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

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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

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     (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

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     (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,

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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

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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

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    (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

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&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

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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
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 "

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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

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          (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

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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

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     (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

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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

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     (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)

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     (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

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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

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&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

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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 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

-------
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

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     (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)

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     (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)

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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

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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 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 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

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     (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

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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

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           (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)

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     (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

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     (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)

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£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 ''

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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

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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/

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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

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     (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,

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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)

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    (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)

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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

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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

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     (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-

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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

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     (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

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    (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

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     (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

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                    (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

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          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

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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, 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 "

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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)

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     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

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     (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

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     (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

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     (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

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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

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     (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

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     (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

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     (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

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     (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

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&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

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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 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 "

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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-

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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

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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

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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-

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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

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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

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     (/) 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

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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

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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
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

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     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

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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 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

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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  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

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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

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     (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

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                           INTRODUCTION
     This  guideline is one of  a  series  of test guidelines  that have been
developed by the Office  of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations

     The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has  developed  this guideline  through  a  process of harmonization  that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared m title 40, 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

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     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

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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

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     (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.

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     (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

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     (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

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     (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

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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
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

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     (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

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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)

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     (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

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     (\) 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

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    (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-

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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,

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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-

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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-

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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-

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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

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                           INTRODUCTION
    This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides  and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations

    The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a  process  of  harmonization  that
blended the testing guidance and requirements  that existed in the Office
of Pollution Prevention and Toxics  (OPPT) and  appeared m Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations  (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization  for Economic Cooperation and Development
(OECD)

    The purpose of harmonizing  these guidelines into a single  set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
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

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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

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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

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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 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-

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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

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     (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-

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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

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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
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     (10) International  Union Against Cancer  Carcmogemcity Testing'
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Against Cancer, Geneva (1969)
                                                                     i
     (11) Leon, B K J  and Laskm, S Number and Species of Experimental
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     (13) Moser V C , McDamel K M , Phillips P M Rat Strain and Stock             "*
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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
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    (16) National Center for Toxicological Research Appendix B Report
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    (17)  Organization  for  Economic  Cooperation and  Development
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    (18)  Page, N P   Chronic  Toxicity and Carcinogemcity Guidelines
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    (27) United States Pharmaceutical Manufacturers Association Guide-
lines for the Assessment of Drug and Medical Device  Safety in  Animals
(1977).

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Geneva(1966)
                                18

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