United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–241

June 1996

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized 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 Animals) 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 Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this test guideline.

Amplitude is the voltage excursion recorded during the process of recording 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 exposure 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 generated 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 clearly toxic effects, but not result in an incidence of fatalities that would preclude 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 ED10, or alternatively, no effects.
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(ii) Subchronic (and chronic) studies. The high dose need not be

greater than 1g/kg. The high dose should result in significant neurotoxic

effects or other clearly toxic effects, but not produce an incidence of fatalities 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 ED10, 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 important 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 requirements of either are violated by the combination.

(7) Study conduct—(i) Choice of nerves. The nerve conduction velocity 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 required. 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 stimulation and recording may be made of any conventional electrode material,

such as stainless steel, although electrodes made of non-polarizing materials 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. (1) 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 electrodes) and should operate from about 10 μA 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.

(3) The recording environment should be enclosed in a Faraday cage

unless electromagnetic field pickup can be shown to be more than 1.5×

the amplifier baseline noise, under recording conditions. The recording

output should be amplified sufficiently 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 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. Response 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 stimulating 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 potential. 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 sensory 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 description of the experimental chambers, programming equipment, data collection devices, and environmental test conditions should be provided.

(ii) Provide a description of the experimental design including procedures 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 maintained 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 variability, e.g., means and standard deviations, and results of statistical analyses.

(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 effects 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 interpretation of the neurotoxicity of the test material should also be included.

(g) References. The following references should be consulted for additional 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 Clinical 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 Wilkins, Baltimore (1980).