OPPTS HARMONIZED TEST GUIDELINES
Series 870
Health Effects
Volume III of III
Guidelines OPPTS 870.6100 - OPPTS 870.7800
August 1998
United States Environmental Protection Agency
Office of Prevention, Pesticides, and Toxic Substances
Washington, D.C. 20460
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Series 870—Health Effects Test Guidelines
OPPTS
Number
870 1000
870 1 100
870 1 200
870 1300
870 2400
870 2500
870 2600
8703100
8703150
870 3200
870 3250
870 3465
870 3700
870 3800
8704100
870 4200
870 4300
8705100
8705140
8705195
8705200
8705250
8705275
870 5300
870 5375
870 5380
870 5385
870 5395
8705450
8705460
8705500
870 5550
870 5575
8705900
870 59 15
3706100
3706200
170 6300
1706500
170 6850
170 6855
170 7200
70 7485
707600
707800
H^^^_^«^^^_
Name
Group A— Acute Toxicity Test Guidelines
Acute loxicity testing-background
Acute oral toxicity
Acute dermal toxicity
Acute inhalation loxicity
Acute eye irritation
Acute dermal irritation
Skin sensitization
Group B— Subchromc Toxicity Test Guidelines
90-Day oral loxicity in rodents
90-Day oral loxicity In nonrodents
21/28-Day dermal toxicity
90-Day dermal toxicity
90-Day inhalation toxiciry
Prenatal developmental toxicity study
Reproduction and fertility ellects
Group C— Chronic Toxicity Test Guidelines
Chronic toxicity
Caranogenicity
Combined chronic toxiaty/caranogenicity
Group D— Genetic Toxicity Test Guidelines
Bacterial reverse mutation lest
Gene mutation in Aspergillus mdulans
Mouse biochemical specific locus test
Mouse visible specific locus lest
Gene mutation In Neurospora crassa
Sex-linked recessive lethal test In Drosophila melanogaster
In vitro mammalian cell gene mutation test
In vitro mammalian chromosome aberration test
Mammalian spermatogomal chromosomal aberration test
Mammalian bone marrow chromosomal aberration test
Mammalian erythrocyle micronudeus test
Rodent dominant lethal assay
Rodent heritable transtocatran assays
Bacterial DNA damage or repair tests
Unscheduled DNA synthesis in mammalian cells in culture
Mitotic gene conversion in Saccharomyces cerevisiae
In vitro sister chromatid exchange assay
n vrvo sister chromatid exchange assay
Group E— Neurotoxlcity Test Guidelines
Acute and 28-day delayed neuroloxicity of organophosphorus substances
•
Neurotoxicity screening battery
Developmental neurotoxeity study
Schedule-controlled operant behavior
Peripheral nerve function
Neurophyswtogy Sensory evoked potentials
Group F— Special Studies Test Guidelines
Companion animal safety
Metabolism and pharmacokmelics
)ermal penetration
mmunoloxicity
Existing Numbers
OPPT
none
798 1175
798 1100
798 1150
798 4500
798 4470
7984100
798 2650
none
none
798 2250
798 2450
798 4900
798 4700
798 3260
798 3300
798 3320
7985100.
5265
798 5140
7985195
798 5200
798 5250
798 5275
7985300
798 5375
7985380
798 5385
798 5395
7985450
7985460
798.5500
798 5550
798 5575
798.5900
7985915
798 6450.
6540.
6560
798 6050.
6200.
.6400
none
798 6500
7986850
798 6855
none
798 7485
none
none
•^^^^^^^^^•^^^^•i
OPP
none
81-1
81-2
81-3
81-4
81-5
81-6
82-1
82-1
82—2
82-3
82-4
83-3
83-4
83-1
83-2
83-5
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
84-2
' 84-2
* 84-2
84-2
84-2
84-2
81-7.
82-5.
82-6
81-6.
82-7.
83-1
83-6
85-5
85-6
none
none
85-1
85-3
85-7
^^"^— ^^^»i
OECD
none
401
402
403
405
404
406
408
409
410
411
413
414
416
452
451
453
471,472
none
none
none
none
477
476
473
483
475
474
478
none
none
482
481
479
none
418. 419
424
none
none
none
none
none
417
none
none
^^^M^^^^^^^^^
EPA Pub
no
712-C-
98-189
98-190
98-192
98-193
98-195
98-196
98-197
98-199
98-200
98-201
98-202
98-204
Qft_pO7
rfW^CW/
98-208
98-210
98-211
98-212
98-247
98-215
98-216
98-217
98-218
98-220
98-221
98-223
98-224
Qft_OOC
vO^££9
98-226
98-227
98-228
98—229
^*^^fcfc^
98-230
98-232
98—234
98-235
98-237
98-238
98— 239
vl^^£w9
98-240
98-241
98-242
98-349
95-244
98-350
98-351
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-237
August 1998
Health Effects Test
Guidelines
OP PTS 870.6100
Acute and 28-Day
Delayed Neurotoxicity of
Organophosphorus
Substances
-------
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. \36,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
Guidelines."
-------
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 ace ylcholinesterase (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 (W./V-diisopropylphosphorodiamido fluoridate) or neuro-
pathic OP ester inhibition.
-------
Organophosphorus induced delayed neurotoxicity (OPLDN) 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
dose.
(e) Test procedures—(1) Animal selection. The adult domestic lay-
ing hen (Callus 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 matenal 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. (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.
(3) Low dose—the low dose should be estimated to be a minimum
effect level, e.g. an ED10, 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-orr/io-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.
(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 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 p.M) plus either (a) buffer or (b) mipafox (50 M.M) 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
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 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.
-------
(li) 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
guideline).
(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.
(0 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-
fects.
(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
(1961).
(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
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 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).
8
-------
&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-98-238
August 1998
Health Effects Test
Guidelines
OPPTS 870.6200
Neurotoxicity Screening
Battery
-------
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. \36,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
Guidelines."
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OPPTS 870.6200 Neurotoxicity screening battery. .
(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 798.6050 Functional Observa-
tional Battery, 798.6200 Motor Activity, and 798.6400 Neuropathology;
and OPP 81-8 Acute Neurotoxicity—Rat, 82-7 90-Day Neurotoxicity—
Rat, and 83-1 Chronic Feeding—Two Species, Rodent and Nonrodent
(Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals, Addendum 10, EPA report 540/09-91-
123, March 1991).
(b) Purpose. This neurotoxicity screening battery consists of a func-
tional observational battery, motor activity, and neuropathology. The func-
tional observational battery consists of noninvasive procedures designed
to detect gross functional deficits in animals and to better quantify behav-
ioral or neurological effects detected in other studies. The motor activity
test uses an automated device that measures the level of activity of an
individual animal. The neuropathological techniques are designed to pro-
vide data to detect and characterize histopathological changes in the central
and peripheral nervous system. This battery is designed to be used in con-
junction with general toxicity studies and changes should be evaluated in
the context of both the concordance between functional neurological and
neuropatholgical effects, and with respect to any other toxicological effects
seen. This test battery is not intended to provide a complete evaluation
of neurotoxicity, and additional functional and morphological evaluation
may be necessary to assess completely the neurotoxic potential of a chemi-
cal.
(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 effective dose.
Motor activity is any movement of the experimental animal.
Neurotoxicity is any adverse effect on the structure or function of
the nervous system related to exposure to a chemical substance.
Toxic effect is an adverse change in the structure or function of an
experimental animal as a result of 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.
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The animals are observed under carefully standardized conditions with suf-
ficient frequency to ensure the detection and quantification of behavioral
and/or neurologic abnormalities, if present. Various functions that could
be affected by neurotoxicants are assessed during each observation period.
Measurements of motor activity of individual animals are made in an auto-
mated device The animals are perfused and tissue samples from the nerv-
ous system are prepared for microscopic examination. The exposure levels
at which significant neurotoxic effects are produced are compared to one
another and to those levels that produce other toxic effects.
(e) Test procedures—(1) Animal selection—(i) Species. In general,
the laboratory rat should be used. Under some circumstances, other spe-
cies, such as the mouse or the dog, may be more appropriate, although
not all of the battery may be adaptable to other species.
(ii) Age. Young adults (at least 42 days old for rats) should be used.
(iii) Sex. Both males and females should be used. Females should
be nulliparous and nonpregnant.
(2) Number of animals. At least 10 males and 10 females should
be used in each dose and control group for behavioral testing. At least
five males and five females should be used in each dose and control group
for terminal neuropathology. If interim neuropathological evaluations are
planned, the number should be increased by the number of animals sched-
uled to be perfused before the end of the study. Animals should be ran-
domly assigned to treatment and control groups.
(3) Control groups, (i) A concurrent (vehicle) control group is re-
quired. Subjects should be treated in the same way as for an exposure
group except that administration of the test substance is omitted. If the
vehicle used has known or potential toxic properties, both untreated or
saline treated and vehicle control groups are required.
(ii) Positive control data from the laboratory performing the testing
should provide evidence of the ability of the observational methods used
to detect major neurotoxic endpoints including limb weakness or paralysis,
tremor, and autonomic signs. Positive control data are also required to
demonstrate the sensitivity and reliability of the activity-measuring device
and testing procedures. These data should demonstrate the ability to detect
chemically induced increases and decreases in activity. Positive control
groups exhibiting central nervous system pathology and peripheral nervous
system pathology are also required. Separate groups for peripheral and
central neuropathology are acceptable (e.g. acrylamide and dimethyl tin).
Permanently injurious substances need not be used for the behavioral tests.
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
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personnel or some other critical element in the testing laboratory has
changed.
(4) Dose level 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 encourage the use
of equally spaced doses and a rationale for dose selection that will maxi-
mally support detection of dose-effect relations. For acute studies, dose
selection may be made relative to the establishment of a benchmark dose
(BD). That is, doses may be specified as successive fractions, e.g.
0.5, 0.25, ...n of the BD. The BD itself may be estimated as the highest
nonlethal dose as determined in a preliminary range-finding lethality study.
A variety of test methodologies may be used for this purpose, and the
method chosen may influence subsequent dose selection. The goal is to
use a dose level that is sufficient to be judged a limit dose, or clearly
toxic.
(i) Acute studies. The high dose need not be greater than 2 g/kg.
Otherwise, the high dose should result in significant neurotoxic effects
or other clearly toxic effects, but not result in an indicence of fatalities
that would preclude a meaningful evaluation of the data. This dose may
be estimated by a BD procedure as described in paragraph (e)(4) of this
guideline, with the middle and low dose levels chosen as fractions of the
BD dose. The lowest dose should produce minimal effect, e.g. an ED 10,
or alternatively, no effects.
(ii) Subchronic and chronic studies. The high dose need not be
greater than 1 g/kg. Otherwise, the high dose level should result in signifi-
cant 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 exposure. 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 recog-
nized that more than one route of exposure may be important and that
these criteria may conflict with one another. In order to save resources,
initially only one route is being required for screening for neurotoxicity.
The route that best meets these criteria should be selected. Dietary feeding
will generally be acceptable for repeated exposures studies.
(6) Combined protocol. The tests described in this screening battery
may be combined with any other toxicity study, as long as none of the
requirements of either are violated by the combination.
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(7) Study conduct—(i) Time of testing. All animals should be
weighed on each test day and at least weekly during the exposure period.
(A) Acute studies. At a minimum, for acute studies observations and
activity testing should be made before the initiation of exposuie, at the
estimated time of peak effect within 8 hours of dosing, and at 7 and 14
days after dosing. Estimation of times of peak effect may be made by
dosing pairs of rats across a range of doses and making regular observa-
tions of gait and arousal.
(B) Subchronic and chronic studies. In a subchronic study, at a min-
imum, observations and activity measurements should be made before the
initiation of exposure and before the daily exposure, or for feeding studies
at the same time of day, during the 4th, 8th, and 13th weeks of exposure.
In chronic studies, at a minimum, observations and activity measurements
should be made before the initiation of exposure and before the daily expo-
sure, or for feeding studies at the same time of day, every 3 months.
(ii) Functional observational battery—(A) General conduct. All
animals in a given study should be observed carefully by trained observers
who are unaware of the animals' treatment, using standardized procedures
to minimize observer variability. Where possible, it is advisable that the
same observer be used to evaluate the animals in a given study. If this
is not possible, some demonstration of interobserver reliability is required.
The animals should be removed from the home cage to a standard arena
for observation. Effort should be made to ensure that variations in the
test conditions are minimal and are not systematically related to treatment.
Among the variables that can affect behavior are sound level, temperature,
humidity, lighting, odors, time of day, and environmental distractions.- Ex-
plicit, operationally defined scales for each measure of the battery are to
be used. The development of objective quantitative measures of the obser-
vational end-points specified is encouraged. Examples of observational
procedures using defined protocols may be found in paragraphs (g)(6),
(g)(8), and (g)(ll) of this guideline. The functional observational battery
should include a thorough description of the subject's appearance, behav-
ior, and functional integrity. This should be assessed through observations
in the home cage and while the rat is moving freely in an open field,
and through manipulative tests. Testing should proceed from the least to
the most interactive with the subject. Scoring criteria, or explicitly defined
scales, should be developed for those measures which involve subjective
ranking.
(B) List of measures. The functional observational battery should
include the following list of measures:
(7) Assessment of signs of autonomic function, including but not lim-
ited to:
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(/) Ranking of the degree of lacrimation and salivation, with a range
of severity scores from none to severe.
(n) Presence or absence of piloerection and exophthalmus.
(//;) Ranking or count of urination and defecation, including polyuna
and diarrhea. This is most easily conducted during the open field assess-
ment.
(iv) Pupillary function such as constriction of the pupil in response
to light or a measure of pupil size. .
(v) Degree of palpebral closure, e.g., ptosis.
(2) Description, incidence, and severity of any convulsions, tremors,
or abnormal motor movements, both in the home cage and the open field.
(3) Ranking of the subject's reactivity to general stimuli such as re-
moval from the cage or handling, with a range of severity scores from
no reaction to hyperreactivity.
(4) Ranking of the subject's general level of activity during observa-
tions of the unperturbed subject in the open field, with a range of severity
scores from unresponsive to hyperactive.
(5) Descriptions and incidence of posture and gait abnormalities ob-
served in the home cage and open field.
(6) Ranking of any gait abnormalities, with a range of severity scores
from none to severe.
(7) Forelimb and hindlimb grip strength measured using an objective
procedure, e.g. that described by Meyer et al. under paragraph (g)(10) of
this guideline
(8) Quantitative measure of landing foot splay; the procedure de-
scribed in paragraph (g)(4) of this guideline is recommended.
(9) Sensorimotor responses to stimuli of different modalities will be
used to detect gross sensory deficits. Pain perception may be assessed by
a ranking or measure of the reaction to a tail-pinch, tail-flick, or hot-plate.
The response to a sudden sound, e.g., click or snap, may be used to assess
audition.
(10) Body weight.
(II) Description and incidence of any unusual or abnormal behaviors,
excessive or repetitive actions (stereotypies), emaciation, dehydration,
hypotonia or hypertonia, altered fur appearance, red or crusty deposits
around the eyes, nose, or mouth, and any other observations that may fa-
cilitate interpretation of the data.
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(C) Additional measures. Other measures may also be included and
the development and validation of new tests is encouraged. Further infor-
mation on the neurobehavioral integrity of the subject may be provided
by:
(/) Count of rearing activity on the open field.
(2) Ranking of righting ability.
(3) Body temperature.
(4) Excessive or spontaneous vocalizations.
(5) Alterations in rate and ease of respiration, e.g., rales or dyspnea.
(6) Sensorimotor responses to visual or proprioceptive stimuli.
(iii) Motor activity. Motor activity should be monitored by an auto-
mated activity recording apparatus. The device used must be capable of
detecting both increases and decreases in activity, i.e., baseline activity
as measured by the device must not be so low as to preclude detection
of decreases nor so high as to preclude detection of increases in activity.
Each device should be tested by standard procedures to ensure, to the ex-
tent possible, reliability of operation across devices and across days for
any one device. In addition, treatment groups must be balanced across
devices. Each animal should be tested individually. The test session should
be long enough for motor activity to approach asymptotic levels by the
last 20 percent of the session for nontreated control animals. All sessions
should have the same duration. Treatment groups should be
counterbalanced across test times. Effort should be made to ensure that
variations in the test conditions are minimal and are not systematically
related to treatment. Among the variables which can affect motor activity
are sound level, size and shape of the test cage, temperature, relative hu-
midity, lighting conditions, odors, use of the home cage or a novel test
cage, and environmental distractions.
(iv) Neuropathology: Collection, processing and examination of
tissue samples. To provide for adequate sampling as well as optimal pres-
ervation of cellular integrity for the detection of neuropathological alter-
ations, tissue should be prepared for histological analysis using in situ per-
fusion and paraffin and/or plastic embedding procedures. Paraffin embed-
ding is acceptable for tissue samples from the central nervous system. Plas-
tic embedding of tissue samples from the central nervous system is encour-
aged, when feasible. Plastic embedding is required for tissue samples from
the peripheral nervous system. Subject to professional judgment and the
type of neuropathological alterations observed, it is recommended that ad-
ditional methods, such as Bodian's or Bielchowsky's silver methods, and/
or glial fibrillary. acidic protein (GFAP) immunohistochemistry be used
in conjunction with more standard stains to determine the lowest dose level
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at which neuropathological alterations are observed. When new or existing
data provide evidence of structural alterations it is recommended that the
GFAP immunoassay also be considered. A description of this technique
can be found in paragraph (g)(l2) of this guideline
(A) Fixation and processing of tissue. The nervous system should
be fixed by in situ perfusion with an appropriate aldehyde fixative. De-
tailed descriptions of vascular perfusions may be found in paragraphs
(g)(7). (g)(13), (g)(19), and (g)(-21) of this guideline. Any gross abnormali-
ties should be noted. Tissue samples taken should adequately represent
all major regions of the nervous system. Detailed dissection procedures
are described in paragraphs (g)(19)(chapter 50), and (g)(13) of this guide-
line. The tissue samples should be postfixed and processed according to
standardized ' published histological protocols under paragraph (g)(l),
(g)(2). (g)(3), (g)(14), (g)(19), or (g)(20) of this guideline. Tissue blocks
and slides should be appropriately identified when stored. Histological sec-
tions should be stained for hematoxylin and eosin (H&E), or a comparable
stain according to standard published protocols under paragraphs (g)(l),
(g)(2), and (g)(14) of this guideline.
(B) Qualitative examination. Representative histological sections
from the tissue samples should be examined microscopically by an appro-
priately trained pathologist for evidence of neuropathological alterations.
The nervous system should be thoroughly examined for evidence of any
treatment-related neuropathological alterations. Particular attention should
be paid to regions known to be sensitive to neurotoxic insult or those
regions likely to be affected based on the results of functional tests. Such
treatment-related neuropathological alterations should be clearly distin-
guished from artifacts resulting from influences other than exposure to the
test substance. Guidance for both regions to be examined and the types
of neuropathological alterations that typically result from toxicant exposure
can be found in paragraph (g)(20) of this guideline. A stepwise examina-
tion 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 then exam-
ined sequentially.
(C) Subjective diagnosis. If any evidence of neuropathological alter-
ations is found in the qualitative examination, then a subjective diagnosis
will be performed for the purpose of evaluating dose-response relation-
ships. All regions of the nervous system exhibiting any evidenceof
neuropathological changes should be included in this analysis. Sections
from all dose groups from each region will be coded and examined in
randomized order without knowledge of the code. The frequency of each
type and severity of each lesion will be recorded. After all samples from
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all dose groups including all regions have been rated, the code will be
broken and statistical analysis performed to evaluate dose-response rela-
tionships. For each type of dose-related lesion observed, examples of dif-
ferent degrees of severity should be described. Photomicrographs of typical
examples of treatment-related regions are recommended to augment these
descriptions. These examples will also serve to illustrate a rating scale.
such as 1+, 2+, and 3+ for the degree of severity ranging from very slight
to very extensive.
(f) Data reporting and evaluation. The final test report must include
the following information:
(1) Description of equipment and test methods. A description of
the general design of the experiment and any equipment used should be
provided. This should include a short justification explaining any decisions
involving professional judgment.
(i) A detailed description of the procedures used to standardize obser-
vations, including the arena and scoring criteria.
(ii) Positive control data from the laboratory performing the test that
demonstrate the sensitivity of the procedures being used. Historical data
may be used if all essential aspects of the experimental 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.
(2) Results. The following information must be arranged by test
group dose level.
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number.
(B) Its body weight and score on each sign at each observation time,
the time and cause of death (if appropriate), total session activity counts,
and intrasession subtotals for each day measured.
(ii) Summary data for each group must include:
(A) The number of animals at the start of the test.
(B) The number of animals showing each observation score at each
observation time.
(C) The mean and standard deviation for each continuous endpoint
at each observation time.
(D) Results of statistical analyses for each measure, where appro-
priate.
8
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(in) All neuropathological observations should be recorded and ar-
ranged by test groups. This data may be presented in the following rec-
ommended format:
(A) Description of lesions for each animal. For each animal, data must
be submitted' showing its identification (animal number, sex, treatment,
dose, duration), a list of structures examined as well as the locations, na-
ture, frequency, and severity of lesions. Inclusion of photomicrographs is
strongly recommended for demonstrating typical examples of the type and
severity of the neuropathological alterations observed. Any diagnoses de-
rived from neurological signs and lesions including naturally occurring dis-
eases or conditions, should be recorded.
(B) Counts and incidence of neuropathological alterations by test
group. Data should be tabulated to show:
(7) The number of animals used in each group and the number of
animals in which any lesion was found.
