United States Prevention, Pesticides EPA712-C-98-223
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5375
In Vitro Mammalian
Chromosome Aberration
Test
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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.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.5375 In vitro mammalian chromosome aberration test.
(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 798.5375 In vitro mam-
malian cytogenetics and OECD 473, In Vitro Mammalian Chromosome
Aberration Test.
(b) Purpose. (1) The purpose of the in vitro chromosome aberration
test is to identify agents that cause structural chromosome aberrations in
cultured mammalian cells (see paragraphs (i)(l), (i)(2), and (i)(3) of this
guideline). Structural aberrations may be of two types, chromosome or
chromatid. With the majority of chemical mutagens, induced aberrations
are of the chromatid type, but chromosome-type aberrations also occur.
An increase in polyploidy may indicate that a chemical has the potential
to induce numerical aberrations. However, this guideline is not designed
to measure numerical aberrations and is not routinely used for that pur-
pose. Chromosome mutations and related events are the cause of many
human genetic diseases and there is substantial evidence that chromosome
mutations and related events causing alterations in oncogenes and tumour-
suppressor genes of somatic cells are involved in cancer induction in hu-
mans and experimental animals.
(2) The in vitro chromosome aberration test may employ cultures of
established cell lines, cell strains or primary cell cultures. The cells used
are selected on the basis of growth ability in culture, stability of the
karyotype, chromosome number, chromosome diversity, and spontaneous
frequency of chromosome aberrations.
(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 aberration is structural chromosome damage ex-
pressed as breakage of single chromatids or breakage and reunion between
chromatids.
Chromosome-type aberration is structural chromosome damage ex-
pressed as breakage, or breakage and reunion, of both chromatids at an
identical site.
Endoreduplication is a process in which after an S period of DNA
replication, the nucleus does not go into mitosis but starts another S period.
The result is chromosomes with 4, 8, 16,...chromatids.
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Gap is an achromatic lesion smaller than the width of one chromatid,
and with minimum misalignment of the chromatid(s).
Mitotic index is the ratio of cells in metaphase divided by the total
number of cells observed in a population of cells; an indication of the
degree of proliferation of that population.
Numerical aberration is a change in the number of chromosomes
from the normal number characteristic of the cells utilized.
Polyploidy is a multiple of the haploid chromosome number (n) other
than the diploid number (i.e., 3n, 4n, and so on).
Structural aberration is a change in chromosome structure detectable
by microscopic examination of the metaphase stage of cell division, ob-
served as deletions and fragments, intrachanges, and interchanges.
(d) Initial considerations. (1) Tests conducted in vitro generally re-
quire the use of an exogenous source of metabolic activation. This meta-
bolic activation system cannot mimic entirely the mammalian in vivo con-
ditions. Care should be taken to avoid conditions which would lead to
positive results which do not reflect intrinsic mutagenicity and may arise
from changes in pH, osmolality, or high levels of cytotoxicity (see para-
graphs (i)(4) and (i)(5) of this guideline).
(2) This test is used to screen for possible mammalian mutagens and
carcinogens. Many compounds that are positive in this test are mammalian
carcinogens; however, there is not a perfect correlation between this test
and carcinogenicity. Correlation is dependent on chemical class and there
is increasing evidence that there are carcinogens that are not detected by
this test because they appear to act through mechanisms other than direct
DNA damage.
(e) Principle of the test method. Cell cultures are exposed to the
test substance both with and without metabolic activation. At predeter-
mined intervals after exposure of cell cultures to the test substance, they
are treated with a metaphase-arresting substance (e.g., Colcemid® or col-
chicine), harvested, stained, and metaphase cells are analysed microscopi-
cally for the presence of chromosome aberrations.
(f) Description of the method—(1) Preparations—(i) Cells. A vari-
ety of cell lines, strains, or primary cell cultures, including human cells,
may be used (e.g., Chinese hamster fibroblasts, human, or other mamma-
lian peripheral blood lymphocytes).
