[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9130]
[Page 330-336]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart H_Health Effects Test Guidelines
Sec. 799.9130 TSCA acute inhalation toxicity.
(a) Scope. This section is intended to meet testing requirements
under section 4 of the Toxic Substances Control Act (TSCA).
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.
(b) Source. The source material used in developing this TSCA test
guideline is the harmonized Office of Prevention, Pesticides, and Toxic
Substances (OPPTS) test guideline 870.1300 (August 1998, final
guideline). These sources are available at the address in paragraph (g)
of this section.
(c) Definitions. The definitions in section 3 of TSCA and the
definitions in 40 CFR Part 792--Good Laboratory Practice Standards apply
to this section. The following definitions also apply to this section.
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 hours 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.
Geometric standard deviation (GSD) is a dimensionless number equal
to the
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ratio between the mass median aerodynamic diameter (MMAD) and either 84%
or 16% of the diameter size distribution (e.g., MMAD = 2 m; 84% = 4 m;
GSD = 4/2 = 2.0.) The MMAD, together with the GSD, describe the particle
size distribution of an aerosol. Use of the GSD may not be valid for
non-lognormally distributed aerosols. (If the size distribution deviates
from the lognormal, it shall be noted).
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 .
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% of animals exposed for a specified time. 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. 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 and test
animal species should also be specified, e.g., 4 hours LC50
in F344.
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% 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 hours,
or where this is not possible due to physical or chemical properties of
the test substance, the maximum attainable concentration where a
particle size distribution having an MMAD between 1 and 4 [micro]m
cannot be maintained, using the procedures described under paragraph (e)
of this section. For fibers, the bivariate distribution of length and
diameter must ensure inhalability. For gases and vapors, the
concentrations need not be greater than 50,000 ppm or 50% of the lower
explosive limit, whichever is lower. If a test at an aerosol or
particulate exposure of 2 mg/L (actual concentration of respirable
substance) for 4 hours or, where this is not feasible, the maximum
attainable concentration, using the procedures described for this study,
produces no observable toxic effects, then a full study using three
concentrations will not be necessary. Similarly, if a test at a gas or
vapor exposure of 50,000 ppm or 50% of the lower explosive limit,
whichever is lower, produces no observable toxic effects, then a full
study using three concentrations will not be necessary.
(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 primarily to
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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 sacrificed. 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 under EPA Good Laboratory Practice Standards at 40 CFR
part 792, subpart f.
(3) Test procedures--(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. (1) 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 investigator should provide justification
and reasoning for the selection.
(2) Health Status. Body weight and feed consumption are not
sufficient indicators of the health status of animals prior to
initiating an inhalation toxicity study. Prior to initiating the study,
animals must be monitored for known viral and bacterial respiratory
pathogens determined by conventional microbiological assays (e.g.,
serology). The animals must be free from pathogens at the start of
exposure.
(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% of the
mean weight of each sex.
(C) Number of animals and sex. (1) At least five experimentally
naive animals are used at each concentration and they must 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. 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 must be nulliparous and nonpregnant.
(3) In acute toxicity tests with animals of a higher order than
rodents, the use of fewer animals per concentration group should be
considered.
(D) Assignment of animals. (1) Each animal must be assigned a unique
identification number. A system to assign animals to test groups and
control groups randomly is required.
(2) Control groups. A concurrent untreated control group is not
necessary. Where a vehicle other than water is used to generate an
appropriate concentration of the test substance in the atmosphere and
historical data are not available or adequate to determine the acute
toxicity of the vehicle, a vehicle control group must be used. The
vehicle control group must be a sham-treated group. Except for treatment
with the test substance, animals in the vehicle control group must be
handled in a manner identical to the test-group animals.
(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. Animals must be housed individually in inhalation chambers
during exposure to aerosols.
(1) Before and after exposure, the temperature of the animal room
should be 22 3 [deg]C and the relative humidity
30-70%.
(2) Where lighting is artificial, the sequence should be 12 hours
light/12 hours dark.
(3) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water.
