748R98001
OPPTS HARMONIZED TEST GUIDELINES
Series 870
Health Effects
Volume II of III
Guidelines OPPTS 870.5100 - OPPTS 870.5915
August 1998
United States Environmental Protection Agency
Office of Prevention, Pesticides, and Toxic Substances
Washington, B.C. 20460
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Series 870—Health Effects Test Guidelines
OPPTS
Number
8TO 1000
870 1 100
870 1200
8701300
870 2400
870 2500
8702600
8703100
870 3150
8703200
870 3250
870 346S
870 3700
870 3800
870 4100
870 4200
870 4300
8705100
870 5140
870 5195
870 5200
8705250
870 5275
8705300
8705375
8703380
870 538S
870 539S
870 5450
8705460
8705500
670 5550
870S575
8705900
8705915
8706100
870 6200
8706300
8706500
870 6850
870 6855
8707200
870 7485
870 7600
870 7800
Name
Group A — Acute Toxicity Tasl Guidelines
Acute toxialy teshrtg-background
Acuie oral toxtcity
Acute dermal loxioly
Acute inhalation loxicity
Acute eye irritation
Acute dermal irritation
Skin sensitizatton
Group 0— Subchronic Toxicity Tesl Guidelines
90 Day oral toxicit/m rodenis
90 Day oral loxicity tn nonrodents
21/28 Day dermal toxicity
90 Day dermal toxtctty
90-Day inhalation toxicity
Prenatal developmental toxicity study
Reproduction and fertility effects
Group C — Chronic Toxicity Test Guidelines
Chronic loxicfty
Caidnogenicity
Combined chronic toxiaty/caranogentcity
Group D— Genetic Toxicity Test Guidelines
Bacterial reverse mutation test
Gene mutation in Aspergiltus ntdulans
Mouse biochemical specific locus test
Mouse vsble specific locus lest
Gene mutation In Neurospora crassa
Sex linked recessive lethal test tn Drosophila melanogaster
In vitro mammalian cell gene mutation test
In vitro mammalian chromosome aberration lest
Mammalian spermatogonial chromosomal aberration lest
Mammalian bone marrow chromosomal aberration test
Mammalian erythrocyte rrncronudeus test
Rodent dominant lethal assay
Rodent heritable Iranstocation assays
Bacterial DNA damage or repair tests
Unscheduled DNA synthesis in mammalian cells m culture
Mitoiic gene conversion in Saccftaromyces ceravfeae
In vitro sister chromatifl exchange assay
In vivo sister chromatid exchange assay
Group E — Neurotoxiclly Test Guidelines
Acute and 28 day delayed neurotoxlcity ol organophosphoms substances
-
Neuroioxicity screening battery
Developmental neuroloxicity study
Schedule-controlled operant behavior
Peripheral nerve function
Neurophysiotogy Sensory evoked potentials
Group F— Special Studies Test Guidelines
Companion animal safety
Metabolism and pharmacokmetics
Dermal penetration
Immunoloxialy
Existing Numbers
OPPT
none
7981175
798 1100
798 1150
7984500
798 4470
7984100
7982650
none
none
798 2250
798 2450
798 4900
798 4700
798 3260
7983300
7983320
7985100
5265
7985140
798S19S
798 5200
798 5250
7985275
798.5300
798 5375
7985380
7985385
7985395
7985450
7985460
7985500
7985550
7985575
798.5900
7985915
7986450
6540
6560
7986050
6200
6400
none
798 6500
7986850
798 6855
none
798 7485
none
none
OPP
none
81-1
81-2
81-3
61-4
81-5
81-6
82-1
82-1
82—2
82-3
82-4
83-3
83-4
83-1
83-2
83-5
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
' 84-2
* 84-2
84-2
84-2
84-2
81-7
82-5
82-6
81-8
82-7
83-1
83-6
85-5
85-6
none
none
85-1
85-3
85-7
OECO
none
401
402
403
405
404
406
408
409
410
411
413
414
416
452
451
453
471 472
none
none
none
none
477
476
473
483
475
474
478
none
none
482
481
479
none
418 419
424
none
none
none
none
none
417
none
none
EPA Pub
no
712-C-
98-189
98-190
98-192
98-193
98-1SS
98-196
98-197
98-199
98-200
98-201
98-202
98-204
98-207
98-208
98-210
98-211
98-212
98-247
98-215
98-216
96-217
98-218
98-220
98-221
98-223
98-224
98-225
98-226
98-227
98-228
98-229
98-230
98-232
98-234
98-235
98-237
98-238
98-239
98-240
98-241
98-242
98-349
95-244
98-350
98-351
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-247
August 1998
Health Effects Test
Guidelines
OPPTS 870.5100
Bacterial Reverse
Mutation Test
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INIRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Offtce 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 m 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 Rodenttcide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Pnntmg Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.5100 Bacterial reverse mutation test.
(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 5100 Eschenchia
coh WP2 and WP2 uvrA reverse mutation assays, OPPTS 40 CFR
798 5265 The salmonella typhimunum reverse mutation assay and OECD
471 and 472, Bactenal Reverse Mutation Test
(b) Purpose. (1) The bacterial reverse mutation test uses ammo-acid
requiring strains of Salmonella typhimunum (S typhimunum) and
Eschenchia coh (E coh) to detect point mutations, which involve substi-
tution, addition or deletion of one or a few DNA base pairs (see references
in paragraphs (g)(l), (g)(2), and (g)(3) of this guideline) The principle
of this bacterial reverse mutation test is that it detects mutations which
revert mutations present in the test strains and restore the functional capa-
bility of the bactena to synthesize an essential ammo acid The revertant
bactena are detected by their ability to grow in the absence of the ammo
acid required by the parent test strain
(2) Point mutations are the cause of many human genetic diseases
and there is substantial evidence that point mutations in oncogenes and
tumour suppressor genes of somatic cells are involved in tumour formation
in humans and experimental animals The bacterial reverse mutation test
is rapid, inexpensive and relatively easy to perform Many of the test
strains have several features that make them more sensitive for the detec-
tion of mutations, including responsive DNA sequences at the reversion
sites, increased cell permeability to large molecules and elimination of
DNA repair systems or enhancement of error-prone DNA repair processes
The specificity of the test strains can provide some useful information on
the types of mutations that are induced by genotoxic agents. A very large
data base of results for a wide variety of structures is available for bacterial
reverse mutation tests and well-established methodologies have been de-
veloped for testing chemicals with different physico-chemical properties,
including volatile compounds.
(c) Definitions. The definitions in section 3 of TSCA and m 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline The following definitions also apply to this test guideline.
Reverse mutation test m either Salmonella typhimunum or
Eschenchia coh detects mutation in an ammo-acid requiring strain (histi-
dme or tryptophan, respectively) to produce a strain independent of an
outside supply of ammo-acid
-------
Base pair substitution mutagens are agents that cause a base change
in DNA In a reversion test this change may occur at the site of the original
mutation, or at a second site in the bacterial genome
Frameshift mutagens are agents that cause the addition or deletion
of one or more base pairs in the DNA, thus changing the reading frame
in the RNA
Initial considerations. (1) The bacterial reverse mutation test uti-
lizes prokaryotic cells, which differ from mammalian cells in such factors
as uptake, metabolism, chromosome structure and DNA repair processes
Tests conducted in vitro generally require the use of an exogenous source
of metabolic activation In vitro metabolic activation systems cannot mimic
entirely the mammalian in vivo conditions The test therefore does not pro-
vide direct information on the mutagenic and carcinogenic potency of a
substance in mammals
(2) The bacterial reverse mutation test is commonly employed as an
creen for genotoxic activity and, in particular, for point mutation-
mau^.i0 activity An extensive data base has demonstrated that many
chemicals that are positive in this test also exhibit mutagenic activity in
other tests There are examples of mutagenic agents which are not detected
by this test, reasons for these shortcomings can be ascnbed to the specific
nature of the endpomt detected, differences in metabolic activation, or dif-
ferences in bioavailability On the other hand, factors which enhance the
sensitivity of the bacterial reverse mutation test can lead to an overestima-
tion of mutagenic activity
(3) The bacterial reverse mutation test may not be appropriate for
the evaluation of certain classes of chemicals, for example highly bacteri-
cidal compounds (e.g, certain antibiotics) and those which are thought
(or known) to interfere specifically with the mammalian cell replication
system (eg., some topoisomerase inhibitors and some nucleoside ana-
logues). In such cases, mammalian mutation tests may be more appro-
priate
(4) Although many compounds that are positive in this test are mam-
malian carcinogens, the correlation is not absolute It is dependent on
chemical class and there are carcinogens that are not detected by this test
because they act through other, non-genotoxic mechanisms or mechanisms
absent in bacterial cells
(e) Test method—(1) Principle, (i) Suspensions of bacterial cells are
exposed to the test substance m the presence and in the absence of an
exogenous metabolic activation system In the plate incorporation method,
these suspensions are mixed with an overlay agar and plated immediately
onto minimal medium In the premcubation method, the treatment mixture
is incubated and then mixed with an overlay agar before plating onto mini-
mal medium For both techniques, after 2 or 3 days of incubation, revertant
-------
colonies are counted and compared to the number of spontaneous revertant
colonies on solvent control plates
(u) Several procedures for performing the bacterial reverse mutation
test have been described Among those commonly used are the plate incor-
poration method (see references in paragraphs (g)(l), (g)(2), (g)(3), and
(g)(4) of this guideline), the premcubation method (see references in para-
graphs (g)(2), (g)(3), (g)(5), (g)(6), (gX7), and (g)(8) of this guideline),
the fluctuation method (see references in paragraphs (g)(9) and (g)(10)
of this guideline), and the suspension method (see reference in paragraph
(g)(ll) of this guideline) Suggestions for modifications for the testing of
gases or vapours have been described (see reference in paragraph (g)(12)
of this guideline)
(in) The procedures described in this guideline pertain primarily to
the plate incorporation and premcubation methods Either of them is ac-
ceptable for conducting experiments both with and without metabolic acti-
vation Some compounds may be detected more efficiently using the
premcubation method These compounds belong to chemical classes that
include short chain aliphatic mtrosamines, divalent metals, aldehydes, azo-
dyes and diazo compounds, pyrolhzidme alkaloids, allyl compounds and
mtro compounds (see reference in paragraph (g)(3) of this guideline). It
is also recognized that certain classes of mutagens are not always detected
using standard procedures such as the plate incorporation method or
premcubation method These should be regarded as "special cases" and
it is strongly recommended that alternative procedures should be used for
their detection The following "special cases" could be identified (to-
gether with examples of procedures that could be used for their detection)
azo-dyes and diazo compounds (see references in paragraphs (g)(3), (g)(5),
(g)(6), and (g)(13) of this guideline), gases and volatile chemicals (see
references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this guide-
line), and glycosides (see references in paragraphs (g)(17) and (g)(18) of
this guideline) A deviation from the standard procedure needs to be sci-
entifically justified
(2) Description—(i) Preparations—(A) Bacteria. (1) Fresh cultures
of bactena should be grown up to the late exponential or early stationary
phase of growth (approximately 109 cells per ml). Cultures in late station-
ary phase should not be used The cultures used in the experiment should
contain a high litre of viable bactena. The litre may be demonstrated eilher
from historical control data on growth curves, or in each assay ihrough
the delermmalion of viable cell numbers by a plating experiment.
(2) The cullure lemperalure should be 37 °C
(3) At least five strains of bactena should be used These should in-
clude four strains of S typhtmunum (TAI535, TA1537 or TA97a or
TA97; TA98, and TA100) that have been shown to be reliable and
-------
reproducibly responsive between laboratories These four S typhtmunum
strains have GC base pairs at the primary reversion site and it is known
that they may not detect certain oxidising mutagens, cross-linking agents
and hydrazmes Such substances may be detected by Ecoli WP2 strains
or 5 nphimurium TA1Q2 (see reference in paragraph (g)(19) of this guide-
line) which have an AT base pair at the primary reversion site Therefore
the recommended combination of strains is
(i) 5 typhimunum TA1535
00 5 typhtmunum TA1537 or TA97 or TA97a
OH) S typhimunum TA98
(iv) S typhimunum 1 MOO
(v) E coli WP2 uvrA, or E coll WP2 uvrA (pKMlOl), or S
typhimunum TA102
In order to detect cross-linking mutagens it may be preferable to include
TA102 or to add a DNA repair-proficient strain of E coli [e g , E.coli WP2
or£a?/
-------
graphs (g)(l) and (g)(2) of this guideline) or a combination of
phenobarbitone and (3-naphthoflavone (see references in paragraphs
(g)(18), (g)(20), and (g)(21) of this guideline) The post-mitochondria!
fraction is usually used at concentrations in the range from 5 to 30 percent
v/v in the S9-mix The choice and condition of a metabolic activation sys-
tem may depend upon the class of chemical being tested In some cases
it may be appropriate to utilize more than one concentration of post-
mitochondnal fraction For azo-dyes and diazo-compounds, using a reduc-
tive metabolic activation system may be more appropnate (see references
in paragraphs (g)(6) and (g)(13) of this guideline)
(D) Test substance/preparation. Solid test substances should be dis-
solved or suspended in appropnate solvents or vehicles and diluted if ap-
propnate pnor to treatment of the bactena Liquid test substances may
be added directly to the test systems and/or diluted pnor to treatment
Fresh preparations should be employed unless stability data demonstrate
the acceptability of storage
(n) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the bactena and the S9 activity (see
reference in paragraph (g)(22) of this guideline) If other than well-known
solvent/vehicles are used, their inclusion should be supported by data indi-
cating their compatibility It is recommended that wherever possible, the
use of an aqueous solvent/vehicle be considered first When testing water-
unstable substances, the organic solvents used should be free of water
(B) Exposure concentrations. (7) Amongst the cntena to be taken
into consideration when determining the highest amount of test substance
to be used are cytotoxicity and solubility in the final treatment mixture.
It may be useful to determine toxicity and insolubility in a preliminary
experiment. Cytotoxicity may be detected by a reduction in the number
of revertant colonies, a cleanng or diminution of the background lawn,
or the degree of survival of treated cultures The cytotoxictty of a sub-
stance may be altered in the presence of metabolic activation systems. In-
solubility should be assessed as precipitation in die final mixture under
the actual test conditions and evident to the unaided eye. The rec-
ommended maximum test concentration for soluble non-cytotoxtc sub-
stances is 5 mg/plate or 5u,l/plate For non-cytotoxic substances that are
not soluble at Smg/plate or 5|il/plate, one or more concentrations tested
should be insoluble in the final treatment mixture Test substances that
are cytotoxic already below Smg/plate or Spl/plate should be tested up
to a cytotoxic concentration The precipitate should not interfere with the
sconng
(2) At least five different analysable concentrations of the test sub-
stance should be used with approximately half log (i e VlO) intervals be-
-------
tween test points for an initial experiment Smaller intervals may be appro-
priate when a concentration-response is being investigated
(J) Testing above the concentration of 5 mg/plate or 5|il/plate may
be considered when evaluating substances containing substantial amounts
of potentially mutagenic impurities
(C) Controls. (7) Concurrent strain-specific positive and negative
(solvent or vehicle) controls, both with and without metabolic activation,
should be included in each assay Positive control concentrations that dem-
onstrate the effective performance of each assay should be selected
(2) For assays employing a metabolic activation system, the positive
Control reference substance(s) should be selected on the basis of the type
of bactena strains used The following chemicals are examples of suitable
positive controls for assays with metabolic activation-
r
Chemical
CAS number
9,10-Dimethylanthracene
7,12-Dimethylbenzanthracene
Congo Red (for the reductive metabolic activation method)
Benzo(a)pyrene
Cyclopnosphamide (monohydrate)
2-Aminoanthracene
781^43-1]
'57-97-6]
573-58-0]
50-32-8]
50-18-0]
6055-19-2J
613-13-8]
2-Arrunoanthracene should not be used as the sole indicator of the efficacy
of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should
also be characterised with a mutagen that requires metabolic activation
by imcrosomal enzymes, e g , benzo(a)pyrene, dimethylbenzanthracene.
(3) For assays performed without metabolic activation system, exam-
ples of strain-specific positive controls are*
al
(a) Sodium azide
b) 2-Nitrofluorene
[c) 9-Aminoacndtne or ICR 1 91
d) Cum«»ne hydroperoxide . . .
e) Mitomycin C
f) N-Ethyl-N-nitro-N-nitrosoguanidine or
4-nrtroquinoline 1 -oxide
{g) Furytfuramide (AF-2)
CAS number
[26628-22-8]
607-57-8]
90-45-9]
17070-45-0]
80-15-9]
50-07-7]
70-25-7]
[56-57-5]
(3688-53-7]
Strain
TAl535andTA100
TA98
TA1537,TA97and
TA97a
TA102
WP2 uvrA and TA1 02
WP2, WP2 uvrA and
WP2 uvrA (pKMlOl)
Ptasmid-contaming
strains
(4) Other appropriate positive control reference substances may be
used The use of chemical class-related positive control chemicals may
be considered, when available
-------
(5) Negative controls, consisting of solvent or vehicle alone, without
test substance, and otherwise treated in the same way as the treatment
groups, should be included In addition, untreated controls should also be
used unless there are historical control data demonstrating that no delete-
rious or mutagenic effects are induced by the chosen solvent
(3) Procedure—(i) Treatment with test substance. (A) For the plate
incorporation method (see references in paragraphs (g)(l), (g)(2), (g)(3),
and (g)(4) of this guideline), without metabolic activation, usually 005
ml or 0 1 ml of the test solutions, 0 1 ml of fresh bacterial culture (contain-
ing approximately 108 viable cells) and 0 5 ml of stenle buffer are mixed
with 2 0 ml of overlay agar For the assay with metabolic activation, usu-
ally 0 5 ml of metabolic activation mixture containing an adequate amount
of post-mitochondnal fraction (m the range from 5 to 30 percent v/v in
the metabolic activation mixture) are mixed with the overlay agar (20
ml), together with the bacteria and test substance/test solution The con-
tents of each tube are mixed and poured over the surface of a minimal
agar plate The overlay agar is allowed to solidify before incubation
(B) For the premcubation method (see references in paragraphs (g)(2),
(g)(3), (g)(5), and (g)(6) of this guideline) the test substance/test solution
is premcubated with the test strain (containing approximately 108 viable
cells) and stenle buffer or the metabolic activation system (0.5 ml) usually
for 20 mm or more at 30-37 °C pnor to mixing with the overlay agar
and pouring onto the surface of a minimal agar plate Usually, 0 05 or
0 1 ml of test substance/test solution, 0 1 ml of bacteria, and 0.5 ml of
S9-mix or stenle buffer, are mixed with 2 0 ml of overlay agar Tubes
should be aerated during pre-mcubation by using a shaker.
(C) For an adequate estimate of variation, tnphcate plating should
be used at each dose level The use of duplicate plating is acceptable when
scientifically justified The occasional loss of a plate does not necessarily
invalidate the assay
(D) Gaseous or volatile substances should be tested by appropnate
methods, such as in sealed vessels (see references in paragraphs (g)(12),
(g)(14), (g)(15), and (g)(16) of this guideline)
(a) Incubation. All plates in a given assay should be incubated at
37 °C for 48-72 hours After the incubation penod, the number of
revertant colonies per plate is counted
(0 Data and reporting—(1) Treatment of results, (i) Data should
be presented as the number of revertant colonies per plate. The number
of revertant colonies on both negative (solvent control, and untreated con-
trol if used) and positive control plates should also be given
-------
fn) Individual plate counts the mean number of revertant colonies
per plate and the standard deviation should be presented for the test sub-
stance and positive and negative (untreated and/or solvent) controls
(111) There is no requirement for verification of a clear positive re-
sponse Equivocal results should be clarified by further testing preferably
using a modification of experimental conditions Negative results need to
be confirmed on a case-by-case basis In those cases where confirmation
of negative results is not considered necessary, justification should be pro-
vided Modification of study parameters to extend the range of conditions
assessed should be considered in follow-up experiments Study parameters
that might be modified include the concentration spacing, the method of
treatment (plate incorporation or liquid premcubation), and metabolic acti-
vation conditions
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a concentration-related
increase over the range tested and/or a reproducible increase at one or
more concentrations in the number of revertant colonies per plate in at
least one strain with or without metabolic activation system (see reference
in paragraph (g)(23) of this guideline) Biological relevance of the results
should be considered first Statistical methods may be used as an aid in
evaluating the test results (see reference in paragraph (g)(24) of this guide-
line) However, statistical significance should not be the only determining
factor for a positive response
(n) A test substance for which the results do not meet the above cn-
tena is considered non-mutagenic in this test
(in) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated
(iv) Positive results from the bacterial reverse mutation test indicate
that a :^ tance induces point mutations by base substitutions or
frameshifts in the genome of either Salmonella typhimunum and/or
Eschenchia coh Negative results indicate that under the test conditions,
the test substance is not mutagemc in the tested species.
(3) Test report The test report should include the following informa-
tion.
(i) Test substance.
(A) Identification data and CAS no , if known
(B) Physical nature and punty
(C) Physicochemical properties relevant to the conduct of the study.
8
-------
(D) Stability of the test substance, if known
(11) Solvent/vehicle
(A) Justification for choice of solvent/vehicle
(B) Solubility and stability of the test substance tn solvent/vehicle,
if known
(in) Strains
(A) Strains used
(B) Number of cells per culture
(C) Strain characteristics
(iv) Test conditions
(A) Amount of test substance per plate (mg/plate or ml/plate) with
rationale for selection of dose and number of plates per concentration
(B) Media used
(C) Type and composition of metabolic activation system, including
acceptability criteria
(D) Treatment procedures
(v) Results
(A) Signs of toxicity
(B) Signs of precipitation
(C) Individual plate counts
(D) The mean number of revertant colonies per plate and standard
deviation
(E) Dose-response relationship, where possible
(F) Statistical analyses, if any
(G) Concurrent negative (solvent/vehicle) and positive control data,
with ranges, means and standard deviations
(H) Historical negative (solvent/vehicle) and positive control data,
with e g., ranges, means and standard deviations
(vi) Discussion of the results
(vn) Conclusion
9 /
-------
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Ames, B N , McCann, J , and Yamasaki, E Methods for Detecting
Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome
Mutagenicity Test Mutation Research 31, 347-364 (1975)
(2) Maron, D M and Ames, B N Revised Methods for the Salmonella
Mutagenicity Test Mutation Research 113, 173-215 (1983)
(3) Gatehouse, D et al Recommendations for the Performance of
Bacterial Mutation Assays Mutation Research 312, 217-233 (1994)
(4) Kier, L.D et al The Salmonella Typhimunum/Mammahan
Microsomal Assay A Report of the U S Environmental Protection Agency
Gene-Tox Program Mutation Research 168, 69-240 (1986)
(5) Yahagi, T et al Mutagenicity of Carcinogen Azo Dyes and Their
Derivatives Cancer Letters 1, 91-96 (1975)
(6) Matsushima, M et al Factors Modulating Mutagenicity Microbial
Tests (Ed) "Norpoth, K H and Garner, R C Short-Term Test Systems for
Detecting Carcinogens (Springer, Berlin-Heidelberg-New York, 1980) pp.
273-285
(7) Gatehouse, D G et al Bacterial Mutation Assays (Ed) Kirkland,
DJ Basic Mutagenicity Tests UKEMS Part 1 Revised (Cambridge Uni-
versity Press, 1990) pp 13-61
(8) Aeschbacher, H U, Wolleb, U, and Porchet, L J Liquid
Premcubation Mutagenicity Test for Foods. Food Safety 8, 167-177
(1987)
(9) Green, M H L , Munel, W J , and Bndges, B A Use of a Sim-
plified Fluctuation Test to Detect Low Levels of Mutagens Mutation Re-
search 38, 33^2 (1976)
(10) Hubbard, S A et al The Fluctuation Test in Bactena. (Ed)
Kilbey, B J , Legator, M , Nichols, W, and Ramel C. Handbook of Muta-
genicity Test Procedures 2nd Edition (Elsevier, Amsterdam-New York-
Oxford, 1984) pp. 141-161
(11) Thompson, ED and Melampy, PJ An Examination of the
Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella
Typhimunum. Environmental Mutagenesis 3,453-465 (1981).
