United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–193

June 1996

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 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. Environmental 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, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized 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 Animals) 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 establishing a dosage regimen in subchronic and other studies and may provide

initial information on the mode of toxic action of a substance. An evaluation 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 Laboratory Practice Standards apply to this test guideline. The following definitions 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 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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LC50 (median lethal concentration) is a statistically derived estimate

of a concentration of a substance that can be expected to cause death during 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 aerodynamic diameter (MMAD) 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 minimize the need for animal testing, the Agency encourages the review of

existing acute toxicity information on mixtures that are substantially similar 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 inadequate, 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 substance, the maximum attainable concentration, using the procedures described under paragraph (e) of this guideline. If no lethality is demonstrated, no further testing for acute inhalation toxicity is needed. 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 method. Several groups of experimental animals are exposed to the test substance 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 available 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 surviving 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 required number of groups.

(ii) Animal selection—(A) Species and strain. Although several

mammalian test species may be used, the preferred species is the rat. Commonly 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 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. (1) At least five experimentally naive animals

are used at each concentration and they should be of one sex. After completion 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 available to demonstrate that animals of the sex tested are markedly more sensitive, 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 control groups randomly is required.

(E) Housing. The 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.
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(1) 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. (1) 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 negative 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 appropriate 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 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:

(1) The rate of air flow should be monitored continuously, but recorded 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 recorded 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 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 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 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.
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(3) During the development of the generating system, particle size

analysis should be performed to establish the stability of aerosol concentrations. 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 μm. 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 μm range.

(4) Temperature and humidity should be monitored continuously and

recorded at least every 30 min. The temperate 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 designated 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 circumstances.

(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 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, respiratory 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 exposure, 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 temperatures 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, microscopic 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 described 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 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

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 abnormalities, 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 information should be reported:
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(1) Test conditions. (i) Description of exposure apparatus including

design, type, dimensions.

(ii) Source of air, system for generating the test article.

(iii) 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:

(i) Airflow rates through the inhalation equipment.

(ii) Temperature and humidity of the air.

(iii) 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).

(vi) 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).

(i) Description of toxic effects including their time of onset, duration,

reversibility, and relationship to concentration.

(ii) Body weights.

(iii) Time of dosing and time of death during or following exposure.

(iv) Concentration-response curves for mortality and other toxic effects (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.

(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.

(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 evaluating the results.

(f) References. The following references should be consulted for additional background material on this test guideline.

(1) American Society for Testing and Materials. Standard Test Method for Estimating Acute Oral Toxicity in Rats. A standard issued under

the fixed designation E 1163–87, under the jurisdiction of ASTM Committee 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 considerations 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 Toxicity. Adopted May 12, 1981.

(6) Organization for Economic Cooperation and Development. OECD

Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method, Endorsed by the Joint Meeting of the Chemicals 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.
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(8) U. S. EPA. Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. 2/1/94. Health Effects Division,

Office of Pesticide Programs.