United States Prevention, Pesticides EPA712-C-96-223
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5375
In Vitro Mammalian
Cytogenetics
'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 870.5375 In vitro mammalian cytogenetics.
(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 798.5375 In Vitro Mam-
malian Cytogenetics and OECD 473 Genetic Toxicology: In Vitro Mam-
malian Cytogenetic Test.
(b) Purpose. The in vitro cytogenetics test is a mutagenicity test sys-
tem for the detection of chromosomal aberrations in cultured mammalian
cells. Chromosomal aberrations may be either structural or numerical.
However, because cytogenetic assays are usually designed to analyze cells
at their first post-treatment mitosis and numerical aberrations require at
least one cell division to be visualized, this type of aberration is generally
not observed in a routine cytogenetics assay. Structural aberrations may
be of two types, chromosome or chromatid.
(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. In vitro cytogenetics assays may
employ cultures of established cell lines, cell strains or primary cell cul-
tures. Cell cultures are exposed to the test substance both with and without
metabolic activation. Following exposure of cell cultures to test substances,
they are treated with a spindle inhibitor (e.g., 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 are analyzed for chromosomal aberrations.
(2) Description. Cell cultures are exposed to test compounds and har-
vested at various intervals after treatment. Prior to harvesting, cells are
treated with a spindle inhibitor (e.g., colchicine or Colcemid®) to accumu-
late cells in ometaphase. Chromosome preparations from cells are made,
stained and scored for chromosomal aberrations.
(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
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for Mycoplasma contamination and may be periodically checked for
karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and
incubation conditions (culture vessels CC>2 concentrations, temperature and
humidity) should be used.
(4) Metabolic activation. Cells should be exposed to test substance
both in the presence and absence of an appropriate metabolic activation
system.
(5) Control groups. Positive and negative (untreated and/or vehicle)
controls both with and without metabolic activation should be included
in each experiment. When metabolic activation is used, the positive control
substance should be known to require such activation.
(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
interfere with cell viability or growth rate. Treatment vessels should be
chosen to ensure that there is no visible interaction, such as etching, be-
tween the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. Multiple concentrations of the test sub-
stance over a range adequate to define the response should be tested. Gen-
erally the highest test substance concentrations tested with and without
metabolic activation should show evidence of cytotoxicity or reduced mi-
totic activity. 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.
(e) Test performance—(1) Established cell lines and strains. 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.
(2) Human lymphocyte cultures. Heparinized or acid-citrate-dex-
trose 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 may also be utilized.
(3) Treatment with test substance. For established cell lines and
strains, cells in the exponential phase of growth should be treated with
test substances in the presence and absence of an exogenous metabolic
activation system. Mitogen-stimulated human lymphocyte cultures may be
treated with the test substance in a similar manner.
(4) Number of cultures. At least two independent cultures should
be used for each experimental point.
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(5) Culture harvest time, (i) For established cell lines and strains,
multiple harvest times are recommended. However, for screening purposes,
a single harvest time may be appropriate. If the test chemical changes
the cell cycle length, the fixation intervals should be changed accordingly.
If a single harvest time is selected, supporting data for the harvest time
should be presented in such a study.
(ii) For human lymphocyte cultures, the substance to be tested may
be added to the cultures at various times after mitogen stimulation so that
there is a single harvest time after the initiation of the cell culture. Alter-
natively, a single treatment may be followed by multiple harvest times.
Harvest time should be extended for those chemicals which induce an ap-
parent cell cycle delay. Because the population of human lymphocytes is
only partially synchronized, a single treatment, at, or close to, the time
when metaphase stages first appear in the culture will include cells in all
phases of the division cycle. Therefore, a single harvest at the time of
second mitosis may be carried out for screening purposes.
(iii) Cell cultures should be treated with a spindle inhibitor, (e.g., col-
chicine or Colcemid®), 1 or 2 h prior to harvesting. Each culture should
be harvested and processed separately for the preparation of chromosomes.
(6) Chromosome preparation. Chromosome preparation involves
hypotonic treatment of the cells, fixation and staining.
(7) Analysis. Slides should be coded before analysis. In human
lymphocytes, only cells containing 46 centromeres should be analyzed. In
established cell lines and strains, only metaphases containing
±2 centromeres of the modal number should be analyzed. Uniform criteria
for scoring aberrations should be used.
(8) Confirmatory tests. When appropriate, a single positive response
should be confirmed by testing over a narrow range of concentrations.
(f) Data and report—(1) Treatment of results. Data should be pre-
sented in a tabular form. Different types of structural chromosomal aberra-
tions should be listed with their numbers and frequencies for experimental
and control groups. Data should be evaluated by appropriate statistical
methods. Gaps or achromatic lesions are recorded separately and not in-
cluded in the total aberration frequency.
(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 structural chromosomal aberrations. An-
other criterion may be based upon detection of a reproducible and statis-
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tically 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 structural chromosomal ab-
errations or a statistically significant and reproducible positive response
at any one of the test points is considered nonmutagenic in this system.
(iii) Biological and statistical significance should be considered to-
gether in the evaluation.
(4) Test evaluation, (i) Positive results in the in vitro cytogenetics
assay indicate that under the test conditions the test substance induces
chromosomal aberrations in cultured mammalian somatic cells.
(ii) Negative results indicate that under the test conditions the test
substance does not induce chromosomal aberrations in cultured mamma-
lian 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 and passage number at time of treatment, and
number of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium,
temperature and CCh concentration.
(iii) Test chemical vehicle, concentration and rationale for the selec-
tion of the concentrations used in the assay, and duration of treatment.
(iv) Details of both the protocol used to prepare the metabolic activa-
tion system and of its use in the assay.
(v) Identity of spindle inhibitor, its concentration, and duration of
treatment.
(vi) Date of cell harvest.
(vii) Positive and negative controls.
(viii) Methods used for preparation of slides for microscopic examina-
tion.
(ix) Number of metaphases analyzed.
(x) Mitotic index where applicable.
(xi) Criteria for scoring aberrations.
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(xii) Type and number of aberrations, given separately for each treat-
ed and control culture, total number of aberrations per group; frequency
distribution of number of chromosomes in established cell lines and
strains.
(xiii) Dose-response relationship, if applicable.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Ames, B.N. et al Methods for detecting carcinogens and mutagens
with the &z/mo«e//a/mammalian-microsome mutagenicity test. Mutation
Research 31:347-364 (1975).
(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, London (1976) pp. 1-29.
(3) Howard, P.N. et al. Chromosomal aberrations induced by jV-meth-
yl-jV-nitro-jV-nitrosoguanidine in mammalian cells. In Vitro 7:359-365
(1972).
(4) Ishidate, M. Jr. and Odashima, S. Chromosome tests with 134
compounds on Chinese hamster cells in vitro: A screening for chemical
carcinogens. Mutation Research 48:337-354 (1975).
(5) Preston, R.J. et al. Mammalian in vivo and in vitro cytogenetic
assays: A report of the Gene-Tox Program. Mutation Research 87:143-
188(1981).
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