(2) The number of animals affected by each different type of lesion,
the locations, frequency, and average grade of each type of lesion.
(3) Evaluation of data. The findings from the screening battery
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 presence or absence, incidence and severity, of any neu-
rotoxic effects. The evaluation should include appropriate statistical analy-
ses, for example, parametric tests for continuous data and nonparametric
tests for the remainder. Choice of analyses should consider tests appro-
priate to the experimental design, including repeated measures. There may
be many acceptable ways to analyze data.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Armed Forces Institute of Pathology. Manual of Histologic Stain-
ing Methods. McGraw-Hill, NY (1968).
(2) Bennet, H.S. et al. Science and art in the preparing tissues embed-
ded in plastic for light microscopy, with special reference to glycol meth-
acrylate, glass knives and simple stains. Stain Technology 51:71-97
(1976).
(3) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining
of large epoxy sections and application to surgical pathology. American
Journal of Clinical Pathology 81:25-29 (1984).
-------
(4) Edwards, P.M and Parker V.H. A simple, sensitive and objective
method for early assessment of acrylarrude neuropathy in rats. Toxicologv
and Applied Pharmacology 40:589-591 (1977).
(5) Finger, F.W. Measuring behavioral activity. In: Methods in
Psychobiology Vol. 2, Ed. R.D. Myers. Academic, NY. pp.1-19 (1972).
(6) Gad, S. A neuromuscular screen for use in industrial toxicology.
Journal of Toxicology and Environmental Health 9:691-704 (1982).
(7) Hayat, M.A. Volume 1. Biological applications. In: Principles and
Techniques of Electron Microscopy. Van Nostrand Reinhold, NY (1970).
(8) Irwin, S. Comprehensive observational assessment: la. A system-
atic quantitative procedure for assessing the behavioral physiological state
of the mouse. Psychopharmacologia 13:222-257 (1968).
(9) Kinnard, E.J. and Watzman, N. Techniques utilized in the evalua-
tion of psychotropic drugs on animals activity. Journal of Pharmaceutical
Sciences 55:995-1012 (1966).
(10) Meyer, O.A. et al. A method for the routine assessment of fore-
and hindlimb grip strength of rats and mice. Neurobehavioral Toxicology
1:233-236(1979).
(11) Moser V.C. et al. Comparison of chlordimeform and carbaryl
using a functional observational battery. Fundamental and Applied Toxi-
cology 11:189-206 (1988).
(12) O'Callaghan, J.P. Quantification of glial fibrillary acidic protein:
comparison of slot-immunobinding assays with a novel sandwich ELISA,
Neurotoxicology and Teratology, 13:275-281 (1991).
(13) Palay, S.L. and Chan Palay, V. Cerebellar Cortex: Cytology and
Organizatio. Springer Verlag, NY (1974).
(14) Pender, M.P. A simple method for high resolution light micros-
copy of nervous tissue. Journal of Neuroscience Methods 15:213-218
(1985).
(15) Ralis, H.M. et al. Techniques in Neurohistology, Butterworths,
London (1973).
(16) Reiter, L.W. Use of activity measures in behavioral toxicology.
Environmental Health Perspectives 26:9-20 (1978).
(17) Reiter, L.W. and MacPhail, R.C. Motor Activity: A survey of
methods with potential use in toxicity testing. Neurobehavorial Toxicology
1—Suppl. 1:53-66(1979).
10
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(18) Robbins, T W. A critique of the methods available for the meas-
urement of spontaneous motor activity. Handbook of Psychopharmacologv
Vol 7 Eds. Iversen. L.L.. Iverson. D.S . Snvder. S.H. Plenum. NY pp
37-82(1977).
(19) Spencer. P.S., Schaumburg, H.H. Eds., Experimental and Clini-
cal Neurotoxicology. Williams and Wilkins, Baltimore (1980).
(20) World Health Organization. Principles and Methods for the As-
sessment of Neurotoxicity Associated with Exposure to Chemicals (Envi-
ronmental Health Criteria 60), World Health Organizations Publications
Center USA, Albany, NY (1986).
(21) Zeman, W. and Innes, J.R. Craigie's Neuroanatomy of the Rat,
Academic, NY (1963).
11
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&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic SubstancGS
(7101)
EPA712-C-S8-239
August 1998
Health Effects Test
Guidelines
OPPTS 870.6300
Developmental
Neurotoxicity Study
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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. \36,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
Guidelines."
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OPPTS 870.6300 Developmental neurotoxicity study.
(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 is OPP 83-6 Developmental Neurotoxicity
Study (Pesticide Assessment Guidelines, Subdivision F~Hazard Evalua-
tipn: Human and Domestic Animals, Addendum 10, EPA report 540/09-
91-123, March 1991).
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a chemical substance or mixture (test substance), determination
of the potential for developmental neurotoxicity is important. This study
is designed to develop data on the potential functional and morphological
hazards to the nervous system which may arise in the offspring from expo-
sure of the mother during pregnancy and lactation.
(c) Principle of the test method. The test substance is administered
to several groups of pregnant animals during gestation and early lactation,
one dose level being used per group. Offspring are randomly selected from
within litters for neurotoxicity evaluation. The evaluation includes observa-
tions to detect gross neurologic and behavioral abnormalities, determina-
tion of motor activity, response to auditory startle, assessment of learning,
neuropathological evaluation, and brain weights. This protocol may be
used as a separate study, as a followup to a standard developmental tox-
icity and/or adult neurotoxicity study, or as part of a two-generation repro-
duction study, with assessment of the offspring conducted on the second
(Fa) generation.
(d) Test procedure—(1) Animal selection—(i) Species and strain.
Testing should be performed in the rat. Because of its differences in timing
of developmental events compared to strains that are more commonly test-
ed in other developmental and reproductive toxicity studies, it is preferred
that the Fischer 344 strain not be used. If a sponsor wishes to use the
Fischer 344 rat or a mammalian species other than the rat, ample justifica-
tion/reasoning for this selection must be provided.
(ii) Age. Young adult (nulliparous females) animals should be used.
(iii) Sex. Pregnant female animals should be used at each dose level.
(iv) Number of animals. (A) The objective is for'a sufficient number
of pregnant rats to be exposed to the test substance to ensure that an ade-
quate number of offspring are produced for neurotoxicity evaluation. At
least 20 litters are recommended at each dose level.
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(B) On postnatal day 4. the size of each litter should be adjusted
by eliminating extra pups by random selection to yield, as nearly as pos-
sible, four male and four females per litter. Whenever the number of pups
of either sex prevents having four of each sex per litter, partial adjustment
(for example, five males and three females) is permitted. Testing is not
appropriate for litters of less than seven pups. Elimination of runts only
is not appropriate. Individual pups should be identified uniquely after
standardization of litters. A method that may be used for identification
can be found under paragraph (f)(t) of this guideline.
(v) Assignment of animals for behavioral tests, brain weights, and
neuropathological evaluations. After standardization of litters, one male
or one female from each litter (total of 10 males and 10 females per dose
group) should be randomly assigned to one of the following tests: Motor
activity, auditory startle, and learning and memory, in weanling and adult
animals. On postnatal day 11, either 1 male or 1 female pup from each
litter (total of 10 males and 10 females per dose group) should be sac-
rificed. Brain weights should be measured in all of these pups and, of
these pups, six per sex per dose should be selected for neuropathological
evaluation. At the termination of the study, either 1 male or 1 female from
each litter (total of 10 males and 10 females per dose group) should be
sacrificed and brain weights should be measured. An additional group of
six animals per sex per dose group (one male or one female per litter)
should be sacrificed at the termination of the study for neuropathological
evaluation.
(2) Control group. A concurrent control group is required. This
group should be a sham-treated group or, if a vehicle is used in administer-
ing the test substance, a vehicle control group. The vehicle should neither
be developmentally toxic nor have effects on reproduction. Animals in
the control group should be handled in an identical manner to test group
animals.
(3) Dose levels and dose selection, (i) At least three dose levels of
the test substance plus a control group (vehicle control, if a vehicle is
used) should be used.
(ii) If the test substance has been shown to be developmentally toxic
either in a standard developmental toxicity study or in a pilot study, the
highest dose level should be the maximum dose which will not induce
in utero or neonatal death or malformations sufficient to preclude a mean-
ingful evaluation of neurotoxicity.
(iii) If a standard developmental toxicity study has not been con-
ducted, the highest dose level, unless limited by the physicochemical na-
ture or biological properties of the substance, should induce some overt
maternal toxicity, but should not result in a reduction in weight gain ex-
ceeding 20 percent during gestation and lactation.
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(iv) The lowest dose-should not produce any grossly observable evi-
dence of either maternal or developmental neurotoxicity.
(v) The intermediate doses should be equally spaced between the
highest and lowest doses used.
(4) Dosing period. Day 0 of gestation is the day on which a vaginal
plug and/or sperm are observed. The dosing period should cover the period
from day 6 of gestation through day-10 postnatally. Dosing should not
occur on the day of parturition in those animals who have not completely
delivered their offspring.
(5) Administration of the test substance. The test substance or vehi-
cle should be administered orally. Other routes of administration may be
acceptable, on a case-by-case basis, with ample justification/reasoning for
this selection. The test substance or vehicle should be administered based
on the most recent weight determination.
(6) Observation of dams, (i) A gross examination of the dams should
be made at least once each day before daily treatment.
(ii) Ten dams per group should be observed outside the home cage
at least twice during the gestational dosing period (days 6-21) and twice
during the lactational dosing period (days 1-10) for signs of toxicity. The
animals should be observed by trained technicians who are unaware of
the animals' treatment, using standardized procedures to maximize inter-
observer reliability. Where possible, it is advisable that the same observer
be used to evaluate the animals in a given study. If this is not possible,
some demonstration of interobserver reliability is required.
(iii) During the treatment and observation periods under paragraph
(d)(6)(ii), observations should include:
(A) Assessment of signs of autonomic function, including but not lim-
ited to:
(7) Ranking of the degree of lacrimation and salivation, with a range
of severity scores from none to severe.
(2) Presence or absence of piloerection and exophthalmus.
(3) Ranking or count of urination and defecation, including polyuria
and diarrhea.
(4} Pupillary function such as constriction of the pupil in responseto
light or a measure of pupil size.
(5) Degree of palpebral closure, e.g., ptosis.
(B) Description, incidence, and severity of any convulsions, tremors,
or abnormal movements.
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(C) Description and incidence of posture and gait abnormalities.
(D) Description and incidence of any unusual or abnormal behaviors.
excessive or repetitive actions (stereotypies), emaciation, dehydration.
hypotoma or hypertoma, altered fur appearance, red or crusty deposits
around the eyes, nose, or mouth, and any other observations that may fa-
cilitate interpretation of the data.
(iv) Signs of toxicity should be recorded as they are observed, includ-
ing the time of onset, degree, and duration.
(v) Animals should be weighed at least weekly and on the day of
delivery and postnatal days 11 and 21 (weaning) and such weights should
be recorded.
(vi) The day of delivery of litters should be recorded and considered
as postnatal day 0.
(7) Study conduct—(i) Observation of offspring. (A) All offspring
should be examined cage-side at least daily for gross signs of mortality
or morbidity.
(B) A total of 10 male offspring and 10 female offspring per dose
group should be examined outside the cage for signs of toxicity on days
4, 11, 21, 35, 45, and 60. The offspring should be observed by trained
technicians, who are unaware of the treatment being used, using standard-
ized procedures to maximize interobserver reliability. Where possible, it
is advisable that the same observer be used to evaluate the animals in
a given study. If this is not possible, some demonstration of interobserver
reliability is required. At a minimum, the end points outlined in paragraph
(d)(6)(iii) of this guideline should be monitored as appropriate for the de-
velopmental stage being observed.
(C) Any gross signs of toxicity in the offspring should be recorded
as they are observed, including the time of onset, degree, and duration.
(ii) Developmental landmarks. Live pups should be counted and
each pup within a litter should be weighed individually at birth or soon
thereafter, and on postnatal days 4, 11, 17, and 21 and at least once every
2 weeks thereafter. The age of vaginal opening and preputial separation
should be determined. General procedures for these determinations may
be found in paragraphs (0(1) and (f)(ll) of this guideline.
(iii) Motor activity. Motor activity should be monitored specifically
on postnatal days 13, 17 21, and 60 (±2 days). Motor activity must be
monitored by an automated activity recording apparatus. The device must
be capable of detecting both increases and decreases in activity, (i.e., base-
line activity as measured by the device must not be so low as to preclude
detection of decreases nor so high as to preclude detection of increases
in activity). Each device should be tested by standard procedures to ensure,
-------
to the extent possible, reliability of operation across devices and across
days for any one device. In addition, treatment groups must be balanced
across devices Each animal should be tested individually. The test session
should be long enough for motor activity to approach asymptotic levels
by the last 20 percent of the session for nontreated control animals. All
sessions should have the same duration. Treatment groups should be
counter-balanced across test times. Activity counts should be collected in
equal time periods of no greater than 10 minutes duration. Efforts should
be made to ensure that variations in the test conditions are minimal and
are not systematically related to treatment. Among the variables that can
affect motor activity are sound level, size and shape of the test cage, tem-
perature, relative humidity, light conditions, odors, use of home cage or
novel test cage, and environmental distractions. Additional information on
the conduct of a motor activity study may be obtained in OPPTS 870.6200.
(iv) Auditory startle test. An auditory startle habituation test should
be performed on the offspring around the time of weaning and around
day 60. Day of testing should be counterbalanced across treated and con-
trol groups. Details on the conduct of this testing may be obtained under
paragraph (f)O) of this guideline. In performing the auditory startle task,
the mean response amplitude on each block of 10 trials (5 blocks of 10
trials per session on each day of testing) should be made. While use of
prepulse inhibition is not a requirement, it is highly recommended. Details
on the conduct of this test may be obtained in paragraph (f)(10) of this
guideline
(v) Learning and memory tests. A test of associative learning and
memory should be conducted around the time of weaning and around day
60. Day of testing should be counterbalanced across treated and control
groups. The same or separate tests may be used at these two stages of
development. Some flexibility is allowed in the choice of tests for learning
and memory in weanling and adult rats. However, the tests must be de-
signed to fulfill two criteria. First, learning must be assessed either as a
change across several repeated learning trials or sessions, or, in tests in-
volving a single trial, with reference to a condition that controls for non-
associative effects of the training experience. Second, the tests should in-
clude some measure of memory (short-term or long-term) in addition to
original learning (acquisition). If the tests of learning and memory reveal
an effect of the test compound, it may be in the best interest of the sponsor
to conduct additional tests to rule out alternative interpretations based on
alterations in sensory, motivational, and/or motor capacities. In addition
to the above two criteria, it is recommended that the test of learning and
memory be chosen on the basis of its demonstrated sensitivity to the class
of compound under investigation, if such information is available in the
literature. In the absence of such information, examples of tests that could
be made to meet the above criteria include: Delayed-matching-to-position,
as described for the adult rat (see paragraph (0(3) of this guideline) and
-------
for the infant rat (see paragraph (f)(9) of this guideline), olfactory condi-
tioning, as described in paragraph (0(13) of this guideline; and acquisition
and retention of schedule-controlled behavior (see paragraphs (f)(4) and
(f)(5) of'this guideline). Additional tests for weanling rats are described
under paragraphs (0(20) and (0(12) of this guideline, and for adult rats
under paragraph (0(16) of this guideline.
(vi) Neuropathology. Neuropathological evaluation should be con-
ducted on animals on postnatal day 11 and at the termination of the study.
At 11 days of age, one male or female pup should be removed from each
litter such that equal numbers of male and female offspring are removed
from all litters combined. Of these, six male and six female pups per dose
group will be sacrificed for neuropathological analysis. The pups will be
killed by exposure to carbon dioxide and immediately thereafter the brains
should be removed, weighed, and immersion-fixed in an appropriate
aldehyde fixative. The remaining animals will be sacrificed in a similar
manner and immediately thereafter their brains removed and weighed. At
the termination of the study, one male or one female from each litter will
be killed by exposure to carbon dioxide and immediately thereafter the
brain should be removed and weighed. In addition, six animals per sex
per dose group (one male or female per litter) should be sacrificed at the
termination of the study for neuropathological evaluation.
Neuropathological analysis of animals sacrificed at the termination of the
study should be performed in accordance with OPPTS 870.6200.
Neuropathological evaluation of animals sacrificed on postnatal day 11 and
at termination of the study should include a qualitative analysis and
semiquantitative analysis as well as simple morphometrics.
(A) Fixation and processing of tissue samples for postnatal day
11 animals. Immediately following removal, the brain should be weighed
and immersion fixed in an appropriate aldehyde fixative. The brains should
be postfixed and processed according to standardized published histo-
logical protocols under paragraphs (f)(6), (0(14), (0(17), and (0(21) of
this guideline. Paraffin embedding is acceptable but plastic embedding is
preferred and recommended. Tissue blocks and slides should be appro-
priately identified when stored. Histological sections should be stained for
hematoxylin and eosin, or a similar stain according to standard published
protocols under paragraphs (0(2), (0(18), and (0(23) of this guideline.
For animals sacrificed at the termination of the study, methods for fixation
and processing of tissue samples are provided in paragraph (e)(4)(iv)(A)
of OPPTS 870.6200.
(B) Qualitative analysis. The purposes of the qualitative examination
are threefold—to identify regions within the nervous system exhibiting evi-
dence of neuropathological alterations, to identify types of neuropatholo-
gical alterations resulting from exposure to the test substance, and to deter-
mine the range of severity of the neuropathological alterations. Representa-
tive histological sections from the tissue samples should be examined mi-
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croscopically by an appropriately trained pathologist for evidence of
neuropathological alteiations The following stepwise procedure is rec-
ommended for the qualitative analysis First, sections from the high dose
group are compared with those of the control group. If no evidence of
neuropathological alterations is found in animals of the high dose group,
no further analysis is required. If evidence of neuropathological alterations
are found in the high dose group, then animals from the intermediate and
low dose group are examined. Subject to professional judgment and the
kind of neuropathological alterations observed, it is recommended that ad-
ditional methods such as Bodian's or Bielchowsky's silver methods and/
or imrnunohistocherrustry for glial fibrillary acid protein be used in con-
junction with more standard stains to determine the lowest dose level at
which neuropathological alterations are observed. Evaluations of postnatal
day 11 pups is described in paragraphs (d)(7)(vi)(B)(7) and (d)(7)(vi)(B)(2)
of this guideline. For animals sacrificed at the termination of the study,
the regions to be examined and the types of alterations that should be
assessed are identified in paragraph (e)(4)(iv)(B) of OPPTS 870.6200.
(7) Regions to be examined. The brains should be examined for any
evidence of treatment-related neuropathological alterations and adequate
samples should be taken from all major brain regions (e.g., olfactory bulbs,
cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus,
midbrain (tectum, tegmentum, and cerebral peduncles), brainstem and cer-
ebellum) to ensure a thorough examination.
(2) Types of alterations. Guidance for neuropathological examination
for indications of developmental insult to the brain can be found in para-
graphs (f)(8) and (0(22) of this guideline. In addition to more typical kinds
of cellular alterations (e.g., neuronal vacuolation, degeneration, necrosis)
and tissue changes (e.g., astrocytic proliferation, leukocytic infiltration, and
cystic formation) particular emphasis should be paid to structural changes
indicative of developmental insult including but not restricted to:
(0 Gross changes in the size or shape of brain regions such as alter-
ations in the size of the cerebral hemispheres or the normal pattern of
foliation of the cerebellum.
(11) The death of neuronal precursors, abnormal proliferation, or ab-
normal migration, as indicated by pyknotic cells or ectopic neurons, or
gross alterations in regions with active proliferative and migratory zones,
alterations in transient developmental structures (e.g., the external germinal
zone of the cerebellum, see paragraph (0(15) of this guideline).
(i/i) Abnormal differentiation, while more apparent with special
stains, may also be indicated by shrunken and malformed cell bodies.
(iv) Evidence of hydrocephalus, in particular enlargement of the ven-
tricles, stenosis of the cerebral aqueduct and general thinning of the cere-
bral hemispheres.
-------
(C) Subjective diagnosis. If any evidence of neuropathological alter-
ations is found in the qualitative examination, then a subjective diagnosis
will be performed for the purpose of evaluating dose-response relation-
ships. All regions of the brain exhibiting any evidence of
neuropathological changes should be included in this analysis. Sections
of each region from all dose groups will be coded as to treatment and
examined in randomized order. The frequency of each type and the sever-
ity of each lesion will be recorded. After all sections from all dose groups
including all regions have been rated, the code will be broken and statis-
tical analyses performed to evaluate dose-response relationships. For each
type of dose related lesion observed, examples of different ranges of sever-
ity should be described. The examples will serve to illustrate a rating scale,
such as 1+, 2+, and 3+ for the degree of severity ranging from very slight
to veryextensive.
(D) Simple morphometric analysis. Since the disruption of devel-
opmental processes is sometimes more clearly reflected in the rate or ex-
tent of growth of particular brain regions, some form of morphometric
analysis should be performed on postnatal day 11 and at the termination
of the study to assess the structural development of the brain. At a mini-
mum, this would consist of a reliable estimate of the thickness of major
layers at representative locations within the neocortex, hippocampus, and
cerebellum. For guidance on such measurements see Rodier and Gramann
under .paragraph (0(19) of this guideline.
(e) Data collection, reporting, and evaluation. The following spe-
cific information should be reported:
(1) Description of test system and test methods. A description of
the general design of the experiment should be provided. This should in-
clude:
(i) A detailed description of the procedures used to standardize obser-
vations and procedures as well as operational definitions for scoring obser-
vations.
