United States Prevention, Pesticides EPA712-C-02-189
Environmental Protection and Toxic Substances December 2002
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.1000
Acute Toxicity Testing
Background
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
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 Internet Web site at http:/
/www.epa.gov/opptsfrs/home/guidelin.htm.
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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) Background. The source material for this revised harmonized test
guideline is OPPTS 870.1000 Acute Toxicity Testing—Background, dated
August 1998.
(b) Purpose. The Agency considers the evaluation of toxicity fol-
lowing short term exposure to a chemical to be an integral step in the
assessment of its toxic potential under the regulatory framework of its pes-
ticide and toxic substances programs. In the assessment and evaluation
of the toxic characteristics of a substance, acute toxicity is generally per-
formed by the probable route of exposure in order to provide information
on health hazards likely to arise from short-term exposure by that route.
For pesticides, the short-term toxicity testing battery consists of acute tox-
icity tests by the oral, dermal, and inhalation routes; skin and eye irritation
testing; and testing for dermal sensitization. Data from an acute study may
serve as a basis for hazard categorization, labeling, or child-resistant pack-
aging and may also serve to designate pesticides which may be applied
only by certified applicators. It may also be an initial step in establishing
a dosage regimen in subchronic and other studies and may provide infor-
mation 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 severity of all abnormalities, including behavioral and clinical abnor-
malities, 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 (g)(l) of this guideline) and
in 40 CFR part 797 in September 1985 for the Office of Pollution Preven-
tion and Toxics (OPPT).
(2) Rejection rate analysis. In 1993, as part of its Pesticide Rejection
Rate Analysis, Agency and industry scientists met to perform a guideline-
by-guideline review of toxicology studies including acute toxicity studies.
The purpose of this guideline-by-guideline 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 (g)(2)
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-
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elation (CMA), Health Canada, and the California Department of Pesticide
Regulation (CDPR) to discuss acceptable methods for the conduct of acute
toxicity studies. The discussions of this meeting were incorporated into
a preliminary Registration Division document titled Conduct of Acute Tox-
icity Studies (see paragraph (g)(3) of this guideline). These documents sup-
plement the acute toxicology guidelines in Subdivision F.
(3) Guideline harmonization. The Series 870 Health Effects test
guidelines have been harmonized between OPP and OPPT and, where pos-
sible, with the Organization for Economic Cooperation and Development
(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.
(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 endpoints 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 incoordination, in addition to the recov-
ery from these effects during the observation period. Both dead and sur-
viving animals are necropsied to evaluate gross anatomical evidence of
organ toxicity. In selected cases, additional testing may be justified to bet-
ter characterize the kinds of abnormalities that have been found in the
organs of the necropsied animals. These sound, scientific practices rep-
resent some of the means which maximize the utility of the data obtained
from a limited number of test animals to achieve a balance between pro-
tecting humans 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 pre-
serving 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-characterized mixtures or products may
not need additional testing if there are sufficient bridging data available
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for meaningful extrapolation. In those cases, classification would be ex-
trapolated from the mixture already tested.
(ii) EPA recommends the Up-and-Down Procedure (UDP), as detailed
in this guideline and adopted by OECD as test Guideline 425 (see para-
graph (g)(4) of this guideline), to access acute oral toxicity. This method
provides a point estimate of lethality and confidence interval. A dedicated
program (AOT425StatPgm) has been developed by EPA to assist labora-
tories in the conduct of this protocol. The Agency strongly recommends
the use of this software package which is available on EPA's Internet Web
site at http://www.epa.gov/oppfeadl/harmonization. Acute oral toxicity
testing may also be performed using the Fixed Dose Method of OECD
Guideline 420 (see paragraph (g)(5) of this guideline) or the Acute Toxic
Class Method of OECD Guideline 423 (see paragraph (g)(6) of this guide-
line). These methods assess lethality within a dose range.
