ASTM E1263-97(2003)
(Guide)Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes
Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes
SIGNIFICANCE AND USE
This guide provides guidelines for the selection of animal species, dosage and sampling conditions, sampling and scoring methods, statistical design, and analysis of genotoxicity assays in which the endpoint measured is the frequency of micronucleated erythrocytes in mammalian bone marrow.
SCOPE
1.1 This guide provides recommended guidelines for performing the mammalian in vivo bone marrow micronucleus assay. Under appropriate test conditions, measurement of the frequency of newly formed micronucleated erythrocytes in bone marrow provides a convenient index of chromosomal damage in nucleated erythrocyte precursor cells. The rationale for the occurrence of micronuclei in conjunction with chromosomal damage has been described previously (1). This guide describes conditions under which the frequency of micronucleated erythrocytes in mammalian bone marrow is an appropriate measure of in vivo chromosomal damage, and provides guidelines for the design and technical execution of assays employing this endpoint.
1.2 The following guidelines for mammalian bone marrow erythrocyte micronucleus assays have been published by organizations concerned with the evaluation of genotoxicity test data. These references should be consulted for recommendations on details not covered in depth by this guide and for requirements of specific organizations or government agencies (2-6).
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:E1263–97 (Reapproved 2003)
Standard Guide for
Conduct of Micronucleus Assays in Mammalian Bone
Marrow Erythrocytes
This standard is issued under the fixed designation E 1263; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope ing the residual RNAwhich remains in the newly-formed cells
for about 2 days after enucleation. Cells that stain uniformly
1.1 This guide provides recommended guidelines for per-
positive for RNA are referred to as polychromatic, or poly-
forming the mammalian in vivo bone marrow micronucleus
chromatophilic, erythrocytes (PCEs). Cells that do not stain
assay. Under appropriate test conditions, measurement of the
positively for RNA are referred to as normochromatic eryth-
frequency of newly formed micronucleated erythrocytes in
rocytes (NCEs). An increase in the frequency of micronucle-
bone marrow provides a convenient index of chromosomal
ated PCEs relative to the vehicle control group indicates that
damage in nucleated erythrocyte precursor cells. The rationale
the test substance induced structural chromosomal damage or
for the occurrence of micronuclei in conjunction with chromo-
lagging chromosomes aneuploidy in the nucleated erythrocytic
somal damage has been described previously (1). This guide
cells.
describes conditions under which the frequency of micronucle-
atederythrocytesinmammalianbonemarrowisanappropriate
3. Significance and Use
measure of in vivo chromosomal damage, and provides guide-
3.1 This guide provides guidelines for the selection of
lines for the design and technical execution of assays employ-
animal species, dosage and sampling conditions, sampling and
ing this endpoint.
scoring methods, statistical design, and analysis of genotoxic-
1.2 The following guidelines for mammalian bone marrow
ity assays in which the endpoint measured is the frequency of
erythrocyte micronucleus assays have been published by orga-
micronucleated erythrocytes in mammalian bone marrow.
nizations concerned with the evaluation of genotoxicity test
data. These references should be consulted for recommenda-
4. Animal Selection and Care
tions on details not covered in depth by this guide and for
4.1 Laboratory species that are suitable for use in this assay
requirements of specific organizations or government agencies
include the mouse (Mus musculus), rat (Rattus rattus), and
(2-6).
Chinese hamster (Cricetulus griseus) (1). Other species prob-
1.3 This standard does not purport to address all of the
ably are equally suitable. If species or strains not previously
safety concerns, if any, associated with its use. It is the
used are employed, it must be established that the preparation
responsibility of the user of this standard to establish appro-
procedure adequately visualizes RNA-containing erythrocytes
priate safety and health practices and determine the applica-
and micronuclei, that potential artifacts such as aggregated
bility of regulatory limitations prior to use.
RNA and mast cell granules do not interfere with the identifi-
2. Summary of Guide cation of micronuclei under the conditions employed, and that
the micronucleus frequency is responsive to known clastogens
2.1 Animals are exposed either acutely or chronically to a
and aneuploidy-inducing agents in that species and strain.
test substance. At predetermined times after or during expo-
4.2 In choosing the species and strain of test animal,
sure, animals are sacrificed and the bone marrow is extracted,
consideration should be given both to the availability of
spread on slides, and stained.The frequency of micronucleated
historical data on the response of that species and strain to
cells among the newly-formed (RNA-containing) erythrocytes
known genotoxins and to the availability of other toxicity data
isdetermined,andthisfrequencyiscomparedamongtreatment
on the same test material in the species and strain chosen.
groups. The newly-formed erythrocytes are identified by stain-
Choice of the same strain to be used in other genotoxicity
assays of the same test material, or in long-term toxicity or
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
carcinogenicity bioassays, has the advantage that the micro-
Surgical Materials and Devices and is the direct responsibility of Subcommittee
nucleus frequency can be directly compared with other end-
F04.16 on Biocompatibility Test Methods.
points. The species for which the largest data base on known
Current edition approved Sept. 10, 2003. Published September 2003. Originally
approved in 1988. Last previous edition approved in 1997 as E 1263 – 97. genotoxins is available is the mouse (1).
