United States Prevention, Pesticides EPA712-C-96-291
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.1350
Acute Inhalation Toxicity
With Histopathology
'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 870.1350 Acute inhalation toxicity with histopathology.
(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. This is a new guideline developed in the Office
of Research and Development.
(b) Purpose. In the assessment and evaluation of the potential human
health effects of chemical substances, it is appropriate to test for acute
inhalation toxic effects. The goals of this test are to characterize the expo-
sure-response relationship for sensitive endpoints following acute exposure
and to characterize toxicologic response following acute high exposures.
The latter is of particular concern in relation to spills and other accidental
releases. This testing is designed to determine the gross pathology resulting
from acute inhalation exposure to a substance. In addition, because toxic
effects on the respiratory tract are of particular concern following inhala-
tion exposure, several indicators of respiratory toxicity consisting of
histopathology on fixed tissue and evaluation of cellular and biochemical
parameters in bronchoalveolar lavage fluid should be employed. This acute
testing should be augmented by the respiratory sensory irritation assay in
mice (ASTM, 1984). The respiratory histopathology consists of specialized
techniques to preserve tissues of the respiratory tract in order to allow
detailed microscopic examination to identify adverse effects of chemical
substances on this organ system. The bronchoalveolar lavage is designed
to be a rapid screening test to provide an early indicator of pulmonary
toxicity by examining biochemical and cytologic endpoints of material
from the lungs of animals exposed to potentially toxic chemical sub-
stances. The mouse respiratory sensory irritation assay is a non-invasive
assay designed to detect sensory irritation during exposure to a chemical
substance (ASTM, 1984). These acute tests are designed to assess the rela-
tionship, if any, between the animals exposed to the test substance, the
incidence and severity of observed abnormalities, gross lesions, body
weight changes, effects on mortality, and any other toxic effects. These
acute tests are not intended to provide a complete evaluation of the
toxicologic effects of a substance, and additional functional and mor-
phological evaluations may be necessary to assess completely the potential
effects produced by a chemical substance. Additional tests may include
longer-term exposures, or more in-depth evaluation of specific organ sys-
tems as indicated by signs of toxicity following acute exposure.
(c) Definitions. The following definitions apply to this test guideline.
Aerodynamic diameter refers to the size of particles or aerosols. It
is the diameter of a sphere of unit density that behaves aerodynamically
(has the same settling velocity in air) as the particle of the test substance.
It is used to compare particles of different size, shape and density, and
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to predict where in the respiratory tract such particles may be primarily
deposited.
Exposure response is the relationship between the exposure con-
centration and the measured response expressed as a group mean in the
case of a continuous variable or as incidence in the case of a quantil vari-
able.
Geometric standard deviation (GSD) is a dimensionless number equal
to the ratio between the mass median aerodynamic diameter (MMAD) and
either 84% or 16% of the diameter size distribution (e.g., MMAD = 2
(im; 84% = 4 (im; GSD = 4/2 = 2.0.) The MMAD, together with the
GSD, describe the particle size distribution of an aerosol.
Lower respiratory tract consists of those structures of the respiratory
tract below the larynx.
The mass geometric mean aerodynamic diameter or the mass median
aerodynamic diameter (MMAD) is the calculated aerodynamic diameter
that divides the particles of an aerosol (a gaseous suspension of fine liquid
or solid particles) in half, based on the weight of the particles. By weight,
50% of the particles will be larger than the MMAD and 50% of the par-
ticles will be smaller than the MMAD.
Particle regional deposition is the region of the respiratory tract
where particles of different sizes deposit. Particles between 5 and 30 (im
mainly deposit in the nasopharyngeal region by inertial impaction, particles
between 1 and 5 (im deposit by sedimentation in the tracheal/bronchial/
bronchiolar/alveolar regions, and particles of 1 (im or less deposit in the
alveolar region by diffusion.
