United States      Prevention, Pesticides     EPA712-C-98-237
          Environmental Protection    and Toxic Substances     August 1998
          Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.6100
          Acute and 28-Day
          Delayed Neurotoxicity of

     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  870.6100  Acute  and  28-day  delayed  neurotoxicity  of
organophosphorus substances.
    (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 798.6450  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, OPP 82-6
Delayed Neurotoxicity of Organophosphorus Substances Following Acute
and 28-Day Exposures (Pesticide Assessment Guidelines,  Subdivision F—
Hazard Evaluation:  Human and Domestic Animals, Addendum 10, EPA
report 540/09-91-123,  March 1991); OECD 418 Delayed Neurotoxicity
of Organophosphorus  Substances  following Acute  Exposure and OECD
419 Delayed Neurotoxicity of Organophosphorus Substances: 28-Day Re-
peated Dose Study.

    (b) Purpose.  In the assessment of organophosphorus substances,
(OPs) studies of delayed neurotoxicity  using the  adult hen as the  test ani-
mal and including behavioral observation of gait, histopathological assess-
ment of brain, peripheral nerve, and spinal cord, and neurochemical assess-
ment 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 Lab-
oratory Practice Standards apply to this test guideline.  The following defi-
nitions 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 mem-
brane-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 sen-
sitive  to mipafox (A^A^-diisopropylphosphorodiamido fluoridate) or neuro-
pathic OP ester inhibition.

     Organophosphorus induced delayed neurotoxicity (OPIDN) is a neu-
rological syndrome in which limb weakness and upper motor neuron spas-
ticity 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 bio-
chemical 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  suffi-
cient 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 re-
sults of the acute  study are completely negative,  that is, there are no de-
layed behavioral or histopathological effects, and  no significant NTE inhi-
bition, 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

     (e) Test procedures—(1) Animal selection. The adult domestic lay-
ing 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 abnor-
malities 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 nor-
mally 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

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.

     (7) 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

     (B) 28-day  study. (7)  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 suffi-
cient 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 mean-
ingful evaluation of the data.
        Low dose — the  low dose should be estimated to be a minimum
effect level, e.g. an ED 10, or alternatively, a NOEL.

     (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-ort/zo-cresyl phos-
phate (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 re-
determinations 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 meas-
urement 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 re-
ceive 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 selec-
tively. The principle of the assay is first  to determine the amount of hy-
drolysis 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.

     (7) 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.

     (2) Materials. This assay requires paraoxon, mipafox, and phenyl val-
erate, all of which can be obtained commercially.
         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 HC1). Meninges and blood
vessels are rapidly removed and the brain is blotted dry, weighed, and  homog-
enized 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 |iM) plus either (a)  buffer or (b)  mipafox (50 |iM)  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-amino-
phenazone) (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
KsFe(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 main-
taining 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 coef-
ficient 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 sam-
ples 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.

     (ii) 21-Day observation. All remaining hens should be carefully ob-
served 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 lim-
ited 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

     (iii) Necropsy and histopathology. (A)  Gross necropsies are rec-
ommended for all survivors  and should include observation of the appear-
ance of the brain and spinal cord.  All animals should be prepared for mi-
croscopic  examination. Tissues should be fixed by whole body perfusion,
with a fixative appropriate for the embedding media. Sections should in-
clude 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  inter-
mediate 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 in-
clude 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.

     (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 be-
havioral, neurochemical, and histopathological effects and of any other ob-
served 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 ef-

     (g) References. The following references should be consulted for ad-
ditional 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 Phar-
macology 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

     (4) Johnson, C.D. and Russell, R.L. A rapid, simple, radiometric assay
for cholinesterase, suitable  for  multiple  determinations. Analytical Bio-
chemistry 64:229-238 (1975).

     (5) Johnson, M.K. Organophosphorus esters causing delayed neuro-
toxic 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).

     (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 Con-
ference on Environmental Hazards of Agrochemicals in Developing Coun-
tries, 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 applica-
tions,  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

     (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 Neu-
ropathy in relation to in vivo inhibition of Neurotoxic Esterase by Neuro-
toxic 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).