United States      Prevention, Pesticides     EPA712-C-96-225
         Environmental Protection    and Toxic Substances     June 1996
         Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.5385
          In Vivo Mammalian
          Cytogenetics Tests:
          Bone Marrow
          Chromosomal Analysis
               "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 870.5385  In vivo mammalian cytogenetics tests: Bone mar-
row chromosomal analysis.
    (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 798.5385 In Vivo Mam-
malian Bone  Marrow Cytogenetics Tests:  Chromosomal Analysis;  OPP
84-2 Mutagenicity Testing (Pesticide Assessment Guidelines, Subdivision
F—Hazard Evaluation; Human and Domestic Animals) EPA report 5407
09-82-025, 1982; and OECD guideline  475 Genetic Toxicology: In Vivo
Mammalian Bone Marrow Cytogenetics Tests—Chromosomal Analysis.

    (b) Purpose. The in vivo bone marrow cytogenetic test is a muta-
genicity test for the detection of structural chromosomal aberrations. Chro-
mosomal aberrations are generally evaluated in first posttreatment mitoses.
With the majority of chemical mutagens, induced aberrations  are of the
chromatid type but chromosome type aberrations also occur.

    (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.

    Chromatid-type aberrations are damage expressed as breakage of sin-
gle chromatids or breakage and/or reunion between chromatids.

    Chromosome-type aberrations are changes which result from damage
expressed  in both sister chromatids at the same time.

    (d) Test  method—(1) Principle. Animals are exposed to test chemi-
cals by appropriate routes and are sacrificed at sequential intervals. Chro-
mosome preparations are made from bone marrow cells. The stained prep-
arations are examined  and metaphase cells are scored for  chromosomal

    (2) Description. The method employs bone marrow of laboratory ro-
dents which have been exposed to test chemicals. Prior to sacrifice, ani-
mals are  further  treated with a spindle inhibitor, (e.g., colchicine  or
Colcemid  ®) to arrest the cells in ometaphase. Chromosome preparations
from the cells are stained and scored for chromosomal aberrations.

    (3) Animal selection—(i) Species and strain. Any appropriate mam-
malian species may be used.  Examples of commonly used rodent species
are rats, mice, and hamsters.

    (ii) Age.  Healthy young adult animals should be used.


     (iii) Number and  sex. At least  five female and five male animals
per experimental and control group  should be used.  Thus, 10 animals
would be sacrificed per time  per group treated with the test compound
if several test times after treatment are included in the experimental sched-
ule. The use of a single  sex or smaller number of animals should be justi-

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

     (4) Control groups—(1) Concurrent controls, (i) Concurrent posi-
tive and negative (vehicle) controls should be included in the assay.

     (ii) Positive controls. A single dose positive control showing a sig-
nificant response at any one time point is adequate. A compound known
to produce chromosomal aberrations in vivo should be employed as the
positive control.

     (5)  Test  chemicals—(i) Vehicle.  When possible,  test chemicals
should be dissolved in isotonic saline or distilled water. Water insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The ve-
hicles  used  should neither interfere with the  test chemical 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., partial inhibition of mitosis) or should be the highest
dose attainable (to  a maximum of 5,000 mg/kg). Additional dose levels
may be used. For determination of dose-response, at least three dose levels
should be used.

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

     (iv) Treatment schedule. In general,  test substances should be ad-
ministered once only. However, based on toxicological  information a re-
peated treatment schedule may be employed.

     (e) Test performance—(1) Generally  the test may be performed
in two assays, (i) Animals should be  treated with the test substance once
at the  selected doses. Samples should be taken at three times after treat-
ment. For rodents, the central sampling interval is 24 h. Since cell cycle
kinetics can be influenced by the  test  substance, one earlier  and one  later
sampling interval adequately spaced within the range of 6 to 48 h should
be applied.  Where  the additional dose levels  are tested in  a subsequent
experiment,  samples should be taken  at the predetermined most  sensitive
interval or, if this is not established,  at the  central sampling time. If the

most sensitive interval is known and documented with data, only this one
time point should be sampled.

