[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: 40CFR799.9135]
[Page 336-343]
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. 799.9135 TSCA acute inhalation toxicity with histopathology.
(a) Scope. This section is intended to meet the testing requirements
under section 4 of the Toxic Substances Control Act (TSCA). 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 exposure-
response relationship for sensitive endpoints following acute exposure
and to characterize toxicologic response following acute high exposures.
The
[[Page 337]]
latter is of particular concern in relation to spills and other
accidental releases. This testing is designed to determine the gross
pathology and histopathology resulting from acute inhalation exposure to
a substance. Because toxic effects on the respiratory tract are of
particular concern following inhalation 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. 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 substances. These acute tests are designed to assess the
relationship, if any, between the animals' exposure to the test
substance and to demonstrate relationship between the animals' exposure
and the incidence and severity of observed abnormalities, including
gross or histopathologic 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 morphological 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 systems as indicated by
signs of toxicity following acute exposure.
(b) Source. This a new section developed by the United States
Environmental Protection Agency.
(c) Definitions. The following definitions apply to this section.
Aerodynamic diameter (dae) refers to the size of
particles. 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 to predict where in the respiratory tract
such particles may be primarily deposited.
Exposure response is the relationship between the exposure
concentration and the measured toxic response, whether expressed as a
group mean standard deviation) in the case of a
continuous variable or as incidence in the case of a quantal variable.
This definiton should not preclude the exploration of other dose metrics
in establishing this relationship.
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
[micro]m; 84% = 4 [micro]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).
Inhalability is the ratio of the number concentration of particles
of a certain aerodynamic diameter, dae, that are inspired
through the nose or mouth to the number concentration of the same
dae present in the inspired volume of ambient air. In humans,
inhalability can exceed 15 [micro]m dae, whereas inhalability
dramatically decreases for particles above 4 [micro]m dae in
small laboratory animals.
Lower respiratory tract consists of those structures of the
respiratory tract below the larynx.
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
particles will be smaller than the MMAD.
Particle regional deposition is the fraction of inhaled particles
that deposits in the specific region of the respiratory tract. The major
mechanisms of particle deposition in the respiratory tract
[[Page 338]]
include impaction, sedimentation, diffusion, interception, and
electrostatic precipitation. The deposition mechanism that is dominant
for a given region depends on the respiratory tract architecture and
ventilation rate of the species and the aerosol particle size and
distribution. The respiratory tract in both humans and various
experimental mammals can be divided into three regions on the basis of
structure, size, and function:
(1) The extrathoracic region or upper respiratory tract that
includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and
larynx.
(2) The tracheobronchial region that includes the trachea, bronchi,
and bronchioles (including the terminal bronchioles).
(3) The alveolar region that includes the respiratory bronchioles
(if present in the species), alveolar ducts, alveolar sacs, and alveoli.
Respiratory effects are any adverse effects on the structure or
functions of the respiratory system related to exposure to a chemical
substance.
Target organ is any organ found to be a target of toxicity in the 4-
hour (hr) high concentration group as a result of:
(1) The initial histopathologic examination (respiratory tract,
liver, kidney, gross lesions); or
(2) The retrospective histopathologic examination of archived organs
triggered by their identification as targets of toxicity in a 90-day
study.
Toxic effects are any adverse changes (a change that is
statistically and 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 and including the larynx.
(d) Principle of the test method. The test substance shall be
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 hrs following exposure, the animals shall be sacrificed and
necropsied, and tissue samples from the respiratory tract and other
major organs will be prepared for microscopic 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-hr test, additional
exposure periods of 1 hr and 8 hrs will be required to determine the
effect of exposure time on the toxicity observed. A 1-hr exposure study
can be elected as an option to provide data suitable for risk assessment
for very short duration exposures as may occur from chemical releases.
In the absence of adequate toxicological data for 1-hr exposure, the
Agency will extrapolate to shorter-term exposures from the 4-hr data on
the basis of concentration alone. This is a conservative method of
extrapolation, consistent with general Agency methods for deriving
criteria for short-term exposure from longer-term studies (a
concentration x time extrapolation would result in higher concentration
for a shorter duration).
(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 concentration level. The females shall be nulliparous and
nonpregnant.
