.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA712–C–96–223 June 1996 

# Health Effects Test Guidelines OPPTS 870.5375 In Vitro Mammalian Cytogenetics

‘‘Public Draft’’

### i 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 theNational 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 proceduresthat 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 theEPA 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.’’

### 1 OPPTS 870.5375 In vitro mammalian cytogenetics. (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 material used in developing this harmonized OPPTS test guideline is OPPT 40 CFR 798.5375 In Vitro Mammalian Cytogenetics and OECD 473 Genetic Toxicology: In Vitro Mammalian Cytogenetic Test. (b) Purpose. The in vitro cytogenetics test is a mutagenicity test system for the detection of chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations may be either structural or numerical. However, because cytogenetic assays are usually designed to analyze cells at their first post-treatment mitosis and numerical aberrations require at least one cell division to be visualized, this type of aberration is generally not observed in a routine cytogenetics assay. Structural aberrations may be of two types, chromosome or chromatid. (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. Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids. Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time. (d) Test method—(1) Principle. In vitro cytogenetics assays mayemploy cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance both with and without metabolic activation. Following exposure of cell cultures to test substances, they are treated with a spindle inhibitor (e.g., colchicine or Colcemid  ) to arrest cells in a metaphase-like stage of mitosis (c -metaphase). Cells are then harvested and chromosome preparations made. Preparations are stained and metaphase cells are analyzed for chromosomal aberrations. (2) Description. Cell cultures are exposed to test compounds and harvested at various intervals after treatment. Prior to harvesting, cells are treated with a spindle inhibitor (e.g., colchicine or Colcemid  ) to accumulate cells in c -metaphase. Chromosome preparations from cells are made, stained and scored for chromosomal aberrations. (3) Cells—(i) Type of cells used in the assay. There are a variety of cell lines or primary cell cultures, including human cells, which may be used in the assay. Established cell lines and strains should be checked

### 2 for Mycoplasma contamination and may be periodically checked for karyotype stability. (ii) Cell growth and maintenance. Appropriate culture media, and incubation conditions (culture vessels CO 2 concentrations, temperature and humidity) should be used. (4) Metabolic activation. Cells should be exposed to test substance both in the presence and absence of an appropriate metabolic activation system. (5) Control groups. Positive and negative (untreated and/or vehicle) controls both with and without metabolic activation should be included in each experiment. When metabolic activation is used, the positive control substance should be known to require such activation. (6) Test chemicals—(i) Vehicle. Test 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. Treatment vessels should bechosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel. (ii) Exposure concentrations. Multiple concentrations of the test substance over a range adequate to define the response should be tested. Generally the highest test substance concentrations tested with and withoutmetabolic activation should show evidence of cytotoxicity or reduced mitotic activity. Relatively insoluble substances should be tested up to the limit of solubility. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis. (e) Test performance—(1) Established cell lines and strains. Prior to use in the assay, cells should be generated from stock cultures, seeded in culture vessels at the appropriate density and incubated at 37 ° C. (2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose whole blood should be added to culture medium containing a mitogen, e.g., phytohemagglutinin (PHA) and incubated at 37 ° C. White cells sedimented by gravity (buffy coat) or lymphocytes which have been purified on a density gradient may also be utilized. (3) Treatment with test substance. For established cell lines and strains, cells in the exponential phase of growth should be treated with test substances in the presence and absence of an exogenous metabolic activation system. Mitogen-stimulated human lymphocyte cultures may be treated with the test substance in a similar manner. (4) Number of cultures. At least two independent cultures should be used for each experimental point.

### 3 (5) Culture harvest time. (i) For established cell lines and strains, multiple harvest times are recommended. However, for screening purposes, a single harvest time may be appropriate. If the test chemical changes the cell cycle length, the fixation intervals should be changed accordingly. If a single harvest time is selected, supporting data for the harvest time should be presented in such a study. (ii) For human lymphocyte cultures, the substance to be tested may be added to the cultures at various times after mitogen stimulation so thatthere is a single harvest time after the initiation of the cell culture. Alternatively, a single treatment may be followed by multiple harvest times.Harvest time should be extended for those chemicals which induce an apparent cell cycle delay. Because the population of human lymphocytes is only partially synchronized, a single treatment, at, or close to, the time when metaphase stages first appear in the culture will include cells in all phases of the division cycle. Therefore, a single harvest at the time of second mitosis may be carried out for screening purposes.(iii) Cell cultures should be treated with a spindle inhibitor, (e.g., colchicine or Colcemid  ), 1 or 2 h prior to harvesting. Each culture should be harvested and processed separately for the preparation of chromosomes. (6) Chromosome preparation. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining. (7) Analysis. Slides should be coded before analysis. In human lymphocytes, only cells containing 46 centromeres should be analyzed. In established cell lines and strains, only metaphases containing ±2 centromeres of the modal number should be analyzed. Uniform criteria for scoring aberrations should be used. (8) Confirmatory tests. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations. (f) Data and report—(1) Treatment of results. Data should be presented in a tabular form. Different types of structural chromosomal aberrations should be listed with their numbers and frequencies for experimental and control groups. Data should be evaluated by appropriate statisticalmethods. Gaps or achromatic lesions are recorded separately and not included in the total aberration frequency. (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 number of structural chromosomal aberrations. Another criterion may be based upon detection of a reproducible and statis-

### 4 tically significant positive response for at least one of the test substance concentrations.(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of structural chromosomal aberrations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.(iii) Biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (i) Positive results in the in vitro cytogenetics assay indicate that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells. (ii) Negative results indicate that under the test conditions the testsubstance does not induce chromosomal aberrations in cultured mammalian somatic cells. (5) Test report. In addition to the reporting recommendations asspecified 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, and number of cell cultures. (ii) Methods used for maintenance of cell cultures including medium, temperature and CO 2 concentration.(iii) Test chemical vehicle, concentration and rationale for the selection of the concentrations used in the assay, and duration of treatment.(iv) Details of both the protocol used to prepare the metabolic activation system and of its use in the assay. (v) Identity of spindle inhibitor, its concentration, and duration of treatment. (vi) Date of cell harvest. (vii) Positive and negative controls.(viii) Methods used for preparation of slides for microscopic examination. (ix) Number of metaphases analyzed. (x) Mitotic index where applicable. (xi) Criteria for scoring aberrations.

### 5(xii) Type and number of aberrations, given separately for each treated and control culture, total number of aberrations per group; frequency distribution of number of chromosomes in established cell lines and strains. (xiii) Dose-response relationship, if applicable. (g) 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) Evans, H.J. Cytological methods for detecting chemical mutagens. Chemical mutagens, principles and methods for their detection, Vol. 4, Ed. A. Hollaender Plenum, New York, London (1976) pp. 1–29. (3) Howard, P.N. et al. Chromosomal aberrations induced by N-methyl-N ′ -nitro-N -nitrosoguanidine in mammalian cells. In Vitro 7:359–365 (1972). (4) Ishidate, M. Jr. and Odashima, S. Chromosome tests with 134 compounds on Chinese hamster cells in vitro: A screening for chemical carcinogens. Mutation Research 48:337–354 (1975). (5) Preston, R.J. et al. Mammalian in vivo and in vitro cytogenetic assays: A report of the Gene-Tox Program. Mutation Research 87:143– 188 (1981).