748R98002
OPPTS HARMONIZED TEST GUIDELINES
Series 870
Health Effects
Volume I of III
Guidelines OPPTS 870.1000 - OPPTS 870.4300
August 1998
United States Environmental Protection Agency
Office of Prevention, Pesticides, and Toxic Substances
Washington, D.C. 20460
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Series 870—Health Effects Test Guidelines
OPPTS
Number
870 1000
870 1100
870 1200
870 1300
870 2400
870 2500
870 2600
870 3100
870 3150
870 3200
870 3250
870 346S
870 3700
870 3800
8704100
870 4200
870 4300
8705100
8705140
8705195
8705200
870 5250
870 5275
870 5300
870 5375
8705380
870.5385
870 5395
870 5450
870 5460
8705500
870 5550
870.5575
8705900
8705915
8706100
8706200
870 6300
8706500
8706850
870 6855
870 7200
870 7485
870 7600
870 7800
Name
Group A— Acute Toxicity Test Guidelines
Acute loxicity testing-background
Acute oral toxicity
Acute dermal toxicity
Acute inhalation toxicity
Acute eye irritation
Acute dermal irritation
Skin sensitization
Group B — Subchronic Toxicity Test Guidelines
90 Day oral loxicity in rodents
90 Day oral toxicity in nonrodents
21/28 Day dermal toxicity
90 Day dermal toxiary
90 Day inhalation toxicity
Prenatal developmental toxicily study
Reproduction and fertility effects
Group C — Chronic Toxicity Test Guidelines
Chronic toxicity
Caranogenicity
Combined chronic toxiaty/carcinogenicify
Group D — Genetic Toxicity Test Guidelines
Bacterial reverse mutation test
Gene mutation in AspergiSus nidulans
Mouse biochemical specific locus test
Moose vtsble specific locus test
Gene mutation in Neurospora crassa
Sex linked recessive lethal test In Drosophila metonogsster
In vitro mammalian celt gene mutation test
In vitro mammalian chromosome aberration lest
Mammalian spermatogorval chromosomal aberration lest
Mammalian bone marrow chromosomal aberration test
Mammalian erythrocyte mlcronudeus lest
Rodent dominant lethal assay
Rodent heritable translocation assays
Bacterial DNA damage or repair tests
Unscheduled DNA synthesis In mammalian cells in culture
Mitolic gene conversion in Saccharomyces cerevisiae
In vitro sister chromatid exchange assay
In vivo sister chromatid exchange assay
Group E— Neurotoxielty Test Guidelines
Acute and 28 day delayed neuroloxicity ol organophosphorus substances
-
Neurotoxiclty screening battery
Developmental neurotoxicity study
Schedule controlled operant behavior
Peripheral nerve function
Neurophysralogy Sensory evoked potentials
Group F — Special Studies Test Guidelines
Companion animal safety
Metabolism and pharmacolonelics
Dermal penetration
Imrnunotoxicity
Existing Numbers
OPPT
none
798 1175
7981100
7981150
798 4500
798 4470
7984100
7982650
none
none
798 2250
798 2450
798 4900
7984700
798 3260
798 3300
798 3320
7985100
5265
7985140
7985195
7985200
798 5250
7985275
798 S300
798 5375
7985380
798 5385
798 5395
7985450
7985460
7985500
798 5550
7985575
7985900
7985915
7986450
6540
6560
798 6050
6200
6400
none
798 6500
798 6850
798 6855
none
798 7485
none
none
OPP
none
81-1
81-2
81-3
81-4
81-5
81-6
83-1
82-1
82—2
82-3
82-4
83-3
83-1
83-1
83-2
83-5
84-2
84-2
34-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
* 84-2
84-2
84-2
84-2
81-7
82-5
82-6
81-8
82-7
83-1
83-6
85-5
85-6
none
none
85-1
85-3
85-7
OECD
none
401
402
403
405
404
406
408
409
410
411
413
414
416
452
451
453
471 472
none
none
none
none
477
476
473
483
475
474
478
none
none
482
481
479
none
418 419
424
none
none
none
none
none
417
none
none
EPA Pub
no
712-C-
98-189
93-190
98-192
98-193
98-195
98-196
98-197
90-139
98-200
98-201
98-202
98-204
98-207
98-208
98-2fO
98-211
98-212
98-247
98-215
98-216
98-217
98-218
98-220
98-221
98-223
98-224
98-225
98-226
98-227
98-228
98-229
98-230
98-232
98-234
98-235
98-237
98-238
98-239
98-240
98-241
98-242
98-349
95-244
98-350
98-351
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United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-189
August 1998
&EPA
Health Effects Test
Guidelines
OPPTS 870.1000
Acute Toxicity Testing
Background
US EPA Headquarters Library
Mail code 3201
1300 Pennsylvania Avenue NW
Washington DC 20460
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed m the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared m publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the US.
Government Pnntng Office, Washington, DC 20402 on disks or paper
copies call (202) 312-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.1000 Acute toxicity testing—background.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) (Reserved]
(b) Purpose. The Agency considers the evaluation of toxicity follow-
ing short term exposure to a chemical to be an integral step in the assess-
ment of its toxic potential under the regulatory framework of its pesticide
and toxic substances programs In the assessment and evaluation of the
toxic characteristics of a substance, acute toxicity is generally performed
by the probable route of exposure in order to provide information on health
hazards likely to anse from short-term exposure by that route For pes-
ticides, the short-term toxicity testing battery consists of acute toxicity tests
by the oral, dermal, and inhalation routes, skin and eye irritation testing;
and testing for dermal sensmzation Data from an acute study may serve
as a basis for hazard categonzation, labeling, or child-resistant packaging
and may also serve to designate pesticides which may be applied only
by certified applicators It is also an initial step in establishing a dosage
regimen in subchromc and other studies and may provide information on
absorption and the mode of toxic action of a substance An evaluation
of acute toxicity data should include the relationship, if any, between the
exposure of animals to the test substance and the incidence and seventy
of all abnormalities, including behavioral and clinical abnormalities, the
reversibility of observed abnormalities, gross lesions, body weight
changes, effects on mortality, and any other toxic effects,
(c) History—(1) Acute toxicity test guidelines. Test guidelines for
acute toxicity were first published by the Agency in October 1982 as part
of Subdivision F of the Pesticide Assessment Guidelines for the Office
of Pesticide Programs (OPP) (see paragraph (f)(4) of this guideline) and
in 40 CFR part 797 in September 1985 for the Office of Toxic Substances
(OPPTS).
(2) Rejection rate analysis. In 1993, as part of its Pesticide Rejection
Rate Analysis, Agency and industry scientists met to perform a guidelme-
by-guidelme review of toxicology studies including acute toxicity studies
The purpose of this guidelme-by-guidelme review was to identify those
factors that most frequently cause toxicology studies required for pesticide
reregistration to be rejected The results were published as the Pesticide
Reregistration Rejection Rate Analysis Toxicology (see paragraph (f)(5)
of this guideline) In 1995, representatives from the Agency met with the
American Crop Protection Association (ACPA), the Chemical Producers
and Distributors Association (CPDA), the Chemical Manufacturers Asso-
ciation (CMA), Health Canada, and the California Department of Pesticide
Regulation (CDPR) to discuss acceptable methods for the conduct of acute
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toxicity studies The discussions of this meeting were incorporated into
a preliminary Registration Division document titled Conduct of Acute Tox-
icity Studies (see paragraph (f)(6) of this guideline) These documents sup-
plement the acute toxicology guidelines in Subdivision F
(3) Guideline harmonization. The Senes 870 Health Effects test
guidelines have been harmonized between OPP and OPPTS and, where
possible, with OECD test guidelines Scientific considerations from both
of the analyses described in paragraph (c)(2) of this guideline have been
incorporated into the revised test guidelines
f
(d) Approaches to the determination of acute toxicity. (1) At
present, the evaluation of chemicals for acute toxicity is necessary for the
protection of public health and the environment The Agency supports
measures dedicated to reducing the use of animals in toxicity testing
When animal testing is required for this purpose, testing should be done
in ways that minimize numbers of animals used and that take full account
of their welfare. To this end, when conducting a test, the Agency stresses
the simultaneous monitoring of several endpomts of toxicity in animals
in a single acute study including sublethal effects as well as lethality.
Dosed animals are observed for abnormal behavioral manifestations such
as increased salivation or muscular incoordmation, in addition to the recov-
ery from these effects during the observation penod Both dead and surviv-
ing animals are necropsied to evaluate gross anatomical evidence of organ
toxicity In selected cases, additional testing may be justified to better char-
acterize the kinds of abnormalities that have been found in the organs
of the necropsied animals These sound, scientific practices represent some
of the means which maximize the utility of the data obtained from a lim-
ited number of test animals to achieve a balance between protecting hu-
mans and the environment, and the welfare and utilization of laboratory
animals
(2) EPA recommends the following means to reduce the number of
animals used to evaluate acute effects of chemical exposure while preserv-
ing its ability to make reasonable judgements about safety
(i) Use of data from structurally related substances or mixtures In
order to minimize the need for animal testing for acute effects, the Agency
encourages the review of existing acute-toxicity information on chemical
substances that are structurally related to the agent under investigation
In certain cases, it may be possible to obtain enough information to make
preliminary hazard evaluations that may reduce the need for further animal
testing for acute effects Similarly, mixtures or formulated products that
are substantially similar to well-charactenzed mixtures or products may
not need additional testing if there are sufficient bridging data available
for meaningful extrapolation In those cases, classification would be ex-
trapolated from the mixture already tested
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(n) Use of appropriate alternative test protocols when available Thus,
for example, acute oral toxicity testing may be performed using the Fixed
Dose Method (OECD Guideline 420, see paragraph (0(1) of this guide-
line), or the Acute Toxic Class Method (OECD Guideline 423, see para-
graph (f)(2) of this guideline), or the Up-and-Down Method (OECD
Guideline 425, see paragraph (f)(3) of this guideline) Abbreviated meth-
ods are not yet available through OECD for acute toxicity by other routes
of exposure
(m) Weight of evidence approaches to dermal and ocular irritation
Several factors should be considered in determining the corrosion and im-
tation potential of chemicals before testing is undertaken Existing human
experience and data and animal observations and data should be the first
line of analysis, as it gives information directly referable to effects on
the skin In some cases, enough information may be available from struc-
turally related compounds to make classification decisions. Likewise, pH
extremes (pH <2 or >11 5) may indicate dermal effects, especially when
buffering capacity is known, although the correlation is not perfect Gen-
erally, such agents are expected to produce significant effects on the skin.
It also stands to reason that if a chemical is extremely toxic by the dermal
route, a dermal irritation/corrosion study may not be needed Likewise,
if there is a lack of any dermal reaction at the limit dose (2,000 mg/kg)
m an acute toxicity study (for which observations of dermal reactions were
made), a dermal irritation/corrosion study again may not be needed. It
should be noted, however, that often acute dermal toxicity and dermal im-
tation/corrosion testing are performed in different species that may differ
in sensitivity In vitro alternatives that have been validated and accepted
may also be used to help make classification decisions
(iv) All of the available information on a chemical should be used
in determining the need for in vivo dermal irritation testing. Although in-
formation might be gained from the evaluation of single parameters within
a tier (e g , caustic alkalies and acids with extreme pH (pH <2 or >11.5)
should be considered as dermal corrosives), there is ment m considenng
the totality of existing information and making an overall weight of evi-
dence determination. This is especially true when there is information
available on some but not all parameters
(v) Use of limit testing For chemicals judged to be relatively non-
toxic, a single group of animals is given a large dose of the agent. If
no lethality is demonstrated, no further testing is pursued The substance
is classified in hazard categories according to the limit dose used (See
the following paragraph for a discussion of toxicity categones under
FIFRA)
(e) Regulatory applications under FIFRA. (1) Precautionary label-
ing provides the pesticide user with a general idea of the potential toxicity,
irritation and sensitization hazard associated with the use of a pesticide
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(see EPA Label Review Manual (paragraph (f)(7) of this guideline) and
40 CFR Part 156—Labeling Requirements for Pesticides and Devices)
Precautionary labeling also identifies the precautions necessary to avoid
exposure as well as any personal protective equipment which should be
used when handling a pesticide and statements of practical treatment in
case of accidental exposure The United States is an active participant in
negotiations to develop a globally harmonized system for classification and
labeling. Planning for the globally harmonized system will be completed
in the year 2000 with implementation to be phased in after planning is
completed This section describes the current system in place for pesticides
m the United States and will be revised and updated when the globally
harmonized system is fully implemented.
(2) Precautionary labeling which includes the signal word, personal
protective equipment, hazard symbol, and statements of practical treatment
is normally determined by six acute toxicity studies and product composi-
tion. The acute oral, acute dermal and acute inhalation studies are used
to determine the LDso of a product via the designated route of exposure
The primary eye irritation and primary skin irritation studies measure the
seventy of irritation or corrosivity caused by a product The dermal sen-
sitization study determines whether a product is capable of causing an al-
lergic reaction With the exception of the dermal sensitization study, each
acute toxicity study is assigned a toxicity category as defined in the table
below All products falling into toxicity categones I-IV must bear a signal
word and in some cases warning symbols
(3) Personal Protective Equipment Personal protective equipment
which includes use of protective clothing, chemical resistant gloves, pro-
tective eye gear, and respiratory protective devices, is determined by the
results of six acute toxicity studies according to toxicity category (see
table). The degree of protection required is graded according to the degree
of acute toxicity and the hazard classification category of the chemical
or product These requirements are set forth in 40 CFR 170 240 in the
Worker Protection Standard
(4) Restricted entry intervals Agricultural products must display a
restricted entry interval A restricted entry interval is the time immediately
following a pesticide application dunng which entry into the treated area
is restricted. Restricted entry intervals are based on the most severe acute
toxicity category assigned to the acute dermal, eye irritation and skin imta-
tion data for all of the active ingredients in a pesticide product. The dura-
tion of restricted entry intervals is based on the seventy of toxicity, with
products classified in category I requiring intervals of 48 hours or more
and products classified in category III or IV requiring intervals of 12
hours
(5) Child-resistant packaging FIFRA establishes standards with re-
spect to pesticide packaging of products intended for use in residential
-------
settings in order to protect children or adults from serious illness or injury
resulting from accidental mgestion or contact with pesticides Criteria for
which pesticides must be distributed or sold in child-resistant packaging
aie based on classification according to the toxicity categories set forth
in the table
(6) Restricted use pesticide The Agency determines whether a pes-
ticide must be applied under the direct supervision of a certified applicator
Such clarification for restricted use is based upon consideration of toxicity
data, including acute toxicity, exposure, and intended use
(7) Biochemical pest control agents are tested in a special tiered pro-
gression The technical grade biochemical pest control agent is always
characterized by acute toxicity tests However, because of their nontoxic
mode of action against the target pest, further testing of the biochemical
pest control agent is normally not required Microbial pest control agents
are tested using the OPPTS Harmonized Test Guidelines Senes 885, Mi-
crobial Pesticide Test Guidelines, for pathogemcity/mfectivity In addition,
all formulations of microbial pest control agents are tested for precaution-
ary labeling using acute toxicity tests in the OPPTS Harmonized Test
Guidelines Senes 870, Health Effects Test Guidelines
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Toxicity Categories
Study
Acute Oral
Acute Dermal
Acute Inhalation
Eye Irritation
Skin irritation
Categoryl
Up to and including 50
mg/kg
Up to and including 200
mg/kg
Up to and including 0 05
mg/liter
Corrosive (irreversible
destruction of ocular
tissue) or cornea!
involvement or irritation
persisting for more than
21 days
Corrosive (tissue
destruction into the
dermis and/or scarring)
Category II
>50 through 500
mg/kg
>200 through
2000 mg/kg
>0 05 through 0 5
mg/liter
Cornea!
involvement or
irritation clearing
in 8-21 days
Severe irritation at
72 hours (severe
erythema or
edema)
Category III
>500 through
5000 mg/kg
>2000 through'
5000 mg/kg
>0 5 through 2
mg/liter
Cornea!
involvement or
irritation clearing
in 7 days or less
Moderate irritation
at 72 hours
(moderate
erythema)
Category IV
>5000 rng/kg
>5000 rng/kg
>2 mg/liter
Minimal effects
clearing in less
than 24 hours
Mild or slight
irritation (no
irritation or slight
erythema)
Study
Dermal Sensittzation
Study results
Product is a sensitizer or is positive
for sensrtization
Study results
Product is not a sensitizer or is
negative for sensitization
(f) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals Guideline 420 Acute Oral Toxicity-
Fixed Done Method Adopted July 17, 1992.
(2) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals Guideline 423* Acute Oral Toxicity-
Acute Toxic Class Method Adopted March 22, 1996
(3) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals Guideline 425 Acute Oral Toxicity-
Up-and-Down Method Approved June 1998
(4) U.S Environmental Protection Agency Pesticide Assessment
Guidelines, Subdivision F Health Effects EPA report 540/09-82-025, Oc-
tober 1982
(5) US Environmental Protection Agency Pesticide Reregistration
Rejection Rate Analysis Toxicology EPA report 738-R-93-004 July
1993
(6) U S Environmental Protection Agency Conduct of Acute Toxicity
Studies EPA report 737-R-97-002 September 1997
(7) U S Environmental Protection Agency Label Review Manual 2nd
Edition EPA report 737-B-96-001 December 1996
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oEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-190
August 1993
Health Effects Test
Guidelines
OPPTS870.1100
Acute Oral Toxicity
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxtc Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U S.C 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC l^etseq)
Final Guideline Release: This guideline is available from the US.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http-//www.epa.gov/epahome/research htm) under the heading "Research-
ers and Scientistsn'est Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870 1 1 00 Acute oral toxicity
(a) Scope — (1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798 1175 Acute Oral Toxicity,
OPP 81-1 (Pesticide Assessment Guidelines, Subdivision F — Hazard Eval-
uation; Human and Domestic Animals) EPA report 540/09-82-025, 1982,
and OECD 401 Acute Oral Toxicity
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a substance, determination of acute oral toxicity is usually an
initial step It provides information on health hazards likely to arise from
short-term exposure by the oral route Data from an acute study may serve
as a basis for classification and labeling It is traditionally a step in estab-
lishing a dosage regimen in subchromc and other studies and may provide
initial information on the mode of toxic action of a substance An evalua-
tion of acute toxicity data should include the relationship, if any, between
the exposure of animals to the test substance and the incidence and seventy
of all abnormalities, including behavioral and clinical abnormalities, the
reversibility of observed abnormalities, gross lesions, body weight
changes, effects on mortality, and any other toxic effects
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792— Good Lab-
oratory Practice Standards apply to this test guideline The following defi-
nitions also apply to this test guideline
Acute oral toxicity is the adverse effects occurring within a short pe-
riod of time after oral administration of either a single dose of a substance
or multiple doses given within a 24-hour period
Dosage is a general term comprising the dose, its frequency, and the
duration of dosing
Dose is the amount of test substance administered. Dose is expressed
as weight of test substance (milligrams, grams) per unit weight of test
animal (e g. milligrams per kilogram)
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
Dose-response is the relationship between the dose and the proportion
of a population sample showing a defined effect
(median lethal dose) is a statistically denved estimate of single
dose of a substance that can be expected to cause death in 50 percent
-------
of animals when administered by the oral route The LDso value is ex-
pressed in terms of weight of test substance per unit weight of test animal
(milligrams per kilogram)
(d) Approaches to the determination of acute toxicity. (1) EPA
recommends the following means to reduce the number of animals used
to evaluate acute effects of chemical exposure while preserving its ability
to make reasonable judgments about safety
(i) Use of appropriate alternative test protocols when available Thus,
for example, acute oral toxicity testing may be performed using the Fixed
Dose Method (OECD Guideline 420, see paragraph (f)(l) of this guide-
line), or the Acute Toxic Class Method (OECD Guideline 423, see
aragraph (f)(2) of this guideline), or the Up-and-Down Method (OECD
Guideline 425, see paragraph (f)(3) of this guideline) Abbreviated meth-
ods are not yet available through OECD for acute toxicity by other routes
of exposure However, OECD is actively revising its approaches to testing
for dermal and ocular irritation and when this is done, the OPPTS guide-
lines will be updated to harmonize with the OECD revisions
(11) Limit test. When data on structurally related chemicals are inad-
equate, a limit test may be considered If rodents are used, a limit dose
of at least 2,000 mg per kilogram of body weight may be administered
to a single group of five males and five females using the procedures
described under paragraph (e) of this guideline If no lethality is dem-
onstrated, no further testing for acute oral toxicity is needed. (Under cur-
rent policy and regulations for pesticide products, precautionary statements
may still be required unless there are data to indicate the LD50 is greater
than 5,000 mg/kg ) If compound-related mortality is produced in the limit
test, further study may need to be considered.
(111) Estimation of acute oral toxicity When further study is war-
ranted, EPA generally supports limiting such tests to those using the lowest
number of animals feasible Given the approval internationally through
OECD of three alternative test methods to the "traditional" acute oral
toxicity test, it is time to reassess the status of acute toxicity testing The
three OECD alternatives include the following The fixed dose procedure
is a refinement of the traditional acute oral test that employs nonlethal
endpomts. In contrast, the acute toxic class and up-and-down procedures
estimate lethality within a dose range and as a point estimate, respectively,
and reduce animal usage m comparison to the "traditional" test.
(A) The up and down procedure as descnbed in OECD Guideline
425 referenced in paragraph (f)(4) of this guideline This method is highly
recommended
(B) The acute toxic class method as descnbed in OECD Guideline
423 and referenced in paragraph (f)(6) of this guideline
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(C) A three-dose method described as the conventional acute toxicity
test under paragraph (e) in this guideline, and in OECD Guideline 401
referenced in paragraph (f)(7) of this guideline
(D) The fixed dose method as described m OECD Guideline 420 and
referenced in paragraph (f)(5) of this guideline
(e) Conventional acute toxicity test—(1) Principle of the test meth-
od. The test substance is administered orally by gavage in graduated doses
to several groups of experimental animals, one dose being used per group
The doses chosen may be based on the results of a range finding test
Subsequently, observations of effects and deaths are made Animals that
die during the test are necropsied, and at the conclusion of the test the
surviving animals are sacrificed and necropsied This guideline is directed
primarily to studies in rodent species but may be adapted for studies in
nonrodents Animals showing severe and endunng signs of distress and
pain may need to be humanely killed Dosing test substances in a way
known to cause marked pain and distress due to corrosive or imtatmg
properties need not be earned out
(2) Substance to be tested. Test, control and reference substances
are discussed in 40 CFR Part 792—Good Laboratory Practice Standards.
(3) Test procedures—(i) Preparations. Healthy young adult animals
are acclimatized to the laboratory conditions for at least 5 days pnor to
the test before the test animals are randomized and assigned to the treat-
ment groups
(ii) Animal selection—(A) Species and strain. Although several
mammalian test species may be used, the rat is the preferred species Com-
monly used laboratory strains should be employed If another species is
used, the tester should provide justification and reasoning for its selection.
(B) Age. Young adult rats between 8- and 12-weeks-old at the be-
ginning of dosing should be used Rabbits should be at least 12 weeks
of age at study initiation The weight variation of animals used in a test
should be within 20 percent of the mean weight for each sex
(C) Number and sex of animals. (7) At least five experimentally
naive rodents are used at each dose level They should all be of the same
sex After completion of the study m one sex, at least one group of five
animals of the other sex is dosed to establish that animals of this sex
are not markedly more sensitive to the test substance The use of fewer
animals may be justified in individual circumstances Where adequate in-
formation is available to demonstrate that animals of the sex tested are
markedly more sensitive, testing in animals of the other sex may be dis-
pensed with An acceptable option would be to test at least one group
of five animals per sex at one or more dose levels to definitively determine
the more sensitive sex pnor to conducting the main study
-------
(2) The females should be nulhparous and nonpregnant
(J) In acute toxicity tests with animals of a highei order than rodents,
the use of smaller numbers should be considered
(D) Assignment of animals. Each animal must be assigned a unique
identification number A system to assign animals to test groups and con-
trol groups randomly is required
(E) Housing. Animals may be group-caged by sex, but the number
of animals per cage must not interfere with clear observation of each ani-
mal. The biological properties of the test substance or toxic effects (e g
morbidity, excitability) may indicate a need for individual caging
(1) The temperature of the experimental animal rooms should be at
22 ± 3 °C for rodents
(2) The relative humidity of the experimental animal rooms should
be 30 to 70 percent
(3) Where lighting is artificial, the sequence should be 12-hours light/
12-hours dark.
(4) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water
(in) Dose levels and dose selection. (A) Three dose levels should
be used, 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 permit an acceptable estimation of the LDso Range
finding studies using single animals may help to estimate the positioning
of dose groups so that no more than three dose levels will be necessary.
An acceptable option for pesticide products would be to set the dose levels
m correlation with the OPP toxicity categories (bracketing) In these cases,
the determination of an LDso may not be necessary
(B) Limit test. This test has been defined and described under para-
graph (d)(2)(ti) of this guideline
(C) Vehicle. Where necessary, the test substance is dissolved or sus-
pended in a suitable vehicle If a vehicle or diluent is needed, it should
not elicit toxic effects itself nor substantially alter the chemical or toxi-
cological properties of the test substance It is recommended that wherever
possible the use of an aqueous solution be considered first, followed by
consideration of a solution in oil (e g , corn oil), and then by consideration
of possible solution in other vehicles Toxic characteristics of nonaqueous
vehicles should be known, and, if not known, should be determined before
the test
-------
(D) 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, except when an aqueous solu-
tipn is used in which case 2 mL/100 g may be administered Either con-
stant volume or constant concentration administration is acceptable when
dosing, provided the following guidance is employed When possible, the
liquid test material should be dosed neat Otherwise, it may be diluted,
using the highest concentration possible, although volumes less than 0 5
mL per animal would not be required Lower dose volumes are acceptable
if they can be accurately administered Solid matenals should be sus-
pended or dissolved in the minimum amount of vehicle and dosed at the
highest concentration possible
(iv) Exposure and exposure duration. (A) Animals should be fasted
prior to test substance administration For the rat, feed should be withheld
overnight, for other rodents with higher metabolic rates a shorter period
of fasting is appropriate
(B) The test substance should be administered in a single dose by
gavage, using a stomach tube or suitable intubation cannula
(C) 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 adminis-
tered in fractions, it may be necessary to provide the animals with food
and water, depending on the length of the dosing period
(D) After the substance has been administered, feed may be withheld
for an additional 3-4 hours
(v) Observation period. Although 14 days is recommended as a min-
imum observation penod, the duration of observation should not be fixed
rigidly It should be determined by the toxic reactions, rate of onset, and
length of recovery penod, and may thus be extended when considered nec-
essary The time at which signs of toxicity appear, their duration, and the
time to death are important, especially if there is a tendency for deaths
to be delayed
(vi) Observation of animals. (A) A careful clinical examination
should be made at least once each day
(B) Additional observations should be made daily, especially in the
early days of the study Appropriate actions should be taken to minimize
loss of animals to the study (e g, necropsy or refrigeration of those ani-
mals found dead and isolation of weak or monbund animals)
(C) Observations should be detailed and carefully recorded, preferably
using explicitly defined scales Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonomic effects such as salivation,
-------
central nervous system effects, including tremors and convulsions, changes
in the level of activity, gait and posture, reactivity to handling or sensory
stimuli altered strength, and stereotypies or bizarre behavior (e g , self-
mutilation, walking backwards)
(D) 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 1
day
(E) The time of death should be recorded as precisely as possible
/
(vu) Gross pathology. (A) At the end of the test, surviving animals
should be weighed and sacrificed
(B) A gross necropsy should be performed on all animals under test
All gross pathology changes should be recorded
(C) If necropsy cannot be performed immediately after a dead animal
is discovered, the animal should be refrigerated (not frozen) at tempera-
tures low enough to minimize autolysis Necropsies should be performed
as soon as practicable, normally within a day or two.
(via) Additional evaluation. 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
(ix) Data and reporting—(A) Treatment of results. Data should
be summarized m tabular form, showing for each test group the number
of animals at the start of the test, body weights, tune of death of individual
animals at different dose levels, number of animals displaying other signs
of toxicity, description of toxic effects, and necropsy findings Any meth-
ods used for calculation of the LDso or any other parameters should be
specified and referenced Methods for parameter estimation are descnbed
under paragraphs (0(1), (0(2), and (0(3) of this guideline
(B) Evaluation of results. An evaluation should include the relation-
ship, if any, between exposure of the animals to the test substance and
the incidence and seventy of all abnormalities, including behavioral and
clinical abnormalities, gross lesions, body weight changes, effects on mor-
tality, and any other toxic effects The LDso value should always be con-
sidered in conjunction with the observed toxic effects and any necropsy
findings The LDso value is a relatively coarse measurement, useful only
as a reference value for classification and labeling purposes, and for an
expression of the lethal potential of the test substance by the mgestion
route Reference should always be made to the experimental animal spe-
cies m which the LDso value was obtained
(C) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J and 40 CFR part 160, subpart J, the
-------
following specific information should be reported The test report should
include
(/) Species, strain, sex, and source of test animals
(2) Method of randomization in assigning animals to test and control
groups
(3) Rationale for selection of species, if other than that recommended
(4) Tabulation of individual and test group data by sex and dose level
(eg number of animals exposed, number of animals showing signs of
toxicity and number of animals that died or were killed during the test)
(0 Description of toxic effects, including their time of onset, duration,
reversibility, and relationship to dose
00 Body weights
(in) Time of dosing and time of death after dosing
(iv) Dose-response curves for mortality and other toxic effects (when
permitted by the method of determination)
(v) Gross pathology findings
(vi) Histopathology findings and any additional clinical chemistry
evaluations, if performed
(5) Description of any pretest conditioning, including diet, quarantine
and treatment for disease
(6) Description of cagmg conditions including Number (or change
in number) of animals per cage, bedding material, ambient temperature
and humidity, photopenod, and identification of diet of test animals
(7) Manufacturer, source, punty, and lot number of test substance
(8) Relevant properties of substance tested including physical state
and pH (if applicable)
(9) Identification and composition of any vehicles (e.g , diluents, sus-
pending agents, and emulsifiers) or other materials used in administering
the test substance
(10) A list of references cited in the body of the report References
to any published literature used in developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu-
ating the results
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline
-------
(1) Chanter, D O and Hey wood, R The LD50 Test Some Consider-
ations of Precision Tautology Letter* 10 303-307 (1982)
(2) Finney, D J Chapter 3—Estimation of the median effective dose
and Chapter 4—Maximum likelihood estimation, Probit Analysis, 3rd ed
Cambridge, London (1971)
(3) Finney, D J The Median Lethal Dose and Its Estimation. Archives
of Toxicology 56 215-218 (1985)
(4) Organization for Economic Cooperation and Development. OECD
Guidelines^ for the Testing of Chemicals OECD Guideline 425 Acute Oral
Toxicity Up-and-Down Procedure, Approved June 1998
(5) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 420. Acute Oral Tox-
icity—Fixed Dose Method, Adopted July 17, 1992
(6) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 423 Acute Oral Toxicity
- Acute Toxic Class Method, Adopted March 22, 1996
(7) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 401 Acute Oral Toxicity,
Adopted February 24, 1987
8
-------
&EPA
United Slates
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-99-192
August 1998
Health Effects Test
Guidelines
OPPTS 870.1200
Acute Dermal Toxicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC 136, etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.1200 Acute dermal toxicity.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798 1100 Acute Dermal Tox-
icity, OPP 81-2 Acute Dermal Toxicity (Pesticide Assessment Guidelines,
Subdivision F—Hazard Evaluation, Human and Domestic Animals) EPA
report 540/09-82-025, 1982, and OECD 402 Acute Dermal Toxicity
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a substance, determination of acute dermal toxicity is useful where
exposure by the dermal route is likely It provides information on health
hazards likely to arise from short-term exposure by the dermal route Data
from an acute study may serve as a basis for classification and labeling
It is an initial step in establishing a dosage regimen in subchronic and
other studies and may provide information on dermal absorption and the
mode of toxic action of a substance by this route An evaluation of acute
toxicity data should include the relationship, if any, between the exposure
of animals to the test substance and the incidence and seventy of all abnor-
malities, including behavioral and clinical abnormalities, the reversibility
of observed abnormalities, gross lesions, body weight changes, effects on
mortality, and any other toxic effects
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions m 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline
Acute dermal toxicity is the adverse effects occurring within a short
time of dermal application of a single dose of a substance or multiple
doses given within a 24-h period
Dosage is a general term comprising the dose, its frequency and the
duration of dosing
Dose is the amount of test substance applied Dose is expressed as
weight of test substance (grams, milligrams) per unit weight of test animal
(e g milligrams per kilogram)
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
Dose-response is the relationship between the dose and the proportion
of a population sample showing a defined effect
-------
(median lethal dose), dermal, is a statistically derived estimate
of a single dose of a substance that can be expected to cause death in
50 percent of treated animals when applied to the skin The LDso value
is expressed in terms of weight of test substance per unit weight of test
animal (milligrams per kilogram)
(d) Approaches to the determination of acute toxicity. (1) EPA
recommends the following means to reduce the number of animals used
to evaluate acute effects of chemical exposure while preserving Us ability
to make reasonable judgments about safety
(i) Using data from substantially similar mixtures In order to mini-
mize the need for animal testing, the Agency encourages the review of
existing acute toxicity information on mixtures that are substantially simi-
lar to the mixture under investigation In certain cases it may be possible
to glean enough information to make preliminary hazard evaluations that
may reduce the need for further animal testing
(11) Limit test When data on structurally related chemicals are inad-
equate, a limit test may be considered If rodents are used, a limit dose
of at least 2,000 mg/kg bodyweight may be administered to a single group
of five males and five females using the procedures described under para-
graph (e) of this guideline If no lethality is demonstrated, no further test-
ing for acute dermal toxicity is needed (Under current policy for pesticide
products, precautionary statements may still be required unless there are
data to indicate the LDso is greater than 5,000 mg/kg ) If compound-related
mortality is produced, further study may need to be considered.
(2) [Reserved]
(e) Conventional acute toxicity test—(1) Principle of the test meth-
od. The test substance is applied dermally m graduated doses to several
groups of experimental animals, one dose being used per group. The doses
chosen may be based on the results of a range finding test. Subsequently,
observations of effects and deaths are made Animals that die during the
test are necropsied, and at the conclusion of the test the surviving animals
are sacrificed and necropsied This guideline is directed primarily to stud-
ies in either rats, rabbits, or guinea pigs but may be adapted for studies
in other species Animals showing severe and enduring signs of distress
and pain may need to be humanely killed Dosing test substances in a
way known to cause marked pain and distress due to corrosive or irritating
properties need not be earned out
(2) Substance to be tested. Test, control, and reference substances
are discussed in 40 CFR Part 792—Good Laboratory Practice Standards
(3) Test procedures—(i) Preparations. Healthy young adult animals
are acclimatized to the laboratory conditions for at least 5 days prior to
-------
the test before the test animals are randomized and assigned to the treat-
ment groups
(n) Animal selection—(A) Species and strain. The rat, rabbit, or
guinea pig may be used The albino rabbit is preferred because of its size,
ease of handling, skin permeability, and extensive data base Commonly
used laboratory strains should be employed If a species other than rats,
rabbits, or guinea pigs is used, the tester should provide justification and
reasoning for Us selection
(B) Age Young adult animals, rats between 8- and 12-weeks-old,
rabbits at least 12-weeks-old, and guinea pigs between 5- and 6-weeks-
old at the beginning of dosing should be used The weight variation of
animals used in a test should be within 20 percent of the mean weight
for each sex
(C) Number and sex of animals. (7) At least five experimentally
naive animals with healthy intact skin are used at each dose level They
should all be of the same sex After completion of the study in one sex,
at least one group of five animals of the other sex is dosed to establish
that animals of this sex are not markedly more sensitive to the test sub-
stance. The use of fewer animals may be justified in individual cir-
cumstances Where adequate information is available to demonstrate that
animals of the sex tested are markedly more sensitive, testing in animals
of the other sex may be dispensed with An acceptable option would be
to test at least one group of five animals per sex at one or more dose
levels to definitively determine the more sensitive sex prior to conducting
the main study
(2) The females should be nulliparous and nonpregnant
(3) In acute toxicity tests with animals of a higher order than those
mentioned above, the use of smaller numbers should be considered
(D) Assignment of animals. Each animal must be assigned a unique
identification number A system to randomly assign animals to test groups
and control groups is required
(E) Housing. Animals should be housed in individual cages
(7) The temperature of the experimental animal rooms should be at
22 ±3 °C for rodents, 20±3 °C for rabbits
(2) The relative humidity of the experimental animal rooms should
be 30 to 70 percent.
