-------
HEALTH EFFECTS TEST GUIDELINES AND
SUPPORT DOCUMENTS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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50272-101
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
"EPA 560/6-82-001
4. Title and Subtitle
Health Effects Test Guidelines
7. Author(s)
3. Recipient's Accession No.
PB82-23298U
5. Report Date
August, 1982
8. Performing Organization Rept. No.
9. Performing Organization Name and Address
Office of Pesticides and Toxic Substances
Office of Toxic Substances (TS-792)
United States Environmental Protection Agency
401 M Street, S.W.
Washington, B.C. 20460
10. Project/Task/Work Unit No.
11. Contract(C) or Grant(G) No.
(C)
(G)
12. Sponsoring Organization Name and Address
13. Type of Report & Period Covered
Annual Report
14.
15. Supplementary Notes
16. Abstract (Limit: 200 words)
These documents constitute a set of 39 health effects test guidelines (and, in some
cases, support documents) that may be cited as methodologies to be used in chemical
specific test rules promulgated under Section 4(a) of the Toxic Substances Control
_Act (TSCA). These guidelines cover testing for general toxicity, specific organ/
tissue toxicity, mutagenicity, neurotoxicity and special studies. The guidelines
will be published in loose leaf form and updates will be made available as changes
are dictated by experience and/or advances in the state-of-the-art.
17. Document Analysis a. Descriptors
b. Identifiers/Open-Ended Terms
c. COSATI Field/Group
REPRODUCED BY
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SPRINGFIELD, V*. 22161
18. Availability Statement
Release unlimited
19. Security Class (This Report)
Unclassified
20. Security Class (This Page)
Unclassified
21. No. of Pages
432
22. Price
(See ANSI-Z39.18)
See fnstructions on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
Department of Commerce
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TABLE OF CONTENTS
Guidelines
I. GENERAL TOXICITY TESTING
Acute Exposure
Dermal Toxicity
Inhalation toxicity
Oral Toxicity
Subchronic Exposure
Dermal Toxicity
Inhalation Toxicity
Oral Toxicity
Chronic Exposure
Chronic Toxicity
Oncogenicity
Combined Chronic Toxicity/
Oncogenicity
II. SPECIFIC ORGAN/TISSUE TOXICITY
Dermal Sensitization
Primary Dermal Irritation
Primary Eye Irritation
Reproduction/Fertility Effects
Teratogenicity
III. MUTAGENICITY
Gene Mutations
Salmonella typhimurium
Escheria coli WP2 and WP2 uvrA
Aspergillus nidulans
Neurospora crassa
Sex Linked Recessive Lethal
Test in Drosophila
melanogaster '
Somatic Cells in Culture
Mouse Specific-Locus Test
Index
HG-Acute-Dermal
HG-Acute-Inhal
HG-Acute-Oral
HG-Subchronic-Dermal
HG- S i/b oh r on i c-1 nh a 1
HG-Subjhronic-Oral
HG-Chronic
HG-Chronic-Onco
HG-Chronic-Combined
HG-Organ/Tissue-Dermal Sensit
HG-Organ/Tissue-Dermal Irrit
HG-Organ/Tissue-Eye Irrit
HG-Organ/Tissue-Repro/Fert
HG-Organ/Tissue-Terato
HG-Gene Muta-S. typhimurium
HG-Gene Muta-E. coli
HG-Gene Muta-A. nidulans
HG-Gene Muta-N. crassa
HG-Gene Muta-Insects
HG-Gene Muta-Somatic Cells
HG-Gene Muta-Mammal
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Guidelines
Index
Chromosomal Effects
In Vitro Mammalian
Cytogenetics
In Vivo Mammalian Bone Marrow
Cytogenetics Tests
Chromosomal Analysis
In Vivo Micronucleus Assay
Heritable Translocation Test in
• Drosophila' melanogaster
Dominant Lethal Ass.ay
Rodent Heritable Translocation
Assay "
DNA Effects
Differential Growth" Inhibition
of Repair Deficient Bacteria:
"Bacterial DNA Damage or
Repair Tests"
Unschedbled DNA Synthesis in
Mammalian Cells in Culture
Mitotic Gene Conversion in
gaccharomyces cerevisi.ae
In Vitro .Sister Chromatid
Exchange Assay
In Vivo Sister Chrpmatid
Exchange Assay
IV. NEUROTOXICITY
Neuropathology
Support Document
Peripheral Nerve Function
Support Document
Motor Activity
Support Document
Acute Delayed Neurotoxicity
of Organophosphorus Substances
Subchronic Delayed Neurotoxicity
erf Organophosp'horus Substances
HG-Chromo-In vitro
HG-Chromo-Bone Marrow
HG-Chromo-Micronuc
HG-Chromo-Insects
HG-Chromo-Dom Lethal
HG-Chromo-Herit Translocat
HG-DNA-Damage/Repair
HG-DNA-Unsched Syn
HG-DNA-Gene Conversion
HG-DNA-Sister-Chrom-In vitro
i
HG-DNA-Sister-Chrom-In vivo
HG-Neuro-Path
HS-Neuro-Path
HG-Neuro-Peri Nerve
HS-Neuro-Peri Nerve
HG-Neuro-Motor Act
HS-Neuro-Motor Act
HG-Neuro-Acute Delayed
HG-Neuro-Subchronic Delayed
V. SPECIAL STUDIES
Metabolism
HG-Spec Stud-Metab
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PREAMBLE
The following guidelines describe methods for performing testing
of chemical substances under the Toxic Substances Control Act
(TSCA). These methods include the state-of-the-art for
evaluating certain properties, processes and effects of
chemical substances. They are intended to provide guidance
to test sponsors in developing test protocols for compliance
with test rules issued under Section 4 of the TSCA. They
may also provide guidance for testing which is unrelated
to regulatory requirements. Support documentation is
included for some of these guidelines. It is expected that
additional guidelines and support documentation will be
incorporated later as the state-of-the-art evolves or the
need for them warrants.
Since these guidelines are divided into three sections which
cover the diverse areas of health effects, environmental
effects and chemical fate testing, there are some differences
in the ways they are presented. These differences are
explained in an introduction prepared for each section.
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I. GENERAL TOXICITY
TESTING
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HG-Acute-Dermal
August, 1982
ACUTE EXPOSURE
DERMAL TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Acute-Dermal
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a substance, determination of acute
dermal toxicity is useful where exposure by the dermal route
is likely. The purpose of an acute dermal study is to
determine the median lethal dose (LD50), its statistical
limits and slope using a single exposure up to a 24-hour
period and a 14-day post-exposure observation period. This
purpose can be accomplished by performing the provisions
contained in this guideline. Data from an acute dermal
toxicity study serves as a basis for classification and
labelling. It is also an initial step in establishing a
dosage regimen in subchronic and other studies. With the
addition of certain other test elements this guideline may
provide information on dermal absorption and the mode of
toxic action of a substance by this route.
II. DEFINITIONS
A. Acute dermal toxicity is the adverse effects occuring
within a short time period following dermal application
of single dose of a test substance.
B. Dosage is a general term comprising the dose, its
frequency and the duration of dosing.
C. Dose is the amount of test substance applied. Dose is
expressed as weight of test substance (g, mg) per unit
weight of test animal (e.g. mg/kg).
D. Dose-effect is the relationship between the dose and
the magnitude of a defined biological effect either in
an individual or in a population sample.
E. Dose-response is the relationship between the dose and
the proportion of a population sample showing a defined
effect. -
F. LD50 (median lethal dose), dermal, is a statistically
derived single dose of a test substance that can be
expected to cause death in 50 percent of treated
animals when applied to the skin. "The LD50 value is
expressed in terms of weight of test substance (g, mg)
per unit weight of test animal (e.g. mg/kg).
-1-
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HG-Acute-Dermal
III. PRINCIPLE OF THE TEST METHOD
The test substance is applied to the skin in graduated doses
to several groups of experimental animals, one dose being
used per group. Subsequently, observations of effects and
deaths are made. Animals which die during the test are
necropsied, and at the conclusion of the test the surviving
animals are sacrificed and necropsied.
IV. LIMIT TEST
If a test at a dose of at least 2000 mg/kg body weight,
using the procedures described for this study, produces no
compound-related mortality, then a full study using three
dose levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
The rat, rabbit or guinea pig may be used. The
albino rabbit is preferred because of its size,
skin permeability and extensive data base.
Commonly used laboratory strains should be
employed. If a species other than the three
indicated above is used, the tester should provide
justification and reasoning for its selection.
2. Age
Young adult animals should be used. The following
weight ranges are suggested to provide animals of
a size which facilitates the conduct of the test:
rats, 200 to 300 g; rabbits 2.0 to 3.0 kg; guinea
pigs 350 to 450 g.
3. Sex
a. Equal numbers of animals of each sex with
healthy intact skin should be used for each
dose level.
b. The females should be nulliparous and non-
pregnant.
4. Numbers
At least 10 animals (5 females and 5 males) at
each dose level should be used.
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HG-Acu te-Dermal
B. Control groups
Neither a concurrent untreated nor vehicle control
group is recommended except when the toxicity of the
vehicle is unknown.
C. Dose levels and dose selection
1. At least three dose levels should be used and
spaced appropriately to produce test groups with a
range of toxic effects and mortality rates. The
data should be sufficient to produce a dose-
response curve and, where possible, permit an
acceptable determination of the LD50.
2. Vehicle
a. When necessary, the test substance is
dissolved or suspended in a suitable
vehicle. It is recommended that whenever
possible the usage of an aqueous solution be
considered first, followed by consideration
of a solution in oil (e.g. corn oil) and then
by possible solution in other vehicles. For
non-aqueous vehicles the toxic
characteristics of the vehicle should be
known, and if not known should be determined
before the test.
b. When testing solids, which may be pulverized
if appropriate, the test substance should be
moistened sufficiently with water or, where
necessary, a suitable vehicle to ensure good
contact with skin. When a vehicle is used,
the influence of the vehicle on penetration
of skin by the test substance should be taken
into account.
D. Exposure duration
The duration of exposure should be 24 hours.
E. Observation period
The observation period should be at least 14 days.
However, the duration of observation should not be
fixed rigidly. It should be determined by the toxic
reactions, rate of onset and length of recovery period,
and may thus be extended when considered necessary.
The time at which signs of toxicity appear and
disappear, their duration and the time of death are
important, especially if there is a tendency for deaths
to be delayed.
-3-
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HG-Acute-Dermal
F. Preparation of animal skin
1. Shortly before testing, fur should be clipped from
the dorsal area of the trunk of the test
animals. Shaving may be employed, but it should
be carried out approximately 24 hours before the
test. Care must be taken to avoid abrading the
skin which could alter its permeability.
2. Not less than 10 percent of the body surface area
should be clear for the application of the test
substance. The weight of the animal should be
taken into account when deciding on the area to be
cleared and on the dimensions of any covering
used.
G. Application of test substance
1. The test substance should be applied uniformly
over an area which is approximately 10 percent of
the total body surface area. With highly toxic
substances the surface area covered may be less,
but as much of the area should be covered with as
thin and uniform a film as possible.
2. The test substance should be held in contact with
the skin with a porous gauze dressing and non-
irritating tape throughout a 24-hour exposure
period. The test site should be further covered
in a suitable manner to retain the gauze dressing
and test substance and ensure that the animals
cannot ingest the test substance. Restrainers may
be used to prevent the ingestion of the test
substance, but complete immobilization is not a
recommended method.
3. At the end of the exposure period, residual test
substance should be removed, where practicable
using water or an appropriate solvent.
H. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily with
appropriate actions taken to minimize loss of
animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
-4-
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HG-Acute-Dermal
3. Cage-side observations should include, but not be
limited to, changes in skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern. Particular
attention should be directed to observations of
tremors, convulsions, salivation, diarrhea,
lethargy, sleep and coma.
4. Individual weights of animals should be determined
shortly before the test substance is applied.
Individual weights should also be taken weekly
thereafter and at death. Changes in weight should
be calculated and recorded when survival exceeds
one day.
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, surviving animals should
be weighed and sacrificed.
I. Gross pathology
Consideration should be given to performing a gross
necropsy of all animals where indicated by the nature
of the toxic effects observed. All gross pathological
changes should be recorded.
J. Histopathology
Microscopic examination of organs showing evidence of
gross pathology in animals surviving 24 hours or more
should also be considered because it may yield useful
information.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, time of death of individual animals at
different dose levels, number of animals displaying
other signs of toxicity, description of toxic effects
and necropsy findings.
-5-
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HG-Acute-Dermal
B. Evaluation of results
The dermal LD50 value should always be considered in
conjunction with the observed toxic effects and any
necropsy findings. The LD50 value is a relatively
coarse measurement, useful only as a reference value
for classification and labelling purposes, and
expressing the possible lethal potential of the test
substance following dermal exposure. Reference should
always be made to the experimental animal species in
which the LD50 value was obtained. An evaluation
should include the relationships, if any, between the
animals' exposure to the test substance and the
incidence and severity of all abnormalities, including
behavioral and clinical abnormalities, gross lesions,
body weight changes, effects on mortality, and any
other toxicological effects.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported.
1. Tabulation of response data by sex and dose level
(i.e. number of animals dying, number of animals
showing signs of toxicity, number of animals
exposed);
2. Description of toxic effects;
3. Time of death after dosing;
4. LD50 value for each sex (intact skin) determined
at 14 days (with the method of determination
specified);
5. Ninety-five percent confidence interval for the
LD50;
6. Dose-mortality curve and slope (where permitted by
the method of determination);
7. Body weight data; and
8. Pathology findings, when performed.
-6-
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HG-Acute-Dermal
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Bliss C.I. 1938. The determination of the dosage
mortality curve from small numbers. Quarterly Journal
Pharm. Pharmacology, 11:192-216.
2. Finney, D.G. 1971. Probit Analysis. Chapter 3—
Estimation of the median effective dose, Chapter 4--
Maximum likelihood estimation. 3rd Edition. London:
Cambridge University Press. 60 pp. 1971.
3. Litchfield J.T., Jr., Wilcoxon, F. 1949. A simplified
method of evaluating dose-effect experiments. Journal
of Pharmacology and Experimental Therapeutics. 96:99-
113.
4. Miller, L.C., Tainter, M.L. 1944. Estimation of the
ED50 and its error by means of logarithmic graph
paper. Proceedings of the Society for Experimental
Biology and Medicine. 57:261-264.
5. NAS. 1977. National Academy of Sciences. Principles
and Methods for Evaluating the Toxicity of Household
Substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
6. Thompson, W.R. 1947. Use of moving averages and
interpolation to estimate median effective dose.
Bacteriological Review. 11:115-141.
7. Weil, C.S. 1952. Tables for convenient calculation of
median effective dose and instructions in their use,
Biometrics, 8:249-263.
8. WHO. 1978. World Health Organization. Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organization. 272 pp.
-7-
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HG-Acute-Inhal
August, 1982
ACUTE EXPOSURE
INHALATION TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
, WASHINGTON, D.C. 20460
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HG-Acute-Inhal
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of an inhalable material, such as a gas,
volatile substance or aerosol/particulate, determination of
acute inhalation toxicity is usually an initial step. It
provides information on health hazards likely to arise from
short term exposure by the inhalation route. The purpose of
an acute inhalation study is to determine the median lethal
dose (LC50), its statistical limits and slope using a single
exposure, usually of 4 hours, and a 14-day post-exposure
observation period. This purpose can be accomplished by
performing the provisions contained in this guideline. Data
from an acute study serves as a basis for classification and
labelling. It is also an initial step in establishing a
dosage regimen in subchronic and other studies. With the
addition of certain other test elements, this guideline may
provide information on the mode of toxic action of a
substance.
II. DEFINITIONS
A. Acute inhalation toxicity is the adverse effects caused
by a substance following a single uninterrupted
exposure by inhalation over a short period of time (24
hours or less) to a substance capable of being inhaled.
B. Aerodynamic diameter applies to the size of particles
of aerosols. It is the diameter of a sphere of unit
density which behaves aerodynamically as the particle
of the test substance. It is used to compare particles
of different size and densities and to predict where in
the respiratory tract such particles may be
deposited. This term is used in contrast to measured
or geometric diameter which is representative of actual
diameters which in themselves cannot be related to
deposition within the respiratory tract.
C. The geometric mean diameter or the median diameter is
the calculated aerodynamic diameter which divides the
particles of an aerosol in half based on the weight of
the particles. Fifty percent of the particles by
weight will be larger than the median diameter and 50
percent of the particles will be smaller than the
median diameter. The median diameter and its geometric
standard deviation is used to statistically describe
the particle size distribution of any aerosol based on
the weight and size of the particles.
-1-
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HG-Acute-Inhal
D. Inhalable diameter refers to that aerodynamic diameter
of a particle which is considered to be inhalable for
the organism. It is used to refer to particles which
are capable of being inhaled and may be deposited
anywhere within the respiratory tract from the trachea
to the alveoli. For man, the inhalable diameter is
considered as 15 micrometers or less.
E. The LC50 (median lethal concentration) is a
statistically derived concentration of a substance that
can be expected to cause death during a limited
exposure interval (usually 4 hours) or within a fixed
time after exposure in 50 percent of animals exposed
when administered by inhalation. The LC50 value is
expressed as weight of test substance (g, mg) per
standard volume of air (e.g. mg/1).
III. PRINCIPLE OF THE TEST METHOD
Several groups of experimental animals are exposed for a
defined period to the test substance in graduated
concentrations, one concentration being used per group.
Subsequently observations of effects and deaths are made.
Animals which die during the test should be necropsied and
at the conclusion of the test surviving animals should be
sacrificed and necropsied as necessary.
IV. LIMIT TEST
If a test at an exposure of 5 mg/1 (actual concentration of
respirable substances) for 4 hours or, where this is not
possible due to physical or chemical properties of the test
substance, the maximum attainable concentration, using the
procedures described for this study, produces no compound-
related mortality, then a full study using three dose levels
might not be necessary.
V. TEST PROCEDURES
A. Animal Selection
1. Species and strain
Although several mammalian test species may be
used the rat is the preferred species. Commonly
used laboratory strains should be used. If
another mammalian species is employed, the tester
should provide justification/reasoning for its
selection.
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HG-Acute-Inhal
2. Age
Young adult animals should be used. The weight
variation of animals used in a test should not
exceed ± 20 percent of the mean weight of each
sex.
3. Sex
a. Equal numbers of animals of each sex should
be used for each dose level.
b. The females should be nulliparous and non-
pregnant.
4. Numbers
At least 10 animals (5 females and 5 males) at
each dose level should be used.
B. Control groups
Where a vehicle is used to help generate an appropriate
concentration of the substance in the atmosphere a
vehicle control group should be used.
C. Dose levels and dose selection
1. At least three exposure concentrations should be
used and spaced appropriately to produce test
groups with a range of toxic effects and mortality
rates. The data should be sufficient to produce a
dose-mortality curve and, where possible, permit
an acceptable determination of an LC50.
2. Where necessary, a suitable vehicle may be added
to the test substance to help generate an
appropriate concentration of the test substance in
the atmosphere. If a vehicle or diluent is
needed, ideally it should not ellicit important
toxic effects itself or substantially alter the
chemical or toxicological properties of the test
substance.
3. In the case of potentially explosive test
substances, care should be taken to avoid
generating explosive concentrations.
4. To establish suitable exposure concentrations, a
trial test is recommended.
-3-
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HG-Acute-Inhal
D. Exposure duration
The duration of exposure should be at least 4 hours
after equilibration of the chamber concentrations.
E. Observation period
The observation period should be at least 14 days.
However, the duration of observation should not be
fixed rigidly. It should be determined by the toxic
reactions, rate of onset and length of recovery period,
and may thus be extended when considered necessary.
The time at which signs of toxicity appear and
disappear, their duration and the time of death are
important, especially if there is a tendency for deaths
to be delayed.
F. Inhalation exposure
1. The animals should be tested with inhalation
equipment designed to sustain a dynamic air flow
of 12 to 15 air changes per hour, ensure an
adequate oxygen content of 19 percent and an
evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize
crowding of the test animals and maximize their
exposure to the test substance. This is best
accomplished by individual caging. As a general
rule to ensure stability of a chamber atmosphere,
the total "volume" of the test animals should not
exceed 5 percent of the volume of the test
chamber. Alternatively, oro-nasal, head-only, or
whole body individual chamber exposure may be
used.
2. A suitable analytical concentration control system
should be used. The rate of air flow should be
adjusted to ensure that conditions throughout the
equipment are essentially the same. Maintenance
of a slight negative pressure inside the chamber
will prevent leakage of the test substance into
the surrounding area.
3. The temperature at which the test is performed
should be maintained at 22°C (* 2°). Ideally, the
relative humidity should be maintained between 40
to 60 percent, but in certain instances (e.g.
tests of aerosols, use of water vehicle) this may
not be practicable.
-4-
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HG-Acute-Inhal
G. Physical measurements
Measurements or monitoring should be made of the
following:
1. The rate of air flow should be monitored
continuously, but should be recorded at least
every 30 minutes.
2. The actual concentrations of the test substance
should be measured in the breathing zone. During
the exposure period the actual concentration of
the test substance should be held as constant as
practicable. Continuous monitoring is
desirable. Measurement of actual concentrations
should be recorded near the beginning, middle, and
end of the exposure period.
3. During the development of the generating system,
particle size analysis should be performed to
establish the stability of aerosol
concentrations. During exposure, analysis should
be made as often as necessary to determine the
consistency of particle size distribution and
homogeneity of the exposure stream.
4. Temperature and humidity should be monitored
continuously but should be recorded at least every
30 minutes.
H. Food and water during exposure period
Food should be withheld during exposure. Water may
also be withheld in certain cases.
I. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily with
appropriate actions taken to minimize loss of
animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
-5-
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HG-Acute-Inhal
3. Cage-side observations should include, but not be
limited to, changes in the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern. Particular
attention should be directed to observation of
tremors, convulsions, salivation,diarrhea,
lethargy, sleep and coma.
4. Individual weights of animals should be determined
shortly before the test substance is
administered. Individual weights should be taken
weekly thereafter and at death. Changes in weight
should be calculated and recorded when survival
exceeds one day.
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, the surviving animals
should be weighed and sacrificed.
J. Gross pathology
Consideration should be given to performing a gross
necropsy of all animals where indicated by the nature
of the toxic effects observed with particular reference
to any changes in the respiratory tract. Where there
are signficant signs of toxicity indicating the
possible involvement of other organs, these should be
examined and all gross pathological changes recorded.
K. Histopathology
Microscopic examination of organs showing evidence of
gross pathology in animals surviving 24 hours or more,
should be considered since it may yield useful
information.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, time of death of individual animals at
different exposure levels, number of animals displaying
other signs of toxicity, description of toxic effects
and necropsy findings.
-6-
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HG-Acute-Inhal
B. The LC50 value should always be considered in
conjunction with the observed toxic effects and any
necropsy findings. The LC50 value is a relatively
coarse measurement, useful only as a reference value
for classification and labelling purposes, and
expressing possible lethal potential of the test
substance following inhalation. Reference should
always be made to the experimental animal species in
which the LC50 value was obtained. An evaluation
should include the relationship, if any, between the
animals' exposure to the test substance and the
incidence and severity of all abnormalities, including
behavioral and clinical abnormalities, gross lesions,
body weight changes, effects on mortality and any other
toxicological effects.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Test conditions
a. Description of exposure apparatus including
design, type, dimensions, source of air,
system for generating particulates and
aerosols, method of conditioning air,
treatment of exhaust air and the method of
housing the animals in a test chamber.
b. The equipment for measuring temperature,
humidity, and particulate aerosol
concentrations and size should be described.
2. Exposure data
These should be tabulated and presented with mean
values and a measure of variablity (e.g. standard
deviation) and should include:
a. Airflow rates through th inhalation
equipment;
b. Temperature and humidity of air;
c. Nominal concentration (total amount of test
substance fed into the inhalation equipment
divided by volume of air);
-7-
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HG-Acute-Inhal
d. Actual concentration in test breathing zone;
and
e. Particle size distribution (e.g. median
aerodynamic diameter of particles with
standard deviation from the mean)
Animal data
a. Tabulation of response data by sex and
exposure level (i.e. number of animals dying,
number of animals showing signs of toxicity,
number of animals exposed);
b. Description of toxic effects;
c. Time of death during or following exposure;
d. LC50 for each sex determined at 14 days (with
method of calculations specified);
e. Ninety-five percent confidence interval for
the LC50;
f. Dose-mortality curve and slope (where
permitted by the method of determination);
g. Body weight data; and
h. Pathology findings, when performed.
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HG-Acute-Inhal
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Bliss, C.I. 1938. The determination of the dosage
mortality curve from small numbers. Quarterly Journal
Pharm. Pharmacology. 11:192-216.
2. Finney, D.G. 1971. Probit Analysis. Chapter 3—
Estimation of the median effective dose, Chapter 4—
Maximum likelihood estimation. 3rd Edition. London:
Cambridge University Press. 60 pp.
3. Litchfield, J.T., Jr., Wilcoxon, F. 1949. A
simplified method of evaluating dose-effect
experiments. Journal of Pharmacology and Experimental
Therapeutics. 96:99-115.
4. Miller, L.C., Tainter, M.L. 1944. Estimation of the
ED50 and its error by means of logarithmic graph
paper. Proceedings of the Society for Experimental
Biology and Medicine. 57:261-264.
5. NAS. 1977. National Academy of Sciences. Principles
and Procedures for Evaluating the Toxicity of Household
Substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Reserach Council, National Academy of
Sciences. 130 pp.
6. Smyth, H.F., Jr., Carpenter, C.P., Weil, C.S.,
Striegel, J.A. 1962. Range finding toxicity data:
List VI. American Industrial Hygiene Association
Journal. 23:95.
7. Thompson, W.R. 1947. Use of moving averages and
interpolation to estimate median effective dose.
Bacteriological Review. 11:115-145.
8. Weil, C.S. 1952. Tables for convenient calculation of
median effective dose and instructions in their use.
Biometrics. 8:249-263.
9. WHO. 1979. World Health Organization. Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organization. 272 pp.
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HG-Acute-Oral
August, 1982
ACUTE EXPOSURE
ORAL TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Acute-Oral
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a substance, determination of acute oral
toxicity is usually an initial step. It provides
information on health hazards likely to arise from a short
term exposure by the oral route. The purpose of an acute
oral study is to determine the median lethal dose (LD50),
its statistical limits and slope using a single exposure up
to a 24-hour period and a 14-day post-exposure observation
period. This purpose can be accomplished by performing the
provisions contained in this guideline. Data from an acute
study serves as a basis for classification and labelling.
It is also an initial step in establishing a dosage regimen
in subchronic and other studies. With the addition of
certain other test elements, this guideline may provide
information on the mode of toxic action of a substance.
II. DEFINITIONS
A. Acute oral toxicity is the adverse effects occurring
within a short time of oral administration of a single
dose of a substance or multiple doses given within 24
hours.
B. Dosage is a general term comprising the dose, its
frequency and the duration of dosage.
C. Dose is the amount of test substance administered.
Dose is expressed as weight of test substance (g, mg)
per unit weight of test animal (e.g. mg/kg).
D. Dose-effect is the relationship between the dose and
the magnitude of a defined biological effect either in
an individual or in a population sample.
E. Dose-response is the relationship between the dose and
the proportion of a population sample showing a defined
effect.
F. LD50 (median lethal dose), oral, is a statistically
derived single dose of a substance that can be expected
to cause death in 50 percent of animals when
administered by the oral route. The LD50 value is
expressed in terms of weight of test substance (g, mg)
per unit weight of test animal (e.g. mg/kg).
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HG-Acute-Oral
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered orally by gavage in
graduated doses to several groups of experimental animals,
one dose being used per group. Subsequently observations of
effects and deaths are made. Animals which die during the
test should be necropsied, and at the conclusion of the test
the surviving animals should be sacrificed and necropsied.
This guideline is directed primarily to studies in rodent
species but may be adapted for studies in non-rodents.
IV. LIMIT TEST
If a test at a dose level of at least 5000 mg/kg body
weight, using the procedures described for the study,
produces no compound-related mortality, then a full study
using three dose levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
Although several mammalian test species may be
used, the rat is the preferred species. Commonly
used laboratory strains should be employed. If
another species is used, the tester should provide
justification and reasoning for its selection.
2. Age
Young adult animals should be used. The weight
variation of animals used in a test should not
exceed ± 20 percent of the mean weight for each
sex.
3. Sex
a. Equal numbers of animals of each sex should
be used for each dose level.
b. The females should be nulliparous and non-
pregnant.
Numbers
At least 10 animals (5 females and 5 males) at
each dose level should be used.
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HG-Acute-Oral
B. Control groups
Neither a concurrent untreated nor vehicle control
group is required except when the toxicity of the
vehicle is unknown.
C. Dose levels and dose selection
1. At least three dose levels should be used and
spaced appropriately to produce test groups with a
range of toxic effects and mortality rates. The
data should be sufficient to produce a dose
response curve and, where possible, permit an
acceptable determination of the LD50.
2. Vehicle
Where necessary, the test substance is dissolved
or suspended in a suitable vehicle. It is
recommended that wherever possible the usage of an
aqueous solution be considered first, followed by
consideration of a solution in oil (e.g. corn oil)
and then by possible solution in other vehicles.
For non-aqueous vehicles the toxic characteristics
of the vehicle should be known, and if not known
should be determined before the test.
3. Volume
The maximum volume of liquid that can be
administered at one time depends on the size of
the test animal. In rodents, the volume should
not exceed 1 ml/100 g body weight. Variability in
test volume should be minimized by adjusting the
concentration to ensure a constant volume at all
dose levels.
.D. Exposure duration
The test substance should be administered over a period
not exceeding 24 hours.
E. Observation period
The observation period should be at least 14 days.
However, the duration of observation should not be
fixed rigidly. It should be determined by the toxic
reactions, rate of onset and length of recovery period,
and may thus be extended when considered necessary.
The time at which signs of toxicity appear and
disappear, their duration and the time to death are
important, especially if there is a tendency for deaths
to be delayed.
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HG-Acute-Oral
F. Exposure
1. The test substance should be administered in a
single dose by gavage, using a stomach tube or
suitable intubation cannula.
2. Animals should be fasted prior to test substance
administration. For the rat, food should be
withheld overnight; for other rodents with higher
metabolic rates a shorter period of fasting is
appropriate.
3. After the substance has been administered, food
may be withheld for an additional 3-4 hours.
4. If a single dose is not possible, the dose may be
given in smaller fractions over a period not
exceeding 24 hours. Where a dose is administered
in fractions, it may be necessary to provide the
animals with food and water depending on the
length of the dosing period.
G. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily with
appropriate actions taken to minimize loss of
animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation of weak or moribund animals).
3. Cage-side observations should include, but not be
limited to,,changes in the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern. Particular
attention should be directed to observation of
tremors, convulsions, salivation, diarrhea,
lethargy, sleep and coma.
4. Individual weights of animals should be determined
shortly before the test substance is administered,
weekly thereafter and at death. Changes in
weights should be calculated and recorded when
survival exceeds one day.
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HG-Acute-Oral
5. The time of death should be recorded as precisely
as possible.
6. At the end of the test, surviving animals should
be weighed and sacrificed.
H. Gross pathology
Consideration should be given to performing a gross
necropsy of all animals where indicated by the nature
of the toxic effects observed. All gross pathology
changes should be recorded.
I. Histopathology
Microscopic examination of organs showing evidence of
gross pathology in animals surviving 24 hours or more
should also be considered because it may yield useful
information.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, time of death of individual animals at
different dose levels, number of animals displaying
other signs of toxicity, description of toxic effects
and necropsy findings.
B. Evaluation of results
The LD50 value should always be considered in
conjunction with the observed toxic effects and any
necropsy findings. The LD50 value is a relatively
coarse measurement useful only as a reference value for
classification and labelling purposes, and for
expressing the possible lethal potential of the test
substance by the injestion route. Reference should
always be made to the experimental animal species in
which the LD50 value was obtained. An evaluation
should include the relationship, if any, between the
animal's exposure to the test substance and the
incidence and severity of all abnormalities, including
behavioral and clinical abnormalities, gross lesions,
body weight changes, effects on mortality, and any
other toxicological effects.
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HG-Acute-Oral
Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Tabulation of response data by sex and dose level
(i.e. number of animals dying; number of animals
showing signs of toxicity; number of animals
exposed);
2. Description of toxic effects;
3. Time of death after dosing;
4. LD50 value for each sex determined at 14 days
(with the method of determination specified);
5. Ninety-five percent confidence interval for the
LD50;
6. Dose-mortality curve and slope (where permitted,
by the method of determination);
7. Body weight data; and
8. Pathology findings, when performed.
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HG-Acute-Oral
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Balazs, T. 1970. "Measurement of acute toxicity," in
"Methods in Toxicology." Edited by G.E. Paget.
Philadelphia: F.A. Davis Co. PP. 49-82.
2. Bliss, C.I. 1938. The determination of the dosage
mortality curve from small numbers. Quarterly Journal
Pharm. Pharmacology. 11:192-216.
3. Finney, D.G. 1971. Probit Analysis. Chapter 3—
Estimation of the median effective dose, Chapter 4—
Maximum likelihood estimation. 3rd Edition. London:
Cambridge University Press. 60 pp.
4. Hunter W.J., Lingk, W., Recht, P. 1979.
Intercomparison Study on the Determination of Single
Administration Toxicity in Rats. Journal Association
of Official Analytical Chemists. 62(4):864-873.
5. Litchfield, J.T., Jr., Wilcoxon, F. 1949. A
simplified method of evaluating dose-effect
experiments, Journal of Pharmacology and Experimental
Therapeutics. 96:99-115.
6. Miller, L.C., Tainter, M.L. 1944. Estimation of the
ED50 and its error by means of logarithmic graph paper,
Proceedings of the Society for Experimental Biology and
Medicine. 57:261-264.
7. NAS. 1977. National Academy of Sciences. Principles
and Procedures for Evaluating the Toxicity of Household
Substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
8. Thompson, W.R. 1947. Use of moving averages and
interpolation to estimate median effective dose.
Bacteriological Review. 11:115-145.
9. Weil, C.S. 1952. Tables for convenient calculation of
median effective dose and instructions in their use.
Biometrics. 8:249-263.
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HG-Subchronic-Dermal
August, 1982
SUBCHRONIC EXPOSURE
DERMAL TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Subchronic-Dermal
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a chemical, the determination of
subchronic dermal toxicity may be carried out after initial
information on toxicity has been obtained by acute
testing. The subchronic dermal study has been designed to
permit the determination of the no-observed-effect level and
toxic effects associated with continuous or repeated
exposure to a test substance for a period of 90 days. The
test is not capable of determining those effects that have a
long latency period for development (e.g., carcinogenicity
and life shortening). It provides information on health
hazards likely to arise from repeated exposure by the dermal
route over a limited period of time. It will provide
information on target organs, the possibilities of
accumulation, and can be of use in selecting dose levels for
chronic studies and for establishing safety criteria for
human exposure.
II. DEFINITIONS
A. Subchronic dermal toxicity is the adverse effects
occurring as a result of the repeated daily exposure of
experimental animals to a chemical by dermal
application for part (approximately 10 percent) of a
life span.
B. Dose in a dermal test is the amount of test substance
applied to the skin (applied daily in subchronic
tests). Dose is expressed as weight of the substance
(g, mg) per unit weight of test animal (e.g. mg/kg).
C. No-effect level/No-toxic-effect level/No-adverse-effect
level/No-observed-effeet level is the maximum dose used
in a test which produces no observed adverse effects.
A no-observed-effect level is expressed in terms of the
weight of a test substance given daily per unit weight
of test animal (mg/kg).
D. Cumulative toxicity is the adverse effects of repeated
doses occuring as a result of prolonged action on, or
increased concentration of the administered test
substance or its metabolites in susceptible tissues.
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HG-Subchronic-Dermal
III. PRINCIPLE OF THE TEST METHOD
The test substance is applied daily to the skin in graduated
doses to several groups of experimental animals, one dose
level per unit group, for a period of 90 days. During the
period of application the animals are observed daily to
detect signs of toxicity. Animals which die during the test
are necropsied, and at the conclusion of the test the
surviving animals are sacrificed and necropsied and
appropriate histopathological examinations carried out.
IV. LIMIT TEST
If a test at one dose level of at least 1000 mg/kg body
weight (expected human exposure may indicate the need for a
higher dose level), using the procedures described for this
study, produces no observable toxic effects and if toxicity
would not be expected based upon data of structurally
related compounds, then a full study using three dose levels
might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
The rat, rabbit or guinea pig may be used although
the albino rabbit is preferred. The albino rabbit
is preferred because of its size, skin
permeability and extensive data base. Commonly
used laboratory strains should be employed. If
another mammalian species is used, the tester
should provide justification/reasoning for its
selection.
2. Age
Young adult animals should be used. The following
weight ranges at the start of the test are
suggested in order to provide animals of a size
which facilitates the conduct of the test: rats,
200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs,
350 to 450 g.
3. _S_ex
a. Equal numbers of animals of each sex with
healthy skin should be used at each dose
level.
b. The females should be nulliparous and non-
pregnant.
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HG-Subchronic-Dermal
4. Numbers
a. At least 20 animals (10 females and 10 males)
should be used at each dose level.
b. If interim sacrifices are planned, the number
should be increased by the number of animals
scheduled to be sacrificed before completion
of the study.
B. Control groups
A concurrent control group is recommended. This group
should be an untreated or sham treated control group
or, if a vehicle is used in administering the test
substance, a vehicle control group. If the toxic
properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control
groups are recommended.
C. Satellite group
A satellite group of 20 animals (10 animals per sex)
may be treated with the high dose level for 90 days and
observed for reversibility, persistence, or delayed
occurence, of toxic effects for a post-treatment period
of appropriate length, normally not less than 28 days.
D. Dose level and dose selection
1. In subchronic toxicity tests, it is desirable to
have a dose-response relationship as well as a no-
observed-toxic-effeet level. Therefore, at least
three dose levels with a control and, where
appropriate, a vehicle control (corresponding to
the concentration of vehicle at the highest
exposure level) should be used. Doses should be
spaced appropriately to produce test groups with a
range of toxic effects and mortality rates. The
data should be sufficient to produce a dose-
response curve.
2. The highest dose level should result in toxic
effects but not produce severe skin irritation or
an incidence of fatalities which would prevent a
meaningful evaluation.
3. The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure, the lowest dose
level should exceed this.
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HG-Subchronic-Dermal
4. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If more
than one intermediate dose is used, the dose
levels should be spaced to produce a gradation of
toxic effects.
5. In the low and intermediate groups and in the
controls the incidence of fatalities should be
low, to permit a meaningful evaluation of the
results.
E. Exposure conditions
The animals are treated with test substance, ideally
for at least 6 hours per day on a 7-day per week basis,
for a period of 90 days. However, based primarily on
practical considerations, application on a 5-day per
week basis is considered to be acceptable.
F. Observation period
1. Duration of observation should be for at least 90
days.
2. Animals in the satellite group scheduled for
follow-up observations should be kept for a
further 28 days without treatment to detect
recovery from, or persistence of, toxic effects.
G. Preparation of animal skin
1. Shortly before testing, fur should be clipped from
the dorsal area of the trunk of the test
animals. Shaving may be employed, but it should
be carried out approximately 24 hours before the
test. Repeat clipping or shaving is usually
needed at approximately weekly intervals. When
clipping or shaving the fur, care should be taken
to avoid abrading the skin, which could alter its
permeability.
2. Not less than 10 percent of the body surface area
should be clear for the application of the test
substance. The weight of the animal should be
taken into account when deciding on the area to be
cleared and on the dimensions of any covering
used.
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HG-Subchronic-Dermal
3. When testing solids, which may be pulverized if
appropriate, the test substance should be
moistened sufficiently with water or, where
necessary, a suitable.vehicle to ensure good
contact with the skin. When a vehicle is used,
the influence of the vehicle on penetration of
skin by the test substance should be taken into
account.
H. Application of the test substance
1. The test substance should be applied uniformly
over an area which is approximately 10 percent of
the total body surface area. With highly toxic
substances, the surface area covered may be less,
but as much of the area should be covered with as
thin and uniform a film as possible.
2. During the exposure period, the test substance
should be held in contact with the skin with a
porous gauze dressing and non-irritating tape.
The test site should be further covered in a
suitable manner to retain the gauze dressing and
test substance and ensure that the animals cannot
ingest the test substance. Restrainers may be
used to prevent the ingestion of the test
substance, but complete immobilization is not a
recommended method.
I. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily with
appropriate actions taken to minimize loss of
animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Signs of toxicity should be recorded as they are
observed, including the time of onset, the degree
and duration.
4. Cage-side observations should include, but not be
limited to, changes in skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern.
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HG-Subchronic-Dermal
5. Animals should be weighed weekly. Food
consumption should also be determined weekly if
abnormal body weight changes are observed.
6. At the end of the study period, all survivors in
the non-satellite treatment groups are
sacrificed. Moribund animals should be removed
and sacrificed when noticed.
J. Clinical examinations
1. The following examinations should be made on at
least 5 animals of each sex in each group:
a. Certain hematology determinations should be
carried out at least three times during the
test period: just prior to initiation of
dosing (baseline data), after approximately
30 days on test and just prior to terminal
sacrifice at the end of the test period.
Hematology determinations which should be
appropriate to all studies: hematocrit,
hemoglobin concentration, erythrocyte count,
total and differential leucocyte count, and a
measure of clotting potential such as
clotting time, prothrombin time,
thromboplastin time, or platelet count.
b. Certain clinical biochemistry determinations
on blood should be carried out at least three
times: just prior to initiation of dosing
(baseline data), after approximately 30 days
on test and just prior to terminal sacrifice
at the end of the test period. Test areas
which are considered appropriate to all
studies: electrolyte balance, carbohydrate
metabolism, and liver and kidney function.
The selection of specific tests will be
influenced by observations on the mode of
action of the substance. Suggested
determinations: calcium, phosphorus,
chloride, sodium, potassium, fasting glucose
(with the period of fasting appropriate to
the species), serum glutamic-pyruvic
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HG-Subchronic-Dermal
transaminase*, serum glutamic oxaloacetic
transaminase**, ornithine decarboxylase,
gamma glutamyl transpeptidase, urea nitrogen,
albumen, blood creatinine, total bilirubin
and total serum protein measurements. Other
determinations which may be necessary for an
adequate toxicological evaluation include:
analyses of lipids, hormones, acid/base
balance, methemoglobin and cholinesterase
activity. Additional clinical biochemistry
may be employed, where necessary, to extend
the investigation of observed effects.
* Now known as serum alanine
aminotransferase.
** Now known as serum aspartate
aminotransferase.
2. The following examinations should be made on at
least 5 animals of each sex in each group:
a. Ophthalmological examination, using an
ophthalmoscope or equivalent suitable
equipment, should be made prior to exposure
to the test substance and at the termination
of the study. If changes in the eyes are
detected all animals should be examined.
b. Urinalysis is not suggested on a routine
basis, but only when there is an indication
based on expected or observed toxicity.
K. Gross necropsy
1. All animals should be subjected to a full gross
necropsy which includes examination of the
external surface of the body, all orifices, and
the cranial, thoracic and abdominal cavities and
their contents.
2. The liver, kidneys, adrenals, brain and gonads
should be weighed wet, as soon as possible after
dissection, to avoid drying.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible future
histopathological examination: normal and treated
skin; all gross lesions; brain - including
sections of medulla/pons, cerebellar cortex and
cerebral cortex; pituitary; thyroid/parathyroid;
thymus; trachea; lungs; heart; (sternum with bone
marrow); salivary glands; liver; spleen; kidneys;
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HG-Subch ironic-Dermal
adrenals; pancreas; gonads; uterus; accessory
genital organs; aorta; gall bladder (if present);
esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder;
representative lymph node; (mammary gland); (thigh
musculature); peripheral nerve; (eye); (femur -
including articular surface); (spinal cord at
three levels - cervical; midthoracic and lumbar);
and (exorbital lachrymal glands).
L. Histopathology
The following histopathology should be performed:
1. Full histopathology on normal and treated skin and
on organs and tissues, listed above, of all
animals in the control and high dose groups.
2. All gross lesions in all animals.
3. Target organs in all animals.
4. The tissues mentioned in brackets (listed above) -
if indicated by signs of toxicity or expected
target organ involvement.
5. Lungs of animals (rodents) in the low and
intermediate dose groups should be subjected to
histopathological examination for evidence of
infection, since this provides a convenient
assessment of the state of health of the animals.
6. When a satellite group is used, histopathology
should be performed on tissues and organs
identified as showing effects in other treated
groups.
VI. DATA AND REPORTING
A. Treatment of results
1. Data should be summarized in tabular form, showing
for each test group the number of animals at the
start of the test, the number of animals showing
lesions, the types of lesions and the percentage
of animals displaying each type of lesion.
2. All observed results, quantitative and incidental,
should be evaluated by an appropriate statistical
method. Any generally accepted statistical method
may be used; the statistical methods should be
selected during the design of the study.
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HG-Subchronic-Dermal
/
B. Evaluation of results
The findings of a subchronic dermal toxicity study
should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the
observed toxic effects and the necropsy and
histopathological findings. The evaluation should
include the relationship between the dose of the test
substance and the presence or absence, the incidence
and severity, of abnormalities, including behavioral
and clinical abnormalities, gross lesions, identified
target organs, body weight changes, effect on mortality
and any other general or specific toxic effects. A
properly conducted subchronic test should provide a
statisfactory estimation of a no-effect level.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported.
1. Group animal data
Tabulation of toxic response data by species,
strain, sex and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of toxicity;
and
c. Number of animals exposed.
2. Individual animal data
a. Time of death during the study or whether
animals survived to termination;
b. Time of observation of each abnormal sign and
its subsequent course;
c. Body weight data;
d. Food consumption data when collected;
e. Hematological tests employed and all results;
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HG-Subchronic-Dermal
f. Clinical biochemistry tests employed and all
results;
g. Necropsy findings;
h. Detailed description of all histopathological
findings; and
i. Statistical treatment of results where
appropriate.
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HG-Subchronic-Dermal
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Draize, J.H. 1959. Third Printing: 1975. "Dermal
toxicity," in "Appraisal of Chemicals in Food, Drugs
and Cosmetics." The Association of Food and Drug
Officials of the United States. PP. 46-59.
2. Fitzhugh, O.G. 1959. Third Printing: 1975.
"Subacute toxicity," in "Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics." The
Association of Food and Drug Officials of the United
States. PP. 26-35.
3. NAS. 1977. National Academy of Sciences. Prinicples
and Procedures for Evaluating the Toxicity of Household
Substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
4. WHO. 1978. World Health Organization. Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organization. 272 pp.
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HG-Subchronic-Inhal
August, 1982
SUBCHRONIC EXPOSURE
INHALATION TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Subchronic-Inhal
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a gas, volatile substance, or
aerosol/particulate, determination of subchronic inhalation
toxicity may be carried out after initial information on
toxicity has been obtained by acute testing. The subchronic
inhalation study has been designed to permit the
determination of the no-observed-effect level and toxic
effects associated with continuous or repeated exposure to a
test substance for a period of 90 days. The test is not
capable of determining those effects that have a long
latency period for development (e.g., carcinogenicity and
life shortening). It provides information on health hazards
likely to arise from repeated exposures by the inhalation
route over a limited period of time. It will provide
information on target organs, the possibilities of
accumulation, and can be of use in selecting dose levels for
chronic studies and for establishing safety criteria for
human exposure. Hazards of inhaled substances are
influenced by the inherent toxicity and by physical factors
such as volatility and particle size.
II. DEFINITIONS
A. Subchronic inhalation toxicity is the adverse effects
occuring as a result of the repeated daily exposure of
experimental animals to a chemical by inhalation for
part (approximately 10 percent) of a life span.
B. Aerodynamic diameter applies to the size of particles
of aerosols. It is the diameter of a sphere of unit
density which behaves aerodynamically as the particle
of the test substance. It is used to compare particles
of different size and densities and to predict where in
the respiratory tract such particles may be
deposited. This term is used in contrast to measured
or geometric diameter which is representative of actual
diameters which in themselves cannot be related to
deposition within the respiratory tract.
C. The geometric mean diameter or the median diameter is
the calculated aerodynamic diameter which divides the
particles of an aerosol in half based on the weight of
the particles. Fifty percent of the particles by
weight will be larger than the median diameter and 50
percent of the particles will be smaller than the
median diameter. The median diameter describes the
particle size distribution of any aerosol based on the
weight and size of the particles.
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HG-Subchronic-Inhal
D. Inhalable diameter refers to that aerodynamic diameter
of a particle which is considered to be inhalable for
the organism. It is used to refer to particles which
are capable of being inhaled and may be deposited
anywhere within the respiratory tract from the trachea
to the alveoli. For man, inhalable diameter is
considered as 15 micrometers or less.
E. Dose is the amount of test substance administered.
Dose is expressed as weight of test substance (g, mg)
per unit weight of test animal (e.g. mg/kg), or as
weight of test substance per unit weight of food or
drinking water.
F. No-effect level/No-toxic-effect level/No-adverse-effect
level/No-observed-effeet level is the maximum dose used
in a test which produces no observed adverse effects.
A no-observed-effect level is expressed in terms of the
weight of a substance given daily per unit weight of
test animal (mg/kg).
G. Cumulative toxicity is the adverse effects of repeated
doses occuring as a result of prolonged action on, or
increased concentration of the administered substance
or its metabolites in susceptible tissues.
III. PRINCIPLE OF THE TEST METHOD
Several groups of experimental animals are exposed daily for
a defined period to the test substance in graduated
concentrations, one concentration being used per group, for
a period of 90 days. During the period of administration,
the animals are observed daily to detect signs of
toxicity. Animals which die during the test are necropsied
and at the conclusion of the test, surviving animals are
sacrificed and necropsied and appropriate histopathological
examinations carried out.
IV. TEST PROCEDURES
A. Animal selection
1. Species and strain
A variety of rodent species may be used although
the rat is the preferred species. Commonly used
laboratory strains should be employed. If another
mammalian species is used, the tester should
provide justification/reasoning for its selection.
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HG-Subchronic-Inhal
Age
Young adult animals should be used. At the
commencement of the study the weight variation of
animals should not exceed ± 20 percent of the mean
weight for each sex.
3. Sex
a. Equal numbers of animals of each sex should
be used at each dose level.
b. Females should be nulliparous and non-
pregnant.
4. Numbers
a. At least 20 animals (10 females and 10 males)
should be used for each test group.
b. If interim sacrifices are planned, the number
of animals should be increased by the number
of animals scheduled to be sacrificed before
the completion of the study.
B. Control groups
A concurrent control group is recommended. This group
should be an untreated or sham treated control group.
Except for treatment with the test substance, animals
in the control group should be handled in a manner
identical to the test group animals. Where a vehicle
is used to help generate an appropriate concentration
of the substance in the atmosphere, a vehicle control
group should be used. If the toxic properties of the
vehicle are not known or cannot be made available, both
untreated and vehicle control groups are recommended.
C. Satellite group
A satellite group of 20 animals (10 animals per sex)
may be treated with the high concentration level for 90
days and observed for reversibility, persistence, or
delayed occurence of toxic effects for a post-treatment
period of appropriate length, normally not less than 28
days.
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HG-Subchronic-Inhal
D. Dose levels and dose selection
1. In subchronic toxicity tests, it is desirable to
have a dose-response relationship as well as a no-
observed-toxic-effeet level. Therefore, at least
three dose levels with a control and, where
appropriate, a vehicle control (corresponding to
the concentration of vehicle at the highest
exposure level) should be used. Doses should be
spaced appropriately to produce test groups with a
range of toxic effects and mortality rates. The
data should be sufficient to produce a dose-
response curve.
2. The highest concentration should result in toxic
effects but not produce an incidence of fatalities
which would prevent a meaningful evaluation.
3. The lowest concentration should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest
concentration should exceed this.
4. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If more
than one intermediate dose level is used, the
concentrations should be spaced to produce a
gradation of toxic effects.
5. In the low and intermediate groups and in the
controls the incidence of fatalities should be
low, to permit a meaningful evaluation of the
results.
6. In the case of potentially explosive test
substances, care should be taken to avoid
generating explosive concentrations.
E. Exposure conditions
The animals are exposed to the test substance, ideally
for 6 hours per day on a 7-day per week basis, for a
period of 90 days. However, based primarily on
practical considerations, exposure on a 5-day per week
basis is considered to be acceptable.
F. Observation period
1. Duration of observation should be for at least 90
days.
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HG-Subchronic-Inhal
2. Animals in a satellite group scheduled for follow-
up observations should be kept for an additional
28 days without treatment to detect recovery from,
or persistence of, toxic effects.
G. Inhalation exposure
1. The animals should be tested in inhalation
equipment designed to sustain a dynamic air flow
of 12 to 15 air changes per hour and ensure an
adequate oxygen content of 19 percent and an
evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize
crowding of the test animals and maximize their
exposure to the test substance. This is best
accomplished by individual caging. As a general
rule, to ensure stability of a chamber atmosphere,
the total "volume" of the test animals should not
exceed 5 percent of the volume of the test
chamber. Oro-nasal or head-only exposure may be
used if it is desirable to avoid concurrent
exposure by the dermal or oral routes.
2. A dynamic inhalation system with a suitable
analytical concentration control system should be
used. The rate of air flow should be adjusted to
', ensure that conditions throughout the equipment
are essentially the same. Maintenance of slight
negative pressure inside the chamber will prevent
leakage of the test substance into the surrounding
areas.
3. The temperature at which the test is performed
should be maintained at 22° C (± 2°). Ideally,
the relative humidity should be maintained between
40 to 60 percent, but in certain instances (e.g.
tests of aerosols, use of water vehicle) this may
not be practicable.
H. Physical measurements
Measurements or monitoring should be made of the
following:
1. The rate of air flow should be monitored
continuously but should be recorded at least every
30 minutes.
