United States      Prevention, Pesticides     EPA712-C-96-260
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.8800
          Transformation of Cells
          in Culture
                'Public Draft"

     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.).

     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:

     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,  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

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-

     (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-

     (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-

     (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:

     (i) Cell type used, including subclone designation and passage num-
ber;  number of cell  cultures; methods used for maintenance of cell cul-

     (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

     (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

     (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

Holland Biomedical Press, Amsterdam, New York, Oxford pp. 177-190

    (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).