P.I 

OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

2) ll-A-19 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:14 AM Sheet 2 of 93 
3037?-1 

ft -oib-crr 

P.2 

REPORT DOCUMENTATION 

PAGE 

t. REPORT HO. 

4. Title and Subtitle 

Pesticide Assessnent Guidelines, Subdivision F, 

Neurotoxicity Test Guidelines 

r. Autho*^ Bill Sette , EPA/HED 

AfTXS 3//S-/9/ 

S. Recipient's Aeeautan No. 

S. Report Oat* 

540/09-91-123 

8. Performing Organization Re rt. No: 

PB91-154617 

9. Performing Organization Nam* and Address 10. Pre|*ct/Ta»k/Work Unit N<». 

U.S. Environmental Protection Agency 

OPP/HED (H7509C) 

401 M Street, S.W. 

Washington, D.C. 20460 

11. ContrecttC* or GrantfG) No. 

to 

(G) 

12. Sponsoring Organization Nam* and Address 

same as #9 

IS. Typa of Report * Parted Covered 

14. 

uui m~\ /g^ m IS. Supplementary Notes rn 

LEPA SSlAf, R DOr.tcc CT 

I C Abetract (Umrt 200 word*) 

These new revised Neurotoxicity Test Guidelines are being added to Subdivision 

F of the Pesticide Assessment Guidelines, vhich provides guidance for registrants 

in the conduct of test to support registration of pesticides under the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA). These guidelines will 

also apply to future testing requirements for industrial chenicals under the 

Ttoxic Substances Act (TSCA). These revised neurotoxicity guidelines have 

been reviewed and approved by the FIFRA Scientific Advisory Panel and made 

available for public cenment. The following guidelines are included in the 

package: (1) Neurotoxicity Screening Battery including Elmctional Observational 

Battery, Motor Activity, and Neuropathology, including an assay for Glial 

Fibrillary Acidic Protein; (2) Delayed Neurotoxicity of Organophosphorous 

Substances following Acute and 28-day Exposures, including an assay for 

Neurotoxic Esterase; (3) Develcpmental Neurotoxicity Study; (4) Schedule-Controlled 

Operant Behavior; and (5) Peripheral Nerve Function. 

17. Document Analysis a. Descriptors 

b. Idanttflars/Opan-Endod Terms 

c. COSATt Field/Group 

I t . Availability Statement 19. Security Class (This Report) 

20. Security Class (This Page) 

21. No. of Pages 

60 

22. Price 

(See ANSt-239.18) See Instructions on Reverse OPTIONAL FORM 272 (*-TD 

(Formerly NTIS-35) 

Department ot Commerce 
P.3 

' \ 

March 1991 

EPA 540/09-91-123 

PB 91-154617 

PESTICIDE ASSESSMENT GUIDELINES 

SUBDIVISION F 

HAZARD EVALUATION: 

HUMAN AND DOMESTIC ANIMALS 

ADDENDUM 10 

NEUROTOXICITY 

SERIES 81, 82, AND 83 

Prepared by: 

William F. Sette, Ph.D. 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 
P.4 

FOREWORD 

These new and revised neurotoxicity guidelines are intended 

to replace and supplement the set of neurotoxicity guidelines 

originally published in the 1982 Pesticide Assessment Guidelines, 

Subdivision F, Hazard Evaluation: Human and Domestic Animals; 

EPA-540/9-82-05; October 1982; National Technical Information 

Service, Springfield, VA 22161. These guidelines have been 

written in coordination with upcoming proposed revisions to the 

Toxicology Data Requirements of Part 158 of Title 4 0 of the Code 

of Federal Regulations (40 CFR 158). These guidelines have 

undergone extensive review within the Agency, public comment, and 

review by the FIFRA Scientific Advisory Panel. 

ACKNOWLEDGMENTS 

The development and revision of these guidelines was due to 

the dedicated efforts -of many people inside and outside EPA, but 

predominantly the members of 2 workgroups. Their efforts are here 

noted and gratefully acknowledged. 

The following people participated in the revision of these 

guidelines as members of the ad hoc Workgroup :Kevin Crofton, 

Karl Jensen, Tina Levine, Robert MacPhail, Suzanne McMaster, 

Virginia Moser, Stephanie Padilla, D. Cooper Rees, Lawrence 

Reiter, Hugh Tilson, and William Sette, chairman. 

The Developmental Neurotoxicity Guideline Workgroup 

included: Zoltan Annau, Angela Auletta, Marlissa Campbell, Neil 

Chernoff, Kevin Crofton, Lynda Erinoff, Earl Grey, Karl Jensen, 

Carole Kimmel, Tina Levine, Robert MacPhail, Suzanne McMaster, 

C.J. Nelson, Myron Ottley, D. Cooper Rees, Larry Reiter, Jennifer 

Seed, William Sette, Mark Stanton, Hugh Tilson, Hal Zenick, and 

Elaine Francis as chairwoman. 
P.5 

Subdivision F 

Neurotoxicity 

Table of Contents 

Foreword, Acknowledgments 1 

Table of Contents 2 

Series 81-7, 82-6 

Delayed Neurotoxicity of Organophosphorus Substances 

Following Acute and 28 Day Exposures 3 

Series 81-8, 82-7, 83-1 

Neurotoxicity Screening Battery 13 

Appendix 1: Guideline for Assaying Glial 

Fibrillary Acidic Protein 28 

Series 83-6 

Developmental Neurotoxicity Study 3 2 

Series 85 

Schedule Controlled Operant Behavior 49 

Peripheral Nerve Function 55 
P.6 

DELAYED NEUROTOXICITY 

OF ORGANOPHOSPHORUS SUBSTANCES 

FOLLOWING ACUTE AND 28 DAY EXPOSURES 

(a) Purpose. In the assessment of organophosphorus 

substances , studies of delayed neurotoxicity using the adult hen 

as the test animal and including behavioral observation of gait, 

histopathological assessment of brain, peripheral nerve, and spinal 

cord, and neurochemical assessment of inhibition of 

acetylcholinesterase(AchE) and neurotoxic esterase (NTE) are needed 

to identify and characterize these potential effects. 

This guideline now requires an acute dosing regimen in hens in 

combination with assays of neurotoxic esterase (and 

acetylcholinesterase) to screen for this effect. Use of data on 

the inhibition of NTE in conjunction with behavioral and 

pathological data offers a number of advantages . It is important 

to recognize that many acute studies can provide equivocal evidence 

of behavioral or pathological effects. Some trixylenyl phosphates 

(Johnson, 1975), for example, are negative, or at best equivocal, 

after acute exposures, yet clearly cause OPIDN after repeated 

exposures. The continuous, rather than descriptive, nature of NTE 

data and the fact that considerable inhibition is generally 

required to produce OPIDN, will help to more convincingly conclude 

that a substance is -negative, based solely on an acute study. 

Conversely, NTE data can also provide a better indication of 

potential delayed neurotoxicity, i.e. if the behavioral and 

histopathological data after an acute exposure are equivocal, and 

the NTE inhibition is significant, then further study is 

appropriate. 

The revision of the 90 day study to a 28 day study is based on 

the idea that these shorter exposures offer savings in animals and 

cost and because 28 days are often closer to the duration of 

exposure applicators may experience than 90 days. In some cases, 

further study may be required to resolve data that are difficult to 

interpret clearly, or to establish more refined dose response 

relations, or to assess the particular use patterns of the 

substance. 

(b) Definitions. (1) Organophosphorus induced delayed 

neurotoxicity (OPIDN) is a neurological syndrome in which limb 

weakness and upper motor neuron spasticity are the predominant 

clinical signs; distal axonopathy of peripheral nerve and spinal 

cord are the correlative pathological signs; and inhibtion and 

aging of neurotoxic esterase in neural tissues are the correlative 

biochemical effects. Clinical signs and pathology first appear 

between 1 and 2 weeks following exposures that typically inhibit 

and subsequently age neurotoxic esterase. 

