United States       Prevention, Pesticides     EPA712-C-96-229
          Environmental Protection    and Toxic Substances     June 1996
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.5500
          Bacterial DMA Damage
          or Repair Tests
                '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 870.5500   Bacterial DMA damage or repair tests.
     (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 798.5500 Differential
Growth Inhibition of Repair Proficient and Repair Deficient Bacteria:  Bac-
terial DNA Damage or Repair Tests and OPP guideline 84-2 Mutagenicity
Testing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evalua-
tion; 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 imply-
ing 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 ab-
sence of specific DNA repair genes. The response is expressed in the  pref-
erential 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

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 den-
sity  should be documented  and should be adequate for the experimental

     (5) Metabolic activation. Bacteria should be exposed to the test sub-
stance both in the presence and absence of an appropriate metabolic activa-
tion  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 posi-
tive  controls are methyl methanesulfonate for polA strains and  mitomycin
C for rec strains.

     (iv) Positive controls to ensure the efficacy of the activation sys-
tem. The positive control reference  substance for tests including a meta-
bolic activation system should be selected on the basis of the type of acti-
vation system used in the test.

     (v) Other positive controls. Other positive control reference sub-
stances 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 concentra-
tion  are cytotoxicity  and solubility. Cytotoxicity of the  test chemical may
be altered in the presence of metabolic activation systems. For freely solu-
ble nontoxic chemicals, the upper test chemical concentration should be
determined on a case by case basis. Because results are expressed as diam-
eters 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 rep-
licates. When appropriate, a positive response should be confirmed by test-
ing over a narrow range of concentrations.

     (f) Test performance—(1) Diffusion assay—(i) Disc diffusion as-
says. 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 dilu-
tions 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 incu-
bated 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 as-
says. Results should be expressed in diameters of zones of growth inhibi-
tion 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 sur-
vival of each  strain or as  a relative  survival (ratio) of the two  strains.

     (B) Results can also be expressed as the concentrations  required to
effect a predetermined survival rate (e.g. DSV, the dose permitting 37 per-
cent survival). These  data are derived from the survival curve. The con-
centration should be expressed as weight per volume, as moles, or as mo-

     (C) Similarly, results  can be expressed  as  minimal inhibitory con-
centration or as minimal  lethal dose. The former is determined by the ab-
sence 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 concentra-
tions during the treatment. Raw  data, prior to transformation, should be
provided. These should include actual quantities measured, e.g., neat num-
bers. For measurement of diffusion, the diameters of the discs  and/or well
should be indicated and the measurements should indicate whether the di-
ameter of the discs and/or well was subtracted. Moreover, mention should
be made as to whether the test chemical gave a sharp,  diffuse, or double-
zone 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 experi-
mental  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 deter-
mining  a positive result,  one of which is a statistically  significant dose-
related preferential inhibition or killing of the repair deficient strain. An-
other criterion may be based upon detection of a reproducible and  statis-
tically significant positive response for at least one of the test  points.

     (ii) A test substance  which does not produce  either a statistically sig-
nificant 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.

     (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) 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 activa-
tion 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 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 31:347-364 (1975).

     (2) Kada, T.  et al.  In vitro and host-mediated rec-assay  procedures
for screening chemical mutagens; and phloxine, a mutagenic red dye de-
tected, 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).