[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.2450]
[Page 145-150]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart C_Subchronic Exposure
Sec. 798.2450 Inhalation toxicity.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a gas, volatile substance, or aerosol/particulate,
determination of subchronic inhalation toxicity may be carried out after
initial information on toxicity has been obtained by acute testing. The
subchronic inhalation study has been designed to permit the
determination of the no-observed-effect level and toxic effects
associated with continuous or repeated exposure to a test substance for
a period of 90 days. The test is not capable of determining those
effects that have a long latency
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period for development (e.g., carcinogenicity and life shortening). It
provides information on health hazards likely to arise from repeated
exposures by the inhalation route over a limited period of time. It will
provide information on target organs, the possibilities of accumulation,
and can be of use in selecting dose levels for chronic studies and for
establishing safety criteria for human exposure. Hazards of inhaled
substances are influenced by the inherent toxicity and by physical
factors such as volatility and particle size.
(b) Definitions. (1) Subchronic inhalation toxicity is the adverse
effects occurring as a result of the repeated daily exposure of
experimental animals to a chemical by inhalation for part (approximately
10 percent) of a life span.
(2) Aerodynamic diameter applies to the size of particles of
aerosols. It is the diameter of a sphere of unit density which behaves
aerodynamically as the particle of the test substance. It is used to
compare particles of different size and densities and to predict where
in the respiratory tract such particles may be deposited. This term is
used in contrast to measured or geometric diameter which is
representative of actual diameters which in themselves cannot be related
to deposition within the respiratory tract.
(3) The geometric mean diameter or the median diameter is the
calculated aerodynamic diameter which divides the particles of an
aerosol in half based on the weight of the particles. Fifty percent of
the particles by weight will be larger than the median diameter and 50
percent of the particles will be smaller than the median diameter. The
median diameter describes the particle size distribution of any aerosol
based on the weight and size of the particles.
(4) Inhalable diameter refers to that aerodynamic diameter of a
particle which is considered to be inhalable for the organism. It is
used to refer to particles which are capable of being inhaled and may be
deposited anywhere within the respiratory tract from the trachea to the
alveoli. For man, inhalable diameter is considered as 15 micrometers or
less.
(5) Dose refers to an exposure level. Exposure is expressed as
weight or volume of test substance per volume of air (mg/l), or as parts
per million (ppm).
(6) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which
produces no observed adverse effects. A no-observed-effect level is
expressed in terms of weight or volume of test substance given daily per
unit volume of air (mg/l or ppm).
(7) Cumulative toxicity is the adverse effects of repeated doses
occuring as a result of prolonged action on, or increased concentration
of the administered test substance or its metabolites in susceptible
tissues.
(c) Principle of the test method. Several groups of experimental
animals are exposed daily for a defined period to the test substance in
graduated concentrations, one concentration being used per group, for a
period of 90 days. During the period of administration, the animals are
observed daily to detect signs of toxicity. Animals which die during the
test are necropsied and at the conclusion of the test, surviving animals
are sacrificed and necropsied and appropriate histopathological
examinations carried out.
(d) Test procedures--(1) Animal selection--(i) Species and strain. A
mammalian species shall be used for testing. A variety of rodent species
may be used, although the rat is the preferred species. Commonly used
laboratory strains shall be employed. If another mammalian species is
used, the tester shall provide justification/ reasoning for its
selection.
(ii) Age. Young adult animals shall be used. At the commencement of
the study the weight variation of animals shall not exceed 20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at
each dose level.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 females and 10 males)
shall be used for each test group. If another mammalian species is
selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4
males and 4 females) shall be used.
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(B) If interim sacrifices are planned, the number of animals shall
be increased by the number of animals scheduled to be sacrificed before
the completion of the study.
