United States Prevention, Pesticides EPA712-C-96-241
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.6850
Peripheral Nerve
Function
"Public Draft'
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the
OPP Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 870.6850 Peripheral nerve function.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA)(7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA)(15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798.6850 Peripheral Nerve
Function and OPP 85-6 Peripheral Nerve Function (Pesticide Assessment
Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Ani-
mals) EPA report 540/09-82-025, 1982.
(b) Purpose. In the assessment and evaluation of the potential human
health effects of substances, it may be necessary to test for
neurophysiological effects. Substances that have been shown to produce
peripheral neuropathy in other neurotoxicity studies (or other
neuropathological changes in peripheral nerves), as well as substances with
a structural similarity to those causing such effects, may be appropriate
to evaluate with this test. This guideline defines procedures for evaluating
certain aspects of the neurophysiological functioning of peripheral nerves.
Our purpose is to evaluate the effects of exposures on the velocity and
amplitude of conduction of peripheral nerves. Any observed effects should
be evaluated in the context of both the concordance between functional
neurological and neuropathological effects and with respect to any other
toxicological effects seen. Additional tests may be necessary to completely
assess the neurophysiological effects of any substance.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.
Amplitude is the voltage excursion recorded during the process of re-
cording the compound nerve action potential. It is an indirect measure of
the number of axons firing.
Conduction velocity is the speed at which the compound nerve action
potential traverses a nerve.
Neurotoxicity is any adverse effect on the structure or function of
the nervous system related to exposure to a chemical substance.
(d) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being used per group.
The peripheral nerve conduction velocity and amplitude are assessed using
electrophysiological techniques. The exposure levels at which significant
neurotoxic effects are produced are compared to one another and to those
levels that cause neuropathological effects and/or other toxic effects.
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(e) Test procedures—(1) Animal selection—(i) Species and strain.
Testing should be performed on a laboratory rodent unless such factors
as the comparative metabolism of the chemical or species sensitivity to
the toxic effects of the test substance, as evidenced by the results of other
studies, dictate otherwise. All animals should have been laboratory-reared
to ensure consistency of diet and environmental conditions across groups
and should be of the same strain and from the same supplier. If this is
not possible, groups should be balanced to ensure that differences are not
systemically related to treatment.
(ii) Age and weight. Young adult animals (at least 60 days old for
rats) must be used. Age (+15 days for rats) must not vary across groups.
Weights should be within ±10 percent of the mean.
(iii) Sex. Either (or both) sexes may be used. Sex must not vary across
groups.
(2) Number of animals. Twenty animals should be exposed to each
level of the test substance and/or control procedure. The goal is to be
able to detect a 10 percent change from normal conduction velocity at
the 5 percent level with 90 percent power.
(3) Control groups, (i) A concurrent control group is required. For
control groups, subjects should be treated in the same way as for an expo-
sure group except that administration of the test substance is omitted.
(ii) Positive control data from the laboratory performing the testing
should provide evidence that the experimental procedures are sensitive to
substances known to affect peripheral nerve function. Positive control data
should be collected at the time of the test study unless the laboratory can
demonstrate the adequacy of historical data for this purpose, i.e. by the
approach outlined in this guideline. Positive control data need only be gen-
erated roughly once every year as long as most conditions remain the
same.
(4) Dose levels and dose selection. At least three doses should be
used in addition to the vehicle control group. The data should be sufficient
to produce a dose-effect curve. The Agency strongly encourages the use
of equally spaced doses and a rationale for dose selection that will enable
detection of dose-effect relations to the highest degree.
(i) Acute studies. The high dose need not be greater than 2 g/kg.
The high dose should result in significant neurotoxic effects or other clear-
ly toxic effects, but not result in an incidence of fatalities that would pre-
clude a meaningful evaluation of the data. The middle and low doses
should be fractions of the high dose. The lowest dose should produce mini-
mal effects, e.g., an ED 10, or alternatively, no effects.
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(ii) Subchronic (and chronic) studies. The high dose need not be
greater than Ig/kg. The high dose should result in significant neurotoxic
effects or other clearly toxic effects, but not produce an incidence of fatali-
ties that would prevent a meaningful evaluation of the data. The middle
and low doses should be fractions of the high dose. The lowest dose should
produce minimal effects, e.g an ED 10, or alternatively, no effects.