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HG-Subchronic-Inhal
2. The actual concentrations of the test substance
should be measured in the breathing zone. During
the exposure period the actual concentrations of
the test substance should be held as constant as
practicable, monitored continuously and measured
at least at the beginning, at an intermediate time
and at the end of the exposure period.
3. During the development of the generating system,
particle size analysis should be performed to
establish the stability of aerosol
concentrations. During exposure, analysis should
be conducted as often as necessary to determine
the consistency of particle size distribution.
4. Temperature and humidity should be monitored
continuously but should be recorded at least every
30 minutes.
I. Food and water during exposure period
Food should be withheld during exposure. Water may
also be withheld in certain cases.
J. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily with
appropriate actions taken to minimize loss of
animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Signs of toxicity should be recorded as they are
observed including the time of onset, the degree
and duration.
4. Cage-side observations should include, but not be
limited to, changes in the skin and fur, eyes and
mucous membranes, respiratory, circulatory,
autonomic and central nervous systems, somatomotor
activity and behavior pattern.
5. Animals should be weighed weekly. Food
consumption should also be determined weekly if
abnormal body weight changes are observed.
6. At the end of the study period all survivors in
the non-satellite treatment groups should be
sacrificed. Moribund animals should be removed
and sacrificed when noticed.
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HG-Subchronic-Inhal
K. Clinical examinations
1. The following examinations should be made on at
least 5 animals of each sex in each group:
a. Certain hematology determinations should be
carried out at least three times during the
test period: just prior to initiation of
dosing (base line data), after approximately
30 days on test and just prior to terminal
sacrifice at the end of the test period.
Hematology determinations which should be
appropriate to all studies: hematocrit,
hemoglobin concentration, erythrocyte count,
total and differential leucocyte count, and a
measure of clotting potential such as
clotting time, prothrombin time,
thromboplastin time, or platelet count.
b. Certain clinical biochemistry determinations
on blood should be carried out at least three
times: just prior to initiation of dosing
(base line data), after approximately 30 days
on test and just prior to terminal sacrifice
at the end of the test period. Clinical
biochemical test areas which are considered
appropriate to all studies: electrolyte
balance, carbohydrate metabolism, and liver
and kidney function. The selection of
specific tests will be influenced by
observations on the mode of action of the
substance. Suggested determinations:
calcium, phosphorus, chloride, sodium,
potassium, fasting glucose (with period of
fasting appropriate to the species), serum
glutamic-pyruvic transaminase*, serum
glutamic-oxaloacetic transaminase**,
ornithine decarboxylase, gamma glutamyl
transpeptidase, urea nitrogen, albumen, blood
creatinine, total bilirubin and total serum
protein measurements. Other determinations
which may be necessary for an adequate
toxicological evaluation include: analyses of
lipids, hormones, acid/base balance,
methemoglobin and cholinesterase activity.
Additional clinical biochemistry may be
employed, where necessary, to extend the
investigation of observed effects.
* Now known as serum alanine
aminotransferase.
** Now known as serum aspartate
aminotransferase.
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HG-Subchronic-Inhal
2. The following examinations should be made on at
least 5 animals of each sex in each group:
a. Ophthalmological examination, using an
ophthalmoscope or equivalent suitable
equipment, should be made prior to exposure
to the test substance and at the termination
of the study. If changes in the eyes are
detected, all animals should be examined.
b. Urinalysis is not recommended on a routine
basis, but only when there is an indication
based on expected or observed toxicity.
L. Gross pathology
1. All animals should be subjected to a full gross
necropsy which includes examination of the
external surface of the body, all orifices and the
cranial, thoracic and abdominal cavities and their
contents.
2. At least the liver, kidneys, adrenals, brain, and
gonads should be weighed wet, as soon as possible
after dissection to avoid drying.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible future
histopathological examination: all gross lesions;
lungs - which should be removed intact, weighed
and treated with a suitable fixative to ensure
that lung structure is maintained (perfusion with
the fixative is considered to be an effective
procedure); nasopharyngeal tissues; brain -
including sections of medulla/pons cerebellar
cortex and cerebral cortex; pituitary;
thyroid/parathyroid; thymus; trachea; heart;
sternum with bone marrow; salivary glands; liver;
spleen; kidneys; adrenals; pancreas; gonads;
uterus; accessory genital organs; aorta; (skin);
gall bladder (if present); esophagus; stomach;
duodenum; jejunum; ileum; cecum; colon; rectum;
urinary bladder; representative lymph node;
(mammary gland); (thigh musculature); peripheral
nerve; (eyes); (femur - cervical, midthoracic and
lumbar); and (exorbital lachrymal glands).
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HG-Subchronic-Inhal
M. Histopathology
The following histopathology should be performed:
1. Full histopathology on the respiratory tract and
other organs and tissues, listed above, of all
animals in the control and high dose groups.
2. All gross lesions in all animals.
3. Target organs in all animals.
4. The tissues mentioned in brackets (listed above)
if indicated by signs of toxicity or target organ
involvement.
5. Lungs of animals in the low and intermediate dose
groups should also be subjected to
histopathological examination, primarily for
evidence of infection since this provides a
covenient assessment of the state of health of the
animals.
6. When a satellite group is used, histopathology
should be performed on tissues and organs
identified as showing effects in other treated
groups.
V. DATA AND REPORTING
A. Treatment of results
1. Data should be summarized in tabular form, showing
for each test group the number of animals at the
start of the test, the number of animals showing
lesions, the types of lesions, the percentage of
animals displaying each type of lesion.
2. All observed results, quantitative and incidental,
should be evaluated by an appropriate statistical
method. Any generally accepted statistical method
may be used; the statistical methods should be
selected during the design of the study.
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HG-Subchronic-Inhal
B. Evaluation of results
The findings of a subchronic inhalation toxicity study
should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the
observed toxic effects and the necropsy and
histopathological findings. The evaluation will
include the relationship between the concentration of
the test substance and duration of exposure, and the
presence or absence, the incidence and severity, of
abnormalities, including behavioral and clinical
abnormalities, gross lesions, identified target organs,
body weight changes, effects on mortality and any other
general or specific toxic effects. A properly
conducted subchronic test should provide a satisfactory
estimation of a no-effect level.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Test conditions
a. Description of exposure apparatus, including
design, type, dimensions, source of air,
system for generating particulates and
aerosols, method of conditioning air,
treatment of exhaust air and the method of
housing animals in a test chamber.
b. The equipment for measuring temperature,
humidity and particulate aerosol
concentrations and size should be described.
2. Exposure data
These should be tabulated and presented with mean
values and measure of variability (e.g. standard
deviation) and should include:
a. Airflow rates through the inhalation
equipment;
b. Temperature and humidity of air;
c. Nominal concentration (total amount of test
substance fed into the inhalation equipment
divided by volume of air);
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HG-Subchronic-Inhal
d. Actual concentration in test breathing zone;
and
e. Particle size distribution (e.g. median
aerodynamic diameter of particles with
standard deviation from the mean).
Group animal data
Tabulation of toxic response data by species,
strain, sex, and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of toxicity;
and
c. Number of animals exposed.
Individual animal data
a. Time of death during the study or whether
animals survived to termination;
b. Time of observation of each abnormal sign and
its subsequent course;
c. Body weight data;
d. Food consumption data when collected;
e. Hematological tests employed and all results;
f. Clinical biochemistry tests employed and all
results;
g. Necropsy findings;
h. Detailed description of all histopathological
findings; and
i. Statistical treatment of results where
appropriate.
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HG-Subchronic-Inhal
VI. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Cage, J.C. 1970. "Experimental Inhalation
Toxicology," in "Methods in Toxicology." Edited by
G.E. Paget. "Philadelphia: F.A. Davis Company. PP.
258-277.
2. Casarett, L.J., Doull, J. 1975. Toxicology: The
Basic Science of Poisons. New York: Macmillan
Publishing Co. Inc. Chapter 9.
3. MacFarland, H.N. 1976. "Respiratory Toxicology," in
"Essays in Toxicology." Edited by W.J. Hayes. New
York: Academic Press. Vol. 7. PP. 121-154.
4. NAS. 1977. National Academy of Sciences. Principles
and Procedures for Evaluating the Toxicity of Household
Substances. Washington, D.C.: A report prepared by
the Committee for the Revision of NAS Publication 1138,
under the auspices of the Committee on Toxicology,
National Research Council, National Academy of
Sciences. 130 pp.
5. WHO. 1978. World Health Organization. Principles and
Methods for Evaluating the Toxicity of Chemicals. Part
I. Environmental Health Criteria 6. Geneva: World
Health Organization. 272 pp.
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HG-Subchronic-Oral
August, 1982
SUBCHRONIC EXPOSURE
ORAL TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Subchronic-Oral
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a chemical, the determination of
subchronic oral toxicity may be carried out after initial
information on toxicity has been obtained by acute
testing. The subchronic oral study has been designed to
permit the determination of the no-observed-effect level
and toxic effects associated with continuous or repeated
exposure to a test substance for a period of 90 days. The
test is not capable of determining those effects that have
a long latency period for development (e.g., carcino-
genicity and life shortening). It provides information on
health hazards likely to arise from repeated exposure by
the oral route over a limited period of time. It will
provide information on target organs, the possibilities of
accumulation, and can be of use in selecting dose levels
for chronic studies and for establishing safety criteria
for human exposure.
II. DEFINITIONS
A. Subchronic oral toxicity is the adverse effects
occurring as a result of the repeated daily exposure
of experimental animals to a chemical by the oral
route for a part (approximately ten percent) of a
life span.
B. Dose is the amount of test substance administered.
Dose is expressed as weight of test substance (g, mg)
per unit weight of test animal (e.g., mg/kg), or as
weight of test substance per unit weight of food or
drinking water.
C. No-effect level/No-toxic-effeet level/No-adverse-
effect level/No-observed-effeet level is the maximum
dose used in a test which produces no observed
adverse effects. A no-observed-effect level is
expressed in terms of the weight of a substance given
daily per unit weight of test animal (mg/kg). When
administered to animals in food or drinking water the
no-observed-effect level is expressed as mg/kg of
food or mg/ml of water.
D. Cumulative toxicity is the adverse effects of
repeated doses occuring as a result of prolonged
action on, or increased concentration of the
administered substance or its metabolites in
susceptible tissue.
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HG-Subchronic-Oral
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered orally in graduated
daily doses to several groups of experimental ianimals, one
dose level per group, for a period of 90 days. During the
period of administration the animals are observed daily to
detect signs of toxicity. Animals which die during the
period of administration are necropsied, and at the
conclusion of the test all surviving histopathological
examinations carried out.
IV. LIMIT TEST
If a test at one dose level of at least 1,000 mg/kg body
weight (expected human exposure may indicate the need for a
higher dose level), using the procedures described for this
study, produces no observable toxic effects and if toxicity
would not be expected based upon data of structurally
related compounds, then a full study using three dose
levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
A variety of rodent species may be used,
although the rat is the preferred species.
Commonly used laboratory strains should be
employed. The commonly used non-rodent species
is the dog, preferably of a defined breed; the
beagle is frequently used. If other mammalian
species are used, the tester should provide
justification/reasoning for their selection.
2. Age
a. General
Young adult animals should be employed.
At the commencement of the study the
weight variation of animals used should
not exceed ± 20 percent of the mean
weight for each sex.
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HG-Subchronic-Oral
b. Rodents
Dosing should begin as soon as possible
after weaning, ideally before the rats
are 6, and in any case, not more than 8
weeks old.
c. Non-rodent
In the case of the dog, dosing should
commence after acclimatization,
preferably at 4-6 months and not later
than 9 months of age.
3. Sex
a. Equal numbers of animals of each sex
should be used at each dose level.
b. The females should be nulliparous and
non-pregnant.
4. Numbers
a. Rodents
At least 20 animals (10 females and 10
males) should be used at each dose level,
b. Non-rodents
At least eight animals (4 females and 4
males) should be used at each dose level,
c. If interim sacrifices are planned, the
number should be increased by the number
of animals scheduled to be sacrificed
before the completion of the study.
B. Control groups
A concurrent control group is recommended. This
group should be an untreated or sham treated control
group or, if a vehicle is used in administering the
test substance, a vehicle control group. If the
toxic properties of the vehicle are not known or
cannot be made available, both untreated and vehicle
control groups are recommended.
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HG-Subchronic-Oral
C. Satellite group
(Rodent) A satellite group of 20 animals (10 animals
per sex) may be treated with the high dose level for
90 days and observed for reversibility, persistence,
or delayed occurrence of toxic effects for a post-
treatment period of appropriate length, normally not
less than 28 days.
D. Dose levels and dose selection
1. In subchronic toxicity tests, it is desirable
to have a dose response relationship as well as
no-observed-toxic-effeet level. Therefore, at
least three dose levels with a control and,
where appropriate, a vehicle control
(corresponding to the concentration of vehicle
at the highest exposure level) should be
used. Doses should be spaced appropriately to
produce test groups with a range ot toxic
effects and mortality rates. The data should
be sufficient to produce a dose response curve.
2. The highest dose level in rodents should result
in toxic effects but not produce an incidence
of fatalities which would prevent a meaningful
evaluation; for non-rodents there should be no
fatalities.
3. The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest dose
level should exceed this.
4. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If
more than one intermediate dose is used, the
dose levels should be spaced to produce a
gradation of toxic effects.
5. For rodents, the incidence of fatalities in low
and intermediate dose groups and in the
controls should be low, to permit a meaningful
evaluation of the results; for non-rodents,
there should be no fatalities.
E. Exposure conditions
The animals are dosed with the test substance ideally
on a 7-day per week basis over a period of 90 days.
However, based primarily on practical considerations,
dosing in gavage or capsule studies on a 5-day per
week basis is considered to be acceptable.
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HG-Subchronic-Oral
F. Observation period
1. Duration of observation should be for at least
90 days.
2. Animals in the satellite group scheduled for
follow-up observations should be kept for a
further 28 days without treatment to detect
recovery from, or persistence of, toxic
effects.
G. Administration of the test substance
1. The test substance may be administered in the
diet or in capsules. In addition, for rodents
it may also be administered by gavage or in the
drinking water.
2. All animals should be dosed by the same method
during the entire experimental period.
3. Where necessary, the test substance is
dissolved or suspended in a suitable vehicle.
If a vehicle or diluent is needed, ideally it
should not elicit important toxic effects
itself nor substantially alter the chemical or
toxicological properties of the test
substance. It is recommended that wherever
possible the usage of an aqueous solution be
considered first, followed by consideration of
a solution of oil and then by possible solution
in other vehicles.
4. For substances of low toxicity, it is important
to ensure that when administered in the diet
the quantities of the test substance involved
do not interfere with normal nutrition. When
the test substance is administered in the diet
either a constant dietary concentration (ppm)
or a constant dose level in terms of the
animals' body weight may be used; the
alternative used should be specified.
5. For a substance administered by gavage or
capsule, the dose should be given at similar
times each day, and adjusted at intervals
(weekly or bi-weekly) to maintain a constant
dose level in terms of animal body weight.
H. Observation of animals
1. A careful clinical examination should be made
at least once each day.
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HG-Subchronic-Oral
2. Additional observations should be made daily
with appropriate actions taken to minimize loss
of animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Signs of toxicity should be recorded as they
are observed including the time of onset,
degree and duration.
4. Cage-side observations should include, but not
be limited to, changes in skin and fur, eyes
and mucous membranes, respiratory, circulatory,
autonomic and central nervous systems,
somatomotor activity and behavior pattern.
5. Measurements should be made weekly of food
consumption or water consumption when the test
substance is administered in the food or
drinking water, respectively.
6. Animals should be weighed weekly.
7. At the end of the 90-day period all survivors
in the non-satellite treatment groups are
sacrificed. Moribund animals should be removed
and sacrificed when noticed.
I. Clinical examinations
1. The following examinations should be made on at
least five animals of each sex in.each group
for rodents and all animals when non-rodents
are used as test animals.
a. Certain hematology determinations should
be carried out at least three times
during the test period: just prior to
initiation of dosing (baseline data),
after approximately 30 days on test and
just prior to terminal sacrifice at the
end of the test period. Hematology
determinations which should be
appropriate to all studies: hematocrit,
hemoglobin concentration, erythrocyte
count, total and differential leucocyte
count, and a measure of clotting
potential such as clotting time,
prothrombin time, thromboplastin time, or
platelet count.
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HG-Subchronic-Oral
Certain clinical biochemistry
determinations should be carried out at
least three times during the test
period: just prior to initiation of
dosing (baseline data), after
approximately 30 days on test and just
prior to terminal sacrifice at the end of
the test period. Clinical biochemical
test areas which are considered
appropriate to all studies: electrolyte
balance, carbohydrate metabolism, and
liver and kidney function. The selection
of specific tests will be influenced by
observations on the mode of action of the
substance. Suggested determinations:
calcium, phosphorus, chloride, sodium,
potassium, fasting glucose (with period
of fasting appropriate to the
species/breed), serum glutamic-pyruvic
transaminase*, serum glutamic oxaloacetic
transaminase**, ornithine decarboxylase,
gamma glutamyl transpeptidase, urea
nitrogen, albumen, blood creatinine,
total bilirubin and total serum protein
measurements. Other determinations which
may be necessary for an adequate
toxicological evaluation include analyses
of lipids, hormones, acid/base balance,
methemoglobin and cholinesterase
activity. Additional clinical
biochemistry may be employed where
necessary to extend the investigation of
observed effects. Non-rodents should be
fasted for a period (not more than 24
hours) before taking blood samples.
* Now knowm as serum alanine
aminotransferase.
** Now known as serum aspartate
aminotransferase.
2. The following examinations should be made on at
least five animals of each sex in each group
for rodents and all animals on test for non-
rodents.
a. Ophthalmological examination, using an
ophthalmoscope or equivalent suitable
equipment, should be made prior to the
administration of the test substance and
at the termination of the study. If
changes in the eyes are detected all
animals should be examined.
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HG-Subchronic-Oral
b. Urinalysis is not recommmended on a
routine basis, but only when there is an
indication based on expected or observed
toxicity.
J. Gross necropsy
1. All animals should be subjected to a full gross
necropsy which includes examination of the
external surface of the body, all orifices, and
the cranial, thoracic and abdominal cavities
and their contents.
2. At least the liver, kidneys, adrenals, and
gonads should be weighed wet, as soon as
possible after dissection to avoid drying. In
addition, for the rodent, the brain; for the
non-rodent, the thyroid with parathyroids also
should be weighed wet.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible
future histopathological examination: all
gross lesions; brain-including sections of
medulla/pons, cerebellar cortex and cerebral
cortex; pituitary; thyroid/parathyroid; thymus;
lungs; trachea; heart; sternum with bone
marrow; salivary glands; liver; spleen;
kidneys/adrenals; pancreas; gonads; uterus;
accessory genital organs; aorta; (skin), (non-
rat gall bladder); esophagus; stomach;
duodenum; jejunum; ileum; cecum; colon; rectum;
urinary bladder; representative lymph node;
(mammary gland), (thigh musculature),
peripheral nerve; (eyes); (femur-including
articular surface);(spinal cord at three levels
- cervical, midthoracic and lumbar); and,
(rodent - exorbital lachrymal glands).
K. Histopathology
The following histopathology should be performed:
1. Full histopathology on the organs and tissues,
listed above, of all rodents in the control and
high dose groups, all non-rodents, and all
rodents that died or were killed during the
study.
2. All gross lesions in all animals.
3. Target organs in all animals.
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HG-Subchronic-Oral
4. The tissues mentioned in brackets (listed
above) if indicated by signs of toxicity or
target organ involvement.
5. Lungs, liver and kidneys of all animals.
Special attention to examination of the lungs
of rodents should be made for evidence of
infection since this provides a convenient
assessment of the state of health of the
animals.
6. When a satellite group is used (rodents),
histopathology should be performed on tissues
and organs identified as showing effects in the
treated groups.
VI. DATA AND REPORTING
A. Treatment of results
1. Data should be summarized in tabular form,
showing for each test group the number of
animals at the start of the test, the number of
animals showing lesions, the types of lesions
and the percentage of animals displaying each
type of lesion.
2. All observed results, quantitative and
incidental, should be evaluated by an
appropriate statistical method. Any generally
accepted statistical methods may be used; the
statistical methods should be selected during
the design of the study.
B. Evaluation of the study results
1. The findings of a subchronic oral toxicity
study should be evaluated in conjunction with
the.findings of preceding studies and
considered in terms of the toxic effects and
the necropsy and histopathological findings.
The evaluation will include the relationship
between the dose of the test substance and the
presence or absence, the incidence and
severity, of abnormalities, including
behavioral and clinical abnormalities, gross
lesions, identified target organs, body weight
changes, effects on mortality and any other
general or specific toxic effects. A properly
conducted subchronic test should provide a
satisfactory estimation of a no-effect level.
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HG-Subchronic-Oral
2. In any study which demonstrates an absence of
toxic effects, further investigation to
establish absorption and bioavailability of the
test substance should be considered.
C. Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Group animal data
Tabulation of toxic response data by species,
strain, sex and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of
toxicity; and
c. Number of animals exposed.
2. Individual animal data
a. Time of death during the study or whether
animals survived to termination;
b. Time of observation of each abnormal sign
and its subsequent course;
c. Body weight data;
d. Food consumption data when collected;
e. Hematological tests employed and all
results;
f. Clinical biochemistry tests employed and
all results;
g. Necropsy findings;
h. Detailed description of all
histopathological findings; and
i. Statistical treatment of results where
appropriate.
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HG-Subchronic-Oral
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. Boyd, E.M. 1972. Predictive Toxicometrics. Chapter
14—Pilot Studies, 15—Uniposal Clinical Parameters,
16—Uniposal Autopsy Parameters. Baltimore:
Williams and Wilkins. 48 pp.
2. Fitzhugh, O.G. 1959. Third Printing: 1975.
"Subacute Toxicity" in "Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics." The
Association of Food and Drug Officials of the United
States. PP. 26-35.
3. FSC. 1978. Food Safety Council. "Subchronic
Toxicity Studies," in "Proposed System for Food
Safety Assessment." Columbia: Food Safety
Council. PP. 83-96
4. NAS. 1977. National Academy of Sciences.
Principles and Procedures for Evaluating the Toxicity
of Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
5. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
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HG-Chronic
August, 1982
CHRONIC EXPOSURE
CHRONIC TOXICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Chronic
I. PURPOSE
The objective of a chronic toxicity study is to determine
the effects of a substance in a mammalian species following
prolonged and repeated exposure. Under the conditions of
the chronic toxicity test, effects which require a long
latency period or which are cumulative should become
manifest. The application of this guideline should
generate data on which to identify the majority of chronic
effects and shall serve to define long term dose-response
relationships. The design and conduct of chronic toxicity
tests should allow for the detection of general toxic
effects, including neurological, physiological,
biochemical, and hematological effects and exposure-related
morphological (pathology) effects.
II. TEST PROCEDURES
A. Animal selection
1. Species and strain
Testing should be performed with two mammalian
species, one a rodent and another a non-
rodent. The rat is the preferred rodent
species and the dog is the preferred non-rodent
species. Commonly used laboratory strains
should be employed. If other mammalian species
are used, the tester should provide
justification/reasoning for their selection.
2. Age
a. Dosing of rats should begin as soon as
possible after weaning, ideally before
the rats are six, but in no case more
than eight weeks old.
b. Dosing of dogs should begin between four
and six months of age and in no case
later than nine months of age.
c. At commencement of the study the weight
variation of animals used should not
exceed * 20 percent of the mean weight
for each sex.
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HG-Chronic
3. Sex
a. Equal numbers of animals of each sex
should be used at each dose level.
b. The females should be nulliparous and
non-pregnant.
4. Numbers
a. For rodents, at least 40 animals (20
females and 20 males) and for non-rodents
(dogs) at least eight animals (four
females and four males) should be used at
each dose level.
b. If interim sacrifices are planned the
number should be increased by the number
of animals scheduled to be sacrificed
during the course of the study.
c. The number of animals at the termination
of the study must be adequate for a
meaningful and valid statistical
evaluation of chronic effects.
B. Control groups
1. A concurrent control group is suggested. This
. group should be an untreated or sham treated
control group or, if a vehicle is used in
administering the test substance, a vehicle
control group. If the toxic properties of the
vehicle are not known or cannot be made
available, both untreated and vehicle control
groups are strongly suggested.
2. In special circumstances such as in inhalation
studies involving aerosols or the use of an
emulsifier of uncharacterized biological
activity in oral studies, a concurrent negative
control group should be utilized. The negative
control group should be treated in the same
manner as all other test animals except that
this control group should not be exposed to
either the test substance or any vehicle.
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HG-Chronic
C. Dose levels and dose selection
1. In chronic toxicity tests, it is necessary to
have a dose-response relationship as well as a
no-observed-toxic-effeet level. Therefore, at
least three dose levels should be used in
addition to the concurrent control group. Dose
levels should be spaced to produce a gradation
of effects.
2. The high dose level in rodents should elicit
some signs of toxicity without causing
excessive lethality; for non-rodents, there
should be signs of toxicity but there should be
no fatalities.
3. The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest dose
level should exceed this even though this dose
level may result in some signs of toxicity.
4. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If
more than one intermediate dose is used, the
dose levels should be spaced to produce a
gradation of toxic effects.
5. For rodents, the incidence of fatalities in low
and intermediate dose groups and in the
controls should be low to permit a meaningful
evaluation of the results. For non-rodents,
there should be no fatalities.
D. Exposure- conditions
The animals are dosed with the test substance ideally
on a 7-day per week basis over a period of at least
12 months. However, based primarily on practical
considerations, dosing on a 5-day per week basis is
considered to be acceptable.
E. Observation period
Duration of observation should be for at least 12
months.
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HG-Chronic
Administration of the test substance
The three main routes of administration are oral,
dermal, and inhalation. The choice of the route of
administration depends upon the physical and chemical
characteristics of the test substance and the form
typifying exposure in humans.
1. Oral studies
a. The animals should receive the test
substance in their diet, dissolved in
drinking water, or given by gavage or
capsule for a period of at least 12
months.
b. If the test substance is administered in
the drinking water, or mixed in the diet,
exposure is continuous.
c. For a diet mixture, the highest
concentration should not exceed 5
percent.
2. Dermal studies
a. The animals are treated by topical
application with the test substance,
ideally for at least 6 hours per day.
b. Fur should be clipped from the dorsal
area of the trunk of the test animals.
Care must be taken to avoid abrading the
skin which could alter its permeability.
c. The .test substance should be applied
uniformly over a shaved area which is
approximately ten percent of the total
body surface area. With highly toxic
substances, the surface area covered may
be less, but as much of the area should
be covered with as thin and uniform a
film as possible.
d. During the exposure period, the test
substance may be held if necessary, in
contact with the skin with a porous gauze
dressing and non-irritating tape. The
test site should be further covered in a
suitable manner to retain the gauze
dressing and test substance and ensure
that the animals cannot ingest the test
substance.
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HG-Chronic
3. Inhalation studies
a. The animals should be tested with
inhalation equipment designed to sustain
a dynamic air flow of 12 to 15 air
changes per hour, ensure an adequate
oxygen content of 19 percent and an
evenly distributed exposure atmosphere.
Where a chamber is used, its design
should minimize crowding of the test
animals and maximize their exposure to
the test substance. This is best
accomplished by individual caging. As a
general rule to ensure stability of a
chamber atmosphere, the total "volume" of
the test animals should not exceed 5
percent of the volume of the test
chamber. Alternatively, oro-nasal, head-
only or whole body individual chamber
exposure may be used.
b. The temperature at which the test is
performed should be maintained at 22°C (±
2°). Ideally, the relative humidity
should be maintained between 40 to 60
percent, but in certain instances (e.g.
tests of aerosols, use of water vehicle)
this may not be practicable.
c. Food and water should be withheld during
each daily six-hour exposure period.
d. A dynamic inhalation system with a
suitable analytical concentration control
system should be used. The rate of air
flow should be adjusted to ensure that
conditions throughout the equipment are
essentially the same. Maintenance of
slight negative pressure inside the
chamber will prevent leakage of the test
substance into the surrounding areas.
G. Observation of animals
1. A careful clinical examination should be made
at least once each day.
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HG-Chronic
2. Additional observations should be made daily
with appropriate actions taken to minimize loss
of animals to the study (e.g. , necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Clinical signs of toxicity including suspected
tumors and mortality should be recorded as they
are observed, including the time of onset, the
degree and duration.
4. Cage-side observations should include, but not
be limited to, changes in skin and fur, eyes
and mucous membranes, respiratory, ciculatory,
autonomic and central nervous systems,
somatomotor activity and behavior pattern.
5. Body weights should be recorded individually
for all animals once a week during the first 13
weeks of the test period and at least once
every four weeks thereafter unless signs of
clinical toxicity suggest more frequent
weighings to facilitate monitoring of health
status.
6. When the test substance is administered in the
food or drinking water, measurements of food or
water consumption, respectively, should be
determined weekly during the first 13 weeks of
the study and then at approximately monthly
intervals unless health status or body weight
changes dictate otherwise.
7. At the end of the study period all survivors
should be sacrificed. Moribund animals should
be removed and sacrificed when noticed.
H. Physical measurements
For inhalation studies, measurements or monitoring
should be made of the following:
1. The rate of air flow should be monitored
continuously, but should be recorded at
intervals of at least once every 30 minutes.
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HG-Chronic
2. During each exposure period the actual
concentrations of the test substance should be
held as constant as practicable, monitored
continuously and measured at least three times
during the test period: at the beginning, at
an intermediate time and at the end of the
period.
3. During the development of the generating
system, particle size analysis should be
performed to establish the stability of aerosol
concentrations. During exposure, analysis
should be conducted as often as necessary to
determine the consistency of particle size
distribution and homogeneity of the exposure
stream.
4. Temperature and humidity should be monitored
continuously, but should be recorded at
intervals of at least once every 30 minutes.
I. Clinical examinations
The following examinations should be made on at least
ten rats of each sex per dose and on all non-rodents.
1. Certain hematology determinations (e.g.,
hemoglobin content, packed cell volume, total
red blood cells, total white blood cells,
platelets, or other measures of clotting
potential) should be performed at termination
and should be performed at three months, six
months and at approximately six-month intervals
thereafter (for studies extending beyond 12
months) on blood samples collected from all
non-rodents and from ten rats per sex of all
groups. These collections should be from the
same animals at each interval. If clinical
observations suggest a deterioration in health
of the animals during the study, a differential
blood count of the affected animals should be
performed. A differential blood count should
be performed on samples from those animals in
the highest dosage group and the controls.
Differential blood counts should be performed
for the next lower group(s) if there is a major
discrepancy between the highest group and the
controls. If hematological effects were noted
in the subchronic test, hematological testing
should be performed at 3, 6, 12, 18 and 24
months for a two year study and at 3, 6 and 12
months for a one year study.
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HG-Chronic
Certain clinical biochemistry determinations on
blood should be carried out at least three
times during the test period: just prior to
initiation of dosing (base line data), near the
middle and at the end of the test period.
Blood samples should be drawn for clinical
chemistry measurements from all non-rodents and
at least ten rodents per sex of all groups; if
possible, from the same rodents at each time
interval. Test areas which are considered
appropriate to all studies: electrolyte
balance, carbohydrate metabolism and liver and
kidney function. The selection of specific
tests will be influenced by observations on the
mode of action of the substance and signs of
clinical toxicity. Suggested chemical
determinations: calcium, phosphorus, chloride,
sodium, potassium, fasting glucose (with period
of fasting appropriate to the species), serum
glutamic-pyruvic transaminase*, serum glutamic
oxaloacetic transaminase**, ornithine
decarboxylase, gamma glutamyl transpeptidase,
blood urea nitrogen, albumen, blood creatinine,
creatinine phosphokinase, total cholesterol,
total bilirubin and total serum protein
measurements. Other determinations which may
be necessary for an adequate toxicological
evaluation include analyses of lipids,
hormones, acid/base balance, methemoglobin and
cholinesterase activity. Additional clinical
biochemistry may be employed where necessary to
extend the investigation of observed effects.
* Now known as serum alanine aminotransferase.
** Now known as serum aspartate
aminotransferase.
3. Urine samples from rodents at the same
intervals as the hematological examinations
(above) should be collected for analysis. The
following determinations should be made from
either individual animals or on a pooled
sample/sex/group for rodents: appearance
(volume and specific gravity), protein,
glucose, ketones, bilirubin occult blood (semi-
quantitatively); and microscopy of sediment
(semi-quantitatively).
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HG-Chronic
4. Ophthalmological examination, using an
ophthalmoscope or equivalent suitable
equipment, should be made prior to the
administration of the test substance and at the
termination of the study. If changes in eyes
are detected all animals should be examined.
J. Gross necropsy
1. A complete gross examination should be
performed on all animals, including those which
died during the experiment or were killed in
moribund conditions.
2. The liver, kidneys, adrenals, brain and gonads
should be weighed wet, as soon as possible
after dissection to avoid drying. For these
organs, at least ten rodents per sex per group
and all non-rodents should be weighed.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible
future histopathological examination: all
gross lesions and tumors; brain - including
sections of medulla/pons, cerebellar cortex,
and cerebral cortex; pituitary;
thyroid/parathyroid; thymus; lungs; trachea;
heart; sternum and/or femur with bone marrow;
salivary glands; liver; spleen; kidneys;
adrenals; esophagus; stomach; duodenum;
jejunum; ileum; cecum; colon; rectum; urinary
bladder; representative lymph nodes; pancreas;
gonads; uterus; accessory genital organs;
femal.e mammary gland; aorta; gall bladder (if
present); skin; musculature; peripheral nerve;
spinal cord at three levels-cervical,
midthoracic, and lumbar; and eyes. In
inhalation studies,, the entire respiratory
tract, including nose, pharynx, larynx, and
paranasal sinuses should be examined and
preserved. In dermal studies, skin from sites
of skin painting should be examined and
preserved.
4. Inflation of lungs and urinary bladder with a
fixative is the optimal method for preservation
of these tissues. The proper inflation and
fixation of the lungs in inhalation studies is
considered essential for appropriate and valid
histopathological examination.
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HG-Chronic
5. If other clinical examinations are carried out,
the information obtained from these procedures
should be available before microscopic
examination, since they may provide significant
guidance to the pathologist.
Histopathology
1. The following histopathology should be
performed:
a. Full histopathology on the organs and
tissues, listed above, of all non-
rodents, of all rodents in the control
and high dose groups and of all rodents
that died or were killed during the
study.
b. All gross lesions in all animals.
c. Target organs in all animals.
d. Lungs, liver and kidneys of all
animals. Special attention to
examination of the lungs of rodents
should be made for evidence of infection
since this provides an assessment of the
state of health of the animals.
2. If excessive early deaths or other problems
occur in the high dose group compromising the
significance of the data, the next dose level
should be examined for complete histopathology.
3. In case the results of an experiment give
evidence of substantial alteration of the
animals' normal longevity or the induction of
effects that might affect a toxic response, the
next lower dose level should be examined fully,
as described above.
4. An attempt should be made to correlate gross
observations with microscopic findings.
III. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form,
showing for each test group the number of
animals at the start of the test, the number of
animals showing lesions, the types of lesions
and the percentage of animals displaying each
type of lesion.
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HG-Chronic
2. All observed results, quantitative and
incidental, should be evaluated by an
appropriate statistical method. Any generally
accepted statistical methods may be used; the
statistical methods should be selected during
the design of the study.
B. Evaluation of study results
1. The findings of a chronic toxicity study should
be evaluated in conjunction with the findings
of preceding studies and considered in terms of
the toxic effects, the necropsy and
histopathological findings. The evaluation
will include the relationship between the dose
of the test substance and the presence,
incidence and severity of abnormalities
(including behavioral and clinical
abnormalities), gross lesions, identified
target organs, body weight changes, effects on
mortality and any other general or specific
toxic effects.
2. In any study which demonstrates an absence of
toxic effects, further investigation to
establish absorption and bioavailability of the
test substance should be considered.
C. Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Group animal data
Tabulation of toxic response data by species,
strain, sex and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of
toxicity; and
c. Number of animals exposed.
2. Individual animal data
a. Time of death during the study or whether
animals survived to termination;
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HG-Chronic
b. Time of observation of each abnormal sign
and its subsequent course;
c. Body weight data;
d. Food and water consumption data, when
collected;
e. Results of ophthalmological examination,
when performed;
f. Hematological tests employed and all
results;
g. Clinical biochemistry tests employed and
all results;
h. Necropsy findings;
i. Detailed description of all
histopathological findings; and
j. Statistical treatment of results, where
appropriate.
In addition, for inhalation studies the following
should be reported:
3. Test conditions
a. Description of exposure apparatus
including design, type, dimensions,
source of air, system for generating
particulates and aerosols, method of
conditioning air, treatment of exhaust
air and the method of housing the animals
in a test chamber.
b. The equipment for measuring temperature,
humidity, and particulate aerosol
concentrations and size should be
described.
4. Exposure data
These should be tabulated and presented with
mean values and a measure of variability (e.g.
standard deviation) and should include:
a. Airflow rates through the inhalation
equipment;
b. Temperature and humidity of air;
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HG-Chronic
c. Nominal concentration (total amount of
test substance fed into the inhalation
equipment divided by volume of air);
d. Actual concentration in test breathing
zone; and
e. Particle size distribution (e.g. median
aerodynamic diameter of particles with
standard deviation from the mean).
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HG-Chronic
IV. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. Benitz, K.F. 1970. "Measurement of Chronic
Toxicity," in "Methods of Toxicology." Edited by
G.E. Paget. Oxford: Blackwell Scientific
Publications. PP. 82-131.
2. D'Aguanno, W. 1974. "Drug Safety Evaluation—Pre-
Clinical Considerations," _iii "Industrial
Pharmacology: Neuroleptics." Edited by S. Fielding
and H. Lai. Mt. Kisco: Futura Publishing Co. Vol.
I. PP. 317-332.
3. Fitzhugh, O.G. 1959. Third Printing: 1975.
"Chronic Oral Toxicity," in "Appraisal of the Safety
of Chemicals in Foods, Drugs and Cosmetics." The
Association of Food and Drug Officials of the United
States. PP. 36-45.
4. Goldenthal, E.I., D'Aguanno, W. 1959. Third
Printing: 1975. "Evaluation of Drugs," _in
"Appraisal of the Safety of Chemicals in Foods,
Drugs, and Cosmetics." The Association of Food and
Drug Officials of the United States. PP. 60-67.
5. NAS. 1977. National Academy of Sciences.
Principles and Procedures for Evaluating the Toxicity
of Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences, 130 pp.
6. NCTR. 1972. National Center for Toxicological
Research. Report of Chronic Studies Task Force
Committee, April 13-21, 1972. Appendix B.
Rockville: National Center for Toxicological
Research. 50 pp.
7. Page, N.P. 1977. Chronic Toxicity and
Carcinogenicity Guidelines, Journal of Environmental
Pathology and Toxicology. 1:161-182.
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HG-Chronic
8. Schwartz, E. 1974. "Toxicology of Neuroleptic
Agents," in "Industrial Pharmacology:
Neuroleptics." Edited by S. Fielding and H. Lai.
Mt. Kisco, Futura Publishing Co. PP. 203-221.
9. USPMA. 1977. United States Pharmaceutical
Manufacturers Association. Guidelines for the
Assessment of Drug and Medical Device Safety in
Animals. 64 pp.
10. WHO. 1975. World Health Organization. Guidelines
for Evaluation of Drugs for Use in Man. WHO
Technical Report Series No. 563. Geneva: World
Health Organization. 59 pp.
11. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
12. WHO. 1966. World Health Organization. Principles
for Pre-Clinical Testing of Drug Safety. WHO
Technical Report Series No. 341. Geneva: World
Health Organization. 22 pp.
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August, 1982
ONCOGENICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
. WASHINGTON, D.C. 20460
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HG-Chronic-Onco
I. PURPOSE
The objective of a long-term oncogenicity study is to
observe test animals for a major portion of their life span
for the development of neoplastic lesions during or after
exposure to various doses of a test substance by an
appropriate route of administration.
II. TEST PROCEDURES
A. Animal selection
1. Species and strain
It is recommended that a compound of unknown
activity should be tested on two mammalian
species. Rats and mice are the species of
choice because of their relatively short life
spans, the limited cost of their maintenance,
their widespread use in pharmacological and
toxicological studies, their susceptibility to
tumor induction, and the availability of inbred
or sufficiently characterized strains.
Commonly used laboratory strains should be
employed. If other species are used, the
tester should provide justification/reasoning
for their selection.
2.
a. Dosing of rodents should begin as soon as
possible after weaning, ideally before
the animals are six, but in no case more
than eight weeks old.
b. At commencement of the study, the weight
variation of animals used should not
exceed ± 20 percent of the mean weight
for each sex.
c. Studies using prenatal or neonatal
animals may be recommended under special
conditions.
3. Sex
a. Equal numbers of animals of each sex
should be used at each dose level.
b. The females should be nulliparous and
non-pregnant.
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4. Numbers
a. For rodents, at least 100 animals (50
females and 50 males) should be used at
each dose level and concurrent control.
b. If interim sacrifices are planned the
number should be increased by the number
of animals scheduled to be sacrificed
during the course of the study.
c. The number of animals at the termination
of the study should be adequate for a
meaningful and valid statistical
evaluation of long term exposure. For a
valid interpretation of negative results,
it is essential that survival in all
groups does not fall below 50 percent at
the time of termination.
B. Control groups
1. A concurrent control group is recommended.
This group should be an untreated or sham
treated control group or, if a vehicle is used
in administering the test substance, a vehicle
control group. If the toxic properties of the
vehicle are not known or cannot be made
available, both untreated and vehicle control
groups are recommended.
2. In special circumstances such as in inhalation
studies involving aerosols or the use of an
emulsifier of uncharacterized biological
activity in oral studies, a concurrent negative
control group should be utilized. The negative
control group should be treated in the same
manner as all other test animals except that
this control group should not be exposed to
either the test substance or any vehicle.
3. The use of historical control data (i.e., the
incidence of tumors and other suspect lesions
normally occuring under the same laboratory
conditions and in the same strain of animals
employed in the test) is desirable for
assessing the significance of changes observed
in exposed animals.
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HG-Chronic-Onco
C. Dose levels and dose selection
1. For risk assessment purposes, at least three
dose levels should be used, in addition to the
concurrent control group. Dose levels should
be spaced to produce a gradation of chronic
effects.
2. The high dose level should elicit signs of
minimal toxicity without substantially altering
the normal life span.
3. The lowest dose should not interfere with
normal growth, development and longevity of the
animal; and it should not otherwise cause any
indication of toxicity. In general, this
should not be lower than ten percent of the
high dose.
4. The intermediate dose(s) should be established
in a mid-range between the high and low doses,
depending upon the toxicokinetic properties of
the chemical, if known.
5. The selection of these dose levels should be
based on existing data, preferably on the
results of subchronic studies.
D. Exposure conditions
The animals are dosed with the test substance ideally
on a seven-day per week basis over a period of at
least 24 months for rats, and 18 months for mice.
However, based primarily on practical considerations,
dosing on a five-day per week basis is considered to
be acceptable.
E. Observations period
It is necessary that the duration of an oncogenicity
test comprise the majority of the normal life span of
the strain of animals to be used. This time period
should not be less than 24 months for rats and 18
months for mice, and ordinarily not longer than 30
months for rats and 24 months for mice. For longer
time periods, and where any other species are used,
consultation with the Agency in regard to the
duration of the test is advised.
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F. Administration of the test substance
The three main routes of administration are oral,
dermal, and inhalation. The choice of the route of
administration depends upon the physical and chemical
characteristics of the test substance and the form
typifying exposure in humans.
1. Oral studies
a. The animals should receive the test
substance in their diet, dissolved in
drinking water, or given by gavage or
capsule for a period of at least 24
months for rats and 18 months for mice.
b. If the test substance is administered in
the drinking water, or mixed in the diet,
exposure should be continuous.
c. For a diet mixture, the highest
concentration should not exceed five
percent.
2. Dermal studies
a. The animals are treated by topical
application with the test substance,
ideally for at least six hours per day.
b. Fur should be clipped from the dorsal
area of the trunk of the test animals.
Care should be taken to avoid abrading
the skin which could alter its
permeability.
c. The test substance should be applied
uniformly over a shaved area which is
approximately ten percent of the total
body surface area. With highly toxic
substances, the surface area covered may
be less, but as much of the area should
be covered with as thin and uniform a
film as possible.
d. During the exposure period, the test
substance may be held, if necessary, in
contact with the skin with a porous gauze
dressing and non-irritating tape. The
test site should be further covered in a
suitable manner to retain the gauze
dressing and test substance and ensure
that the animals cannot ingest the test
substance.
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3. Inhalation studies
a. The animals should be tested with
inhalation equipment designed to sustain
a dynamic air flow of 12 to 15 air
changes per hour, ensure an adequate
oxygen content of 19 percent and an
evenly distributed exposure atmosphere.
Where a chamber is used, its design
should minimize crowding of the test
animals and maximize their exposure to
the test substance. This is best
accomplished by individual caging. As a
general rule to ensure stability of a
chamber atmosphere, the total "volume" of
the test animals should not exceed five
percent of the volume of the test
chamber. Alternatively, oro-nasal, head-
only, or whole body individual chamber
exposure may be used.
b. The temperature at which the test is
performed should be maintained at 22°C (*
2°). Ideally, the relative humidity
should be maintained between 40 to 60
percent, but in certain instances (e.g.
tests of aerosols, use of water vehicle)
this may not be practicable.
c. Food and water should be withheld during
each daily six-hour exposure period.
d. A dynamic inhalation system with a
suitable analytical concentration control
system should be used. The rate of air
flow should be adjusted to ensure that
conditions throughout the equipment are
essentially the same. Maintenance of
slight negative pressure inside the
chamber will prevent leakage of the test
substance into the surrounding areas.
G. Observation of animals
1. A careful clinical examination should be made
at least once each day.
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2. Additional observations should be made daily
with appropriate actions take to minimize loss
of animals to the study (e.g. necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Clinical signs and mortality should be recorded
for all animals. Special attention should be
paid to tumor development. The time of onset,
location, dimensions, appearance and
progression of each grossly visible or palpable
tumor should be recorded.
4. Body weights should be recorded individually
for all animals once a week during the first 13
weeks of the test period and at least once
every four weeks thereafter unless signs of
clinical toxicity suggest more frequent
weighings to facilitate monitoring of health
status.
5. When the test substance is administered in the
food or drinking water, measurements of food or
water consumption, respectively, should be
determined weekly during the first 13 weeks of
the study and then at approximately monthly
intervals unless health status or body weight
changes dictate otherwise.
6. At the end of the study period all survivors
are sacrificed. Moribund animals should be
removed and sacrificed when noticed.
H. Physical measurements
For inhalation studies, measurements or monitoring
should be made of the following:
1. The rate of air flow should be monitored
continuously, but should be recorded at
intervals of at least once very 30 minutes.
2. During each exposure period the actual
concentrations of the test substance should be
held as constant as practicable, monitored
continuously and measured at least three times
during the test period: at the beginning, at
an intermediate time and at the end of the
period.
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3. During the development of the generating
system, particle size analysis should be
performed to establish the stability of aerosol
concentrations. During exposure, analyses
should be conducted as often as necessary to
determine the consistency of particle size
distribution and homogeneity of the exposure
stream.
4. Temperature and humidity should be monitored
continuously, but should be recorded at
intervals of at least once every 30 minutes.
I. Clinical examinations
At 12 months, 18 months and at sacrifice, a blood
smear should be obtained from all animals. A
differential blood count should be performed on blood
smears from those animals in the highest dosage group
and the controls. If these data, or data from the
pathological examiniation indicate a need, then the
12 and 18 month blood smears from other dose levels
should also be examined. Differential blood counts
should be performed for the next lower group(s) only
if there is a major discrepancy between the highest
group and the controls. If clinical observations
suggest a deterioration in health of the animals
during the study, a differential blood count of the
affected animals should be performed.
J. Gross necropsy
1. A complete gross examination should be
performed on all animals, including those which
died during the experiment or were killed in
moribund conditions.
2. The following organs and tissues or
representative samples thereof, should be
preserved in a suitable medium for possible
future histopathological examination: all
gross lesions and tumors of all animals should
be preserved; brain - including sections of
medulla/pons, cerebellar cortex and cerebral
cortex; pituitary; thyroid/parathyroid; thymus;
lungs; trachea; heart; spinal cord at three
levels - cervical, midthoracic and lumbar;
sternum and/or femur with bone marrow; salivary
glands; liver; spleen; kidneys; adrenals;
esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder;
representative lymph nodes; pancreas; gonads;
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uterus; accessory genital organs; female
mammary gland; skin; musculature; peripheral
nerve; and eyes. In special studies such as
inhalation studies, the entire respiratory
tract should be preserved, including nasal
cavity, pharynx, larynx and paranasal
sinuses. In dermal studies, skin from sites of
skin painting should be examined and preserved.
3. Inflation of lungs and urinary bladder with a
fixative is the optimal method for preservation
of these tissues. The proper inflation and
fixation of the lungs in inhalation studies is
a necessary requirement for appropriate and
valid histopathological examination.
4. If other clinical examinations are carried out,
the information obtained from these procedures
should be available before microscopic
examination, since they may provide significant
guidance to the pathologist.
K. Histopathology
1. The following histopathology should be
performed:
a. Full histopathology on organs and tissues
listed above of all animals in the
control and high dose groups and all
animals that died or were killed during
the study.
b. All gross lesions in all animals.
c. Target organs in all animals.
2. If a significant difference is observed in
hyperplastic, pre-neoplastic or neoplastic
lesions between the highest dose and control
groups, microscopic examination should be made
on that particular organ or tissue of all
animals in the study;
3. If excessive early deaths or other problems
occur in the high dose group, compromising the
significance of the data, the next lower dose
level should be examined for complete
histopathology.
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HG-Chronic-Onco
4. In case the results of an experiment give
evidence of substantial alteration of the
animals' normal longevity or the induction of
effects that might affect a neoplastic
response, the next lower dose level should be
examined, fully as described above.
5. An attempt should be made to correlate gross
observations with microscopic findings.
III. DATA AND REPORTING
A. Treatment of results
1. Data should be summarized in tabular form,
showing for each test group the number of
animals at the start of the test, the number of
animals showing lesions, the types of lesions
and the percentage of animals displaying each
type of lesion.
2. All observed results, quantitative and
incidental, should be evaluated by an
appropriate statistical method. Any generally
accepted statistical methods may be used; the
statistical methods should be selected during
the design of the study.
B. Evaluation of study results
1. The findings of an oncogenic toxicity study
should be evaluated in conjunction with the
findings of preceding studies and considered in
terms of the toxic effects, the necropsy and
histopathological findings. The evaluation
should include the relationship between the
dose of the test substance and the presence,
incidence and severity of abnormalities
(including behavioral and clinical
abnormalities), gross lesions, identified
target organs, body weight changes, effects on
mortality and any other general or specific
toxic effects.
2. In any study which demonstrates an absence of
toxic effects, further investigation to
establish absorption and bioavailability of the
test substance should be considered.
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Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Group animal data
Tabulation of toxic response data by species,
strain, sex and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of
toxicity; and
c. Number of animals exposed.
2. Individual animal data
a. Time of death during the study or whether
animals survived to termination;
b. Time of observation of each abnormal sign
and its subsequent course;
c. Body weight data;
d. Food and water consumption data, when
collected;
e. Results of ophthalmological examination,
when performed;
f. Hematological tests employed and all
results;
g. Clinical biochemistry tests employed and
all results;
h. Necropsy findings;
i. Detailed description of all
histopathological findings;
j. Statistical treatment of results, where
appropriate; and
k. Historical control data, if taken into
account.
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In addition, for inhalation studies the following
should be reported:
3. Test conditions
a. Description of exposure apparatus
including design, type, dimensions,
source of air, system for generating
particulates and aerosols, method of
conditioning air, treatment of exhaust
air and the method of housing the animals
in a test chamber.
b. The equipment for measuring temperature,
humidity, and particulate aerosol
concentrations and size should be
described.
4. Exposure data
These should be tabulated and presented with
mean values and a measure of variability (e.g.
standard deviation) and should include:
a. Airflow rates through the inhalation
equipment;
b. Temperature and humidity of air;
c. Nominal concentration (total amount of
test substance fed into the inhalation
equipment divided by volume of air);
d. Actual concentration in test breathing
zone; and
e. Particle size distribution (e.g. median
aerodynamic diameter of particles with
standard deviation from the mean).
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HG-Chronic-Onco
IV. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. DHEW. 1975. Department of Health and Welfare. The
Testing of Chemicals for Carcinogenicity,
Mutagenicity, Teratogenicity. Canada: The Honorable
marc Lalonde, Minister of Health and Welfare.
Department of Health and Welfare. 183 pp.
2. Food and Drug Administration Advisory Committee on
Protocols for Safety Evaluation: Panel on
Carcinogenesis. 1971. Report on Cancer Testing in
the Safety of Food Additives and Pesticides.
Toxicology and Applied Pharmacology. 20:419-438.
3. IUCC. 1969. International Union Against Cancer.
"Carcinogenicity Testing," in "IUCC Technical Report
Series, Vol. 2," Edited by I. Berenblum. Geneva:
International Union Against Cancer. 56 pp.
4. Leong, B.K.J. , Laskin, S. 1975. Number and Species
of Experimental Animals for Inhalation
Carcinogenicity Studies. Cincinnati: Paper
presented at Conference on Target Organ Toxicity.
Sept., 1975.
5. NAS. 1977. National Academy of Sciences.
Prinicples and Procedures for Evaluating the Toxicity
of Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
6. NCI. 1976. National Cancer Institute. Report of
the Subtask Group on Carcinogen Testing to the
Interagency Collaborative Group on Environmental
Carcinogenesis. Bethesda: United States National
Cancer Institute. 24 pp.