(12) Araki, A et al Improved Method for Mutagenicity Testing of
Gaseous Compounds by Using a Gas Sampling Bag Mutation Research
307, 335-344 (1994)
10
-------
(13) Pnval, M J et al Mutagemcity of Benzidme and Benzidme-Con-
gener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay
Mutation Research 136, 33-47 (1984)
{14)Zeiger, E et al Salmonella Mutagemcity Tests V Results from
the Testing of 311 Chemicals Environmental and Molecular Mutagenesis
19 2-141(1992)
(15) Simmon, V , Kauhanen, K , and Tardiff, R G Mutagemc Activ-
ity of Chemicals Identified in Drinking Water (Ed) Scott, D, Badges,
B , and Sobels, F Progress m Genetic Toxicology (Elsevier, Amsterdam,
1977) pp 249-258
(16) Hughes, TJ et al Vaporization Technique to Measure Muta-
gemc Activity of Volatile Organic Chemicals in the Ames/Salmonella
Assay Environmental Mutagenesis 9, 421-441 (1987)
(17) Matsushima, T et al Mutagemcity of the Naturally Occurring
Carcinogen Cycasm and Synthetic Methylazoxy Methane Conjugates in
Salmonella Typhimunurn Cancer Research 39, 3780-3782 (1979)
(18) Tamura, G et al Fecalase A Model for Activation of Dietary
Glycosides to Mutagens by Intestinal Flora Proceedings of the National
Academy of Sciences USA 77,4961^965 (1980)
(19) Wilcox, P et al Comparison of Salmonella Typhimunum TA
102 With Eschenchia Coh WP2 Tester Strains Mutagenesis 5, 285-291
(1990)
(20) Matsushima, T et al A Safe Substitute for Polychlonnated
Biphenyls as an Inducer of Metabolic Activation Systems (Ed) F.J de
Serres et al In Vitro Metabolic Activation in Mutagenesis Testing
(Elsevier, North Holland, 1976) pp 85-88
(21) Elliott, B.M et al Alternatives to Aroclor 1254-Induced S9 in
In Vitro Genotoxicity Assays Mutagenesis 7, 175-177 (1992)
(22) Maron, D , Katzenellenbogen, J., and Ames, B.N Compatibility
of Organic Solvents With the Salmonella/Microsome Test Mutation Re-
search 88, 343-350 (1981)
(23) Claxton, L.D et al Guide for the Salmonella Typhimunum/
Mammalian Microsome Tests for Bacterial Mutagemcity Mutation Re-
search 189, 83-91 (1987)
(24) Mahon, GAT et al Analysis of Data from Microbial Colony
Assays UKEMS Sub-Committee on Guidelines for Mutagemcity Testing
Part II. (Ed) Kirkland, D J Statistical Evaluation of Mutagemcity Test
Data (Cambridge University Press, 1989) pp 28-65
11
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-21S
August 1998
Health Effects Test
Guidelines
OPPTS 870.5140
Gene Mutation in
Aspergillus nidulans
-------
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 m 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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC. 136,etseq)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5140 Gene mutation in Aspergillus nidulans
(a) Scbpe—(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 \eq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source matenals used in developing this har-
monized OPPTS test guideline are OPPT 40 CFR 7985140 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 (A nidulans) is a eukaryotic fungus
which has been developed to detect and study a variety of genetic phenom-
ena including 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 populations The methionine and 2-thioxanthme forward muta-
tion systems can be used to detect mutations in A nidulans
(c) Definitions. The definitions in section 3 of TSCA and m 40 CFR
Part 792—Good'Laboratory Practice Standards (GLP) apply to this test
guideline The following definition also applies to this test guideline.
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 aflatoxm BI
(e) Test method—(1) Principle. Conidia are exposed to the test
chemical both with and without metabolic activation and plated on selec-
tive 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 control 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-
thioxanthme systems the haplotd Glascow biAl, meth Gl strain is the most
commonly used strain although other strains may be appropnate Any
translocation-free strain which produces green colonies on tmoxanthme
free medium and yellow colonies on medium containing thioxanthme may
be used in the thioxanthme system
-------
(it) 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
(in) 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, comdial chains broken up, mycehal debns 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 a test sub-
stance both m 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 appropnate vehicle and then
further diluted in vehicle for use m the assay.
(11) 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 appropnate, a positive response should be confirmed by
using a narrow range of test concentrations
(f) Test Performance—(1) Treatment Germinating or quiescent
conidia m liquid suspension should be exposed to the test chemical at
-------
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 centnfugation 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 methiomne system,
conidia should be plated on methionine deficient medium for mutant selec-
tion and on medium supplemented with methionine to determine survival
(n) Thioxanthine system. (A) For the 2-thioxanthme system, treated
conidia should be plated on nitrogen-free glucose and salts minimal me-
dium containing 2-thioxanthme
(B) After incubation, green colonies should be counted and isolated
by restreakmg 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 endpomt (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.
(n) Fifteen to twenty plates per concentration are preferred for the
2-thioxanthme 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 m
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 appropnate
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
-------
be based upon detection of a reproducible and statistically significant posi-
tive response for at least one of the test points
(a) 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 nonmutagemc in this system
(in) Both biological and statistical significance should be considered
together in the evaluation
(4) Test evaluation, (i) Positive results from the methionme and 2-
thioxanthine systems in A mdulans indicate that, under the test conditions,
the test substance causes gene (point) mutations in the DNA of this orga-
nism caused by base-pair changes and small deletions in the genome
(11) Negative results indicate that under the test conditions the test
chemical is not mutagenic in A mdulans
(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
(u) Test chemical vehicle, doses used, rationale for dose selection,
and toxicity data
(in) Method used for preparation of comdia
(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 tunes and temperature
(vu) Positive and negative controls
(vm) 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 Sa/m0/ie//a/marnmalian-microsome mutagemcity test. Mutation
Research 31.347-364 (1975)
(2) Kafer, E et al Aspergillus mdulans' systems and results of tests
for chemical induction of mitotic segregation and mutation. I Diploid and
-------
duplication assay systems a report of the U S EPA s Gene-Tox Program
Mutation Research 98 1-48 (1982)
(3) Munson, RJ and Goodhead, DT 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 mdulans 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)
-------
£EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-9S-216
August 1998
Health Effects Test
Guidelines
OPPTS 870.5195
Mouse Biochemical
Specific Locus Test
-------
INI RODUCTION
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 m 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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC l^etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 5195 Mouse biochemical specific locus test.
(a) Scope—(I) 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 798.5195 Mouse bio-
chemical specific locus test 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. The mouse biochemical specific locus test (MBSL) may
be used to detect and quantitate mutations originating in the germ line
of a mammalian species
(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
Biochemical specific locus mutation is a genetic change resulting from
a DNA lesion causing alterations in proteins that can be detected by
electrophoretic methods
Germ line comprises the cells in the gonads of higher eukaryotes,
which are the earners of the genetic information for the species.
(d) Test method—(1) Principle. The principle of the MBSL is that
heritable damage to the genome can be detected by electrophoretic analysis
of proteins in the tissues of the progeny of mice treated with germ cell
mutagens
(2) Description. For technical reasons, males rather than females are
generally treated with the test chemical Treated males are then mated to
untreated females to produce FI progeny Both blood and kidney samples
are taken from progeny for electrophoretic analysis. Up to 33 loci can
be examined by starch-gel electrophoresis and broad-range isoelectric fo-
cussing. Mutants are identified by variations from the normal
electrophoretic pattern Presumed mutants are bred to confirm the genetic
nature of the change
(3) Animal selection—(i) Species and strain. Mice should be used
as the test species Although the biochemical specific locus test could be
performed in a number of inbred strains, in the most frequently used cross,
C57BL/6 females are mated to DBA/2 males to produce (C57BL/6 x
DBA/2) FI progeny
(ii) Age. Healthy, sexually mature (at least 8 weeks old) animals
should be used for treatment and breeding
-------
(in) Number. A decision on the minimum number of treated animals
should take into account possible effects of the test chemical on the fertil-
ity of the treated animals Other considerations should include
(A) The production of concurrent spontaneous controls
(B) The use of positive controls
(C) The power of the test
(4) Control groups—(i) Concurrent controls. An appropriate num-
ber of concurrent control loci should be analyzed in each experiment
These should be partly derived from matings of untreated animals (from
5 to 20 percent of the treated matings), although some data on control
loci can be taken from the study of the alleles transmitted from the un-
treated parent in the experimental cross However, any laboratory which
has had no poor experience with the test should produce a spontaneous
control sample of about 5,000 progeny animals and a positive control sam-
ple (using 100 mg/kg ethylmtrosourea) of at least 1,200 offspnng
(u) Historical controls. Long-term, accumulated spontaneous control
data (currently, 1 mutation in 1,200,000 control loci screened) are available
for comparative purposes
(5) Test chemicals—(i) Vehicle. When possible, test chemicals
should be dissolved or suspended in distilled water or buffered isotonic
saline Water-insoluble chemicals should be dissolved or suspended m ap-
propriate vehicles The vehicle used should neither interfere with the test
chemical nor produce major toxic effects Fresh preparations of the test
chemical should be employed
(n) Dose levels Usually, only one dose need be tested. This should
be the maximum tolerated dose (MTD), the highest dose tolerated without
toxic effects. Any temporary sterility induced due to elimination of
spermatogoma at this dose must be of only moderate duration, as deter-
mined by a return of males to fertility within 80 days after treatment For
evaluation of dose-response, it is recommended that at least two dose lev-
els be tested.
(m) Route of administration. Acceptable routes of administration
include, but are not limited to, gavage, inhalation, and mixture with food
or water, and mtrapentoneal or intravenous injections
(e) Test performance—(1) Treatment and mating. Male DBA/2
mice should be treated with the test chemical and mated to virgin
C57BL/6 females immediately after cessation of treatment Each treated
male should be mated to new virgin C57BL/6 females each week Each
pairing will continue for a week until the next week's mating is to begin
This mating schedule permits sampling of all post-spermatogomal stages
of germ-cell development during the first 7 weeks after exposure
-------
Spermatogonial stem cells are studied thereafter Repeated mating cycles
should be conducted until sufficient offspring have been obtained to meet
the power criterion of the assay for spermatogonial stern cells
(2) Examination of offspring—(i) Birth and weaning. Offspring
should be examined at birth and at weaning for externally detectable
changes in morphology and behavior, these could be due to dominant
mutations Such characteristics may include, but are not limited to, vari-
ations in coat color, appearance of eyes, size (in which case weighing
of variant animals and httermates should be earned out), fur texture, etc
Gross changes in external form and behavior should also be sought Scru-
tiny of such visible characteristics of all animals should be made during
all subsequent manipulations of the animals
(n) Tissue sampling Blood (about 0 1 mL) and one kidney should
be removed from progeny mice under anesthesia Both tissues are then
prepared for analysis by electrophoresis
(in) Electrophoresis The gene products of 6 loci should be analyzed
in the blood sample by broad-range isoelectnc focusing and of 27 loci
in the kidney sample by starch-gel electrophoresis and enzyme-specific
staining Details on these procedures are included in paragraphs (h)(2) and
(h)(3) of this guideline
(iv) Mutant identification Presumptive electrophoretic mutants
should be identified by variation from the normal electrophoretic banding
patterns Reruns of all variant samples should be performed to confirm
the presence of altered banding patterns Samples from parents of progeny
exhibiting banding pattern variations should be assayed to determine
whether the variant was induced by the experimental treatment or was
pre-existing. All treatment-induced variants are bred to determine the ge-
netic nature of the change.
(f) Data and reports—(1) Treatment of results. Data should be pre-
sented in tabular form and should permit independent analysis of cell
stage-specific effects, and dose-dependent phenomena The data should be
recorded and analyzed in such a way that clusters of identical mutations
are clearly identified The individual mutants detected should be thor-
oughly descnbed In addition, concurrent positive control data (if em-
ployed) and spontaneous control data should also be tabulated These con-
current controls should be added to, as well as compared with, the histori-
cal control data
(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 response, one of which is a statistically significant dose-
related increase in the frequency of electrophoretic mutations Another en-
-------
tenon may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of these test points
(11) A test chemical which does not produce a statistically significant
increase in the frequency of electrophoretic mutations over the spontaneous
frequency, or a statistically significant and reproducible positive response
for at least one of the test points, is considered nonmutagemc in this sys-
tem, provided that the sample size is sufficient to exclude a biologically
significant increase in mutation frequency
(m) Biological and statistical significance should be considered to-
gether in the evaluation
(4) Test evaluation (i) Positive results in the MBSL indicate that,
under the test conditions, the test chemical induces heritable gene
mutations in a mammalian species
(11) Negative results indicate that, under the test conditions, the test
chemical does not induce heritable gene mutations in a mammalian spe-
cies
(5) Test report In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, the following specific information
should be reported*
(i) Strain, age, and weight of animals used, numbers of animals of
each sex in experimental and control groups
(11) Test chemical vehicle, doses used, rationale for dose selection,
and toxicity data, if available
(m) Route and duration of exposure
(iv) Mating schedule
(v) Number of loci screened for both treated and spontaneous data
(vi) Cntena for scoring mutants
(vii) Number of mutants found/locus
(viu) Loci at which mutations were found
(ix) Use of concurrent negative and positive controls
(x) Dose-response relationship, if applicable
(g) Additional requirements. Testing facilities conducting the mouse
biochemical specific locus test in accordance with this section should, in
addition to adhering to the provisions of 40 CFR 792 190 and 792 195,
obtain, adequately identify and retain for at least 10 years, acceptable 35-
mm photographs (and their negatives) of the stained isoelectnc-focusmg
-------
columns and the stained starch-gels obtained following analjses of blood
and kidney preparations respectively, from mutant nuce, their siblings,
and their parents
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Johnson, FM et al The detection of mutants in mice by
electrophoresis Results of a model induction experiment with
procarbazme Genetics 97 113-124 (1981)
(2) Johnson, F M and Lewis, S E Mutation rate determinations based
on electrophoretic analysis of laboratory mice Mutation Research 82 125-
135(1981)
(3) Johnson, F M and Lewis, S E Electrophoretically detected ger-
minal mutations induced by ethylmtrosourea in the mouse Proceedings
of the National Academy of Sciences 78 3138-3141 (1981)
(4) Lewis, S E et al Dominant visible and electrophoretically ex-
pressed mutations induced in male mice exposed to ethylene oxide by in-
halation Environmental Mutagenesis 8 867-872 (1986)
-------
vvEPA
United Stales
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101)
EPA712-C-98-217
August 1998
Health Effects Test
Guidelines
OPPTS 870.5200
Mouse Visible Specific
Locus Test
-------
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 Rodenucide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 5200 Mouse visible specific locus test
(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 5200 Mouse visible
specific locus test and OPP 84-2 Mutagemcity Testing (Pesticide Assess-
ment Guidelines, Subdivision F—Hazard Evaluation, Human and Domes-
tic Animals) EPA report 540/09-82-025, 1982
(b) Purpose. The mouse visible specific locus test (MSLT) may be
used to detect and quantitate mutations in the germ line of a mammalian
species
(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
Germ line is the cells in the gonads of higher eukaryotes which are
the earners of the genetic information for the species
Visible specific locus mutanon is a genetic change that alters factors
responsible for coat color and other visible characteristics of certain mouse
strains
(d) Test method—(1) Principle, (i) The principle of the MSLT is
to cross individuals who differ with respect to the genes present at certain
specific loci, so that a genetic alteration involving the standard gene at
any one of these loci will produce an offspring detectably different from
the standard heterozygote The genetic change may be detectable by var-
ious means, depending on the loci chosen to be marked
(11) Three variations of the method currently exist for detecting newly
arising point mutations in mouse germ cells
(A) The visible specific locus test using either five or seven loci.
(B) The biochemical specific locus test using up to 20 enzymes
(C) The test for mutations at histocompatibihty loci
(in) Of the three tests, the visible specific locus test has been most
widely used m assessing genetic hazard due to environmental agents. It
is the method descnbed m this guideline
(2) Description. For technical reasons, males rather than females are
generally treated with the test agent Treated males are then mated to fe-
males which are genetically homozygous for certain specific visible marker
-------
loci Offspring are examined in the next generation for evidence that a
new mutation has arisen
(3) Animal selection—(i) Species and strain. Mice should be used
as the test species Male mice should be either (CaHxlOOFi or
(101xC3H)Fi hybrids Females should be T stock virgins
(n) Age. Healthy sexually mature animals should be used
(in) Number. A decision on the minimum number of treated animals
should take into account the spontaneous variation of the biological charac-
terization being evaluated Other considerations should include
(A) The use of either historical or concurrent controls
(B) The power of the test
(C) The minimal rate of induction required
(D) The use of positive controls
(E) The level of significance desired
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups
(4) Control groups—(i) Concurrent controls. The use of positive
or spontaneous controls is left to the discretion of the investigator. How-
ever, any laboratory which has had no prior expenence with the test
should, at its first attempt, produce a negative control sample of 20,000
and a positive control, using 100 mg/kg 1-ethylnitrosourea, in a sample
of 5,000 off spring
(u) Historical controls. Long-term, accumulated spontaneous control
data of 43/801,406 are available for comparative purposes
(5) Test chemicals—
-------
(in) Route of administration. Acceptable routes of administration
include gavage, inhalation, admixture with food or water, and IP or IV
injections
(e) Test performance—(1) Treatment and mating Hybrid FI
(C3Hxl01) or (101xC3H) male mice should be treated with the test sub-
stance and immediately mated to virgin T stock females Each treated male
should be mated to a fresh group of two to four virgin females each week
for 7 weeks, after which he should be returned to the first group of females
and rotated through the seven sets of females repeatedly This mating
schedule generally permits sampling of all postspermatagonial stages of
germ cell development during the first 7 weeks and rapid accumulation
of data for exposed spermatagonial stem cells thereafter Repeated mating
cycles should be conducted until the entire spermatogomal cycle has been
evaluated and enough offspring have been obtained to meet the power
criterion of the assay
(2) Examination of offspring, (i) Offspring may be examined at (or
soon after) birth but must be examined at about 3 weeks of age at which
time the numbers of mutant and nonmutant offspnng in each Utter should
be recorded
(11) Nonmutant progeny should be discarded Mutant progeny should
be subjected to genetic tests for verification
(f) Data and report—(1) Treatment of results. Data should be pre-
sented in tabular form and should permit independent analysis of cell-stage
specific effects and dose-dependent phenomena. The data should be re-
corded and analyzed in such a way that clusters of identical mutations
are clearly identified The individual mutants detected should be thor-
oughly descnbed. In addition, concurrent positive and negative control
data, if they are available, should be tabulated so that it is possible to
differentiate between concurrent (when available) and long-term accumu-
lated mutation frequencies
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods
(3) Interpretation of results. (0 There are several criteria for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of specific locus mutations Another en-
tenon may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test points.
(11) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of specific locus mutations
or a statistically significant and reproducible positive response at any one
of the test points is considered nonmutagenic in this system
-------
(ui) Both biological and statistical significance should be considered
together in the evaluation
(4) Test evaluation, (i) Positive results in the MSLT indicate that
under the test conditions the test substance induces heritable gene
mutations in the test species
(11) Negative results indicate that under the test conditions the test
substance does not induce heritable gene mutations in the test species
(5) Test report In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, the following specific information
should be reported
(i) Strain, age, and weight of animals used, number of animals of
each sex in experimental and control groups
(11) Test chemical vehicle, doses used, rationale for dose selection,
and toxicity data
(iii) Route and duration of exposure
(iv) Mating schedule
(v) Time of examination for mutant progeny
(vi) Criteria for sconng mutants
(vu) Use of concurrent or negative controls
(vui) Dose response relationship, if applicable
(g) Additional requirements. Testing facilities conducting the mouse
visible specific locus test in accordance with this section should, in addi-
tion to adhering to the provisions of 40 CFR 792 190 and 792.195 obtain,
and retain for at least 10 years, acceptable 35-mm color photographs (and
their negatives) demonstrating the visible mutations observed in mutant
animals and the lack of such mutations in their siblings and parents
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Russell, L.B et al. The mouse specific locus test with agents other
than radiations interpretation of data and recommendations for future
work A report of the U S EPA's Gene-Tox Program Mutation Research
86.329-354(1981)
(2) [Reserved]
4
-------
vvEPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-218
August 1998
Health Effects Test
Guidelines
OPPTS 870.5250
Gene Mutation in
Neurospora crassa
-------
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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC 136, etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www.epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5250 Gene mutation m 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 Mutagemcity 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 (N crassa) is a eukaryouc fungus
which has been developed to detect and study a variety of genetic phenom-
ena including chemically induced mutagenesis N crassa can be used to
detect both forward and reverse gene mutation These mutations are de-
tected by biochemical or morphological changes in the treated population
The most commonly used mutation assay in N 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
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 noncolomal strains of N crassa grown on media containing sorbose
as well as glucose. Under these conditions, colonies are formed and repro-
ducible colonial morphology results Adenme-requinng 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 m this assay The use of other
strains may also be appropriate
(11) 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
(111) Media. Fne'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 hours to select colonies with spreading growth pat-
terns in which myceha cover the entire flask Flasks should be incubated
at 23-25°C and those with bright orange conidia selected for preparation
of comdial suspensions Suspensions should be diluted for use in distilled
water
(5) Metabolic activation. Conidia should be exposed to a 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
(n) 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 nongrowmg
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
(11) 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
(111) 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 appropnate
statistical techniques
(3) Interpretation of results, (i) There are several catena 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
(11) 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.
(in) Both biological and statistical significance should be considered
together in the evaluation
-------
(4) Test evaluation, (i) Positive results from the ad-3 system in N
crassa indicate that, under the test conditions, the test substance causes
mutations in the DNA of this organism
(n) Negative results indicate that under the test conditions the test
substance is not mutagenic in N 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
(11) Test chemical vehicle, doses used, and rationale for dose selec-
tion
(111) 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 m the assay
(vu) Dose-response relationship, if applicable
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Brockman, RE. and de Serres, FJ. Induction of ad-3 mutants
of Neurospora crassa by 2-aminopunne Genetics 48' 597-604 (1963)
(2) de Serres, FJ 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 aflatoxm
Bi to metabolites mutagenic in Neurospora crassa. Mutation Research
37:27-32 (1976)
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-220
August 1998
Health Effects Test
Guidelines
OPPTS 870.5275
Sex-Linked Recessive
Lethal Test in Drosophila
melanogaster
-------
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
(7US.C 136, etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132. This guideline is also available electronically
m PDF (portable document format) from EPA's World Wide Web site
(http-//www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines."
-------
OPPTS 870.5275 Sex-linked recessive lethal test in Drosophiia
melanogaster.
(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 5275 Sex-hnked
recessive lethal test in Drosophiia melanogaster and OECD 477 Genetic
Toxicology Sex-Linked Recessive Lethal Test in Drosophiia
melanogaster
(b) Purpose. The sex-linked recessive lethal (SLRL) test using
Drosophiia melanogaster (D melanogaster) detects the occurrence of
mutations, both point mutations and small deletions, in the germ line of
the insect This test is a forward mutation assay capable of screening for
mutations at about 800 loci on the X-chromosome. This represents about
80 percent of all X-chromosome loci The X-chromosome represents ap-
proximately one-fifth of the entire haploid 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 definitions also apply to this test guideline
Lethal mutation is a change m the genome which, when expressed,
causes death to the earner.
Recessive mutation is a change m the genome which is expressed
in the homozygous or hemizygous condition
Sex-hnked genes are present on the sex (X or Y) chromosomes. Sex-
linked genes in the context of this guideline refer only to those located
on the X-chromosome
(d) Reference substances. These may include, but need not be lim-
ited to, ethyl methanesulfonate or Af-mtrosodimethylamme
(e) Test method—(1) Principle. Mutations in the X-chromosome of
D melanogaster are phenotypically expressed in males carrying the mutant
gene. When the mutation is lethal in the hemizygous condition, its pres-
ence is inferred from the absence of one class of male offspring out of
the two that are normally produced by a heterozygous female. The SLRL
test takes advantage of these facts by means of specially marked and ar-
ranged chromosomes
(2) Description. Wild-type males are treated and mated to appropriate
females Female offspnng are mated individually to their brothers, and
in the next generation the progeny from each separate dose are scored
for phenotypically wild-type males Absence of these males indicates that
-------
a sex-lmked recessive lethal mutation has occurred in a germ cell of the
PI male
(3) Drosophila stocks. Males of a well-defined wild type stock and
females of the Muller-5 stock may be used Other appropriately marked
female stocks with multiple inverted X-chromosomes may also be used
(4) Control groups—(i) Concurrent controls. Concurrent positive
and negative (vehicle) controls should be included in each experiment
(n) Positive controls. Examples of positive controls include ethyl
methanesulfonate and Af-mtrosodimethylamine
(in) Other positive controls. Other positive control reference sub-
stances may be used
(iv) Negative controls. Negative (vehicle) controls should be in-
cluded The size of the negative (vehicle) control group should be deter-
mined by the availability of appropriate laboratory historical control data.