The boldface numbers in parentheses refer to the list of references at the end of
this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1263–97 (2003)
4.3 Animalsshouldbeobtainedfromarecognizedsourceof 6. Dose Selection
laboratory animals and should be acclimated to laboratory
6.1 The doses to be employed should be selected on the
conditions prior to use. Upon arrival, the age, sex, weight, and
basis of either toxicity data obtained in the same laboratory or
health of each animal should be documented. Only healthy
published toxicity data, if available. Preliminary range-finding
animals should be used. Animal care and housing should
experiment(s) should employ a minimum of two animals per
conform to prevailing guidelines for the country and institution
dose group and should use the solvent and route of exposure to
where the work is conducted. General information on guide-
be employed in the final experiment. The highest dose level
lines for animal care and use can be obtained from the
should be chosen to meet one or more of the following criteria
American Association for Accreditation of Laboratory Animal
in the experiment carried out with the full test group:
Care. For any given experiment, all animals should be from
6.1.1 It should cause a marked and significant increase in
the same source and should be approximately the same age
the micronucleus frequency in the target cell population.
(within one week for young adults). In the absence of special
6.1.2 Itshouldproduceastatisticallysignificantsuppression
requirements for a particular age and sex, young adults of both
of the frequency of RNA-positive erythrocytes.
sexes are recommended. Data from each sex should be
6.1.3 It should cause compound-related signs of toxicity or
analyzed independently.
significantly reduce survival.
6.1.4 It should be the maximum practical dose that can be
5. Route of Administration and Choice of Vehicle
administered. The maximum practical dose of a nontoxic test
material is determined by the physical bulk and solubility.
5.1 The choice of exposure route depends on the objective
Testing at such a maximum dose level has been referred to as
of the experiment. The objective of most micronucleus assays
a “limit test” in OECD and EPA/TSCAtesting guidelines.This
is to determine if the test substance induces types of chromo-
dosewillvarywithtestagent,butwillgenerallybeintherange
somal damage known to result in the formation of micronuclei.
5 to 10 g/kg for acute oral or intraperitoneal (i.p.) administra-
In this case, it is desirable to choose a route of administration
tion (3, 5).
and a vehicle that maximize the dose delivered to the target
6.2 The doses employed should include a minimum of two,
tissue. For this purpose, intraperitoneal and oral routes have
and preferably three, doses, at least one of which does not
been used most commonly, although others may also be
severely reduce the frequency of RNA-positive erythrocytes
appropriate. In other cases, the objective may be to evaluate
(the frequency should be at least 10 to 20 % of the control
specificallyinvivoactivityunderconditionsbaseduponknown
value) and which does not significantly reduce the survival of
exposure routes in man. In such cases, the appropriate route is
the test animals. The rationale for selecting test doses has
the one that provides the best experimental model of the
previously been discussed in the U.S. Environmental Protec-
expected exposure route in man.
tion Agency Gene-Tox Program report on the bone marrow
5.2 The choice of a solvent or vehicle is influenced by
polychromatic erythrocyte assay (1) and by Salamone and
several factors, including the chemical nature and solubility of
Heddle (9). Because the maximum cytogenetic effect is likely
the test substance, its toxicity to the test organism, and the
to be found at doses near the maximum tolerated dose (MTD),
route of exposure. In all cases care must be taken to ensure that
thelowerdosesshouldbespacedatrelativelysmallincrements
the vehicle selected will not produce measurable toxicity or 1 1
below the highest dose (for example, no more than ⁄2 and ⁄4 of
interfere with the normal uptake and metabolism of the test
the upper dose).