(3) Where lighting is artificial, the sequence should be 12-h light/
12-h dark
-------
(4) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water
(m) Dose levels and dose selection. (A) 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 permit an acceptable estimation of the median
lethal dose Range finding studies using single animals may help to esti-
mate the positioning of the dose groups so that no more than three dose
levels will be necessary An acceptable option for pesticide products would
be to set the dose levels m correlation with the OPP toxicity categories
(bracketing). In these cases, the determination of an LDso may not be nec-
essary
(B) Limit test This test is described under paragraph (d)(2)(u) of this
guideline
(C) Vehicle Solids should be pulverized when possible The test sub-
stance should be moistened sufficiently with water or, where necessary,
a suitable vehicle to ensure good contact with skin If a vehicle or diluent
is needed, it should not elicit toxic effects itself nor substantially alter
the chemical or lexicological properties of the test substance. In addition,
the influence of the vehicle on penetration of skin by the test substance
should be taken into account It is recommended that wherever possible
the use of an aqueous solution be considered first, followed by consider-
ation of a solution m oil (e g corn oil), and then by consideration of pos-
sible solution in other vehicles For nonaqueous vehicles the toxic charac-
teristics of the vehicle should be known, and if not known should be deter-
mined before the test Acceptable alternative vehicles include gum arable,
ethanol and water, carboxymethyl cellulose, glycerol, propylene glycol,
PEG vegetable oil, and mineral oil as long as the vehicle is not imtatmg
and the inability to use water or saline is justified in the report
(iv) Exposure and exposure duration. The test substance should be
administered over a penod of 24 h
(v) Preparation of animal skin. Fur should be clipped from the dor-
sal area of the trunk of the test animals Shaving may be employed, but
it should be earned out at least 24 h before dosing Care must be taken
to avoid abrading the skin, which would alter its permeability
(vi) Application of test substance. (A) The test substance should
be applied uniformly over a shaved or clipped area which is approximately
10 percent of the body surface area The area starting at the scapulae
(shoulders) to the wing of the ileum (hip bone) and half way down the
flank on each side of the animal should be shaved or clipped. Liquid test
materials should be undiluted if possible With highly toxic substances,
the surface area covered may be less, but as much of the area as possible
should be covered with as thin and uniform a film as practical The test
-------
material is not removed until 24 h after application In the case where
less than 10 percent of the surface area is covered an approximation of
the exposed areas should be determined
(B) The test substance should be held in contact with the skin with
a porous gauze dressing (<8 ply) and nonirritatmg tape throughout a
24-h 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 Restramers may be used to
prevent the ingestion of the test substance, but complete immobilization
is not a recommended method Although a semiocclusive dressing is pre-
ferred, an occlusive dressing will also be acceptable
(C) At the end of the exposure penod, residual test substance should
be removed where practicable using water or an appropnate solvent
(vn) Observation period. Although 14 days is recommended as a
minimum observation period, the duration of observation should not be
fixed ngidly It should be determined by the toxic reactions, rate of onset,
and length of recovery penod and may thus be extended when considered
necessary The time at which signs of toxicity appear, their duration, and
the time to death are important, especially if there is a tendency for deaths
to be delayed
(vin) Observation of animals. (A) A careful clinical examination
should be made at least once each day
(B) Additional observations should be made daily, especially in the
early days of the study Appropnate actions should be taken to minimize
loss of animals to the study (e g necropsy or refrigeration of those animals
found dead and isolation of weak or monbund animals).
(C) Observations should be detailed and carefully recorded, preferably
using explicitly defined scales Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonomic effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
in the level of activity, gait and posture, reactivity to handling or sensory
stimuli, altered strength, and stereotypies or bizarre behavior (e g self-
mutilation, walking backwards)
(D) 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
(E) The time of death should be recorded as precisely as possible
(ix) Gross pathology. (A) At the end of the test, surviving animals
should be weighed and sacnficed
-------
(B) A gross necropsy should be performed on all animals under test
All gross pathology changes should be recorded
(C) If necropsy cannot be performed immediately after a dead animal
is discovered, the animal should be refrigerated (not frozen) at tempera-
tures low enough to minimize autolysis Necropsies should be performed
as soon as practicable, normally within a day or two
(x) Additional evaluations. Microscopic examination of organs
showing evidence of gross pathology in animals surviving 24 h or more
should also be considered because it may yield useful information
(xi) 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, body weights, 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. Any meth-
ods used for calculation of the LDso or any other parameters should be
specified and referenced Methods for parameter estimation are described
under paragraphs (0(1). (0(2), and (0(3) of this guideline
(B) Evaluation of results. An evaluation should include the relation-
ship, if any, between exposure of the animals to the test substance and
the incidence and seventy of all abnormalities, including behavioral and
clinical abnormalities, gross lesions, body weight changes, effects on mor-
tality, and any other toxic effects The LDso value should always be con-
sidered in conjunction with the observed toxic effects and any necropsy
findings. The LDso value is a relatively coarse measurement, useful only
as a reference value for classification and labeling purposes, and for an
expression of the lethal potential of the test substance by the ingestion
route Reference should always be made to the experimental animal spe-
cies m which the LD5o value was obtained
(C) Test report In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J and 40 CFR part 160, subpart J, the
following specific information should be reported The test report should
include
(/) Species, strain, sex, and source of test animals
(2) Method of randomization in assigning animals to test and control
groups
(J) Rationale for selection of species, if other than that recommended
(4) Tabulation of individual and test group data by sex and dose level
(e g number of animals exposed, number of animals showing signs of
toxicity and number of animals that died or were killed during the test)
-------
(0 Description of toxic effects, including their time of onset, duration,
reversibility, and relationship to dose
(«) Body weights
(in) Time of dosing and time of death after dosing
(iv) Dose-response curves for mortality and other toxic effects (when
permitted by the method of determination)
(v) Gross pathology findings
f
(vi) Histopathology findings and any additional clinical chemistry
evaluations, if performed
(5) Description of any pre-test conditioning, including diet, quarantine
and treatment for disease
(6) Description of caging conditions including Number (or change
in number) of animals per cage, bedding material, ambient temperature
and humidity, photopenod, and identification of diet of test animals
(7) Manufacturer, source, punty, and lot number of test substance
(8) Relevant properties of substance tested including physical state
and pH (if applicable)
(9) Identification and composition of any vehicles (e g , diluents, sus-
pending agents, and emulsifiers) or other materials used in administering
the test substance
(10) A list of references cited m the body of the report. References
to any published literature used m developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu-
ating the results.
(f) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Chanter, D O and Hey wood, R , The LDso Test Some Consider-
ations of Precision, Toxicology Letters 10 303-307 (1982)
(2) Fmney, D J Chapter 3—Estimation of the median effective dose
and Chapter 4-Maximum likelihood estimation, Probit Analysis, 3rd ed
Cambridge, London (1971)
(3) Fmney, D J The Median Lethal Dose and Its Estimation Archives
of Toxicology 56215-218(1985)
(4) Organization for Economic Cooperation and Development OECD
Guidelines for the Testing of Chemicals Final Draft OECD Guideline 425
-------
Acute Oral Toxicity Up-and-Down Procedure to be adopted in the Tenth
Addendum to the OECD Guidelines for the Testing of Chemicals
(5) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 420 Acute Oral Tox-
ic ity—Fixed Dose Method Adopted July 17, 1992
(6) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 423 Acute Oral Tox-
icity—Acute Toxic Class Method Adopted March 22,1996
(7) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 402 Acute Dermal Tox-
icity Adopted- February 24, 1987
8
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-193
August 1998
Health Effects Test
Guidelines
OPPTS 870.1300
Acute Inhalation Toxicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed m the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC I36,etseq)
Final Guideline Release: This guideline is available from the U S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.1300 Acute inhalation toxicity
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used m developing this har-
monized OPPTS test guideline are 40 CFR 798 1150 Acute Inhalation
Toxicity, OPP 81-3 Acute Inhalation Toxicity-Rat(Pesticide Assessment
Guidelines, Subdivision F—Hazard Evaluation, Human and Domestic Ani-
mals) EPA report 540/09-82-025, 1982, and OECD guideline 403 Acute
Inhalation Toxicity
(b) Purpose. Determination of acute toxicity is usually an initial step
in the assessment and evaluation of the toxic characteristics of a substance
that may be inhaled such as a gas, volatile substance, or aerosol/particle
It provides information on health hazards likely to arise from short-term
exposure by the inhalation route Data from an acute study may serve as
a basis for classification and labeling It is traditionally a step in establish-
ing a dosage regimen in subchronic and other studies and may provide
initial information on the mode of toxic action of a substance An evalua-
tion of acute toxicity data should include the relationship, if any, between
the animals' exposure to the test substance and the incidence and seventy
of all abnormalities, including behavioral and clinical abnormalities, the
reversibility of observed abnormalities, gross lesions, body weight
changes, effects on mortality, and any other toxic effects
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline The following defi-
nitions also apply to this test guideline
Acute inhalation toxicity is the adverse effect caused by a substance
following a single uninterrupted exposure by inhalation over a short period
of time (24 h or less) to a substance capable of being inhaled
Aerodynamic equivalent diameter is defined as the diameter of a unit-
density sphere having the same terminal settling velocity as the particle
in question, whatever its size, shape, and density It is used to predict
where in the respiratory tract such particles may be deposited
Concentration is expressed as weight of the test substance per unit
volume of air, e g milligrams per liter
Inhalable diameter refers to that aerodynamic diameter of a particle
which is considered to be mhalable for the organism under study It is
used to refer to particles which are capable of being inhaled and deposited
anywhere within the respiratory tract
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(median lethal concentration) is a statistically derived estimate
of a concentration of a substance that can be expected to cause death dur-
ing exposure or within a fixed time aftei exposure in 50 percent of animals
exposed for a specified time The LCso value is expressed as weight of
test substance per unit volume of air (milligrams per liter) or parts per
million For clarity, the exposure duration should also be specified, e g
4-h LC50
Mass median aerodynamic diameter (MMAD) is the median aero-
dynamic diameter and, along with the geometric standard deviation, is used
to describe the particle size distribution of any aerosol statistically, based
on the weight and size of the particles Fifty percent of the particles by
weight will be smaller than the median diameter and 50 percent of the
particles will be larger
(d) Approaches to the determination of acute toxicity. (1) EPA
recommends the following means to reduce the number of animals used
to evaluate acute effects of chemical exposure while preserving its ability
to make reasonable judgments about safety
(i) Using data from substantially similar mixtures In order to mini-
mize the need for animal testing, the Agency encourages the review of
existing acute toxicity information on mixtures that are substantially simi-
lar to mixtures under investigation In certain cases, it may be possible
to get enough information to make preliminary hazard evaluations that may
reduce the need for further animal testing
(n) Limit test When data on structurally related chemicals are inad-
equate, a limit test may be considered In the limit test, a single group
of five males and five females is exposed to 2 mg/L for 4 h, or where
this is not possible due to physical or chemical properties of the test sub-
stance, the maximum attainable concentration, using the procedures de-
scribed under paragraph (e) of this guideline If no lethality is dem-
onstrated, no further testing for acute inhalation toxicity is needed If
compound-related mortality is produced, further study may need to be con-
sidered
(2) [Reserved]
(e) Conventional acute toxicity test—(1) Principle of the test meth-
od. Several groups of experimental animals are exposed to the test sub-
stance in graduated concentrations for a defined period, one concentration
being used per group When a vehicle other than water is used to help
generate an appropnate concentration of the substance in the atmosphere,
a vehicle control group should be used when historical data are not avail-
able or adequate to determine the acute inhalation toxicity of the vehicle
Subsequently, observations of effects and death are made Animals that
die during the test are necropsied and at the conclusion of the test surviv-
ing animals are sacnficed and necropsied This guideline is directed pn-
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manly to studies in rodent species but may be adapted for studies in non-
rodents Animals showing severe and enduring signs of distress and pain
may need to be humanely killed Dosing test substances in a way known
to cause marked pain and distress due to corrosive or imtatmg properties
need not be earned out
(2) Substance to be tested. Test, control, and reference substances
are discussed in 40 CFR part 792, subpart F (Good Laboratory Practice
Standards)
(3) Test procedure — (i) Preparation. Healthy young adult animals
are acclimatized to the laboratory conditions for at least 5 days pnor to
the test Before the test, animals are randomized and assigned to the re-
quired number of groups
(u) Animal selection — (A) Species and strain. Although several
mammalian test species may be used, the preferred species is the rat Com-
monly used laboratory strains should be employed If another mammalian
species is used, the tester should provide justification and reasoning for
its selection
(B) Age. Young adult rats between 8-12 weeks old at the beginning
of dosing, should be used The weight variation m animals or between
groups used m a test should not exceed ±20 percent of the mean weight
of each sex
(C) Number and sex. (1) At least five experimentally naive animals
are used at each concentration and they should be of one sex After com-
pletion of the study m one sex, at least one group of five animals of the
other sex is exposed to establish that animals of this sex are not markedly
more sensitive to the test substance The use of fewer animals may be
justified in individual circumstances Where adequate information is avail-
able to demonstrate that animals of the sex tested are markedly more sen-
sitive, testing in animals of the other sex is not required An acceptable
option would be to test at least one group of five animals per sex at one
or more dose levels to definitively determine the more sensitive sex pnor
to conducting the main study
(2) Females should be nulliparous and nonpregnant.
In acute toxicity tests with animals of a higher order than rodents,
the use of smaller numbers should be considered
(D) Assignment of animals. Each animal must be assigned a unique
identification number A system to assign animals to test groups and con-
trol groups randomly is required
(E) Housing. The animals may be group-caged by sex, but the num-
ber of animals per cage must not interfere with clear observation of each
animal The biological properties of the test substance or toxic effects (e g
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morbidity, excitability) may indicate a need for individual caging Animals
must be housed individually in inhalation chambers during exposure to
aerosols
(/) Before and after exposure, the temperature of the animal room
should be 22 ±3 °C and the relative humidity 30-70 percent
(2) Where lighting is artificial, the sequence should be 12 h light/
12 h dark.
(3) For feeding, conventional laboratory' diets may be used with an
unlimited supply of drinking water
(F) Equipment. (7) The animals should be tested with inhalation
equipment designed to sustain a dynamic air flow of at least 10 air changes
per hour, an adequate oxygen content of at least 19 percent, and uniform
conditions throughout the exposure chamber Maintenance of slight nega-
tive pressure inside the chamber will prevent leakage of the test substance
into the surrounding areas It is normally not necessary to measure cham-
ber oxygen concentration if airflow is adequate
(2) The selection of a dynamic inhalation chamber should be appro-
priate for the test article and test system Where a whole body chamber
is used to expose animals to an aerosol, individual housing must be used
to minimize crowding of the test animals and maximize their exposure
to the test substance 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 It is recommended, but not required, that nose-only
or head-only exposure be used for aerosol studies in order to minimize
oral exposures due to animals licking compound off their fur The animals
should be acclimated and heat stress minimized
(G) Physical measurements. Measurements or monitoring should be
made of the following*
(/) The rate of air flow should be monitored continuously, but re-
corded at least 3 times during the exposure
(2) The actual concentrations of the test substance should be measured
in the breathing zone During the exposure period, the actual concentra-
tions of the test substance shall be held as constant as practicable and
monitored continuously or intermittently depending on the method of anal-
ysis Chamber concentration may be measured using gravimetric or analyt-
ical methods as appropriate. If tnal run measurements are reasonably con-
sistent (±10 percent for liquid aerosol, gas, or vapor, ±20 percent for dry
aerosol), then two measurements should be sufficient If measurements are
not consistent, three to four measurements should be taken Whenever the
test article is a formulation, the analytical concentration must be reported
for the total formulation, and not just for the active ingredient (AI) If,
-------
for example, a formulation contains 10 percent AI and 90 percent tnerts,
a chamber analytical limit concentration of 2 mg/L would consist of 0 2
mg/L of the AI It is not necessary to analyze inert ingredients provided
the mixture at the animal's breathing zone is analogous to the formulation,
the grounds for this conclusion must be provided in the study report If
there is some difficulty in measuring chamber analytical concentration due
to precipitation, nonhomogeneous mixtures, volatile components, or other
factors, additional analyses of inert components may be necessary
(3) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concentra-
tions The MMAD particle size range should be between 1-4 (im The
particle size of hygroscopic materials should be small enough when dry
to assure that the size of the swollen particle will still be within the 1-
4 fim range Measurements of aerodynamic particle size in the animal's
breathing zone should be measured during a trial run If MMAD values
for each exposure level are within 10 percent of each other, then two meas-
urements dunng the exposures should be sufficient If pretest measure-
ments are not within 10 percent of each other, three to four measurements
should be taken
(4) Temperature and humidity should be monitored continuously and
recorded at least 3 times dunng exposure The temperature at which the
test is performed should be maintained at 22 ±2 °C The relative humidity
should be maintained between 30 and 70 percent humidity, but in certain
instances (tests of aerosols) this may not be practicable
(111) Exposure duration and levels. (A) Shortly before exposure, the
animals are weighed and then exposed to the test concentration m the des-
ignated apparatus for 4 h after equilibration of the chamber concentrations
Other durations may be needed to meet specific requirements. Food should
be withheld dunng exposure Water may also be withheld m certain cir-
cumstances
(B) Exposure levels. Three concentration levels should be used and
spaced appropriately to produce a concentration-response curve and permit
an estimation of the median lethal concentration Range-finding studies
using single animals may help to estimate the positioning of the test groups
so that no more than three concentration levels will be necessary An ac-
ceptable option for pesticide products would be to set the dose levels m
correlation with the OPP toxicity categones (bracketing) In these cases,
the determination of an LDso may not be necessary
(iv) Observation period. The observation period should be at least
14 days However, the duration of observation should not be fixed ngidly
It should be determined by the toxic reactions, rate of onset, and length
of recovery penod, and thus may be extended when considered necessary
The time at which signs of toxicity appear, their duration, and the time
-------
of death are important, especially if there is a tendency for deaths to be
delayed
(v) Observation of animals (A) A careful clinical examination
should be made at least once each day
(B) Additional observations should be made daily with appropnate
actions taken to minimize loss of animals to the study, e g. necropsy or
refngeration of those animals found dead and isolation of weak or mori-
bund animals
(C) Observations should be detailed and carefully recorded, preferably
using explicitly defined scales Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonormc effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
m the level of activity, gait and posture, reactivity to handling or sensory
stimuli, altered strength, and stereotypies or bizarre behavior (e g , self mu-
tilation, walking backwards)
(D) Individual weights of animals should be determined pnor to expo-
sure, weekly after exposure, and at death Changes in weights should be
calculated and recorded when survival exceeds 1 day
(E) The tune of death should be recorded as precisely as possible
(vi) Gross pathology. (A) At the end of the test, surviving animals
should be weighed and sacnficed
(B) A gross necropsy should be performed on all animals under test,
with particular reference to any changes in the respiratory tract. All gross
pathology changes should be recorded
(C) If necropsy cannot be performed immediately after a dead animal
is discovered, the animal should be refrigerated (not frozen) at tempera-
tures low enough to minimize autolysis Necropsies should be performed
as soon as possible, normally within a day or two
(via) Additional evaluations. In animals surviving 24 h or more, mi-
croscopic examination of organs showing evidence of gross pathology
should be considered since it may yield useful information.
(ix) 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, body weights, 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 Any
method used for calculation of the LC50 or any other parameters should
be specified and referenced Methods for parameter estimation are de-
scribed under paragraphs (0(1), (0(2), and (0(3) of this guideline
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(B) Evaluation of results. The LCso value should be considered in
conjunction with the observed toxic effects and the necropsy findings The
LCso value is a relatively coarse measurement useful only for classification
and labeling purposes and an expression of the lethal potential of the test
substance following inhalation Reference should always be made to the
experimental animal species and exposure duration in which the LCso
value was obtained An evaluation should include the relationship, if any,
between exposure of animals to the test substance and the incidence and
seventy of all abnormalities including behavioral and clinical abnormali-
ties, gross lesions, body weight changes, mortality, and other toxic effects
(C) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J and 40 CFR 160, subpart J, the following
specific information should be reported
(1) Test conditions (/) Description of exposure apparatus including
design, type, dimensions
00 Source of air, system for generating the test article
(in) Equipment for measuring temperature, humidity, particle size,
and actual concentration
(iv) Treatment of exhaust air and the method of housing the animals
in a test chamber when this is used
(2) Exposure data These should be tabulated and presented with
mean values and a measure of variability (e g standard deviation) and
should include
0) Airflow rates through the inhalation equipment
00 Temperature and humidity of the air
(in) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air)
(tv) Actual (analytical or gravimetric) concentration in test breathing
zone
(v) Particle size distribution, and calculated MMAD and geometric
standard deviation (GSD)
(vO Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable), and the efforts taken
to comply with these aspects of the guidelines
(3) Species, strain, sex, and source of test animals
(4) Method of randomization in assigning animals to test and control
groups
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(5) Rationale for selection of species, if other than that recommended
(6) Tabulation of individual and test group data by sex and exposure
level (e g number of animals exposed, number of animals showing signs
of toxicity and number of animals that died or were killed during the test)
(i) Description of toxic effects including their time of onset, duration,
reversibility, and relationship to concentration
(«) Body weights
(m) T^ime of dosing and time of death during or following exposure
(iv) Concentration-response curves for mortality and other toxic ef-
fects (when permitted by the method of determination)
(v) Gross pathology findings
(vi) Histopathology findings and any additional clinical chemistry
evaluations if performed
(7) Description of any pretest conditioning, including diet, quarantine
and treatment for disease
(8) Descnption of cagmg conditions, including- number (or change
in number) of animals per cage, bedding matenal, ambient temperature
and humidity, photopenod, and identification of diet of test animals
(9) Manufacturer (source), lot number, and punty of test substance
(10) Identification and composition of any vehicles (e.g , diluents,
suspending agents, and emulsifiers) or other materials used in administer-
ing the test substance
(11) A list of references cited in the body of the report References
to any published literature used in developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu-
ating the results
(f) References. The following references should be consulted for ad-
ditional background matenal on this test guideline
(1) Chanter, D.O. and Heywood, R The LDso test: some consider-
ations of precision Toxicology Letters 10-303-307(1982)
(2) Fmney, D G Chapter 3—Estimation of the median effective dose,
Chapter 4—Maximum likelihood estimation Probit Analysis 3rd Ed
(Cambridge, London (1971)
(3) Fmney, D J The Median Lethal Dose and Its Estimation, Archives
of Toxicology 56 215-218 (1985)
8
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(4) Organization for Economic Cooperation and Development OECD
Guidelines for the Testing of Chemicals Final Draft OECD Guideline 425
Acute Oral Toxicity Up-and-Down Procedure to be adopted in the Tenth
Addendum to the OECD Guidelines for the Testing of Chemicals
(5) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 403 Acute Inhalation Tox-
icity. Adopted May 12, 1981
(6) Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals Guideline 420 Acute Oral Tox-
icity—Fixed Dose Method Adopted July 17, 1992
(7) Organization for Economic Cooperation and Development OECD
Guidelines for Testing of Chemicals Guideline 423 Acute Oral Tox-
icity—Acute Toxic Class Method Adopted March 22, 1996
(8) U S EPA Interim Policy for Particle Size and Limit Concentra-
tion Issues in Inhalation Toxicity Studies 2/1/94 Health Effects Division,
Office of Pesticide Programs
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&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-9&-195
August 1998
Health Effects Test
Guidelines
OPPTS 870.2400
Acute Eye Irritation
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.SC I36,etseq)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
m PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines."
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OPPTS 870 2400 Acute eye irritation.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et <:eq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source matenals used in developing this har-
monized OPPTS test guideline are OPPTS 798 4500 Primary Eye Irrita-
tion, OPP 81-4 Acute Eye Irritation—Rabbit (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation, Human and Domestic Animals)
EPA report 540/09-82-025, 1982, and OECD 405 Acute Eye Irritation/
Corrosion
(b) Purpose. (1) In the assessment and evaluation of the toxic charac-
teristics of a substance, determination of the irritant and/or corrosive ef-
fects 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
(2) Data on primary eye irritation are required by 40 CFR 158 340
to support the registration of each manufacturing-use product and end-use
product (See § 158 50 to determine whether these data must be submitted
and which purity/grade of the test substance should be tested)
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
Eye corrosion is the production of irreversible tissue damage in the
eye following application of a test substance to the anterior surface of
the eye
Eye irritation is the production of reversible changes m the eye fol-
lowing the application of a test substance to the anterior surface of the
eye.
(d) Principle of the test method. The substance to be tested is ap-
plied in a single dose to one of the eyes m each of several experimental
animals, the untreated eye is used to provide control information The de-
gree 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 irreversibihty of the effects observed The period of
observation should be at least 72 h, but need not exceed 21 days Animals
showing severe and enduring signs of distress and pain may need to be
killed in a humane fashion
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(e) Initial considerations. (1) 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 Buffer capac-
ity should also be taken into account
(2) 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.
(3) Results from well validated and accepted in vitro test systems
may serve ta identify corrosives or irritants such that the test material need
not be tested in vivo
(f) Test procedures—(1) Animal selection—(i) Species and strain.
A variety of experimental animals has been used, but it is recommended
that testing should be performed using healthy adult albino rabbits. Com-
monly used laboratory strains should be used If another mammalian spe-
cies is used, the tester should provide justification/reasoning for its selec-
tion.
(n) Number of animals. A single animal should be considered if
marked effects are anticipated If the results of this test in one animal
suggest the test substance to be a severe imtant (reversible effect) or corro-
sive (irreversible effect) to the eye using the procedure described, further
tests may not need to be performed In cases other than a single animal
test, at least three animals should be used Occasionally, further testing
in additional animals may be appropriate to clarify equivocal responses
(2) 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 1 sec from a distance of 10 cm directly m 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 For volatile substances,
the dose may be estimated by weighing the container before and after
use.
(3) Examination of eyes prior to test Both eyes of each experi-
mental animal provisionally selected for testing should be examined within
24 h before testing starts by the same procedure to be used during the
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test examination Animals showing eye irritation, ocular defects, or pre-
existing corneal injury should not be used
(4) Application of the test substance (i) The test substance should
be placed in the conjunctival sac of one eye of each animal after gently
pulling the lower hd away from the eyeball The lids are then gently held
together for about 1 sec 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 poor to
instillation of the test substance The type and concentration of the local
anesthetic, should be carefully selected to ensure that no significant dif-
ferences in' reaction to the test substance will result from its use. The con-
trol eye should be similarly anesthetized
(u) The eyes of the test animals should not be washed out for
24 h following instillation of the test substance At 24 h, a washout may
be used if considered appropriate This is to show whether washing with
water palliates or exacerbates irritation
(m) For some substances shown to be irritating by this test, additional
testing using animals with eyes washed soon after instillation of the sub-
stance may be indicated Half a minute after instillation, the eyes of the
animals are washed with water for 30 sec, using a volume and velocity
of flow which will not cause injury
(5) Observation period. The duration of the observation penod is
at least 72 h, and should not be fixed rigidly, but should be sufficient
to evaluate fully the reversibility or irreversibility of the effects observed
The observation penod normally need not exceed 21 days after instillation
(6) Clinical examination and scoring, (i) The eyes should be exam-
ined at 1, 24, 48, and 72 h If there is no evidence of irritation at 72
h, the study may be ended Extended observation (eg at 7 and 21 days)
may be necessary if there is persistent corneal involvement or other ocular
irritation in order to determine the progress of the lesions and their revers-
ibility or irreversibility In addition to the observations of the cornea, ins
and conjunctivae, any other lesions which are noted should be recorded
and reported. The grades of ocular reaction using the following table
should be recorded at each examination
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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 ins clearly visible *1
Easily discernible translucent area, details of tris slightly obscured *2
Nacrous area, no details or ins visible, size of pupil barely discernible *3
Opaque cornea, ins not discernible through the opacity *4
ins
Normal 0
Markedly deepened rugae, congestion, swelling moderate circumcorneal hy-
peremia, or injection, any of these or combination of any thereof, ins still re-
acting to light (sluggish reaction is positive) '1
No reaction to lighl, hemorrhage, gross destruction (any or all of these) *2
Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and
ins)
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, cnmson color, individual vessels not easily discernible *2
Diffuse beefy red *3
Chemosis (refers to (ids and/or nictitating membranes)
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 grades
(11) Examination of reactions can be facilitated by use of a binocular
loupe, hand slit-lamp, biomicroscope, or other suitable device. After re-
cording the observations at 24 h, the eyes of any or all rabbits may be
further examined with the aid of fluorescem.
(m) The grading of ocular responses is subject to various interpreta-
tions 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.
(g) Data and reporting—(1) Data summary. Data should be sum-
marized in tabular form, showing for each individual animal the irritation
scores at observation time up until reversal (nonposiuve grades) or 21 days
when the test is concluded, a description of the degree and nature of irnta-
tion, the presence of senous lesions and any effects other than ocular
which were observed
(2) Evaluation of the results. The ocular 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 imtant 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
-------
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, the following specific information
should be reported
(i)Species, strain, sex, age, and source of test animal
(11) Rationale for selection of species (if species is other than the spe-
cies preferred
(1111) Tabulation of irritant/corrosive response data for each individual
animal at each observation time point (eg 1, 24, 48, and 72 h until revers-
ibility of lesions or termination of the test)
(iv) Descnption of any lesions observed
(v) Narrative descnption of the degree and nature of imtation or cor-
rosion observed
(vi) Descnption of the method used to score the irritation at 1, 24,
48, and 72 h (e.g hand slit-lamp, biomicroscope, fluorescein stain)
(vu) Descnption of any nonocular effects noted
(vm) Descnption of any pre-test conditioning, including diet, quar-
antine, and treatment of disease
(ix) Descnption of caging conditions including number (and any
change m number) of animals per cage, bedding matenal, ambient tem-
perature and humidity, photopenod, and identification of diet of test ani-
mal
(x) Manufacturer, source, punty, and lot number of test substance
(xi) Physical nature, and, where appropnate, concentration and pH
value for the test substance
(xu) Identification, composition, and charactenstics of any vehicles
(e g , diluents, suspending agents, emulsifiers, and anesthetics) or other
matenals used in admimstenng the test substance
(xm) A list of references cited in the body of the report, i.e, ref-
erences to any published literature used in developing the test protocol,
performing the testing, making and interpreting observations, and compil-
ing and evaluating the results
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Buehler, E V and Newmann, E A A Comparison of Eye Imtation
in Monkeys and Rabbits Toxicology and Applied Pharmacology 6 701-
710(1964)
-------
(2) Draize, J H Dermal Toxicity Appraisal of the Safety of Chemicals
in Foods, Drugs and Cosmetics The Association of Food and Drug Offi-
cials of the United States (1959) 3rd printing 1975, pp 49-52
(3) Draize, J H et al Methods for the study of irritation and toxicity
of substances applied topically to the skin and mucous membranes Jour-
nal of Pharmacology and Experimental Therapeutics 83 377-390 (1944)
(4) Loorms, T A Essentials of Toxicology Lea and Febicer, Philadel-
phia 3rd ed 1978 pp. 226-232
(5) Kay, J H and Calandra, J C , Interpretation of eye irritation tests
Journal of the Society of Cosmetic Chemists 13 281-289 (1962)
(6) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances A report prepared by
the Committee for the revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
(7) World Health Organization Part I Environmental Health Criteria
6 Principles and Methods for Evaluating the Toxicity of Chemicals World
Health Organization, Geneva (1978)
-------
vvEPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-196
August 1998
Health Effects Test
Guidelines
OPPTS 870.2500
Acute Dermal Irritation
-------
INTRODUCTION
This guideline is one of a senes of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, 'Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870 2500 Acute dermal irritation
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798 4470 Primary Dermal Im-
tation, OPP 81-5 Primary Dermal Irritation (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation, Human and Domestic Animals)
EPA repor} 540/09-82-025, 1982, and OECD 404 Acute Dermal Irritation/
Corrosion
(b) Purpose. Determination of the irritant and/or corrosive effects on
skin of mammals is useful in the assessment and evaluation of the toxic
characteristics of a substance where exposure by the dermal route is likely
Information derived from this test serves to indicate the existence of pos-
sible hazards likely to arise from exposure of the skin to the test substance
Data on primary dermal irritation are required by 40 CFR part 158 to
support the registration of each manufacturing-use product and end-use
product. See specifically § § 158 50 and 158 340 to determine whether
these data must be submitted
(c) Definitions. The definitions m section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
Dermal corrosion is the production of irreversible tissue damage in
the skin following the application of the test substance.
Dermal irritation is the production of reversible inflammatory
changes in the skin following the application of a test substance.
Pharmacological effect means any chemically induced physiological
changes m the test animal
Target organ means any organ of a test animal showing evidence
of an effect of chemical treatment
(d) Principle of the test methods. (1) 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 (except when severe irritation/corro-
sion is suspected and the stepwise procedure is used (see paragraph
(f)(l)(m) of this guideline)) The degree of irritation is read and scored
at specified intervals and is further descnbed to provide a complete evalua-
tion of the effects The duration of the study should be sufficient to permit
a full evaluation of the reversibility or irreversibihty of the effects ob-
served but need not exceed 14 days
-------
(2) When testing solids (which may be pulverized if considered nec-
essary), 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
(e) Initial considerations. (1) 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 corro-
sive properties
/
(2) It is unnecessary to test matenals which have been shown to be
highly toxic (LDso less than 200 mg/kg) by the dermal route or have been
shown not to produce irritation of the skin at the limit test dose level
of 2000 mg/kg body weight
(3) It may not be necessary to test in vivo matenals for which corro-
sive properties are predicted on the basis of results from well validated
and accepted in vitro tests If an in vitro test is performed before the m
vivo test, a description or reference to the test, including details of the
procedure, must be given together with results obtained with the test and
reference substances
(4) It may not be necessary to test matenals for which corrosive po-
tential is predicted from structure-activity relationships
(f) Test procedures—(1) Animal selection—(i) Species and strain.
The albino rabbit is recommended as the preferred species. If another
mammalian species is used, the tester should provide justification/reason-
ing for its selection
(11) Number of animals. At least three healthy adult animals (either
sex) should be used unless justification/reasoning for using fewer animals
is provided. It is recommended that a stepwise procedure be used to expose
one animal, followed by additional animals to clarify equivocal responses.
