United States Prevention, Pesticides EPA712-C-96-321
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Toxicology Test
Guidelines
OPPTS 885.3500
Cell Culture
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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.).
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), internet: http://
fedbbs.access.gpo.gov, 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 885.3500 Cell 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.}.
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 152A-16.
(b) Purpose. Cell culture tests provide information on the ability of
viral pest control agents to infect, replicate in, transform or cause toxicity
in, mammalian cell lines.
(c) Definitions The following definitions apply to this guideline:
Cytopathic effects (CPE) are any host cell damage or injury resulting
from infection of the cell by a virus. These effects can be morphological
or biochemical, and include but are not limited to, cell growth, attachment,
morphology, nucleus size and shape, and cellular processes such as
macromolecular synthesis.
Most infectious form (MIF) is the form or preparation of virus that
gives optimal infection in the susceptible cell culture or organism. For
non-occluded viruses, the MIF is the purified virus or purified tissues ob-
tained from an infected host. For occluded viruses (e.g. baculoviruses,
cytoplasmic polyhedrosis viruses, entomopox viruses) the MIF for cell cul-
ture or injection into an organism is extracellular virus found in cell culture
medium or in infectious hemolymph. The MIF for susceptible insect hosts
for infection by natural routes (feeding) is the viral occlusion body.
Transformation is the detectable modification of a host cell phenotype
induced by the presence of viral nucleic acid. Transformed cells are con-
sidered to be infected by the virus.
Viral infectivity is the ability of viral genes to became established
in a host cell genome, or the ability of viral genes to be expressed in
a host cell (resulting in the production of virus-encoded nucleic acids).
Viral toxicity is the ability of a virus to inflict injury or damage to
a host cell, where infection by, and/or replication of the virus are not nec-
essarily required. Toxicity can also be the ability of non-viral components
of a preparation to inflict injury or damage to a host cell.
(d) Test standards—(1) Substance to be tested. The purest, most
infectious (MIF) of the virus should be used. Preparations of insect viruses
should be free of insect hemolymph, unless it has been determined that
the hemolymph is not toxic to the cell cultures used. The inoculum should
be titered by the most sensitive assay available, and in the most permissive
host system (cell culture or, if not available, host organism). For testing
in the model systems, a minimum of five plaque-forming units (PFU) per
cell is required when a plaque assay for the virus is available, or 7x the
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LD50 units when a plaque assay for the virus is not available. If fewer
units per cell or organism are used, justification/reasoning must be pro-
vided for using the lesser amount.
(2) Cell cultures. The following cells are recommended—one human
line (e.g., WI38), one primary cell type (e.g. foreskin), one primate contin-
uous line (e.g. monkey CV-1 or BSC-1), primary Syrian hamster embryo
(SHE) cells (to provide data for the cell transformation assay, described
under paragraph (d)(5) of this guideline). One other cell line is to be se-
lected to evaluate potential concerns intrinsic to the specific viral pest con-
trol agent, and its intended use. Justification/reasoning must be provided
for the selection of this latter cell line.
(3) Toxicity evaluation. Efficiency-of-plating tests should be per-
formed with each cell line. For each cell line, approximately 200 cells
are plated on each of 30 dishes. At 24 h postplating, 10 dishes per cell
line are exposed to approximately 106 units of the virus. Appropriate verte-
brate cell culture medium is added to 10 different dishes, and, if applica-
ble, 10 dishes per cell line are exposed to invertebrate medium only. At
1 h postexposure, all cultures are fed with the appropriate vertebrate cell
culture medium, and are incubated until control cultures have colonies con-
sisting of at least 25 cells/colony. All cultures are fixed and stained, and
colonies are enumerated.
(4) Infectivity evaluation, (i) Subconfluent cultures (containing ap-
proximately 2 x 105 cells on 25 cm2 dishes) of each cell line are exposed
to >1 x 106 units of the viral pest control agent. Appropriate controls in-
clude subconfluent cultures that receive no treatment, and those that are
exposed to virus-free inoculation medium. At 7 days and at 14 days
postinoculation, cells are to be subcultured.
(ii) Cell cultures are observed daily for 21 days postinoculation for
appearance of CPE.
(iii) Cultures are quantitatively assayed for the virus on days 1, 2,
5, 7, 14, and 21 postinoculation.
(A) Cells (entire culture, or >2 x 105 cells) are to be assayed in trip-
licate for viral antigen and nucleic acid.
(B) Cell culture fluid from replicate cultures is to be assayed for in-
fectious virus, using an appropriate susceptible host model system.
(iv) Assays for fate of input virus and for presence of viral proteins
and nucleic acid.
(A) The enzyme linked immunosorbent assay (ELISA), dot-
immunobinding assay (see paragraph (g)(9) of this guideline, Hawkes et
al., 1982, and Ram et al., 1982), protein blot immunoassay (Western trans-
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fer) (see paragraph (g)(7) of this guideline and Volkman and Goldsmith,
1982) or similar assays are recommended for protein determination.
(B) The dot hybridization procedure (see paragraphs (g)(l), (g)(2),
and (g)(6) of this guideline), Southern hybridization procedure (see para-
graphs (g)(7) and (g)(8) of this guideline) or other similar assay are rec-
ommended for protein determination.
(v) To serve as controls, for each cell culture, cells inoculated with
a preparation of the inactivated test virus should be analyzed as described
for the active test virus, and for each series of tests, the inoculum should
be tested in the permissive cell line or host organism as a positive control
and for direct reference to the data obtained from the vertebrate cell lines.