(2) Neuropathy target esterase (NTE) or neurotoxic 

esterase is a membrane-bound protein that hydrolyzes phenyl 

valerate. The inhibition and "aging" of the phosphorylated NTE, 

i.e., the covalent binding of the OP to the enzyme, is highly 

correlated with the initiation of OPIDN. Not all O-Ps that inhibit 
P.7 

Delayed Neuro 

NTE cause OPIDN, but all O-Ps that cause OPIDN inhibit NTE. 

(3) NTE activity is operationally defined as the phenyl 

valerate hydrolytic activity resistant to paraoxon but sensitive to 

mipafox or neuropathic O-P ester inhibition. 

(c) Principle of the test method. The test sequence consists 

of acute and 28 day exposure studies. Any significant effects on 

behavior(delayed effects), histopathology, or inhibition of NTE in 

the acute study are sufficient cause to conduct the 28 day study. 

The test substance is administered orally to domestic hens that in 

some cases have been protected from acute cholinergic effects. The 

animals are observed for at least 21 days for gait changes and 

other signs. Neurochemical examination of selected neural tissues 

is undertaken on some animals at some time(s) after exposure. 

Histopathology of brain, spinal cord, and peripheral nerve are 

performed at the termination of 21 day observation periods. 

If the results of the acute study are completely negative, that is, 

there are no delayed behavioral effects, and no histopathological 

effects, and no significant NTE inhibition, then the 28 day study 

is not required. Otherwise, the 28 day study should be conducted. 

In the 28 day study, 3 exposure levels are used to describe the 

dose response curve sufficiently to estimate a reference dose. 

(d) Test Procedures. (1) Animal selection. The adult domestic 

laying hen (Gallus gallus domesticus), aged 8 to 14 months, is 

recommended. Standard size breeds and strains should be employed. 

Healthy young adult hens free from interfering viral diseases and 

medication and without abnormalities of gait should be acclimatized 

to the laboratory conditions for at least 5 days prior to 

randomization and assignment to treatment and control groups. 

(2) Housing and feeding conditions. Cages or enclosures 

which are large enough to permit free mobility of the hens and easy 

observation of gait should be used. Where the lighting is 

artificial, the sequence should be 12 hours light, 12 hours dark. 

Appropriate diets should be administered as well as an unlimited 

supply of drinking water. The hens should be weighed weekly. Any 

moribund hens should be removed and sacrificed. 

(3) Route of Administration. Dosage of test substance 

should normally be by the oral route, preferably by gavage. Liquids 

may be given neat or dissolved in an appropriate vehicle such as 

corn oil; solids should be dissolved if at all possible since large 

doses of solids in gelatin capsules may significantly impair 

absorption. Dermal exposures may be the most significant route of 

exposure for applicators and for non-food uses and there may be 

important differences in toxicity by this route. Conduct of these 

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

(4) Study Design. (i) General. An important 

consideration for the design of these studies is prediction of 

activity based on the structure of the material and the published 

literature. Some materials, e.g. phosphinates, are known to inhibit 
P.8 

Delayed Neuro 

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 3 exposure groups are required in 

addition to the vehicle control group. Ideally, the data should be 

sufficient to produce a dose-effect curve. We strongly encourage 

the use of equally spaced doses and a rationale for dose selection 

that will maximally support detection of dose-effect relations. 

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 rigidly followed. 

(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 a hen LD50 has been established, then this, 

of course, may be used, although some verification may be prudent. 

If the rat LD50 is known, it may serve as the starting point of 

estimation. A preliminary lethality study in unprotected hens may 

be conducted to estimate the acute high dose. A variety of test 

methodologies may be used to estimate the unprotected lethal dose 

of test materials. Of course, the method of estimate of the lethal 

dose may influence the subsequent dosage estimates. 

From the preliminary data, if cholinergic signs were seen very soon 

after dosing, prophylaxis using atropine may be appropriate. 

Atropine (20 mg/kg, s.c, up to every 2 hours) should be used to 

prevent death from acute cholinergic effects. 

(B) 28 Day Study. (1) Levels of test substances 

greater than 1 g/kg need not be tested. 

(2) High dose. The high dose selected 

should be estimated to be sufficient to cause OPIDN or be a maximum 

tolerated dose based on the acute data, but not result in an 

incidence of fatalities that would prevent a meaningful evaluation 

of the data. 

(3) Low dose. The low dose should be 

estimated to be a minimum effect level, e.g., an ED10, or 

alternatively, a no effect level. 

(4) The intermediate dose level should be 

equally spaced between the high and low doses. 

(5) Intermediate responses in NTE i.e., 
P.9 

Delayed Neuro 

greater than 15% and less than 70%, can be crudely extrapolated as 

if the dose-response were a simple first order relationship. That 

is, if a certain dose caused 50% inhibition, then twice that dose 

might cause 75%. 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 3 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 

Triorthocresyl phosphate (TOCP), is required for both acute and 2 8 

day studies. This group may be a concurrent or historical control 

group.(This should also include at least 3 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 6 survivors for histopathology and 3 hens for 

NTE measurement are treated in a manner identical to the treated 

groups, except that -administration of the test substance is 

omitted. When protective agents are used, all members of the dose 

groups and vehicle controls should receive the same treatment. 

(5) Study Conduct, (i) Biochemical measurements. (A)NTE 

Assay. The test method is a differential assay of the ability of 

neural tissue, following O-P exposure, to selectively hydrolyze a 

phenyl valerate substrate. The principle of the assay is: first, 

to determine the amount of hydrolysis that occurs in the presence 

of a non-neurotoxic inhibitor, paraoxon, (a), which is intended to 

occupy irrelevant sites; 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 0-P ester and the activity of (b) is therefore reduced. 

(1) Three hens from each group should be 

sacrificed at 48 hours 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 (diethyl 4-nitrophenyl phosphate) , mipafox (N, N_!_-

diisopropylphosphorodiamido fluoridate) , and phenyl valerate. They 

all can be obtained commercially. 

(3) The assay has four stages: 

Preparation of tissue; differential preincubation; hydrolysis of 
Delayed Neuro 

substrate; and measurement of product. The quotations that follow 

are from Johnson (1977) as corrected or modified in Johnson (1982) . 

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

acceptable methods have been used. They primarily involve minor 

technical modification (Sprague et al. 1981; Soliman et al. 1982). 

(a) Preparation of tissue. "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° with HCL) . Meninges and 

blood vessels are rapidly removed and the brain is blotted dry, 

weighed, and homogenized thoroughly in ice-cold buffer(at a volume 

of at least 1:30, w/v), using a high-speed rotating perspex pestle 

with not more than 0.25 mm difference in diameter between pestle 

and tube." 

(b) Differential preincubation. 

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

are pre-incubated in Tris/EDTA buffer pH 8 at 3 7° for exactly 2 0 

minutes with paraoxon (40 to 100 uM) plus either (a) buffer or (b) 

mipafox (50uM) in a final volume of 2 ml." 

(c) Hydrolysis of substrate. "After 

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

incubation is continued for exactly 15 minutes. 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 sulphate (1-2% 

w/v) in buffer containing 4-aminoantipyrine (otherwise known as 4-

aminophenazone) (0.25 percent)." 

(d) Measurement of product. This 

assay is based on the colorimetric determination of liberated 

phenol. 

(1) "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." 

(2) "A nontissue blank, kept to 

10 percent of the paraoxon tube value by maintaining the substrate 

phenol fee, should be included in each group of assay tubes. 

Typical control.absorbance values would be 0.8 for paraoxon, 0.3 5 

for paraoxon and mipafox and 0.07 for the blank. Colour 

development takes (1-2 min) in solutions stopped with sodium 

dodecyl sulphate. The extinction coefficient of phenol under these 

conditions is 15,600 at a wavelength of 490 nm. NTE activity is 

represented by the difference in absorbance obtained from samples 

incubated under conditions (a) and (b) respectively." 