(5) Test chemicals—(0 Vehicle. Test chemicals should be dissolved
in water Compounds which are insoluble in water may be dissolved or
suspended m appropriate vehicles (e g , a mixture of ethanol and Tween-
60 or 80) and then diluted in water or saline poor to administration The
use of dimethylsulfoxide as a vehicle should be avoided
(n) Dose levels. For the initial assessment of mutagemcity, it is suffi-
cient to test a single dose of the test substance for screening purposes
This dose should be the maximum tolerated dose, or that which produces
some indication of toxicity, or should be the highest dose attainable. For
dose-response purposes, at least three additional dose levels should be
used
(111) Route of administration. Exposure may be oral, by injection
or by exposure to gases or vapors Feeding of the test compound may
be done in sugar solution When necessary, substances may be dissolved
m 0 7 percent NaCl solution and injected into the thorax or abdomen.
(f) Test performance—(1) Treatment and mating. Wild-type males
(3 to 5 days old) should be treated with the test substance and mated
individually to an appropriate number of virgin females from the Muller-
5 stock or females from another appropriately marked (with multiply-in-
verted X-chromosomes) stock The females should be replaced with fresh
virgins every 2 to 3 days to cover the entire germ cell cycle The offspring
of these females are scored for lethal effects corresponding to the effects
on mature sperm, mid or late stage spermatids, early spermatids, spermato-
cytes and spermatogoma at the time of treatment
(2) FI matings. Heterozygous FI females from the above crosses
should be allowed to mate individually (i e, one female per vial) with
-------
their brothers In the F2 generation, each culture should be scored for the
absence of wild-type males If a culture appears to have arisen from an
FI female carrying a lethal in the parental X-chromosome (i e, no males
with the treated chromosome are observed), daughters of that female with
the same genotype should be tested to ascertain if the lethality is repeated
m the next generation
(3) Number of matings. (i) The test should be designed with a pre-
determined sensitivity and power The number of flies in each group
should reflect these defined parameters The spontaneous mutant frequency
observed in the appropriate control group will strongly influence the num-
ber of treated chromosomes that must be analysed to detect substances
which show mutation rates close to those of the controls.
(u) Test results should be confirmed in a separate experiment
(g) Data and report—(1) Treatment of results. Data should be tab-
ulated to show the number of chromosomes tested, the number of nonfer-
tile males and the number of lethal chromosomes at each exposure con-
centration and for each mating period for each male treated Numbers of
clusters of different size per male should be reported
(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 sex-lined recessive lethals. Another en-
tenon may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test points
(n) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of sex-hnked recessive lethals
or a statistically significant and reproducible positive response at any one
of the test points is considered non-mutagenic in this system
(iii) Both biological and statistical significance should be considered
together in the evaluation
(4) Test evaluation (i) Positive results m the SLRL test in D
melanogaster indicate that under the test conditions the test agent causes
mutations in germ cells of this insect
(11) Negative results indicate that under the test conditions the test
substance is not mutagenic in D melanogaster
(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) Drosophila stock used in the assay, age of insects, number of
males treated, number of stenle males, number of F2 cultures established,
number of ¥2 cultures without progeny
(11) Test chemical vehicle, treatment and sampling schedule, exposure
levels, toxicity data, negative (vehicle) and positive controls, if appropriate
(m) Criteria for sconng lethals
(iv) Number of chromosomes tested, number of chromosomes scored,
number of chromosomes carrying a lethal mutation
(v) Historical control data, if available
(vi) Dose-response relationship, if applicable
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Sobels, F.H and Vogel, E The capacity of Drosophila for detect-
ing relevant genetic damage Mutation Research 41 95-106 (1976).
(2) Wurgler F E et al Drosophila as assay system for detecting ge-
netic changes Handbook of mutagemcity test procedures. Eds. Kilbey,
B J , Legator, M, Nichols, W, Ramel, C Elsevier/North Holland Bio-
medical, Amsterdam (1977) pp 335-373
-------
vvEPA
United States
Environmental Protection
Agency
Prevention Pestcides
and Toxic Substances
(7101)
EPA712-C-9&-221
August 1998
Health Effects Test
Guidelines
OPPTS 870.5300
In Vitro Mammalian Cell
Gene Mutation Test
-------
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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC 136, etseq)
t
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(httpV/www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 5300 In vitro mammalian cell gene mutation test.
(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 5300 Detection of
gene mutations in somatic cells in culture and OECD 476, In Vitro Mam-
malian Cell Gene Mutation Test
(b) Introduction. The in vitro mammalian ceil gene mutation test
can be used to detect gene mutations induced by chemical substances. Suit-
able cell lines include L5178Y mouse lymphoma cells, the CHO, AS52
and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid
cells (see reference in paragraph (g)(l) of this guideline) In these cell
lines the most commonly-used genetic endpomts measure mutation at thy-
nudme kinase (TK) and hypoxanthine-guanine phosphonbosyl transferase
(HPRT), and a transgene of xanthme-guanme phosphonbosyl transferase
(XPRT) The TK, HPRT and XPRT mutation tests detect different spectra
of genetic events The autosomal location of TK and XPRT may allow
the detection of genetic events (e g, large deletions) not detected at the
HPRT locus on X-chromosomes (see references in paragraphs (g)(2),
(g)(3), (g)(4),(g)(5), and (g)(6) of this guideline)
(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
Base pair substitution mutagens are substances which cause substi-
tution of one or several base pairs in the DNA
Forward mutation is a gene mutation from the parental type to the
mutant form which gives nse to an alteration or a loss of the enzymatic
activity or the function of the encoded protein
Frameshift mutagens are substances which cause the addition or dele-
tion of single or multiple base pairs in the DNA molecule.
Mutant frequency ts the number of mutant cells observed divided by
the number of viable cells
Phenotypic expression time is a penod during which unaltered gene
products are depleted from newly mutated cells
Relative suspension growth is an increase in cell number over the
expression penod relative to the negative control
Relative total growth is an increase in cell number over time com-
pared to a control population of cells, calculated as the product of suspen-
-------
sion growth relative to the negative control times cloning efficiency rel-
ative to negative control
Survival is the cloning efficiency of the treated cells when plated at
the end ot the treatment period, survival is usually expressed in relation
to the survival of the control cell population
Viability is the cloning efficiency of the treated cells at the time of
plating in selective conditions after the expression period
(d) Initial considerations. (1) In the in vitro mammalian cell gene
mutation test, cultures of established cell lines or cell strains can be used
The cells used are selected on the basis of growth ability in culture and
stability of the spontaneous mutation frequency Tests conducted m vitro
generallv require the use of an exogenous source of metabolic activation
This metabolic activation system cannot mimic entirely the mammalian
in vivo conditions Care should be taken to avoid conditions which would
lead to results not reflecting intrinsic mutagenicity Positive results which
do not reflect intrinsic mutagenicity may arise from changes in pH, osmo-
lality or high levels of cytotoxicity (see reference in paragraph (g)(7) of
this guideline)
This test is used to screen for possible mammalian mutagens and
carcinogens Many compounds that are positive in this test are mammalian
carcinogens, however, there is not a perfect correlation between this test
and carcinogemcity Correlation is dependent on chemical class and there
is increasing evidence that there are carcinogens that are not detected by
this test because they appear to act through other, non-genotoxic mecha-
nisms or mechanisms absent in bacterial cells (see reference in paragraph
(g)(6) of this guideline).
(e) Test method — (1) Principle, (i) Cells deficient in thymidme ki-
nase (TK) due to the mutation TK+A •> TK-'- are resistant to the cytotoxic
effects of the pyrimidme analogue tnfluorothymidine (TFT) Thymidme
kinase proficient cells are sensitive to TFT, which causes the inhibition
of cellular metabolism and halts further cell division. Thus mutant cells
are able to proliferate in the presence of TFT, whereas normal cells, which
contain thymidme kinase, are not. Similarly, cells deficient in HPRT or
XPRT are selected by resistance to 6-thioguanme (TG) or 8-azaguanme
(AG) The properties of the test substance should be considered carefully
if a base analogue or a compound related to the selective agent is tested
in any of the mammalian cell gene mutation tests. For example, any sus-
pected selective toxicity by the test substance for mutant and non-mutant
cells should be investigated. Thus, performance of the selection system/
agent must be confirmed when testing chemicals structurally related to
the selective agent (see reference in paragraph (g)(8) of this guideline).
(11) Cells in suspension or monolayer culture are exposed to the test
substance, both with and without metabolic activation, for a suitable period
-------
of time and subcultured to determine cytotoxicity and to allow phenotypic
expression prior to mutant selection (see references in paragraphs (g)(9),
(g)(10), (g)(l 1), (g)(12), and (g)(13) of this guideline) Cytotoxicity is usu-
ally determined by measuring the relative cloning efficiency (survival) or
relative total growth of the cultures after the treatment period The treated
cultures are maintained in growth medium for a sufficient period of time.
characteristic of each selected locus and cell type, to allow near-optimal
phenotyptc expression of induced mutations Mutant frequency is deter-
mined by seeding known numbers of cells m medium containing the selec-
tive agent to detect mutant cells, and in medium without selective agent
to determine the cloning efficiency (viability) After a suitable incubation
time, colonies are counted The mutant frequency is derived from the num-
ber of mutant colonies in selective medium and the number of colonies
in non-selective medium
(2) Description—(i) Preparations—(A) Cells. (/) A variety of cell
types are available for use in this test including subclones of L5178Y,
CHO, CHO-AS52, V79, or TK6 cells Cell types used in this test should
have a demonstrated sensitivity to chemical mutagens, a high cloning effi-
ciency and. a stable spontaneous mutant frequency Cells should be
checked for Mycoplasma contamination and should not be used if contami-
nated
(2) The test should be designed to have a predetermined sensitivity
and power The number of cells, cultures and concentrations of test sub-
stance used should reflect these defined parameters (see reference in para-
graph (g)(14) of this guideline) The minimal number of viable cells sur-
viving treatment and used at each stage in the test should be based on
the spontaneous mutation frequency A general guide is to use a cell num-
ber which is at least ten tunes the inverse of the spontaneous mutation
frequency However, it is recommended to utilise at least 106 cells Ade-
quate historical data on the cell system used should be available to indicate
consistent performance of the test
(B) Media and culture conditions. Appropnate culture media and
incubation conditions (culture vessels, temperature, CO^ concentration and
humidity) should be used Media should be chosen according to the selec-
tive systems and cell type used in the test It is particularly important that
culture conditions should be chosen that ensure optimal growth of cells
during the expression penod and colony forming ability of both mutant
and non-mutant cells
(C) Preparation of cultures. Cells are propagated from stock cul-
tures, seeded m culture medium and incubated at 37 °C. Prior to use in
this test, cultures may need to be cleansed of pre-existing mutant cells.
(D) Metabolic activation. Cells 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 co-factor-supplemented
post-mitochondria! fraction (S9) prepared from the livers of rodents treated
with enzyme-inducing agents such as Aroclor 1254 (see references in para-
graphs (g)(15), (g)(16), (g)(17), and (g)(18) of this guideline) or a com-
bination of phenobarbitone and p-naphthoflavone (see references in para-
graphs (g)(19) and (g)(20) of this guideline) The post-mitochondnal frac-
tion is usually used at concentrations in the range from 1-10 percent
v/v in the final test medium The choice and condition of a metabolic
activation system may depend upon the class of chemical being tested
In some cases it may be appropriate to utilize more than one concentration
of post-mitochondnal fraction A number of developments, including the
construction of genetically engineered cell lines expressing specific activat-
ing enzymes, may provide the potential for endogenous activation The
choice of the cell lines used should be scientifically justified (e g , by the
relevance of the cytochrorne P450 isoenzyme to the metabolism of the
test substance)
(E) Test substance/preparations. Solid test substances should be dis-
solved or suspended in appropriate solvents or vehicles and diluted if ap-
propriate prior to treatment of the cells Liquid test substances may be
added directly to the test systems and/or diluted prior to treatment. Fresh
preparations should be employed unless stability data demonstrate the ac-
ceptability of storage
(u) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the cells and the S9 activity. If other
than well-known solvent/vehicles are used, their inclusion should be sup-
ported by data indicating their compatibility It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle be considered first.
When testing water-unstable substances, the organic solvents used should
be free of water Water can be removed by adding a molecular sieve
(B) Exposure concentrations. (7) Among the criteria to be consid-
ered when determining the highest concentration are cytotoxicity and solu-
bility in the test system and changes in pH or osmolality
(2) Cytotoxicity should be determined with and without metabolic ac-
tivation in the main experiment using an appropnate indicator of cell integ-
rity and growth, such as relative cloning efficiency (survival) or relative
total growth It may be useful to determine cytotoxicity and solubility in
a preliminary experiment.
(3) At least four analysable concentrations should be used. Where
there is cytotoxicity, these concentrations should cover a range from the
maximum to little or no toxicity, this will usually mean that the concentra-
tion levels should be separated by no more than a factor between 2 and
If the maximum concentration is based on cytotoxicity then it should
-------
result in approximately 10-20 percent but not less than 10 percent relative
survival (relative cloning efficiency) or relative total growth For relatively
non-cytotoxic compounds the maximum concentration should be
5 mg/ml, 5(il/ml, or 0 01 M whichever is the lowest
(4) Relatively insoluble substances should be tested up to or beyond
their limit of solubility under culture conditions Evidence of insolubility
should be determined m the final treatment medium to which cells are
exposed. It may be useful to assess solubility at the beginning and end
of the treatment, as solubility can change dunng the course of exposure
in the test system due to presence of cells, S9, serum etc Insolubility
can be detected by using the unaided eye The precipitate should not inter-
fere with the scoring
(C) Controls. (7) Concurrent positive and negative (solvent or vehi-
cle) controls both with and without metabolic activation should be included
in each experiment When metabolic activation is used the positive control
chemical should be one that requires activation to give a mutagenic re-
sponse
(2) Examples of positive control substances include
Metabolic Activation condition
Absence of exogenous meta-
bolic activation
Presence of exogenous meta-
bolic activation
Locus
HPRT
TK (small and
large colonies)
XPRT
HPRT
TK (small and
large colonies)
XPRT
Chemical
Ethyl methanesulfonate
Ethylnitrosourea
Methyl methanesulfonate
Ethyl methanesulfonate
Ethylnitrosourea
3-Methylcholanthrene
N-Nitrosodimethylamine
7 , 1 2-Dimethylbenzantnracene
Cyclophosphamide
(monohydrate)
Benzo(a)pyrene
3-Methylcholanthrene
N-Nrtrosodimethylamme
(for high levels ol S-9)
Benzo(a)pyrene
CAS number
[62-50-0]
[759-73-9}
66-27-3]
62-50-0]
759-73-9]
56-49-5]
62-75-9
57-97-6
50-18-0
[6055-1 S
SO-32-8
56-49-5
62-75-9
-21)
[50-32-8]
(3) Other appropriate positive control reference substances may be
used, eg, if a laboratory has a historical data base on 5-Bromo 2'-
deoxyundine [CAS no 59-14-3], this reference substance could be used
as well The use of chemical class-related positive control chemicals may
be considered, when available
(4) Negative controls, consisting of solvent or vehicle alone in the
treatment medium, and treated in the same way as the treatment groups
should be included In addition, untreated controls should also be used
unless there are historical control data demonstrating that no deleterious
or mutagenic effects are induced by the chosen solvent
-------
(3) Procedure—(i) Treatment with test substance. (A) Proliferating
cells should be exposed to the test substance both with and without meta-
bolic activation Exposure should be for a suitable period of time (usually
3 to 6 hours is effective) Exposure time may be extended over one or
more cell cycles
(B) Either duplicate or single treated cultures may be used at each
concentration tested When single cultures are used, the number of con-
centrations should be increased to ensure an adequate number of cultures
for analysis (e g , at least eight analysable concentrations) Duplicate nega-
tive (solvent) control cultures should be used
(C) Gaseous or volatile substances should be tested by appropnate
methods, such as in sealed culture vessels (see references in paragraphs
(g)(21) and (g)(22) of this guideline)
(11) Measurement of survival, viability, and mutant frequency. (A)
At the end of the exposure penod, cells should be washed and cultured
to determine survival and to allow for expression of the mutant phenotype
Measurement of cytotoxicity by determining the relative cloning efficiency
(survival) or relative total growth of the cultures is usually initiated after
the treatment penod
(B) Each locus has a defined minimum time requirement to allow
near optimal phenotypic expression of newly induced mutants (HPRT and
XPRT require at least 6-8 days, and TK at least 2 days) Cells are grown
in medium with and without selective agent(s) for determination of num-
bers of mutants and cloning efficiency, respectively The measurement of
viability (used to calculate mutant frequency) is initiated at the end of
the expression tune by plating in non-selective medium
(C) If the test substance is positive in the L5178Y TK+'-test, colony
sizing should be performed on at least one of the test cultures (the highest
positive concentration) and on the negative and positive controls If the
test substance is negative in the L5178Y TK+/-test, colony sizing should
be penormed on the negative and positive controls In studies using
TK6TK+'-, colony sizing may also beperformed
(0 Data and reporting—(1) Treatment of results, (i) Data should
include cytotoxicity and viability determination, colony counts and mutant
frequencies for the treated and control cultures. In the case of a positive
response in the L5178Y TK+/'test, colonies are scored using the criteria
of small and large colonies on at least one concentration of the test sub-
stance (highest positive concentration) and on the negative and positive
control. The molecular and cytogenetic nature of both large and small col-
ony mutants has been explored m detail (see references in paragraphs
(g)(23) and (g)(24) of this guideline) In the TK+'-test, colonies are scored
using the criteria of normal growth (large) and slow growth (small) colo-
nies (see reference in paragraph (g)(25) of this guideline) Mutant cells
-------
that have suffered the most extensive genetic damage have prolonged dou-
bling times and thus form small colonies This damage typically ranges
in scale from the losses of the entire gene to karyotypically visible chro-
mosome aberrations The induction of small colony mutants has been asso-
ciated with chemicals that induce gross chromosome aberrations (see ref-
erence in paragraph (g)(26) of this guideline) Less senously affected mu-
tant cells grow at rates similar to the parental cells and form large colonies
(n) Survival (relative cloning efficiencies) or relative total growth
should be given Mutant frequency should be expressed as number of mu-
tant cells per number of surviving cells
(111) Individual culture data should be provided Additionally, all data
should be summarized in tabular form
(iv) There is no requirement for verification of a clear positive re-
sponse Equivocal results should be clarified by further testing preferably
using a modification of experimental conditions Negative results need to
be confirmed on a case-by-case basis In those cases where confutation
of negative results is not considered necessary, justification should be pro-
vided Modification of study parameters to extend the range of conditions
assessed should be considered in follow-up experiments for either equivo-
cal or negative results Study parameters that might be modified include
the concentration spacing, and the metabolic activation conditions.
(2) Evaluation and interpretation of results, (i) There are several
catena for determining a positive result, such as a concentration-related,
or a reproducible increase in mutant frequency Biological relevance of
the results should be considered first Statistical methods may be used as
an aid in evaluating the test results Statistical significance should not be
the only determining factor for a positive response
(ii) A test substance, for which the results do not meet the above
criteria is considered non-mutagenic in this system.
(in) Although most studies will give clearly positive or negative re-
sults, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
(iv) Positive results for an in vitro mammalian cell gene mutation
test indicate that the test substance induces gene mutations in the cultured
mammalian cells used A positive concentration-response that is reproduc-
ible is most meaningful Negative results indicate that, under the test con-
ditions, the test substance does not induce gene mutations in the cultured
mammalian cells used
tion
(3) Test report The test report should include the following mforma-
-------
(i) Test substance
(A) Identification data and CAS no , if known
(B) Physical nature and punty
(C) Physicochenucal properties relevant to the conduct of the study
(D) Stability of the test substance
(n) Solvent/vehicle
(A) Justification for choice of vehicle/solvent
(B) Solubility and stability of the test substance in solvent/vehicle,
if known
(in) Cells.
(A) Type and source of cells
(B) Number of cell cultures
(C) Number of cell passages, if applicable.
(D) Methods for maintenance of cell cultures, if applicable
(E) Absence of Mycoplasma
(iv) Test conditions
(A) Rationale for selection of concentrations and number of cell cul-
tures including e g, cytotoxicity data and solubility limitations, if avail-
able.
(B) Composition of media, CC>2 concentration.
(C) Concentration of test substance
(D) Volume of vehicle and test substance added
(E) Incubation temperature
(F) Incubation time
(G) Duration of treatment
(H) Cell density during treatment
(I) Type and composition of metabolic activation system including
acceptability criteria
(J) Positive and negative controls
8
-------
(K) Length of expression period (including number ot cells seeded,
and subcultures and feeding schedules, if appropriate)
(L) Selective agent(s)
(M) Criteria for considering tests as positive, negative or equivocal
(N) Methods used to enumerate numbers of viable and mutant cells
(O) Definition of colonies of which size and type are considered (in-
cluding criteria for "small" and "large" colonies, as appropriate)
(v) Results
(A) Signs of toxicity
(B) Signs of precipitation
(C) Data on pH and osmolality during the exposure to the test sub-
stance, if determined
(D) Colony size if scored for at least negative and positive controls.
(E) Laboratory's adequacy to detect small colony mutants with the
L5178Y TK+/- system, where appropriate
(F) Dose-response relationship, where possible.
(G) Statistical analyses, if any
(H) Concurrent negative (solvent/vehicle) and positive control data
(I) Historical negative (solvent/vehicle) and positive control data with
ranges, means, and standard deviations.
(J) Mutant frequency
(vi) Discussion of the results
(vn) Conclusion
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Moore, M M , DeManm, D M , DeSerres, F J , and Tmdall, K R
(Eds) Banbury Report 28 Mammalian Cell Mutagenesis, Cold Spring
Harbor Laboratory (New York, New York, 1987)
(2) Chu, E.HY and Mailing, HV Mammalian Cell Genetics II
Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells
In Vitro, Proceedings of the National Academy of Science USA, 61, 1306-
1312(1968)
-------
(3) Liber, H L and Thilly, W G Mutation Assay at the Thymidme
Kinase Locus in Diploid Human Lyrnphoblasts Mutation Research 94,
467^85 (1982)
(4) Moore, MM et al Differential Mutant Quantitation at the Mouse
Lymphoma TK and CHO HGPRT Loci Mutagenesis 4, 394^03 (1989).
(5) Aaron, C S and Stankowski, Jr, L F Comparison of the AS52/
XPRT and the CHO/HPRT Assays Evaluation of Six Drug Candidates
Mutation Research 223, 121-128 (1989)
(6) Aaron, C S et al Mammalian Cell Gene Mutation Assays Work-
ing Group Report Report of the International Workshop on Standardiza-
tion of Genotoxicity Test Procedures Mutation Research 312, 235-239
(1994)
(7) Scott, D et al Genotoxicity Under Extreme Culture Conditions.
A report from ICPEMC Task Group 9 Mutation Research 257, 147-204
(1991)
(8) Chve, D. et al. Specific Gene Mutations in L5178Y Cells in Cul-
ture. A Report of the U S Environmental Protection Agency Gene-Tox
Program Mutation Research 115,225-251 (1983).
(9) Li, A P et al A Review and Analysis of the Chinese Hamster
Ovary/Hypoxanthine Guanine Phosphonbosyl Transferase System to De-
termine the Mutagemcity of Chemical Agents A Report of Phase III of
the U.S. Environmental Protection Agency Gene-Tox Program Mutation
Research 196, 17-36 (1988)
(10) Li, A.P. et al A Guide for the Performance of the Chinese Ham-
ster Ovary Cell/Hypoxanthme-Guanine Phosphonbosyl Transferase Gene
Mutation Assay Mutation Research 189, 135-141 (1987)
(11) Liber, H.L., Yandell, DW, and Little, J.B A Comparison of
Mutation Induction at the tk and hprt Loci in Human Lymphoblastoid
Cells, Quantitative Differences are Due to an Additional Class of
Mutations at the Autosomal TK Locus Mutation Research 216, 9-17
(1989)
(12) Stankowski, L F Jr, Tmdall, K R , and Hsie, A W Quantitative
and Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced
Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Mu-
tation in AS52 Cells. Mutation Research 160, 133-147 (1986).