7. NCTR. 1972. National Center,,for Toxicological
Research. Report of Chronic Studies Task Force
Committee, Appendix B. April 13-21. Rockville:
National Center for Toxicological Research. 50 pp.
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8. Page, N.P. 1977. Chronic Toxicity and
Carcinogenicity Guidelines. Journal Environmental
Pathology and Toxicology. 1:161-182.
9. Page, N.P. 1977. "Concepts of a Bioassay Program in
Environmental Carcinogenesis," _in_ "Advances in Modern
Toxicology." Edited by Kraybill and Mehlman.
Washington, D.C.: Hemisphere Publishing
Corporation. Volume 3. PP. 87-171.
10. Sontag, J.M., Page, N.P., Saffiotti, U. 1976.
Guidelines for Carcinogen Bioassay in Small
Rodents. Bethesda: United States Cancer Institute,
Division of Cancer Control and Prevention,
Carcinogenesis Bioassay Program. NCI-CS-TR-1. 65
pp.
11. United States Pharmaceutical Manufacturers
Association. 1977. Guidelines for the Assessment of
Drug and Medical Device Safety in Animals. 64 pp.
12. WHO. 1969. World Health Organization. Principles
for the Testing and Evaluation of Drugs for
Carcinogenicity. WHO Technical Report Series No.
426. Geneva: World Health Organization. 26 pp.
13. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
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HG-Chronic-Combined
August, 1982
COMBINED CHRONIC TOXICITY/ONCOGENICITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Chronic-Combined
I. PURPOSE
The objective of a combined chronic toxicity/oncogenicity
study is to determine the effects of a substance in a
mammalian species following prolonged and repeated
exposure. The application of this guideline should
generate data which identify the majority of chronic and
oncogenic effects and determine dose-response
relationships. The design and conduct should allow for the
detection of neoplastic effects and a determination of
oncogenic potential as well as general toxicity, including
neurological, physiological, biochemical, and hematological
effects and exposure-related morphological (pathology)
effects.
II. TEST PROCEDURES
A. Animal selection
1. Species and strain
Preliminary studies providing data on acute,
subchronic, and metabolic responses should have
been carried out to permit an appropriate
choice of animals (species and strain). As
discussed in other guidelines, the mouse and
rat have been most widely used for assessment
of oncogenic potential, while the rat and dog
have been most often studies for chronic
toxicity. The rat is the species of choice for
combined chronic toxicity and oncogenicity
studies. The provisions of this guideline are
designed primarily for use with the rat as the
test species. If other species are used, the
tester should provide justification/ reasoning
for their selection. The strain selected
should be susceptible to the oncogenic or toxic
effect of the class of substances being tested,
if known, and provided it does not have a
spontaneous background too high for meaningful
assessment. Commonly used laboratory strains
should be employed.
2. Age
a. Dosing of rats should begin as soon as
possible after weaning, ideally before
the rats are six, but in no case more
than eight weeks old.
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HG-Chronic-Combined
b. At commencement of the study, the weight
variation of animals used should exceed
4r 20 percent of the mean weight for each
sex.
c. Studies using prenatal or neonatal
animals may be recommended under special
conditions.
3. Sex
a. Equal numbers of animals of each sex
should be used at each dose level.
b. The females should be nulliparous and
non-pregnant.
4. Numbers
a. At least 100 rodents (50 females and 50
males) should be used at each dose level
and concurrent control for those groups
not intended for early sacrifice. At
least 40 rodents (20 females and 20
males) should be used for satellite dose
group(s) and the satellite control
group. The purpose of the satellite
group is to allow for the evaluation of
pathology other than neoplasia.
b. If interim sacrifices are planned, the
number of animals should be increased by
the number of animals scheduled to be
sacrificed during the course of the
s t udy.
c. The number of animals at the termination
of each phase of the study should be
adequate for a meaningful and valid
statistical evaluation of long term
exposure. For a valid interpretation of
negative results, it is essential that
survival in all groups does not fall
below 50 percent at the time of
termination.
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HG-Chronic-Combined
B. Control groups
1. A concurrent control group (50 females and 50
males) and a satellite control group (20
females and 20 males) are recommended. These
groups should be untreated or sham treated
control groups or, if a vehicle is used in
administering the test substance, vehicle
control groups. If the toxic properties of the
vehicle are not known or cannot be made
available, both untreated and vehicle control
groups are recommended. Animals in the
satellite control group should be sacrificed at
the same time the satellite test group is
terminated.
2. In special circumstances such as inhalation
studies involving aerosols or the use of an
emulsifier of uncharacterized biological
activity in oral studies, a concurrent negative
control group should be utilized. The negative
control group should be treated in the same
manner as all other test animals, except that
this control group should not be exposed to the
test substance or any vehicle.
3. The use of historical control data (i.e., the
incidence of tumors and other suspect lesions
normally occuring under the same laboratory
conditions and in the same strain of animals
employed in the test) is desirable for
assessing the significance of changes observed
in exposed animals.
C. Dose levels and dose selection
1. For risk assessment purposes, at least three
dose levels should be used, in addition to the
concurrent control group. Dose levels should
be spaced to produce a gradation of effects.
2. The highest dose level in rodents should elicit
signs of toxicity without substantially
altering the normal life span due to effects
other than tumors.
3. The lowest dose level should produce no
evidence of toxicity. Where there is a usable
estimation of human exposure, the lowest dose
level should exceed this even though this dose
level may result in some signs of toxicity.
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HG-Chronic-Combined
4. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If
more than one intermediate dose is used the
dose levels should be spaced to produce a
gradation of toxic effects.
5. For rodents, the incidence of fatalities in low
and intermediate dose groups and in the
controls should be low to permit a meaningful
evaluation of the results.
6. For chronic toxicological assessment, a high
dose treated satellite and a concurrent control
satellite group should be included in the study
design. The highest dose for satellite animals
should be chosen so as to produce frank
toxicity, but not excessive lethality, in order
to elucidate a chronic toxicological profile of
the test substance. If more than one dose
level is selected for satellite dose groups,
the doses should be spaced to produce a
gradation of toxic effects.
D. Exposure conditions
The animals are dosed with the test substance ideally
on a seven-day per week basis over a period of at
least 24 months for rats, and 18 months for mice and
hamsters, except for the animals in the satellite
groups which should be dosed for 12 months.
E. Observation period
It is necessary that the duration of the oncogenicity
test comprise the majority of the normal life span of
the animals to be used. It has been suggested that
the duration of the study should be for the entire
lifetime of all animals. However, a few animals may
greatly exceed the average lifetime and the duration
of the study may be unnecessarily extended and
complicate the conduct and evaluation of the study.
Rather, a finite period covering the majority of the
expected life span of the strain is preferred since
the probability is high that, for the great majority
of chemicals, induced tumors will occur within such
an observation period. The following guidelines are
recommended:
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1. Generally, the termination of the study should
be at 18 months for mice and hamsters and 24
months for rats; however, for certain strains
of animals with greater longevity and/or low
spontaneous tumor rate, termination should be
at 24 months for mice and hamsters and at 30
months for rats. For longer time periods, and
where any other species are used, consultation
with the Agency in regard to duration of the
test is advised.
2. However, termination of the study is acceptable
when the number of survivors of the lower doses
or control group reach 25 percent. In the case
where only the high dose group dies prematurely
for obvious reasons of toxicity, this should
not trigger termination of the study.
3, The satellite groups and the concurrent
satellite control group should be retained in
the study for at least 12 months. These groups
should be scheduled for sacrifice for an
estimation of test-substance-related pathology
uncomplicated by geriatric changes.
E. Administration of the test substance
The three main routes of administration are oral,
dermal, and inhalation. The choice of the route of
administration depends upon the physical and chemical
characteristics of the test substance and the form
typifying exposure in humans.
1. Oral studies
a. The animals should receive the test
substance in their diet, dissolved in
drinking water, or given by gavage or
capsule for a period of at least 24
months for rats and 18 months for mice
and hamsters.
b. If the test substance is administered in
the drinking water, or mixed in the diet,
exposure is continuous.
c. For a diet mixture, the highest
concentration should not exceed five
percent.
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2. Dermal studies
a. The animals are treated by topical
application with the test substance,
ideally for at least six hours per day.
b. Fur should be clipped from the dorsal
area of the trunk of the test animals.
Care should be taken to avoid abrading
the skin which could alter its
permeability.
c. The test substance should be applied
uniformly over a shaved area which is
approximately ten percent of the total
body surface area. With highly toxic
substances, the surface area covered may
be less, but as much of the area should
be covered with as thin and uniform a
film as possible.
d. During the exposure period, the test
substance may be held, if necessary, in
contact with the skin with a porous gauze
dressing and non-irritating tape. The
test site should be further covered in a
suitable manner to retain the gauze
dressing and test substance and ensure
that the animals cannot ingest the test
substance.
3. Inhalation studies
a. The animals should be tested with
inhalation equipment designed to sustain
a dynamic air flow of 12 to 15 air
changes per hour, ensure an adequate
oxygen content of 19 percent and an
evenly distributed exposure atmosphere.
Where a chamber is used, its design
should minimize crowding of the test
animals and maximize their exposure to
the test substance. This is best
accomplished by individual caging. As a
general rule, to ensure stability of a
chamber atmosphere, the total "volume" of
the test animals should not exceed five
percent of the volume of the test
chamber. Alternatively, oro-nasal, head-
only, or whole body individual chamber
exposure may be used.
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HG-Chronic-Combined
b. The temperature at which the test is
performed should be maintained at 22°C
(* 2°). Ideally, the relative humidity
should be maintained between 40 to 60
percent, but in certain instances (e.g.,
tests of aerosols, use of water vehicle)
this may not be practicable.
c. Food and water should be withheld during
each daily six-hour exposure period.
d. A dynamic inhalation system with a
suitable analytical concentration control
system should be used. The rate of air
flow should be adjusted to ensure that
conditions throughout the equipment are
essentially the same. Maintenance of
slight negative pressure inside the
chamber will prevent leakage of the test
substance into the surrounding areas.
F. Observation of animals
1. A careful clinical examination should be made
at least once each day.
2. Additional observations should be made daily
with appropriate actions taken to minimize loss
of animals to the study (e.g., necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
3. Clinical signs and mortality should be recorded
for all animals. Special attention should be
paid to tumor development. The time of onset,
location, dimensions, appearance and
progression of each grossly visible or palpable
tumor should be recorded.
4. Body weights should be recorded individually
for all animals once a week during the first 13
weeks of the test period and at least once
every four weeks thereafter, unless signs of
clinical toxicity suggest more frequent
weighings to facilitate monitoring of health
status.
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HG-Chronic-Combined
5. When the test substance is administered in the
food or drinking water, measurements of food or
water consumption, respectively, should be
determined weekly during the first 13 weeks of
the study and then at approximately monthly
intervals unless health status or body weight
changes dictate otherwise.
6. At the end of the study period all survivors
are sacrificed. Moribund animals should be
removed and sacrificed when noticed.
G. Physical measurements
For inhalation studies, measurements or monitoring
should be made of the following:
1. The rate of air flow should be monitored
continuously, but should be recorded at
intervals of at least once every 30 minutes.
2. During each exposure period the actual
concentrations of the test substance should be
held as constant as practicable, monitored
continuously and measured at least three times
during the test period: at the beginning, at
an intermediate time and at the end of the
period.
3. During the development of the generating
system, particle size analysis should be
performed to establish the stability of aerosol
concentrations. During exposure, analyses
should be conducted as often as necessary to
determine the consistency of particle size
distribution and homogeneity of the exposure
stream.
4. Temperature and humidity should be monitored
continuously, but should be recorded at
intervals of at least once every 30 minutes.
H. Clinical examinations
1. The following examinations should be made on at
least 20 rodents of each sex per dose level:
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HG-Chronic-Combined
a. Certain hematology determinations (e.g.,
hemoglobin content, packed cell volume,
total red blood cells, total white blood
cells, platelets, or other measures of
clotting potential) should be performed
at termination and should be performed at
three months, six months and at
approximately six-month intervals
thereafter (for those groups on test for
longer than 12 months) on blood samples
collected from 20 rodents per sex of all
groups. These collections should be from
the same animals at each interval. If
clinical observations suggest a
deterioration in health of the animals
during the study, a differential blood
count of the affected animals should be
performed. A differential blood count
should be performed on samples from those
animals in the highest dosage group and
the controls. Differential blood counts
should be performed for the next lower
group(s) if there-is a major discrepancy
between the highest group and the
controls. If hematological effects were
noted in the subchronic test,
hematological testing should be performed
at 3, 6, 12, 18 and 24 months for a two
year study.
b. Certain clinical biochemistry
determinations on blood should be carried
out at least three times during the test
period: just prior to initiation of
dosing (baseline data), near the middle
and at the end of the test period. Blood
samples should be drawn for clinical
measurements from at least ten rodents
per sex of all groups; if possible, from
the same rodents at each time interval.
Test areas which are considered
appropriate to all studies: electrolyte
balance, carbohydrate metabolism and
liver and kidney function. The selection
of specific tests will be influenced by
observations on the mode of action of the
substance and signs of clinical
toxicity. Suggested chemical
determinations: calcium, phosphorus,
chloride, sodium, potassium, fasting
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HG-Chronic-Combined
glucose (with period of fasting
appropriate to the species), serum
glutamic-pyruvic transaminase*, serum
glutamic oxaloacetic transaminase**,
ornithine decarboxylase, gamma glutamyl
transpeptidase, blood urea nitrogen,
albumen, creatinine phosphokinase, total
cholesterol, total bilirubin and total
serum protein measurements. Other
determinations which may be necessary for
an adequate toxicological evaluation
include analyses of lipids, hormones,
acid/base balance, methemoglobin and
cholinesterase activity. Additional
clinical biochemistry may be employed
where necessary to extend the
investigation of observed effects.
* Now known as serum alanine
aminotransferase.
** Now known as serum aspartate
aminotransferase.
2. The following should be performed on at least
ten rodents of each sex per dose level:
a. Urine samples from the same rodents at
the same intervals as hematological
examination above, should be collected
for analysis. The following
determinations should be made from either
individual animals or on a pooled
sample/sex/group for rodents: appearance
(volume and specific gravity), protein,
glucose, ketones, bilirubin, occult blood
(seml-quantitatively) and microscopy of
sediment (semi-quantitatively).
b. Ophthalmological examination, using an
ophthalmoscope or equivalent suitable
equipment, should be made prior to the
administration of the test substance and
at the termination of the study. If
changes in the eyes are detected all
animals should be examined.
I. Gross necropsy
1. A complete gross examination should be
performed on all animals, including those which
died during the experiment or were killed in
moribund conditions.
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HG-Chronic-Corabined
2. The liver, kidneys, adrenals, brain and gonads
should be weighed wet, as soon as possible
after dissection to avoid drying. For these
organs, at least ten rodents per sex per group
should be weighed.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible
future histopathological examination: all
gross lesions and tumors; brain - including
sections of medulla/pons, cerebellar cortex,
and cerebral cortex; pituitary; thyroid/
parathyroid; thymus; lungs; trachea; heart;
sternum and/or femur with bone marrow; salivary
glands; liver; spleen; kidneys; adrenals;
esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder;
representative lymph nodes; pancreas; gonads;
uterus; accessory genital organs; female
mammary gland; aorta; gall bladder (if
present); skin; musculature; peripheral nerve;
spinal cord at three levels - cervical,
midthoracic, and lumbar; and eyes. In
inhalation studies, the entire respiratory
tract, including nose, pharynx, larynx and
paranasal sinuses should be examined and
preserved. In dermal studies, skin from sites
of skin painting should be examined and
preserved.
4. Inflation of lungs and urinary bladder with a
fixative is the optimal method for preservation
of these tissues. The proper inflation and
fixation of the lungs in inhalation studies is
considered essential for appropriate and valid
histopathological examination.
5. If other clinical examinations are carried out,
the information obtained from these procedures
should be available before microscopic
examination, since they may provide significant
guidance to the pathologist.
J. Histopathology
1. The following histopathology should be
performed:
a. Full histopathology on the organs and
tissues, listed above, of all non-
rodents, of all rodents that died or
where killed during the study.
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HG-Chronic-Combined
b. All gross lesions in all animals.
c. Target organs in all animals.
d. Lungs, liver and kidneys of all
animals. Special attention to
examination of the lungs of rodents
should be made for evidence of infection
since this provides an assessment of the
state of health of the animals.
2. If excessive early deaths or other problems
occur in the high dose group compromising the
significance of the data, the next dose level
should be examined for complete histopathology.
3. In case the results of the experiment give
evidence of substantial alteration of the
animals' normal longevity or the induction of
effects that might affect a toxic response, the
next lower dose level should be examined as
described above.
4. An attempt should be made to correlate gross
observations with microscopic findings.
III. DATA AND REPORTING
A. Treatment of results
1. Data should be summarized in tabular form,
showing for each test group the number of
animals at the start of the test, the number of
animals showing lesions, the types of lesions
and the percentage of animals displaying each
type of lesion.
2. All observed results, quantitative and
incidental, should be evaluated by an
appropriate statistical method. Any generally
accepted statistical methods may be used; the
statistical methods should be selected during
the design of the study.
B. Evaluation of study results
1. The findings of a combined chronic
toxicity/oncogenicity study should be evaluated
in conjunction with the findings of preceding
studies and considered in terms of the toxic
effects, the necropsy and histopathological
findings. The evaluation will include the
relationship between the dose of the test
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HG-Chronic-Combined
substance and the presence, incidence and
severity of abnormalities (including behavioral
and clinical abnormalities), gross lesions,
identified target organs, body weight changes,
effects on mortality and any other general or
specific toxic effects.
2. In any study which demonstrates an absence of
toxic effects, further investigation to
establish absorption and bioavailability of the
test substance should be considered.
3. In order for a negative test to be acceptable,
it should meet the following criteria: no more
than ten percent of any group is lost due to
autolysis, cannibalism, or management problems;
and survival in each group is no less than 50
percent at 18 months for mice and hamsters and
at 24 months for rats.
C. Test report
In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice
Standards [Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Group animal data
Tabulation of toxic response data by species,
strain, sex and exposure level for:
a. Number of animals dying;
b. Number of animals showing signs of
toxicity; and
c. Number of animals exposed.
2. Individual animal data
a. Time of death during the study or whether
animals survived to termination;
b. Time of observation of each abnormal sign
and its subsequent course;
c. Body weight data;
d. Food and water consumption data, when
collected;
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HG-Chronic-Combined
e. Results of ophthalmological examination,
when performed;
f. Hematological tests employed and all
results;
g. Clinical biochemistry tests employed and
all results;
h. Necropsy findings;
i. Detailed description of all
histopathological findings;
j. Statistical treatment of results where
appropriate; and
k. Historical control data, if taken into
account.
In addition, for inhalation studies the following
should be reported:
3. Test conditions
a. Description of exposure apparatus
including design, type, dimensions,
source of air, system for generating
particulates and aerosols, method of
conditioning air, treatment of exhaust
air and the method of housing the animals
in a test chamber.
b. The equipment for measuring temperature,
humidity, and particulate aerosol
concentrations and size should be
described.
4. Exposure data
These should be tabulated and presented with
mean values and a measure of variability (e.g.
standard deviation) and should include:
a. Airflow rates through the inhalation
equipment;
b. Temperature and humidity of air;
c. Nominal concentration (total amount of
test substance fed into the inhalation
equipment divided by volume of air);
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HG-Chronic-Combined
d. Actual concentration in test breathing
zone; and
e. Particle size distribution (e.g. median
aerodynamic diameter of particles with
standard deviation from the mean).
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HG-Chronic-Combined
IV. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of information on
which this section is based. They should not be considered the
only source of information on test performance, however.
1. Benitz, K.F. 1970. "Measurement of Chronic
Toxicity," in "Methods of Toxicology." Edited by
G.E. Paget. Oxford: Blackwell Scientific
Publications. PP. 82-131.
2. D'Aguanno, W. 1974. "Drug Safety Evaluation—Pre-
Clinical Considerations," in "Industrial
Pharmacology: Neuroleptics." Edited by S. Fielding
and H. Lai. Mt. Kisco: Futura Publishing Co. Vol.
I. PP. 317-332.
3. DHEW. 1975. Department of Health and Welfare. The
Testing of Chemicals for Carcinogenicity,
Mutagenicity, Teratogenicity. Canada: The Honorable
marc Lalonde, Minister of Health and Welfare.
Department of Health and Welfare. 183 pp.
4. Fitzhugh, O.G. 1959. Third Printing: 1975.
"Chronic Oral Toxicity," in "Appraisal of the Safety
of Chemicals in Foods, Drugs and Cosmetics." The
Association of Food and Drug Officials of the United
States. PP. 36-45.
5. Food and Drug Administration Advisory Committee on
Protocols for Safety Evaluation: Panel on
Carcinogenesis. 1971. Report on Cancer Testing in
the Safety of Food Additives and Pesticides.
Toxicology and Applied Pharmacology. 20:419-438.
6. Goldenthal, E.I>, D'Aguanno, W. 1959. Third
Printing: 1975. "Evaluation of Drugs," in
"Appraisal of the Safety of Chemicals in Foods,
Drugs, and Cosmetics." The Association of Food and
Drug Officials of the United States. PP. 60-67.
7. IUCC. 1969. International Union Against Cancer.
"Carcinogenicity Testing," in "IUCC Technical Report
Series, Vol. 2," Edited by I. Berenblum. Geneva:
International Union Against Cancer. 56 pp.
8. Leong, B.K.J., Laskin, S. 1975. Number and Species
of Experimental Animals for Inhalation
Carcinogenicity Studies. Cincinnati: Paper
presented at Conference on Target Organ Toxicity.
Sept., 1975.
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HG-Chronic-Combined
9. NAS. 1977. National Academy of Sciences.
Prinicples and Procedures for Evaluating the Toxicity
of Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
10. NCI. 1976. National Cancer Institute. Report of
the Subtask Group on Carcinogen Testing to the
Interagency Collaborative Group on Environmental
Carcinogenesis. Bethesda: United States National
Cancer Institute. 24 pp.
11. NCTR. 1972. National Center for Toxicological
Research. Report of Chronic Studies Task Force
Committee, Appendix B. April 13-21. Rockville:
National Center for Toxicological Research. 50 pp.
12. Page, N.P. 1977. Chronic Toxicity and
Carcinogenicity Guidelines. Journal Environmental
Pathology and Toxicology. 1:161-182.
13. Page, N.P. 1977. "Concepts of a Bioassay Program in
Environmental Carcinogenesis," in "Advances in Modern
Toxicology." Edited by Kraybill and Mehlman.
Washington, D.C.: Hemisphere Publishing
Corporation. Volume 3. PP. 87-171.
14. Schwartz, E. 1974. "Toxicology of Neuroleptic
Agents," in "Industrial Pharmacology:
Neuroleptics." Edited by S. Fielding and H. Lai.
Mt. Kisco, Futura Publishing Co. PP. 203-221.
15. Sontag, J.M., Page, N.P., Saffiotti, U. 1976.
Guidelines for Carcinogen Bioassay in Small
Rodents. Bethesda: United States Cancer Institute,
Division of Cancer Control and Prevention,
Carcinogenesis Bioassay Program. NCI-CS-TR-1. 65
PP.
16. USPMA. 1977. United States Pharmaceutical
Manufacturers Association. Guidelines for the
Assessment of Drug and Medical Device Safety in
Animals. 64 pp.
17. WHO. 1969. World Health Organization. Principles
for the Testing and Evaluation of Drugs for
Carcinogenicity. WHO Technical Report Series No.
426. Geneva: World Health Organization. 26 pp.
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HG-Chronic-Combined
18. WHO. 1975. World Health Organization. Guidelines
for Evaluation of Drugs for Use in Man. WHO
Technical Report Series No. 563. Geneva: World
Health Organization. 59 pp.
19. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
20. WHO. 1966. World Health Organization. Principles
for Pre-Clinical Testing of Drug Safety. WHO
Technical Report Series No. 341. Geneva: World
Health Organization. 22 pp.
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II. SPECIFIC ORGAN/TISSUE
TOXICITY
-------
HG-Organ/Tissue-Dermal Sensit
August, 1982
DERMAL SENSITIZATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Organ/Tissue-Dermal Sensit
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a substance, determination of its
potential to provoke skin sensitization reactions is
important. Information derived from tests for skin
sensitization serves to identify the possible hazard to a
population repeatedly exposed to a test substance. While
the desirability of skin sensitization testing is
recognized, there are some real differences of opinion about
the best method to use. The test selected should be a
reliable screening procedure which should not fail to
identify substances with significant allergenic potential,
while at the same time avoiding false negative results.
II. DEFINITIONS
A. Skin sensitization (allergic contact dermatitis) is an
immunologically mediated cutaneous reaction to a
substance. In the human, the responses may be
characterized by pruritis, erythema, edema, papules,
vesicles, bullae or a combination of these. In other
species the reactions may differ and only erythema and
edema may be seen.
B. Induction period is a period of at least one week
following a sensitization exposure during which a
hypersensitive state is developed.
C. Induction exposure is an experimental exposure of a
subject to a test substance with the intention of
inducing a hypersensitive state.
D. Challenge exposure is an experimental exposure of a
previously treated subject to a test substance
following an induction period, to determine whether
the subject will react in a hypersensitive manner.
III. PRINCIPLE OF THE TEST METHOD
Following initial exposure(s) to a test substance, the
animals are subsequently subjected, after a period of not
less than one week, to a challenge exposure with the test
substance to establish whether a hypersensitive state has
been induced. Sensitization is determined by examining the
reaction to the challenge exposure and comparing this
reaction to that of the initial induction exposure.
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HG-Organ/Tissue-Dermal Sensit
IV. TEST PROCEDURES
A. Any of the following seven test methods is considered
to be acceptable. It is realized, however, that the
methods differ in their probability and degree of
reaction to sensitizing substances.
1. Freund's complete adjuvant test.
2. Guinea pig maximization test;
3. Split adjuvant technique;
4. Buehler test;
5. Open epicutaneous test;
6. Mauer optimization test.
7. Footpad technique in guinea pig.
B. Removal of hair is by clipping, shaving, or possibly
by depilation, depending on the test method used.
C. Animal selection
1. Species and strain
The young adult guinea pig is the preferred
species. Commonly used laboratory strains
should be employed. If other species are used,
the tester should provide
justification/reasoning for their selection.
2. Number and sex
a. The number and sex of animals used will
depend on the method employed.
b. The females should be nulliparous and non-
pregnant.
D. Control animals
1. Periodic use of a positive control substance
with an acceptable level of reliability for the
test system selected is recommended;
2. Animals may act as their own controls or groups
of induced animals can be compared to groups
which have received only a challenge exposure.
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HG-Organ/Tissue-Dermal Sensit
E. Dose levels
The dose level will depend upon the method selected.
F. Observation of animals
1. Skin reactions should be graded and recorded
after the challenge exposures at the time
specified by the methodology selected. This is
usually 24, 48, and 72 hours. Additional
notations should be made as necessary to fully
describe unusual responses;
2. Regardless of method selected, initial and
terminal body weights should be recorded.
G. Procedures
The procedures to be used are those described by the
methodology chosen.
V. DATA AND REPORTING
A. Data should be summarized in tabular form, showing for
each individual animal the skin reaction, results of
the induction exposure(s) and the challenge
exposure(s) at times indicated by the method chosen.
As a minimum, the erythema and edema should be graded
and any unusual finding should be recorded.
B. Evaluation of the results
The evaluation of results will provide information on
the proportion of each group that became sensitized
and the extent (slight, moderate, severe) of the
sensitization reaction in each individual animal.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. A description of the method used and the
commonly accepted name;
2. Information on the positive control study;
including positive control used, method used and
time conducted;
3. The number and sex of the test animals;
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HG-Organ/Tissue-Dermal Sensit
4. Species and strain;
5. Individual weights of the animals at the start
of the test and at the conclusion of the test;
6. A brief description of the grading system; and
7. Each reading made on each individual animal.
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HG-Organ/Tissue-Dermal Sensit
VI. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Buehler, E.V. 1965. Delayed Contact Hypersensitivity
in the Guinea Pig. Archives Dermatology. 91:171.
2. Draize, J.H. 1955. Dermal Toxicity. Food Drug
Cosmetic Law Journal. 10:722-732.
3. Klecak, G. 1977. "Identification of Contact
Allergens: Predictive Tests in Animals," in "Advances
in Modern Toxicology: Dermatology and
Pharmacology." Edited by F.N. Marzulli and H.I.
Maibach. Washinton, D.C.: Hemisphere Publishing
Corporation. 4:305-339.
4. Klecak, G., Geleick, H., Grey, J.R. 1977. Screening
of Fragrance Materials for Allergenicity in the Guinea
Pig. 1. Comparison of Four Testing Methods. Journal
of the Society of Cosmetic Chemists. 28:53-64.
5. Magnusson, B., Kligman, A.M. 1973. The
Identification of Contact Allergens by Animal Assay.
The Guinea Pig Maximization Test. The Journal of
Investigative Dermatology. 52:268-276.
6. Maguire, H.C. 1973. The Bioassay of Contact
Allergens in the Guinea Pig. Journal of the Society
of Cosmetic Chemists. 24:151-162.
7. Maurer, T., Thomann, P., Weirich, E.G., Hess, R.
1975. "The Optimization Test in the Guinea Pig. A
Method for the Predictive Evaluation of the Contact
Allergenicity of Chemicals. Agents and Actions.
Basel: Birkhauser Verlag. Vol 5/2. PP. 174-149.
8. Maurer, T., Thomann, P., Weirich, E.G., Hess, R.
1975. "The Optimization Test in the Guinea Pig: A
Method for the Predictive Evaluation of the Contact
Allergenicity of Chemicals," in "International
Congress Series Excerpta Medica No. 376. Vol. 203."
PP. 203-205.
— 5—
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HG-Organ/Tissue-Dermal Irrit
August, 1982
PRIMARY DERMAL IRRITATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCE
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Organ/Tissue-Dermal Irrit
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a substance, determination of the
irritant and/or corrosive effects on skin of mammals is an
important initial step. Information derived from this test
serves to indicate the existence of possible hazards likely
to arise from exposure of the skin to the test substance.
II. DEFINITIONS
A. Dermal irritation is the production of reversible
inflammatory changes in the skin following the
application of a test substance.
B. Dermal corrosion is the production of irreversible
tissue damage in the skin following the application of
the test substance.
III. PRINCIPLE OF THE TEST METHOD
A. The substance to be tested is applied in a single dose
to the skin of several experimental animals, each
animal serving as its own control. The degree of
irritation is read and scored at specified intervals
and is further described to provide a complete
evaluation of the effects. The duration of the study
should be sufficient to permit a full evaluation of
the reversibility or irreversibility of the effects
observed but need not exceed 14 days.
B. When testing solids (which may be pulverized if
considered necessary), the test substance should be
moistened sufficiently with water or, where necessary,
a suitable vehicle, to ensure good contact with the
skin. When vehicles are used, the influence of the
vehicle on irritation of skin by the test substance
should be taken into account. Liquid test substances
are generally used undiluted.
C. Strongly acidic or alkaline substances, for example
with a demonstrated pH of 2 or less, or 11.5 or
greater, need not be tested for primary dermal
irritation, owing to their predictable corrosive
properties.
D. The testing of materials which have been shown to be
highly toxic by the dermal route is unnecessary.
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HG-Organ/Tissue-Dermal Irrit
IV. TEST PROCEDURES
A. Animal selection
1. Species and strain
The albino rabbit is recommended as the
preferred species. If another mammalian species
is used, the tester should provide
justification/reasoning for its selection.
2. Number of animals
At least 6 healthy adult animals should be used
unless, justification/reasoning for using fewer
animals is provided.
B. Control animals
Separate animals are not recommened for an untreated
control group. Adjacent areas of untreated skin of
each animal may serve as a control for the test.
C. Dose level
A dose of 0.5 ml of liquid or 5 mg of solid or semi-
solid is applied to the test site.
D. Preparation of animals' skins
Approximately 24 hours before the test, fur should be
removed from the test area by clipping or shaving from
the dorsal area of the trunk of the animals. Care
should be taken to avoid abrading the skin. Only
animals with healthy intact skin should be used.
E. Application of the test substance
1. The recommended exposure duration is 4-hours.
Longer exposure may be indicated under certain
conditions (e.g. expected pattern of human use
and exposure). At the end of the exposure
period, residual test substance should generally
be removed, where practicable, using water or an
appropriate solvent, without altering the
existing response or the integrity of the
epidermis.
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HG-Organ/Tissue-Dermal Irrit
2. The test substance should be applied to a small
area (approximately 6 cm2) of skin and covered
with a gauze patch, which is held in place with
non-irritating tape. In the case of liquids or
some pastes, it may be necessary to apply the
test substance to the gauze patch and then apply
that to the skin. The patch should be loosely
held in contact with the skin by means of a
suitable semi-occlusive dressing for the
duration of the exposure period. However, the
use of an occlusive dressing may be considered
appropriate in some cases. Access by the animal
to the patch and resultant ingestion/inhalation
of the test substance should be prevented.
F. Observation period
The duration of the observation period should be at
least 72 hours, but should not be rigidly fixed. It
should be sufficient to fully evaluate the
reversibility or irreversiblity of the effects
observed. It need not exceed 14 days after
application.
G. Clinical examination and scoring
After removal of the patch, animals should be examined
for signs of erythema and edema and the responses
scored within 30-60 minutes, and then at 24, 48 and 72
hours after patch removal.
Dermal irritation should be scored and .recorded
according to the grades in Table 1, below. Further
observations may be needed, as necessary, to establish
reversibility. In addition to the observation of
irritation, any lesions and other toxic effects should
be fully described.
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HG-Organ/Tissue-Dermal Irrit
Table 1; Evaluation of Skin Reaction
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) .... 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar
formation (injuries in depth) . 4
Maximum possible 4
Edema Formation Value
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well defined by
definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter
and extending beyond area of exposure ... 4
Maximum possible,
-4-
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HG-Organ/Tissue-Dermal Irrit
V. DATA AND REPORTING
A. Data should be summarized in tabular form, showing for
each individual animal the irritation scores for
erythema and edema at 30 to 60 minutes, 24, 48 and 72
hours after patch removal, any lesions, a description
of the degree and nature of irritation, corrosion or
reversibility, and any other toxic effects observed.
B. Evaluation of results
The dermal irritation scores should be evaluated in
conjunction with the nature and reversibility or
otherwise of the responses observed. The individual
scores do not represent an absolute standard for the
irritant properties of a material. They should be
viewed as reference values which are only meaningful
when supported by a full description and evaluation of
the observations. The use of an occlusive dressing is
a severe test and the results are relevant to very few
likely human exposure conditions.
C. Test report
In addition to the reporting recommendations as
specified in the EPA Good Laboratory Practice
Standards [Subpart J, Part 792, Chapter I of Title
40. Code of Federal Regulations] the following
specific information should be reported:
1. Physical nature and, where appropriate,
concentration, and pH value for the test
substance;
2. Species and strain;
3. Tabulation of irritation response data for each
individual animal for each observation time
period (e.g. 30 to 60 minutes, 24, 48, 72 hours
after patch removal);
4. Description of any lesions observed;
5. Narrative description of the degree and nature
of irritation observed; and
6. Description of any toxic effects other than
dermal irritation.
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HG-Organ/Tissue-Derma.l Irrit
VI. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Draize, J.H. 1959. Third Printing: 1975. "Dermal
Toxicity," in "Appraisal of the Safety of Chemicals in
Foods, Drugs and Cosmetics." Association of Food and
Drug Officials of the United States. PP. 46-59.
2. Draize, J.H. , Woodward, G., Calvery, H.O. 1944.
Methods for the Study of Irritation and Toxicity of
Substances Applied Topically to the Skin and Mucous
Membranes. Journal of Pharmacology Experiment
Therapeutics. 83:377-390.
3. Marzulli, F.N., Maibach, H.I. 1977.
"Dermatotoxicology and Pharmacology," in "Advances in
Modern Toxicology." Vol. 4. New York: Hemisphere
Publishing Corporation.
4. NAS. 1978. National Academy of Sciences.
Priniciples and Procedures for Evaluating the Toxicity
of Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, Under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
5. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
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HG-Organ/Tissue-Eye Irrit
August, 1982
PRIMARY EYE IRRITATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Organ/Tissue-Eye Irrit
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of a substance, determination of the
irritant and/or corrosive effects on eyes of mammals is an
important initial step. Information derived from this test
serves to indicate the existence of possible hazards likely
to arise from exposure of the eyes and associated mucous
membranes to the test substance.
II. DEFINITIONS
A. Eye irritation
The production of reversible changes in the eye
following the application of a test substance to the
anterior surface of the eye.
B. Eye corrosion
The production of irreversible tissue damage in the
eye1 following application of a test substance to the
anterior surface of the eye.
III. PRINCIPLE OF THE TEST METHOD
A. The substance to be tested is applied in a single dose
to one of the eyes in each of several experimental
animals; the untreated eye is used to provide control
information. The degree of irritation/corrosion is
evaluated and scored at specified intervals and is
fully described to provide a complete evaluation of
the effects. The duration of the study should be
sufficient to permit a full evaluation of the
reversibility or irreversibility of the effects
observed but need not exceed 21 days.
B. Strongly acidic or alkaline substances, for example,
with a demonstrated pH of 2 or less, or 11.5 or
greater, need not be tested owing to their predictable
corrosive properties.
C. Materials which have demonstrated definite corrosion
or severe irritation in a dermal study need not be
further tested for eye irritation. It may be presumed
that such substances will produce similarly severe
effects in the eyes.
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HG-Organ/Tissue-Eye Irrit
IV. TEST PROCEDURES
A. Animal selection
1. Species and strain
A variety of experimental animals have been
used, but it is recommended that testing should
be performed using healthy adult albino
rabbits. Commonly used laboratory strains
should be used. If another mammalian species is
used, the tester should provide
justification/reasoning for its selection.
2. Number of. animals
At least 6 animals should be used, unless
justification/reasoning for using fewer animals
is provided.
B. Dose level
For testing liquids, a dose of 0.1 ml is
recommended. In testing solids, pastes, and
particulate substances, the amount used should have a
volume of 0.1 ml, or a weight of not more than 100 mg
(the weight must always be recorded). If the test
material is solid or granular, it should be ground to
a fine dust. The volume of particulates should be
measured after gently compacting them (e.g. by tapping
the measuring container). To test a substance
contained in a pressurized aerosol container, the eye
should be held open and the test substance
administered in a single burst of about one second
from a distance of 10 cm directly in front of the
eye. The dose may be estimated by weighing the
container before and after use. Care should be taken
not to damage the eye. Pump sprays should not be used
but instead the liquid should be expelled and 0.1 ml
collected and instilled into the eye as described for
liquids.
C. Examination of eyes prior to test
Both eyes of each experimental animal provisionally
selected for testing should be examined within 24
hours before testing starts by the same procedure to
be used during the test examination. Animals showing
eye irritation, ocular defects or pre-existing corneal
injury should not be used.
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HG-Organ/Tissue-Eye Irrit
D. Application of the test substance
1. The test substance should be placed in the
conjunctival sac of one eye of each animal after
gently pulling the lower lid away from the
eyeball. The lids are then gently held together
for about one second in order to limit loss of
the material. The other eye, which remains
untreated, serves as a control. If it is
thought that the substance may cause extreme
pain, local anesthetic may be used prior to
instillation of the test substance. The type
and concentration of the local anesthetic should
be carefully selected to ensure that no
significant differences in reaction to the test
substance will result from its use. The control
eye should be similarly anesthetized.
2. The eyes of the test animals should not be
washed out for 24 hours following instillation
of the test substance. At 24 hours, a washout
may be used if considered appropriate.
E. Observation period
The duration of the observation period is at least 72
hours, but should not be fixed rigidly. It should be
sufficient to evaluate fully the reversibility or
irreversibility of the effects observed. It normally
need not exceed 21 days after instillation.
F. Clinical examination and scoring
1. The eyes should be examined at 1, 24, 48, and 72
hours. If there is no evidence of irritation at
72 hours, the study may be ended. Extended
observation may be necessary if there is
persistent corneal involvement or other ocular
irritation in order to determine the progress of
the lesions and their reversibility or
irreversibility. In addition to the
observations of the cornea, iris and
conjunctivae, any other lesions which are noted
should be recorded and reported. The grades of
ocular reaction using Tabl"e I should be recorded
at each examination.
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HG-Organ/Tissue-Eye Irrit
Table I: Grades for Ocular Lesions
Cornea
Opacity: degree of density (area most dense
taken for reading). No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than
slight dulling of normal luster), details of
iris clearly visible 1*
Easily discernible translucent area, details
of iris slightly obscured 2*
Nacrous area, no details or iris visible,
size of pupil barely discernible 3*
Opaque cornea, iris not discernible through
the opacity 4*
Iris
Normal 0
Markedly deepened rugae, congestion, swelling
moderate circumcorneal hyperemia, or injection,
any of these or combination of any thereof, iris still
reacting to light (sluggish reaction is positive). ... 1*
No reaction to light, hemorrhage, gross destruction
(any or all of these) 2*
Conjunctivae
Redness (refers to palpebral and bulbar
conjunctivae, cornea and iris).
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) ... 1
Diffuse, crimson color, individual vessels not
easily discernible 2*
Diffuse beefy red chemosis: lids and/or
nictitating membranes 3*
No swelling 0
Any swelling above normal (includes nictitating
membranes) 1
Obvious swelling with partial eversion of lids 2*
Swelling with lids about half closed 3*
Swelling with lids more than half closed 4*
*Starred figures indicate positive effect
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HG-Organ/Tissue-Eye Irrit
2. Examination of reactions can be facilitated by
use of a binocular loupe, hand slit-lamp,
biomicroscope, or other suitable device. After
recording the observations at 24 hours, the eyes
of any or all rabbits may be further examined
with the aid of fluorescein.
3. The grading of ocular responses is subject to
various interpretations. To promote
harmonization and to assist testing laboratories
and those involved in making and interpreting
the observations, an illustrated guide in
grading eye irritation should be used. (Such an
illustrated guide is in use in the United States
and can be obtained form the Consumer Product
Safety Commission, Washington, D.C. 20207)
V. DATA AND REPORTING
A. Data should be summarized in tabular form, showing for
each individual animal the irritation scores at the
designated observation time; a description of the
degree and nature of irritation; the presence of
serious lesions and any effects other than ocular
which were observed.
B. Evaluation of the results
The ocular irritation scores should be evaluated in
conjunction with the nature and reversiblity or
otherwise of the responses observed. The individual
scores do not represent an absolute standard for the
irritant properties of a material. They should be
viewed as reference values and are only meaningful
when supported by a full description and evaluation of
the observations.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Physical nature and, where appropriate,
concentration and pH value for the test
substance;
2. Species and strain;
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HG-Organ/Tissue-Eye Irrit
3. Tabulation of irritant/corrosive reponse data
for each individual animal at each observation
time point (e.g. 1, 24, 48, and 72 hours);
4. Description of any lesions observed;
5. Narrative description of the degree and nature
of irritation or corrosion observed;
6. Description of the method used to score the
irritation at 1, 24, 48 and 72 hours (e.g. hand
slit-lamp, biomicroscope, fluorescein); and
7. Description of any non-ocular effects noted.
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HG-Organ/Tissue-Eye Irrit
VI. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Buehler, E.V., Newmann, E.A. 1964. A Comparison of
Eye Irritation in Monkeys and Rabbits. Toxicology and
Applied Pharmacology. 6:701-710.
2. Draize, J.H. 1959. Third Printing 1975. "Dermal
Toxicity," in "Appraisal of the Safety of Chemicals in
Foods, Drugs and Cosmetics." The Association of Food
and Drug Officials of the United States. PP. 49-52.
3. Draize, J.H., Woodward, G., Calvery, H.O. 1944.
Methods for the Study of Irritation and Toxicity of
Substances Applied Topically to the Skin and Mucous
Membranes. Journal of Pharmacology and Experimental
Therapeutics. 83:377-390.
4. Loomis, T.A. 1974. Essentials of Toxicology. Second
Edition. Philadelphia: Lea and Febicer. pp. 207-
213.
5. NAS. 1977. National Academy of Sciences. Principles
and Procedures for Evaluating the Toxicity of
Household Substances. Washington, D.C.: A report
prepared by the Committee for the revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
6. United States Federal Hazardous Substances Act
Regulations. Title 16, Code of Federal Regulations.
38 FR 27012, Sept. 27, 1973; 38 FR 30105, Nov. 1,
1973.
7. WHO. 1978. World Health Organization. Principles
and Methods for Evaluating the Toxicity of
Chemicals. Part I. Environmental Health Criteria
6. Geneva: World Health Organization. 272 pp.
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HG-Organ/Tissue-Repro/Fert
August, 1982
REPRODUCTION AND FERTILITY EFFECTS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Organ/Tissue-Repro/Fert
I. PURPOSE
This guideline for two-generation reproduction testing is
designed to provide general information concerning the
effects of a test substance on gonadal function, conception,
parturition, and the growth and development of the
offspring. The study may also provide information about the
effects of the test substance on neonatal morbidity,
mortality, and preliminary data on teratogenesis and serve
as a guide for subsequent tests.
II. PRINCIPLE OF THE TEST METHOD
The test substance is administered to parental (P^) animals
prior to their mating, during the resultant pregnancies, and
through the weaning of their F-^ offspring. The substance is
then administered to selected FI offspring during their
growth into adulthood, mating, and production of an F2
generation, up until the F2 generation .is 21 days old.
III. TEST PROCEDURES
A. Animal selection
1. Species and strain
The rat is the preferred species. If another
mammalian species is used, the tester should
provide justification/reasoning for its
selection. Strains with low fecundity should
not be used.
2. Age
Parental (P^ animals should be about 8 weeks
old at the start of dosing.
3. Sex
a. For an adequate assessment of fertility,
both males and females should be studied.
b. The females should be nulliparous and non-
pregnant.
4. Number of animals
Each test and control group should contain at
least 20 males and a sufficient number of
females to yield at least 20 pregnant females at
or near term.
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HG-Organ/Tissue-Repro/Fert
B. Control groups
1. A concurrent control group is recommended. This
group should be an untreated or sham treated
control group or if a vehicle is used in
administering the test substance, a vehicle
control group.
2. If a vehicle is used in administering the test
substance, the control group should receive the
vehicle in the highest volume used.
3. If a vehicle or other additive is used to
facilitate dosing, it should not interfer with
absorption of the test substance or produce
toxic effects.
-.*'
C. Dose levels and dose selection
1. At least three dose levels and a concurrent
control should be used.
2. The highest dose level should induce toxicity
but not mortality in the parental (PI) animals.
3. The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest dose
should exceed this.
4. The intermediate dose level(s) should produce
minimal observable toxic effects. If more than
one intermediate dose is used, dose levels
should be spaced to produce a gradation of toxic
effects.
5. The incidence of fatalities in low and
intermediate dose groups and in the controls
should be low to permit meaningful evaluation of
the results.
D. Exposure conditions
The animals should be dosed with the test substance,
ideally, on a seven-days per week basis using the
testing schedule presented in Table I.
1. Table I contains the dosing, mating, delivery,
and sacrifice schedule for animals on test.
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HG-Organ/Tissue-Repro/Pert
a. Daily dosing of the parental (Pi) males
and females should begin when they are
about 8 weeks old. For both sexes, dosing
should be continued for at least eight
weeks before the mating period.
b. Dosing of P^ males should continue through
the three week mating period. At the end
of the mating period, P ± males should be
sacrificed and examined. Dosing of the FI
males saved for mating should continue
from the time they are weaned through the
period they are mated with the F-^ females
(11 weeks). F^ males may be sacrificed
after the F1 mating period.
c. Daily dosing of the PI females should
continue through the three week mating
period, pregnancy, and to the weaning of
the F^ offspring at three weeks after
delivery. Dosing of the FI females saved
for mating should continue from the time
they are weaned, through the period they
are mated with the F1 males (11 weeks),
pregnancy, and to the weaning of the F2
offspring.
2. All animals are sacrificed as scheduled.
a. All Pi males should be sacrificed at the
end of the three week mating period.
b. FI males selected for mating should be
sacrificed at the end of the three week
period of the F^ mating.
c. F! males and females not selected for
mating should be sacrificed when weaned.
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HG-Organ/Tissue-Repro/Fert
Table 1. Approximate Dosing and Breeding Schedule
Weeks
on
Study
0
8-10
11-14
Pl
Fl F2
Dosing of Pi male
and females begin.
P-^ mating
Dosing of
period.
P, males F-, born and litter sizes
14-17
ends at week 23.
Sacrifice P-i males.
Dosing of P-^
females ends.
P-^ females are
sacrificed.
randomly adjusted to 8
pups each.
F-, weaned; Dosing of F-,
females beings.
F-, offspring not selected
for mating are sacrificed,
25-28
F^ mating; Dosing of Fj
males ends at week 36.
FT males are sacrificed
28-31
Remaining
are
Fl
sacrificed.
females
?2 born and litter
sizes randomly
adjusted to 8 pups
each.
Fj offspring
sacrificed.
are
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HG-Organ/Tissue-Repro/Pert
d. The parental females should be sacrificed
upon weaning of their F^ offspring.
e. FI dams and their F2 offspring are
sacrificed when the offspring are 21 days
of age.
E. Observation period
Duration of observation should be for at least 28
weeks from dosing of PI animals to sacrifice of F2
offspring at weaning.
F. Administration of the test substance
1. Oral studies
a. When administered by gavage or capsule,
the dosage administered to each animal
prior to mating should be based on the
individual animal's body weight and
adjusted weekly. During pregnancy the
dosage should be based on the body weight
at Day 0 and 6 of pregnancy.
b. It is recommended that the test substance
be administered in the diet or drinking
water.
c. If the test substance is administered in
the drinking water, or mixed in the diet,
exposure is continuous.
d. For a diet mixture, the highest
concentration should not exceed five
percent.
2. If the dermal or the inhalation route of
administration is used, the tester should
provide justification and reasoning for its
selection.
G. Mating procedure
1. Parental
a. For each mating, each female should be
placed with a single male from the same
dose level until pregnancy occurs or three
weeks have elapsed. Paired matings should
be clearly identified and mixed matings
with other males avoided.
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HG-Organ/Tissue-Repro/Pert
b. Those pairs that fail to mate should be
evaluated to determine the cause of the
apparent infertility. This may involve
such procedures as additional
opportunities to mate with proven fertile
males or females, histological examination
of the reproductive organs, and
examination of the estrus or spermatogenic
cycles.
c. Each day, the females should be examined
for presence of sperm or vaginal plugs.
Day 0 of pregnancy is defined as the day
vaginal plugs or sperm are found.
2. FT cross
a. For mating the FX offspring, one male and
one female are randomly selected from each
litter for cross mating with another pup
of the same dose level at weaning, but
different litter, to produce the F2
generation.
b. F-^ males and females not selected for
mating are sacrificed upon weaning.
3. Special housing
Near parturition, pregnant animals should be
caged separately in delivery or maternity cages
and provided with nesting materials.
4. Standardization of litter sizes
a. On day 4 after birth, the size of each
litter should be adjusted by eliminating
extra pups by random selection to yield,
as nearly as possible, 4 males and 4
females per litter.
b. Whenever the number of male or female pups
prevents having 4 of each sex per litter,
partial adjustment (for example, 5 males
and 3 females) is permitted. Adjustments
are not appropriate for litters of less
than 8 pups.
c. Elimination of runts only is not
appropriate.
d. Adjustments of the F2 litters is conducted
in the same manner.
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HG-Organ/Tissue-Repro/Fert
H. Observation of animals
1. A careful clinical examination should be made at
least once each day. Pertinent behavioral
changes, signs of difficult or prolonged
parturition, food consumption and all signs of
toxicity, including mortality, should be
recorded. These observations should be reported
for each individual animal.
2. The duration of gestation should be calculated
from Day 0 of pregnancy.
3. Each litter should be examined as soon as
possible after delivery for the number of pups,
stillbirths, live births, and the presence of
gross anomalies. Dead pups and pups sacrificed
at day 4 should be preserved and studied for
possible defects and cause of death. Live pups
should be counted and litters weighed, by
weighing each individual pup at birth, or soon
thereafter, and on days 4, 7, 14 and 21 after
parturition.
4. Physical or behavioral abnormalities observed in
the dams or offspring should be recorded.
5. Pi males and females should be weighed on the
first day of dosing and weekly thereafter. FI
litters should be weighed at birth, or soon
thereafter, and on days 4, 7, 14 and 21. In all
cases, litter weights should be calculated from
the weights of the individual pups.
I. Gross necropsy
1. A complete gross examination should be performed
on all animals, including those which died
during the experiment or were killed in moribund
conditions.
2. Special attention should be directed to the
organs of the reproductive system.
3. The following organs and tissues, or
representative samples thereof, should be
preserved in a suitable medium for possible
future histopathological examination: vagina;
uterus; ovaries; testes; epididymus; seminal
vesicles; prostate; and, target organ(s) of all
P! and FI animals selected for mating.
_ *7 —
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HG-Organ/Tissue-Repro/Fert
J. Histopathology
1. The following histopathology should be
performed:
a. Full histopathology on the organs listed
above for all high dose, and control PI
and F-^ animals selected for mating.
b. Organs demonstrating pathology in these
animals should then be examined in animals
from the other dose groups.
c. Microscopic examination should be made of
all tissues showing gross pathological
changes.
IV. DATA AND REPORTING
A. Treatment of Results
Data should be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, the number of animals pregnant, the types of
change and the precentage of animals displaying each
type of change.
B. Evaluation of study results
1. An evaluation of test results, including the
statistical analysis, based on the clinical
findings, the gross necropsy findings, and the
microscopic results, should be made and
supplied. This should include an evaluation of
the relationship, or lack thereof, between the
animals' exposure to the test substance and the
incidence and severity of all abnormalities.
