United States       Prevention, Pesticides     EPA712-C-96-235
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.5915
          In Vivo Sister Chromatid
          Exchange 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 870.5915   In vivo sister chromatid exchange 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 materials used in developing this har-
monized OPPTS  test guideline are OPPT 40 CFR 798.5915 In Vivo Sister
Chromatid Exchange Assay and OPP 84-2 Mutagenicity Testing (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation; Human  and
Domestic Animals) EPA report 540/09-82-025, 1982.

    (b) Purpose. The sister  chromatid exchange (SCE)  assay  detects the
ability of a chemical to enhance the exchange of DNA between two sister
chromatids of a  duplicating chromosome. The test may be performed in
vitro using cultured mammalian cells or in vivo using nonmammalian or
mammalian tissues. The most commonly used assays employ bone marrow
or lymphocytes from mammalian species such as mice,  rats, or hamsters.
Human lymphocytes may also be used.

    (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.

    Sister  chromatid exchanges are reciprocal  interchanges of the two
chromatid arms within a single chromosome. These exchanges  are visual-
ized during the metaphase portion of the cell cycle and presumably require
enzymatic incision, translocation and ligation of at least two DNA helices.

    (d) Test method—(1) Principle. Animals  are exposed to test sub-
stance  by   appropriate   routes   followed   by  administration   of
bromodeoxyuridine  (BrdU).   A  spindle  inhibitor (e.g., colchicine  or
Colcemid®) is administered prior to sacrifice. After sacrifice, tissue is ob-
tained and metaphase preparations made, stained and scored for  SCE.

    (2) Description. The method described here  employs bone marrow
of laboratory  rodents exposed to test chemicals.  After treatment with test
chemical,  animals  are further treated with BrdU and, prior to sacrifice,
with a spindle inhibitor (e.g., colchicine or  Colcemid®)  to arrest cells in
ometaphase.  After sacrifice, chromosome preparations from bone marrow
cells are made, stained and scored for SCE.

    (3) Animal selection—(i) Species and strain. Any appropriate mam-
malian species may be used. Examples of commonly used rodent species
include mice,  rats, 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.  The use of a single
sex or different number of animals should be justified.

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

     (4) Control  groups—(i) Concurrent controls. Current positive and
negative (vehicle) controls should be included in the assay.

     (ii) Positive  controls.  A compound know to produce SCE 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-
hicle used  should 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 or that
producing some indication  of toxicity as evidenced by animal morbidity
(including death)  or target cell toxicity. The  LD50  is a suitable  guide.
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 of administration are
IP or oral. Other routes may be appropriate.

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

     (e) Test  performance—(1) Treatment. Animals should be treated
with test chemical followed by administration of BrdU. BrdU may be ad-
ministered by multiple IP injections, by continuous tail  vein infusion or
by subcutaneous  implantation of tablets. Animals should be treated with
a spindle inhibitor (e.g. colchicine or Colcemid®) 2  h prior to sacrifice.
After sacrifice, bone marrow should be extracted and slides made and pre-
pared for SCE evaluation.

     (2) Staining  method.  Staining of slides  to reveal SCEs  can be per-
formed  according to any  of several protocols.  However, the fluorescence
plus Giemsa method is recommended.

     (3) Number  of cells scored. 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, providing scores for both the number of  SCE for
each metaphase and the number of SCE per  chromosome for each meta-
phase. 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 SCE. Another criterion may be based
upon detection of a reproducible and statistically significant positive re-
sponse for at 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 SCE or a  statistically sig-
nificant and reproducible positive response at any one of the test points
is considered not to induce rearrangements of DNA segments in this sys-

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

     (4) Test evaluation, (i) Positive results in the in vivo SCE assay indi-
cate that under the test conditions the test substance  induces  reciprocal
interchanges in the bone marrow of the test species.

     (ii)  Negative results indicate that under the test conditions  the test
substance does not induce reciprocal  interchanges 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  level used,  rationale  for dose selec-
tion, toxicity data, negative and positive controls.

     (iii) Route  and schedule  of administration of both test chemical and

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

     (v) Time of sacrifice after administration  of BrdU.

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

     (vii) Criteria for scoring SCE.

     (viii) Dose-response relationship, if applicable.

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

     (1) Allen, J.W. et,al. Bromodeoxyuridine  tablet  methodology for in
vivo studies of DNA synthesis. Somatic Cell Genetics 4:393-405  (1978).

     (2) Allen, J.W.  et al. Simplified technique for  in vivo  analysis of
sister  chromatid exchanges  using  5-bromodeoxyuridine  tablets.  Cyto-
genetics Cell Genetics 18:231-237 (1977).

     (3) Latt, S.A. et  al.  Sister chromatid exchanges: A report of the U.S.
EPA Gene-Tox Program. Mutation Research 87:17-62 (1981).