(in) Stepwise exposure of animals A single rabbit may be used if
it is suspected that the test matenal might produce severe imtation/ corro-
sion. Three test patches are applied concurrently or sequentially to the ani-
mal. The first patch is removed after 3 mm If no senous skin reaction
is observed, the second patch is removed after 1 hour If observations indi-
cate that exposure can be continued humanely, the third patch is removed
after 4 hours and the responses graded If a corrosive effect is observed
after either 3 mm or 1 hour of exposure, the test is immediately terminated
by removal of the remaining patches If a corrosive effect is observed after
an exposure of up to 4 hours, then further animal testing is not required
If no corrosive effect is observed in one animal after a 4-hour exposure,
the test is completed using two additional animals, each with one patch
-------
only, for an exposure period of 4 hours If it is expected that the test
substance will not produce severe imtancy or corrosion, the test may be
started using three animals, each receiving one patch for an exposure pe-
riod of 4 hours
(2) Control animals. Separate animals are not recommended for an
untreated control group Adjacent areas of untreated skin of each animal
may serve as a control for the test
(3) Dose level. A dose of 0 5 mL of liquid or 500 mg of solid or
semisohd is applied to the test site
/
(4) Preparation of test area. Approximately 24 h 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
(5) Application of the test substance, (i) The recommended expo-
sure duration is normally 4 hours unless corrosion is observed (see para-
graph (f)(l)(m) of this guideline) 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 appropnate solvent,
without altering the existing response or the integrity of the epidermis
(u) When vehicles are used, the influence of the vehicle on irritation
of skin by the test substance should by taken into account If a vehicle
is used, it should not alter the absorption, distribution, metabolism, reten-
tion or the chemical properties of the test substance nor should it enhance,
reduce, or alter its toxic characteristics Although water or saline is the
preferred agent to be used for moistening dry test materials, other agents
may be used providing the use is justified Acceptable alternatives include
gum arable, ethanol and water, carboxymethyl cellulose, polyethylene gly-
col, glycerol, vegetable oil, and mineral oil
(in) The test substance should be applied to a small area (approxi-
mately 6 cm2) of skin and covered with a gauze patch, which is held
in place with nommtating tape In the case of liquids or some pastes,
it may be necessary to apply the test substance to the gauze patch and
apply that to the skin The patch should be loosely held in contact with
the skin by means of a suitable semiocclusive dressing for the duration
of the exposure penod Access by the animal to the patch and resultant
ingestion/mhalation of the test substance should be prevented
(6) Observation period. The duration of the observation penod need
not be ngidly fixed It should be sufficient to fully evaluate the reversibil-
ity or irreversibihty of the effects observed It need not exceed 14 days
after application
-------
(7) Clinical examination and scoring, (i) After removal of the patch,
animals should be examined for signs of erythema and edema and the
responses scored within 30-60 rmn, and at 24, 48, and 72 hours after patch
removal
(n) Dermal irritation should be scored and recorded according to the
grades in the following Table 1 Further observations may be needed, as
necessary, to establish reversibility In addition to the observation of imta-
tion, any lesions and other toxic effects should be fully described
Table 1 —Evaluation of Skin Reaction
Value
Erythema and Eschar Formation
No erythema
Very slight erythema (barely perceptible)
Well-defined erythema
Moderate to severe erythema
Severe erythema (beet redness) to slight eschar formation (injuries in depth)
Maximum possible
Edema Formation
No edema
Very slight edema (barely perceptible)
Slight edema (edges of area well defined by definite raising)
Moderate edema (raised approximately 1 mm)
Severe edema (raised more than 1 mm and extending beyond area of exposure
Maximum possible
0
1
2
3
4
4
0
1
2
3
4
4
(g) Data and reporting—(1) Data summary. Data should be sum-
marized in tabular form, showing for each individual animal the irritation
scores for erythema and edema at 30 to 60 mm, and 24, 48, and 72 hours
after patch removal, any other dermal lesions, a description of the degree
and nature of irritation, corrosion and reversibility, and any other toxic
effects observed
(2) Evaluation of results. The dermal irritation scores should be eval-
uated in conjunction with the nature and reversibility or otherwise of the
responses observed The individual scores do not represent an absolute
standard for the imtant 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
(3) Test report In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion should be reported'
(i) Species, strain, sex, age and source of test animal
(n) Rationale for selection of species (if species is other than the spe-
cies preferred or required by OPP's toxicology data requirements for pes-
ticide registration)
-------
(in) Tabulation of erythema and edema data and any other dermal
lesions/responses for each individual animal at each observation time point
(e g 30-60 minutes and 24, 48, and 72 hours until end of test/reversibil-
ity)
(iv) Descnption of any lesions observed
(v) Narrative description of the degree and nature of irritation or cor-
rosion observed
(vi) Descnption of any systemic effects observed
i
(vu) Descnption of any pre-test conditioning, including diet, quar-
antine and treatment of disease
(vin) Descnption of caging conditions including number (and any
change in number) of animals per cage, bedding matenal, ambient tem-
perature and humidity, photopenod, and identification of diet of test ani-
mal.
(ix) Manufacturer, source, punty, and lot number of test substance.
(x) Physical nature, and, where appropnate, concentration and pH
value for the test substance
(xi) Identification and composition of any vehicles (e g , diluents, sus-
pending agents, and emulsifiers) or other matenals used in administering
the test substance
(xii) A list of references cited in the body of the report, i e , references
to any published literature used in developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu-
ating the results
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Bermes, E.W et al Chapter 2 Statistics, normal value and quality
control Fundamentals of Clinical Chemistry Tietz, N , ed WB Saunders,
Philadelphia (1976)
(2) Dharan, M Total Quality Control m the Chemical Laboratory
CV. Mosby St Louis (1977)
(3) Dixon, W J ed Biomedical Computer Programs (BMD) 2nd edi-
tion, University of California Press Los Angeles (1970)
(4) Draize, J H Dermal Toxtcity Appraisal of the Safety of Chemicals
in Foods. Drugs and Cosmetics Association of Food and Drug Officials
of the United States (1959, 3rd pnntmg 1975) pp 46-59
-------
(5) Draize, J H et al Methods for the Study of Irritation and Toxicity
of Substances Applied Topically to the Skin and Mucous Membranes,
Journal of Pharmacolog\ and Experimental Therapeutics 83 377-390
(1944)
(6) Feigenbaum, A V Total Quality Control Engineering and Man-
agement McGraw-Hill, New York (1961)
(7) Galen, R S and S R Gambino Beyond Normality (The Predictive
Value and Efficiency of Medical Diagnosis) Wiley, New York (1975)
(8) Inborn, S L , ed , Quality Assurance Practices for Health Labora-
tories. American Public Health Association Washington, D C 20036
(1978).
(9) Marzulh, F N and Maibach, H I Dermatotoxicology and Phar-
macology, Advances in Modern Toxicology Vol 4 (New York: Hemi-
sphere Publishing Corp, 1977)
(10) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances A report prepared by
the Committee for the Revision of NAS Publication 1138, Under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC, (1978)
(12) US EPA, Atlas of Dermal Lesions, Office of Pesticides and
Toxic Substances, Report 20T-2004, August 1990
(13) World Health Organization Part I Environmental Health Catena
6, Principles and Methods for Evaluating the Toxicity of Chemicals Gene-
va, World Health Organization (1978)
(14) Young, J R et al Classification as corrosive or imtant to skin
of preparations containing acidic or alkaline substances without testing on
animals. Toxicology In Vitro. 2,19 (1988)
-------
£EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-197
August 1993
Health Effects Test
Guidelines
OPPTS 870.2600
Skin Sensitization
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be subrmtted'to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines ''
-------
OPPTS 870.2600 Skin sensitization.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal [nsecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are the OPPT 40 CFR 798 4100 Dermal
Sensitization, OPP 81-6 Dermal Sensitization (Pesticide Assessment
Guidelines, Subdivision F—Hazard Evaluation, Human and Domestic Ani-
mals) EPA report 540/09-82-025, 1982, and OECD 406 Skm Sensitiza-
tion
(b) Purpose. Determination of the potential to cause or elicit skm-
sensitization reactions (allergic contact dermatitis) is an important element
in evaluating a substance's toxicity Information denved from skm-sen-
sitization tests serves to identify possible hazards to a population exposed
repeatedly to a test substance The test selected should identify substances
with significant allergenic potential and minimize false negative results
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline The following definitions also apply to this test guideline
Challenge exposure is an experimental exposure of a previously treat-
ed subject to a test substance following an induction period, to determine
if the subject will react in a hypersensitive manner
Induction exposure is an experimental exposure of a subject to a test
substance with the intention of inducing a hypersensitive state
Induction period is a period of a least 1 week following an induction
exposure during which a hypersensitive state may develop
Skin sensitization (allergic contact dermatitis) is an immunologically
mediated cutaneous reaction to a substance In the human, the responses
may be characterized by pruntis, 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
(d) Principle of the test method. Following initial exposure to a test
substance, the animals are subjected, after a period of not less than
1 week, to a challenge exposure with the test substance to establish wheth-
er a hypersensitive state has been induced Sensitization is determined by
examining the reaction to the challenge exposure and comparing this reac-
tion with that of the initial induction exposure The test animals are ini-
tially exposed to the test substance by intradermal and/or epidermal appli-
cation (induction exposure) Following a rest period of 10 to 14 days (the
induction period), during which an immune response may develop, the
-------
animals are exposed to a challenge dose The extent and degree of skin
reaction to the challenge exposure is compared with that demonstrated by
control animals that undergo sham treatment during induction and then
receive the challenge exposure
(e) Test procedures. (1) Any of the following test methods is consid-
ered to be acceptable
(i) Buehler test
(11) Guinea-pig maximization test (GPMT)
/
(111) Other.
(A) Open epicutaneous test
(B) Maurer optimization test
(C) Split adjuvant technique
(D) Freund's complete adjuvant test
(E) Draize sensitization test
(2) The GPMT of Magnusson and Kligman, which uses adjuvant, and
the nonadjuvant Buehler test are given preference over other methods. Al-
though strong preference is given to either the Buehler test or the GPMT,
it is recognized that other tests may give useful results If other tests are
used, the tester should provide justification/reasoning for their use, meth-
ods and protocols must be provided, and each test should include a positive
and a negative control group
(0 Screening tests. The mouse ear swelling test (MEST) (see para-
graphs (0(9), (0(10), (0(11), and (0(12) of this guideline) or the local
(auricular) lymph node assay (LLNA) (see paragraphs (0(13), (0(14),
(0(15), and (0(16) of this guideline) in the mouse may be used as screen-
ing tests to detect moderate to strong sensitizers. If a positive result is
seen in either assay, the test substance may be designated a potential sen-
sitizer, and it may not be necessary to conduct a further test in guinea
pigs If the LLNA or MEST does not indicate sensitization, the test sub-
stance should not be designated a nonsensitizer without confirmation in
an accepted test using guinea pigs
(g) Animal selection—(1) Species and strain. The young adult guin-
ea pig is preferred Commonly used laboratory strains should be employed
If other species are used, the tester should provide justification/reasoning
for their selection
(2) Housing and feeding. The temperature of the experimental ani-
mal room should be 20 ±3 °C with the relative humidity 30-70 percent.
Where the lighting is artificial, the sequence should be 12 h light/
-------
12 h dark Conventional laboratory diets may be used with an unlimited
supply of drinking water It is essential that guinea pigs receive an ade-
quate amount of ascorbic acid
(3) Number and sex. The number and sex will depend on the method
chosen Either sex may be used in the Buehler test and the GPMT If
females are used, they should be nulliparous and not pregnant The Buehler
test recommends using a minimum of 20 animals in the treatment and
at least 10 as controls At least 10 animals in the treatment group and
5 in the control group should be used with the GPMT, with the stipulation
that if it is' not possible to conclude that the test substance is a sensitizer
after using fewer than 20 test and 10 control guinea pigs, the testing of
additional animals to give a total of at least 20 test and 10 control animals
is strongly recommended
(4) Control animals, (i) The sensitivity and reliability of the experi-
mental technique used should be assessed every 6 months m naive animals
by the use of positive control substances known to have mild-to-moderate
skin-sensitizing properties In a properly conducted test, a response of at
least 30 percent m an adjuvant test and at least 15 percent in a nonadjuvant
test should be expected for mild-moderate sensittzers Preferred substances
are hexylcmnamic aldehyde (CAS No 101-86-0), mercaptobenzothiazole
(CAS No 149-30-4), benzocame (CAS No 94-09-7), dimtro-chloro-ben-
zene (CAS No 97^00-7), or DER 331 epoxy resin There may be cir-
cumstances where, given adequate justification, other control substances
meeting the above cntena may be used
(11) Depending upon the test selected, animals may be used as their
own controls, but usually there will be a separate group of sham-treated
animals that are exposed to the test substance only after the induction pe-
riod, whose reactions are compared to those of the animals that have re-
ceived both induction and challenge exposures Control groups which pro-
vide the best design should be used Some cases may best be served by
both naive and vehicle control groups
(5) Dose levels. The dose level will depend on the test method se-
lected In the Buehler test, the concentration of the induction dose should
be high enough to cause mild irritation, and the challenge dose should
use the highest non-imtatmg concentration In the GPMT, the concentra-
tion of the induction dose should be well tolerated systemically, and should
be high enough to cause mild-to-moderate skin irritation, the GPMT chal-
lenge dose should use the highest non-imtatmg concentration
(6) Observation of animals, (i) Skin reactions should be graded and
recorded after the challenge exposures at the time specified by the meth-
odology selected This is usually at 24 and 48 hours Additional notations
should be made as necessary to fully describe unusual responses
-------
(a) Regardless of the test method selected, initial and terminal body
weights should be taken and recorded
(7) Procedures. The procedures to be used are those described by
the test method chosen Brief summaries are given here, but the tester
should refer to the original literature for more complete guidance on con-
ducting the Buehler test (under paragraphs (i)(l), (0(2), (i)(3), and (i)(4)
of this guideline) or the GPMT (under paragraphs (i)(5), (0(6), (i)(7), and
(i)(8) of this guideline)
(i) The Buehler test uses topical administration via a closed patch
on days 0, 6-8, and 13-15 for induction, with topical challenge of the
untreated flank for 6 hours on day 27-28 Readings are made approxi-
mately 24 hours after removing the challenge patch, and again 24 hours
after that If the results are equivocal, the animals may be rechallenged
one week later, using either the original control group or a new control
group for comparison See paragraphs (i)(l), (0(2), (0(3), and (0(4) of
this guideline
(11) The GPMT uses intradermal injection with and without Freund's
complete adjuvant (FCA) for induction, followed on days 5-8 by topical
irritation/induction, followed by topical challenge for 24 hours on day 20-
22 Readings are made approximately 24 hours after removal of the chal-
lenge dose, and again after another 24 hours As with the Buehler test,
if the results are equivocal, the animals may be rechallenged 1 week later
If only 10 animals were used initially and gave equivocal results, the use
of an additional 10 experimental and 5 control animals is strongly rec-
ommended. See paragraphs (0(5), (0(6), (0(7), and (0(8) of this guideline
(iii) Blind reading of both test and control animals is recommended.
(iv) Removal of the test material should be accomplished with water
or an appropnate solvent, without altering the existing response or the in-
tegrity of the epidermis
(v) Hair is removed from the site of application by clipping, shaving,
or possibly by depilation, depending on the test selected.
(h) Data and reporting. Data should be summarized in tabular form,
showing for each individual animal the skin reaction, results of the induc-
tion exposure, and the challenge exposure at times indicated by the method
chosen. As a minimum, the erythema and edema should be graded and
any unusual finding should be recorded
(1) 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
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(2) Test report In addition to the reporting requirements as specified
under 40 CFR part 158 (for pesticides) and 40 CFR part 792, subpait
J (for toxic substances), the following specific information should be re-
ported
(i) A description of the method used and the commonly accepted
name
(u) Information on the positive control study, including the positive
control substance used, the method used, and the time conducted
(m) The number, species, strain, age, source, and sex of the test ani-
mals
(iv) Individual weights of the animals at the start of the test and at
the conclusion of the test.
(v) A brief descnption of the grading system
(vi) Each reading made on each individual animal
(vn) The chemical identification and relevant physicochemical prop-
erties of the test substance
(vm) The vehicles used for induction and challenge, and justification
for their use, if other than water or physiological saline Any material that
might reasonably be expected to react with or enhance or retard absorption
of the test substance should be reported
(ix) The total amount of test substance applied for induction and chal-
lenge, and the technique of application m each case
(x) Descnption of any pre-test conditioning, including diet, quarantine
and treatment of disease
(xi) Descnption of caging conditions including number (and any
change m number) of animals per cage, bedding material, ambient tem-
perature and humidity, photopenod, and identification of diet of test ani-
mal.
(xii) Histopathological findings, if any
(xui) Discussion of results
(xiv) Manufacturer, source, punty, and lot number of test substance
(xv) Physical nature, and, where appropnate, concentration and pH
value for the test substance
(xvi) A list of references cited in the body of the report, i e, ref-
erences to any published literature used in developing the test protocol,
-------
performing the testing, making and interpreting observations, and compil-
ing and evaluating the results
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Buehler, E V Delayed contact hypersensitivity in the guinea pig,
Archives of Dermatology 91- 171-177 (1965)
(2) Ritz, H L and Buehler, E V Planning, conduct and interpretation
of guinea pig sensitization patch tests in Current Concepts in Cutaneous
Toxicity, ed V Drill and P Lazar Academic, New York, NY pp. 25-
42 (1980)
(3) Buehler, E V Occlusive patch method for skin sensitization in
guinea pigs the Buehler method Food and Chemical Toxicology 32.97-
101 (1994)
(4) Buehler, E V Prospective testing for delayed contact
hypersensitivity in guinea pigs the Buehler method, in Methods in
Immunotoxicology, Vol 2, ed G Burleson, A Munson, and J Dean
Wiley, NY, pp. 343-356 (1995)
(5) Magnusson, B and Kligman, A M The identification of contact
allergens by animal assay The guinea pig maximization test. Journal of
Investigative Dermatology 52 268-276 (1969)
(6) Magnusson, B and Kligman, A M Allergic contact dermatitis in
the guinea pig Charles C Thomas, Springfield, IL (1970)
(7) Magnusson, B Identification of contact sensitizers by animal
assay Contact Dermatology 6 46 (1980)
(8) Magnusson, B et al Determination of skin sensitization potential
of chemicals. Predictive testing in guinea pigs Arbete och Halsa 26(E)
(1979)
(9) Gad, S C et al Development and validation of an alternative der-
mal sensitization test* the mouse ear swelling test (MEST) Toxicology
and Applied Pharmacology 84 93-114 (1986)
(10) Maisey, J and Miller, K , Assessment of the ability of mice fed
on Vitamm-A supplemented diet to respond to a variety of potential con-
tact sensitizers Contact Dermatitis 15 17-23(1986)
(11) Thorne, PS et al, The nonmvasive mouse ear swelling assay
I Refinements for detecting weak contact sensitizers Fundamental and
Applied Toxicology 17 790-806 (1991)
-------
(12) Thorne, PS et al The noninvasive mouse ear swelling assay
II Testing the contact sensitizing potency of fragrances Fundamental and
Applied Toxicology 17 807-820 (1991)
(13) Kimber, I et al The munne local lymph node assay for identi-
fication of contact allergens a preliminary evaluation of in situ measure-
ment of lymphocyte proliferation Contact Dermatitis 21 215-220 (1989)
(14) Kimber, I et al Identification of contact allergens using the mu-
nne local lymph node assay comparisons with the Buehler Occluded Patch
Test in guinea pigs Journal of Applied Toxicology 10 173-180 (1990)
(15) Kimber, I et al The munne local lymph node assay results
of an mterlaboratory tnal Toxicology Letters 55 203-213 (1991)
(16) Basketter, DA et al Interlaboratory evaluation of the local
lymph node assay with 25 chemicals and comparison with guinea pig test
data Toxicology Methods 1 30^3 (1991)
-------
A EPA
United States
Environmental Protection
Agency
Prevention Pesticides
arid Toxic Substances
(7101)
EPA712-C-98-199
August 1998
Health Effects Test
Guidelines
OPPTS 870.3100
90-Day Oral Toxicity in
Rodents
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INTRODUCTION
This guideline is one of a senes of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.3100 90-Day oral toxicity in rodents.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798 2650 Oral Toxicity, OPP
82-1 90-Day Oral—Two Species, Rodent and Nonrodent, and OECD 408
Subchronic Oral Toxicity—Rodent 90-Day.
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a chemical, the determination of subchronic oral toxicity may be
earned out after initial information on toxicity has been obtained by acute
testing The subchronic oral study has been designed to permit the deter-
mination of the no-observed-effect level (NOEL) and toxic effects associ-
ated with continuous or repeated exposure to a test substance for a period
of 90 days This study is not capable of determining those effects that
have a long latency period for development (e g , carcmogemcity and life
shortening) Extrapolation from the results of this study to humans is valid
only to a limited degree However, it can useful m providing information
on health hazards likely to arise from repeated exposure by the oral route
over a limited period of time, such as 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
(c) Definitions. The definitions in section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline
Cumulative toxicity is the adverse effects of repeated dosesoccumng
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissue.
Dose in a subchronic oral study is the amount of test substance ad-
ministered daily via the oral route (gavage, drinking water or diet) for
a period of 90 days Dose is expressed as weight of the test substance
(grams, milligrams) per unit body weight (BW) of test animal (milligram
per kilogram) or as weight of the test substance in parts per million in
food or drinking water per day
No-observed-effect-level (NOEL) is the maximum dose used in a
study which produces no adverse effects The NOEL is usually expressed
in terms of the weight of a test substance given daily per unit weight
of test animal (milligrams per kilogram per day)
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 10 percent) of the test animal's
life span.
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) 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 or if toxic effects would not be expected based
upon data of structurally related compounds, then a full study using three
dose levels might not be necessary
(e) Test procedures—(1) Animal selection—(i) 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
(n) Age/weight (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization
(B) Dosing of rodents should generally begin no later than 8-9 weeks
of age
(C) At the commencement of the study the weight variation of ani-
mals used should be within 20 percent of the mean weight for each sex.
(at) Sex. Equal numbers of animals of each sex should be used at
each dose level, and the females should be nulhparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 males and 10 females)
at each dose level
(B) If interim sacnfices are planned, the number should be increased
by the number of animals scheduled to be sacrificed before the completion
of the study
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(D) Each animal should be assigned a unique identification number
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal The biological properties of the test substance or toxic effects
(e g , morbidity, excitability) may indicate a need for individual caging.
(B) The temperature of the experimental animal rooms should be at
22 ± 3 °C
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(C) The relative humidity of the experimental animal rooms should
be 50 ± 20 percent
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark
(E) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test For feeding, conventional laboratory diets may be
used with an unlimited supply of drinking water
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
penod of quarantine An acclimation penod of at least five days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle If a vehicle or dilu-
ent is needed, the vehicle should not elicit toxic effects or substantially
alter the chemical or lexicological 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 in oil and then
solution in other vehicles
(11) If possible, one lot of the test substance tested should be used
throughout the duration of the study and the research sample should be
stored under conditions that maintain its punty and stability. Prior to the
initiation of the study, there should be a characterization of the test sub-
stance, including the punty of the test compound and, if technically fea-
sible, the names and quantities of contaminants and impurities
(in) If the test or control substance is to be incorporated into feed
or another vehicle, the penod during which the test substance is stable
in such a mixture should be determined pnor to the initiation of the study
Its homogeneity and concentration should be determined pnor to the initi-
ation of the study and penodically dunng the study Statistically random-
ized samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation, and storage procedures are being followed, and that the
appropnate concentration of the test or control substance is contained in
the mixture
(3) Control groups. A concurrent control group is required This
group should be an untreated or sham-treated control group or, if a vehicle
is used in administenng 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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(4) 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 occurrence of toxic effects for a
post-treatment period of appropriate length, normally not less than 28 days
In addition, a control group of 20 animals (10 animals of each sex) should
be added to the satellite study
(5) Dose levels and dose selection, (i) In subchronic toxicity tests,
it is desirable to determine a dose-response relationship as well as a
NOEL Therefore, at least three dose levels plus a control and, where ap-
propriate, a vehicle control (corresponding to the concentration of vehicle
at the highest dose level) should be used Doses should be spaced appro-
priately to produce test groups with a range of toxic effects The data
should be sufficient to produce a dose-response curve
(n) The highest dose level should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful eval-
uation
(m) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest dose level should produce no evidence of toxicity
(6) Administration of the test substance, (i) If the test substance
is administered by gavage, the animals are dosed with the test substance
on a 7-day per week basis for a period of at least 90 days However,
based primarily on practical considerations, dosing by gavage on a 5-day
per week basis is acceptable If the test substance is administered in the
drinking water, or mixed in the diet, then exposure should be on a 7-
day per week basis
(u) All animals should be dosed by the same method during the entire
experimental period
(in) 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 (parts per million) or
a constant dose level in terms of body weight should be used, the alter-
native used should be specified
(iv) For a substance administered by gavage, the dose should be given
at approximately the same time each day, and adjusted at intervals (weekly
or biweekly) to maintain a constant dose level in terms of body weight
(7) Observation period, (i) The animals should be observed for a
period of 90 days
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(n) Animals in the satellite group (if used) scheduled for follow-up
observations should be kept for at least 28 days further without treatment
to detect recovery from, or persistence of, toxic effects
(8) Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality Appropriate actions should
be taken to minimize loss of animals to the study (e g , necropsy or refrig-
eration of those animals found dead and isolation or sacrifice of weak
or moribund animals) General clinical observations should be made at
least once a day, preferably at the same time each day, taking into consid-
eration the, peak period of anticipated effects after dosing The clinical
condition of the animal should be recorded
(n) A careful clinical examination should be made at least once pnor
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals These observations should
be made outside the home cage, preferably in a standard arena, and at
similar times on each occasion Effort should be made to ensure that vari-
ations in the observation conditions are minimal Observations should be
detailed and carefully recorded, preferably using scoring systems, explic-
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes m skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonomic activity (eg., lacrunation,
piloerection, pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e g, excessive grooming, repetitive circling) or
bizarre behavior (e g , self-mutilation, walking backwards) should be re-
corded
(m) Once, near the end of the exposure period and in any case not
earlier than m week 11, assessment of motor activity, gnp strength, and
sensory reactivity to stimuli of different types (e g, visual, auditory, and
propnoceptive stimuli) should be conducted Further details of the proce-
dures that could be followed are described in the references listed under
paragraphs (h)(2), (h)(5), (h)(6), (h)(7), (h)(8), and (h)(H) of this guide-
line
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vi) Measurements of food consumption and water consumption, if
dnnkmg water is the exposure route, should be made weekly
-------
(vn) Individual weights of animals should be determined shortly be-
fore the test substance is administered, weekly thereafter, and at death
(vni) Moribund animals should be removed and sacrificed when no-
ticed and the time of death should be recorded as precisely as possible
(ix) At termination, all survivors in the treatment and control groups
should be sacrificed
(9) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex in
each group The hematoiogy and clinical chemistry parameters should be
examined at terminal sacnfice at the end of the study Overnight fasting
of the animals prior to blood sampling is recommended Overall, there
is a need for a flexible approach in the measures examined, depending
on the observed or expected effects from a chemical, and in the frequency
of measures, depending on the duration of potential chemical exposures
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombin time or
activated partial thromboplastin time
(ii) Clinical chemistry (A) Parameters which are considered appro-
priate to all studies are 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 clini-
cal toxicity
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total
protein and albumin More than 2 hepatic enzymes, (such as alanme
ammotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl transpeptidase) should also be meas-
ured Measurements of addtional enzymes (of hepatic or other origin) and
bile acids, may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobm, and
chohnesterases
(111) Optionally, the following unnalysis determinations could be per-
formed during the last week of the study using timed unne volume collec-
-------
tion appearance, volume, osmolahty or specific gravity, pH, protein, glu-
cose and blood/blood cells
(10) Ophthalmological examination. Ophthalmological examina-
tions using an ophthalmoscope or an equivalent device should be made
on all animals prior to the administration of the test substance and on
all high dose and control groups at termination If changes m the eyes
are detected, all animals in the other dose groups should be examined
(11) Gross necropsy, (i) All animals should be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all onfices, and the cranial, thoracic and abdominal cavities and their
contents
(11) The liver, kidneys, adrenals, testes, epididymides, ovaries, uterus,
thymus, spleen, brain, and heart should be trimmed and weighed wet, as
soon as possible after dissection
(m) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present)
(B) Nervous system—brain (including sections of medulla/pons, cere-
bellum and cerebrum), pituitary, peripheral nerve (sciatic or ubial, pref-
erably in close proximity to the muscle), spinal cord (three levels: cervical,
mid-thoracic and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose
(E) Cardiovascular/hemopoieuc system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen, thymus
(F) Urogemtal system—kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland
(G) Others—all gross lesions and masses, skin
(13) Histopathology. (i) The following histopathology should be per-
formed
(A) Full histopathology on the organs and tissues, listed under para-
graph (e)(12)(m) of this guideline, of ail rodents in the control and high
dose groups, and all rodents that died or were killed dunng the study
-------
(B) All gross lesions m all animals
(C) Target tissues in all animals
(D) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing effects in the treated
groups
(11) 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
/
(m) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours poor
to tnrnrning
(f) Data and reporting—(1) Treatment of results, (i) Data should
be summanzed m 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
(u) When applicable, all observed results, qualitative and quantitative,
should be evaluated by an appropriate and generally accepted statistical
method. Any generally accepted statistical methods may be used, the sta-
tistical methods, including significance cntena, should be selected during
the design of the study
(2) Evaluation of study results. The findings of a subchronic oral
toxicity study should be evaluated in conjunction with the findings of pre-
ceding studies and considered m terms of the toxic effects and the ne-
cropsy and histopathological findings The evaluation will include the rela-
tionship between the dose of the test substance and the presence or ab-
sence, the incidence and seventy, 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 NOEL It also can indicate the need for an additional
longer-term study and provide information on the selection of dose levels
(3) Test report In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92-
64-12367-9), the following specific information should be reported-
(i) Test substance characterization should include
8
-------
(A) Chenrucal identification
(B) Lot or batch number
(C) Physical properties
(D) Punty/impunties
(u) Identification and composition of any vehicle used
(m) Test system should contain data on
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age including body weight data and sex
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation penod
(iv) Test procedure should include the following data
(A) Method of randomization used
(B) Full description of experimental design and procedure
(C) Dose regimen including levels, methods, and volume
(v) Test results should include
(A) Group animal data Tabulation of toxic response data by species,
strain, sex and exposure level for
(/) Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(1) Date of death during the study or whether animals survived to
termination
(2) Date of observation of each abnormal sign and its subsequent
course
(J) Body weight data
(4) Feed and water (if collected) consumption data
9
-------
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water
(6) Results of ophthalmological examination
(7) Results of hematological tests performed
(8) Results of clinical chemistry tests performed
(9) Results of unnalysis, if performed
(JO) Necropsy findings, including absolute and relative (to body
weight) organ weight data
(11) Detailed description of all histopathological findings.
(12) Statistical treatment of results, where appropriate.
(g) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with GLP regula-
tions
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Boyd, EM Chapter 14 Pilot Studies, 15 Umposal Clinical Pa-
rameters, 16 Umposal Autopsy Parameters Predictive Toxicometrics Wil-
liams and Wilkins, Baltimore (1972)
(2) Crofton K M, Howard J L, Moser V C , Gill M.W , Letter L.W ,
Tilson H A., MacPhail, R C Interlaboratory Comparison of Motor Activity
Experiments Implication for Neurotoxicological Assesments
Neurotoxicol Teratol 13, 599-609 (1991)
(3) Fitzhugh, OG Subacute Toxicity, Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics The Association of Food and
Drug Officials of the United States (1959, 3rd Printing 1975) pp 260935.
(4) Food Safety Council Subchromc Toxicity Studies, Proposed Sys-
tem for Food Safety Assessment (Columbia- Food Safety Council, 1978)
pp 830996
(5) Gad S C A Neuromuscular Screen for Use m Industrial Toxi-
cology Journal of Toxicology and Environmental Health 9, 691-704
(1982)
(6) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals Environmental Health Criteria Document No. 60 (1986)
10
-------
(7) Meyer O A , Tilson H A , Byrd W C , Riley M T A Method for
the Routine Assessment of Fore- and Hind-Limb Gnp Strength of Rats
and Mice Neurobehav Toxicol 1,233-236 (1979)
(8) Moser V C , McDaniel K M , Phillips P M Rat Strain and Stock
Comparisons using a Functional Observational Battery Baseline Values
and Effects of Armtraz Toxicol Appl Pharmacol 108, 267-283 (1991)
(9) National Academy of Sciences Principles and Procedures for
] Evaluating the Toxicity of Household Substances, A report prepared by
j the Committee for the Revision of NAS Publication 1138, under the aus-
i pices of th'e Committee on Toxicology, National Research Council, Na-
, tional Academy of Sciences, Washington, DC (1977)
{ (10) Organization for Economic Cooperation and Development.
j OECD Guidelines for Testing of Chemicals Guideline 408 Subchronic
Oral Toxicity-Rodent 90-day Study, Adopted May 12,1981
1 (11) Tupper, D E , Wallace RB Utility of the Neurologic Examina-
tion in Rats Acta Neurobiol Exp 40, 999-1003 (1980)
(12) United States Environmental Protection Agency Office of Test-
ing and Evaluation Proposed Health Effects Test Standards for Toxic Sub-
stances Control Act Test Rules. 40 CFR Part 772. Standard for Develop-
ment of Test Data Subpart D FEDERAL REGISTER Vol 44, pp 27350-
27362
(13) Wemgand K, Brown G , Hall R. et al Harmonization of Animal
Clinical Pathology Testing in Toxicity and Safety Studies Fundam &
Appl Toxicol 29 198-201 (1996)
(14) World Health Organization Guidelines for Evaluation of Drugs
for Use in Man, WHO Technical Report Series No 563. (Geneva: World
Health Organization, 1975)
(15) World Health Organization Part I Environmental Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals (Ge-
neva- World Health Organization, 1978)
(16) World Health Organization Principles for Pre-Clmical Testing
of Drug Safety, WHO Technical Report Series No 341 (Geneva World
Health Organization, 1966)
11
-------
United States Prevention Pesticides EPA712-C-98-200
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3150
90-Day Oral Toxicity in
Nonrodents
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed m the Office
of Pollution Prevention and Toxics (OPPT) and appeared in title 40, chap-
ter I, subchapter R of the Code of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of the Na-
tional Technical Information Service (NTIS) and the guidelines published
by the Organization for Economic Cooperation and Development (OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC 136, etseq).
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
m PDF (portable document format) from EPA's World Wide Web site
(http //www epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.3150 90-day oral toxicity in nonrodents.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline is OPP 82-1 90-Day Oral—Two Species,
Rodent and Nonrodent (Pesticide Assessment Guidelines, Subdivision F—
Hazard Evaluation, Human and Domestic Animals) EPA report 540/09-
82-025, 1982, and OECD 409 Subchromc Oral Toxicity—Nonrodent 90-
Day
(b) Purpose. The determination of subchronic oral toxicity in the as-
sessment and evaluation of the toxic characteristics of a chemical may
be earned 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 (NOEL) 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 carcmogemcity and
life shortening) Extrapolation from the results of this study to humans
is valid only to a limited degree However, it can be useful in providing
information on health hazards likely to anse from repeated exposure by
the oral route over a limited period of tune It provides information on
target organs, the possibilities of accumulation, and can be of use in select-
ing dose levels for chronic studies and for establishing safety criteria for
human exposure
(c) Definitions. The definitions in section 3 of TSCA and m 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline The following definitions also apply to this test guideline.
Cumulative toxicity is the adverse effects of repeated doses occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissue
Dose in an oral subchronic study is the amount of test substance
administered daily via the oral route (gavage, capsules, diet or drinking
water) for 90 days Dose is expressed as weight of test substance (grams,
milligrams) per unit weight of test animal (e.g. milligrams per kilogram),
or as weight of test substance per unit weight of food or drinking water
per day
No-observed-effect-level (NOEL) is the maximum dose used in a test
which produces no observed adverse effects A NOEL is expressed in
terms of the weight of a substance given daily per unit weight of test
animal (milligrams per kilogram)
-------
Subchromc oral toKicity 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 of the test animal s Itfe span
Target organ is any organ of a test animal showing evidence of an
effect induced by the test substance
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (BW) (expected human exposure may indicate the need for
a higher dose level), using the procedures described for this study, pro-
duces no observable toxic effects and if toxicity would not be expected
based upon data of structurally related compounds, a full study using three
dose levels might not be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian nonrodent species should be used for testing The com-
monly used nonrodent 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 his or her selection.
(u) Age/weight (A) Young adult animals should be used.
(B) In the case of the dog, dosing should commence after acclimation,
preferably at 4 to 6 months and not later than 9 months of age.
(C)At the commencement of the study the weight variation of animals
used should be within 20 percent of the mean weight for each sex.
(in) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level
(B) The females should be nulliparous and nonpregnant
(iv) Numbers. (A) At least eight animals (four females and four
males) should be used at each dose level
(B) If interim sacnfices are planned, the number should be increased
by the number of animals scheduled to be sacrificed before the completion
of the study.
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(D) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues and microscopic slides
should be identified by reference to the animal's unique number
(v) Husbandry. (A) Caging and environmental conditions should be
appropriate to the nonrodent species However, it is recommended that
dogs are housed individually The number of animals per cage must not
interfere with a clear observation of each animal
-------
(B) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water The choice of diet may be influenced
by the need to ensure a suitable admixture of the test substance when
administered by this method
(C) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested for impurities that might influence the outcome
of the test For feeding, conventional laboratory diets may be used with
an unlimited supply of drinking water
(D) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
penod of quarantine An acclimation period of at least 5 days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle If a vehicle or dilu-
ent is needed, the vehicle should not elicit toxic effects or substantially
alter the chemical or lexicological properties of the test substance It is
recommended that whenever possible the usage of an aqueous solution
be considered first, followed by consideration of a solution of oil and then
solution m other vehicles
(li) If possible, one lot of the test substance tested should be used
throughout the duration of the study and the research sample should be
stored under conditions that maintain its purity and stability Pnor to the
initiation of the study, there should be characterization of the test sub-
stance, including the punty of the test compound and, if technically fea-
sible, the names and quantities of contaminants and impurities
(in) If the test or control substance is to be incorporated into feed
or another vehicle, the penod during which the test substance is stable
in such a mixture should be determined prior to the initiation of the study.
