[Code of Federal Regulations]
[Title 40, Volume 30]
[Revised as of July 1, 2004]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5375]
[Page 195-197]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart F_Genetic Toxicity
Sec. 798.5375 In vitro mammalian cytogenetics.
(a) Purpose. The in vitro cytogenetics test is a mutagenicity test
system 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
analyse 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.
(b) Definitions. (1) Chromosome-type aberrations are changes which
result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of
single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. In vitro cytogenetics assays may
employ cultures of established cell lines, cell strains or primary cell
cultures. 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 (c-metaphase). 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
harvested at various intervals after treatment. Prior to harvesting,
cells are treated with a spindle inhibitor (e.g., colchicine or
Colcemid) to accumulate cells in c-metaphase. 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 for Mycoplasma contamination and may be periodically checked for
karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and
incubation conditions (culture vessels CO2 concentrations,
temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall 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 shall be included in
each experiment. When metabolic activation is used, the positive control
substance shall 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 shall not
interfere with cell viability or growth rate. Treatment vessels should
be chosen to ensure that there is no visible interaction, such as
etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. Multiple concentrations of the test
substance over a range adequate to define the response should be tested.
Generally the highest test substance concentrations tested with and
without metabolic activation should show evidence of cytotoxicity or
reduced mitotic 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
[deg]C.
[[Page 196]]
(2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose
whole blood should be added to culture medium containing a mitogen,
e.g., phytohemagglutinin (PHA) and incubated at 37 [deg]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 shall 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 shall be
used for each experimental point.
(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. Alternatively, a single treatment may be followed by multiple
harvest times. Harvest time should be extended for those chemicals which
induce an apparent 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 shall be treated with a spindle inhibitor,
(e.g., colchicine or Colcemid[reg]), 1 or 2 hours prior to harvesting.
Each culture shall 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 shall be coded before analysis. In human
lymphocytes, only cells containing 46 centromeres shall be analyzed. In
established cell lines and strains, only metaphases containing 2 centromeres of the modal number shall be analyzed.
Uniform criteria for scoring aberrations shall be used.
(8) Confirmatory tests. When appropriate, a single positive response
shall be confirmed by testing over a narrow range of concentrations.
(f) Data and report--(1) Treatment of results. Data shall be
presented in a tabular form. Different types of structural chromosomal
aberrations shall 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 included 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
determining a positive result, one of which is a statistically
significant dose-related increase in the number of structural
chromosomal aberrations. Another criterion may be based upon detection
of a reproducible and statistically significant positive response for at
least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of structural
chromosomal aberrations or a statistically significant and reproducible
positive response at any one of the test points is considered
nonmutagenic 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
cytogenetics assay indicate that under the test conditions the test
substance induces chromosomal aberrations in cultured mammalian somatic
cells.
[[Page 197]]
(ii) Negative results indicate that under the test conditions the
test substance does not induce chromosomal aberrations 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
information shall be reported:
(i) Cells used, density and passage number 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
selection of the concentrations used in the assay, duration of
treatment.
(iv) Details of both the protocol used to prepare the metabolic
activation 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
examination.
(ix) Number of metaphases analysed.
(x) Mitotic index where applicable.
(xi) Criteria for scoring aberrations.
(xii) Type and number of aberrations, given separately for each
treated 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. For additional background information on this test
guideline the following references should be consulted.
(1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting
carcinogens and mutagens with the Salmonella/ 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 (New York, London: Plenum Press,
1976) pp. 1-29.
(3) Howard, P.N., Bloom, A.D., Krooth, R.S. ``Chromosomal
aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine in mammalian
cells,'' In Vitro 7:359-365 (1972).
(4) Ishidate, M. Jr., 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., Au, W., Bender, M.A., Brewen, J.G., Carrano,
A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., ``Mammalian in
vivo and in vitro cytogenetic assays: A report of the Gene-tox
Program,'' Mutation Research, 87:143-188 (1981).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]