(v) 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 shall be monitored for known viral and bacterial respiratory
pathogens determined by conventional microbiological assays (e.g.,
serology). The animals shall be free from pathogens at the start of
exposure.
[[Page 339]]
(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. 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 atmosphere, a vehicle control group shall be used.
If the 4- and 8-hr exposure studies are conducted concurrently, a
concurrent 8-hr sham-exposed control group may serve as the control
group for both the 4-hr and the 8-hr exposure studies, provided there is
adequate historical control data showing no changes in histopathology or
bronchoalveolar lavage of controls exposed for 4 and 8 hrs. Similarly,
if the optional 1-hr exposure study is conducted concurrently with the
4- and/or 8-hr study, the concurrent control group for those studies may
also be used for the 1-hr study, provided adequate historical control
data show no changes in histopathology or bronchoalveolar lavage between
controls exposed for these time periods.
(4) Concentration level and concentration selection. For the 4-hr
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 produce an exposure-response 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 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-adverse-
effects level (NOAEL).
(i) Limit concentration. For aerosols and particles, the high
concentrations need not be greater than 2 mg/L, or concentrations that
cannot maintain a particle size distribution having an MMAD between 1
and 4 [micro]m (i.e., a particle size that permits inhalability and
deposition throughout the respiratory tract). 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 hrs 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.
(ii) 8-hr study and optional 1-hr study. If the 8-hr study is
triggered, three concentrations shall be tested. These concentrations
should allow for the determination of an effect level and a NOAEL. If
the option to perform a 1-hr study is elected, three concentrations
shall be selected and tested in a similar manner.
(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 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 hr 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.
[[Page 340]]
(ii) Environmental conditions. The temperature at which the test is
performed shall 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.
(iii) Exposure periodicity. For acute testing, the exposure design
shall enable 4 hrs of exposure to the target concentrations, as defined
by an average of 5% for gases and vapors and
15% for particles and aerosols. If triggered by
the results of the 4-hr exposure, additional testing shall be conducted
in a comparable manner using an 8-hr exposure period.
(6) Physical measurements. Measurements or monitoring shall be made
of the following:
(i) Chemical purity of the test material shall be analyzed.
(ii) The rate of airflow shall be monitored continuously, but shall
be recorded at least every 30 minutes.
(iii) 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
practical, 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. Well-
established and published monitoring methods should be used where
available. If no standard methods are available, then accuracy and
precision information must be supplied.
(iv) During the development of the generating system, appropriate
particle size analysis shall be performed to establish the stability of
the aerosol. During exposure, analysis should be conducted as often as
necessary to determine the consistency of particle size distribution.
The particle size distribution shall have an MMAD between 1 and 4
[micro]m. The particle size of hygroscopic materials shall be small
enough when dry to assure that the size of the particle at saturation
will still have an MMAD between 1 and 4 [micro]m. Characterization for
fibers shall include the bivariate distribution of length and diameter;
this distribution must ensure inhalability.
(v) If the test substance is present in a mixture, the mass and
composition of the entire mixture, as well as the principal compound,
shall be measured.
(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 hrs following exposure to allow
expression 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
gross necropsy 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
thereof, shall 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; 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 shall also be preserved in a
suitable medium.
(10) Histopathology. The following histopathology shall be
performed:
[[Page 341]]
(i) Full histopathology shall be performed on the respiratory tract,
liver and kidney of all animals in the control and high concentration
groups. The histopathology of the respiratory tract is described under
paragraph (e)(11) of this section.
(ii) All gross lesions which differ from controls in frequency,
distribution, type, or severity in all concentration groups.
(iii) Target organs in all animals, as indicated by the observations
in the high concentration group in this study. Histopathologic
examination of target organs in animals at all concentration levels
(rather than only to the extent necessary to define the NOAEL) can
support the application of exposure-response analyses such as the
benchmark concentration approach.
(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)
should be elevated in high concentration animals of the 4-hr acute study
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 respiratory bronchioles (if present), 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. The lungs shall be infused with a fixative while in
an inflated state of fixed pressure.
(iii) The upper respiratory tract shall be examined for
histopathologic lesions. This examination shall use a minimum of four
sections located as specified under paragraphs (e)(11)(iii)(A) through
(e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule
shall be conducted. The method described by the reference under
paragraph (h)(11) of this section should be given consideration. The use
of additional sections shall be left to the discretion of the study
pathologist, but consideration should be given to additional sections as
recommended in the reference under paragraph (h)(8) of this section to
ensure adequate evaluation of the entire upper respiratory tract,
particularly the nasopharyngeal meatus. The following transverse
sections shall be examined:
(A) Immediately posterior to the upper incisor teeth.