(2) Control groups. A concurrent control group is required. This
group shall be an untreated or sham-treated control 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 atmosphere, a vehicle control group shall be used. If the toxic
properties of the vehicle are not known or cannot be made available,
both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high concentration level for 90 days and
observed for reversibility, persistence, or delayed occurrence of toxic
effects for a post-treatment period of appropriate length, normally not
less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity
tests, it is desirable to have a concentration-response relationship as
well as a no-observed-toxic-effect level. Therefore, at least 3
concentration levels with a control and, where appropriate, a vehicle
control (corresponding to the concentration of vehicle at the highest
exposure level) shall be used. Concentrations should be spaced
appropriately to produce test groups with a range of toxic effects. The
data should be sufficient to produce a concentration-response curve.
(ii) The highest concentration should result in toxic effects but
not produce an incidence of fatalities which would prevent a meaningful
evaluation.
(iii) The lowest concentration should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the
lowest concentration should exceed this.
(iv) Ideally, the intermediate concentration level(s) should produce
minimal observable toxic effects. If more than one intermediate
concentration level is used, the concentrations should be spaced to
produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the
incidence of fatalities should be low, to permit a meaningful evaluation
of the results.
(vi) In the case of potentially explosive test substances, care
should be taken to avoid generating explosive concentrations.
(5) Exposure conditions. The animals should be exposed to the test
substance, ideally for 6 hours per day on a 7-day per week basis, for a
period of 90 days. However, based primarily on practical considerations,
exposure on a 5-day-per-week basis for 6 hours per day is the minimum
acceptable exposure period.
(6) Observation period. (i) Duration of observation shall be for at
least 90 days.
(ii) Animals in a satellite group scheduled for followup
observations should be kept for at least 28 days further without
treatment to detect recovery from, or persistence of, toxic effects.
(7) Inhalation exposure. (i) The animals shall be tested in
inhalation equipment designed to sustain a minimum dynamic air flow of
12 to 15 air changes per hour and ensure an adequate oxygen content of
19 percent and an evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize crowding of the test animals
and maximize their exposure to the test substance. This is best
accomplished by individual caging. To ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals shall not exceed 5
percent of the volume of the test chamber. Oronasal or head-only
exposure may be used if it is desirable to avoid concurrent exposure by
the dermal or oral routes.
(ii) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall be adjusted to ensure that
conditions throughout the exposure chamber are essentially the same.
Maintenance of slight negative pressure inside the chamber will prevent
leakage of the test substance into surrounding areas.
(iii) The temperature at which the test is performed should be
maintained
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at 22 [deg]C (2[deg]). Ideally, the relative
humidity should be maintained between 40 to 60 percent, but in certain
instances (e.g., tests of aerosols, use of water vehicle) this may not
be practicable.
(8) Physical measurements. Measurements or monitoring shall be made
of the following:
(i) The rate of air flow shall be monitored continuously and
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
concentrations of the test substance shall be held as constant as
practicable, monitored continuously or intermittently depending on the
method of analysis, and recorded at least at the beginning, at an
intermediate time, and at the end of the exposure period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analysis
shall be conducted as often as necessary to determine the consistency of
particle size distribution.
(iv) Temperature and humidity shall be monitored continuously but
shall be recorded at least every 30 minutes.
(9) Feed and water during exposure period. Feed shall be withheld
during exposure. Water may also be withheld during exposure.
(10) Observation of animals. (i) Each animal shall be observed daily
and, if necessary, handled to appraise its physical condition.
(ii) 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 or sacrifice
of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed
including the time of onset, the degree, and duration.
(iv) Cage-side observations should include, but not be limited to,
changes in the skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be
determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period all survivors in the
nonsatellite treatment groups shall be sacrificed. Moribund animals
shall be removed and sacrificed when noticed.