(ii) Positive control data from the laboratory performing the test that
demonstrate the sensitivity of the procedures being used. These data do
not have to be from studies using prenatal exposures. However, the labora-
tory must demonstrate competence in evaluation effects in neonatal ani-
mals perinatally exposed to chemicals and establish test norms for the ap-
propriate age group.
(iii) Procedures for calibrating and ensuring the equivalence of de-
vices and the balancing of treatment groups in testing procedures.
(iv) A short justification explaining any decisions involving profes-
sional judgement.
8
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(2) Results. The following information must be arranged by each
treatment and control group:
(0 In tabular form, data for each animal must be provided showing:
(A) Its identification number and the litter from which it came.
(B) Its body weight and score on each developmental landmark at
each observation time.
(C) Total session activity counts and intrasession subtotals on each
day measured.
(D) Auditory startle response amplitude per session and intrasession
amplitudes on each day measured.
(E) Appropriate data for each repeated trial (or session) showing ac-
quisition and retention scores on the tests of learning and memory on each
day measured.
(F) Time and cause of death (if appropriate); any neurological signs
observed; a list of structures examined as well as the locations, nature,
frequency, and extent of lesions; and brain weights.
(ii) The following data should also be provided, as appropriate:
(A) Inclusion of photomicrographs demonstrating typical examples of
the type and extent of the neuropathological alterations observed is rec-
ommended.
(B) Any diagnoses derived from neurological signs and lesions, in-
cluding naturally occurring diseases or conditions, should also be recorded.
(iii) Summary data for each treatment and control group must include:
(A) The number of animals at the start of the test.
(B) The body weight of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
•(D) The number of animals showing each abnormal sign at each ob-
servation time.
(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous endpoint
at each observation time. These will include body weight, motor activity
counts, auditory startle responses, performance in learning and memory
tests, regional brain weights and whole brain weights (both absolute and
relative).
-------
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion,
the location, frequency and average grade of each type of lesion foi each
animal.
(I) The values of all morphometric measurements made for each ani-
mal listed by treatment group.
(3) Evaluation of data. An evaluation of test results must be made.
The evaluation should include the relationship between the doses of the
test substance and the presence or absence, incidence, and extent of any
neurotoxic effect. The evaluation should include appropriate statistical
analyses. The choice of analyses should consider tests appropriate to the
experimental design and needed adjustments for multiple comparisons. The
evaluation should include the relationship, if any, between observed
neuropathological and behavioral alterations.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter,
L.W., Sobotka, T.J., Tilson, H. A., and Nelson, B.K. Collaborative behav-
ioral teratolgy study: Protocol design and testing procedures.
Neurobehavioral Toxicology and Teratology 7:579-586 (1985).
(2) Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H. Science
and art in preparing tissues embedded in plastic for light microscopy, with
special reference to glycol methacrylate, glass knives and simple stains.
Stain Technology 51:71-97 (1976).
(3) Bushnell, P.J. Effects of delay, intertrial interval, delay behavior
and trimethyltin on spatial delayed response in rats. Neurotoxicology and
Teratology 10:237-244 (1988).
(4) Campbell, B.A. and Haroutunian, V. Effects of age on long-term
memory:Retention of fixed interval responding. Journal of Gerontology
36:338-341 (1981).
(5) Cory-Slechta, D.A., Weiss, B., and Cox, C. Delayed behavioral
toxicity of lead with increasing exposure concentration. Toxicology and
Applied Pharmacology 71:342-352 (1983).
(6) Di Sant Agnese, P. A. and De Mesy Jensen, K.L. Dibasic staining
of large epoxy tissue sections and application to surgical pathology. Amer-
ican Journal of Clinical Pathology 81:25-29 (1984).
(7) U.S. Environmental Protection Agency. Neurotoxicity Screening
Battery. In: Pesticide Assessment Guidelines, Subdivision F, Addendum
10. EPA 540/09-91-123. NTIS PB 91-154617. (1991).
10
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(8) Fnede. R. L Developmental Neuropathology Springer-Verlag,
New York, pp 1-23. 297-313, 326-351. (1975).
(9) Green, R.J. and Stanton, M.E. Differential ontogeny of working
memory and reference memory in the rat. Behavioral Neuroscience
103.98-105(1989).
(10) Ison. J.R. Reflex modification as an objective test for sensory
processing following toxicant exposure. Neurobehavioral Toxicology and
Teratology 6:437^45 (1984).
(11) Korenbrot, C.C.. Huhtaniemi, I.T., and Weiner, R.I. Preputial
separation as an external sign of pubertal development in the male rat.
Biology of Reproduction 17:298-303 (1977).
(12) Krasnegor, N.A., Blass, E.M., Hofer, M.A., and Smotherman,
W.P. (eds.) Perinatal Development: A Psychobiological Perspective. Aca-
demic Press, Orlando, pp. 11-37, 145-167. (1987).
(13) Kucharski, D. and Spear, N.E. Conditioning of aversion to an
odor paired with peripheral shock in the developing rat. Developmental
Psychobiology 17:465^179 (1984).
(14) Luna, L. G. (editor). Manual of Histologic Staining Methods of
the Armed Forces Institute of Pathology. (Third Edition). McGraw-Hill,
New York. pp. 1-31. (1968).
(15) Miale, I. L. and Sidman, R.L. An autoradiographic analysis of
histogenesis in the mouse cerebellum. Experimental Neurology. 4:277-296
(1961).
(16) Miller, D.B. and Eckerman, D.A. Learning and memory meas-
ures. In: Neurobehavioral Toxicology , Z. Annau (ed). Johns Hopkins Uni-
versity Press, Baltimore, pp. 94-149 (1986).
(17) Pender, M.P. A simple method for high resolution light micros-
copy of nervous tissue. Journal of Neuroscience Methods. 15:213-218
(1985).
(18) Ralis, H.M., Beesley, R.A., and Ralis, Z.A. Techniques in
Neurohistology. Butterworths, London, pp. 57-145. (1973).
(19) Rodier, P.M. and Gramann, W.J. Morphologic effects of inter-
ference with cell proliferation in the early fetal period. Neurobehavioral
Toxicology 1:129-135(1979).
(20) Spear, N.E. and Campbell, B.A. (eds.) Ontogeny of Learning
and Memory. Erlbaum, New Jersey, pp. 101-133, 157-224. (1979).
(21) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H.
Neuropathological methods for the detection of neurotoxic disease. In: Ex-
11
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penmental and Clinical Neurotoxicology. Spencer. PS. and Schaumburg,
H H (eds.). Williams and Wilkms, Baltimore, pp. 743-757. (1980)
(22) Suzuki, K. Special vulnerabilities of the developing nervous sys-
tem to toxic substances. In: Experimental and Clinical Neurotoxicology.
Spencer, P.S. and Schaumburg, H.H. (eds.). Williams and Wilkins, Balti-
more, pp. 48-61 (1980).
(23) Luna, L.G. (Editor). Manual of Histologic Staining Methods of
the Armed Forces Institute of Pathology. (Third Edition). McGraw-Hill,
New York. pp. 32-46. (1968).
12
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oEPA
United States
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101)
EPA712-C-98-240
August 1998
Health Effects Test
Guidelines
OPPTS 870.6500
Schedule-Controlled
Operant Behavior
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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. 136, et seq.).
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
Guidelines."
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OPPTS 870.6500 Schedule-controlled operant behavior.
(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 798.6500 Schedule-Controlled
Operant Behavior and OPP 85-5 Schedule-Controlled Operant Behavior
(Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals, 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 functional neu-
rotoxic effects. Substances that have been observed to produce neurotoxic
signs in other toxicity studies (e.g. central nervous system (CNS) depres-
sion or stimulation), as well as substances with a structural similarity to
neurotoxicants affecting performance, learning, or memory may be appro-
priate to evaluate with this test. This guideline defines procedures for con-
ducting studies of schedule-controlled operant behavior, one way of evalu-
ating the rate and pattern of a class of learned behavior (under paragraphs
(g)UX (g)(4), (g)(5), and (g)(6) of this guideline. The purpose of the guide-
line is to evaluate the effects of acute and repeated exposures on the rate
and pattern of responding under schedules of reinforcement. 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 lexicological effects seen. Operant behavior tests may be also
used to evaluate many other aspects of behavior (under paragraph (g)(3)
of this guideline). Additional tests may be necessary to completely assess
the effects of any substance on learning, memory, or behavioral perform-
ance.
(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.
Behavioral toxicity is any adverse change in. the functioning of the
organism with respect to its environment in relation to exposure to a chem-
ical substance.
ED is effective dose.
Neurotoxicity is -any adverse effect on the structure or function of
the nervous system related to exposure to a chemical substance.
Operant, operant behavior, operant conditioning: An operant is a
class of behavioral responses which changes or operates on the environ-
-------
ment in the same way. Operant behavior is further distinguished as behav-
ior which is modified by its consequences. Operant conditioning is the
experimental procedure used to modify some class of behavior by rein-
forcement or punishment.
Schedule of reinforcement specifies the relation between behavioral
responses and the delivery of reinforcers", such as food or water (under
paragraph (g)(2) of this guideline). For example, a fixed ratio (FR) sched-
ule requires a fixed number of responses to produce a reinforcer (e.g. FR
30). Under a fixed interval (FI) schedule, the first response after a fixed
period of time is reinforced (e.g. FI 5 min).
(d) Principle of the test method. Experimental animals are trained
to perform under a schedule of reinforcement and measurements of their
operant behavior are made. Several doses of the test substance are adminis-
tered according to the experimental design (between groups or within sub-
jects) and the duration of exposure (acute or repeated). Measurements of
the operant behavior are repeated. For use of this test to study learning,
animals may be trained following exposure. A descriptive and statistical
evaluation of the data is made to evaluate the nature and extent of any
changes in behavior in relation to exposures to the test substance. Com-
parisons are made between any exposures that influence the behavior and
exposures that have neuropathological effects or effects on other targets
of the chemical.
(e) Test procedures—(1) Experimental design. These test proce-
dures may be used to evaluate the behavior of experimental animals receiv-
ing either acute or repeated exposures. For acute exposure studies, either
within-subject or between-groups experimental designs may be used. For
repeated exposure studies, between-groups designs should be used, but
within-subject comparisons (preexposure and postexposure) are rec-
ommended and encouraged.
(2) Animal selection—(i) Species. For most studies the laboratory
mouse or rat is recommended. Standard strains should be used. Under
some circumstances other species may be recommended.
(ii) Age. Experimental animals should be young adults. Rats or mice
should be at least 14 and 6 weeks old, respectively, prior to exposure.
(iii) Sex. Because of the labor-intensive nature of this testing, only
one sex may be used. If data on the test chemical indicate that one sex
is more sensitive to the test substance, or if it receives greater exposure,
that sex should be used. If females are used, they should be virgins.
(iv) Experimental history. Animals should be experimentally and
chemically naive.
-------
(3) Number of animals. Eight to ten animals should be exposed to
each level of the test substance and/or control procedure.
(4) Control groups. (0 A concurrent control group or control ses-
sions (according to the design of the study) are 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 operant behavior. Both increases and decreases
in response rate should be demonstrated. Data based on acute exposures
will be adequate. Permanently injurious substances need not be used. His-
torical data may be used if the essential aspects of the experimental proce-
dure remain the same. Periodic updating of positive control data is rec-
ommended. New positive control data should also be collected when per-
sonnel or some other critical element in the testing laboratory has changed.
(5) Dose levels and dose selection. At least three doses should be
used in addition to the vehicle control group (or sessions for within subject
studies). Ideally, the data should be sufficient to produce a dose-effect
curve. The Agency strongly encourage the use of equally spaced doses
and a rationale for dose selection that will maximally support detection
of dose-effect relations.
(i) Acute studies. The high dose need not be greater than 2 g/kg.
Otherwise, 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.
(ii) Subchronic (and chronic) studies. The high dose need not be
greater than 1 g/kg. Otherwise, the high dose should result in significant
neurotoxic effects or other clearly toxic effects, but not produce an inci-
dence 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.
(6) Route of exposure. 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. It should be recognized that for many
materials 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.
-------
(7) Combined protocol. The tests described in this screening battery
may be combined with any other toxicity study, as long as none of the
requirements of either are violated by the combination.
(8) Study conduct—(i) Apparatus. Behavioral responses and the de-
livery of reinforcers should be controlled and monitored by automated
equipment located so that its operation does not provide unintended cues
or otherwise interfere with the ongoing behavior. Individual chambers
should be sound attenuated to prevent disruptions of behavior by external
noise. The response manipulanda, feeders, and any stimulus devices should
be tested before each session; these devices should be calibrated periodi-
cally.
(ii) Chamber assignment. Concurrent treatment groups should be
balanced across chambers. Each subject should be tested in the chamber
to which it is initially assigned.
(iii) Schedule of food availability. (A) If a nonpreferred positive
reinforcer is used, all subjects should be placed on a schedule of food
availability until they reach a fixed percentage, e.g. 80 to 90 percent, of
their ad libitum body weight, or kept at a fixed weight and fed after each
session.
(B) Subjects must be trained until they display demonstrable stability
in performance across days prior to exposure. One simple and useful cri-
terion is a minimum number of sessions on the schedule and no systematic
trend during the 5 days before exposure.
(iv) Time, frequency, and duration of testing—(A) Time of testing.
All experimental animals should be tested at the same time of day and
with respect to the time of exposure. For acute studies, testing should be
performed when effects are estimated to peak, which may be estimated
from data on the functional observational battery, motor activity, or from
pilot studies. For subchronic studies, subjects should be tested prior to
daily exposure in order to assess cumulative effects.
(6) Frequency of testing. The maintenance of stable operant behav-
ior normally will require regular and frequent (e.g. 5 days a week) testing
sessions. Animals should be weighed on each test day.
(C) Duration of testing. Experimental sessions should be long
enough to reasonably see the effects of exposure, but brief enough to be
practical. Under most circumstances, a session length of 30-40 min should
be adequate.
(v) Schedule selection. The schedule of reinforcement chosen should
generate response rates that may increase or decrease as a function of ex-
posure. Many schedules of reinforcement can do this: A single schedule
maintaining a moderate response rate, FI schedules, which engender a van-
-------
ety of response rates in each interval, or multiple schedules, where dif-
ferent components may maintain high and low response rates.
(f) Data reporting and evaluation. The final test report must include
the following information:
(1) Description of equipment and test methods, (i) A descnption
of the experimental chambers, programming equipment, data collection de-
vices, and environmental test conditions should be provided. Procedures
for calibrating devices should also be described.
(ii) A description of the experimental design including procedures for
balancing treatment groups and the stability criterion should be provided.
(iii) Positive control data from the laboratory performing the test that
demonstrates the sensitivity of the schedule used should be provided. His-
torical data may be used if all essential aspects of the experimental proto-
col are the same. Historical control data can be critical in the interpretation
of study findings. The Agency encourages submission of such data to fa-
cilitate the rapid and complete review of the significance of effects seen.
(2) Results. Data for each animal should be arranged in tabular form
by test group including the animal identification number, body weight,
preexposure rate and patterns of responding, changes in response rate and
patterns produced by the chemical, and group data for the same variables,
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 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 the data.
(iii) Citations from the 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 ad-
ditional background material on this test guideline.
(1) Dews, P.B. Assessing the Effects of Drugs, In Methods in
Psychobiology, Vol. 2, (Ed.) R.D. Myers. Academic, NY. pp. 83-124
(1972).
-------
(2) Ferster, C.B. and Skinner, B.F. Schedules of Reinforcement Ap-
pleton-Century-Crofts, NY (1957).
(3) Laties, V.G. How Operant Conditioning Can Contribute to Behav-
ioral Toxicology, Environmental Health Perspectives 28:29-35 (1978).
(4) National Academy of Sciences. Principles for Evaluating Chemi-
cals in the Environment. National Academy of Sciences, Washington, DC
(1975).
(5) National Academy of Sciences. Principles and Procedures for
Evaluating the Toxicity of Household Substances. National Academy of
Sciences, Washington, DC (1977).
(6) National Academy of Sciences. Strategies to determine needs and
priorities for toxicity testing. Appendix 3B. Reference Protocol Guidelines
for Neurobehavioral Toxicity Tests 2:123-129 (1982).
-------
&EPA
United States
Environmental Protection
Agency
Prevention. Pesticides
andToxic Substances
(7101)
EPA 712rC-98-241
August 1998
Health Effects Test
Guidelines
OPPTS 870.6850
Peripheral Nerve
Function
-------
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. 136, 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
Guidelines."
-------
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
lexicological 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
nerve.
(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
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 elec-
trodes) and should operate from about 10 JO.A 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 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.
(0 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 m
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) 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 Electrom\ograph\. Williams and Wil-
kms. Baltimore (1980).
(5) U.S. Environmental Protection Agency. Guidelines for
Neurotoxicity Risk Assessment. FEDERAL REGISTER 63 FR 26926-26954
May 14, 1998.
-------
United States Prevention, Pesticides EPA712-C-98-242
Environmental Protection and Toxic Substances' August 1998
Agency (7101) .
&EPA Health Effects Test
Guidelines
OPPTS 870.6855
Neurophysiology:
Sensory Evoked
Potentials
-------
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. 136, 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
Guidelines."
-------
OPPTS 870.6855 Neurophysiology: sensory evoked potentials.
(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 teq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. This guideline was developed jointly between-the
Office of Pesticide Programs and the Office of Pollution Prevention and
Toxics in cooperation with the Office of Research and Development of
the Environmental Protection Agency. The source material used in devel-
oping this harmonized guideline is 40 CFR 798.6855 Neurophysiology:
sensory evoked potentials.
(b) Purpose. The techniques in this guideline are designed to detect
and characterize changes in the sensory aspects of nervous system function
that result from exposure to chemical substances. The techniques involve
neurophysiological measurements from adult animals and are sensitive to
changes in the function of auditory, somatosensory (body sensation) and
visual sensory systems. These procedures can be used in two ways:
(1) To detect sensory dysfunction produced by compounds in the ab-
sence of relevant information.
(2) When there are reasons to expect that particular sensory functions
are specifically sensitive to the test compound. The procedures employed
during a particular study will be selected on a case-by-case basis depend-
ing on information available at the time of the study design, signs of tox-
icity observed during the study, and/or the purpose of the study. It will
be the responsibility of those submitting to justify the selection of a spe-
cific test from the categories of electrophysiological tests available. The
tests are adaptable so that they may be used in sum or in part, and either
alone or in conjunction with other tests including: A functional observa-
tional battery, motor activity, neuropathology, and general toxicity studies.
These studies may involve acute, subchronic, or chronic exposures.
(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.
Neurotoxicity is any adverse effect on the structure or function of
the nervous system related to exposure to a chemical substance.
Toxic effect is any adverse change in structure or function of an exper-
imental animal as a result of exposure to a chemical substance.
(d) Principle of the test method. The test substance is administered
to several groups of experimental animals, each group receiving a different
dose level. Electrodes for recording brain electrical activity are temporarily
-------
or permanently affixed to the test animals. After electrodes are in place
and. if appropriate surgical recovery, stimuli for the visual, auditory or
somatosensory sensory systems are presented to the test animals, and the
resulting brain electrical potentials are recorded. The electrical potentials
recorded from animals treated with the test compound are compared to
those recorded from control animals. The results are interpreted regarding
the extent to which treatment with the test compound altered the normal
function of the sensory systems tested.
(e) Test procedure—(1) Animal selection—(i) Species and strain.
Testing should generally be performed in the laboratory rat, preferably a
pigmented strain. Albino strains of animals have abnormalities of the vis-
ual and auditory systems (see paragraphs (g)(4), (g)(5), and (g)(14) of this
guideline), including: The absence of pigment from the retinal pigment
epithelial layer, high incidence of spontaneous retinal pathologies, prob-
lems of photogenic retinopathy (under paragraph (g)(8) of this guideline),
abnormal pattern visual evoked potentials (under paragraph (g)(l) of this
guideline), lack of normal pigment in the stria vascularis of the cochlea
(see paragraph (g)(18) of this guideline) and differential susceptibility to
ototoxic noise and drugs from pigmented strains (under paragraphs (g)(2)
and (g)(3) of this guideline). However, it is recognized that under some
circumstances, use of another species or an albino strain may be justified.
If another species or an albino strain is used, the user must submit jus-
tification.
(ii) Age. Animals should be young adults (42-120 days of age, for
rats) at the start of exposure. Implantation of chronic electrodes in rats
younger than 60 days of age is not advised.
(iii) Sex. (A) In order to reduce the number of animals used, only
one sex is required. Male rats may be preferred because there is more
existing data on them. If, on the other hand, females are known or expected
to be more sensitive to the test agent, they may be used. The user should
provide justification for the sex selected.
(B) If females are used, they should be nulliparous and nonpregnant.
(2) Number of animals. A final sample size of 'at least 10 animals
should be used in each dose and control group. The number of animals
to be used should be based on appropriate statistical methods and an allow-
ance for attrition due to anticipated problems such as loss of electrode
preparations, etc. Note that the rate of attrition should be estimated based
on the experience of the laboratory performing the testing. If interim
neurophysiological evaluations are planned in long-term dosing studies,
it may be advisable to include an additional number of animals sufficient
for the interim studies. Animals should be randomly assigned to treatment
and control groups. If groups are not randomly assigned, justification must
be provided.
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(3) Control groups, (i) A concurrent ("sham" exposure) vehicle
control group is required. Subjects should be treated in the same way as
an exposure group except that administration of the test substance is omit-
ted If the vehicle used has known or potential neuroactive properties, both
untreated and vehicle-control groups are required.
