United States       Prevention, Pesticides     EPA712-C-96-230
          Environmental Protection    and Toxic Substances     June 1996
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.5550
          Unscheduled DMA
          Synthesis in Mammalian
          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 870.5550   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

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

     (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

     (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

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

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

     (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.


     (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

     (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.

     (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