(3) "Under standard conditions 

NTE hydrolyzes about 2400 nanomoles of substrate/min/g of cortex, 

550 for spinal cord, and 100 for sciatic nerve." 

(B) AChE measures . Assay of 
P.11 

Delayed Neuro 

acetylcholinesterase in the brains of the same birds (Johnson and 

Russell, 1975; or Ellman et al. , 1961) shall also be performed.. 

The level of AChE inhibition is a useful index of lethal potency 

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

useful for subsequent dose selection. 

(ii) 21 Dav observation. All remaining hens should 

be carefully observed at least once daily for a period of at least 

21 days and signs of toxicity recorded, including the time of 

onset, degree, and duration. Observations should include, but not 

be limited to, behavioral abnormality, locomotor ataxia, and 

paralysis. At least twice a week the hens should be taken outside 

the cages and subjected to a period of forced motor activity, such 

as ladder climbing, in order to enhance the observation of minimal 

responses. A rating scale of at least four levels should be used to 

grade ataxia, e.g. Roberts et al. (1983). 

(iii) Necropsy and Histopathology. Gross necropsies 

are recommended for all survivors and should include observation of 

the appearance of the brain and spinal cord. All animals shall be 

prepared for microscopic examination. Tissues shall be fixed by 

whole body perfusion, with a fixative appropriate for the embedding 

media. Sections should include medulla oblongata, spinal cord, and 

peripheral nerves. The- spinal cord sections should be taken from 

the rostral cervical, the midthoracic, and the lumbo-sacral 

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. 

For 28 day studies, a stepwise examination of tissue samples is 

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

dose group are first compared with those of the control group. If 

no neuropathological alterations are observed in samples from the 

high dose group, subsequent analysis is not required. If 

neuropathological alterations are observed in samples from the high 

dose group, samples from the intermediate and low dose groups are 

then examined sequentially. 

(e) Data reporting and evaluation. (1) Test report. In 

addition to any other applicable reporting requirements, the final 

test report must include the following information: 

(i) Toxic response data by group with a description 

of clinical signs; the criteria for the grading system for ataxia 

and any other scales should be defined. 

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

or whether it survived to termination. 

(iii) The day of the first occurrence of each 

abnormal sign and its subsequent course including its degree. 

(iv) Body weight data. 

(v) Necropsy findings for each animal, including a 

description of the appearance of the brain and the spinal cord. 

(vi) Biochemical data for each animal assessed, 

including absorbance values for each animal tested, and blank 

8 
Delayed Neuro 

sample data. 

(vii) A detailed description of all 

histopathological findings. 

•(viii) Statistical treatment of results, where 

appropriate. 

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

tabular form, showing for each test group the number of animals at 

the start of the test, the number of animals showing lesions or 

effects, the types of lesions or effects and the percentage of 

animals displaying each type of lesion or effect. 

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

neurotoxicity studies should be evaluated in terms of the incidence 

and severity of behavioral, neurochemical, and histopathological 

effects and of any other observed effects in the treated and 

control groups, as well as any information known or available to 

the authors, such as published studies. For a variety of results 

seen, further studies may be necessary to characterize these 

effects. 

(f) References. For additional background information on this 

test guideline the following references should be consulted: 

Caroldi, S., Lotti, M. "Neurotoxic Esterase in Peripheral Nerve: 

Assay Inhibition, and Rate of Resynthesis." Toxicology and Applied 

Pharmacology. 62, 498-501 (1982). 

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). 

Ellman G.L., Courtney, K.D., Andres, v., and Featherstone, R.M. "A 

new and rapid colorimetric determination of acetylcholinesterase 

activity. Biochem. Pharmacol. 7:88-95.(1961) 

Johnson, CD . and Russell, R.L. "A rapid, simple, radiometric assay 

for cholinesterase, suitable for multiple determinations. Anal. 

Biochem. 64: 229-238 (1975J .... 

Johnson, M.K. "Organophosphorus esters causing delayed neurotoxic 

effects: Mechanism of action and structure/activity studies", 

Archives of Toxicology 34:259-288.(1975a) 

Johnson, M.K. "The delayed neuropathy caused by some 

organophosphorus esters:Mechanism and challenge". Crit.Rev.Toxicol. 

3:289-316.(1975b) 

Johnson, M.K. "Improved Assay of Neurotoxic Esterase for Screening 

Organophosphates for Delayed Neurotoxicity Potential," Archives of 

Toxicology. 37, 113-115 (1977). 
P.13 

~ 1 

Delayed Neuro 

Johnson, M.K. "Delayed neurotoxicity tests of organophosphorus 

esters: a proposed protocol integrating neuropathy target 

esterase(NTE) assays with behaviour and histopathology tests to 

obtain more information more quickly from fewer animals," 

Proceedings of the International Conference on Environmental 

Hazards of Agrochemicals in Developing Countries. Alexandria, 

Egypt, November 8-12, 1983; Volume I, pp. 474-493. 

Johnson, M.K. "The target for initiation of delayed neurotoxicity 

by organophosphorus esters: biochemical studies and toxicological 

applications", E.Hodgson, J.R. Bend,R.M.Philpot, eds., Reviews in 

Biochem• Toxicol. 4,141-212. Elsevier, New York(1982) 

Johnson, M.K., Richardson, R.J. "Biochemical Endpoints: 

Neurotoxic Esterase Assay." Neurotoxicology. 4(2):311-320 (1983). 

Kayyali, U.S., Moore, T., Randall, J.C and Richardson, R.J. 

"Neurotoxic Esterase Assay: Corrected wavelength and Extinction 

Coefficient. The Toxicologist. 6:1 #292, 73 (1989) 

Roberts, N.L., Fairley, C , and Phillips, c Screening acute 

delayed and subchronic neurotoxicity studies in the hen: 

Measurements and evaluations of clinical signs following 

administration of TOCP. Neurotoxicology. 4, 263-270. 

Soliman, S.A., Linder, R., Farmer, J., Curley, A. "Species 

Susceptibility to Delayed Toxic Neuropathy in relation to in vivo 

inhibition of Neurotoxic Esterase by Neurotoxic Organophosphorus 

Ester," Journal of Toxicology and Environmental Health. 9, 189-197 

(1982) . 

Sprague, G.L., Sandvik, L.L., Bickford, A.A. "Time course for 

neurotoxic esterase activity in hens given multiple diisopropyl 

fluorophosphate injections," Neurotoxicology. 2, 523-532 (1981). 

10 
P.14 

Delayed Neuro 

TABLE 1 

STUDY DESIGN 

# OF HENS NTE/TIME BEHAVIOR/PATHOLOGY 

ACUTE STUDY 

POSITIVE CONTROLS 

VEHICLE CONTROLS 

DOSE 

28 DAY STUDY 

POSITIVE CONTROLS 

VEHICLE CONTROLS 

HIGH DOSE 

LOW DOSE 

MID DOSE 

9 

9 

9 

9 

9 

9 

9 

9 

HOURS 

3/48 

3/48 

3/48 

HOURS 

3/48 

3/48 

3/48 

3/48 

3/48 

6 

6 

6 

6 

6 

6 

6 

6 

11 
P.15 

Delayed Neuro 

NOTES 

1. Substances" appropriate for testing are uncharged esters, 

thioesters, or anhydrides of organophosphoric, organophosphonic, or 

organophosphoramidic acids or of the related phosphorothioic, 

phosphonothioic, or phosphorthioamidic acids. 

2. Two commenters questioned the validity or usefulness of NTE 

measurements. 

The Agency believes that this assay will be both valid and 

useful and that this is the consensus of the scientific community 

. Only 2 of roughly 30 commenters questioned the addition of this 

assay. The SAP clearly endorsed this approach. The published 

literature amply demonstrates that inhibition of this protein is 

necessary but not sufficient for the initiation of OPIDN, and that 

it is highly correlated with the other signs of OPIDN, i.e. gait 

changes, and central-peripheral distal axonopathy. The assay itself 

was reviewed by the SAP in 1987 and has since been reviewed again 

after minor revision both in ORD and by a number of reviewers as 

well as the SAP. We believe that this assay possesses therefore, 

concurrent, predictive, and content validity. While inhibition of 

NTE is not, per se, an adverse effect, it is not being used as the 

sole basis of such assertions. 

