United States       Prevention, Pesticides     EPA712-C-96-226
          Environmental       and Toxic Substances     June 1996
          Protection Agency      (7101)
&EPA    Health Effects Test
          OPPTS 870.5395
          In Vivo Mammalian
          Cytogenetics Tests:
          Micron ucleus Assay
                "Public Draft'

     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

     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.).

     Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that  need to  be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access  Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public    Docket    at    (703)    305-5805    or     by    e-mail:

     To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency,  401  M  St.  SW.,  Washington, DC 20460. In  person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted  electronically by  sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.

     Final  Guideline Release: This guideline is available  from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin  Board.   By  modem   dial   202-512-1387,   telnet   and  ftp:
fedbbs.access.gpo.gov (IP,  or  call 202-512-0132 for disks
or paper copies.  This  guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access  Gopher
(gopher.epa.gov) under the heading  "Environmental Test Methods and

OPPTS 870.5395  In vivo mammalian cytogenetics tests: Erythrocyte
micronucleus assay.
    (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 material used in developing this har-
monized OPPTS test guideline is OPPT 40 CFR 798.5395 In Vivo Mam-
malian Bone  Marrow Cytogenetics Tests: Micronucleus Assay; OPP 84-
2 Mutagenicity  Testing  (Pesticide Assessment Guidelines, Subdivision F—
Hazard Evaluation; Human and Domestic Animals) EPA report 540/09-
82-025, 1982;  and OECD  474 Genetic Toxicology:  Micronucleus Test.

    (b) Purpose.  The  micronucleus test is a  mammalian in vivo test
which  detects damage of the chromosomes or mitotic apparatus by chemi-
cals. Polychromatic erythrocytes in the  bone marrow of rodents are used
in this  assay.  When the erythroblast develops into an erythrocyte the main
nucleus is extruded and may leave a micronucleus in the cytoplasm. The
visualization of micronuclei is facilitated  in these cells because they lack
a nucleus. Micronuclei  form under normal conditions. The assay is based
on  an  increase  in  the  frequency of  micronucleated polychromatic
erythrocytes in bone marrow of treated animals.

    (c) Definition. 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 definition also applies to this test guideline.

    Micronuclei are small particles  consisting  of  acentric fragments  of
chromosomes or entire  chromosomes, which lag behind at anaphase  of
cell division.  After telophase, these fragments may not be included in the
nuclei  of daughter cells and form single or multiple micronuclei in the

    (d) Test  method—(1) Principle, (i) Animals are exposed  to test sub-
stance  by an  appropriate route. They are  sacrificed, the bone marrow ex-
tracted, and smear preparations made and stained. Polychromatic erythro-
cytes are scored for micronuclei under the microscope.

    (ii) Micronuclei may also be detected  in other test systems:

    (A) Tissue culture.

    (B) Plants.

    (C) Blood  smears.

    (D) Fetal tissues.

    (E) Meiotic cells.

    (F) Hepatic cells.

    (2) Description. The method employs bone marrow  of laboratory
mammals which are exposed to test substances.

    (3) Animal  selection—(i)  Species  and  strain.  Mice  are  rec-
ommended. However, any appropriate mammalian species may be used.

    (ii) Age. Young adult animals shall be used.

    (iii) Number and sex.  At least five female and five male animals
per experimental and control group shall be used. Thus, 10 animals would
be sacrificed  per time per group if several test times after treatment were
included in the experimental schedule. The use of a single sex or a smaller
number of animals should be justified.

    (iv) Assignment to  groups. Animals shall be randomized and as-
signed to treatment and control groups.

    (4) Control  groups—(i)  Concurrent  controls.  Concurrent positive
and negative (vehicle) controls  shall be included in each assay.

    (ii) Positive controls.  A  compound known to produce micronuclei
in vivo shall be employed as the positive control.

    (5) Test chemicals—(i) Vehicle. When appropriate  for the route of
administration, solid and liquid test substances should be dissolved or sus-
pended in distilled water or isotonic saline. Water insoluble chemicals may
be dissolved  or suspended in appropriate vehicles. The vehicle used shall
neither interfere with the test compound nor produce toxic effects. Fresh
preparations of the test compound should be employed.

    (ii) Dose levels.  For an initial assessment,  one dose of the test sub-
stance may be used,  the dose  being the maximum tolerated dose (to  a
maximum  of  5,000 mg/kg)   or  that  producing  some indication  of
cytotoxicity, e.g. a change in the ratio of polychromatic to normochromatic
erythrocytes.  Additional dose  levels may be used. For  determination of
dose response, at least three dose levels shall be used.

    (iii) Route of administration. The usual routes of administration are
IP or oral.  Other routes may be appropriate.

    (iv) Treatment schedule.  Test substances should generally be admin-
istered only once. However, based upon toxicological information a re-
peated treatment schedule may be employed.

