United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–237

June 1996

Health Effects Test

Guidelines

OPPTS 870.6100

Delayed Neurotoxicity of

Organophosphorus

Substances Following

Acute and 28–Day

Exposure

‘‘Public Draft’’
i

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 810.6100 Delayed neurotoxicity of organophosphorus substances following acute and 28–day exposure.

(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 798.6450 NTE Neurotox

Assay, 40 CFR 798.6540 Acute Delayed Neurotoxicity of

Organophosphorus Compounds, 40 CFR 798.6560 Subchronic Delayed

Neurotoxicity of Organophosphorus Compounds; OPP 81–7 Delayed

Neurotoxicity (Acute)—Hen, OPP 82–5 Subchronic Neurotoxicity: 90–

Day Study, OPP 82–6 28–Day Delayed Neurotoxicity—Hen (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Animals) EPA report 540/09–82–025, 1982; OECD 418 Acute Delayed Neurotoxicity of Organophosphorus Substances and OECD 419

Subchronic Delayed Neurotoxicity of Organophosphorus Substances—90–

Day.

(b) Purpose. In the assessment of organophosphorus substances,

(OPs) studies of delayed neurotoxicity using the adult hen as the test animal and including behavioral observation of gait, histopathological assessment of brain, peripheral nerve, and spinal cord, and neurochemical assessment of inhibition of acetylcholinesterase (AChE) and neurotoxic esterase

(NTE) are needed to identify and characterize these potential effects.

(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.

ED is the effective dose.

LD50 is the median lethal dose.

Neuropathy target esterase (NTE) or neurotoxic esterase is a membrane-bound protein that hydrolyzes phenyl valerate. The inhibition and

‘‘aging’’ of the phosphorylated NTE, i.e. the covalent binding of the OP

to the enzyme, is highly correlated with the initiation of organophosphorus

induced delayed neurotoxicity (OPIDN). Not all OPs that inhibit NTE

cause OPIDN, but all OPs that cause OPIDN inhibit NTE.

NOEL is the no-observed-effect-level.

NTE activity is operationally defined as the phenyl valerate hydrolytic

activity resistant to paraoxon (diethyl 4-nitrophenyl phosphate) but sensitive to mipafox (N,N′-diisopropylphosphorodiamido fluoridate) or neuropathic OP ester inhibition.
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Organophosphorus induced delayed neurotoxicity (OPIDN) is a neurological syndrome in which limb weakness and upper motor neuron spasticity are the predominant clinical signs; distal axonopathy of peripheral

nerve and spinal cord are the correlative pathological signs, and inhibition

and aging of neurotoxic esterase in neural tissues are the correlative biochemical effects. Clinical signs and pathology first appear between 1 and

2 weeks following exposures that typically inhibit and subsequently age

neurotoxic esterase.

(d) Principle of the test method. The test sequence consists of acute

and 28–day exposure studies. Any significant effects on behavior (delayed

effects), histopathology, or inhibition of NTE in the acute study are sufficient cause to conduct the 28–day study. The test substance is administered

orally to domestic hens that in some cases have been protected from acute

cholinergic effects. The animals are observed for at least 21 days after

the last dose for gait changes and other signs. Neurochemical examination

of selected neural tissues is undertaken on some animals at some times

after exposure. Histopathology of brain, spinal cord, and peripheral nerve

are performed at the termination of 21–day observation periods. If the results of the acute study are completely negative, that is, there are no delayed behavioral or histopathological effects, and no significant NTE inhibition, the 28–day study is not required. Otherwise, the 28–day study

should be conducted. In the 28–day study, three exposure levels are used

to describe the dose response curve sufficiently to estimate a reference

dose.

(e) Test procedures—(1) Animal selection. The adult domestic laying hen (Gallus gallus domesticus), aged 8 to 14 months, is recommended.

Standard size breeds and strains should be employed. Healthy young adult

hens free from interfering viral diseases and medication and without abnormalities of gait should be acclimatized to the laboratory conditions for

at least 5 days prior to randomization and assignment to treatment and

control groups.

(2) Housing and feeding conditions. Cages or enclosures which are

large enough to permit free mobility of the hens and easy observation

of gait should be used. Where the lighting is artificial, the sequence should

be 12 h light/12 h dark. Appropriate diets should be administered as well

as an unlimited supply of drinking water. The hens should be weighed

weekly. Any moribund hens should be removed and sacrificed.

(3) Route of administration. Dosage of test substance should normally be by the oral route, preferably by gavage. Liquids may be given

neat or dissolved in an appropriate vehicle such as corn oil; solids should

be dissolved if at all possible since large doses of solids in gelatin capsules

may significantly impair absorption. Dermal exposures may be the most

significant route of exposure for applicators and for nonfood uses and there
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may be important differences in toxicity by this route. Conduct of these

studies by this route may be appropriate and should be considered.