(ii) Media and culture conditions. Appropriate culture media, and
incubation conditions (culture vessels, CCh concentration, temperature and
humidity) should be used in maintaining cultures. Established cell lines
and strains should be checked routinely for stability in the modal chro-
mosome number and the absence of Mycoplasma contamination and
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should not be used if contaminated. The normal cell-cycle time for the
cells and culture conditions used should be known.
(iii) Preparation of cultures—(A) Established cell lines and
strains. Cells are propagated from stock cultures, seeded in culture me-
dium at a density such that the cultures will not reach confluency before
the time of harvest, and incubated at 37 °C.
(B) Lymphocytes. Whole blood treated with an anti-coagulant (e.g.,
heparin) or separated lymphocytes obtained from healthy subjects are
added to culture medium containing a mitogen (e.g., phytohemagglutinin)
and incubated at 37 °C.
(iv) Metabolic activation. Cells should be exposed to the test sub-
stance both in the presence and absence of an appropriate metabolic activa-
tion system. The most commonly used system is a co-factor-supplemented
post-mitochondrial fraction (S9) prepared from the livers of rodents treated
with enzyme-inducing agents such as Aroclor 1254 (see paragraphs (i)(6),
(i)(7), (8)(i), and (i)(9) of this guideline), or a mixture of phenobarbitone
and p-naphthoflavone (see paragraphs (i)(10), (i)(ll), and (i)(12) of this
guideline). The post-mitochondrial fraction is usually used at concentra-
tions in the range from 1-10 percent v/v in the final test medium. The
condition of a metabolic activation system may depend upon the class of
chemical being tested. In some cases, it may be appropriate to utilize more
than one concentration of post-mitochondrial fraction. A number of devel-
opments, including the construction of genetically engineered cell lines
expressing specific activating enzymes, may provide the potential for en-
dogenous activation. The choice of the cell lines used should be scientif-
ically justified (e.g., by the relevance of the cytochrome P450 isoenzyme
for the metabolism of the test substance).
(v) Test substance/preparation. Solid test substances should be dis-
solved or suspended in appropriate solvents or vehicles and diluted, if ap-
propriate, prior to treatment of the cells. Liquid test substances may be
added directly to the test systems and/or diluted prior to treatment. Fresh
preparations of the test substance should be employed unless stability data
demonstrate the acceptability of storage.
(2) Test conditions—(i) Solvent/vehicle. The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the cells and the S9 activity. If other
than well-known solvent/vehicles are used, their inclusion should be sup-
ported by data indicating their compatibility. It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle be considered first.
When testing water-unstable substances, the organic solvents used should
be free of water. Water can be removed by adding a molecular sieve.
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(ii) Exposure concentrations. (A) Among the criteria to be consid-
ered when determining the highest concentration are cytotoxicity, solu-
bility in the test system, and changes in pH or osmolality.
(B) Cytotoxicity should be determined with and without metabolic
activation in the main experiment using an appropriate indication of cell
integrity and growth, such as degree of confluency, viable cell counts, or
mitotic index. It may be useful to determine cytotoxicity and solubility
in a preliminary experiment.
(C) At least three analyzable concentrations should be used. Where
cytotoxicity occurs, these concentrations should cover a range from the
maximum to little or no toxicity; this will usually mean that the concentra-
tions should be separated by no more than a factor between 2 and VlO.
At the time of harvesting, the highest concentration should show a signifi-
cant reduction in degree of confluency, cell count or mitotic index, (all
greater than 50 percent). The mitotic index is only an indirect measure
of cytotoxic/cyto static effects and depends on the time after treatment.
However, the mitotic index is acceptable for suspension cultures in which
other toxicity measurements may be cumbersome and impractical. Infor-
mation on cell-cycle kinetics, such as average generation time (AGT),
could be used as supplementary information. AGT, however, is an overall
average that does not always reveal the existence of delayed subpopula-
tions, and even slight increases in average generation time can be associ-
ated with very substantial delay in the time of optimal yield of aberrations.
For relatively non-cytotoxic compounds the maximum concentration
should be 5 (ig/ml, 5mg/ml, or 0.01M, whichever is the lowest.