(F) Inhalation equipment. (1) Animals can be exposed to the
substance by either a nose-only procedure or in a
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whole-body exposure chamber. Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into the
surrounding areas. The nose-only exposure procedure is recommended for
studies of aerosols to minimize exposures confounding resultant from
test substance ingestion due to test animal fur licking following
exposures. Animals must be acclimated to the nose-only exposure chamber
prior to study and heat stress minimized during testing.
(2) Inhalation chambers. The animals must be tested in inhalation
equipment designed to sustain a dynamic airflow for nose-only exposures
of at least 300 ml/minute/animal or an airflow for whole-body exposures
of at least 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 must 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.
(3) Environmental conditions. The temperature at which the test is
performed must be maintained at 22 [deg]C (2
[deg]C). Ideally, the relative humidity should be maintained between 40%
and 60%, but in certain instances (e.g., tests using water as a
vehicle), this may not be practical.
(G) Physical measurements. Measurements or monitoring must be made
of the following:
(1) Chemical purity of the test material must be analyzed. If the
test substance is present in a mixture, the mass and composition of the
entire mixture, as well as the principal compound, must be measured. If
there is some difficulty in measuring chamber analytical concentration
due to precipitation, nonhomogeneous mixtures, volatile components, or
other factors, additional analyses of components may be necessary.
(2) The rate of air flow should be monitored continuously, and must
be recorded at least every 30 minutes during the exposure period.
(3) The actual concentrations of the test substance must be measured
in the breathing zone. During the exposure period, the actual
concentrations of the test substance must be held as constant as
practicable, monitored continuously or intermittently depending on the
method of analysis, and recorded at least three times (i.e., at the
beginning, at an intermediate time, and at the end) during the exposure
period. Chamber concentration may be measured using gravimetric or
analytical methods as appropriate. If trial run measurements are
reasonably consistent (10% for liquid aerosol,
gas, or vapor; 20% for dry aerosol), then a
minimum of two measurements are sufficient. If measurements are not
consistent, then a minimum of four measurements should be taken.
(4) During the development of the generating system, particle size
analysis must 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 distribution.
The MMAD particle size range should be between 1-4 [micro]m. The
particle size of hygroscopic materials must be small enough when dry to
assure that the size of the swollen particle will still be within the 1-
4 [micro]m MMAD range. Characterization for fibers must include the
bivariate distribution of length and diameter; this distribution must
ensure inhalability. Measurements of aerodynamic particle size in the
animal's breathing zone must be measured during a trial run. If MMAD
values for each exposure level are within 10% of each other, then a
minimum of two measurements during the exposures should be sufficient.
If pretest measurements are not within 10% of each other, then a minimum
of four measurements should be taken.
(5) Temperature and humidity must be monitored continuously, and
must be recorded at least every 30 minutes.
(iii) Exposure duration and concentration levels. (A) Exposure
duration. Shortly before exposure, the animals are weighed and then
exposed to the test target concentration in the designated apparatus for
4 hour exposure period after equilibration of the chamber
concentrations. The target concentration is defined by an average of 5%
for gases and vapors and 15% for
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particles and aerosols. The animals are weighed again at the conclusion
of the exposure period to determine body weight change. Other durations
may be needed to meet specific requirements. Food must be withheld
during exposure. Water may also be withheld in certain circumstances.
(B) Exposure concentration levels. At least three concentration
levels and a vehicle control group, if required (see paragraph
(e)(3)(ii)(D)(2) of this section), must be used. The concentration
levels should be spaced appropriately to produce a concentration-
response curve and permit an estimation of the median lethal
concentration (LC50). 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 maximally support detection of concentration-
response relationship. The high concentration should be clearly toxic or
a limit concentration, 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-effects level (NOEL).
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.
(C) When the physical and chemical properties of the test substance
show a low flash point or the test substance is otherwise known or
thought to be explosive, care must be taken to avoid exposure level
concentrations that could result in an exposure chamber explosion during
the test.
(iv) Observation period. The observation period must 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, the duration of
the signs observed, and the time of death must be recorded and are
important, especially if there is a tendency for delayed effects.
(v) Observation of animals. (A) A careful clinical examination must
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 must 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 activity, gait and posture,
reactivity to handling or sensory stimuli, altered strength, and
stereotypies or bizarre behavior (e.g., self mutilation, walking
backwards).