    (e) Test  performance—(1) Treatment and sampling times, (i) Ani-
mals shall be treated with the test substance once at the highest tolerated
dose.  Sampling times should coincide with the maximum responses of the
assay  which  varies with  the test substance. Therefore, using  the highest
dose,  bone marrow samples should be taken at  least three times, starting

not earlier than  12 h after treatment, with appropriate intervals following
the first sample but not extending beyond 72 h. When other doses are
used sampling shall be at the maximum sensitive period, or, if that is not
known, approximately 24  h after treatment. Other  appropriate sampling
times may be used  in addition.  If the most sensitive interval is known
and documented with data, only this one time point need be sampled.

     (ii) If a repeated treatment schedule is used, samples shall be taken
at least three times,  starting not earlier than 12 h after the last treatment
and at appropriate intervals following the  first sample, but not extending
beyond 72 h.

     (iii) Bone marrow shall be obtained immediately after sacrifice. Cells
shall be prepared, put on slides, spread as a smear and stained.

     (2) Analysis. Slides  shall be coded before microscopic analysis. At
least 1,000 polychromatic erythrocytes per animal shall be scored for the
incidence of micronuclei. The ratio  of polychromatic to  normochromatic
erythrocytes should be determined for each animal by counting a total of
200 erythrocytes. To ensure consistency with OECD and other guidelines,
1,000 polychromatic  erythrocytes are  recommended. Additional informa-
tion  may  be  obtained  by scoring  normochromatic  erythrocytes  for

     (f) Data and report—(1) Treatment of results. Criteria  for scoring
micronuclei shall be given. Individual data shall be presented in a tabular
form including positive and negative (vehicle) controls and experimental
groups. The number of polychromatic erythrocytes scored, the number of
micronucleated polychromatic  erythrocytes,  the  percentage of micronu-
cleated cells, the number  of micronucleated normochromatic erythro-
cytes, and, if applicable, the percentage of micronucleated erythrocytes and
the ratio of normochromatic to polychromatic erythrocytes shall be listed
separately  for each  experimental and control animal. Absolute numbers
shall be included if percentages are reported.

     (2) Statistical evaluation. Data should be evaluated by  appropriate
statistical methods.

     (3) Interpretation of results, (i) There are several criteria for deter-
mining a positive response, one of which is a statistically  significant dose-
related  increase  in  the  number  of  micronucleated  polychromatic
erythrocytes. Another criterion may be based upon detection of a reproduc-
ible and statistically significant positive response for at  least  one of the
test substance concentrations.

     (ii) A test substance which does not produce either a statistically sig-
nificant dose-related increase  in the  number  of micronucleated poly-
chromatic erythrocytes or a statistically significant and reproducible posi-

tive response  at any one of the test points is  considered  nonmutagenic
in this system.

    (iii) Both biological and statistical significance should  be considered
together in the evaluation.

    (4) Test evaluation, (i) The results of the  micronucleus test provide
information on the ability of a chemical to induce micronuclei in poly-
chromatic erythrocytes of the test species under the conditions of the test.
This damage may have been the result of chromosomal damage or damage
to the mitotic apparatus.

    (ii) Negative  results indicate that  under the test conditions the test
substance does not produce micronuclei in the bone marrow of the test

    (5) Test  report. In addition  to the reporting recommendations  as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion shall be reported:

    (i)  Species, strain,  age, weight, number, and sex of animals in each
treatment and control group.

    (ii) Test chemical vehicle, dose levels used, rationale for dose  selec-

    (iii) Rationale for and  description  of treatment and  sampling sched-
ules, toxicity data,  negative and positive controls.

    (iv) Details of the protocol used for slide preparation.

    (v) Criteria for identifying micronucleated erythrocytes.

    (vi) Dose-response relationship, if applicable.

    (g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

    (1) Cihak, R.  Evaluation of benzidine by the micronucleus test. Muta-
tion Research 67: 383-384 (1979).

    (2) Cole, R.J. et al.  Short-term tests for transplacentally active car-
cinogens.   1.  Micronucleus formation   in  fetal  and  maternal  mouse
erythroblasts. Mutation Research 80: 141-157 (1981).

    (3) Kliesch,  U. et al. Micronucleus  test  and bone-marrow  chro-
mosome  analysis.  A comparison of 2  methods in  vivo for evaluating
chemically induced chromosomal alterations. Mutation Research  80: 321-

    (4) Matter, B. and Schmid, W. Trenimon-induced chromosomal dam-
age in bone-marrow cells of six mammalian species,  evaluated by the
micronucleus test. Mutation Research 12: 417-425 (1971).

    (5) Schmid, W. The micronucleus test. Mutation Research 31:9-15

    (6) Schmid, W.  The micronucleus test for cytogenetic analysis. Chem-
ical Mutagens, Principles  and Methods for their  Detection.  Vol.  4.
Hollaender A,  (Ed.) Plenum New York and  London.  (1976) pp. 31-53.

    (7) Heddle, J.A. et al. The induction of micronuclei as a measure
of genotoxicity. A report of the U.S. Environmental Protection Agency
Gene-Tox Program. Mutation Research 123: 61-118 (1983).