2. In any study which demonstrates an absence of
toxic effects, further investigation to
establish absorption and bioavaliability of the
test substance should be considered.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Toxic response data by sex and dose, including
fertility indices, length of gestation;
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HG-Organ/Tissue-Repro/Pert
2. Species and strain;
3. Time of death during the study or whether
animals survived to termination;
4. Toxic or other effects on reproduction,
offspring, or postnatal growth;
5. Time of observation of each abnormal sign and
its subsequent course;
6. Body weight data for Plr Flf and F2 animals;
7. Necropsy findings;
8. Detailed description of all histopathological
findings; and
9. Statistical treatment of results where
appropriate.
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HG-Organ/Tissue-Repro/Fert
V. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Clermont, Y., Perry, B. 1957. Quantitative Study of
the Cell Population of the Seminiferous Tubules in
Immature Rats. American Journal of Anatomy. 100:241-
267.
2. Goldenthal, E.I. 1966. Guidelines for Reproduction
Studies for Safety Evaluation of Drugs for Human
Use. Washington, D.C.: Drug Review Branch, Division
of Toxicological Evaluation, Bureau of Science, Food
and Drug Administration.
3. Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson,
I.W. 1973. Fertility and Sterility. New York:
American Elsevier Publishing Co., Inc.
4. Oakberg, E.F. 1956. Duration of Spermatogenesis in
the Mouse and Timing of Stages of the Cycle of the
Seminiferous Epithelium. American Journal of
Anatomy. 9:507-516.
5. Roosen-Runge, E.G. 1962. The Process of
Spermatogenesis in Mammals. Biological Review.
37:343-377.
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HG-Organ/Tissue-Terato
August, 1982
TERATOGENICITY STUDY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Organ/Tissue-Terato
I. PURPOSE
The teratogenicity study is designed to determine the
potential of the test substance to induce structural and/or
other abnormalities in the fetus which may arise from
exposure of the mother during pregnancy.
II. DEFINITIONS
A. Teratogenicity is the property of a chemical that
causes permanent structural or functional
abnormalities during the period of embryonic
development.
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered in graduated doses, for
at least that part of the pregnancy covering the period of
organogenesis, to several groups of pregnant experimental
animals, one dose level being used per group. Shortly
before the expected date of delivery, the pregnant females
are sacrificed, the uteri removed, and the contents examined
for embryonic or fetal deaths, and live fetuses.
IV. LIMIT TEST
If a test at a dose of at least 1000 mg/kg body weight,
using the procedures described for this study, produces no
observable embryo toxicity or teratogenicity, then a full
study using three dose levels might not be necessary.
V. TEST PROCEDURES
A. Animal selection
1. Species and strain
Testing should be performed in at least 2
mammalian species. The preferred species are
the rat and the rabbit. If other mammalian
species are used, the tester should provide
justification/reasoning for their selection.
Commonly used laboratory strains should be
employed. The strain should not have low
fecundity and should preferably be characterized
for its sensitivity to teratogens.
2. Age
Young adult animals should be used.
-1-
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HG-Organ/Tissue-Terato
3. Sex
Pregnant female animals should be used at each
dose level.
4. Number of animals
At least 20 pregnant rats, mice or hamsters or
12 pregnant rabbits are recommended at each dose
level. The objective is to ensure that
sufficient pups are produced to permit
meaningful evaluation of the teratogenic
potential of the test substance.
B. Control group
A concurrent control group is recommended. This group
should be an untreated or sham treated control group,
or, if a vehicle is used in administering the test
substance, a vehicle control group. Except for
treatment with the test substance, animals in the
control group(s) should be handled in an identical
manner to test group animals.
C. Dose levels and dose selection
1. At least 3 dose levels with a control and, where
appropriate, a vehicle control, should be used.
2. If a vehicle is used, its toxicological
properties should be characterized. The vehicle
should neither be teratogenic nor have effects
on reproduction.
3. To select the appropriate dose levels, a pilot
or trial study may be advisable. It is not
always necessary to carry out a trial study in
pregnant animals. Comparison of the results
from a trial study in non-pregnant, and the main
study in pregnant animals will demonstrate if
the test substance is more toxic in pregnant
animals. If a trial study is carried out in
pregnant animals, the dose producing embryonic
or fetal lethalities should be determined.
4. Unless limited by the physical/chemical nature
or biological properties of the substance, the
highest dosage level should induce some overt
maternal toxicity such as slight weight loss,
but not more than 10 percent maternal deaths.
-2-
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HG-Organ/Tissue-Terato
5. The lowest dose level should not produce any
evidence of maternal toxicity. Where there is a
usable estimation of human exposure the lowest
level should not exceed this.
6. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If
more than one intermediate dose is used, the
dose levels should be spaced to produce a
gradation of toxic effects.
7. In the low and intermediate dose groups and in
the control groups, incidence of fatalities
should be low, to permit a meaningful evaluation
of the results.
D. Observation period
Day 0 in the test is the day on which a vaginal plug
and/or sperm are observed. The dose period should
cover the period of major organogenesis. This may be
taken as days 6-15 for rat and mouse, 6-14 for
hamster, or 6-18 for rabbit.
E. Administration of test substance
The test substance or vehicle is usually administered
orally, by oral intubation unless the chemical or
physical characteristics of the test substance or
pattern of human exposure suggest a more appropriate
route of administration.
F. Exposure conditions
The female test animals are treated with the test
substance daily throughout the appropriate treatment
period. When given by gavage, the dose may be based
on the weight of the females at the start of substance
administration, or, alternatively, in view of the
rapid weight gain which takes place during pregnancy,
the animals may be weighed periodically and the dosage
based on the most recent weight determination.
G. Observation of animals
1. A careful clinical examination should be made at
least once each day.
2. Additional observations should be made daily
with appropriate actions taken to minimize loss
of animals to the study (e.g., necropsy or
refrigeration of those animals found dead and
isolation or sacrifice of weak or moribund
animals).
-3-
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HG-Organ/Ti ssue-Terato
3. Signs of toxicity should be recorded as they are
observed, including the time of onset, the
degree and duration.
4. During the treatment and observation periods,
cage-side observations should include, but not
be limited to: changes in skin and fur, eye and
mucous membranes, as well as respiratory,
circulatory, autonomic and central nervous
systems, somatomotor activity and behavioral
pattern.
5. Measurements should be made weekly of food
consumption for those animals in a dosed-feeding
study.
6. Animals should be weighed at least weekly.
7. Females showing signs of abortion or permature
delivery should be sacrificed and subjected to a
thorough macroscopic examination.
H. Gross necropsy
1. At the time of sacrifice or death during the
study, the dam should be examined
macroscopically for any structural abnormalities
or pathological changes which may have
influenced the pregnancy.
2. Immediately after sacrifice or death, the uterus
should be removed and the contents examined for
embryonic or fetal deaths and the number of
viable fetuses. It is usually possible to
estimate the time of death in utero where this
has occurred.
3. The number of corpora lutea should be
determined.
4. The sex of the fetuses should be determined and
they should be weighed individually, the weights
recorded, and the mean fetal weight derived.
5. Following removal, each fetus should be examined
externally.
-4-
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HG-Organ/Tissue-Terato
VII. REFERENCES
The following references may be helpful in developing
acceptable procotols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. DHEW. 1975. Department of Health and Welfare. The
Testing of Chemicals for Carcinogenicity, Mutagenicity
and Teratogenicity. Canada: The Honorable Marc
Lalonde, Minister of Health and Welfare, Department of
Health and Welfare. 183 pp.
2. NAS. 1977. National Academy of Sciences. Principles
and Procedures for Evaluating the Toxicity of
Household Substances. Washington, D.C.: A report
prepared by the Committee for the Revision of NAS
Publication 1138, under the auspices of the Committee
on Toxicology, National Research Council, National
Academy of Sciences. 130 pp.
3. WHO. 1967. World Health Organization. Principles
for the Testing of Drugs for Teratogenicity. WHO
Technical Report Series No. 364. Geneva: World
Health Organization. 18 pp.
-7-
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HG-Organ/Tissue-Terato
6. For rats, mice and hamsters, one-third to one-
half of each litter should be prepared and
examined for skeletal anomalies, and the
remaining part of each litter should be prepared
and examined for soft tissue anomalies using
appropriate methods.
7. For rabbits, each fetus should be examined by
careful dissection for visceral anomalies and
then examined for skeletal anomalies.
VI. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form, showing for
each test group; the number of animals at the start of
the test, the number of pregnant animals, the number
and percentages of live fetuses and the number of
fetuses with any soft tissue or skeletal
abnormalities.
B. Evaluation of results
The findings of a teratogenicity study should be
evaluated in terms of the observed effects and the
dose levels producing effects. It is necessary to
consider the historical teratogenicity data on the
species/strain tested. A properly conducted
teratogenicity study should provide a satisfactory
estimation of a no-effect level.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Toxic response data by dose;
2. Species and strain;
3. Time of death during the study or whether
animals survived to termination;
4. Time of observation of each abnormal sign and
its subsequent course;
-5-
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HG-Organ/Tissue-Terato
5. Food and body weight data;
6. Pregnancy and litter data; and
7. Fetal data (live/dead, sex, soft tissue and
skeletal defects, resorptions).
-6-
-------
III. MUTAGENICITY
-------
HG-Gene Muta-S_._ typhimurium
August, 1982
THE SALMONELLA TYPHIMURIUM REVERSE
MUTATION ASSAY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Gene Muta-S_._ typhimurium
I. PURPOSE
The Salmonella typhimurium histidine (his) reversion system
is a microbial assay which measures his~ ) his reversion
induced by chemicals which cause base changes or frameshift
mutations in the genome of this organism.
II. DEFINITIONS
A. A reverse mutation assay in Salmonella typhimurium
detects mutation in a gene of a histidine requiring
strain to produce a histidine independent strain of this
organism.
B. Base pair mutagens are agents which cause a base change
in the DNA. In a reversion assay, this change may occur
at the site of the original mutation or at a second site
in the chromosome.
C. Frameshift mutagens are agents which cause the addition
or deletion of single or multiple base pairs in the DNA
molecule.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, sodium azide,
2-nitrofluorene, 9-aminoacridine or 2-aminoanthracene.
IV. TEST METHOD
A. Principle
Bacteria are exposed to test chemical with and without a
metabolic activation system and plated onto minimal
medium. After a suitable period of incubation,
revertant colonies are counted and compared to the
number of spontaneous revertants in an untreated and/or
vehicle control culture.
B. Description
Several methods for performing the test have been
described. Among those used are:
1. the direct plate incorporation method,
2. the preincubation method,
3. the suspension method, and
4. the gradient plate method.
-------
HG-Gene Muta-S. typhimurium
The procedure described here is for the direct plate
incorporation method.
C. Strain selection
1. Designation
At the present time four strains, TA 1535, TA 1537,
TA 98 and TA 100 should be used. The use of strain
TA 1538 is left to the discretion of the
investigator. Other strains may be utilized when
appropriate.
2. Preparation and storage
Recognized methods of stock culture preparation and
storage should be used. The requirement of
histidine for growth should be demonstrated for
each strain. Other phenotypic characteristics
should be checked using such methods as crystal
violet sensitivity and resistance to ampicillin.
Spontaneous reversion frequency should be in the
range expected either as reported in the literature
or as established in the laboratory by historical
control values.
3. Bacterial growth
Fresh cultures of bactertia should be grown up to
the late exponential or early stationary phase of
growth (approximately 10-lO^cells per ml).
D< Metabolic acti. vatjloji
Bacteria should be exposed to the test substance both in
the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducing agents. The use of other species, tissues or
techniques may also be appropriate.
E. Con t ro 1 g :roups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls should be included in each
experiment. Positive controls should insure both
strain responsiveness and efficacy of the metabolic
activation system.
-------
HG-Gene Muta-jS^ typhimurium
2. Strain specific positive controls
Strain specific positive controls should be
included in the assay. Examples of strain specific
positive controls are as follows:
a. Strain TA 1535, TA 100, sodium azide;
b. TA 98, 2-nitrofluorene;
c. TA 1537, 9-aminoacridine.
3. Positive controls to ensure the efficacy
of the activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test. 2-Aminoanthracene is an
example of a positive control compound in tests
using postmitochondrial fractions from the livers
of rodents treated with enzyme inducing agents such
as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may be
used.
F. Test chemicals
1. 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.
2. Exposure concentrations
a. The test should initially be performed over a
broad range of concentrations. When
appropriate, a positive response should be
confirmed by testing over a narrow range of
concentrations. Among the criteria to be
taken into consideration for determining the
upper limits of test chemical concentration
are cytotoxicity and solubility. Cytotoxicity
of the test chemical may be altered in the
presence of metabolic activation systems.
-------
HG-Gene Muta-j3_._ typhimurium
Toxicity may be evidenced by a reduction in
the number of spontaneous revertants, a
clearing of the background lawn or by the
degree of survival of treated cultures.
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.
b. Generally, a maximum of 5 mg/plate for pure
substances is considered acceptable. At least
5 different amounts of test substance should
be tested with adequate intervals between test
points.
V. TEST PERFORMANCE
A.. Direct plate incorporation method
For this test without metabolic activation, test
chemical and 0.1 ml of a fresh bacterial culture should
be added to 2.0 ml of overlay agar. For tests with
metabolic activation, 0.5 ml of activation mixture
containing an adequate amount of postmitochondrial
fraction should be added to the agar overlay after the
addition of test chemical and bacteria. Contents of
each tube should be mixed and poured over the surface of
a selective agar plate. Overlay agar should be allowed
to solidify before incubation. At the end of the
incubation period, revertant colonies per plate should
be counted.
B. Other methods
Other methods may also be appropriate.
C. Media
An appropriate selective medium with an adequate overlay
agar should be used.
D. Incubation conditions
All plates within a given experiment should be incubated
for the same time period. This incubation period should
be for 48-72 hours at 37 C.
-------
HG-Gene Muta-j3_._ typhimurium
Number of cultures
All plating should be done at least in duplicate. All
results should be confirmed in an independent
experiment.
VI. DATA AND REPORT
A. Treatment of results
Data should be presented as number of revertant colonies
per plate for each replicate and dose. The numbers of
revertant colonies on both negative (untreated and/or
vehicle) and positive control plates should also be
presented. Individual plate counts, the mean number of
revertant colonies per plate and standard deviation
should be presented for test chemical and positive and
negative (untreated and/or vehicle) controls.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating the results of this test. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
revertants. Another criterion, may be based upon
detection of a reproducible and statistically
significant positive response for at least one of
the test substance concentrations. However, the
final decision must be based upon good scientific
judgement.
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of revertants nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
-------
HG-Gene Muta-S_._ typhimurium
D. Test eyaluation
1. Positive results from the S_. typhimurium reverse
mutation assay indicate that the test substance
induces point mutations by base changes or
frameshifts in the genome of this organism.
2. Negative results indicate that under the test
conditions the test substance is not mutagenic in
S. typhimurium.
E. Test report
The test report should include the following
information:
1. bacterial strain used;
2. details of the protocol used for metabolic
activation;
3. dose levels and rationale for selection of dose;
4. positive and negative controls;
5. individual plate counts, mean number of revertant
colonies per plate, standard deviation;
6. dose-response relationship, if applicable;
7. statistical evaluation;
8. discussion of results; and
9. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
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HG-Gene Muta-S. typhimurium
2. de Serres FJ, Shelby MD. 1979. The Salmonella
mutagenicity assay: recommendations. Science 203:563-
565.
3. McMahon RE, Clive JC, Thompson CZ. 1979. Assay of 855
test chemicals in ten tester strains using a new
modification of the Ames test for bacterial mutagens.
Cancer Res 39:682-693.
4. Thompson ED, Melampy PJ. 1981. An examination of the
quantitative suspension assay for mutagenesis with
strains of Salmonella typhimurium. Environmental
Mutagenesis 3:453-465.
5. Vogel HJ, Bonner DM. 1956. Acetylornithinase of E.
cpli; partial purification and some properties. J Biol
Chem 218:97-106.
-------
HG-Gene Muta-E_._ coli
August, 1982
THE ESCHERICHIA COLI WP2 AND
WP2 UVrA REVERSE MUTATION ASSAYS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Gene Muta-E. coli
PURPOSE
The J2_. coli tryptophan (trp) reversion system is a microbial
assay which measures trp~ >trp reversion induced by
chemicals which cause mutations in the genome of this
organism.
II. DEFINITION
A reverse mutation assay in E_. coli detects mutation in a
gene of a tryptophan requiring strain to produce a
tryptophan independent strain of this organism.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to,
4-nitroquinoline oxide, methyl methanesulfonate, or
2-aminoanthracene.
IV. TEST METHOD
A. Principle
Bacteria are exposed to test chemical with and without
metabolic activation and plated onto minimal medium.
After a suitable period of incubation, revertant
colonies are counted and compared to the number of
spontaneous revertants in an untreated and/or vehicle
control culture.
B. Description
Several methods for performing the test have been
described. Among those used are:
1. the direct plate incorporation method,
2. the preincubation method,
3. the treat and plate method, and
4. the modified fluctuation test.
The procedure described here is for the direct plate
incorporation method.
-------
HG-Gene Muta-E_._ coli
C. Strain selection
1. Designation
At the present time, three strains, WP2, WP2 uvrA
and WP2 uyrA/pKMlOl should be used. Other strains
may be utilized when appropriate.
2. Preparation and storage
Recognized methods of stock culture preparation and
storage should be used. The requirement of
tryptophan for growth should be demonstrated for
each strain. Other phenotypic characteristics
should be checked using such methods as sensitivity
to mitomycin C and resistance to ampicillin.
Spontaneous reversion frequency should be in the
range expected either as reported in the literature
or as established in the laboratory by historical
control values.
3. Bacterial growth
Fresh cultures of bacteria should be grown up to
the late exponential or early stationary phase of
growth (approximately 108-109 cells per ml).
D. Metabolic activation
Bacteria should be exposed to the test substance both in
the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducing agents. The use of other species, tissues or
techniques may also be appropriate.
E. Control groups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls should be included in each
experiment.
2. Direct acting positive controls
Examples of positive controls for assays performed
without metabolic activation include methyl
methanesulfonate and 4-nitroquinoline oxide.
-------
HG-Gene Muta-E. coli
3. Positive controIs to ens ure t he e ff i ca cy of the
act i v a 11 o ri^ s y sit em
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test. 2-aminoanthracene is an
example of a positive control compound in tests
using postmitochondrial fractions from the livers
of rodents treated with enzyme inducing agents such
as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may be
used.
F. Test chemicals
1. 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.
2. Exposure concentrations
a. The test should initially be performed over a
broad range of concentrations. When
appropriate, a positive response should be
confirmed by testing over a narrow range of
concentrations. Among the criteria to be
taken into consideration for determining the
upper limits of test chemical concentration
are cytotoxicity and solubility. Cytotoxicity
of the test chemical may be altered in the
presence of metabolic activation systems.
Toxicity may be evidenced by a reduction in
the number of spontaneous revertants, a
clearing of the background lawn or by the
degree of survival of treated 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.
-------
HG-Gene Muta-E. coli
Generally, a maximum of 5 ing/plate for pure
substances is considered acceptable. At least
5 different amounts of test substance should
be tested with adequate intervals between the
test points.
V. TEST PERFORMANCE
A. Direct plate incorporation method
For this test without metabolic activation, test
chemical and 0.1 ml of a fresh bacterial culture should
be added to 2.0 ml of overlay agar. For tests with
metabolic activation, 0.5 ml of activation mixture
containing an adequate amount of postmitochondrial
fraction should be added to the overlay agar after the
addition of test chemical and bacteria. Contents of
each tube should be mixed and poured over the surface of
a selective agar plate. Overlay agar should be allowed
to solidify before incubation. At the end of the
incubation period, revertant colonies per plate should
be counted.
B. Other methods
Other methods may also be appropriate.
C. Media
An appropriate selective medium with an adequate overlay
agar should be used.
D. Incubation conditions
All plates in a given experiment should be incubated for
the same time period. This incubation period should be
for 48-72 hours at 37 C.
E. Number of cultures
All plating should be done at least in duplicate. All
results should be confirmed in an independent
experiment.
-------
HG-Gene Muta-E. coli
VI. DATA AND REPORT
A. Treatment of results
Data should be presented as number of revertant colonies
per plate for each replicate and dose. The numbers of
revertant colonies on both negative (untreated and/or
vehicle) and positive control plates should also be
presented. Individual plate counts, the mean number of
revertant colonies per plate and standard deviation
should be presented for test chemical and positive and
negative (untreated and/or vehicle) controls.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating the results of this test. . Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
revertants. Another criterion may be based upon
detection of a reproducible and statistically
significant positive response for at least one of
the test substance concentrations. However, the
final decision must be based upon good scientific
judgement.
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of revertants nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
D. Test evaluation
1. Positive results from the E. coli reverse mutation
assay indicate that the test substance induces
mutations in the genome of this organism.
2. Negative results indicate that under the test
conditions the test substance is not mutagenic in
E. coli.
-------
HG-Gene Muta-E. coli
3. The E. coli reverse mutation assay may be
espe"cTally suited to testing some classes of
chemicals such as hydrazines, nitrofurans and
nitrosamines.
E* Test report
The test report should include the following
information:
1. bacterial strain used;
2. details of the protocol used for metabolic
activation,
3. dose levels and rationale for selection of dose;
4. positive and negative controls;
5. individual plate counts, mean number of revertant
colonies per plate, standard deviation;
6. dose-response relationship, if applicable;
7. statistical evaluation;
8. discussion of the results; and
9. interpretation of the results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Brusick DJ, Simmon VF, Rosenkranz HS, Ray VA, Stafford
RS. 1980. An evaluation of the Escherichia coli WP2
and WP2 uvrA reverse mutation assay.Mutation Research
76:169-190.
3. Green MHL, Muriel WJ. 1976. Mutagen testing using trp+
in Escherichia coli. Mutation Research 38:3-32.
-------
HG-Gene Muta-E. coli
4. Vogel HJ, Bonner DM. 1956. Acetylornithinase of E.
coli; partial purification and some properties. J Biol
Chem 218:97-106.
-------
HG-Gene Muta-A^_ nidulans
August, 1982
GENE MUTATION IN ASPERGILLUS NIDULANS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Gene Muta-A. nidulans
I. PURPOSE
Aspergillus nidulans is a eukaryotic fungus which has been
developed to detect and study a variety of genetic phenomena
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-thioxanthine forward mutation systems can be
used to detect mutations in A. nidulans.
II. DEFINITION
A forward mutation is a gene mutation from the wild (parent)
type to the mutant condition.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, ethyl
methanesulfonate, cyclophosphamide or aflatoxin B-.
IV. TEST METHOD
A. Principle
Conidia are exposed to test chemical both with and
without metabolic activation and plated on selective
medium to determine changes in colonial morphology or
nutritional requirements. At the end of a suitable
incubation period, mutant colonies are counted and
compared to the number of spontaneous mutants in an
untreated control culture. Simultaneous determination
of survival permits calculation of mutation frequency.
B. Description
Tests for mutation in A. nidulans are performed in
liquid suspension. Treated conidia are plated on
selective medium to determine changes in nutritional
requirements or colonial morphology.
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HG-Gene Muta-A_._ nidulans
C. Strain selection
1. Designation
For the methionine and 2-thioxanthine systems the
haploid Glascow biAl; meth Gl strain is the most
commonly used strain although other strains may be
appropriate. Any translocation-free strain which
produces green colonies on thioxanthine free medium
and yellow colonies on medium containing
thioxanthine may be used in the thioxanthine
system.
2. 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.
3. Media
Any medium which supports growth and a
characteristic colonial morphology may be used in
the assay.
D. Preparation of conidia
Prior to chemical treatment, conidia from 4-5 single
colonies of the appropriate strain are grown at 37 C on
complete medium. At the end of the incubation period,
conidia are collected, conidial chains broken up,
mycelial debris removed and conidia concentrated prior
to removal of the germination inhibitory substance.
Germination inhibitory substance should be removed by
Tween 80 or diethyl ether.
E. Metabolic activation
Conidia should be exposed to test substance both in t.he
presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducing agents. The use of other species, tissues or
techniques may also be appropriate.
-------
HG-Gene Muta-A. nidulans
F. Control groups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls should be included in each
experiment.
2. Direct acting positive controls
Ethyl methanesulfonate is an example of a positive
control for experiments without metabolic
activation.
3. Positive controls to ensure the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test. Cyclophosphamide and
aflatoxin B-^ are examples of positive controls in
tests using postmitochondrial fractions from livers
,of rodents treated with enzyme inducing agents such
as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may be
used.
G. Test chemicals
1. 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.
2. Exposure concentrations
Effective concentrations and treatment times should
be determined in a preliminary assay. Each test
should include five treatment points, two at fixed
concentrations for different time periods, and
three at varying concentrations for fixed periods
of time. The test should initially be performed
over a broad range of concentrations. When
appropriate, a positive response should be
confirmed by using a narrow range of test
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HG-Gene Muta-A. nidulans
concentrations. Among the criteria to be taken
into consideration for determining the upper limits
of test chemical concentration are cytotoxicity and
solubility. Cytotoxicity of the test chemical may
be altered in the presence of a metabolic
activation system. 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.
V. TEST PERFORMANCE
A. Treatment
Germinating or quiescent conidia in liquid suspension
should be exposed to the test chemical at 37 C under
conditions of yellow light and controlled pH and oxygen
tension. At the end of the exposure period, treatment
should be terminated by repeated centrifugation and
washing of the conidia or by dilution. Chemical
neutralization of the test agent may also be used but is
not recommended.
B. Media
1. Methionine system
For the methionine system, condidia should be
plated on methionine deficient medium for mutant
selection and on medium supplemented with
methionine to determine survival.
2. Thioxanthine system
For the 2-thioxanthine system, treated conidia
should be plated on nitrogen-free glucose and salts
minimal medium containing 2-thioxanthine. After
incubation, green colonies should be counted and
isolated by restreaking. The isolated colonies
should be classified on the basis of genetic
criteria. Yellow, wild-type colonies will grow on
the same plate. This permits concurrent
determination of survival and an estimation of
mutation frequency.
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HG-Gene Muta-A. nidulans
C. Determination of mutation frequency and viability
In both systems, mutation frequency and viability should
be determined immediately before and immediately after
chemical treatment.
D. Incubation conditions
All incubations should be at 37 C. Incubation time will
vary depending upon system and endpoint (mutation or
viability) being determined.
E. Number of cultures
1. At least 10 independent plates per concentration
with no more than 20 colonies per plate should be
used in the methionine system.
2. Fifteen to 20 plates per concentration are
preferred for the 2-thioxanthine system.
VI. DATA AND REPORT
A. Treatment of results
Individual plate counts for test substance and controls
should be presented for both mutation induction and
survival. The mean number of colonies per plate and
standard deviation should also be presented. Data
should be presented in tabular form indicating, as
applicable, numbers of colonies counted, and numbers and
classification of mutants identified. Sufficient detail
should be provided for verification of survival and
mutation frequencies.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating this test. Categorical data techniques are
preferred to compare treatment with control. Modeling
may be appropriate for evaluating dose dependent
response. Choice of analyses giving probabilistic
conclusions should consider tests appropriate to the
experimental design and needed adjustments for multiple
comparisons.
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HG-Gene Muta-A. nidulans
C. Interpretation of results^
1. There are several criteria for determining 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 positive response for at least one of
the test points. However, the final decision must
be based upon good scientific judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
mutant colonies nor a statistically significant and
reproducible positive response at any one of the
test points is considered nonmutagenic in this
system. Again, the final decision must be based
upon good scientific judgement.
D. Test evaulation
1. Positive results from the methionine and 2-
thioxanthine systems in A. nidulans indicate that
the test substance causes gene(point) mutations in
the DNA of this organism caused by base pair
changes and small deletions in the genome.
2. Negative results indicate that under the test
conditions the test chemical is not mutagenic in A.
nidulans.
E* Test report
The test report should include the following
information:
1. strain of organism used in the assay;
2. test chemical vehicle, doses used and rationale for
dose selection, toxicity data;
3. method used for preparation of conidia;
4. treatment conditions, including length of exposure
and method used to stop treatment;
5. details of the protocol used for metabolic
activation;
6. incubation times and temperature;
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HG-Gene Muta-A^ nidulans
7. positive and negative' controls;
8. dose-response relationship, if applicable;
9. statistical evaluation;
10. discussion of results; and
11. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Kafer E, Scott BR, Dorn GL, Stafford RS. 1982.
Aspergillus nidulans; 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.
3. Munson RJ, Goodhead DT. 1977. Relation between induced
mutation frequency and cell survival: a theoretical
approach and an examination of experimental data for
eukaryotes. Mutation Research 42:145-159.
4. Scott BR, Dorn GL, Kafer E, Stafford RS. 1982.
Aspergillus^ nidulans; systems and results of tests for
mitotic segregation and mutation. II. Haploid assay
systems and overall response of all systems: a report
of the U.S. EPA's Gene-Tox Program. Mutation Research
98:49-94.
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HG-Gene Muta-N_._ crassa
August, 1982
GENE MUTATION IN NEUROSPORA CRASSA
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Gene Muta-N. crassa
I. PURPOSE
Neurospora crassa is a eukaryotic fungus which has been
developed to detect and study a variety of genetic phenomena
including chemically induced mutagenesis. Jfl. crassa can be
used to detect both forward and reverse gene mutation. These
mutations are detected 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.
II. DEFINITION
A forward mutation is a gene mutation from the wild (parent)
type to the mutant condition.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, ethyl- or
methyl methanesulfonate.
IV. TEST METHOD
A. 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.
B. Description
Forward mutations at the ad-3 locus can be detected
using noncolonial strains of N. crassa grown on media
containing sorbose as well as glucose. Under these
conditions, colonies are formed and reproducible
colonial morphology results. Adenine-requiring mutants
which accumulate a reddish-purple pigment can be readily
identified and counted.
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HG-Gene Muta-NN_ crassa
C. S t r a i n s e 1 e c t icm
1. Designation
At the present time, heterokaryon 12 is recommended
for use in this assay. The use of other strains
may also be appropriate.
2. 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.
3. Media
Erie's No. 3 minimal medium or Westgaard's
Synthetic medium with 1.5% agar or any medium known
to support growth and characteristic colonial
morphology may be used in the assay.
D* Preparation of conidia
Stock cultures should be grown on minimal medium to
select for single colonies with noncolonial
morphology. Single colony isolates then should be
inoculated into agar flasks and incubated at 35 C for 48
hrs to select colonies with spreading growth patterns in
which mycelia cover the entire flask. Flasks should be
incubated at 23-25 C and those with bright orange
conidia selected for preparation of conidial
suspensions. Suspensions should be diluted for use in
distilled water.
E. Me t a bolie a ct i v a t i o n
Conidia should be exposed to test substance both in the
presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducers. The use of other species, tissues or
techniques may also be appropriate.
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HG-Gene Muta-N. crassa
F. Control groups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls should be included in each
experiment.
2. Direct acting positive controls
Examples of positive controls for experiments
without metabolic activation include ethyl- or
methyl methanesulfonate.
3. Ppsi tive controls to ensure the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test.
4. Other positive controls
Other positive control reference substances may
also be used.
G. Test chemicals
1. 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.
2. Exposure concentrations
The test should initially be performed 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.
When appropriate, a positive response should be
confirmed by testing over a narrow range of
concentrations. Among the criteria to be taken
into consideration for determining the upper limits
of test chemical concentration are cytotoxicity and
solubility. Cytotoxicity of the test chemical may
be altered in the presence of metabolic activation
systems. For toxic chemicals, the highest
concentration tested should not reduce survival
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HG-Gene Muta-N. crassa
below 10% 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. Each test should include five
treatment points; two at fixed concentrations for
different time periods, and three at varying
concentrations for fixed periods of time.
V. TEST PERFORMANCE
A. Treatment
1. Growing or nongrowing conidia should be exposed to
the test chemical with and without metabolic
activation. At the end of the exposure period,
treatment should be terminated by chemical
quenching. The quenching solution may contain 0.1%
sodium thiosulfate.
2. Conidia should then be plated on the appropriate
media to determine mutation induction and
viability. At the end of the incubation period,
colonies should be scored for viability and
mutation induction.
3. Mutants should be classified according to color and
morphology.
4. Both mutation frequency and viability should be
determined both immediately before and immediately
after chemical treatment.
B. Incubation conditions
All plates in a given test should be incubated for the
same time period. This incubation period may be from
2-7 days at 30 C.
C. Number of cultures
Generally, fifteen to 20 individual plates per
concentration should be used.
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HG-Gene Muta-N^_ crassa
VI. DATA AND REPORT
A. Treatment of results
Individual plate counts for test substance and controls
should be presented for both mutation 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 mutation frequencies.
B. Statistical evaluation
Several statistical techniques are acceptable in
. evaluating the results of this test. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining 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 positive response for at least one of
the test points. However, the final decision must
be based upon good scientific judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
mutant colonies nor a statistically significant and
reproducible positive response at any one of the
test points is considered nonmutagenic in this
system. Again, the final decision must be based
upon good scientific judgement.
D. Test evaluation
1. Positive results from the ad-3 system in N. crassa
indicate that the test substance causes mutations
in the DNA of this organism.
2. Negative results indicate that under the test
conditions the test substance is not mutagenic in
N. crassa.
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HG-Gene Muta-N. crassa
E. Test report
The test report should include the following
information:
1. strain of organism used in the assay;
2. test chemical vehicle, doses used and rationale for
dose selection;
3. method used for preparation of conida;
4. treatment conditions, including length of exposure
and method used to stop treatment;
5. incubation times and temperature;
6. details of the protocol used for metabolic
activation;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and
10. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Brockman HE, de Serres FJ. 1963. Induction of ad-3
mutants of Neurospqra crassa by 2-aminopurine. Genetics
48: 597-604.
2. de Serres FJ, Mailing HV. 1971. 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. In: Chemical
mutagens: principles and methods for their detection,
Vol. 2. Hollaender A, ed. New York and London: Plenum
Press, pp. 311-342.
3. Matzinger PK, Ong T-M. 1976. In vitro activation of
to roetabolites mutageni
ation Research 37:27-32.
aflatoxin Bl to roetabolites mutagenic in Neurospora
crassa. Mutati
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HG-Gene Muta-Insects
August, 1982
SEX-LINKED RECESSIVE LETHAL
TEST IN DROSOPHILA MELANOGASTER
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Gene Muta-Insects
I. PURPOSE
The sex-linked recessive lethal (SLRL) test using Drosophila
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% of all X-chromosome
loci. The X-chromosome represents approximately one-fifth of
the entire haploid genome.
II. DEFINITIONS
A. Lethal mutation is a change in the genome which, when
expressed, causes death to the carrier.
B. Recessive mutation is a change in the genome which is
expressed in the homozygous or hemizygous condition.
C. Sex-Linked 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.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, ethyl
methanesulfonate or N-nitroso-dimethylamine.
IV. TEST METHOD
A. Principle
Mutations in the X-chromosome of JD. me 1 a nog a s t e r are
phenotypically expressed in males carrying the mutant
gene. When the mutation is lethal in the hemizygous
condition, its presence 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 arranged chromosomes.
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HG-Gene Muta-Insects
B. Description
Wild-type males are treated and mated to appropriate
females. Female offspring 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-linked recessive lethal mutation has occurred in a
germ cell of the PI male<
C« Prosophila 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 multiply inverted X-
chromosomes may also be used.
D. Control groups
1. Concurrent controls
Concurrent positive and negative (vehicle) controls
should be included in each experiment.
2. Positive controls
Examples of positive controls include ethyl
methanesulfonate and N-nitroso-dimethylamine.
3. Other positive controls
Other positive control reference substances may be
used.
4. Negative controls
Negative (vehicle) controls should be included.
However, if appropriate laboratory historical
control data are available, concurrent controls may
not be necessary.
E. Test chemicals
1. Vehicle
Test chemicals should be dissolved in water.
Compounds which are insoluble in water may be
dissolved or suspended in appropriate vehicles
(e.g., a mixture of ethanol and Tween-60 or 80) and
then diluted in water or saline prior to
administration. Dimethylsulfoxide should be
avoided as a vehicle.
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HG-Gene Muta-Insects
2. Dose levels^
For the initial assessment of mutagenicity, it may
be sufficient to test a single dose of the test
substance. This dose should be the maximum
tolerated dose or that which produces some
indication of toxicity. If the test is being used
to verify mutagenic activity, at least two
additional exposure levels should be used.
3« Route of admini_s_t_r_at_ip_n_
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 in 0.7% NaCl solution
and injected into the thorax or abdomen.
V. TEST PERFORMANCE
A. Treatmen t and mating
Wild-type males (3-5 days old) should be treated with
the test substance and mated individually to an excess
of virgin females from the Muller-5 stock or females
from another appropriately marked (with multiply-
inverted X-chromosomes) stock. The females should be
replaced with fresh virgins every 2-3 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, spermatocytes and spermatogonia at the
time of treatment.
B. Fl matings
Heterozygous F-, 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 F-^ female
carrying a lethal in the parental X-chromsome (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 in
the next generation.
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HG-Gene Muta-Insects
C. Number of matings
1. The test should be designed with a predetermined
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 number of treated chromosomes that must be
analysed to detect substances which show mutation
rates close to those of the controls.
2. Test results should be confirmed in a separate
experiment.
VI. DATA AND REPORT
A. Treatment of results
Data should be tabulated to show the number of
chromosomes tested/ the number of nonfertile males and
the number of lethal chromosomes at each exposure
concentration and for each mating period for each male
treated. Numbers of clusters of different size per male
should be reported.
B. Statisti cal evaluation
Several statistical techniques are acceptable in
evaluating sex-linked recessive lethal tests.
Clustering of recessive lethals originating from one
male should be considered and evaluated in an
appropriate statistical manner. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C» Interpretation of res_u_l_t_s_
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
sex-lined recessive lethals. Another criterion may
be based upon detection of a reproducible and
statistically significant positive response for at
least one of the test points. However, the final
decision must be based upon good scientific
judgement.
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HG-Gene Muta-Insects
2. A test substance producing neither a statistically
significant dose-related increase in the number of
sex-linked recessive lethals nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
D. Test eyaj.uatioil
1. Positive results in the SLRL test in D.
me 1 anogajster indicate that the test agent causes
mutations in germ cells of this insect.
2. Negative results indicate that under the test
conditions the test substance is not mutagenic in
D. me1anogaster.
E. Test report
The test report should include the following
information:
1. Drosophila stock used in the assay, age of insects,
number of males treated, number of sterile males,
number of F2 cultures established, number of F2
cultures without progeny;
2. test chemical vehicle, treatment and sampling
schedule, exposure levels, toxicity data, negative
(vehicle) and positive controls, if appropriate;
3. criteria for scoring lethals;
4. number of chromosomes tested, number of chromosomes
scored, numer of chromosomes carrying a lethal
mutation;
5. historical control data, if available;
6. dose-response relationship, if applicable;
7. statistical evaluation;
8. discussion of results; and
9. interpretation of results.
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HG-Gene Muta-Insects
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of information
on which this section is based. They should not be
considered the only source of information on test
performance, however.
1. Sobels FH, Vogel E. 1976. The capacity of Drosophila
for detecting relevant genetic damage. Mutation
Research 41:95-106.
2. Wurgler FE, Sobels FH, Vogel E. 1977. Drosophila as
assay system for detecting genetic changes. In:
Handbook of mutagenicity test procedures. Kilbey BJ,
Legator M, Nichols W, Ramel C, eds. Amsterdam:
Elsevier/North Holland Biomedical Press, pp. 335-373.
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HG-Gene Muta-Somatic Cells
August, 1982
DETECTION OF GENE MUTATIONS IN
SOMATIC CELLS IN CULTURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Gene Muta-Somatic Cells
I. PURPOSE
Mammalian cell culture systems may be used to detect
mutations induced by chemical substances. Widely used cell
lines include L5178Y mouse lymphoma cells and the CHO and
V-79 lines of Chinese hamster cells. In these cell lines the
most commonly used systems measure mutation at the thymidine
kinase (TK, L5178Y cells), hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT, CHO and V-79 cells) and
Na+/K+ ATPase (V-79) loci. The TK and HGPRT mutational
systems detect base pair mutations, frameshift mutations and
small deletions; the Na+/K+ ATPase system detects base pair
mutations only.
II. DEFINITIONS
A. A forward mutation assay in mammalian cells detects a
gene mutation from the parent type to the mutant
condition which is due to a change in an enzymatic or
functional protein.
B. Frameshift mutagens are agents which cause the addition
or deletion of single or multiple base pairs in the DNA
molecule.
C. Phenotypic expression time is a period during which
unaltered gene products are depleted from newly mutated
cells.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, ethyl
methanesulfonate, N-nitroso-dimethylamine, 2-
acetylaminofluorene, 7,12-dimethylbenzanthracene or
hycanthone.
IV. TEST METHOD
A. Principle
Cells are exposed to test agent both with and without
metabolic activation for a suitable period of time and
subcultured to determine cytotoxicity and allow
phenotypic expression prior to mutant selection. Cells
with altered Na+/K+ ATPase are selected by ouabain.
Cells deficient in TK or HGPRT are unable to convert
certain nucleosides or their analogues to nucleotides.
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HG-Gene Muta-Somatic Cells
Bromodeoxyuridine (BrdU), trifluorothymidine (TFT),
azaguanine (AG) and thioguanine (TG) nucleotides are
lethal to the parental cell at concentrations which are
nonlethal to mutant cells. Mutant cells are, therefore,
capable of proliferation in the presence of these
agents.
B. Description
Cells in suspension or monolayer culture are exposed to
the test substance, both with and without a metabolic
activation system, for a defined period of time.
Cytotoxic effects of treatment are determined by
measuring the colony forming abilities or growth rates
of the cultures after the treatment period. Treated
cultures are maintained in growth medium for a
sufficient period of time - characteristic of each
selected locus - to allow near-optimal phenotypic
expression of induced mutations. The cultures are
analyzed for mutant frequency at the end of the
expression time by seeding known numbers of cells in
medium with and without the selective agent. After a
suitable incubation time, cell colonies are counted.
The number of mutant colonies in selection medium is
adjusted by the number of colonies in nonselection
medium to derive the mutant frequency.
C. Cells
1. Type of ce 11 s u s ed i n the assay
A variety of cell lines are available for use in
this assay. These include subclones of L5178Y, CHO
cells or V-79 cells with a demonstrated sensitivity
to chemical mutagens, a high cloning efficiency and
a low spontaneous mutation frequency.
2. Cell growth and maintenance
Appropriate growth media, chosen according to
selective system and cell type used in the assay,
C02 concentrations, temperature and humidity should
be used in maintaining cultures. Established cell
lines should be periodically checked for Mycoplasma
contamination. It is also desirable to check the
cells periodically for karyotype stability.
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HG-Gene Muta-Somatic Cells
D. Metabolic activation
Cells should be exposed to test substance both in
the presence and absence of an appropriate
metabolic activation system. Examples of such
activation systems include cofactor supplemented
postmitochondrial fractions prepared from the
livers of mammals treated with enzyme inducers and
primary cultures of mammalian hepatocytes. The use
of other tissues or techniques may also be
appropriate.
E. Control groups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls with and without metabolic
activation should be included in each experiment.
2. Direct acting positive controls
Examples of positive controls for assays without
metabolic activation include ethyl methanesulfonate
or hycanthone.
3. Positive controls to ensure the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test. N-nitroso-dimethylamine
and 2-acetylaminofluorene are examples of positive
control compounds in tests using postmitochondrial
fractions from the livers of rodents treated with
enzyme inducing agents such as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may be
used.
F. Test chemicals
1. Vehicle
Test chemicals and positive control reference
substances may be prepared in growth medium or
dissolved or suspended in appropriate vehicles and
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HG-Gene Muta-Somatic Cells
then further diluted in growth medium for use in
the assay. Final concentration of the vehicle
should not affect cell viability.
2. E xpos u re co n ce n t r a t i o n s
Multiple concentrations of the test substance,
based upon cytotoxicity, and over a range adequate
to define the response should be tested. These
concentrations, with and without metabolic
activation, should yield a concentration-related
toxic effect. The highest concentration tested
should produce a low level of survival; survival in
the lowest concentration tested should approximate
survival in the negative (untreated and/or vehicle)
control. Relatively insoluable substances should
be tested up to the limits of solubility. For
freely soluble nontoxic substances, the upper test
substance concentration should be determined on a
case by case basis.
V. TEST PERFORMANCE
A. Mouse lymphgma L5178Y cells
Prior to exposure to test substance, cells with a low
spontaneous mutation frequency should be centrifuged and
resuspended in medium at the appropriate cell density.
Cells should be exposed to test substance both with and
without metabolic activation. Exposure should generally
be limited to 4 hours. At the end of the incubation
period, cells should be washed free of test substance,
suspended in medium, diluted to the appropriate cell
density and incubated for expression of mutant
phenotype. At the end of the expression time, cells
should be grown in soft agar cloning medium in the
presence and absence of selective agent. At the end of
a suitable incubation period, cells should be counted
and the numbers of viable and mutant colonies
determined.
B. CHO cells
1. Prior to exposure to test chemical, cells should be
plated at the appropriate cell density and
incubated at 37 C until cells are attached to the
culture vessel. Cells should be exposed to test
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HG-Gene Muta-Somatic Cells
substance both with and without metabolic
activation. For experiments with metabolic
activation by postmitochondrial fractions, the
exposure time should generally be limited to 5
hr. For experiments without metabolic activation,
exposure time may be 5 hr or may be extended over
the CHO doubling time.
2. At the end of the treatment period, cells should be
removed from the culture vessel, a portion diluted
to appropriate concentrations, plated and incubated
at 37 C. At the end of the incubation period,
colonies should be fixed, stained and counted to
determine cytotoxicity.
3. For expression of mutant phenotype, the remaining
cells should be subcultured an appropriate number
of times prior to growth in medium containing the
selective agent. TG is recommended as the
selective agent. Cloning efficiency prior to
selection should be determined by growth in medium
free of the selective agent. Medium should not be
changed during the selection period. After an
appropriate incubation period, colonies should be
fixed, stained and counted for mutant selection and
cloning efficiency.
C. V-79 cells
1. Prior to exposure to test substance, cells should
be removed from the culture vessel, centrifuged,
and resuspended in medium at the appropriate cell
density and grown in suspension or monolayer
culture. In either case, cells should be exposed
to test substance with and without metabolic
activation for a suitable period of time. For
experiments with metabolic activation by post-
mitochondrial fractions, exposure should generally
be limited to 2 hr. For experiments with cell
mediated activation systems, exposure may be
extended to 18-24 hr. At the end of the treatment
period, cells should be washed free of test
chemical and subcultured for expression of the
mutant phenotype.
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HG-Gene Muta-Somatic Cells
2. At the end of the expression period, cells should
be seeded in selective medium for determination of
number of mutants and in medium free of selective
agent for determination of viable colonies. At the
end of the incubation period, cells should be
fixed, stained and counted to determine the number
of mutant and viable colonies.
D. Number of cultures
At least two replicate experiments are recommended. In
each, a minimum of two independent cultures per
experimental point should be used.
VI. DATA AND REPORT
A. Treatment of results
Individual plate counts for test substance and control
should be presented for both mutation induction and
survival. The mean number of colonies per plate and
standard deviation should also be presented. Data
should be presented in tabular form giving survival (and
cloning efficiencies) as a percentage of the control
levels. Mutation frequency should be expressed as
number of mutants per number of clonable cells. If the
vehicle control or other controls appear to be toxic,
this should be indicated. Sufficient detail should be
provided for verification of survival and mutation
frequencies.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating this test. Choice of analyses should ,
consider tests appropriate to the experimental design
and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining 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 positive response for at least one of
the test points. However, the final decision must
be based upon good scientific judgement.
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HG-Gene Muta-Somatic Cells
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of mutant colonies nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
D. Test evaluation
1. Positive results in experiments with cells in
culture indicate that the test substance causes
mutation in cultured mammalian somatic cells.
2. Negative results indicate that under the test
conditions, the test substance is not mutagenic for
cultured mammalian somatic cells.
E. Test report
The test report should include the following
information:
1. cells used, passage number at time of treatment,
number of cell cultures;
2. methods used for maintenance of cell cultures,
including medium, temperature and C02
concentration;
3. test chemical vehicle, concentration and rationale
for selection of concentrations of test substance
used in the assay;
4. details of the protocol used for metabolic
activation;
5. cell density at treatment, duration of treatment,
and cloning media;
6. positive and negative controls;
7. selective agent used;
8. expression period (including numbers of cells
seeded and subculture and feeding schedules, if
appropriate);
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HG-Gene Muta-Soraatic Cells
9. methods used to enumerate numbers of viable and
mutant cells;
10. dose-response relationship, if applicable;
11. statistical evaluation;
12. discussion of results; and
13. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Amacher DE, Paillet SC, Ray V. 1979. Point mutations
at the thymidine kinase locus in L5178Y mouse lymphoma
cells. I. Application to genetic toxicology testing.
Mutation Research 64:391-406.
2. Amacher DE, Paillet SC, Turner GN, Ray VA, Salsburg
VA. 1980. Point mutations at the thymidine kinase
locus in L5178Y mouse lymphoma cells. II. Test
validation and interpretation. Mutation Research
72:447-474.
3. Bradley MO, Bhuyan B, Francis MC, Langenback R, Peterson
A, Huberman E. 1981. Mutagenesis by chemical agents in
V-79 Chinese hamster cells: a review and analysis of
the literature: a report of the Gene-Tox Program.
Mutation Research 87:81-142.
4. Clive D, Johnson KO, Spector JFS, Batson AG, Brown MM.
1979. Validation and characterization of the L5178Y
TK+/- mouse lymphoma mutagen assay system. Mutation
Research 59:61-108.
5. Clive D, Spector JFS. 1975. Laboratory procedures for
assessing specific locus mutations at the TK locus in
cultured L5178Y mouse lymphoma cells. Mutation Research
31:17-29.
8
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HG-Gene Muta-Somatic Cells
Hsie AW, Casciano DA, Couch DB, Krahn DF, O'Neill JP,
Whitfield BL. 1981. The use of Chinese hamster ovary
cells to quantify specific locus mutation and to
determine mutagenicity of chemicals: a report of the
U.S. EPA's Gene-Tox Program. Mutation Research 86:193-
214.
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HG-Gene Muta-Mammal
August, 1982
THE MOUSE SPECIFIC LOCUS TEST
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Gene Muta-Mammal
I. PURPOSE
The mouse specific locus test (MSLT) may be used to detect
and quantitate mutations in the germ line of a mammalian
species.
II. DEFINITIONS
A. A visible specific locus mutation is a genetic change
that alters factors responsible for coat color and other
visible characteristics of certain mouse strains.
B. The germ line is the cells in the gonads of higher
eukaryotes which are the carriers of the genetic
information for the species.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
1. 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 various means, depending on
the loci chosen to be marked.
2. 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 5
or 7 loci;
b. the biochemial specific locus test using up to
20 enzymes; and
c. the test for mutations at histocompatibility
loci.
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HG-Gene Muta-Mammals
3. Of the three tests, the visible specific locus test
has been most widely used in assessing genetic
hazard due to environmental agents.
B. Description
For technical reasons, males rather than females are
generally treated with the test agent. Treated males
are then mated to females 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.
C. Animal selection
1. Species and strain
Mice are recommended as the test species. Male
mice should be either (C3H X 101 )F-L or (101 X
C-jHJFj^ hybrids. Females should be T stock virgins.
2. Age
Healthy sexually mature animals should be used.
3. Number
A decision on the minimum number of treated animals
should take into account the spontaneous variation
of the biological characterization being
evaluated. Other considerations should include:
a. the use either historical or concurrent
controls;
b. the power of the test;
c. the minimal rate of induction required;
d. the use of positive controls; and
e. the level of significance desired.
4 . Assignment to groups
Animals should be randomized and assigned to
treatment and control groups.
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HG-Gene Muta-Manunals
D. Control groups
1. Concurr ent co nt rpls
No positive or spontaneous controls are recommended
as concurrent parts of the MSLT. Any laboratory
which has had no prior experience 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-ethyl-nitrosourea, in a sample of
5000 offspring.
2. Historical co n t r o 1 s
Long term, accumulated spontaneous control data of
43/801,406 are available for comparative purposes
and should be used.
E. Test chemicals
1. Vehicle
When possible, test chemicals should be dissolved
or suspended in isotonic saline buffered
appropriately, if needed, for stability. Insoluble
chemicals should be dissolved or suspended in
appropriate vehicles. The vehicle used should
neither interfere with the test compound nor
produce major toxic effects. Fresh preparations of
the test chemical should be employed.
2. Dose levels
Usually, only one dose level need be tested. This
should be the highest dose tolerated without toxic
effects, provided that any temporary sterility
induced due to elimination of spermatagonia is of
only moderate duration, as determined by a return
of males to fertility within 80 days after
treatment.
3. Route of administration
The route of administration should be chosen by the
investigator based upon the nature of the test
chemical. Acceptable routes of administration
include gavage, inhalation, admixture with food or
water, and IP or IV injections.
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HG-Gene Muta-Mammals
V. TEST PERFORMANCE
A. Tre a tme n_t_ a nd ma t i ng
Hybrid F-j^ (C3H X 101 or 101 X C3H) male mice should be
treated with the test substance and immediately mated to
virgin T stock females. Each treated male should be
mated to a fresh group of 2-4 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.
B. Examination of offspring
Offspring may be examined at (or soon after) birth but
must be examined at about 3 week of age at which time
the numbers of mutant and nonmutant offspring in each
litter should be recorded. Nonmutant progeny should be
discarded. Mutant progeny should be subjected to
genetic tests for verification.
VI• DATA AND REPORT
A. Treatment of results
Data should be presented 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 thoroughly
described. In addition, 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, accumulated mutation
frequencies. Statistical comparison should be made
between experimental groups, and between experimental
groups and long term, accumulated controls. Comparison
should also be made between experimental groups and
concurrent controls when such data are available.
B. S t a t i s t i ca 1 eya 1 uati cm
Several statistical techniques are acceptable in
evaluating results of this test. For small numbers of
mutations, exact tests are preferred.