(5) Cell transformation assay, (i) The ability of the viral pest control
agent to transform primary Syrian hamster embryo (SHE) cells is to be
determined, using an appropriate assay system. If other test systems are
used, justification/rationale must be presented to show that the alternate
systems are appropriate.
(ii) Transformation of SHE cells with Simian adenovirus 7 (SAV 7)
serves as the positive control. SHE cells treated with cell culture medium
alone, and SHE cells treated with a killed preparation of the inactivated
viral pest control agent serve as appropriate negative controls. The inac-
tivation procedure must be demonstrated as effective in preventing trans-
formation. An efficiency of plating test with SHE cells is considered an
appropriate toxicity control.
(iii) If the data show that the test virus modifies the cell phenotype,
cells from cultures derived from morphologically transformed colonies are
to be inoculated into hamsters, and tumorigenesis in the host animal is
to be evaluated.
(iv) This assay may not be required if, in the infectivity evaluations
(under paragraph (d)(4) of this guideline), it is conclusively demonstrated
that viral nucleic acid is not persistent in any of the test cell lines em-
ployed.
(e) Data reporting and evaluation. The following information
should be provided for each test:
(1) CPE in the cell monolayers. (i) The appearance of CPE should
be described in such a way that virus-induced cell destruction is differen-
tiated from nonspecific effects.
(ii) Cultures should be inspected with the aid of a microscope to pro-
vide evidence of CPE that should be recorded as:
(A) 1+ = suggestive of virus-induced morphologic changes.
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(B) 2+ = definitive morphologic changes.
(C) 3+ = more than 50 percent cell degeneration.
(D) 4+ = complete cell destruction.
(iii) The TCID50 value calculated by an appropriate statistical meth-
od. For computation of the infectivity results, only cultures showing a
>2+ CPE are considered to be infected.
(2) Toxicity evaluation, (i) Details of all procedures used, including
appropriate reagents and materials, and assay sensitivities and limitations.
(ii) Efficiency of plating data of cultures receiving virus, and cultures
receiving vertebrate media (control cultures) and invertebrate media.
(iii) Assessment of mitotic process prevention or of interference with
chromosomal replication, as indicated for example, by significant reduc-
tions in efficiency of plating.
(3) Assay of culture fluid. Details of procedures used, including a
discussion of all data that indicate viral replication.
(4) Data from assays of input viruses, (i) Details of procedures used
for detection of viral antigens and nucleic acids and their persistence in
culture, including appropriate reagents and materials, and assay sensitivi-
ties and limitations.
(ii) Intracellular concentration of viral antigens and viral nucleic
acids, reported as a function of cell number (e.g., viral genome number/
cell).
(5) Cell transformation assay, (i) Details of the protocols used for
the cell transformation assay, and reference to the assay used, if published.
(ii) Control value data, including deficiency of plating results.
(iii) Tumorigenesis data in test animals, if this study is required.
(6) General information to be provided for all tests, (i) The source
of each cell line used.
(ii) Evidence for lack of adventitious agents in cell lines.
(iii) Information on genetic stability of continuous cell lines, and on
donors of primary cells.
(f) Tier progression. The requirement for further studies as indicated
by the data from these studies also will be based on data from the other
test requirements in this tier.
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(1) If the data show that the viral pest control agent is not cytotoxic,
nor does it infect, replicate in, or transform any cell culture, no further
testing is required.
(2) If the data show that the viral pest control agent preparation is
toxic to any of the test cell cultures, but does not infect, replicate in, or
transform any of the cell cultures:
(i) The toxic components of the preparation may have to be identified.
(ii) An acute toxicity study may be required with the toxic compo-
nents.
(3) If the viral pest control agent infects any of the test cell cultures,
reproductive and fertility effects, oncogenicity, immunodeficiency, and pri-
mate infectivity/pathogenicity studies may be required.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Bishop, D.H.L. The application of RNA finger printing and se-
quencing to viral diagnosis. Current Topics in Microbiology and
Immunolology 104:259-271 (1983).
(2) Brandsma, J. and G. Miller. Nucleic acid spot hybridization: rapid
quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA.
Proceedings of the National Academy of Science 77:6851-6855 (1980).
(3) Casto, B.C. Adenovirus transformation of hamster embryo cells.
Journal of Virology 2:376-383 (1968).
(4) Hames, B.D. and S.J. Higgens. (Eds.) Nucleic acid hybridization.
A practical approach. IRL Press, Washington, DC (ISBN 0-947946-23-
3) (1985).
(5) Heidelberberger 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).
(6) Kafatos, F.C., et al., Determination of nucleic acid sequence
homologies and relative concentrations by a dot hybridization procedure.
Nucleic Acids Research 7:1541-1552 (1979).
(7) Smith, G. and M.D. Summers, Application of a novel
radioimmunoassay to identify baculovirus structural proteins that share
interspecies antigenic determinants. Journal of Virology 39:125-137
(1981).
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(8) Southern, E., Detection of specific sequences among DNA frag-
ments separated by gel electrophoresis. Journal of Molecular Biology
98:503 (1975).
(9) Tijssen, P., Laboratory techniques in biochemistry and molecular
biology. Practice and theory of enzyme immunoassays. Elsevier (ISBN 0-
444-80633-4) (1985).
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