12 
P.16 

NEUROTOXICITY SCREENING BATTERY1 

(a) Purpose. In the assessment and evaluation of the 

potential human health effects of chemical substances, it is 

appropriate to test for neurotoxic effects. This neurotoxicity 

screening battery consists of a functional observational battery, 

motor activity, and neuropathology. The functional observational 

battery consists of non-invasive procedures designed to detect 

gross functional deficits in animals and to better quantify 

behavioral 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 provide data to 

detect and characterize histopathological changes in the central 

and peripheral nervous system. This battery is designed to be 

used in conjunction 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 chemical. 

(b) Definitions. 

(1) Neurotoxicity is any adverse effect on the 

structure or function of the nervous system related to exposure 

to a chemical substance. 

(2) A toxic effect is an adverse change in the 

structure or function of an experimental animal as a result of 

exposure to a chemical substance. 

(3) Motor activity is any movement of the experimental 

animal. 

(c) Principle of the test method. The test substance is 

administered to several groups of experimental animals, one dose 

being used per group. The animals are observed under carefully 

standardized conditions with sufficient 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 automated device. The animals are perfused and tissue 

samples from the nervous 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. 

(d) Test procedures. (1) Animal selection, (i) Species. In 

general, the laboratory rat should be used. Under some 

circumstances, other species, such as the mouse or the dog, may 

be more appropriate, although not all of the battery may be 

13 
P.17 

Neuro Screen 

adaptable to other species. 

(ii) Age. Young adults (at least 42 days old for 

rats) shall be used. 

(iii) Sex. (A) Both males and females shall be 

used. 

(B) Females shall be nulliparous and 

nonpregnant. 

(2) Number of animals. At least ten males and ten 

females shall be used in each dose and control group for 

behavioral testing . At least five males and five females shall 

be used in each dose and control group for terminal 

neuropathology. If interim neuropathological evaluations are 

planned, the number shall be increased by the number of animals 

scheduled to be perfused before the end of the study. Animals 

shall be. randomly assigned to treatment and control groups. 

(3) Control groups, (i) A concurrent (vehicle) control 

group is required. Subjects shall 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 shall provide evidence of the ability of 

the observational methods used to detect major neurotoxic 

endpoints including limb weakness or paralysis (e.g., repeated 

exposure to acrylamide),tremor (e.g., pp'DDT), and autonomic 

signs (e.g.,carbaryl). Positive control data are also required to 

demonstrate the sensitivity and reliability of the activitymeasuring 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 

trimethyl tin). Positive control data shall be collected at the 

time of the test study unless the laboratory can demonstrate the 

adequacy of historical data for this purpose, i.e., by the 

approach outlined in this guideline . 

(4) Dose level and dose selection . At least 3 doses 

shall be used in addition to the vehicle control group. Ideally, 

the data should be sufficient to produce a dose-effect curve. We 

strongly encourage the use of equally spaced doses and a 

rationale for dose selection that will maximally 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. 1/2, 1/4, of the BD. 

The BD itself may be estimated as the highest non-lethal dose as 

determined in a preliminary range-finding lethality study. A 

variety of test methodologies may be used for this purpose, and 

14 
P.18 

Neuro Screen 

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 shall 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 benchmark dose procedure as described above, with the middle 

and low dose levels chosen as fractions of the benchmark dose. 

The lowest dose shall produce minimal effect, e.g. an ED10, or 

alternatively, no effects. 

(ii) Subchronic (and Chronic) Studies. The high 

dose need not be greater than lg/Kg. Otherwise, the high dose 

level shall result in significant neurotoxic effects or other 

clearly toxic effects, but not produce an incidence of fatalities 

that would prevent a meaningful evaluation of the data. The 

middle and low doses should be fractions of the high dose. The 

lowest dose shall 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 recognized 

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. 

(7) Study conduct, (i) Time of testing. All animals 

shall 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 shall be made before 

the initiation of exposure, at the estimated time of peak effect 

within 8 hours of dosing, and at 7 and 14 days after dosing. 

Estimation of time(s) of peak effect may be made by dosing pairs 

of rats across a range of doses and making regular observations 

of gait and arousal . 

(B) Subchronic (and Chronic) Studies . In a 

subchronic study, at a minimum, observations and activity 

measurements shall 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 

15 
P.19 

Neuro Screen 

chronic studies, at a minimum, observations and activity 

measurements shall be made before the initiation of exposure and 

before the daily exposure, 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 shall be observed 

carefully by trained observers who are unaware of the animal's 

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 inter-observer 

reliability is required. The animals shall 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. Explicit, operationally defined scales for each 

measure of the battery are to be used. The development of 

objective quantitative measures of the observational end-points 

specified is encouraged. Examples of observational procedures 

using defined protocols may be found in Irwin (1968), Gad (1982), 

and Moser et al. (1988). The functional observational battery 

shall include a thorough description of the subject's appearance, 

behavior, and functional integrity. This shall be assessed 

through: observations in the home cage; 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, shall be 

developed for those measures which involve subjective ranking. 

(B) List of measures. The functional 

observational battery shall include the following list of 

measures. 

(1) Assessment of signs of autonomic 

function, including but not limited to: 

a) ranking of the degree of 

lacrimation and salivation, with a range of severity scores from 

none to severe; 

b) presence or absence of 

piloerection and exophthalmus; 

c) ranking or count of urination 

and defecation, including polyuria and diarrhea. This is most 

easily conducted during the open field assessment. 

d) pupillary function such as 

constriction of the pupil in response to light or a measure of 

pupil size; 

e) degree of palpebral closure, 

e.g., ptosis. 

(2) Description, incidence, and severity 

of any convulsions, tremors, or abnormal motor movements, both in 

16 
P.20 

Neuro screen 

the home cage and the open field. 

(3) Ranking of the subject's reactivity 

to general stimuli such as removal from the cage or handling, 

with a range of severity scores from no reaction to 

hyperreactivity. 

(4) Ranking of the subject's arousal 

level or state of alertness during observations of the 

unperturbed subject in the open field, with a range of severity 

scores from coma to hyperalertness . 

(5) Descriptions and incidence of 

posture and gait abnormalities observed 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 ai^. (1979) . 

(8) Quantitative measure of landing foot 

splay ; the procedure described by Edwards and Parker (1977) 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. 

(11) 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 facilitate 

interpretation of the data. 

(C) Additional measures. Other measures may 

also be included and the development and validation of new tests 

is encouraged. Further information on the neurobehavioral 

integrity of the subject may be provided by: 

(1) 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 shall be 

monitored by an automated activity recording apparatus. The 

device used must be capable of detecting both increases and 

17 
P.21 

Neuro Screen 

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 shall 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 shall be tested 

individually. The test session shall be long enough for motor 

activity to approach asymptotic levels by the last 20 percent of 

the session for non-treated control animals. All sessions shall 

have the same duration. Treatment groups shall be counterbalanced across test times (See endnote 6). 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 humidity, 

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 preservation of cellular integrity 

for the detection of neuropathological alterations, tissue shall 

be prepared for histological analysis using in situ perfusion and 

paraffin and/or plastic embedding procedures. Paraffin embedding 

is acceptable for tissue samples from the central nervous system. 

Plastic embedding of tissue samples from the central nervous 

system is encouraged, 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 

additional methods, such as Bodian's or Bielchowsky's silver 

methods, and/or GFAP immunohistochemistry be used in conjunction 

with more standard stains to determine the lowest dose level at 

which neuropathological alterations are observed. When such 

special stains indicate evidence of structural alterations it is 

recommended that the GFAP radioimmunoassay also be performed, 

particularly when additional animals are available for use in the 

radioimmunoassay XS e e Appendix 1, Guideline for GFAP 

radioimmunoassay) . 