Respiratory effects are any adverse effects on the structure or func-
tions of the respiratory system related to exposure to a chemical substance.
Sensory airway irritation is any sign that a chemical substance is
stimulating the nerves of the respiratory tract as identified by the char-
acteristic pause during expiration in the respiratory sensory irritation test.
Toxic effects are any adverse changes (a change that is statistically
or biologically significant) in the structure or function of an experimental
animal as a result of exposure to a chemical substance.
Upper respiratory tract consists of those structures of the respiratory
tract above the larynx.
(d) Principle of the test method. The test substance is administered
to several groups of experimental animals; one concentration level and
duration being used per group. Bronchoalveolar lavage shall be used to
evaluate early effects on the respiratory system by examining changes in
the content of the lavage fluid of the lung. At 24 hours following exposure,
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the animals shall be sacrificed and necropsied, and tissue samples from
the respiratory tract and other major organs will be prepared for micro-
scopic examination. The exposure levels at which significant toxic effects
on the respiratory organ system are produced are compared to those levels
that produce other toxic effects. As triggered by the results of the 4-hour
test, additional exposure periods of 1 hour and 8 hours will be required
to determine the effect of exposure time on the toxicity observed.
(e) Test procedures—(1) Animal selection—(i) Species. In general,
the laboratory rat and mouse should be used. Under some circumstances,
other species, such as the hamster or guinea pig, may be more appropriate,
and if these or other species are used, justification should be provided.
(ii) Strain. If rats and mice are used, the use of the F344 rat and
the B6C3F1 mouse is preferred to facilitate comparison with existing data.
(iii) Age. Young adults shall be used. The weight variation of animals
used in a test should not exceed + 20 % of the mean weight for each
species.
(iv) Sex. Equal numbers of animals of each sex shall be used for
each dose level. The females shall be nulliparous and nonpregnant.
(v) Health status. Body weight and feed consumption are not suffi-
cient indicators of the health status of animals prior to initiating an inhala-
tion toxicity study. Prior to initiating the study, animals shall be monitored
for known pathogenic rodent viruses as determined by conventional
microbiological assays (e.g., serology). In addition, a few animals shall
be sacrificed prior to exposure, lung lavage fluid shall be evaluated for
polymorphonuclear leukocytes (PMNs), and biochemical parameters shall
be evaluated as in the lung lavage test described in paragraph (e)(12) of
this section to assure the health of the animals. PMNs should be less than
1% of lavage cells. The animals shall be free from pathogens at the start
of exposure.
(2) Number of animals. At least five males and five females shall
be used in each concentration/duration and control group. Animals shall
be randomly assigned to treatment and control groups.
(3) Control groups. A concurrent control group is required for all
tests. The control group shall be a sham treated group. Except for treatment
with the test substance, animals in the control group shall be handled in
a manner identical to the test group animals. Where a vehicle is used to
help generate an appropriate concentration of the substance in the atmos-
phere, a vehicle control group shall be used.
(4) Concentration level and concentration selection. For the 4-hour
study, at least three concentrations shall be used in addition to the control
group. Ideally, the data generated from the test should be sufficient to
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produce an exposure-effect curve. The concentrations can either be linearly
or logarithmically spaced depending on the anticipated steepness of the
concentration-response curve. A rationale for concentration selection
should be provided to indicate that the selected concentrations will maxi-
mally support detection of concentration-effect relationship. The high con-
centration should be clearly toxic or a limit dose, but should not result
in an incidence of fatalities that would preclude a meaningful evaluation
of the data. The lowest concentration should define a no-observed-adverse-
effects level (NOAEL).
(i) Limit dose. The high concentrations need not be greater than 5
mg/L, or for aerosol and particulate concentrations, concentrations that
cannot maintain a size distribution less than or equal to 4 (im (MAD)
(i.e., a particle size distribution that permits deposition throughout the res-
piratory tract). If a test at an exposure of 5 mg/L (actual concentration
of respirable substance) for 4 hours or, where this is not feasible, the maxi-
mum attainable concentration, using the procedures described for this
study, produces no observable toxic effects, then a full study using three
dose levels will not be necessary.