(D) For relatively insoluble substances that are not toxic at concentra-
tions lower than the insoluble concentration, the highest dose used should
be a concentration above the limit of solubility in the final culture medium
at the end of the treatment period. In some cases (e.g., when toxicity oc-
curs only at higher than the lowest insoluble concentration) it is advisable
to test at more than one concentration with visible precipitation. It may
be useful to assess solubility at the beginning and the end of the treatment,
as solubility can change during the course of exposure in the test system
due to presence of cells, S9, serum etc. Insolubility can be detected by
using the unaided eye. The precipitate should not interfere with the scor-
ing.
(iii) Controls. (A) Concurrent positive and negative (solvent or vehi-
cle) controls both with and without metabolic activation should be included
in each experiment. When metabolic activation is used, the positive control
chemical should be the one that requires activation to give a mutagenic
response.
(B) Positive controls should employ a known clastogen at exposure
levels expected to give a reproducible and detectable increase over back-
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ground which demonstrates the sensitivity of the test system. Positive con-
trol concentrations should be chosen so that the effects are clear but do
not immediately reveal the identity of the coded slides to the reader. Exam-
ples of positive-control substances include:
Metabolic activation condition
Absence of exogenous metabolic activation
Presence of exogenous metabolic activation
Chemical
Methyl methanesulfonate
Ethyl methanesulfonate
Ethylnitrosourea
Mitomycin C
4-Nitroquinoline-N-Oxide
Benzo(a)pyrene
Cyclophosphamide
(monohvdrate)
CAS number
66-27-3]
62-50-0
759-73-S
50-07-7
56-57-5:
50-32-8
50-1 8-0:
([6055-1 £
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(C) Other appropriate positive control substances may be used. The
use of chemical class-related positive-control chemicals may be consid-
ered, when available.
(D) Negative controls, consisting of solvent or vehicle alone in the
treatment medium, and treated in the same way as the treatment cultures,
should be included for every harvest time. In addition, untreated controls
should also be used unless there are historical-control data demonstrating
that no deleterious or mutagenic effects are induced by the chosen solvent.
(g) Procedure—(1) Treatment with test substance, (i) Proliferating
cells are treated with the test substance in the presence and absence of
a metabolic-activation system. Treatment of lymphocytes should com-
mence at about 48 hours after mitogenic stimulation.
(ii) Duplicate cultures should be used at each concentration, and are
strongly recommended for negative/solvent control cultures. Where mini-
mal variation between duplicate cultures can be demonstrated (see para-
graphs (i)(13) and (i)(14) of this guideline), from historical data, it may
be acceptable for single cultures to be used at each concentration.
(iii) Gaseous or volatile substances should be tested by appropriate
methods, such as in sealed culture vessels (see paragraphs (i)(15) and
(i)(16) of this guideline).
(2) Culture harvest time. In the first experiment, cells should be
exposed to the test substance both with and without metabolic activation
for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell-
cycle length after the beginning of treatment (see paragraph (i)(12) of this
guideline). If this protocol gives negative results both with and without
activation, an additional experiment without activation should be done,
with continuous treatment until sampling at a time equivalent to about
1.5 normal cell-cycle lengths. Certain chemicals may be more readily de-
tected by treatment/sampling times longer than 1.5 cycle lengths. Negative
results with metabolic activation need to be confirmed on a case-by-case
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basis. In those cases where confirmation of negative results is not consid-
ered necessary, justification should be provided.
(3) Chromosome preparation. Cell cultures should be treated with
Colcemid® or colchicine usually for 1 to 3 hours prior to harvesting. Each
cell culture should be harvested and processed separately for the prepara-
tion of chromosomes. Chromosome preparation involves hypotonic treat-
ment of the cells, fixation and staining.
(4) Analysis, (i) All slides, including those of positive and negative
controls, should be independently coded before microscopic analysis. Since
fixation procedures often result in the breakage of a proportion of meta-
phase cells with loss of chromosomes, the cells scored should therefore
contain a number of centromeres equal to the modal number ±2 for all
cell types. At least 200 well-spread metaphases should be scored per con-
centration and control equally divided amongst the duplicates, if applica-
ble. This number can be reduced when high numbers of aberrations are
observed.