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HG-Gene Muta-Mammals
Evidence of intraclass correlation (within litters or
sires) indicates an adjusted analysis should be
considered. Choice of analyses should consider tests
appropriate to the experimental design and needed
adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
specific locus mutations. Another criterion may be
based upon detection of a reproducible and
statistically significant positive response for at
least one of the test points. However, the final
decision must be based upon good scientific
judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
specific locus mutations nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
D. Test evaluation
1. Positive results in the MSLT indicate that the test
substance induces heritable gene mutations in the
test species.
2. Negative results indicate that under the test
conditions the test substance does not induce
heritable gene mutations in the test species.
E. Test report
The test report should include the following
information:
1. strain, age and weight of animals used, number of
animals of each sex in experimental and control
groups;
2. test chemical vehicle, doses used and rationale for
dose selection, toxicity data;
3. route and duration of exposure;
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HG-Gene Muta-Mammals
4. mating schedule;
5. time of examination for mutant progeny;
6. criteria for scoring mutants;
7. use of concurrent or negative controls;
8. dose response relationship, if applicable;
9. statical evaluation;
10. discussion of results; and
11. interpretation of results.
VII. REFERENCES
The following reference may be helpful in developing
acceptable protocols, and provides a background of
information on which this section is based. It should not
be considered the only source of information on test
performance, however.
1. Russell LB, Selby PB, von Halle E, Sheridan W, Valcovic
L. 1981. The mouse specific locus test with agents
other than radiations: interpretation of data and
recommendations for future work: a report of the Gene-
Tox Program. Mutation Research 86:329-354.
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HG-Chromo-In Vitro
August, 1982
IN VITRO MAMMALIAN CYTOGENETICS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Chromo-In Vitro
I. PURPOSE
The in vitro cytogenetics test is a short term mutagenicity
test system for the detection of chromosomal aberrations in
cultured mammalian cells. Chromosomal aberrations may be
either structural or numerical. However, because cytogenetic
assays are usually designed to analyse cells at their first
post-treatment mitosis and numerical aberrations require at
least one cell division to be visualized, this type of
aberration is generally not observed in a routine
cytogenetics assay. Structural aberrations may be of two
types: chromosome or chromatid. Chromosome-type aberrations
are induced when a compound acts in the G-, phase of the cell
cycle. Chromatid-type aberrations are induced when a
chemical acts in the S or G2 phase of the cell cycle. The
majority of chemicals, including those which act in G^,
induce only chromatid-type aberrations because the damage,
although induced in G-^, does not become manifest until S
phase. Radiation and radiomimetic agents, however, induce
damage in all phases of the cell cycle.
II. DEFINITIONS
A. Chromosome-type aberrations are changes which result
from damage expressed in both sister chromatids at the
same locus.
B. Chromatid-type aberrations result from damage expressed
as breakage of single chromatids or breakage and/or
reunion between chromatids.
C. Numerical aberrations are variations of the normal
chromosome number characteristic of the cells used in
the assay.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
In vitro cytogenetics assays may employ cultures of
established cell lines, cell strains or primary cell
cultures. Cell cultures are exposed to the test
substance both with and without metabolic activation.
Following exposure of cell cultures to test substances,
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HG-Chromo-In Vitro
they are treated 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 are analyzed for chromosomal
aberrations.
B. Description
Cell cultures are exposed to test compounds and
harvested at various intervals after treatment. Prior
to harvesting, cells are treated with colchicine or
Colcemid® to accumulate cells in c-metaphase.
Chromosome preparations from cells are made, stained and
scored for chromosomal aberrations.
C. Cells
1. 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.
2- Cell growth and maintenance
Appropriate growth media, CO2 concentration,
temperature and humidity should be used in
maintaining cultures. Established cell lines and
strains should be periodically checked for
Mycoplasma contamination. It is also desirable to
check the cells periodically for karyotype
stability.
D. Metabolic activation
1. Cells should be exposed to test substance both in
the presence and absence of an appropriate
metabolic activation system. Examples of such
systems include cofactor supplemented
postmitochondrial fractions prepared from the
livers of mammals treated with enzyme inducers and
primary cultures of mammalian, hepatocytes. The use
of other tissues or techniques may also be
appropriate.
2. It is recognized that the use of metabolic
activation systems in in vitro cytogenetics assays
may present problems of cytotoxicity to the test
system. If a chemical gives a negative result when
tested without metabolic activation, every attempt
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HG-Chromo-In Vitro
should be made to test it with metabolic activation
in this system. If this is not feasible because of
technical difficulties with metabolic activation
systems, it is recommended that the chemical be
retested in an in vivo cytogenetics assay.
E. Co nt ro1 g r gups
1* Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls both with and without metabolic
activation should be included in each experiment.
2. Pi re c t act ing pos i t i ve con tro1s
For tests without metabolic activation, a compound
known to produce chromosomal aberrations in vitro
without the use of such a system should be used as
the positive control.
3. Positive controls to ensure the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test.
F. Test chemicals
1. Vehicle
Test chemicals and positive control reference
substances may be prepared in growth medium or
dissolved or suspended in appropriate vehicles and
then further diluted in growth medium for use in
the assay. Final concentration of the vehicle
should not affect cell viability.
2. Exposure concentrations
Multiple concentrations of the test substance over
a range adequate to define the response should be
tested. The highest test substance concentration
with and without metabolic activation should
suppress mitotic activity by approximately 50%.
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.
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HG-Chromo-In Vitro
V. TEST PERFORMANCE
A. Established cell lines and strains
Prior to use in the assay, cells should be generated
from stock cultures, seeded in culture vessels at the
appropriate density and incubated at 37 C.
B. Human lymphocyte cultures
Heparinized or acid-citrate-dextrose whole blood should
be added to culture medium containing a mitogen, e.g.
phytohemagglutinin (PHA) and incubated at 37 C. White
cells sedimented by gravity (buffy coat) may also be
utilized as may lymphocytes which have been purified on
a density gradient.
C. Treatment with test substance
For established cell lines and strains, cells in the
exponential phase of growth should be treated with test
substances in the presence and absence of a metabolic
activation system. Mitogen-stimulated human lymphocyte
cultures may be treated with the test substance in a
similar manner.
D. Number of cultures
At least two independent cultures should be used for
each experimental point.
E. Culture harvest time
For established cell lines and strains multiple harvest
times are recommended. If the test chemical changes the
cell cycle length, the fixation intervals should be
changed accordingly. For human lymphocyte cultures, the
substance to be tested may be added to the cultures at
various times after mitogen stimulation so that there is
a single harvest time after the initiation of the cell
culture. Alternatively, a single treatment may be
followed by multiple harvest times. Harvest time should
be extended for those chemicals which induce an apparent
cell cycle delay. For screening purposes, a single
harvest time, eg at 24 hours, may be appropriate for
established cell lines and strains. Because the
population of human lymphocytes is only partially
synchronized, a single treatment, at, or close to, the
time when metaphase stages first appear in the culture
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HG-Chromo-In Vitro
will include cells in all phases of the division
cycle. Therefore, a single harvest at the time of
second mitosis may be carried out for screening
purposes. Cell cultures are treated with
colchicine or Colcemid® one or two hours prior to
harvesting. Each culture is harvested and
processed separately for the preparation of
chromosomes.
F. Chromosome preparation
Chromosome preparation involves hypotonic treatment of
the cells, fixation and staining.
G. Analysis
Slides should be coded before analysis. The number of
cells to be analysed should be based upon the
spontaneous control frequency, defined sensitivity and
the power chosen for the test before analysis. In human
lymphocytes, only cells containing 46 centromeres should
be analysed. In established cell lines and strains only
metaphases containing _+_ 2 centromeres of the modal
number should be analysed. Uniform criteria for scoring
aberrations should be used.
VI. DATA AND REPORT
A. Treatment of results
Data should be presented in a tabular form. Different
types of structural chromosomal aberrations should be
listed with their numbers and frequencies for
experimental and control groups. Data should be
evaluated by appropriate statistical methods. Gaps or
achromatic lesions are recorded separately and not
included in the total aberration frequency.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating this test. Choice of analyses should
consider tests appropriate to the experimental design
and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
structural chromosomal aberrations.
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HG-Chromo-In Vitro
Another criterion may be based upon detection of a
reproducible and statistically significant positive
response for at least one of the test substance
concentrations. However, the final decision must
be based upon good scientific judgement.
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of structural chromosomal aberrations
nor a statistically significant and reproducible
positive response at any one of the test points is
considered nonmutagenic in this system. Again, the
final decision must be based upon good scientific
judgement.
D. Test evaluation
1. Positive results in the in vitro cytogenetics assay
indicate that the test substance induces
chromosomal aberrations in cultured mammalian
somatic cells.
2. Negative results indicate that under the test
conditions the test substance does not induce
chromosomal aberrations in cultured mammalian
somatic cells.
E. Test report
The test report should include the following
information:
1. cells used, density and passage number at time of
treatment, number of cell cultures;
2. methods used for maintenance of cell cultures
including medium, temperature and C02
concentration;
3. test chemical vehicle, concentration and rationale
for the selection of the concentrations used in the
assay, duration of treatment;
4. details of the protocol used for metabolic
activation;
5. duration of treatment with and concentrations of
colchicine or Colcemid® used;
6. time of cell harvest;
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HG-Chromo-In Vitro
7. positive and negative controls;
8. methods used for preparation of slides for
microscopic examination;
9. number of metaphases analysed;
10. mitotic index;
11. criteria for scoring aberrations;
12. type and number of aberrations, given separately
for each treated and control culture, frequency
distribution of number of chromosomes in
established cell lines and strains;
13. dose-response relationship, if applicable;
14. statistical evaluation^
15. discussion of results; and
16. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Evans HJ. 1976. Cytological methods for detecting
chemical mutagens. In: Chemical mutagens, principles
and methods for their detection, Vol.4. Hollaender A,
ed. New York, London: Plenum Press, pp. 1-29.
3. Howard PN, Bloom AD, Krooth RS. 1972. Chromosomal
aberrations induced by N-methyl-N'-nitro-N-
nitrosoguanidine in mammalian cells. In Vitro 7:359-
365.
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HG-Chromo-In Vitro
4. Ishidate M Jr, Odashima S. 1975. Chromosome tests with
134 compounds on Chinese hamster cells in vitro: a
screening for chemical carcinogens. Mutation Research
48:337-354.
5. Preston RJ, Au W, Bender MA, Brewen JG, Carrano AV,
Heddle JA, McFee AF, Wolff S, Wassom JS. 1981.
Mammalian in vivo and in vitro cytogenetic assays: a
report of the Gene-Tox Program. Mutation Research
87:143-188.
8
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HG-Chrorao-Bone Marrow
August, 1982
IN VIVO MAMMALIAN BONE MARROW
CYTOGENETICS TESTS: CHROMOSOMAL ANALYSIS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Chromo-Bone Marrow
I. PURPOSE
The in vivo cytogenetics assay tests for the ability of a
chemical to induce chromosomal aberrations in mammalian
species. Chromosomal aberrations may be either structural or
numerical. However, because cytogenetics assays are designed
to analyse cells at their first post-treatment mitosis and
numerical aberrations require at least one cell division to
be visualized, this type of aberration is not generally
observed in a routine cytogenetics assay. Structural
aberrations may be of two types: chromosome or chromatid.
Chromosome-type aberrations are induced when a compound acts
in the G-, phase of the cell cycle. Chromatid-type
aberrations are induced when a chemical acts in the S or 62
phase of the cell cycle. The majority of chemicals,
including those which act in G^, induce only chromatid-type
aberrations because the damage, although induced in G-^, does
not become manifest until S phase. Radiation and
radiomimetic agents, however, induce damage in all phases of
the cell cycle.
II. DEFINITIONS
A. Chromosome-type aberrations are changes which result
from damage expressed in both sister chromatids at the
same time.
B. Chromatid-type aberrations are damage expressed as
breakage of single chromatids or breakage and/or reunion
between chromatids.
C. Numerical aberrations are variations of the normal
chromosome number characteristic of the cells utilized.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
Animals are exposed to test chemicals by appropriate
routes and are sacrificed at sequential intervals.
Chromosome preparations are made from bone marrow
cells. The stained preparations are examined under a
microscope and metaphase cells are scored for
chromosomal aberrations.
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HG-Chromo-Bone Marrow
B. Description
The method employs bone marrow of laboratory rodents
exposed to test chemicals. Animals are further treated,
prior to sacrifice, with colchicine or Colcemid® to
arrest the cells in c-metaphase. Chromosome
preparations from the cells are made, stained and scored
for chromosomal aberrations.
C. Animal selection
1. Species and strain
Any appropriate mammalian species may be used.
Examples of commonly used rodent species include
rats, mice, Chinese, Syrian or Armenian hamsters.
2. Age
Healthy young adult animals should be used.
3» 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.
4. Ass ignment to groups
Animals should be randomized and assigned to
treatment and control groups.
5. Housing and feeding conditions
Animals may be caged in groups by sex or
individually; the number of animals per cage should
not interfere with clear observation of each
animal. Appropriate diet and drinking water should
be supplied ad libitum. Temperature, humidity and
light cycles should be controlled as dictated by
good animal husbandry procedures.
D. Control groups
1. Con current con tro1s
Concurrent positive and negative (vehicle) controls
should be included in the assay.
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HG-Chromo-Bone Marrow
Positive cntro3.s
A single dose positive control showing a
significant response at any one time point is
adequate. A compound known to produce chromosomal
aberrations in vivo should be employed as the
positive control.
E. Te s t chemi ca 1 s
1. Vehicle
When possible, test chemicals should be dissolved
in isotonic saline. 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 compound should be
employed.
Dose levels
At least three dose levels should be used. The
highest dose tested should produce some indication
of toxicity as evidenced by animal morbidity
(including death) or target cell toxicity. The
LD5Q is a suitable guide.
3. Route of administration
The route of administration should be chosen by the
investigator based upon the nature of the test
chemical. The usual routes of administration are
IP or oral. However, other routes may be
appropriate when indicated by scientific evidence.
4. Treatment schedule
Test substances should generally be administered
only once. However, based upon pharmacpkinetic
information a repeated treatment schedule may be
employed. The repeated treatment schedule can only
be applied if the test substance does not exhibit
cytotoxic effects in the bone marrow at the doses
used.
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HG-Chromo-Bone Marrow
V. TEST PERFROMANCE
A. Treatment and sampling times
Test compounds should be administered acutely (1
dose). Repeated exposures may be used when
pharmacokinetic or other toxicological information
indicates the chemical is active only after repeated
administration. For acute exposure, treated and
negative control animals should be sacrificed at .times
after treatment which adequately evaluate G±, S, and G2
phases of the cell cycle. Since cell cycle kinetics can
be influenced by the test substance, three sampling
times appropriately spaced within the range of 6 to 48
hours should be used. Sampling times after repeated
dosages should adequately assess effects at different
stages of the cell cycle.
®
B. Administration of colchicine or Colcemid
Prior to sacrifice, animals should be injected^ IP with
an appropriate dose of colchicine or Colcemid to arrest
cells at c-metaphase.
c* Preparation of slides
Following sacrifice, the bone marrow should be aspirated
from the femur, exposed to hypotonic solution, and
fixed. The cells should then be spread on slides and
stained. Chromosome preparations should be made
following standard procedures.
D. Analysis
The number of cells per animal to be analysed should be
based upon the spontaneous control frequency and defined
power and sensitivity of the test. Toxicity tests for
dose selection or historical control data may provide
approximate background frequencies for sample size
determinations. Uniform criteria should be used for
scoring aberrations. Slides should be coded before
microscopic analysis.
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HG-Chromo-Bone Marrow
VI. DATA AND REPORT
A. Treatment o£ re su11s
Data should be presented in a tabular form. Different
types of structural chromosomal abnormalities should be
listed with their numbers and frequencies for each cell
of each animal in all experimental and control groups.
Gaps or achromatic lesions should be recorded separately
and included in the total aberration frequency.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating this test. Choice of analyses should
consider tests appropriate to the experimental design
and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
structural chromosomal aberrations. Another
criterion may be based upon detection of a
reproducible and statistically significant positive
response for at least one of the test points.
However, the final decision must be based upon good
scientific judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
chromosomal aberrations nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
D. Test evaluation
1. Positive results in the in vivo bone marrow
cytogenetics assay demonstrate the ability of the
test substance to induce chromosomal aberrations in
the bone marrow of the test species.
2. Negative results indicate that under the test
conditions, the test substance does not induce
chromosomal aberrations in the bone marrow of the
test species.
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HG-Chromo-Bone Marrow
E. Test report
The test report should include the following
information:
1. species, strain, age, weight,.number and sex of
animals in each treatment and control group;
2. test chemical vehicle, dose levels used, rationale
for dose selection;
3. route of administration, treatment and sampling
schedules, toxicity data, negative and positive
controls;
4. details of treatment with colchicine or Colcemid®;
5. details of the protocol used for chromosome
preparation, number of metaphases scored per
animal, type and number of aberrations given
separately for each treated and control animal;
6. criteria for scoring aberrations;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and
10. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Adler ID, Ramarao G, Epstein SS. 1971. In vivo
cytogenetic effects of trimethyl-phosphate and of TEPA
on bone marrow cells of male rats. Mutation Research
13:263-273.
2. Evans HJ. 1976. Cytological methods for detecting
chemical mutagens. In: Chemical mutagens: principles
and methods for their detection, Vol. 4. Hollaender A,
ed. New York and London: Plenum Press, pp. 1-29.
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HG-Chromo-Bone Marrow
Kilian JD, Moreland FE, Benge MC, Legator MS, Whorton EB
Jr. 1977. A collaborative study to measure
intralaboratory variation with the in vivo bone marrow
metaphase procedure. In: Handbook of mutagenicity test
procedures. Kilby BJ, Legator M, Nichols C, Ramel C,
eds. Amsterdam: Elsevier/North Holland Biomedical
Press, pp. 243-260.
Preston JR, Au W, Bender MA, Brewen JG, Carrano AV,
Heddle JA, McFee AF, Wolff S, Wassom J. 1981.
Mammalian in vivo and in vitro cytogenetics assays:
report of the Gene-Tox Program. Mutation Research
87:143-188.
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HG-Chromo-Micronuc
August, 1982
IN VIVO MAMMALIAN BONE MARROW CYTOGENETICS TESTS;
MICRONUCLEUS ASSAY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Chromo-Mi cronuc
I. PURPOSE
The micronucleus test is a mammalian in vivo test which
detects damage of the chromosomes or mitotic apparatus by
chemicals. Polychromatic erythrocytes in the bone marrow of
rodents are used in this assay. When the erythroblast
develops into an erythrocyte the ,main nucleus is extruded and
may leave a micronucleus in the cytoplasm. The visualization
of micronuclei is facilitated in these cells because they
lack a nucleus. Micronuclei form under normal conditions.
The assay is based on an increase in the frequency of
micronucleated polychromatic erythrocytes in bone marrow of
treated animals.
II. DEFINITION
Micronuclei are small particles consisting of acentric
fragments of chromosomes or entire chromosomes, which lag
behind at anaphase of cell division. After telophase, these
fragments may not be included in the nuclei of daughter cells
and form single or multiple micronuclei in the cytoplasm.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
Animals are exposed to test substance by an appropriate
route. They are sacrificed, the bone marrow extracted
and smear preparations made and stained. Polychromatic
erythrocytes are scored for micronuclei under the
microscope.
B. Description
The method employs bone marrow of laboratory mammals
which are exposed to test substances.
C. Animal selection
1. Specie^ and strain
Mice are recommended. However, any appropriate
mammalian species may be used.
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HG-Chromo-Micronuc
2. Age
Healthy young adult animals should be used.
3. Number and sex
At least five female and five male animals per
experimental and control group should be used.
Thus, 10 animals would be sacrificed per time per
group if several test times after treatment were
included in the experimental schedule. The use of
a single sex or a different number of animals
should be justified.
4. Ass ig nme nt_to groups
Animals should be randomized and assigned to
treatment and control groups.
5. Housing and feeding conditions
Animals may be caged in groups by sex or
individually; the number of animals per cage should
not interfere with clear observation of each
animal. Appropriate diet and drinking water should
be supplied ad libitum. Temperature, humidity and
light cycles should be controlled as dictated by
good animal husbandry procedures.
D. Control groups
1. Concurrent controls
Concurrent positive and negative (vehicle) controls
should be included in each assay.
2- Positive contro1s
A compound known to produce micronuclei in vivo
should be employed as the positive control.
E* Te s t ch e mi ca 1 s
1. Vehicle
Solid and liquid test substances should be
dissolved or suspended in isotonic saline.
Insoluble chemicals may be dissolved or suspended
in appropriate vehicles. The vehicle used should
neither interfere with the test compound nor
produce toxic effects. Fresh preparations of the
test compound should be employed.
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HG-Chromo-Mi cronuc
2. Dose levels
At least three dose levels should be used. The
highest dose tested should be the maximum tolerated
dose or that producing some indication of toxicity
such as that evidenced by a change in the ratio of
polychromatic to normochromatic enythrocytes.
3. Route of administration
The route of administration should be chosen by the
investigator based upon the nature of the test
chemical. The usual routes of administration are
IP or oral. However, other routes may be
appropriate when indicated by scientific evidence.
4. T re ait me n t s ch e d ul e
Test substances should generally be administered
only once. However, based upon pharmocokinetic
information a repeated treatment schedule may be
employed. The repeated treatment schedule can only
be applied if the test substance does not exhibit
cytotoxic effects in the bone marrow at the doses
used.
V. TEST PERFORMANCE
A. Treatment and sampling times
1. Animals should be treated with the test substance
once. Sampling times should coincide with the
maximum response of the assay which varies with the
test substance. Therefore, bone marrow samples
should be taken at least three times, starting not
earlier than 12 hours after treatment, with
appropriate intervals following the first sample
but not extending beyond 72 hours.
2. If pharmacokinetic and metabolic information
indicate a repeated treatment schedule, repeated
dosing may be used and samples should be taken at
least three times, starting not earlier than 12
hours after the last treatment and at appropriate
intervals following the first sample, but not
extending beyond 72 hours. In either case, if the
maximum sensitive period is not known, at least one
sample should be taken at approximately 24 hours
after treatment.
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HG-Chromo-Micronuc
3. Bone marrow is obtained from both femurs of freshly
killed animals. Cells are prepared, put on slides,
spread as a smear and stained.
B. ANALYSIS
Slides should be coded before microscopic analysis. At
least 1000 polychromatic erthyrocytes per animal should
be scored for the incidence of micronuclei. The ratio
of polychromatic to normochromatic erythrocytes should
be determined for each animal by counting a total of
1000 erythrocytes. Additional information may be
obtained by scoring normochromatic erythrocytes for
micronuclei.
VI. DATA AND REPORT
A. Treatment of results
Criteria for scoring micronuclei should be given.
Individual data should be presented in a tabular form
including positive and negative (vehicle) controls and
experimental groups. The number of polychromatic
erythrocytes scored, the number of micronucleated
polychromatic erythrocytes, the percentage of
micronucleated cells, the number of micronucleated
normochromatic erythrocytes, and, if applicable, the
percentage of micronucleated erythrocytes and the ratio
of normochromatic to polychromatic erythrocytes should
be listed separately for each experimental and control
animal. Absolute numbers should be included if
percentages are reported.
B. S t a t i s tic a1 e ya 1uat i on
Several statistical techniques are acceptable in
evaluating the results of this test. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive response, one of which is a statistically
significant dose related increase in the number of
micronucleated polychromatic erythrocytes. Another
criterion may be based upon detection of a
reproducible and statistically significant positive
response for at least one of the test substance
concentrations. However, the final decision must
be based upon good scientific judgement.
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HG-Chromo-Micronuc
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of micronucleated polychromatic
erythrocytes nor a statistically significant and
reproducible positive response at any one of the
test points is considered nonmutagenic in this
system. Again, the final decision must be based
upon good scientific judgement.
D. Test evaluation
1. The results of the micronucleus test provide
information on the ability of a chemical to induce
micronuclei in polychromatic erythrocytes of the
test species which may have been the result of
chromosomal damage or damage to the mitotic
apparatus.
2. Negative results indicate that under the test
conditions the test substance does not produce
micronuclei in the bone marrow of the test species.
E. Test report
The test report should include the following
information:
1. species, strain, age, weight, number and sex of
animals in each treatment and control group;
2. test chemical vehicle, dose levels used, rationale
for dose selection;
3. rationale for and description of treatment and
sampling schedules, toxicity data, negative and
positive controls;
4. details of the protocol used for slide preparation;
5. criteria for identifying micronucleated
erythrocytes;
6. dose-response relationship if applicable;
7. statistical evaluation;
8. discussion of results; and
9. interpretation of results.
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HG-Chromo-Mi cronuc
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should
not be considered the only source of information on test
performance, however.
1. Cihak R. 1979. Evaluation of benzidine by the
micronucleus test. Mutation Research 67:383-384.
2. Cole RJ, Taylor N, Cole J, Arlett CF. Short-term tests
for transplacentally active carcinogens. 1.
Micronucleus formation in fetal and maternal mouse
erythroblasts. 1981. Mutation Research 80:141-157.
3. Kliesch U, Danford N, Adler ID. 1981. Micronucleus
test and bone-marrow chromosome analysis. A comparison
of 2 methods in vivo for evaluating chemically induced
chromosomal alterations. Mutation Research 80:321-332.
4. Matter B, Schmid W. 1971. Trenimon-induced chromosomal
damage in bone-marrow cells of six mammalian species,
evaluated by the micronucleus test. Mutation Research
12:417-425.
5. Schmid W. 1975. The micronucleus test. Mutation
Research 31:9-15.
6. Schmid W. 1976. The micronucleus test for cytogenetic
analysis. In: Chemical mutagens, principles and
methods for their detection, Vol. 4. Hollaender A,
ed. New York and London: Plenum Press, pp. 31-53.
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HG-Chromo-Insects
August, 1982
HERITABLE TRANSLOCATION TEST IN
DROSOPHILA MELANOGASTER
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Chromo-Insects
I. PURPOSE
The heritable translocation test in Prosophi1a measures the
induction of chromosomal translocations in germ cells of
insects. Stocks carrying genetic markers on two or more
chromosomes are used to follow the assortment of chromosomes
in meiosis. The F-^ male progeny of treated parents are
individually mated to females and the Fo progeny phenotypes
are scored. The observed spectrum of phenotypes is used to
determine the presence or absence of a translocation. This
is usually indicated by a lack of independent assortment of
genes on different chromosomes.
II. DEFINITIONS
A. Chromosome mutations are chromosomal changes resulting
from breakage and reunion of chromosomes. Chromosomal
mutations are also produced through nondisjunction of
chromosomes during cell division.
B. Reciprocal translocations are chromosomal translocations
resulting from reciprocal exchanges between two or more
chromosomes.
C. Heritable translocations are reciprocal translocations
transmitted from parent to the succeeding progeny.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, ethyl
methanesulfonate or N-dimethyl-nitrosamine.
IV. TEST METHOD
A. Principle
The method is based on the principle that balanced
reciprocal chromosomal translocations can be induced by
chemicals in the germ cells of treated flies and that
these translocations are detected in the F2 progeny
using genetic markers (mutations). Different mutations
may be used as genetic markers and two or more of the
four chromosomes may be genetically marked for inclusion
in this test.
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HG-Chromo-Insects
B. Description
Wild-type males are treated with chemical and bred with
females of known genetic markers. The F-^ males are
collected and individually bred with virgin females of
the female parental stock. The resulting F2 progeny are
scored. Putative translocation carriers are confirmed
with an F^ cross.
1• Illustrative example
The following example serves to illusrate the
method. Males carrying genes for red eye color on
chromosomes II and III are bred with females of
white eye color carrying alleles for brown (bw) on
the second chromosome and scarlet (st) and pink
(pp) on the third chromosome. The F, male progeny
are bred with virgin females of the female parental
stock and the resulting F2 progeny are examined for
eye color phenotypes. If there is no translocation
in the F^ male, then the resulting F2 progeny will
have four eye color phenotypes: red, white, orange,
and brown. If the F, male carries a translocation
between chromosomes II and III, only red and white-
eye phenotypes are obtained in the F2 generation.
This happens because the F-, translocation
heterozygote produces two Balanced (carrying either
the parental or the translocated configuration of
markers) and two unbalanced gametes. The
unbalanced gametes (carrying one normal and one
translocated chromosome) are unable to develop into
normal individuals in the F2 generation.
C. Drosophila stocks
Wild-type males and females of the genotype bwtstrpp
(white eyes) may be used in the heritable translocation
test. Other appropriately marked Drosophila stocks may
also be used.
D* Control groups
1: Concurrent controls
Concurrent positive and negative (vehicle) controls
should be included in each experiment.
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HG-Chromo-Insects
2. Positive controls
Examples of positive controls include ethyl
methanesulfonate and N-nitroso-dimethylamine.
3. Other positive controls
Other positive control reference substances may be
used.
4. Negative cont ro1s
Negative (vehicle) controls should be included.
However, if appropriate laboratory historical
control data are available, concurrent controls may
not be necessary.
E* Test chemicals
1. Vehicle
Test chemicals should be dissolved in water.
Compounds which are insoluble in water may be
dissolved or suspended in appropriate vehicles
(e.g., a mixture of ethanol and Tween-60 or 80),
and then diluted in water or saline prior to
administration. Dimethylsulfoxide should be
avoided as a vehicle.
2. Dose levels
For the initial assessment of mutagenicity, it may
be sufficient to test a single dose of the test
substance. This dose should be the maximum
tolerated dose or that which produces some
indication of toxicity. If the test is being used
to verify mutagenic activity, at least two
additional exposure levels should be used.
3. Route of administration
Exposure may be oral, by injection or by exposure
to gases or vapours. Feeding of the test compound
may be done in sugar solution. When necessary,
substances may be dissolved in 0.7% NaCl solution
and injected into the thorax or abdomen.
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HG-Chromo-Insects
V. TEST PERFORMANCE
A. Pi mating
1. In the primary screen of a chemical, it is enough
to sample one germ cell stage, either mature sperm
or spermatids (for indirect acting mutagens).
Other stages may be sampled if needed, i.e. when
mature germ cells give a positive result and data
from earlier germ cells are needed for the purpose
of risk assessment. Thus, the treated males may be
mated only once for a period of 3 days to sample
sperm or transferred every 2-3 days to cover the
entire germ cell cycle.
2. Mass matings may be performed because the control
rate for translocations in the available literature
is very low (near 0) and clustered events are
extremely rare. Mated females may be aged for 2
weeks in order to recover an enhanced incidence of
translocation due to the storage effect. The
females are then allowed to lay eggs and F, males
are collected for test mating.
B. Fl mating
FjL males should be bred with virgin females of the
parental female stock. Since each F-, male represents
one treated gamete of the male parent, the F^ males have
to be mated individually to virgin females. Each F-^
male should be mated to three females to ensure
sufficient progeny.
C. Scoring the F2 generation
F2 cultures (each representing 1 F-^ male tested) should
be scored for the presence or absence of phenotype
variations (linkage of markers) from the expected
types. The test should be designed with a predetermined
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 number of
treated chromosomes that must be analysed to detect
substances which show mutation rates close to those of
the controls. A positive test should be confirmed by F?
mating trials.
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HG-Chromo-Insects
D. Number of replicate experiments
Replicate experiments are usually performed for each
dose of the compound tested. If a chemical is a potent
inducer of translocations, one experiment may be
sufficient. Otherwise two or three replicate
experiments should be done.
VI. DATA AND REPORT
A. Treatment of results
Data should be tabulated to show the number of
translocations and the number of fertile F^ males at
each exposure for each germ cell stage sampled.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating Drosophila heritable translocation tests.
Choice of analyses should consider tests appropriate to
the experimental design (including replicate
experiments) and needed adjustments for multiple
comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
heritable translocations. Another criterion may be
based upon detection of a reproducible and
statistically significant positive response for at
least one of the test points. However, the final
decision must be based upon good scientific
judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
heritable translocations nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
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HG-Chromo-Insects
D. Test evaluation
1. Positive results in the heritable translocation
test in Drosophila indicate that the test substance
causes chromosome damage in germ cells of this
insect.
2. Negative results indicate that under the test
conditions the test substance does not cause
chromosomal damage in JD. me 1 a nog aster.
E. Test report
The test report should include the following
information:
1. Drosphila stock used in the assay, age of insects,
number of males treated, number of F2 cultures
established, number of replicate experiments;
2. test chemical vehicle, treatment and mating
schedule, exposure levels, toxicity data, dose and
route of exposure;
3. positive and negative (vehicle) controls;
4. historical control data, if available;
5. number of chromosomes scored;
6. criteria for scoring mutant chromosomes;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and
10. interpretation of results.
VII. REFERENCES
The following reference may be helpful in developing
acceptable protocols, and provides a background of
information on which this section is based. It should not
be considered the only source of information on test
performance, however.
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HG-Chromo-Insects
Wurgler FE, Sobels FHf Vogel E. 1979. Drosophila as
assay system for detecting genetic changes. In:
Handbook of mutagenicity test procedures. Kilby BJ,
Legator M, Nichols W, Ramel C, eds. Amsterdam:
Elsevier/North Holland Biomedical Press, pp. 335-374.
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HG-Chromo-Dom Lethal
August, 1982
RODENT DOMINANT LETHAL ASSAY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HGrChromo-Dom Lethal
I. PURPOSE
Dominant lethal (DL) effects caiise embryonic death. Dominant
lethals are generally accepted/to be the result of
chromosomal damage (structural and numerical anomalies).
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 may
also be the result of toxic effects.
II. DEFINITION
A dominant lethal mutation is one occurring in a germ cell,
which does not cause dysfunction of the germ cell, but which
kills the fertilized^egxr~or developing embryo.
III. REFERENCE SUBSTANCES ;
These may include, but need not be limited to,
triethylenemelamine, cyclophosphamide or ethyl
methanesulfonate. •
IV. TEST METHOD
A. Principle
Generally, male animals are exposed to the test
substance and mated to untreated virgin females. The
females are sacrificed after an appropriate period of
time and the contents of the uteri are examined to
determine the numbers of live and dead embryos. The
ratio of dead to live embryos from the treated group
compared to the ratio of dead to live embryos from the
control group is used as a measure of dominant
lethality.
B. Description
Several treatment protocols are available. The most
widely-used require single administration of the test
substance or treatment on five consecutive days. Other
treatment schedules may be used if justified by the
investigator.
c* Animal selection
1. Species
Rats or^mi.ce^are recommended as the test species.
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HG-Chromo-Dom Lethal
2. Age
\
v
Healthy sexually mature animals should be used.
\
3. Number
N
A decision on the minimum number of treated males
should be based on the number of females to which
the male is mated, on average litter size and on
practical constraints such^as number of dose
levels.
4. Assignment to groups ;
Animals should be randomized and assigned to
treatment and control groups.
/
D. Control g roups \
!• Concurrent controls
Concurrent positive and negative (vehicle) controls
should be included in each experiment.
2. Po s i t i ye co n t ro1s
Any compound known to induce DL in the species
being tested is acceptable as a positive control
reference substance. Triethylenemelamine,
cyclophosphamide and ethyl methanesulfonate are
examples of positive controls.
E. Test chemicals
1. Vehicle
When possible, test substances should be dissolved
or suspended in isotonic saline. 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.
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HG-Chromo-Dom Lethal
2. Dose levels
At least three dose levels should be used. The
highest dose tested should lead to signs of
toxicity or reduced fertility but should not induce
death or complete sterility. This dose may be
determined in a preliminary experiment or may be
available from other studies on the toxicity of the
agent. Nontoxic chemicals should be tested up to 5
g/kg on a single administration and up to 1
g/kg/day on repeated administration.
3. Route of ad mi n is t rat ion
The route of administration should be chosen by the
investigator based upon the nature of the test
chemical. The usual routes of administration are
oral or by IP injection. Other routes of
administration may be used.
V. TEST PERFORMANCE
A. Treatment
Male animals should be treated by the chosen route for
the selected time interval.
B. Mating
At the end of the treatment period, each male should be
mated to 1 or 2 virgin or nulliparous females. Females
should be left with the males for at least the duration
of one estrus cycle or until mating has occurred as
determined by the presence of sperm in the vagina or by
the presence of a vaginal plug. At the end of the
mating period, females should be removed and replaced
with 1 or 2 additional females. The mating schedule
should be governed by the treatment schedule and should
sample the entire spermatogenic cycle.
C. .Sacri_f ice_
Females should be sacrificed at approximately
midpregnancy and uterine contents examined to determine
number of pregnant females and number of live and dead
implants. It is recommended that females not be
sacrificed later than the seventeenth day of
pregnancy. The determination of number of corpora lutea
and estimation of preimplantation loss are left to the
discretion of the investigator.
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HG-Chromo-Dom Lethal
VI. DATA AND REPORT
A. Tr e a tme n t of re suit s
Data should be tabulated 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, dose level and week of
mating, the frequencies of live implants and of dead
implants should be enumerated. If the data are recorded
as early and late deaths, the tables should make that
clear. If preimplantation loss is estimated, it should
be reported. Preimplantation loss can be calculated as
a discrepancy between the number of corpora lutea and
the number of implants or as a reduction in the average
number of implants per uterus in comparision with
control matings.
B. Statist i cal eyalua t ion
Several statistical techniques are acceptable in
evaluating the results of DL assays. The male is
considered the experimental unit. One technique is an
analysis of variance which includes males, females,
weeks, and doses as variables. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation pj results^
1. There are several criteria for determining 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 positive response for at least one of
the test points. However, the final decision must
be based upon good scientific judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
dominant lethals nor a statistically significant
and reproducible positive response at any one of
the test points is considered nonmutagenic in this
system. Again, the final decision must be based
upon good scientific judgement.
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HG-Chromo-Dom Lethal
D* Test evaluation
1. A positive DL assay suggests the possible
genotoxicity of the test substance in the germ
cells of the test species.
2. Negative results suggest that under the conditions
of the test the test substance may not be genotoxic
in the germ cells of the test species.
E. Test Report
The test report should include the following
information:
1. species, strain and age of animals used, number of
animals of each sex in experimental and control
groups;
2. test chemical vehicle, dose levels tested and
rationale for dosage selection, negative and
positive controls, toxicity data;
3. route and duration of exposure;
4. mating schedule;
5. method used to determine that mating has occured;
6. time of sacrifice;
7. criteria for scoring dominant lethals;
8. dose-response relationship, if applicable;
9. statistical evaluation;
10. discussion of results, and
11. interpretation of results.
VII. References
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
-------
HG-Chromo-Dom Lethal
1. Brewen JGf Payne HS, Jones KP, Preston RJ. 1975.
Studies on chemically induced dominant lethality. I.
The cytogenetic basis of MMS-induced dominant lethality
in post-meiotic germ cells. Mutation Research 33:239-
250.
2. Ehling UH, Machemer L, Buselmaier E, Dycka D, Frohberg
H, Kratochvilova J, Lang R, Lorke D, Muller D, Pheh Jf
Rohrborn G, Roll R, Schulze-Schencking M, Wiemann H.
1978. 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.
-------
HG-Chromo-Herit Translocat
August, 1982
RODENT HERITABLE TRANSLOCATION ASSAYS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Chromo-Herit Translocat
I. PURPOSE
This test detects transmitted chromosomal damage which
manifests as balanced reciprocal translocations in progeny
descended from parental males treated with chemical
mutagens.
II. DEFINITIONS
A. A heritable translocation is one in which end segments
of nonhomologous chromosomes are involved in a
reciprocal exchange.
B. Diakinesis and metaphase 1 are stages of meiotic
prophase scored cytologically for the presence of
multivalent chromosome association characteristic of
translocation carriers.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
When a balanced reciprocal translocation is induced in a
parental male germ cell/ the resulting progeny is a
translocation heterozygote.
1. Basis for fertili ty screening
Male translocation heterozygotes may be completely
sterile. This class consists of two types of sex-
autosome translocations:
a. translocations between autosomes in which at
least one of the breaks occurs close to one
end of a chromosome; and,
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HG-Chromo-Herit Translocat
b. those that carry multiple translocations. The
majority of male translocation heterozygotes
are semisterile - they carry one or (rarely)
two translocations. The degree of
semisterility is dependent upon the
proportions of balanced and unbalanced
(duplication-deficiency) gametes produced in
the ejaculate as a function of meiotic
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.
2. Basis for cytological screening
The great majority of male translocation
heterozygotes can be identified cytologically
through analysis of diakinesis metaphase I
spermatocytes. Translocation heterozygotes are
characterized 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
spermatogonial and somatic cell metaphase
preparations.
B. Description
Essentially, two methods have been used to screen for
translocation heterozygosity; one method uses a mating
sequence to identify sterile and semisterile males
followed by cytological examination of these male
individuals; the other method deletes the mating
sequence altogether and all F-^ 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 confirmation as translocation heterozygotes.
C. Animal selection
1. Species
The mouse is the species generally used, and is
recommended.
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HG-Chromo-Herit Translocat
2. Age
Healthy sexually mature animals should be used.
3. Number
a. The number of male animals necessary is
determined by the following factors:
(1) the use of either historical or
concurrent controls;
(2) the power of the test;
(3) the minimal rate of induction required;
(4) whether positive controls are used and;
(5) the level of significance desired.
b. At least 300 progeny per dose should be
tested.
4. Assignment to groups
Animals should be randomized and assigned to
treatment and control groups.
D. Control groups
1. Concurrent controls
No concurrent positive 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 chemical.
2. 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.
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HG-Chromo-Herit Translocat
E. Test chemicals
1. Vehicle
Solid and liquid test substances should be
dissolved or suspended in isotonic saline.
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.
2. Dose levels
At least two dose levels should be used. The
highest dose level should result in toxic effects
but should not produce an incidence of fatalities
which would prevent a meaningful evaluation.
3. Route of administration
The route of administration should be chosen by the
investigator based upon the nature of the test
chemical. Acceptable routes of administration
include oral, inhalation, admixture with food or
water, and IP or IV injection.
V. TEST PERFORMANCE
A. Treatment and mating
The animals should be dosed with the test substance 7
days/week over a period of 35 days. After treatment,
each male should be caged with 2 untreated females for a
period 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 are recorded. All male progeny should be
weaned and all female progeny should be discarded.
B. Testing for translocation heterozygosity
i
When males are sexually mature, testing for
translocation heterozygosity should begin. One of two
methods should be used; the first method involves
mating, determining those animals which are sterile or
semisterile and subsequent cytological analysis of
suspect progeny; the other method does not involve
mating and determining sterility or semisterility; all
progeny are examined cytologically.
-------
HG-Chromo-Herit Translocat
1. Determination of sterility or s_emisterility
a. Convent ional me thod
Females are mated, usually three females for
each male, and each female is killed at
midpregnancy. Living and dead implantations
are counted. Criteria for determining normal
and semisterile males are usually established
for each new strain because the number of dead
implantations varies considerably among
strains.
b. Sequential method
Males to be tested are caged individually with
females and the majority of the presumably
normal males are identified on the basis of a
predetermined size of 1 or 2 litters.
Breeding pens are examined daily on weekdays
beginning 18 days after pairing. Young are
discarded immediately after they are scored.
Males that sire a litter whose size is the
same as or greater than the minimum set for a
translocation-free condition are discarded
with their litter. If the litter size is
smaller than the predetermined number, a
second litter 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.
2« Cytological analysis
For cytological analysis of suspected semisteriles,
the air-drying technique is used. Observation of
at least 2 diakinesis-metaphase 1 cells with
mutivalent association constitutes the required
evidence for the presence of a translocation.
Sterile males are examined by one of two methods,
those with testes of normal size and sperm in the
epididymis are examined by the same techniques used
for semisteriles. Animals with small testes are
examined by squash preparations or, alternatively,
by examination of mitotic metaphase preparations.
If squash preparations do not yield diakinesis-
metaphase 1 cells, analysis of spermtogonia or bone
marrow for the presence of unusually long or short
chromosomes should be performed.
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HG-Chromo-Herit Translocat
VI. DATA AND REPORT
A. Treatment of results
1. 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 steriles
and semisteriles (if the fertility test is used),
the number of cytogenetically confirmed
translocation heterozygotes (if the fertility test
is used, report the number of confirmed steriles
and confirmed partial steriles), the translocation
rate, and either the standard error of the rate or
the upper 95% confidence limit on the rate.
2. 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.
B. S tat i sit i ca 1 ey a 1 ua t ion
Several statistical techniques are acceptable in
evaluating results of this test. For small numbers of
mutations, exact tests are preferred. Choice of
analyses should consider the nature of the controls,
concurrent or historical, and needed adjustments for
multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related increase in the number of
heritable translocations. Another criterion may be
based upon detection of a reproducible and
statistically significant positive response for at
least one of the test points. However, the final
decision must be based upon good scientific
judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
heritable translocations nor a statistically
significant and reproducible positive response at
any one of the test points is considered non-
mutagenic in this system. Again, the final
decision must be based upon good scientific
judgement.
-------
HG-Chromo-Herit Translocat
D. Test evaluation
1. Positive results in the heritable translocation
assay indicate that the test substance causes
heritable chromosomal damage in the test species.
2. Negative results indicate that under the test
conditions the test substance does not cause
heritable chromosomal damage in the test species.
E. Test report
The test report should include the following
information:
1. species, strain, age, weight and number of animals
of each sex in each group;
2. test chemical vehicle, route and schedule of
administration, toxicity data;
3. dosing regimen, doses tested and rationale for
dosage selection;
4. mating schedule, number of females mated to each
male;
5. the use of historical or concurrent controls;
6. screening procedure including the decision criteria
used and the method by which they were determined;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and,
10. interpretation of results.
-------
HG-Chromo-Herit Translocat
VII. REFERENCES
The following reference may be helpful in developing
acceptable protocols, and provides a background of
information on which this section is based. It should not
be considered the only source of information on test
performance, however.
1. Generoso WM, Bishop JB, Goslee DG, Newell GW, Sheu C-J,
von Halle E. 1980. Heritable translocation test in
mice. Mutation Research 76:191-215.
8
-------
HG-DNA-Damage/Repair
August, 1982
DIFFERENTIAL GROWTH INHIBITION OF
REPAIR PROFICIENT AND REPAIR DEFICIENT
BACTERIA: "BACTERIAL DNA DAMAGE
OR REPAIR TESTS"
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-DNA-Damage/Repair
I. PURPOSE
Bacterial DNA damage or repair tests measure DNA damage
which is expressed as differential cell killing or growth
inhibition of repair deficient bacteria in a set of repair
proficient and deficient strains. These tests do not measure
mutagenic events per se. They are used as an indication of
the interaction of a chemical with genetic material implying
the potential for genotoxicity.
II. DEFINITION
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.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to,
chloramphenicol or methyl methanesulfonate.
IV. TEST METHOD
A. 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 or absence
of specific DNA repair genes. The response is expressed
in the preferential inhibition of growth or the
preferential killing of the DNA repair deficient strain
since it is incapable of removing certain chemical
lesions from its DNA.
B. Description
Several methods for performing the test have been
described. Those described here are:
1. tests performed on solid medium (diffusion tests);
and
2. tests performed in liquid culture (suspension
tests).
-------
HG-DNA-Daraage/Repair
C. Strain selection
1. Designation
At the present time, Escherichia coli polA
(W3110/p3478) or Bacillus subtilis rec (H17/M45)
pairs are recommended. Other pairs may be utilized
when appropriate.
2. 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.
D. Bacterial growth
Good microbiological techniques should be used to grow
fresh cultures of bacteria. The phase of growth and
cell density should be documented and should be adequate
for the experimental design.
E. Metabolic activation
Bacteria should be exposed to the test substance both in
the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducing agents. The use of other species, tissues or
techniques may also be appropriate.
F. Control groups
1. Concurrent controls
Concurrent positive, negative, and vehicle controls
should be included in each assay.
2« Negative controls
The negative control should show nonpreferential
growth inhibition (i.e., should affect both strains
equally). Chloramphenicol is an example of a
negative control.
-------
HG-DNA-Damage/Repair
3. Genotype specific conj^rc?ls
Examples of genotype specific positive controls are
methyl methanesulfonate for pplA strains and
mitomycin C for rec strains.
4. Positive controls to ensutre the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test.
5. Other positive controls
Other positive control reference substances may be
used.
G. Test chemicals
1. Vehicle
Test chemicals and positive and negative control
reference substances should be dissolved in an
appropriate vehicle and then further diluted in
vehicle for use in the assay.
2. Exposure conceritrations
The test should initially be performed over a broad
range of concentrations. When appropriate, a
positive response should be confirmed by testing
over a narrow range of concentrations. Among the
criteria to be taken into consideration for
determining the upper limits of test chemical
concentration are cytotoxicity and solubility.
Cytotoxicity of the test chemical may be altered in
the presence of metabolic activation systems. For
freely soluble nontoxic chemicals, the upper test
chemical concentration should be determined on a
case by case basis. Because results are expressed
as diameters of zones of growth inhibition in the
diffusion test, it is most important that the
amounts of chemical on the disc (or in the wells)
are exact replicates.
-------
HG-DNA-Damage/Repair
V. TEST PERFORMANCE
A. Diffusion assays
1. Disc diffusion assays
Disc diffusion assays may be performed in two ways:
a. a single strain of bacteria may be added to an
agar overlay or spread on the surface of the
agar and the test chemical placed on a filter
disc on the surface of the agar or;
b. DNA repair proficient and DNA repair deficient
bacteria may be streaked in a line on the
surface of the agar of the same plate and a
disc saturated with test chemical placed on
the surface of the agar in contact with the
streaks.
• - 2• Well diffusion assays
In well diffusion assays, bacteria may be either
added to the agar overlay or spread onto the
surface of the agar. A solution of the test
chemical is then placed into a well in the agar.
B. S ui spension ass ays
1. A bacterial suspension may be exposed to the test
chemical and the number of surviving bacteria
determined (as colony-forming units) either as a
function of time of treatment or as a function of
the concentration of test agent.
2. Nonturbid suspensions of bacteria may be exposed to
serial dilutions of the test agent and a minimal
inhibitory concentration for each strain
determined, as evidenced by the presence or absence
of visible growth after a period of incubation.
»
3. Paired bacterial suspensions (usually with some
initial turbidity) may be treated with a single
dose of the chemical. Positive results are
indicated by a differential inhibition in the rate
of increase of turbidity of the paired cultures.
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HG-DNA-Damage/Repair
C. Number of ^ultujres
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.
D. Incubat ion cond it ions
All plates in a given test should be incubated for the
same time period. This incubation period should be for
18-24 hrs at 37 C.
VI. DATA AND REPORT
A. Treatment of results
!• Diffusion assays
Results should be expressed in diameters of zones
of growth inhibition in millimeters or as areas
derived therefrom as mm . Dose response data, if
available, should be presented using the same
units.
2. Liquid suspension assays
a. Survival data can be presented as dose
responses, preferably as percentage of
survivors or fractional survival of each
strain or as a relative survival (ratio) of
the two strains.
b. Results can also be expressed as the
concentrations required to effect a
predetermined survival rate (e.g, D^, the
dose permiting 37% survival). These data are
derived from the survival curve. The
concentration should be expressed as weight
per volume, as moles, or as molarity.
c. Similarly, results can be expressed as minimal
inhibitory concentration or as minimal lethal
dose. The former is determined by the absence
of visible growth in liquid medium and the
latter is determined by plating dilutions onto
semisolid media.
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HG-DNA-Damage/Repair
3. In all tests, concentrations must be given as the
final concentrations during the treatment. Raw
data, prior to transformation, should be
provided. These should include actual quantities
measured, e.g., neat numbers. For measurement of
diffusion, the diameters of the discs and/or well
should be indicated and the measurements should
indicate whether the diameter of the discs and/or
well was subtracted. Moreover, mention should be
made as to whether the test chemical gave a sharp,
diffuse, or double-zone of growth inhibition. If
it is the latter, the investigator should indicate
whether the inner or the outer zone was measured.
4. Viability data should be given as the actual plate
counts with an indication of the dilution used and
the volume plated or as derived titers (cells per
ml). Transformed data alone in the absence of
experimental data are not acceptable (i.e, ratios,
differences, survival fraction).
B. Statistical evaluation
Such standard bioassay analyses as are used for
antibiotic data may be used. These consist of fitting
either logit or probit models of the survival data and
determining whether the difference in slopes is
significantly different from zero. For data where the
measurement is zone of inhibition, standard regression
analyses may be used. Nonparametric analyses may be
appropriate with small numbers of replicates.
c• Interpretation of results
1. There are several criteria for determining a
positive result, one of which is a statistically
significant dose-related preferential inhibition or
killing of the repair deficient strain. Another
criterion may be based upon detection of a
reproducible and statistically significant positive
response for at least one of the test points.
However, the final decision must be based upon good
scientific judgement.
2. A test substance producing neither a statistically
significant dose-related preferential inhibition or
killing of the repair deficient strain nor a
statistically significant and reproducible positive
response at any one of the test points is
considered nonmutagenic in this system. Again, the
final decision must be based upon good scientific
judgement.
-------
HG-DNA-Damage/Repair
D. Test evaluation
DNA damage tests in bacteria do not measure DNA repair
per se nor do they measure mutations. They measure DNA
damage which is expressed as cell killing or growth
inhibition. A positive result in a DNA damage test in
the absence of a positive result in another system is
difficult to evaluate in the absence of a better data
base.
E. Test Report
The test report should include the following
information:
1. bacterial strains used;
2. phase of bacterial cell growth at time of use in
the assay;
3. media composition;
4. details of the protocol used for metabolic
activation;
5. treatment protocol, including doses used and
rationale for dose selection, positive and negative
controls;
6. method used for determination of degree of cell
kill;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and
10. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
-------
HG-DNA-Damage/Repa i r
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Kada T, Sadie Y, Tutikawa K. 1972. In vitro and host-
mediated "rec-assay" procedures for screening chemical
mutagens; and phloxine, a mutagenic red dye detected.
