United States       Prevention, Pesticides      EPA712-C-98-241
          Environmental Protection    and Toxic Substances      August 1998
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.6850
           Peripheral Nerve

     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

     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.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

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, Addendum 10, EPA report 540/09-91-123, March 1991).

     (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.

     (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 (42-120  days old for rats)
should be used.

     (iii) Sex. In order to reduce the number of animals used, and because
of the labor-intensive nature of this testing, only one  sex  may be  used.
If data indicate that one sex is more sensitive to the test substance,  or
if it receives greater exposure, it may be preferred. If females are  used,
they should be virgins.

     (2) Number of animals. At least 10 animals should be used in  each
test and control group. The number of animals to be used should be based
on  appropriate statistical methods  and  an allowance for attrition due to
anticipated problems, such as loss due to anesthesia, etc. Animals should
be  randomly assigned to treatment and control groups.  If not, some jus-
tification is required.

     (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 or procedures known to affect peripheral nerve  function. Per-
manently injurious substances need not be used. Temperature change could
be used as a positive control procedure without causing permanent injury
to the  animals. Historical data may be used if the essential  aspects of the
experimental procedure  remain  the same. Periodic updating of positive
control data  is recommended. New positive control data should  also be
collected when personnel or some other critical element in the testing lab-
oratory has changed.

     (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.

     (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, bio-
availability, 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 impor-
tant  and that these  criteria may  conflict with one another. The route that
best  meets these criteria should  be selected.  Dietary  feeding will be gen-
erally 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

     (ii) Preparation. (A) In vivo testing of anesthetized  animals  is re-
quired. A barbiturate  or an inhalation anesthetic such as isoflorane is ap-
propriate.  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.

     (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

     (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 testing environment should be isolated from extraneous light
and noise and controlled for temperature. Enclosure in a Faraday cage can
help reduce 50 Hz noise. The recording output should be amplified suffi-
ciently to render the compound action potential 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 electrode-preparation interface
produces, and operate without significant current leakage through the re-
cording 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 inter-
stimulus  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)
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-
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-

     (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) Guidance for  interpretation of peripheral nerve function data is
described under paragraph (g)(5) of this guideline.

     (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).

    (4) Johnson, E.W. Practical Electromyography. Williams and Wil-
kins, Baltimore (1980).

    (5)  U.S.   Environmental  Protection   Agency.   Guidelines  for
Neurotoxicity Risk Assessment. FEDERAL REGISTER 63  FR 26926-26954,
May 14, 1998.