ASTM E1263-97(2008)
(Guide)Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes (Withdrawn 2015)
Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes (Withdrawn 2015)
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 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
WITHDRAWN RATIONALE
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.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this guide was withdrawn in November 2014.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1263 − 97(Reapproved 2008)
Standard Guide for
Conduct of Micronucleus Assays in Mammalian Bone
Marrow Erythrocytes
This standard is issued under the fixed designation E1263; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope exposure, animals are sacrificed and the bone marrow is
extracted, spread on slides, and stained. The frequency of
1.1 This guide provides recommended guidelines for per-
micronucleated cells among the newly-formed (RNA-
forming the mammalian in vivo bone marrow micronucleus
containing) erythrocytes is determined, and this frequency is
assay. Under appropriate test conditions, measurement of the
compared among treatment groups. The newly-formed eryth-
frequency of newly formed micronucleated erythrocytes in
rocytes are identified by staining the residual RNA which
bone marrow provides a convenient index of chromosomal
remains in the newly-formed cells for about 2 days after
damage in nucleated erythrocyte precursor cells. The rationale
enucleation. Cells that stain uniformly positive for RNA are
for the occurrence of micronuclei in conjunction with chromo-
2 referred to as polychromatic, or polychromatophilic, erythro-
somal damage has been described previously (1). This guide
cytes (PCEs). Cells that do not stain positively for RNA are
describes conditions under which the frequency of micronu-
referred to as normochromatic erythrocytes (NCEs). An in-
cleated erythrocytes in mammalian bone marrow is an appro-
crease in the frequency of micronucleated PCEs relative to the
priate measure of in vivo chromosomal damage, and provides
vehicle control group indicates that the test substance induced
guidelines for the design and technical execution of assays
structural chromosomal damage or lagging chromosomes
employing this endpoint.
aneuploidy in the nucleated erythrocytic cells.
1.2 The following guidelines for mammalian bone marrow
erythrocyte micronucleus assays have been published by orga-
3. Significance and Use
nizations concerned with the evaluation of genotoxicity test
3.1 This guide provides guidelines for the selection of
data. These references should be consulted for recommenda-
animal species, dosage and sampling conditions, sampling and
tions on details not covered in depth by this guide and for
scoring methods, statistical design, and analysis of genotoxic-
requirements of specific organizations or government agencies
ity assays in which the endpoint measured is the frequency of
(2-6).
micronucleated erythrocytes in mammalian bone marrow.
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
4. Animal Selection and Care
standard.
4.1 Laboratory species that are suitable for use in this assay
1.4 This standard does not purport to address all of the
include the mouse (Mus musculus), rat (Rattus rattus), and
safety concerns, if any, associated with its use. It is the
Chinese hamster (Cricetulus griseus) (1). Other species prob-
responsibility of the user of this standard to establish appro-
ably are equally suitable. If species or strains not previously
priate safety and health practices and determine the applica-
used are employed, it must be established that the preparation
bility of regulatory limitations prior to use.
procedure adequately visualizes RNA-containing erythrocytes
and micronuclei, that potential artifacts such as aggregated
2. Summary of Guide
RNA and mast cell granules do not interfere with the identifi-
2.1 Animals are exposed either acutely or chronically to a
cation of micronuclei under the conditions employed, and that
test substance. At predetermined times after or during the micronucleus frequency is responsive to known clastogens
and aneuploidy-inducing agents in that species and strain.
4.2 In choosing the species and strain of test animal,
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
consideration should be given both to the availability of
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
historical data on the response of that species and strain to
F04.16 on Biocompatibility Test Methods.
Current edition approved Aug. 1, 2008. Published August 2008. Originally
known genotoxins and to the availability of other toxicity data
approved in 1988. Last previous edition approved in 2003 as E1263 – 97 (2003).
on the same test material in the species and strain chosen.
DOI: 10.1520/E1263-97R08.
