United States      Prevention, Pesticides     EPA712-C-96-218
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.5250
          Gene Mutation in
          Neurospora crassa
                '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 870.5250  Gene mutation in Neurospora crassa.
    (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 798.5250 Gene Mutation
in Neurospora crassa and OPP 84-2 Mutagenicity Testing (Pesticide As-
sessment Guidelines, Subdivision F—Hazard Evaluation; Human and Do-
mestic Animals) EPA report 540/09-82-025, 1982.

    (b) Purpose.  Neurospora crassa is  a eukaryotic fungus which has
been developed to detect and study a variety of genetic phenomena includ-
ing chemically induced mutagenesis. TV. crassa can be used to detect both
forward and reverse gene mutation. These mutations are detected by bio-
chemical or morphological  changes in the treated  population. The  most
commonly  used mutation assay in TV. crassa  measures  forward mutation
in the ad-3  region of the genome.

    (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 definition also applies to this test guideline.

    A forward mutation is a gene mutation from the wild (parent) type
to the mutant condition.

    (d) Reference substances. These may include, but need not be lim-
ited to, ethyl or methyl methanesulfonate.

    (e) Test method—(1) Principle. The detection of forward mutations
at the  ad-3 locus in either  homokaryons  or heterokaryons may be used.
However, use of two component heterokaryons  is recommended because
of the  greater range of mutations which can be  recovered.  In either  case,
the test relies on  the identification of purple  (mutant) colonies among a
large number of white  (wild-type) colonies.  A representative  sample  of
purple  colonies can be recovered and thoroughly analyzed genetically.

    (2) Description. Forward mutations at the ad-3 locus can be detected
using noncolonial strains of TV. crassa grown on media containing sorbose
as well as glucose. Under these conditions, colonies are formed and repro-
ducible colonial morphology results. Adenine-requiring mutants which ac-
cumulate a reddish-purple pigment can be readily identified and counted.

    (3)  Strain  selection—(i)  Designation.  At  the   present  time,
heterokaryon 12 is recommended for  use in this assay. The use of other
strains may also be 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.

     (iii) Media.  Frie's No. 3 minimal medium or Westgaard's Synthetic
medium with  1.5 percent agar or any medium known to support growth
and characteristic colonial morphology may be used in the assay.

     (4) Preparation of conidia. Stock cultures should be grown on mini-
mal  medium to  select for single colonies with noncolonial morphology.
Single colony isolates then should be inoculated into agar flasks and incu-
bated at 35  °C for 48 h to select colonies with spreading growth patterns
in which  mycelia cover the entire  flask. Flasks  should be incubated at
23-25 °C and those  with bright orange  conidia selected for preparation
of conidial suspensions. Suspensions should be diluted for use in distilled

     (5) Metabolic activation. Conidia should be exposed to  test sub-
stance both in the presence and absence of an appropriate metabolic activa-
tion  system.

     (6) Control groups.  Concurrent positive and negative (untreated and/
or vehicle) controls both with and without metabolic activation should be
included in each experiment.

     (7) Test chemicals—(i) Vehicle.  Test chemicals and positive control
reference substances should be dissolved in an appropriate vehicle and then
further diluted in vehicle for use in the  assay.

     (ii) Exposure  concentrations.  (A)  The test  should initially be per-
formed over a broad range of concentrations selected on the basis of  a
preliminary  assay. Effective treatment times should also be selected in the
preliminary  assay.

     (B) Among the criteria to be taken into consideration for determining
the upper limits of test chemical concentration are cytotoxicity and solu-
bility.  Cytotoxicity  of the test chemical  may be  altered in the presence
of metabolic  activation systems.  For toxic  chemicals,  the  highest  con-
centration tested should not reduce survival below  10 percent of that seen
in the  control  cultures. Relatively insoluble chemicals  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.

     (C) Each test should include five treatment points; two at fixed con-
centrations for different time periods,  and three at varying concentrations
for fixed periods of time.

     (D)  When appropriate, a positive response should be  confirmed by
testing over  a narrow range of concentrations.

     (f) Test performance—(1) Treatment, (i) Growing or nongrowing
conidia should be exposed to the test chemical with and without metabolic
activation.  At the end of the exposure period, treatment should be termi-
nated by chemical quenching.  The quenching  solution may contain 0.1
percent sodium thiosulfate.

     (ii) Conidia should then be plated on the appropriate  media to deter-
mine mutation induction and viability. At the end of the incubation period,
colonies should be scored for viability and mutation induction.

     (iii) Mutants should be classified according to color and morphology.

     (iv) Both mutation frequency and viability should be determined both
immediately before and immediately after chemical treatment.

     (2) Incubation conditions. All plates in a given test  should be incu-
bated for the same  time period. This incubation period may be from 2
to 7  days at 30 °C.

     (3) Number of cultures. Generally, 15 to 20  individual plates per
concentration should be used.

     (g) Data and  report—(1) Treatment  of  results.  Individual plate
counts for  test substance  and controls should be  presented for both muta-
tion  induction and  survival. The mean number of colonies per plate  and
standard deviation should be presented. Data should be presented in tabular
form indicating, as applicable, numbers of colonies  counted, numbers of
mutants identified and classification of mutants  (e.g., color segregants).
Sufficient detail should be provided for verification of survival and muta-
tion  frequencies.

     (2) Statistical evaluation. Data should  be  evaluated by appropriate
statistical techniques.

     (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 number of mutant colonies. Another criterion may
be based upon detection of a reproducible and statistically significant posi-
tive response for at least one of the test points.

     (ii) A  test substance  which does not  produce either a  statistically sig-
nificant dose-related increase in the number of mutant colonies or a statis-
tically significant  and reproducible positive  response  at  any one of the
test points is considered nonmutagenic in this system.

     (iii) Both biological  and statistical significance should be considered
together in the evaluation.

     (4) Test evaluation, (i) Positive results from the ad-3 system in TV.
crassa indicate that, under the test conditions, the test substance causes
mutations in the DNA of this organism.

     (ii) Negative  results  indicate that under the test  conditions the test
substance is not mutagenic in TV. crassa.

     (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) Strain of organism used in the assay.

     (ii) Test chemical vehicle, doses used,  and rationale for dose selec-

     (iii) Method used for preparation of conidia.

     (iv) Treatment conditions, including  length of exposure and method
used to stop treatment.

     (v) Incubation times and temperature.

     (vi) Details of both the protocol used to prepare the metabolic activa-
tion system and of its use in the assay.

     (vii) Dose-response relationship, if applicable.

     (h) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1) Brockman, H.E.  and de Serres,  F.J.  Induction of ad-3  mutants
of Neurospora crassa by 2-aminopurine. Genetics  48: 597-604  (1963).

     (2) de Serres, F.J. and Mailing, H.V.  Measurement of recessive lethal
damage over the entire genome and at two specific loci in the ad-3 region
of a  two-component  heterokaryon  of  Neurospora  crassa.  Chemical
mutagens:  principles  and  methods for  their detection.  Vol.   2,  Ed.
Hollaender, A. Plenum, New York and London (1971) pp. 311-342.

     (3) Matzinger, P.K. and  Ong, T-M.  In vitro activation of aflatoxin
BI to  metabolites mutagenic  in Neurospora crassa. Mutation Research
37:27-32 (1976).