United States Prevention, Pesticides EPA712-C-96-230
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5550
Unscheduled DMA
Synthesis in Mammalian
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 870.5550 Unscheduled DMA synthesis in mammalian 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 materials used in developing this har-
monized OPPTS test guideline are OPPT 40 CFR 798.5550 Unscheduled
DNA Synthesis in Mammalian Cells in Culture and OECD guideline 482
Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Syn-
thesis in Mammalian Cells in Vitro.
(b) Purpose. Unscheduled DNA synthesis (UDS) in mammalian cells
in culture measures the repair of DNA damage induced by a variety of
agents including chemicals, radiation and viruses. UDS may be measured
in both in vitro and in vivo systems.
(c) Definition. 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 definition also applies to this test guideline.
Unscheduled DNA synthesis in mammalian cells in culture is the in-
corporation of tritium-labeled thymidine (3 H-TdR) into the DNA of cells
which are not in the S phase of the cell cycle.
(d) Reference substances. These may include, but need not be lim-
ited to, 7,12-dimethylbenzanthracene, 2-acetylaminofluorene, 4-nitroquino
line oxide or A^-dimethylnitrosamine.
(e) Test method—(1) Principle. Mammalian cells in culture, either
primary cultures of rodent hepatocytes or established cell lines, are ex-
posed to the test agent. Established cell lines are treated both with and
without metabolic activation. UDS is measured by the uptake of 3 H-TdR
into the DNA of non-S phase cells. Uptake may be determined by
autoradiography or by liquid scintillation counting (LSC) of DNA from
treated cells.
(2) Description—(i) Autoradiography. For autoradiography,
coverslip cultures of cells are exposed to test chemical in medium contain-
ing 3 H-TdR. At the end of the treatment period, cells are fixed, dipped
in autoradiographic emulsion, and exposed at 4 °C. At the end of the expo-
sure period, cells are stained and labeled nuclei are counted either manu-
ally or with an electronic counter. Established cell lines should be treated
both with and without metabolic activation.
(ii) LSC determinations. For LSC determinations of UDS, confluent
cultures of cells are treated with test chemical both with and without meta-
bolic activation. At the end of the exposure period, DNA is extracted from
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the treated cells. Total DNA content is determined and extent of 3H-TdR
incorporation is determined by scintillation counting.
(3) Cells—(i) Type of cells used in the assay. (A) A variety of cell
lines or primary cell cultures, including human cells, may be used in the
assay.
(B) Established cell lines should be checked for Mycoplasma contami-
nation and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media and
incubation conditions (culture vessels CCh concentration), temperature,
and humidity should be used.
(4) Metabolic activation, (i) A metabolic activation system is not
used with primary cultures of rodent hepatocytes.
(ii) Established cell lines should be exposed to test substance both
in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Concurrent positive and negative (untreated and/
or vehicle) controls both with and without metabolic activation as appro-
priate should be included in each experiment.
(6) Test chemicals—(i) Vehicle. Test chemicals and positive control
reference substances may be prepared in culture media or dissolved or
suspended in appropriate vehicles prior to treatment of the cells. Final con-
centration of the vehicle should not interfere with cell viability or growth
rate.
(ii) Exposure concentrations. Multiple concentrations of test sub-
stance, based upon cytotoxicity and over a range adequate to define the
response, should be used. For cytotoxic chemicals, the first dose to elicit
a cytotoxic response in a preliminary assay should be the highest dose
tested. Relatively insoluble compounds should be tested up to the limits
of solubility. For freely soluble nontoxic chemicals, the upper test chemical
concentration should be determined on a case by case basis.
(f) Test performance—(1) Primary cultures of rodent hepatocytes.
Freshly isolated rodent hepatocytes should be treated with chemical in me-
dium containing 3H-TdR. At the end of the treatment period, cells should
be drained of medium, rinsed, fixed, dried and attached to microscope
slides. Slides should be dipped in autoradiographic emulsion, exposed at
4 °C for an appropriate length of time, developed, stained and counted.
(2) Established cell lines—(i) Autoradiographic techniques. The
techniques for treatment of established cell lines are the same as those
for primary cultures of rodent hepatocytes except that cells must not enter
S phase prior to treatment. Entry of cells into S phase may be blocked
by several methods (e.g. by growth in medium deficient in arginine or
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low in serum or by treatment with chemical agents such as hydroxyurea).
