United States Prevention, Pesticides EPA712-C-96-260
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8800
Morphologic
Transformation of Cells
in Culture
'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 870.8800 Morphologic transformation of cells in culture.
(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 795.285 Morphologic
Transformation of Cells in Culture.
(b) Purpose. In vitro assays for cellular transformation are
semiquantitative assays for the ability of chemical agents to transform
(morphologically alter) cells in culture. Such transformation is associated
with certain phenotypic changes such as loss of contact inhibition and the
ability to form colonies in soft agar medium. The process by which these
changes occur is assumed to be closely related to the process of in vivo
carcinogenesis. Morphologically transformed cells appear as foci of dense,
piled-up, altered cells on an underlying monolayer of normal cells. Three
types of foci have been recognized. Type III foci appear to be most closely
correlated with in vivo tumor formation. The ultimate criterion for mor-
phologic transformation is the ability of the transformed cells to induce
tumors when inoculated into appropriate hosts. Not all cells which appear
to be morphologically transformed are capable of tumor formation. In gen-
eral, there is reasonably good correlation between in vitro transformation
and in vivo oncogenesis, although the correlation varies depending on the
system being studied. These systems are believed to be reasonably good
predictors of in vivo activity, and positive results are viewed as potential
indications of in vivo carcinogenesis.
(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.
Morphologic transformation is the acquisition of certain phenotypic
characteristics, most notably loss of contact inhibition and loss of anchor-
age dependence, which are often but not always associated with the ability
to induce tumors in appropriate hosts.
Type III foci of transformed cells are multilayered aggregations of
densely staining cells with random orientation and criss-cross arrays at
the periphery of the aggregate. They appear as dark stained areas on a
light staining background monolayer which is one-cell thick.
(d) Test method—(1) Principle, (i) Three systems for detecting
chemically induced morphologic transformation have been described—sys-
tems which employ cell lines (cells with an indefinite lifespan), systems
which employ cell strains (cells with a finite or limited lifespan), and sys-
tems which detect the interaction between chemicals and oncogenic vi-
ruses.
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(ii) This study should employ an established cell line for detection
of morphologic transformaton.
(2) Description. Cells in culture are exposed to the test substance,
both with and without metabolic activation, for a defined period of time.
Cytotoxicity is determined by measuring the colony-forming ability and
growth rate of the cultures after the treatment period. At the end of the
treatment period, cultures are maintained in growth medium for a sufficient
period of time to allow near-optimal expression of transformed foci.
(3) Cells, (i) Balb/c-3T3 mouse cells originally obtained from clone
A-31 or its derivatives should be used in the assay. Cells should be
checked for mycoplasm contamination prior to use in the assay and may
be checked for karyotype.
(ii) Appropriate culture media and incubation conditions (culture ves-
sels, CO2 concentrations, temperature, and humidity) should be used.
(4) Metabolic activation. Cells should be exposed to test substance
both in the presence and absence of a metabolic activation system. The
metabolic activation system should be derived from primary cultures of
rat hepatocytes.
(5) Control groups. Positive and negative (untreated and vehicle)
controls should be included in each experiment. 3-Methylcholanthrene is
an example of a positive control for experiments without metabolic activa-
tion. Dimethylnitrosamine is an example of a positive control in experi-
ments with metabolic activation.
(6) Test chemicals—(i) Vehicle. Test agents should be dissolved in
serum-complete culture medium prior to treatment of the cells.
(ii) Exposure concentrations. Several concentrations (usually at least
four) of the test substance should be used. These should be selected on
the basis of a preliminary cytotoxicity assay performed both with and with-
out metabolic activation. The highest concentration should produce a low
level of survival (approximately 10 to 20 percent), and the survival in
the lowest concentration should approximate that of the negative control.
(e) Test performance. (1) Cells should be exposed to the test sub-
stance both with and without metabolic activation. Exposure should be
for 72 h for experiments without metabolic activation and for 48 h for
experiments with metabolic activation unless different exposure times are
justified by the investigator.
(2) At the end of the exposure period, cells should be washed and
cultured to determine viability and to allow for expression of trans-
formation.