(13) Turner, NT , Batson, AG, and Chve, D Procedures for the
L5178/TK+/-> TK>'-Mouse Lymphoma Cell Mutagemcity Assay (Eds)
Kilbey, B J. et al Handbook of Mutagemcity Test Procedures (Elsevier
Science Publishers, New York, 1984) pp 239-268
10
-------
(14) Aden, C F et al Mammalian Cell Gene Mutation Assays Based
Upon Colony Formation (Ed ) Kirkland, D J Statistical Evaluation of Mu-
tagemcity Test Data (Cambridge University Press, 1989) pp 66-101
(15) Abbondandolo, A et al Induction of 6-Thioguanme-Resistant
Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Acti-
vated Dimethylnitrosamme Mutation Research 46, 365-373 (1977).
(16) Ames, B N , McCann, J , and Yamasaki, E Methods for Detect-
ing Carcinogens and Mutagens with the Salmonella/Mammahan-
Microsome Mutagemcity Test Mutation Research 31, 347-364 (1975)
(17) Clive, D et al Validation and Characterization of the
L5178Y/TK+/-Mouse Lymphoma Mutagen Assay System Mutation Re-
search 59, 61-108 (197F9)
(18) Maron, D M and Ames, B N Revised Methods for the Sal-
monella Mutagemcity Test Mutation Research 113, 173, 215 (1983)
(19) Elliott, B M et al Alternatives to Aroclor 1254-Induced S9 in
In Vitro Genotoxicity Assays Mutagenesis 7, 175-177 (1992)
(20) Matsushima, T et al A Safe Substitute for Polychlortnated
Biphenyls as an Inducer of Metabolic Activation Systems (Eds ) de Serres,
F J , Fouts, J R , Bend, J R , and Philpot, R M In Vitro Metabolic Activa-
tion in Mutagenesis Testing (Elsevier, North-Holland, 1976) pp 85-88
(21) Krahn, DF, Barsky, FC, and McCooey, K.T CHO/HGPRT
Mutation Assay Evaluation of Gases and Volatile Liquids. (Eds.) Tice,
R R , Costa, D L, and Schaich, K M Genotoxic Effects of Airborne
Agents (New York, Plenum, 1982) pp 91-103
(22) Zamora, P O et al Evaluation of an Exposure System Using
Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens
in the CHO/HGPRT Mutation Assay Environmental Mutagenesis 5, 795-
801 (1983).
(23) Applegate, M L et al Molecular Dissection of Mutations at the
Heterozygous Thymidme Kinase Locus in Mouse Lymphoma Cells Pro-
ceedings of the National Academy of Science USA, 87, 51-55 (1990)
(24) Moore, MM et al Analysis of Tnfluorothymidme-Resistant
(TFTr) Mutants of L5178Y/TK+/-Mouse Lymphoma Cells Mutation Re-
search 151, 161-174 (1985)
(25) Yandell, D W , Dryja, T P , and Little J B Molecular Genetic
Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in
Human Cells Mutation Research 229, 89-102 (1990)
(26) Moore, M M and Doerr, C L Comparison of Chromosome Ab-
erration Frequency and Small-Colony TK-Deficient Mutant Frequency in
11
-------
L5178Y/TK-(-/-372C Mouse Lymphoma Cells Mutagenesis 5, 609-614
(1990)
12
-------
&EPA
United States
Envrronmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-223
August 1998
Health Effects Test
Guidelines
OPPTS 870.5375
In Vitro Mammalian
Chromosome Aberration
Test
-------
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
USC. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Pnnting Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http.//www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5375 In vitro mammalian chromosome aberration test.
(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 5375 In vitro mam-
malian cytogenetics and OECD 473, In Vitro Mammalian Chromosome
Aberration Test
(b) Purpose (1) The purpose of the in vitro chromosome aberration
test is to identify agents that cause structural chromosome aberrations in
cultured mammalian cells (see paragraphs (i)(l), (0(2), and (i)(3) of this
guideline) Structural aberrations may be of two types, chromosome or
chromatid With the majority of chemical mutagens, induced aberrations
are of the chromatid type, but chromosome-type aberrations also occur
An increase in polyploidy may indicate that a chemical has the potential
to induce numerical aberrations However, this guideline is not designed
to measure numerical aberrations and is not routinely used for that pur-
pose Chromosome mutations and related events are the cause of many
human genetic diseases and there is substantial evidence that chromosome
mutations and related events causing alterations in oncogenes and tumour-
suppressor genes of somatic cells are involved in cancer induction in hu-
mans and experimental animals
(2) The m vitro chromosome aberration test may employ cultures of
established cell lines, cell strains or primary cell cultures. The cells used
are selected on the basis of growth ability in culture, stability of the
karyotype, chromosome number, chromosome diversity, and spontaneous
frequency of chromosome aberrations
(c) Definitions. The definitions in section 3 of TSCA and m 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 aberration is structural chromosome damage ex-
pressed as breakage of single chromatids or breakage and reunion between
chromatids
Chromosome-type aberration is structural chromosome damage ex-
pressed as breakage, or breakage and reunion, of both chromatids at an
identical site
Endoreduplication is a process in which after an S period of DNA
replication, the nucleus does not go into mitosis but starts another S period
The result is chromosomes with 4, 8, 16, chromatids
-------
Gap is an achromatic lesion smaller than the width of one chromatid,
and with minimum misalignment of the chromatid(s)
Mitonc index is the ratio of cells in metaphase divided by the total
number of cells observed in a population of cells, an indication of the
degree of proliferation of that population
Numerical aberration is a change in the number of chromosomes
from the normal number characteristic of the cells utilized
Polyploidy is a multiple of the haploid chromosome number (n) other
than the diploid number (i e , 3n, 4n, and so on)
Structural aberration is a change in chromosome structure detectable
by microscopic examination of the metaphase stage of cell division, ob-
served as deletions and fragments, mtrachanges, and interchanges
(d) Initial considerations (1) Tests conducted in vitro generally re-
quire the use of an exogenous source of metabolic activation This meta-
bolic activation system cannot mimic entirely the mammalian in vivo con-
ditions Care should be taken to avoid conditions which would lead to
positive results which do not reflect intrinsic mutagemcity and may anse
from changes in pH, osmolahty, or high levels of cytotoxicity (see para-
graphs (i)(4) and (i)(5) of this guideline)
(2) This test is used to screen for possible mammalian mutagens and
carcinogens Many compounds that are positive in this test are mammalian
carcinogens, however, there is not a perfect correlation between this test
and carcmogenicity. Correlation is dependent on chemical class and there
is increasing evidence that there are carcinogens that are not detected by
this test because they appear to act through mechanisms other than direct
DNA damage
(e) Principle of the test method. Cell cultures are exposed to the
test substance both with and without metabolic activation At predeter-
mined intervals after exposure of cell cultures to the test substance, they
are treated with a metaphase-arrestmg substance (e.g., Colcemid® or col-
chicine), harvested, stained, and metaphase cells are analysed microscopi-
cally for the presence of chromosome aberrations
(f) Description of the method—(1) Preparations—(i) Cells. A vari-
ety of cell lines, strains, or primary cell cultures, including human cells,
may be used (e.g , Chinese hamster fibroblasts, human, or other mamma-
lian peripheral blood lymphocytes)
(u) Media and culture conditions. Appropriate culture media, and
incubation conditions (culture vessels, COa concentration, temperature and
humidity) should be used in maintaining cultures. Established cell lines
and strains should be checked routinely for stability in the modal chro-
mosome number and the absence of Mycoplasma contamination and
-------
should not be used if contaminated The normal cell-cycle time for the
cells and culture conditions used should be known
(111) Preparation of cultures—(A) Established cell lines and
strains Cells are propagated from stock cultures, seeded m culture me-
dium at a density such that the cultures will not reach confluency before
the time of harvest, and incubated at 37 °C
(B) Lymphocytes Whole blood treated with an anti-coagulant (e g ,
hepann) or separated lymphocytes obtained from healthy subjects are
added to culture medium containing a rmtogen (e g , phytohemagglutinin)
and incubated at 37 °C
(iv) Metabolic activation Cells should be exposed to the test sub-
stance both in the presence and absence of an appropnate metabolic activa-
tion system The most commonly used system is a co-factor-supplemented
post-mitochondnal fraction (S9) prepared from the livers of rodents treated
with enzyme-inducing agents such as Aroclor 1254 (see paragraphs (i)(6),
(0(7), (8)(i), and (i)(9) of this guideline), or a mixture of phenobarbitone
and p-naphthoflavone (see paragraphs (0(10), (0(11), and (0(12) of this
guideline). The post-mitochondnal fraction is usually used at concentra-
tions in the range from 1-10 percent v/v in the final test medium The
condition of a metabolic activation system may depend upon the class of
chemical being tested In some cases, it may be appropnate to utilize more
than one concentration of post-mitochondnal fraction A number of devel-
opments, including the construction of genetically engineered cell lines
expressing specific activating enzymes, may provide the potential for en-
dogenous activation The choice of the cell lines used should be scientif-
ically justified (e g , by the relevance of the cytochrome P450 isoenzyme
for the metabolism of the test substance)
(v) Test substance/preparation Solid test substances should be dis-
solved or suspended in appropnate solvents or vehicles and diluted, if ap-
propnate, pnor to treatment of the cells Liquid test substances may be
added directly to the test systems and/or diluted pnor to treatment Fresh
preparations of the test substance should be employed unless stability data
demonstrate the acceptability of storage
(2) Test conditions—(i) Solvent/vehicle The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the cells and the S9 activity If other
than well-known solvent/vehicles are used, their inclusion should be sup-
ported by data indicating their compatibility It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle be considered first
When testing water-unstable substances, the organic solvents used should
be free of water Water can be removed by adding a molecular sieve
-------
(u) Exposure concentrations (A) Among the catena to be consid-
ered when determining the highest concentration are cytotoxicity, solu-
bility in the test system and changes in pH or osrnolality
(B) Cytotoxicity should be determined with and without metabolic
activation in the main experiment using an appropriate indication of cell
integrity and growth, such as degree of confluency, viable cell counts, or
mitotic index It may be useful to determine cytotoxicity and solubility
in a preliminary experiment
(C) At least three analyzable concentrations should be used Where
cytotoxicity occurs, these concentrations should cover a range from the
maximum to little or no toxicity, this will usually mean that the concentra-
tions should be separated by no more than a factor between 2 and VlO.
At the time of harvesting, the highest concentration should show a signifi-
cant reduction in degree of confluency, cell count or mitotic index, (all
greater than 50 percent) The mitotic index is only an indirect measure
of cytotoxic/cytostatic effects and depends on the tune after treatment.
However, the mitotic index is acceptable for suspension cultures in which
other toxicity measurements may be cumbersome and impractical Infor-
mation on cell-cycle kinetics, such as average generation time (ACT),
could be used as supplementary information ACT, however, is an overall
average that does not always reveal the existence of delayed subpopula-
tions, and even slight increases in average generation time can be associ-
ated with very substantial delay in the time of optimal yield of aberrations.
For relatively non-cytotoxic compounds the maximum concentration
should be 5 ng/ml, 5mg/ml, or 0 01M, whichever is the lowest.
(D) For relatively insoluble substances that are not toxic at concentra-
tions lower than the insoluble concentration, the highest dose used should
be a concentration above the limit of solubility in the final culture medium
at the end of the treatment penod In some cases (e.g , when toxicity oc-
curs only at higher than the lowest insoluble concentration) it is advisable
to test at more than one concentration with visible precipitation. It may
be useful to assess solubility at the beginning and the end of the treatment,
as solubility can change during the course of exposure in the test system
due to presence of cells, S9, serum etc Insolubility can be detected by
using the unaided eye The precipitate should not interfere with the scor-
ing
(111) Controls (A) Concurrent positive and negative (solvent or vehi-
cle) controls both with and without metabolic activation should be included
in each expenment When metabolic activation is used, the positive control
chemical should be the one that requires activation to give a mutagenic
response.
(6) Positive controls should employ a known clastogen at exposure
levels expected to give a reproducible and detectable increase over back-
4
-------
ground which demonstrates the sensitivity of the test system Positive con-
trol concentrations should be chosen so that the effects are clear but do
not immediately reveal the identity of the coded slides to the reader Exam-
ples of positive-control substances include
Metabolic activation condition
Absence ol exogenous metabolic activation
Presence of exogenous metabolic activation
Chemical
Methyl methanesulfonate
Ethyl methanesulfonate
Ethylnilrosourea
Mitomycm C
4-Nrtroquinolme-N-Oxide
Benzo(a)pyrene
Cyclophosphamide
(monohydrate)
CAS number
66-27-3
62-50-0
759-73-
50-07-7
56-57-5
50-32-8
50-18-0
([6055-1 £
>}
-2))
(C) Other appropriate positive control substances may be used The
use of chemical class-related positive-control chemicals may be consid-
ered, when available
(D) Negative controls, consisting of solvent or vehicle alone in the
treatment medium, and treated in the same way as the treatment cultures,
should be included for every harvest time In addition, untreated controls
should also be used unless there are historical-control data demonstrating
that no deletenous or mutagenic effects are induced by the chosen solvent
(g) Procedure—(1) Treatment with test substance (i) Proliferating
cells are treated with the test substance in the presence and absence of
a metabolic-activation system Treatment of lymphocytes should com-
mence at about 48 hours after mitogemc stimulation
(n) Duplicate cultures should be used at each concentration, and are
strongly recommended for negative/solvent control cultures Where mini-
mal variation between duplicate cultures can be demonstrated (see para-
graphs (0(13) and (i)(14) of this guideline), from historical data, it may
be acceptable for single cultures to be used at each concentration.
(m) Gaseous or volatile substances should be tested by appropriate
methods, such as in sealed culture vessels (see paragraphs (0(15) and
(0(16) of this guideline)
(2) Culture harvest time In the first experiment, cells should be
exposed to the test substance both with and without metabolic activation
for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell-
cycle length after the beginning of treatment (see paragraph (0(12) of this
guideline) If this protocol gives negative results both with and without
activation, an additional experiment without activation should be done,
with continuous treatment until sampling at a tune equivalent to about
1 5 normal cell-cycle lengths Certain chemicals may be more readily de-
tected by treatment/sampling times longer than 1 5 cycle lengths. Negative
results with metabolic activation need to be confirmed on a case-by-case
-------
basis In those cases where confirmation of negative results is not consid-
ered necessary, justification should be provided
(3) Chromosome preparation Cell cultures should be treated with
Colcerrud® or colchicme usually for 1 to 3 hours prior to harvesting Each
cell culture should be harvested and processed separately for the prepara-
tion of chromosomes Chromosome preparation involves hypotonic treat-
ment of the cells, fixation and staining
(4) Analysis (i) All slides, including those of positive and negative
controls, should be independently coded before microscopic analysis Since
fixation procedures often result in the breakage of a proportion of meta-
phase cells with loss of chromosomes, the cells scored should therefore
contain a number of centromeres equal to the modal number ±2 for all
cell types At least 200 well-spread metaphases should be scored per con-
centration and control equally divided amongst the duplicates, if applica-
ble This number can be reduced when high numbers of aberrations are
observed.
(11) Though the purpose of the test is to detect structural chromosome
aberrations, it is important to record polyploidy and endoreduplication
when these events are seen
(h) Data and reporting—(1) Treatment of results (i) The experi-
mental unit is the cell, and therefore the percentage of cells with structural
chromosome aberration(s) should be evaluated Different types of struc-
tural chromosome aberrations should be listed with their numbers and fre-
quencies for experimental and control cultures Gaps are recorded sepa-
rately and reported but generally not included in the total aberration fre-
quency
(11) Concurrent measures of cytotoxicity for all treated and negative
control cultures in the main aberration expenment(s) should also be re-
corded.
(in) Individual culture data should be provided Additionally, all data
should be summarized in tabular form
(iv) There is no requirement for verification of a clear positive re-
sponse Equivocal results should be clarified by further testing preferably
using modification of experimental conditions The need to confirm nega-
tive results has been discussed in paragraph (g)(2) of this guideline. Modi-
fication of study parameters to extend the range of conditions assessed
should be considered in follow-up experiments Study parameters that
might be modified include the concentration spacing and the metabolic
activation conditions
(2) Evaluation and interpretation of results (i) There are several
criteria for determining a positive result, such as a concentration-related
-------
increase or a reproducible increase in the number of cells with chro-
mosome aberrations Biological relevance of the results should be consid-
ered first Statistical methods may be used as an aid in evaluating the
test results (see paragraphs (0(3) and (0(13) of this guideline) Statistical
significance should not be the only determining factor for a positive re-
sponse
(11) An increase in the number of polyploid cells may indicate that
the test substance has the potential to inhibit mitotic processes and to in-
duce numencal chromosome aberrations An increase in the number of
cells with endoreduphcated chromosomes may indicate that the test sub-
stance has the potential to inhibit cell-cycle progression (see paragraphs
(i)(17) and (i)(18) of this guideline)
(111) A test substance for which the results do not meet the criteria
in paragraphs (h)(2)(i) and (h)(2)(u) of this guideline is considered non-
mutagenic in this system
(iv) Although most experiments will give clearly positive or negative
results, m rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of tunes the experiment is repeated
(v) Positive results from the in vitro chromosome aberration test indi-
cate that the test substance induces structural chromosome aberrations in
cultured mammalian somatic cells. Negative results indicate that, under
the test conditions, the test substance does not induce chromosome aberra-
tions in cultured mammalian somatic cells
(3) Test report The test report should include the following informa-
tion
(i) Test substance
(A) Identification data and CAS no , if known
(B) Physical nature and punty
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known
(11) Solvent/vehicle
(A) Justification for choice of solvent/vehicle
(B) Solubility and stability of the test substance in solvent/vehicle,
if known
(m) Cells.
(A) Type and source of cells
-------
(B) Karyotype features and suitability of the cell type used
(C) Absence of M\copla$ma, if applicable
(D) Information on cell-cycle length
(E) Sex of blood donors, whole blood or separated lymphocytes,
mitogen used
(F) Number of passages, if applicable
(G) Methods for maintenance of cell cultures if applicable.
(H) Modal number of chromosomes
(iv) Test conditions
(A) Identity of metaphase arresting substance, its concentration and
duration of cell exposure
(B) Rationale for selection of concentrations and number of cultures
including, e g , cytotoxicity data and solubility limitations, if available
(C) Composition of media, CC>2 concentration if applicable.
(D) Concentration of test substance
(E) Volume of vehicle and test substance added
(F) Incubation temperature
(G) Incubation time
(H) Duration of treatment
(I) Cell density at seeding, if appropriate
(J) Type and composition of metabolic activation system, including
acceptability criteria.
(K) Positive and negative controls
(L) Methods of slide preparation
(M) Cntena for scoring aberrations
(N) Number of metaphases analyzed
(O) Methods for the measurements of toxicity
(P) Cntena for considenng studies as positive, negative or equivocal
(v) Results
8
-------
(A) Signs of toxicity e g , degree of confluency cell-cycle data cell
counts, nutotic index
(B) Signs of precipitation
(C) Data on pH and osmolahty of the treatment medium, if deter-
mined
(D) Definition for aberrations, including gaps
(E) Number of cells with chromosome aberrations and type of chro-
mosome aberrations given separately for each treated and control culture
(F) Changes in ploidy if seen
(G) Dose-response relationship, where possible
(H) Statistical analyses, if any
(I) Concurrent negative (solvent/vehicle) and positive control data
(J) Historical negative (solvent/vehicle) and positive control data, with
ranges, means and standard deviations
(vi) Discussion of the results
(vn) Conclusion.
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Evans, HJ Cytological Methods for Detecting Chemical
Mutagens Chemical Mutagens, Principles and Methods for their Detec-
tion, Vol 4, Hollaender, A Ed Plenum Press, New York and London,
pp 1-29(1976)
(2) Ishidate, M Jr and Sofum, T The In Vitro Chromosomal Aberra-
tion Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture.
Progress in Mutation Research, Vol 5, Ashby, J et al, Eds Elsevier
Science Publishers, Amsterdam-New York-Oxford, pp. 427-432 (1985)
(3) Galloway, S M et al Chromosome aberration and sister chromatid
exchanges in Chinese hamster ovary cells Evaluation of 108 chemicals
Environmental and Molecular Mutagenesis 10 (suppl 10), 1-175 (1987)
(4) Scott, D et al Genotoxicity under Extreme Culture Conditions.
A report from ICPEMC Task Group 9 Mutation Research 257, 147-204
(1991)
(5) Monta, T et al Clastogemcity of Low pH toVanous Cultured
Mammalian Cells Mutation Research 268, 297-305 (1992)
-------
(6) Ames, B N , McCann, J and Yamasaki, E Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian Microsome
Mutagenicity Test Mutation Research 31, 347-364 (1975)
(7) Maron, D M and Ames, B N Revised Methods for the Salmonella
Mutagenicity Test Mutation Research 113, 173-215 (1983)
(8) Natarajan, AT et al Cytogenetic Effects of Mutagens/Carcmo-
gens after Activation in a Microsomal System In Vitro, I Induction of
Chromosome Aberrations and Sister Chromatid Exchanges by
Diethylnitrosamine (DEN) and Dimethylmtrosamine (DMN) in CHO Cells
m the Presence of Rat-Liver Microsomes Mutation Research 37, 83-90
(1976)
(9) Matsuoka, A., Hayashi, M and Ishidate, M, Jr Chromosomal
Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro Muta-
tion Research 66, 277-290 (1979)
(10) Elliot, B M et al Report of UK Environmental Mutagen Society
Working Party Alternatives to Aroclor 1254-mduced S9 in In Vitro
Genotoxicity Assays Mutagenesis 7, 175-177 (1992)
(11) Matsushima, T et al A Safe Substitute for Polychlonnated
Biphenyls as an Inducer of Metabolic Activation Systems de Serres, F J ,
Fouts, J R , Bend, J R and Philpot, R M Eds In Vitro Metabolic Activa-
tion in Mutagenesis Testing, Elsevier, North-Holland, pp 85-88 (1976).
(12) Galloway, S M et al Report from Working Group on In Vitro
Tests for Chromosomal Aberrations Mutation Research 312, 241-261
(1994)
(13) Richardson, C et al Analysis of Data from In Vitro Cytogenetic
Assays Statistical Evaluation of Mutagenicity Test Data Kirkland, D.J.,
Ed. Cambridge University Press, Cambridge, pp 141-154 (1989)
(14) Soper, K..A and Galloway S M Replicate Flasks are not Nec-
essary for In Vitro Chromosome Aberration Assays in CHO.Cells Muta-
tion Research 312,139-149 (1994)
(15) Krahn, D.F, Barsky, FC and McCooey, KT CHO/HGPRT
Mutation Assay Evaluation of Gases and Volatile Liquids Tice, R R,
Costa, D.L, Schaich, K.M Eds Genotoxic Effects of Airborne Agents.
New York, Plenum, pp 91-103 (1982)
(16) Zamora, PO et al Evaluation of an Exposure System Using
Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens
in the CHO/HGPRT Mutation Assay Environmental Mutagenesis 5, 795-
801 (1983)
10
-------
(17) Locke-Huhle, C Endoreduplication in Chinese hamster cells dur-
ing alpha-radiation induced G2 arrest Mutation Research 119, 403-413
(1983)
(18) Huang, Y Change, C and Trosko, JE Aphidicohn - induced
endoreduphcation in Chinese hamster cells Cancer Research 43 136?-
1364(1983)
11
-------
United Slates prevention Pesticides EPA712-C-98-224
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5380
Mammalian
Spermatogonial
Chromosome Aberration
Test
-------
INTKODUC PION
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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.SC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies' call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 5380 Mammalian spermatogomal chromosome aberration
test.
(a) Scope—(I) 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 is OECD 483, Mammalian Spermatogomal
Chromosome Aberration Test
(b) Purpose (1) The purpose of the in vivo mammalian
spermatogomal chromosome aberration test is to identify those substances
that cause structural aberrations in mammalian spermatogomal cells (see
paragraphs (i)(l), (i)(2), (0(3), (i)(4), and (i)(5) of this guideline). Struc-
tural aberrations may be of two types, chromosome or chromatid. With
the majority of chemical mutagens, induced aberrations are of the chro-
matid type, but chromosome-type aberrations also occur This guideline
is not designed to measure numencal aberrations and is not routinely used
for this purpose Chromosome mutations and related events are the cause
of many human genetic diseases
(2) This test measures chromosome events m spermatogomal germ
cells and is, therefore, expected to be predictive of induction of inheritable
mutations in germ cells
(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
Chromand-type aberration is structural chromosome damage ex-
pressed as breakage of single chromatids or breakage and reunion between
chromatids.