Its homogeneity and concentration should be determined prior to the initi-
ation of the study and periodically dunng the study Statistically random-
ized samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation and storage procedures are being followed, and that the
appropriate concentration of the tost or control substance is contained in
the mixture.
(3) Control groups. A concurrent control group is required 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 required
-------
(4) Satellite group. A satellite group of eight animals (four 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
In addition, a control group of 8 animals (4 animals per sex) should be
added to the satellite study
(5) Dose levels and dose selection, (i) In subchromc toxicity tests,
it is desirable to have a dose response relationship as well as a NOEL
Therefore, at least three dose levels with a control and, where appropriate,
a vehicle control (corresponding to the concentration of vehicle at the high-
est exposure level) should be used Doses should be spaced appropnately
to produce test groups with a range of toxic effects. The data should be
sufficient to produce a dose-response curve
(u) The highest dose level should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful eval-
uation
(111) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest dose level should not produce any evidence of tox-
icity.
(6) Administration of the test substance, (i) The test substance may
be administered in the diet, drinking water, by gavage or in capsules Ideal-
ly, if the test substance is administered by gavage or in capsules, the ani-
mals should be dosed with the test material on a 7-day per week basis
for a period of at least 90 days However, based primarily on practical
considerations, dosing by gavage or with capsules on a 5-day per week
basis is acceptable If the test substance is administered m the drinking
water or mixed in the diet, then exposure should be on a 7-day per week
basis.
(u) All animals should be dosed by the same method during the enure
experimental period
(iii) 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 (parts per million) or
a constant dose level in terms of the body weight of the animals should
be used, the alternative used should be specified
(iv) For a substance administered by gavage or capsules, the dose
should be given at approximately the same time each day, and adjusted
at intervals (weekly or biweekly) to maintain a constant dose level m terms
of animal body weight
-------
(7) Observation period, (i) Duration of observation should be for
at least 90 days
(it) Animals in the satellite group (if used) scheduled for follow-up
observations should be kept for at least 28 days further without treatment
to detect recovery from, or persistence of, toxic effects
(8) Observation of animals, (i) Each animal should be observed
twice daily for morbidity and mortality Appropriate actions should be
taken to minimize loss of animals to the study (e g , necropsy or refrigera-
tion of those animals found dead and isolation or sacrifice of weak or
monbund animals ) General clinical observations should be made at least
once a day, preferably at the same time each day, taking into consideration
the peak period of anticipated effects after dosing The clinical condition
of the animal should be recorded
(n) A careful clinical examination should be made prior to the initi-
ation of treatment and at least once weekly dunng treatment Detailed ob-
servations should be made on all animals These observations should be
made, where practical, outside the home cage in a standard arena and pref-
erably at similar times on each occasion Effort should be made to ensure
that variations in the observation conditions are minimal Signs of toxicity
should be carefully recorded, including tune of onset, degree and duration.
Observations should include, but not be limited to, changes in skin, fur,
eyes, mucous membranes, occurrence of secretions and excretions, and au-
tonomic activity (e g , lacnmation, piloerection, pupil size, unusual res-
piratory pattern). Changes m level of activity, gait, posture, altered
strength, and response to handling as well as the presence of clonic or
tonic movements, stereotypies (e g, excessive grooming, repetitive cir-
cling) or bizarre behavior (e g , self-mutilation) should be recorded
(m) Measurements of feed consumption and water consumption,
when drinking water is the exposure route, should be made weekly
(iv) Animals should be weighed shortly before the test substance is
administered and weekly dunng the treatment period
(v) Monbund animals should be removed and sacnficed when noticed
and the time of death should be recorded as precisely as possible
(vi) At the end of the 90-day penod all survivors in the nonsatellite
control and treatment groups should be sacnficed
(9) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex in
each group The hematology and clinical chemistry parameters should be
examined pnor to treatment, either at monthly intervals or midway through
the treatment penod, and at the end of the treatment penod on all groups
of animals, including concurrent controls Overnight fasting of the animals
-------
prior to blood sampling is recommended Overall, there is a need for a
flexible approach m the measures examined, depending on the observed
or expected effects from a chemical, and in the frequency of measures,
depending on the duration of potential chemical exposures
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombm time and
activated partial thromboplastin time
(u) Clinical chemistry (A) Clinical biochemistry test areas which are
considered appropriate to all studies are 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
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, calcium, phosphorus, chloride, glucose, total cholesterol,
urea nitrogen, creatmine, total protein, total bilirubin, and albumin Sug-
gested hepatic enzymes include alanine armnotransferase, aspartate
ammotransferase, alkaline phosphatase, sorbitol dehydrogenase, and
gamma glutamyl transpeptidase Measurements of addtional enzymes (of
hepatic or other origin) and bile acids may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
rettculocyte counts and bone marrow cytology may be indicated
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include fasting
tnglycendes, hormones, methemoglobm, and chohnesterases
(m) Unnalysis should be performed pnor to treatment, midway
through treatment and at the end of the study using timed urme collection
Unnalysis determinations include* appearance, volume, osmolahty or spe-
cific gravity, pH, protein, glucose, and blood/blood cells
(10) Ophthalmological examination. Ophthalmological examination,
using an ophthalmoscope or equivalent suitable equipment, should be
made on all animals pnor to the administration of the test substance and
at termination of the study, preferably m all animals but at least the high
dose and control groups If changes in the eyes are detected, all animals
in the other dose groups should be examined
(11) Gross necropsy, (i) All animals should be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all onfices, and the cranial, thoracic, and abdominal cavities and
their contents
-------
(u) At least the liver (with gall bladder), kidneys, adrenals, testes,
epididymides, ovaries, uterus thyroid (with parathyroid), thymus, spleen,
brain, and heart should be weighed wet as soon as possible after dissection
to avoid drying
(m) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibia,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow
(and/or a fresh aspirate), lymph nodes (preferably one lymph node cover-
ing the route of administration and another one distant from the route of
administration to cover systemic effects), spleen, thymus.
(F) Urogemtal system—kidneys, unnary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovanes, female mammary gland
(G) Other—all gross lesions and masses, skin
(12) Histopathology. The following histopathology should be per-
formed.
(i) Full histopathology on the organs and tissues, listed in paragraph
(eXll)(m) of this guideline, in at least all animals in the control- and
high-dose groups. The examination should be extended to all animals in
all dosage groups if treatment-related changes are observed in the high-
dose group
(A) All gross lesions in all animals
(B) Target organs in all animals
(C) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing effects in the treated
group
(n) If excessive early deaths or other problems occur m the high dose
group compromising the significance of the data, the next dose level
should be examined for complete histopathology
-------
(ui) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours prior
to trimming
(f) Data and reporting—(1) Treatment of results, (i) 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
(11) When applicable, all numerical results should be evaluated by
an appropriate and generally acceptable statistical method Any generally
accepted statistical methods may be used, the statistical methods should
be selected during the design of the study
(2) Evaluation of the study results, (i) The findings of a subchromc
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 seventy 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 subchromc test should provide a satisfactory
estimation of a NOEL It can also indicate the need for an additional
longer-term study and provide information on selection of dose levels.
(3) Test report In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J (Good Laboratory Practice Standards),
40 CFR part 160 and the OECD Principles of GLP (ISBN 92-64-12367-
9) the following specific information should be reported-
(i) Test substance characterization should include
(A) Chemical identification
(B) Lot or batch numbers
(C) Physical properties
(D) Punty/impunties
(n) Identification and composition of any vehicle used
(m) Test system should contain data on
8
-------
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age, including body weight data and sex
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation period
(iv) Test procedure should include the following data
(A) Method of randomization used
(B) Full description of experimental design and procedure
(C) Dose regime including levels, method, and volume
(v) Test results should include
(A) Group animal data Tabulation of toxic response data by species,
strain, sex, and exposure level for
(/) Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(7) Date of death during the study or whether animals survived to
termination
(2) Date of observation of each abnormal sign and its subsequent
course.
(5) Body weight data
(4) Feed and water (when collected) consumption data
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water
(6) Ophthalmological examination data
(7) Hematological tests employed and all results
(5) Clinical biochemistry tests employed and all results
(9) Unnalysis tests employed and all results
9
-------
(10) Necropsy findings, including absolute and relative (to body
weight) organ weight data
(//) Detailed description of all histopathological findings
(12) Statistical treatment of results, where appropriate
(h) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with GLP regula-
tions
(i) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Boyd, E M Chapter 14—Pilot Studies, 15—Umposal Clinical Pa-
rameters, 16—Umposal Autopsy Parameters, in Predictive Toxicometncs
Williams and Wilkms, Baltimore, MD (1972)
(2) Fitzhugh, O G 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 (1959, 3rd Printing 1975) pp 26-35
(3) Food Safety Council Subchromc Toxicity Studies, in Proposed
System for Food Safety Assessment. Food Safety Council, Columbia
(1978) pp 83-96
(4) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances, a report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
(5) Wemgand K, Brown G , Hall R et al Harmonization of Animal
Clinical Pathology Testing in Toxicity and Safety Studies Fundam. &
Appl ToxicoL 29 198-201 (1996)
(6) World Health Organization Part I Environmental Health Catena
6, in Principles and Methods for Evaluating the Toxicity of Chemicals
World Health Organization, Geneva (1978)
10
-------
&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-201
August 1998
Health Effects Test
Guidelines
OPPTS 870.3200
21/28-Day Dermal
Toxicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in title 40, chap-
ter I, subchapter R of the Code of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared m publications of the Na-
tional Technical Information Service (NTIS) and the guidelines published
by the Organization for Economic Cooperation and Development (OECD)
The purpose of harmonizing these guidelines into a single set of
XDPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7USC I36,etseq )
Final Guideline Release: This guideline is available from the U S
Government Pnntmg Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
-------
OPPTS 870.3200 21/28-Day dermal toxicity.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are OPP 82-2 21-Day Dermal (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation, Human and
Domestic Animals) EPA report 540/09-82-025, 1982, and OECD guide-
line 410 Repeated Dose Dermal Toxicity 21-28 Day
(b) Purpose. A 21/28 day repeated dose dermal study will provide
information on possible health hazards likely to anse from repeated dermal
exposure to a test substance for a period of 21/28 days This study is
not capable of determining those effects that have a long latency period
for development (e g , carcmogemcity and life shortening) Extrapolation
from the results of this study to humans is valid only to a limited degree.
It can, however, provide useful information on the degree of percutaneous
absorption, target organs, the possibilities of accumulation, and can be of
use in selecting dose levels for longer-term studies and for establishing
safety cntena for human exposure
(c) Definitions. The definitions m section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline
Cumulative toxicity is the adverse effect of repeated doses occurring
as a result of prolonged action or increased concentration of the adminis-
tered test substance or its metabolites in susceptible tissues
Dose in a 21/28 day repeated dose dermal study is the amount of
test substance applied daily to the skin for 21/28 days Dose is expressed
as weight of the test substance (grams, milligrams), or as weight of the
test substance per unit body weight of test animal (milligrams per kilo-
gram), or as weight of test substance per unit of surface area (milligrams
per square centimeter)
No-observed-effect level (NOEL) is the maximum dose used in a
study which produces no adverse effects The NOEL level is expressed
in terms of the weight of a test substance given daily per unit weight
of test animal (milligrams per kilogram per day)
Repeated dose dermal study is used to embrace the toxic effects asso-
ciated with repeated doses of a chemical over part of a life span of the
test animal Dosing periods lying between a single dose and 10 percent
of life span are often referred to as subacute The term subacute is semanti-
cally incorrect To distinguish such a dosing period from the classical sub-
-------
chronic period, it may be described as short-term repeated dose study
Study durations have been 14, 21, or 28 days
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) 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 guideline, produces
no observable toxic effects, or if toxic effects would not be expected based
upon data on structurally related compounds, a full study using three dose
levels might not be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian species should be used for testing The rat, rabbit, or guinea
pig may be used. Commonly used laboratory strains should be employed
If other mammalian species are used, the tester should provide justifica-
tion/reasoning for their selection When a subchromc dermal study is con-
ducted as a preliminary to a chronic study, the same species and strain
should be used in both studies
(n) Age. (A) Testing should be started with young healthy animals
as soon as possible after weaning and acclimatization
(B) Dosing should generally begin in guinea pigs between 5-6 weeks
of age, in rats between 8-9 weeks of age, and in rabbits at least 12 weeks
old
(C) At the commencement of the study, the weight variation of ani-
mals used should not exceed 20 percent of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex with healthy skin
should be used at each dose level The females should be nulliparous and
nonpregnant except for specially designed studies
(iv) Numbers. (A) For studies where the data will form the definitive
basis for a nsk assessment, e g, where a NOEL for nsk assessment is
needed, 10 animals/sex/dose will be needed For screening studies, 5 am-
mals/sex/dose will generally be sufficient
(B) If interim sacnfices are planned, the number should be increased
by the number of animals scheduled to be sacnficed before completion
of the study
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(D) Each animal must be assigned a unique identification number
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to an animal's unique number
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(v) Husbandry. (A) Animals should be housed in individual cages
(B) The temperature of the experimental animal rooms should be at
22 ± 3 °C for rodents and 20 ± 3 °C for rabbits
(C) The relative humidity of the experimental animal rooms should
be 30 to 70 percent
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark
(E) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test. For feeding, conventional laboratory diets may be
used with an unlimited supply of drinking water
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine An acclimation period of at least 5 days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle If a vehicle or dilu-
ent is needed, the vehicle should not elicit toxic effects or substantially
alter the chemical or toxicological properties of the test substance. It is
recommended that, whenever possible, the usage of an aqueous solution
be considered first, followed by consideration of a solution of oil and then
solution of other vehicles
(ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability Pnor to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound and if technically feasible, the
name and quantities of unknown contaminants and impurities.
(111) If the test substance is dissolved or suspended in a vehicle, the
penod dunng which the test substance is stable in such a mixture should
be determined pnor to the initiation of the study Its homogeneity and
concentration should be determined pnor to the initiation of the study and
periodically during the study Statistically randomized samples of the mix-
ture should be analyzed to ensure that proper mixing, formulation, and
storage procedures are being followed, and that the appropriate concentra-
tion of the test or control substance is contained in the mixture
(3) Control groups. A concurrent control group is required This
group should be an untreated or sham-treated control group or, if a vehicle
is used in the application of the test substance, a vehicle control group
-------
If the toxic properties of the vehicle are not known or not available, both
untreated/sham-treated and vehicle control groups are required
(4) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high-dose level for 21/28 days and observed
for reversibility, persistence, or delayed occurrence of toxic effects for a
post-treatment period of 14 days In addition a control group of 20 animals
(10 animals of each sex) should be added to the satellite study
(5) Dose levels and dose selection, (i) For the repeated dose study,
it is desirable to determine a dose-response relationship as well as a
NOEL Therefore, at least three dose levels plus a Control and, where ap-
propriate, a vehicle control (corresponding to the concentration of vehicle
at the highest-dose level) group should be used Doses should be spaced
appropriately to produce test groups with a range of toxic effects The
data should be sufficient to produce a dose-response curve
(u) The highest-dose level should result in toxic effects but not
produce severe skin irritation or an incidence of fatality which would pre-
vent a meaningful evaluation If application of the test substance produces
severe skin irritation, the concentration may be reduced, although this may
result in a reduction in, or absence of, other toxic effects at the high-
dose level. If the skm has been badly damaged early in the study, it may
be necessary to terminate the study and undertake a new one at lower
concentrations
(111) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest-dose level should not produce any evidence of toxic
effects
(6) Preparation of animal skin. Fur should be clipped from not less
than 10 percent of the body surface area shortly before testing for applica-
tion of the test substance In order to dose approximately 10 percent of
the body surface, the area starting at the scapulae (shoulders) to the wing
of the ileum (hipbone) and half way down the flank on each side of the
animal should be shaved. Shaving should be earned out approximately
24 hours before the test. Repeated 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 per-
meability)
(7) Preparation of test substance, (i) Liquid test substances are gen-
erally used undiluted, except as indicated m paragraph (e)(5)(u) of this
guideline.
(n) Solids should be pulverized when possible The substance should
be moistened sufficiently with water or, when necessary, a suitable vehicle
-------
to ensure good contact with the skin When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account
(111) The volume of application should be kept constant, e g less than
300 |iL for the rat, different concentrations of test solution should be pre-
pared for different dose levels
(8) Administration of test substance, (i) The duration of exposure
should be at least for 21/28 days
(11) The animals should be treated with test substance for at least 6
h/day on a 7-day per week basis However, based on practical consider-
ations, application on a 5-day per week basis is acceptable Dosing should
be conducted at approximately the same time each day
(m) The test substance should be applied uniformly over the treatment
site
(iv) The surface area covered may be less for highly toxic substances
As much of the area should be covered with as thin and uniform a film
as possible
(v) During the exposure period, the test substance should be held in
contact with the skin with a, porous gauze dressing (less than or equal
to 8 ply) The test site should be further covered with nonimtatmg tape
to retain the gauze dressing and the test substance and to ensure that the
animals cannot ingest the test substance Restramers may be used to pre-
vent the ingestion of the test substance, but complete immobilization is
not recommended The test substance may be wiped from the skin after
the six-hour exposure period to prevent mgesuon
(9) Observation period. The animals should be observed for a penod
of 21/28 days. Animals in the satellite group, (if one is used) scheduled
for follow-up observations should be kept for at least 14 days further with-
out treatment to assess reversibility
(10) Observation of animals, (i) Observations should be made at
least twice each day for morbidity and mortality Appropriate actions
should be 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)
(11) A careful clinical examination should be made at least once pnor
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals These observations should
be made outside the home cage, preferably in a standard arena, and at
similar times on each occasion Effort should be made to ensure that vari-
ations m the observation conditions are minimal Observations should be
detailed and carefully recorded, preferably using scoring systems, exphc-
-------
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes in skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonomic activity (e g, lacnmation,
piloerection, pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of dome or tonic
movements, stereotypies (e g , excessive grooming, repetitive circling) or
bizarre behavior (e g, self-mutilation, walking backwards) should be re-
corded.
(111) Once, near the end of the exposure penod and in any case not
earlier than in week 3/4, assessment of motor activity, gnp strength, and
sensory reactivity to stimuli of different types (e g, visual, auditory, and
propnoceptive stimuli) should be conducted Further details of the proce-
dures that could be followed are descnbed in the references listed under
paragraphs (h)(D, (h)(3), (h)(4), (h)(5), (h)(6), and (h)(9) of this guideline
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits.
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vi) Individual weights of animals should be determined shortly be-
fore the test substance is administered, weekly thereafter, and at death
(vn) Food consumption should also be determined weekly if abnormal
body weight changes are observed
(vm) Monbund animals should be removed and sacnficed when no-
ticed The tune of death should be recorded as precisely as possible.
(ix) At termination, all survivors in the treatment groups should be
sacnficed.
(11) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex m
each group. The hematology and clinical chemistry parameters should be
examined at terminal sacrifice at the end of the study. Overnight fasting
of the animals prior to blood sampling is recommended Overall, there
is a need for a flexible approach in the measures examined, depending
on the observed or expected effects from a chemical, and in the frequency
of measures, depending on the duration of potential chemical exposures
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
-------
count, and a measure of clotting potential, such as prothrombin time or
activated partial thromboplastin time
(u) Clinical chemistry (A) Parameters which are considered appro-
priate to all studies are 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 clini-
cal toxicity
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, glucose, total cholesterol, urea nitrogen, creatmine, total
protein and albumin More than 2 hepatic enzymes, (such as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl transpeptidase) should also be meas-
ured Measurements of addtional enzymes (of hepatic or other ongm) and
bile acids, may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobm, and
chohnesterases.
(in) Optionally, the following unnalysis determinations could be per-
formed dunng the last week of the study using timed unne volume collec-
tion appearance, volume, osmolality or specific gravity, pH, protein, glu-
cose, and blood/blood cells
(12) Ophthalmological examination. Ophthalmological examina-
tions using an ophthalmoscope or an equivalent device should be made
on all animals pnor to the administration of the test substance and on
all high-dose and control groups at termination If changes in the eyes
are detected, all animals m the other dose groups should be examined
(13) Gross necropsy, (i) 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
(u) The liver, brain, kidneys, spleen, adrenals, testes, epididymides,
uterus, ovaries, thymus, and heart should be trimmed and weighed wet,
as soon as possible after dissection
(m) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination
(A) Digestive system
-------
(/) Salivary glands
(2) Esophagus
(3) Stomach
(4) Duodenum
(5) Jejunum
(6) Ileum
(7) Cecum
(8) Colon
(9) Rectum.
(10) Liver
(11) Pancreas
(12) Gall bladder (when present)
(B) Nervous system
(/) Brain (multiple sections, including cerebrum, cerebellum, and me-
dulla/pons)
(2) Pituitary
(3) Peripheral nerve(sciatic or tibial, preferably m close proximity to
the muscle)
(4) Spinal cord (three levels, cervical, mid-thoracic, and lumbar)
(5) Eyes (retina, optic nerve)
(C) Glandular system
(1) Adrenals
(2) Parathyroids
(3) Thyroids
(D) Respiratory system
(/) Trachea
(2) Lung.
(3) Pharynx
(4) Larynx
8
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(5) Nose
(E) Cardiovascular/Hematopoieitic system
(/) Aorta
(2) Heart
(5) Bone marrow (and/or fresh aspirate)
(4) Lymph nodes (preferably one lymph node covering the route of
administration and another one distant from the route of administration
to cover systemic effects)
(5) Spleen
(6) Thymus
(F) Urogemtal system
CO Kidneys
(2) Unnary bladder
(3) Prostate.
(4) Testes.
(5) Epididyrmdes
(6) Seminal vesicles
(7) Uterus
(5) Ovanes
(9) Female mammary gland
(G) Others
(/) All gross lesions and masses
(2) Skin (both treated and adjacent untreated areas)
(14) Histopathology. (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tissues, listed under para-
graph (e)(13)(ui) of this guideline, of all animals in the control and high-
dose groups
(B) All gross lesions in all animals
(C) Target organs in all animals
9
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(D) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing toxic effects in the
treated groups
(11) 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
(111) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designed for microscopic examination should
be fixed in 10 percent buffered formalin or a recognized suitable fixative
as soon as necropsy is performed and no less than 48 hours prior to dim-
ming
(f) Data and reporting—(1) Treatment of results, (i) Data should
be summarized in tabular form, showing for each test group—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
(ii) When applicable, all observed results, quantitative and qualitative,
should be evaluated by an appropriate statistical method Any generally
accepted statistical method should be used, the statistical methods includ-
ing significance criteria should be selected dunng the design of the study
(2) Evaluation of study results. The findings of a 21/28 day dermal
toxicity study should be evaluated m conjunction with the findings of pre-
ceding studies and considered m terms of toxic effects, and the necropsy
and histopathological findings The evaluation should include the relation-
ship between the dose of the test substance, the incidence and seventy
of abnormalities including behavioral and clinical abnormalities, gross le-
sions, identified target organs, body weight changes, effect on mortality
and any other general or specific toxic effects A properly conducted 21/
28 day dermal toxicity study should provide information on the effects
of repeated application of a substance and a satisfactory estimation of a
NOEL. It also can indicate the need for an additional longer-term study
and provide information on the selection of dose levels
(3) Test report In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92-
64-12367-9), the following specific information should be reported
(i) Test substance characterization should include
(A) Chemical identification
(B) Lot or batch numbers
10
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(C) Physical properties
(D) Punty/impunties
(E) Identification and composition of any vehicle if used
(n) Test system should contain data on
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age including body weight data and sex
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation period
(111) Test procedure should include the following data1
(A) Method of randomization used
(B) Full description of experimental design and procedure
(C) Dose regime including levels, method, and volume
(iv) Test results should include
(A) Group animal data Tabulation of toxic response data by species,
strain, sex, and exposure level for
(1) Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(7) Date of death during the study or whether animals survived to
termination
(2) Date of observation of each abnormal sign and its subsequent
course
(3) Body weight data
(4) Feed consumption data, when collected
(5) Results of ophthalmological examination
(6) Results of the hematology tests performed
11
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(7) Results of the clinical chemistry tests performed
(5) Results of urmalysis, when performed
(9) Results of the observations made
(10) Necropsy findings, including absolute and relative organ weight
data
(11) Detailed description of all histopathologicai findings.
(12) Statistical treatment of results, where appropriate
(g) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with GLP regula-
tions
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline
(1) Crofton K M , Howard J L , Moser V C , Gill M W , Leiter L W ,
Tilson H A , MacPhail, R C Interlaboratory Comparison of Motor Activity
Experiments Implication for Neurotoxicological Assessments
Neurotoxicol Teratol 13, 599-609 (1991)
(2) Draize, JH Dermal toxicity, Appraisal of Chemicals in Food,
Drugs and Cosmetics The Association of Food and Drug Officials of the
United States, 3rd pnntmg 1975 pp 46-59 (1959)
(3) Gad S C. A Neuromuscular Screen for Use in Industrial Toxi-
cology Journal of Toxicology and Environmental Health 9, 691-704
(1982)
(4) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals. Environmental Health Catena Document No 60. (1986)
(5) Meyer O A , Tilson H A , Byrd W C , Riley M T A Method for
the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats
and Mice Neurobehav Toxicology 1,233-236 (1979)
(6) Moser V C , McDamel K M, Phillips P M Rat Strain and Stock
Comparisons using a Functional Observational Battery Baseline Values
and Effects of Amitraz Toxicology and Applied Pharmacology 108, 267-
283(1991)
(7) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
12
-------
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
(8) Organization for Economic Cooperation and Development Guide-
lines for Testing of Chemicals, Section 4—Health Effects, Part 410, Re-
peated Dose Toxicity Study, Pans (1981)
(9) Tupper, D E , Wallace R B Utility of the Neurologic Examination
mRats Acta Neurobwloical Exp 40,999-1003(1980)
(10) Wemgand K , Brown G , Hall R el al Harmonization of Animal
Clinical Pathology Testing in Toxicity and Safety Studies Fundamental
and Applied Toxicology 29198-201 (1996)
(11) World Health Organization Part I Environmental Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals
(World Health Organization, Geneva) (1978)
13
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A EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-202
August 1998
Health Effects Test
Guidelines
OPPTS 870.3250
90-Day Dermal Toxicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC 136, etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(httpV/www epa gov/epahome/research htm) under the heading ' 'Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines ''
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OPPTS 870 3250 90-Day dermal toxicity
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798 2250 Dermal Toxicity,
OPP 82-3 90-Day Dermal (Pesticide Assessment Guidelines, Subdivision
F—Hazard Evaluation, Human and Domestic Animals) EPA report 540/
09-82-025, 1982, and OECD 411 Subchromc Dermal Toxicity 90-Day
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a chemical, the determination of subchronic dermal toxicity may
be earned 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 (NOEL) and toxic effects
associated with continuous or repeated exposure to a test substance for
a period of 90 days This study is not capable of determining those effects
that have a long latency period for development (e g , carcinogemcity and
life shortening) Extrapolation from the results of this study to humans
is valid only to a limited degree It can, however, provide useful informa-
tion on the degree of percutaneous absorption, target organs, the possibili-
ties of accumulation, and can be of use in selecting dose levels for chronic
studies and for establishing safety catena for human exposure
(c) Definitions. The definitions in section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part 792~Good Lab-
oratory Practice Standards apply to this test guideline The following defi-
nitions also apply to this test guideline
Cumulative toxicity is the adverse effect of repeated doses occumng
as a result of prolonged action or increased concentration of the adminis-
tered test substance or its metabolites in susceptible tissues
Dose in a subchronic dermal study is the amount of test substance
applied daily to the skin for 90 days Dose is expressed as weight of the
test substance (grams, milligrams), per unit body weight of test animal
(milligrams per kilogram), or as weight of the test substance per unit of
surface area (milligrams per square centimeter) per day
No-observed-effect level (NOEL) is the maximum dose used in a study
which produces no adverse effects The NOEL is expressed m terms of
the weight of a test substance given daily per unit weight of test animal
(milligrams per kilogram per day)
Subchronic dermal toxicity is the adverse effects occumng as a result
of the repeated daily exposure of expenmental animals to a chemical by
the dermal route for a part of the test animal's life span
-------
Target otgan is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) 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 guideline, produces
no observable toxic effects or if toxic effects would not be expected based
upon data on structurally related compounds, a full study using three dose
levels might not be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian species should be used for testing The rat, rabbit, or guinea
pig may be used Commonly used laboratory strains should be employed
If other mammalian species are used, the tester should provide justifica-
tion/reasoning for their selection When a subchronic dermal study is con-
ducted as a preliminary to a chronic dermal study, the same species and
strain should be used in both studies
(11) Age/weight (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization
(B) Dosing should generally begin in guinea pigs between 5-6 weeks
of age, in rats between 8-9 weeks of age, and in rabbits at least 12 weeks
old
(C) At the commencement of the study, the weight variation of ani-
mals used should be within 20 percent of the mean weight for each sex.
(111) Sex. Equal numbers of animals of each sex with healthy skin
should be used at each dose level The females should be nulliparous and
nonpregnant except for specially designed studies
(iv) Numbers. (A) At least 20 animals (10 animals per sex) 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
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(D) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the animal's unique number
(v) Husbandry. (A) Animals should be housed m individual cages.
(B) The temperature of the experimental animal rooms should be at
22 ± 3 °C
-------
(C) The relative humidity of the experimental animal rooms should
be 50 ± 20 percent
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark
(E) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test For feeding, conventional laboratory diets may be
used with an unlimited supply of dnnkmg water
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine An acclimation period of at least five days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle If a vehicle or dilu-
ent is needed, the vehicle should not elicit toxic effects or substantially
alter the chemical or toxicological properties of the test substance. It is
recommended that, whenever possible, the usage of an aqueous solution
be considered first, followed by consideration of a solution of oil and then
solution of other vehicles
(11) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability Prior to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound and if technically feasible, the
name and quantities of unknown contaminants and impurities
(111) If the test substance is dissolved or suspended in a vehicle, the
period during which the test substance is stable in such a mixture should
be determined prior to the initiation of the study Its homogeneity and
concentration should be determined prior to the initiation of the study and
periodically during the study Statistically randomized samples of the mix-
ture should be analyzed to ensure that proper mixing, formulation, and
storage procedures are being followed, and that the appropriate concentra-
tion of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required This
group should be an untreated or sham-treated control group or, if a vehicle
is used in the application of the test substance, a vehicle control group.
If the toxic properties of the vehicle are not known or not available, both
untreated/sham-treated and vehicle control groups are required
-------
(4) 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 occurrence of toxic effects for a
post-treatment period of appropriate length, normally not less than 28 days
In addition a control group of 20 animals (10 animals per sex) should
be added to the satellite study
(5) Dose levels and dose selection, (i) In subchromc toxicity tests,
it is desirable to determine a dose-response relationship as well as a
NOEL Therefore, at least three dose levels plus a control and, where ap-
propriate, a vehicle control (corresponding to the concentration of vehicle
at the highest dose level) group should be used Doses should be spaced
appropriately to produce test groups with a range of toxic effects. The
data should be sufficient to produce a dose-response curve.
(ti) The highest dose level should elicit signs of toxicity but not
produce severe skin irritation or an incidence of fatality which would pre-
vent a meaningful evaluation If application of the test substance produces
severe skin irritation, the concentration may be reduced, although this may
result in a reduction m, or absence of, other toxic effects at the high dose
level. If the skin has been badly damaged early in the study, it may be
necessary to terminate the study and undertake a new one at lower con-
centrations
(m) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest dose level should not produce any evidence of toxic
effects.
(6) Preparation of animal skin. Shortly before testing, fur should
be clipped from not less than 10 percent of the body surface area for appli-
cation of the test substance In order to dose approximately 10 percent
of the body surface, the area starting at the scapulae (shoulders) to the
wing of the ileum (hipbone) and half way down the flank on each side
of the animal should be shaved Shaving should be earned out approxi-
mately 24 hours before dosing Repeated clipping or shavmg is usually
needed at approximately weekly intervals When clipping or shavmg the
fur, care should be taken to avoid abrading the skin which could alter
its permeability
(7) Preparation of test substance, (i) Liquid test substances are gen-
erally used undiluted, except as indicated in paragraph (e)(5)(u) of this
guideline
(u) Solids should be pulverized when possible The substance should
be moistened sufficiently with water or, when necessary, a suitable vehicle
to ensure good contact with the skin When a vehicle is used, the influence
-------
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account
(in) The volume of application should be kept constant, e g , less than
300 jiL for the rat, different concentrations of test solution should be pre-
pared for different dose levels
(8) Administration of test substance, (i) The duration of exposure
should be at least for 90 days
(u) Ideally, the animals should be treated with test substance for at
least 6 h/day on a 7-day per week basis However, based on practical
considerations, application on a 5-day per week basis is acceptable Dosing
should be conducted at approximately the same time each day
(111) The test substance should be applied uniformly over the treatment
site.
(iv) The surface area covered may be less for highly toxic substances
As much of the area should be covered with as thin and uniform a film
as possible
(v) During the exposure period, the test substance should be held in
contact with the skin with a porous gauze dressing (less than or equal
to 8 ply) The test site should be further covered with nonimtatmg tape
to retain the gauze dressing and the test substance and to ensure that the
animals cannot ingest the test substance Restramers may be used to pre-
vent the ingestion of the test substance, but complete immobilization is
not recommended The test substance may be wiped from the skin after
the six-hour exposure penod to prevent ingestion
(9) Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality Appropriate actions should
be taken to minimize loss of animals to the study (e g , necropsy or refrig-
eration of those animals found dead and isolation or sacrifice of weak
or moribund animals) General clinical observations should be made at
least once a day, preferably at the same time each day, taking into consid-
eration the peak penod of anticipated effects after dosing The clinical
condition of the animal should be recorded
(n) A careful clinical examination should be made at least once pnor
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals These observations should
be made outside the home cage, preferably in a standard arena, and at
similar times on each occasion Effort should be made to ensure that vari-
ations in the observation conditions are minimal Observations should be
detailed and carefully recorded, preferably using scoring systems, explic-
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes in skin, fur, eyes, mucous membranes, occurrence
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of secretions and excretions and autonormc activity (e g , lacnmation,
piloerection. pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e g , excessive grooming, repetitive circling) or
bizarre behavior (eg., self-mutilation, walking backwards) should be re-
corded
(m) Once, near the end of the exposure period and in any case not
earlier than in week 11, assessment of motor activity, gnp strength, and
sensory reactivity to stimuli of different types (e g, visual, auditory, and
propnoceptive stimuli) should be conducted Further details of the proce-
dures that could be followed are descnbed in the references listed under
paragraphs (h)(l), (h)(3), (h)(4), (h)(5), (h)(6), and (h)(9) of this guideline
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vi) Individual weights of animals should be determined shortly be-
fore the test substance is administered, weekly thereafter, and at death.
(vu) Food consumption should also be determined weekly if abnormal
body weight changes are observed
(vm) Moribund animals should be removed and sacrificed when no-
ticed and the time of death should be recorded as precisely as possible
(ix) At termination, all survivors in the control and treatment groups
should be sacrificed
(10) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex in
each group The hematology and clinical chemistry parameters should be
examined at terminal sacrifice at the end of the study Overnight fasting
of the animals prior to blood sampling is recommended. Overall, there
is a need for a flexible approach m the measures examined, depending
on the observed or expected effects from a chemical, and in the frequency
of measures, depending on the duration of potential chemical exposures
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombin time or
activated partial thromboplastin time
-------
(u) Clinical chemistry (A) Parameters which are considered appro-
priate to all studies are 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 clini-
cal toxic ity
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, glucose, total cholesterol, urea nitrogen, creatmme, total
protein and albumin More than 2 hepatic enzymes, (such as alamne
aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl transpeptidase) should also be meas-
ured Measurements of additional enzymes (of hepatic or other origin) and
bile acids, may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobm, and
chohnesterases
(ui) Optionally, the following unnalysis determinations could be per-
formed during the last week of the study using timed urine volume collec-
tion appearance, volume, osmolality or specific gravity, pH, protein, glu-
cose and blood/blood cells
(11) Ophthalmological examination. Using an ophthalmoscope or
an equivalent device, Ophthalmological examinations should be made on
all animals pnor to the administration of the test substance and on all
high dose and control groups at termination If changes in the eyes are
detected, all animals in the other dose groups should be examined.