Choice of the same strain to be used in other genotoxicity
The boldface numbers in parentheses refer to the list of references at the end of
this guide. assays of the same test material, or in long-term toxicity or
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1263 − 97 (2008)
carcinogenicity bioassays, has the advantage that the micro- withcertaintestsubstances (7)andoneunconfirmedreportthat
nucleus frequency can be directly compared with other end- DMSO increases the frequency of chromosomal aberrations in
points. The species for which the largest data base on known the rat (8).
genotoxins is available is the mouse (1).
6. Dose Selection
4.3 Animalsshouldbeobtainedfromarecognizedsourceof
6.1 The doses to be employed should be selected on the
laboratory animals and should be acclimated to laboratory
basis of either toxicity data obtained in the same laboratory or
conditions prior to use. Upon arrival, the age, sex, weight, and
published toxicity data, if available. Preliminary range-finding
health of each animal should be documented. Only healthy
experiment(s) should employ a minimum of two animals per
animals should be used. Animal care and housing should
dose group and should use the solvent and route of exposure to
conform to prevailing guidelines for the country and institution
be employed in the final experiment. The highest dose level
where the work is conducted. General information on guide-
should be chosen to meet one or more of the following criteria
lines for animal care and use can be obtained from the
in the experiment carried out with the full test group:
American Association for Accreditation of Laboratory Animal
6.1.1 It should cause a marked and significant increase in
Care. For any given experiment, all animals should be from
the micronucleus frequency in the target cell population.
the same source and should be approximately the same age
6.1.2 Itshouldproduceastatisticallysignificantsuppression
(within one week for young adults). In the absence of special
of the frequency of RNA-positive erythrocytes.
requirements for a particular age and sex, young adults of both
6.1.3 It should cause compound-related signs of toxicity or
sexes are recommended. Data from each sex should be
significantly reduce survival.
analyzed independently.
6.1.4 It should be the maximum practical dose that can be
administered. The maximum practical dose of a nontoxic test
5. Route of Administration and Choice of Vehicle
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—Avehicle or solvent control
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
solvent control relative to the spontaneous frequency in un-
treated animals, so that any effect of the vehicle or solvent is
Available from American Association for Accreditation of Laboratory Animal
Care (AAALAC), 5283 Corporate Dr., Suite 203, Frederick, MD 21703–2879. known.
E1263 − 97 (2008)
7.3 Positive Control Substance—A positive control the mouse, this minimum time between treatment and appear-
substance, that is, a substance known to induce micronuclei in ance of micronuclei is about 5 h (10). For most chemicals,
bone marrow, should be included with each experiment to substantial increases in the micronucleus frequency have not
confirm that all features of the protocol have been carried out been found earlier than 9 to 12 h after treatment. Since the life
correctly. The positive control agent preferably should be one span of the RNA-positive erythrocyte within the bone marrow
that is chemically related to the test substance and preferably has been reported to be between 10 and 30 h in the mouse and
administered by the same route as the test article. In addition, rat (for review, see (9)), any micronucleated RNA-positive
theagentordoseshouldbechosentoproduceamildorweakly erythrocytesformedwillremaininthebonemarrowforatleast
positive result. This provides a better evaluation of the sensi- 10 to 12 h. It is therefore not necessary to sample earlier than
tivity of the assay than does the use of a high dose of a potent 19 to 24 h after the first treatment.
clastogenwhichwouldalmostalwaysbedetectedregardlessof 9.3.2 Duetodifferencesbetweentestagentsinthetimeafter
whether or not the sensitivity of the assay were optimal. treatmentatwhichthepeakfrequencyofmicronucleioccurs,it
is important that two or more samples be taken if only one or
8. Number of Animals/Sex
two treatments are given.Available data indicate that this peak
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,
9. Treatment and Sampling Schedule
the period during which micronucleated PCEs are observable
may be longer than 24 to 36 h (9). However, the micronu-
9.1 The main requirement of the treatment/sampling sched-
cleated PCE frequency usually is not constant during this
ule is to obtain at least one sample at or near the time of the
period, 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 treatme
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