Chromosome-type aberration is structural chromosome damage ex-
pressed as breakage, or breakage and reunion, of both chromatids at an
identical site
Gap is an achromatic lesion smaller than the width of one chromatid,
and with minimum misalignment of the chromatids
Numencal aberration is a change in the number of chromosomes
from the normal number characteristic of the animals utilized
Polyploidy is a multiple of the haploid chromosome number (n) other
than the diploid number (i e , 3n, 4n, and so on)
Structural aberration is a change in chromosome structure detectable
by microscopic examination of the metaphase stage of cell division, ob-
served as deletions, intrachanges or interchanges
-------
(d) Initial considerations (1) Rodents are routinely used in this test
This in vivo cytogenetic test detects chromosome aberrations in
spermatogomal mitoses Other target cells are not the subject of this guide-
line
(2) To detect chromatid-type aberrations in spermatogomal cells, the
first mitotic cell division following treatment should be examined before
these lesions are lost in subsequent cell divisions Additional information
from treated spermatogomal stem cells can be obtained by meiotic chro-
mosome analysis for chromosome-type aberrations at diakinesis-metaphase
I when the treated cells become spermatocytes.
(3) This in vivo test is designed to investigate whether somatic cell
mutagens are also active in germ ceils In addition, the spermatogonial
test is relevant to assessing mutagemcity hazard in that it allows consider-
ation of factors of m vivo metabolism, pharmacokmetics, and DNA-repair
processes
(4) A number of generations of spermatogonia are present in the testis
with a spectrum of sensitivity to chemical treatment Thus, the aberrations
detected represent an aggregate response of treated spermatogomal cell
populations, with the more numerous differentiated spermatogomal cells
predominating Depending on their position within the testis, different gen-
erations of spermatogonia may or may not be exposed to the general cir-
culation, because of the physical and physiological Sertoh cell barrier and
the blood-testis barrier
(5) If there is evidence that the test substance, or a reactive
metabolite, will not reach the target tissue, it is not appropriate to use
this test
(e) Principle of the test method Animals are exposed to the test
substance by an appropriate route of exposure and are sacrificed at appro-
priate times after treatment Prior to sacrifice, animals are treated with
a metaphase-arresting agent (e g , colchicme or Colcemid®). Chromosome
preparations are then made from germ cells and stained, and metaphase
cells are analyzed for chromosome aberrations
(f) Description of the method—(1) Preparations—(i) Selection of
animal species Male Chinese hamsters and mice are commonly used
However, males of other appropnate mammalian species may be used.
Commonly used laboratory strains of healthy young-adult animals should
be employed At the commencement of the study the weight variation of
animals should be minimal and not exceed ±20 percent of the mean
weight
(u) Housing and feeding conditions. The temperature in the experi-
mental animal room should be 22 °C ( ±3 °C) Although the relative hu-
midity should be at least 30 percent and preferably not exceed 70 percent
-------
other than during room cleaning, the aim should be 50-60 percent Light-
ing should be artificial, the sequence being 12 hours tight, 12 hours dark
For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water The choice of diet may be influenced by the
need to ensure a suitable admixture of a test substance when administered
by this method Animals may be housed individually, or be caged in small
groups
(m) Preparation of the animals Healthy young-adult males should
be randomly assigned to the control and treatment groups Cages should
be arranged in such a way that possible effects due to cage placement
are minimized The animals are identified uniquely The animals are accli-
mated to the laboratory conditions for at least 5 days prior to the start
of the study
(iv) Preparation of doses Solid test substances should be dissolved
or suspended in appropriate solvents or vehicles and diluted, if appropnate,
prior to dosing of the animals Liquid test substances may be dosed di-
rectly or diluted pnor to dosing Fresh preparations of the test substance
should be employed unless stability data demonstrate the acceptability of
storage
(2) Test conditions—(i) Solvent/vehicle The solvent/vehicle should
not produce toxic effects at the dose levels used and should not be sus-
pected of chemical reaction with the test substance If other than well-
known solvents/vehicles are used, their inclusion should be supported with
reference data indicating their compatibility It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle should be considered
first.
(ii) Controls (A) Concurrent positive and negative (solvent/vehicle)
controls should be included in each test Except for treatment with the
test substance, animals in the control groups should be handled in an iden-
tical manner to animals in the treated groups
(B) Positive controls should produce structural chromosome aberra-
tions in vivo m spermatogomal cells when administered at exposure levels
expected to give a detectable increase over background Positive control
doses should be chosen so that the effects are clear but do not immediately
reveal the identity of the coded slides to the reader It is acceptable that
the positive control be administered by a route different from the test sub-
stance and sampled at only a single tune In addition, the use of chemical
class-related positive control chemicals may be considered, when available
Examples of positive control substances include.
-------
Chemical
CAS number
Cyclophosphamide (monohydrate)
Cyclohexylamine
Mitomycin C
Monomeric acrylamide
Tnethylenemeiamine
[50-18-0
(6055-19-2)}
108-91-8]
50-07-7)
79-06-1]
51-18-3]
(C) Negative controls, treated with solvent or vehicle alone, and oth-
erwise treated in the same way as the treatment groups, should be included
for every sampling time, unless acceptable inter-animal variability and fre-
quency of cells with chromosome aberrations are demonstrated by histori-
cal control data In addition, untreated controls should also be used unless
there are histoncal or published control data demonstrating that no delete-
rious or mutagenic effects are induced by the chosen solvent/vehicle.
(g) Procedure—(1) Number of animals Each treated and control
group should include at least five analyzable males
(2) Treatment schedule (i) Test substances are preferably adminis-
tered once or twice (i e as a single treatment or as two treatments) Test
substances may also be administered as a split dose, i e two treatments
on the same day separated by no more than a few hours, to facilitate ad-
ministering a large volume of material Other dose regimens should be
scientifically justified
(ii) In the highest dose group, two sampling times after treatment
should be used Since cell cycle kinetics can be influenced by the test
substance, one early and one late sampling time are used around 24 and
48 hours after treatment For doses other than the highest dose, a sampling
time of 24 hours or 1 5 cell cycle length after treatment should be taken,
unless another sampling time is known to be more appropnate for detec-
tion of effects (see paragraph (i)(6) of this guideline)
(m) In addition, other sampling tunes may be used For example, in-
the case of chemicals which may induce chromosome lagging, or may
exert S-mdependent effects, earlier sampling times may be appropnate (see
paragraph (i)(l) of this guideline)
(iv) The appropriateness of a repeated treatment schedule needs to
be identified on a case-by-case basis Following a repeated treatment
schedule the animals should then be sacrificed 24 hours (1.5 cell-cycle
length) after the last treatment Additional sampling tunes may be used
where appropnate
(v) Pnor to sacnfice, animals are injected intrapentoneally with an
appropnate dose of a metaphase arresting substance (e g, Colcemid® or
colcrucme) Animals are sampled at an appropnate interval thereafter For
-------
mice this interval is approximately 3-5 hours, tor Chinese hamsters this
interval is approximately 4-5 hours
(3) Dose levels If a range finding study is performed because there
are no suitable data available, it should be performed in the same labora-
tory, using the same species, strain, and treatment regimen to be used in
the main study (see paragraph (i)(7) of this guideline) If there is toxicity,
three-dose levels are used for the first sampling time These dose levels
should cover a range from the maximum to little or no toxicity At the
later sampling time only the highest dose needs to be used The highest
dose is defined as the dose producing signs of toxicity such that higher-
dose levels, based on the same dosing regimen, would be expected to
produce lethality Substances with specific biological activities at low non-
toxic doses (such as hormones and rmtogens) may be exceptions to the
dose-setting criteria and should be evaluated on a case-by-case basis The
highest dose may also be defined as a dose that produces some indication
of toxicity in the spermatogomal cells (e g, a reduction in the ratio of
spermatogonial mitoses to first and second meiotic metaphases, this reduc-
tion should not exceed 50 percent)
(4) Limit test If a test at one dose level of at least 2,000 mg/kg
body weight/day using a single treatment, or as two treatments on the same
day, produces no observable toxic effects, and if genotoxicity would not
be expected based upon data from structurally related substances, then a
full study using three-dose levels may not be considered necessary Ex-
pected human exposure may indicate the need for a higher dose level to
be used in the limit test
(5) Administration of doses The test substance is usually adminis-
tered by gavage using a stomach tube or a suitable intubation cannula,
or by mtrapentoneal injection Other routes of exposure may be acceptable
where they can be justified The maximum volume of liquid that can be
administered by gavage or injection at one tune depends on the size of
the test animal The volume should not exceed 2 ml/lOOg body weight.
The use of volumes higher than these must be justified Except for irritat-
ing or corrosive substances, which will normally reveal exacerbated effects
with higher concentrations, variability in test volume should be minimized
by adjusting the concentration to ensure a constant volume at all dose
levels
(6) Chromosome preparation Immediately after sacrifice, cell sus-
pensions should be obtained from one or both testes, exposed to hypotonic
solution and fixed The cells should be then spread on slides and stained.
(7) Analysis For each animal at least 100 well-spread metaphases
should be analyzed (i e a minimum of 500 metaphases per group) This
number could be reduced when high numbers of aberrations are observed
All slides, including those of positive and negative controls, should be
-------
independently coded before microscopic analysis Since fixation proce-
dures often result in the breakage of a proportion of metaphases with loss
of chromosomes, the cells scored should contain a number of centromeres
equal to the number 2n±2
(h) Data and reporting—(1) Treatment of results (i) Individual
animal data should be presented in tabular form The experimental unit
is the animal For each animal the number of cells with structural chro-
mosome aberration(s) and the number of chromosome aberrations per cell
should be evaluated Different types of structural chromosome aberrations
should be listed with their numbers and frequencies for treated and control
groups. Gaps are recorded separately and reported but generally not in-
cluded in the total aberration frequency
(11) If mitosis as well as meiosts is observed, the ratio of
spermatogonial mitoses to first and second meiotic metaphases should be
determined as a measure of cytotoxicity for all treated and negative control
animals in a total sample of 100 dividing cells per animal to establish
a possible cytotoxic effect If only mitosis is observed, the mitosis index
should be determined in at least 1,000 cells for each animal
(2) Evaluation and interpretation of results (i) There are several
catena for determining a positive result, such as a dose-related increase
in the relative number of cells with chromosome aberrations or a clear
increase in the number of cells with aberrations in a single-dose group
at a single-sampling tune Biological relevance of the results should be
considered first. Statistical methods may be used as an aid in evaluating
the test results (see paragraph (i)(8) of this guideline). Statistical signifi-
cance should not be the only determining factor for a positive response
Equivocal results should be clarified by further testing preferably using
a modification of experimental conditions
(n) A test substance for which the results do not meet the catena
in paragraph (h)(2)(i) of this guideline is considered nonmutagemc in this
test
(in) Although most experiments will give clearly positive or negative
results, m rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
(iv) Positive results from the in vivo spermatogonial chromosome ab-
erration test indicate that a substance induces chromosome aberrations in
the germ cells of the species tested Negative results indicate that, under
the test conditions, the test substance does not induce chromosome aberra-
tions in the germ cells of the species tested
(v) The likelihood that the test substance or its metabolites reach the
target tissue should be discussed
-------
(3) Test report The test report should include the following informa-
tion
(i) Test substance
(A) Identification data and CAS No , if known
(B) Physical nature and punty
(C) Physicochenucal properties relevant to the conduct of the study
(D) Stability of the test substance, if known
(li) Solvent/vehicle
(A) Justification for choice of vehicle
(B) Solubility and stability of the test substance m solvent/vehicle,
if known
(ui) Test animals
(A) Species/strain used
(B) Number and age of animals
(C) Source, housing conditions, diet, etc
(D) Individual weight of the animals at the start of the test, including
body weight range, mean, and standard deviation for each group.
(iv) Test conditions
(A) Data from range finding study, if conducted
(B) Rationale for dose level selection
(C) Rationale for route of administration
(D) Details of test substance preparation
(E) Details of the administration of the test substance
(F) Rationale for sacrifice times
(G) Conversion from diet/dnnking water test substance concentration
(ppm) to the actual dose (mg/kg body weight/day), if applicable
(H) Details of food and water quality
(I) Detailed description of treatment and sampling schedules
(J) Methods for measurement of toxicity
7
-------
(K) Identity of metaphase arresting substance its concentration and
duration of treatment
(L) Methods of slide preparation
(M) Criteria for scoring aberrations
(N) Number of cells analyzed per animal
(0) Criteria for considering studies as positive, negative, or equivocal
(v) Results
(A) Signs of toxicity
(B) Mitotic index
(C) Ratio of spermatogomal mitoses cells to first and second meiotic
metaphases
(D) Type and number of aberrations, given separately for each animal
(E) Total number of aberrations per group
(F) Number of cells with aberrations per group
(G) Dose-response relationship, where possible
(H) Statistical analyses, if any.
(I) Concurrent negative control data
(J) Historical negative control data with ranges, means, and standard
deviations
(K) Concurrent positive control data
(L) Changes in ploidy, if seen.
(vi) Discussion of the results
(vii) Conclusion.
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Adier, ID Clastogemc Potential in Mouse Spermatogoma of
Chemical Mutagens Related to Their Cell-Cycle Specifications Genetic
Toxicology of Environmental Chemicals, Part B Genetic Effects and Ap-
plied Mutagenesis, Ramel, C , Lambert, B and Magnusson, J (Eds) Liss,
New York, pp 477-484 (1986)
8
-------
(2) Adler I D Cytogenetic Tests in Mammals Mutagemcity Testing
a Practical Approach Ed S Venitt and J M Parry IRL Press, Oxford,
Washington DC, pp 275-306 (1984)
(3) Evans, E P, Breckon, G and Ford, C E An Air-Drying Method
for Meiotic Preparations from Mammalian Testes Cytogenetics and Cell
Genetics 3, 289-294 (1964)
(4) Richold, M In Vivo Cytogenetics Assays D J Kirkland (Ed)
Basic Mutagemcity Tests, UKEMS Recommended Procedures UKEMS
Subcommittee on Guidelines for Mutagemcity Testing Report Part I re-
vised Cambridge University Press, Cambridge, New York, Port Chester,
Melbourne, Sydney, pp 115-141 (1990)
(5) Yamamoto, K and Kikuchi, Y A New Method for Preparation
of Mammalian Spermatogonial Chromosomes Mutation Research 52,
207-209(1978)
(6) Adler ID et al International Workshop on Standardisation of
Genotoxicity Test Procedures Summary Report of the Working Group on
Mammalian Germ Cell Tests Mutation Research 312, 313-318 (1994).
(7) Fielder, R J et al Report of British Toxicology Society/UK Envi-
ronmental Mutagen Society Working Group Dose setting in In Vivo Muta-
gemcity Assays Mutagenesis 7, 313-319 (1992)
(8) Lovell, D.P et al Statistical Analysis of In Vivo Cytogenetic As-
says. D J Kirkland (Ed) Statistical Evaluation of Mutagemcity Test Data
UKEMS Sub-Committee on Guidelines for Mutagemcity Testing, Report,
Part III. Cambridge University Press, Cambridge, New York, Port Chester,
Melbourne, Sydney, pp 184-232 (1989)
-------
United States Prevention Pesticides EPA712-C-98-225
Environmental Pro lection and Toxic Substances August 1998
Agency <7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5385
Mammalian Bone
Marrow Chromosome
Aberration Test
-------
INTRODUCI ION
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 tn 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
(7 U S C. 136, et seq )
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines."
-------
OPPTS 870.5385 Mammalian bone marrow chromosome aberration
test
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenucide 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 5385 In vivo mam-
malian cytogenetics test Bone marrow chromosomal analysis and OECD
475, Mammalian Bone Marrow Chromosome Aberration Test
«
(b) Purpose. The mammalian in vivo chromosome aberration test is
used for the detection of structural chromosome aberrations induced by
test compounds in bone marrow cells of animals, usually rodents (see ref-
erences in paragraphs (g)(l), (g)(2), (g)(3), and (g)(4) of this guideline)
Structural chromosome aberrations may be of two types, chromosome or
chromatid An increase in polyploidy may indicate that a chemical has
the potential to induce numerical aberrations With the majonty of chemi-
cal mutagens, induced aberrations are of the chromatid-type, but chro-
mosome-type aberrations also occur Chromosome mutations and related
events are the cause of many human-genetic diseases and there is substan-
tial evidence that chromosome mutations and related events causing alter-
ations in oncogenes and tumour-suppressor genes are involved in cancer
in humans and experimental systems
(c) Definitions. The following definitions apply to the guideline
Chromatid-type aberration is structural chromosome damage ex-
pressed as breakage of single chromatids or breakage and reunion between
chromatids
Chromosome-type aberration is structural chromosome damage ex-
pressed as breakage, or breakage and reunion, of both chromatids at an
identical site.
Endoreduphcation is a process in which after an S period of DNA
replication, the nucleus does not go into mitosis but starts another S penod
The result is chromosomes with 2,4,8,. chromatids.
Gap is an achromatic lesion smaller than the width of one chromatid,
and with minimum misalignment of the chromatids.
Numerical aberration is a change in the number of chromosomes
from the normal number characteristic of the animals utilized
Polyploidy is a multiple of the haploid chromosome number (n) other
than the diploid number (i e , 3n, 4n, and so on)
-------
Structural aberration is a change in chromosome structure detectable
by microscopic examination of the metaphase stage of cell division, ob-
served as deletions and fragments, mtrachanges or interchanges
(d) Initial considerations. (1) Rodents are routinely used in this test
Bone marrow is the target tissue in this test, since it is a highly
vasculanzed tissue, and it contains a population of rapidly cycling cells
that can be readily isolated and processed Other species and target tissues
are not the subject of this guideline
(2) This chromosome aberration test is especially relevant to assessing
mutagemc hazard in that it allows consideration of factors of in vivo me-
tabolism, pharmacokmetics, and DNA-repair processes although these may
vary among species and among tissues An in vivo test is also useful for
further investigation of a mutagemc effect detected by an in vitro test
(3) If there is evidence that the test substance, or a reactive
metabolite, will not reach the target tissue, it is not appropriate to use
this test
(e) Test method—(1) Principle. Animals are exposed to the test sub-
stance by an appropriate route of exposure and are sacrificed at appropriate
times after treatment. Pnor to sacrifice, animals are treated with a meta-
phase-arrestmg agent (& g , colchicme or Colcemid®) Chromosome prep-
arations are then made from the bone marrow cells and stained, and meta-
phase cells are analyzed for chromosome aberrations
(2) Description—(i) Preparations—(A) Selection of animal species.
Rats, mice, and Chinese hamsters are commonly used, although any appro-
priate mammalian species may be used Commonly used laboratory strains
of healthy young-adult animals should be employed At the commence-
ment of the study, the weight variation of animals should be minimal and
not exceed ±20 percent of the mean weight of each sex
• (B) Housing and feeding conditions. The temperature m the experi-
mental animal room should be 22 °C (±3 °C) Although the relative humid-
ity should be at least 30 percent and preferably not exceed 70 percent
other than during room cleaning, the aim should be 50-60 percent Light-
ing should be artificial, the sequence being 12 hours light, 12 hours dark.
For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water The choice of diet may be influenced by the
need to ensure a suitable admixture of a test substance when administered
by this method Animals may be housed individually, or be caged in small
groups of the same sex
(C) Preparation of the animals. Healthy, young-adult animals should
be randomly assigned to the control and treatment groups Cages should
be arranged in such a way that possible effects due to cage placement
-------
are minimized The animals are identified uniquely The animals are accli-
mated to the laboratory conditions for at least 5 days
(D) Preparation of doses. Solid test substances should be dissolved
or suspended in appropriate solvents or vehicles and diluted, if appropriate,
prior to dosing of the animals Liquid-test substances may be dosed di-
rectly or diluted pnor to dosing Fresh preparations of the test substance
should be employed unless stability data demonstrate the acceptability of
storage
(11) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not produce toxic effects at the dose levels used, and should not be sus-
pected of chemical reaction with the test substance If other than well-
known solvents/vehicles are used, their inclusion should be supported with
data indicating their compatibility It is recommended that wherever pos-
sible, the use of an aqueous solvent/vehicle should be considered first.
(B) Controls. (7) Concurrent positive and negative (solvent/vehicle)
controls should be included for each sex in each test Except for treatment
with the test substance, animals in the control groups should be handled
in an identical manner to the animals in the treated groups
(2) Positive controls should produce structural chromosome aberra-
tions m vivo at exposure levels expected to give a detectable increase over
background Positive control doses should be chosen so that the effects
are clear but do not immediately reveal the identity of the coded slides
to the reader It is acceptable that the positive control be administered
by a route different from the test substance and sampled at only a single
tune The use of chemical class related positive control chemicals may
be considered, when available Examples of positive control substances
include.
Chemical
Triethylenemelamme
Ethyl methanesulphonate
Ethyl nitrosourea
Mitomycin C
Cyciophosphamtde (monohydrate)
CAS number
[51-18-3
62-50-0
[759-73-9
[50-07-7
50-18-0
([6055-19-2]
(3) Negative controls, treated with solvent or vehicle alone, and other-
wise treated in the same way as the treatment groups, should be included
for every sampling time, unless acceptable mter-ammal variability and fre-
quencies of cells with chromosome aberrations are available from histori-
cal control data. If single sampling is applied for negative controls, the
most appropriate time is the first sampling tune In addition, untreated
controls should also be used unless there are historical or published control
data demonstrating that no deletenous or mutagemc effects are induced
by the chosen solvent/vehicle
-------
(3) Procedure—(0 Number and sex of animals. Each treated and
control group should include at least five analyzable animals per sex If
at the time of the study there are data available from studies in the same
species and using the same route of exposure that demonstrate that there
are no substantial differences in toxicity between sexes, then testing m
a single sex will be sufficient Where human exposure to chemicals may
be sex specific, as for example with some pharmaceutical agents, the test
should be performed with animals of the appropriate sex
(n) Treatment schedule. (A) Test substances are preferably adminis-
tered as a single treatment Test substances may also be administered as
a split dose, i.e two treatments on the same day separated by no more
than a few hours, to facilitate administering a large volume of material
Other dose regimens should be scientifically justified
(B) Samples should be taken at two separate times following treat-
ment on one day For rodents, the first sampling interval is 1 5 normal
cell-cycle length (the latter being normally 12-18 hours) following treat-
ment Since the time required for uptake and metabolism of the test sub-
stance as well as its effect on cell-cycle kinetics can affect the optimum
time for chromosome aberration detection, a later sample collection 24
hours after the first sample time is recommended If dose regimens of
more than 1 day are used, one sampling time at 1 5 normal cell-cycle
lengths after the final treatment should be used
(C) Prior to sacrifice, animals should be injected mtrapentoneally
with an appropriate dose of a metaphase arresting agent (e g., Colcemid®
or colchicme). Animals are sampled at an appropriate interval thereafter.
For mice this interval is approximately 3-5 hours, for Chinese hamsters
this interval is approximately 4-5 hours Cells should be harvested from
the bone marrow and analysed from chromosome aberrations.
(m) Dose levels. If a range finding study is performed because there
are no suitable data available, it should be performed in the same labora-
tory, using the same species, strain, sex, and treatment regimen to be used
in the mam study (see reference in paragraph (g)(5) of this guideline)
If there is toxicity, three-dose levels should be used for the first sampling
tune. These dose levels should cover a range from the maximum to little
or no toxicity At the later sampling time only the highest dose needs
to be' used. The highest dose is defined as the dose producing signs of
toxicity such that higher dose levels, based on the same dosing regimen,
would be expected to produce lethality Substances with specific biological
activities at low non-toxic doses (such as hormones and rmtogens) may
be exceptions to the dose-setting criteria and should be evaluated on a
case-by-case basis The highest dose may also be defined as a dose that
produces some indication of toxicity in the bone marrow (e.g , greater than
50 percent reduction m mitotic index)
-------
(iv) Limit test If a test at one dose level of at least 2,000 mg/kg
body weight using a single treatment or as two treatments on the same
day produces no observable toxic effects, and if genotoxicity would not
be expected based on data from structurally related compounds then a
full study using three-dose levels may not be considered necessary For
studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/
day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for
treatment longer than 14 days Expected human exposure may indicate
the need for a higher-dose level to be used in the limit test
(v) Administration of doses. The test substance is usually adminis-
tered by gavage using a stomach tube or a suitable intubation cannula,
or by mtrapentoneal injection Other routes of exposure may be acceptable
where they can be justified The maximum volume of liquid that can be
administered by gavage or injection at one time depends on the size of
the test animal The volume should not exceed 2 ml/lOlDg body weight
The use of volumes higher than these must be justified Except for imtat-
mg or corrosive substances which will normally reveal exacerbated effects
with higher concentrations, variability in test volume should be minimised
by adjusting the concentration to ensure a constant volume at all dose
levels.