(12) Gross necropsy, (i) All animals should be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all onfices, and the cranial, thoracic and abdominal cavities and their
contents
(11) The liver, brain, kidneys, spleen, adrenals, testes, epididymides,
uterus, ovaries, thymus and heart should be trimmed and weighed wet,
as soon as possible after dissection
(111) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present)
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(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons) pituitary, peripheral nerve (sciatic or tibial,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen, thymus
(F) Urogemtal system—kidneys, urinary bladder, prostate, testes,
epididyrrudes, seminal vesicle(s), uterus, ovaries, female mammary gland
(G) Other—all gross lesions and masses, skin (both treated and adja-
cent untreated areas)
(13) Histopathology. (i) The following histopathology should be per-
formed
(A) Full histopathology on the organs and tissues, listed under para-
graph (e)(12)(iii) of this guideline, of all animals m 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
(D) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing toxic effects in the
treated groups
(a) If excessive early deaths or other problems occur m the high dose
group compromising the significance of the data, the next dose level
should be examined for complete histopathology
(ui) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed m 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours prior
to trimming
(f) Data and reporting—(1) Treatment of results, (i) Data should
be summarized m tabular form, showing for each test group, number of
animals at the start of the test, the number of animals showing lesions,
8
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the types of lesions and the percentage of animals displaying each type
of lesion
(a) When applicable, all observed results, qualitative and quantitative,
should be evaluated by an appropriate and generally acceptable statistical
method Any, generally accepted statistical method should be used, the
statistical methods including significance cntena should be selected dunng
the design of the study.
(2) Evaluation of study results. The findings of a subchromc dermal
toxicity study should be evaluated in conjunction with the findings of pre-
ceding studies and considered in terms of toxic effects and the necropsy
and histopathological findings The evaluation should include the relation-
ship between the dose of the test substance, the incidence and seventy
of abnormalities including behavioral and clinical abnormalities, gross le-
sions, identified target organs, body weight changes, effect on mortality,
and any other general or specific toxic effects A properly conducted 90-
day subchromc dermal study should provide information on the effects
of repeated application of a substance and a satisfactory estimation of a
NOEL It also can indicate the need for an additional longer-term study
and provide information on the selection of dose levels.
(3) Test report In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92-64-
12367-9), the following specific information should be reported-
(i) Test substance characterization should include
(A) Chemical identification
(B) Lot or batch numbers.
(C) Physical properties
(D) Punty/unpunties
(11) Identification and composition of any vehicle if used
(111) Test system should contain data on
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age including body weight data and sex
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation period
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(iv) Test procedure should include the following data
(A) Method of randomization used
(B) Full description of experimental design and procedure
(C) Dose regime including levels, method, and volume
(v) Test results should include
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for
(1) Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(/) Date of death during the study or whether animals survived to
termination
(2) Date of observation of each abnormal sign and its subsequent
course
(3) Body weight data
(4) Feed consumption data, when collected
(5) Results of ophthalmological examination
(6) Results of hematological tests performed
(7) Results of clinical chemistry tests performed
(5) Results of unnalysis, when performed
(9) Results of observations made
(10) Necropsy findings, including absolute and relative (to body
weight) organ weight data
(11) Detailed descnption of all histopathological findings
(12) Statistical treatment of results, where appropriate.
(g) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with GLP regula-
tions
10
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(h) References. The following references should be consulted for
background information on this test guideline
(I) Crofton K M , Howard J L , Moser V C , Gill M W , Letter L W ,
Tilson H A , MacPhail, R C Interlaboratory Comparison of Motor Activity
Experiments Implication for Neurotoxicological Assessments
NeurotoKicoi Teratol 13,599-609 (1991)
(2) Draize, J H Dermal toxicity Appraisal of Chemicals in Food,
Drugs and Cosmetics The Association of Food and Drug Officials of the
United States (1959) 3rd pnntmg 1975 pp 46-59
(3) Gad S C A Neuromuscular Screen for Use in Industrial Toxi-
cology Journal of Toxicology and Environmental Health 9, 691-704
(1982)
(4) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals Environmental Health Criteria Document No 60 (1986)
(5) Meyer O A , Tilson H A , Byrd W C , Riley M.T A Method for
the Routine Assessment of Fore- and Hind-Limb Gnp Strength of Rats
and Mice Neurobehav Toxicol 1,233-236 (1979)
(6) Moser V C , McDaniel K M, Phillips P M Rat Strain and Stock
Comparisons using a Functional Observational Battery Baseline Values
and Effects of Anutraz Toxicol Appl Pharmacol 108, 267-283 (1991)
(7) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
(8) Organization for Economic Cooperation and Development Guide-
lines for Testing of Chemicals, Section 4-Health Effects, Part 411 Sub-
chronic Toxicity Studies, Pans, 1981
(9) Tupper, D E , Wallace R B Utility of the Neurologic Examination
in Rats. Acta Neurobiol Exp 40, 999-1003 (1980)
(10) United States Environmental Protection Agency Office of Test-
ing and Evaluation Proposed Health Effects Test Standards for Toxic Sub-
stances Control Act Test Rules 40 CFR Part 772 Standard for Develop-
ment of Test Data Subpart D FEDERAL REGISTER Vol. 44, No 91. Pp
27350-27362.
(11) Wemgand K, Brown G, Hall R et al (1996) Harmonization of
Animal Clinical Pathology Testing in Toxicity and Safety Studies
Fundam & Appl Toxicol 29 198-201
11
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(12) World Health Organization Part I Environmental Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals (Ge-
neva World Health Organization, 1978)
(13) World Health Organization Guidelines for Evaluation of Drugs
for Use in Man, WHO Technical Report Senes No 563 (Geneva World
Health Organization, 1975)
(14) World Health Organization Principles for Pre-Chmcal Testing
of Drug Safety, WHO Technical Report Senes No 341 (Geneva. WHO
1966)
12
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&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-204
August 1998
Health Effects Test
Guidelines
OPPTS 870.3465
90-Day Inhalation
Toxicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared m title 40, chap-
ter I, subchapter R of the Code of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of the Na-
tional Technical Information Service (NTIS) and the guidelines published
by the Organization for Economic Cooperation and Development (OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7US.C \36,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.3465 90-Day inhalation toxicity.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, el seq) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source matenals used in developing this har-
monized OPPTS test guideline are 40 CFR 798 2450 Inhalation Toxicity,
OPP 82-^ 90-Day Inhalation—Rat (Pesticide Assessment Guidelines,
Subdivision F—Hazard Evaluation, Human and Domestic Animals) EPA
report 540/09-82-025, 1982, and OECD 413 Subchromc Inhalation Tox-
icity 90-Day
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a gas, volatile substance, or aerosol/paniculate, determination of
subchronic inhalation toxicity may be earned 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-Ievel (NOEL) and toxic effects associated with continuous or re-
peated exposure to a test substance for a period of 90 days This study
is not capable of determining those effects that have a long latency period
for development (e g , carcmogemcity and life shortening) Extrapolation
from the results of this study to humans is valid only to a limited degree.
It can, however, provide useful information on health hazards likely to
arise from repeated exposures by the inhalation route over a limited penod
of time It will provide information on target organs and the possibilities
of accumulation, and can be of use in selecting concentration levels for
chronic studies and establishing safety criteria for human exposure Haz-
ards of inhaled substances are influenced by the inherent toxicity and by
physical factors such as volatility and particle size.
(c) Definitions. The definitions in section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline
Aerodynamic equivalent diameter is defined as the diameter of a unit
density sphere having the same terminal settling velocity as the particle
in question, whatever its size, shape, and density It is used to predict
where in the respiratory tract such particles may be deposited
Concentration in a subchronic inhalation study is the amount of test
substance administered via inhalation for a penod of 90 days Concentra-
tion is expressed as weight of the test substance per unit volume of air
(milligrams per liter or parts per million)
Cumulative toxicity is the adverse effects of repeated concentration
occurring as a result of prolonged action on, or increased concentration
of the administered test substance or its metabolites in susceptible tissues
-------
Inhalable diameter refers to that aerodynamic diameter of a particle
which is considered to be mhalable for the organism It is used to refer
to particles which are capable of being inhaled and may be deposited any-
where within the respiratory tract
No-observed-effect-level (NOEL) is the maximum concentration used
in a study which produces no adverse effects
Mass median aerodynamic diameter (MMAD) is the median aero-
dynamic diameter and along with the geometric standard deviation (GSD)
is used to describe the particle size distribution of any aerosol statistically
based on the weight and size of the particles Fifty percent of the particles
by weight will be smaller than the median diameter and 50 percent of
the particles will be larger
Subchromc inhalation toxicity is the adverse effects occurring 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
(d) Limit test If exposure at a concentration of 1 mg/L (expected
human exposure may indicate the need for a higher concentration), or
where this is not possible due to physical or chemical properties of the
test substance, the maximum attainable concentration produces no observ-
able toxic effects, then a full study using three concentrations might not
be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian species shall be used for testing 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 shall provide justification/reasoning for its selection
(n) Age/weight (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8-9 weeks
of age
(C) At the commencement of the study the weight variation of ani-
mals used shall not exceed ± 20 percent of the mean weight for each
sex
(in) Sex. (A) Equal numbers of animals of each sex shall be used
at each concentration
(B) Females shall be nulhparous and nonpregnant
(iv) Numbers. (A) At least 20 animals (10 females and 10 males)
should be used for each test group
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(B) If interim sacrifices are planned, the number of animals shall be
increased by the number of animals scheduled to be sacrificed before the
completion of the study
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(D) Each animal shall be assigned a unique identification number
Dead animals, their preserved organs and tissues, and microscopic slides
shall be identified by reference to the animal1 s unique number
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal. The biological properties of the test substance or toxic effects
(e.g , morbidity, excitability) may indicate a need for individual caging
Animals must be housed individually in inhalation chambers during expo-
sure to aerosols
(B) The temperature of the experimental animal rooms should be at
22 ± 3 °C
(C) The relative humidity of the experimental animal rooms should
be 30*70 percent
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark
(E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test. For feeding, conventional laboratory diets may be
used with an unlimited supply of drinking water
(F) The study should not be initiated until animals have been allowed
a penod of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
penod of quarantine An acclimation penod of at least 5 days is rec-
ommended
(2) Control and test substances, (i) Whenever it is necessary to for-
mulate the test substance with a vehicle for aerosol generation, the vehicle
ideally should not elicit toxic effects or substantially alter the chemical
or lexicological properties of the test substance
(11) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability Pnor to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the punty of the test substance and, if technically feasible, the
name and quantities of unknown contaminants and impurities
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(3) Control groups. A concurrent control group is required This
group shall be an untreated or sham-treated control group Except for treat-
ment with the test substance, animals in the control group shall be handled
in a manner identical to the test group animals Where a vehicle other
than water is used to generate a substance, 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 required
(4) 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 occurrence of toxic ef-
fects for a post-treatment period of appropriate length, normally not less
than 28 days In addition, a control group of 20 animals (10 animals of
each sex) should be added to the satellite study
(5) Concentration levels and concentration selection, (i) In sub-
chronic toxicity tests, it is desirable to have a concentration-response rela-
tionship as well as a NOEL Therefore, at least three concentration levels
plus a control and, where appropriate, a vehicle control (corresponding
to the concentration of vehicle at the highest exposure level) shall be used
Concentrations should be spaced appropnately to produce test groups with
a range of toxic effects The data should be sufficient to produce a con-
centration-response curve
(n) The highest concentration should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful eval-
uation
(in) The intermediate concentrations should be spaced to produce a
gradation of toxic effects
(iv) The lowest concentration should produce no evidence of toxicity
(v) In the case of potentially explosive test substances, care should
be taken to avoid generating explosive concentrations
(6) Administration of the test substance. Animals should be ex-
posed to the test substance for 6 h per day on a 7-day per week basis
for a period of at least 90 days Based primarily on practical consider-
ations, exposure for 6 h per day on a 5-day per week basis is acceptable
(7) Observation period. The animals should be observed for a period
of 90 days Animals in the satellite group (if used) scheduled for follow-
up observations should be kept for at least 28 days further without treat-
ment to assess reversibility
(8) Exposure specifications, (i) The animals shall be tested in dy-
namic inhalation equipment designed to sustain a minimum airflow of 10
air changes per hour, an adequate oxygen content of at least 19 percent,
and uniform conditions throughout the exposure chamber Maintenance of
-------
slight negative pressure inside the chamber will prevent leakage of the
test substance into the surrounding areas It is not normally necessary to
measure chamber oxygen concentration if airflow is adequate
(n) The selection of a dynamic inhalation chamber should be appro-
priate for the test substance and test system Where a whole body chamber
is used to expose animals to an aerosol, individual housing must be used
to minimize crowding of the test animals and maximize their exposure
to the test substance To ensure stability of a chamber atmosphere, the
total volume occupied by the test animals shall not exceed 5 percent of
the volume of the test chamber It is recommended, but not required, that
nose-only or head-only exposure be used for aerosol studies in order to
minimize oral exposures due to animals licking compound off their fur
Heat stress should be minimized
(m) The temperature at which the test is performed should be main-
tained at 22 ±2 °C The relative humidity should be maintained between
40-60 percent, but in certain instances (e g , use of water vehicle) this
may not be practicable
(9) Physical measurements. Measurements or monitoring shall be
made of the following
(i) The rate of airflow shall be monitored continuously but recorded
at least three times dunng the exposure
(n) The actual concentrations of the test substance shall be measured
m the animal's breathing zone Dunng the exposure period, the actual con-
centrations of the test substance shall be held as constant as practicable
and monitored continuously or intermittently depending on the method of
analysis. Chamber concentration may be measured using gravimetric or
analytical methods as appropriate If trial run measurements are reasonably
consistent (±10 percent for liquid aerosol, gas, or vapor, ±20 percent for
dry aerosol), then two measurements should be sufficient If measurements
are not consistent, three to four measurements should be taken Whenever
the test substance is a formulation, or it is necessary to formulate the test
substance with a vehicle for aerosol generation, the analytical concentra-
tion must be reported for the total formulation, and not just for the active
ingredient (AI) If, for example, a formulation contains 10 percent AI and
90 percent inerts, a chamber analytical limit concentration of 2 mg/L
would consist of 0 2 mg/L of the AI It is not necessary to analyze inert
ingredients provided the mixture at the animal's breathing zone is analo-
gous to the formulation, the grounds for this conclusion must be provided
in the study report If there is some difficulty in measuring chamber analyt-
ical concentration due to precipitation, nonhomogeneous mixtures, volatile
components, or other factors, additional analyses of inert components may
be necessary
-------
(in) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol concentra-
tions with respect to particle size The MMAD particle size range should
be between 1-3 fim The particle size of hygroscopic matenals should
be small enough when dry to assure that the size of the swollen particle
will still be within the 1-3 |tim range Measurements of aerodynamic par-
ticle size in the animal's breathing zone should be measured during a trial
run If MMAD values for each exposure level are within 10 percent of
each other, then two measurements during the exposures should be suffi-
cient. If pretest measurements are not within 10 percent of each other,
three to four measurements should be taken
(iv) Temperature and humidity shall be monitored continuously and
recorded at least three times during an exposure
(10) Feed and water during exposure period. Feed shall be with-
held during exposure Water may also be withheld during exposure
(11) Observation of animals, (i) During and following exposure, ob-
servations are made and recorded systematically, individual records should
be maintained for each animal It is not always possible to observe animals
during exposure in a whole-body chamber
(u) Observations shall be made at least twice each day for morbidity
and mortality Appropnate actions should be 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).
(in) General clinical observations should be made at least once a day,
preferably at the same time each day, taking into consideration the peak
penod of anticipated effects after dosing The clinical condition of the ani-
mal should be recorded
(iv) A careful clinical examination should be made at least once prior
to the initiation of treatment (to allow for within subject comparisons) and
once weekly dunng treatment in all animals These observations should
be made outside the home cage, preferably m a standard arena, and at
similar tunes on each occasion Effort should be made to ensure that vari-
ations in the observation conditions are minimal Observations should be
detailed and carefully recorded, preferably using sconng systems, explic-
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes in skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonomic activity (eg., lacrimation,
piloerection, pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of clomc or tonic
movements, stereotypies (e g, excessive grooming, repetitive circling) or
bizarre behavior (e g , self-mutilation, walking backwards) should be re-
corded.
-------
(v) Once, near the end of the exposure period and in any case not
earlier than in week 11 assessment of motor activity, grip strength, and
sensory reactivity to stimuli of different types (e g , visual, auditory, and
propnoceptive stimuli) should be conducted Further details of the proce-
dures that could be followed are described in the references listed under
paragraphs (h)(3), (h)(4), (h)(5), (h)(7), (h)(8), and (h)(ll) of this guide-
line
(vi) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other" studies and the daily clinical observations did not reveal any func-
tional deficits
(vu) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vui) Individual weights of animals shall be determined shortly before
the test substance is administered, and weekly thereafter
(ix) Food consumption shall also be determined weekly if abnormal
body weight changes are observed
(x) Moribund animals should be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible
(xi) At termination, all survivors in the treatment groups shall be sac-
rificed
(12) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex in
each group The hematology and clinical chemistry parameters should be
examined at terminal sacrifice at the end of the study Overnight fasting
of the animals pnor to blood sampling is recommended Overall, there
is a need for a flexible approach m the measures examined, depending
on the observed or expected effects from a chemical, and in the frequency
of measures, depending on the duration of potential chemical exposures
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombm time or
activated partial thromboplastm time
(11) Clinical chemistry (A) Parameters which are considered appro-
priate to all studies are 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 clini-
cal toxicity
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(B) The recommended clinical chemistry determinations are potas-
sium, sodium, glucose, total cholesterol, urea nitrogen, creatmme, total
protein and albumin More than 2 hepatic enzymes, (such as alanme
aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol
dehydrogenase, or gamma glutamyl transpeptidase) should also be meas-
ured. Measurements of addtional enzymes (of hepatic or other ongin) and
bile acids, may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobin, and
chohnesterases
(m) Optionally, the following unnalysis determinations could be per-
formed during the last week of the study using tuned unne volume collec-
tion- appearance, volume, osmolality or specific gravity, pH, protein, glu-
cose, and blood/blood cells
(13) Ophthalmological examination. Ophthalmological examina-
tions using an ophthalmoscope or an equivalent device should be made
on all animals pnor to the administration of the test substance and on
all high dose and control groups at termination If changes in the eyes
are detected, all animals in the other dose groups should be examined
(14) Gross necropsy, (i) All animals shall be subjected to a full gross
necropsy which includes examination of the external surface of the body,
all onfices and the cranial, thoracic, and abdominal cavities and their con-
tents.
(n) The liver, kidneys, adrenals, testes, epididymides, ovanes, uterus,
thymus, spleen, brain, and heart shall be trimmed and weighed wet, as
soon as possible after dissection to avoid drying
(m) The following organs and tissues, or representative samples there-
of, shall be preserved m a suitable medium for possible future
histopathological examination
(A) Digestive system
(1) Salivary glands
(2) Esophagus
(5) Stomach
(4) Duodenum
(5) Jejunum.
8
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(6) Ileum
(7) Cecum
(8) Colon
(9) Rectum
(10) Liver
(//) Pancreas
(12) Gallbladder (where present)
(B) Nervous system
(/) Brain (including sections of medulla/pons, cerebellum, and cere-
brum).
(2) Pituitary
(3) Peripheral nerve (sciatic or tibtal, preferably in close proximity
to the muscle)
(4) Spinal cord (three levels cervical, mid-thoracic, and lumbar).
(5) Eyes (retina, optic nerve)
(C) Glandular system
(]) Adrenals
(2) Parathyroids
(3) Thyroids
(D) Respiratory system
(1) Trachea
(2) Lung
(3) Pharynx
(4) Larynx
(5) Nose
(E) Cardiovascular/hematopoietic system
(1) Aorta
(2) Heart
(3) Bone marrow (and/or fresh aspirate)
9
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(4) Lymph nodes (preferably one node covering the route of adminis-
tration and another one distant from the route of administration)
(5) Spleen
(6) Thymus
(F) Urogemtal system
(7) Kidneys
(2) Urinary bladder
(3) Prostate
(4) Testes
(5) Epididymides
(6) Seminal vesicle(s)
(7) Uterus
(8) Ovanes
(9) Female mammary gland
(G) Other
(7) Lacnmal gland
(2) Skin
(3) All gross lesions and masses
(15) Histopathology. (i) The following histopathology shall be per-
formed-
(A) Full histopathology on the respiratory tract and other organs and
tissues, listed under paragraph (e)(15)(m) of this guideline, of all animals
in the control and high exposure 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
(D) Lungs of all animals Special attention to examination of the res-
piratory tract should be made for evidence of infection as this provides
a convenient assessment of the state of health of the animals
(E) When a satellite group is used, histopathology shall be performed
on tissues and organs identified as showing effects in the treated groups
10
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(n) If excessive early deaths or other problems occur in the high expo-
sure group compromising the significance of the data, the next concentra-
tion should be examined for complete histopathology
(in) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours pnor
to trimming
(f) Data and reporting—(1) Treatment of results, (i) Data shall
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
(11) When applicable, all observed results, quantitative and qualitative,
should be evaluated by an appropriate statistical method. Any generally
accepted statistical method may be used, the statistical methods including
significance criteria should be selected during the design of the study
(2) Evaluation of study results. The findings of the subchronic inha-
lation toxicity study should be evaluated in conjunction with the findings
of preceding studies and considered m terms of the observed toxic effects
and the necropsy and histopathological findings The evaluation will in-
clude the relationship between the concentration of the test substance and
duration of exposure, and the presence or absence, the incidence and sever-
ity, 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 sub-
chronic test should provide a satisfactory estimation of a no-effect level.
It also can indicate the need for an additional longer-term study and pro-
vide information on the selection of concentrations
(3) Test report In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J and
40 CFR part 160, and the OECD principles of GLP (ISBN 92-64-12367-
9) the following specific information shall be reported
(i) Test substance characterization shall include
(A) Chemical identification
(B) Lot or batch number
(C) Physical properties
(D) Punty/impunties
11
-------
(E) Identification and composition of any vehicle used
(u) Test system information shall include
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age, sex, and body weight data
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation period
(m) Test procedure information shall include
(A) Method of randomization used
(B) Full description of experimental design and procedure
(C) Exposure regimen including levels, methods, and volume
(D) Test conditions The following exposure conditions must be re-
ported-
(/) Description of exposure apparatus including design, type, volume,
source of air, system for generating aerosols, method of conditioning air,
treatment of exhaust air and the method of housing the animals in a test
chamber.
(2) The equipment for measuring temperature, humidity, and particu-
late aerosol concentrations and size should be descnbed
(E) Exposure data These shall be tabulated and presented with mean
values and a measure of variability (e g, standard deviation) and should
include
(/) Airflow rates through the inhalation equipment
(2) Temperature and humidity of air
(3) Actual (analytical or gravimetric) concentration in the breathing
zone.
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air)
(5) Particle size distribution, calculated mass median aerodynamic di-
ameter (MMAD) and geometnc standard deviation (GSD)
12
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(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines
(iv) Test results (A) Group animal data Tabulation of toxic response
data by species, strain, sex, and exposure level for
Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(1) Time of death during the study or whether animals survived to
termination
(2) Time of observation of each abnormal sign and its subsequent
course.
(3) Body weight data
(4) Feed consumption data, when collected
(5) Results of ophthalmological examination
(6) Results of hematological tests performed
(7) Results of clinical chemistry tests performed
(5) Results of urmalysis, if performed
(9) Results of observations made
(10) Necropsy findings, including absolute and relative organ weight
data.
(11) Detailed description of all histopathological findings.
(12) Statistical treatment of results, where appropriate
(g) Quality control. A system shall be developed and maintained to
assure and document adequate performance of laboratory staff and equip-
ment The study must be conducted in compliance with GLP regulations
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Cage, J C Experimental Inhalation Toxicology, Methods in Toxi-
cology Ed GE Paget (Philadelphia FA Davis Co , 1970) pp 258-277
13
-------
(2) Casarett, LJ and DoulL J Chapter 9, Toxicology The Basic
Science of Poisons (New York Macnullan Publishing Co Inc , 1975).
(3) Crofton K M , Howard J L , Moser V C , Gill M W , Leiter L W ,
Tilson H A , MacPhail, R C Interlaboratory Comparison of Motor Activity
Experiments Implication for Neurotoxicological Assessments
Neurotoxicol Teratal 13,599-609 (1991)
(4) Gad S C A Neuromuscular Screen for Use in Industrial Toxi-
cology Journal of Toxicology and Environmental Health 9, 691-704
(1982)
(5) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals Environmental Health Cntena Document No 60 (1986)
(6) MacFarland, H N Respiratory Toxicology, Essays in Toxicology
Ed W.J Hayes Vol 7 (New York Academic Press, 1976) pp. 121-154.
(7) Meyer O A , Tilson H A , Byrd W C , Riley M T. A Method for
the Routine Assessment of Fore- and Hind-Limb Gnp Strength of Rats
and Mice Neurobehav Toxicol 1,233-236 (1979)
(8) Moser V C , McDaniel K M , Phillips P.M Rat Strain and Stock
Comparisons using a Functional Observational Battery. Baseline Values
and Effects of Amitraz Toxicol Appl Pharmacol 108, 267-283 (1991)
(9) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances, a report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
(10) Organization for Economic Cooperation and Development
Guidelines for testing of chemicals, Section 4—Health Effects, Part—413
Subchromc Inhalation Toxicity Studies (Pans, 1981)
(11) Tupper, D E , Wallace R B Utility of the Neurologic Examina-
tion in Rats Acta. Neurobiol Exp 40, 999-1003 (1980)
(12) US Environmental Protection Agency, Office of Testing and
Evaluation. Proposed health effects test standards for toxic substances con-
trol act test rules, 40 CFR Part 772 Standard for Development of Test
Data SubpartD FEDERAL REGISTER (44 FR 27350-27362)
(13) U S Environmental Protection Agency, Office of Pesticide Pro-
grams, Health Effects Division Interim policy for particle size and limit
concentration issues in inhalation toxicity studies (February 1, 1994)
14
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(14) Wemgand K, Brown G, Hall R et al Harmonization of Animal
Clinical Pathology Testing in Toxicity and Safety Studies Fundam &
Appl Toxtcol 29 198-201 (1996)
(15) World Health Organization Parti Environmental Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals (Gene-
va World Health Organization, 1978)
(16) World Health Organization Principles for pre-chnical testing of
drug safety WHO Technical Report Series No 341 (Geneva World Health
Organization, 1966)
15
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&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-9S-207
August 1998
Health Effects Test
Guidelines
OPPTS 870.3700
Prenatal Developmenta
Toxicity Study
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC 136,etseq)
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
m ASCII and PDF (portable document format) from EPA's World Wide
Web site (http //www epa gov/epahome/research htm) under the heading
''Researchers and Scientists/Test Methods and Guidehnes/OPPTS Har-
monized Test Guidelines ''
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OPPTS 870 3700 Prenatal developmental towcity study
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ), as amended by the Food
Quality Protection Act (FQPA)(Pub L 104-170), and the Toxic Sub-
stances Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPPT guideline under 40 CFR
798 4900, OPP guideline 83-3, and OECD guideline 414
(b) Purpose. This guideline for developmental toxicity testing is de-
signed to provide general information concerning the effects of exposure
of the pregnant test animal on the developing organism, this may include
death, structural abnormalities, or altered growth and an assessment of ma-
ternal effects For information on testing for functional deficiencies and
other postnatal effects, the guidelines for the two-generation reproductive
toxicity study and the developmental neurotoxicity study should be con-
sulted
(c) Good laboratory practice standards. The study should be con-
ducted in accordance with the laboratory practices stipulated in 40 CFR
Part 160 (FIFRA) and 40 CFR Part 792 (TSCA)—Good Laboratory Prac-
tice Standards.
(d) Principle of the test method. The test substance is administered
to pregnant animals, at least from implantation to one day pnor to the
expected day of parturition Shortly before the expected date of delivery,
the pregnant females are terminated, the utenne contents are examined,
and the fetuses are processed for visceral and skeletal evaluation
(e) Test procedures—(1) Animal selection—(i) Species and strain.
It is recommended that testing be performed in the most relevant species,
and that laboratory species and strains which are commonly used m pre-
natal developmental toxicity testing be employed The preferred rodent
species is the rat and the preferred non-rodent species is the rabbit
(n) Age. Young adult animals should be used
(111) Sex. Nulliparous female animals should be used at each dose
level Animals should be mated with males of the same species and strain,
avoiding the mating of siblings, if parentage is known Day 0 in the test
is the day on which a vaginal plug and/or sperm are observed m the rodent
or that insemination is performed or observed in the rabbit
(iv) Animal care. Animal care and housing should be m accordance
with the recommendations contained in the DHHS/PHS NIH Publication
No 86-23, 1985, Guidelines for the Care and Housing of Laboratory Ani-
mals, or other appropriate guidelines
-------
(v) Number of animals. Each test and control group should contain
a sufficient number of animals to yield approximately 20 animals with
implantation sites at necropsy
(2) Administration of test and control substances—(i) Dose levels
and dose selection. (A) At least three-dose levels and a concurrent control
should be used Healthy animals should be randomly assigned to the con-
trol and treatment groups, m a manner which results in comparable mean
body weight values among all groups The dose levels should be spaced
to produce a gradation of toxic effects Unless limited by the physical/
chemical nature or biological properties of the test substance, the highest
dose should be chosen with the aim to induce some developmental and/
or maternal toxic ity but not death or severe suffenng. In the case of mater-
nal mortality, this should not be more than approximately 10 percent The
intermediate dose levels should produce minimal observable toxic effects.
The lowest dose level should not produce any evidence of either maternal
or developmental toxicity (i e, the no-observed-adverse-effect level,
NOAEL) or should be at or near the limit of detection for the most sen-
sitive endpomt Two- or four-fold intervals are frequently optimal for spac-
ing the dose levels, and the addition of a fourth test group is often pref-
erable to using very large intervals (e g , more than a factor of 10) between
dosages
(B) It is desirable that additional information on metabolism and
pharmacokinetics of the test substance be available to demonstrate the ade-
quacy of the dosing regimen This information should be available poor
to testing
(C) The highest dose tested need not exceed 1,000 mg/kg/day by oral
or dermal administration, or 2 mg/L (or the maximum attainable concentra-
tion) by inhalation, unless potential human exposure data indicate the need
for higher doses If a test performed at the limit dose level, using the
procedures descnbed for this study, produces no observable toxicity and
if an effect would not be expected based upon data from structurally relat-
ed compounds, then a full study using three-dose levels may not be consid-
ered necessary.
(11) Control group. (A) A concurrent control group should be used
This group should be a sham-treated control group or a vehicle-control
group if a vehicle is used in administering the test substance
(B) The vehicle control group should receive the vehicle m the high-
est volume used
(C) If a vehicle or other additive is used to facilitate dosing, consider-
ation should be given to the following characteristics Effects on the ab-
sorption, distribution, metabolism, or retention of the test substance, effects
on the chemical properties of the test substance which may alter its toxic
-------
characteristics, and effects on the food or water consumption or the nutri-
tional status of the animals
(111) Route of administration (A) The test substance or vehicle is
usually administered orally by intubation
(B) If another route of administration is used, for example, when the
route of administration is based upon the principal route of potential
human exposure, the tester should provide justification and reasoning for
its selection, and appropriate modifications may be necessary Further in-
formation on dermal or inhalation exposure is provided under paragraphs
(h)(12), (h)(28), and (h)(29) of this guideline Care should be taken to
minimize stress on the maternal animals For materials administered by
inhalation, whole-body exposure is preferable to nose-only exposure due
to the stress of restraint required for nose-only exposure
(C) The test substance should be administered at approximately the
same time each day
(D) When administered by gavage or dermal application, the dose
to each animal should be based on the most recent individual body weight
determination
(iv) Dosing schedule. At minimum, the test substance should be ad-
ministered daily from implantation to the day before cesarean section on
the day pnor to the expected day of parturition Alternatively, if prelimi-
nary studies do not indicate a high potential for preimplantation loss, treat-
ment may be extended to include the entire penod of gestation, from fer-
tilization to approximately 1 day pnor to the expected day of termination
(0 Observation of animals—(1) Maternal, (i) Each animal should
be observed at least once daily, considering the peak period of anticipated
effects after dosing. Mortality, monbundity, pertinent behavioral changes,
and all signs of overt toxicity should be recorded at this cageside observa-
tion. In addition, thorough physical examinations should be conducted at
the same time maternal body weights are recorded.
(11) Animals should be weighed on day 0, at termination, and at least
at 3-day intervals during the dosing penod
(111) Food consumption should be recorded on at least 3-day intervals,
preferably on days when body weights are recorded
(iv) Termination schedule (A) Females should be terminated imme-
diately pnor to the expected day of delivery
(B) Females showing signs of abortion or premature delivery pnor
to scheduled termination should be killed and subjected to a thorough mac-
roscopic examination
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(v) Gross necropsy At the time of termination or death during the
study, the dam should be examined rnacroscopically for any structural ab-
normalities or pathological changes which may have influenced the preg-
nancy Evaluation of the dams during cesarean section and subsequent fetal
analyses should be conducted without knowledge of treatment group in
order to minimize bias
(vi) Examination of uterine contents (A) Immediately after termi-
nation or as soon as possible after death, the uten should be removed
and the pregnancy status of the animals ascertained Uten that appear non-
gravid should be further examined (eg by ammonium sulfide staining)
to confirm the nonpregnant status
(B) Each gravid uterus (with cervix) should be weighed Gravid uter-
ine weights should not be obtained from dead animals if autolysis or de-
composition has occurred
(C) The number of corpora lutea should be determined for pregnant
animals
(D) The uterine contents should be examined for embryonic or fetal
deaths and the number of viable fetuses The degree of resorption should
be described in order to help estimate the relative time of death of the
conceptus
(2) Fetal, (i) The sex and body weight of each fetus should be deter-
mined
(11) Each fetus should be examined for external anomalies.
(in) Fetuses should be examined for skeletal and soft tissue anomalies
(e g variations and malformations or other categories of anomalies as de-
fined by the performing laboratory)
(A) For rodents, approximately one-half of each litter should be pre-
pared by standard techniques and examined for skeletal alterations, pref-
erably bone and cartilage The remainder should be prepared and examined
for soft tissue anomalies, using appropnate serial sectioning or gross dis-
section techniques It is also acceptable to examine all fetuses by careful
dissection for soft tissue anomalies followed by an examination for skeletal
anomalies
(B) For rabbits, all fetuses should be examined for both soft tissue
and skeletal alterations The bodies of these fetuses should be evaluated
by careful dissection for soft-tissue anomalies, followed by preparation and
examination for skeletal anomalies An adequate evaluation of the internal
structures of the head, including the eyes, brain, nasal passages, and
tongue, should be conducted for at least half of the fetuses
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(g) Data and reporting—(1) Treatment of results. Data should be
reported individually and summarized in tabular form, showing for each
test group the types of change and the number of darns, fetuses, and litters
displaying each type of change
(2) Evaluation of study results. The following should be provided
(i) Maternal and fetal test results, including an evaluation of the rela-
tionship, or lack thereof, between the exposure of the animals to the test
substance and the incidence and seventy of all findings
(u) Criteria used for categorizing fetal external, soft tissue, and skele-
tal anomalies
(111) When appropriate, historical control data to enhance interpreta-
tion of study results Historical data (on litter incidence and fetal incidence
within litter), when used, should be compiled, presented, and analyzed in
an appropnate and relevant manner In order to justify its use as an analyt-
ical tool, information such as the dates of study conduct, the strain and
source of the animals, and the vehicle and route of administration should
be included
(iv) Statistical analysis of the study findings should include sufficient
information on the method of analysis, so that an independent reviewer/
statistician can reevaluate and reconstruct the analysis In the evaluation
of study data, the litter should be considered the basic unit of analysis
(v) In any study which demonstrates an absence of toxic effects, fur-
ther investigation to establish absorption and bioavailabihty of the test sub-
stance should be considered
(3) Test report In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, and 40 CFR part 160, subpart J, the
following specific information should be reported Both individual and
summary data should be presented.