(4) Study design—(i) General. An important consideration for the

design of these studies is prediction of activity based on the structure of

the material and the published literature. Some materials, e.g.

phosphinates, are known to inhibit NTE, but not to be capable of aging

and thus are not expected to cause OPIDN. Many materials have structural

features that will permit inhibition and aging, i.e. ester linkages, and are

of potential concern. Published data are available for many materials and

may be very useful for many aspects of the design and interpretation of

these studies.

(ii) Dose levels and selection. For the acute study, a single exposure

group is required. The acute dose level should be chosen to maximize

the amount of material given to the hens, particularly in cases where some

activity is expected. For the 28–day study, at least three exposure groups

are required in addition to the vehicle control group. Ideally, the data

should be sufficient to produce a dose-effect curve. The use of equally

spaced doses and a rationale for dose selection that will maximally support

detection of dose-effect relations is strongly encouraged. The rationale for

dose selection chosen by the investigator should be explicitly stated. The

following guidance for dose selection is somewhat complex and is not

intended to be followed rigidly.

(A) Acute study. Selection of the dose level for the acute study may

be based on a limit dose or lethal doses and other available data, e.g.

on NTE inhibition.

(1) Levels of test substances greater than 2 g/kg need not be tested.

(2) Lethal doses. Either an LD50 or an approximate lethal dose

(ALD) in the hen may be used to determine the acute high dose. If, from

the preliminary data, cholinergic signs are seen very soon after dosing,

prophylaxis using atropine may be appropriate. Atropine (20 mg/kg, s.c.,

up to every 2 h) should be used to prevent death from acute cholinergic

effects.

(B) 28–day study. (1) Levels of test substances greater than 1 g/

kg need not be tested.

(2) High dose—the high dose selected should be estimated to be sufficient to cause OPIDN or be a maximum tolerated dose based on the acute

data, but not result in an incidence of fatalities that would prevent a meaningful evaluation of the data.

(3) Low dose—the low dose should be estimated to be a minimum

effect level, e.g. an ED10, or alternatively, a NOEL.
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(4) The intermediate dose level should be equally spaced between

the high and low doses.

(5) Intermediate responses in NTE i.e. greater than 15 percent and

less than 70 percent, can be crudely extrapolated as if the dose-response

were a simple first order relationship. That is, if a certain dose caused

50 percent inhibition, twice that dose might cause 75 percent inhibition.

Such extrapolation is very crude but can be useful in giving some guidance

for dose estimation.

(iii) Numbers of animals. Exposure groups should be large enough

to provide six survivors for both behavioral observations and

histopathology. At least three hens are required for determination of NTE

in each dose or control group and at each time point.

(iv) Control groups. A positive control group of at least six hens

treated with a known delayed neurotoxicant, such as tri-ortho-cresyl phosphate (TOCP), is required for both acute and 28–day studies. This group

may be a concurrent or historical control group. (This should also include

at least three hens assessed for biochemical measurements.) Periodic

redeterminations of the sensitivity of the assays is suggested, for historical

control data, i.e. when some essential element of the test conduct by the

performing laboratory has changed. A concurrent control group sufficient

to provide six survivors for histopathology and three hens for NTE measurement are treated in a manner identical to the treated groups, except

that administration of the test substance is omitted. When protective agents

are used, all members of the dose groups and vehicle controls should receive the same treatment.

(5) Study conduct—(i) Biochemical measurements—(A) NTE

assay. The test method is a differential assay of the ability of neural tissue,

following OP exposure, to hydrolyze a phenyl valerate substrate selectively. The principle of the assay is first to determine the amount of hydrolysis that occurs in the presence of a nonneurotoxic inhibitor, paraoxon,

(a), which is intended to occupy irrelevant sites, and second to determine

the activity in the presence of paraoxon and a known neuropathic inhibitor,

mipafox, (b). NTE activity is the difference between (a) and (b), that is,

the proportion of activity inhibited only by mipafox. Thus, the ‘‘mipafox

site’’ is already occupied following exposure to a neuropathic OP ester

and the activity of (b) is therefore reduced.

(1) Three hens from each group should be sacrificed at 48 h after

the last dose. Depending on the duration of acute signs as an indication

of the disposition of the test material, the time for sacrifice for NTE and

AchE assessment may be chosen at a different time to optimize detection

of effects. Both the brain and spinal cord should be prepared for assay

of NTE. Perform duplicate assays of NTE in brain and spinal cord of

three birds from each group and control group.
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(2) Materials. This assay requires paraoxon, mipafox, and phenyl valerate, all of which can be obtained commercially.