(vi) Chromosome preparation. Immediately after sacrifice, bone
marrow should be obtained, exposed to hypotonic solution and fixed The
cells should be then spread on slides and stained
(vu) Analysis. (A) The mitotic index should be determined as a meas-
ure of cytotoxicity in at least 1,000 cells per animal for all treated animals
(including positive controls) and untreated negative control animals
(B) At least 100 cells should be analyzed for each animal. This num-
ber could be reduced when high numbers of aberrations are observed All
slides, including those of positive and negative controls, should be inde-
pendently coded before microscopic analysis Since slide preparation pro-
cedures often result m the breakage of a proportion of metaphases with
loss of chromosomes, the cells scored should therefore contain a number
of centromeres equal to the number 2n ±2.
(f) Data and reporting—(1) Treatment of results. Individual animal
data should be presented in tabular form The experimental unit is the
animal For each animal the number of cells scored, the number of aberra-
tions per cell and the percentage of cells with structural chromosome
aberration(s) should be evaluated Different types of structural chro-
mosome aberrations should be listed with their numbers and frequencies
for treated and control groups Gaps should be recorded separately and
reported but generally not included in the total aberration frequency If
there is no evidence for a difference in response between the sexes, the
data may be combined for statistical analysis
-------
(2) Evaluation and interpretation of results. (0 There are several
cntena for determining a positive result, such as a dose-related increase
in the relative number of cells with chromosome aberrations or a clear
increase in the number of cells with aberrations in a single-dose group
at a single-sampling time Biological relevance of the results should be
considered first Statistical methods may be used as an aid in evaluating
the test results (see reference in paragraph (g)(6) of this guideline) Statis-
tical significance should not be the only determining factor for a positive
response Equivocal results should be clarified by further testing preferably
using a modification of expenmental conditions
(11) An increase in polyploidy may indicate that the test substance
has the potential to induce numerical chromosome aberrations An increase
in endoreduphcation may indicate that the test substance has the potential
to inhibit cell-cycle progression (see references in paragraphs (g)(7) and
(g)(8) of this guideline)
(111) A test substance for which the results do not meet the cntena
in paragraphs (f)(2)(i) and (f)(2)(u) of this guideline is considered non-
mutagenic m this test
(iv) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of experiments performed
(v) Positive results from the in vivo chromosome aberration test indi-
cate that a substance induces chromosome aberrations in the bone marrow
of the species tested Negative results indicate that, under the test condi-
tions, the test substance does not induce chromosome aberrations in the
bone marrow of the species tested
(vi) The likelihood that the test substance or its metabolites reach
the general circulation or specifically the target tissue (e g , systemic tox-
icity) should be discussed
(3) Test report The test report should include the following informa-
tion
(i) Test substance*
(A) Identification data and CAS No , if known
(B) Physical nature and punty
(C) Physicochemical properties relevant to the conduct of the study
(D) Stability of the test substance, if known
(11) Solvent/vehicle
-------
(A) Justification for choice of vehicle
(B) Solubility and stability of the test substance in solvent/vehicle,
if known
(in) Test animals
(A) Species/strain used
(B) Number, age, and sex of animals
(C) Source, housing conditions, diet, etc
(D) Individual weight of the animals at the start of the test, including
body weight range, mean, and standard deviation for each group
(iv) Test conditions
(A) Positive and negative (vehicle/solvent) controls
(B) Data from range-finding study, if conducted
(C) Rationale for dose level selection
(D) Details of test substance preparation
(E) Details of the administration of the test substance
(F) Rationale for route of administration
(G) Methods for verifying that the test substance reached the general
circulation or target tissue, if applicable
(H) Conversion from diet/dnnking water test substance concentration
parts per million (ppm) to the actual dose (mg/kg body weight/day), if
applicable
(I) Details of food and water quality
(J) Detailed description of treatment and sampling schedules.
(K) Methods for measurement of toxicity
(L) Identity of metaphase arresting substance, its concentration and
duration of treatment
(M) Methods of slide preparation
(N) Criteria for scoring aberrations
(O) Number of cells analysed per animal
(P) Criteria for considering studies as positive, negative or equivocal
(v) Results
7
-------
(A) Signs of toxictty
(B) Muotic index
(C) Type and number of aberrations, given separately for each animal
(D) Total number of aberrations per group with means and standard
deviations
(E) Number of cells with aberrations per group with means and stand-
ard deviations
(F) Changes in ploidy, if seen
(G) Dose-response relationship, where possible
(H) Statistical analyses, if any
(I) Concurrent negative control data
(J) Historical negative control data with ranges, means and standard
deviations.
(K) Concurrent positive control data
(vi) Discussion of the results
(vn) Conclusion
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Adler, I.D Cytogenetic Tests in Mammals Mutagemcity Testing:
A Practical Approach (Eds ) S Venitt and J M Parry (IRL Press, Oxford,
Washington DC, pp 275-306 (1984)
(2) Preston, R.J et al Mammalian In Vivo Cytogenetic Assays: Anal-
ysis of Chromosome Aberrations in Bone Marrow Cells Mutation Re-
search 189,157-165 (1987)
(3) Richold, M. et al In Vivo Cytogenetic Assays (Ed ) D J. Kirkland
Basic Mutagemcity Tests, UKEMS Recommended Procedures. UKEMS
Subcommittee on Guidelines for Mutagemcity Testing Report. Part I re-
vised (Cambridge University Press, Cambridge, New York, Port Chester,
Melbourne, Sydney, pp 115-141(1990)
(4) Tice, R.R et al Report from the Working Group on the In Vivo
Mammalian Bone Marrow Chromosomal Aberration Test Mutation Re-
search 312, 305-312 (1994)
(5) Fielder, R J et al Report of Bntish Toxicology Society/UK Envi-
ronmental Mutagen Society Working Group Dose Setting in In Vivo Mu-
tagemcity Assays Mutagenesis 7, 313-319 (1992)
8
-------
(6) Lovell, DP et al Statistical Analysis of In Vivo Cytogenetic As-
says UKEMS Sub-Committee on Guidelines for Mutagenicity Testing
Report Part III Statistical Evaluation of Mutagenicity Test Data (Ed ) D
J Kirkland (Cambridge University Press, Cambridge, pp 184-232 (1989)
(7) Locke-Huhle, C Endoreduphcation in Chinese Hamster Cells
During Alpha-Radiation Induced G2 Arrest Mutation Research 119 403-
413(1983)
(8) Huang, Y et al Aphidicolm-Induced Endoreduphcation in Chi-
nese Hamster Cells Cancer Research 43, 1362-1364 (1983)
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-226
August 1998
Health Effects Test
Guidelines
OPPTS 870.5395
Mammalian Erythrocyte
MicronucleusTest
-------
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
(7 USC I36,etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www.epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines."
-------
OPPTS 870 5395 Mammalian erythrocyte micronucleus test.
(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 5385 In vivo mam-
malian bone marrow cytogenetics test Micronucleus assay and OECD
474, Mammalian Erythrocyte Micronucleus Test
(b) Purpose. (1) The mammalian in vivo micronucleus test is used
for the detection of damage induced by the test substance to the chro-
mosomes or the rrutotic apparatus of erythroblasts by analysis of
erythrocytes as sampled in bone marrow and/or peripheral blood cells of
animals, usually rodents
(2) The purpose of the micronucleus test is to identify substances
that cause cytogenetic damage which results in the formation of
micronuclei containing lagging chromosome fragments or whole chro-
mosomes.
(3) When a bone marrow erythroblast develops into a polychromatic
erythrocyte, the main nucleus is extruded, any micronucleus that has been
formed may remain behind in the otherwise anucleated cytoplasm. Visual-
ization of micronuclei is facilitated in these cells because they lack a mam
nucleus An increase in the frequency of micronucleated polychromatic
erythrocytes in treated animals is an indication of induced chromosome
damage
(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
Centromere (kmetochore) is a region of a chromosome with which
spindle fibers are associated during cell division, allowing orderly move-
ment of daughter chromosomes to the poles of the daughter cells
Micronuclei are small nuclei, separate from and additional to the main
nuclei of cells, produced dunng telophase of mitosis (meiosis) by lagging
chromosome fragments or whole chromosomes
Normochromatic erythrocyte is a mature erythrocyte that lacks
nbosomes and can be distinguished from immature, polychromatic
erythrocytes by stains selective for nbosomes
Polychromatic erythrocyte is a immature erythrocyte, in an intermedi-
ate stage of development, that still contains nbosomes and therefore can
be distinguished from mature, normochromatic erythrocytes by stains se-
lective for nbosomes
-------
(d) Initial considerations. (1) The bone marrow of rodents is rou-
tinely used in this test since polychromatic erythrocytes are produced in
that tissue The measurement of micronucleated immature (polychromatic)
erythrocytes in peripheral blood is equally acceptable in any species in
which the inability of the spleen to remove micronucleated erythrocytes
has been demonstrated, or which has shown an adequate sensitivity to de-
tect agents that cause structural or numerical chromosome aberrations
Micronuclei can be distinguished by a number of catena These include
identification of the presence or absence of a kinetochore or centromenc
DNA in the rmcronuclei The frequency of micronucleated immature (pol-
ychromatic) erythrocytes is the principal endpomt The number of mature
(normochromatic) erythrocytes in the penpheral blood that contain
rmcronuclei among a given number of mature erythrocytes can also be
used as the endpomt of the assay when animals are treated continuously
for 4 weeks or more This mammalian in vivo micronucleus test is espe-
cially relevant to assessing mutagenic hazard m that it allows consideration
of factors of in vivo metabolism, pharmacokmetics, and DNA-repair proc-
esses although these may vary among species, among tissues and among
genetic endpomts An m vivo assay is also useful for further investigation
of a mutagenic effect detected by an in vitro system
(2) If there is evidence that the test substance, or a reactive
metabolite, will not reach the target tissue, it is not appropnate to use
this test
i
(e) Test method—(1) Principle. Animals are exposed to the test sub-
stance by an appropriate route If bone marrow is used, the animals are
sacrificed at appropnate times after treatment, the bone marrow extracted,
and preparations made and stained (see references in paragraphs (g)(l),
(g)(2), and (g)(3) of this guideline) When penpheral blood is used, the
blood is collected at appropnate tunes after treatment and smear prepara-
tions are made and stained (see references m paragraphs (g)(3), (g)(4),
(g)(5), and (g)(6) of this guideline) For studies with penpheral blood, as
little time as possible should elapse between the last exposure and cell
harvest. Preparations are analyzed for the presence of rmcronuclei.
(2) Description—(i) Preparations—(A) Selection of animal species.
Mice or rats are recommended if bone marrow is used, although any ap-
propnate mammalian species may be used When penpheral blood is used,
mice are recommended However, any appropnate mammalian species
may be used provided it is a species in which the spleen does not remove
micronucleated erythrocytes or a species which has shown an adequate
sensitivity to detect agents that cause structural or numerical chromosome
aberrations Commonly used laboratory strains of healthy young animals
should be employed At the commencement of the study, the weight vari-
ation of animals should be minimal and not exceed ±20 percent of the
mean weight of each sex
-------
(B) Housing and feeding conditions. The temperature in the experi-
mental animal room should be 22 °C (±3 °C) Although the relative humid-
ity should be at least 30 percent and preferably not exceed 70 percent
other than during room cleaning, the aim should be 50-60 percent Light-
ing should be artificial, the sequence being 12 hours light, 12 hours dark
For feeding, conventional laboratory diets may be used with an unlimited
supply of drinking water The choice of diet may be influenced by the
need to ensure a suitable admixture of a test substance when administered
by this route Animals may be housed individually, or caged in small
groups of the same sex
(C) Preparation of the animals. Healthy young adult animals should
be randomly assigned to the control and treatment groups The animals
are identified uniquely The animals are acclimated to the laboratory condi-
tions for at least five days Cages should be arranged in such a way that
possible effects due to cage placement are minimized
(D) Preparation of doses. Solid test substances should be dissolved
or suspended in appropriate solvents or vehicles and diluted, if appropriate,
pnor to dosing of the animals Liquid-test substances may be dosed di-
rectly or diluted pnor to dosing Fresh preparations of the test substance
should be employed unless stability data demonstrate the acceptability of
storage.
(u) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not produce toxic effects at the dose levels used, and should not be sus-
pected of chemical reaction with the test substance If other than well-
known solvents/vehicles are used, their inclusion should be supported with
reference data indicating their compatibility It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle should be considered
first.
(B) Controls. (7) Concurrent positive and negative (solvent/vehicle)
controls should be included for each sex in each test Except for treatment
with the test substance, animals in the control groups should be handled
in an identical manner to animals of the treatment groups
(2) Positive controls should produce rrucronuclei m vivo at exposure
levels expected to give a detectable increase over background. Positive-
control doses should be chosen so that the effects are clear but do not
immediately reveal the identity of the coded slides to the reader. It is ac-
ceptable that the positive control be administered by a route different from
the test substance and sampled at only a single time In addition, the use
of chemical class-related positive control chemicals may be considered,
when available Examples of positive control substances include.
-------
Chemical
Ethyl methanesulphonate
Ethyl mtrosourea
Mitomycin C
Cyclophosphamide (monohydrate)
Triethylenemelamme
CAS number
[62-50-0
{759-73-9
[50-07-7
[50-18-0
([6055-1 9-2]'
[51-18-3
(J) Negative controls, treated with solvent or vehicle alone, and other-
wise treated in the same way as the treatment groups should be included
for every sampling time, unless acceptable inter-animal variability and fre-
quencies of cells with rrucronuclei are demonstrated by historical-control
data If single sampling is applied for negative controls, the most appro-
priate time is the first sampling tune In addition, untreated controls should
also be used unless there are historical- or pubhshed-control data dem-
onstrating that no deleterious or mutagenic effects are induced by the cho-
sen solvent/vehicle
(4) If peripheral blood is used, a pre-treatment sample may also be
acceptable as a concurrent negative control, but only in the short peripheral
blood studies (e g , one-three treatment(s)) when the resulting data are in
the expected range for the historical control
(3) Procedure—(i) Number and sex of animals. Each treated and
control group should include at least 5 analyzable animals per sex (see
reference in paragraph (g)(7) of this guideline) If at the tune of the study
there are data available from studies in the same species and using the
same route of exposure that demonstrate that there are no substantial dif-
ferences between sexes in toxicity, then testing in a single sex will be
sufficient. Where human exposure to chemicals may be sex specific, as
for example with some pharmaceutical agents, the test should be per-
formed with animals of the appropnate sex
(11) Treatment schedule. (A) No standard treatment schedule (i.e
1, 2, or more treatments at 24 hour intervals) can be recommended. The
samples from extended dose regimens are acceptable as long as a positive
effect has been demonstrated for this study or, for a negative study, as
long as toxicity has been demonstrated or the limit dose has been used,
and dosing continued until the time of sampling Test substances may also
be administered as a split dose, i e , two treatments on the same day sepa-
rated by no more than a few hours, to facilitate administering a large vol-
ume of material
(B) The test may be performed in two ways.
(7) Animals should be treated with the test substance once. Samples
of bone marrow should be taken at least twice, starting not earlier man
24 hour after treatment, but not extending beyond 48 hours after treatment
with appropnate mterval(s) between samples The use of sampling times
earlier than 24 hours after treatment should be justified Samples of penph-
-------
eral blood should be taken at least twice, starting not earlier than 36 hours
after treatment, with appropriate intervals following the first sample, but
not extending beyond 72 hours When a positive response is recognized
at one sampling time, additional sampling is not required
(2) If two or more daily treatments are used (e g two or more treat-
ments at 24 hour intervals), samples should be collected once between
18 and 24 hours following the final treatment for the bone marrow and
once between 36 and 48 hours following the final treatment for the periph-
eral blood (see reference in paragraph (g)(8) of this guideline)
(C) Other sampling times may be used in addition, when relevant
(m) Dose levels. If a range finding study is performed because there
are no suitable data available, it should be performed in the same labora-
tory, using the same species, strain, sex, and treatment regimen to be used
in the mam study (see reference in paragraph (g)(9) of this guideline)
If there is toxicity, three-dose levels should be used for the first sampling
time These dose levels should cover a range from the maximum to little
or no toxicity At the later sampling time only the highest dose needs
to be used The highest dose is defined as the dose producing signs of
toxicity such that higher dose levels, based on the same dosing regimen,
would be expected to produce lethality Substances with specific biological
activities at low non-toxic doses (such as hormones and mitogens) may
be exceptions to the dose-setting criteria and should be evaluated on a
case-by-case basis The highest dose may also be defined as a dose that
produces some indication of toxicity in the bone marrow (e g. a reduction
m the proportion of immature erythrocytes among total erythrocytes in
the bone marrow or peripheral blood).
(iv) Limit test If a test at one dose level of at least 2,000 mg/kg
body weight using a single treatment, or as two treatments on the same
day, produces no observable toxic effects, and if genotoxicity would not
be expected based upon data from structurally related substances, then a
full study using three-dose levels may not be considered necessary. For
studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/
day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for
treatment longer than 14 days Expected human exposure may indicate
the need for a higher-dose level to be used m the limit test
(v) Administration of doses. The test substance is usually adminis-
tered by gavage using a stomach tube or a suitable intubation cannula,
or by mtrapentoneal injection Other routes of exposure may be acceptable
where they can be justified The maximum volume of liquid that can be
administered by gavage or injection at one time depends on the size of
the test animal The volume should not exceed 2 ml/lOOg body weight.
The use of volumes higher than these must be justified. Except for irritat-
ing or corrosive substances which will normally reveal exacerbated effects
-------
with higher concentrations, variability in test volume should be minimized
by adjusting the concentration to ensure a constant volume at all dose
levels
(vi) Bone marrow/blood preparation. Bone marrow cells should be
obtained from the femurs or tibias immediately following sacrifice Cells
should be removed from femurs or tibias, prepared and stained using estab-
lished methods Peripheral blood is obtained from the tail vein or other
appropnate blood vessel Blood cells are immediately stained supravitally
(see references in paragraphs (g)(4), (g)(5), and (g)(6) of this guideline)
or smear preparations are made and then stained The use of a DNA-spe-
cific stain (e g acridme orange (see reference in paragraph (g)(10) of this
guideline) or Hoechst 33258 plus pyronm-Y (see reference in paragraph
(g)(ll) of this guideline) can eliminate some of the artifacts associated
with using a non-DNA-specific stain This advantage does not preclude
the use of conventional stains (e g, Giemsa) Additional systems (e g cel-
lulose columns to remove nucleated cells (see reference in paragraph
(g)(12) of this guideline) can also be used provided that these systems
have been shown to adequately work for micronucleus preparation in the
laboratory
(vn) Analysis. The proportion of immature among total (immature
+ mature) erythrocytes is determined for each animal by counting a total
of at least 200 erythrocytes for bone marrow and 1,000 erythrocytes for
peripheral blood (see reference in paragraph (g)(13) of this guideline) All
slides, including those of positive and negative controls, should be inde-
pendently coded before microscopic analysis. At least 2,000 immature
erythrocytes per animal should be scored for the incidence of
micronucleated immature erythrocytes Additional information may be ob-
tained by sconng mature erythrocytes for micronuclei. When analyzing
slides, the proportion of immature erythrocytes among total erythrocytes
should not be less than 20 percent of the control value. When animals
are treated continuously for 4 weeks or more, at least 2,000 mature
erythrocytes per animal can also be scored for the incidence of
micronuclei Systems for automated analysis (image analysis) and cell sus-
pensions (flow cytometry) are acceptable alternatives to manual evaluation
if appropnately justified and validated
(f) Data and reporting—(1) Treatment of results. Individual animal
data should be presented in tabular form. The experimental unit is the
animal The number of immature erythrocytes scored, the number of
micronucleated immature erythrocytes, and the number of immature
among total erythrocytes should be listed separately for each animal ana-
lyzed. When animals are treated continuously for 4 weeks or more, the
data on mature erythrocytes should also be given if it is collected. The
proportion of immature among total erythrocytes and, if considered appli-
cable, the percentage of micronucleated erythrocytes should be given for
each animal If there is no evidence for a difference in response between
-------
the sexes, the data from both sexes may be combined foi statistical analy-
sis
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a dose-related increase
in the number of rrucronucleated cells or a clear increase in the number
of rmcronucleated cells in a single-dose group at a single-sampling time
Biological relevance of the results should be considered first Statistical
methods may be used as an aid in evaluating the test results (see references
in paragraphs (g)(14) and (g)(15) of this guideline) Statistical significance
should not be the only determining factor for a positive response. Equivo-
cal results should be clarified by further testing preferably using a modi-
fication of experimental conditions
(11) A test substance for which the results do not meet the criteria
in paragraph (f)(2)(i) of this guideline is considered non-mutagenic in this
test
(in) Although most experiments will give clearly positive or negative
results, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance Results may remain equivocal or
questionable regardless of the number of tunes the experiment is repeated
Positive results in the micronucleus test indicate that a substance induces
micronuclei which are the result of chromosomal damage or damage to
the nutotic apparatus m the erythroblasts of the test species. Negative re-
sults indicate that, under the test conditions, the test substance does not
produce micronuclei in the immature erythrocytes of the test species
(iv) The likelihood that the test substance or its metabolites reach
the general circulation or specifically the target tissue (e g systemic tox-
icity) should be discussed
(3) Test report The test report should include the following informa-
tion-
(i) Test substance
(A) Identification data and CAS No , if known
(B) Physical nature and punty
(C) Physiochemical properties relevant to the conduct of the study
(D) Stability of the test substance, if known
(u) Solvent/vehicle
(A) Justification for choice of vehicle
(B) Solubility and stability of the test substance in the solvent/vehicle,
if known
-------
(in) Test animals
(A) Species/strain used
(B) Number, age, and sex of animals
(C) Source, housing conditions, diet, etc
(D) Individual weight of the animals at the start of the test, including
body weight range, mean, and standard deviation for each group
(iv) Test conditions
(A) Positive and negative (vehicle/solvent) control data
(B) Data from range-finding study, if conducted
(C) Rationale for dose-level selection
(D) Details of test substance preparation
(£) Details of the administration of the test substance
(F) Rationale for route of administration
(G) Methods for verifying that the test substance reached the general
circulation or target tissue, if applicable
(H) Conversion from diet/dnnkmg water test substance concentration
parts per million (ppm) to the actual dose (mg/kg body weight/day), if
applicable
(I) Details of food and water quality
(J) Detailed description of treatment and sampling schedules
(K) Methods of slide preparation
(L) Methods for measurement of toxicity
(M) Criteria for sconng rmcronucleated immature erythrocytes
(N) Number of cells analyzed per animal.
(O) Criteria for considering studies as positive, negative, or equivocal.
(v) Results
(A) Signs of toxicity.
(B) Proportion of immature erythrocytes among total erythrocytes
(C) Number of micronucleated immature erythrocytes, given sepa-
rately for each animal
8
-------
(D) Mean ± standard deviation of micronucleated immature
erythrocytes per group
(E) Dose-response relationship, where possible
(F) Statistical analyses and method applied
(G) Concurrent and historical negative-control data
(H) Concurrent positive-control data
(vi) Discussion of the results
(vn) Conclusion
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Heddle, J A et al A Rapid In vivo Test for Chromosomal Dam-
age Mutation Research 18, 187-190(1973)
(2) Schmid, W The Micronucleus Test Mutation Research 31, 9-
15 (1975)
(3) Mavournm, K H et al The In vivo Micronucleus Assay in Mam-
malian Bone Marrow and Peripheral Blood A report of the U S. Environ-
mental Protection Agency Gene-Tox Program Mutation Research 239,
29-80 (1990)
(4) Hayashi, M et al The Micronucleus Assay with Mouse Peripheral
Blood Reticulocytes Using Acndme Orange-Coated Slides Mutation Re-
search 245, 245-249 (1990)
(5) The Collaborative Study Group for the Micronucleus Test Micro-
nucleus Test with Mouse Peripheral Blood Erythrocytes by Acndine Or-
ange Supravital Staining The Summary Report of the 5th Collaborative
Study by CSGMT/JEMS MMS Mutation Research 278, 83-98 (1992).
(6) The Collaborative Study Group for the Micronucleus Test
(CSGMT/JEMMS MMS, The Mammalian Mutagenesis Study Group of
the Environmental Mutagen Society of Japan) Protocol recommended for
the short-term mouse peripheral blood micronucleus test Mutagenesis 10,
153-159 (1995)
(7) Hayashi, M et al In vivo Rodent Erythrocyte Micronucleus
Assay Mutation Research 312, 293-304 (1994)
(8) Higashikum, N and Sutou, S An optimal, generalized sampling
time of 30 V- 6 h after double dosing in the mouse peripheral blood micro-
nucleus test. Mutagenesis 10, 313-319 (1995).