Mutation Research 16:165-174.
3. Leifer Z, Kada T, Mandel M, Zeiger E, Stafford R,
Rosenkranz HS. 1981. An evaluation of bacterial DNA
repair tests for predicting genotoxicity and
carcinogenicity: a report of the U.S. EPA's Gene-Tox
Program. Mutation Research 87:211-297.
4. Slater EE, Anderson MD, Rosenkranz HS. 1971. Rapid
detection of mutagens and carcinogens. Cancer Res
31:970-973.
8
-------
HG-DNA-Unsched Syn
I. 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.
II. DEFINITION
In this guideline, unscheduled DNA synthesis in mammalian
cells in culture is defined as the incorporation of tritium
labelled thymidine (3H-TdR) into the DNA of cells which are
not in the S phase of the cell cycle.
III. REFERENCE SUBSTANCES
These may include, but need not be limited to, 7,12-
dimethylbenzanthracene, 2-acetylaminofluorene, 4-
nitroquinoline oxide or N-dimethyl-nitrosamine.
IV. TEST METHOD
A. Principle
Mammalian cells in culture, either primary rat
hepatocytes or established cell lines, are exposed to
the test agent. Established cell lines are treated both
with and without metabolic activation. UDS is measured
by the uptake of 3n-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.
B. Description
1. Autorad ipgraphy
For autoradiography, coverslip cultures of cells
are exposed to test chemical in medium containing
JH-TdR. At the end of the treatment period, cells
are fixed, dipped in autoradiographic emulsion, and
exposed at 4 C. At the end of the exposure period,
cells are stained and labeled nuclei are counted
either manually or with an electronic counter.
Established cell lines should be treated both with
and without metabolic activation.
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HG-DNA-Unsched Syn
2. LSC det e rmination s
For LSC determinations of UDS, confluent cultures
of cells are treated with test chemical both with
and without metabolic activation. At the end of
the exposure period, DNA is extracted from the
treated cells. Total DNA content is determined
biochemically and extent of 3H-TdR incorporation is
determined by scintillation counting.
C. Cells
!• Type of ce1Is used in the assay
Primary cultures of rat hepatocytes or established
cell lines (e.g., human diploid fibroblasts) may be
used in the assay.
2• Cell growth and maintenance^
Appropriate growth media CG>2 concentration,
temperature and humidity should be used in
maintaining cultures. Established cell lines
should be periodically checked for Mycoplasma
contamination. It is also desirable to check~ the
cells periodically for karyotype stability.
D. Metabolic activation
1. A metabolic activation system is not used with
primary hepatocyte cultures.
2. Established cell lines should be exposed to test
substance both in the presence and absence of an
appropriate metabolic activation system. The most
commonly used systems are cofactor supplemented
postmitochondrial fractions prepared from the
livers of mammals treated with enzyme inducers.
The use of other tissues or techniques may also be
appropriate.
E* Control g roups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls with and without metabolic
activation should be included in each experiment.
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HG-DNA-Unsched Syn
August, 1982
UNSCHEDULED DNA SYNTHESIS IN MAMMALIAN
CELLS IN CULTURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-DNA-Unsched Syn
2» Positive controls for rat hepatocyte assays
Examples of positive controls for the rat
hepatocyte assay include 7,12-
dimethylbenzanthracene or 2-acetylaminofluorene.
3. Positive controls for assays with established
cell lines
a. Direct acting positive controls
4-Nitroguinoline oxide is an example of a
positive control for both the autoradiographic
and LSC assays performed without metabolic
activation.
k* Positive controls to ensure the efficacy of
the activation system
The positive control reference substance for
tests including a metabolic activation system
should be selected on the basis of the type of
activation system used in the test. For both
autoradiographic and LSC assays, N-dimethyl-
nitrosamine is an example of a positive
control compound in tests using
postmitochondrial fractions from the livers of
rodents treated with enzyme inducing agents
such as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may be
used.
F. Test chemicals
1. Vehicle
Test chemicals and positive control reference
substances may be prepared in growth medium or
dissolved or suspended in appropriate vehicles and
then further diluted in growth medium for use in
the assay. Final concentration of the vehicle
should not affect cell viability.
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HG-DNA-Unsched Syn
2. Exposure concentrations
Multiple concentrations of test substance, based
upon cytotoxicity, and over a range adequate to
define the response should be used. Generally, at
least five exposure concentrations covering a 2-log
range should be tested. 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.
V. TEST PERFORMANCE
A. Primary rat hepatocytes
Freshly isolated rat hepatocytes should be treated with
chemical in medium containing 3n-TdR. At the end of the
treatment period, cells should be drained of medium,
rinsed, fixed, dried and attached to microscope
slides. Slides should be dipped in autoradiographic
emulsion, exposed at 4 C for an appropriate length of
time, developed, stained and counted.
B. Established cell lines
1. Autoradipgraphic techniques
The techniques for treatment of established cell
lines are the same as those for primary rat
hepatocytes except that cells must not enter S
phase prior to treatment. Entry of cells into S
phase may be blocked by growth in arginine
deficient medium, by growth in medium low in serum
content or by the use of hydroxyurea. Tests should
be done both in the presence and absence of a
metabolic activation system.
2. LSC measurement of UPS
Prior to treatment with test agent, entry of cells
into S phase should be blocked as described
above. Cells should be exposed to the test
chemical in medium containing 3n-TdR. At the end
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HG-DNA-Unsched Syn
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.
c* Acceptable ba ckgrqund f req_uen_cie_s_
1. Autoradiographic determinations
In determining UDS in cells in culture, S phase
nuclei in both treated and control populations are
not counted. 3n-TdR incorporation in the cytoplasm
should be determined by counting three nucleus-
sized areas in the cytoplasm of each cell
counted. The value of 3n-TdR incorporation in the
cytoplasm should be subtracted from the number of
grains found over the cell nucleus to give the net
incorporation rate. In solvent treated control
cultures, net incorporation into the nucleus should
be less than 1.
2. LSC determinations
Historical background incorporation rates of 3n_TdR
into untreated established cell lines should be
established for each laboratory.
D. Number o f ce11s coun ted
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.
E. Number of cultures
Six independent cultures at each concentration and
control should be used in LSC UDS determinations.
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HG-DNA-Unsched Syn
VI. DATA AND REPORT
A • Treatment of results
1. Autorad iograph i c de terminat ions
For autoradiographic determinations, once
untransformed data are recorded, background counts
should be subtracted to give the correct nucleatf
grain count. Values should be reported as net
grains per nucleus. Mean, median and mode may be
used to describe the distribution of net grains per
nucleus.
2. LSC determinations
For LSC determinations, 3H-TdR incorporation should
be reported as dpm/ug DNA. Average dpm/ug DNA with
standard deviation or standard error of the mean
may be used to describe distribution of
incorporation in these studies.
B. Statistical evaluation
Several statistical techniques are acceptable in
evaluating this test. Choice of analyses should
consider experimental design and adjustments needed for
multiple comparisons.
C. Interpretation of results
1. There are several criteria for determining 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 at least one of the test points.
However, the final decision must be based upon good
scientific judgement.
2. A test substance which produces neither a
statistically significant dose-related increase in
the incorporation of 3H-TdR into treated cells nor
a statistically significant and reproducible
positive response at any one of the test points is
considered nonmutagenic in this system. Again, the
final decision must be based upon good scientific
judgement.
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HG-DNA-Unsched Syn
D. Test evaluation
1. Positive results in the UDS assay indicate that the
test substance may have the potential to cause DNA
damage in cultured mammalian somatic cells.
2. Negative results indicate that under the test
conditions the test substance may not have the
potential to cause DNA damage in cultured mammalian
somatic cells.
E. Test report
The test report should include the following
information:
1. cells used, density and passage number at time of
treatment, number of cell cultures;
2. methods use for maintenance of cell cultures
including medium, temperature and C02
concentration;
3. test chemical vehicle, concentration and rationale
for selection of concentrations used in the assay;
4. details of the protocol used for metabolic
activation;
5. treatment protocol;
6. positive and negative controls;
7. protocol used for autoradiography;
8. details of the method used to block entry of cells
into S phase;
9. details of the methods used for DNA extraction and
determination of total DNA content in LSC
determinations;
I
10. historical background incorporation rates of 3H-TdR
in untreated cell lines;
11. dose-response relationship, if applicable;
12. statistical evaluation;
13. discussion of results; and
14. interpretation of results.
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HG-DNA-Unsched Syn
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonel1a/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Rasmussen RE, Painter RB. 1966. Radiation-stimulated
DNA synthesis in cultured mammalian cells. J Cell Biol
29:11-19.
3. Stich HF, San PPS, Lam KJ, Koropatnick DJ, Lo LW,
Laishes BA. 1976. DNA fragmentation and DNA repair as
an in vitro and in vivo assay for chemical
procarcinogens, carcinogens and carcinogenic nitrosation
products. In: Screening tests in chemical
carcinogenesis. Bartsch H, Tomatis L, eds. Lyon: IARC
Scientific Publications, No. 12, pp. 617-636.
4. Williams GM. 1976. Carcinogen-induced DNA repair in
primary rat liver cell cultures: a possible screen for
chemical carcinogens. Cancer Letters 1:231-236.
5. Williams GM. 1977. Detection of chemical carcinogens
by unscheduled DNA synthesis in rat liver primary cell
cultures. Cancer Res 37:1845-1851.
8
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HG-DNA-Gene Conversion
August, 1982
MITOTIC GENE CONVERSION IN
SACCHAROMYCES CEREVISIAE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-DNA-Gene Conversion
I. PURPOSE
The mitotic gene conversion assay in the yeast, Saccharomyces
cerevisiae, measures the conversion of differentially
inactive alleles to wild-type alleles by mutagenic agents.
Heteroallelic 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
heteroallelic diploids grow only in medium supplemented with
a specific nutrient such as tryptophan. When gene conversion
occurs, a fully active wild-type phenotype is produced from
these inactive alleles through intragenic recombination.
These wild-type colonies grow on a medium lacking the
specific nutritional requirement (selective medium).
II. DEFINITIONS
A. Mitotic gene conversion is detected by the change of
inactive alleles of the same gene to wild-type alleles
through intragenic recombination in mitotic cells.
B. Heteroallelic diploids are diploid strains of yeast
carrying two different, inactive alleles of the same
gene locus causing a nutritional requirement.
III. REFERENCE SUBSTANCES
These may include but need not be limited to, hydrazine
sulfate or 2-acetylaminofluorene.
IV. TEST METHOD
A. Principle
The method is based on the fact that heteroallelic
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 mutagenic
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).
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HG-DNA-Gene Conversion
B. Description^
Heteroallelic diploid strains such as D7, requiring 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 medium as a result of gene conversion are
scored.
C. Strain selection
1. Designation
At the present timef S. cerevisiae strain D7 is
recommended for use in thTs assay. The use of
other strains may also be appropriate.
2• Preparatig^n 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.
3. Media
YEP glucose medium enriched with the appropriate
growth factors may be used for cell growth and
maintenance. Other media may also be appropriate.
D. Selection gf!___cultures
Cells should be grown with aeration in liquid medium
enriched with growth factors to early stationary
phase. Cells should then be seeded on selective medium
to determine the rate of spontaneous conversion.
Cultures with a high rate of spontaneous conversion
should be discarded.
E. Metabolic activation
Cells should be exposed to test chemical both in the
presence and absence of an appropriate metabolic
activation system. The most commonly used system is a
cofactor supplemented postmitochondrial fraction
prepared from the livers of rodents treated with enzyme
inducing agents. The use of other species, tissues or
techniques may also be appropriate.
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HG-DNA-Gene Conversion
F. Co n t r o 1 g JT g u p s
1. Con cu r rent con t rpis
Concurrent positive and negative (untreated and/or
vehicle) controls should be included in each
expermient.
2. Direct acting positive controls
Hydrazine sulfate is an example of a positive
control for experiments without metabolic
activation.
3. Positive controls to ensure the efficacy of the
activation system
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test. 2-Acetylaminofluorene is
an example of a positive control compound in tests
using postmitochondrial fractions from the livers
of rodents treated with enzyme inducing agents such
as Aroclor-1254.
4. Other positive controls
Other positive control reference substances may
also be used.
G. Test chemicals
1. 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. Dimethylsulfoxide should be avoided
as a vehicle.
2. Expos ur e con <;ent r a tl pns
The test should initially be performed over a broad
range of concentrations. When appropriate, a
positive response should be confirmed by using a
narrow range of concentrations. Among the criteria
to be taken into consideration for determining the
upper limits of test chemical concentration are
cytotoxicity and solubility. Cytotoxicity of the
test chemical may be altered in the presence of
-------
HG-DNA-Gene Conversion
metabolic activation systems. For cytotoxic
chemicals, the highest dose tested should not
reduce survival to less than 10% of that seen in
the untreated 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.
V. TEST PERFORMANCE
A. Treatment
Cultures should be treated in liquid suspension.
Resting cells should be treated in buffer; growing cells
should be treated in a synthetic medium. Cultures with
low spontaneous convertant frequencies should be
centrifuged, washed and resuspended 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 centrifuged, 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.
B. Number of cultures
At least six individual plates per treatment
concentration and control should be used.
C. Incubation conditions
All plates in a given experiment should be incubated for
the same time period. This incubation period may be
from 4-6 days at 28 C.
VI. DATA AND REPORT
A. 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
4
-------
HG-DNA-Gene Conversion
of viable and convertant colonies scored, survival
frequency and convertant frequencies for each treatment
and control 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.
B. Statisti ca1 e va1ua t ion
Several statistical techniques are acceptable in
evaluating this test. Choice of analyses should
consider tests appropriate to the experimental design
and needed adjustments for multiple comparisons.
^• Interpretation of results
1. There are several criteria for determining 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 positive response for at least one of
the test points. However, the final decision must
be based upon good scientific judgement.
2. A test substance producing neither a statistically
significant dose-related increase in the number of
gene conversions nor a statistically significant
and reproducible positive response at any one of
the test points is considered nonmutagenic in this
system. Again, the final decision must be based
upon good scientific judgement.
D* Test evaluation^
1. Positive results in this assay indicate that the
test chemical causes mitotic gene conversion in the
yeast J3_. cerevisiae.
2. Negative results indicate that under the test
conditions the test chemical does not cause mitotic
gene conversion in S_. cere vis _iae_.
E. Test report
The test report should include the following
information:
1. strain of organism used in the assay;
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HG-DNA-Gene Conversion
2. test chemical vehicle, doses used and rationale for
dosage selection;
3. method used to select cultures;
4. treatment protocol including cell density at
treatment and length of exposure to test substance;
5. details of the protocol used for metabolic
activation;
6. incubation times and temperatures;
7. dose-response relationship, if applicable;
8. statistical evaluation;
9. discussion of results; and
10. interpretation of results.
VII. REFERENCES
The following references may be helpful in developing
acceptable protocols, and provide a background of
information on which this section is based. They should not
be considered the only source of information on test
performance, however.
1. Ames BN, McCann J, Yamasaki E. 1975. Methods for
detecting carcinogens and mutagens with the
Salmonella/mammalian-microsome mutagenicity test.
Mutation Research 31:347-364.
2. Callen DP, Philpot RM. 1975. Cytochrome P-450 and the
activation of promutagens in Saccharomyces cerevisiae.
Mutation Research 45:309-324.
3. Zimmermann FK. 1979. Procedures used in the induction
of mitotic recombination and mutation in the yeast
SaccharpmYces cerevisiae. In: Handbook of mutagenicity
t es t proceclures~. Kilby BJ, Legator M, Nicols W, Ramel
C, eds. Amsterdam: Elsevier/North Holland Biomedical
Press, pp. 119-134.
4. Zimmermann FK, Kern R, Rosenberger H. 1975. A yeast
strain for simultaneous detection of induced mitotic
crossing over, mitotic gene conversion and reverse
mutation. Mutation Research 28:381-388.
-------
HG-DNA-Sister Chrom-In Vitro
August, 1982
IN VITRO SISTER CHROMATID EXCHANGE ASSAY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-DNA-Sister Chrom-In Vitro
I. 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.
II. DEFINITION
Sister chromatid exchanges represent reciprocal interchanges
of the two chromatid arms within a single chromosome. These
exchanges are visualized during the metaphase portion of the
cell cycle and presumably require enzymatic incision,
translocation and ligation of at least two DNA helices.
III. REFERENCE SUBSTANCES
Not applicable.
IV. TEST METHOD
A. Principle
Following exposure of cell cultures to test chemicals,
they are allowed to replicate in the presence of
bromodeoxyuridine (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.
B. Description
In vitro SCE assays may employ monolayer or suspension
cultures of established cell lines, cell strains or
primary cell cultures. Cell cultures are exposed to
test chemical and are allowed to replicate in the
presence of BrdU. Prior to harvest, cells are treated
with Colcemid® or colchicine to accumulate cells in c-
metaphase. Chromosome preparations from cells are made,
stained and analyzed for SCEs.
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HG-DNA-Sister Chrom-In Vitro
C. Cells
1. Type of cells used in the assay
There are a variety of cell lines (e.g., Chinese
hamster cells) or primary cell cultures, including
human cells, which may be used in the assay.
2. Cell growth and maintenance
Appropriate growth media, C02 concentrations,
temperature, and humidity should be used in
maintaining cultures. Established cell lines and
strains should be periodically checked for
Mycpplasma contamination. It is also desirable to
check the cells periodically for karyotype
stability.
D. Metabolic activation
1. Cells should be exposed to test chemical both in
the presence and absence of an appropriate
metabolic activation system. The most commonly
used systems include cofactor supplemented post-
mi tochondrial fractions prepared from the livers of
mammals treated with enzyme inducers and primary
cultures of mammalian hepatocytes. The use of
other tissues or techniques may also be
appropriate.
2. It is recognized that the use of metabolic
activation systems in in vitro SCE assays may
present problems of cytotoxicity to the test
system. If a chemical gives a negative result when
tested without metabolic activation, every attempt
should be made to test it with metabolic activation
in this system. If this is not feasible because of
technical difficulties with metabolic activation
systems, it is recommended that the chemical be
retested in an in vivo SCE assay.
E. Control g roups
1. Concurrent controls
Concurrent positive and negative (untreated and/or
vehicle) controls, with and without metabolic
activation, should be included in each assay.
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HG-DNA-Sister Chrom-In Vitro
2- Direct acting positive controls
For tests without metabolic activation, a compound
known to produce SCE in vitro without the use of
such a system should be used as the positive
control.
3. Posi t ive controls to ensure the efficacy of the
actiyat ion sys tem
The positive control reference substance for tests
including a metabolic activation system should be
selected on the basis of the type of activation
system used in the test.
F. Test chemicals
1. Vehicle
Test chemicals and positive control reference
substances may be prepared in growth medium or
dissolved or suspended in appropriate vehicles and
then further diluted in growth medium for use in
the assay. Final concentration of the vehicle
should not affect cell viability.
2. Expo sure con ce n trat ions
Multiple concentrations of the test substance over
a range adequate to define the response should be
tested. When appropriate, a positive response
should be confirmed by using a narrow range of test
concentrations. Among the criteria to be taken
into consideration for determining the upper lir-'.ts
of test chemical concentration are cytotoxicity and
solubility. Cytotoxicity of the test substance may
be altered in the presence of metabolic activation
systems. Cytotoxicity may be evidenced by a large
(e.g. 75%) decrease in the number of cells that
have divided twice in the presence of BrdU or a
significant increase in the frequency of structural
chromosomal aberrations. 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.
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HG-DNA-Sister Chrom-In Vitro
V. TEST PERFORMANCE
A. .Established cell lines and strains
1. Prior to use in the assay, cells should be
generated from stock cultures, seeded in culture
vessels at the appropriate density and incubated at
37 C. Numbers of fibroblast cells seeded should be
adjusted so that the cell monolayer is not more
than 50% confluent at the time of harvest.
2. 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.
For tests with postmitochrondrial metabolic
activation systems, concentration of the post-
mitochondrial fraction should generally be limited
to 10% for cells in monolayer culture. Serum
content of the media during treatment with post-
mi tochondrial fractions generally should be reduced
to 2%. At the end of the exposure period, 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 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 colchicine or Colcemid 2 hr prior
to harvesting.
B. Human lymphocyte cultures
1. For preparation of human lymphocyte cell cultures,
heparinized or acid-citrate-dextrose treated whole
blood should be added to culture medium containing
a mitogen, e.g., phytohemagglutinin (PHA) and
incubated at 37 C. White cells sedimented by
gravity (buffy coat) may also be utilized as may
lymphocytes which have been purified on a density
gradient such as Ficoll-Hypaque.
2. Cells should be exposed to the test chemical during
at least two time intervals, e.g. GQ and S.
Exposure during the GQ phase of the cell cycle
should be accomplished by adding the test substance
prior to addition of mitogen. After GQ exposure,
the cells may be washed and then cultured in the
absence of the chemical. As an alternative
procedure, the cells may be exposed during or after
-------
HG-DNA-Sister Chrom-In Vitro
the first S phase (approximately 24-30 hrs after
raitogen stimulation), washed, and then recultured
in the absence of the chemical. Cells may then be
fixed and stained and SCEs determined.
C. 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.
D. Staining method
Staining of slides to reveal SCEs can performed
according to any of several protocols. However, the
fluorescence plus Giemsa method is recommended.
E. Number of cultures
At least two independent cultures should be used for
each experimental point.
F. Analysis
Slides should be coded before analysis. The number of
cells to be analysed should be based upon the
spontaneous control frequency and defined sensitivity
and the power of the test chosen before analysis. In
human lymphocytes, only cells containing 46 centromeres
should be analysed. In established cell lines and
strains, only metaphases containing ± 2 centromeres of
the modal number should be analysed. Uniform criteria
for scoring SCEs should be used.
VI. DATA AND REPORT
A. Treatment of results
Data should be presented in tabular form, providing
scores for both the number of SCEs for each metaphase
and the number of SCEs per chromosome for each
metaphase.
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HG-DNA-Sister Chrom-In Vitro
B. Statistical eyaluatiori
Several statistical techniques are acceptable in
evaluating the results of this test. Choice of analyses
should consider tests appropriate to the experimental
design and needed adjustments for multiple comparisons.
C. Interpretation of results
1. There are several criteria for determing a positive
result, one of which is a statistically significant
dose-related increase in the number of sister
chromatid exchanges. Another criterion may be.
based upon detection of a reproducible and
statistically signficiant positive response for at
least one of the test substance concentrations.
However, the final decision must be based upon good
scientific judgement.
2. A test substance which produces neither a
statistically significant dose-related increase in
the number of sister chormatid exchanges nor a
statistically significant and reproducible positive
response at any one of the test points is
considered nonmutagenic in this system. Again, the
final decision must be based upon good scientific
judgement.
D. Test evaluation^
1. Positive results in the in vitro SCE assay indicate
that the test substance induces chromosomal
alterations in cultured mammalian somatic cells.
2. Negative results indicate that under the test
conditions the test substance does not induce
chromosomal alterations in cultured mammalian
somatic cells.
E. Test report
The test report should include the following
information:
1. cells used, density at time of treatment, number of
cell cultures;
2. methods used for maintenance of cell cultures
including medium, temperature and C02
concentration;
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HG-DNA-Sister Chrom-In Vitro
3. test chemical vehicle, concentration and rationale
for the selection of the concentrations of test
chemical used in the assay, duration of treatment;
4. details of the protocol used for metabolic
activation;
5. growth period in BrdU; duration of treatment with
and concentrations of colchicine or Colcemid® used;
6. time of cell harvest;
7. positive and negative controls;
8. method used to prepare slides for SCE
determination;
9. criteria for scoring SCEs;
10. details of the protocol used for growth and
treatment of human cells if used in the assay;
11. dose-response relationship, if applicable;
12. statistical evaluation;
13. discussion of results; and
14. interpretation of results.
VII. REFERENCES
The following reference may be helpful in developing
acceptable protocols, and provides a background of
information on which this section is based. It should not
be considered the only source of information on test
performance, however.
1. Latt SA, Allen J, Bloom SE, Carrano A, Falke E, Kram D,
Schneider E, Schreck R, Tice R, Whitfield B, Wolff S.
1981. Sister chromatid exchanges: a report of the U.S.
EPA's Gene-Tox Program. Mutation Research 87:17-62.
-------
IV. NEUROTOXICITY
-------
HG-Neuro-Path
August, 1982
NEUROPATHOLOGY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Neuro-Path
I. PURPOSE
The techniques in this guideline are designed to develop data on
morphologic changes in the nervous system for chemical substances and
mixtures subject to such testing under the Toxic Substances Control
Act. The data will detect and characterize morphologic changes, if and
when they occur, and determine a no-effect level for such changes.
Neuropathological evaluation should be complemented by other
neurotoxicity studies, e.g. behavioral and neurophysiological studies.
Neuropathological evaluation may be done following acute, subchronic or
chronic exposure.
II. DEFINITIONS
Neurotoxicity or a neurotoxic effect is an adverse change in the
structure or function of the nervous system following exposure to a
chemical agent.
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered to several groups of experimental
animals, one dose being used per group. The animals are sacrificed and
tissues in the nervous system are examined grossly and prepared for
microscopic examination. Starting with the highest dosage level,
tissues are examined under the light microscope for morphologic changes,
until a no effect level is determined. In cases where light microscopy
-------
HG-Neuro-Path
has revealed neuropathology, the no effect level may be confirmed by
electron microscopy.
W. TEST PROCEDURE
A. Animal Selection.
1. Species and Strain.
Testing should be performed in the species being used in other
tests for neurotoxicity. This will generally be the
laboratory rat. The choice of species shall take into
consideration such factors as the comparative metabolism of
the chemical and species sensitivity to the toxic effects of
the test substance, as evidenced by the results of other
studies, the potential for combined studies, and the
availability of other toxicity data for the species.
2. Age.
Animals shall be young adults ( * 150-200 gm for rats) at the
start of exposure.
3. Sex.
Both sexes should be used unless it is demonstrated that one
sex is refractory to the effects.
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HG-Neuro-Path
B. Number of Animals.
A minimum of six animals per group shall be used. It is
recommended that ten animals per group be used.
C. Control Groups.
1. A concurrent control group(s) is (are) required. This group
must be an untreated control group or, if a vehicle is used in
administering the test substance, a vehicle control group. If
the vehicle used has a known or potential toxic property, both
untreated and vehicle control groups are required.
2. A satellite group of animals may be treated with the high
level for 90 days and observed for reversibility, persistence,
or delayed occurrence of toxic effects for a post-treatment
period of appropriate length, normally not less than 28 days.
D. Dose Levels and Dose Selection.
At least three dose-level groups (in addition to control group(s))
shall be used and spaced appropriately to produce a range of toxic
effects. The data should be sufficient to produce a dose response
curve.
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1. Highest Dose
The highest dose level in rodents should result in neurotoxic
effects but not produce an incidence of fatalities which would
prevent a meaningful evaluation.
2. Lowest Dose
The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human
exposure the lowest dose level should exceed this.
3. Intermediate Dose(s)
Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose
is used, the dose levels should be spaced to produce a
gradation of toxic effects.
E. Duration of Testing.
The exposure duration will be specified in the test rule. This
will generally be 90 days exposure.
F. Route of Mministration.
The test substance shall be administered by a route specified in
the test rule. This will generally be the route most closely
approximating the route of human exposure. The exposure protocol
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shall conform to that outlined in the appropriate acute or
subchronic toxicity guideline.
G. Cgmbined Protocol.
The tests described herein may be combined with any other toxicity
study/ as long as none of the requirements of either are violated
by the combination.
H. Study Conduct.
1. Observation of Animals.
All toxicological (e.g. weight loss) and neurological signs
(e.g. motor disturbance) shall be recorded frequently enough
to observe any abnormality, and not less than weekly.
2. Sacrifice of Animals.
a. General.
The goal of the techniques outlined for sacrifice of
animals and preparation of tissues is preservation of
tissue morphology to simulate the living state of the
cell.
b. Perfusion Technique.
Animals shall be perfused J.n situ by a generally
recognized technique. For fixation suitable for light or
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electron microscopy, saline solution followed by buffered
2.5% glutaraldehyde or buffered 4.0% paraformaldehyde, is
recommended. While some minor modifications or
variations in procedures are used in different
laboratories, a detailed and standard procedure for
vascular perfusion may be found in the text by Zeman and
Innes (1963), Hayat (1970) and by Spencer and Schaumburg
(1980). A more sophisticated technique is described by
Palay and Chan-Palay (1974).
c. Rsmoval of Brain and Cord.
After perfusion, the bony structure (cranium and
vertebral column) should be exposed. Animals should then
be stored in fixative-filled bags at 4°C for 8-12
hours. The cranium and vertebral column shall be removed
carefully by trained technicians without physical damage
of the brain and cord. Detailed dissection procedures
may be found in the text by Palay and Chan-Palay
(1974). After removal, simple measurement of the size
(length and width) and weight of the whole brain
(cerebrum, cerebellum, pons-medulla) should be made. Any
abnormal coloration or discoloration of the brain and
cord should also be noted and recorded.
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d. Sampling.
Unless a given test rule specifies otherwise, cross-
sections of the following areas shall be examined: the
forebrain, the center of the cerebrum, the midbrain, the
cerebellum and pons, and the medulla oblongata; the
spinal cord at cervical and lumbar swellings (Cj-Cg and
L1~L4^; Gasserian ganglia, dorsal root ganglia (C3-C6,
L1~L4^' ^orsal an^ ventral root fibers (C^-Cg, L-pL^),
proximal sciatic nerve (mid-thigh and sciatic notch),
sural nerve (at knee), and tibial nerve (at knee). Other
sites and tissue elements (e.g. gastrocnemius muscle)
should be examined if deemed necessary. Any observable
gross changes shall be recorded.
3. Specimen Storage.
Tissue samples from both the central and peripheral nervous
system shall be further immersion fixed and stored in
appropriate fixative (e.g. 10% buffered formalin for light
microscopy; 2.5% buffered gluteraldehyde or 4.0% buffered
paraformaldehyde for electron microscopy) for future
examination. The volume of fixative versus the volume of
tissues in a specimen jar shall be no less than 25:1. All
stored tissues should be washed with buffer for at least 2
hours prior to further tissue processing.
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4. Histopathology Examination.
a. Fixation.
Tissue specimens stored in 10% buffered formalin may be
used for this purpose. All tissues must be immersion
fixed in fixative for at least 48 hours prior to further
tissue processing.
b. Dehydration.
All tissue specimens should be washed for at least one
hour with water or buffer, prior to dehydration. (A
longer washing time is needed if the specimens have been
stored in fixative for a prolonged period of time).
Dehydration can be performed with increasing
concentration of graded ethanols up to absolute
alcohol.
c. Clearing and Bnbedding^
After dehydration, tissue specimens shall be cleared with
xylene and embedded in paraffin or paraplast. Multiple
tissue specimens (e.g. brain, cord, ganglia) may be
embedded together in one single block for sectioning.
All tissue blocks should be labelled showing at least the
experiment number, animal number, and specimens
embedded.
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d. Sectioning.
Tissue sections, 5-6 microns in thickness, shall be
prepared from the tissue blocks and mounted on standard
glass slides. It is recommended that several additional
sections be made from each block at this time for
possible future needs for special stainings. All tissue
blocks and slides should be filed and stored in properly
labelled files or boxes.
e. Histopcttholpgiqal ^chniques.
Although the information available for a given chanical
substance may dictate test-rule specific changes, the
following general testing sequence is proposed for
gathering histopathological data:
(1) General Staining.
A general staining procedure shall be performed on
all .tissue specimens in the highest treatment
group. Hematoxylin and eosin (H&E) shall be used
for this purpose. The staining shall be
differentiated properly to achieve bluish nuclei
with pinkish background.
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(2) Special Stains.
Based on the results of the general staining,
selected sites and cellular components shall be
further evaluated by the use of specific
techniques. If H&E screening does not provide such
information, a battery of stains shall be used to
assess the following components in all required
sampling: neuronal body (e.g. Einarson's
gallocyanin), axon (e.g. Bodian), myelin sheath
(e.g. Kluver's Luxol Fast Blue) and neurofibrils
(e.g. Bielchosky). In addition, peripheral nerve
fiber teasing shall be used. Detailed staining
methodology is available in standard
histotechnological manuals such as AFIP (1968),
Ralis et al. (1973), and Chang (1979). The nerve
fiber teasing technique is discussed in Spencer and
Schaumberg (1980). A section of normal tissue
shall be included in each staining to assure that
adequate staining has occurred. Any changes shall
be noted and representative photographs shall be
taken. If a lesion(s) is observed, the special
techniques shall be repeated in the next lower
treatment group until no further lesion is
detectable.
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(3) Alternative Technique.
If the anatomical locus of expected neuro-pathology
is well-defined, epoxy-embedded sections stained
with toluidine blue may be used for small sized
tissue samples. This technique obviates the need
for special stains for cellular components.
Detailed methodology is available in Spencer and
Schaumberg (1980).
(4) Electron Microscopy.
Based on the results of light microscopic
evaluation, specific tissue sites which reveal a
lesion(s) shall be further evaluated by electron
microscopy in the highest treatment group which
does not reveal any light microscopic lesion. If a
lesion is observed, the next lower treatment group
shall be evaluated until no significant lesion is
found. Detailed methodology is available in Hayat
(1970).
f. Examination.
(1) General.
All stained microscopic slides shall be examined
with a standard research microscope. Examples of
cellular alterations (e.g., neuronal vacuolation,
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degeneration, and necrosis) and tissue changes
(e.g., gliosis, leukocytic infiltration, and cystic
formation) shall be recorded and photographed.
(2) Electron Microcopy.
Since the size of the tissue samples that can be
examined is very small, at least 3-4 tissue blocks
from each sampling site must be examined. Tissue
sections must be examined with a transmission
electron microscope. Three main categories of
structural changes must be considered:
(a) Neuronal body.
The shape and position of the nucleus and
nucleolus as well as any change in the
chromatin patterns shall be noted. Within
the neuronal cytoplasm, cytoplasmic
organelles such as mitochondria, lysosomes,
neurotubules, neurofilaments, microfilaments,
endoplasmic reticulum and polyribosomes
(Nissl substance), Golgi complex, and
secretory granules shall be examined.
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(b) Neuronal processes.
The structual integrity or alterations of
dendrites, axons (myelinated and
unmyelinated), myelin sheaths, and synapses
shall be noted.
(c) Supporting oells.
Attention must also be paid to the number and
structural integrity of the neuroglial
elements (oligodendrocytes, astrocytes, and
microglia) of the central nervous system, and
the Schwann cells, satellite cells, and
capsule cells of the peripheral nervous
system. Any changes in the endothelial cells
and ependymal lining cells shall also be
noted whenever possible. The nature,
severity, and frequency of each type of
lesion in each specimen must be recorded.
Representative lesions must be photographed.
V. EATA COLLECTION, REPORTING, AND EVALUATION
In addition to information meeting the requirements stated in the EPA
Good Laboratory Practice Standards [Subpart J, Part 792, Chapter I of
Title 40 Code of Regulations], the following specific information should
be reported:
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A. Description of Test System and Test Methods
A description of the general design of the experiinent including a
short justification explaining any decisions where professional
judgement is involved such as fixation technique and choice of
stains.
B. Results
All observations shall be recorded and arranged by test groups.
This data may be presented in the following recommended format:
1. Description of Signs and Lesions for Each Animal.
For each animal, data must be submitted showing its
identification (animal number, treatment, dose, duration),
neurologic signs, location(s), nature of, frequency, and
severity of lesion(s). A commonly-used scale such as 1+, 2+,
3+, and 4+ for degree of severity ranging from very slight to
extensive may be used. Any diagnoses derived from neurologic
signs and lesions including naturally occurring diseases or
conditions, should also be recorded.
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2. Counts and Incidence of Lesions, by Test Group.
Data shall be tabulated to show: a. The number of animals
used in each group, the number of animals displaying specific
neurologic signs, and the number of animals in which any
lesion was found; b. The number of animals affected by each
different type of lesion, the average grade of each type of
lesion, and the frequency of each different type and/or
location of lesion.
3. Evaluation of Data.
An evaluation of the data based on gross necropsy findings and
microscopic pathology observations shall be made and
supplied. The evaluation shall include the relationship, if
any, between the animal's exposure to the test substance and
the frequency and severity of the lesions observed. The
evaluation of dose-response, if existent, for various groups
shall be given, and a description of statistical method must
be presented. The evaluation of neuropathology data should
include, where applicable, an assessment in conjunction with
other neurotoxicity studies.
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REFERENCES
AFIP. 1968. Manual of histologic staining methods. New York: McGraw-Hill
Chang LW. 1979. A color atlas and manual for applied histochemistry.
Springfield, IL: Charles C. Thomas.
Hayat MA. 1970. Principles and techniques of electron microscopy, Vol. 1.
Biological applications. New York: Van Nostrand Reinhold.
Palay SL, Chan-Palay V. 1974. Cerebellar cortex: cytology and organiza-
tion. New York: Springer-Verlag.
Ralis HM, Beesley RA, Ralis ZA. 1973. Techniques in neurohistology. London:
Butterworths.
Spencer PS, Schaumburg HH (eds). 1980. Experimental and clinical
neurotoxioology. Baltimore: Williams and Wilkins.
Zeman W, JRM Innes JRM. 1963. Craigie's neuroanatomy of the rat. New York:
Academic.
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August, 1982
NEURDPATHODOGY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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TABLE OF CONTENTS
PAGE
I. NEURQPATHOLOGY IN TOXICOLOGY 1
A. Nervous system as a target organ 1
B. Neuropathology (morphology) as a tool in
detecting neurotoxicity 2
C. Basic objective of the test standard in
neuropathology 3
II. RATIONALES FOR STUDY DESIGN 3
A. Species 3
B. Sex and age 4
C. Number of animals 4
D. Dose and duration of testing 4
E. Route of exposure 5
F. Technical personnel and
pathologist requirements 6
III. RATIONALES FOR STUDY CONDUCT 6
A. Proper techniques in animal
sacrifice and tissue handling 6
B. Vascular perfusion 7
C. Fixative of choice 9
D. Gross examination of brain and cord 10
1. Brain weight and size 10
2. Gross appearance of the brain and cord. 10
E. Specimen storage 11
F. General histopathological evaluation 12
1. Tissue sampling 13
2. Tissue processing 13
3. Paraffin/hematoxylin-eosin (H & E)
or epoxy-toluidine blue technique
as a general screening method 14
4. Data collection, evaluation, and
reporting 15
IV. SPECIFIC DETECTION AND EVALUATION TECHNIQUES ... 15
A. Need for special techniques 15
B. General objective of
the special techniques 17
C. Methods selected for specific
evaluation of neural changes 18
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D. When to perform 19
E. Test scheme 20
F. Economy of the special evaluation methods . 24
V. ELECTRON MICROSCOPY 25
A. Objective 25
B. Limitations and advantages 26
C. When to perform 27
D. Test scheme 28
E. Elements to be examined 29
REFERENCES 30
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I. NEUROPATHOLOGY IN TOXICOLOGY
A. Nervous System as a Target Organ
Many chemical compounds such as acetylethyl tetramethyl tetralin
(AETT), acrylamide, heavy metals, carbon monoxide,
organophosphorous compounds, to name a few, are found to have toxic
effects on the nervous system. Because of the unique structure and
functional properties of the nervous system, neurotoxioology is
becoming a specialized area of investigation (Spencer and
Schaumberg 1980).
Since the nervous system either dominates or influences most
functions of the organism and is not, in a general sense, capable
of full regeneration after severe injury, any toxic assault on the
nervous system may create a significant and long-lasting impact on
the health and function of the organism. Therefore, the
identification and understanding of neurotoxicants are of primary
importance.
Toxic substances can affect various aspects of the nervous system
(biochemical, physiological, morphological, and behavioral),
inducing changes in sensory, motor, cognitive or emotional
function. These various disciplines may be used to detect and
evaluate the impact of substances on the nervous system. The
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guideline for neuropathology focuses on selected procedures for
detection and evaluation of morphological changes in the nervous
system.
B. Neuropathology (Morphology) as a Tool in Detecting Neurotoxicity
Cells in the nervous system/ like cells in other systems, can
function properly only upon total structural integrity. Unlike
most other cells in the body of the organisms, nervous-system cells
exhibit a highly specialized architecture: neurons, myelinating
cells (oligodendrocytes and Schwann cells), and astrocytes each
possess one or more long cellular processes which depend for their
maintenance on synthetic activities carried out at a remote site in
the cell and the transport of nutrients over long distances. The
specialized architectural design of nervous system cells thus
provides much greater vulnerability to toxic attack than cells of
other systems (Spencer and Schaumburg 1980).
Because the nervous system has a functional reserve and exhibits a
certain degree of plasticity, functional disturbance in the form of
neurological signs or behavioral changes may not be manifest until
a significant amount of structural damage has been incurred.
Depletion of the structural reserve of the nervous system may make
the organism more vulnerable to subsequent toxic attack, or to the
effects of abnormal metabolism or aging. Therefore, early
morphological alterations in the nervous system, with or without
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functional deficits, represent a significant and sensitive
indication of neural damage.
C. Basic Objective of the Guideline in Neurppothplogy
The basic goal of the techniques developed for evaluation of
morphological changes is to preserve the tissue morphology to
simulate the living state of the cell. The objective of this
guideline is to present a standard approach for sensitive detection
and systematic evaluation of morphological changes in various
components of the nervous tissues and cells: neuronal cell body,
axons, dendrites, myelin sheaths, nerve fibers, and subcellular
organelles.
II. Rationales for Study Design
A. Species
The species being studied for functional neurotoxicity assessment
should also be evaluated for morphological changes. Morphological
data generated in this guideline, therefore, can then be correlated
with data generated in other studies. Rodents will generally be
the species of choice because (a) They have been used as animal
models for the investigations of the local or systemic toxicities
of chemical substances. The information generated on the
pharmacokinetics (e.g., distribution, excretion, etc.) and target
organ toxicities (e.g., on kidney, liver, etc.) of the
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chemicals may enhance the understanding of the toxic impacts on the
nervous system, (b) Pure bred (genetically known) strains are
available, which enhances the reproducibility of the experiments.
The comparative metabolism of the chemical and species sensitivity
to the toxic effects of the test substance are also important
factors for consideration.
B. Sex and Age
Toxic susceptibility or sensitivity is known to vary with age and
sex of the animals. Young adults should be used because of their
fully matured nervous system (unless the experiment is designed to
study toxic impacts on a system which may decline with age). If
scientific judgement deems data from males and females is
warranted, both should be used.
C. Number of Animals
A minimum of six animals per group is recommended because this will
provide enough tissues for acceptable evaluation.
D. Dose and Duration of Testing
At least three dosage groups (high, medium, and low) should be
tested. It is recommended that the high dose selected should not
be acutely lethal to the animals (neuropathology usually takes time
to develop) but should induce some observable neurological signs
which may be helpful in determining the location(s) of the
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lesion(s). The low dose level selected hopefully will not induce
overt signs for about 90 days. This "no observed effect level" is
important for the evaluation of the risk of long-term, low dose
exposure to the substance. Recommendations in this standard are
also consistent with those recommended in the federal guidelines
for other toxicity studies. Since neuropathology is an integral
part of the overall neurotoxicology study, the general schene for
animal treatment (dose levels, duration of exposure, etc.) should
be in line with those recommended for other aspects of
neurotoxicological study so that data generated can support,
complement, and supplement other studies.
E. Route of Exposure
The route of exposure used should be consistent across neuro- and
other toxicity studies to facilitate comparison of results.
Different routes of exposure will result in different rates of
absorption and excretion of the test substance and thus influence
the toxic effect of the substance. All routes of exposure
(inhalation, injection, intubation, etc.) have been used by
different investigators to induce neurotoxicity. However, the
selection of the method of administration should be dictated by the
nature of the test substance and the metabolism (if known) of the
test substance as well as the route of human exposure.
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6. Technical Personnel and Pathologist Requirements
The general requirements for personnel are discussed in the EPA
Good Laboratory Practice Standards [Subpart B, Part 792, Chapter I
of Title 40, Code of Federal Regulations]. It is further
recommended in this test guideline that the pathologist in charge
of the project not only should have been trained basically in
pathology (M.D., D.V.M., D.O. or Ph.D. in Experimental Pathology),
but also should have special training or experience in
neuropathology. This is recommended because neuropathology is an
extremely specialized field and often has its own technical
"language." A lack of formal training or experience in this area
will severely limit the investigator's ability in directing the
experiments and in interpreting the findings. For projects that
involve electron microscopy, the pathologist should also be well
aware of the techniques of electron microscopy and well-trained in
the interpretation and data analysis of ultrastructures of nervous
tissues. A general electron microscopist, cytologist, or
neuroanatomist will not be able to manage the complexity of
neuropathology at the ultrastructural level.
III. RATIONALES FOR THE STUDY CONDUCT
A. Proper Techniques in Animal Sacrifice^ andI Tissue Handling
Improper sacrifice and tissue handling techniques frequently
produce irreversible artifacts in the nervous tissues. Therefore,
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all techniques should be performed by trained laboratory personnel
who are experienced in this area of research. Although there are
various techniques which are equally acceptable for
neuromorphological research, they all share similar basic
principles. The techniques and methodologies which are recommended
in this test standard represent those proven to be reliable,
reproducible, cost efficient, and easy to perform.
The purpose of anatomic pathology is to make morphological
evaluation of disease (pathological) states of the cells and
tissues. Thus, the same general principles and techniques for
morphological research as those employed by anatomists should be
followed to ensure proper morphological preservation.
B. Vascular Perfusion
Nervous tissues, particularly those of the CNS, are subjected to
rapid anoxic and autolytic changes after death of the animals.
Rapid fixation by means of vascular perfusion will help to prevent
such undesirable changes. Perfusion fixation also serves to harden
the nervous tissue and reduce the chances of artifact occurrence
during brain and cord removal.
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During the past decade, procedures for proper fixation and
preservation of nervous tissues have undergone considerable changes
and modification. For good morphological preservation, all animals
should be perfused for both light and electron microscopy. The
available data indicate that the quality of preservation of the
fine structure is affected not only by the characteristics of the
fixative, but also by the method of applying the fixative to the
tissue. Comparative studies have shown that the rate of
penetration of a fixative is influenced by various conditions
prevalent before and during fixation. It appears that under _iti
vivo conditions, the rate and depth of fixation are increased by
perfusion fixation. For example, rat kidneys fixed in vivo by
flooding with 1% osmium tetroxide were penetrated at a rate of
about 20 urn per minute (Maunsbach et al. 1962), whereas similar
tissues fixed ir\_ vitro were penetrated at a rate of only about 8 urn
per minute (Rhodin, 1954). The above tissues fixed in_ vivo also
showed good fixation to a depth of 100 to 200 urn compared with only
30 to 40 urn depth achieved through in_ vitro fixation. This
increased rate of penetration is probably related to an increased
tubular flow of the fixative in the living kidneys.
The method employed in in vivo fixation is perfusion of a suitable
fixing agent through the vascular channels. This method
essentially involves a rapid and uniform penetration of a fixative
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into all parts of the tissue, prior to any injury to the tissue by
anoxia or direct handling. Fixation by perfusion is expected to
stabilize the tissue against diffusion and translocation of
cellular substances, which may occur during fixation by
immersion. The application of this method is thus especially
useful for tissues that are exceptionally sensitive to the effects
of oxygen deprivation and physical handling. Neural tissues,
especially the central nervous system are satisfactorily fixed only
by perfusion methods. Under optimal fixation conditions, an
artifact-free nervous system tissue can be obtained.
Fixative of Choice
Although 10% buffered formalin is an excellent general fixative for
light microscopy, it has a very high aldehyde content and hardens
the tissues too much for electron microscopy. Phosphate-buffered
2.5% glutaraldehyde or 4.0% paraformaldehyde having an osmolality
of about 600-800 mOsM at pH 7.4 is excellent for perfusion of brain
tissues. Furthermore, tissues perfused with these fixatives can be
subjected to further immersion fixation in 10% buffered formalin or
in the same fixative; thus permitting both light and electron
microscopy investigation.
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A mixture of parafontaldehyde and glutaraldehyde (modified
Karnovsky) is also an excellent perfusate and fixative for the
nervous system. However, it requires more labor and cost in its
preparation and does not pose a significant advantage over
glutaraldehyde or paraformaldehyde alone.
D. Gross Examination of Brain and Cord
1. Brain Weight and Size.
An edematous (swelling) condition (e.g., acute lead poisoning)
may be induced under some neurotoxic conditions. Simple
weighing and measuring of the removed brain may generate this
data. Such procedures can be done within one minute and are
of no cost to the investigator. However, the data collected
may be of great value.
2. Gross Appearance of the Brain and Cord.
Simple observation of the gross appearance of the brain and
cord may yield invaluable pathological information, such as
petechial hemorrhage (e.g., in carbon monoxide poisoning) or
area of necrosis. This observation can be made during
brain/cord removal and cutting and imposes no significant time
or cost to the investigator.
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The brain should be cut at various levels for both gross and
light microscopic examination. Frontal plane cuttings which
can reveal various internal structures of the brain on both
hemispheres are recommended.
The cervical and lumbar segments of the cord (C^-Cg, L^-^)
are selected for special attention because these are the most
prominent areas of the cord controlling the input/output of
the upper and lower extremities. Cross sections of the cord
will reveal internal structures of both sides of the cord.
E. Specimen Storage
The usual practice in the preparation of biological materials for
microscopy is to fix, process, and embed tissues immediately after
they are excised. However, in some cases due to unavoidable
circumstances, it is not possible to process the specimens
immediately after they are killed or excised from the source, so
they must be stored for a period of time. The necessity of storage
may arise when it poses a possibility that special detection tech-
niques (e.g., for myelin damage) or electron microscopy may be
needed after the general histopathological evaluation (H & E or
toluidine blue) is performed.
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Half of the tissue samples can be fixed and stored in 10% buffered
formalin (for light microscopy) and the other half of the specimen
can be fixed and stored in buffered glutaraldehyde or
paraformaldehyde (for possible electron microscopy inves-
tigation). Sabatini et al. (1963) pointed out that tissues can be
stored in various aldehydes up to several months without any
significant adverse effect on the fine structure. Yamamoto and
Rosario (1967) also reported that tissue can be stored in buffered
formaldehyde (paraformaldehyde) for over one year without
undesirable structural changes. Actually, tissues previously well
fixed with aldehydes can be transferred for storage in cold buffer,
containing sucrose, up to several months.
Washing the stored tissues with a buffer solution (for electron
microscopy) or with water (for light microscopy) prior to tissue
processing is a critical factor in obtaining good sectioning.
F. General Histopathological Evaluation
•Hie basic objective of this part of the examination is to detect
any morphological lesions in cells and tissues within the
resolution power of the light microscope by means of a general
staining method.
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1. Tissue Sampling.
The frontal cuttings of the brain and the cross sections of
the cord (C^-Cg, L^-L^) prepared during gross examination can
be subjected to embedding and light microscopic examination.
Those tissues from the peripheral nervous system which are
known to be vulnerable to neurotoxicants are also included in
the screening. All these tissues comprise a fairly good
general representation of both the central and peripheral
nervous systems. This general tissue survey is required only
for the investigation of pptential neurotoxicants whose toxic
effects are still unknown. For known neurotoxicants, only
selected pertinent tissues are needed for the study.
2. Tissue Processing.
All the basic procedures for tissue processing (fixation,
dehydration, clearing, embedding, and sectioning) are fairly
standard. No special justifications are needed.
It is recommended that multiple tissues can be embedded in one
single paraffin block for sectioning. This practice is found
to be efficient not only in labor effort (sectioning and
staining), but also in storage space (tissue blocks and
slides) as well as in material costs. It also saves the
pathologist's time in examining multiple tissues in one single
microscopic slide.
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3. Paraffjji/Henatpxylin-Eksin (H & E) or Epoxy/rpluidine Blue
Technique as General Screening Method.
H & E is recognized as one of the most useful techniques for
general tissue screening in pathology. With paraffin
embedding, not only larger tissue samples (e.g., entire cross
sections of the rodent brain) can be embedded and sectioned,
multiple tissues (e.g., brain, cord, ganglia, etc.) can also
be embedded together in one single block for sectioning and
staining. Paraffin embedded sections can also be subjected to
special staining techniques when that becomes necessary. It
is therefore a good, reliable, and cost-efficient technique.
Epoxy embedded sections stained with toluidine blue can be
substituted for the H & E technique as a rapid, routine
screening method under certain conditions (Spencer and
Schaumburg 1980). This procedure is actually a "thick"
section preparation for electron microscopy. Sections can be
screened with light microscope and the tissue block further
trimmed for sectioning. However, this technique can be
applied only to a relatively small area of tissue sample and
is therefore not very useful unless the precise site of the
toxic action is known. Thus, this approach may not be
appropriate for the study of unknown neurotoxicants. Tissues
subjected to epoxy embedding also require osmium tetroxide
14
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HS-Neuro-Path
fixation and staining and cannot be subjected to further
staining with other special neurohistochemical methods.
Because it requires epoxy embedding and osmium tetroxide
staining, it is also more costly than the basic H & E
method. However, very thin (1.0 micron) sections can be
generated from the epoxy embedded tissues which provide much
higher resolution power than the paraffin sections. Thus the
epoxy/toluidine blue method will be a more sensitive method
than the routine H & E method and can detect histological
lesions more readily. Because of the sensitivity of this
method, the need of special detection techniques (stains) may
be alleviated.
4. Data Collection, Evaluation and Reporting^.
The general guideline implemented in this test standard is
consistent with those provided in previously published
guidelines. It promotes clear and efficient data collection,
note-keeping, and morphological data evaluation and
statistical analysis.
IV. SPECIFIC DEFECTION AND EVALUATION TECHNIQUES
A. Need for Special Techniques
It has recently been stated by Dr. J. B. Cavanagh, the eminent
British neuropathologist that "special techniques are needed to
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show the cellular details of damage to brain and nerve. An 'H &
E1, while adequate for most general pathology, is insufficient to
supply the answers to neuropathological problems. The special
techniques and the special knowledge are reasons for the separation
of neuropathology frcrn its parent field." (Spencer and Schaumburg
1980).