(5) Route of administration. Selection of route may be based on
several criteria including, the most likely route of human exposure,
bioavailability, the likelihood of observing effects, practical difficulties,
and the likelihood of producing nonspecific effects. For many materials,
it should be recognized that more than one route of exposure may be im-
portant and that these criteria may conflict with one another. The route
that best meets these criteria should be selected. Dietary feeding will be
generally be acceptable for repeated exposure studies.
(6) Combined protocol. The test described in this guideline may be
combined with any other toxicity study, as long as none of the require-
ments of either are violated by the combination.
(7) Study conduct—(i) Choice of nerves. The nerve conduction ve-
locity test must assess the properties of both sensory and motor nerve
axons separately. Either a hind limb (e.g. tibial) or tail (e.g. ventral caudal)
nerve must be chosen. Response amplitude may be measured in a mixed
nerve.
(ii) Preparation. (A) In vivo testing of anesthetized animals is re-
quired. A barbiturate anesthetic is appropriate. Care should be taken to
ensure that all animals are administered an equivalent dosage and that the
dosage is not excessive. If dissection is used, extreme caution must be
observed to avoid damage to either the nerve or the immediate vascular
supply.
(B) Both core and nerve temperature must be monitored and kept
constant (±0.5 °C) during the study. Monitoring of skin temperature is
adequate if it can be demonstrated that the skin temperature reflects the
nerve temperature in the preparation under use. Skin temperature should
be monitored with a needle thermistor at a constant site, the midpoint of
the nerve segment to be tested.
(iii) Electrodes—(A) Choice of electrodes. Electrodes for stimula-
tion and recording may be made of any conventional electrode material,
such as stainless steel, although electrodes made of non-polarizing mate-
rials are preferable. If surface electrodes are used, care must be taken to
ensure that good electrical contact is achieved between the electrode and
the tissue surface. All electrodes must be thoroughly cleaned following
each application.
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(B) Electrode placement. Electrode placement must be constant with
respect to anatomical landmarks across animals (e.g. a fixed number of
millimeters from the base of the tail). Distances between electrodes used
to calculate conduction velocity must be measurable to ±0.5 mm. The
recording electrodes should be as far from the stimulating electrodes as
possible. A 40 mm separation is adequate in the caudal tail nerve of the
rat.
(C) Recording conditions. (7) The animal should be grounded at
about midpoint between the nearest stimulating and recording electrodes.
With the preamplifier set at its maximal band width, the stimulus artifact
should have returned to baseline before any neural response to be used
in the analysis is recorded.
(2) The electrical stimulator must be isolated from ground. Biphasic
or balanced pair stimuli to reduce polarization effects are acceptable. A
constant current stimulator is preferred (and required for polarized elec-
trodes) and should operate from about 10 (iA to about 10 mA. If a constant
voltage stimulator is used, it should operate to 250V. All equipment should
be calibrated with respect to time, voltage, and temperature.
The recording environment should be enclosed in a Faraday cage
unless electromagnetic field pickup can be shown to be more than 1.5x
the amplifier baseline noise, under recording conditions. The recording
output should be amplified sufficiently to render the compound action po-
tential easily measurable with an oscilloscope. The amplifier should pass
signals between 2.0 Hz and 4 kHz without more than a 3dB decrement.
The preamplifier must be capacitatively coupled or, if direct coupled to
the first stages, must be able to tolerate any DC potentials which the elec-
trode-preparation interface produces, and operate without significant cur-
rent leakage through the recording electrodes.
(4) A hard copy for all waveforms or averaged waveforms from
which measurements are derived, and for all control recording required
by this standard must be available. Hard copies must include a time and
voltage calibration signal.
(iv) Procedure — (A) General. Stimulation should occur at an
interstimulus interval significantly below the relative refractory period for
the nerve under study. Stimulus intensity should be increased gradually
until the response amplitude no longer increases. At this point the maximal
stimulus current is determined. An intensity 25-50 percent (a fixed value
in a given study) above the maximal intensity so determined should be
used for determining response peak latency and response amplitude. Re-
sponse peak latency may be read off the oscilloscope following single
sweeps or determined by an average of a fired number of responsors. The
baseline-to-peak height technique (under paragraph (g)(2) of this guideline)
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is acceptable for determination of the nerve compound action potential
amplitude, but in this case, at least 16 responses must be averaged.
