United States Prevention, Pesticides EPA712-C-96-234
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5900
In Vitro Sister Chromatid
Exchange Assay
'Public Draft"
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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.).
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:
guidelines@epamail.epa.gov.
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 162.140.64.19), 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
Guidelines."
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OPPTS 870.5900 In vitro 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 798.5900 In Vitro Sister
Chromatid Exchange Assay and OECD 479 Genetic Toxicology: In Vitro
Sister Chromatid Exchange Assay in Mammalian Cells.
(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, for example, rodent or human cells, or in vivo using mam-
mals, for example, rodents such as mice, rats and hamsters.
(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 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. Following exposure of cell cultures
to test chemicals, they are allowed to replicate in the presence of
bromodeoxyuridine (BrdU), followed by treatment with colchicine or
colcemid to arrest cells in a metaphase-like stage of mitosis (ometaphase).
Cells are then harvested and chromosome preparations made. Preparations
are stained and metaphase cells analyzed for SCEs.
(2) Description. In vitro SCE assays may employ monolayer or sus-
pension cultures of established cell lines, cell strains, or primary cell cul-
tures. Cell cultures are exposed to test chemical and are allowed to rep-
licate in the presence of BrdU. Prior to harvest, cells are treated with a
spindle inhibitor (e.g. Colchicine or Colcemid #) to accumulate cells in
ometaphase. Chromosome preparations from cells are made, stained and
analyzed for SCEs.
(3) Cells—(i) Type of cells used in the assay. There are a variety
of cell lines or primary cell cultures, including human cells, which may
be used in the assay. Established cell lines and strains should be checked
for Mycoplasma contamination and may be periodically checked for
karyotype stability.
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(ii) Cell growth and maintenance. Appropriate culture media and
incubation conditions (culture vessels, temperature, humidity, and CCh
concentration) should be used.
(4) Metabolic activation. Cells should be exposed to test chemical
both in the presence and absence of an appropriate metabolic activation
system.
(5) Control groups—Concurrent controls. Positive and negative
(untreated and/or vehicle) controls, with and without metabolic activation,
should be included in each experiment.
(6) Test chemicals—(i) Vehicle. Test substances may be prepared
in culture media or dissolved or suspended in appropriate vehicles prior
to treatment of the cells. Final concentration of the vehicle should not
reduce cell viability or growth rate.
(ii) Exposure concentrations. Multiple concentrations of the test sub-
stance over a range adequate to define the response should be tested.
Among the criteria to be taken into consideration for determining the upper
limits of test chemical concentration are cytotoxicity and solubility.
Cytotoxicity of the test substance may be altered in the presence of meta-
bolic activation systems. Cytotoxicity may be evidenced by a large (e.g.,
75 percent) decrease in the number of cells that have divided twice in
the presence of BrdU. Relatively insoluble substances should be tested
up to the limit of solubility. For freely soluble nontoxic chemicals, the
upper test chemical concentration should be determined on a case by case
basis. When appropriate, a positive response should be confirmed by using
a narrow range of test concentrations.
(e) Test performance—(1) Established cell lines and strains, (i)
Prior to use in the assay, cells should be generated from stock cultures,
seeded in culture vessels at the appropriate density and incubated at
37 °C.
(ii) Cell lines and strains should be treated with test chemical both
with and without metabolic activation when they are in the exponential
stage of growth. At the end of the exposure period, cells should be washed
and incubated for two replication cycles in medium containing BrdU. After
BrdU is added, the cultures should be handled in darkness, under "safe"
(e.g., darkroom) lights, or in dim light from incandescent lamps to mini-
mize photolysis of BrdU containing DNA. At the end of the BrdU incuba-
tion period, cells should be fixed and stained for SCE determination. Cul-
tures should be treated with a spindle inhibitor (e.g., colchicine or
Colcemid®) 2 h prior to harvesting.
(2) Human lymphocyte cultures, (i) For preparation of human lym-
phocyte cell cultures, heparinized or acid-citrate-dextrose treated whole
blood should be added to culture medium containing a mitogen, e.g.,
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phytohemagglutinin (PHA) and incubated at 37 °C. White cells sedimented
by gravity (buffy coat) or lymphocytes which have been purified on a
density gradient such as Ficoll-Hypaque may also be utilized.