(3) The assay has four stages: Preparation of tissue, differential

preincubation, hydrolysis of substrate, and measurement of product. The

quotations that follow are from Johnson under paragraph (g)(7) of this

guideline as corrected or modified in paragraph (g)(9) of this guideline.

His is the best known method for conduct of this assay. Other acceptable

methods, which primarily involve minor technical modification, have been

used (see paragraphs (g)(13) and (g)(14) of this guideline).

* * * the whole brain (is) removed and cooled in ice-cold buffer (50 mM

Tris/0.2 mM EDTA adjusted to pH 8.0 at 25 °C with HCl). Meninges and blood

vessels are rapidly removed and the brain is blotted dry, weighed, and homogenized thoroughly in ice-cold buffer (at a volume of at least 1:30, W/V), using

a high-speed rotating perspex pestle with not more than 0.25 mm difference

in diameter between pestle and tube.

Paired samples of homogenate (equivalent to about 6.0 mg tissue) are

preincubated in Tris/EDTA buffer pH 8 at 37 °C for exactly 20 min with

paraoxon (40 to 100 μM) plus either (a) buffer or (b) mipafox (50 μM) in a

final volume of 2 mL.

After preincubation, dispersion (2 mL) of phenyl valerate is added and the

incubation is continued for exactly 15 min. The dispersion is prepared by adding

a solution of Triton X–100 (0.03 percent in water) (30 vol) to a solution of

phenyl valerate (15 or 20 mg/mL) in redistilled dimethylformamide (1 vol) and

mixing thoroughly (by swirling): other solvents give less satisfactory dispersions.

Reaction is stopped by adding 2 mL of sodium dodecyl sulfate (1–2 percent

W/V) in buffer containing 4-aminoantipyrine (otherwise known as 4-aminophenazone) (0.25 percent).

This assay is based on the colorimetric determination of liberated phenol.

The coupling of phenol liberated in the assay with the aminoantipyrine may be

performed at any convenient time after quenching the enzyme: 1 mL of

K3Fe(CN)6 (0.4 percent in water) is added and the stable red colour is read

at 490 nm.

A nontissue blank, kept to 10 percent of the paraoxon tube value by maintaining the substrate phenol-free, should be included in each group of assay

tubes. Typical control absorbance values would be 0.8 for paraoxon, 0.35 for

paraoxon and mipafox and 0.07 for the blank. Colour development takes

1–2 min in solutions stopped with sodium dodecyl sulphate. The extinction coefficient of phenol under these conditions is 15,600 at a wavelength of 490 nm.

NTE activity is represented by the difference in absorbance obtained from samples incubated under conditions (a) and (b) respectively.

Under standard conditions NTE hydrolyzes about 2,400 nmol substrate/

min/g of cortex, 550 for spinal cord, and 100 for sciatic nerve.

(B) AChE measures. Assay of acetylcholinesterase in the brains of

the same birds (according to paragraphs (g)(3) and (g)(4) of this guideline)

should also be performed. The level of AChE inhibition is a useful index

of lethal potency and the ratio of lethal potency to NTE inhibitory potency

can be useful for subsequent dose selection.
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(ii) 21–Day observation. All remaining hens should be carefully observed at least once daily for a period of at least 21 days until 21 days

after the last dose and signs of toxicity recorded, including the time of

onset, degree, and duration. Observations should include, but not be limited to, behavioral abnormality, locomotor ataxia, and paralysis. At least

twice a week the hens should be taken outside the cages and subjected

to a period of forced motor activity, such as ladder climbing, in order

to enhance the observation of minimal responses. A rating scale of at least

four levels should be used to grade ataxia (see paragraph (g)(12) of this

guideline).

(iii) Necropsy and histopathology. (A) Gross necropsies are recommended for all survivors and should include observation of the appearance of the brain and spinal cord. All animals should be prepared for microscopic examination. Tissues should be fixed by whole body perfusion,

with a fixative appropriate for the embedding media. Sections should include medulla oblongata, spinal cord, and peripheral nerves. The spinal

cord sections should be taken from the rostral cervical, the midthoracic,

and the lumbosacral regions. Section of the proximal regions of both of

the tibial nerves and their branches should be taken. Sections should be

stained with appropriate myelin- and axon-specific stains.

(B) For 28–day studies, a stepwise examination of tissue samples is

recommended. In such a stepwise examination, sections from the high dose

group are first compared with those of the control group. If no

neuropathological alterations are observed in samples from the high dose

group, subsequent analysis is not required. If neuropathological alterations

are observed in samples from the high dose group, samples from the intermediate and low dose groups are examined sequentially.