Tests should be done both in the presence and absence of a metabolic
activation system.
(ii) LSC measurement of UDS. Prior to treatment with test agent,
entry of cells into S phase should be blocked as described in paragraph
(f)(2)(i) of this guideline. Cells should be exposed to the test chemical
in medium containing 3H-TdR. At the end of the incubation period, DNA
should be extracted from the cells by hydrolysis with perchloroacetic acid
or by other acceptable methods. One aliquot of DNA is used to determine
total DNA content; a second aliquot is used to measure the extent of 3H-
TdR incorporation.
(3) Acceptable background frequencies—(i) Autoradiographic de-
terminations. Net incorporation of 3HTdR into the nucleus of solvent
treated control cultures should be less than 1.
(ii) LSC determinations. Historical background incorporation rates
of 3H-TdR into untreated established cell lines should be established for
each laboratory.
(4) Number of cells counted. A minimum of 50 cells per culture
should be counted for autoradiographic UDS determinations. Slides should
be coded before being counted. Several widely separated random fields
should be counted on each slide. Cytoplasm adjacent to the nuclear areas
should be counted to determine spontaneous background.
(5) Number of cultures. Six independent cultures at each concentra-
tion and control should be used in LSC UDS determinations.
(g) Data and report—(1) Treatment of results—(i)
Autoradiographic determinations. For autoradiographic determinations,
once untransformed data are recorded, background counts should be sub-
tracted to give the correct nuclear grain count. Values should be reported
as net grains per nucleus. Mean, median, and mode may be used to de-
scribe the distribution of net grains per nucleus.
(ii) LSC determinations. For LSC determinations, 3 H-TdR incorpo-
ration should be reported as disintegrations per minute per microgram of
DNA. Average disintegrations per minute per microgram of DNA with
standard deviation or standard error of the mean may be used to describe
distribution of incorporation in these studies.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results, (i) There are several criteria for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the incorporation of 3H-TdR into treated cells. Another
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criterion may be based upon detection of a reproducible and statistically
significant positive response for a least one of the test points.
(ii) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the incorporation of 3H-TdR into treated
cells or a statistically significant and reproducible positive response at any
one of the test points is considered not to induce UDS in the test system.
(iii) Both biological and statistical significance should be considered
together in the evaluation.
(4) Test evaluation, (i) Positive results in the UDS assay indicate
that under the test conditions the test substance may induce DNA damage
in cultured mammalian somatic cells.
(ii) Negative results indicate that under the test conditions the test
substance does not induce DNA damage 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 informa-
tion should be reported:
(i) Cells used, density and passage number at time of treatment, num-
ber of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium,
temperature, and CCh concentration.
(iii) Test chemical vehicle, concentration and rationale for selection
of concentrations used in the assay.
(iv) Details of both the protocol used preparation of the metabolic
activation system and its use in the assay.
(v) Treatment protocol.
(vi) Positive and negative controls.
(vii) Protocol used for autoradiography.
(viii) Details of the method used to block entry of cells into S phase.
(ix) Details of the methods used for DNA extraction and determina-
tion of total DNA content in LSC determinations.
(x) Historical background incorporation rates of 3H-TdR in untreated
cell lines.
(xi) Dose-response relationship, if applicable.
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(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens
with the &z/mo«e//a/mammalian-microsome mutagenicity test, Mutation
Research 31:34''-364 (1975).
(2) Rasmussen, R.E. and Painter, R.B. Radiation-stimulated DNA
synthesis in cultured mammalian cells. Journal of Cell Biology 29:11-19
(1966).
(3) Stich, H.F. et al. DNA fragmentation and DNA repair as an in
vitro and in vivo assay for chemical procarcinogens, carcinogens and car-
cinogenic nitrosation products, Screening tests in chemical carcinogenesis.
Eds. Bartsch, H., Tomatis, L. IARC Scientific, Lyon, No. 12 (1976) pp.
617-636.
(4) Williams, G.M. Carcinogen-induced DNA repair in primary rat
liver cell cultures: a possible screen for chemical carcinogens. Cancer Let-
ters 1:231-236(1976).
(5) Williams, G.M. Detection of chemical carcinogens by unsched-
uled DNA synthesis in rat liver primary cell cultures. Cancer Research
37:1845-1851(1977).
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