(ii) Though the purpose of the test is to detect structural chromosome
aberrations, it is important to record polyploidy and endoreduplication
when these events are seen.
(h) Data and reporting—(1) Treatment of results, (i) The experi-
mental unit is the cell, and therefore the percentage of cells with structural
chromosome aberration(s) should be evaluated. Different types of struc-
tural chromosome aberrations should be listed with their numbers and fre-
quencies for experimental and control cultures. Gaps are recorded sepa-
rately and reported but generally not included in the total aberration fre-
quency.
(ii) Concurrent measures of cytotoxicity for all treated and negative
control cultures in the main aberration experiment(s) should also be re-
corded.
(iii) Individual culture data should be provided. Additionally, all data
should be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive re-
sponse. Equivocal results should be clarified by further testing preferably
using modification of experimental conditions. The need to confirm nega-
tive results has been discussed in paragraph (g)(2) of this guideline. Modi-
fication of study parameters to extend the range of conditions assessed
should be considered in follow-up experiments. Study parameters that
might be modified include the concentration spacing and the metabolic
activation conditions.
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a concentration-related
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increase or a reproducible increase in the number of cells with chro-
mosome aberrations. Biological relevance of the results should be consid-
ered first. Statistical methods may be used as an aid in evaluating the
test results (see paragraphs (i)(3) and (i)(13) of this guideline). Statistical
significance should not be the only determining factor for a positive re-
sponse.
(ii) An increase in the number of polyploid cells may indicate that
the test substance has the potential to inhibit mitotic processes and to in-
duce numerical chromosome aberrations. An increase in the number of
cells with endoreduplicated chromosomes may indicate that the test sub-
stance has the potential to inhibit cell-cycle progression (see paragraphs
(i)(17) and (i)(18) of this guideline).
(iii) A test substance for which the results do not meet the criteria
in paragraphs (h)(2)(i) and (h)(2)(ii) of this guideline is considered non-
mutagenic in this system.
(iv) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance. Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
(v) Positive results from the in vitro chromosome aberration test indi-
cate that the test substance induces structural chromosome aberrations in
cultured mammalian somatic cells. Negative results indicate that, under
the test conditions, the test substance does not induce chromosome aberra-
tions in cultured mammalian somatic cells.
(3) Test report. The test report should include the following informa-
tion.
(i) Test substance.
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle.
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle,
if known.
(iii) Cells.
(A) Type and source of cells.
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(B) Karyotype features and suitability of the cell type used.
(C) Absence of Mycoplasma, if applicable.
(D) Information on cell-cycle length.
(E) Sex of blood donors, whole blood or separated lymphocytes,
mitogen used.
(F) Number of passages, if applicable.
(G) Methods for maintenance of cell cultures if applicable.
(H) Modal number of chromosomes.
(iv) Test conditions.
(A) Identity of metaphase arresting substance, its concentration and
duration of cell exposure.
(B) Rationale for selection of concentrations and number of cultures
including, e.g., cytotoxicity data and solubility limitations, if available.
(C) Composition of media, CCh concentration if applicable.
(D) Concentration of test substance.
(E) Volume of vehicle and test substance added.
(F) Incubation temperature.
(G) Incubation time.
(H) Duration of treatment.
(I) Cell density at seeding, if appropriate.
(J) Type and composition of metabolic activation system, including
acceptability criteria.
(K) Positive and negative controls.
(L) Methods of slide preparation.
(M) Criteria for scoring aberrations.
(N) Number of metaphases analyzed.
(O) Methods for the measurements of toxicity.
(P) Criteria for considering studies as positive, negative or equivocal.
(v) Results.
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(A) Signs of toxicity, e.g., degree of confluency, cell-cycle data, cell
counts, mitotic index.
(B) Signs of precipitation.
(C) Data on pH and osmolality of the treatment medium, if deter-
mined.
