[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: 40CFR798.6400]
[Page 217-221]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart G_Neurotoxicity
Sec. 798.6400 Neuropathology.
(a) Purpose. The techniques in this guideline are designed to
develop data on morphologic changes in the nervous system for chemical
substances and mixtures subject to such testing under the Toxic
Substances Control Act. The data will detect and characterize
morphologic changes, if and when they occur, and determine a no-effect
level for such changes. Neuropathological evaluation should be
complemented by other neurotoxicity studies, e.g. behavioral and
neurophysiological studies. Neuropathological evaluation may be done
following acute, subchronic or chronic exposure.
(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse
change in the structure or function of the nervous system following
exposure to a chemical agent.
(c) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being used per
group. The animals are sacrificed 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 effect level is
determined. In cases where light microscopy has revealed neuropathology,
the no effect level may be confirmed by electron microscopy.
(d) Test procedure--(1) Animal selection--(i) Species and strain.
Testing shall be performed in the species being used in other tests for
neurotoxicity. This will generally be the laboratory rat. 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.
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(ii) Age. Animals shall be young adults (150-200 gm for rats) at the
start of exposure.
(iii) Sex. Both sexes shall be used unless it is demonstrated that
one sex is refractory to the effects.
(2) Number of animals. A minimum of six animals per group shall be
used. The tissues from each animal shall be examined separately. It is
recomse (iv)mended that ten animals per group be used.
(3) Control groups. (i) A concurrent control group(s) is (are)
required. This group must be an untreated control group or, if a vehicle
is used in administering the test substance, a vehicle control group. If
the vehicle used has a known or potential toxic property, both untreated
and vehicle control groups are required.
(ii) A satellite group of animals may be treated with the high 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. At least 3 doses, equally spaced
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log
unit shall be used in addition to a zero dose or vehicle administration.
The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear behavioral effects or
(B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-
dependent effects at two dose levels or (B) no effects at two dose
levels, respectively.
(5) Duration of testing. The exposure duration will be specified in
the test rule. This will generally be 90 days exposure.
(6) Route of administration. The test substance shall be
administered by a route specified in the test rule. This will generally
be the route most closely approximating the route of human exposure. The
exposure protocol shall conform to that outlined in the appropriate
acute or subchronic toxicity guideline.
(7) Combined protocol. 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.
(8) 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) Sacrifice of animals--(A) General. The goal of the techniques
outlined for sacrifice of animals and preparation of tissues is
preservation of tissues 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) under
paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of
this section, and by Spencer and Schaumburg (1980) under paragraph
(f)(6) of this section. A more sophisticated technique is described by
Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.
(C) Removal of brain and cord. After perfusion, the bonystructure
(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 (f)(4) 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. Unless a given test rule specifies otherwise, cross-
sections of the following areas shall be examined: The forebrain, the
center of the cerebrum, the midbrain, the cerebellum
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and pons, and the medulla oblongata; the spinal cord at cervical and
lumbar swelling (C3-C6 and L1-
L4); Gasserian ganglia, dorsal root ganglia (C3-
C6, L1-L4), dorsal and ventral root
fibers (C3-C6, L\1\-L4), proximal
sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and
tibial nerve (at knee). Other sites and tissue elements (e.g.,
gastrocnemius muscle) should be examined if deemed necessary. Any
observable gross changes shall be recorded.
(iii) Specimen storage. Tissue samples from both the central and
peripheral 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.
(iv) 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. Although the information available
for a given chemical substance may dictate test-rule specific changes,
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) Special stains. Based on the results of the general staining,
selected sites and cellular components shall be further evaluated by the
use of specific techniques. If H&E screening does not provide such
information, a battery of stains shall be used to assess the following
components in all appropriate required samples: neuronal body (e.g.,
Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g.,
Kluver's Luxol Fast Blue) and neurofibrils (e.g., Bielchosky). In
addition, peripheral nerve fiber teasing shall be used. Detailed
staining methodology is available in standard histotechnological manuals
such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al.
(1973) under paragraph (f)(5) of this section, and Chang (1979) under
paragraph (f)(2) of this section. The nerve fiber teasing technique is
discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) 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.
(3) Alternative technique. If the anatomical locus of expected
neuro-pathology is well-defined, epoxy-embedded sections stained with
toluidine blue may be used for small sized tissue samples. This
technique obviates the need
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for special stains for cellular components. Detailed methodology is
available in Spencer and Schaumberg (1980) under paragraph (f)(6) of
this section.
(4) Electron microscopy. Based on the results of light microscopic
evaluation, specific tissue sites which reveal a lesion(s) shall be
further evaluated by electron microscopy in the highest treatment group
which does not reveal any light microscopic lesion. If a lesion is
observed, the next lower treatment group shall be evaluated until no
significant lesion is found. Detailed methodology is available in Hayat
(1970) under paragraph (f)(3) of this section.
(F) Examination--(1) General. 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.
(2) Electron microscopy. Since the size of the tissue samples that
can be examined is very small, at least 3 to 4 tissue blocks from each
sampling site must be examined. Tissue sections must be examined with a
transmission electron microscope. Three main categories of structural
changes must be considered:
(i) Neuronal body. The shape and position of the nucleus and
nucleolus as well as any change in the chromatin patterns shall be
noted. Within the neuronal cytoplasm, cytoplasmic organelles such as
mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments,
endoplasmic reticulum and polyribosomes (Nissl substance), Golgi
complex, and secretory granules shall be examined.
(ii) Neuronal processes. The structural integrity or alterations of
dendrites, axons (myelinated and unmyelinated), myelin sheaths, and
synapses shall be noted.
(iii) Supporting cells. Attention must also be paid to the number
and structural integrity of the neuroglial elements (oligodendrocytes,
astrocytes, and microglia) of the central nervous system, and the
Schwann cells, satellite cells, and capsule cells of the peripheral
nervous system. Any changes in the endothelial cells and ependymal
lining cells shall also be noted whenever possible. The nature,
severity, and frequency of each type of lesion in each specimen must be
recorded. Representative lesions must be photographed and labeled
appropriately.
(e) Data collection, reporting, and evaluation. In addition to
information meeting the requirements stated under 40 CFR part 792
subpart J, the following specific information shall be reported:
(1) Description of test system and test methods. 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.
(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, 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, should
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;
(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 substance and the
frequency and severity of the lesions observed.
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(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 should include, where
applicable, an assessment in conjunction with other neurotoxicity
studies performed (eg. electrophysiological, behavioral, neurochemical).
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-
Hill (1968).
(2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry.
(Springfield, IL: Charles C. Thomas, 1979).
(3) Hayat, M.A. ``Vol. 1. Biological applications,'' Principles and
techniques of electron microscopy. (New York: Van Nostrand Reinhold,
1970)
(4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and
Organization. (New York: Springer-Verlag, 1974).
(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in
Neurohistology. (London: Butterworths, 1973).
(6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical
Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat.
(New York: Academic, 1963).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]