United States Prevention, Pesticides EPA712-C-98-226
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5395
Mammalian Erythrocyte
Micron ucleus Test
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.5395 Mammalian erythrocyte micronucleus test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are OPPT 40 CFR 798.5385 In vivo mam-
malian bone marrow cytogenetics test: Micronucleus assay and OECD
474, Mammalian Erythrocyte Micronucleus Test.
(b) Purpose. (1) The mammalian in vivo micronucleus test is used
for the detection of damage induced by the test substance to the chro-
mosomes or the mitotic apparatus of erythroblasts by analysis of
erythrocytes as sampled in bone marrow and/or peripheral blood cells of
animals, usually rodents.
(2) The purpose of the micronucleus test is to identify substances
that cause cytogenetic damage which results in the formation of
micronuclei containing lagging chromosome fragments or whole chro-
mosomes.
(3) When a bone marrow erythroblast develops into a polychromatic
erythrocyte, the main nucleus is extruded; any micronucleus that has been
formed may remain behind in the otherwise anucleated cytoplasm. Visual-
ization of micronuclei is facilitated in these cells because they lack a main
nucleus. An increase in the frequency of micronucleated polychromatic
erythrocytes in treated animals is an indication of induced chromosome
damage.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
Centromere (kinetochore) is a region of a chromosome with which
spindle fibers are associated during cell division, allowing orderly move-
ment of daughter chromosomes to the poles of the daughter cells.
Micronuclei are small nuclei, separate from and additional to the main
nuclei of cells, produced during telophase of mitosis (meiosis) by lagging
chromosome fragments or whole chromosomes.
Normochromatic erythrocyte is a mature erythrocyte that lacks
ribosomes and can be distinguished from immature, polychromatic
erythrocytes by stains selective for ribosomes.
Polychromatic erythrocyte is a immature erythrocyte, in an intermedi-
ate stage of development, that still contains ribosomes and therefore can
be distinguished from mature, normochromatic erythrocytes by stains se-
lective for ribosomes.
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(d) Initial considerations. (1) The bone marrow of rodents is rou-
tinely used in this test since polychromatic erythrocytes are produced in
that tissue. The measurement of micronucleated immature (polychromatic)
erythrocytes in peripheral blood is equally acceptable in any species in
which the inability of the spleen to remove micronucleated erythrocytes
has been demonstrated, or which has shown an adequate sensitivity to de-
tect agents that cause structural or numerical chromosome aberrations.
Micronuclei can be distinguished by a number of criteria. These include
identification of the presence or absence of a kinetochore or centromeric
DNA in the micronuclei. The frequency of micronucleated immature (pol-
ychromatic) erythrocytes is the principal endpoint. The number of mature
(normochromatic) erythrocytes in the peripheral blood that contain
micronuclei among a given number of mature erythrocytes can also be
used as the endpoint of the assay when animals are treated continuously
for 4 weeks or more. This mammalian in vivo micronucleus test is espe-
cially relevant to assessing mutagenic hazard in that it allows consideration
of factors of in vivo metabolism, pharmacokinetics, and DNA-repair proc-
esses although these may vary among species, among tissues and among
genetic endpoints. An in vivo assay is also useful for further investigation
of a mutagenic effect detected by an in vitro system.
(2) If there is evidence that the test substance, or a reactive
metabolite, will not reach the target tissue, it is not appropriate to use
this test.
(e) Test method—(1) Principle. Animals are exposed to the test sub-
stance by an appropriate route. If bone marrow is used, the animals are
sacrificed at appropriate times after treatment, the bone marrow extracted,
and preparations made and stained (see references in paragraphs (g)(l),
(g)(2), and (g)(3) of this guideline). When peripheral blood is used, the
blood is collected at appropriate times after treatment and smear prepara-
tions are made and stained (see references in paragraphs (g)(3), (g)(4),
(g)(5), and (g)(6) of this guideline). For studies with peripheral blood, as
little time as possible should elapse between the last exposure and cell
harvest. Preparations are analyzed for the presence of micronuclei.
(2) Description—(i) Preparations—(A) Selection of animal species.