(i) Species and strain
(11) Maternal toxic response data by dose, including but not limited
to
(A) The number of animals at the start of the test, the number of
animals surviving, the number pregnant, and the number aborting
(B) Day of death dunng the study or whether animals survived to
termination
(C) Day of observation of each abnormal clinical sign and its subse-
quent course
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(D) Body weight and body weight change data, including body weight
change adjusted for gravid uterine weight
(E) Food consumption and, if applicable, water consumption data
(F) Necropsy findings, including gravid uterine weight
(in) Developmental endpomts by dose for litters with implants, in-
cluding
(A) Corpora lutea counts
(B) Implantation data, number and percent of live and dead fetuses,
and resorptions (early and late)
(C) Pre- and postimplantation loss calculations
(iv) Developmental endpomts by dose for litters with live fetuses,
including
(A) Number and percent of live offspring
(B) Sex ratio
(C) Fetal body weight data, preferably by sex and with sexes com-
bined
(D) External, soft tissue, and skeletal malformation and variation data
The total number and percent of fetuses and litters with any external, soft
tissue, or skeletal alteration, as well as the types and incidences of individ-
ual anomalies, should be reported
(v) The numbers used in calculating all percentages or indices.
(vi) Adequate statistical treatment of results
(vn) A copy of the study protocol and any amendments should be
included.
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Ahverti, V L et al The extent of fetal ossification as an index
of delayed development in teratogenicity studies in the rat Teratology
20237-242(1979)
(2) Barrow, M V and W J Taylor A rapid method for detecting mal-
formations in rat fetuses Journal of Morphology 127 291-306 (1969)
(3) Burdi, A R Toluidme blue-alizarin red S staining of cartilage and
bone in whole-mount skeltons in vitro Stain Technolology 40 45-48
(1965)
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(4) Edwards, J A The external development of the rabbit and rat em-
bryo In Advances in Teratolog\ (ed D H M Woolam) Vol 3 Academic,
NY (1968)
(5) Fritz, H Prenatal ossification in rabbits as indicative of fetal matu-
rity Teratology 11313-320(1974)
(6) Fntz, H and R Hess Ossification of the rat and mouse skeleton
in the perinatal period Teratology 3 331-338 (1970)
(7) Gibson, J P etal Use of the rabbit in teratogemcity studies Toxi-
cology and Applied Pharmacology 9 398^08 (1966)
(8) Inouye, M Differential staining of cartilage and bone in fetal
mouse skeleton by alcian blue and alizarin red S Congenital Anomalies
16(3) 171-173 (1976)
(9) Igarashi, E. el al Frequence of spontaneous axial skeletal vari-
ations detected by the double staining technique for ossified and cartilagi-
nous skeleton in rat fetuses Congenital Anomalies 32 381-391 (1992)
(10) Kaufman, M (ed ) The Atlas of Mouse Development Academic
Press, London (1993)
(11) Kimmel, C A et al Skeletal development following heat expo-
sure in the rat Teratology 47 229-242 (1993)
(12) Kimmel, C A and EZ Francis Proceedings of the workshop
on the acceptability and interpretation of dermal developmental toxicity
studies Fundamental and Applied Toxicology 14 386-398 (1990)
(13) Kimmel, C A and C Trammell A rapid procedure for routine
double staining of cartilage and bone in fetal and adult animals Stain
Technology 56 271-273 (1981)
(14) Kimmel, CA and JG Wilson Skeletal deviation m rats mal-
formations or variations^ Teratology 8 309-316 (1973)
(15) Man, MC et al Comparison of single and double staining for
evaluation of skeletal development the effects of ethylene glycol (EG)
in CD rats Teratology 37 476 (1988)
(16) Marr, MC et al Developmental stages of the CD (Sprague-
Dawley) rat skeleton after maternal exposure to ethylene glycol. Teratol-
ogy 46-169-181 (1992)
(17) McLeod, MJ Differential staining of cartilage and bone m
whole mouse fetuses by Alcian blue and alizarin red S Teratology 22 299-
301 (1980)
-------
(18) Monie, I W et al Dissection procedures for rat fetuses permit-
ting alizarin red staining of skeleton and histological study of viscera Sup-
plement to Teratology Workshop Manual, pp 163-173 (1965)
(19) Organization for Economic Cooperation and Development, No
414 Teratogemcity, Guidelines for Testing of Chemicals [C(83)44
(Final)] (1983)
(20) Salewski (Koeln), VE Faerbermethode zum makroskopischen
nachweis von implantations stellen am uterus der ratte Naunyn-
Schmeidebergs Archiv fur Pharmakologie und Expenmentelle Pathologie
247 367 (1964)
(21) Spark, C and A B Dawson The order and time of appearance
of centers of ossification in the fore and hind limbs of the albino rat, with
special reference to the possible influence of the sex factor American
Journal of Anatomy 41 411-445 (1928)
(22) Staples, R E Detection of visceral alterations in mammalian
fetuses Teratology 9(3) A37-A38 (1974)
(23) Staples, R E and V L Schnell Refinements in rapid clearing
technique in the KOH—alizarin red S method for fetal bone Stain Tech-
nology 39 61-63 (1964)
(24) Strong, R M The order time and rate of ossification of the albino
rat (mus norvegicus albinus) skeleton American Journal of Anatomy 36
313-355(1928)
(25) Stuckhardt, J L and S M Poppe Fresh visceral examination of
rat and rabbit fetuses used in teratogemcity testing Teratogenesis, Car-
cinogenesis, and Mutagenesis 4 181-188 (1984)
(26) U S Environmental Protection Agency Guideline 83-3
Teratogencity Study Pesticide Assessment Guidelines, Subdivision F.
Hazard Evaluation. Human and Domestic Animals Office of Pesticides
and Toxic Substances, Washington, DC, EPA-540/9-82-025 (1982)
(27) U S Environmental Protection Agency Subpart E—Specific
Organ/Tissue Toxicity, 40 CFR 798 4900 Developmental Toxicity Study
(28) US Environmental Protection Agency Health Effects Test
Guidelines, OPPTS 870 3200, 21/28-Day Dermal Toxicity, July 1998
(29) U S Environmental Protection Agency Health Effects Test
Guidelines, OPPTS 870 3465, 90-Day Inhalation Toxicity, July 1998
(30) U S Environmental Protection Agency Guidelines for Devel-
opmental Toxicity Risk Assessment FEDERAL REGISTER (56 FR 63798-
63826, Decembers, 1991)
8
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(31) Van Julsmgha, EB and CG Bennett A dissecting procedure
for the detection of anomalies in the rabbit foetal head In Methods in
Prenatal Toxicology (eds D Neubert, HJ Merker, and T E Kwasigroch)
University of Chicago, Chicago, IL, pp 126-144 (1977)
(32) Walker, D G andZT Wirtschafter The Genesis of the Rat Skel-
eton Thomas, Springfield, IL (1957)
(33) Whitaker, J and D M Dix Double-staining for rat foetus skele-
tons in teratological studies Laboratory Animals 13 309-310 (1979)
(34) Wilson, J G Embryological considerations in teratology In
"Teratology Principles and Techniques" (ed JG Wilson and J
Warkany) University of Chicago, Chicago, IL, pp 251-277 (1965)
(35) Wilson, J G and F C Fraser, ed Handbook of Teratology, Vol
4 Plenum, NY (1977)
(36) Yasuda, M and T Yuki Color Atlas of Fetal Skeleton of the
Mouse, Rat, and Rabbit Ace Art Co , Osaka, Japan (1996)
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&EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-208
August 1998
Health Effects Test
Guidelines
OPPTS 870.3800
Reproduction and
Fertility Effects
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed m the Office
of Pollution Prevention and Toxics (OPPT) and appeared m Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) .and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC I36,etseq)
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in ASCII and PDF (portable document format) from EPA's World Wide
Web site (http //www epa gov/epahome/research.htm) under the heading
"Researchers and Scientists/Test Methods and Guidelmes/OPPTS Har-
monized Test Guidelines''
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OPPTS 870.3800 Reproduction and fertility effects
(a) Scope—(1) Applicability This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ), as amended by the Food
Quality Protection Act (FQPA)(Pub L 104-170) and the Toxic Sub-
stances Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPPT guideline under 40 CFR
798 4700, OPP guideline 83-4, and OECD guideline 416
(b) Purpose. This guideline for two-generation reproduction testing
is designed to provide general information concerning the effects of a test
substance on the integrity and performance of the male and female repro-
ductive systems, including gonadal function, the estrous cycle, mating be-
havior, conception, gestation, parturition, lactation, and weaning, and on
the growth and development of the offspring The study may also provide
information about the effects of the test substance on neonatal morbidity,
mortality, target organs in the offspnng, and preliminary data on prenatal
and postnatal developmental toxicity and serve as a guide for subsequent
tests Additionally, since the study design includes in utero as well as post-
natal exposure, this study provides the opportunity to examine the suscepti-
bility of the immature/neonatal animal For further information on func-
tional deficiencies and developmental effects, additional study segments
can be incorporated into the protocol, utilizing the guidelines for devel-
opmental toxicity or developmental neurotoxicity
(c) Good laboratory practice standards. The study should be con-
ducted in accordance with the laboratory practices stipulated in 40 CFR
Part 160 (FIFRA) and 40 CFR Part 792 (TSCA)—Good Laboratory Prac-
tice Standards
(d) Principle of the test method. The test substance is administered
to parental (P) animals poor to and during their mating, during the result-
ant pregnancies, and through the weaning of their Fl offspnng The sub-
stance is then administered to selected Fl offspnng dunng their growth
into adulthood, mating, and production of an F2 generation, until the F2
generation is weaned
(e) Test procedures—(1) Animal selection—(i) Species and strain.
The rat is the most commonly used species for testing If another mamma-
lian species is used, the tester should provide justification/reasoning for
its selection, and appropnate modifications will be necessary Healthy pa-
rental animals, which have been acclimated to laboratory conditions for
at least 5 days and have not been subjected to previous experimental proce-
dures, should be used Strains of low fecundity should not be used
(11) Age. Parental (P) animals should be 5 to 9 weeks old at the start
of dosing The animals of all test groups should be of uniform weight,
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age, and parity as nearly as practicable, and should be representative of
the species and strain under study
(in) Sex. (A) For an adequate assessment of fertility, both males and
females should be studied
(B) The females should be nulliparous and nonpregnant
(iv) Animal care. Animal care and housing should be in accordance
with the recommendations contained in DHHS/PHS NIH Publication No
86-23, 1985, Guidelines for the Care and Use of Laboratory Animals,
or other appropriate guidelines
(v) Number of animals. Each control group should contain a suffi-
cient number of mating pairs to yield approximately 20 pregnant females
Each test group should contain a similar number of mating pairs
(vi) Identification of animals. Each animal should be assigned a
unique identification number For the P generation, this should be done
before dosing starts For the Fl generation, this should be done for animals
selected for mating, in addition, records indicating the Utter of ongm
should be maintained for all selected Fl animals
(2) Administration of test and control substances—(i) Dose levels
and dose selection. (A) At least three-dose levels and a concurrent control
should be used. Healthy animals should be randomly assigned to the con-
trol and treatment groups, in a manner which results in comparable mean
body weight values among all groups The dose levels should be spaced
to produce a gradation of toxic effects Unless limited by the physical/
chemical nature or biological properties of the test substance, the highest
dose should be chosen with die aim to induce some reproductive and/
or systemic toxicity but not death or severe suffering. In the case of paren-
tal mortality, this should not be more than approximately 10 percent. The
intermediate dose levels should produce minimal observable toxic effects.
The lowest dose level should not produce any evidence of either systemic
or reproductive toxicity (i e, the no-observed-adverse-effect level,
NOAEL) or should be at or near the limit of detection for the most sen-
sitive endpomt Two- or four-fold intervals are frequently optimal for spac-
ing the dose levels, and the addition of a fourth test group is often pref-
erable to using very large intervals (e g , more than a factor of 10) between
dosages.
(B) It is desirable that additional information on metabolism and
pharmacokmetics of the test substance be available to demonstrate the ade-
quacy of the dosing regimen This information should be available pnor
to testing
(C) The highest dose tested should not exceed 1,000 mg/kg/day (or
20,000 ppm in the diet), unless potential human exposure data indicate
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the need for higher doses If a test performed at the limit dose level, using
the procedures described for this study, produces no observable toxicity
and if an effect would not be expected based upon data from structurally
related compounds, then a full study using three dose levels may not be
considered necessary
(11) Control group. (A) A concurrent control group should be used
This group should be an untreated or sham treated group or a vehicle-
control group if a vehicle is used in administering the test substance
(B) If a vehicle is used m administering the test substance, the control
group should receive the vehicle in the highest volume used
(C) If a vehicle or other additive is used to facilitate dosing, consider-
ation should be given to the following characteristics Effects on the ab-
sorption, distribution, metabolism, or retention of the test substance, effects
on the chemical properties of the test substance which may alter its toxic
characteristics, and effects on the food or water consumption or the nutri-
tional status of the animals
(D) If a test substance is administered m the diet and causes reduced
dietary intake or utilization, the use of a pair-fed control group may be
considered necessary
(m) Route of administration. (A) The test substance is usually ad-
ministered by the oral route (diet, drinking water, or gavage).
(B) If administered by gavage or dermal application, the dosage ad-
ministered to each animal pnor to mating and during gestation and lacta-
tion should be based on the individual animal body weight and adjusted
weekly at a minimum
(C) If another route of administration is used, for example, when the
route of administration is based upon the principal route of potential
human exposure, the tester should provide justification and reasoning for
its selection, and appropnate modifications may be necessary Further in-
formation on dermal or inhalation exposure is provided under paragraphs
(g)(18) and (g)(19) of this guideline Care should be taken to minimize
stress on the maternal animals and their litters during gestation and lacta-
tion
(D) All animals should be dosed by the same method during the ap-
propnate experimental period
(iv) Dosing schedule. (A) The animals should be dosed with the test
substance on a 7-days-a-week basis
(B) Daily dosing of the parental (P) males and females should begin
when they are 5 to 9 weeks old Daily dosing of the Fl males and females
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should begin at weaning For both sexes (P and Fl), dosing should be
continued for at least 10 weeks before the mating period
(C) Daily dosing of the P and Fl males and females should continue
until termination
(3) Mating procedure—(i) Parental. (A) For each mating, each fe-
male should be placed with a single randomly selected male from the same
dose level (11 mating) until evidence of copulation is observed or either
3 estrous periods or 2 weeks has elapsed Animals should be separated
as soon as possible after evidence of copulation is observed If mating
has not occurred after 2 weeks or 3 estrous periods, the animals should
be separated without further opportunity for mating Mating pairs should
be clearly identified in the data
(B) Vaginal smears should be collected daily and examined for all
females during mating, until evidence of copulation is observed.
(C) Each day, the females should be examined for presence of sperm
or vaginal plugs Day 0 of pregnancy is defined as the day a vaginal plug
or sperm are found
(11) Fl mating. For mating the Fl offspnng, at least one male and
one female should be randomly selected from each litter for mating with
another pup of the same dose level but different litter, to produce the F2
generation
(111) Second mating. In certain instances, such as poor reproductive
performance in the controls, or in the event of treatment-related alterations
in litter size, the adults may be remated to produce an Fib or F2b litter
If production of a second litter is deemed necessary in either generation,
the dams should be remated approximately 1-2 weeks following weaning
of the last Fla or F2a Utter
(iv) Special housing. After evidence of copulation, animals that are
presumed to be pregnant should be caged separately in delivery or mater-
nity cages. Pregnant animals should be provided with nesting materials
when parturition is near
(v) Standardization of litter sizes. (A) Animals should be allowed
to Utter normally and rear their offspnng to weaning Standardization of
litter sizes is optional.
(B) If standardization is performed, the following procedure should
be used On day 4 after birth, the size of each litter may be adjusted by
eliminating extra pups by random selection to yield, as nearly as possible,
four males and four females per litter or five males and five females per
litter Selective elimination of pups, i e based upon body weight, is not
appropnate Whenever the number of male or female pups prevents having
four (or five) of each sex per litter, partial adjustment (for example, five
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males and three females, or four males and six females) is acceptable
Adjustments are not appropnate for litters of eight pups or less
(4) Observation of animals—(i) Parental. (A) Throughout the test
period, each animal should be observed at least once daily, considering
the peak penod of anticipated effects after dosing Mortality, moribundity,
pertinent behavioral changes, signs of difficult or prolonged parturition,
and all signs of overt toxicity should be recorded at this cageside examina-
tion In addition, thorough physical examinations should be conducted
weekly on each animal
(B) Parental animals (P and Fl) should be weighed on the first day
of dosing and weekly thereafter Parental females (P and Fl) should be
weighed at a minimum on approximately gestation days 0, 7, 14, and 2 \,
and dunng lactation on the same days as the weighing of litters
(C) Dunng the premating and gestation periods, food consumption
should be measured weekly at a minimum Water consumption should be
measured weekly at a minimum if the test substance is administered in
the water
(D) Estrous cycle length and pattern should be evaluated by vaginal
smears for all P and Fl females dunng a minimum of 3 weeks pnor to
mating and throughout cohabitation, care should be taken to prevent the
induction of pseudopregnancy
(E) For all P and Fl males at termination, sperm from one tesus and
one epididyrms should be collected for enumeration of homogemzation-
resistant spermatids and cauda epididymal sperm reserves, respectively. In
addition, sperm from the cauda epididymis (or vas deferens) should be
collected for evaluation of sperm motihty and sperm morphology
(1) The total number of homogemzation-resistant testicular sperm and
cauda epididymal sperm should be enumerated (see paragraphs (g)(3) and
(g)(13) of this guideline) Cauda sperm reserves can be denved from the
concentration and volume of sperm in the suspension used to complete
the qualitative evaluations, and the number of sperm recovered by subse-
quent mincing and/or homogenizing of the remaining cauda tissue Enu-
meration in only control and high-dose P and Fl males may be performed
unless treatment-related effects are observed; in that case, the lower dose
groups should also be evaluated
(2) An evaluation of epididymal (or vas deferens) sperm motihty
should be performed Sperm should be recovered while minimizing dam-
age (refer to paragraph (g)(13) of this guideline), and the percentage of
progressively motile sperm should be determined either subjectively or ob-
jectively For objective evaluations, an acceptable counting chamber of
sufficient depth can be used to effectively combine the assessment of mo-
tihty with sperm count and sperm morphology When computer-assisted
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motion analysis is performed (refer to paragraph (g)(13) of this guideline),
the derivation of progressive motihty relies on user-defined thresholds for
average path velocity and straightness or linear index If samples are
videotaped, or images otherwise lecorded, at the time of necropsy, subse-
quent analysis of only control and high-dose P and Fl males may be per-
formed unless treatment-related effects are observed, in that case, the
lower dose groups should also be evaluated In the absence of a video
or digital image, all samples in all treatment groups should be analyzed
at necropsy
(3) A morphological evaluation of an epididymal (or vas deferens)
sperm sample should be performed Sperm (at least 200 per sample) should
be examined as fixed, wet preparations (refer to paragraphs (g)(7) and
(g)(13) of this guideline) and classified as either normal (both head and
midpiece/tail appear normal) or abnormal Examples of morphologic sperm
abnormalities would include fusion, isolated heads, and misshapen heads
and/or tails Evaluation of only control and high-dose P and Fl males may
be performed unless treatment-related effects are observed, in that case,
the lower dose groups should also be evaluated
(11) Offspring. (A) Each litter should be examined as soon as possible
after delivery (lactation day 0) to establish the number and sex of pups,
stillbirths, live births, and the presence of gross anomalies Pups found
dead on day 0 should be examined for possible defects and cause of death
(B) Live pups should be counted, sexed, and weighed individually
at birth, or soon thereafter, at least on days 4, 7, 14, and 21 of lactation,
at the time of vaginal patency or balanopreputial separation, and at termi-
nation
(C) The age of vaginal opening and preputial separation should be
determined for Fl weanlings selected for mating If there is a treatment-
related effect in Fl sex ratio or sexual maturation, anogemtal distance
should be measured on day 0 for all F2 pups
(5) Termination schedule, (i) All P and Fl adult males and females
should be terminated when they are no longer needed for assessment of
reproductive effects
(11) Fl offspring not selected for mating and all F2 offspring should
be terminated at comparable ages after weaning
(6) Gross necropsy, (i) At the time of termination or death during
the study, all parental animals (P and Fl) and when litter size permits
at least three pups per sex per litter from the unselected Fl weanlings
and the F2 weanlings should be examined macroscopically for any struc-
tural abnormalities or pathological changes Special attention should be
paid to the organs of the reproductive system
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(u) Dead pups or pups that are terminated in a moribund condition
should be examined for possible defects and/or cause of death
(ui) At the time of necropsy, a vaginal smear should be examined
to determine the stage of the estrous cycle The uten of all cohabited fe-
males should be examined, in a manner which does not compromise
histopathological evaluation, for the presence and number of implantation
sites
(7) Organ weights, (i) At the time of termination, the following or-
gans of all P and Fl parental animals should be weighed-
(A) Uterus (with oviducts and cervix), ovaries
(B) Testes, epididymides (total weights for both and cauda weight
for either one or both), seminal vesicles (with coagulating glands and their
fluids), and prostate
(C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and known
target organs
(11) For Fl and F2 weanlings that are examined macroscopically, the
following organs should be weighed for one randomly selected pup per
sex per litter
(A) Brain
(B) Spleen and thymus
(8) Tissue preservation. The following organs and tissues, or rep-
resentative samples thereof, should be fixed and stored in a suitable me-
dium for histopathological examination
(i) For the parental (P and Fl) animals
(A) Vagina, uterus with oviducts, cervix, and ovanes
(B) One testis (preserved in Bourns fixative or comparable preserva-
tive), one epididyrms, seminal vesicles, prostate, and coagulating gland
(C) Pituitary and adrenal glands
(D) Target organs, when previously identified, from all P and Fl ani-
mals selected for mating
(E) Grossly abnormal tissue
(11) For Fl and F2 weanlings selected for macroscopic examination.
Grossly abnormal tissue and target organs, when known
(9) Histopathology—(i) Parental animals. Full histopathology of the
organs listed in paragraph (e)(8)(i) of this guideline should be performed
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for ten randomly chosen high dose and control P and Fl animals per sex,
for those animals that were selected for mating Organs demonstrating
treatment-related changes should also be examined for the remainder of
the high-dose and control animals and for all parental animals in the low-
and mid-dose groups Additionally, reproductive organs of the low- and
mid-dose animals suspected of reduced fertility, e g , those that failed to
mate, conceive, sire, or deliver healthy offspring, or for which estrous cy-
chcity or sperm number, motihty, or morphology were affected, should
be subjected to histopathological evaluation Besides gross lesions such
as atrophy or tumors, testicular histopathological examination should be
conducted in order to to identify treatment-related effects such as retained
spermatids, missing germ cell layers or types, multmucleated giant cells,
or sloughing of spermatogenic cells into the lumen (refer to paragraph
(g)(ll) of this guideline) Examination of the intact epididymis should in-
clude the caput, corpus, and cauda, which can be accomplished by evalua-
tion of a longitudinal section, and should be conducted in order to identify
such lesions as sperm granulomas, leukocytic infiltration (inflammation),
aberrant cell types within the lumen, or the absence of clear cells in the
cauda epididymal epithelium The postlactational ovary should contain pri-
mordial and growing follicles as well as the large corpora lutea of lacta-
tion Histopathological examination should detect qualitative depletion of
the primordial follicle population A quantitative evaluation of primordial
follicles should be conducted for Fl females, the number of animals, ovar-
ian section selection, and section sample size should be statistically appro-
priate for the evaluation procedure used
Examination should include enumeration of the number of primordial fol-
licles, which can be combined with small growing follicles (see paragraphs
(g)(l) and (g)(2) of this guideline), for comparison of treated and control
ovaries
(11) Weanlings. For Fl and F2 weanlings, histopathologtcal examina-
tion of treatment-related abnormalities noted at macroscopic examination
should be considered, if such evaluation were deemed appropriate and
would contribute to the interpretation of the study data.
(f) Data and reporting—(1) Treatment of results. Data should be
reported individually and summarized in tabular form, showing for each
test group the types of change and the number of animals displaying each
type of change
(2) Evaluation of study results, (i) An evaluation of test results, in-
cluding the statistical analysis, should be provided This should include
an evaluation of the relationship, or lack thereof, between the exposure
of the animals to the test substance and the incidence and seventy of all
abnormalities
8
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(n) When appropriate, historical control data should be used to en-
hance interpretation of study results Historical data, when used, should
be compiled, presented, and analyzed in an appropriate and relevant man-
ner In order to justify its use as an analytical tool, information such as
the dates of study conduct, the strain and source of the animals, and the
vehicle and route of administration should be included
(m) Statistical analysis of the study findings should include sufficient
information on the method of analysis, so that an independent reviewer/
statistician can reevaluate and reconstruct the analysis
(iv) In any study which demonstrates an absence of toxic effects, fur-
ther investigation to establish absorption and bioavailabihty of the test sub-
stance should be considered
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J and 40 CFR part 160, subpart J, the
following specific information should be reported Both individual and
summary data should be presented
(i) Species and strain
(u) Toxic response data by sex and dose, including indices of mating,
fertility, gestation, birth, viability, and lactation, offspring sex ratio,
precoital interval, including the number of days until mating and the num-
ber of estrous periods until mating, and duration of gestation calculated
from day 0 of pregnancy The report should provide the numbers used
in calculating all indices
(m) Day (week) of death dunng the study or whether animals sur-
vived to termination, date (age) of litter termination
(iv) Toxic or other effects on reproduction, offspring, or postnatal
growth
(v) Developmental milestone data (mean age of vaginal opening and
preputial separation, and mean anogenital distance, when measured)
(vi) An analysis of P and Fl females cycle pattern and mean estrous
cycle length.
(vu) Day (week) of observation of each abnormal sign and its subse-
quent course
(vm) Body weight and body weight change data by sex for P, Fl,
and F2 animals.
(ix) Food (and water, if applicable) consumption, food efficiency
(body weight gain per gram of food consumed), and test matenal con-
sumption for P and Fl animals, except for the period of cohabitation
-------
(\) Total cauda epididymal sperm number, homogemzation-resistant
testis spermatid number, number and percent of progressively motile
sperm, number and percent of morphologically normal sperm, and number
and percent of sperm with each identified anomaly
(xi) Stage of the estrous cycle at the time of termination for P and
Fl parental females
(XH) Necropsy findings
(xm) Implantation data and postimplantation loss calculations for P
and Fl parental females
(xiv) Absolute and adjusted organ weight data
(xv) Detailed description of all histopathological findings
(xvi) Adequate statistical treatment of results
(xvu) A copy of the study protocol and any amendments should be
included
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Bolon, B et al Differential follicle counts as a screen for chemi-
cally induced ovarian toxicity in mice results from continuous breeding
bioassays Fundamental and Applied Toxicology 39 1-10(1997)
(2) Bucci, T J et al The effect of sampling procedure on differential
ovarian follicle counts Reproductive Toxicology 11(5) 689-696 (1997)
(3) Gray, L E et al A dose-response analysis of methoxychlor-m-
duced alterations of reproductive development and function m the rat Fun-
damental and Applied Toxicology 12 92-108 (1989)
(4) Heindel, J J and R E Chapin, (eds) Part B Female Reproductive
Systems, Methods in Toxicology, Academic, Orlando, FL (1993)
(5) Hemdel, J J et al Histological assessment of ovarian follicle num-
ber in mice as a screen of ovarian toxicity In Growth Factors and the
Ovary, A N. Hirshfield (ed ), Plenum, NY, pp 421-426 (1989)
(6) Korenbrot, C C et al Preputial separation as an external sign of
pubertal development m the male rat Biology of Reproduction 17:298-
303(1977)
(7) Linder, RE et al Endpomts of spermatoxicity m the rat after
short duration exposures to fourteen reproductive toxicants Reproductive
Toxicology 6 491-505 (1992)
10
-------
(8) Manson, J M and Y J Kang Test methods for assessing female
reproductive and developmental toxicology In Principles and Methods
of Toxicology, A W Hayes (ed ), Raven, New York (1989)
(9) Organization for Economic Cooperation and Development, No
416 Two Generation Reproduction Toxicity Study, Guidelines for Testing
of Chemicals [C(83)44 (Final)] (1983)
(10) Pederson, T and H Peters Proposal for classification of oocytes
and follicles in the mouse ovary Journal of Reproduction and Fertility
17 555-557 (1988)
(11) Russell, LD et al Histological and Histopathological Evalua-
tion of the Testis, Cache River, Clearwater, FL (1990).
(12) Sadleir, R M F S Cycles and seasons, In Reproduction m Mam-
mals' I Germ Cells and Fertilization, C R Auston and R V Short (eds ),
Cambridge, NY (1979)
(13) Seed, J , R E Chapm, E D Clegg, L A Dostal, R H Foote, M E
Hum, G R Klinefelter, S L Makns, S D Perreault, S Schrader, D Seyler,
R Sprando, K A Treinen, D N R Veeramachanem, and L D Wise Meth-
ods for assessing sperm motihty, morphology, and counts in the rat, rabbit,
and dog a consensus report Reproductive Toxicology 10(3) 237-244
(1996).
(14) Smith, B J et al Comparison of random and serial sections in
assessment of ovarian toxicity Reproductive Toxicology 5 379-383 (1991)
(15) Thomas, J A Toxic responses of the reproductive system. In'
Casarett and Doull's Toxicology, MO Amdur, J Doull, and C.D
Klaassen (eds ), Pergamon, NY (1991)
(16) U.S Environmental Protection Agency OPP Guideline 83-4
Reproductive and Fertility Effects Pesticide Assessment Guidelines, Sub-
division F, Hazard Evaluation Human and Domestic Animals Office of
Pesticides and Toxic Substances, Washington, DC, EPA-540/9-82-025
(1982)
(17) US Environmental Protection Agency Subpart E—Specific
Organ/Tissue Toxicity, 40 CFR 798 4700 Reproduction and Fertility Ef-
fects
(18) US Environmental Protection Agency Health Effects Test
Guidelines, OPPTS 870 3250, 90-Day Dermal Toxicity, July 1998
(19) US Environmental Protection Agency Health Effects Test
Guidelines, OPPTS 870 3465, 90-Day Inhalation Toxicity, July 1998
11
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(20) U S Environmental Protection Agency Reproductive Toxicity
Risk Assessment Guidelines Federal Register 61 FR 56274-56322
(1996)
(21) Working, PK and M Hurtt Computerized videomicrographic
analysis of rat sperm motihty Journal of Andrology 8330-337 (1987)
(22) Zemck, H et al Assessment of male reproductive toxicity a
risk assessment approach In Principles and Methods ofToxicohg\, A W
Hayes (ed.), Raven, NY (1994)
12
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£EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-210
August 1938
Health Effects Test
Guidelines
OPPTS 870.4100
ChronicToxicity
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in title 40, chap-
ter I, subchapter R of the Code of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of the Na-
tional Technical Information Service (NTIS) and the guidelines published
by the Organization for Economic Cooperation and Development (OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S Environ-
mental Protection Agency under the Toxic Substances Control Act (15
USC 2601) and the Federal Insecticide, Fungicide and Rodenttcide Act
(7 USC I36,etseq.)
Final Guideline Release: This guideline is available from the U.S
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
m PDF (portable document format) from EPA's World Wide Web site
(http //www.epa.gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.4100 Chronic toxicity.
(a) Scope—(1) Applicability This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798 3260 Chronic Toxicity,
OPP 83-1 Chronic Feeding—Two Species, Rodent and Nonrodent (Pes-
ticide Assessment Guidelines, Subdivision F—Hazard Evaluation, Human
and Domestic Animals) EPA report 540/09-82-025, 1982, and OECD 452
Chronic Toxicity studies
(b) 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 A chronic toxicity study should generate data from
which to identify the majority of chronic effects and 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 neu-
rological, physiological, biochemical, and hematological effects and expo-
sure-related- morphological (pathological) effects
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline The following definitions also apply to this test guideline.
Chrome toxicity is the adverse effects occurring as a result of the
repeated daily exposure of experimental animals to a chemical by the oral,
dermal, or inhalation routes of exposure
Cumulative toxicity is the adverse effects of repeated doses occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissue
Dose in a chronic toxicity study is the amount of test substance ad-
ministered daily via the oral, dermal or inhalation routes for a period of
at least 12 months Dose is expressed as weight of the test substance
(grams, milligrams) per unit body weight (BW) of test animal (milligram
per kilogram), or as weight of the test substance in parts per million (ppm)
in food or dnnkmg water per day For inhalation exposure, dose is ex-
pressed as weight of the test substance per unit volume of air (milligrams
per liter) or as parts per million per day For dermal exposure, dose is
expressed as weight of the test substance (grams, milligrams) per unit body
weight of the test animal (milligrams per kilogram) or as weight of the
substance per unit of surface area (milligrams per square centimeter) per
day
No-observed-effect-level (NOEL) is the maximum dose used in a
study which produces no adverse effects The NOEL is usually expressed
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in terms of the weight of a test substance given daily per unit weight
of test animal (milligrams per kilogram per day)
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
BW (expected human exposure may indicate the need for a higher dose
level), using the procedures described for this study, produces no observ-
able toxic effects and if toxicity would not be expected based upon data
of structurally related compounds, a full study using three dose levels
might not be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain
Testing should be performed with two mammalian species, one a rodent
and the other a nonrodent The rat is the preferred rodent species and the
dog is the preferred nonrodent species Commonly used laboratory strains
should be employed If other mammalian species are used, the tester
should provide justification/reasoning for their selection
(a) Age/weight (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8 weeks
of age.
(C) Dosing of dogs should begin between 4 and 6 months of age
and in no case later than 9 months of age
(D) At commencement of the study, the weight variation of animals
used should be within 20 percent of the mean weight for each sex.
(E) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(in) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level
(B) Females should be nulhparous and nonpregnant
(iv) Numbers. (A) For rodents, at least 40 animals (20 males and
20 females) and for nonrodents (dogs) at least 8 animals (4 females and
4 males) should be used at each dose level and concurrent control group.
(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 ef-
-------
fects The Agency must be notified if excessive early deaths or other prob-
lems are encountered that might compromise the integrity of the study
(D) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required
(E) Each animal should be assigned a unique identification number
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Rodents may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal The biological properties of the test substance or toxic effects
(e g , morbidity, excitability) may indicate a need for individual caging.
Rodents should be housed individually in dermal studies and dunng expo-
sure in inhalation studies Caging should be appropriate to the nonrodent
species However, it is recommended that dogs are housed individually
(B) The temperature of the experimental animal rooms should be at
22 ± 3 °C
(C) The relative humidity of the experimental animal rooms should
be 50 ± 20 percent
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark.
(E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test. Animals should be fed and watered ad libitum with
food replaced at least weekly
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
penod of quarantine An acclimation penod of at least 5 days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu-
ent is needed it should not elicit toxic effects itself nor substantially alter
the chemical or lexicological properties of the test substance It is rec-
ommended that wherever possible the use of an aqueous solution be the
first choice, followed by consideration of solution in oil, and finally, solu-
tion in other vehicles
(11) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Pnor to the initiation
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of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound, and, if technically feasible, the
names and quantities of contaminants and impurities
(m) If the test or control substance is to be incorporated into feed
or another vehicle, the period dunng which the test substance is stable
in such a mixture should be determined prior to the initiation of the study
Its homogeneity and concentration should be determined prior to the initi-
ation of the study and periodically dunng the study Statistically random-
ized samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation, and storage procedures are being followed, and that the
appropriate concentration of the test or control substance is contained in
the mixture
(3) Control groups. A concurrent control group is required 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 required
(4) Satellite group. A satellite group of 40 animals (20 animals per
sex) for rodents and 8 animals (4 animals per sex) for nonrodents may
be treated with the high-dose level for 12 months and observed for revers-
ibility, persistence, or delayed occurrence of toxic effects for a post-treat-
ment of appropriate length, normally not less than 28 days. In addition,
a control group of 40 animals (20 animals per sex) for rodents and 8 ani-
mals (4 animals per sex) for nonrodents should be added to the satellite
study.
