United States Prevention, Pesticides EPA712-C-96-193
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.1300
Acute Inhalation Toxicity
"Public Draft'
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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.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
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 material 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 diameter is defined as the diameter of a unit-density
sphere having the same terminal settling velocity as the particle in ques-
tion, 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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LC50 (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 LC50 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 is the geometric mean aero-
dynamic diameter (MMAD) and, along with the geometric standard devi-
ation, is used to describe the particle size distribution of any aerosol statis-
tically, 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 per-
cent 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
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, 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 animals, 6-10 weeks old on receipt and be-
tween 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.
(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.
morbidity, excitability) may indicate a need for individual caging.
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(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.
(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, 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 rec-
ommended, 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 every 30 min.
(2) The actual concentrations of the test substance should be measured
in the breathing zone. Measurement of actual concentrations should be re-
corded near the beginning, middle, and end of the exposure period. During
the exposure period, the actual concentration of the test substance should
be held as constant as practicable. Whenever the test article is a formula-
tion, 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 inerts 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 conclu-
sion 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, addi-
tional analyses of inert components may be necessary.
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(3) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concentra-
tions. During exposure, analysis should be made as often as necessary to
determine the consistency of particle size distribution. 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.
(4) Temperature and humidity should be monitored continuously and
recorded at least every 30 min. The temperate at which the test is per-
formed 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.
(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
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 motor activity, gait and posture, reactivity to handling or
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sensory stimuli, grip strength, and stereotypies or bizarre behavior (e.g.,
self mutilation, 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
no later than 16 h after death.
(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 LC50 or any other parameters should
be specified and referenced. Methods for parameter estimation are de-
scribed under paragraphs (f)(2), (f)(3), and (f)(4) of this guideline.
(B) Evaluation of results. The LC50 value should be considered in
conjunction with the observed toxic effects and the necropsy findings. The
LC50 value is a relatively coarse measurement useful only for classifica-
tion 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
LC50 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 abnor-
malities, 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. The following specific informa-
tion should be reported:
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(7) Test conditions. (/) Description of exposure apparatus including
design, type, dimensions.
(if) Source of air, system for generating the test article.
(fff) 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:
(/) Airflow rates through the inhalation equipment.
(ff) 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).
(v/) 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.
(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).
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(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.
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.
(10) A list of references cited in the body of the report. References
to any published literature used in developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu-
ating the results.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Society for Testing and Materials. Standard Test Meth-
od for Estimating Acute Oral Toxicity in Rats. A standard issued under
the fixed designation E 1163-87, under the jurisdiction of ASTM Commit-
tee E-35 on Pesticides and is the direct responsibility of Subcommittee
E3526 on Safety to Man, Philadelphia, PA (1987).
(2) Chanter, D.O. and Heywood, R. The LD50 test: some consider-
ations of precision. Toxicology Letters 10:303-307 (1982).
(3) Finney, D.G. Chapter 3 — Estimation of the median effective dose,
Chapter 4 — Maximum likelihood estimation. Probit Analysis. 3rd Ed.
(Cambridge, London. (1971).
(4) Finney, D.J. The Median Lethal Dose and Its Estimation, Archives
of Toxicology 56:215-218 (1985).
(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, Endorsed by the Joint Meeting of the Chemi-
cals Group and Management Committee, 20th-22nd November, 1991,
ENV/EPOC(92)15.
(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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(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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