United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-98-190
August 1998
Health Effects Test
Guidelines
OPPTS 870.1100
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 Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Filial Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.1100 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 EvhI-
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 or. 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 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 exposure of animals to the test substance and the incidence and severity
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 pprio J.
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.
LDso (median lethal dose) is a statistically derived estimate of single
dose of a substance that can be expected to cause death in 50 percent
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of animals when administered by the oral route. The LD50 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 (0(1) 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 CZCD 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.
(ii)	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.
(iii)	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
endpoints. 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 in comparison to the "traditional" test.
(A)	The up and down procedure as described in OECD Guideline
425 referenced in paragraph (f)(4) of this guideline. This method is highly
recommended.
(B)	The acute toxic class method as described 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 in 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 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 carried out.
(2)	Substance to be tested. Test, control and reference substances
are discussed in 40 CFR Part 792—Good Laboratory Practice Standards.
(3)	Test procedures—(i) Preparations. Healthy young adult animals
are acclimatized to the laboratory conditions for at least 5 days prior to
the test before the test animals are randomized and assigned to the treat-
ment groups.
(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. (/) At least five experimentally
naive rodents aie 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 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 prior to conducting the main study.
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(2) The females should be nulliparous and nonpregnant.
(5) 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. 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.
(/) 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.
(5) Where lighting is artificial, the sequence should be 12-hours light/
12-hours dark.
(4) For feeding, conventional lauoratory diets may be used with an
unlimited supply of drinking water.
(iii) 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 LD50. 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
in correlation with the OPP toxicity categories (bracketing). In these cases,
the determination of an LD50 may not be necessary.
(B)	Limit test. This test has been defined and described under para-
graph (d)(2)(ii) 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 substanoo. 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-
tion 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 materials 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 period, the duration of observation should not be fixed
rigidly. It should be determined by the toxic reactions, rate of onset, and
length of recovery period, and may thus be extended when considered 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 moribund 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.
(vii)	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.
(viii)	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 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 LD50 or any other parameters should be
specified and referenced. Methods for parameter estimation are described
under paragraphs (f)(1), (f)(2), and (f)(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 severity of all abnormalities, including behavioral and
clinical abnormalities, gross lesions, body weight changes, effects on mor-
tality, and any other toxic effects. The LD50 value should always be con-
sidered in conjunction with the observed toxic effects and any necropsy
findings. The LD50 value is a relatively coarse measurement, useful only
as a reference value for classification and 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 in which the LD50 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:
(7) 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
(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).
(J) Description of toxic effects, including their time of onset, duration,
reversibility, and relationship to dose.
(ii) Body weights.
{Hi) Time of dosing and time of death after dosing.
O'v) 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 caging conditions including: Number (or change
in number) of animals per cage, bedding material, ambient temperature
and humidity, photoperiod, and identification of diet of test animals.
(7)	Manufacturer, source, purity, 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 Heywood, R. The LD50 Test: Some Consider-
ations of Precision. Toxicology Letters 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.
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