Mice or rats are recommended if bone marrow is used, although any ap-
propriate mammalian species may be used. When peripheral blood is used,
mice are recommended. However, any appropriate mammalian species
may be used provided it is a species in which the spleen does not remove
micronucleated erythrocytes or a species which has shown an adequate
sensitivity to detect agents that cause structural or numerical chromosome
aberrations. Commonly used laboratory strains of healthy young animals
should be employed. At the commencement of the study, the weight vari-
ation of animals should be minimal and not exceed ±20 percent of the
mean weight of each sex.
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(B) Housing and feeding conditions. The temperature in the experi-
mental animal room should be 22 °C (±3 °C). Although the relative humid-
ity should be at least 30 percent and preferably not exceed 70 percent
other than during room cleaning, the aim should be 50-60 percent. Light-
ing should be artificial, the sequence being 12 hours light, 12 hours dark.
For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water. The choice of diet may be influenced by the
need to ensure a suitable admixture of a test substance when administered
by this route. Animals may be housed individually, or caged in small
groups of the same sex.
(C) Preparation of the animals. Healthy young adult animals should
be randomly assigned to the control and treatment groups. The animals
are identified uniquely. The animals are acclimated to the laboratory condi-
tions for at least five days. Cages should be arranged in such a way that
possible effects due to cage placement are minimized.
(D) Preparation of doses. Solid test substances should be dissolved
or suspended in appropriate solvents or vehicles and diluted, if appropriate,
prior to dosing of the animals. Liquid-test substances may be dosed di-
rectly or diluted prior to dosing. Fresh preparations of the test substance
should be employed unless stability data demonstrate the acceptability of
storage.
(ii) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not produce toxic effects at the dose levels used, and should not be sus-
pected of chemical reaction with the test substance. If other than well-
known solvents/vehicles are used, their inclusion should be supported with
reference data indicating their compatibility. It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle should be considered
first.
(B) Controls. (7) Concurrent positive and negative (solvent/vehicle)
controls should be included for each sex in each test. Except for treatment
with the test substance, animals in the control groups should be handled
in an identical manner to animals of the treatment groups.
(2) Positive controls should produce micronuclei in vivo at exposure
levels expected to give a detectable increase over background. Positive-
control doses should be chosen so that the effects are clear but do not
immediately reveal the identity of the coded slides to the reader. It is ac-
ceptable that the positive control be administered by a route different from
the test substance and sampled at only a single time. In addition, the use
of chemical class-related positive control chemicals may be considered,
when available. Examples of positive control substances include:
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Chemical
Ethyl methanesulphonate
Ethyl nitrosourea
Mitomycin C
Cyclophosphamide (monohydrate)
Triethvlenemelamine
CAS number
[62-50-0]
[759-73-9]
[50-07-7]
[50-1 8-0]
([6055-19-2])
[51-18-31
(3) Negative controls, treated with solvent or vehicle alone, and other-
wise treated in the same way as the treatment groups should be included
for every sampling time, unless acceptable inter-animal variability and fre-
quencies of cells with micronuclei are demonstrated by historical-control
data. If single sampling is applied for negative controls, the most appro-
priate time is the first sampling time. In addition, untreated controls should
also be used unless there are historical- or published-control data dem-
onstrating that no deleterious or mutagenic effects are induced by the cho-
sen solvent/vehicle.
(4) If peripheral blood is used, a pre-treatment sample may also be
acceptable as a concurrent negative control, but only in the short peripheral
blood studies (e.g., one-three treatment(s)) when the resulting data are in
the expected range for the historical control.
(3) Procedure—(i) Number and sex of animals. Each treated and
control group should include at least 5 analyzable animals per sex (see
reference in paragraph (g)(7) of this guideline). If at the time of the study
there are data available from studies in the same species and using the
same route of exposure that demonstrate that there are no substantial dif-
ferences between sexes in toxicity, then testing in a single sex will be
sufficient. Where human exposure to chemicals may be sex specific, as
for example with some pharmaceutical agents, the test should be per-
formed with animals of the appropriate sex.
(ii) Treatment schedule. (A) No standard treatment schedule (i.e.
