United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–229

June 1996

Health Effects Test

Guidelines

OPPTS 870.5500

Bacterial DNA Damage

or Repair Tests

‘‘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 870.5500 Bacterial DNA damage or repair tests.

(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 798.5500 Differential

Growth Inhibition of Repair Proficient and Repair Deficient Bacteria: Bacterial DNA Damage or Repair Tests and OPP guideline 84–2 Mutagenicity

Testing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Animals) EPA report 540/09–82–025, 1982.

(b) Purpose. Bacterial DNA damage or repair tests measure DNA

damage which is expressed as differential cell killing or growth inhibition

of repair deficient bacteria in a set of repair proficient and deficient strains.

These tests do not measure mutagenic events per se. They are used as

an indication of the interaction of a chemical with genetic material implying the potential for genotoxicity. Tests for differential growth inhibition

of repair proficient and repair deficient bacteria measure differences in

chemically induced cell killing between wild-type strains with full repair

capacity and mutant strains deficient in one or more of the enzymes which

govern repair of damaged DNA.

(c) Reference substances. These may include, but need not be limited

to, chloramphenicol or methyl methanesulfonate.

(d) Test method—(1) Principle. The tests detect agents that interact

with cellular DNA to produce growth inhibition or killing. This interaction

is recognized by specific cellular repair systems. The assays are based

upon the use of paired bacterial strains that differ by the presence of absence of specific DNA repair genes. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair

deficient strain since it is incapable of removing certain chemical lesions

from its DNA.

(2) Description. Several methods for performing the test have been

described. Those described here are:

(i) Tests performed on solid medium (diffusion tests).

(ii) Tests performed in liquid culture (suspension tests).

(3) Strain selection—(i) Designation. At the present time,

Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45)

pairs are recommended. Other pairs may be utilized when appropriate.

(ii) Preparation and storage. Stock culture preparation and storage,

growth requirements, method of strain identification and demonstration of
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appropriate phenotypic requirements should be performed using good

microbiological techniques and should be documented.

(4) Bacterial growth. Good microbiological techniques should be

used to grow fresh cultures of bacteria. The phase of growth and cell density should be documented and should be adequate for the experimental

design.

(5) Metabolic activation. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor supplemented

postmitochondrial fraction prepared from the livers of rodents treated with

enzyme inducing agents. The use of other species, tissues, or techniques

may also be appropriate.

(6) Control groups—(i) Concurrent controls. Concurrent positive,

negative, and vehicle controls should be included in each assay.

(ii) Negative controls. The negative control should show

nonpreferential growth inhibition (i.e. should affect both strains equally).

Chloramphenicol is an example of a negative control.

(iii) Genotype specific controls. Examples of genotype specific positive controls are methyl methanesulfonate for polA strains and mitomycin

C for rec strains.

(iv) Positive controls to ensure the efficacy of the activation system. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test.

(v) Other positive controls. Other positive control reference substances may be used.

(e) Test chemicals—(1) Vehicle. Test chemicals and positive and

negative control reference substances should be dissolved in an appropriate

vehicle and then further diluted in vehicle for use in the assay.

(2) Exposure concentrations. The test should initially be performed

over a broad range of concentrations. Among the criteria to be taken into

consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may

be altered in the presence of metabolic activation systems. For freely soluble nontoxic chemicals, the upper test chemical concentration should be

determined on a case by case basis. Because results are expressed as diameters of zones of growth inhibition in the diffusion test, it is most important

that the amounts of chemical on the disc (or in the wells) are exact replicates. When appropriate, a positive response should be confirmed by testing over a narrow range of concentrations.
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(f) Test performance—(1) Diffusion assay—(i) Disc diffusion assays. Disc diffusion assays may be performed in two ways:

(A) A single strain of bacteria may be added to an agar overlay or

spread on the surface of the agar and the test chemical placed on a filter

disc on the surface of the agar.

(B) DNA repair proficient and DNA repair deficient bacteria may

be streaked in a line on the surface of the agar of the same plate and

a disc saturated with test chemical placed on the surface of the agar in

contact with the streaks.

(ii) Well diffusion assays. In well diffusion assays, bacteria may be

either added to the agar overlay or spread onto the surface of the agar.