(B) Motor nerve. Motor conduction velocity may be measured from
a mixed nerve by recording the muscle action potential which follows the
compound action potential of the nerve. The stimulus intensity should be
adjusted so that the amplitude of the muscle action potential is
supramaximal. Measurement of the latency from stimulation to the onset
of the compound muscle action potential gives a measure of the conduction
time of the motor nerve fibers. To calculate the conduction velocity, the
nerve must be stimulated sequentially in two places each with the same
cathode-anode distance, and with the cathode located toward the recording
electrode. The cathode to cathode distance between the two sets of stimu-
lating electrodes should be divided by the difference between the two
latencies of muscle action potential in order to obtain conduction velocity.
Placement of electrodes should be described; site of nerve stimulation may
differ from point of entry through skin.
(C) Sensory nerve. The somatosensory evoked potential may be used
to determine the sensory nerve conduction velocity in a mixed nerve. The
cathode should be placed proximally at the two stimulation locations with
the same cathode-anode distances. The recording electrodes are placed on
the skull. The conduction velocity is calculated by dividing the distance
between the two stimulating cathodes by the difference between the two
latencies of the largest primary peak of the somatosensory evoked poten-
tial. Between 64 and 123 responses should be averaged. The stimulation
frequency should be about 0.5 Hz. Stimulus intensity should be the same
as that used for determining the motor conduction velocity. Should the
peak of the somatosensory response be so broad that it cannot be replicated
with an accuracy of less than 5 percent of the latency difference observed,
then a point on the rising phase of the potential should be chosen, e.g.
at a voltage that is 50 percent of the peak voltage. Alternatively, the sen-
sory nerve conduction velocity can be obtained from a purely sensory
nerve or from stimulation of the dorsal rootlets of a mixed nerve, using
two recording electrode pairs.
(f) Data collection, reporting, and evaluation. The final test report
must include the following information:
(1) Description of equipment and test methods, (i) Give a descrip-
tion of the experimental chambers, programming equipment, data collec-
tion devices, and environmental test conditions should be provided.
(ii) Provide a description of the experimental design including proce-
dures for balancing treatment groups.
(iii) Positive control data from the laboratory performing the test
which demonstrate the sensitivity of the procedure being used should be
provided. Historical data may be used if all essential aspects of the experi-
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mental protocol are the same. Historical control data can be critical in
the interpretation of study findings. The Agency encourages submission
of such data to facilitate the rapid and complete review of the significance
of effects seen.
(iv) Include hard copies of waveforms from which measurements
were made as well as control recordings.
(v) Provide voltage and time calibration referable to the standards
of the National Institute of Standards and Technology (NIST) or to other
standards of accuracy sufficient for the measurements used.
(vi) Include data demonstrating that nerve temperature was main-
tained constant throughout the recording period.
(2) Results. Data for each animal should be arranged in tabular form
by test group, including the animal identification number, body weight,
nerve conduction velocity, and amplitude. Group summary data should
also be reported, including standard measures of central tendency and vari-
ability, e.g., means and standard deviations, and results of statistical analy-
ses.
(3) Evaluation of data, (i) The findings should be evaluated in the
context of preceding and/or concurrent toxicity studies and any correlated
functional and histopathological findings. The evaluation should include
the relationship between the doses of the test substance and the incidence
and magnitude of any observed effects, i.e. dose-effect curves for any ef-
fects seen.
(ii) The evaluation should include appropriate statistical analyses.
Choice of analyses should consider tests appropriate to the experimental
design, including repeated measures. There may be many acceptable ways
to analyze data.
(iii) Any known citations from the open literature related to the inter-
pretation of the neurotoxicity of the test material should also be included.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline:
(1) Aminoff, M.J. (Ed.) Electrodiagnosis in Clinical Neurology.
Churchill Livingstone, NY (1980).
(2) Daube, J. Nerve Conduction Studies. In: Electrodiagnosis in Clini-
cal Neurology. M.J. Aminoff (Ed.) Churchill Livingstone, NY. Pp. 229-
264 (1980).
(3) Glatt, A.F. et al. Testing of peripheral nerve function in chronic
experiments in rats. Pharmacology and Therapeutics 5:539-534 (1979).
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(4) Johnson, E.W. Practical Electromyography. Williams and Wil-
kins, Baltimore (1980).
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