(A) Fixation and Processing of Tissue. The 

nervous system shall be fixed by in situ perfusion with an 

appropriate aldehyde fixative. Detailed descriptions of 

vascular perfusions may be found in Zeman and Innes (1963), Hayat 

(1970), Spencer and Schaumburg (1980), and Palay and Chan Palay 

(1974). Any gross abnormalities should be noted. Tissue 

samples taken shall adequately represent all major regions of the 

nervous system. Detailed dissection procedures are described in 

chapter 50 of Spencer and Schaumburg (1980) and in Palay and Chan 

Palay (1974). The tissue samples should be postfixed and 

18 
P.22 

Neuro Screen 

processed according to standardized published histological 

protocols such as AFIP (1968), WHO (1986), Spencer and 

Schaumburg (1980), Bennet et al. (1976), Di Sant Agnese and De 

Mesy Jensen (1984), or Pender (1985). Tissue blocks and 

slides shall be appropriately identified when stored. 

Histological sections shall be stained for hematoxylin and 

eosin (H&E), or a comparable stain according to standard 

published protocols such as AFIP(1968), Ralis et al. (1973), or 

Bennet et al. (1976). 

(B) Qualitative Examination . 

Representative histological sections from the tissue samples 

shall be examined 

microscopically by an appropriately 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 

distinguished 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 WHO 

(1986). A stepwise examination of tissue samples is recommended. 

In such a stepwise examination, sections from the high dose group 

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

neuropathological alterations are observed in samples from the 

high dose group, subsequent analysis is not required. If 

neuropathological alterations are observed in samples from the 

high dose group, samples from the intermediate and low dose 

groups are then examined sequentially. 

(C) Subjective Diagnosis . If any evidence 

of neuropathological alterations is found in the qualitative 

examination, then a subjective diagnosis will be performed 

for the purpose of evaluating dose-response relationships. 

All regions of the nervous system exhibiting any evidence 

of neuropathological changes shall 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 all dose groups including all regions have been rated, 

the code will be broken and statistical analysis performed 

to evaluate dose-response relationships. For each type of 

dose-related lesion observed, examples of different degrees 

of severity shall 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 

19 
P.23 

Neuro Screen 

the degree of severity ranging from very slight to very 

extensive. 

(e) 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 

j udgment. 

(i) A detailed description of the procedures used 

to standardize observations, including the arena and scoring 

criteria. Procedures for calibrating and assuring the equivalence 

of activity devices and balancing treatment groups should also be 

described. 

(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. We encourage 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 intra-session 

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 appropriate. 

(iii) All neuropathological observations shall 

be recorded and arranged by test groups. This data may be 

presented in the following recommended 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 

location(s), nature, frequency, and severity of lesion(s). 

Inclusion of photomicrographs is strongly recommended for 

20 
P.24 

Neuro Screen 

demonstrating typical examples of the type and severity of the 

neuropathological alterations observed is strongly recommended. 

Any diagnoses derived from neurological signs and lesions 

including naturally occurring diseases or conditions, shall be 

recorded. 

(B) Counts and incidence of neuropathological 

alterations by test group. Data shall be tabulated to show: 

(1) 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 shall include the 

relationship between the doses of the test substance and the 

presence or absence, incidence and severity, of any neurotoxic 

effects. The evaluation should include appropriate statisticalanalyses, for example, parametric tests for continuous data and 

non-parametric tests for the remainder. Choice of analyses 

should consider tests appropriate to the experimental design, 

including repeated measures. There may be many acceptable ways to 

analyze data. Statistical analysis comparing total activity 

counts of treatment vs control animals at each measured time must 

be made and supplied. The report must include dose-effect curves 

for observations, motor activity expressed as activity counts, 

and any gross necropsy findings and lesions observed. 

(f) References. For additional background information on 

this test guideline the following references should be consulted: 

AFIP. Manual of Histologic Staining Methods New York: McGraw 

Hill, 1968. 

Bennet, H.S., Wyrick, A.D., Lee, S.W., McNeil, J.H. "Science and 

art in the 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). 

Di Sant Agnese, P.A., De Mesy Jensen, K. "Dibasic staining of 

large epoxy sections and application to surgical pathology" 

American Journal of Clinical pathology 81: 25-29 (1984). 

Edwards, P.M., Parker V.H. "A simple, sensitive and objective 

method for early assessment of acrylamide neuropathy in rats," 

Toxicology and Applied Pharmacology. 40: 589-591 (1977). 

Finger, F.W. "Measuring behavioral activity," Methods in 

Psychobiology Vol. 2 Ed. R.D. Myers . New York: Academic Press. 

21 
V 

Neuro Screen 

pp.1-19 (1972). 

Gad, S. A neuromuscular screen for use in industrial toxicology. 

J.Toxicol. Environ. Health 9:691-704 (1982) 

Hayat, M.A. "Volume 1. Biological applications." Principles and 

Techniques of Electron Microscopy. New York, Van Nostrand 

Reinhold. (1970). 

Irwin, S. "Comprehensive observational assessment: Ia. A 

systematic quantitative procedure for assessing the behavioral 

physiological state of the mouse," Psychopharmacologia. 13: 222-

257 (1968). 

Kinnard, E.J. and Watzman, N. "Techniques utilized in the 

evaluation of psychotropic drugs on animals activity." Journal of 

Pharmaceutical Sciences. 55: 995-1012 (1966). 

Meyer, O.A., Tilson, H.A., Byrd, W.C, and Riley, W.T. A method 

for the routine assessment of fore- and hindlimb grip strength of 

rats and mice. Neurobehav. Toxicol. 1:233-236 (1979) 

Moser V.C., Mc Cormick J.P., Creason J.P., and MacPhail R.C. 

Comparison of chlordimeform and carbaryl using a functional 

observational battery. Fund. Appl. Toxicol. 11:189-206 (1988). 

Palay, S.L., Chan Palay, V. Cerebellar Cortex: Cytology and 

Organization New York: Springer Verlag. (1974). 

Pender, M.P. "A simple method for high resolution light 

microscopy of nervous tissue" Journal of Neuroscience Methods 15: 

213-218 (1985). 

Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in 

Neurohistology London: Butterworths. (197 3). 

Reiter, L.W. "Use of activity measures in behavioral toxicology," 

Environmental Health Perspectives. 26: 9-20 (1978). 

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). 

Robbins, T.W. "A critique of the methods available for the 

measurement of spontaneous motor activity." Handbook of 

Psvchopharmacology. Vol 7. Eds. Iversen, L.L., Iverson, D.S., 

Snyder, S.H. New York: Plenum Press, pp. 37-82 (1977). 

Spencer, P.S., Schaumburg, H.H. (eds) Experimental and Clinical 

Neurotoxicology Baltimore: Williams and Wilkins (1980). 

22 
P.26 

Neuro Screen 

WHO, Principles and Methods for the Assessment of 

Neurotoxicity Associated with Exposure to Chemicals 

(Environmental Health Criteria 60) World Health 

Organizations Publications Center USA, Albany, New York 

(1986) 

Zeman, W., Innes, J.R. Craigie's Neuroanatomy of the Rat New 

York: Academic Press. (1963). 

NOTES 

1. This version of these Neurotoxicity Test Guidelines represents 

a joint effort of the Offices of Pesticide Programs (OPP) and the 

Office of Toxic Substances (OTS), in cooperation with many 

scientists in the Office of Research and Development (ORD), to 

develop a common set of guidelines for their testing 

requirements. This OPTS version grew out of the set of guidelines 

developed and eventually published by OTS (50 FR 39397 9/27/85; 

amended at 52 FR 19082, 5/20/87). The revisions were initiated by 

an ad hoc Workgroup of scientists from these 3 offices. They were 

presented for review by the Scientific Advisory Panel of OPP and 

made available for public comment for 2 months. Over 30 groups 

and individuals submitted comments. Many sections of these 

neurotoxicity guidelines have been revised to take account of 

these comments. The rationale for some of these general revisions 

is provided here. What earlier were separate guidelines for the 

functional observational battery, motor activity, and 

neuropathology have now been combined into this single guideline 

both for efficiency and because they were designed to be used 

together. 