    (ii) If a repeated treatment  schedule is used  at the selected doses,
samples should be taken 6 and 24 h after the last treatment; other sampling
times  may be used if justified. Where the  additional dose levels are tested
in a subsequent experiment, samples should be taken at the predetermined
most  sensitive interval or,  if this is not established, at 6 h after  the last

    (2) Administration of spindle inhibitor. Prior to sacrifice,  animals
should be injected IP with  an appropriate  dose  of a spindle inhibitor (e.g.,
colchicine or Colcemid®) to arrest cells in  ometaphase.

    (3) Preparation of slides. Immediately after sacrifice, the bone mar-
row should  be obtained, exposed to hypotonic solution, and  fixed. The
cells should then be spread on slides and stained.  Chromosome  prepara-
tions should be made following standard procedures.

    (4) Analysis. The number of cells to be analyzed  per animal should
be based upon the number of animals used, the negative control frequency,
the predetermined sensitivity, and the power chosen for the test. Slides
should be coded before microscopic analysis.

    (f) Data and report—(1) Treatment of results. Data should be pre-
sented in tabular form for both cells and animals. Different types of struc-
tural chromosomal aberrations  should be  listed with their numbers and a
mean  frequency per  cell for each animal in all  treated and control groups.
Gaps  (achromatic  lesions) should be recorded separately and not included
in the total abberration frequency. Differences among animals within each
group  should be  considered before making comparisons between treated
and control groups.

    (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 result, one of  which is a statistically significant  dose-
related increase in the number of structual chromosomal aberrations or
abnormal metaphase figures. Another criterion may  be  based upon detec-
tion of a reproducible  and statistically significant positive response for a
least one of the test points.

    (ii) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of chromosomal  aberrations
or abnormal metaphase figures or a statistically significant and reproduc-
ible positive  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) Positive results in the in vivo bone marrow
cytogenetics assay indicate that under the test conditions the test substance
induces chromosomal aberrations in the bone marrow of the  test species.

     (ii) Negative results  indicate that under the test conditions, the test
substance does not induce chromosomal aberrations in the bone marrow
of the test species.

     (5) Test  report.  In addition  to  the  reporting recommendations  as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion should 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) Route of administration, treatment and sampling schedules, tox-
icity data, negative and positive controls.

     (iv) Identity of spindle-inhibitor,  its  concentration  and  duration  of

     (v) Details of the protocol used for chromosome preparation, number
of cells scored per animal, type and number of aberrations given separately
for  each treated and control animal.

     (vi) Mitotic index, where applicable.

     (vii) Criteria for scoring aberrations.

     (viii) Number and frequency of aberrant cells per animal in each treat-
ment and control  groups.

     (ix) Total number of aberrations per group.

     (x) Number of cells with aberrations per group.

     (xi) Dose-response relationship, if applicable.

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

     (1) Adler, I.D.  et al., In  vivo  cytogenetic  effects of trimethyl phos-
phate and of TEPA on bone marrow cells of male rats, Mutation Research
13:263-273 (1971).

    (2) Evans, H.J. Cytological methods for detecting chemical mutagens,
Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4.
Ed. A. Hollaender. Plenum, New York and London. (1976) pp. 1-29.

    (3) Kilian, J.D. et al. A collaborative study to measure intralaboratory
variation with the  in vivo bone morrow metaphase procedure. Handbook
of mutagenicity  test procedures. Eds. Kilby, B.J., Legator, M. Nichols,
C.,  Ramel,  D.  Elsevier/North Holland  Biomedical Press,  Amsterdam
(1977) 243-260.

    (4) Preston, J.R. et  al., Mammalian in vivo and vitro cytogenetics
assays: Report of the Gene-Tox Program. Mutation Research 87:143-188