A solution of the test chemical is then placed into a well in the agar.

(2) Suspension assays. (i) A bacterial suspension may be exposed

to the test chemical and the number of surviving bacteria determined (as

colony-forming units) either as a function of time of treatment or as a

function of the concentration of test agent.

(ii) Nonturbid suspensions of bacteria may be exposed to serial dilutions of the test agent and a minimal inhibitory concentration for each

strain determined, as evidenced by the presence or absence of visible

growth after a period of incubation.

(iii) Paired bacterial suspensions (usually with some initial turbidity)

may be treated with a single dose of the chemical. Positive results are

indicated by a differential inhibition in the rate of increase of turbidity

of the paired cultures.

(3) Number of cultures. When using a plate diffusion procedure,

at least two independent plates should be used at each dilution. In liquid

suspension assays, at least two independent specimens for determination

of the number of viable cells should be plated.

(4) Incubation conditions. All plates in a given test should be incubated for the same time period. This incubation period should be for 18

to 24 h at 37 °C.

(g) Data and report—(1) Treatment of results—(i) Diffusion assays. Results should be expressed in diameters of zones of growth inhibition in millimeters or as areas derived therefrom as square millimeters.

Dose-response data, if available, should be presented using the same units.

(ii) Liquid suspension assays. (A) Survival data can be presented

as dose responses, preferably as percentage of survivors or fractional survival of each strain or as a relative survival (ratio) of the two strains.
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(B) Results can also be expressed as the concentrations required to

effect a predetermined survival rate (e.g. D37, the dose permitting 37 percent survival). These data are derived from the survival curve. The concentration should be expressed as weight per volume, as moles, or as molarity.

(C) Similarly, results can be expressed as minimal inhibitory concentration or as minimal lethal dose. The former is determined by the absence of visible growth in liquid medium and the latter is determined by

plating dilutions onto semisolid media.

(iii) In all tests, concentrations must be given as the final concentrations during the treatment. Raw data, prior to transformation, should be

provided. These should include actual quantities measured, e.g., neat numbers. For measurement of diffusion, the diameters of the discs and/or well

should be indicated and the measurements should indicate whether the diameter of the discs and/or well was subtracted. Moreover, mention should

be made as to whether the test chemical gave a sharp, diffuse, or doublezone of growth inhibition. If it is the latter, the investigator should indicate

whether the inner or the outer zone was measured.

(iv) Viability data should be given as the actual plate counts with

an indication of the dilution used and the volume plated or as derived

titers (cells per milliliter). Transformed data alone in the absence of experimental data are not acceptable (i.e, ratios, differences, survival fraction).

(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 preferential inhibition or killing of the repair deficient strain. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.

(ii) A test substance which does not produce either a statistically significant dose-related preferential inhibition or killing of the repair deficient

strain or a statistically significant and reproducible positive response at

any one of the test points is considered not to interact with the genetic

material of the organisms used in assay.

(iii) Both biological and statistical significance should be considered

together in the evaluation.

(4) Test evaluation. DNA damage tests in bacteria do not measure

DNA repair per se nor do they measure mutations. They measure DNA

damage which is expressed as cell killing or growth inhibition. A positive

result in a DNA damage test in the absence of a positive result in another

system is difficult to evaluate in the absence of a better data base.
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(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) Bacterial strains used.

(ii) Phase of bacterial cell growth at time of use in the assay.

(iii) Media composition.

(iv) Details of both the protocol used to prepare the metabolic activation system and its use in the assay.

(v) Treatment protocol, including doses used and rationale for dose

selection, positive and negative controls.

(vi) Method used for determination of degree of cell kill.

(vii) 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) Kada, T. et al. In vitro and host-mediated rec-assay procedures

for screening chemical mutagens; and phloxine, a mutagenic red dye detected, Mutation Research 16:165–174 (1972).

(3) Leifer, Z. et al. An evaluation of bacterial DNA repair tests for

predicting genotoxicity and carcinogenicity: A report of the U.S. EPA’s

Gene-Tox Program. Mutation Research 87:211–297 (1981).

(4) Slater, E.E. et al. Rapid detection of mutagens and carcinogens.

Cancer Research 31:970–973 (1971).