2. The Agency recognizes that tests of motor acitivity alone do 

not provide a complete evaluation of the effects of a chemical on 

the nervous system. However, the automated test of motor activity 

will provide an objective assessment of neurobehavioral function, 

as well as the only measurement of habituation, which is an 

indication of the organism's ability to adapt to its environment. 

3. The power calculations to determine group size for motor 

activity have been deleted. Group sizes of ten/sex will be 

sufficient for well designed and executed studies. Poorly 

designed and/or executed studies may be judged invalid. The 

original intent was to allow for flexibility in the use of 

devices with different operating characteristics (larger variance 

in measures necessitate larger group sizes). Comments were mostly 

negative, based on concerns about perceived uncertainty of the 

adequacy of a sample size until a study was complete. 

23 
P.27 

Neuro Screen 

4. Positive control data need only be generated approximately 

once every year as long as most conditions remain the same. Each 

observer should conduct such studies as part of their training. 

The untreated control data from training studies could then be 

submitted as part of the laboratory's historical control data. 

5. The adequacy of the high dose for hazard identification is 

one of the most critical issues of experimental design. The high 

dose criteria in these guidelines now include both a limit dose 

and " significant neurotoxic effects or other clearly toxic 

effects". 

These revised criteria are very similar to those for 

systemic studies and are intended by this to facilitate combined 

studies. 

We now also include some guidance for dose selection in 

acute studies based on a benchmark dose criterion and fractions 

thereof. 

This is intended to provide an example of one operational means 

of establishing a set of acute doses and to reduce the number of 

animals used for estimates of lethal doses. 

These 3 dose studies are intended to both identify effects 

of exposure and to estimate dose levels without adverse effect. 

We have added language to encourage greater emphasis on obtaining 

dose response data, e.g., equally spaced doses and lesser 

emphasis on a low dose totally without effect. This was done for 

2 reasons. First, the presence or absence of dose-related changes 

can be critical in the evaluation of effects of exposure. Second, 

various methods using an ED10 as the basis for estimating 

reference doses are increasingly discussed by many authors. 

We strongly urge sponsors to seek guidance from the Agency 

before initiating their studies and to provide a rationale for 

dose selection in these studies. 

Several commenters were concerned about the interpretation 

of effects seen at levels where other significant toxicity was 

present and questioned the need for or efficiency of testing at 

such doses. First, in OPP, the 90 day Neurotoxicity Study is 

intended as a screen prior to inclusion of neurotoxicity tests in 

chronic studies. In OTS, subchronic non-oncogenicity studies are 

generally considered sufficient for evaluating chronic toxicity. 

Thus the doses of a 90 day neurotoxicity study for either office 

should be maximized to encourage the identification of chronic 

effects. 

Further, concurrent toxicity does not obviate the need to 

identify other kinds of effects that may be more important in 

different situations, either for other groups or after different 

exposure regimens. Ultimately, all of the targets of a toxicant 

may be important for identifying affected individuals or be the 

critical effect under a variety of exposure conditions or in 

different groups. 

24 
P.28 

Neuro Screen 

6. The term benchmark dose as defined here is for the purpose of 

dose selection and should not be confused with the use of this 

term for the purpose of establishing a common dose such as an 

ED10 for extrapolation in risk assessment. 

7. The time of peak effect refers to the time within 

approximately 8 hours after dosing and was intended to help 

maximize detection of effects of acute exposure. 

8. For repeated exposure studies using routes other than through 

the diet, the intent of testing before the daily dose is to 

minimize the impact of that day's dose. We recognize that for 

some materials, residual material from preceding dose or doses 

may be the source of observed effects, but these are part of the 

effects of concern. In some studies, examination of animals 

following exposure may help to further describe the duration of 

such effects. 

9. Not all rats must be tested in one day, but time of testing 

should be balanced across groups, and for any other potential 

confounds, e.g., sex. 

10. Measures of reactivity refer to the subject's reaction to 

some external stimulus, e.g., removal from the cage or handling, 

while arousal or state of alertness refers to the behavior of the 

undisturbed subject observed in the open field. This is often 

described more technically as the distinction between respondent 

and operant behaviors. 

11. Landing foot splay and grip strength do not measure the same 

function. These tests are viewed as complementary, and having 

both will aid in the interpretation of data. 

12. The goal of the procedures outlined for the preparation and 

processing of tissue samples is to optimally preserve tissue 

morphology for microscopic examination. The higher resolution 

obtainable in plastic embedded tissue is considered to optimize 

the detection of a number of types of lesions, particularly in 

the peripheral nervous system. In contrast, paraffin-embedded 

material is more amenable to sampling large regions of the 

nervous system and is considered optimal for a variety of special 

stains that may be useful in characterizing neuropathological 

alterations. Several organizations felt the requirement for 

separate animals for plastic and paraffin-embedding of tissue 

samples was excessive. Furthermore, commentors presented views 

that differences in plastic and paraffin techniques did not 

25 
Neuro Screen 

require the use of separate animals and that in an appropriate 

tiered evaluation scheme both plastic and paraffin-embedding 

could be used with tissue samples from the same animals. The 

requirement for separate animals is now eliminated, with paraffin 

being acceptable and plastic being recommended. 

13. The higher resolution obtainable in plastic embedded tissue 

is considered to optimize the detection of a number of types of 

lesions, particularly in the peripheral nervous system. Paraffin 

embedded material is more amenable to sampling large regions of 

the nervous system and is considered optimal for a variety of 

special stains that may be useful in characterizing 

neuropathological alterations. 

14. Although, EPA believes that the GFAP assay has been shown to 

be sensitive to the neurotoxic effects of agents in both the 

adult and developing nervous systems, this assay has been deleted 

at this time. However, the Agency will continue using this assay 

experimentally and encourages others to do so, as well, in an 

effort to obtain additional validation of its use as a means to 

assess the neurotoxic potential of agents. In addition, if GFAP 

immunohistochemistry is used as a special stain in the 

neuropathology segment of the testing protocol and evidence of a 

glial response to toxicant injury is observed, application of the 

radioimmunoassay is encouraged in order to provide objective, 

quantitative dose-response data. 

15. The Agency received some comments that the list of specified 

regions of the nervous system to be examined was inadequate, 

while others felt it was too detailed. Moreover, comments were 

received that argued that the list of potential types of 

neuropathological alterations also was too restricted. These 

lists were intended to serve as guidance. Since they appear to 

be subject to misinterpretation, the requirement for a thorough 

examination of the nervous system for any evidence of 

neuropathological alteration is now explicitly stated. In 

addition, a list of all structures examined is required in the 

final report. The requirement for examination of more than one 

section per region, however, has now been deleted. 

16. The purpose of the semi-quantitative analysis is to evaluate 

the relationship between the incidence and severity of the 

neuropathological alterations and the exposure. Since the rating 

scale is by necessity subjective, it is necessary to ensure that 

any bias resulting from the previous qualitative examination of 

the tissue samples is minimized. Several organizations commented 

26 
P.30 

Neuro Screen 

that blind evaluation of tissue sections should not be required. 

In the initial qualitative analysis, in which the types of 

lesions and regions affected are first identified, blind 

evaluation is not required. However, in the semi-quantitative 

analysis in which the dose-response relationship is evaluated, it 

is imperative that the evaluation be as objective as possible. 

Moreover, since the semi-quantitative analysis focuses on a 

limited number of regions for lesions previously described in the 

qualitative analysis, blind reading is required to ensure 

objectivity. Thus, it is required that the subjective rating of 

the severity and incidence be performed without knowledge of 

treatment.. 