1, 2, or more treatments at 24 hour intervals) can be recommended. The
samples from extended dose regimens are acceptable as long as a positive
effect has been demonstrated for this study or, for a negative study, as
long as toxicity has been demonstrated or the limit dose has been used,
and dosing continued until the time of sampling. Test substances may also
be administered as a split dose, i.e., two treatments on the same day sepa-
rated by no more than a few hours, to facilitate administering a large vol-
ume of material.
(B) The test may be performed in two ways:
(7) Animals should be treated with the test substance once. Samples
of bone marrow should be taken at least twice, starting not earlier than
24 hour after treatment, but not extending beyond 48 hours after treatment
with appropriate interval(s) between samples. The use of sampling times
earlier than 24 hours after treatment should be justified. Samples of periph-
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eral blood should be taken at least twice, starting not earlier than 36 hours
after treatment, with appropriate intervals following the first sample, but
not extending beyond 72 hours. When a positive response is recognized
at one sampling time, additional sampling is not required.
(2) If two or more daily treatments are used (e.g. two or more treat-
ments at 24 hour intervals), samples should be collected once between
18 and 24 hours following the final treatment for the bone marrow and
once between 36 and 48 hours following the final treatment for the periph-
eral blood (see reference in paragraph (g)(8) of this guideline).
(C) Other sampling times may be used in addition, when relevant.
(iii) Dose levels. If a range finding study is performed because there
are no suitable data available, it should be performed in the same labora-
tory, using the same species, strain, sex, and treatment regimen to be used
in the main study (see reference in paragraph (g)(9) of this guideline).
If there is toxicity, three-dose levels should be used for the first sampling
time. These dose levels should cover a range from the maximum to little
or no toxicity. At the later sampling time only the highest dose needs
to be used. The highest dose is defined as the dose producing signs of
toxicity such that higher dose levels, based on the same dosing regimen,
would be expected to produce lethality. Substances with specific biological
activities at low non-toxic doses (such as hormones and mitogens) may
be exceptions to the dose-setting criteria and should be evaluated on a
case-by-case basis. The highest dose may also be defined as a dose that
produces some indication of toxicity in the bone marrow (e.g. a reduction
in the proportion of immature erythrocytes among total erythrocytes in
the bone marrow or peripheral blood).
(iv) Limit test. If a test at one dose level of at least 2,000 mg/kg
body weight using a single treatment, or as two treatments on the same
day, produces no observable toxic effects, and if genotoxicity would not
be expected based upon data from structurally related substances, then a
full study using three-dose levels may not be considered necessary. For
studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/
day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for
treatment longer than 14 days. Expected human exposure may indicate
the need for a higher-dose level to be used in the limit test.
(v) Administration of doses. The test substance is usually adminis-
tered by gavage using a stomach tube or a suitable intubation cannula,
or by intraperitoneal injection. Other routes of exposure may be acceptable
where they can be justified. The maximum volume of liquid that can be
administered by gavage or injection at one time depends on the size of
the test animal. The volume should not exceed 2 ml/lOOg body weight.
The use of volumes higher than these must be justified. Except for irritat-
ing or corrosive substances which will normally reveal exacerbated effects
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with higher concentrations, variability in test volume should be minimized
by adjusting the concentration to ensure a constant volume at all dose
levels.
(vi) Bone marrow/blood preparation. Bone marrow cells should be
obtained from the femurs or tibias immediately following sacrifice. Cells
should be removed from femurs or tibias, prepared and stained using estab-
lished methods. Peripheral blood is obtained from the tail vein or other
appropriate blood vessel. Blood cells are immediately stained supravitally
(see references in paragraphs (g)(4), (g)(5), and (g)(6) of this guideline)
or smear preparations are made and then stained. The use of a DNA-spe-
cific stain (e.g. acridine orange (see reference in paragraph (g)(10) of this
guideline) or Hoechst 33258 plus pyronin-Y (see reference in paragraph
(g)(ll) of this guideline) can eliminate some of the artifacts associated
with using a non-DNA-specific stain. This advantage does not preclude
the use of conventional stains (e.g., Giemsa). Additional systems (e.g. cel-
lulose columns to remove nucleated cells (see reference in paragraph
(g)(12) of this guideline) can also be used provided that these systems
have been shown to adequately work for micronucleus preparation in the
laboratory.