(ii) 1-Hour study. If triggered, three concentrations shall be tested.
These concentrations should allow for the determination of an effect level
and a no-observable-adverse-effect-level.
(iii) 8-Hour study. If triggered, three concentrations shall be tested.
These concentrations should allow for the determination of an effect level
and a no-observable-adverse-effect-level.
(5) Inhalation exposure. Animals can be exposed to the substance
by either a nose only procedure or in a whole body exposure chamber.
(i) Inhalation chambers. The animals shall be tested in inhalation
equipment designed to sustain a dynamic air flow of 12 to 15 air changes
per hour and ensure an adequate oxygen content of at least 19% and an
evenly distributed exposure atmosphere. Where a whole body chamber is
used, its design shall minimize crowding by providing individual caging.
As a general rule, to ensure stability of a chamber atmosphere, the total
"volume" of the test animals should not exceed 5% of the volume of
the test chamber.
(ii) Environmental conditions. The temperature at which the test is
performed shall be maintained at 22 °C ( ±2 °C). Ideally, the relative hu-
midity should be maintained between 40% and 60%, but in certain in-
stances (e.g., tests using water as a vehicle), this may not be practical.
(iii) Exposure periodicity. For acute testing, the animals shall be ex-
posed to the test substance for at least 4 hours after the chamber has
reached equilibrium. If triggered by the results of the 4 hour exposure,
additional testing shall be conducted using 1 and 8 hour exposure periods.
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(6) Physical measurements. Measurements or monitoring shall be
made of the following:
(i) The rate of air flow shall be monitored continuously, but shall
be recorded at least every 30 minutes.
(ii) The actual concentrations of the test substance shall 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, mon-
itored continuously, and recorded at least at the beginning, at an intermedi-
ate time, and at the end of the exposure period.
(iii) During the development of the generating system, where appro-
priate, particle size analysis shall be performed to establish the stability
of aerosol concentrations. During exposure, analysis should be conducted
as often as necessary to determine the consistency of particle size distribu-
tion.
(iv) Chemical purity, where appropriate, shall be verified after the
compound is aerosolized, preferably by sampling in the animals' breathing
zone prior to initiating the study. This is to ensure that purity has not
changed from the stock material to the breathing zone, and that degrada-
tion products have not formed.
(v) If the compound is present in a mixture, the mass and composition
of the entire mixture, as well as the principal compound, shall be meas-
ured.
(vi) Temperature and humidity shall be monitored continuously, but
shall be recorded at least every 30 minutes.
(7) Food and water during exposure period. Food shall be withheld
during exposure. Water may also be withheld in certain cases.
(8) Observation period. The bronchoalveolar lavage and respiratory
pathology shall be conducted 24 hours following exposure to allow expres-
sion of signs of toxicity. There is concern that some latency time will
be required to allow migration of cells and macromolecules into the lungs
following exposure, and that some pathology may require macromolecular
synthesis or degradation before cell damage develops.
(9) Gross pathology, (i) All animals shall be subjected to a full ne-
cropsy which includes examination of orifices and the cranial, thoracic,
and abdominal cavities and their contents.
(ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads shall
be weighed wet, as soon as possible after dissection to avoid drying.
(iii) The following organs and tissues, or representative samples there-
of, shall be preserved in a suitable medium for possible future
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histopathological examination: All gross lesions; brain-including sections
of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/
parathyroid; thymus; heart; sternum with bone marrow; salivary glands;
liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs
(epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall
bladder (if present); esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh
musculature; peripheral nerve; spinal cord at three levels cervical,
midthoracic, and lumbar; and eyes. Tissue preservation for the respiratory
tract is described in paragraph (e)(ll) of this section.