17. The data and analyses supplied in the report must be 

evaluated by Agency risk assessors. Thus, the report must be 

sufficiently detailed for the Agency to evaluate the quality of 

the study. Since no list of regions to be examined is outlined 

in the guideline, a list of regions examined must be supplied 

with the report. Similarly, an adequate description of lesions -

observed must be supplied also. The Agency received comments 

that the requirement of photomicrographs to document 

neuropathological alterations was extremely costly. The Agency 

has decided to recommend, rather than require, the use of 

photomicrographs to aid in the description of typical examples of 

treatment-related lesions. 

27 
P.31 

Neuro Screen 

APPENDIX 1 

GUIDELINE FOR ASSAYING GLIAL FIBRILLARY ACIDIC PROTEIN 

(a) Purpose. Chemical-induced injury of the nervous system 

is associated with astrocytic hypertrophy at the site of damage 

(see O'Callaghan, 1988). Assays of glial fibrillary acid protein 

(GFAP), the major intermediate filament protein of astrocytes, 

can be used to document this response. To date, a diverse 

variety of chemical insults known to be injurious to the central 

nervous system have been shown to increase GFAP. Moreover, 

increases in GFAP can be seen at dosages below those necessary to 

produce cytopathology as determined by routine Nissl stains 

(standard neuropathology). Thus, it appears that assays of GFAP 

represent a sensitive approach for documenting the existence and 

location of chemical-induced injury of the central nervous 

system. 

(b) Principle of the test method. This guideline will 

describe the conduct of a radioimmunoassay for measurement of the 

amount of GFAP in the brain of exposed and control animals. It 

is based on modifications (O'Callaghan & Miller 1985, O'Callaghan 

1987, O'Callaghan and Miller, 1988) of the dot-immunobinding 

procedure described by Jahn et al. (1984). Briefly, samples are 

assayed for total protein, diluted in dot-immunobinding buffer, 

and applied to nitrocellulose sheets. The spotted sheets are 

then fixed, blocked, washed, and incubated in anti-GFAP and 

[1251] Protein A. Bound protein A is then quantified by gamma 

spectrometry. In lieu of purified protein standards, standard 

curves are constructed from dilution of a single control sample. 

By comparing the immunoreactivity of individual samples (both 

control and treated groups) with that of the sample used to 

generate the standard curve, the relative immunoreactivity of 

each sample is obtained. The immunoreactivity of the control 

groups is normalized to 100% and all data are expressed as a 

percentage of control. This biochemical test is intended to be 

used in conjunction with behavioral and pathological studies as 

part of the screening battery that includes the functional 

observational battery, motor activity and histopathology. 

(c) Test procedure. (1) Animal selection, (i) Species and 

strain. Test shall be performed in the species being used in 

other tests for neurotoxicity. This will generally be the 

laboratory rat. 

(ii) Age. Based on the other concurrent testing 

young adult rats shall be used. 

(iii) Number of animals. At least 6 animals per 

dose shall be used. 

(2) Materials: [1251] Protein A (2-10 uCi/ug), Antisera 

to GFAP, Nitrocellulose paper (0.1 or 0.2 um pore size), a sample 

28 
P.32 

Neuro Screen 

application template (optional, e.g. Minifold II, Schleicher & 

Schuell, Keene NH), plastic incubation trays. 

(3) Study conduct, (i) Tissue Preparation. Animals are 

killed by decapitation 72 hours after the last dose. The brain 

is excised from the skull. The following six regions are then 

dissected freehand: cerebellum, cerebral cortex, hippocampus, 

striatum, thalamus/hypothalamus, and the rest of the brain. 

Each region is then weighed and homogenized in 10 volumes of 

hot (70-90 degrees C) 1% (w/v) sodium dodecyl sulfate (SDS). 

Homogenization is best achieved through sonic disruption. A 

motor driven pestle inserted into a tissue grinding vessel 

is a suitable alternative. The homogenized samples can then 

be stored frozen at -70 C for at least 4 years without loss 

of GFAP content. 

(ii) Total Protein Assay. Aliquots of the tissue 

samples are assayed for total protein using the method of Smith 

et. al. (1985). This assay is available in kit form (Pierce 

Chemical Company, Rockford, IL). 

(iii) Sample Preparation. Dilute tissue samples' 

in sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHC03), 2 mM 

MgC12), 5 mM Hepes, pH 7.4, 0.7% Triton X-100) to a final 

concentration of 0.25 mg total protein per ml (5 ug/20 ul). 

(iv) Preparation of Standard Curve. Dilute a 

single control sample in sample buffer to give at least five 

standards, between 1 and 10 ug total protein per 20 ul. The 

suggested values of total protein per 20 ul sample buffer are: 

1.25, 2.50, 3.25, 5.0, 6.25, 7.5, 8.75, and 10.0 ug. 

(v) Preparation of Nitrocellulose Sheets. 

Nitrocellulose sheets of 0.1 or 0.2 micron pore size are rinsed 

by immersion in distilled water for 5 minutes and then air dried. 

(vi) Sample Application. Samples can be spotted 

onto the nitrocellulose sheets free-hand or with the aid of a 

template. For free-hand application, draw a grid of squares 

approximately 2 cm by 2 cm on the nitrocellulose sheets 

using a soft pencil. Spot 5-10 ul portions to the center of 

each square for a total sample volume of 20 ul. For 

template aided sample application a washerless microliter 

capacity sample application manifold is used. Position the 

nitrocellulose sheet in the sample application device as 

recommended by the manufacturer and spot a 20 ul sample in 

one application. Do not wet the nitrocellulose or any 

support elements prior to sample application. Do not apply 

vacuum during or after sample application. After spotting 

samples (using either method), let the sheets air dry. The 

sheets can be stored at room temperature for several days 

after sample application. 

(vii) Standard Incubation Conditions. These 

conditions have been described by Jahn et al. (1984). All steps 

are carried out at room temperature on a flat shaking platform 

(one complete excursion every 2-3 seconds). For best results do 

29 
P.33 

Neuro Screen 

not use rocking or orbital shakers. Perform the following 

steps in enough solution to cover the nitrocellulose sheets 

to a depth of 1 cm. 

(A) Incubate 20 minutes in fixer (25% v/v 

isopropanol, 10% v/v acetic acid). 

(B) Discard fixer, wash several times in 

deionized water to eliminate the fixer, and then incubate for 5 

minutes in Tris-buffered saline (TBS, 200 mM NaCl, 60 mM Tris-HCl 

pH 7.4). 

(C) Discard TBS and incubate 1 hour in 

blocking solution (0.5% gelatin (w/v) in TBS. 

(D) Discard blocking solution and incubate 

for 2 hours in antibody solution (anti-GFAP antiserum diluted to 

the desired dilution in blocking solution containing 0.1% Triton 

X-100). Serum antibovine GFAP, which cross reacts with GFAP from 

rodents and humans, can be obtained commercially (e.g. Dako 

Corp.) and used at a dilution of 1:500. 

(E) Discard antibody solution, wash in 4 

changes of TBS for 5 minutes each time. Then wash in TBS for 10" 

minutes. 

(F) Discard TBS and incubate in blocking 

solution for 30 minutes. 

(G) Discard blocking solution and incubate 

for 1 hour in Protein A solution ([1251]-labeled Protein A 

diluted in blocking solution containing 0.1% Triton X-100, 

sufficient to produce 2000 cpm per 10 ul of protein A solution). 

(H) Remove protein A solution (it can be 

reused once). Wash in 0.1% Triton X-100 in TBS (TBSTX) for 5 

minutes, 4 times. Then wash in TBSTX for 2-3 hours for 4 

additional times. An overnight wash in a larger volume can be 

used to replace the last 4 washes. 

(I) Hang sheets up to dry, cut out squares or 

spots and count radioactivity in a gamma counter. 

(viii) Expression of data. Compare radioactivity 

counts for samples obtained from control and treated animals with 

counts obtained from the standard curve. By comparing the 

immunoreactivity (counts) of each sample with that of the 

standard curve, the relative amount of GFAP in each sample 

can be determined and expressed as a percent of control. 

