United States Prevention, Pesticides EPA712-C-98-193
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA 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 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 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 870.1300 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 in 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 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 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 inhalable 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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LC5o (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 after 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.
(ii) 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 appropriate 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 sacrificed and necropsied. This guideline is directed pri-
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marily 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 irritating properties
need not be carried 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 prior to
the test. Before the test, animals are randomized and assigned to the re-
quired number of groups.
(ii) 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 in animals or between
groups used in a test should not exceed + 20 percent of the mean weight
of each sex.
(C) Number and sex. (7) At least five experimentally naive animals
are used at each concentration and they should be of one sex. After com-
pletion of the study in 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 prior
to conducting the main study.
(2) Females should be nulliparous and nonpregnant.
(3) In acute toxicity tests with animals of a higher order than rodents,
the use of smaller numbers should be considered.
(D) Assignment of animals. Each animal must be assigned a unique
identification number. A system to assign animals to test groups and con-
trol groups randomly is required.
(E) Housing. The animals may be group-caged by sex, but the num-
ber of animals per cage must not interfere with clear observation of each
animal. The biological properties of the test substance or toxic effects (e.g.
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morbidity, excitability) may indicate a need for individual caging. Animals
must be housed individually in inhalation chambers during exposure to
aerosols.
(7) 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:
(7) 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 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 article is a formulation, the analytical concentration must be reported
for the total formulation, and not just for the active ingredient (AI). If,
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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 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.
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 (im 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 during 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 during 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.
(iii) Exposure duration and levels. (A) Shortly before exposure, the
animals are weighed and then exposed to the test concentration in 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 during exposure. Water may also be withheld in 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 in
correlation with the OPP toxicity categories (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 rigidly.
It should be determined by the toxic reactions, rate of onset, and length
of recovery period, and thus may be extended when considered necessary.
The time at which signs of toxicity appear, their duration, and the time
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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 appropriate
actions taken to minimize loss of animals to the study, e.g. necropsy or
refrigeration of those animals found dead and isolation of weak or 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, 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 mu-
tilation, walking backwards).
(D) Individual weights of animals should be determined prior to expo-
sure, weekly after exposure, 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.
(vi) 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,
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.
(viii) 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 LCso or any other parameters should
be specified and referenced. Methods for parameter estimation are de-
scribed under paragraphs (f)(l), (f)(2), and (f)(3) of this guideline.
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(B) Evaluation of results. The LC50 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
severity 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.
(7) Test conditions. (/) Description of exposure apparatus including
design, type, dimensions.
(//) Source of air, system for generating the test article.
(fff) Equipment for measuring temperature, humidity, particle size,
and actual concentration.
(fv) 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:
(/) Airflow rates through the inhalation equipment.
(if) Temperature and humidity of the air.
(///) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(iv) Actual (analytical or gravimetric) concentration in test breathing
zone.
(v) Particle size distribution, and calculated MMAD and geometric
standard deviation (GSD).
(vf) 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).
(/) Description of toxic effects including their time of onset, duration,
reversibility, and relationship to concentration.
(//) Body weights.
(///) Time 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) 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.
(9) Manufacturer (source), lot number, and purity of test substance.
(Iff) 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 material 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) Finney, D.G. Chapter 3—Estimation of the median effective dose,
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).
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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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