(B) At the incisor papilla.
(C) At the second palatal ridge.
(D) At the level of the first upper molar teeth.
(iv) The laryngeal mucosa shall be examined for histopathologic
changes. Sections of the larynx to be examined include the epithelium
covering the base of the epiglottis, the ventral pouch, and the medial
surfaces of the vocal processes of the arytenoid cartilages.
(12) Bronchoalveolar lavage. (i) Animals can be exposed to the
substance by either a nose-only procedure or in a whole-body exposure
chamber.
(ii) Care should be taken that the method used to kill the animal
results 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 approximately 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 [deg]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 shall
be performed to determine macrophage activity. Assay methods described
in the references under paragraphs (h)(1) and (h)(3) of this section may
be used.
(13) Combined protocol. The tests described may be combined with any
other toxicity study, as long as none of
[[Page 342]]
the requirements of either are violated by the combination.
(f) Triggered testing. If no adverse effects are seen in the 4-hr
study as compared with controls, no further testing is necessary. If the
4-hr study shows positive effects in histopathology or the
bronchoalveolar lavage, an 8-hr study shall be conducted. Only those
tissues showing positive results in the 4-hr study must be pursued in
the follow-up 8-hr study. Similarly, if the option to perform a 1-hr
study is exercised, only those tissues showing positive results in the
4-hr study shall be pursued.
(g) Data reporting and evaluation. The final test report shall
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.
(i) Description of exposure apparatus, including design, type,
dimensions, source of air, system for generating particles, 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,
humidity, and particulate aerosol concentration and size.
(iii) Exposure data shall be tabulated and presented with mean
values and 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 air.
(D) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by the volume of air).
(E) Actual concentration in test breathing zone.
(F) Particle size distribution (e.g., MMAD with GSD) and the
bivariate distribution of fiber length and diameter, where appropriate.
(2) Results--(i) General group animal data. The following
information shall 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 alterations
by 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.
(v) Results of the bronchoalveolar lavage study. Data shall be
tabulated 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 representative 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
[[Page 343]]
evaluation shall include the relationship between the concentrations of
the test substance and the presence 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 concentration-response curves for the
bronchoalveolar lavage and tables reporting observations at each
concentration level for necropsy findings and gross, general, and
respiratory system histopathology.
(h) Reference. 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., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Burleson, G.R., Fuller, L.B., M[eacute]nache, M.G., and Graham,
J.A. Poly (I): poly (C)-enhanced alveolar peritoneal macrophage
phagocytosis: Quantification by a new method utilizing fluorescent
beads. Proceedings of the Society of Experimental Biology and Medicine.
184:468-476 (1987).
(2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.)
Toxicology of the Lung. (Raven Press, New York, 1993) pp. i-xii, 1-30.
(3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary
defenses against streptococcal infection in rats and mice following O3
exposure: Differences in disease susceptibility and neutrophil
recruitment. Toxicology and Applied Pharmacology. 123:211-218 (1993).
(4) 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 approaches
for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid
analysis. Fundamental and Applied Toxicology. 5:451-458 (1985).
(5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung
damage. Environmental Health Perspectives. 56:115-129 (1984).
(6) 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
Pharmacology. 50:123-136 (1979).
(7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition.
Concepts in Inhalation Toxicology. (Taylor and Francis, Washington, DC,
1995) pp.i-xxiv, 1-24, 441-470.
(8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T.
Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions
in Rats and Mice. Toxicologic Pathology. 22:353-372 (1994).
(9) Phalen, R.F. (Ed) Methods in Inhalation Toxicology. (CRC Press,
Boca Raton, FL, 1997) pp. i-xii, 1-12.
(10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and
Grumbein, S.L. Histologic methods and interspecies variations in the
laryngeal histology of F344/N rats and B6C3F1 mice. Toxicology and
Pathology. 20:44-51 (1992).
(11) Young, J.T. Histopathologic examination of the rat nasal
cavity. Fundamental and Applied Toxicology. 1:309-312 (1981).