(D) Individual weights of animals must be determined pre-exposure
and post-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
must be weighed, sacrificed and a gross necropsy must be performed on
all animals under test, with particular reference to any changes in the
respiratory tract. All gross pathology changes must be recorded.
(1) The gross necropsy must include examination of orifices and the
cranial, thoracic, and abdominal cavities, and contents.
(2) At least the lungs, liver, kidneys, adrenals, brain, and gonads
should be weighed wet, as soon as possible after dissection to avoid
drying.
(3) Optionally, the following organs and tissues, or representative
samples thereof, may be preserved in a suitable medium for possible
future 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;
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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. Respiratory tract
tissues should be perfusion preserved in a suitable medium.
(B) If necropsy cannot be performed immediately after a dead animal
is discovered during the observation period, the animal should be
refrigerated (not frozen) at temperatures low enough to minimize
autolysis. Necropsies should be performed as soon as possible after
death (normally within 24 to 48 hours).
(vii) Additional evaluations. In animals surviving 24 hours or more,
microscopic examination of organs showing evidence of gross pathology
should be considered since it may yield useful information on the nature
of acute toxic effects.
(f) Data and reporting--(1) Treatment of results. Data must 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. The method used for calculation of the
LC50 or any other parameters must be specified and
referenced. Some acceptable methods for parameter estimation are
described in the references described in paragraphs (g)(1), (g)(2), and
(g)(3) of this section.
(2) Evaluation of results. The LC50 value should be
considered in conjunction with the observed toxic effects and the
necropsy findings. The 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.
(3) Test report. In addition to the reporting requirements specified
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J, the following specific information must be reported. The test report
shall include:
(i) Test conditions. (A) Description of exposure apparatus including
design, type, dimensions.
(B) Source of air, system for generating the test article as
particle, aerosol, gas, or vapor.
(C) Method for conditioning air, equipment for measuring
temperature, humidity, particle size or particulate aerosol
concentration size, and actual concentration.
(D) Treatment of exhaust air and the method of housing the animals
in a test chamber when this is used.
(ii) Exposure data. The exposure data must be tabulated and
presented with mean values and a measure of variability (e.g., standard
deviation) and should include:
(A) Chemical purity of the test material.
(B) Airflow rates through the inhalation equipment.
(C) Temperature and humidity of the air.
(D) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(E) Actual (analytical or gravimetric) concentration in test
breathing zone.
(F) Particle size distribution (calculated MMAD and GSD) and the
bivariate distribution of fiber length and diameter, where appropriate.
(G) 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 this section.
(iii) Species, strain, sex, and source of test animals.
(iv) Method of randomization in assigning animals to test and
control groups.
(v) Rationale for selection of species, if other than that
recommended.
(vi) Results. Tabulation of individual and test group data by sex
and exposure concentration level (e.g., number of animals exposed,
number of animals showing signs of toxicity and number of animals that
died or were sacrificed during the test).
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(A) Description of toxic effects including time of onset, duration,
reversibility, and relationship to the exposure concentration levels.
(B) Pre-exposure and post-exposure body weight change in animals,
and weight change during the observation period.
(C) Time of dosing and time of death during or following exposure.
(D) Concentration-response curves for mortality and other toxic
effects (when permitted by the method of determination).
(E) Gross pathology necropsy findings in the test animals and
vehicle control animals, if included. Data must be tabulated to show the
counts and incidence of gross alterations observed for each group tested
and the number of animals affected by each type of lesion along with the
location and frequency of each type of lesion.
(F) Histopathology findings and any additional evaluations (e.g.,
clinical chemistry), if performed.
(vii) Description of any pretest conditioning, including diet,
quarantine and treatment for disease.
(viii) 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.
(ix) Manufacturer (source), lot number, and purity of test
substance.
(x) Identification and composition of any vehicles (e.g., diluents,
suspending agents, and emulsifiers) or other materials , if used in
administering the test substance.
(xi) 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.
(g) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available for inspection at the TSCA Nonconfidential
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Chanter, D.O. and Heywood, R. The LD50 test: some
considerations 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).
(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
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. Adopted: July 17, 1992.
(7) Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity
Acute Toxic Class Method. Adopted: March 22, 1996.
(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.
[65 FR 78776, Dec. 15, 2000]