(5) Dose levels and dose selections, (i) In chronic toxicity tests, it
is desirable to determine a dose-response relationship as well as a NOEL
Therefore, at least three dose levels with a control group and, where appro-
priate, a vehicle control (corresponding to the concentration of the vehicle
at the highest exposure level) should be used Dose levels should be spaced
to produce a gradation of effects A rationale must be provided for the
doses selected
(n) The highest-dose level should elicit signs of toxicity without sub-
stantially altering the normal life span of the animal The highest dose
should be determined based on the findings from a 90-day study to ensure
that the dose used is adequate to assess the chronic toxicity of the test
substance Thus, the selection of the highest dose to be tested is dependent
upon changes observed in several lexicological parameters in subchronic
studies The highest dose tested need not exceed 1,000 mg/kg/day If der-
mal application of the test substance produces severe skin irritation, then
it may be necessary either to terminate the study and choose a lower high-
dose level or to reduce the dose level Gross criteria for defining severe
irritation would include ulcers, fissures, exudate/crust(eschar), dead tissue,
-------
or anything leading to destruction of the functional integrity of the epider-
mis (eg caking, open sores, fissunng, eschar) Histological criteria for
defining severe irritation would include folhcular and interfolhcular crust
microulcer, mild/moderate degeneration/necrosis, moderate/marked epi-
dermal edema, marked dermal edema, and marked inflammation
(in) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest-dose level should produce no evidence of toxicity
(6) 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
(i) Oral studies. Ideally, the animals should be dosed by gavage or
with capsules on a 7-day per week basis for a period of at least 12 months.
However, based primarily on practical considerations, dosing by gavage
or capsules on a 5-day per week schedule is acceptable If the test sub-
stance is administered via in the drinking water or mixed in the diet, expo-
sure should be on a 7-day per week basis
(ii) Dermal studies. (A) Preparation of animal skin Shortly before
testing, fur should be clipped from not less than 10 percent of the body
surface area for application of the test substance In order to dose approxi-
mately 10 percent of the body surface, the area starting at the scapulae
(shoulders) to the wmg of the ileum (hipbone) and half way down the
flank on each side of the animal should be shaved Shaving should be
earned out approximately 24 hours before dosing Repeated clipping or
shaving is usually needed at approximately weekly intervals. When clip-
ping or shaving the fur, care should be taken to avoid abrading the skin
which could alter its permeability
(B) Preparation of test substance Liquid test substances are generally
used undiluted, except as indicated in paragraph (e)(5)(n) of this guideline.
Solids should be pulverized when possible The substance should be moist-
ened sufficiently with water or, when necessary, with a suitable vehicle
to ensure good contact with the skin When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account The volume of application should be
kept constant, e g less than 100 uJL for the mouse and less than 300 uL
for the rat Different concentrations of test solution should be prepared
for different dose levels
(C) Administration of test substance The duration of exposure should
be at least for 12 months Ideally the animals should be treated with test
substance for at least 6 h/day on a 7-day per week basis However, based
on practical considerations, application on a 5-day per week basis is ac-
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ceptable Dosing should be conducted at approximately the same time each
day The test substance should be applied uniformly over the treatment
site The surface area covered may be less for highly toxic substances
As much of the area should be covered with as thin and uniform a film
as possible For rats, the test substance may be held in contact with the
skm \\ ith a porous gauze dressing and nommtatmg tape if necessary The
test site should be further covered in a suitable manner to retain the gauze
dressing plus test substance and to ensure that the animals cannot ingest
the test substance. The application site should not be covered when the
mouse is the species of choice The test substance may be wiped from
the skin after the six-hour exposure period to prevent mgestion
(111) Inhalation studies (A) The animals should be exposed to the
test substance for 6 h/day on a 7-day per week basis, for a period of at
least 12 months However, based primarily on practical considerations, ex-
posure for 6 hours per day on a 5-day per week basis is acceptable.
(B) The animals should be tested in dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19 percent, and uniform conditions
throughout the exposure chamber Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into surround-
ing areas It is not normally necessary to measure chamber oxygen con-
centration if airflow is adequate
(C) The selection of a dynamic inhalation chamber should be appro-
priate for the test substance and test system When a whole body chamber
is used, individual housing must be used to minimize crowding of the
test animals and maximize their exposure to the test substance To ensure
stability of a chamber atmosphere, the total volume occupied by the test
animals should not exceed 5 percent of the volume of the test chamber
It is recommended, but not required, that nose-only or head-only exposure
be used for aerosol studies in order to minimize oral exposures due to
animals licking compound off their fur The animals should be acclimated
and heat stress minimized
(D) The temperature at which the test is performed should be main-
tained at 22 ± 2 °C The relative humidity should be maintained between
40-60 percent, but in certain instances (e g, use of water vehicle) this
may not be practicable
(E) The rate of air flow should be monitored continuously but re-
corded at least three times during the exposure
(F) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 mm
(G) The actual concentrations of the test substance should be meas-
ured m the breathing zone During the exposure period, the actual con-
-------
centrations of the test substance should be held as constant as practicable,
monitored continuously or intermittently depending on the method of anal-
ysis Chamber concentration may be measured using gravimetric or analyt-
ical methods, as appropriate If trial run measurements are reasonably con-
sistent (± 10 percent for liquid aerosol, gas, or vapor, ± 20 percent for
dry aerosol), then two measurements should be sufficient If measurements
are not consistent, three to four measurements should be taken Whenever
the test substance is a formulation, or it is necessary to formulate the test
substance with a vehicle for aerosol generation, the analytical concentra-
tion must be reported for the total formulation, and not just for the active
ingredient (AI). If, for example, a formulation contains 10 percent AI and
90 percent inerts, a chamber of analytical limit concentration of
2 mg/L would consist of 0 2 mg/L of the AI It is not necessary to analyze
inert ingredients provided the mixture at the animal's breathing zone is
analogous to the formulation, the grounds for this conclusion must be pro-
vided in the study report If there is some difficulty measunng chamber
analytical concentration due to precipitation, nonhomogeneous mixtures,
volatile components, or other factors, additional analysis of inert compo-
nents may be necessary
(H) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concentra-
tions with respect to particle size. The mass median aerodynamic diameter
(MMAD) particle size range should be between 1-3 ^irn. The particle size
of hygroscopic materials should be small enough when dry to assure that
the size of the swollen particle will still be within the 1-3 p.m range Meas-
urements of aerodynamic particle size in the animal's breathing zone
should be measured during a tnal run. If MMAD values for each exposure
level are within 10 percent of each other, then two measurements during
the exposures should be sufficient If pretest measurements are not within
10 percent of each other, three to four measurements should be taken
(I) Feed should be withheld during exposure Water may also be with-
held during exposure
(7) Observation period, (i) Animals should be observed for a period
of at least 12 months
(u) Animals in a satellite group (if used) scheduled for follow-up ob-
servations should be kept for at least 28 days further without treatment
to detect recovery from, or persistence of, toxic effects
(8) Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality Appropriate actions should
be taken to minimize loss of animals to the study (e g , necropsy or refrig-
eration of those animals found dead and isolation or sacrifice of weak
or moribund animals) General clinical observations should be made at
least once a day, preferably at the same time each day, taking into consid-
-------
eration the peak period of anticipated effects after dosing The clinical
condition of the animal should be recorded
(a) A careful clinical examination should be made at least once prior
to the initiation of treatment (to allow for within subject comparisons) and
once weekly dunng treatment in all animals These observations should
be made outside the home cage, preferably in a standard arena, and at
similar times on each occasion Effort should be made to ensure that vari-
ations in the observation conditions are minimal. Observations should be
detailed and carefully recorded, preferably using scoring systems, explic-
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes in skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonomic activity (e g , lacnmation,
piloerection, pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of clomc or tonic
movements, stereotypies (e g, excessive grooming, repetitive circling) or
bizarre behavior (e g , self-mutilation, walking backwards) should be re-
corded.
(in) Once, near the end of the first year of the exposure period and
in any case not earlier than in month 11, assessment of motor activity,
grip strength, and sensory reactivity to stimuli of different types (e g , vis-
ual, auditory, and propnoceptive stimuli) should be conducted in rodents
Further details of the procedures that could be followed are described in
the references listed under paragraphs (h)(2), (h)(6), (h)(8), (h)(9), (h)(10),
and (h)(17) of this guideline
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits.
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vi) Body weights should be recorded individually for all animals
once pnor to the administration of the test substance, once a week dunng
the first 13 weeks of study and at least once every 4 weeks thereafter,
unless signs of clinical toxicity suggest more frequent weighing to facili-
tate momtonng of health status
(vii) Measurements of feed consumption should be determined weekly
dunng the first 13 weeks of the study and at approximately monthly inter-
vals thereafter unless health status or body weight changes dictate other-
wise. Measurements of water consumption should be determined at the
same intervals if the test substance is administered in the dnnktng water
8
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(via) Moribund animals should be removed and sacrificed when no-
ticed and the time of death should be recorded as precisely as possible
All survivors should be sacrificed at the end of the study period
(9) Clinical pathology. Hematology, clinical chemistry, and unnal-
ysis should be performed on 10 rats per sex per group, and on all non-
rodents In rodents, the parameters should be examined at approximately
6 month intervals during the conduct of the study and at termination If
possible, these collections should be from the same animals at each inter-
val In nonrodents, the parameters should be examined once or twice prior
to initiation of treatment, at 6-month intervals during the conduct of the
study, and at termination If hematological and biochemical effects were
seen m the subchronic study, testing should also be performed at 3 months
Overnight fasting of animals pnor to blood sampling is recommended
(i) Hematology. The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombm time or
activated partial thromboplastm time.
(it) Clinical chemistry. (A) Parameters which are considered appro-
priate to all studies are 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 clini-
cal toxicity
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, calcium (nonrodent), phosphorus (nonrodent), chloride
(nonrodent), glucose, total cholesterol, urea nitrogen, creatmine, total pro-
tein, total bilirubm (nonrodent), and albumin More than two hepatic en-
zymes, (such as alanme anunotransferase, aspartate aminotransferase, alka-
line phosphatase, sorbitol dehydrogenase, or gamma glutamyl
transpeptidase) should also be measured Measurements of addtional en-
zymes (of hepatic or other origin) and bile acids, may also be useful
(C) If a test chemical has an effect on the hematopoietic system,
rettculocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobm, and
cholmesterases
(m) Urinalysis. Unnalysis for rodents should be performed at the
end of the study using timed urine collection Unnalysis for nonrodents
should be performed pnor to treatment, midway through treatment and
at the end of the study using timed unne collection Unnalysis determma-
-------
tions include appearance, volume, osmolahty or specific gravity, pH, pro-
tein, glucose, and blood/blood cells
(10) Ophthalmological examination. Examinations should be made
of all animals using an ophthalmoscope or equivalent device prior to the
administration of the test substance and at termination of the study on
10 rats of each sex in the high-dose and control groups and preferably
in all nonrodents, but at least the control and high-dose groups should
be examined If changes m eyes are detected, all animals should be exam-
ined
(11) Gross necropsy, (i) All animals should be subjected to a full
sross necropsy which includes examination of the external surface of the
oody, all orifices, and the cranial, thoracic and abdominal cavities and their
contents
(11) At least the liver, kidneys, adrenals, testes, epididymides, ovaries,
uterus, nonrodent thyroid (with parathyroid), spleen, brain, and heart
should be weighed wet as soon as possible after dissection to avoid drying
The lungs should be weighed if the test substance is administered by the
inhalation route
(m) The following organs and tissues, or representative samples there-
of, should be preserved m a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present).
(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial,
preferably m close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen
(F) Urogemtal system—kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovanes, female mammary gland
(G) Other—all gross lesions and masses, skin
(iv) In inhalation studies, the entire respiratory tract, including nose,
pharynx, larynx, and paranasal sinuses should be examined and preserved
10
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In dermal studies, skin from treated and adjacent control skin sites should
be examined and preserved
(v) 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
(vi) Information from clinical pathology and other m-hfe data should
be considered before microscopic examination, since they may provide sig-
nificant guidance to the pathologist
(12) Histopathology. (i) The following histopathology should be per-
formed
(A) Full histopathology on the organs and tissues (listed under para-
graph (e)(ll)(m) of this guideline) of all rodents and nonrodents in the
control and high-dose groups, and all rodents and nonrodents that died
or were killed during the study The examination should be extended to
all animals in all dosage groups if treatment-related changes are observed
in the high-dose group
(B) All gross lesions m all animals
(C) Target tissues in all animals
(11) If the results show substantial alteration of the animal's normal
life span, or other effects that might compromise the significance of the
data, the next lower levels should be examined fully as described in para-
graph (e)(12)(i) of this guideline
(hi) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours pnor
to trimming.
(0 Data and reporting—(1) Treatment of results. (0 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.
(11) When applicable, all observed results (quantitative and qualitative)
should be evaluated by an appropriate statistical method Any generally
accepted statistical methods may be used, the statistical methods including
significance criteria should be selected during the design of the study
11
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(2) Evaluation of study results. The findings of a chronic toxicity
study should be evaluated tn conjunction with the findings of preceding
studies and considered in terms of the toxic effects as well as 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
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J, 40 CFR part 160, and the OECD Prin-
ciples of GLP (ISBN 92-64-12367-9), the following specific information
should be reported'
(i) Test substance characterization should include
(A) Chemical identification
(B) Lot or batch number
(C) Physical properties
(D) Punty/impunties
(u) Identification and composition of any vehicle used
(in) Test system should contain data on
(A) Species and strain of animals used and rationale for selection
if other than that recommended
(B) Age including body weight data and sex
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(D) Identification of animal diet
(E) Acclimation period
(iv) Test procedure should include the following data'
(A) Method of randomization used
(B) Full descnption of experimental design and procedure
(C) Dose regimen including levels, methods, and volume
(v) Test results
(A) Group animal data Tabulation of toxic response data by species,
strain, sex and exposure level for
12
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(7) Number of animals exposed
(2) Number of animals showing signs of toxicity
(3) Number of animals dying
(B) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(7) Time of death dunng the study or whether animals survived to
termination.
(2) Time of observation of each abnormal sign and its subsequent
course
(3) Body weight data
(4) Feed and water (if collected) consumption data
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water
(6) Results of ophthalmological examinations
(7) Results of hematological tests performed
(8) Results of clinical chemistry tests performed
(9) Unnalysis tests performed and results
(10) Results of observations made
(11) Necropsy findings, including absolute and relative (to body
weight) organ weight data
(12) Detailed descnption of all histopathological findings
(13) Statistical treatment of results, where appropriate
(iv) In addition, for inhalation studies the following should be re-
ported
(A) Test conditions The following exposure conditions must be re-
ported'
(1) Descnption of exposure apparatus including design, type, dimen-
sions, source of air, system for generating paniculate and aerosols, method
of conditioning air, treatment of exhaust air and the method of housing
the animals in a test chamber
(2) The equipment for measuring temperature, humidity, and particu-
late aerosol concentrations and size should be described
13
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(B) Exposure data These data should be tabulated and presented with
mean values and a measure of variability (e g , standard deviation) and
should include
(/) Airflow rates through the inhalation equipment
(2) Temperature and humidity of air
(3) Actual (analytical or gravimetric) concentration in the breathing
zone
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air)
(5) Particle size distribution, calculated MMAD, and geometric stand-
ard deviation (GSD)
(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines
(g) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment. The study must be conducted m compliance with GLP regula-
tions as described by the Agency (40 CFR parts 160 and 792), and the
OECD principles of GLP (ISBN 92-64-12367-9)
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline
(1) Bemtz, KF Measurement of Chronic Toxicity Methods ofToxi-
cology. Ed. G E. Paget Blackweil, Oxford pp 82-131(1970).
(2) Crofton K.M , Howard J L, Moser V C , GUI M.W , Leiter L W ,
Tilson H A., MacPhail, R C Interlaboratory Comparison of Motor Activity
Experiments* Implication for Neurotoxicological Assesments
Neurotoxicol Teratol 13,599-609 (1991)
(3) D'Aguanno, W Drug Safety Evaluation—Pre-CImical Consider-
ations Industrial Pharmacology Neuroteptic Vol I, Ed. S Fielding and
H Lai Futura, Mt Kisco, NY pp 317-332(1974)
(4) Fitzhugh, 0 G Chronic Oral Toxicity, Appraisal of the Safety
of Chemicals m Foods, Drugs and Cosmetics The Association of Food
and Drug Officials of the United States pp 36-45 (1959, 3rd Printing
1975)
(5) Food Safety Council Proposed System for Food Safety Assess-
ment Prepared by the Scientific Committee, Food Safety Council Food
and Cosmetic Toxicology Vol 16, Supplement 2 (December 1978)
14
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(6) Gad S C A Neuromuscular Screen for Use in Industrial Toxi-
cology Journal of Toxicology and Environmental Health 9, 691-704
(1982)
(7) Goldenthal, El and D'Aguanno, W Evaluation of Drugs, Ap-
praisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics The
Association of Food and Drug Officials of the United States pp 60-67
(1959, 3rd Printing 1975)
(8) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals Environmental Health Catena Document No 60. (1986)
(9) Meyer O A, Tilson H A , Byrd W C , Riiey M T A Method for
the Routine Assessment of Fore- and Hind-Limb Gnp Strength of Rats
and Mice. Neurobehav Toxicol 1,233-236 (1979)
(10) Moser V C , McDamel K M , Phillips P M Rat Strain and Stock
Comparisons using a Functional Observational Battery: Baseline Values
and Effects of Armtraz Toxicol Appl PharmacoL 108, 267-283 (1991)
(11) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances,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, Washington, DC (1977)
(12) National Center for Toxicological Research Appendix B, Report
of Chronic Studies Task Force Committee, Apnl 13-21, 1972 National
Center for Toxicological Research, Rockville, MD (1972)
(13) Organization for Economic Cooperation and Development.
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452
Chronic Toxicity Studies, Pans (1981)
(14) Page, NP Chronic Toxicity and Carcinogemcity Guidelines.
Journal of Environmental Pathology and Toxicology 11 161-182 (1977)
(15) Schwartz, E Toxicology of Neuroleptic Agents. Industrial Phar-
macology. Neuroleptic S Fielding and H Lai Futura, Mt Kisco, NY
pp. 203-221 (1974)
(16) Toxicity and Clinical Tnal Subcommittee, Committee on Safety
of Medicine (November 1977)
(17) Tupper, D E , Wallace R B Utility of the Neurologic Examina-
tion in Rats Acta Neurobwl Exp 40, 999-1003 (1980)
(18) United States Pharmaceutical Manufacturers Association Guide-
lines for the Assessment of Drug and Medical Device Safety in Animals
(1977)
15
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(19) Wemgand K, Brown G, Hall R et al (1996) Harmonization
of Animal Clinical Pathology Testing in Toxicity and Safety Studies
Fundam andAppl Toxicol 29 198-201
(20) World Health Organization (WHO) Guidelines for Evaluation
of Drugs for Use in Man WHO Technical Report Series No 563
(21) World Health Organization (WHO) Part I Environmental Health
Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemi-
cals WHO, Geneva (1978)
(22) World Health Organization (WHO) Principles for Pre-Climcal
Testing of Drug Safety, WHO Technical Report Series No 341 WHO,
Geneva (1966)
16
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EPA
United States
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA712-C-98-211
August 1998
Health Effects Test
Guidelines
OPPTS 870.4200
Carcinogenicity
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared m Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S C 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 USC I36,etseq )
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelmes/OPPTS Harmonized Test
Guidelines "
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OPPTS 870.4200 Carcmogenicity.
(a) Scope—(1) Applicability This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used m developing this har-
monized OPPTS test guideline are 40 CFR 798 3300 Oncogemcity, OPP
83-2 Carcmogenicity—Two Species, Rat and Mouse Preferred (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation, Human and
Domestic Animals) EPA report 540/09-82^025, 1982, and OECD 451
Carcmogenicity Studies
(b) Purpose. The objective of a long-term Carcmogenicity study is
to observe test animals for a major portion of their life span for develop-
ment of neoplastic lesions dunng or after exposure to various doses of
a test substance by an appropriate route of administration
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this guide-
line The following definitions also apply to this guideline
Carcmogenicity is the development of neoplastic lesions as a result
of the repeated daily exposure of experimental animals to a chemical by
the oral, dermal, or inhalation routes of exposure
Cumulative toxicity is the adverse effects of repeated dose occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissues.
Dose in a Carcmogenicity study is the amount of test substance ad-
ministered via the oral, dermal or inhalation routes for a period of up to
24 months Dose is expressed as weight of the test substance (grams, milli-
grams) per unit body weight of test animal (milligram per kilogram), or
as weight of the test substance in parts per million (ppm) in food or drink-
ing water When exposed via inhalation, dose is expressed as weight of
the test substance per unit volume of air (milligrams per liter) or as parts
per million
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) Test procedures—(1) Animal selection—(i) Species and strain.
Testing should be performed on two mammalian species Rats and mice
are the species of choice because of their relatively short life spans, limited
cost of maintenance, widespread use in pharmacological and toxicologicai
studies, susceptibility to tumor induction, and the availability of inbred
or sufficiently characterized strains Commonly used laboratory strains
-------
should be used If other mammalian species are used, the tester should
provide justification/reasoning for their selection
(11) Age/weight. (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age
(C) At commencement of the study, the weight variation of animals
used should be within 20 percent of the mean weight for each sex
(D) Studies using prenatal or neonatal animals may be recommended
under special conditions
(m) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level
(B) Females should be nulhparous and nonpregnant
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females)
should be used at each dose level and concurrent control group.
(B) If interim sacrifices are planned, the number should be increased
by the number of animals scheduled to be sacrificed dunng the course
of the study.
(C) For a meaningful and valid statistical evaluation of long term
exposure and for a valid interpretation of negative results, the number of
animals in any group should not fall below 50 percent at 15 months in
mice and 18 months in rats Survival in any group should not fall below
25 percent at 18 months in mice and 24 months in rats
(D) The use of adequate randomization procedures for the proper allo-
cation of animals to test and control groups is required to avoid bias
(E) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must "not interfere with clear observation of
each animal The biological properties of the test substance or toxic effects
(e.g, morbidity, excitability) may indicate a need for individual caging
Animals should be housed individually in dermal studies and dunng expo-
sure in inhalation studies
(B) The temperature of the experimental animal rooms shouid be at
22 ± 3 °C
(C) The relative humidity of the experimental animal rooms should
be 50 ± 20 percent.
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(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark
(E) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure uniform distribution and ade-
quacy of nutntional requirements of the species tested and for impurities
that might influence the outcome of the test Animals should be fed and
watered ad libitum with food replaced at least weekly
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine An acclimation penod of at least five days is rec-
ommended
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu-
ent is needed, it should not elicit toxic effects itself It is recommended
that wherever possible the use of an aqueous solution be considered first,
followed by consideration of solution in oil, and finally solution in other
vehicles
(11) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Pnor to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the punty of the test compound, and, if possible, the name and
quantities of contaminants and impurities
(in) If the test or control substance is to be incorporated into feed
or another vehicle, the penod during which the test substance is stable
in such a mixture should be determined pnor to the initiation of the study
Its homogeneity and concentration should be determined pnor to the initi-
ation of the study and periodically dunng the study Statistically random-
ized samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation, and storage procedures are being followed, and that the
appropnate concentration of the test or control substance is contained in
the mixture.
(3) Control groups. A concurrent control group (50 males and 50
females) is required. This group should be untreated or if a vehicle is
used m adrmnistenng the test substance, a vehicle control group. If the
toxic properties of the vehicle are not known, both untreated and vehicle
control groups are required
(4) Dose levels and dose selection, (i) For nsk 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. A rationale for the doses selected must be provided
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(u) The highest-dose level should elicit signs of toxicity without sub-
stantially altering the normal life span due to effects other than tumors
The highest dose should be determined based on the findings from a 90-
day study to ensure that the dose used is adequate to asses the carcinogenic
potential of the test substance Thus, the selection of the highest dose to
be tested is dependent upon changes observed in several toxicological pa-
rameters in subchronic studies The highest dose tested need not exceed
i,000mg/kg/day
(111) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest-dose level should produce no evidence of toxicity
(v) For skin carcmogemcity studies, when toxicity to the skin is a
determining factor, the highest dose selected should not destroy the func-
tional integrity of the skm, the intermediate doses should be a minimally
irritating dose, and the low dose should be the highest nommtatmg dose
(vi) The criteria for selecting the dose levels for sbn carcinogemcity
studies, based on gross and histopathologic dermal lesions, are as follows
(A) Gross catena for reaching the high dose
(/) Erythema (moderate)
(2) Scaling.
(3) Edema (mild)
(4) Alopecia.
(5) Thickening
(B) Histologic catena for reaching the high-dose1
(./) Epidermal hyperplasia
(2) Epidermal hyperkeratosis
(3) Epidermal parakeratosis
(4) Adnexal atrophy/hyperplasia
(5) Fibrosis.
(6) Spongiosis (minimal-mild)
(7) Epidermal edema (minimal-mild)
(5) Dermal edema (minimal-moderate)
(9) Inflammation (moderate)
4
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(C) Gross criteria for exceeding the high-dose
(/) Ulcers, fissures
(2) Exudate/crust (eschar)
(3) nonviable (dead) tissues
(4) Anything leading to destruction of the functional integrity of the
epidermis (e g , caking, fissunng, open sores, eschar)
(D) Histologic catena for exceeding the high-dose
(/) Crust (interfolhcular and folhcular)
(2) Microulcer
(3) Degeneration/necrosis (mild to moderate)
(4) Epidermal edema (moderate to marked)
(5) Dermal edema (marked)
(6) Inflammation (marked)
(5) 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.
(i) Oral studies. If the test substance is administered by gavage, the
animals are dosed with the test substance on a 7-day per week basis for
a penod of at least 18 months for mice and hamsters and 24 months for
rats However, based primarily on practical considerations, dosing by ga-
vage on a 5-day per week basis is acceptable If the test substance is
administered m the drinking water or mixed in the diet, then exposure
should be on a 7-day per week basis
(u) Dermal studies. (A) The animals should be treated with the test
substance for at least 6 h/day on a 7-day per week basis for a penod
of at least 18 months for mice and hamsters and 24 months for rats How-
ever, based primanly on practical considerations, application on a 5-day
per week basis is acceptable Dosing should be conducted at approximately
the same time each day
(B) Fur should be clipped weekly from the dorsal area of the trunk
of the test animals Care should be taken to avoid abrading the skm which
could alter its permeability A minimum of 24 hours should be allowed
for the skin to recover before the next dosing of the animal
(C) Preparation of test substance Liquid test substances are generally
used undiluted, except as indicated in paragraph (e)(4)(vi) of this guideline
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Solids should be pulverized when possible The substance should be moist-
ened sufficiently with water or, when necessary, with a suitable vehicle
to ensure good contact with the skin When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account The volume of application should be
kept constant, e g less than 100 u.L for the mouse and less than 300 |iL
for the rat. Different concentrations of test solution should be prepared
for different dose levels
(D) The test substance should be applied uniformly over a shaved
area which is approximately 10 percent of the total body surface area
In order to dose approximately 10 percent of the body surface, the area
starting at the scapulae (shoulders) to the wing of the ileum (hipbone)
and half way down the flank on each side of the animal should be shaved.
With highly toxic substances, the surface area covered may be less, but
as much of the area as possible should be covered with as thin and uniform
a film as practical
(E) Dunng the exposure period, the application site should not be
covered when mice or hamsters are the species of choice. For rats, the
test substance may be held m contact with the skin with a porous gauze
dressing and nonimtatmg tape if necessary 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 The test sub-
stance may be wiped from the skin after the 6-hour exposure to prevent
mgestion
(ui) Inhalation studies. (A) The animals should be exposed to the
test substance for 6 h/day on a 7-day per week basis, for a period of at
least 18 months in mice and 24 months in rats However, based primarily
on practical considerations, exposure for 6 h/day on a 5-day per week
basis is acceptable
(B) The animals should be tested m dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19 percent, and uniform conditions
throughout the exposure chamber Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into surround-
ing areas.
(C) The selection of a dynamic inhalation chamber should be appro-
priate for the test substance and test system Where a whole body chamber
is used to expose animals to an aerosol, individual housing must be used
to minimize crowding of the test animals and maximize their exposure
to the test substance To ensure stability of a chamber atmosphere, the
total volume occupied by the test animals should not exceed 5 percent
of the volume of the test chamber It is recommended, but not required,
that nose-only or head-only exposure be used for aerosol studies in order
-------
to minimize oral exposures due to animals licking compound off their fur
Heat stress should be minimized
(D) The temperature at which the test is performed should be mam-
tamed at 22 ± 20 °C 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
(E) The rate of air flow should be monitored continuously but re-
corded at least three times dunng exposure
(F) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 minutes
(G) The actual concentrations of the test substance should be meas-
ured in the breathing zone Dunng the exposure period, the actual con-
centrations of the test substance should be held as constant as practicable,
monitored continuously or intermittently depending on the method of anal-
ysis. Chamber concentrations may be measured using gravimetric or ana-
lytical methods as appropriate If trial run measurements are reasonably
consistent (± 10 percent for liquid aerosol, gas, or vapor, ± 20 percent
for dry aerosol), the two measurements should be sufficient If measure-
ments are not consistent, then three to four measurements should be taken
Whenever the test substance is a formulation, or it is necessary to formu-
late the test substance with a vehicle for aerosol generation, the analytical
concentration must be reported for the total formulation, and not just for
the active ingredient (AI) If, for example, a formulation contains 10 per-
cent AI and 90 percent merts, a chamber of analytical limit concentration
of 2 mg/L would consist of 0 2 mg/L of the AI It is not necessary to
analyze inert ingredients provided the mixture at the animal's breathing
zone is analogous to the formulation, the grounds for this conclusion must
be provided in the study report If there is some difficulty measuring cham-
ber analytical concentration due to precipitation, nonhomogeneous mix-
tures, volatile components, or other factors, additional analysis of inert
components may be necessary
(H) Dunng the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concentra-
tions with respect to particle size Measurement of aerodynamic particle
size in the animals's breathing zone should be measured dunng a tnal
run. If MM AD values for each exposure level are within 10 percent of
each other, then two measurements during the exposures should be suffi-
cient If pretest measurements are not within 10 percent of each other,
three to four measurements should be taken The mass median aero-
dynamic diameter (MMAD) particle size range should be between 1-3
fim The particle size of hygroscopic materials should be small enough
when dry to assure that the size of the swollen particle will still be within
the 1-3 Jim range
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(I) Feed should be withheld during exposure Water may also be with-
held dunng exposure
(6) Observation period. It is necessary that the duration of the car-
cmogemcity study comprise the majority of the normal life span of the
strain of animals 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 penods, and where any
other species are used, consultation with the Agency in regard to the dura-
tion of the study is advised
(7) Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality Appropnate actions should
be taken to minimize loss of animals from the study (e g, necropsy or
refrigeration of those animals found dead and isolation or sacrifice of weak
or monbund animals)
(«) A careful clinical examination should be made at least once week-
ly Observations should be detailed and carefully recorded, preferably
using explicitly defined scales Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonormc effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
m the level of activity, gait and posture, reactivity to handling or sensory
stimuli, altered strength and stereotypies or bizarre behavior (eg., self-
mutilation, walking backwards)
(in) Body weights should be recorded individually for all animals
once pretreatment, once a week dunng the first 13 weeks of the study
and at least once every 4 weeks, thereafter, unless signs of clinical toxicity
suggest more frequent weighing to facilitate momtonng of health status
(iv) Measurements of feed consumption should be determined weekly
dunng the first 13 weeks of the study and at approximately monthly inter-
vals thereafter unless health status or body weight changes dictate other-
wise Measurements of water consumption should be determined at the
same intervals if the test substance is administered in the drinking water.
(v) Monbund animals should be removed and sacnficed when noticed
and the time of death should be recorded as precisely as possible. At the
end of the study penod, all survivors should be sacnficed
(8) Clinical pathology. At 12 months, 18 months, and at terminal
sacnflce, 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 from the terminal sacnfice If
these data, or data from the pathological examination indicate a need, then
the 12- and 18-month blood smears should also be examined Differential
blood counts should be performed for the next lower groups if there is
8
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a major discrepancy between the highest group and the controls It clinical
observations suggest a deterioration in health of the animals during the
study, a differential blood count of the affected animals should be per-
formed
(9) Gross necropsy, (i) A complete gross examination should be per-
formed on all animals, including those that died during the experiment
or were killed m a moribund condition
(n) At least the liver, kidneys, adrenals, testes, epididymides, ovanes,
uterus, spleen, brain and heart should be weighed wet as soon as possible
after dissection to avoid drying The lungs should be weighed if the test
substance is administered by the inhalation route The organs should be
weighed from interim sacrifice animals as well as from at least 10 animals
per sex per group at terminal sacrifice
(iii) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present)
(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or Ubial,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen
(F) Urogenital system—kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovanes, female mammary gland
(G) Other—all gross lesions and masses, skin
(iv) In inhalation studies, the entire respiratory tract, including nose,
pharynx, larynx, and paranasal sinuses should be examined and preserved
In dermal studies, skin from treated and adjacent control skin sites should
be examined and preserved
(v) 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 essential for appropriate and valid
histopathological examination
(vi) Information from clinical pathology, and other in-life data should
be considered before microscopic examination, since they may provide sig-
nificant guidance to the pathologist
(10) Histopathology. (i) The following histopathology should be per-
formed'
(A) Full histopathology on the organs and tissues listed under para-
graph (d)(9)(m) of this guideline of all animals in the control and high-
dose groups and all animals that died or were killed during the study
(B) AH gross lesions in all animals
(C) Target organs in all animals
(11) If the results show substantial alteration of the animal's normal
life span, the induction of effects that might affect a neoplastic response,
or other effects that might compromise the significance of the data, the
next lower dose levels should be examined as described under paragraph
(d)(10)(i) of this guideline
(111) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours pnor
to trimming
(e) Data and reporting—(1) Treatment of results, (i) 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.
(11) When applicable, all observed results (quantitative and qualitative)
should be evaluated by an appropriate statistical method Any generally
accepted statistical methods may be used, the statistical methods including
significance criteria should be selected during the design of the study
(2) Evaluation of study results, (i) The findings of a carcmogenicity
study should be evaluated in conjunction with the findings of previous
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
seventy of abnormalities (including behavioral and clinical abnormalities),
10
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gross lesions, identified target organs, body weight changes, effects on
mortality, and any other general or specific toxic effects
(n) In any study which demonstrates an absence of toxic effects, fur-
ther investigation to establish absorption and bioavailabhty of the test sub-
stance should be considered
(111) In order for a negative test to be acceptable, it should meet the
following cntena No more than 10 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 15 months for mice and 18 months
for rats Survival should not fall below 25 percent at 18 months for mice
and 24 months for rats
(iv) The use of historical control data from an appropriate time penod
from the same testing laboratory (i e, the incidence of tumors and other
suspect lesions normally occurring under the same laboratory conditions
and in the same strain of animals employed m the test) is helpful for as-
sessing the significance of changes observed in the current study.
(3) Test report (i) In addition to the reporting requirements as speci-
fied under 40 CFR part 792, subpart J, 40 CFR part 160, and the OECD
Principles of GLP (ISBN 92-64-12367-9), the following specific informa-
tion should be reported
(A) Test substance characterization should include
(/) Chemical identification
(2) Lot or batch number
(3) Physical properties
(4) Punty/impunties
(5) Identification and composition of any vehicle used
(B) Test system should contain data on
(/) Species and strain of animals used and rationale for selection if
other than that recommended
(2) Age including body weight data and sex.
(3) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(4) Identification of animal diet
(5) Acclimation penod
(C) Test procedure should include the following data
11
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(/) Method of randomization used
(2) Full descnption of experimental design and procedure
(3) Dose regimen including levels, methods, and volume
(4) Test results, (i) Group animal data Tabulation of toxic response
data by species, strain, sex and exposure level for
(A) Number of animals exposed
(B) Number of animals showing signs of toxicity
(C) Number of animals dying
(u) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(A) Time of death dunng 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) Feed and water consumption data, when collected
(E) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or dnnking water
(F) Results of clinical pathology when performed
(G) Necropsy findings including absolute/relative organ weight data.
(H) Detailed descnption of all histopathological findings
(I) Statistical treatment of results where appropriate
(J) Historical control data.
