 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 or
not the test agent was negative, positive, or equivocal
under the conditions of the assay.
B.7. Authorized signatures and GLP compliance
statement (if necessary)
Signatures of study director, responsible staff and
management, are included. A GLP compliance state-
ment along with quality assurance unit’s statement is
also included.
References
[1] D. Brusick, Principles of Genetic Toxicology, 2nd edn.,
Plenum, New York, 1987.
[2] G. Krishna, R. Fiedler, J.C. Theiss, Simultaneous evaluation
of clastogenicity, aneugenicity, and toxicity in the mouse
micronucleus assay using immunofluorescence, Mutat. Res.
282 (1992) 159–304.
166 G. Krishna, M. Hayashi / Mutation Research 455 (2000) 155–166
[3] B.M. Miller, H.F. Zitzelsberger, H.U. Weier, I.-D. Adler,
Classification of micronuclei in mouse erythrocytes:
immunofluorescent staining using CREST antibodies
compared to in situ hybridization with biotinylated gamma
satellite DNA, Mutagenesis 6 (1991) 297–302.
[4] W. Schmid, The micronucleus test, Mutat. Res. 31 (1975)
9–15.
[5] M. Hayashi, R.R. Tice, J.T. MacGregor, D. Anderson, D.H.
Blakey, M. Kirsch-Volders, F.B. Oleson Jr., F. Pacchierotti, F.
Romagna, H. Shimada, S. Sutou, B. Vannier, In vivo rodent
erythrocyte micronucleus assay, Mutat. Res. 312 (1994) 293–
304.
[6] L. Muller, Y. Kikuchi, G. Probst, L. Schechtman, H.
Shimada, T. Sofuni, D. Tweats, ICH-harmonized guidances on
genotoxicity testing of pharmaceuticals: evolution, reasoning
and impact, Mutat. Res. 436 (1999) 195–225.
[7] OECD, 1997, Guideline for the testing of chemicals.
mammalian erythrocyte micronucleus test. Guideline 474,
July, 1997.
[8] G. Krishna, G. Urda, J. Theiss, Principles and practices of
integrating genotoxicity evaluation into routine toxicology
studies: a pharmaceutical industry perspective, Environ. Mol.
Mutagen. 32 (1998) 115–120.
[9] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, I. Adler,
D.H. Blakey, S.D. Dertinger, G. Krishna, T. Morita,
A. Russo, S. Sutou, In vivo rodent erythrocyte micronucleus
assay: II. Some aspects of protocol design including
repeated treatments, integration with toxicity testing, and
automated scoring, Env. Mol. Mutagen. 35 (2000) 234–
252.
[10] F. Romagna, C.D. Staniforth, The automated bone marrow
micronucleus test, Mutat. Res. 213 (1989) 91–104.
[11] G. Krishna, J. Theiss, Concurrent analysis of cytogenetic
damage in vivo: a multiple endpoint-multiple tissue approach,
Environ. Mol. Mutagen. 25 (1995) 314–320.
[12] K. Criswell, G. Krishna, D. Zielinski, G. Urda, J. Theiss,
P. Juneau, M. Bleavins, Use of acridine orange in: flow
cytometric evaluation of erythropoietic cytotoxicity, Mutat.
Res. 414 (1998) 49–61.
[13] K. Criswell, G. Krishna, D. Zielinski, G. Urda, J. Theiss,
P. Juneau, M. Bleavins, Use of acridine orange in: flow
cytometric evaluation of micronuclei induction, Mutat. Res.
414 (1998) 63–75.
[14] G. Krishna, K. Criswell, D. Zielinski, G. Urda, P. Juneau,
M. Bleavins, J. Theiss, Validation of micronucleus evaluation
in the rat using flow cytometry with five model compounds,
Environ. Mol. Mutagen. 31 (1998) 46.
[15] M. Hayashi, T. Sofuni, M. Ishidate Jr., An application of
acridine orange fluorescent staining to the micronucleus test,
Mutat. Res. 120 (1983) 241–247.