Indeed, while H & E is an excellent general stain, it is a very
non-specific dye, staining nuclei blue and all other tissue
components pink. Therefore, unless the lesions involved are of
obvious or extensive nature, paraffin/H & E method may fail to
detect changes involving the complex structures and all the tissue
components of the nervous system. Special techniques are useful to
selectively detect and precisely diagnose changes in the various
cellular and tissue components in the nervous system; axons,
dendrites, Nissl patterns of the neurons, myelin sheath, etc.
However, if epoxy/toluidine blue method is employed, the high
sensitivity of this method will enable rapid detection of the
lesions without further special techniques.
Contrary to many beliefs, most of the special neurohistochemical
techniques are actually quite inexpensive and easy to perform.
Most can be done with paraffin sections. Thus additional sections
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can simply be obtained from the tissue block when sections for H &
E staining are being made. The "additional" time for an
experienced technician to cut one section (for H & E staining)
versus a ribbon of several sections (for special stains) is
probably 2-3 minutes per block. The chemicals required for these
special stains are readily available and are not particularly high
priced. Furthermore/ multiple slides as well as multiple tissues
on one slide can be stained together at the same time in one
staining rack; thus the additional labor time is also not
excessive.
B. General Objective of the Special Techniques
The objective of this segment of the study is to use well
established and reliable neurohistochemical methods to identify and
better define specific structural damages of the nervous tissues
(neuronal body, axon, myelin sheath, and peripheral nerve fiber),
which may go undetected or may be difficult to determine by H & E
staining method.
Other tissue elements such as dendrites, glial cells, endothelial
cells, neuromuscular junction, and skeletal muscles may change
either as a direct toxic impact or as secondary degeneration to the
toxic substance. These elements can be examined with general
screening methods (H & E or epoxy/toluidine blue). Special
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HS-Neuro-Path
techniques for these elements are usually too difficult and costly
for basic laboratories and will not be included in this guideline.
C. Methods for Specific Evaluation of Neural Changes
Special techniques are fairly "routine" in many well established
neuropathology laboratories. They have been used and proven to be
very helpful in detecting many changes involving the nervous
system, such as: early destruction of neuronal Nissl substances
(gallocyanin stain) as seen in methylmercury poisoning (Chang and
Hartmann 1972), axonal changes (Bodian stain) as observed in IDPN
intoxication (Chou and Hartmann 1964), segmental demyelination
(nerve fiber teasing technique) as seen in lead poisoning
(Fullerton, 1966) and peripheral nerve fiber changes as seen in
acetyl ethyl tetramethyl tetralin intoxication (Spencer et al.
1980), neurofibrillary changes (Bielchowsky stain) as seen in
aluminum intoxication (Klatzo et al. 1965), and primary or
secondary destruction of myelin sheaths (Kluyer's Ijaxol Fast Blue
Stain) as seen in human subacute myelcopticoneuropathy (Shiraki
1977) and in clioquinol intoxication (Ikuta et al. 1977). Many of
these lesions may go undetected or unidentified (particularly in
mild pathological situations) by H & E method. Therefore, special
techniques, when used properly, play an important role in
neuropathology evaluation.
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HS-Neuro-Path
Positive control slides are needed for each of the special stains
to guard against false-positive or false-negative results. These
slides must be done together with the test tissues in each staining
procedure to assure the validity of the technique.
D. When to Perform
As stated earlier, because of the high resolution and sensitivity
of the epoxy/toluidine blue method, special stains may not be
needed for animal tissues screened by that technique. However,
special techniques have meritorious value for paraffin embedded
sections.
Most neurotoxicants are fairly site and action specific, and may be
characterized according to their primary or major toxic
consequences: neuronopathy (e.g. methylmercury on cerebellar
granule cells and dorsal root ganglia; trimethyltin on hippocampal
neurons), axonopathy (e.g. acrylamide on distal axons; IDPN on
proximal axons), and myelinopathy (e.g. lead on Schwann myelin
sheaths; triethyltin on central myelin). Therefore, probably only
one or two special techniques will be employed for a given study on
known neurotoxicants, e.g., for peripheral nerve degeneration
(e.g., acrylamide poisoning), nerve fiber teasing technique will
suffice. In the situation of proximal axonal swelling as seen in
IDPN intoxication, a combined staining of Bodian/LFB will be enough
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HS-Neuro-Path
to detect small axonal swellings (low dose condition) which may go
undetected by H & E method and to demonstrate the "swollen, round
structures" are myelinated axons and not swollen dendrites
(dendritic torpedoes) or tangential cuts of neuronal bodies.
Secondary or other minor lesions produced by a neurotoxic compound
should be noted wi€h H & E stained sections and do not require
detailed special technique screening.
For testing of a potential neurotoxicant whose toxic action is not
known, a very high dose or acute exposure group should be included
in hope of inducing a more dramatic lesion which can be easily
detected by H & E staining method. Once the general nature and
site of lesion is known, epoxy/toluidine blue method can be
performed on these isolated tissues, or appropriate special
techniques for paraffin preparations can then be selected
accordingly. If there is absolutely no clue that can be obtained
either from the H & E stained sections at any dose level, from the
general chemical structure and characterization of the test
compound, or from the neurological symptomology as to the probable
general nature of the toxic impact, the entire battery of special
techniques will have to be used. Situations as such are very rare
indeed, because if a chemical compound is neurotoxic, some form of
morphological lesion will usually be produced in high dose
situations. The implementation of this requirement of using
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HS-Neuro-Path
the entire battery of special techniques for paraffin preparations
in the test standard is necessary and serves as a safeguard for
such rare, but possible, situations.
E. Ttest Scheme
T\K> test schemes can be suggested: One is used for known
neurotoxicants, the other for suspected neurotoxicants. The H & E
stain may be used for both test schemes to the limit of its
capacity to detect a lesion. Then the true "no effect level" maybe
confirmed by examining the H & E "no effect" tissue with more
sensitive, special stains. Alternatively, the epoxy/toluidine blue
technique may be used for known neurotoxicants or after H & E has
been used to identify the general nature and site of the lesion.
A summary of these test schemes is presented in the flow charts
(see Figures 1 and 2).
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HS-Neuro-Path
FIGURE 1 GENERAL TEST SCHEME FOR "KNCWN" NEUROTOXICANT
General Screen on
selected, specific target tissues
I
H & E
Epoxy/Toluidine Blue
Identify highest treatment group
which demonstrates no H & E lesions
Identify highest treatment group.
which demonstrates no lesions .
1
Select appropriate special
technique(s)
If no further
lesion can be
detected
If lesion(s) is observed,
repeat special techniques
in next lower treatment group
until no further lesion is
detectable
Electron Microscopy
on specific tissue sites of
highest treatment group which
does not reveal any light microscopy (LM) lesion
. I
If no morphological lesion,
terminate experiment
If lesion is observed,
repeat EM survey in the
next lower treatment group
until no significant lesion
is found
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HS-Neuro-Path
FIGURE 2 GENERAL TEST SCHEME FDR "SUSPECTED" NcUROTCKICANT
General H & E screening on
all recommended tissue samples
Identify the general nature
and site of the lesion(s)
If no lesions can be identified
Proceed as for "known"
neurotoxicant (Figure 1)
Perform the entire battery of
special techniques on all tissues
on the highest treatment group
I
I
If no lesion is
identified, terminate
experiment
If some lesion(s)
detected, proceed
as for "known"
neurotoxicant (Figure 1}
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HS-Neuro-Path
F. Economy of The_ Special Evaluation Methods
It is the intent of this guideline to obtain maximal information
with minimal but detailed screening. Data generated should be
useful not just to demonstrate that some form of morphological
lesion exists, but also be able to accurately define the nature of
the lesion, to provide morphological support and correlation with
other studies (e.g., behavioral and neurophysiological), and to
elucidate the "safety" level (no observable lesion) of the test
substance in terms of morphological judgment. Thus, with all the
efforts in economizing (both cost and effort) the investigation in
mind, the test scheme suggested represents a carefully designed
approach to cover all possible situations. Granted, some of the
procedures will rarely be used, but they are implemented to
safeguard a neurotoxic compound passing the screening undetected.
A chemical compound can be confidently declared as producing no
observable light microscopy lesions at a given dose level if and
only if both H & E and selected special technique(s) failed to
reveal any morphological abnormality at that dose level.
Special stains will certainly increase the number of slides to be
cut, stained, and read. As stated earlier, preparation of the
additional sections will not increase the technician's time
significantly. Time involved for special staining differs with the
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HS-Neuro-Path
stains involved. However, multiple slides with multiple tissues
per slide can be stained together to reduce the labor time and
chemical cost. Since only one or two special techniques will prob-
ably be required for one treatment group under normal situations,
the additional slides generated will not be exceedingly great. A
well trained pathologist should be able to read a slide under 2-3
minute's time (particularly for special stains which define the
lesion very sharply). The additional time for slide reading is
therefore also within reason. Compared to other screening methods
(e.g., behavioral studies), pathology involves much less time and
cost.
V. ELECTRON MCIR06COPY
A. Objective
The basic objective of this portion of the test standard is to
establish a standard approach in using the most modern and
sensitive diagnostic tool available to provide morphological
information on the existence or non-existence of ill-effects of a
compound at given dose level(s) on specific tissues.
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HS-Neuro-Path
B. Limitations; and advantages
Because of the sraallness of the tissue size which can be examined,
it is impractical to use M as a general screening tool. But
because of its high resolution power/ it can be used to screen and
detect subtle morphological lesions (subcellular changes) even at
very low dosage levels on specific areas of selected tissue samples
which are known to have light microscopic lesions at much higher
dose levels. In other words, it can be used to determine whether a
"no observable lesion" situation by light microscopic criteria is
truly free of any morphological toxic change or it is merely due to
the "insensitivity" of the light microscopic resolution. The
conclusion of no morphological (pathological) change can be
confidently drawn if and only if no detectible structural change
can be found even with the most sensitive instrument (EM)
available.
The functional significance of subtle morphological changes (e.g.,
mitochondrial swelling or synaptic abnormality) cannot be
determined by morphological techniques. Such observation only
provides morphological information on structural changes in the
organelles, cells or tissues in the nervous system under certain
toxic conditions. Although such changes may or may not exert an
immediate or apparent functional deficit to the animal (as stated
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HS-Neuro-Path
earlier, the nervous system is an extremely plastic organ which can
compensate for many functional deficits despite significant
structural damage), such information is extremely important in
revealing the early toxic impact of the chemical in question.
Physiological and/or behavioral studies would help to elucidate the
functional aspect of the organism following toxic exposures.
The only possible way to increase the "sensitivity" of electron
microscopy is probably by means of morphometric analysis
(quantitative morphology) where extremely subtle changes in the
number, size, or distribution of cells or organelles can be
estimated as a consequence of toxic influence on the biological
system. Because this technique may be too time consuming and
costly to average investigators, it is not included in this
guideline.
c* When to Perform
Because of the small tissue size that can be examined by electron
microscopy, EM should not be used as a "general" screening tool,
but rather, should be limited only to screen specific areas on
selected tissue samples where lesions are known by light microscopy
to occur at higher dose levels. EM study is not needed in any test
group(s) demonstrating IM observable pathology. Since the precise
tissue site(s) of the lesion(s) will be very well defined by light
microscopy at higher dosage levels, usually only one or two
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HS-Neuro-Path
selected tissue sites are needed for EM survey at low dose
situations. Thus, the actual workload for EM work is really not
very much for any given test.
D. Test Scheme
For investigation of substances with known toxic impact, select
tissue samples from the highest dose group where no light
microscopic lesion is detected should be first subjected to EM
study. If EM lesions are observed, tissues fron the next lower
dose group should also be examined. However, if no EM lesion is
found, no further EM study is needed.
A similar approach may be exercised for the study of potential
neurotoxicants. EM is performed on specific and selected tissues
if and only if IM lesions can be demonstrated at higher dose
levels. No EM study is necessary if no detectible LFl lesion, both
by H & E and by special histochemical stains, is observed at all
dose levels studied.
A general test scheme is provided as flow charts (see Figure 1 and
2).
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HS-Neuro-Path
E. Elements to be Examined
The elements recommended for examination are all basic
cellular/tissue components of the nervous system.
Since specific structural differences of some elements/organelles
may be species related, control animals must be used at all times
as reference tissues.
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HS-Neuro-Path
REFERENCES
AFIP. 1968. Manual of histologic staining methods. McGraw-Hill.
Chang LW. 1979. A color atlas and manual for applied histochemistry.
Springfield, IL: Charles C. Thomas.
Chang LW, Hartmann HH. 1972. Ultrastructural studies of the nervous system
after mercury intoxication. I. Pathological changes in the nerve cell
bodies. Acta Neuropath. 20:122-138.
Chou SM, Hartmann HH. 1964. Axonal lesions and waltzing syndrome of the IDPN
administered rats. Acta Neuropath. 3:438-450.
Fullerton PM. 1966. Chronic peripheral neuropathy produced by lead poisoning
in guinea pigs. J. Neuropath. Exp. Neurol. 25:214-236.
Hayat MA. 1970. Principles and techniques of electron microscopy, Vol. 1.
Biological applications. New York: Van Nostrand Reinhold Co.
Ikuta F, Atsumi T, Makifuchi T, Sato T, Ubaki T. 1977. Neuropathology of
subacute myelcopticoneuropathy (clioquinol intoxication) in humans and
experimental animals. In: Neurotoxioology. Roizin, L, Shiraki, H, and
Grcevic, N., eds. New York: Raven, pp. 353-360.
Klatzo I, Wisniewski HM, Streicher E. 1975. Experimental production of
neurofibrillary degeneration. I. Light microscopic observations. J.
Neuropath. Exp. Neurol. 24:187-199.
Maunsbach AB, Madden SC, Latta H. 1962. Variations in fine structure of
renal tubular epithelium under different conditions of fixation. J. Ultrastr.
Res. 6:511-530.
Palay SL, Chan-Palay V. 1974. Cerebellar cortex: cytology and organiza-
tion. New York: Springer-Verlag.
Ralis HM, Beesley RA, Ralis ZA. 1973. Techniques in neurohistology.
London: Butterworths.
Rhodin T. 1954. Correlation of Ultrastructural organization and function in
normal and experimentally changed proximal tubule cells of the mouse kidney.
Stockholm: Diss. Karnol. Inst.
Riley JN, fofolker DW. 1978. Morphological alterations in hippocampus after
long term alcohol consumption in mice. Science 201:646-648.
Sabatini DD, Bensch K, Barnett RT. 1963. Cytochemistry and electron
microscopy. The preservation of cellular structure and enzymatic activity by
aldehyde fixation. J. Cell Biol. 17:19-58.
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Shiraki H. 1977. Neuropathology of subacute myeloopticoneuropathy in humans
with special reference to experimental whole body autoradiographic studies
using labeled quinoform compounds. In: Neurotoxioology. Roizin, L, Shiraki,
H, and Grcevic N., eds. New York: Raven Press, pp. 327-344.
Spencer PS, Foster GV, Sterman AB, Horoupian D. 1980. Acetyl ethyl
tetramethyl tetralin. In: Experimental and clinical neurotoxicology.
Spencer, PS and Schaumburg, HH. eds. Baltimore: Williams & Wilkins., pp. 296-
308.
Spencer PS, Schaumburg HH (eds). 1980. Experimental and clinical
neurotoxicology. Baltimore: Williams and Wilkins.
Thompson SW. 1966. Selected histochemical and histopatnological methods.
Springfield: Charles C. Thomas.
Yamamoto I, Tosario B. 1967. Buffered formalin for primary fixation and
preservation of tissue for a long time. Proc. 25th Ann. Meeting, Electron
Micros. Soc. Am. Baton Rouge, LA: Claitor's Publishing Division, p. 24-25.
Zeman W, Innes JRM. 1963. Craigie's neuroanatomy of the rat. New York:
Academic.
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HG-Neuro-Peri Nerve
August, 1982
PERIPHERAL NERVE FUNCTION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Neuro-Peri Nerve
I. PURPOSE
The techniques in this standard are designed to develop
data on neurophysiological changes in the nervous system
for chemical substances and mixtures subject to such
testing under the Toxic Substances Control Act. The data
will characterize the neurophysiological changes, if and
when they occur and determine dose-effect. The EPA will
use these data to assess the risk of neurotoxic effects
these chemical may present to human health.
II. DEFINITIONS
A. Neurotoxicity or a neurotoxic effect is an adverse
change in the structure or function of the nervous
system following exposure to a chemical agent.
B. Conduction velocity is the speed at which the
compound nerve action potential traverses a nerve.
C. Amplitude is the voltage excursion recorded during
the process of recording the compound nerve action
potential. It is an indirect measure of the number
of axons firing.
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HG-Neuro-Peri Nerve
D. Chronaxy is the minimum stimulus pulse duration
required to produce a response at twice the rheobase
current. It is an indirect measure of the number of
axons firing.
E. Rheobase is the lowest current capable of producing a
response. It is determined with stimulus pulses so
long that further increase in their duration do not
lower the current required to produce a given
increment in the response.
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered to several groups of
experimental animals, one dose being used per group. The
peripheral nerve conduction velocity, amplitude and
chronaxy are assessed using electrophysiological
techniques. A dose-effect function is determined.
IV. TEST PROCEDURE
A. An ima1 Se1e ction
1. Species and Strain
Testing should be performed on a laboratory
rodent unless such factors as the comparative
metabolism of the chemical or species
sensitivity to the toxic effects of the test
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HG-Neuro-Peri Nerve
substance, as evidenced by the results of other
studies, dictate otherwise. All animals should
have been laboratory-reared to ensure
consistency of diet and environmental conditions
across groups and should be of the same strain
and from the same supplier. If this is not
possible, groups shall be balanced to ensure
that differences are not systematically related
to treatment.
2. Age
Young adult animals (at least 60 days for rats)
must be used. Age (_+_ 15 days for rats) must not
vary across groups.
3. Sex
Either sex may be used. Sex must not vary
across groups.
B. Number of An ima 1 s
Sufficient numbers of animals shall be used to detect
a 10% change from normal conduction velocity at the
5% level with 90% power. Generally, 20 animals/group
will satisfy this requirement.
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HG-Neuro-Peri Nerve
C. Control Groups
1. A concurrent control group is required. This group
must be an untreated group, or, if a vehicle is used
in administering the test substance, a vehicle
control group. If the toxic properties of the
vehicle are not known or cannot be made available,
both untreated and vehicle control groups are
required.
2. A positive control group is required to demonstrate
the sensitivity of the testing procedure. At least
three doses of a reference substance shall be used.
The doses shall produce graded changes in at least
one electrophysiological end point. Acute
administration is sufficient.
3. A satellite group may be treated with the high dose
level for 90 days and observed for reversibility,
persistence, or delayed occurrence of toxic effects
for a post-treatment period of appropriate length,
normally not less than 28 days.
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HG-Neuro-Peri Nerve
D. Dose Levels and Dose Selection
At least three dose level groups (in addition to the
control group(s)) shall be used and spaced
appropriately to producer a range of toxic effects.
The data should .be sufficient to produce a dose
response curve.
1. Highest Dose
The highest dose level in rodents should result
in toxic effects but not produce an incidence of
fatalities which would prevent a meaningful
evaluation.
2. Lowest Dose
The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest dose
level should exceed this.
3. Intermediate Dose(s)
Ideally, the intermediate dose level(s) should
produce miminal observable toxic effects. If
more than one intermediate dose is used, the
dose levels should be spaced to produce a
gradation of toxic effects.
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HG-Neuro-Peri Nerve
E. Duration of Testing
The exposure duration will be specified in the test
rule. This will generally be 90 days exposure.
F. Route of Administration
The test substance shall be administered by a route
specified in the test rule. This will usually be the
route most closely approximating the route of human
exposure. The exposure protocol shall conform to
that outlined in the appropriate acute or subchronic
toxicity guideline.
G. Combined Protocol
The tests described herein may be combined with any
other toxicity study, as long as none of the
requirements of either are violated by the
combination.
H. Study Conduct
1. Choice of Nerve(s)
The nerve conduction velocity test must
separately assess the properties of both sensory
and motor nerve axons. Either a hind limb
(e.g., tibial) or tail (e.g., ventral caudal)
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HG-Neuro-Peri Nerve
nerve must be chosen. Response amplitude and
chronaxy may be measured in a mixed nerve.
2. Preparation
a> In vivo testing of anesthetized animals is
required. A barbiturate anesthetic is
appropriate. Care should be taken to
ensure that all animals are administered an
equivalent dosage and that the dosage is
not excessive. If dissection is used,
extreme caution must be observed to avoid
damage to either the nerve or the immediate
vascular supply.
b. Both core and nerve temperature must be
monitored and kept constant (+0.5°C) during
the study. Monitoring of skin temperature
is adequate if it can be demonstrated that
the skin temperature reflects the nerve
temperature in the preparation under use.
Skin temperature should be monitored with a
needle thermistor at a constant site/ the
midpoint of the nerve segment to be tested.
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HG-Neuro-Peri Nerve
Electrodes.
(1) Choice of Electrodes.
Electrodes stimulation and recording
may be made of any conventional
electrode material, such as stainless
steel, although electrodes for non-
polarizing materials are
preferable. If surface electrodes
are used, care must be taken to
ensure that good electrical contact
is achieved between the electrode and
the tissue surface. Following each
application, any electrode must be
thoroughly cleaned.
(2) Electrodle PI.acemenit.
Electrode placement must be constant
with respect to anatomical landmarks
across animals (e.g. a fixed number
of mm from the base of the tail).
Distances between electrodes used to
calculate conduction velocity must be
measurable to _+_ 0.5mm. The recording
electrodes should be as far from the
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HG-Neuro-Peri Nerve
stimulating electrodes as possible.
A 40 mm separation is adequate in the
caudal tail nerve of the rat.
(3) Recording Conditions.
The animal should be grounded at
about the midpoint between the
nearest stimulating and recording
electrodes. The recording conditions
must be such that the stimulus
artifact has returned to baseline
before any neural response is
recorded which is used in the
analysis, under condition of maximal
band width of the preamplifier.
d. The electrical stimulator must be isolated
from ground. For conduction velocity and
response amplitude determinations, biphasic
or balanced pair stimuli to reduce
polarization effects are acceptable. For
measurement of chronaxy the stimuli
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HG-Neuro-Peri Nerve
should be square wave pulses, the duration
of which may be varied (usually 0.01-1.0
msec). A constant current stimulator is
preferred (and required for polarizable
electrodes) and should operate from about
10 uA to about 10 mA. If a constant
voltage stimulator is used, it should
operate to 250V. All equipment shall be
calibrated with respect to time, voltage,
and temperature.
e. The recording environment should be
enclosed in a Faraday cage unless
electromagnetic field pick-up can be shown
to be more than 1.5 times the amplifer
baseline noise, under recording
conditions. The recording output should be
amplified sufficiently to render the
compound action potential easily
measureable with an oscilloscope. The
amplifier should pass signals between 2.0
Hz and 4 kHz without more than a 3dB
decrement. The preamplifer must be
capacitatively coupled or, if direct
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HG-Neuro-Peri Nerve
coupled to the first stages, must be able
to tolerate any DC potentials which the
electrode-preparation interface produces,
and to operate without significant current
leakage through the recording electrodes.
f. A hard copy must be available for all
waveforms or averaged waveforms from which
measurements are derived, and for all
control recording required by this
standard. Hard copies must include a time
and voltage calibration signal.
3. Procedure
General
(1) Nerve response peak latency and
amplitude. Stimulation should occur
at inter-stimulus interval
significantly below the relative
refractory period for the nerve under
study. Stimulus intensity should be
increased gradually until the
response amplitude no longer
increases. At this point the
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HG-Neuro-Peri Nerve
"maximal" stimulus current is
determined. An intensity 25-50% (a
fixed value in a given study) above
the maximal intensity so determined
should be used for determining
response peak latency and response
amplitude. Response peak latency may
be read off the oscilloscope
following single sweeps or determined
by an average of a fired number of
responses. The baseline-to-peak
height technique (Daube, 1980) is
acceptable for determination of the
nerve compound action potential
amplitude, but in this case, at least
16 responses must be averaged.
(2) Determination of Chronaxy.
Chronaxy is defined as the minimal
stimulus pulse duration required to
produce a response at twice the
rheobase current. To determine
chronaxy, rheobase current must first
be determined. The rheobase current
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HG-Neuro-Peri Nerve
is the lowest current capable of
producing the response, and is
determined with stimulus pulses so
long (usually 1.0 msec for nerve
responses) that further increases in
their duration do not lower the
current required to produce a given
increment in the response. Once the
rheobase current is determined the
value obtained is doubled. Further
stimulation occurs at this higher
current level, but with the pulse
duration shortened below that which
elicits the increment in the
response. The pulse duration is then
gradually lengthened until the
original response recurs, that pulse
duration is defined as the
chronaxy. Such a determination
should be made at two levels of
stimulation, one near the nerve
threshold, and one near the maximal
stimulus strength.
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b. Motor Nerve
Motor conduction velocity may be measured
from a mixed nerve by recording the muscle
action potential, which follows the compound
action potential of the nerve. The
stimulus intensity is adjusted so that the
amplitude of the muscle action potential is
supramaximal. Measurement of the latency
from stimulation to the onset of the
compound muscle action potential gives a
measure of the conduction time of the motor
nerve fibers. To calculate the conduction
velocity, the nerve must be stimulated
sequentially in two places each with the
same cathode-anode distance, and with the
cathode located toward the recording
electrode. The cathode to cathode distance
between the two sets of stimulating
electrodes is divided by the difference
between the two latencies of muscle action
potential in order to obtain conduction
velocity. Placement of electrodes shall be
described-site of nerve stimulation may
differ from point of entry through skin.
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c. Sensory Nerve
The somatosensory evoked potential may be
used to determine the sensory nerve
conduction velocity in a mixed nerve. The
cathode is placed proximally at the two
stimulation locations with the same
cathode-anode distances. The recording
electrodes are placed on the skull. The
conduction velocity is calculated by
dividing the distance between the two
stimulating cathodes by the difference
between the two latencies of the largest
priminary peak of the somatosensory evoked
potential. Between 64 and 128 responses
should be averaged. The stimulation
frequency should be about 0.5 Hz. Stimulus
intensity should be the same as that used
for determining the motor conduction
velocity. Should the peak of the
somatosensory response be so broad that it
cannot be replicated with an accuracy of
less than 5% of the latency difference
observed, then a point on the rising phase
of the potential should be chosen, e.g. at
a voltage 50% of the peak voltage.
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Alternatively, the sensory nerve conduction
velocity can be obtained from a purely
sensory nerve or from stimulation of the
dorsal rootlets of a mixed nerve, using two
recording electrode pairs.
V. DATA COLLECTION, REPORTING AND EVALUATION
In addition to information meeting the requirements stated
in the EPA Good Laboratory Practice Standards [Subpart J,
Part 792, Chapter I of Title 40 Code of Regulations], the
following specific information should be reported:
A. Description of Test System and Test Methods
1. Positive control data from the laboratory
performing the test which demonstrate the
sensitivity of the procedure being used.
2. Hard copies of waveforms from which measurements
were made as well as control recordings.
3. Voltage and time calibration referable to the
standards of the Bureau of Standards or to other
standards of accuracy sufficient for the
measurements used.
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4. Data demonstrating that nerve temperature was
maintained constant throughout the recording
period.
B. Results
The following information must be arranged by test
group (dose level):
1. In tabular form, data must be provide showing
for each animal:
a. Its identification number;
•*)
b. Body weight, nerve conduction velocity,
amplitude and chronaxy.
2. Group summary data should also reported.
C. Eva1uation of Data
An evaluation of the test results (including their
statistical analysis) must be made and supplied.
This submission must include dose-effect curves for
conduction velocity, amplitude and chronaxy and a
description of statistical methods. Deviation from
conventional parametric techniques must be justified,
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REFERENCES
Aminoff, M.J. (Ed). 1980. Electrodiagnosis in Clinical
Neurology. New York: Churchill Livingstone.
Daube, J. 1980. Nerve conduction studies. In:
Electrodiagnosis j.n^ Clinical Neurology. Aminoff MJ (ed).
New York: Churchill Livingstone, pp. 229-264.
Glatt, A.F, H.N. Talaat and W.P. Koella 1979. Testing of
peripheral nerve function in chronic experiments in rats.
Pharmac. Ther. 5:539-543.
Johnson, E.W. 1980. Practical Electromyography. Baltimore:
Williams and Wilkins.
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August, 1982
PERIPHERAL NERVE FUNCTION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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TABLE OF CONTENTS
Pages
I. INTRODUCTION: NEED FOR STUDIES OF PERIPHERAL 1
Nerve Function
II. RATIONALES FOR STUDY DESIGN 12
A. Choice of Subjects 12
B. Choice of Nerve(s) for Test 13
C. Number of Animals 14
D. Preparation 16
1. General 16
2. Anesthesia 17
3. Temperature 18
4. Electrodes 19
III. METHODS OF STUDY CONDUCT 27
A. Procedure for Determining Conduction
Velocity and Amplitude 27
1. General 27
2. Motor Nerve 31
3. Sensory Nerve 33
B. Procedure for Determining of Chronaxy 37
REFERENCES 39
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INTRODUCTION; NEED FOR STUDIES OF PERIPHERAL
NERVE FUNCTION
The vertebrate nervous system has both central and
peripheral components. The peripheral nervous system is
composed of those axons, dendrites, cell bodies and
accessory organs not present in the brain or spinal cord.
Because of their immediate accessibility, considerable
information about the characteristics of peripheral nerves
is available, including their anatomy, physiology and
response to toxic insult. Many texts are available which
describe the general properties of the peripheral nervous
system (e.g. Waxman 1978). A few general points are
important for orientation purposes and these will be
described briefly below.
Peripheral nerves consist of heterogeneous bundles of axons
from nerve cells. Most peripheral nerves contain both
sensory and motor axons of a variety of diameters from
nerve cells in different areas of the body. Information
transmission along nerves is by way of action potentials in
the axons. In the normal (physiological) condition, axons
conduct action potentials in one direction
(orthodromically) which is towards the central nervous
system for sensory axons, and away from the cell body for
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motor axons. The larger diameter axons conduct action
potentials more rapidly (i.e. have faster conduction
velocities) than the small ones. All but the smallest
axons have a myelin sheath, which is a fatty external
insulation interrupted at regular intervals by nodes of
Ranvier. Action potential conduction in myelinated nerves
is saltatory, appearing to jump from one node of Ranvier to
the next. Saltatory conduction is more rapid than
conduction in unmyelinated nerves. Since conduction
velocity is related to axon diameter (e.g. Cullhein and
Ulfhake 1979), depending in some preparations upon a
general geometric factor shown by Mirolli and Talbott
(1972) to be equal to /A/P, where A is the cross-sectional
area and P is the perimeter, and since axon diameter of any
particular nerve cell tends to decrease with distance from
the cell body of origin (largely due to branching)
(Quillian 1956), conduction velocity is not constant along
the entire length of the axon.
The ability of an axon to conduct an action potential is
contingent upon the integrity of its membrane and the
maintenance of ionic concentration differences between the
inside and outside of the axon (i.e. across the
membrane). Maintenance of the ionic concentration
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differences depends in part upon the integrity of energy
metabolism in the membrane (Hodgkin and Keynes 1955). The
greater speed of myelinated nerve conduction is obviously
contingent upon the integrity of the myelin sheath and the
node of Ranvier.
A number of substances have been demonstrated to produce
peripheral neuropathy. Among these substances are organic
solvents such as n-hexane (e.g., Scelsi et al. 1980),
carbon disulfide (Vigliani 1954), methyl-n-butyl ketone
(e.g. Spencer et al. 1975) and acrylamide (LeQuesne
1980). A delayed form of peripheral neuropathy is produced
I
by some organophosphate esters (Barnes and Denz 1953).
Peripheral neuropathy has also been reported following
lithium intoxication (Uchigata et al. 1981), alcoholism
(e.g. Ballantyne et al. 1980), diabetes (e.g. Sharma and
Thomas 1974), dapsone exposure (Roller et al. 1977),
disulfiram (Moddel et al. 1978), and1 nitrous oxide exposure
(Layzer et al. 1978).
Not all agents which produce peripheral neuropathies do so
by the same mechanism. Recent efforts to classify
neurotoxic disease according to the cellular target site
(Spencer and Schaumburg 1980) have identified three basic
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types of toxicants: those which affect the cell body
(neuronopathy); those which affect the peripheral processes
of the cell (axonopathy) and those which affect the myelin
sheath covering the axons of some cells (myelinopathy).
Most of the toxicants producing peripheral neuropathy have
the axon as the primary target, although some produce
axonal damage which is secondary to neuronopathy (e.g.
doxorubicin, Cho et al. 1980). Substances producing
primary toxicity in axons may selectively affect either the
distal axon (e.g. hexacarbons, carbon disulfide, acrylamide
and TOCP, Spencer and Schaumburg 1980) or the proximal axon
(e.g. J3/JJ1- iminodipropionitrile; Spencer and Schaumburg
1980). Substances which preferentially attack myelin also
produce peripheral neuropathy (e.g. hexachlorophene;
Towfighi et al. 1973).
Unfortunately it is not yet possible to predict the type of
toxic effect from the chemical structure of the compound.
For example, two structurally similar alkyltin compounds
produce strikingly different toxic effects. Triethyltin
produces a myelinopathy (Torack et al. 1970), while
trimethyltin produces a neuronopathy (Brown et al. 1979).
Tests for detection of peripheral nerve dysfunction must
therefore be sufficiently sensitive and generalized to
detect these different types of effects.
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A growing number of studies have used the well developed
science of electrodiagnosis to detect neuropathy. The
techniques available in this science have been used in
neurological clinics for some time, but have been applied
less frequently to detection of neurotoxicity in
experimental animals.
The techniques of electrodiagnosis allow the direct
measurement of activity in nerves. Production of changes
in electrical potential during activity is a general
property of axons. Details of the properties of these
axons are available in any physiology text (e.g.
Mountcastle 1968). Briefly, it can be stated that an axon
responds to a stimulus in an all-or-none way. When
stimulated, either via postsynaptic potentials from its
cell body, generator potentials at its terminals, or an
electrical stimulus applied in the vicinity of the axon
itself, an axon either transmits or does not transmit an
action potential i.e. responds in an all-or-none-fashion.
Barring toxic or extrinsic interference or stimulation in
the relative refractory period, once an action potential is
triggered in a particular axon, its conduction properties
are exactly like all other action potentials which have
been triggered in that axon: it has the same speed and
amplitude.
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Since nerves are made up of bundles of axons with different
thresholds for production of an action potential, and with
different conduction velocities, an electrically stimulated
nerve does not produce an all-or-nothing response in the
same sense that an axon does. The action potential
produced by stimulation of a nerve is called the compound
action potential, because it contains the compounded
activity of the axons within it. Following any particular
stimulus, none, some, or all of the axons in the nerve
might produce action potentials. Whether a particular axon
produces an action potential depends upon its thresold, its
proximity to the stimulating electrode, and whether the
axon is in a refractory period from any previous
stimulation. In a given experiment, when stimulation is at
a low rate (e.g. less than 25 hz) and electrode position is
unchanged, the only variable which should alter the
presence or absence of an action potential from a
particular axon is stimulus intensity. The compound action
potential recorded from a nerve represents the summated
activity of all the axons stimulated by the stimulus. As
stimulus intensity increases, more axons will reach their
thresholds for action potentials, and thus the amplitude of
the response may be expected to increase. When the
stimulus is sufficiently intense to have activated all
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axons in the nerve, further increases in intensity will no
longer increase the amplitude of the response.
The latency from the time of supramaximal stimulation to
the time at which a compound action potential is recorded
from the nerve depends upon the distance between the
stimulating and recording electrodes (assuming both are
close to the nerve) and the conduction velocity of the
stimulated axons in the nerve. Rapidly conducting axons
have lower thresholds than slowly conducting axons and,
therefore, at low stimulus intensities only the more
rapidly conducting axons will be recorded. Whether a
particular axon's action potential contributes to the
compound action potential is in part contingent upon
whether the recording electrode is close enough to detect
it. An action potential may occur in part of the nerve
which is relatively distant from the recording
electrodes. In that case, its contribution to the
amplitude of the compound action potential will be small.
If it is a relatively rapidly conducting action potential,
its peak will occur before the peak of the compound action
potential. Thus the ascending slope of the compound action
potential is composed of the ascending slope of action
potentials which peak before, shortly after, and at the
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same time as the peak of the compound action potential.
The peak of the compound action potential represents the
time at which the most fibers are active at the recording
site. As such, measurement of the latency from stimulus to
peak of the compound action potential reflects conduction
velocity in the population of axons best represented in the
nerve (Hursh 1939). To assess the most rapidly conducting
axons, latencies must be calculated to the onset of the
compound action potential.
The duration of the compound action potential also depends
upon the stimulus intensity. As higher stimulus
intensities are used, fibers with a wider range of
conduction velocity are stimulated and therefore at the
recording electrodes some responses arrive relatively early
(large low threshold fibers relatively distant from the
stimulus), while most arrive relatively late (small higher
threshold fibers). The net effect of intensity increases
is prolongation of the recorded response.
Any process which alters the distribution of fiber types
within a nerve (i.e. selectively destroys large or small
fibers) will obviously shift the conduction velocity of the
whole nerve response. Equally obvious is that, as fibers
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are destroyed, the amplitude of the compound nerve action
potential will decrease in proportion to the destroyed
fibers' normal contribution to the compound action
potential. Finally, any process which alters the membrane
properties of the axon will change the stimulus
requirements for elicitation of a maximal response.
Assessment of the functional integrity of the peripheral
nervous system with the techniques of electrodiagnosis
(i.e. neurophysiologically), may take many forms. Among
the techniques which have been utilized are dissection of
single axons (e.g. Mitolo-Chieppa and Carratu 1980),
assessment of refractory period (Lowitzsch et al. 1981,
Hopf and Eysholdt 1978), assessment of the extent to which
axons and nerves can follow trains of stimuli which occur
at high rates (Lehmann and Tachmann 1974), measurement of
peripheral nerve reflexes such as the H reflex and
responses such as the F response (Lachman et al. 1980),
accomodation indices (Quevedo et al. 1980), and use of
collision techniques for selectively blocking activity of
some nerve axons to study others (Kimura 1976). Some of
these techniques may become useful in assessment of toxic
neuropathy in the future. The present standard, however,
focuses upon three measures of the functional integrity of
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the nerve which have demonstrated utility in both the
neurological clinic and in detection of toxic neuropathy:
conduction velocity, response amplitude, and chronaxy.
Conduction velocity is the speed at which action potential
are conducted along axons. As indicated earlier,
conduction velocity of an axon is dependent upon the
diameter and myelin sheath of the axon, and conduction
velocity of a nerve is dependent upon which axons in the
nerve are stimulated. Conduction velocity is usually
measured in such a way that the activity of the fastest
conducting axons is assessed. Changes in conduction
velocity which occur following exposure to toxic agents
producing an axonopathy are reliable, but usually not
large, often ranging from 10% to 30% of control values
(Gilliatt 1973). On the other hand, demyelination produces
large decrements (>50%) in conduction velocity (McDonald
1963). It is therefore reasonable to assume that if large
decrements in conduction velocity have occured, either the
nerve contains a large number of demyelinated axons, or the
population of large diameter axons in the nerve has been
greatly reduced.
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As indicated earlier, the amplitude of the recorded
compound action potential is related to the number of axons
in the nerve which have activated. The only way to assess
amplitude is to ensure that all of the axons capable of
producing action potentials are in fact responding.
Otherwise, differences in amplitude between groups may
simply reflect differing thresholds of stimulation for the
different populations of axons. One may ensure that all
excitable axons in the nerve are stimulated when further
increases in stimulating current fail to produce increases
in response amplitude. It is generally recognized that
decreases in amplitude of the compound nerve action
potential occur in both axonopathy and myelinopathy. The
two conditions are most easily differentiated by observing
the concommitantly greater reduction in conduction velocity
which occurs with myelinopathy (Daube 1980).
Chronaxy determination in muscle is a routine procedure in
clinical electromyography and is useful for detecting
denervation (Rogoff 1980). Determination of chronaxy in
peripheral nerves is less common, but allows assessment of
excitability of the nerve. A number of natural (e.g.
tetrodotoxin), and man made (e.g. DDT), toxicants affect
membrane properties of neurons.
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Alterations in chronaxy may be expected to aid in detection
of these effects, however the precise nature of the effect
will not be discernible from an altered chronaxy. Detailed
characterization of toxic effects would have to be carried
out by other techniques, such as voltage clamping of the
membrane (Narahashi 1980).
Aside from their clinical utility, the techniques of
electrodiagnosis have demonstrated sensitivity to
neurotoxicity. Deficits in conduction properties of rat
peripheral nerves have been demonstrated following exposure
to a variety of substances, including acrylamide (Boyes
1980), n-hexane (Robert 1981), carbon monoxide (Petajan et
al. 1976), hexachlorophene (Maxwell and Le Quesne 1979),
and carbon disulfide (Seppalainen and Haltia 1980).
II. RATIONALES FOR STUDY DESIGN
A. Choice of Subjects
Few a^ priori reasons can be developed for requiring
the use of a particular species for these tests. Rats
and mice have particular advantages since they are
relatively cheap and available. Since a moderately
large sample size (e.g., 20 subjects/group) is
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required to detect changes this may make the use of
rodents generally preferable. Organophosphate
compounds (e.g., TOCP) known to produce delayed
axonopathies in some species do not produce delayed
neurotoxicity in rodents. The effects of
organophosphate compounds may only be detected using
hens or other species of known sensitivity to that
. type of.toxicant. . Due to the ease with which long
nerves are studied compared to short nerves, many
investigators may choose large animals such as cats.
Use of the cat may make in vitro recordings from the
sural nerve (pure sensory) more feasible.
Regardless of the species chosen, it is important to
use animals from the same strain, of the same gender,
and of the same age (+_ 15 d for rats), since these
variables have been shown to influence nerve
conduction properties (Miyoshi and Goto 1973, Glatt et
al. 1979, Hegmann 1975).
B. Choice of Nerve(s) for Test
The nerve conduction test should assess the properties
of at least one sensory nerve and one motor nerve.
Many peripheral nervous system disturbances related to
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toxicants are first detected in sensory nerves (e.g.
Schuchmann and Braddom 1980). Since long nerves are
generally affected earlier than short nerves ( e.g.,
Barnes and Denz 1953) (perhaps owing to their larger
diameter), either a hindlimb or tail nerve should be
chosen. The caudal tail nerve of the rat is probably
the easiest with which to work, and the description of
methods which follow is therefore oriented towards the
use of this nerve. The methods described are
sufficiently general that they may be easily adapted
to different nerves in different species.
C. Number of Animals
Details of the experimental design will depend upon
whether a particular study is to involve acute or
repeated exposures. It is not the purpose of this
standard to specify precise exposure parameters or
number of groups to be used. However, in any study it
will be necessary to include an untreated (vehicle
treated) and a positive control group, i.e., a
compound and dosage known to affect peripheral nerve
function. The number of subjects to be tested in each
group must be considered. The methods described so
far have specified conditions that would allow
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differences in conduction velocity on the order of 10%
to be detected. In the rat caudal tail nerve, 2 m/sec
represents about 10% of the normal conduction velocity
(100 day old rats, temperature controlled at 37°C-
Miyoshi and Goto 1973, Glatt et al. 1979). Using the
rough rule of thumb that means whose standard errors
do not overlap are probably significantly different,
it is clear that a 10% alteration in conduction
velocity will not be detected unless each standard
error is less than 5% of the mean. Thus, in order to
detect a 4 m/sec difference in conduction velocity,
enough subjects should be tested to ensure that the
standard error of the mean is less than 5%. In actual
practice (e.g. Miyoshi and Goto, 1973), a group size
between n = 10 and n = 25 should achieve such a
standard error, but the actual group size must depend
on the variability achieved in the individual
laboratory. A greater standard error (^10%) is
acceptable for amplitude measurements, and should be
achieved with the same group size as required for
conduction velocity measurements.
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Statistical analysis of the acquired data should be by
conventional parametric techniques, presumably
analysis of variance.
D. Preparation
1. General
In vivo testing of nerve function is desirable
to ensure comparability in chronic, subchronic,
and acute studies. Numerous studies have
indicated the feasibility of serial testing of
the rat caudal nerve (e.g., Glatt et al., 1979,
Miyoshi and Goto* 1973, and Rebert, 1981).
Although it is possible to adequately perform
tests using isolated nerves, many more subjects
may be required since no within-subject designs
would be possible. Variability is apparently
not decreased by using in situ dissected nerve
preparations (e.g., compare Rasminsky et al.
1978, with Glatt et al. 1979). In using the in_
vivo preparation, use of anesthetized animals is
recommended. Use of the unanesthetized
preparation would be preferable except that the
procedure produces discomfort in the animal
which can be avoided by anesthesia.
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Unanesthetized preparations should be used only
when it is presumed that the experiment will be
compromised by the anesthetic, or it can be
demonstrated that the animal is not in distress.
2. Anesthesia
General anesthetics are known to block nerve
conduction, but depending upon the anesthetic
there is a varying degree of safety between the
concentration which produces general anesthesia
and the concentration which produces blockade of
nerve conduction (Barker 1975). This margin of
safety is especially large for the barbiturate
anesthetics. In the rat caudal tail nerve, no
change in conduction velocity occurs following
20 mg/kg sodium pentobarbital. A 3.5 m/sec
decrease in velocity occurs following 40 mg/kg
but no further decline is evident at 60 mg/kg
(Glatt et al. 1979). Therefore, barbiturate
anesthetic is appropriate. Care should be taken
to ensure that (1) all animals are administered
an equivalent dosage, and (2) the dosage
administered is not more than is necessary to
maintain a level of general anesthesia.
Investigators should be alerted to the
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possibility that the appropriate dosage may be
influenced by the toxic effects of the test
substance. For rats and mice, a combination of
sodium pentobarbital and chloral hydrate
(Chloropent, Fort Dodge) is effective at 3.5
ml/kg body weight (i.p.). A dosage of 50 mg/kg
sodium pentobarbital with 0.1 mg/kg atropine
pre-treatment is also adequate. Some
anesthetics, such as urethane, seem to offer an
advantage since their depression of peripheral
reflexes is less than barbiturates, and the
level of anesthesia is apparently stable for a
longer period of time than sodium
pentobarbital. Urethane, unfortunately,
produces liver damage and therefore renders the
session terminal for the subject.
3. Temperature
Temperature is a critical variable which must be
monitored and maintained during the
experiment. Conduction velocity may drop as
much as 2.4 m/sec for every C° drop in
temperature (Davis et al. 1975, Braddom and
Schuchmann 1980). Further, it is well known
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that some many anesthetics impair
thermoregulation (see Goodman and Oilman
1980). An acceptable study must demonstrate
that temperature has been adequately
controlled. An effective way to do this is to
immerse the tail (if the tail nerve is used) or
the nerve (if a dissected in vivo or in vitro
preparation is used) in a warm mineral oil (or
other non-conducting fluid) bath, maintained at
constant temperature. The tail (or nerve) must
remain in the solution during testing, and
should be in the solution for at least 5 min
(Miyoshi and Goto 1973) before testing is begun,
to allow warming up to the constant temperature
of the bath.
4. Electrodes
Electrodes for stimulation and recording may be
made of any conventional electrode material. An
effective electrode for recording from the rat
tail nerve is the needle electrode commercially
available from a number of suppliers. Different
electrode materials have different impedances,
and this may affect response amplitude. If the
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electrode type remains constant within an
experiment, there will be no variation in
response amplitude as a result of changing
electrode types. If surface electrodes are used
in place of needle electrodes, care must be
taken to ensure that good electrical contact is
achieved between the electrode and the tissue
surface. The tissue should be cleaned and an
electrolyte gel applied to the tissue-electrode
junction. The use of surface electrodes is more
difficult when bath immersion is used for
heating, since leakage of the bath fluid into
the tissue-electrode junction will significantly
reduce conductivity. If surface electrodes are
used, the investigator must be able to
demonstrate that good electrical contact was
maintained. An acceptable method for
accomplishing this is measurement of tissue
impedance. In addition, the DC potential from
the electrodes should be monitored unless the
amplifier is known to be able to tolerate
electrode offsets of up to 800 mV, e.g. by
capacitative coupling (Schmitt and Almasi 1971,
Patterson 1978). Following each application,
any electrode used must be thoroughly cleaned.
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Electrode placement and the accurate measurement
of interelectrode distances is critical to the
success of the method. A scheme must be devised
to ensure that electrodes are placed in the same
location from one animal to the next. One
successful method is to use a plastic block
grooved on the underside to form a trough which
matches the contour of the tail, with holes
penetrating the groove at precisely measured
intervals. The holes, which should be just
large enough to accept a needle electrode, may
be conveniently spaced at 5 mm distances in a
line which tracks the length of the tail at
approximately the midpoint between the midline
and the lateral surface. Electrodes should also
always be inserted to a common depth in the
tail. Although conduction velocity can be
calculated whenever the distance between the
stimulating and recording electrodes is known,
it is important to keep this distance constant
across animals. As the distance between the
recording and stimulating electrodes increases,
the compound action potential waveform becomes
broader due to between-fiber differences in the
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conduction velocity of fibers within the nerve
(Gasser, 1943). Consequently, failure to keep a
constant distance between animals may yield
erroneous estimates of both velocity and
amplitude of the compound action potential.
In order to achieve an accurate assessment of
conduction velocities, it is necessary to place
the recording electrodes as far away from the
stimulating electrodes as possible. If the
stimulating and recording electrodes are too
close, then the measuring equipment may not be
sensitive enough to resolve small differences in
peak latency between groups. An adequate
distance between the most distal stimulating
electrode nearest the recording electrode (which
should be the cathode) and the nearest recording
electrode is 40 mm. This distance allows
assessment of conduction velocities which may
differ by 1 m/sec if the recording device can
i>
detect differences of 0.1 msec. However, this
determination of conduction velocity assumes
that the point of stimulation is accurately
known. Under some conditions the stimulating
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point can be several millimeters from the
cathode. A more accurate method utlizes the
latency between two recording electrode pairs
(with a single stimulation pair), while a
somewhat less accurate, but acceptable method,
uses the latency difference between a single
recording pair and two stimulating pairs. The
latter method is required when a response adds
an indeterminate delay to the conduction
latency, as in the muscle response and the
somatosensory evoked response. Since conduction
velocity measurements for both motor and sensory
fibers must be obtained according to the
guideline, the use of two stimulating pairs will
generally be used in determining conduction
velocity in a mixed nerve by means of a single
recording and single stimulating pair will be
redundant. Such a recording configuration will
be useful in determining action potential
amplitude, and peak latency in a mixed nerve, as
described in Section III. A. 1. The distance
between the anodal and cathodal electrodes
should be about 5.0 mm and remain constant
throughout the experiment and should be the same
for all pairs of stimulating electrodes used on
23
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HS-Neuro-Peri Nerve
a given nerve. The animal should be grounded at
midpoint between stimulating and recording
electrodes.
To reduce artifacts, the electrical stimulator
used to elicit the compound action potentials
should be isolated from ground. The stimuli
should be square wave pulses of constant
duration; usually between 0.01 and 1.0 msec
duration. Pulses which are 0.1 msec are
convenient for routine use since they are not
long enough to produce a large artifact, but are
long enough such that the stimulus intensity
does not have to be excessive in order to
achieve a supramaximal stimulus. Current flow,
not voltage, is the effective simulus for nerve
tissue (Ranck 1975) and thus a more precise
description of the stimulus is provided when a
constant current rather than a constant voltage
is used. Furthermore, polarization of
electrodes can change current flow from a
constant voltage stimulator. Therefore, a
constant current stimulator is desirable. The
current range over which a stimulator should
24
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HS-Neuro-Peri Nerve
operate is from about 10 uA to about 10 mA. If
a constant voltage stimulator is used instead of
a constant current stimulator, it should operate
up to 250 V in order to provide sufficient
current to excite abnormal nerves (Daube 1980)
and should be provided with a series resistor
across which a differential amplifier can
measure current.
The recording environment, in addition to
including a constant temperature apparatus
(described above), may need to be enclosed in a
Faraday cage. A grounded Faraday cage will
significantly reduce electrical interference
from other sources (Wolbarsht 1964).
The recording electrodes should be lead, via a
shielded cable, to the input of a differential
amplifier. The gain of the amplifier should be
sufficient to render the compound action
potential easily measurable with an oscilloscope
(i.e., produce a peak deflection of 4-5 cm) and
at the same time be within the range of
appropriate input signal for a signal averager
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HS-Neuro-Peri Nerve
or summator. The amplifier should pass signals
between 2.0 Hz and 4kHz without more than a 3dB
decrement. A signal averager or computer is
necessary to accurately measure the compound
action potential amplitude. A large variety of
such devices are available. The only constraint
is that the device must be capable of sampling
at a rate sufficient to detect small alterations
in conduction time. A minimum sampling rate is
20 usec/point, or 50 kHz, for conduction
distances as short as 40 mm. At this sample
rate, a change in conduction velocity from 40
m/sec to 39 m/sec would be detectable. For
nerves with faster conduction velocities, this
combination of sample rate and segment length
would not detect alterations of 1 m/sec.
Hard copies must be available for representative
compound action potentials from which
measurements are included in a study. These may
take the form of oscilloscope tracing
photographs, X-Y plotter outputs from a signal
averager, or any other suitable device. Hard
copies should contain a time and amplitude
calibration signal.
