United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–230

June 1996

Health Effects Test

Guidelines

OPPTS 870.5550

Unscheduled DNA

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 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 870.5550 Unscheduled DNA synthesis in mammalian 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 materials used in developing this harmonized 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 Synthesis 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 incorporation 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 limited to, 7,12-dimethylbenzanthracene, 2-acetylaminofluorene, 4-nitroquino

line oxide or N-dimethylnitrosamine.

(e) Test method—(1) Principle. Mammalian cells in culture, either

primary cultures of rodent hepatocytes or established cell lines, are exposed 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 containing 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 exposure period, cells are stained and labeled nuclei are counted either manually 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 metabolic 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 contamination and may be periodically checked for karyotype stability.

(ii) Cell growth and maintenance. Appropriate culture media and

incubation conditions (culture vessels CO2 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 appropriate 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 concentration of the vehicle should not interfere with cell viability or growth

rate.

(ii) Exposure concentrations. Multiple concentrations of test substance, 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 medium 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 3HTdR incorporation.

(3) Acceptable background frequencies—(i) Autoradiographic determinations. 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 concentration 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 subtracted to give the correct nuclear grain count. Values should be reported

as net grains per nucleus. Mean, median, and mode may be used to describe the distribution of net grains per nucleus.

(ii) LSC determinations. For LSC determinations, 3 H-TdR incorporation 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 determining a positive result, one of which is a statistically significant doserelated 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 significant 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 information should 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 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 determination 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 additional background material on this test guideline.

(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens

with the Salmonella/mammalian-microsome mutagenicity test, Mutation

Research 31:347–364 (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 carcinogenic 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 Letters 1:231–236 (1976).

(5) Williams, G.M. Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Research

37:1845–1851 (1977).