Section

[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

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.

(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

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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.

(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.

[[Page 197]]

(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.

(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]