[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.66]
[Page 564-567]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
Subpart F--Testing Requirements for Registration
Sec. 79.66 Neuropathology assessment.
(a) Purpose. (1) The histopathological and biochemical techniques in
this guideline are designed to develop data in animals on morphologic
changes in the nervous system associated with repeated inhalation
exposures to motor vehicle emissions. These tests are not intended to
provide a detailed evaluation of neurotoxicity. Neuropathological
evaluation should be complemented by other neurotoxicity studies, e.g.
behavioral and neurophysiological studies and/or general toxicity
testing, to more completely assess the neurotoxic potential of an
exposure.
(2) [Reserved]
(b) Definition. Neurotoxicity (NTX) or a neurotoxic effect is an
adverse change in the structure or function of the nervous system
following exposure to a chemical substance.
(c) Principle of the test method. (1) Laboratory rodents are exposed
to one of several concentration levels of a test atmosphere for at least
six hours daily over a period of 90 days. At the end of the exposure
period, the animals are anaesthetized, perfused in situ with fixative,
and tissues in the nervous system are examined grossly and prepared for
microscopic examination. Starting with the highest dosage level, tissues
are examined under the light microscope for morphologic changes, until a
no-observed-adverse-effect level is determined. In cases where light
microscopy has revealed neuropathology, the NOAEL may be confirmed by
electron microscopy.
(2) The tests described herein may be combined with any other
toxicity study, as long as none of the requirements of either are
violated by the combination. Specifically, this assay may be combined
with a subchronic toxicity study, pursuant to provisions in Sec. 79.62.
(d) Limit test. If a test at one dose level of the highest
concentration that can be achieved while maintaining a particle size
distribution with a mass median aerodynamic diameter (MMAD) of 4
micrometers ([mu]m) or less, using the procedures described in paragraph
(a) of this section, produces no observable toxic effects and if
toxicity would not be expected based upon data of structurally related
compounds, then a full study using three dose levels might not be
necessary. Expected human exposure though may indicate the need for a
higher dose level.
(e) Test procedures--(1) Animal selection--(i) Species and strain.
Testing shall be performed in the species being used in other NTX tests.
A standard strain of laboratory rat is recommended. The choice of
species shall take into consideration such factors as the comparative
metabolism of the chemical and species sensitivity to the toxic effects
of the test substance, as evidenced by the results of other studies, the
potential for combined studies, and the availability of other toxicity
data for the species.
(ii) Age. Animals shall be at least ten weeks of age at the start of
exposure.
(iii) Sex. Both sexes shall be used unless it is demonstrated that
one sex is refractory to the effects of exposure.
(2) Number of Animals. A minimum of ten animals per group shall be
used. The tissues from each animal shall be examined separately.
(3) Control Groups. (i) A concurrent control group, exposed to
clean, filtered air only, is required.
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(ii) The laboratory performing the testing shall provide positive
control data, e.g., results from repeated acrylamide exposure, as
evidence of the ability of their histology procedures to detect
neurotoxic endpoints. Positive control data shall be collected at the
time of the test study unless the laboratory can demonstrate the
adequacy of historical data for the planned study.
(iii) A satellite group of 10 female and 10 male test subjects shall
be treated with the highest concentration level for the duration of the
exposure and observed thereafter for reversibility, persistence, or
delayed occurrence of toxic effects during a post-treatment period of
not less than 28 days.
(4) Inhalation exposure. (i) All data developed within this study
shall be in accordance with good laboratory practice provisions under
Sec. 79.60.
(ii) The general conduct of this study shall be in accordance with
the vehicle emissions inhalation exposure guideline in Sec. 79.61.
(5) Study conduct--(i) Observation of animals. All toxicological
(e.g., weight loss) and neurological signs (e.g., motor disturbance)
shall be recorded frequently enough to observe any abnormality, and not
less than weekly.