(iii) Weight of evidence approaches to dermal and ocular irritation.
Several factors should be considered in determining the corrosion and irri-
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)
in an acute toxicity study (for which observations of dermal reactions were
made), a dermal irritation/corrosion study again may not be needed for
labeling purposes. It should be noted, however, that often acute dermal
toxicity and dermal irritation/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 deci-
sions.
(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 merit in considering
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
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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 categories 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
(see EPA Label Review Manual (paragraph (g)(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. A globally harmonized system for classifica-
tion and labeling has been approved through the United Nations. Imple-
mentation will be phased in by United Nations countries, with schedules
to be announced. This section describes the current system in place for
pesticides in 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 LD50 of a product via the designated route of exposure.
The primary eye irritation and primary skin irritation studies measure the
severity 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 categories 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 during 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 irrita-
tion data for all of the active ingredients in a pesticide product. The dura-
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tion of restricted entry intervals is based on the severity of toxicity, with
products classified in category I requiring intervals of 48 hours or more
and products classified in category III or IV requiring intervals of 12
hours.
(5) Child-resistant packaging. FIFRA establishes standards with re-
spect to pesticide packaging of products intended for use in residential
settings in order to protect children or adults from serious illness or injury
resulting from accidental ingestion or contact with pesticides. Criteria in
40 CFR part 157 for which pesticides must be distributed or sold in child-
resistant packaging are 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 Series 885, Mi-
crobial Pesticide Test Guidelines, for pathogenicity/infectivity. In addition,
all formulations of microbial pest control agents are tested for pre-
cautionary labeling using acute toxicity tests in the OPPTS Harmonized
Test Guidelines Series 870, Health Effects Test Guidelines.
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Toxicity Categories
Study
Acute Oral
Acute Dermal
Acute Inhalation
Eye Irritation
Skin irritation
Category 1
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 corneal
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
Corneal
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
Corneal
involvement or
irritation clearing
in 7 days or less
Moderate irritation
at 72 hours
(moderate
erythema)
Category IV
>5000 mg/kg
>5000 mg/kg
>2 mg/liter
Minimal effects
clearing in less
than 24 hours
Mild or slight
irritation (no
irritation or slight
erythema)
Study
Dermal Sensitization
Study results
Product is a sensitizer or is positive
for sensitization
Study results
Product is not a sensitizer or is
negative for sensitization
(f) Regulatory applications under TSCA. (i) Acute oral toxicity
data are used to provide a basic understanding of acute effects and to
serve as a starting point for human hazard and risk assessments focused
on occupational and general population exposures.
(ii) Acute oral toxicity testing is included in testing menus to obtain
basic or "screening level" information on certain chemicals. These in-
clude higher volume/higher exposure new chemicals where TSCA section
5(e) "exposure-based" testing authorities are used to obtain a basic level
of hazard and environmental fate information; and High Production Vol-
ume existing chemicals (i.e., those produced and/or imported at or above
1 million Ibs/yr) information data set.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) U.S. Environmental Protection Agency. Pesticide Assessment
Guidelines, Subdivision F: Health Effects. EPA report 540/09-82-025, Oc-
tober 1982.
(2) U.S. Environmental Protection Agency. Pesticide Reregistration
Rejection Rate Analysis: Toxicology. EPA report 738-R-93-004. July
1993.
(3) U.S. Environmental Protection Agency. Conduct of Acute Toxicity
Studies. EPA report 737-R-97-002. September 1997.
(4) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals. Guideline 425: Acute Oral Toxicity-
Up-and-Down Method. Approved: December 2001.
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(5) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity-
Fixed Done Method. Adopted: December 2001.
(6) Organization for Economic Cooperation and Development, OECD
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity-
Acute Toxic Class Method. Adopted: December 2001.
(7) U.S. Environmental Protection Agency. Label Review Manual 2nd
Edition. EPA report 737-B-96-001. December 1996.
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