(vii) Analysis. The proportion of immature among total (immature
+ mature) erythrocytes is determined for each animal by counting a total
of at least 200 erythrocytes for bone marrow and 1,000 erythrocytes for
peripheral blood (see reference in paragraph (g)(13) of this guideline). All
slides, including those of positive and negative controls, should be inde-
pendently coded before microscopic analysis. At least 2,000 immature
erythrocytes per animal should be scored for the incidence of
micronucleated immature erythrocytes. Additional information may be ob-
tained by scoring mature erythrocytes for micronuclei. When analyzing
slides, the proportion of immature erythrocytes among total erythrocytes
should not be less than 20 percent of the control value. When animals
are treated continuously for 4 weeks or more, at least 2,000 mature
erythrocytes per animal can also be scored for the incidence of
micronuclei. Systems for automated analysis (image analysis) and cell sus-
pensions (flow cytometry) are acceptable alternatives to manual evaluation
if appropriately justified and validated.
(f) Data and reporting—(1) Treatment of results. Individual animal
data should be presented in tabular form. The experimental unit is the
animal. The number of immature erythrocytes scored, the number of
micronucleated immature erythrocytes, and the number of immature
among total erythrocytes should be listed separately for each animal ana-
lyzed. When animals are treated continuously for 4 weeks or more, the
data on mature erythrocytes should also be given if it is collected. The
proportion of immature among total erythrocytes and, if considered appli-
cable, the percentage of micronucleated erythrocytes should be given for
each animal. If there is no evidence for a difference in response between
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the sexes, the data from both sexes may be combined for statistical analy-
sis.
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a dose-related increase
in the number of micronucleated cells or a clear increase in the number
of micronucleated cells in a single-dose group at a single-sampling time.
Biological relevance of the results should be considered first. Statistical
methods may be used as an aid in evaluating the test results (see references
in paragraphs (g)(14) and (g)(15) of this guideline). Statistical significance
should not be the only determining factor for a positive response. Equivo-
cal results should be clarified by further testing preferably using a modi-
fication of experimental conditions.
(ii) A test substance for which the results do not meet the criteria
in paragraph (f)(2)(i) of this guideline is considered non-mutagenic in this
test.
(iii) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance. Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
Positive results in the micronucleus test indicate that a substance induces
micronuclei which are the result of chromosomal damage or damage to
the mitotic apparatus in the erythroblasts of the test species. Negative re-
sults indicate that, under the test conditions, the test substance does not
produce micronuclei in the immature erythrocytes of the test species.
(iv) The likelihood that the test substance or its metabolites reach
the general circulation or specifically the target tissue (e.g. systemic tox-
icity) should be discussed.
(3) Test report. The test report should include the following informa-
tion:
(i) Test substance:
(A) Identification data and CAS No., if known.
(B) Physical nature and purity.
(C) Physiochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle.
(B) Solubility and stability of the test substance in the solvent/vehicle,
if known.
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(iii) Test animals:
(A) Species/strain used.
(B) Number, age, and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weight of the animals at the start of the test, including
body weight range, mean, and standard deviation for each group.
(iv) Test conditions:
(A) Positive and negative (vehicle/solvent) control data.
(B) Data from range-finding study, if conducted.
(C) Rationale for dose-level selection.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for route of administration.
(G) Methods for verifying that the test substance reached the general
circulation or target tissue, if applicable.
(H) Conversion from diet/drinking water test substance concentration
parts per million (ppm) to the actual dose (mg/kg body weight/day), if
applicable.
(I) Details of food and water quality.
(J) Detailed description of treatment and sampling schedules.
(K) Methods of slide preparation.
(L) Methods for measurement of toxicity.
(M) Criteria for scoring micronucleated immature erythrocytes.
(N) Number of cells analyzed per animal.
(O) Criteria for considering studies as positive, negative, or equivocal.
(v) Results:
(A) Signs of toxicity.
(B) Proportion of immature erythrocytes among total erythrocytes.
(C) Number of micronucleated immature erythrocytes, given sepa-
rately for each animal.
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(D) Mean + standard deviation of micronucleated immature
erythrocytes per group.
(E) Dose-response relationship, where possible.
(F) Statistical analyses and method applied.
(G) Concurrent and historical negative-control data.