(ii) Positive control groups exhibiting functional changes in the sen-
sory systems to be tested are required in order to demonstrate the capabil-
ity of the laboratory performing the testing to conduct the procedures. Sep-
arate setups for each sensory system are acceptable, but not necessary.
In addition, for each sensory modality in vehicle or untreated control group
data, it should be demonstrated that the mean of an amplitude-sensitive
dependent measure increases monotomcally as a function of stimulus in-
tensity as defined in paragraph (e)(7)(v)(D) of this guideline. Historical
data may be used if the essential aspects of the experimental procedure
remain the same. Periodic updating of positive control data is rec-
ommended. New positive control data should also be collected when per-,
sonnel or some other critical element in the testing laboratory has changed.
(4) Dose levels and dose selection, (i) At least three dose levels
should be used in addition to the vehicle control group. Ideally, the data
should be sufficient to produce a dose-effect curve. We encourage the use
of equally spaced doses on a logarithmic scale, and a rationale for dose
selection that will enable detection of dose-effect relations to the highest
degree possible. For acute studies, dose selection may be made relative
to the establishment of a benchmark dose (BD). That is, doses may be
specified as successive fractions, e.g. 0.5, 0.25 of the BD. The BD itself
may be estimated as the highest nonlethal dose as determined in a prelimi-
nary range-finding study. A variety of test methodologies may be used
to determine a BD, and the method chosen may influence subsequent dose
selection. The goal is to use a dose level that is sufficient to be judged
a limit dose, or clearly toxic. Alternatively, the BD may be specified as
a dose of the test compound producing clear neurotoxic effects in previous
studies.
(ii) Acute studies. The high dose need not be greater than 2 g/kg.
Otherwise, 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. This dose may
be estimated by a BD procedure as described above, with the middle and
low dose levels chosen as fractions of the BD. The lowest dose should
produce minimal effects or, alternatively, no effects.
(iii) Subchronic and chronic studies. The high dose need not be
greater than 1 g/kg/day. Otherwise, the high dose level should result in
significant neurotoxic effects or other clearly toxic effects, but not produce
an incidence of fatalities that would preclude a meaningful evaluation of
-------
the data. The middle and low dose should be fractions of the high dose.
The low dose should produce rrunimal effects, or alternatively, no effects.
(5) Route of exposure. Selection of route may be based on several
criteria: The most likely route of human exposure, the greater likelihood
of observing effects, the practical difficulties, the likelihood of producing
nonspecific effects, and existing data regarding the test compound. -Be-
cause more than one route of exposure may be important for many mate-
rials, these criteria may conflict with one another. The route that best meets
these criteria should be selected.
(6) Combined protocol. The tests described herein may be combined
with any other toxicity study, as long as none of the requirements of either
is violated by the combination.
(7) Study conduct—(i) Preparation of animals for recording. (A)
For electrophysiological recording it usually will be necessary to implant
animals with chronic in-dwelling electrodes using stereotaxic surgical pro-
cedures. In some circumstances, acute attachment of temporary electrodes
may be acceptable if criteria for humane treatment of animals, for record-
ing without undue anesthesia, and for data acceptability detailed below
can be met. Chronic implantation of electrodes will require surgical anes-
thesia and surgical techniques appropriate for the species as outlined in
current laboratory animal care guidelines under paragraph (g)(17) of this
guideline. Standard animal surgical practices should be followed as out-
lined in a number of standard references (e.g. paragraph (g)(ll) of this
guideline). Once anesthetized, animals are usually placed in a stereotaxic
device in order to position the head firmly. The stereotaxic device should
be designed to prevent trauma to the tympanic membranes and, for audi-
tory studies, tympanic membranes should be examined after removal from
the stereotaxic device.
(B) Care should be taken during surgery to prevent drying of the
cornea through means such as regular application of fluids such as mineral
oil, saline, or artificial tear solution. Once the animal is positioned in the
stereotaxic device and the implantation site is prepared, the electrodes
should be positioned with reference to standard skull markings and/or to
published brain atlas coordinates (see paragraph (g)(12) or (g)(13) of this
guideline).
a
(C) For recording potentials which are generated in sensory cortex,
the recording or active electrodes are to be positioned as close as possible
to the brain sites generating the response. For "far-field" potentials such
as the brainstem auditory evoked potential, which are conducted through
cranial tissues from sites relatively distant to the recording electrodes, the
location of the recording electrodes should be such as to provide good
resolution of the major waveform components, but need not be as close
as possible to the generator sites. The site of the reference electrode for
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diffeiential recordings should be indifferent with respect to stimulus-
evoked electrical activity to the extent possible. A ground electrode should.
also be included The electrodes should be made of a material that is not
toxic to neural tissue. The electrodes should be made of a nonpolanzable
material, such as silver-silver chloride, if potentials of a frequency less
than approximately 1 Hz are to be reported. However, extra caution should
be exercised in this case to avoid toxic effects of such electrodes. Elec-
trodes should be described as to composition, size, shape, and position.
(D) Wound sites should be treated and closed so as to prevent infec-
tions and to protect the integrity of the electrodes. Following surgery, elec-
trode impedance should be measured in order to verify that electrode con-
nections are intact and functional. Following surgery, animals should be
given sufficient time to recover from the anesthetic and surgical trauma
prior to testing; ordinarily a period of one week is recommended. Prior
to testing, the wound site should be inspected for signs of infection or
inflammation, and any animal showing such signs should be removed from
the experiment.
(E) For acute studies, it is necessary to perform surgery prior to ad-
ministering the test compound. In repeated dosing, it may be necessary
to implant the electrodes during the course of treatment with the test
compound due to the limited time which electrode preparations may be
expected to remain intact. It may be advisable to prevent exposure to the
test compound on the day of surgery due to possible interactions of the
effects of the test compound and the anesthetic agent. The time between
surgical implantation of electrodes and testing should be equal for all ani-
mals or, if unequal, balanced across the treatment groups. Animals losing
electrode preparations between time of surgery and testing should be re-
moved from the study and not submitted to another surgery. At termination
of the study, postmortem examination of each animal should be used to
determine if the electrodes were incorrectly positioned, or if the presence
of epidural electrodes damaged the underlying neural tissue. If either of
these two conditions is found, the data from the involved animal should
be discarded.
(ii) Testing environment (A) Electrophysiological testing should be
conducted in a chamber or room which is isolated from extraneous light
and noise and controlled for temperature. Background noise, light, and
room temperature should be reported. Where possible, testing should be
conducted without the use of restraint or anesthetics. The use of restraint
may be necessary when a specific orientation to, or distance from, the
stimulation equipment is required, or when movement of the animal would
interfere with recording.
(B) For auditory testing of unrestrained, unanesthetized animals,
acoustic stimuli should be of equal sound pressure level wherever the ani-
mals' ears may be placed during data acquisition. The animal enclosure
-------
should be acoustically transparent to sound in the frequency band of the
stimulus spectrum. The use of anesthetic is permissible when consider-
ations of animal discomfort prohibit testing awake animals, and when it
can be reasonably expected that the effects of the test agent would not
be substantially different under anesthesia. If recording is to be performed
on anesthetized animals, body temperature should be maintained within
a normal range during testing. In addition, if recording from anesthetized
animals, the power spectrum of the spontaneous electroencephalogram
(EEG) should be measured in order to control depth of anesthesia. Position
of the stimulation device or devices relative to the appropriate sensory
organs should be specified.
(iii) Electrophysiological recording. (A) Electrophysiological re-
cording procedures should follow generally accepted practice such as is
found in standard reference texts (under paragraph (g)(9) or (g)(16) of
this guideline). Typically, it is appropriate to differentially record between
active and reference electrodes using an amplifier with high common mode
rejection and high input impedance relative to that of the electrodes.
(B) Electrical shielding and grounding should be used to eliminate
activity in the electrophysiological recording at the frequency of the power
lines reflecting inductive noise. Electrophysiological amplifiers and filter
bandpass settings must be appropriate to the signal being measured. For
example, ac-coupling is appropriate for most applications, but de-coupling
should be used if steady potentials are to be measured. Analog electrical
activity is typically digitized using an analog-to-digital converter operating
at a rate at least twice, preferable higher, the highest frequency passing
the input filters. Analog or digital filtering of data is appropriate to im-
prove signal-to-noise ratios. If using analog filtering, the decay functions
of the high and low bands of the filters should be specified, and filtering
parameters should be selected to avoid amplitude reductions or phase shifts
in the frequency bands of the signal to be measured.
(iv) Signal averaging. (A) Signal averaging of the input data syn-
chronized with the stimulation is appropriate to increase the signal-to-noise
ratio. The number of trials averaged should be sufficient to yield reliable
data, and a waveform in control animals for which the maximum ampli-
tude measure to be reported at the minimum intensity stimulus is at least
50 percent greater in amplitude than the recording noise level. The dura-
tion of the sampling epoch should be sufficient to encompass all major
components of transient evoked potentials. Automated artifact rejection
routines may be employed to reject occasional spurious data provided that
no greater than 50 percent of the original trials are rejected for any given
waveform, and all final averaged waveforms for a given stimulus condition
are based on an equal number of trials.
(B) The data for cases in which greater than 50 percent of the trials
are rejected due to artifacts should be discarded, and the recording condi-
-------
tions improved, if possible, to allow more suitable data collection The
recording noise level should be determined in order to illustrate the signal-
to-noise ratio. Acceptable methods to do so include: examining a portion
of the data preceding the presentation of the stimulus, but following the
last of the evoked activity from the previous stimulus in transient evoked
potentials; averaging an equal number of trials as the recording session
but without presentation of the eliciting stimulus (for visual stimuli this
should be accomplished by temporarily blocking the test subjects view
of the stimulus with an opaque material); averaging alternate trials of in-
verted polarity during the standard recording session, or reaveraging the
original raw data from the standard recording session over a similar num-
ber of trials in a manner nonsynchronous with the eliciting stimulus. The
voltage gain and temporal response properties of the electrophysiological
recording equipment should be calibrated for each experiment, or more
frequently as needed. The same recording conditions should be used on
all animals tested.
(v) Stimulation. The selection of stimulation parameters will depend
upon the specific goals of the study. Tests should include measures of
visual, auditory and somatosensory systems, unless there are reasons to
limit testing to particular aspects of sensory function. The testing of mul-
tiple sensory systems in the same animal has been demonstrated (see para-
graphs (g)(10) and (g)(15) of this guideline). The order of presentation
of the different tests to individual subjects be either random, balanced
across treatment groups, or fixed. If the test order is fixed, justification
for the test order should be provided.
(A) Visual stimuli. Visual testing should include separate tests in-
volving light flashes and patterned stimuli. Visual pattern testing should
employ stimuli with a sinusoidal spatial luminance profile, and should in-
clude a range of pattern sizes which encompass the low, middle and high
spatial frequency ranges of the contrast sensitivity function of the test spe-
cies. Techniques for recording visual evoked potentials using both flashed
(under paragraph (g)(6) of this guideline) and patterned stimuli (under
paragraph (g)(l) of this guideline) are available for laboratory rats.
(B) Auditory stimuli. Auditory testing should include a stimulus of
broadband frequency characteristics, such as a click. In addition, pure tone
stimuli reflecting the low, middle and high frequency portions of the
audiometric function of the test species should be used. Techniques for
recording auditory evoked potentials using both click and pure tone stimuli
(under paragraph (g)(15) of this guideline) are available for laboratory rats.
(C) Somatosensory stimuli. Somatosensory testing should include
electrical stimuli delivered to the tail or distal portions of the lower extrem-
ities. Techniques for recording somatosensory evoked potentials are avail-
able for laboratory rats (under paragraph (g)(15) of this guideline).
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(D) Stimulus levels. (7) For studies designed to detect a change in
sensory function produced by a compound for which little is known, it
is sufficient to use a single stimulus level for each visual, auditory or
somatosensory evoked potential. Justification for the stimulus level se-
lected should be provided. For studies designed to characterize a sensory
effect, at least three different levels of flash intensity, pattern contrast,
acoustic click stimulus sound pressure level or somatosensory stimulus
current are required for each stimulus condition. The stimulus levels
should be chosen on the basis of prior experience and/or pilot studies.
(2) In control animals, the low level should be near the response
threshold, but large enough to produce a response amplitude at least 50
percent greater than the recording noise level. Specification of the high
stimulus level varies with stimulus type. For flashed visual stimuli the high
stimulus level should either produce a maximal response, or be the maxi-
mum output available from a conventional stimulator (e.g. Grass model
PS-22 photic stimulator). For patterned visual stimuli the high stimulus
level should either produce a maximal response, or be below the highest
level of contrast within the linear range of the input-voltage/stimulus-con-
trast calibration function of the stimulus screen. For somatosensory stimuli
the high stimulus level should either produce a maximal response, or be
at or below a current which produces minimal reflexive muscle movement
or other indications of discomfort. For acoustic stimuli the high stimulus
level should be below approximately 80 decibels sound pressure level in
order to avoid production of temporary or permanent threshold shifts.
(3) For all types of stimuli, the middle stimulus level should produce
a response intermediate between high and low stimulus levels. The phys-
ical and temporal parameters of stimuli should be calibrated against known
standards for each experiment using commonly accepted procedures.
(4) Visual stimulus luminance and contrast, or for flashes integrated
power, should be calibrated with an appropriate radiometer or photometer
against a known standard. Acoustic stimuli should be calibrated for level
using equipment which meets standards of the American Nattonal Stand-
ards Institute (ANSI) for sound level meters. Stimuli should be measured
and reported as peak levels, maximum root mean square (max RMS), or
"peak equivalent" sound pressure levels. In addition, the polarity of the
electrical stimulus and the transduction system for acoustic stimuli should
be reported.
(vi) Measurement (A) Dependent measures should be taken from
each evoked potential which are sensitive to changes of both amplitude
(voltage) and latency (time after stimulus onset) for transient evoked po-
tentials or phase for steady-state evoked potentials. Enough measures
should be taken to adequately reflect the shape of the evoked potential
in control animals.
8
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(B) A variety of measurement schemes may be acceptable, provided
they are specified a priori, do not ignore major portions of the waveform.
yield ratio-scale values, and meet the criterion for positive controls speci-
fied in paragraph (e)(3)(u) of this guideline. If the measurement scheme
involves inspection of each waveform by an operator and scoring of the
waveform, the criteria for scoring should be objective and stated.
(C) The experiment also must be conducted so that the experimental
personnel are unaware of the treatment of individual animals at the time
of scoring. Measurement schemes that minimize the scoring by personnel
of each response are preferred. The same data scoring procedure must be
used on all subjects.
(D) Previously collected data demonstrating selective effects of the
test compound may be used to restrict the number of test parameters and/
or the number of measured endpoints in order to examine more restricted
hypotheses. Colonic temperature should be measured at the time of each
electrophysiological recording. Body weight should be measured on each
test day.
(vii) Acute. Testing should be timed to include the estimated time
of peak effect.
(viii) Repeated dosing. Testing should be conducted after the com-
pletion of dosing with the test compound when it can be expected that
transient effects of the final treatment have dissipated. Additional testing
may be conducted during the course of treatment in order to provide infor-
mation on the emergence of toxic effects.
(0 Data reporting and evaluation. The following should be re-
ported:
(1) Description of the test methods. This must include:
(i) Positive control data from the laboratory performing the test which
demonstrate the sensitivity of the procedure being used. Historical control
data can be critical in the interpretation of study findings. We require sub-
mission of such data to facilitate the rapid review of the significance of
the observed effects.
(ii) Procedures for calibrating the stimulation and recording equip-
ment and balancing the groups.
/
(2) Results. The following must be arranged by test group (dose
level).
(i) In tabular form, data must be provided showing for each animal:
(A) Its identification number.
(B) Body weight for each day tested.
-------
(C) Values of each evoked potential dependent measure
(D) Body temperature of the animal at the time of acquisition of each
evoked potential.
(iii) Group summary data should also be reported. Data reporting
should include in tabular form measures of central tendency and variability
for each combination of stimulus conditions and treatment with the test
compound, and the statistical significance level for effects of treatment
with the test compound associated with each set of values. The final sam-
ple size should be reported along with reasons for excluded or missing
data. The noise level should be reported. Graphic presentation of the data,
or portions thereof, may also be included. Also, samples of typical individ-
ual animal evoked potential waveforms illustrating all stimulus conditions
and the noise level, or preferably group mean waveforms of the same,
should be included.
(3) Evaluations of the data—(i) Data analysis. (A) Numerical data
analysis should include a measure of central tendency, such as mean, and
a measure of variability, such as standard error of the mean, for each stim-
ulus and dose treatment combination, and for each dependent variable.
(B) Statistical analysis should test the null hypothesis of no statis-
tically significant overall effect of treatment with the test compound across
stimulus conditions for each type of sensory evoked potential. In addition,
statistical tests for interactions between treatment with the test compound
and the manipulation of the stimulus parameters should be performed and
the results reported. The choice of statistical analysis should consider the
experimental design and address the problem of adjustments for multiple
statistical analyses.
(ii) Interpretation. The report should include an interpretation of the
neurotoxicological significance of the findings, and relate the
neurophysiological results to those of other neurotoxicological results, and
to other data to the extent possible. Guidance for interpretation of sensory
evoked potential data is described under paragraph (g)(18) of this guide-
line.
(g) References. The following references should be consulted for ad-
ditional background information on this guideline.
(1) Boyes, W. K. and R. S. Dyer. Pattern reversal visual evoked po-
tentials in awake rats. Brain Research Bulletin 10:817-823 (1983).
(2) Conlee J.W. et al. Differential susceptibility to noise-induced per-
manent threshold shift between albino and pigmented guinea pigs. Hearing
Research 23:81-91 (1986).
10
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(3) Conlee J W. et al. Differential susceptibility to gentairucin
ototoxicity between albino and pigmented guinea pigs Hearing Research
41-43-52(1989)
(4) Creel. D. Inappropriate use of albino animals in research. Phar-
macology and Bioochemical Behavior 12:969-977 (1980).
(5) Creel, D. Albinism and evoked potentials: Factors in the selection
of infrahuman models in predicting the human response to neurotoxic
agents. Neurobehavioral Toxicology and Teratology 6:447-453 (1984).
(6) Dyer, R. S. and H. S. Swartzwelder. Sex and strain differences
in the visual evoked potentials of albino and hooded rats. Pharmacology
and Biochemical Behavior 9:301-306 (1978).
(7) Herr, D.W. and Boys, W.K. Electrophysiological analyses of com-
plex brain systems; Sensory evoked potentials and their generators. In:
Neurotoxicology Approaches and Methods, L.W. Chang and W. Slikker,
eds. Academic Press, NY, 205-221 (1955)
(8) Heywood, R. and C. Gopinath. Morphological assessment of vis-
ual dysfunction. Toxicology and Pathology 18:204-217 (1990).
(9) International Federation of Societies for Electroencephalography
and Clinical Neurophysiology. Recommendations for the Practice of Clini-
cal Neurophysiology. Elsevier Science, Amsterdam, The Netherlands
(1983).
(10) Mattsson, J. L. and R. R. Albee. Sensory evoked potentials in
neurotoxicology. Neurotoxicology and Teratology 10:435-443 (1988).
(11) Myers, R.D. Methods in Psychobiology. Academic Press, NY
(1971).
(12) Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordi-
nates. Academic Press, Sydney (1982).
(13) Pelligrino, L. J. et al. A Stereotaxic Atlas of the Rat Brain, 2nd
ed., Plenum, NY (1979).
(14) Prieur, D. J. Albino animals: Their use and misuse in biomedical
research. Comparative Pathology Bulletin XIV(3):l-4 (1982).
(15) Rebert, C. S. Multisensory evoked potentials in experimental and
applied neurotoxicology. Neurobehavioral Toxicology and Teratology
5:659-671 (1983).
(16) Thompson, R.F. Methods in Physiological Psychology, Vol 1:
Bioelectric Recording Techniques; Parts A, B and C. Academic Press, NY
(1973 and 1974).
11
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(17) U.S. Department of Health and Human Services. Public Health
Service, National Institutes of Health, Publication No. 85-23, Guide for
the care and use of laboratory animals. Revised 1985.
(18) U.S. Environmental Protection Agency. Guidelines for
Neurotoxicity Risk Assessment. FEDERAL REGISTER 63 FR 26926-26954,
May 14, 1998.
(19) Witkop, C. J. et al. (eds), The Metabolic Basis of Inherited Dis-
eases, McGraw-Hill, NY, pp. 301-346 (1983).
12
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a EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances'
, (7101)
EPA712-C-98-349
August 1998
Health Effects Test
Guidelines
OPPTS 870.7200
Companion Animal
Safety
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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.).
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
Guidelines."
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OPPTS 870.7200 Companion animal safety.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 81-6 Domestic Animal Safety Test-
ing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals) EPA report 540/09-82-025, 1982.
(b) Purpose. (1) Companion animal safety studies are intended to
demonstrate that pesticide formulations for the treatment of external pests
on domestic animals have an adequate margin of safety if the products
are misused (overused). Data from companion animal safety studies also
serve as a basis for product labeling. This guideline is intended to promote
uniform review of data and to assure consistency and fairness in the re-
quirements for these studies. Although not a toxicity study of the type
required for pesticide registration, a companion animal safety study is most
comparable to an acute dermal toxicity study.
(2) This guideline also serves the purpose of providing harmonization
between the Environmental Protection Agency and the Center for Veteri-
nary Medicine in the Food and Drug Administration (FDA), which is also
responsible for conducting target animal safety studies.