(ii) The following is a minimal list of measures that shall be
noted:
(A) Body weight;
(B) Subject's reactivity to general stimuli such as removal from the
cage or handling;
(C) Description, incidence, and severity of any convulsions,
tremors, or abnormal motor movements in the home cage;
(D) Descriptions and incidence of posture and gait abnormalities
observed in the home cage; and
(E) Description and incidence of any unusual or abnormal behaviors,
excessive or repetitive actions (stereotypies), emaciation, dehydration,
hypotonia or hypertonia, altered fur appearance, red or crusty deposits
around the eyes, nose, or mouth, and any other observations that may
facilitate interpretation of the data.
(iii) Sacrifice of animals--(A) General. The goal of the techniques
outlined for sacrifice of animals and preparation of tissues is
preservation of tissue morphology to simulate the living state of the
cell.
(B) Perfusion technique. Animals shall be perfused in situ by a
generally recognized technique. For fixation suitable for light or
electronic microscopy, saline solution followed by buffered 2.5 percent
glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended.
While some minor modifications or variations in procedures are used in
different laboratories, a detailed and standard procedure for vascular
perfusion may be found in the text by Zeman and Innes (1963), Hayat
(1970), and Spencer and Schaumburg (1980) under paragraph (g) of this
section. A more sophisticated technique is described by Palay and Chan-
Palay (1974) under paragraph (g) of this section. In addition, the lungs
shall be instilled with fixative via the trachea during the fixation
process in order to preserve the lungs and achieve whole-body fixation.
(C) Removal of brain and cord. After perfusion, the bony structure
(cranium and vertebral column) shall be exposed. Animals shall then be
stored in fixative-filled bags at 4 deg.C for 8-12 hours. The cranium
and vertebral column shall be removed carefully by trained technicians
without physical damage of the brain and cord. Detailed dissection
procedures may be found in the text by Palay and Chan-Palay (1974) under
paragraph (g) of this section. After removal, simple measurement of the
size (length and width) and weight of the whole brain (cerebrum,
cerebellum, pons-medulla) shall be made. Any abnormal coloration or
discoloration of the brain and cord shall also be noted and recorded.
(D) Sampling. Cross-sections of the following areas shall be
examined: The forebrain, the center of the cerebrum, the midbrain, the
cerebellum, and the medulla oblongata; the spinal cord at the cervical
swelling (C3-C6), and proximal sciatic nerve (mid-
thigh and sciatic notch) or tibial nerve (at knee). Other sites and
tissue elements (e.g., gastrocnemius muscle) shall be examined if deemed
necessary. Any observable gross changes shall be recorded.
(iv) Specimen storage. Tissue samples from both the central and
peripheral
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nervous system shall be further immersion fixed and stored in
appropriate fixative (e.g., 10 percent buffered formalin for light
microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered
paraformaldehyde for electron microscopy) for future examination. The
volume of fixative versus the volume of tissues in a specimen jar shall
be no less than 25:1. All stored tissues shall be washed with buffer for
at least 2 hours prior to further tissue processing.
(v) Histopathology examination--(A) Fixation. Tissue specimens
stored in 10 percent buffered formalin may be used for this purpose. All
tissues must be immersion fixed in fixative for at least 48 hours prior
to further tissue processing.
(B) Dehydration. All tissue specimens shall be washed for at least 1
hour with water or buffer, prior to dehydration. (A longer washing time
is needed if the specimens have been stored in fixative for a prolonged
period of time.) Dehydration can be performed with increasing
concentration of graded ethanols up to absolute alcohol.
(C) Clearing and embedding. After dehydration, tissue specimens
shall be cleared with xylene and embedded in paraffin or paraplast.
Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded
together in one single block for sectioning. All tissue blocks shall be
labelled showing at least the experiment number, animal number, and
specimens embedded.
(D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall
be prepared from the tissue blocks and mounted on standard glass slides.
It is recommended that several additional sections be made from each
block at this time for possible future needs for special stainings. All
tissue blocks and slides shall be filed and stored in properly labeled
files or boxes.
(E) Histopathological techniques. The following general testing
sequence is proposed for gathering histopathological data:
(1) General staining. A general staining procedure shall be
performed on all tissue specimens in the highest treatment group.
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining
shall be differentiated properly to achieve bluish nuclei with pinkish
background.