(11) Clinical examinations. (i) The following examinations shall be
made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least
two times during the test period on all groups of animals including
concurrent controls: After 30 days of test and just prior to terminal
sacrifice at the end of the test period. Hematology determinations which
are appropriate to all studies: Hematocrit, hemoglobin concentration,
erythrocyte count, total and differential leukocyte count, and a measure
of clotting potential such as clotting time, prothrombin time,
thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least two times during the test period on all groups of
animals including concurrent controls: After 30 days of test and just
prior to terminal sacrifice at the end of the test period. Clinical
biochemistry test areas which are considered appropriate to all studies:
Electrolyte balance, carbohydrate metabolism, and liver and kidney
function. The selection of specific tests will be influenced by
observations on the mode of action of the substance. Suggested
determinations: calcium, phosphorus, chloride, sodium, potassium,
fasting glucose (with period of fasting appropriate to the species),
serum glutamic-pyruvic transaminase, (now known as serum alanine
aminotransferase), serum glutamic-oxaloacetic transaminase (now known as
serum aspartate aminotransferase), ornithine decarboxylase, gamma
glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total
bilirubin, and total serum protein measurements. Other determinations
which may be necessary for an adequate toxicological evaluation include:
Analyses of lipids, hormones, acid/base
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balance, methemoglobin, and cholinesterase activity. Additional clinical
biochemistry may be employed, where necessary, to extend the
investigation of observed effects.
(ii) The following examinations shall be made on high dose and
control groups. If changes in the eyes are detected, all animals shall
be examined:
(A) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, shall be made prior to exposure to the
test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when
there is an indication based on expected and/or observed toxicity.
(12) Gross pathology. (i) All animals shall be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices and the cranial, thoracic, and abdominal cavities and
their contents.
(ii) At least the liver, kidneys, adrenals, brain, and gonads shall
be weighed wet, as soon as possible after dissection to avoid drying. In
addition, for the rodent, the brain; for the non-rodent, the thyroid
with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions; lungs--which should be
removed intact, weighed, and treated with a suitable fixative to ensure
that lung structure is maintained (perfusion with the fixative is
considered to be an effective procedure); nasopharyngeal tissues;
brain--including sections of medulla/pons cerebellar cortex and cerebral
cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum
with bone marrow; salivary glands; liver; spleen; kidneys; adrenals;
pancreas; gonads; uterus; accessory genital organs (epididymis,
prostate, and, if present, seminal vesicles); aorta; (skin); gall
bladder (if present); esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder; representative lymph node;
(mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur--
including articular surface); (spinal cord at three levels--cervical,
midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(13) Histopathology. The following histopathology shall be
performed:
(i) Full histopathology on the respiratory tract and other organs
and tissues, listed above, of all animals in the control and high dose
groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated
by signs of toxicity or target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose
groups shall also be subjected to histopathological examination,
primarily for evidence of infection since this provides a convenient
assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be
performed on tissues and organs identified as showing effects in the
treated groups.
(e) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions, and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods should be
selected during the design of the study.
(2) Evaluation of results. The findings of the subchronic inhalation
toxicity study should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the observed toxic effects
and the necropsy and histopathological findings. The evaluation will
include the relationship between the concentration of the test substance
and duration of exposure, and the presence or absence, the incidence and
severity, of abnormalities, including behavioral and clinical
abnormalities, gross lesions, identified target organs, body weight
changes, effects on mortality and any other general or specific toxic
effects. A properly
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conducted subchronic test should provide a satisfactory estimation of a
no-effect level.
(3) Test report. In addition to the reporting requirements as
specified under EPA Good Laboratory Practice Standards, 40 CFR part 792,
subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus,
including design, type, dimensions, source of air, system for generating
particulates and aerosols, method of conditioning air, treatment of
exhaust air, and the method of housing animals in a test chamber.
(B) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean
values and measure of variability (e.g., standard deviation) and shall
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 volume of air).
(D) Actual concentration in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of
particles with standard deviation from the mean).
(iii) Group animal data. Tabulation of toxic response data by
species, strain, sex, and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(iv) Individual animal data. (A) Date of death during the study or
whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Cage, J.C. ``Experimental Inhalation Toxicology,'' Methods in
Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-
277.
(2) Casarett, L.J., Doull, J. ``Chapter 9.'' Toxicology: The Basic
Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).
(3) MacFarland, H.N. ``Respiratory Toxicology,'' Essays in
Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp.
121-154.
(4) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' a report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19073, May 20, 1987;
52 FR 26150, July 13, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May
16, 1989]