(3) This guideline is limited to products for use on dogs and cats
due to the high volume use of products in these two species. The guidance
is based on professional experience, documentation in the scientific lit-
erature, and policy and procedures of other agencies involved in regulatory
veterinary medicine, (see Target Animal Safety Guidelines for New Ani-
mal Drugs, under paragraph (i)(3) of this guideline.)
(4) The guideline addresses data requirements for the safety assess-
ment of products applied directly to animals. Products used to treat exter-
nal pests on domestic animals include, but are not limited to collars,
sprays, dips, shampoos, and spot treatments. Due to differences in methods
of application, specific testing procedures for individual products are de-
pendent on label claims. Labeled uses also impact on the duration of treat-
ment and on the age and species of test animal used in a companion animal
safety study.
(5) The studies conducted to satisfy this guideline should not be mis-
taken for toxicity studies, as their intent is not to establish a no-observed-
effect-level (NOEL), but to provide assurance that an adequate margin of
safety exists.
(6) Studies conducted to satisfy companion animal safety guidelines
should be conducted in compliance with 40 CFR part 792 and 40 CFR
-------
160 (Good Laboratory Practice Standards) and a statement of compliance
should be contained within the final report.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
part 792 apply to this test guideline. The following definitions also apply
to this test guideline.
Acute dermal toxicity is the adverse effect occurring during or follow-
ing a dermal exposure to a single dose of a test substance or to repeat
applications to achieve dose. For purposes of companion animal safety
studies, the test substance is always the final formulation of a pesticide
product.
Adverse effect is an undesirable effect reflected in the animal by alter-
ations in structure, function, or behavior. .
Companion animal will be limited to dogs and cats for the purpose
of this guideline.
Companion animal safety study is a study conducted for the purpose
of establishing an adequate margin of safety and not a NOEL of toxicity.
Dosage is a term comprising the dose, its frequency, and the duration
of dosing.
Dose is the amount of test substance administered and is expressed
in weight of test substance per unit weight of the test animal (e.g. milli-
grams per kilogram).
Margin of safety is the difference between the effective dose (rec-
ommended dose) and the toxic dose. The companion animal safety study
guideline does not require the determination of a toxic dose but rather
the establishment of an adequate margin of safety.
Max/tax is a product containing multiple toxicants at the maximum
levels which would be anticipated in formulated topical products.
Vehicle is the end-use product formulation, i.e. the inert ingredients
without the active ingredients.
(d) Principle of the test method. (1) The design of a companion
animal safety study should reflect the product label, i.e. the method of
administration, species and age group, frequency of application, etc., used
in the study should be identical to that of the end-use product. The test
formulation should be applied to several groups of experimental animals
at the label recommended dose and multiples of this dose (3X and 5X
the recommended dose).
(2) If the product label states that a treatment can be repeated, the
companion animal safety study should also include a repeat treatment.
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(3) Observations and measurements of possible treatment-related ef-
fects should be reported for 14 days posttreatment. Animals that die or
are sacrificed in a moribund state should be subjected to a necropsy in
an attempt to arrive at the cause of death. Routine sacrifice or necropsy
is not required for surviving animals.
(e) Substance to be tested. (1) The end-use product should be tested
at the recommended dose (IX). For exaggerated doses (3X and 5X the
recommended dose), products specifically prepared for this type of study
that contain higher concentrations (3X and 5X) of the active ingredient
are preferred. See additional discussion under paragraph (g)(3)(iii) of this
guideline.
(2) Because of the practice of combining several pesticides in one
product, a procedure has been proposed whereby maximum concentrations
of multiple active ingredients have been used to determine the margin of
safety of end-use products. This practice has been referred to as the max/
tox procedure.
(f) Limit test. If a test at one dose level of at least 5X the rec-
ommended dose, using the procedures described for the study, produces
no evidence of treatment-related toxicity, a full study using a minimum
of three dose levels may not be necessary.
(g) Test procedures—(1) Animal selection—(i) Species. The species
recommended for treatment on the product label should be included in
the study. Studies should be performed on healthy dogs and cats represent-
ative of the classes of dogs and cats (size, weight range, sex, or age) for
which the product is intended.
(ii) Age. The age of animals in the study is dependant upon label
claims. If only adults (6 months or older) are the targeted population of
animals to receive treatment, adults only will suffice. However, if a prod-
uct is registered for use on pediatric animals (i.e. puppies and kittens),
the label should state a minimum age for this group, for example, "Do
not use on puppies (kittens) less than eight weeks of age". Consequently,
the product should be tested in 8 week-old animals in the companion ani-
mal safety study.
(iii) Sex. (A) Equal numbers of animals of each sex are recommended
for each dosage level.
(B) Females should be nonpregnant.
(iv) Numbers. At least six animals per sex should be used at each
dosage level.
(v) Pretreatment. Animals should be vaccinated, dewormed, and ac-
climated for 2 weeks prior to the initiation of the study. They should be
examined by a veterinarian and their suitability should be ascertained prior
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to inclusion in the study. Animals should be free of infectious diseases
which could complicate the interpretation of the study results.
(2) Control group. A concurrent vehicle control group is rec-
ommended. Negative (untreated) controls may occasionally be employed
to determine whether adverse effects are'due to the inert ingredients in
a formulation.
(3) Dosing—(i) Dose levels. The dose levels of the end-use product
to be tested should include control (OX), IX, 3X, and 5X the recommended
dose. The targeted adequate margin of safety is 5X. Consideration will
be given to products with less than a 5X margin of safety, depending on
the severity of clinical signs of toxicity (e.g. transient, non-life-threatening
signs). The route of administration should be the proposed label route.
(ii) Vehicle. The vehicle control should be administered at a 5X level.
The vehicle should contain the inert ingredients at the maximum levels
that would appear in the 5X formulation.
(iii) Methods of achieving exaggerated doses. It is preferred that
formulations be revised to include exaggerated amounts of the active ingre-
dient. How'ever, if this cannot be achieved because of volume constraints
or the physical properties of the ingredients (active or inert), multiple treat-
ments at frequent intervals during the same dosing period will be accept-
able.
(A) Spray, dip or shampoo formulations may be applied at the rec-
ommended dose at hourly intervals to achieve an exaggerated dose, i.e.
three times for a 3X dose and five times for a 5X dose. Other methods
to achieve exaggerated doses will be considered on a case-by-case basis.
(B) Multiple collars may be worn simultaneously by the experimental
animals to achieve an exaggerated dose.
(4) Observation period. The observation period should be at least
14 days following the last treatment. The time at which clinical signs of
toxicity appear and disappear should be recorded. The duration of the ob-
servation period should not be rigid, but should be determined by the toxic
reactions, the rate of onset and the length of the recovery period.
(5) Administration, (i) The route of administration for these products
should be by the topical or dermal route. The product should be applied
in accordance with the label directions. No clipping of the hair or prepara-
tion of the skin is required unless such directions appear on the label.
(ii) If a single dose is not possible, multiple treatments at frequent
intervals during the same dosing period will be acceptable.
(iii) If the product label recommends repeat treatments, multiple treat-
ments should be included in a companion animal safety study. When re-
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peat treatments are required, products will be handled on a case-by-case
basis with the retreatment interval being driven by label claims and instruc-
tions for use For fleas collars, a one month duration of study may be
sufficient for well-characterized products. Several months may be required
for new chemical entities or collars containing multiple active ingredients.
Exceptions would include:
(A) Single ingredient diluted products intended for use on dogs only,
where chronic studies in the intended species exist with the technical prod-
uct.
(B) Products with retreatment intervals of 30 days or more.
(C) Products with retreatment intervals of 14 to 30 days which have
no observed toxicity following exposure to a 5X dose level.
It is advisable that when complex issues arise, protocols be submitted in
advance for review and comment.
(6) Observations of animals, (i) Careful clinical observations should
be conducted at hourly intervals on the day of treatment for at least 4
h after the last treatment and twice daily thereafter for the duration of
the observation period.
(ii) If adverse reactions are observed, the observation period on the
day of treatment should be extended to a time at which no further adverse
reactions are observed.
(iii) Observations should include, but not be limited to, changes in
skin and fur, eyes and mucous membranes, respiratory system, circulatory
system, autonomic and central nervous system, somatomotor activity, and
behavior pattern. Particular attention should be directed to observations
of central nervous system signs (seizures, tremors, salivation), vomiting
and diarrhea.
(iv) The following provides a more complete list of possible observa-
tions.
(A) Ocular: Corneal opacities, nystagmus, pupillary changes,
blepharospasm, blindness, iritis, chemosis, photophobia, congestion,
blanching, discharge, and conjunctivitis.
(B) Equilibrium: Unsteadiness (walking or standing), incoordination,
ataxia or paresis, and abnormal reflexes.
(C) Muscular disturbances: Localized or generalized tremors, lip
drooping and/or salivation, paralysis, atony, and atrophy.
(D) Behavior (mental attitude): Anxious, apprehensive, circling, co-
matose, depressed, sedated, restless, panting, convulsions, and aggression.
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(E) Integument: Alopecia, haircoat condition, status of hydration, pru-
ritus, and erythema.
(F) Gastrointestinal: Consistency of stools, propulsive diarrhea, hyper-
active gut, abdominal muscle tenseness, and vomiting.
(G) Cardiovascular: Heart rate, heart rhythm, and color of mucous
membranes.
(H) Appetite/general health: Body weight, feed consumption, and
water consumption.
(I) Respiratory: Respirations/minute, dyspnea, respiratory sounds,
color of mucous membranes, nasal discharge, apnea.
(v) Individual body weights should be measured twice during the ac-
climation period, with the second measurement being taken immediately
prior to the initiation of treatment, and on day-7 and day-14 of the obser-
vation period.
(vi) Individual food consumption should be measured on a daily basis.
(vii) The time of death should be reported for animals dying or sac-
rificed moribund. Gross necropsies should be conducted to determine the
cause of death; abnormal organs/tissues should be examined histologically.
(viii) Clinical pathology should be assessed prior to treatment, 24 h
posttreatment and, if altered, on day-7. Clinical pathology examinations
are required to determine the possibility of a treatment-related effect on
hematology and clinical chemistry parameters, regardless of whether or
not clinical signs of toxicity are evident. The following examinations
should be made:
(A) Hematology; Hemoglobin, mean corpuscular volume, hemato-
crit.rnean corpuscular hemoglobin, red blood cell count, mean corpuscular
hemoglobin concentration, white blood cell count, white blood cell dif-
ferential count, prothrombin time, and activated partial thromboplastic
time.
(B) Clinical chemistry. Glucose, creatinine, sodium, chloride, potas-
sium, phosphorus, total protein, albumin, globulin, calcium, blood urea ni-
trogen, alkaline phosphatase, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and total and direct bilirubin.
(C) If the compound is a known cholinesterase inhibitor, plasma, and
red cell cholinesterase should be examined pretreatment, and at 6 to 8,
24, and 48 h, posttreatment.
(h) Data and reporting—(1) Treatment of results, (i) Data should
be summarized in tabular form, showing, for each test group:
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(A) The number, age and sex of the animals at the start of the study.
Results from adult and pediatnc animals should be separated
(B) The number of animals displaying clinical signs of toxicity. a
description of the observations and the time of onset.
(C) Individual and group mean values for body weight, food con-
sumption, and clinical pathology measurements.
(D) Time and cause of death, if known, for animals which died or
were sacrificed in a moribund condition.
(ii) All observed results should be evaluated by an appropriate statis-
tical method that was selected during the design of the study.
(2) Evaluation of study results. The evaluation should include the
relationship between the dose of the test substance and the presence or
absence, the incidence and severity, of abnormalities, including behavioral
abnormalities, clinical abnormalities, body weight changes, effects on mor-
tality and cause of death, identified target organ, and any other general
or specific acute toxic effect.
(3) Test report. In addition to information required under 40 CFR
part 792, subpart J, and 40 CFR part 160, subpart J, the test report sum-
mary should include the following information.
(i) Toxic response and other effects data by sex and dose.
(ii) Species and breed used.
(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination.
(B) Time of observation of each abnormal sign and its subsequent
course.
(C) Food consumption.
(D) Body weight data.
(E) Hematology tests employed and the results.
(F) Clinical chemistry tests employed and the results.
(G) Necropsy findings on animals that died or were sacrificed in a
moribund condition.
(H) Detailed description of histological findings on animals that died
or were sacrificed in a moribund condition.
(I) Statistical treatment of results.
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(J) Other observations and/or findings.
(i) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Hoskins, J.D. Veterinary- Pediatrics. Dogs and Cats From Birth
to Six Months. Saunders, Philadelphia (1990).
(2) Jones, B.D. Liver and Pancreatic Disease. Presented to D.C.
Academy of Veterinary Medicine, Fairfax, VA, June 9, 1994.
(3) Target Animal Safety Guidelines for New Animal Drugs. Prepared
by the Office of New Animal Drug Evaluation, Center for Veterinary Med-
icine, Food and Drug Administration, June 1989.
8
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&EPA
United States
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101) •
EPA712-C-98-244
August 1998
Health Effects Test
Guidelines
OPPT.S 870.7485
Metabolism and
Pharmacokineticb
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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. 136, et seq.).
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
Guidelines."
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OPPTS 870.7485 Metabolism and pharmacokinetics
(a) Scope—(I) 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 materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798.7485 Metabolism and
pharmacokinetics; OPP 85-1 Metabolism Study (Pesticide Assessment
Guidelines, Subdivision F - Hazard Evaluation: Human and Domestic Ani-
mals, EPA report 540/09-82-025, October 1982); and OECD Guideline
417 Toxicokinetics.
(b) Purpose. (1) Testing of the disposition of a test substance is de-
signed to obtain adequate information on its absorption, distribution, bio-
transformation, and excretion and to aid in understanding the mechanism
of toxicity. Basic pharmacokinetic parameters determined from these stud-
ies will also provide information on the potential for accumulation of the
test substance in tissues and/or organs and the potential for induction of
biotransformation as a result of exposure to the test substance. These data
can be used to assess the adequacy and relevance of the extrapolation of
animal toxicity data (particularly chronic toxicity and/or carcinogenicity
data) to human risk assessment.
(2) Metabolism data can also be used to assist in determining whether
animal toxicity studies have adequately addressed any toxicity concerns
arising from exposure to plant metabolites, and in the setting of tolerances,
if any, for those metabolites in raw agricultural commodities.
(c) Definitions. The following definitions apply to this guideline:
Metabolism (biotransformation) is the sum of the processes by which
a foreign chemical is subjected to chemical change by living organisms.
LOEL is the lowest observable effect level.
NOEL is the no observable effect level.
Pharmacokinetics is the quantitation and determination of the time
course and dose dependency of the absorption, distribution, biotrans-
formation, and excretion of chemicals.
(d) Good laboratory practice standards. The pharmacokinetics and
metabolism tests outlined in this guideline are to conform to the laboratory
practices stipulated in 40 CFR parts 160 and 792 (Good Laboratory Prac-
tice Standards).
(e) Test Procedures. Test procedures presented below utilize a tier
system to minimize the use of resources and to allow flexibility in the
conduct of metabolism studies. The proposed tier system will consist of
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a basic data set (Tier 1) and additional studies (Tier 2) which may be
requested based upon the existing toxicology data base and/or the results
of Tier 1 testing which are found to impact upon the risk assessment proc-
ess. For Tier 1 testing, the oral route will typically be required, however,
if the use pattern results in other types of exposure, other routes (dermal
and/or inhalation) may be required for initial testing of the disposition of
a chemical substance. The registrant should justify the route of exposure
to the Agency. Complete descriptions of the test procedures for these other
routes of exposure can be found under paragraph (i) of this guideline. Ex-
cept in unusual circumstances, the tiered approach to metabolism testing
should apply to all listed routes of exposure.
(1) Pilot studies. The use of pilot studies is recommended and en-
couraged for the selection of experimental conditions for the
pharmacokinetics and metabolism studies (mass balance, analytical proce-
dures, dose-finding, excretion of CCb, etc.).
(2) Animal selection—(i) Species. The rat shall normally be used
for testing because it has been used extensively for metabolic and toxi-
cological studies. The use of other or additional species may be required
if critical toxicology studies demonstrate evidence of significant toxicity
in these species or if metabolism is shown to be more relevant to humans
in the test species.
(ii) Strain. Adult animals of the strain used or proposed to be used
for the determination of adverse health effects associated with the test sub-
stance.
(3) Material to be tested—(i) Test substance. (A) A radiolabeled
test substance using 14C should be used for all material balance and
metabolite identification aspects of the study. Other radioactive and stable
isotopes may be used, particularly if the element is responsible for or is
a part of the toxic portion of the compound. If it can be demonstrated
that the material balance and metabolite identification requirements can
be met using unlabeled test substance, then radiolabeled compound need
not be used. If possible, the radiolabel should be located in a core portion
of the molecule which is metabolically stable (it is not exchangeable, is
not removed metabolically as CCh, and does not become part of the one-
carbon pool of the organism). Labeling of multiple sites of the molecule
may be necessary to follow the metabolic fate of the compound.
(B) The label should follow the test compound and/or its major
metabolites until excreted. The radiopurity of the radioactive test substance
shall be the highest attainable for a particular test substance (ideally it
should be greater than 95 percent) and reasonable effort should be made
to identify impurities present at or above 2 percent. The purity, along with
the identity of major impurities which have been identified, shall be re-
ported. For other segments of the study, nonradioactive test substance may
-------
be used if it can be demonstrated that the analytical specificity and sen-
sitivity of the method used with nonradioactive test substance is equal to
or greater than that which could be obtained with the radiolabeled test
substance. The radioactive and nonradioactive test substances shall be ana-
lyzed using an appropriate method to establish purity and identity. Addi-
tional guidance will be provided in chemical specific test rules to assist
in the definition and specifications of test substances composed of mixtures
and methods for determination of purity.
(ii) Administration of test substance. Test substance should be dis-
solved or suspended homogeneously in a vehicle usually employed for
acute administration. A rationale for the choice of vehicle should be pro-
vided. The customary method of administration will be by oral gavage;
however, administration by gelatin capsule or as a dietary mixture may
be advantageous in specific situations. Verification of the actual dose ad-
ministered to each animal should be provided.
(4) Tier testing, (i) The multiplicity of metabolic parameters that im-
pact the outcome of toxicological evaluations preclude the use of a univer-
sal study design for routine toxicological evaluation of a test substance.
The usefulness of a particular study design depends upon the biological
activity of a compound and circumstances of exposure. For these reasons,
a tiered system is proposed for evaluation of the metabolism/kinetic prop-
erties of a test substance.
(ii) The first tier data set is a definitive study by the appropriate route
of exposure conducted in male rats to determine the routes and rate of
excretion and to identify excreted metabolites. First tier data will also pro-
vide basic information for additional testing (Tier 2) if such testing is con-
sidered necessary. In the majority of cases, Tier 1 data are expected to
satisfy regulatory requirements for biotransformation and pharmacokinetic
data on test chemicals.
(iii) Second tier testing describes a variety of metabolism/kinetic ex-
periments which address specific questions based on the existing toxi-
cology data base and/or those results of Tier 1 testing impacting signifi-
cantly on the risk assessment process. For conduct of these studies, indi-
vidualized protocols may be necessary. Protocols for these studies, if re-
quired, can be developed as a cooperative effort between Agency and in-
dustry scientists.
(f) Tier 1 data requirements (minimum data set). At this initial
level of testing, biotransformation and pharmacokinetic data from a single
low dose group will be required. This study will determine the rate and
routes of excretion and the type of metabolites generated.
(1) Number and sex of animals. A minimum of four male young
adult animals will be required for Tier 1 testing. The use of both sexes
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may be required in cases where there is evidence to support significant
sex-related differences in toxicity.
(2) Dose selection, (i) A single dose is required for each route of
exposure. The dose should be nontoxic, but high enough to allow for
metabolite identification in excreta. If no other toxicity data are available
for selection of the low dose, a dose identified as a fraction of the LD50
(as determined from acute toxicity studies) may be used. The magnitude
of the dose used in Tier 1 studies should be justified in the final report.
(ii) For test substances of low toxicity a maximum dose of
1,000 mg/kg should be used; chemical-specific considerations may neces-
sitate a higher maximum dose and will be addressed in specific test rules.
(3) Measurements—(i) Excretion. (A) Data obtained from this sec-
tion (percent recovery of administered dose from urine, feces, and expired
air) will be used to determine the rate and extent of excretion of test chem-
ical, to assist in establishing mass balance, and will be used in conjunction
with pharmacokinetic parameters to determine the extent of absorption.
The quantities of radioactivity eliminated in the urine, feces, and expired
air shall be determined separately at appropriate time intervals.
(B) If a pilot study has shown that no significant amount of radio-
activity is excreted in expired air, then expired air need not be collected
in the definitive study.
(C) Each animal is to be placed in a separate metabolic unit for col-
lection of excreta (urine, feces and expired air). At the end of each collec-
tion period, the metabolic units are to be rinsed with appropriate solvent
to ensure maximum recovery of radiolabel. Excreta collection shall be ter-
minated at 7 days, or after at least 90 percent of the administered dose
has been recovered, whichever occurs first. The total quantities of radio-
activity in urine are to be determined at 6, 12, and 24 h on day 1 of
collection, and daily thereafter until study termination, unless pilot studies
suggest alternate or additional time points for collection. The total quan-
tities of radioactivity in feces should be determined on a daily basis begin-
ning at 24 h post-dose, and daily thereafter until study termination. The
collection of CCb and other volatile materials may be discontinued when
less than 1 percent of the administered dose is found in the exhaled air
during a 24-h collection period.
