United States Prevention, Pesticides EPA712-C-96-215
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5140
Gene Mutation in
Aspergillus nidulans
'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 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.).
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:
guidelines@epamail.epa.gov.
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 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 870.5140 Gene mutation in Aspergillus nidulans.
(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 798.5140 Gene
mutations in Aspergillus nidulans and OPP 84-2 Mutagenicity Testing
(Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals) EPA report 540/09-82-025, 1982.
(b) Purpose. Aspergillus nidulans is a eukaryotic fungus which has
been developed to detect and study a variety of genetic phenomena includ-
ing chemically induced mutagenesis. A. nidulans can be used to detect
both forward and reverse gene mutation. These mutations are detected by
changes in colonial morphology or nutritional requirements in treated pop-
ulations. The methionine and 2-thioxanthine forward mutation systems can
be used to detect mutations in A. nidulans.
(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 methanesulfonate, cyclophosphamide, or aflatoxin BI.
(e) Test method—(1) Principle. Conidia are exposed to test chemical
both with and without metabolic activation and plated on selective medium
to determine changes in colonial morphology or nutritional requirements.
At the end of a suitable incubation period, mutant colonies are counted
and compared to the number of spontaneous mutants in an untreated con-
trol culture. Simultaneous determination of survival permits calculation of
mutation frequency.
(2) Description. Tests for mutation in A. nidulans are performed in
liquid suspension. Treated conidia are plated on selective medium to deter-
mine changes in nutritional requirements or colonial morphology.
(3) Strain selection—(i) Designation. For the methionine and
2-thioxanthine systems the haploid Glascow biAl; meth Gl strain is the
most commonly used strain although other strains may be appropriate. Any
translocation-free strain which produces green colonies on thioxanthine
free medium and yellow colonies on medium containing thioxanthine may
be used in the thioxanthine system.
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(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. Any medium which supports growth and a characteristic
colonial morphology may be used in the assay.
(4) Preparation of conidia. Prior to chemical treatment, conidia from
four to five single colonies of the appropriate strain are grown at 37 °C
on complete medium. At the end of the incubation period, conidia are
collected, conidial chains broken up, mycelial debris removed and conidia
concentrated prior to removal of the germination inhibitory substance. Ger-
mination inhibitory substance should be removed by Tween 80 or diethyl
ether.
(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) 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.
(C) 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 a metabolic activation system. Relatively insoluble chemicals should
be tested up to the limits of solubility. For freely soluble nontoxic chemi-
cals, the upper test chemical concentration should be determined on a case
by case basis.
(D) When appropriate, a positive response should be confirmed by
using a narrow range of test concentrations.
(f) Test Performance—(1) Treatment. Germinating or quiescent
conidia in liquid suspension should be exposed to the test chemical at
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37 °C under conditions of yellow light and controlled pH and oxygen ten-
sion. At the end of the exposure period, treatment should be terminated
by repeated centrifugation and washing of the conidia or by dilution.
Chemical neutralization of the test agent may also be used but is not rec-
ommended.
(2) Media—(i) Methionine system. For the methionine system,
conidia should be plated on methionine deficient medium for mutant selec-
tion and on medium supplemented with methionine to determine survival.
(ii) Thioxanthine system. (A) For the 2-thioxanthine system, treated
conidia should be plated on nitrogen-free glucose and salts minimal me-
dium containing 2-thioxanthine.
(B) After incubation, green colonies should be counted and isolated
by restreaking. The isolated colonies should be classified on the basis of
genetic criteria. Yellow, wild-type colonies will grow on the same plate.
This permits concurrent determination of survival and an estimation of
mutation frequency.
(3) Determination of mutation frequency and viability. In both sys-
tems, mutation frequency and viability should be determined immediately
before and immediately after chemical treatment.
(4) Incubation conditions. All incubations should be at 37 °C. Incu-
bation time will vary depending upon system and endpoint (mutation or
viability) being determined.
(5) Number of cultures, (i) At least 10 independent plates per con-
centration with no more than 20 colonies per plate should be used in the
methionine system.
(ii) Fifteen to twenty plates per concentration are preferred for the
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2-thioxanthine system
(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 also be presented. Data should be presented in
tabular form indicating, as applicable, numbers of colonies counted, and
numbers and classification of mutants identified. Sufficient detail should
be provided for verification of survival and mutation frequencies.
(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 increase in the number of mutant colonies. Another criterion may
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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 methionine and
2-thioxanthine systems in 4. nidulans indicate that, under the test condi-
tions, the test substance causes gene (point) mutations in the DNA of this
organism caused by base pair changes and small deletions in the genome.
(ii) Negative results indicate that under the test conditions the test
chemical is not mutagenic in A. nidulans.
(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 selection,
toxicity data.
(iii) Method used for preparation of conidia.
(iv) Treatment conditions, including length of exposure and method
used to stop treatment.
(v) Details of both the protocol used to prepare the metabolic activa-
tion system and of its use in the assay.
(vi) Incubation times and temperature.
(vii) Positive and negative controls.
(viii) Dose-response relationship, if applicable.
(h) 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) Kafer, E. et al. Aspergillus nidulans: systems and results of tests
for chemical induction of mitotic segregation and mutation. I. Diploid and
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duplication assay systems: a report of the U.S. EPA's Gene-Tox Program,
Mutation Research 98:1-48 (1982).
(3) Munson, R.J. and Goodhead, D.T. Relation between induced mu-
tation frequency and cell survival: a theoretical approach and an examina-
tion of experimental data for eukaryotes. Mutation Research 42:145-159
(1977).
(4) Scott, B.R. et al. Aspergillus nidulans: systems and results of tests
for mitotic segregation and mutation. II. Haploid assay systems and overall
response of all systems: a report of the U.S. EPA's Gene-Tox Program.
Mutation Research 98:49-94 (1982).
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