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HS-Neuro-Peri Nerve
III. METHODS OF STUDY CONDUCT
A. Procedure for Determining Conduction Velocity and
Amplitude
1. General
In a typical experiment, the procedure should be
as follows. All equipment is turned on,
checked, and calibrated. The constant
temperature bath is heated to the appropriate
temperature. The animal (whose group should be
blind to the testing technician if possible) is
anesthetized, the tail (or nerve) is placed on
the recording trough, and the electrodes are
inserted. At this point, a check should be made
of the adequacy of recording. The tail is then
immersed in the mineral oil bath and allowed to
warm for at least 5 min (Glatt et al., 1979).
When the tail has warmed, the stimulator is
turned on and may be used at any convenient rate
(e.g., 2-3 hz) which is significantly below the
relative refractory period of the nerve. The
current is gradually increased until the nerve
27
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HS-Neuro-Peri Nerve
response is noted on an oscilloscope. The nerve
response should be easily detectable before a
muscle response becomes apparent. If it is not,
the electrode may be repositioned. However,
preceding statements regarding electrode
position still apply. If a question exists
whether a response is the result of nerve or
muscle activity, the muscle activity may be
fatigued with a high frequency (e.g., 500 hz)
stimulus. This procedure is only useful for
identification purposes, not data collection.
High rates of stimulation would cause stimuli to
fall within the relative refractory period of
the nerve, and therefore reduce amplitude. As
the nerve response becomes apparent, the
stimulus intensity is gradually increased until
the response amplitude no longer increases.
This value of the stimulus is the maximal
intensity. The intensity used for studying
conduction velocity and response amplitude
should be set at some fixed ratio, e.g. 25%-50%
above the maximal intensity. For determining
conduction velocity, the response latency may
either be read off the oscilloscope or,
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HS-Neuro-Peri Nerve
preferably, determined from the averager. An
average of 16 responses should be sufficient to
produce a reliable estimate of both the latency
and amplitude of the compound action
potential. In clinical electromyography, the
latency for motor conduction velocity studies is
usually taken to the onset of the compound
muscle action potential wave while the latency
for sensory conduction velocity studies is
usually taken to the peak of the compound nerve
action potential wave (Schuchmann and Braddom
1980). Peak latencies provide an index of the
conduction velocity of the average fiber in the
bundle, while onset latencies provide an index
of the conduction velocity of the fastest fibers
in the bundle. Given the occasionally small
size of the compound action potential, it may be
more expedient to consistently measure the peak
latency than the onset latency or to use as the
end point voltage at one-half the peak
voltage. This measurement is less dependent
upon the sensitivity of the recording
equipment. It should be noted that measurements
29
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HS-Neuro-Peri Nerve
derived from the compound nerve action potential
in a mixed nerve include both sensory and motor
fibers. The conduction velocity is calculated
by dividing the conduction distance (from most
distal stimulating to most proximal recording
electrodes) by the conduction time (peak
latency-onset of stimulus artifact).
Two approaches to measuring the amplitude of the
compound action potential wave are possible.
Either the peak height may be measured from the
pre-spike baseline, or the area under the wave
may be measured from onset to return to
baseline. In either case, it is necessary to
make the measurements in a reproducible and
accurate manner. Many averagers have a
capability of providing a digital output
corresponding to a cursor location on the
displayed waveform. With these instruments, the
peak height is easily determined as the
difference in voltage between the peak and the
baseline. The peak height gives an estimate of
the number of axons with average conduction
30
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HS-Neuro-Peri Nerve
velocities in the fiber bundle or nerve
measured. The area under the wave gives a
measure of the total number of functional axons
within the nerve. This area may be measured
either by a version of the polar planimeter, or
by electronic means using either a computer or
attachments available on special purpose
averagers. Given the added information provided
by performing measurements of area, it is
recommended as the standard technique. Bipolar
recording techniques may produce responses whose
amplitude and areas are difficult to measure
(i.e. biphasic). In these cases the negative
pole of the differential amplifier may be
grounded. If chosen for one preparation, this
monopolar configuration should be maintained
throughout the experiment.
2. Motor Nerve
In most cases, it is appropriate to
differentiate between the sensory and motor
components of the nerve response. Since the
compound action potential of the mixed nerve
recorded as described above confounds the
orthodromic motor fiber and antidromic
31
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HS-Neuro-Peri Nerve
sensory fiber responses, it is of minimal value
as a measure of conduction velocity although it
can provide adequate information on nerve
response amplitude, peak latency, and
chronaxy. However, extraction of the motor
component of the mixed nerve is a simple
matter. The muscle response which follows the
compound action potential of the nerve results
only from activity of the motor fibers in the
nerve. Thus, measurement of the latency from
stimulation to the onset of the muscle response
gives a measure of the conduction time of the
motor fibers. This measure is confounded with
the synaptic delay between the arrival of the
action potentials at the terminals of the axons
and the contraction of the muscle. To calculate
the conduction velocity, therefore, the nerve
must be stimulated in two places. The distance
between the two sets of stimulating electrodes
(the cathode to cathode distance) is then
divided by the difference between the two
latencies in order to obtain the conduction
velocity. In these studies, the stimulus is
32
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HS-Neuro-Peri Nerve
adjusted so that the amplitude of the muscle
action potential is supramaximal. The amplitude
of the muscle action potential is measured by
the same means as described for the mixed nerve
action potential. Under these conditions, the
distance between the proximal and distal
stimulating cathodes must be at least 20 mm for
sweep sample rates as slow as 50 kHz.
3. Sensory Nerve
Two approaches to recording the conduction
velocity of the sensory nerve response are
acceptable. In the first, a purely sensory
nerve, or the sensory branch of a mixed nerve,
is stimulated and the antidromic action
potential is recorded a fixed distance from the
stimulus as described above. This direct
recording method is conceptually the simplest.
However, it requires careful dissection and is
especially difficult in rodents since the sural
nerve (the most appropriate nerve upon which to
do the study) is quite short. An acceptable
procedure is to stimulate the sural nerve and
33
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HS-Neuro-Peri Nerve
record from the sciatic nerve trunk in the
pelvis. If this procedure is used, great care
must be taken in measuring the conduction
distance. The sural nerve of cats is a more
suitable preparation upon which to perform this
study by the direct recording method. However,
the time required to perform a successful
dissection without damage to the nerve is
greater and cats are more expensive.
An alternative approach may be easily used in
the rodent. The tail nerve is prepared as for
motor nerve determinations, except that the
cathode is placed proximally instead of distally
at the two stimulation locations. The recording
electrodes are placed on the skull for recording
the somatosensory evoked potential. The
somatosensory evoked potential represents
activity along a number of tracts and across a
number of synapses. However, the difference
between the latency to the onset of the first
peak of the response, when recorded following
stimulation at two tail locations 40 mm apart
34
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HS-Neuro-Peri Nerve
should give an accurate measurement of the
conduction time between the two stimulating
electrodes and will reflect only sensory
activity (Giblin 1980). The conduction velocity
is calculated by dividing the distance between
the two stimulating cathodes by the difference
between the two latencies. Should the peak of
the somatosensory response be so broad that it
cannot be replicate with an accuracy of less
than 5% of the latency difference observed, then
a point on the rising phase of the potential
should be chosen, e.g. at a voltage 50% of the
peak voltage. Alternatively, the sensory nerve
conduction velocity can be obtained from a
purely sensory nerve or from stimulation of the
dorsal rootlets of a mixed nerve, using two
recording electrode pairs to obtain the
conduction velocity by difference in the
latency.
In using the somatosensory evoked potential to
determine the sensory nerve conduction velocity,
it is necessary to average more than 16
responses. A reasonable number of responses to
35
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HS-Neuro-Peri Nerve
average is between 64 and 128. The stimulation
frequency must be slowed to about 0.5 hz for
these studies since more rapid stimulation will
alter the properties of the first wave of the
somatosensory evoked potential. The electrodes
for recording the somatosensory evoked potential
may be either small stainless steel screws
threaded into the skull (0-80 or 00-90), or
electrode wires applied to the surface of the
skull. If the latter approach is taken, care
must be taken to ensure that good electrical
contact is maintained between the skull and the
electrode. An electrolyte gel applied in a
small dab at the electrode tip is sufficient for
this purpose. Precise electrode location is not
critical as long as it is the same from one
animal to the next. A useful configuration for
this experiment in rats is to place the active
electrode 2 mm posterior and 1 mm lateral to
bregma on the side contralateral to the
stimulation electrodes. The reference electrode
may be place 5 mm anterior to bregma along the
midline.
36
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HS-Neuro-Peri Nerve
B. Procedure for Determination of Chronaxy
Muscle chronaxy determination is a routine procedure
in clinical electrodiagnosis. It is sensitive to
denervation, and may detect alterations at an earlier
stage than nerve amplitude or velocity. Nerve chronaxy
measurements allow assessment of the excitability of
the nerve and may also detect pyramidal tract
dysfunction (Petty and Johnson 1980).
When peripheral nerve is stimulated by a square wave
pulse, the amplitude of the current required to
produce a given response, be it the smallest
detectable or maximal response, depends upon the
duration of the square wave. In general, as the pulse
duration becomes longer, the required current
intensity becomes less until finally it is noted that
increasing pulse durations no longer lower the
stimulating current required to produce a given
increment in the response. The curve generated by
this procedure a strength-duration curve. The
asymptote current for long duration pulses is known as
rheobase. To determine chronaxy, the rheobase current
is doubled. Further stimulation occurs at this
37
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HS-Neuro-Peri Nerve
highest current level, but with the pulse duration
shortened below that which elicits the increment in
the response. The pulse durations is then gradually
lengthened until the original response recurs, and
that pulse duration is defined as the chronaxy. All
of the considerations which apply to performance of
conduction velocity and nerve amplitude tests also
apply to determination of chronaxy. The two
procedures may be carried out upon the same
preparation, thereby minimizing subject and personnel
costs. Although completion of the strength-duration
curves may be more sensitive than chronaxy, the
resulting data are more difficult to evaluate
statistically and are not required at this time.
38
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HS-Neuro-Peri Nerve
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43
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HG-Neuro-Motor Act
August, 1982
MOTOR ACTIVITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
-------
HG-Neuro-Motor Act
I. PURPOSE
A. General
In the assessment and evaluation of the toxic
characteristics of a substance, determination of the
effects of administration of the substance on motor
activity is useful when neurotoxicity is suspected.
B. Acute Motor Activity Test
The purpose of the acute motor activity test is to
determine whether changes in motor activity occur at
acute exposure levels below those which cause
systemic toxicity. This test is an initial step in
determining the potential of a substance to produce
acute neurotoxicity and in establishing a dosage
regimen for subchronic testing. Data from an acute
motor activity test may also serve as a basis for
screening members of a class of substances for known
neurotoxicity, prior to the initiation of more
complex subchronic neurotoxicity testing.
c* S ub chron i c Mo tor Act i v i ty Test
The purpose of the subchronic motor activity test is
to determine whether the repeated administration of a
suspected neurotoxicant results in changes in motor
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HG-Neuro-Motor Act
activity at exposure levels below those which cause
systemic toxicity. This test is an initial step in
determining the potential of a substance to produce
subchronic neurotoxicity.
II. DEFINITIONS
A. Neurotoxicity is the adverse effect on the structure
or function of the central and/or peripheral nervous
system related to exposure to a chemical substance.
B. Motor activity is any movement of the experimental
animal.
C. A toxic effect is an adverse change in the structure
or function of an experimental animal as a result of
exposure to a chemical substance.
PRINCIPLE OF THE TEST METHOD
The test substance is administered to several groups of
experimental animals, one dose being used per group.
Measurements of motor activity are made. The exposure
levels at which significant changes in motor activity are
produced is compared to those levels which produce toxic
effects not originating in the central and/or periperal
nervous system.
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HG-Neuro-Motor Act
IV. TEST PROCEDURES
A. Animal Selection
1. Species and Strain
Testing shall be performed in a laboratory rat
or mouse. The choice of species should take
into consideration such factors as the
comparative metabolism of the chemical and
species sensitivity to the toxic effects of the
test substance, as evidenced by the results of
other studies, the potential for combined
studies, and the availability of other toxicity
data for the species.
2. Age
For acute exposures, animals should be sexually
mature. For repeated exposures, weanling
animals should be used.
3. Sex
a. Equal numbers of animals of each sex are
required for each dose level for the motor
activity test.
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HG-Neuro-Motor Act
b. The females should be nulliparous and non-
pregnant.
B. Number of Animals
Animals shall be randomly assigned to test and
control groups. Each test or control group must be
designed to contain a sufficient number of animals at
the completion of the study to detect a 40% change in
activity of the test groups relative to the control
group with 90% power at the 5% level. For most
designs, calculations can be made according to Dixon
and Massey (1957), Neter and Wasserman (1974), Sokal
and Rohlf (1969), or Jensen (1972).
C. Control Groups
1. A concurrent control group is required. This
group must be an untreated group, or, if a
vehicle is used in administering the test
substance, a vehicle control group. If the
toxic properties of the vehicle are not known or
cannot be made available, both untreated and
vehicle control groups are required.
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HG-Neuro-Motor Act
2. Positive control data are required to demonstrate the
sensitivity and reliability of the activity measuring
device and testing procedure. D-amphetamine shall be
used to demonstrate increases in motor activity.
Chlorpromazine shall be used to demonstrate
decreases. At least three doses of each reference
substance shall be used. Acute administration of the
reference substance is sufficient. Positive control
data shall be collected at the time of the test study
unless the laboratory can demonstrate the adequacy of
historical data for this purpose.
3. A satellite group may be treated with the high dose
level for 90 days and observed for reversibility,
persistence or delayed occurrence of toxic effects
for a post-treatment period of appropriate length,
normally not less than 28 days.
D. Dose Levels and Dose Selection
1. General
At least three dose levels (in addition to the
control group(s)) shall be used and spaced
appropriately to produce a range of toxic effects.
The data should be sufficient to produce a dose
response curve, permit an acceptable determination
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HG-Neuro-Motor Act
of the ED50 for raotor activity changes, and allow a
comparison of doses affecting motor activity to those
producing other toxic effects.
2. Subchronic
a. The highest dose level should result in toxic
effects but not produce an incidence of
fatalities which would prevent a meaningful
evaluation.
b. The lowest dose level should not produce any
evidence of toxicity. Where there is a usable
estimation of human exposure the lowest dose
level should exceed this.
c. Ideally, the intermediate dose level(s) should
produce minimal observable toxic effects. If
more than one intermediate dose is used, the
dose level should be spaced to produce a
gradation of toxic effects.
E. Duration of Testing
The duration of exposure will be specifed in the test
rule.
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HG-Neuro-Motor Act
F. Route of A^dministration
The test substance shall be administered by the
method specified in the test rule. This will usually
be the route most closely approximating the route of
human exposure. The exposure protocol shall conform
to that outlined in the appropriate acute or
subchronic toxicity study guideline.
G. Combined Protocol
The tests described herein may be combined with any
other toxicity study, as long as none of the
requirements of either are violated by the
combination.
H. Situdy Conduct
1. General
Motor activity must be monitored by an automated
activity recording apparatus. The device used
must be capable of detecting both increases and
decreases in activity, i.e. baseline activity as
measured by the device must not be so low as to
preclude decreases nor so .high as to preclude
increases. Each device shall be tested by a
standard procedure to ensure, to the extent
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HG-Neuro-Motor Act
possible, reliability of operation across devices and
across days for any one device. In addition,
treatment groups must be balanced across devices.
Each animal shall be tested individually. The test
session shall be long enough for motor activity to
approach asymptotic levels by the last 20% of the
/
session for most treatments and animals. All
sessions should have the same duration. Treatment
groups shall be counter-balanced across test times.
Effort should be made to ensure that variations in
the test conditions are minimal and are not
systematically related to treatment. Among the
variables which can affect motor activity are sound
level, size and shape of the test cage, temperature,
relative humidity, lighting conditions, odors, use of
home cage or novel test cage and environmental
distractions. Tests shall be executed by an
appropriately trained individual.
2. Acute
Testing shall be timed to include the time of
peak signs.
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HG-Neuro-Motor Act
3. Subchronic
All animals shall be tested prior to initiation
of exposure and at 30 _+2, 60 +2 and 90 +2 days
during the exposure period. Testing shall occur
prior to the daily exposure. Animals shall be
weighed on each test day and at least once
weekly during the exposure period.
V. DATA REPORTING AND EVALUATION
In addition to the reporting requirements specified in the
EPA Good Laboratory Practice Standards [Subpart J, Part
792, Chapter I of Title 40. Code of Federal Regulations]
the final test report must include the following
information:
A. Description of System and Test Methods
1. Positive control data from the laboratory
performing the test which demonstrate the
sensitivity of the procedure being used.
2. Procedures for calibrating and assuring the
equivalence of devices and balancing treatment
groups.
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HG-Neuro-Motor Act
B. Results
The following information must be arranged by test
group (dose level).
1. In tabular form, data must be provided showing
for each animal:
a. Its identification number;
b. Body weight, total session activity counts,
and intrasession subtotals for each date
measured.
2. Group summary data should also be reported.
C. Evaluation of Data
An evaluation of the test results (including
statistical analysis comparing total activity counts
at the end of exposure of treatment vs control
animals must be made and supplied. This submission
must include dose-effect curves for motor activity
expressed as activity counts.
10
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HG-Neuro-Motor Act
REFERENCES
Dixon WJ, Massey EJ. 1957. Introduction to Statistical
Analysis, 2nd Edition. New York: McGraw-Hill.
Finger FW. 1972. Measuring behavoral activity. In: Methods in
Psychobiology, Vol. 2. Myers RD, ed. New York: Academic, pp. 1-
19.
Jensen DR. 1972. Some simultaneous raultivariate procedures
using Retelling's T2 Statistics. Biometrics. 28:39-53.
Kinnard EJ and Watzman N. 1966. Techniques utilized in the
evaluation of psychotropic drugs on animals activity. J.
Pharmac. Sci. 55:995-1012.
Neter J and Wasserman W. 1974. Applied Linear Statistical
Models. Homewood, Richard D. Irwin, Inc.
Reiter LE 1978 Use of activity measures in behavioral
toxicology. Environmental Health Perspectives 26:9-20.
Reiter LW and MacPhail RC. 1979. Motor Activity: A survey of
methods with potential use in toxicity testing. Neurobehav.
Toxicol. 1: Suppl. 1, 53-66.
Robbins TW. 1977. A critique of the methods available for the
measurement of spontaneous motor activity. In: Handbook of
Psychopharmacology. Vol 7. Iversen LL, Iversen DS, Snyder SH.
eds. New York: Plenum, pp.37-82.
Sokal RP and Rohlf EJ. 1969. Biometry. San Francisco: W.H.
Freeman and Co.
11
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HS-Neuro-Motor Act
August, 1982
MOTOR ACTIVITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HS-Neuro-Motor Act
TABLE OF CONTENTS
Pages
I. INTRODUCTION 1
II. RATIONALES FOR STUDY DESIGN 2
A. Species 2
B. Age 3
C. Number of Animals 3
D. Dose Selection 4
E. Route of Exposure 5
III. RATIONALES FOR STUDY CONDUCT 6
A. Apparatus 6
B. Time of Testing 7
C. Environmental Variables 7
D. Session Length 8
E. Frequency of Testing 8
F. Individual Measurement 9
REFERENCES 10
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HS-Neuro-Motor Act
I. INTRODUCTION
Motor activity has been extensively studied in both
behavioral pharmacology and behavioral toxicology (Reiter
1978, Reiter and MacPhail 1979, Irwin 1968, Kinnard and
Watzman 1966). There are several reasons for this
popularity: 1. Motor activity occurs naturally. All
animals, including man, explore their environment. 2. The
functional state of the nervous system is often reflected
in amounts of motor activity and represents behavior which
is relevant to the animal's survival (Reiter 1978). 3.
Studies of motor activity indicate that it is sensitive to
the effects of a wide variety of agents (Dews 1953,
Fibiger and Campbell 1971, Fibiger and Campbell 1971,
Irwin 1968, Kellogg and Lundborg 1972, Norton et al. 1976,
Segal 1975, Silverman and Williams 1975, Vasko et al.
1979, Waldeck, 1974, 1975, Ahlenius et al. 1973, Anden et
al. 1973, Campbell and Mabry 1973, Costa et al. 1972,
Creese and Iverson 1973, Erinoff et al. 1979, Foldes and
Costa 1975, Jacobs et al. 1974, Sulser et al. 1968,
Weissman et al. 1966). 4. Measurement of motor activity
is relatively easy; no training or deprivation techniques
are necessary. There are many commercially available
devices which give a reliable quantification of motor
activity (Reiter and MacPhail 1979, Kinnard and Watzman
1966, Robbins 1977).
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HS-Neuro-Motor Act
II. RATIONALES FOR STUDY DESIGN
A. Species
Rodents have been widely used and extensively studied
in behavioral pharmacology and toxicology. There are
many devices capable of measuring the motor activity
of rats and mice. The history of the development of
psychoactive drugs indicates that the motor activity
of rats and mice are predictive of psychoactive
potential in humans (Irwin 1968, Kinnard and Watzman
1966, Dews 1953, Turner 1965). Because rats are the
preferred rodent species specified in the subchronic
toxicity guidelines, their use in behavioral studies
will facilitate combined studies and aid in the
integration of data. However, mice may be
preferable, particularly if the metabolic and/or
toxicity data indicate that they are a more
appropriate choice and if considerable data exist on
the chemical's effects in mice. More data are
available on motor activity in mice. They are more
easily handled, require less space, and in a
subchronic study, the change of the size relationship
of mouse to testing equipment is not as drastic as
with rats. In addition mice are generally less
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HS-Neuro-Motor Act
costly. Since a sufficient data base has not been
established for larger mammals, their use is
discouraged.
B. Age
Practical limitations (i.e. weaning) preclude
exposure before Day 21. For subchronic studies,
exposure should not start later than approximately
Day 42, corresponding to the subchronic toxicity
guidelines. This includes the normal adult life span
and is maximally cost effective.
C- Number of Animals
The sensitivity of the test will depend on both the
group size and the normal variability of the test
system (Sokal and Rohlf 1969). Baseline motor
activity can be quite variable (a coefficient of
variation of 25% is not uncommon). Thus, a group
size of 10 will allow detection of about a 40% change
in activity at the 5% level with 90% certainty. A
group size greater than 34 would be required to
detect a 20% change with the same confidence and
certainly. For substances which affect motor
activity, a 40% change will be adequate for
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HS-Neuro-Motor Act
determining whether the dose range which affects
activity is well below the generally toxic range.
D. Dose Selection
The standards for selection of dose levels are
designed to optimize exposure conditions to reveal a
X
toxic response. Three doses are minimially
acceptable for this:
The highest dose allows the characterization of the
response when it occurs; the lowest dose provides
information on no-effect level; the middle dose
identifies that a dose-response relationship
exists. Since changes in motor activity may be
secondary to systemic toxicity, it is important that
we be able to determine the relative doses at which
behavioral and systemic effects occur. If behavioral
changes only occur when there is evidence of systemic
toxicity, we might not want to require the investment
of resources to pursue the behavioral effects
further. To answer this question it is best that the
lowest dose used be at the threshold of behavioral
effects.
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HS-Neuro-Motor Act
E. Route of Exposure
i • • ••
EPA is generally requiring that test substances be
administered by the route that duplicates or most
closely simulates the major known or expected route
by which human exposure occurs. This is the accepted
method because results are generally directly
amenable to evaluation in terms of potential human
; health hazards.
i
However, if humans are exposed via several routes,
the major route of exposure may not be the most
important determinant. In this case, EPA may
, consider the most important determinant to be the
route which is anticipated to be the most sensitive
in terms of repeated exposure toxicity. In deciding
on route of expousre, EPA will consider not only
human use or exposure but also specific properties of
the chemical including its absorption, distribution
and metabolism.
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HS-Neuro-Motor Act
III. RATIONALES FOR STUDY CONDUCT
A. Apparatus
The use of automated activity measurement devices is
proposed. These devices have been widely used and
validated and are more readily standardized and
cheaper to perform than subjective rating scales.
(Reiter and MacPhail 1979, Robbins 1977, Kinnard and
Watzmann 1966). The use of commercially available
devices is encouraged. It is important that an
activity-measuring device detects both increases and
decreases in activity should they occur; some tilt-
cages (stabilimeters) engender a low baseline
activity such that decreases would be difficult to
detect. It should be noted that many types of field
detector devices are relatively new and have not been
subjected to as wide a validation. However their
major problem has been the recording of many types of
activity rather than specifically locomotor activity
(Reiter and MacPhail 1979). While this is a problem
for students of locomotion per se, it is less so when
the major objective is toxicity screening (Reiter and
MacPhail 1979). The Agency sees no reason to
prohibit the use of such devices as long as
differences in operational characteristics can be
controlled for.
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HS-Neuro-Motor Act
B* Time of Testing
The time of testing with respect to the light-dark
cycle is an important variable that influences
activity (Robbins 1977). Locomotor activity is
subject to control by an apparently endogenous
rhythm, the precise hormonal and neurochemical
correlates of which are still largely unknown
(Reinberg and Halberg 1971). Many contradictory
findings on activity levels after drug and chemical
exposures might be due to a lack of standardization
of this factor. Therefore, the guideline specifies
that treatment groups must be balanced across test
times.
During repeated exposure, testing shall occur before
exposure to eliminate acute effects of the test
substance.
C. Environmental Variables
Many environmental variables affect motor activity.
Poor control of these variables will result in
.increased control group variability, thus
necessitating larger sample sizes. The variables
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HS-Neuro-Motor Act
listed in the guideline are among those that should
be controlled to ensure the reliability of the
testing procedure.
D* Sess ion Leng th
Motor activity is typically very high when the animal
first enters the test environment and decreases with
time. To minimize the influence of presession
handling and other external variables, the session
should be long enough for motor activity to have
stabilized. This will also tend to equalize the
measurements across different types of apparatus,
which may differ with respect to the time for such
stability to be reached.
E. Frequency of Testing
For repeated exposure protocols, monthly testing is
proposed as a way to obtain data on the effects of
duration of exposure without excessive investment of
facilities or labor. Animals should be tested before
the initial exposure to obtain a baseline level.
This will provide a check on the randomization of
treatment groups with respect to activity.
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HS-Neuro-Motor Act
F• Individual Measurement
Motor activity and the effects of chemical agents on
such activity may differ when animals are tested in
groups rather than individually (Watzman et al 1966,
Reiter 1978). For purposes of toxicity screening,
the variability induced by group measurement would
complicate the interpretation of the data.
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HS-Neuro-Motor Act
REFERENCES
Ahlenius S, Anden NE, Engel J. 1973. Restoration of locomotor
activity in mice by low 1-dopa doses after suppression by ot-
raethyl-tyrosine but not by reserpine. Brain Res. 62:189-199.
Anden NE, Strombom U, Sevensson TH. 1973. Dopamine and
noradrenaline receptor stimulation: Reversal of reserpine-
induced suppression of motor activity. Psychopharmacology
29:289-298.
Campbell BA and Mabry PD. 1973. The role of catecholamines in
behavioral arousal during ontogenesis. Psychopharmacology
31:253-264.
Costa E, Groppetti A, Naimzada MK. 1972. Effects of ( + )-
amphetamine on the turnover rate of brain catecholamines and
motor activity. Br. J. Pharmac. 44:742-751.
Creese I and Iversen SD. 1973. Blockage of amphetamine-induced
motor stimulation and stereotypy in the adult rat following
neonatal treatment with 6-hydroxydopamine. Brain Res. 55:369-
382.
Dews PB. 1953. The measurement of the influence of drugs on
voluntary activity in mice. Br. J. Pharmac. Chemother. 8:46-48.
Erinoff L, MacPhail RC, Heller A, Seiden LS. 1979. Age-
dependent effects of 6-hydroxydopamine on locomotor activity in
the rat. Brain Res. 64:195-205.
Fibiger HC and Campbell BA. 1971. The effect of para-
chlorophenylalanine on spontaneous locomotor activity in the
rat. Neuropharmacology 10:25-32.
Finger FW. 1972. Measuring behavioral activity. In: Methods
in Psychobiology/ Vol. 2. R. D. Myers, RD. ed. New York:
Academic Press, pp. 1-19.
Foldes A and Costa E. 1975. Relationship of brain monoamine and
locomotor activity in rats. Biochem. Pharmac. 24:1617-1621.
10
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Irwin S. 1968. Comprehensive observational assessment: la. A
systematic, quantitative procedure for assessing the behavioral
and physiologic state of the mouse. Psychopharmacology 13:222-
257.
Irwin S, Slabok M, Thomas G. 1958. Individual differences: I
Correlation between control locomotor activity and sensitivity to
stimulant and depressant drugs. J. Pharmac. exp. Ther. 123:206-
211.
Jacobs BL; Trimbach C, Eubanks EE, Trulson M. 1975. Hippocampal
mediation of raphe lesion and PCPA-induced hyperactiviy in the
rat. Brain Res. 94:253-261.
Kellogg C and Lundborg P. 1965. Ontogenic variations in
responses to 1-dopa and monoamine receptor-stimulating agents.
Psychoparmacology 23:597-598.
Kinnard EJ and Watzman N. 1966. Techniques utilized in the
evaluation of psychotropic drugs on animals activity. J.
Pharmac. Sci. 55:995-1012.
Norton S, Mullenix P, Culver B. 1976. Comparison of the
structure of hyperactive behavior in rats after brain demage from
x-irradiation, carbon monoxide and pallidal lesions. Brain
Res. 116:49-67.
Reinberg A and Halberg F. 1971. Circadiah chronopharmacology.
Ann. Rev. Parmacol 11:455-492.
Reiter LE. 1978. Use of activity measures in behavioral
toxicology. Environmental Health Perspectives 26:9-20.
Reiter LW and MacPhail RC. 1979. Motor Activity: A survey of
methods with potential use in toxicity testing. Neurobehav.
Toxicol. IrSuppl. 1, 53-66.
Reiter LW; Anderson GE; Laskey JW, Cahill DF. 1975.
Developmental and behavioral changes in the rat during chronic
exposure to lead. Envir. Hlth Prespect. 12:119-123.
Robbins TW. 1977. A critique of the methods available for the
measurement of spontaneous motor activity. In: Handbook of
Psychopharmacology. Vol 7, edited by L. L. Iversenu, Iversen SD,
Snyder SH. New York: Plenum Press, pp. 37-82.
Segal DS. 1975. Behavioral characterization of d- and 1-
amphetamine: neurochemical implications. Science 190:475-477.
Silverman AP and Williams H. 1975. Behaviour of rats exposed to
trichloroethylene vapour. Br. J. Ind. Med. 32:308-315.
11
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Skinner BF. 1933. The measurement of spontaneous activity. J.
gen. Psychol. 9:3-24.
Sokal RR and Rohlf FJ. 1969. Biometry. San Francisco: W.H.
Freeman and Co.
Stromberg V and Waldeck B. 1973. Behavioral and biochemical
interaction between caffeine and L-dopa. J. Pharm. Pharmac.
25:302-308.
Sulser F, Owens ML, Norvich NR, Dingell JV. 1968. The relative
role of storage and synthesis of brain norepinephrine in the
psychomotor stimulation evoked by amphetamine or by desipramine
and tetrabenazine. Psychopharmacology 12:322-332.
Turner RA. 1965. Screening Methods in Pharmacology. New York:
Academic Press, pp. 24-34.
Vasko MR, Lutz MP, Domino EF. 1974. Structure-activity
relations of some indolealkylamines in comparison to
phenethylamines on motor activity and acquisition of avoidance
behavior. Psychopharmacology 36:49-58.
Waldeck B. 1974. Modification of caffeine-induced locomotor
stimulation by a cholinergic mechanism. J. Neural Transm.
35:197-205.
Waldeck B. 1975. On the interaction between caffeine and
barbiturates with respect to locomotor activity and brain
catecholamines. Acta pharmac. toxic. 36:172-180.
Watzman N, Barry H, Kinnard WJ, Buckley JP. 1966. Comparison of
different photobeam arrangements for measuring spontaneous
activity in mice. J. Pharmaceut. Sci. 55:907-909.
Weissman A, Koe BK, Tenen SS. 1966. Antiamphetamine effects
following inhibition of tyrosine hydroxylase. J. Pharmac. exp.
Ther. 151:339-352.
12
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HG-Neuro-Acute Delayed
August, 1982
ACUTE DELAYED NEUROTOXICITY
OF ORGANOPHOSPHORUS SUBSTANCES
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Neuro-Acute Delayed
I. PURPOSE
Organophosphorus substances should be considered as
candidates for delayed neurotoxicity studies using the adult
hen as the test animal. This test has certain limitations,
e.g. in predicting effects from repeated exposures. These
limitations may be minimized by conducting an adjunct test
in which inhibition and aging of neurotoxic esterase of hen
neural tissue are measured.
II. DEFINITIONS
Acute delayed neurotoxicity is a prolonged, delayed-onset
locomotor ataxia resulting from single administration of the
test substance, repeated once if necessary.
III. PRINCIPLE OF THE TEST METHOD
The test substance is administered orally in a single dose
to domestic hens (Callus gallus domesticus) which have been
protected from acute cholinergic effects, when
appropriate. The animals are observed for at least 21 days
for delayed neurotoxicity, with redosing and observation for
another 21 days if no effects or equivocal responses are
seen. The animals are observed daily for behavioral
abnormalities, locomotor ataxia and paralysis.
Histopathological examination of selected neural tissues is
undertaken on all animals surviving the initial cholinergic
phases.
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HG-Neuro-Acute Delayed
IV. TEST PROCEDURES
A. Animal .Selection
The adult domestic laying hen, aged between 8-14
months, is recommended. Standard size breeds and
strains should be employed.
B. Number of
A sufficient number of hens should be utilized so that
at least six survive the observation period.
C. Control Groups
1. General
Appropriate control groups should be used. These
should include a positive control group of at
least two hens treated with a known delayed
neurotoxicant and a concurrent control group of at
"least six hens treated in a manner identical to
the treated group, except that administration of
the test substance and any protective agents is
omitted.
2. Re f erence Subs tances
A substance which is known to produce acute
delayed neurotoxicity should be used as a positive
control. Examples of such substances are tri-
orthocresyl phosphate (TOCP) and leptophos.
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HG-Neuro-Acute Delayed
D. Housing and Feeding Conditions
Cages or enclosures which are large enough to permit
free mobility of the hens and easy observation of gait
should be used. Where the lighting is artificial, the
sequence should be 12 hours light, 12 hours dark.
Appropriate diets should be administered as well as an
unlimited supply of drinking water.
E. Dose Level
The selected dose level of the test substance should
not be less than the unprotected LD50 dose. Atropine
or another non-interfering protective agent may be used
to prevent death due to acute cholinergic effects.
Doses of test substance higher than 5000 mg/kg of body
weight need not be tested.
F. Dose Selection
A preliminary LD50 test using an appropriate number of
animals, dosages and dose groups, as recommended in
Test Guideline HG-Acute-Oral, should be performed in
unprotected hens to establish the dose level to be used
in this test. Healthy young adult hens free from
interfering viral diseases and medication and without
abnormalities of gait should be acclimatized to the
laboratory conditions for at least five days prior to
randomization and assignment to treatment and control
groups.
G. Route of Administration
Dosing with the test substance should normally be by
the oral route using gavage, gelatine capsules, or a
comparable method.
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HG-Neuro-Acute Delayed
H. Study Conduct
I '
1. General
The test or control substance should be
administered and observations begun. All hens
should be carefully observed at least once daily
for a period of at least 21 days and signs of
toxicity recorded, including the time of onset,
degree and duration. Observations should include,
but not be limited to, behavioral abnormality,
locomotor ataxia and paralysis. At least twice a
week the hens should be taken outside the cages
and subjected to a period of forced motor
activity, such as ladder climbing, in order to
enhance the observation of minimal responses. If
neurotoxic responses are not observed or if
equivocal responses are seen, then the dose should
be administered again and the animals observed for
an additional 21 days. The hens should be weighed
weekly. Any moribund hens should be removed and
sacrificed.
2. Pathology
a. Gross Necropsy
In the presence of clinical signs of delayed
neurotoxicity useful information may be
provided by gross necropsy.
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HG-Neuro-Acute Delayed
k* Hi s topa tholggy
All animals should be subjected to
microscopic examination. Tissues should be
fixed in situ, preferably using perfusion
techniques. Sections should include medulla
oblongata, spinal cord and peripheral
nerves. The spinal cord sections should be
taken from the upper cervical bulb, the mid-
thoracic and the lumbo-sacral regions.
Section of the proximal region of the tibial
nerve and its branches should be taken.
Sections should, be stained with appropriate
myelin and axon-specific stains.
V. DATA REPORTING AND EVALUATION
A. Test Report
In addition to the reporting requirements specified in
the EPA Good Laboratory Practice Standards [Subpart J,
Part 792, Chapter I of Title 40. Code of Federal
Regulations] the final test report must include the
following information:
1. Toxic response data by group with a description of
clinical manifestations of nervous sytem damage;
where a grading system is used the criteria should
be defined; ,,
i *
2. For each animal, time of death during the study or
whether it survived to termination;
3. The day of observation of each abnormal sign and
its subsequent course;
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HG-Neuro-Acute Delayed
4. Body weight data;
5. Necropsy findings for each animal, when performed;
6. A detailed description of all histopathological
findings;
7. Statistical treatment of results, where
appropriate.
B. Treatment of Results
Data may be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, the number of animals showing lesions or
effects, the types of lesions or effects and the
percentage of animals displaying each type of lesion or
effect.
C. Evaluation of Results
The findings of an acute delayed neurotoxicity study
should be evaluated in terms of the incidence and
severity of neurotoxic effects and of any other
observed effects and histopathological findings in the
treated and control groups.
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HG-Neuro-Acute Delayed
REFERENCES
Abou-Donia MB. 1981. Organophosphorus ester-induced delayed
neurotoxicity. Ann. Rev. Pharraacol. Toxicol. 21:511-548.
Abou-Donia MB, Pressing SH. 1976. Delayed neurotoxicity from
continuous low-dose oral administration of leptophos to hens.
Toxicol. Appl. Pharmacol. 38:595-608.
Baron RL (ed). 1976. Pesticide Induced Delayed Neurotoxicity.
Proceedings of a Conference, February 19-20, 1976, Washington,
D.C. Washington, D.C.: U.S. Environmental Protection Agency.
EPA-600/1-76-025.
"i
Cavanaugh JB. 1973. Peripheral neuropathy caused by chemical
agents. CRC Critical Reviews of Toxicity. 2:365-417.
Johannsen FR, Wright PL, Gordon DE, Levinskas GL, Radue RW,
Graham PR. 1977. Evaluation of delayed neurotoxicity and dose-
response relationship of phosphate esters in the adult hen.
Toxicol. Appl. Pharmacol. 41:291-304.
Johnson MK. 1975. Organophosphorus esters, causing delayed
neurotoxic effects: mechanism of action and structure/activity
studies. Arch. Toxicol. 34:259-288.
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HG-Spec-Stud-Metab
August, 1982
METABOLISM
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Spec-Stud-Me tab
I. PURPOSE
A. Data from studies on the absorption, distribution,
excretion and metabolism of a test chemical are
desirable to aid in the evaluation of test results
from other toxicology studies and in the extrapolation
of data from animals to man. Such studies should be
done on each chemical of toxicological concern. The
concern may be predicated on the level and type of
toxicity observed (or anticipated) and by the
magnitude of potential human exposure to the
chemical. The main purpose of metabolism studies is
to produce data which fortify the understanding of the
safety of the chemical in consideration of its
intended uses and anticipated human exposure. In
addition to the general reasons stated above, a
metabolism study may be performed for the following
purposes:
1. To determine the amount and rate of absorption
of the test chemical at different dose levels;
2. To determine the pattern of distribution of the
test chemical among tissues, organs and fluid
compartments at different dose levels, after
single and repeated dosages;
3. To identify and, to the extent possible,
quantify significant metabolites;
4. To characterize route(s) and rate(s) of
excretion;
5. To determine any possible bioaccumulation
(bioretention) of the test substance and/or
metabolites; and
6. To determine absorption, metabolism, excretion
and distribution as a function of single or
repeated doses. For certain chemicals,
metabolism studies may not adequately define all
of these.
II. DEFINITIONS
A. Bioaccumulation (bioretention) is the uptake and, at
least temporary, storage of a chemical by an exposed
animal. The chemical can be retained in its original
form and/or as modified by enzymatic and non-enzymatic
reactions in the body.
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HG-Spec-Stud-Metab
III. TEST PROCEDURES
A. Animal selection
1. Species
The preferred species is the rat. If another
mammalian species is used, the tester should
provide justification/reasoning for its
selection. Commonly used laboratory strains
should be employed. Preliminary studies may be
performed in several species to develop
information on comparative metabolism.
Information derived from preliminary studies may
help in the selection of species for subsequent
toxicity tests.
2. Age
Young adult animals should be used. For
specific purposes, a comparative study using
very young animals may provide information about
the effects of age on metabolism.
3. Sex
a. Equal numbers of animals of each sex
should be used at each dose level.
b. Females should be nulliparous and non-
pregnant.
4. Numbers
At least 8 animals (4 females and 4 males)
should be used at each dose level.
B. Dose levels and dose selection
1. At least 2 dose levels should be used.
2. The low dose should correspond to a no-effect-
level.
3. The upper dose should produce toxic or
pharmacologic signs, but not severe effects or a
high incidence of mortality which would prevent
a meaningful evaluation.
4. The determination of absorption, tissue
distribution and elimination should be studied
as a function of single or repeated doses.
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HG-Spec-Stud-Metab
5. The conclusive identification of a chemical, and
its metabolites, requires the use of suitable
analytical methods.
C. Observation period
Animals should be kept in individual metabolism cages
for 7 days after the radioactive dose or until 95
percent of the administered dose is excreted
(whichever occurs first), at which time all of the
animals should be killed.
D. Administration of the test substance
1. The study should be done using the oral route
(capsule or gavage). If another route of
administration is used, the tester should
provide justification/reasoning for its
selection. When vehicles are used, attention
should be given to the possibility that they may
interfere with the kinetics of the test
chemical.
2. Labeled test material
a. Single dose testing should be performed
with an analytically pure grade of the
active ingredient, usually in an
isotopically labeled form.
b. Labeled compound may not be required if
sufficiently selective and sensitive
physical-chemical tests for identifying
the compound and its metabolites are
used. The label may be radioactive such
as 14cf 35s, and 36C1 or stable such as
N and l^O. in some cases, more than one
label per molecule may be advantageous.
Labels should be placed in positions that
may be expected to follow the "core" of
the molecule or significant portions
thereof. If possible, one should avoid
placing labels such as l^c in positions
from which it may be expected to enter the
carbon pool of the test animal. Use of
readily exchangeable labeling, should be
avoided. In addition, some animals should
receive repetitive doses of nonlabeled
chemical substance (analytical grade).
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HG-Spec-Stud-Metab
3. The following 4 groups of animals should be
studied:
a. Group A animals shall each receive a
single intravenous dose of the labeled
test substance at the low dose. If it is
not possible to dissolve the test
substance in physiological saline or
water, this group should be omitted.
b. Group B animals should each receive a
single oral dose of the labeled test
substance at the low dose.
c. Group C animals should each receive a
series of single daily oral doses of the
nonlabeled test substance (by capsule or
intubation) over a period of at least 14
days, followed at 24 hours after the last
dose by a single oral dose (by capsule or
intubation) of the labeled test
substance. Each dose should be at the low
dose level.
d. Group D animals should each receive a
single oral dose (by capsule or
intubation) of the labeled test substance
at the high dose level.
E. Observation of animals
1. Distribution
Concentration and quantity of test chemicals in
the tissues and organs should be measured at the
time of sacrifice.
2. Metabolism
For determining the extent of biotransformation,
urine samples and fecal extracts should be
analyzed by suitable techniques. Major
metabolites of the chemical should be identified
by appropriate methods. It is also important to
determine the metabolite pattern of the test
chemical after repeated dosages.
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HG-Spec-Stud-Me tab
3. Excretion
When determining excretion of the test chemical
by laboratory animals, the use of individual
metabolism cages is recommened for collection of
urine and fecal samples. The concentration of
test chemical and major metabolites in urine,
feces and in expired air should be measured at
several time points after exposure (i.e., 4, 8,
12 and 24 hours) and daily thereafter, until
approximately 95 percent of the administered
dose has been excreted or until 7 days after
dosing.
4. In the rat, quantities of label in urine, feces
and expired air should be measured at
appropriate intervals (i.e, 4, 8, 12, and 24
hours, 1.5, 2, 3, 4, 5, 6, and 7 days)
throughout the study for all animals. However,
if a preliminary study shows no volatile labeled
materials are exhaled during the period of zero
to 24 hours after dosing, such evidence may be
submitted in lieu of measuring label in the
expired air for this study. In the dog,
quantities of label in urine and feces should be
measured at appropriate intervals (i.e., every 6
hours for the first 48 hours after dosing and
every 12 hours for the remaining 5 days)
throughout the study for all animals.
For all animals in groups B, C, and D, the
quantity of label in tissues and organs should
be measured at sacrifice by suitable methods
with particular attention to bone, brain, fat,
gonads, heart, kidney, liver, lungs, muscle,
spleen, tissues which displayed pathology (in
this or prior studies), and residual carcass.
5. Urine and feces from all groups should be
analyzed by suitable methods in order to
determine the extent of absorption and
biotransformation and to identify the
metabolites. An assay method for detection of
each major metabolite may be requested by the
Agency.
IV. DATA AND REPORTING
A. Treatment of results
Data should be summarized in tabular form.
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HG-Spec-Stud-Metab
B. Evaluation of results
All observed results, quantitative or incidental,
should be evaluated by an appropriate statistical
method.
C. Test report
In addition to the reporting requirements as specified
in the EPA Good Laboratory Practice Standards [Subpart
J, Part 792, Chapter I of Title 40. Code of Federal
Regulations] the following specific information should
be reported:
1. Quantity of isotope, together with percent
recovery of the administered dose, in feces,
urine, and the following tissues and organs of
animals in all groups: Bone, brain, fat,
gonads, heart, kidney, liver, lung, blood,
muscle, spleen, tissues which displayed
pathology (in this or prior studies), and
residual carcass;
2. Percent absorption. If possible by the oral
route in groups B, C, and D.
3. A full description of the sensitivity and
precision of all procedures used to produce the
data; and
4. Information on the degree (i.e., specific
activity for a radiolabel) and site(s) of
labeling of the test substance.
5. Counting efficacy data should be made available
to the Agency upon request; and
6. Species and strain.
V. ADDITIONAL METABOLISM STUDIES
Additional, more specific studies may be required to clarify
important points. Some areas for possible further study
include: Identification of tissue residues; binding by
macromolecules in the blood, liver, gonads and other
tissues; placental transfer; entrance into breast milk;
biotransformation by specific organs, tissues and cell
fractions; and^bsorption by dermal or inhalation routes of
exposure. Plasma binding studies may be conducted, usually
in vitro with plasma. Placental transfer of a chemical
substance may be determined by dosing pregnant rodents with
chemicals and assaying their fetuses for the chemical.
Additional species may be utilized as the rat and dog differ
significantly in metabolic pattern.
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All.S. GOVERNMENT PRINTING OFFICE. 1982-360-997/2209
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HG-Neuro-Subchronic Delayed
August, 1982
SUBCHRONIC DELAYED NEUROTOXICITY OF
ORGANOPHOSPHORUS SUBSTANCES
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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HG-Neuro-Subchronic Delayed
I. PURPOSE
In the assessment and evaluation of the toxic
characteristics of organophosphorus substances the
determination of subchronic delayed neurotoxicity may be
carried out, usually after initial information on delayed
neurotoxicity has been obtained by acute testing or by the
demonstration of inhibition and aging of neurotoxic esterase
in hen neural tissue. The subchronic delayed neurotoxicity
test provides information on possible health hazards likely
to arise from repeated exposures over a limited period of
time. It will provide information on dose response and can
provide an estimate of a no-effect level which can be of use
for establishing safely criteria for exposure.
II. DEFINITIONS
Subchronic delayed neurotoxicity is a prolonged, delayed-
onset locomotor ataxia resulting from repeated daily
administration of the test substance.
III. PRINCIPLE OF THE TEST METHOD
Multiple dose levels of the test substance are administered
orally to domestic hens (Callus gallus domesticus) for 90
days. The animals are observed at least daily for
behavioral abnormalities, locomotor ataxia and paralysis.
Histopathological examination of selected neural tissues is
undertaken at the termination of the test period.
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HG-Neuro-Subchronic Delayed
IV. TEST PROCEDURES
A. An ima1 Sele ct ion
The adult domestic laying hen, aged between 8-14
months, is recommended. Standard size breeds and
strains should be employed.
B. Number of Animals
Ten hens should be used for each treatment and control
group.
C. Control Group
1. General
A concurrent control group should be used. This
group should be treated in a manner identical to
the treated group, except that administration of
the test substance is omitted.
2. Reference Substances
If a positive control is used, a substance which
is known to produce delayed neurotoxicity should
be employed. Examples of such substances are tri-
orthocresyl phosphate (TOCP) and leptophos.
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HG-Neuro-Subchronic Delayed
D. Hpusing a nd Fe ed i rig Co ndi t i o ns
Cages or enclosures which are large enough to permit
free mobility of the hens and easy observation of gait
should be used. Where the lighting is artificial, the
sequence should be 12 hours light, 12 hours dark.
Appropriate diets should be administered as well as an
unlimited supply of drinking water.
E. Dose Levels
At least three dose levels should be used in addition
to the control group(s). The highest dose level should
result in toxic effects, preferably delayed
neurotoxicity, but not produce an incidence of
fatalities which would prevent a meaningful
evaluation. The lowest dose level should not produce
any evidence of toxicity.
F. Route of Administration
Oral dosing each day for at least five days per week
should be carried out, preferably by gavage or
administration of gelatine capsules-
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HG-Neuro-Subchronic Delayed
G. Study Conduct
1. General
Healthy young adult hens free from interfering
viral diseases and medication and without
abnormalities of gait should be acclimatized to
the laboratory conditions for at least five days
prior to randomization and assignment to treatment
and control groups. The test or control substance
should be administered and observations begun.
All hens should be carefully observed at least
once daily throughout the test period. Signs of
toxicity should be recorded, including the time of
onset, degree and duration. Observations should
include, but not be limited to, behavioural
abnormality, locomotor ataxia and paralysis. At
least once a week the hens should be taken outside
the cages and subjected to a period of forced
motor activity, such as ladder climbing, in order
to enhance the observation of minimal responses.
The hens should be weighed weekly. Any moribund
hens should be removed and sacrificed.
2. Pathology
a. Gross Necropsy
In the presence of clinical signs of delayed
neurotoxicity useful information may be
provided by gross necropsy.
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HG-Neuro-Subchronic Delayed
b. Hi stppathp1pgy
Tissues from all animals should be fixed in
situ, using perfusion techniques. Sections
should include medulla oblongata, spinal
cord and peripheral nerves. The spinal cord
sections should be taken from the upper
cervical bulb, the mid-thoracic and lumbo-
sacral regions. Sections of the proximal
region of the tibial nerve and its branches
and of the sciatic nerve should be taken.
Sections should be stained with appropriate
myelin and axon-specific stains.
Microscopic examination should be carried
out on all hens in the control and high-dose
groups. Microscopic examination should also
be carried out on hens in the low and
intermediate dose groups when there is
evidence of effects in the high-dose group.
V. DATA REPORTING AND EVALUATION
A. Test Report
In addition to the reporting requirements specified in
the EPA Good Laboratory Practice Standards [Subpart J,
Part 192, Chapter I of Title 40. Code of Federal
Regulations] the final test report must include the
following information:
1. Toxic response data by group with a description of
clinical manifestations of nervous sytem damage;
where a grading system is used the criteria should
be defined;
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HG-Neuro-Subchronic Delayed
2. For each animal, time of death during the study or
whether it survived to termination;
3. The day of observation of each abnormal sign and
its subsequent course;
4. Body weight data;
5. Necropsy findings for each animal, when performed;
6. A detailed .description of all histopathological
findings;
7. Statistical treatment of results, where
appropriate.
B. Treatment of Results
Data may be summarized in tabular form, showing for
each test group the number of animals at the start of
the test, the number of animals showing lesions or
effects, the types of lesions or effects and the
percentage of animals displaying each type of lesion or
effect.
All observed results should be evaluated by an
appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods
should be selected during the design of the study.
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HG-Neuro-Subchronic Delayed
C. Evaluation of Re s ul t s
The findings of a subchronic delayed neurotoxicity
study should be evaluated in conjunction with the
findings of preceding studies and considered in terras
of the incidence and severity of observed neurotoxic
effects and any other observed effects and
histopathological findings in the treated and control
groups. A properly conducted subchronic test should
provide a satisfactory estimation of a no-effect level
based on lack of clinical signs and histopathological
changes.
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HG-Neuro-Subchronic Delayed
REFERENCES
Abou-Donia MB. 1981. Organophosphorus ester-induced delayed
neurotoxicity. Ann. Rev. Pharmacol. Toxicol. 21:511-548.
Abou-Donia MB, Dressing SH. 1976. Delayed neurotoxicity from
continuous low-dose oral administration of leptophos to hens.
Toxicol. Appl. Pharmacol. 38:595-608.
Baron RL (ed). 1976. Pesticide Induced Delayed Neurotoxicity.
Proceedings of a Conference, February 19-20, 1976, Washington,
D.C. Washington, D.C.: U.S. Environmental Protection Agency.
EPA-600/1-76-025.
Cavanaugh JB. 1973. Peripheral neuropathy caused by chemical
agents. CRC Critical Reviews of Toxicity. 2:365-417.
Johannsen FR, Wright PL, Gordon DE, Levinskas GL, Radue RW,
Graham PR. 1977. Evaluation of delayed neurotoxicity and dose-
response relationship of phosphate esters in the adult hen.
Toxicol. Appl. Pharmacol. 41:291-304.
Johnson MK. 1975. Organophosphorus esters causing delayed
neurotoxic effects: mechanism of action and structure/activity
studies. Arch. Toxicol. 34:259-288.
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