(ii) Tissue distribution. At the termination of the Tier 1 study, the
following tissues should be collected and stored frozen: Liver, fat, gastro-
intestinal tract, kidney, spleen, whole blood, and residual carcass. If it is
determined that a significant amount of the administered dose is unac-
counted for in the excreta, then data on the percent of the total (free and
bound) radioactive dose in these tissues as well as residual carcass will
be requested. Additional tissues shall be included if there is evidence of
target organ toxicity from subchronic or chronic toxicity studies. For other
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routes of exposure, specific tissues may also be required, such as lungs
m inhalation studies and skin in dermal studies. Certain techniques cur-
rently at various stages of development, e.g. quantitative whole-body
autoradiography, may prove useful in determining if a test substance con-
centrates in certain organs or in determining a specific pattern of distribu-
tion within a given tissue. The use of such techniques is encouraged, but
not required, and may be employed to limit the number of tissues collected
to those shown to contain a measurable amount of radioactivity.
(iii) Metabolism. Excreta shall be collected for identification and
quantitation of unchanged test substance and metabolites as described
under paragraph (f)(3)(i) of this guideline. Pooling of excreta to facilitate
metabolite identification within a given dose group is acceptable. Profiling
of metabolites from each time period is recommended. However, if lack
of sample and/or radioactivity precludes this, pooling of urine as well as
pooling of feces across several time points is acceptable. Appropriate qual-
itative and quantitative methods shall be used to assay urine, feces, and
expired air from treated animals. Reasonable efforts should be made to
identify all metabolites present at 5% or greater of the administered dose
and to provide a metabolic scheme for the test chemical. Compounds
which have been characterized in excreta as comprising 5 percent or great-
er of the administered dose should be identified. If identification at this
level is not possible, a justification/explanation should be provided in the
final report. Identification of metabolites representing less than 5 percent
of the administered dose might be requested if such data are needed for
risk assessment of the test chemical. Structural confirmation should be pro-
vided whenever possible. Validation of the methods used in metabolite
identification should be included.
(g) Tier 2 data requirements. Studies at the Tier 2 level are designed
to answer questions about the disposition of test chemicals based on the
existing toxicology data base and/or results of Tier 1 testing which may
have a significant impact on the risk assessment for the test chemical.
Such studies may address questions regarding absorption, persistence, or
distribution of the test chemical, or a definitive alteration in the metabolic
profile occurring with dose which may be of lexicological concern. At
the Tier 2 level, only those studies which address a specific concern are
required, and will be conducted according to mutual agreement between
the registrant and the Agency. Flexibility will be allowed in the design
of specific experiments as warranted by technological advances in this
field.
(1) Absorption, (i) If the extent of absorption cannot be established
from Tier 1 studies, or where greater than 20 percent of the administered
dose is present in feces, a study to determine the extent of absorption
will be required. This can be accomplished either through intravenous ad-
ministration of test material and measurement of radioactivity in excreta
-------
or after oral administration of test material and measurement of radioactiv-
ity in bile.
(ii) For the intravenous study, a single dose (not to exceed the oral
dose used in Tier 1) of test chemical using an appropriate vehicle should
be administered in a suitable volume (e.g. 1 rnL/kg) at a suitable site to
at least three male rats (both sexes might be used if warranted). The dis-
position of the test chemical should be monitored for oral dosing as out-
lined in paragraph (f)(3)(i) of this guideline. Metabolite identification will
not be required for this study.
(iii) If a biliary excretion study is chosen the oral route of administra-
tion may be requested. In this study, the bile ducts of at least three male
rats (or of both sexes, if warranted) should be appropriately cannulated
and a single dose of the test chemical should be administered to these
rats. Following administration of the test chemical, excretion of radioactiv-
ity in bile should be monitored as long as necessary to determine if a
significant percentage of the administered dose is excreted via this route.
(2) Tissue distribution time course, (i) A time course of tissue dis-
tribution in selected tissues may be required to aid in the determination
of a possible mode of toxic action. This concern may arise from evidence
of extended half-life or possible accumulation of radioactivity in specific
tissues. The selection of tissues for this type of study will be based upon
available evidence of target organ toxicity and/or carcinogenicity, and the
number of time points required will be based upon pharmacokinetic infor-
mation obtained from Tier 1 data. Flexibility will be allowed in the selec-
tion of time points to be studied.
(ii) For this type of study, three rats per time point will be adminis-
tered an appropriate oral dose of test chemical, and the time course of
distribution monitored in selected tissues. Only one sex may be required,
unless target organ toxicity is observed in sex-specific organs. Assessment
of tissue distribution will be made using appropriate techniques for assess-
ment of total amount distributed to tissue and for assessment of metabolite
distribution.
(3) Plasma kinetics. The purpose of this experiment is to obtain esti-
mates of basic pharmacokinetic parameters (half-life, volume of distribu-
tion, absorption rate constant, area under the curve) for the test substance.
Kinetic data may be required if the data can be used to resolve issues
about bioavailability and to clarify whether clearance is saturated in a
dose-dependent fashion. For this experiment a minimum of three rats per
group is required. At least two doses will be required, usually the NOEL
and LOEL from the critical toxicology study. Following administration
of test substance, samples should be obtained from each animal at suitable
time points appropriate sampling methodology. Total radioactivity present
(or total amount of chemical, for nonradioactive materials) should be ana-
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lyzed in whole blood and plasma using appropriate methods, and the
blood/plasma ratio should be calculated.
(4) Induction, (i) Studies addressing possible induction of biotrans-
formation may be requested under one or more of the following conditions:
(A) Available evidence indicates a relationship between induced me-
tabolism and enhanced toxicity.
(B) The available toxicity data indicate a nonlinear relationship be-
tween dose and metabolism.
(C) The results of Tier 1 metabolite identification studies show identi-
fication of a potentially toxic metabolite.
(D) Induction can plausibly be invoked as a factor in such effects
where status may depend on the level of inducible enzymes present. Sev-
eral in vivo and in vitro methods are available for assessment of enzyme
induction, and the experiments which best address the issue at hand can
be determined between Agency and industry scientists. If induction is dem-
onstrated, the relationship of this phenomenon to toxicity observed from
subchronic and/or chronic toxicity studies will need to be addressed.
(iii) If lexicologically significant alterations in the metabolic profile
of the test chemical are observed through either in vitro or in vivo experi-
ments, characterization of the enzyme(s) involved (for example, Phase I
enzymes such as isozymes of the Cytochrome P450-dependent mono-
oxygenase system, Phase II enzymes such as isozymes of sulfotransferase
or uridine diphosphate glucuronosyl transferase, or any other relevant en-
zymes) may be requested. This information will help establish the rel-
evance of the involved enzyme(s) to human risk, as it is known that certain
isozymes are present in animal species which are not present in humans,
and vice versa.
(5) Physiologically-based modeling. Traditional methods of model-
ing have been used to determine kinetic parameters associated with drug
and xenobiotic disposition, but have assumed a purely mathematical con-
struct of mammalian organisms in their operation. On the other hand, more
recent models which take into account the physiological processes of the
animal have been used with success in defining biological determinants
of chemical disposition as well as the relationship between tissue dose
and tissue response. These so-called physiologically-based models, also
allow for cross-species extrapolation which is often necessary in the risk-
assessment process. The use of physiologically-based modeling as an ex-
perimental tool for addressing specific issues related to biotransformation
and pharmacokinetics of a test substance is encouraged. Information as
derived from physiologically-based modeling experiments may aid in the
comparison of biotransformation and pharmacokinetics of a test substance
between animal species and humans, and in the assessment of risk under
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specific exposure conditions. At the discretion of the Agency, or by mutual
agreement, results of physiologically based pharmacokmetic (PBPK) stud-
ies with parent compound may be submitted in lieu of other studies, if
it is determined that such data would provide adequate information to sat-
isfy this guideline.
(h) Reporting of study results. In addition to the reporting require-
ments specified in 40 CFR part 792, subpart J, the completed study (Tier
1 or Tier 2) should be presented in the following format:
(1) Title/Cover Page. Title page and additional requirements (require-
ments for data submission, good laboratory practice, statements of data
confidentiality claims and quality assurance) if relevant to the study report,
should precede the content of the study formatted below. These require-
ments are to be found in 40 CFR parts 158 and 160 or parts 790, 792,
and 799.
(2) Table of Contents. A concise listing is to precede the body of
the report, containing all essential elements of the study and the page and
table number where the element is located in the final report of the study.
Essential elements of the Table of Contents should include a summary,
an introduction, the materials and methods section, results, discussion/ con-
clusions, references, tables, figures, appendices, and key subsections as
deemed appropriate. The Table of Contents should include the page num-
ber of each of these elements.
(3) Body of the report. The body of the report shall include informa-
tion required under this guideline, organized into sections and paragraphs
as follows:
(i) Summary. This section of the study report is to contain a summary
and analysis of the test results and a statement of the conclusions drawn
from the analysis. This section should highlight the nature and magnitude
of metabolites, tissue residue, rate of clearance, bioaccumulation potential,
sex differences, etc. The summary should be presented in sufficient detail
to permit independent evaluation of the findings.
(ii) Introduction. This section of the report should include the objec-
tives of the study, guideline references, regulatory history, if any, and a
rationale.
(iii) Materials and methods. This section of the report is to include
detailed descriptions of all elements including:
(A) Test substance. (1) This section should include identification of
the test substance—chemical name, molecular structure, qualitative and
quantitative determination of its chemical composition, and type and quan-
tities of any impurities whenever possible.
8
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(2) This section should also include information on physical prop-
erties including physical state, color, gross solubility and/or partition coef-
ficient, and stability.
(3) The type or description of any vehicle, diluents, suspending
agents, and emulsifiers or other materials used in administering the test
substance should be stated.
(4) If the test substance is radiolabeled, information on the following
should be included in this subsection: The type of radionuclide, position
of label, specific activity, and radiopurity.
(B) Test animals. This section should include information on the test
animals, including: Species, strain, age at study initiation, sex, body
weight, health status, and animal husbandry.
(C) Methods. This subsection should include details of the study de-
sign and methodology used. It should include a description of:
(7) How the dosing solution was prepared and the type of solvent,
if any, used.
(2) Number of treatment groups and number of animals per group.
(3) Dosage levels and volume.
(4) Route of administration.
(5) Frequency of dosing.
(6) Fasting period (if used).
(7) Total radioactivity per animal.
(£) Animal handling.
(9) Sample collection.
(10) Sample handling.
(77) Analytical methods used for separation.
(72) Quantitation and identification of metabolites.
(13) Other experimental measurements and procedures employed (in-
cluding validation of test methods for metabolite analysis).
(D) Statistical analysis. If statistical analysis is used to analyze the
study findings, then sufficient information on the method of analysis and
the computer program employed should be included so that an independent
reviewer/statistician can reevaluate and reconstruct the analysis. Presen-
tation of models should include a full description of the model to allow
independent reconstruction and validation of the model.
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(iv) Results. All data should be summarized and tabulated with appro-
priate statistical evaluation and placed in the text of this section. Radio-
activity counting data should be summarized and presented as appropriate
for the study, typically as disintegrations per minute and microgram or
milligram equivalents, although other units may be used. Graphic illustra-
tions of the findings, reproduction of representative chromatographic and
spectrometric data, and proposed metabolic pathways and molecular struc-
ture of metabolites should be included in this section. In addition the fol-
lowing information is to be included in this section if applicable:
(A) Justification for modification of exposure conditions, if applica-
ble.
(B) Justification for selection of dose levels for pharmacokmetic and
metabolism studies.
(C) Description of pilot studies used in the experimental design of
the pharmacokinetic and metabolism studies, if applicable.
(D) Quantity and percent recovery of radioactivity in urine, feces,
and expired air, as appropriate. For dermal studies, include recovery data
for treated skin, skin washes, and residual radioactivity in the covering
apparatus and metabolic unit as well as results of the dermal washing
study.
(E) Tissue distribution reported as percent of administered dose and
microgram equivalents per gram of tissue.
(F) Material balance developed from each study involving the assay
of body tissues and excreta.
(G) Plasma levels and pharmacokinetic parameters after administra-
tion by the relevant routes of exposure.
(H) Rate and extent of absorption of the test substance after adminis-
tration by the relevant routes of exposure.
(I) Quantities of the test substance and metabolites (reported as per-
cent of the administered dose) collected in excreta.
(J) Individual animal data.
(v) Discussion and conclusions. (A) In this section the authors)
should:
(7) Provide a plausible explanation of the metabolic pathway for the
test chemical.
(2) Emphasize species and sex differences whenever possible.
10
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(J) Discuss the nature and magnitude of metabolites, rates of clear-
ance, bioaccumulation potential, and level of tissue residues as appropriate.
(B) The author(s) should be able to derive a concise conclusion that
can be supported by the findings of the study.
(vi) Optional sections. The authors may include additional sections
such as appendices, bibliography, tables, etc.
(i) Alternate routes of exposure for Tier 1 testing—(1) Dermal—
(i) Dermal treatment. One (or more if needed) dose levels of the test
substance shall be used in the dermal portion of the study. The low dose
level should be selected in accordance with paragraph (0(2) of this guide-
line. The dermal doses shall be dissolved, if necessary, in a suitable vehicle
and applied in a volume adequate to deliver the doses. Shortly before test-
ing, fur is to be clipped from the dorsal area of the trunk of the test ani-
mals. Shaving may be employed, but it should be carried out approxi-
mately 24 h before the test. When clipping or shaving the fur, care should
be taken to avoid abrading the skin, which could alter its permeability.
Approximately 10 percent of the body surface should be cleared for appli-
cation of the test substance. With highly toxic substances, the surface area
covered may be less than approximately 10 percent, but as much of the
area as possible is to be covered with a thin and uniform film. The same
nominal treatment surface area shall be used for all dermal test groups.
The dosed areas are to be protected with a suitable covering which is
secured in place. The animals shall be housed separately.
(ii) Dermal washing study. (A) A washing experiment is to be con-
ducted to assess the removal of the applied dose of the test substance
by washing the treated skin area with a mild soap and water. A single
dose shall be applied to two animals in accordance with paragraph (f)(2)
of this guideline. After application (2 to 5 min) the treated areas of the
animals shall be washed with a mild soap and water. The amounts of test
substance recovered in the washes shall be determined to assess the effec-
tiveness of removal by washing.
(B) Unless precluded by corrosiveness, the test substance shall be ap-
plied and kept on the skin for a minimum of 6 h. At the time of removal
of the covering, the treated area shall be washed following the procedure
as outlined in the dermal washing study. Both the covering and the washes
shall be analyzed for residual test substance. At the termination of the
studies, each animal shall be sacrificed and the treated skin removed. An
appropriate section of treated skin shall be analyzed to determine residual
radioactivity.
(2) Inhalation. A single (or more if needed) concentration of test
substance shall be used in this portion of the study. The concentration
should be selected in accordance with paragraph (f)(2) of this guideline.
Inhalation treatments are to be conducted using a "nose-cone" or "head-
11
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only" apparatus to prevent absorption by alternate routes of exposure. If
other inhalation exposure conditions are proposed for use in a chemical-
specific test rule, justification for the modification must be documented.
A single exposure over a defined period shall be used for each group—
a typical exposure is 4—6 h.
12
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rxEPA
United States
Environmental Protection
Agency
Prevention. Pesticides
and Toxic Substances
(7101)
EPA712-C-98-350
August 1998
Health Effects Test
Guidelines
OPPTS 870.7600
Dermal Penetration
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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 arid 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 purpos'e 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. 136, 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
Guidelines."
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OPPTS 870.7600 Dermal penetration.
(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. This guideline was developed in the Office of Pes-
ticide Programs of and published as OPP 85-3 Dermal Absorption Studies
of Pesticides (Pesticide Assessment Guidelines, Subdivision F—Hazard
Evaluation; Human and Domestic Animals) EPA report 540/09-82-025,
1982. Dermal absorption studies are not routinely required.
(b) Discussion. (1) Dermal absorption studies are complex kinetic
studies which, in and of themselves, provide no information on the biologi-
cal activity (toxicity) of a compound. Dermal absorption studies may be
required on an individual basis for compounds having a serious toxic effect
identified by oral or inhalation studies, for which a significant route of
human exposure is dermal and for which the assumption of 100 percent
dermal absorption does not produce an adequate margin of exposure
(MOE). That is, a risk assessment must be performed to determine the
need for a dermal absorption study. Dermal absorption studies cannot sub-
stitute for general dermal toxicity studies of up to 90 days of dosing
(OPPTS 870.3200 and OPPTS 870.3250), which must be performed by
the dermal route to assess direct toxicity to the skin (and systemic toxicity
as well).
(2) It is recommended that studies showing significant lexicological
effects, first identified by the oral or inhalation route, be repeated by the
dermal route where practical, rather than performing a dermal absorption
study. Examples are studies of organ, system, or physiological process spe-
cific short term toxicity such as developmental toxicity, immunotoxicity,
or neurotoxicity studies. Low-effect and no-effect doses from such studies
can be used directly in the calculation of and MOE without the necessity
of kinetic evaluations. This approach is preferred since the dermal route
may produce differences in distribution, metabolism, storage, and excretion
from the oral or inhalation routes which can produce qualitative as well
as quantitative differences in the toxicity of a compound. Such differences
are not easily identified by kinetic or metabolism studies and can have
significant effects on the systemic toxic response.
(3) Information on dermal exposure is necessary in order to determine
doses and durations of exposure to be evaluated in the dermal absorption
study. It is expected that this information will have been gathered in order
to perform the risk assessment and to make the basic decision as to wheth-
er the study is required.
(4) An oral kinetic study in the rat is strongly recommended in order
to allow full utilization of the dermal kinetic study in risk assessment.
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It is recommended that the oral kinetic study be performed before the der-
mal absorption study is undertaken. The study must determine the portion
(percent) of the oral dose that is absorbed and the rate of absorption, the
rate of tissue distribution, the routes and rate of excretion and be sufficient
to allow half-life determinations for each of the rates.
(c) Purpose. Data from dermal absorption studies allow the Agency
to make risk determinations in cases where the toxic effect has been deter-
mined by the oral or inhalation route in the experimental animal and the
exposure to humans is by the dermal route. Such determinations may vary
in degree of complexity ranging from the 100 percent assumption used
to justify performing the dermal absorption study to a complete kinetic
analysis. A complete kinetic analysis essentially enables an investigator
to convert oral or inhalation low-effect and no-effect doses into dermal
low-effect and no-effect doses, thus allowing the calculation of an MOE
or risk for systemic toxic effects which have not been or cannot be tested
practically by the dermal route. The analysis can be performed in several
ways. The first involves calculating the maximum systemic doses produced
by the oral (or inhalation) no-effect and low-effect doses and calculating
dermal doses that will produce the same maximum systemic doses. The
calculated dermal no-effect and low-effect values are then compared with
the dermal exposures from the risk assessment. In a second method the
dermal exposure is converted to a maximum systemic dose which is com-
pared with the maximum systemic doses equivalent to the oral no-effect
and low-effect doses.
(d) Material to be tested—(1) Compound. The compound should
be of known chemical purity and radiolabeled, usually with 14C, in a posi-
tion which is part of the "core" of the compound. The label should follow
the compound and its major metabolites until excreted. To the extent prac-
tical, the label should not be exchangable nor should it be metabolically
removed to C02 or become part of the one-carbon pool of the organism.
Other radioactive isotopes such as 35S, 36C1, and 113Sn or stable isotopes
such as 15N and 18O may be used, particularly if the element is responsible
for, or is a part of, the toxic portion of the compound. Labeled compound
may not be required if a sufficiently selective and sensitive physical/chemi-
cal test for identifying the compound is used. The specificity and sensitiv-
ity of the test must be demonstrated in the biological systems (organs,
tissues, and body fluids) being analyzed and a report of this validation
must be included in the report of the dermal absorption study. Use of
a physical/chemical test may require the use of control (untreated) animals
if background interference with the procedure is observed.
(2) Vehicle/solvent. The vehicle system used should duplicate that
under which field exposure occurs. The basic vehicle is usually the mate-
rial used in the commercial formulation (formulation blank). Dilutions are
made with the field vehicle, usually water, to produce a solution or suspen-
sion. In cases where exposure is to the chemical and not a formulation
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or dilution thereof, a neutral suspending agent which does not interact with
the test chemical or affect the permeability of the skin, such as
carboxymethylcellulose. may be used. However, organic solvents or spe-
cial solubilizing/suspending agents must not be used. Formulations applied
dry (dusts, granulars, etc.) should be moistened with water to assist in
the application a quantitative dose. This moisture mimics to some measure
the presence of perspiration in field use.
(3) Dose preparation. Dose solutions/suspensions should be prepared
so that sufficient quantities of radiolabel are present in each dose to allow
sufficient sensitivity for that dose. The sensitivity required for a dose de-
pends upon how small a quantity of the test compound is considered nec-
essary to produce a toxic effect. If it is not possible to detect a significantly
small fraction of that quantity, it will not be possible to determine if the
quantity absorbed allows an acceptable MOE or produces a lexicologically
acceptable risk. The dosing solutions/suspensions may be obtained by any
practical and reliable method that the laboratory can devise. Predictability
of dose preparation is most important—it is critical to know the actual
concentrations obtained and that the dosing material is homogeneous so
that the actual dose administered is known. It is not critical that the nomi-
nal dose be precisely administered.
(e) Test procedure—(1) Variation of procedure. The basic study
described is designed to cover the entire range of doses and durations of
exposure expected for a pesticide designed for a wide variety of uses. It
is frequently possible to cover the use pattern at risk for a particular pes-
ticide with a lesser number of doses and durations of exposure. In the
case of pesticides having a limited pattern of use, Registrants may, after
consultation with the Agency, perform only those doses and durations of
exposure that are applicable to the use pattern which is being considered
for risk assessment.