(2) Peripheral nerve teasing. Peripheral nerve fiber teasing shall
be used. Detailed staining methodology is available in standard
histotechnological manuals such as AFIP (1968), Ralis et al. (1973), and
Chang (1979) under paragraph (g) of this section. The nerve fiber
teasing technique is discussed in Spencer and Schaumberg (1980) under
paragraph (g) of this section. A section of normal tissue shall be
included in each staining to assure that adequate staining has occurred.
Any changes shall be noted and representative photographs shall be
taken. If a lesion(s) is observed, the special techniques shall be
repeated in the next lower treatment group until no further lesion is
detectable.
(F) Examination. All stained microscopic slides shall be examined
with a standard research microscope. Examples of cellular alterations
(e.g., neuronal vacuolation, degeneration, and necrosis) and tissue
changes (e.g., gliosis, leukocytic infiltration, and cystic formation)
shall be recorded and photographed.
(f) Data collection, reporting, and evaluation. In addition to
information meeting the requirements stated under 40 CFR 79.60 and
79.61, the following specific information shall be reported:
(1) Description of test system and test methods. (i) A description
of the general design of the experiment shall be provided. This shall
include a short justification explaining any decisions where
professional judgment is involved such as fixation technique and choice
of stains; and
(ii) Positive control data from the laboratory performing the test
that demonstrate the sensitivity of the procedures being used.
Historical data may be used if all essential aspects of the experimental
protocol are the same.
(2) Results. All observations shall be recorded and arranged by test
groups. This data may be presented in the following recommended format:
(i) Description of signs and lesions for each animal. For each
animal, data must be submitted showing its identification (animal
number, treatment,
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dose, duration), neurologic signs, location(s) nature of, frequency, and
severity of lesion(s). A commonly-used scale such as 1+, 2+, 3+, and 4+
for degree of severity ranging from very slight to extensive may be
used. Any diagnoses derived from neurologic signs and lesions including
naturally occurring diseases or conditions, shall also be recorded;
(ii) Counts and incidence of lesions, by test group. Data shall be
tabulated to show:
(A) The number of animals used in each group, the number of animals
displaying specific neurologic signs, and the number of animals in which
any lesion was found; and
(B) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each
different type and/or location of lesion.
(iii) Evaluation of data. (A) An evaluation of the data based on
gross necropsy findings and microscopic pathology observations shall be
made and supplied. The evaluation shall include the relationship, if
any, between the animal's exposure to the test atmosphere and the
frequency and severity of any lesions observed; and
(B) The evaluation of dose-response, if existent, for various groups
shall be given, and a description of statistical method must be
presented. The evaluation of neuropathology data shall include, where
applicable, an assessment in conjunction with any other neurotoxicity
studies, electrophysiological, behavioral, or neurochemical, which may
be relevant to this study.
(g) References. For additional background information on this test
guideline, the following references should be consulted.
(1) 40 CFR 798.6400, Neuropathology.
(2) AFIP Manual of Histologic Staining Methods. (New York: McGraw-
Hill (1968).
(3) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry.
(Springfield, IL: Charles C. Thomas, 1979).
(4) Dunnick, J.K., et.al. Thirteen-week Toxicity Study of N-Hexane
in B6C3F1 Mice After Inhalation Exposure (1989) Toxicology, 57, 163-172.
(5) Hayat, M.A. ``Vol. 1. Biological applications,'' Principles and
techniques of electron microscopy. (New York: Van Nostrand Reinhold,
1970).
(6) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and
Organization. (New York: Springer-Verlag, 1974).
(7) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in
Neurohistology. (London: Butterworths, 1973).
(8) Sette, W. ``Pesticide Assessment Guidelines, Subdivision F,
Neurotoxicity Test Guidelines.'' Report No. 540/09-91-123 U.S.
Environmental Protection Agency 1991 (NTIS PB91-154617).
(9) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical
Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(10) Zeman, W., Innes, J.R.M. Craigie's Neuroanatomy of the Rat.
(New York: Academic, 1963).
[59 FR 33093, June 27, 1994, as amended at 63 FR 63793, Nov. 17, 1999]