United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–260

June 1996

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized 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 morphologic 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 general, 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 anchorage 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—systems which employ cell lines (cells with an indefinite lifespan), systems

which employ cell strains (cells with a finite or limited lifespan), and systems which detect the interaction between chemicals and oncogenic viruses.
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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 vessels, 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 activation. Dimethylnitrosamine is an example of a positive control in experiments 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 without 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 substance 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 transformation.
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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 determining a positive result, one of which is a statistically significant concentration-related increase in the number of transformed foci. Another criterion may be based upon the 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 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 information should be reported:
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(i) Cell type used, including subclone designation and passage number; number of cell cultures; methods used for maintenance of cell cultures.

(ii) Rationale for selection of concentrations and number of cultures.

(iii) Test conditions: Composition of media, CO2 concentration, concentration 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 period (including number of cells seeded and subculture and feeding schedules, if appropriate).

(iv) Methods used to enumerate numbers of viable cells and transformed foci.

(v) Dose-response relationship, where possible.

(g) References. The following references should be consulted for additional 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 transformation by carcinogens using a clone derived from Balb-3T3. International Journal of Cancer 12:463–473 (1973).

(3) Reznikoff, C.A. et al. Quantitative and qualitative studies of chemical 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 inhibition 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 Junction, NJ. pp. 133–180 (1981).

(6) Sivak, A. and Tu, A.S. Factors influencing neoplastic transformation 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 unscheduled DNA synthesis in rat liver primary cell culture. Cancer Research

37:1845–1851, (1977).