United States      Prevention, Pesticides     EPA712-C-98-223
          Environmental Protection    and Toxic Substances     August 1998
          Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.5375
          In Vitro Mammalian
          Chromosome Aberration

     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.).

     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

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

    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.

     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

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.

     (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-

     (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

     (B)  Positive  controls  should employ a known  clastogen at exposure
levels expected to give  a reproducible and detectable increase over back-

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
Methyl methanesulfonate 	
Ethyl methanesulfonate
Mitomycin C 	
CAS number

50-1 8-0:
([6055-1 £
     (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

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

     (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-

     (ii) Concurrent measures of cytotoxicity for all  treated and negative
control  cultures  in the main aberration experiment(s) should also be re-

     (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

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-

     (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-

     (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.

     (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.


     (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-

     (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

     (5) Morita,  T. et al.  Clastogenicity of Low pH  to Various  Cultured
Mammalian Cells. Mutation Research 268, 297-305 (1992).

    (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

    (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

    (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).


    (17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells dur-
ing alpha-radiation induced G2 arrest.  Mutation Research 119, 403-413

    (18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin - induced
endoreduplication in  Chinese  hamster cells. Cancer Research 43, 1362-
1364 (1983).