(10) Histopathology. The following histopathology shall be per-
formed:
(i) Full histopathology shall be performed on the respiratory tract,
liver and kidney of all animals in the control and high dose groups. The
histopathology of the respiratory tract is described in paragraph (e)(ll)
of this section.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals, as indicated by the observations
in the high dose group in this study.
(iv) Archived organs (identified as targets of toxicity from results of
the 90-day study, if a 90-day study is required for this substance) in high
dose animals of the 4-hour acute study should be evaluated to determine
if they are also targets of acute toxicity.
(11) Respiratory tract histopathology. (i) Representative sections of
the respiratory tract shall be examined histologically. These shall include
the trachea, major conducting airways, alveolar region, terminal and res-
piratory bronchioles, alveolar ducts and sacs, and interstitial tissues.
(ii) Care shall be taken that the method used to kill the animal does
not result in damage to the tissues of the upper or lower respiratory tract.
(iii) Four sections of the nasopharyngeal tissue shall be examined for
histopathologic lesions. This shall include sections through the nasal cav-
ity, and examination of the squamous, transitional, respiratory, and olfac-
tory epithelia. The types and locations of lesions shall be reported using
diagrams of the nose.
(iv) The laryngeal mucosa shall be examined for histopathologic
changes. Sections of the larynx to be examined include the epithelium cov-
ering the base of the epiglottis, the ventral pouch, and the medial surfaces
of the vocal processes of the arytenoid cartilages.
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(12) Bronchoalveolar lavage. (i) Animals can be exposed to the sub-
stance by either a nose only procedure or in a whole body exposure cham-
ber.
(ii) Care should be taken that the method used to kill the animal re-
sults in minimum changes in the fluid of the lungs of the test animals.
(iii) At the appropriate time, the test animals shall be killed and the
heart-lung including trachea removed in bloc. Alternatively, lungs can be
lavaged in situ. If the study will not be compromised, one lobe of the
lungs may be used for lung lavage while the other is fixed for histologic
evaluation. The lungs should be lavaged using physiological saline. The
lavages shall consist of two washes, each of which consists of approxi-
mately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total lung volume.
Additional washes merely tend to reduce the concentrations of the material
collected. The lung lavage fluid shall be stored on ice at 5 °C until assayed.
(iv) The following parameters shall be determined in the lavage fluid
as indicators of cellular damage in the lungs: Total protein, cell count,
and percent leukocytes. In addition, a phagocytosis assay using the proce-
dure of Burleson (Burleson et al., 1987; Gilmour and Selgrade, 1993),
paragraphs (h)(l) and (h)(2) of this section, shall be performed to deter-
mine macrophage activity.
(13) Combined protocol. The tests described may be combined with
any other toxicity study, as long as none of the requirements of either
are violated by the combination.
(f) Triggered testing. If no adverse effects are seen in the 4-hour
study as compared with controls, no further testing is necessary. If the
4-hour study shows positive effects in histopathology of the
bronchoalveolar lavage, a 1-hour study and an 8-hour study shall be con-
ducted. Only those systems showing positive results in the 4-hour study
must be pursued in the follow-up 1-hour and 8-hour studies.
(g) Data reporting and evaluation. The final test report must include
the following information:
(1) Description of equipment and test methods. A description of
the general design of the experiment and any equipment used shall be
provided. This shall include a short justification explaining any deviations
from protocol.
(i) Description of exposure apparatus, including design, type, dimen-
sions, source of air, system for generating particulates, aerosols, gasses,
and vapors, method of conditioning air, treatment of exhaust air, and the
method of housing animals in a test chamber.
(ii) Description of the equipment for measuring temperature, humid-
ity, and particulate aerosol concentration and size.
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(iii) Exposure data shall be tabulated and presented with mean values
and measure of variability (e.g., standard deviation) and should include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature and humidity of air.
(C) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by the volume of air).
(D) Actual concentration in test breathing zone.