-------
(9) Fielder, R J et al Report of British Toxicology Society/UK Envi-
ronmental Mutagen Society Working Group Dose Setting in In vivo Muta-
gemcity Assays Mutagenesis 7, 313-319 (1992)
(10) Hayashi, M et al An Application of Acndme Orange Fluores-
cent Staining to the Micronucleus Test Mutation Research 120, 241-247
(1983)
(11) MacGregor, J T et al A Simple Fluorescent Staining Procedure
for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and
Pyronm Y Mutation Research 120, 269-275 (1983)
(12) Romagna, F and Stamforth, CD The automated bone marrow
micronucieus test Mutation Research 213, 91-104 (1989)
(13) Gollapudi, B and McFadden, LG Sample size for the esti-
mation of polychromatic to normochromatic erythrocyte ratio in the bone
marrow micronucieus test. Mutation Research 347, 97-99 (1995)
(14) Richold, M et al In vivo Cytogenetics Assays, In: D J Kirkland
(Ed) Basic Mutagemcity Tests, UKEMS Recommended Procedures
UKEMS Subcommittee on Guidelines for Mutagemcity Testing Report
Part I revised Cambridge University Press, Cambridge, New York, Port
Chester, Melbourne, Sydney, pp 115-141 (1990)
(15) Lovell, D P et al Statistical Analysis of In vivo Cytogenettc
Assays In DJ. Kirkland (Ed ) Statistical Evaluation of Mutagemcity Test
Data UKEMS Sub-Committee on Guidelines for Mutagemcity Testing,
Report, Part III Cambridge University Press, Cambridge, New York, Port
Chester, Melbourne, Sydney, pp 184-232 (1989)
(16) Heddle, J A et al The Induction of Micronuclei as a Measure
of Genotoxicity Mutation Research 123 61-118(1983).
(17) MacGregor, JT et al Guidelines for the Conduct of Micro-
nucleus Assays in Mammalian Bone Marrow Erythrocytes Mutation Re-
search 189 103-112(1987)
(18) MacGregor, JT et al The In vivo Erythrocyte Micronucieus
Test Measurement at Steady State Increases Assay Efficiency and Permits
Integration with Toxicity Studies Fundamental and Applied Toxicology
14 513-522(1990)
(19) MacGregor, JT et al Micronuclei in Circulating Erythrocytes:
A Rapid Screen for Chromosomal Damage During Routine Toxicity Test-
ing in Mice In. Developments in Science and Practice of Toxicology. Ed
A.W Hayes, R C Schnell and T.S Miya, Elsevier, Amsterdam, pp 555-
558 (1983)
10
-------
c, EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-9&-227
August 1998
Health Effects Test
Guidelines
OPPTS 870.5450
Rodent Dominant Lethal
Assay
-------
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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www.epa gov/epahome/research htm) under the heading ' 'Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5450 Rodent dominant lethal assay.
(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 5450 Rodent domi-
nant Lethal assay and OECD 478 Genetic Toxicology Rodent Dominant
Lethal Assay
(b) Purpose. Dominant lethal (DL) effects cause embryonic or fetal
death Induction of a dominant lethal event after exposure to a chemical
substance indicates that the substance has affected germinal tissue of the
test species Dominant lethals are generally accepted to be the result of
chromosomal damage (structural and numencal anomalies) but gene
mutations and toxic effects cannot be excluded
(c) Definitions. The definitions m 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.
Dominant lethal mutanon is one occurring in a germ cell which does
not cause dysfunction of the gamete, but which is lethal to the fertilized
egg or developing embryo
(d) Reference substances. These may include, but need not be lim-
ited to, tnethylenemelamme, cyclophosphamide, or ethyl
memanesulfonate
(e) Test method—(1) Principle. Generally, male animals are exposed
to the test substance and mated to untreated virgin females The various
germ cell stages can be tested separately by the use of sequential mating
intervals The females are sacnficed after an appropriate period of time
and the contents of the uten are examined to determine the numbers of
implants and live and dead embryos The calculation of the dominant lethal
effect is based on comparison of the live implants per female m the treated
group to the live implants per female in the control group. The increase
of dead implants per female in the treated group over the dead implants
per female in the control group reflects the post-implantation loss. The
post-implantation loss is calculated by determining the ratio of dead to
total implants from the treated group compared to the ratio of dead to
total implants from the control group Pre-implantation loss can be esti-
mated on the basis of corpora lutea counts or by comparing the total im-
plants per female in treated and control groups
(2) Description, (i) Several treatment schedules are available The
most widely used requires single administration of the test substance.
-------
Other treatment schedules, such as treatment on five consecutive days, may
be used if justified by the investigator
(11) Individual males are mated sequentially to virgin females at ap-
propriate intervals The number of matmgs following treatment is governed
by the treatment schedule and should ensure that germ cell maturation
is adequately covered Females are sacrificed in the second half of preg-
nancy and the uterine contents examined to determine the total number
of implants and the number of live and dead embryos
(3) Animal selection—(i) Species. Rats or mice are generally used
as the test species Strains with low background dominant lethality, high
pregnancy frequency, and high implant numbers are recommended
(11) Age. Healthy, sexually mature animals should be used.
(111) Number. An adequate number of animals should be used taking
into account the spontaneous variation of the biological characteristics
being evaluated The number chosen should be based on the predetermined
sensitivity of detection and power of significance For example, in a typical
experiment, the number of males in each group should be sufficient to
provide between 30 and 50 pregnant females per mating interval
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups
(4) Control groups—(i) Concurrent controls. Generally concurrent
positive and negative (vehicle) controls should be included in each experi-
ment When acceptable positive control results are available from experi-
ments conducted recently (within the last 12 months) in the same labora-
tory, these results can be used instead of a concurrent positive control
(n) Positive controls. Positive control substances should be used at
a dose which demonstrates the test sensitivity
(5) Test chemicals—(i) Vehicle. When possible, test substances
should be dissolved or suspended in isotonic saline or distilled water
Water-insoluble chemicals may be dissolved or suspended in appropnate
vehicles The vehicle used should neither interfere with the test chemical
nor produce toxic effects Fresh preparations of the test chemical should
be employed.
(11) Dose levels. Normally, three dose levels should be used The
highest dose should produce signs of toxicity (e g., slightly reduced fertil-
ity and slightly reduced body weight) However, in an initial assessment
of dominant lethality a single high dose may be sufficient Nontoxic sub-
stances should be tested at 5 g/kg or, if this is not practicable, then as
the highest dose attainable
-------
(in) Route of administration. The usual routes of administration are
oral or by IP injection Other routes may be appropuate
(f) Test performance. (1) Individual males are mated sequentially
at appropriate predetermined intervals to one or two virgin females Fe-
males should be left with the males for at least the duration of one estrus
cycle or alternatively until mating has occurred as determined by the pres-
ence of sperm in the vagina or by the presence of a vaginal plug
(2) The number of matmgs following treatment should be governed
by the treatment schedule and should ensure that germ cell maturation
is adequately covered
(3) Females should be sacrificed in the second half of pregnancy and
utenne contents examined to determine the number of implants and live
and dead embryos The ovanes may be examined to determine the number
of corpora lutea
(g) Data and report—(1) Treatment of results. Data should be tab-
ulated to show the number of males, the number of pregnant females, and
the number of nonpregnant females Results of each mating, including the
identity of each male and female, should be reported individually For each
female, the dose level and week of mating and the frequencies of live
implants and of dead implants should be enumerated If the data are re-
corded as early and late deaths, the tables should make that clear If pre-
plantation loss is estimated, it should be reported Pre-implantation loss
can be calculated as the difference between the number of corpora lutea
and the number of implants or as a reduction in the average number of
implants per female in comparison with control matmgs
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods Differences among animals within the control and
treatment groups should be considered before making comparisons be-
tween treated and control groups
(3) Interpretation of results, (i) There are several cntena for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of dominant lethals Another criterion may
be based upon detection of a reproducible and statistically significant posi-
tive response for at least one of the test points
(n) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of dominant lethals or a statis-
tically significant and reproducible positive response at any one of the
test points is considered nonmutagenic in this system
(111) Both biological and statistical significance should be considered
together in the evaluation
-------
(4) Test evaluation, (i) A positive DL assay suggests that under the
test conditions the test substance may be genotoxic in the germ cells of
the treated sex of the test species
(u) A negative result suggests that under the conditions of the test
the test substance may not be genotoxic in the germ cells of the treated
sex of the test species
(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) Species, strain, age, and weights of animals used, number of ani-
mals of each sex in experimental and control groups
(u) Test substance, vehicle used, dose levels and rationale for dosage
selection, negative (vehicle) and positive controls, and experimental obser-
vations, including signs of toxicity
(111) Route and duration of exposure
(iv) Mating schedule
(v) Methods used to determine that mating has occurred (where appli-
cable)
(vi) Criteria for scoring dominant lethals including the number of
early and late embryonic deaths
(vii) Dose-response relationship, if applicable
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) B re wen, JG et al Studies on chemically induced dominant
lethality I The cytogenetic basis of MMS-mduced dominant lethality in
post-meiotic germ cells. Mutation Research 33.239-250 (1975).
(2) Ehlmg, U H et al Standard protocol for the dominant lethal test
on male mice Set up by the Work Group Dominant lethal mutations of
the ad hoc Committee Chemogenetics Archives of Toxicology 39:173-185
(1978)
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-228
August 1998
Health Effects Test
Guidelines
OPPTS 870.5460
Rodent Heritable
Translocation Assays
-------
[N PRODUCTION
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 m 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
USC. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36tetseq)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
m PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 5460 Rodent heritable translocation assays.
(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 5460 Rodent hen-
table translocation assays and OECD guideline 485 Genetic Toxicology
Mouse Heritable Translocation Assay
(b) Purpose. This test detects transmitted chromosomal damage
which manifests as balanced reciprocal translocations in progeny de-
scended from parental males treated with chemical mutagens.
(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
Diakmesis and metaphase I are stages of meiotic prophase scored
cytologically for the presence of multivalent chromosome association char-
acteristic of translocation earners
Heritable translocanon is one in which distal segments of non-
homologous chromosomes are involved in a reciprocal exchange.
(d) Test method—(1) Principle. When a balanced reciprocal
translocation is induced in a parental male germ cell, the resulting progeny
is translocation heterozygote
(i) Basis for fertility screening. Male translocation heterozygotes
may be completely sterile This class consists of two types of
translocations
(A) Translocations between non-homologous chromosomes in which
at least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations The majonty of male
translocation heterozygotes are semistenle—they carry one or (rarely) two
translocations The degree of semistenhty is dependent upon the propor-
tions of balanced and unbalanced (duplication-deficiency) gametes pro-
duced in the ejaculate as a function of meiouc segregation Balanced and
unbalanced sperm are equally capable of fertilizing an egg. Balanced
sperm lead to viable progeny Unbalanced sperm result in early embryonic
lethality.
(li) Basis for cytological screening. The great majonty of male
translocation heterozygotes can be identified cytologically through analysis
of diakmesis metaphase I spermatocytes Translocation heterozygotes are
charactenzed by the presence of multivalent chromosome association such
-------
as a ring or chain of four chromosomes held together by chiasmata in
paired homologous regions Some translocation carriers can be identified
by the presence of extra long and/or extra short chromosomes in
spermatogomal and somatic cell metaphase preparations
(2) Description. Essentially, two methods have been used to screen
for translocation heterozygosity—one method uses a mating sequence to
identify stenle and semistenle males followed by cytological examination
of suspect male individuals, the other method deletes the mating sequence
altogether and all Fj male progeny are examined cytologically for presence
of translocation In the former approach, the mating sequence serves as
a screen which eliminates most fully fertile animals for cytological con-
firmation as translocation heterozygotes
(3) Animal selection—(i) Species. The mouse is the species generally
used and is recommended
(n) Age. Healthy sexually mature animals should be used
(in) Number. The number of male animals necessary is determined
by the following factors
(A) The use of either historical or concurrent controls
(B) The power of the test
(C) The minimal rate of induction required
(D) Whether positive controls are used
(E) The level of significance desired
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups
(4) Control groups—(i) Concurrent controls. No concurrent posi-
tive or negative (vehicle) controls are recommended as routine parts of
the heritable translocation assay However, investigators not experienced
in performing translocation testing should include a substance known to
produce translocations in the assay as a positive control reference chemi-
cal.
(li) Historical controls. At the present time, historical control data
must be used in tests for significance When statistically reliable historical
controls are not available, negative (vehicle) controls should be used
(5) Test chemicals—(i) Vehicle. When appropriate for the route of
administration, solid and liquid test substances should be dissolved or sus-
pended in distilled water or isotonic saline Water-insoluble chemicals may
be dissolved or suspended in appropriate vehicles The vehicle used should
-------
neither interfere with the test chemical nor produce toxic effects Fresh
preparations of the test chemical should be employed
(n) Dose levels At least two dose levels should be used The highest
dose level should result m toxic effects (which should not produce an inci-
dence of fatalities which would prevent a meaningful evaluation) or should
be the highest dose attainable or 5 g/kg body weight
(m) Route of administration. Acceptable routes of administration
include oral, inhalation, admixture with food or water, and IP or IV injec-
tion
(e) Test performance—(1) Treatment and mating. The animals
should be dosed with the test substances 7 days per week over a period
of 35 days After treatment, each male should be caged with two untreated
females for a penod of 1 week At the end of 1 week, females should
be separated from males and caged individually. When females give birth,
the day of birth, litter size, and sex of progeny should be recorded All
male progeny should be weaned, and all female progeny should be dis-
carded
(2) Testing for translocation heterozygosity. When males are sexu-
ally mature, testing for translocation heterozygosity should begin One of
two methods should be used, the first method involves mating, determining
those FI progeny which are stenle or semistenle and subsequent
cytological analysis of suspect progeny, the other method does not involve
mating and determining sterility or semistenhty, all progeny are examined
cytologically.
(0 Determination of sterility or semisterility—(A) Conventional
method. Females are mated, usually three females for each male, and each
female is killed at irudpregnancy. Living and dead implantations are count-
ed. Criteria for determining normal and semistenle males are usually estab-
lished for each new strain because the number of dead implantations vanes
considerably among strains
(B) Sequential method. Males to be tested are caged individually
with females and the majonty of the presumably normal males are identi-
fied on the basis of a predetermined size of one or two Utters Breeding
pens are examined daily on weekdays beginning 18 days after pairing
Young are discarded immediately after they are scored Males that sire
a htter whose size is the same as or greater than the minimum set for
a translocation-free condition are discarded with their htter. If the litter
size is smaller than the predetermined number, a second htter is produced
with the same rule applying Males that cannot be classified as normal
after production of a second litter are tested further by the conventional
method or by cytological confirmation of translocation
-------
(u) Cytological analysis. For cytological analysis of suspected
senustenles, the air-drying technique is used Observation of at least two
diakmesis-metaphase 1 cells with mutivalent association constitutes the re-
quired evidence for the presence of a translocation Sterile males are exam-
ined by one of two methods, those with testes of normal size and sperm
in the epididyrms are examined by the same techniques used for
semistenles Animals with small testes are examined by squash prepara-
tions or, alternatively, by examination of rrutotic metaphase preparations
If squash preparations do not yield diakmesis-metaphase 1 cells, analysis
of spermatogoma or bone marrow for the presence of unusually long or
short chromosomes should be performed
(f) Data and report—(1) Treatment of results, (i) Data should be
presented in tabular form and should include the number of animals at
risk, the germ cell stage treated, the number of partial stenles and
sermsteriles (if the fertility test is used), the number of cytogenetically
confirmed translocation heterozygotes (if the fertility test is used, report
the number of confirmed stenles and confirmed partial stenies), the
translocation rate, and either the standard error of the rate or the upper
95 percent confidence limit on the rate
(u) These data should be presented for both treated and control
groups Historical or concurrent controls should be specified, as well as
the randomization procedure used for concurrent controls
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods
(3) Interpretation of results, (i) There are several catena for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of hentable translocations Another cntenon
may be based upon detection of a reproducible and statistically significant
positive response for at least one of the test points
(u) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of hentable translocations or
a statistically significant and reproducible positive response at any one of
the test points is considered nonmutagenic in this system.
(ni) Both biological and statistical significance should be considered
together in the evaluation
(4) Test evaluation, (i) Positive results in the hentable translocation
assay indicate that under the test conditions the test substance causes heri-
table chromosomal damage in the test species
(ii) Negative results indicate that under the test conditions the test
substance does not cause hentable chromosomal damage in the test spe-
cies
-------
(5) Test report. In addition to the reporting lecommendations as
specified under 40 CFR part 792 subpart J, the following specific informa-
tion should be reported
(i) Species, strain, age, weight, and number of animals of each sex
in each group
(u) Test chemical vehicle, route and schedule of administration, and
toxicity data
(m) Dosing regimen, doses tested, and rationale for dosage selection.
(iv) Mating schedule and number of females mated to each male
(v) The use of historical or concurrent controls
(vi) Screening procedure including the decision criteria used and the
method by which they were determined
(vn) Dose-response relationship, if applicable
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Generoso, W M et al Rentable translocation test in mice Muta-
tion Research 76 191-215 (1980)
(2) [Reserved]
-------
oEPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-229
August 1998
Health Effects Test
Guidelines
OPPTS 870.5500
Bacterial DNA Damage
or Repair Tests
-------
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 m 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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7US.C \36,etseq)
Final Guideline Release: This guideline is available from the U S
Go eminent Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines."
-------
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 Mutagemcity
Testing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evalua-
tion, Human and Domestic Animals) EPA report 540/09-82-025, 1982
(b) Purpose Bactenal 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)
(n) Tests performed in liquid culture (suspension tests)
(3) Strain selection—(i) Designation. At the present tune,
Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45)
pairs are recommended. Other pairs may be utilized when appropriate
(u) 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
design
(5) Metabolic activation Bactena should be exposed to the test sub-
stance both in the presence and absence of an appropnate metabolic activa-
tion system The most commonly used system is a cofactor supplemented
postmitochondnal fraction prepared from the livers of rodents treated with
enzyme inducing agents The use of other species, tissues, or techniques
may also be appropnate
(6) Control groups—(i) Concurrent controls. Concurrent positive,
negative, and vehicle controls should be included in each assay
(n) Negative controls The negative control should show nonpref-
erential growth inhibition (i e , should affect both strains equally) Chlor-
amphemcol is an example of a negative control
(111) Genotype specific controls Examples of genotype specific posi-
tive controls are methyl methanesulfonate for polA strains and mitomycm
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 appropnate
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 cnteria 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 m the diffusion test, it is most important
that the amounts of chemical on the disc (or in the wells) are exact rep-
licates When appropnate, a positive response should be confirmed by test-
ing over a narrow range of concentrations
-------
(0 Test performance—(1) Diffusion assay—(0 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 bactena 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, bactena 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 bactenal suspension may be exposed
to the test chemical and the number of surviving bactena determined (as
colony-forming units) either as a function of time of treatment or as a
function of the concentration of test agent
(n) Nonturbid suspensions of bactena 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 penod of incubation
(m) Paired bactenal suspensions (usually with some initial turbidity)
may be treated with a single dose of the chemical Positive results are
indicated by a differential inhibition m 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 penod This incubation penod should be for 18
to 24 hours 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 denved therefrom as square millimeters.
Dose-response data, if available, should be presented using the same units
(11) 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, 037, 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-
lanty
(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 serrusohd media
(in) 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
tilers (cells per nuUiliter) 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.
(n) 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
(m) 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 m another
system is difficult to evaluate m 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
(11) Phase of bacterial cell growth at time of use in the assay
(111) 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
(vu) 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 Salmonella/mammalian-microsome mutagemcity 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 phloxme, 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 carcmogemcity 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)
-------
&EPA
United Stales
Environmental Protection
Agency
Prevention Pesticides
and Toxtc Substances
(7101)
EPA712-C-98-230
August 1998
Health Effects Test
Guidelines
OPPTS 870.5550
Unscheduled DMA
Synthesis In Mammalian
Cells in Culture
-------
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 m 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
(7 USC. I36,etseq.)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5550 Unscheduled DMA synthesis in mammalian cells in
culture.
(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 5550 Unscheduled
DNA synthesis in mammalian cells in culture and OECD guideline 482
Genetic Toxicology DNA Damage and Repair, Unscheduled DNA Syn-
thesis in Mammalian Cells in Vitro
(b) Purpose. Unscheduled DNA synthesis (UDS) in mammalian cells
in culture measures the repair of DNA damage induced by a variety of
agents including chemicals, radiation and viruses UDS may be measured
in both in vitro and in vivo systems
(c) Definition. 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
Unscheduled DNA synthesis in mammalian cells in culture is the in-
corporation of tntium-labeled thymidme (3H-TdR) into the DNA of cells
which are not in the S phase of the cell cycle
(d) Reference substances. These may include, but need not be lim-
ited to, 7,12-dimethylbenzanthracene, 2-acetyIaminofluorene, 4-
nitroquinohne oxide or W-dimetnylnitrosamine
(e) Test method—(1) Principle. Mammalian cells in culture, either
primary cultures of rodent hepatocytes or established cell lines, are ex-
posed to the test agent. Established cell lines are treated both with and
without metabolic activation UDS is measured by the uptake of 3H-TdR
into the DNA of non-S phase cells. Uptake may be determined by
autoradiography or by liquid scintillation counting (LSC) of DNA from
treated cells
(2) Description—(i) Autoradiography. For autoradiography,
coverslip cultures of cells are exposed to test chemical in medium contain-
ing 3H-TdR. At the end of the treatment penod, cells are fixed, dipped
in autoradiographic emulsion, and exposed at 4 °C At the end of the expo-
sure penod, cells are stained and labeled nuclei are counted either manu-
ally or with an electronic counter Established cell lines should be treated
both with and without metabolic activation.
(ii) LSC determinations For LSC determinations of UDS, confluent
cultures of cells are treated with test chemical both with and without meta-
bolic activation At the end of the exposure penod, DNA is extracted from
-------
the treated cells Total DNA content is determined and extent of 3H-TdR
incorporation is determined by scintillation counting
(3) Cells—(i) Type of cells used in the assay. (A) A variety of cell
lines or primary cell cultures, including human cells, may be used in the
assay
(B) Established cell lines should be checked for Mycoplasma contami-
nation and may be periodically checked for karyotype stability
(11) Cell growth and maintenance Appropnate culture media and
incubation conditions (culture vessels CCh concentration), temperature,
and humidity should be used
(4) Metabolic activation (i) A metabolic activation system is not
used with primary cultures of rodent hepatocytes
(n) Established cell lines should be exposed to test substance both
in the presence and absence of an appropnate metabolic activation system
(5) Control groups Concurrent positive and negative (untreated and/
or vehicle), controls both with and without metabolic activation as appro-
priate should be included m each experiment
(6) Test chemicals—(i) Vehicle. Test chemicals and positive control
reference substances may be prepared in culture media or dissolved or
suspended in appropnate vehicles prior to treatment of the cells Final con-
centration of the vehicle should not interfere with cell viability or growth
rate
(11) Exposure concentrations Multiple concentrations of test sub-
stance, based upon cytotoxicity and over a range adequate to define the
response, should be used For cytotoxic chemicals, the first dose to elicit
a cytotoxic response in a preliminary assay should be the highest dose
tested Relatively insoluble compounds 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
(f) Test performance—(1) Primary cultures of rodent hepatocytes.
Freshly isolated rodent hepatocytes should be treated with chemical in me-
dium containing 3H-TdR At the end of the treatment period, cells should
be drained of medium, nnsed, fixed, dned, and attached to microscope
slides Slides should be dipped in autoradiographic emulsion, exposed at
4 °C for an appropnate length of time, developed, stained, and counted
(2) Established cell lines—(0 Autoradiographic techniques. The
techniques for treatment of established cell lines are the same as those
for pnmary cultures of rodent hepatocytes except that cells must not enter
S phase poor to treatment Entry of ceils into S phase may be blocked
by several methods (eg, by growth in medium deficient in argmine or
-------
low in serum or by treatment with chemical agents such as hydroxyurea)
Tests should be done both in the presence and absence of a metabolic
activation system
(11) LSC measurement of UDS Prior to treatment with test agent,
entry of cells into S phase should be blocked as descnbed in paragraph
(f)(2)(i) of this guideline Cells should be exposed to the test chemical
in medium containing 3H-TdR At the end of the incubation period, DNA
should be extracted from the cells by hydrolysis with perchloroacetic acid
or by other acceptable methods One aliquot of DNA is used to determine
total DNA content, a second aliquot is used to measure the extent of 3H-
TdR incorporation
(3) Acceptable background frequencies—(i) Autoradiographic de-
terminations Net incorporation of 3H-TdR into the nucleus of solvent
treated control cultures should be less than 1
(n) LSC determinations Historical background incorporation rates
of 3H-TdR into untreated established cell lines should be established for
each laboratory
(4) Number of cells counted A minimum of 50 cells per culture
should be counted for autoradiographic UDS determinations. Slides should
be coded before being counted Several widely separated random fields
should be counted on each slide Cytoplasm adjacent to the nuclear areas
should be counted to determine spontaneous background.