(2) Test animal—(i) Species and strafti. The laboratory rat is the
required species because this guideline has been designed and validated
for the rat. Other animal species have been considered and rejected. It
is recommended that the rats be of the same strain as those used for the
metabolism and toxicology studies on the test compound. The rat is not
intended as a model of absorption through the human skin but rather as
a test system for dermal absorption. It is possible to use the absorption
rates determined as a modest overestimate of human dermal absorption
or to perform a kinetic evaluation as described in paragraph (c) of this
guideline for risk assessment purposes.
(ii) Age. Young adult animals should be used.
(iii) Sex. The male rat should be used. The choice of a single sex
allows consistency and comparison from study to study.
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(iv) Numbers. A total of 24 animals is used at each dose level. This
is based on four animals per dose per exposure duration, the minimal ex-
perimental unit. Studies using fewer exposure durations will use fewer ani-
mals per dose but the same number (four) per duration of exposure.
(3) Dose levels and dose selection, (i) Four doses are recommended;
at least three doses should be used. Experience has shown that absorption
is not dose-linear—as the dose per unit area increases the flux increases
to a lesser degree. Some compounds have shown saturation of the absorp-
tion process (no increase of flux with dose). Thus, the quantity and rate
of absorption determined at one dose is generally not valid at a dose which
is greater or less by more than approximately one-half of a log unit. Com-
pounds that are caustic or are dermal irritants should be tested only at
doses which do not show such effects. Direct dermal toxicity is considered
an unacceptable risk and compounds which show such effects are regulated
using the low-effect and no-effect doses for direct dermal toxicity.
(ii) Doses should be at log intervals (i.e. 1.0, 0.1, 0.01 and
0.001 mg/cm2) and span the range of doses expected in field exposure.
The numbers are examples—the interval is the important factor. The maxi-
mum practical dose is on the order of 1 mg/cm2—larger doses tend to
fall off the skin or exceed saturation of the absorption process. When only
three doses are qiven the highest dose should be on the order of
0.1 mg/cm2. When different forms of the compound are tested (salts,
esters, etc.), comparative doses should be equimolar to the doses of the
parent compound.
(iii) Doses must be determined on the basis of quantity per unit area
of exposed skin (expressed as milligrams per square centimeter, not on
the basis of quantity per unit of body weight (milligrams per kilogram).
In this study the skin is being dosed to determine its permeability to the
test compound which is dependent on the dose to the skin. The test animal
per se is not being dosed.
(iv) The maximum dose volume should not exceed 10 uL/cm2. Larger
volumes of liquid have been found to flow on the skin, and produce uneven
dose distribution on the dosed area.
(4) Duration of exposure. In the full study four animals per dose
are exposed for durations of 0.5, 1, 2, 4, 10, and 24 h. For an abbreviated
study, designed for a single exposure scenario, the recommended minimal
durations of exposure are 1, 10, and 24 h. The evaluation with time is
recommended since experience has shown that skin deposition (wash-re-
sistant) and penetration are rarely linear with time, the greatest variation
occurring in the first 1 or 2 h of exposure.
(5) Administration of the test substance—(i) Animal preparation.
The back and shoulders of the rats are clipped free of hair and the area
wiped with acetone 24 h prior to dosing. A soap and water wash may
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be used but care must be taken to nnse the area throughly to remove resid-
ual soap. A soap and water wash is generally harder on the skin than
an acetone wipe. Clipping and wipe produce a standardized skin condition
at the time of dosing. Use of a shorter interval between skin wipe and
dosing has been shown to increase dermal penetration. Animals showing
damage to the skin at the tune of dosing should not be used.
(ii) Dose application. The-measured dose of the compound is applied
to a measured area of the rat's skin of no less than 10 cm2. Because most
dose forms are suspensions, this minimal area is necessary for even spread-
ing. The material is spread evenly until a film is formed over the applica-
tion site. The spreader should be checked for retention of material and
the actual dose applied should be determined by subtraction of retained
material from the total dose. Particular attention must be paid to determin-
ing the actual dose applied.
(iii) Site protection. The application area must be covered to prevent
loss of compound through falling off, being nibbed off, or being licked.
The cover must allow air circulation over the application site to allow
normal evaporation of surface water from the skin. A combination cover
(protective device) consisting of a spacer (a rubber, plastic or glass rectan-
gle, square, or ring glued to the skin) to outline the application site and
a filter paper or gauze cover glued to it is recommended. The spacer should
be impervious to the test solution. The paper or gauze should not be in
direct contact with the test material or the skin. See paragraph (g)(5) of
this guideline for information on volatile chemicals.
(6) Animal processing. The treated animals are placed individually
in metabolism cages. Expired air should be collected if a metabolism study
shows that label is expired. Total urine and feces are collected separately
(a single collection for the entire duration of exposure). At the exposure
intervals (0.5, 1, 2, 4, 10 and 24 h for the standard study) four animals
per dose are anesthetized, exposed skin is washed with a mild soap/deter-
gent solution followed by several water rinses, to mimic human washing,
and the protective device is removed. The skin at the exposure site must
be washed before it is removed from the animal; contact of the wash solu-
tion with the cut edge or undersurface of the skin has been shown to
produce artifactual binding of test compound. Liquid detergent designed
for dishwashing is suggested for the wash solution. The animals are killed
and a blood sample collected from the heart or post cava. Residual urine
is collected from the bladder and added to the collected urine. The exposed
skin, selected organs (if part of the experimental design), and the remain-
der of the animal (carcass) are collected and prepared for determination
of the quantity of compound therein. See paragraph (g)(4) of this guideline
for recommendations on organ/tissue collection.
(7) Sample analysis. A total material balance must be obtained for
each animal. Total compound must be determined in each of the following
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samples: Urine, feces, blood (for consistency, it is recommended that blood
mass be assumed equal to 7 percent of body weight (see paragraphs (i)(l)
and (i)(2) of this guideline), wash from the skin, material in or on the
protective device, material remaining in or on the washed skin, material
in selected organs (if collected), and residue in the carcass. Concentration
of the test compound should be determined in the blood and any organ
samples collected.
(0 Data and reporting—(1) Data to be reported. The study report
should include the following information/data derived from the experi-
mental procedure on all animals in all groups:
(i) The method of determination of the limit of sensitivity and the
limit of sensitivity for each sample type in each dose group.
(ii) The actual quantity of test compound administered to each animal
and the mean for each experimental group of four animals.
(iii) Count (disintegrations per minute per gram), quantity of isotope,
quantity of compound and percent of actual dose administered in each
sample for each experimental animal in each experimental group and the
mean of those values for each experimental group of four animals.
(iv) The concentration, quantity, and percent of dose of test compound
in the blood and all tissues analyzed for each animal and the mean of
that value for each experimental group of four animals.
(v) Mass balance totals for each animal in each experimental group
and for the means for each experimental group.
(vi) Determination of the quantity and the percent absorbed for each
animal and for the group (mean). The quantity absorbed is that portion
of the dose which enters the systemic compartment of the organism. The
quantity in/on the skin is localized in the epidermis (mainly the stratum
comeum) and is not available for systemic distribution and toxicity until
it enters the vascular dermis. This determination is based on the following
distribution of the administered dose:
(A) Not absorbed—quantity in skin wash, and on the protective cover.
(B) Absorbable—quantity in/on the washed skin.
(C) Absorbed—quantity in the urine, cage wash, feces, expired air
(if present), blood, organs (if collected), and the remaining carcass.
(2) Final report format and content. The final report should contain
the items that are listed.
(i) Cover page and regulatory documentation. A cover/title page
and additional documentation (i.e., requirements for data submission, good
laboratory practices (GLP) statement and statement of data confidentiality
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claims), if relevant to the study report, must precede the content of the
study format. These requirements are described in 40 CFR parts 158 and
160.
(ii) Table of contents. The table of contents should include a listing
of the elements of the final report such as the summary, an introduction,
the materials and methods, results, discussion, bibliography, tables, figures,
appendices and key subsections as appropriate.
(iii) Body of the report. This item should include such detail that
the reviewer can assess the quality of the study and conformity to the
dermal absorption guidelines or an approved variation of the guidelines.
It should contain the following sections:
(A) Summary. The test report should contain a summary including
a brief description of the study protocol, chemical used, the animals tested,
and the highlights of the results of the study. Any deviations from the
intended protocols should be noted.
(B) Introduction. Include the objectives of the study and the Guideline
reference. The overall experimental design should be explained.
(C) Materials and methods. (7) Test substance. An identification of
the material tested and vehicles used to include the following:
(i) Test material used in test (chemical name, CAS No.)
(/'/') Properties of the test substance: Chemical structure, form,
radiolabeled, technical label-position, source, radiopurity, lot number,
source, purity, lot number purity, state (liquid or solid), ionization constant
(if applicable), pH (if applicable), solubility in various solvents (if known),
octanol/water partition coefficient (if known)
(HI) Vehicles used (if a formulation vehicle is used, it must be identi-
fied but its confidential composition need not be included in the report),
source, lot number.
(2) Test animals. An identification of the experimental animals used
to include the following: Species and strain, sex, source, body weight
range, pretest condition, housing conditions.
(3) Experimental design. The experimental design, as performed in
the study, must be described in complete detail. A step-by-step description
of the entire study in sufficient detail to allow precise understanding of
how the study was performed is required. The description should include,
but not necessarily be limited to, the following: Doses used, number of
animals per dose group, duration of exposure, preparation of the applica-
tion site, area of the application site, dose preparation, dose application,
dose quantitation, method of protecting the application site, urine, feces
and expired air collection, termination method, sample collection methods,
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skin wash, protective device, skin at application site, blood, individual or-
gans (if collected), residual carcass, urine, feces.
(4) Evaluation procedures. A detailed description of the methodology
of the study to include, but not necessarily be limited to, the following:
Method of assignment of animals to test groups, method of verification
of radiopurity, method of determination of actual dose method of sample
collection, storage and analysis, detailed secondary procedures, such as
sample analysis, should be included in the appendices.
(5) Deviation from protocol. Deviations from the protocol or from
an approved variation thereof must be described along with the rationale
for the changes.
(D) Results. This section should provide a narrative summary of the
results of the study. Data generated are best presented in summary tables
and figures included in paragraph (h) of this guideline.
(E) Discussion. (Optional) This section should provide an assessment
of the results of the study, an interpretation of the observations and should
try to explain unexpected findings. The impact of any deviation from the
guideline protocol should be discussed.
(F) Bibliography. (Optional) Complete citations of any documents re-
ferred to in the report. Referenced documents may include previous re-
ports, correspondence to and from the Agency, publications in the tech-
nical literature, and Agency guidelines. Each citation must be sufficiently
complete so as to allow identification and retrieval of the document.
(G) Tables/Figures. These tables and figures should summarize and
illustrate the results of the study by presenting mean values for each exper-
imental group of four animals. Examples are given at the end of this guide-
line.
(H) Appendixes. These should include individual animal data, analyt-
ical methods, results of analysis of the test substance and the dose formula-
tion, protocol, sample calculations and other information as appropriate.
(g) Additional dermal absorption studies. Additional, more specific
studies may be necessary to clarify important points raised in toxicity, me-
tabolism or kinetic studies of a compound. The studies presented below
provide a beginning or outline for designing compound-specific studies
but the individual studies must be designed specifically for the test
compound and the toxicology or kinetic issues of concern. Consultation
with the Agency before performing such a study is strongly recommended.
The individual sample collections/special treatments may, where practical,
be combined with or added to the basic study. Additional studies are as
follows:
8
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(1) Significant quantity of residue remaining on the washed skin.
(i) For some compounds the washed skin has been found to retain signifi-
cantly more material than was absorbed during the exposure period. In
some cases the difference between absorbed and retained material is suffi-
cient to convert an acceptable risk into an unacceptable risk if all the re-
tained material is considered as absorbed. This study is designed to deter-
mine the fate of that residual material, in particular the portion absorbed
and the rate at which it is absorbed.
(ii) Selected dose levels, as determined from the risk assessment, are
administered to groups of four animals. At 10 h one group from each
dose is terminated as in paragraphs (e)(6) and (e)(7) of this guideline. In
the remaining groups the skin is washed at 10 h, the wash sample collected
for analysis and the groups carried for one or more additional days, up
to 14 days, in metabolism cages. A minimum of 14 days has been sug-
gested by absorption data and a maximum of 21 days has been suggested
by comified epidermal turnover time in the rat. Suggested exposure dura-
tions, per group of four rats, are 10 h, and 1, 2, 7, 14, and 21 days. Urine
and fecal samples are collected for 24 h intervals. Expired air is collected
if labeled material is expired. At termination samples are collected from
each animal as in paragraphs (e)(6) and (e)(7) of this guideline. Samples
should include any organs/tissues collected in the original study.
(2) Determination of metabolites. Experience has shown that both
qualitative and quantitative production of metabolites of foreign com-
pounds can vary significantly with route of administration. When testing
compounds for which it has been determined that specific metabolites are
of toxicological concern, urine and fecal samples collected in the basic
dermal absorption study may be analyzed for the specific metabolites and
their proportionate production. It may be necessary to dose additional ani-
mals to provide sufficient excreta samples. Blood concentration of label
in dermal absorption studies has shown that compound concentration is
usually too small to allow metabolite identification.
(3) Blood/plasma kinetics study, (i) This study is designed to pro-
vide data to be compared with blood/plasma concentrations at effect and
no-effect doses by the route by which the toxic effect was originally identi-
fied (usually oral). The same species must be used for oral and dermal
determinations. When using these data it is not necessary to obtain and
utilize absorption and excretion parameters in determining MOEs by dif-
ferent routes of administration. Kinetic comparisons of the effect of route
on biological activity are performed by comparing concentrations of active
chemical at the active site following administration by different routes.
This information is extremely hard to obtain, so that obtaining it for any
chemical other than the most important of human drugs is usually imprac-
tical. The most practical surrogate data are blood/plasma concentrations
following different dosing routes. An oral blood/plasma kinetics study
must be performed, using at least the effect and no-effect doses from the
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critical toxicology study and data on blood/plasma concentration with time
as well as volume of distnbution and disappearance parameters (half-lives)
obtained before the dermal kinetic study is performed.
(ii) The test compound must be radiolabeled and the radioactivity in
each dose must be sufficient to detect the same minimal concentration
in the blood/plasma. That is, the limit of detection for each dose must
be the same in mass of test material per unit mass of blood/plasma. The
limit of detection must be at least one-hundreth of the maximum blood/
plasma concentration observed following the no-effect oral dose to allow
detection of a hundredfold MOE. A proportionately smaller limit of detec-
tion is necessary for a proportionately larger MOE. If the appropriate limit
of detection cannot be practically obtained the study should not be per-
formed.
(iii) Dermal doses should be selected to bracket the doses (expressed
as milligrams per square centimer) reported for the exposures at risk and
should be at log intervals. Dose preparation, application and protection
of the active site should follow the procedures in the basic dermal absorp-
tion study. The only samples collected for analysis are dosing material
(to determine concentration, homogeneity, and dose applied) and blood
(to determine blood/plasma concentration with dose and time). Analysis
of both whole blood and plasma concentrations is recommended. The rat
has been found to bind many chemicals to the erythrocyte and these data
will allow detection of this process which can have significant effects on
the apparent whole blood kinetics of the bound chemical.
(iv) A minimum of four animals should be used for each dose-dura-
tion-of-exposure data point. Depending upon the size of the blood sample
collected and the time between collections, it may be possible to collect
more than one sample from each animal. However, because of the limited
blood volume in the rat, it is recommended that individual groups of four
rats be used for each exposure (blood sample collection) period.
(v) It is impossible to predict the timing of blood sample collection
following dermal dosing that will be necessary to define the blood/plasma
concentration curve for a particular chemical. Therefore, a preliminary
study is recommended. Use the highest dose proposed for the study, one
or two animals per dose, and collection intervals of 1, 2, 4, 10 and
24 h. Sample collection time can be adjusted from these data to better
fit the expected blood/plasma concentration curve. It must be possible to
identify the peak blood/plasma concentration and characterize the curve
leading to and following it. Note: If blood/plasma concentration is below
the limit of detection for this high dose, it will not be necessary to run
lower doses. No detectable radioactivity can be expected at the lower doses
and the MOE will be derived from the limit of detection following the
high dermal dose.
10
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(4) Organ/tissue collection, (i) If a target organ for the toxic effect
of the test compound has been identified, it may be useful to determine
test compound concentration, with time and dose, in that tissue following
dermal absorption. Such information can used directly in risk assessment
by comparing the maximum concentration in the target organ following
dermal administration with the maximum concentration following the ef-
fect and no-effect doses by the route used in the critical toxicology tests.
Because of the possibility of saturation of absorption by the dermal route,
it may be impossible to reach a toxic concentration in the target organ
by dermal dosing indicating that there is no risk of concern associated
with dermal exposure.
(ii) Organs should be collected during, as part of, the basic dermal
absorption study. Both concentration and total amount of test material in
the organ (and/or tissue sample) must be obtained; concentration for com-
parison between organs and blood in order to detect evidence of bio-
accumulation in a particular organ and total amount as part of the material
balance for the dermal absorption study. Organs/tissues suggested for col-
lection are: The target organs, liver and kidney as metabolic/excretory or-
gans; heart, lungs, spleen, gonads, adrenals, pancreas, as discrete critical
organs; brain as indicative of the effect of the blood brain barrier; muscle
' as a fast storage tissue; and fat as a slow storage tissue.
(5) Volatile compounds, (i) Dermal absorption studies of volatile
compounds can be compromised by inhalation of the vapor, condensation
of the vapor in the metabolism cage with ingestion and condensation of
the vapor on additional areas of the skin. Use of an impermeable cover
on the protective device to counter these effects will produce unrealistic
absorption data. A cover on the protective device consisting of filter paper
impregnated with activated charcoal (or a material such as XAD4
amberlite resin) has been shown to be effective in trapping vaporized or-
ganic test material. A preliminary in vitro test is recommended to deter-
mine the effectiveness of activated charcoal or resin for a particular
compound. For such a test the spacer is glued to a glass plate, a small
measured dose of test material placed within and covered with the charcoal
or resin cover. This model is placed in a metabolism cage maintained at
rat body temperature with a trap on the air outflow to determine whether
any test material escapes the charcoal. After 1 to 2 h the distribution of
the dose is determined from glass plate, spacer, charcoal or resin cover,
metabolism cage wash, and trap. If the dose is confined to the glass plate,
spacer and cover, the study can proceed. If material is found in the cage
wash and/or the trap it may not be possible to perform the study and advice
should be sought from the Agency.
(ii) The experimental design of the dermal absorption study should
follow that of the basic dermal absorption study or an appropriate variation
thereof. A trap may be necessary on the air outflow of the metabolism
11
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cage if the test compound is excreted by respiration. It should also be
determined if the test material evaporates from the excreta.
(6) Infinite dose studies, (i) This study is designed to address dermal
absorption of the test compound while swimming or bathing, during which
the individual is exposed dermally to a constant concentration of pesticide
from an essentially unlimited source. This is a very tricky study and should
be designed in close consultation with the Agency.
(ii) In this study a reservoir is glued to the skin of the rat, filled
with a water solution of the test compound and covered with an impervious
cover. The exposure area is defined while the dose is varied by varying
the concentration of test compound. Concentrations tested should be at
log intervals and selected to bracket the expected field exposure. Exposure
durations should be chosen to match time in the water for the swimmer
or bather.
(iii) Test compound, test animal, animal preparation, animal process-
ing, and sample analysis should be essentially the same as in the basic
dermal absorption study or an appropriate variation thereof. Results of this
type of study are expressed as flux (mass per unit area per unit time).
(h) Explanatory Documentation. For explanatory documentation of
the Guideline write, Public Docket and Freedom of Information Section,
Field Operations Division, Office of Pesticide Programs, Environmental
Protection Agency, Washington, DC 20460, or call (703) 305-5805. Ask
for OPP-00369 (no charge).
(i) The following references should be consulted for additional back-
ground material on this test guideline.
(1) Laboratory Animal Medicine. Fox, J.G., Cohen, B.J., and Loew,
P.M., eds. Academic (1984).
(2) The Laboratory Rat. Baker, H.J., Lindsey, J.R. and Weisbroth,
S.H., eds. Volume I, Biology and Diseases. Academic, p. 108 (1979).
(3) Zendzian, R.P. Skin Penetration Method Suggested for Environ-
mental Protection Agency Requirements. Journal of the American College
of Toxicology 8:829-835 (1989).
12
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&EPA
United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-98-351
August 1998
Health Effects Test
Guidelines
OPPTS 870.7800
Immunotoxicity
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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. 136, et seq.).
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
Guidelines."
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OPPTS 870.7800 Immunotoxicity.
(a) Scope—(I) 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. This is a new guideline developed in the Office
of Pesticide Programs.
\
(b) Purpose. This guideline is intended to provide information on
suppression of the immune system which might occur as a result of re-
peated exposure to a test chemical. While some information on potential
immunotoxic effects may be obtained from hematology, lymphoid organ
weights and histopathology (usually done as part of routine toxicity test-
ing), there are data which demonstrate that these endpoints alone are not
sufficient to predict immunotoxicity (Luster et ai, 1992, 1993 see para-
graphs (j)(8) and (j)(9) of this guideline). Therefore, the tests described
in this guideline are intended to be used along with data from routine
toxicity testing, to provide more accurate information on risk to the im-
mune system. The tests in this guideline do not represent a comprehensive
assessment of immune function.