(E) Particle size distribution (e.g., MMAD with GSD), where appro-
priate.
(2) Results—(i) General group animal data. The following informa-
tion must be arranged by test group exposure level.
(A) Number of animals exposed.
(B) Number of animals dying.
(C) Number of animals showing overt signs of toxicity.
(D) Pre- and post-exposure body weight change in animals, and
weight change during the observation period.
(ii) Counts and incidence of gross alterations observed at necropsy
in the test and control groups. Data shall be tabulated to show:
(A) The number of animals used in each group and the number of
animals in which any gross lesions were found.
(B) The number of animals affected by each different type of lesion,
and the locations and frequency of each type of lesion.
(iii) Counts and incidence of general histologic alterations in the
test group. Data shall be tabulated to show:
(A) The number of animals used in each group and the number of
animals in which any histopathologic lesions were found.
(B) The number of animals affected by each different type of lesion,
and the locations, frequency, and average grade of each type of lesion.
(iv) Counts and incidence of respiratory histopathologic alter-
ations by the test group. The nasal lesions report shall be accompanied
by diagrams of the locations of the lesions. Data shall be tabulated to
show:
(A) The number of animals used in each group and the number of
animals in which any lesion was found.
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(B) The number of animals affected by each different type of lesion,
and the locations, frequency, and average grade of each type of lesion.
(v) Results of the bronchoalveolar lavage study. Data shall be tab-
ulated to show:
(A) The amount of administered lavage fluid and recovered lavage
fluid for each test animal.
(B) The magnitude of change of biochemical and cytologic indices
in lavage fluids at each test concentration for each animal.
(C) Results shall be quantified as amount of constituent/mL of lavage
fluid. This assumes that the amount of lavage fluid recovered is a rep-
resentative sample of the total lavage fluid.
(3) Evaluation of data. The findings from this acute study should
be evaluated in the context of preceding and/or concurrent toxicity studies
and any correlated functional findings. The evaluation shall include the
relationship between the concentrations of the test substance and the pres-
ence or absence, incidence, and severity of any effects. The evaluation
should include appropriate statistical analyses, for example, parametric
tests for continuous data and non-parametric tests for the remainder.
Choice of analyses should consider tests appropriate to the experimental
design, including repeated measures. The report must include concentra-
tion-effect curves for the bronchoalveolar lavage and tables reporting ob-
servations at each concentration level for necropsy findings and gross, gen-
eral, and respiratory system histopathology.
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Burleson, G.R., Fuller, L.B., Me'nache, M.G., and Graham, J.A.
"Poly (I): poly (C)-enhanced alveolar peritoneal macrophage phagocyto-
sis: Quantification by a new method utilizing fluorescent beads." Proceed-
ings of the Society of Experimental Biology and Medicine. 184:468-476
(1987).
(2) Gilmour, G.I., and Selgrade, M.K. "A comparison of the pul-
monary defenses against streptococcal infection in rats and mice following
O3 exposure: Differences in disease susceptibility and neutrophil recruit-
ment." Toxicology and Applied Pharmacology. 123:211-218(1993).
(3) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones,
R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. "New ap-
proaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage
fluid analysis." Fundamental and Applied Toxicology. 5:451-458 (1985).
(4) Henderson, R.F. "Use of bronchoalveolar lavage to detect lung
damage." Environmental Health Perspectives. 56:115-129 (1984).
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(5) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J.
"Early damage indicators in the lung. III. Biochemical and cytological
response of the lung to inhaled metal salts." Toxicology and Applied Phar-
macology. 50:123-136 (1979).
(6) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and
Grumbein, S.L. "Histologic methods and interspecies variations in the la-
ryngeal histology of F344/N rats and B6C3F1 mice." Toxicology and Pa-
thology. 20:44-51 (1992).
(7) Young, J.T. "Histopathologic examination of the rat nasal cav-
ity. '' Fundamental and Applied Toxicology. 1:309-312 (1981).
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