substance at the dose employed. In particular, the vehicle
should not alter the spontaneous micronucleus frequency. If
7. Controls
possible, it is desirable to use isotonic saline for parenteral
7.1 VehicleorSolventControl—Avehicleorsolventcontrol
administration and water or isotonic saline for oral adminis-
shall be included for each sampling condition (dose, time, sex)
tration. For oral administration of organic substances not
in each experiment. Animals are treated with the solvent or
readily soluble in aqueous solution, a pharmaceutical grade of
vehicle in the absence of the test substance. The quantity of
corn or other vegetable oil may be used. Vegetable oil is less
solvent or vehicle administered should be equivalent to the
suitable for intraperitoneal administration because it is poorly
maximum given to the animals receiving the test substance.
absorbed from the peritoneal cavity. Other acceptable choices
This control helps discriminate any test-substance effect from
of vehicle include carboxymethylcellulose or suspension in
any that may have been induced by the solvent.
gum arabic. Dimethylsulfoxide (DMSO) is an effective solvent
7.2 Untreated Control—The use of untreated animals is
for a wide range of substances and has frequently been used in
generally not necessary during routine testing. It is important,
experiments with mice, although there are a few reports of
however, that each laboratory determine the frequency of
foreign intermediates being produced by interaction of DMSO
micronucleated cells in animals treated with the vehicle or
withcertaintestsubstances (7)andoneunconfirmedreportthat
solvent control relative to the spontaneous frequency in un-
DMSO increases the frequency of chromosomal aberrations in
treated animals, so that any effect of the vehicle or solvent is
the rat (8).
known.
7.3 Positive Control Substance—A positive control sub-
stance, that is, a substance known to induce micronuclei in
bone marrow, should be included with each experiment to
American Association for Accreditation of Laboratory Animal Care, 208A
North Cedar Rd., New Lenox, IL 60451. confirm that all features of the protocol have been carried out
E1263–97 (2003)
correctly. The positive control agent preferably should be one been reported to be between 10 and 30 h in the mouse and rat
that is chemically related to the test substance and preferably (for review, see (9)), any micronucleated RNA-positive eryth-
administered by the same route as the test article. In addition, rocytes formed will remain in the bone marrow for at least 10
theagentordoseshouldbechosentoproduceamildorweakly to 12 h. It is therefore not necessary to sample earlier than 19
positive result. This provides a better evaluation of the sensi- to 24 h after the first treatment.
tivity of the assay than does the use of a high dose of a potent 9.3.2 Duetodifferencesbetweentestagentsinthetimeafter
clastogenwhichwouldalmostalwaysbedetectedregardlessof treatmentatwhichthepeakfrequencyofmicronucleioccurs,it
whether or not the sensitivity of the assay were optimal. is important that two or more samples be taken if only one or
two treatments are given.Available data indicate that this peak
8. Number of Animals/Sex
frequency usually occurs between 24 and 48 h after treatment,
8.1 It is desirable to have data for both sexes. For routine
but that in certain cases it may occur as late as 72 h after
screening, both sexes should be tested using a minimum of five
treatment (9). The interval between samples should be shorter
animals of each sex at each test dose. If a positive result is
than the time it would take a clastogen-affected cell population
obtained in one sex, a test agent may be classified as active
to pass through the scorable stage of erythropoiesis. This time
without data from the other sex, but both sexes must be tested
period is approximately 24 to 36 h in mice and rats. Since a
to verify a negative result. clastogen may affect more than a single erythroblast cell cycle,
the period during which micronucleated PCEs are observable
9. Treatment and Sampling Schedule
may be longer than 24 to 36 h (9). However, the micronucle-
9.1 The main requirement of the treatment/sampling sched-
ated PCE frequency usually is not constant during this period,
ule is to obtain at least one sample at or near the time of the
but rises to a maximum and then declines. Because it is
maximum incidence of micronucleated cells among the RNA-
desirable to sample as near as possible to the time of the
positive erythrocytes in bone marrow. The time of maximum
maximum micronucleated PCE frequency, it is recommended
incidence varies with the test agent, dose, and treatment
that the time between samples not exceed approximately 24 h.
schedule.
9.3.3 Based on these considerations, the following sampling
9.2 Treatment Schedule:
schedules are recommended for experiments with mice and
9.2.1 Treatmentprotocolsusingsingle,double,andmultiple
rats.
treatments have been reported (9). Although each of these
9.3.3.1 If one treatment is employed, a minimum of three
treatment schedules has been reported to be advantageous with
samples should be obtained between 20 and 72 h after the
specific test agents, there is insufficient evidence at present to
treatment.
support the exclusive use of a specific treatment schedule for
9.3.3.2 If two treatments are employed, a minimum of two
all test substances.Accordingly, the choice of single, multiple,
samples should be obtained between 20 and 48 h after the last
or continuous dosing protocol
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