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(3) At the end of the incubation period (generally 4 to 6 weeks),
cells should be fixed and stained, and the number of transformed (Type
III) foci should be enumerated.
(4) All results should be confirmed in an independent experiment if
a single, statistically significant positive effect is produced at one dose
point without a dose response. A positive response should be confirmed
by testing over a narrow range of concentrations.
(5) Tumorigenic potential of isolated morphologically transformed
foci may be determined by inoculation into suitable hosts.
(f) Data and report—(1) Treatment of results, (i) Data should be
presented in tabular form. Individual colony counts for the treated and
control groups should be presented for both transformation and survival.
(ii) Survival and cloning efficiencies should be given as a percentage
of the controls. Transformation should be expressed as a number of foci
per dish, the number of dishes with transformed foci, and the number of
transformed foci per number of surviving cells.
(2) Interpretation of results, (i) There are several criteria for deter-
mining a positive result, one of which is a statistically significant con-
centration-related increase in the number of transformed foci. Another cri-
terion may be based upon the detection of a reproducible and statistically
significant positive response for at least one of the test substance con-
centrations.
(ii) A test substance which does not produce either a statistically sig-
nificant concentration-related increase in the number of transformed foci
or a statistically significant and reproducible positive response at any one
of the test points is considered to be negative in this system.
(iii) Both biological and statistical significance should be considered
together in the evaluation.
(3) Test evaluation, (i) Positive results for an in vitro mammalian
cell transformation assay indicate that, under the test conditions, the test
substance induces morphologic transformation in the cultured mammalian
cells used.
(ii) Negative results indicate that, under the test conditions, the test
substance does not induce morphologic transformation in the cultured
mammalian cells used.
(4) 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:
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(i) Cell type used, including subclone designation and passage num-
ber; number of cell cultures; methods used for maintenance of cell cul-
tures.
(ii) Rationale for selection of concentrations and number of cultures.
(iii) Test conditions: Composition of media, CCh concentration, con-
centration of test substance, vehicle, incubation temperature, incubation
time, duration of treatment, cell density during treatment, type of metabolic
activation system, positive and negative controls, length of expression pe-
riod (including number of cells seeded and subculture and feeding sched-
ules, if appropriate).
(iv) Methods used to enumerate numbers of viable cells and trans-
formed foci.
(v) Dose-response relationship, where possible.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Heidelberger, C. et al. Cell transformation by chemical agents—
a review and analysis of the literature: a report of the U.S. Environmental
Protection Agency Gene-Tox Program. Mutation Research 114:283-385
(1983).
(2) Kakunaga, T. A quantitative system for assay of malignant trans-
formation by carcinogens using a clone derived from Balb-3T3. Inter-
national Journal of Cancer 12:463-473 (1973).
(3) Reznikoff, C.A. et al. Quantitative and qualitative studies of chem-
ical transformation of cloned C3H mouse embryo cells sensitive to
postconfluence inhibition of cell division. Cancer Research 33:3239-3249
(1973).
(4) Reznikoff, C.A. et al. Establishment and characterization of a
cloned line of C3H mouse embryo cells sensitive to postconfluence inhibi-
tion of division. Cancer Research 33:3231-3238 (1973).
(5) Sivak, A. et al. Balb/c-3T3 cells as target cells for chemically
induced neoplastic transformation. In: Advances in modern environmental
toxicology, mammalian cell transformation by chemical carcinogens, Vol.
I. Mishra, N., Dunkel, V., Mehlman, M., eds. Senate Press, Princeton Junc-
tion, NJ. pp. 133-180(1981).
(6) Sivak, A. and Tu, A.S. Factors influencing neoplastic trans-
formation by chemical carcinogens in Balb/c-3T3 cells. In: The predictive
value of short-term screening tests in carcinogenicity evaluation. Williams,
G.M., Kroes, R., Waaijers, H.W., Van de Poll, K.W., eds. Elsevier/North
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Holland Biomedical Press, Amsterdam, New York, Oxford pp. 177-190
(1980).
(7) Williams, G.M. Detection of chemical carcinogens by unsched-
uled DNA synthesis in rat liver primary cell culture. Cancer Research
37:1845-1851, (1977).
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