(c) Definitions. The following definitions apply to this guideline.
Antibodies or immunoglobulins (Ig) are part of a large family of
glycoprotein molecules. They are produced by B cells in response to anti-
gens, and bind specifically to the eliciting antigen. The different classes
of immunoglobulins involved in immunity are IgG, IgA, IgM, IgD, and
IgE. Antibodies are found in extracellular fluids, such as serum, saliva,
milk, and lymph. Most antibody responses are T cell-dependent, that is,
functional T and B lymphocytes, as well as antigen-presenting cells (usu-
ally macrophages), are required for the production of antibodies.
CD is the abbreviation for cluster of differentiation, and refers to mol-
ecules expressed on the cell surface. These molecules are useful as distinct
CD molecules are found on different populations of cells of the immune
system. Antibodies against these cell surface markers (e.g., CD4, CDS)
are used to identify and quantitate different cell populations.
Immunotoxicity refers to the ability of a test substance to suppress
immune responses that could enhance the risk of infectious or neoplastic
disease, or to induce inappropriate stimulation of the immune system, thus
contributing to allergic or autoimmune disease. This guideline only ad-
dresses potential immune suppression.
Natural Killer (NK) cells are large granular lymphocytes which non-
specifically lyse cells bearing tumor or viral antigens. NK cells are up-
regulated soon after infection by certain microorganisms, and are thought
to represent the first line of defense against viruses and tumors.
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T and B cells are lymphocytes which are activated in response to
specific antigens (foreign substances, usually proteins). B cells produce
antigen-specific antibodies (see the definition for "antibodies or
immunoglobuhns"), and subpopulations of T cells are frequently needed
to provide help for the antibody response. Other types of T cell participate
in the direct destruction of cells expressing specific foreign (tumor or in-
fectious agent) antigens on the cell surface.
(d) Principles of the test methods. (1) In order to obtain data on
the functional responsiveness of major components of the immune system
to a T cell dependent antigen, sheep red blood cells (SRBC), rats and/
or mice1 must be exposed to the test and control substances for at least
twenty eight days.2 The animals must be immunized by intravenous or
intraperitoneal injection of SRBCs approximately four days (depending on
the strain of animal) prior to the end of the exposure. At the end of the
exposure period, either the plaque forming cell (PFC) assay or an enzyme
linked immunosorbent assay (ELISA) must be performed to determine the
effects of the test substance on the splenic anti-SRBC (IgM) response or
serum anti-SRBC IgM levels, respectively.
(2) In' the event the test substance produces significant suppression
of the anti-SRBC response, expression of phenotypic markers for major
lymphocyte populations (total T and total B), and T cell subpopulations
(T helpers (CD4) and T cytotoxic/suppressors (CDS)), as assessed by flow
cytometry, may be performed to determine the effects of the test substance
on either splenic or peripheral-blood lymphocyte populations and T cell
subpopulations. When this study is performed, the appropriate monoclonal
antibodies for the species being tested should be used. If the test substance
has no significant effect on the anti-SRBC assay, a functional test for NK
cells may be performed to test for a chemical's effect on non-specific im-
munity.3 For tests performed using cells or sera from blood (ELISA or
flow cytometry), it is not necessary to destroy the animals, since immuni-
zation with SRBCs at twenty eight days is not expected to markedly affect
the results of other assays included in subchronic or longer-term studies
(Ladies et al., 1995 see paragraph (j)(7) of this guideline). The necessity
to perform either a quantitative analysis of the effects of a chemical on
the numbers of cells in major lymphocyte populations and T Cell sub-
populations by flow cytometry, or a splenic NK cell activity assay to as-
sess the effects of the test compound on non-specific immunity should
1 If absorption/distnbution/metabolism/excretion (ADME) data are similar between
species, then either rats or mice may be used for the test compound in question. If
such data are lacking, both species should be used.
2 Because there is a fairly rapid turnover of many of the cells in the immune
system, twenty eight days is considered sufficient for the purposes of the anti-SRBC
tests.
3 When these optional tests are included, the phenotypic or NK cell analyses may
be performed at twenty eight days of exposure, or at a later timepoint if ADME data
suggest that a longer exposure is more appropriate.
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be determined on a case-by-case basis, depending upon the outcome of
the anti-SRBC assay.
(e) Limit test. If a test at one dose level of at least 1.000 mg/kg
body weight (or 2 mg/L for inhalation route of exposure) using the proce-
dures described for this study produces no observable toxic effects or if
toxic effects would not be expected based upon data of structurally related
compounds, then a full study using three dose levels might not be nec-
essary. Expected human exposure may indicate the need for a higher dose
level.
(f) Test procedures—(I) Animal selection—(i) Species and strain.
These tests are intended for use in rats and/or mice. Commonly used lab-
oratory strains should be employed.4 All test animals should be free of
pathogens, internal and external parasites. Females should be nulliparous
and nonpregnant. The species, strain and source of the animals must be
identified.
(ii) Age/weight (A) Young, healthy animals- should be employed. At
the commencement of the study, the weight variation of the animals used
should not exceed ± twenty percent of the mean weight for each sex.
(B) Dosing should begin when the test animals are between six and
eight weeks old. .
(iii) Sex. Either sex may be used in the study; if one sex is known
or believed to be more sensitive to the test compound, then that sex should
be used.
(iv) Numbers. (A) At least eight animals should be included in each
dose and control group. The number of animals tested should yield suffi-
cient statistical power to detect a twenty percent change based upon the
interanimal variation which may be encountered in these assays.
(B) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(C) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals may be group-caged by sex, but the
number of animals per cage must not interfere with clear observation of
each animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual caging.
4 The study director should be aware of strain differences in response to SRBC.
For example, if the B6C3Fi hybrid mouse is used in the PFC assay, a response of
800-1,000 PFC/106 spleen cells in control mice should be the minimally acceptable
PFC response.
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(B) The temperature of the experimental animal rooms should be at
22 ± 3°C.
(C) The relative humidity of the experimental animal rooms should
be between thirty and seventy percent.
(D) Where lighting is artificial, the sequence should be 12 hours light,
12 hours dark.
(E) Control and test animals should be maintained on the same type
of bedding and receive feed from the same lot. The feed should be ana-
lyzed to assure adequacy of nutritional requirements of the species tested
and for impurities that might influence the outcome of the test. Rodents
should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until the animals have been
allowed an adequate period of acclimatization or quarantine to environ-
mental conditions. The period of acclimatization should be at least one
week in duration.
(2) Control and test substances, (i) Where necessary, the test sub-
stance should be dissolved or suspended in a suitable vehicle. Ideally, if
a vehicle or diluent is needed, it should not elicit toxic effects or substan-
tially alter the chemical or toxicological properties of the test substance.
It is recommended that an aqueous solution should be used. If solubility
is a problem a solution in oil may be used. Other vehicles may be consid-
ered, but only as a last resort.
(ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Prior to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound and if technically feasible, the
name and quantities of any known contaminants and impurities.
(iii) If the test or positive control substance is to be incorporated into
feed or another vehicle, the period during which the test substance is stable
in such a mixture should be determined prior to the initiation of the study.
Its homogeneity and concentration must also be determined prior to the
initiation of the study and periodically during the study. Statistically ran-
domized samples of the mixture should be analyzed to ensure that proper
mixing, formulation, and storage procedures are being followed, and that
the appropriate concentration of the test or control substance is contained
in the mixture.
(3) Control groups, (i) A concurrent, vehicle-treated control group
is required.
(ii) A separate untreated control group is required if the toxicity of
the vehicle is unknown.
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(lii) A positive control group with a known immunosuppressant (e g.,
cyclophosphamide) is useful in the interpretation of the results or verifica-
tion of the assay sensitivity, and should be included in the study. When
used, a group of at least eight animals should be given the immuno-
suppressive chemical.
(4) Dose levels, (i) In repeated-dose toxicity tests, it is desirable to
have a dose-response relationship and a no observed immunotoxic effect
level. Therefore, at least three dose levels and a negative control should
be used, unless a Limit test is performed as specified in paragraph (e)
of this guideline.
(ii) The highest dose level should not produce significant stress, mal-
nutrition, or fatalities, but ideally should produce some measurable sign
of general toxicity (e.g., a ten percent loss of body weight).
(iii) The lowest dose level ideally should not produce any evidence
of immunotoxicity.
(5) Administration of the test substance, (i) The test substance, ve-
hicle, or positive control substance shall be administered for at least twenty
eight days for the anti-SRBC assay. The route of administration of the
test material will usually be oral; however, this should be determined by
the likely route of occupational or indoor exposure. Therefore, under cer-
tain conditions, the dermal or inhalation route of exposure may be more
relevant for the study. All animals should be dosed by the same method
during the entire experimental period.
(ii) If the test substance is administered by gavage, the animals are
dosed with the test substance ideally on a seven-days-per-week basis.
However, based primarily on practical considerations, dosing by gavage
on a five-days-per-week basis is acceptable. If the test substance is admin-
istered in the drinking water, or mixed directly into the diet, then exposure
should be on a seven-days-per-week basis.
(A) For substances of low toxicity, it is important to ensure that when
administered in the diet, the quantities of the test substance involved do
not interfere with normal nutrition. When the test substance is administered
in the diet, either a constant dietary concentration in parts per million
(ppm) or a constant dose level in terms of the animal's body weight should
be used; the alternative used must be specified.
(B) For a substance administered by gavage, the dose should be given
at approximately the same time each day, and adjusted at intervals (weekly
for mice, twice per week for rats) to maintain a constant dose level in
terms of the animal's body weight.
(iii) If the test substance is administered dermally, refer to the Health
Effects Test Guidelines, OPPTS 870.3250, Subchronic Dermal Toxicity,
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paragraphs (e)(5). (e)(6), (e)(7). and (e)(8) for the procedures to be used.
The exposure time for the anti-SRBC test should be at least twenty eight
days.
(iv) If the test substance is administered by the inhalation route, refer
to the Health Effects Test Guidelines, OPPTS 870.3465, Subchronic Inha-
lation Toxicity, paragraphs (e)(2), (e)(3), (e)(6), (e)(8), (e)(9), and (e)(10)
for the procedures to be used. The exposure time for the anti-SRBC test
should be at least twenty eight days.
(6) Observation period. Duration of the observation period should
be at least twenty eight days.
(7) Observation of animals, (i) Observations should be made at least
once each day for morbidity and mortality. Appropriate actions should be
taken to minimize loss of animals to the study (e.g., necropsy of those
animals found dead and isolation or euthanasia of weak or moribund ani-
mals).
(ii) A careful clinical examination should be made at least once a
week. Observations should be detailed and carefully recorded, preferably
using explicitly defined scales. Observations should include, but not be
limited to: evaluation of skin and fur, eyes,and mucous membranes; res-
piratory and circulatory effects; autonomic effects, such as salivation;
central nervous system effects, including tremors and convulsions, changes
in the level of motor activity, gait and posture, reactivity to handling or
sensory stimuli, grip strength, and stereotypes or bizarre behavior (e.g.,
self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed, includ-
ing the time of onset, degree and duration.,
(iv) Food and water consumption should be determined weekly.
(v) Animals should be weighed immediately prior to dosing, weekly
(twice per week for rats) thereafter, and just prior to euthanasia.
(vi) Any moribund animals should be removed and euthanized when
first noticed. Necropsies should be conducted on all moribund animals,
and on all animals that die during the study.
(vii) The spleen and thymus should be weighed in all animals at the
end of the study.
(g) Immunotoxicity tests—(1) Functional tests. Either a splenic
PFC assay or an ELISA must be used to determine the response to antigen
administration.
(i) Antibody plaque-forming cell- (PFC) assay. The Jerne and
Nordin antibody plaque-forming cell assay, as modified by Cunningham
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(1965) (see paragraph (j)(2) of this guideline) or as described in detail
by Holsapple (1995) (see paragraph (j)(4) of this guideline), should be
used to demonstrate the effects of exposure (at least twenty eight days)
to a test substance on antibody-producing cells from the spleen. The fol-
lowing points should be adhered to when conducting this assay:
(A) The T cell-dependent antigen, SRBC, should be injected intra-
venously or intrapentoneally, usually at twenty four days after the first
dosing with the test substance.5 Although the optimum response time is
usually four days after immunization, some strains of test animal may de-
viate from this time point. Hence, the strain to be used must be evaluated
for the optimum day for PFC formation after immunization.
(B) The activity of each new batch of complement must be deter-
mined. For any given study, the SRBCs should be from a single sheep,
or pool of sheep, for which the shelf life and dose for optimum response
has been determined.
(C) Modifications of the above-cited PFC assay exist (for example,
see Ladies et a/., 1994 or Temple, et al. 1993 in paragraphs (j)(5), (j)(6),
and (j)UO) of this guideline) and may prove useful; however, the complete
citation should be made for the method used, any modifications to the
method should be reported, and the source and, where appropriate, the
activity or purity of important reagents should be given. Justification or
rationale should be provided for each protocol modification.
(D) Samples must be randomized and coded for PFC analysis, so that
the analyst is unaware of the treatment group of each sample examined.
(E) Spleen cell viability should be determined.
(F) The numbers of IgM PFC per spleen, and the number of IgM
PFC per 106 spleen cells must be reported.
(ii) Immunoglobulin quantification. Enzyme-Linked
Immunosorbent Assay (ELISA). As an alternative to a PFC assay, the ef-
fects of the test substance on the antibody response to antigen may be
determined by an ELISA (see Temple, et al, 1993, and Ladies et al.,
1994 in paragraphs (j)(5), (j)(6), and (j)(10) of this guideline for a compari-
son between the PFC and ELISA assays for immunotoxicity assessment).
Test animals must be immunized with SRBCs as for the PFC assay. IgM
liters in the serum of each test animal must be determined (usually four
days after immunization). As with the PFC assay, the optimum dose of
SRBCs and optimum time for collection of the sera must be determined
for the species and strain of animal to be tested. Detailed methods are
described by Temple et al. (1995) (see paragraph 0)00 of this guideline).
s If ^ SRBCs are administered by the mtrapentoneal route, the study director
should be aware that a low percentage of animals may not respond because the antigen
was accidentally injected into the intestinal tract.
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(in) Natural killer (NK) cell activity. The methods in Djeu, et al.
(1995) (see paragraph (j)(3) of this guideline) may be used to demonstrate
the effects of at least twenty eight days of exposure to a test substance
on spontaneous cytotoxic activity. In this assay, splenocytes from treated
and untreated test animals are incubated with 51Cr-labeled YAC-1
lymphoma cells. The amount of radiolabel released from the target cells
after incubation with the effector cells for four hours is used as a measure
of NK cytolysis. The following points should be adhered to when using
the NK cell assay:
(A) Assay controls should be included to account for spontaneous
release of radiolabel from target cells in the absence of effector cells, and
also for the determination of total release of radiolabel.
(B) Target cells other than YAC-1 lymphoma cells may be appro-
priate for use in the assay. In all cases, target cell viability should be
determined.
(C) Modifications of the protocol exist that may prove useful. How-
ever, complete citation must be made to the method used. Modifications
must be reported, and where appropriate, the source, activity, and/or purity
of the reagents should be given. Justification or rationale must be provided
for each protocol modification.
. (2) Enumeration of splenic or peripheral blood total B cells, total
T cells, and T cell subpopulations. The phenotypic analysis of total B
cell, total T cell, and T cell subpopulations from the spleen or peripheral
blood by flow cytometry should be performed after at least twenty eight
days of dosing; this may be performed at a later timepoint, if ADME data
suggest that a longer exposure is more appropriate. If an exposure period
longer than twenty eight days is used, then these tests may be performed
in conjunction with subchronic (ninety day oral, dermal, or inhalation) tox-
icity studies, when these studies are required. Methods are described by
Ladies and Loveless (1994), and Comacoff et al. (1995) (see paragraphs
(j)(l) and (j)(5) of this guideline).
(h) Data and reporting—(1) Treatment of results—(i) Data should
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 effects,
the types of effects and the percentage of animals displaying each type
of effect.
(ii) All observed results, quantitative and incidental, should be evalu-
ated by an appropriate statistical method. Any generally accepted statistical
methods may be used; the statistical methods including significance criteria
should be selected during the design of the study.
(2) Evaluation of study results. The findings of an immunotoxicity
study should be evaluated in conjunction with the findings of preceding
8
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studies and considered in terms of other toxic effects. The evaluation
should include the relationship between the dose of the test substance and
the presence or absence, and the incidence and severity of abnormalities,
including behavioral and clinical abnormalities, gross lesions, identified
target organs, body weight changes, effects on mortality and any other
general or specific toxic effects. A properly conducted test should provide
a satisfactory estimation of a no-observed-effect level. It also may indicate
the need for an additional study and provide information on the selection
of dose levels.
(3) Test report. In addition to the reporting requirements as specified
under EPA Good Laboratory Practice Standards, 40 CFR part 792, Subpart
J, 40 CFR part 160, and the OECD principles of GLP (ISBN 92-64-
12367-9), the following specific information should be reported:
(i) The test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(E) Identification and composition of any vehicle used.
(ii) The test system should contain data on:
(A) Species, strain, and rationale for selection of animal species, if
other than that recommended.
(B) Age, body weight data, and sex.
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
(D) When inhalation is the route of exposure, a description of the
exposure equipment and data should be included (refer to the Health Ef-
fects Test Guidelines, OPPTS 870.3465 Subchronic Inhalation Toxicity,
paragraphs (f)(3)(iii)(D), and (f)(3)(iii)(E)).
(E) Identification of animal diet.
(iii) The test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
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(iv) Test results—(A) Group animal data. Tabulation of toxic re-
sponse data by species, strain, sex and exposure level for:
(7) Number of animals exposed.
(2) Number of animals showing signs of toxicity.
(3) Number of animals dying.
(B) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(C) Date of death during the study or whether animals survived to
termination.
(D) Date of observation of each abnormal sign and its subsequent
course.
(E) Absolute and relative spleen and thymus weight data are required.
(F) Feed and water consumption data, when collected.
(G) Results of immunotoxicity tests.
(H) Necropsy findings of animals that were found moribund and
euthanized or died during the study.
(I) Statistical treatment of results, where appropriate.
(i) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment. The study should be conducted in compliance with the Good
Laboratory Practice (GLP) regulations as described by the Agency (40
CFR parts 160 and 792) and the OECD principles of GLP (ISBN 92-
64-12367-9).
(j) References. The following references should be consulted for ad-
ditional background information on this test guideline:
(1) Comacoff, J.B., Graham, C.S., and LaBrie, T.K. 1995. Phenotypic
identification of peripheral blood mononuclear leukocytes by flow
cytometry as an adjunct to immunotoxicity evaluation. In Methods in
Immunotoxicology (G.R. Burleson, J.H. Dean, and A.E. Munson, Eds.),
Vol. 1, pp 211-226, Wiley-Liss, Inc., New York.
(2) Cunningham, A.J. 1965. A method of increased sensitivity for
detecting single antibody-forming cells. Nature 207:1106-1107.
(3) Djeu, Julie Y. 1995. Natural Killer Activity. In Methods in
Immunotoxicology (G.R. Burleson, J.H. Dean, and A.E. Munson, Eds.) pp
437^149.
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(4) Holsapple, M P. 1995. The plaque-forming cell (PFC) response
in Immunotoxicology: An approach to monitoring the primary effector
function -of B lymphocytes. In Methods in Immunotoxicology (G.R.
Burleson, J.H. Dean, and A.E. Munson, Eds.), Vol. 1, pp. 71-108, Wiley-
Liss, Inc., New York.
(5) Ladies, G.S. and Loveless, S.E. 1994. Cell surface marker analysis
of splenic lymphocyte populations of the CD rat for use in
immunotoxicological studies. Toxicol. Methods 4: 77-91.
(6) Ladies, G.S., Smith, C., Heaps, K., and Loveless, S.E. 1994. Eval-
uation of the humoral immune response of CD rats following a 2-week
exposure to the pesticide carbaryl by the oral, dermal, or inhalation routes.
J. Toxicol. Environ. Health 42:143-156.
(7) Ladies., G.S., Smith, C., Heaps, K., Elliot, G.S., Slone, T.W., and
Loveless, S.E. 1995. Possible incorporation of an immunotoxicological
functional assay for assessing humoral immunity for hazard identification
purposes in rats on standard toxicology study. Toxicology 96:225-238.
(8) Luster, M.I., Portier, C., Pait, D.G., White, K.L., Jr., Gennings,
C., Munson, A.E., and Rosenthal, G.J. 1992. Risk assessment in
immunotoxicology I. Sensitivity and predictability of immune tests.
Fundam. Appl. Toxicol. 18:200-210.
(9) Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J. Germolec.
D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W.,
White, D.L., Munson, A.E., and Comment, C.E. 1993. Risk Assessment
in Immunotoxicology II. Relationships Between Immune and Host Resist-
ance Tests. Fundam. Appl. Toxicol. 21:71-82.
(10) Temple, L., T. T. Kawabata, A. E. Munson, and K. L. White,
Jr. 1993. Comparison of ELISA and plaque-forming cell assays for meas-
uring the humoral immune response to SRBC in rats and mice treated
with benzo[a}pyrene or cyclophosphamide. Fundam. Appl. Toxicol.
21:412-419.
(11) Temple, L., Butterworth, L., Kawabata, T.T., Munson, A.E., and
White, K.L. 1995. ELISA to Measure SRBC Specific Serum IgM: Method
and Data Evaluation. In Methods in Immunotoxicology (G.R. Burleson,
J.H. Dean, and A.E. Munson, Eds.), Vol. 1, pp 137-157, Wiley-Liss, Inc.,
New York.
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