(5) Number of cultures Six independent cultures at each concentra-
tion and control should be used in LSC UDS determinations
(g) Data and report—(1) Treatment of results—(i)
Autoradiographic determinations. For autoradiographic determinations,
once untransformed data are recorded, background counts should be sub-
tracted to give the correct nuclear grain count Values should be reported
as net grains per nucleus Mean, median, and mode may be used to de-
scribe the distribution of net grains per nucleus
(11) LSC determinations For LSC determinations, 3H-TdR incorpo-
ration should be reported as disintegrations per minute per microgram of
DNA. Average disintegrations per minute per microgram of DNA with
standard deviation or standard error of the mean may be used to descnbe
distribution of incorporation in these studies
(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 incorporation of 3H-TdR into treated cells. Another
-------
criterion may be based upon detection of a reproducible and statistically
significant positive response for a least one of the test points
(a) A test substance which does not produce either a statistically sig-
nificant dose-related increase m the incorporation of 3H-TdR into treated
cells or a statistically significant and reproducible positive response at any
one of the test points is considered not to induce UDS in the test system
(m) Both biological and statistical significance should be considered
together in the evaluation
(4) Test evaluation, (i) Positive results in the UDS assay indicate
that under the test conditions the test substance may induce DNA damage
in cultured mammalian somatic cells
(n) Negative results indicate that under the test conditions the test
substance does not induce DNA damage m cultured mammalian somatic
cells.
(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) Cells used, density and passage number at time of treatment, num-
ber of cell cultures
(n) Methods used for maintenance of cell cultures including medium,
temperature, and CC>2 concentration
(in) Test chemical vehicle, concentration, and rationale for selection
of concentrations used in the assay
(iv) Details of both the protocol used preparation of the metabolic
activation system and its use in the assay.
(v) Treatment protocol
(vi) Positive and negative controls
(vu) Protocol used for autoradiography
(viii) Details of the method used to block entry of cells into S phase.
(ix) Details of the methods used for DNA extraction and determina-
tion of total DNA content in LSC determinations
(x) Historical background incorporation rates of 3H-TdR in untreated
cell lines
(xi) Dose-response relationship, if applicable
4
-------
(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 Salmonella/mammahan-microsome mutagemcity test Mutation
Research 31 347-364 (1975)
(2) Rasmussen, R E and Painter, R B Radiation-stimulated DNA
synthesis in cultured mammalian cells Journal of Cell Biology 29 11-19
(1966)
(3) Stich, H F et al DNA fragmentation and DNA repair as an in
vitro and in vivo assay for chemical procarcmogens, carcinogens and car-
cinogenic nitrosation products, Screening tests in chemical carcinogenesis
Eds Bartsch, H , Tomatis, L IARC Scientific, Lyon, No 12 (1976) pp
617-636
(4) Williams, G M Carcinogen-induced DNA repair in primary rat
liver cell cultures a possible screen for chemical carcinogens Cancer Let-
ters 1 231-236(1976)
(5) Williams, G M Detection of chemical carcinogens by unsched-
uled DNA synthesis in rat liver primary cell cultures Cancer Research
37 1845-1851(1977)
-------
United States Prevention Pesticides EPA712-C-98-P3?
Environmental Protection and Toxic Substances August *
(7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5575
Mitotic Gene Conversion
in Saccharomyces
cerevisiae
-------
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
USC. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.SC 136, etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www.epa gov/epahome/research htm) under the heading ' 'Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.5575 Mitotic gene conversion in Saccharomyces
cerevisiae.
(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 m developing this har-
monized OPPTS test guideline are OPPT 40 CFR 798.5575 Mitotic gene
gene conversion in Saccharomyces cerevisiae and OPP 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. The rrutotic gene conversion assay in the yeast,
Saccharomyces cerevisiae (S cerevisiae), measures the conversion of dif-
ferentially inactive alleles to wild-type alleles by mutagemc agents
Heteroallehc diploid yeast strains carry two different inactive alleles of
the same gene locus The presence of these alleles causes a nutritional
requirement, e g , these heteroallehc diploids grow only in medium supple-
mented with a specific nutrient such as tryptophan When gene conversion
occurs, a fully active wild-type phenotype is produced from these inactive
alleles through mtragenic recombination These wild-type colonies grow
on a medium lacking the specific nutritional requirement (selective me-
dium)
(c) Definitions. The definitions m 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.
Heteroallehc diploids are diploid strains of yeast carrying two dif-
ferent, inactive alleles of the same gene locus causing a nutritional require-
ment
Mitotic gene conversion is detected by the change of inactive alleles
of the same gene to wild-type alleles through mtragenic recombination
in mitotic cells
(d) Reference substances. These may include, but need not be lim-
ited to, hydrazine sulfate or 2-acetylaminofluorene
(e) Test method—(1) Principle, The method is based on the fact
that heteroallehc diploid yeast strains carry two inactive alleles of the same
gene locus making them dependent on a specific nutritional requirement
(e g., tryptophan) for their survival Treatment of such strains with muta-
gemc agents can cause conversion of these alleles back to the wild-type
condition which allows growth on a medium lacking the required nutrient
(selective medium)
-------
(2) Description. Heteroallehc diploid strains such as D7, requmng
a specific nutrient in the medium are treated with test chemical with and
without metabolic activation and plated on a selective medium lacking the
required nutrient The wild-type colonies that grow on the selective me-
dium as a result of gene conversion are scored
(3) Strain selection—(i) Designation. At the present time, S
cerevisiae strain D7 is recommended for use in this assay The use of
other strains may also be appropriate
(u) Preparation and storage. Stock culture preparation and storage,
growth requirements, method of strain identification and demonstration of
appropnate phenotypic requirements should be performed using good
microbiological techniques and should be documented
(111) Media. YEP glucose medium enriched with the appropnate
growth factors may be used for cell growth and maintenance Other media
may also be appropnate
(4) Selection of cultures. Cells should be grown with aeration in liq-
uid medium enriched with growth factors to early stationary phase Cells
should then be seeded on selective medium to determine the rate of sponta-
neous conversion Cultures with a high rate of spontaneous conversion
should be discarded
(5) Metabolic activation. Cells should be exposed to test chemical
both in the presence and absence of an appropnate metabolic activation
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 appropnate vehicle and then
further diluted in vehicle for use in the assay. Dimethylsulfoxide should
be avoided as a vehicle
(u) Exposure concentrations. (A) The test should initially be per-
formed over a broad range of concentrations. Among the cntena to be
taken into consideration for determining the upper limits of test chemical
concentration are cytotoxicity and solubility Cytotoxicity of the test chem-
ical may be altered in the presence of metabolic activation systems For
cytotoxic chemicals, the highest dose tested should not reduce survival
to less than 10 percent of that seen in the untreated control cultures. Rel-
atively insoluble chemicals should be tested up to the limits of solubility
For freely soluble nontoxic chemicals, the upper test chemical concentra-
tion should be determined on a case-by-case basis
-------
(B) When appropriate, a positive response should be confirmed by
using a narrow range of concentrations
(0 Test performance—(1) Treatment. Cultures should be treated
in liquid suspension Resting ceils should be treated in buffer, growing
cells should be treated in a synthetic medium Cultures with low sponta-
neous convertant frequencies should be centnfuged, washed and resus-
pended in liquid at the appropriate density Cells should be exposed to
test chemical both in the presence and absence of a metabolic activation
system Independent tubes should be treated for each concentration At
the end of the treatment period, cells should be centnfuged, washed and
resuspended in distilled water prior to plating on selective medium for
convertant selection and on complete medium to determine survival At
the end of the incubation period, plates should be scored for survival and
the presence of convertant colonies
(2) Number of cultures. At least six individual plates per treatment
concentration and control should be used
(3) Incubation conditions. All plates m a given expenment should
be incubated for the same time penod This incubation period may be
from 4 to 6 days at 28 °C
(g) Data and report—(1) Treatment of results. Individual plate
counts for test substance and control should be presented for both
convertants and survivors The mean number of colonies per plate and
standard deviation should also be presented Data should be presented in
tabular form indicating numbers of viable and convertant colonies scored,
survival frequency and convertant frequencies for each treatment and con-
trol culture Conversion frequencies should be expressed as number of
convertants per number of survivors Sufficient detail should be provided
for verification of survival and convertant frequencies
(2) Statistical evaluation. Data should be evaluated by appropnate
statistical methods
(3) Interpretation of results, (i) There are several cntena for deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of gene convertants 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 gene conversions or a statis-
tically significant and reproducible positive response at any one of the
test points is considered nonmutagemc in this system
(in) Both biological and statistical significance should be considered
together in the evaluation
-------
(4) Test evaluation, (i) Positive results in this assay indicate that
under the test conditions the test chemical causes irutotic gene conversion
in the yeast S cerevisiae
(11) Negative results indicate that under the test conditions the test
chemical does not cause mitotic gene conversion in S cerevisiae
(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
(n) Test chemical vehicle, doses used, and rationale for dosage selec-
tion
(m) Method used to select cultures
(iv) Treatment protocol including cell density at treatment and length
of exposure to test substance
(v) Details of both the protocol used to prepare the metabolic activa-
tion system and its use in the assay
(vi) Incubation times and temperatures
(vn) 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 Salmonella/mammalian-microsome mutagemcity test Mutation
Research 31 347-364 (1975)
(2) Callen, D F and Philpot, R M Cytochrome P-450 and the activa-
tion of promutagens in Saccharomyces cerevisiae Mutation Research
45.309-324 (1975)
(3) Zimmermann, F K Procedures used in the induction of mitotic
recombination and mutation in the yeast Saccharomyces cerevisiae. Hand-
book of mutagemcity test procedures Eds Kilby, B.J, Legator, M,
Nicols, W , Ramel, C Elsevier/North Holland Biomedical Press, Amster-
dam (1979) pp. 119-134.
(4) Zimmermann, F.K et al A yeast strain for simultaneous detection
of induced mitotic crossing over, mitotic gene conversion and reverse mu-
tation Mutation Research 28*381-388 (1975)
-------
a EPA
United Slates
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-9B-234
August 1998
Health Effects Test
Guidelines
OPPTS 870.5900
In Vitro Sister Chromatid
Exchange Assay
-------
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
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines."
-------
OPPTS 870.5900 In vitro sister chromatid exchange assay
(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 5900 In vitro sister
chromatid exchange assay and OECD 479 Genetic Toxicology. In Vitro
Sister Chromatid Exchange Assay in Mammalian Cells
(b) Purpose. The sister chromatid exchange (SCE) assay detects the
ability of a chemical to enhance the exchange of DNA between two sister
chromatids of a duplicating chromosome The test may be performed in
vitro, using, for example, rodent or human cells, or in vivo using mammals,
for example, rodents such as mice, rats and hamsters
(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
Sister chromatid exchanges are reciprocal interchanges of the two
chromatid arms within a single chromosome These exchanges are visual-
ized dunng the metaphase portion of the cell cycle and presumably require
enzymatic incision, translocation and hgation of at least two DNA helices.
(d) Test method—(1) Principle. Following exposure of cell cultures
to test chemicals, they are allowed to replicate in the presence of
bromodeoxyuridme (BrdU), followed by treatment with 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 analyzed for SCEs
(2) Description. In vitro SCE assays may employ monolayer or sus-
pension cultures of established cell lines, cell strains, or primary cell cul-
tures. Cell cultures are exposed to test chemical and are allowed to rep-
licate in the presence of BrdU Pnor to harvest, 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
analyzed for SCEs
(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
for Mycoplasma contamination and may be periodically checked for
karyotype stability
-------
(n) Cell growth and maintenance. Appropnate cultuie media and
incubation conditions (culture vessels, temperature, humidity, and CCb
concentration) should be used
(4) Metabolic activation. Cells should be exposed to test chemical
both in the presence and absence of an appropnate metabolic activation
system
(5) Control groups—Concurrent controls. Positive and negative
(untreated and/or vehicle) controls, with and without metabolic activation,
should be included in each experiment
(6) Test chemicals—(i) Vehicle. Test substances may be prepared
in culture media or dissolved or suspended in appropnate vehicles pnor
to treatment of the cells Final concentration of the vehicle should not
reduce cell viability or growth rate
(11) Exposure concentrations. Multiple concentrations of the test sub-
stance over a range adequate to define the response should be tested
Among the cntena to be taken into consideration for determining the upper
limits of test chemical concentration are cytotoxicity and solubility.
Cytotoxicity of the test substance may be altered in the presence of meta-
bolic activation systems Cytotoxicity may be evidenced by a large (e g ,
75 percent) decrease in the number of cells that have divided twice in
the presence of BrdU 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. When appropnate, a positive response should be confirmed by using
a narrow range of test concentrations
(e) Test performance—(1) Established cell lines and strains, (i)
Pnor to use in the assay, cells should be generated from stock cultures,
seeded in culture vessels at the appropnate density and incubated at
37 °C
(n) Cell lines and strains should be treated with test chemical both
with and without metabolic activation when they are in the exponential
stage of growth At the end of the exposure penod, cells should be washed
and incubated for two replication cycles in medium containing BrdU After
BrdU is added, the cultures should be handled in darkness, under "safe"
(e g , darkroom) lights, or in dim light from incandescent lamps to mini-
mize photolysis of BrdU containing DNA At the end of the BrdU incuba-
tion penod, cells should be fixed and stained for SCE determination Cul-
tures should be treated with a spindle inhibitor (e.g., colchicme or
Colcemid ®) 2 hours pnor to harvesting
(2) Human lymphocyte cultures, (i) For preparation of human lym-
phocyte cell cultures, hepanmzed or acid-citrate-dextrose treated whole
blood should be added to culture medium containing a mitogen, e.g,
-------
phytohemagglutinm (PHA) and incubated at 37 °C White cells sedimented
by gravity (buffy coat) or lymphocytes which have been purified on a
density gradient such as Ficoll-Hypaque may also be utilized
(n) Cells should be exposed to the test chemical dunng at last two
time intervals, e g , Go and S Exposure during the Go phase of the cell
cycle should be accomplished by adding the test substance prior to addition
of rmtogen Exposure dunng or after the first S phase may be accom-
plished by exposing cells 24-30 hours after mitosis, under "safe" (eg,
darkroom) lights, or in dim light from incandescent lamps to minimize
photolysis of BrdU containing DNA At the end of the BrdU incubation
period, cells should be fixed and stained for SCE determination Cultures
should be treated with a spindle inhibitor (e g., colchicine or Colcemid®)
2 hours prior to harvesting
(3) Human lymphocyte cultures, (t) For preparation of human lym-
phocyte cell cultures, hepannized or acid-citrate-dextrose treated whole
blood should be added to culture medium containing a mitogen, e g,
phytohemagglutinm (PHA) and incubated at 37 °C White cells sedimented
by gravity (buffy coat) or lymphocytes which have been purified on a
density gradient such as Ficoll-Hypaque may also be utilized
(11) Cells should be exposed to the test chemical dunng at least two
time intervals, e g, Go and S Exposure dunng the Go phase of the cell
cycle should be accomplished by adding the test substance pnor to addition
of mitogen. Exposure dunng or after the first S phase may be accom-
plished by exposing cells 24-30 hours after mitogen stimulation After
exposure, cells should be washed and then cultured in the absence of the
chemical
(4) Culture harvest time. A single harvest time, one that yields an
optimal percentage of second division metaphases, is recommended If
there is reason to suspect that this is not a representative sampling time
(which may occur for short-lived, cycle specific chemicals), then additional
harvest times should be selected
(5) Staining method. Staining of slides to reveal SCEs can be per-
formed according to any of several protocols However, the fluorescence
plus Giemsa method is recommended
(6) Number of cultures. At least two independent cultures should
be used for each expenmental point
(7) Analysis. Slides should be coded before analysis The number
of cells to be analyzed should be based upon the spontaneous control fre-
quency and defined sensitivity and the power of the test chosen 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 Uni-
form criteria for scoring SCEs should be used
(0 Data and report—(I) Treatment of results Data should be pre-
sented in tabular form, providing scores for both the number of SCEs for
each metaphase and the number of SCEs per chromosome for each meta-
phase
(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 ts a statistically significant dose-
related increase m the number of sister chromatid exchanges Another cri-
terion may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test substance concentra-
tions
(») A test substance which produces neither a statistically significant
dose-related increase in the number of sister chromatid exchanges nor a
statistically, significant and reproducible positive response at any one of
the test points is considered not to induce rearrangements of segments of
DNA in this system
(m) Both biological and statistical significance should be considered
together m the evaluation
(4) Test evaluation, (i) Positive results in the in vitro SCE assay
indicate that under the test conditions the test substance induces reciprocal
chromatid interchanges in cultured mammalian somatic cells
(n) Negative results indicate that under the test conditions the test
substance does not induce reciprocal chromatid interchanges in cultured
mammalian somatic cells
(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) Cells used, density at time of treatment, number of cell cultures.
00 Methods used for maintenance of cell cultures including medium,
temperature, and CC«2 concentration
(m) Test chemical vehicle, concentration and rationale for the selec-
tion of the concentrations of test chemical used in the assay, duration of
treatment
(iv) Details of both the protocol used preparation of the metabolic
activation system and its use in the assay
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(v) Growth period in BrdU, identity of spindle inhibitor, its concentra-
tion and duration of treatment
(vi) Time of cell harvest
(vn) Positive and negative controls
(via) Method used to prepare slides for SCE determination
(ix) Catena for scoring SCEs
(x) Details of the protocol used for growth and treatment of human
cells if used in the assay
(xi) Dose-response relationship, if applicable
(g) References. The following references should be consulted for ad-
ditional background matenal on this test guideline
(1) Latt, S A et al Sister chromatid exchanges a report of the U.S
EPA's Gene-Tox Program Mutation Research 87 17-62 (1981)
(2) [Reserved]
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&EPA
United Slates
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-23S
August 1998
Health Effects Test
Guidelines
OPPTS 870.5915
In Vivo Sister Chromatid
Exchange Assay
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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 m 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
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.SC tt6,etseq)
Final Guideline Release: This guideline is available from the U S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and ScientistsH'est Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.5915 In vivo sister chromatid exchange assay.
(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 798.5915 In vivo sister
chromatid exchange assay and OPP 84-2 Mutagemcity Testing (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation, Human and
Domestic Animals) EPA report 540/09-82-025, 1982
(b) Purpose. The sister chromatid exchange (SCE) assay detects the
ability of a chemical to enhance the exchange of DNA between two sister
chromatids of a duplicating chromosome The test may be performed in
vitro using cultured mammalian cells or in vivo using nonmammalian or
mammalian tissues The most commonly used assays employ bone marrow
or lymphocytes from mammalian species such as mice, rats, or hamsters
Human lymphocytes may also be used
(c) Definition. 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
Sister chromatid exchanges are reciprocal interchanges of the two
chromatid arms within a single chromosome. These exchanges are visual-
ized dunng the metaphase portion of the cell cycle and presumably require
enzymatic incision, translocation and ligation of at least two DNA helices.
(d) Test method—(1) Principle. Animals are exposed to test sub-
stance by appropriate routes followed by administration of
bromodeoxyundme (BrdU) A spindle inhibitor (e g, colchicme or
Colcemid®) is administered pnor to sacrifice After sacrifice, tissue is ob-
tained and metaphase preparations made, stained, and scored for SCE
(2) Description. The method descnbed here employs bone marrow
of laboratory rodents exposed to test chemicals. After treatment with test
chemical, animals are further treated with BrdU and, pnor to sacrifice,
with a spindle inhibitor (e g , colchicme or Colcemid®) to arrest cells m
c-metaphase After sacrifice, chromosome preparations from bone marrow
cells are made, stained, and scored for SCE ,
(3) Animal selection—(i) Species and strain. Any appropriate mam-
malian species may be used Examples of commonly used rodent species
include mice, rats, and hamsters
(11) Age. Healthy, young adult animals should be used
1
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(in) Number and sex. At least five female and five male animals
per experimental and control group should be used The use of a single
sex or different number of animals should be justified
(iv) Assignment to groups. Animals should be randomized and as-
signed to treatment and control groups
(4) Control groups—(i) Concurrent controls. Current positive and
negative (vehicle) controls should be included in the assay
(11) Positive controls. A compound know to produce SCE in vivo
should be employed as the positive control
(5) Test chemicals—(i) Vehicle. When possible, test chemicals
should be dissolved in isotonic saline or distilled water Water insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The ve-
hicle used should neither interfere with the test compound nor produce
toxic effects Fresh preparations of the test compound should be employed
(n) Dose levels. For an initial assessment, one dose of the test sub-
stance may be used, the dose being the maximum tolerated dose or that
producing some indication of toxicity as evidenced by animal morbidity
(including death) or target cell toxicity The LDso is a suitable guide Addi-
tional dose levels may be used For determination of dose-response, at
least three dose levels should be used
(111) Route of administration. The usual routes of administration are
IP or oral Other routes may be appropnate
(iv) Treatment schedule. In general, test substances should be ad-
ministered only once However, based upon lexicological information a
repeated treatment schedule may be employed
(e) Test performance—(1) Treatment Animals should be treated
with test chemical followed by administration of BrdU. BrdU may be ad-
ministered by multiple IP injections, by continuous tail vein infusion or
by subcutaneous implantation of tablets Animals should be treated with
a spindle inhibitor (e g, colchicme or Colcemid®) 2 hours prior to sac-
rifice After sacrifice, bone marrow should be extracted and slides made
and prepared for SCE evaluation
(2) Staining method. Staining of slides to reveal SCEs can be per-
formed according to any of several protocols However, the fluorescence
plus Giemsa method is recommended
(3) Number of cells scored. The number of cells to be analyzed per
animal should be based upon the number of animals used, the negative
control frequency, the predetermined sensitivity and the power chosen for
the test Slides should be coded before microscopic analysis
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(f) Data and report—(1) Treatment of results. Data should be pre-
sented in tabular form, providing scores for both the number of SCE for
each metaphase and the number of SCE per chromosome for each rneta-
phase Differences among animals within each group should be considered
before making comparisons between treated and control groups
(2) Statistical evaluation. Data should be evaluated by appropnate
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 SCE Another criterion may be based
upon detection of a reproducible and statistically significant positive re-
sponse for at least one of the test points
(n) A test substance which does not produce either a statistically sig-
nificant dose-related increase in the number of SCE or a statistically sig-
nificant and reproducible positive response at any one of the test points
is considered not to induce rearrangements of DNA segments in this sys-
tem
(m) Both biological and statistical significance should be considered
in the evaluation
(4) Test evaluation, (i) Positive results in the m vivo SCE
assaymdicate that under the test conditions the test substance induces re-
ciprocal interchanges m the bone marrow of the test species
(11) Negative results indicate that under the test conditions the test
substance does not induce reciprocal interchanges in the bone marrow of
the test species
(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) Species, strain, age, weight, number, and sex of animals in each
treatment and control group.
(ii) Test chemical vehicle, dose level used, rationale for dose selec-
tion, toxicity data, negative and positive controls
(m) Route and schedule of administration of both test chemical and
BrdU
(iv) Identity of spindle inhibitor, its concentration and duration of
treatment
(v) Time of sacrifice after administration of BrdU
(vi) Details of the protocol used for slide preparation
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(vn) Criteria for scoring SCE
(vm) Dose-response iclationship if applicable
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Allen, J W et al Bromodeoxyundme tablet methodology for in
vivo studies of DNA synthesis Somatic Cell Genetics 4 393^05 (1978)
(2) Allen, J W et al Simplified technique for in vivo analysis of
sister chromatid exchanges using 5-bromodeoxyundme tablets Cyto-
genetics Cell Genetics 18 231-237 (1977)
(3) Latt, S A et al Sister chromatid exchanges A report of the U.S.
EPA Gene-Tox Program Mutation Research 87 17-62 (1981).
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