(hi) In addition, for inhalation studies the following should be re-
ported:
(A) Test conditions The following exposure conditions must be re-
ported
(/) Descnption of exposure apparatus including design, type, dimen-
sions, source of air, system for generating paniculate and aerosols, method
of conditioning air, treatment of exhaust air and the method of housing
the animals in a test chamber
(2) The equipment for measunng temperature, humidity, and panicu-
late aerosol concentrations and size should be descnbed
12
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(B) Exposure data These should be tabulated and presented with
mean values and a measure of variability (eg standard deviation) and
should include
(7) Airflow rates through the inhalation equipment
(2) Temperature and humidity of air
(j) Actual (analytical or gravimetric) concentration in the breathing
zone
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air)
(5) Particle size distribution, calculated MMAD and geometric stand-
ard deviation (GSD)
(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines
(f) Quality assurance. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with the GLP reg-
ulations as described by the Agency (40 CFR parts 160 and 792) and
the OECD Principles of GLP (ISBN 92-64-12367-9)
(g) References. The following references should be consulted for ad-
ditional background information on this guideline:
(1) Benitz, KF Measurement of Chronic Toxicity Methods of Toxi-
cology Ed G E Paget Blackwell, Oxford pp 82-131 (1970)
(2) D'Aguanno, W Drug Safety Evaluation—Pre-Clinical Consider-
ations. Industrial Pharmacology Neuroleptic Vol I Ed S Fielding and
H Lai. Futura, Mt Kisco, NY pp 317-332(1974)
(3) Department of Health and Welfare The Testing of Chemicals for
Carcmogenicity, Mutagemcity, Teratogemcity Minister of Health and
Welfare Department of Health and Welfare, Canada (1975)
(4) Fitzhugh, O.G Chronic Oral Toxicity, 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 (1959, 3rd Printing
1975)
(5) Food Safety Council Proposed System for Food Safety Assess-
ment Prepared by the Scientific Committee, Food Safety Council Food
and Cosmetic Toxicology , Vol 16, Supplement 2 (December 1978).
13
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(6) Goldenthal, E I and D'Aguanno, W Evaluation of Drugs, Ap-
praisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics The
Association of Food and Drug Officials of the United States pp 60-67
(1959, 3rd Printing 1975)
(7) International Union Against Cancer Carcmogemcity Testing
UCC Technical Report Series, Vol 2 Ed I Berenblum International
Union Against Cancer, Geneva (1969)
(8) Leon, B K J and Laskin, S Number and Species of Experimental
Animals for Inhalation Carcmogemcity Studies Paper presented at Con-
ference on Target Organ Toxicity Cincinnati, OH (September 1975)
National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences Washington, DC (1977)
(10) National Cancer Institute Report of the Subtask Group on Car-
cinogen Testing to the Interagency Collaborative Group on Environmental
Carcmogenesis United States National Cancer Institute Bethesda, MD
(1976)
(11) National Center for Toxicological Research Appendix B, Report
of Chronic Studies Task Force Committee, Apnl 13-21, 1972 National
Center for Toxicological Research, Rockville, MD (1972)
(12) Organization for Economic Cooperation and Development
Guidelines for Testing of Chemicals, Section 4-HeaIth Effects, Part 451
Carcmogemcity Studies, Pans (1981)
(13) Page, NP Chronic Toxicity and Carcmogemcity Guidelines.
Journal of Environmental Pathology and Toxicology 11:161-182 (1977)
(14) Page, NP Concepts of a Bioassay Program in Environmental
Carcmogenesis, Advances m Modern Toxicology Vol 3, Ed Kraybill and
Mehlman Hemisphere, Washington, DC pp 87-171(1977)
(15) Schwartz, E Toxicology of Neuroleptic Agents. Industrial Phar-
macology. Neuroleptics S Fielding and H Lai Futura, Mt Kisco, NY
pp 203-221 (1974)
(16) Sontag, J M et al Guidelines for Carcinogen Bioassay in Small
Rodents NCI-CS-TR-l United States Cancer Institute, Division of Cancer
Control and Prevention, Carcmogenesis Bioassay Program Bethesda, MD
(17) Summary of the EPA Workshop on Carcmogenesis Bioassay via
the Dermal Route EPA Report 50/6-89-002, 50/6-89-003 Washington,
DC
14
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(18) The Atlas of Dermal Lesions, EPA Report 20t-2004, U S Envi-
ronmental Protection Agency, Washington, DC
(19) Toxicity and Clinical Tnal Subcommittee, Committee on Safety
of Medicine, November, 1977
(20) United States Environmental Protection Agency Office of Test-
ing and Evaluation Proposed health effects test standards for toxic sub-
stances control act test rules 40 CFR Part 772 Standard for Development
of Test Data. Subpart D Chronic Health Effects FEDERAL REGISTER No
91 (44 FR 27350-27362)
(21) United States Pharmaceutical Manufacturers Association Guide-
lines for the Assessment of Drug and Medical Device Safety in Animals
(1977)
(22) World Health Organization (WHO) Guidelines for Evaluation
of Drugs for Use in Man WHO Technical Report Series No. 563 (WHO),
Geneva(1975)
(23) World Health Organization (WHO) Part I Environmental Health
Criteria 6 Principles and Methods for Evaluating the Toxicity of Chemi-
cals (WHO), Geneva (1978)
(24) World Health Organization (WHO) Principles for Pre-CHnical
Testing of Drug Safety WHO Technical Report Series No 341 (WHO),
Geneva(1966)
15
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&EPA
United Slates
Environmental Protection
Agency
Prevention Pesticides
and Toxic Substances
(7101)
EPA 712-C-9 8-212
August 1998
Health Effects Test
Guidelines
OPPTS 870.4300
Combined Chronic
Toxicity/Carcinogenicity
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INTRODUCTION
This guideline is one of a senes of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations
The Office of Prevention, Pesticides and Toxic "Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD)
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U S.C. 2601) and the Federal Insecticide, Fungicide and Rodenttcide Act
(7U.SC. 136, et seq )
Final Guideline Release: This guideline is available from the U S
Government Printing Office, Washington, DC 20402 on disks or paper
copies call (202) 512-0132 This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http //www epa gov/epahome/research htm) under the heading "Research-
ers and Scientists/Test Methods and Guidehnes/OPPTS Harmonized Test
Guidelines''
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OPPTS 870 4300 Combined chronic toxicity/carcmogemcity.
(a) Scope—(1) Applicability. This guideline is intended to-meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U S C 136, et seq ) and the Toxic Substances
Control Act (TSCA) (15 U S C 2601)
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798 3320 Combined Chronic
Toxicity/Oncogemcity, OPP 83-5 Combined Chronic Toxicity/
Oncogenicity (Pesticide Assessment Guidelines, Subdivision F—Hazard
Evaluation, Human and Domestic Animals) EPA report 540/09-82-025,
1982, and OECD 453 Combined Chronic Toxicity/Carcmogemcity Stud-
ies
(b) Purpose. The objective of a combined chronic toxicUy/carcmo-
gemcity 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 majonty of chronic and
carcmogemcity effects and determine dose-response relationships The de-
sign and conduct should allow for the detection of neoplastic effects and
a determination of the carcinogenic potential as well as general toxicity,
including neurological, physiological, biochemical, and hematological ef-
fects and exposure-related morphological (pathology) effects
(c) Definitions. The definitions in section 3 of TSCA and the defini-
tions in 40 CFR Part 792—Good Laboratory Practice Standards (GLP)
apply to this guideline The following definitions also apply to this guide-
line
Carcmogemcity is the development of neoplastic lesions as a result
of the repeated daily exposure of experimental animals to a chemical by
the oral, dermal, or inhalation routes of exposure
Chrome toxicity is the adverse effects occurring as a result of the
repeated daily exposure of experimental animals to a chemical by the oral,
dermal, or inhalation routes of exposure
Cumulative toxicity is the adverse effects of repeated dose occurring
as a result of prolonged action on, or increased concentration of, the ad-
ministered test substance or its metabolites in susceptible tissues
Dose in a combined chronic toxicity/carcmogemcity study is the
amount of test substance administered via the oral, dermal, or inhalation
routes for a period of up to 24 months Dose is expressed as weight of
the test substance per unit body weight of test animal (milligrams per kilo-
gram), or as weight of the test substance m parts per million (ppm) in
food or drinking water When exposed via inhalation, dose is expressed
as weight of the test substance per unit volume of air (milligrams per
liter) or as parts per million per day For dermal application, dose is ex-
-------
pressed as weight of the test substance (grams, milligrams) per unit body
weight, of the test animal (milligrams per kilogram) or as weight of the
substance per unit surface area (milligrams per square centimeter) per day
No-observed-effect-level (NOEL) is the maximum dose used in a
study which produces no observed adverse effects The NOEL is usually
expressed m terms of the weight of a test substance given daily per unit
weight of test animal (milligrams per kilogram per day)
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance
(d) 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
oaervable toxic effects or if toxic effects would not be expected based
upon data of structurally related compounds, then a full study using three
dose levels might not be necessary
(e) Test procedures—(1) Animal selection—(i) Species and strain.
Preliminary studies providing data on acute, subchronic, and metabolic re-
sponses should have been earned out to permit an appropriate choice of
"'iimals (species and strain) As discussed in other guidelines, the mouse
and rat have been most widely used for assessment of carcinogenic poten-
tial, while the rat and dog have been most often studied for chronic tox-
icity For the combined chronic toxicity/carcmogemcity study via the oral
and inhalation routes, the rat is the species of choice and for the dermal
route, the mouse is species of choice If other species are used, the tester
should provide justification/reasoning for their selection The strain se-
lected should be susceptible to the carcinogenic or toxic effect of the class
of substances being tested, if known, and provided it does not have a spon-
taneous background incidence too high for meaningful assessment Com-
mc iy used laboratory strains should be employed.
(n) Age/weight (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization
(B) Dosing should generally begin no later than 8 weeks of age
(C) At commencement of the study, the weight variation of animals
used should be within 20 percent of the mean weight for each sex
(D) Studies using prenatal or neonatal animals may be recommended
under special conditions
(m) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level
(B) Females should be nulhparous and nonpregnant
-------
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females)
should be used at each dose level and concurrent control group At least
20 additional rodents (10 males and 10 females) should be used for sat-
ellite dose groups and the satellite control group The purpose of the sat-
ellite group is to allow for the evaluation of chronic toxicity after 12
months of exposure to the test substance
(B) For a meaningful and valid statistical evaluation of long term
exposure and for a valid interpretation of negative results, the number of
animals in any group should not fall below 50 percent at 15 months in
mice and 18 months in rats Survival in any group should not fall below
25 percent at 18 months in mice and 24 months in rats
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(D) Each animal should be assigned a unique identification number
Dead animals (and their preserved organs) and tissues, and microscopic
slides should be identified by reference to the unique numbers assigned
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal The biological properties of the test substance or toxic effects
(e g , morbidity, excitability) may indicate a need for individual caging
Rodents should be housed individually m dermal studies and during expo-
sure in inhalation studies
(B) The temperature of the experimental animal rooms should be at
22±3°C
(C) The relative humidity of the experimental animal rooms should
be 50 ± 20 percent.
(D) Where lighting is artificial, the sequence should be 12 hours light/
12 hours dark.
(E) Control and test animals should be fed from the same batch and
lot The feed should be analyzed to assure uniform distribution and ade-
quacy of nutritional requirements of the species tested and for impurities
that might influence the outcome of the test Animals should be fed and
watered ad libitum with food replaced at least weekly
(F) The study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine An acclimation penod of at least five days is rec-
ommended
(2) Control and test substances (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle If a vehicle or dilu-
-------
ent is needed, it should not elicit toxic effects itself nor substantially alter
the chemical or toxicological properties of the test substance It is rec-
ommended that wherever possible the usage of an aqueous solution be
considered first, followed by consideration of a solution in oil, and finally
solution in other vehicles
(n) One lot of the test substance should be used throughout the dura-
tion of the study if possible, and the research sample should be stored
under conditions that maintain its purity and stability Pnor to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the punty of the test compound, and, if possible, the name and
quantities of contaminants and impurities
(m) If the test or control substance is to be incorporated into feed
or another vehicle, the period during which the test substance is stable
in such a mixture should be determined prior to the initiation of the study
Its homogeneity and concentration should be determined prior to the initi-
ation of the study and periodically dunng the study Statistically random-
L.d samples of the mixture should be analyzed to ensure that proper mix-
ing, formulation, and storage procedures are being followed, and that the
appropriate concentration of the test or control substance is contained in
the mixture
(3) Control groups. A concurrent control group is required. 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 required.
(4) Dose levels and dose selection, (i) For nsk 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. A rationale for the doses selected must be provided
(u) The highest dose level in rodents should elicit signs of toxicity
without substantially altenng the normal life span due to effects other than
tumors The highest dose should be determined based on the findings from
a 90-day study to ensure that the dose used is adequate to assess the chron-
ic toxicity and the carcinogenic potential of the test substance Thus, the
selection of the highest dose to be tested is dependent upon changes ob-
served in several toxicological parameters in subchronic studies. The high-
est dose tested need not exceed 1,000 mg/kg/day
(m) The intermediate-dose levels should be spaced to produce a gra-
dation of toxic effects
(iv) The lowest-dose level should produce no evidence of toxicity
4
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(v) For skin carcmogemcity studies, when toxictty to the skin is a
determining factor, the highest dose selected should not destroy the func-
tional integrity of the skin, the intermediate doses should be a minimally
irritating dose and the low dose should be the highest nommtating dose
(vi) The criteria for selecting the dose levels for skin carcmogemcity
studies, based on gross and histopathologic dermal lesions, are as follows
(A) Gross criteria for reaching the high dose
(7) Erythema (moderate)
(2) Scaling
(J) Edema (mild)
(4) Alopecia
(5) Thickening
(B) Histologic catena for reaching the high dose
(7) Epidermal hyperplasia
(2) Epidermal hyperkeratosis
(3) Epidermal parakeratosis
(4) Adnexal atrophy/hyperplasia
(5) Fibrosis
(6) Spongiosis (minimal-mild)
(7) Epidermal edema (minimal-mild)
(8) Dermal edema (minimal-moderate)
(9) Inflammation (moderate)
(C) Gross criteria for exceeding the high dose
(1} Ulcers-fissures, exudate/crust (eschar)tnonviable (dead) tissues
(2) Anything leading to destruction of the functional integrity of the
epidermis (e g , caking, fissunng, open sores, eschar)
(D) Histologic catena for exceeding the high-dose
(7) Crust (mterfollicular and folhcular)
(2) Microulcer
(3) Degeneration/necrosis (mild to moderate)
5
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(4) Epidermal edema (moderate to marked)
(5) Dermal edema (marked)
(6) Inflammation (marked)
(5) Administration of the test substance. The three mam 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
(0 Oral studies. If the test substance is administered by gavage, the
animals are dosed with the test substance on a 7-day per week basis for
a period of at least 18 months for mice and hamsters and 24 months for
rats However, based primarily on practical considerations, dosing by ga-
vage on a 5-day per week basis is acceptable If the test substance is
administered in the drinking water or mixed m the diet, then exposure
should be on a 7-day per week basis
(11) Dermal studies. (A) Preparation of animal skin Shortly before
testing, fur should be clipped from not less than 10 percent of the body
surface area for application of the test substance In order to dose approxi-
mately 10 percent of the body surface, the area starting at the scapulae
(shoulders) to the wing of the ileum (hipbone) and half way down the
flank on each side of the animal should be shaved Shaving should be
earned out approximately 24 hours before dosing Repeated clipping or
shaving is usually needed at approximately weekly intervals When clip-
ping or shaving the fur, care should be taken to avoid abrading the skin
which could alter its permeability
(B) Preparation of test substance Liquid test substances are generally
used undiluted, except as indicated in paragraph (e)(4)(vi) of this guideline
Solids should be pulverized when possible The substance should be moist-
ened sufficiently with water or, when necessary, with a suitable vehicle
to ensure good contact with the skin When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account The volume of application should be
kept constant, e g less than 100 uL for the mouse and less than 300 p.L
for the rat Different concentrations of test solution should be prepared
for different dose levels
(C) Administration of test substance The duration of exposure should
be at least 18 months for mice and hamsters and 24 months for rats. Ideal-
ly, the animals should be treated with test substance for at least 6 h/day
on a 7-day per week basis However, based on practical considerations,
application on a 5-day per week basis is acceptable. Dosing should be
conducted at approximately the same time each day The test substance
should be applied uniformly over the treatment site The surface area cov-
ered may be less for highly toxic substances As much of the area should
-------
be covered with as thin and uniform a film as possible For rats, the test
substance may be held in contact with the skin with a porous gauze dress-
ing and nommtating tape if necessary The test site should be further cov-
ered in a suitable manner to retain the gauze dressing plus test substance
and to ensure that the animals cannot ingest the test substance The appli-
cation site should not be covered when the mouse is the species of choice
The test substance may be wiped from the skin after the 6-hour exposure
period to prevent mgestion
(in) Inhalation studies. (A) The animals should be exposed to the
test substance, for 6 h/day on a 7-day per week basis, for a period of
at least 18 months in mice and 24 months in rats However, based pri-
marily on practical considerations, exposure for 6 h/day on a 5-day per
week basis is acceptable
(B) The animals should be tested in dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19 percent, and uniform conditions
throughout the exposure chamber Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into surround-
ing areas It is not normally necessary to measure chamber oxygen con-
centration if airflow is adequate
(C) The selection of a dynamic inhalation chamber should be appro-
priate for the test substance and test system Where a whole body chamber
is used, individual housing must be used to minimize crowding of the
test animals and maximize their exposure to the test substance To ensure
stability of a chamber atmosphere, the total volume occupied by the test
animals should not exceed 5 percent of the volume of the test chamber.
It is recommended, but not required, that nose-only or head-only exposure
be used for aerosol studies in order to minimize oral exposures due to
animals licking compound off their fur The animals should be acclimated
and heat stress minimized
(D) The temperature at which the test is performed should be main-
tained at 22 ± 2 °C 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
(E) The rate of air flow should be monitored continuously but re-
corded at least three times dunng the exposure
(F) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 minutes
(G) The actual concentrations of the test substance shall be measured
in the animal's breathing zone Dunng the exposure period, the actual con-
centrations of the test substance shall be held as constant as practicable
and monitored continuously or intermittently depending on the method of
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analysis Chamber concentration may be measured using gravimetric or
analytical methods as appropriate If trial run measurements are reasonably
consistent (± 10 percent for liquid aerosol, gas, or vapor, ± 20 percent
for dry aerosol), then two measurements should be sufficient If measure-
ments are not consistent, three to four measurements should be taken
Whenever the test substance is a formulation, or it is necessary to formu-
late the test substance with a vehicle for aerosol generation, the analytical
concentration must be reported for the total formulation and not just for
the active ingredient (AI) If, for example, a formulation contains 10 per-
cent AI and 90 percent metis, a chamber analytical limit concentration
of 2 mg/L would consist of 0.2 mg/L of the AI It is not necessary to
analyze inert ingredients provided the mixture at the animal's breathing
zone is analogous to the formulation; the grounds for this conclusion must
be provided in the study report If there is some difficulty in measuring
chamber analytical concentration due to precipitation, nonhomogeneous
mixtures, volatile components, or other factors, additional analyses of inert
components may be necessary
(H) Dunng the development of the generating system, particle size
nnalysis should be performed to establish the stability of aerosol concentra-
tions with respect to particle size The mass median aerodynamic diameter
(MMAD) particle size range should be between 1-3 Jim The particle size
of hygroscopic materials should be small enough when dry to assure that
the size of the swollen particle will still be within the 1-3 jj.m range Meas-
urements of aerodynamic particle size in the animal's breathing zone
should be measured during a trial run If MMAD values for each exposure
level are within 10 percent of each other, then two measurements during
the exposures should be sufficient If pretest measurements are not within
10 percent of each other, three to four measurements should be taken
(I) Feed should be withheld during exposure Water may also be with-
held during exposure
(6) Observation period, (i) This tune penod 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 study is advised
(u) Animals in a satellite group to assess chronic toxicity should be
observed for 12 months
(T\ Observation of animals, (i) Observations should be made at least
twice each day for morbidity and mortality Appropriate actions should
be taken to minimize loss of animals to the study (e g , necropsy or refrig-
eration of those animals found dead and isolation or sacrifice of weak
or moribund animals) General clinical observations should be made at
least once a day, preferably at the same time each day, taking into consid-
8
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eration the peak period of anticipated effects after dosing The clinical
condition of the animal should be recorded
(11) A careful clinical examination should be made at least once pnor
to the initiation of treatment (to allow for within subject comparisons) and
once weekly during treatment in all animals These observations should
be made outside the home cage, preferably in a standard arena, and at
similar times on each occasion Effort should be made to ensure that vari-
ations in the observation conditions are minimal Observations should be
detailed and carefully recorded, preferably using scoring systems, explic-
itly defined by the testing laboratory Signs noted should include, but not
be limited to, changes in skin, fur, eyes, mucous membranes, occurrence
of secretions and excretions and autonormc activity (e g , lacnmation,
piloerection, pupil size, unusual respiratory pattern) Changes in gait, pos-
ture and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e g , excessive grooming, repetitive circling) or
bizarre behavior (e g , self-mutilation , walking backwards) should be re-
corded
(m) Once, near the end of the first year of the exposure period and
in any case not earlier than in month 11, assessment of motor activity,
grip strength, and sensory reactivity to stimuli of different types (e g , vis-
ual, auditory, and proprioceptive stimuli) should be conducted in rodents
Further details of the procedures that could be followed are described in
the references listed under paragraphs (h)(2), (h)(7), (h)(9), (h)(12),
(h)(13), and (h)(25) of this guideline
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any func-
tional deficits
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would significantly
interfere with functional test performance
(vi) Body weights should be recorded individually for all animals
once pnor to administration of the test substance, once a week during the
first 13 weeks of the study and at least once every 4 weeks thereafter
unless signs of clinical toxicity suggest more frequent weighing to facili-
tate monitoring of health status
(VH) Measurements of feed consumption should be determined weekly
dunng the first 13 weeks of the study and then at approximately monthly
intervals unless health status or body weight changes dictate otherwise
Measurements of water consumption should be determined at the same
intervals if the test matenal is administered in drinking water
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(vin) Moribund animals should be removed and sacrificed when no-
ticed and the time ot death should be recorded as precisely as possible
At the end of the study period, all survivors should be sacnficed Animals
in the satellite gioup should be sacrificed after 12 months of exposure
to the test substance (interim sacrifice)
(8) Clinical pathology. Hematology, clinical chemistry and
unnalyses should be performed from 10 animals per sex per group The
parameters should be examined at approximately 6 month intervals during
the first 12 months of the study If possible, these collections should be
from the same animals at each interval If hematological and biochemical
effects are seen in the subchronic study, testing should also be performed
at 3 months Overnight fasting of animals pnor to blood sampling is rec-
ommended
(i) Hematology The recommended parameters are red blood cell
count, hemoglobin concentration, hematocnt, mean corpuscular volume,
mean corpuscular hemoglobin, and mean corpuscular hemoglobin con-
centration, white blood cell count, differential leukocyte count, platelet
count, and a measure of clotting potential, such as prothrombm time or
activated partial thromboplastm time
(11) Clinical chemistry (A) Parameters which are considered appro-
priate to all studies are electrolyte balance, carbohydrate metabolism, and
liver and kidney function The selection of specific tests will be influenced
oy observations on the mode of action of the substance and signs of clini-
cal toxicity.
(B) The recommended clinical chemistry determinations are potas-
sium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total
protein, and albumin More than two hepatic enzymes, (such as alanine
aminotransferase, aspartate armnotransferase, alkaline phosphatase, sorbitol
dehvdrogenase, or gamma glutamyl transpeptidase) should also be meas-
^. Measurements of addtional enzymes (of hepatic or other origin) and
bile acids, may also be useful
(m) If a test chemical has an effect on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated
(iv) Other determinations that should be earned out if the test chemi-
cal is known or suspected of affecting related measures include calcium,
phosphorus, fasting tnglycendes, hormones, methemoglobin, and
chohnesterases
(v) Unnalyses Unnalysis for rodents should be performed at the end
of the first year of the study using timed urine collection Unnalysis deter-
minations include appearance, volume, osmolahty or specific gravity, pH,
protein, glucose, and blood/blood cells
10
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(9) Ophthalmological examination. Examinations should be made
on all animals using an ophthalmoscope or an equivalent device prior to
the administration of the test substance and at termination of the study
on 10 animals per sex in the high-dose and control groups If changes
in eyes are detected, all animals should be examined
(10) Gross necropsy, (i) A complete gross examination should be
performed on all animals, including those which died during the experi-
ment or were killed in a moribund condition
(u) At least, the liver, kidneys, adrenals, testes, epididyrmdes, ovanes,
uterus, spleen, brain, and heart should be trimmed and weighed wet, as
soon as possible after dissection to avoid drying The lungs should be
weighed if the test substance is administered by the inhalation route The
organs should be weighed from interim sacrifice animals as well as from
at least 10 animals per sex per group at terminal sacrifice
(in) The following organs and tissues, or representative samples there-
of, should be preserved m a suitable medium for possible future
histopathological examination
(A) Digestive system—salivary glands, esophagus, stomach, duode-
num, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder
(when present)
(B) Nervous system—brain (multiple sections, including cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic, and lumbar), eyes (retina, optic nerve)
(C) Glandular system—adrenals, parathyroid, thyroid
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
the route of administration and another one distant from the route of ad-
ministration to cover systemic effects), spleen
(F) Urogenital system—kidneys, urinary bladder, prostate, testes,
epididymides, seminal vesicle(s), uterus, ovanes, female mammary gland
(G) Other—all gross lesions and masses, skin
(iv) In inhalation studies, the entire respiratory tract, including nose,
pharynx, larynx, and paranasal sinuses should be examined and preserved
In dermal studies, skin from treated and adjacent control skin sites should
be examined and preserved
(v) Inflation of lungs and unnary bladder with a fixative is the optimal
method for preservation of these tissues The proper inflation and fixation
11
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of the lungs in inhalation studies is essential for appropriate and valid
histopathological examination
(vi) Information from clinical pathology and other m-hfe data should
be considered before microscopic examination, since these data may pro-
vide significant guidance to the pathologist
(12) Histopathology. (i) The following histopathology should be per-
formed
(A) Full histopathology on the organs and tissues, listed under para-
graph (e)(10)(iu) of this guideline of all animals in the control and high
dose groups and of all animals that died or were killed dunng the study
(B) All gross lesions in all animals
(C) Target organs in all animals
(11) If the results show substantial alteration of the animal's normal
life span, the induction of effects that might affect a neoplastic response,
or other effects that might compromise the significance of the data, the
next lower levels should be examined fully as descnbed in paragraph
(e)(12)(i) of this guideline
(111) An attempt should be made to correlate gross observations with
microscopic findings
(iv) Tissues and organs designated for microscopic examination
should be fixed in 10 percent buffered formalin or a recognized suitable
fixative as soon as necropsy is performed and no less than 48 hours prior
to trimming.
(f) Data and reporting—(1) Treatment of results, (i) 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
(it) When applicable, all observed results, quantitative and qualitative,
should be evaluated by an appropriate statistical method Any generally
accepted statistical methods may be used, the statistical methods including
significance catena should be selected dunng the design of the study
(2) Evaluation of study results, (i) The findings of a combined
chronic toxicity/carcmogenicity study should be evaluated in conjunction
with the findings of previous studies and considered m 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 seventy of abnormalities (including behavioral and
clinical abnormalities), gross lesions, identified target organs, body weight
12
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changes effects on mortality and any other general or specific toxic ef-
fects
(u) In any study which demonstrates an absence of toxic effects fur-
ther investigation to establish absorption and bioavailabhty of the test sub-
stance should be considered
(111) In order for a negative test to be acceptable, it should meet the
following catena—no more than 10 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 15 months for mice and 18 months
for rats Survival should not fall below 25 percent at 18 months for mice
and 24 months for rats
(iv) The use of historical control data from an appropnate time period
from the same testing laboratory (i e, the incidence of tumors and other
suspect lesions normally occurring under the same laboratory conditions
and in the same strain of animals employed in the test) is helpful for as-
sessing the significance of changes observed in the current study.
(3) Test report (i) In addition to the reporting requirements as speci-
fied under 40 CFR part 792, subpart J, 40 CFR part 160, and the OECD
Principles of GLP (ISBN 92-64-12367-9), the following specific informa-
tion should be reported
(A) Test substance characterization should include
(]) Chemical identification
(2) Lot or batch number
(3) Physical properties
(4} Punty/impunties
(5) Identification and composition of any vehicle used
(B) Test system should contain data on
(7) Species and strain of animals used and rationale for selection if
other than that recommended
(2) Age including body weight data and sex
(3) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods
(4) Identification of animal diet
(5) Acclimation penod
(C) Test procedure should include the following data
13
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(/) Method of randomization used
(2) Full description of experimental design and procedure
(3) Dose regimen including levels, methods, and volume
(4) Test results, (i) Group animal data Tabulation of toxic response
data by species, strain, sex, and exposure level for
(A) Number of animals exposed
(B) Number of animals showing signs of toxicity
(C) Number of animals dying
(11) Individual animal data Data should be presented as summary
(group mean) as well as for individual animals
(A) Time of death dunng the study or whether animals survived to
termination
(B) Time of observation of each abnormal sign and us subsequent
course
(C) Body weight data.
(D) Feed and water consumption data, when collected
(E) Achieved dose (milligrams/kilogram body weight) as a time-
weighed average is the test substance is administered in the diet or drink-
ing water
(F) Results of ophthalmological examination, when performed
(G) Results of hematological tests performed
(H) Results of clinical chemistry tests performed
(I) Results of unnalysis tests performed
(J) Results of observations made
(K) Necropsy findings including absolute/relative organ weight data.
(L) Detailed desc option of all histopathological findings
(M) Statistical treatment of results where appropriate
(N) Historical control data
(m) In addition, for inhalation studies the following should be re-
ported
14
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(A) Test conditions The following exposure conditions must be re-
ported
(/) Description of exposure apparatus including design, type, dimen-
sions, source of air system for generating particulates and aerosols, meth-
od of conditioning air treatment of exhaust air and the method of housing
the animals in a test chamber
(2) The equipment for measuring temperature, humidity, and panicu-
late aerosol concentrations and size should be descnbed
(B) Exposure data These should be tabulated and presented with
mean values and a measure of variability (eg standard deviation) and
should include
(/) Airflow rates through the inhalation equipment
(2) Temperature and humidity of air
(3) Actual (analytical or gravimetric) concentration in the breathing
zone
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air)
(5) Particle size distribution, and calculated mass median aerodynamic
diameter (MMAD) and geometric standard deviation (GSD)
(6) Explanation as to why the desired chamber concentration and/
or particle size could not be achieved (if applicable) and the efforts taken
to comply with this aspect of the guidelines
(g) Quality assurance. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment The study must be conducted in compliance with the GLP reg-
ulations as descnbed by the Agency (40 CFR parts 160 and 792) and
the OECD Pnnciples of GLP (ISBN 92-64-12367-9)
(h) References. The following references should be consulted for ad-
ditional background information on this guideline
(1) Benitz, KF Measurement of Chrome Toxicity Methods of Toxi-
cology Ed GE Paget Blackwell, Oxford pp 82-131(1970)
(2) Crofton K M , Howard J L , Moser V C , Gill M W , Letter L.W ,
Tilson H A , MacPhail, R C Interlaboratory Companson of Motor Activity
Experiments Implication for Neurotoxicotogical Assesments.
Neurotoxicol Teratol 13,599-609 (1991)
15
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(3) D'Aguanno W Drug Safety Evaluation—Pre-Clinical Consider-
ations Industrial Pharmacology Neuroleptic Vol I Ed S Fielding and
H Lai Futura,Mt Kisco, NY pp 317-332(1974)
i
(4) Department of Health and Welfare The Testing of Chemicals jor
Carcmogemcity, Mutagemcin, Teratogemcity Minister of Health and
Welfare Department of Health and Welfare, Canada (1975)
(5) Fitzhugh, 0 G Chronic Oral Toxicity, Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics The Association of Food and
Drug Officials of the United States pp 36^5 (1959, 3rd Printing 1975)
(6) Food Safety Council Proposed system for food safety assessment
Prepared by the scientific committee, Food Safety Council Food and Cos-
metic Toxicology, Vol 16, Supplement 2 (December 1978)
(7) Gad S C A Neuromuscular Screen for Use in Industrial Toxi-
cology J Toxicol Environ Health, 9, 691-704 (1982)
(8) Goldenthal, E I and D'Aguanno, W Evaluation of Drugs, Ap-
praisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics The
Association of Food and Drug Officials of the United States pp. 60-67
(1959, 3rd Printing 1975)
(9) International Programme on Chemical Safety Principles and
Methods for the Assessment of Neurotoxicity Associated with Exposure
to Chemicals Environmental Health Criteria Document No 60 (1986)
(10) International Union Against Cancer Carcmogemcity Testing'
UCC Technical Report Series, Vol 2, Ed I Berenblum International Union
Against Cancer, Geneva (1969)
i
(11) Leon, B K J and Laskm, S Number and Species of Experimental
Animals for Inhalation Carcmogemcity Studies Paper presented at Con-
ference on Target Organ Toxicity Cincinnati, Ohio (September 1975).
(12) Meyer O A , Tilson H A , Byrd W C , Riley M T A Method
for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats
and Mice Neurobehav Toxicol 1,233-236 (1979)
(13) Moser V C , McDamel K M , Phillips P M Rat Strain and Stock "*
Comparisons using a Functional Observational Battery Baseline Values
ana Effects of Armtraz Toxicol Appl Pharmacol 108, 267-283 (1991)
(14) National Academy of Sciences Principles and Procedures for
Evaluating the Toxicity of Household Substances, A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977)
16
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(15) National Cancer Institute Report of the Subtask Group on Car-
cinogen Testing to the Interagency Collaborative Group on Environmental
Carcmogenesis United States National Cancer Institute, Bethesda, MD
(1976)
(16) National Center for Toxicological Research Appendix B Report
of Chronic Studies Task Force Committee, Apnl 13-21, 1972 National
Center for Toxicological Research, Rockville, MD (1972)
(17) Organization for Economic Cooperation and Development
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453
Combined Chronic Toxicity/Carcmogemcity Studies, Pans (1981)
(18) Page, N P Chronic Toxicity and Carcinogemcity Guidelines
Journal of Environmental Pathology and Toxicology 11 161-182 (1977)
(19) Page, NP Concepts of a Bioassay Program in Environmental
Carcmogenesis, Advances in Modern Toxicology Vol 3, Ed Kraybill and
Mehlman. Hemisphere, Washington, D C pp 87-171 (1977)
(20) Schwartz, E Toxicology of Neuroleptic Agents, Industrial Phar-
macology Neuroleptics S Fielding and H Lai Futura, Mt Kisco, NY
pp. 203-221 (1974)
(21) Son tag, J M et al Guidelines for Carcinogen Bioassay in Small
Rodents NCI-CS-TR-1 (Bethesda United States Cancer Institute, Division
of Cancer Control and Prevention, Carcmogenesis Bioassay Program.
(22) Summary of the EPA Workshop on Carcmogenesis Bioassay via
the Dermal Route EPA Report 50/6-89-002, 50/6-89-003 Washington,
DC
(23) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S Envi-
ronmental Protection Agency, Washington, D C
(24) Toxicity and Clinical Trial Subcommittee, Committee on Safety
of Medicine. (November, 1977)
(25) Tupper, D E , Wallace R B Utility of the Neurologic Examina-
tion in Rats. Acta. Neurobiol Exp 40, 999-1003 (1980)
(26) United States Environmental Protection Agency Office of Test-
ing and Evaluation Proposed Halth Effects Test Standards for Toxic Sub-
stances Control Act Test Rules 40 CFR Part 772 Standard for Develop-
ment of Test Data Subpart D Chronic Health Effects FEDERAL REGISTER
Vol.44, No91 pp 27350-27362
(27) United States Pharmaceutical Manufacturers Association Guide-
lines for the Assessment of Drug and Medical Device Safety in Animals
(1977).
17
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(28) Wemgand, K Brown, G , Hall, R et al Harmonization of Ani-
mal Clinical Pathology Testing in Toxicity and Safety Studies Fundam
&Appl Toucol 29 198-201 (1996)
(29) World Health Organization (WHO) Guidelines for Evaluation
of Drugs for Use in Man, WHO Technical Report Series No 563 WHO,
Geneva(1975)
(30) World Health Organization (WHO) Part I Environmental Health
Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemi-
cals WHO, Geneva (1978)
(31) World Health Organization (WHO) Principles for Pre-Chmcal
Testing of Drug Safety, WHO Technical Report Senes No 341 WHO,
Geneva(1966)
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