(H) Concurrent positive-control data.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Heddle, J.A. et al. A Rapid In vivo Test for Chromosomal Dam-
age. Mutation Research 18, 187-190 (1973).
(2) Schmid, W. The Micronucleus Test. Mutation Research 31, 9-
15 (1975).
(3) Mavournin, K.H. et al. The In vivo Micronucleus Assay in Mam-
malian Bone Marrow and Peripheral Blood. A report of the U.S. Environ-
mental Protection Agency Gene-Tox Program. Mutation Research 239,
29-80 (1990).
(4) Hayashi, M. et al. The Micronucleus Assay with Mouse Peripheral
Blood Reticulocytes Using Acridine Orange-Coated Slides. Mutation Re-
search 245, 245-249 (1990).
(5) The Collaborative Study Group for the Micronucleus Test. Micro-
nucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Or-
ange Supravital Staining: The Summary Report of the 5th Collaborative
Study by CSGMT/JEMS.MMS. Mutation Research 278, 83-98 (1992).
(6) The Collaborative Study Group for the Micronucleus Test
(CSGMT/JEMMS.MMS, The Mammalian Mutagenesis Study Group of
the Environmental Mutagen Society of Japan). Protocol recommended for
the short-term mouse peripheral blood micronucleus test. Mutagenesis 10,
153-159(1995).
(7) Hayashi, M. et al. In vivo Rodent Erythrocyte Micronucleus
Assay. Mutation Research 312, 293-304 (1994).
(8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling
time of 30 +/- 6 h after double dosing in the mouse peripheral blood micro-
nucleus test. Mutagenesis 10, 313-319 (1995).
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(9) Fielder, R.J. et al. Report of British Toxicology Society/UK Envi-
ronmental Mutagen Society Working Group: Dose Setting in In vivo Muta-
genicity Assays. Mutagenesis 7, 313-319 (1992).
(10) Hayashi, M. et al. An Application of Acridine Orange Fluores-
cent Staining to the Micronucleus Test. Mutation Research 120, 241-247
(1983).
(11) MacGregor, J.T. et al. A Simple Fluorescent Staining Procedure
for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and
Pyronin Y. Mutation Research 120, 269-275 (1983).
(12) Romagna, F. and Staniforth, C.D. The automated bone marrow
micronucleus test. Mutation Research 213, 91-104 (1989).
(13) Gollapudi, B. and McFadden, L.G. Sample size for the esti-
mation of polychromatic to normochromatic erythrocyte ratio in the bone
marrow micronucleus test. Mutation Research 347, 97-99 (1995).
(14) Richold, M. et al. In vivo Cytogenetics Assays, In: D.J.Kirkland
(Ed.) Basic Mutagenicity Tests, UKEMS Recommended Procedures.
UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report.
Part I revised. Cambridge University Press, Cambridge, New York, Port
Chester, Melbourne, Sydney, pp. 115-141 (1990).
(15) Lovell, D.P. et al. Statistical Analysis of In vivo Cytogenetic
Assays In: D.J. Kirkland (Ed.) Statistical Evaluation of Mutagenicity Test
Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing,
Report, Part III. Cambridge University Press, Cambridge, New York, Port
Chester, Melbourne, Sydney, pp. 184-232 (1989).
(16) Heddle, J.A. et al. The Induction of Micronuclei as a Measure
of Genotoxicity. Mutation Research 123: 61-118 (1983).
(17) MacGregor, J.T. et al. Guidelines for the Conduct of Micro-
nucleus Assays in Mammalian Bone Marrow Erythrocytes. Mutation Re-
search 189: 103-112(1987).
(18) MacGregor, J.T. et al. The In vivo Erythrocyte Micronucleus
Test: Measurement at Steady State Increases Assay Efficiency and Permits
Integration with Toxicity Studies. Fundamental and Applied Toxicology
14: 513-522(1990).
(19) MacGregor, J.T. et al. Micronuclei in Circulating Erythrocytes:
A Rapid Screen for Chromosomal Damage During Routine Toxicity Test-
ing in Mice. In: Developments in Science and Practice of Toxicology. Ed.
A.W. Hayes, R.C. Schnell and T.S. Miya, Elsevier, Amsterdam, pp. 555-
558 (1983).
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