(ii) Cells should be exposed to the test chemical during at last two
time intervals, e.g. Go and S. Exposure during the Go phase of the cell
cycle should be accomplished by adding the test substance prior to addition
of mitogen. Exposure during or after the first S phase may be accom-
plished by exposing cells 24-30 h after mitosis, under "safe" (e.g. dark-
room) lights, or in dim light from incandescent lamps to minimize photoly-
sis of BrdU containing DNA. At the end of the BrdU incubation period,
cells should be fixed and stained for SCE determination. Cultures should
be treated with a spindle inhibitor (e.g. colchicine or Colcemid®) 2 h prior
to harvesting.
(3) Human lymphocyte cultures, (i) For preparation of human lym-
phocyte cell cultures, heparinized or acid-citrate-dextrose treated whole
blood should be added to culture medium containing a mitogen, e.g.,
phytohemagglutinin (PHA) and incubated at 37 °C. White cells sedimented
by gravity (buffy coat) or lymphocytes which have been purified on a
density gradient such as Ficoll-Hypaque may also be utilized.
(ii) Cells should be exposed to the test chemical during at least two
time intervals, e.g., Go and S. Exposure during the Go phase of the cell
cycle should be accomplished by adding the test substance prior to addition
of mitogen. Exposure during or after the first S phase may be accom-
plished by exposing cells 24-30 h after mitogen stimulation. After expo-
sure, cells should be washed and then cultured in the absence of the chemi-
cal.
(4) Culture harvest time. A single harvest time, one that yields an
optimal percentage of second division metaphases, is recommended. If
there is reason to suspect that this is not a representative sampling time
(which may occur for short-lived, cycle specific chemicals), then additional
harvest times should be selected.
(5) 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.
(6) Number of cultures. At least two independent cultures should
be used for each experimental point.
(7) Analysis. Slides should be coded before analysis. The number
of cells to be analyzed should be based upon the spontaneous control fre-
quency and defined sensitivity and the power of the test chosen before
analysis. In human lymphocytes, only cells containing 46 centromeres
should be analyzed. In established cell lines and strains, only metaphases
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containing ±2 centromeres of the modal number should be analyzed. Uni-
form criteria for scoring SCEs should be used.
(f) Data and report—(1) Treatment of results. Data should be pre-
sented in tabular form, providing scores for both the number of SCEs for
each metaphase and the number of SCEs per chromosome for each meta-
phase.
(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 sister chromatid exchanges. Another cri-
terion may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test substance concentra-
tions.
(ii) A test substance which produces neither a statistically significant
dose-related increase in the number of sister chromatid exchanges nor a
statistically significant and reproducible positive response at any one of
the test points is considered not to induce rearrangements of segments of
DNA 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 vitro SCE assay
indicate that under the test conditions the test substance induces reciprocal
chromatid interchanges in cultured mammalian somatic cells.
(ii) Negative results indicate that under the test conditions the test
substance does not induce reciprocal chromatid interchanges in cultured
mammalian somatic cells.
(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) Cells used, density at time of treatment, number of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium,
temperature, and CO2 concentration.
(iii) Test chemical vehicle, concentration and rationale for the selec-
tion of the concentrations of test chemical used in the assay, duration of
treatment.
(iv) Details of both the protocol used preparation of the metabolic
activation system and its use in the assay.
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(v) Growth period in BrdU; identity of spindle inhibitor, its concentra-
tion and duration of treatment.
(vi) Time of cell harvest.
(vii) Positive and negative controls.
(viii) Method used to prepare slides for SCE determination.
(ix) Criteria for scoring SCEs.
(x) Details of the protocol used for growth and treatment of human
cells if used in the assay.
(xi) Dose-response relationship, if applicable.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Latt, S.A. et al. Sister chromatid exchanges: a report of the U.S.
EPA's Gene-Tox Program. Mutation Research 87:17-62 (1981).
(2) [Reserved]
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