(f) Data reporting and evaluation—(1) Test report. In addition to

any other applicable reporting requirements, the final test report must include the following information:

(i) Toxic response data by group with a description of clinical signs;

the criteria for the grading system for ataxia and any other scales should

be defined.

(ii) For each animal, time of death during the study or whether it

survived to termination.

(iii) The day of the first occurrence of each abnormal sign and its

subsequent course including its degree.

(iv) Body weight data.

(v) Necropsy findings for each animal, including a description of the

appearance of the brain and the spinal cord.
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(vi) Biochemical data for each animal assessed, including absorbance

values for each animal tested, and blank sample data.

(vii) A detailed description of all histopathological findings.

(viii) Statistical treatment of results, where appropriate.

(2) Treatment of results. Data may be summarized in tabular form,

showing for each test group the number of animals at the start of the test,

the number of animals showing lesions or effects, the types of lesions

or effects and the percentage of animals displaying each type of lesion

or effect.

(3) Evaluation of results. The findings of these delayed neurotoxicity

studies should be evaluated in terms of the incidence and severity of behavioral, neurochemical, and histopathological effects and of any other observed effects in the treated and control groups, as well as any information

known or available to the authors, such as published studies. For a variety

of results seen, further studies may be necessary to characterize these effects.

(g) References. The following references should be consulted for additional background information on this test guideline:

(1) Caroldi, S. and Lotti, M. Neurotoxic Esterase in Peripheral Nerve:

Assay Inhibition, and Rate of Resynthesis. Toxicology and Applied Pharmacology 62: 498–501 (1982).

(2) Davis, C.S. and Richardson, R.J. Organophosphorus compounds.

In: Experimental and Clinical Neurotoxicology, P.S. Spencer and H.H.

Schaumberg, Eds., Williams and Wilkins, Baltimore, pp. 527–544 (1980).

(3) Ellman G.L. et al. A new and rapid colorimetric determination

of acetylcholinesterase activity. Biochemical Pharmacology 7:88–95

(1961).

(4) Johnson, C.D. and Russell, R.L. A rapid, simple, radiometric assay

for cholinesterase, suitable for multiple determinations. Analytical Biochemistry 64:229–238 (1975).

(5) Johnson, M.K. Organophosphorus esters causing delayed neurotoxic effects: Mechanism of action and structure/activity studies. Archives

of Toxicology 34:259–288 (1975)

(6) Johnson, M.K. The delayed neuropathy caused by some

organophosphorus esters: Mechanism and challenge. Critical Reviews in

Toxicology 3:289–316 (1975)

(7) Johnson, M.K. Improved Assay of Neurotoxic Esterase for

Screening Organophosphates for Delayed Neurotoxicity Potential. Archives

of Toxicology 37: 113–115 (1977).
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(8) Johnson, M.K. Delayed neurotoxicity tests of organophosphorus

esters: a proposed protocol integrating neuropathy target esterase (NTE)

assays with behaviour and histopathology tests to obtain more information

more quickly from fewer animals, Proceedings of the International Conference on Environmental Hazards of Agrochemicals in Developing Countries, Alexandria, Egypt, November 8–12, 1983; Volume I, pp. 474–493.

(9) Johnson, M.K. The target for initiation of delayed neurotoxicity

by organophosphorus esters: biochemical studies and toxicological applications, E. Hodgson, J.R. Bend, and R.M. Philpot, eds., Reviews in

Biochememical Toxicology 4, 141–212. Elsevier, NY (1982).

(10) Johnson, M.K. and Richardson, R.J. Biochemical Endpoints:

Neurotoxic Esterase Assay. Neurotoxicology 4:311–320 (1983).

(11) Kayyali, U.S. et al. Neurotoxic Esterase (NTE) Assay: Optimized

conditions based on detergent-induced shifts in the phenol/ 4-

aminoantipyrine chromophore spectrum. Journal of Analytical Toxicology

15:86-89.

(12) Roberts, N.L. et al. Screening acute delayed and subchronic

neurotoxicity studies in the hen: Measurements and evaluations of clinical

signs following administration of TOCP. Neurotoxicology 4:263–270.

(13) Soliman, S.A. et al. Species Susceptibility to Delayed Toxic Neuropathy in relation to in vivo inhibition of Neurotoxic Esterase by Neurotoxic Organophosphorus Ester. Journal of Toxicology and Environmental

Health 9:189–197 (1982).

(14) Sprague, G.L. et al. Time course for neurotoxic esterase activity

in hens given multiple diisopropyl fluorophosphate injections.

Neurotoxicology 2:523–532 (1981).