(D) Definition for aberrations, including gaps.
(E) Number of cells with chromosome aberrations and type of chro-
mosome aberrations given separately for each treated and control culture.
(F) Changes in ploidy if seen.
(G) Dose-response relationship, where possible.
(H) Statistical analyses, if any.
(I) Concurrent negative (solvent/vehicle) and positive control data.
(J) Historical negative (solvent/vehicle) and positive control data, with
ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Evans, H.J. Cytological Methods for Detecting Chemical
Mutagens. Chemical Mutagens, Principles and Methods for their Detec-
tion, Vol. 4, Hollaender, A. Ed. Plenum Press, New York and London,
pp. 1-29 (1976).
(2) Ishidate, M. Jr. and Sofuni, T. The In Vitro Chromosomal Aberra-
tion Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture.
Progress in Mutation Research, Vol. 5, Ashby, J. et al., Eds. Elsevier
Science Publishers, Amsterdam-New York-Oxford, pp. 427-432 (1985).
(3) Galloway, S.M. et al. Chromosome aberration and sister chromatid
exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals.
Environmental and Molecular Mutagenesis 10 (suppl. 10), 1-175 (1987).
(4) Scott, D. et al.. Genotoxicity under Extreme Culture Conditions.
A report from ICPEMC Task Group 9. Mutation Research 257, 147-204
(1991).
(5) Morita, T. et al. Clastogenicity of Low pH to Various Cultured
Mammalian Cells. Mutation Research 268, 297-305 (1992).
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(6) Ames, B.N., McCann, J. and Yamasaki, E. Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian Microsome
Mutagenicity Test. Mutation Research 31, 347-364 (1975).
(7) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella
Mutagenicity Test. Mutation Research 113, 173-215 (1983).
(8) Natarajan, A.T. et al. Cytogenetic Effects of Mutagens/Carcino-
gens after Activation in a Microsomal System In Vitro, I. Induction of
Chromosome Aberrations and Sister Chromatid Exchanges by
Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells
in the Presence of Rat-Liver Microsomes. Mutation Research 37, 83-90
(1976).
(9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal
Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Muta-
tion Research 66, 277-290 (1979).
(10) Elliot, B.M. et al. Report of UK Environmental Mutagen Society
Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro
Genotoxicity Assays. Mutagenesis 7, 175-177 (1992).
(11) Matsushima, T. et al. A Safe Substitute for Fob/chlorinated
Biphenyls as an Inducer of Metabolic Activation Systems, de Serres, F.J.,
Fouts, J.R., Bend, J.R. and Philpot, R.M. Eds. In Vitro Metabolic Activa-
tion in Mutagenesis Testing, Elsevier, North-Holland, pp. 85-88 (1976).
(12) Galloway, S.M. et al. Report from Working Group on In Vitro
Tests for Chromosomal Aberrations. Mutation Research 312, 241-261
(1994).
(13) Richardson, C. et al. Analysis of Data from In Vitro Cytogenetic
Assays. Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J.,
Ed. Cambridge University Press, Cambridge, pp. 141-154 (1989).
(14) Soper, K.A. and Galloway S.M. Replicate Flasks are not Nec-
essary for In Vitro Chromosome Aberration Assays in CHO Cells. Muta-
tion Research 312, 139-149 (1994).
(15) Krahn, D.F., Barsky, F.C. and McCooey, K.T. CHO/HGPRT
Mutation Assay: Evaluation of Gases and Volatile Liquids. Tice, R.R.,
Costa, D.L., Schaich, K.M. Eds. Genotoxic Effects of Airborne Agents.
New York, Plenum, pp. 91-103 (1982).
(16) Zamora, P.O. et al. Evaluation of an Exposure System Using
Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens
in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis 5, 795-
801 (1983).
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(17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells dur-
ing alpha-radiation induced G2 arrest. Mutation Research 119, 403-413
(1983).
(18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin - induced
endoreduplication in Chinese hamster cells. Cancer Research 43, 1362-
1364 (1983).
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