(d) Data Reporting and Evaluation. (1) Test Report. The 

final test report shall include the following information: 

(i) Body weight and brain region weights at time 

of sacrifice for each subject tested. 

(ii) Indication of whether each subject survived 

to sacrifice or time of death. 

(iii) Data from control animals and blank samples. 

(iv) Statistical evaluation of results. 

(2) Evaluation of Results, (i) Results shall be 

evaluated in terms of the extent of change in the amount of GFAP 

30 
P.34 

Neuro Screen 

as a function of treatment and dose. GFAP assays (of any brain 

region) from 6 samples typically result in a standard error of 

the mean of +/~ 5%. Chemical-induced increase of GFAP of 115% of 

control are likely to be statistically significant. 

(ii) Results of this assay shall be compared to 

and evaluated with behavioral and histopathological data. 

fe) References. For additional background information on 

this test guideline the following references should be consulted:: 

Brock, T.O. and O'Callaghan, J.P. 1987. Quantitative changes 

in the synaptic vesicle proteins, synapsin I and p38 and the 

astrocyte specific protein, glial fibrillary acidic protein, 

are associated with chemical-induced injury to the rat 

central nervous system. J. Neurosci. 7:931-942 

Jahn, R., Schiebler, W. Greengard, P. 1984. A quantitative 

dot-immunobinding assay for protein using nitrocellulose 

membrane filters. Proc. Natl. Acad. Sci. U.S.A. 81:1684-

1687. 

O'Callaghan, J. P. 1988. Neurotypic and gliotypic protein as 

biochemical markers of neurotoxicity. Neurotoxicol. Teratol. 

10:445-452. 

O'Callaghan, J. P. and Miller, D. B. 1988. Acute exposure of 

the neonatal rat to triethyltin results in persistent 

changes in neurotypic and gliotypic proteins. J. Pharmacol. Exp. 

Ther. 244:368-378. 

O'Callaghan, J. P. and Miller, D. B. 1985. Cerebellar 

hypoplasia in the Gunn rat is associated with quantitative 

changes in neurotypic and gliotypic proteins. J. Pharmacol. Exp. 

Ther. 234:522-532. 

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A. K. 

Gartner, F. H., Provenzano, M.D., Fujimoto, E. K. , Goeke, 

N.M. Olson, B.J., Klenk, D.C. 1985. Measurement of protein 

using bicinchoninic acid. Annal. Biochem. 150:76-85 

31 
P.35 

DEVELOPMENTAL NEUROTOXICITY STUDY1 

(a) Purpose. In the assessment and evaluation of the toxic 

characteristics 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 exposure of 

the mother during pregnancy and lactation. 

(b) 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 observations 

to detect gross neurologic and behavioral abnormalities, 

determination 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 

follow-up to a standard developmental toxicity and/or adult 

neurotoxicity study, or as part of a two-generation reproductionstudy, with assessment of the offspring conducted on the F2 

generation. 

(c) 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 tested 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 

justification/reasoning for this selection must be provided. 

(ii) Age. Young adult (nulliparous females) 

animals shall be used. 

(iii) Sex. Pregnant female animals shall 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 adequate number of offspring are 

produced for neurotoxicity evaluation. At least 20 litters are 

recommended at each dose level. For behavioral tests, one female 

and one male pup per litter shall be randomly selected and 

assigned to one of the tests. 

(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 possible, 4 male and 4 females 

per litter. Whenever the number of pups of either sex prevents 

having four of each sex per litter, partial adjustment (for 

example, 5 males and 3 females) is permitted. Testing is not 

appropriate for litters of less than 7 pups. Elimination of 

runts only is not appropriate. Individual pups should be 

32 
P.36 

Devel Neuro 

identified uniquely after standardization of litters. A method 

that may be used for identification can be found in Adams et al. 

(1985) . 

(C) Assignment of animals for behavioral 

tests, brain weights, and neuropathological evaluations. After 

standardization of litters, one male and one female from each 

litter shall 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 one male 

or one female pup from each litter (total of 10 males and 10 

females/dose group) shall be sacrificed. Brain weights shall be 

measured in all of these pups and, of these pups, 6/sex/dose 

shall be selected for neuropathological evaluation. At the 

termination of the study, either one male or one female from each 

litter (total of 10 males and 10 females/dose group) shall be 

sacrificed and brain weights shall be measured. An additional 

group of 6 animals/sex/dose group (one male or one female per 

litter) shall be sacrificed at the termination of the study for. 

neuropathological evaluation. 

(2) Control groups. A concurrent control group(s) is 

(are) required. This group shall be a sham-treated group or, if 

a vehicle is used in administering the test substance, a vehicle 

control group. The vehicle shall neither be developmentally 

toxic nor have effects on reproduction. Animals in the control 

group(s) shall be handled in an identical manner to test group 

animals. 

(3) Dose levels and dose selection, (i) At least 3 

dose levels of the test substance plus a control group (vehicle 

control, if a vehicle is used) shall 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 shall be the 

maximum dose which will not induce in utero or neonatal death or 

malformations sufficient to preclude a meaningful evaluation of 

neurotoxicity . 

(iii) If a standard developmental toxicity study 

has not been conducted, the highest dose level, unless limited by 

the physico-chemical nature or biological properties of the 

substance, shall induce some overt maternal toxicity, but shall 

not result in a reduction in weight gain exceeding 2 0% during 

gestation and lactation. 

(iv) The lowest dose should not produce any 

grossly observable evidence of either maternal or developmental 

neurotoxicity. 

(v) The intermediate dose(s) shall 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 shall cover the period from day 6 of gestation through day 

10 postnatally. Dosing should not occur on the day of 

33 
P.37 

Devel Neuro 

parturition in those animals who have not completely delivered 

their offspring. 

(5) Administration of the test substance. The test 

substance or vehicle shall 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 shall be administered at the same time 

each day. The animals shall be weighed periodically and the 

dosage to be administered based on the most recent weight 

determination. 

(6) Observation of dams, (i) A gross examination of the 

dams shall be made at least once each day before daily treatment. 

The animals shall be observed by trained technicians, who are 

unaware of the animal's 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 

inter-observer reliability is required. 

(ii) During the treatment and observation 

periods, observations shall include: 

(A) Assessment of signs of autonomic 

function, including but.not limited to: 

(1) 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 response to 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. 

(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, hypotonia or hypertonia, 

altered fur appearance, red or crusty deposits around the eyes, 

nose, or mouth, and any other observations that may facilitate 

interpretation of the data. 

(iii) Signs of toxicity shall be recorded as they 

are observed, including the time of onset, degree, and duration. 

(iv) Animals shall be weighed at least weekly and 

on the day of delivery and postnatal days 11 and 21 (weaning); 

such weights shall be recorded. 

(v) The day of delivery of litters shall be 

34 
P.38 

Devel Neuro 

recorded and considered as postnatal day 0. 

(7) Study conduct, (i) Observation of offspring. 

(A) All offspring shall be examined cage-side 

at least daily for gross signs of mortality or morbidity. 

(B) All offspring shall be examined outside 

the cage for gross signs of toxicity whenever they are weighed or 

removed from their cages for behavioral testing. The offspring 

shall 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. At a minimum, the end points 

outlined in paragraph (6) (ii) shall be monitored as appropriate 

for the developmental stage being observed. 

(C) Any gross signs of toxicity in the 

offspring shall be recorded as they are observed, including the 

time of onset, degree, and duration. 

(ii) Developmental landmarks. Live pups shall be 

counted and each pup within a litter shall be weighed 

individually at birth or soon thereafter, and on postnatal days 

4, 11, 17, 21 and at least once every two weeks thereafter. The 

age of vaginal opening and preputial separation shall be 

determined. General procedures for these determinations may be 

found in Adams et al. (1985), and Korenbrot et al. 

(1977)respectively. 

(iii) Motor activity. Motor activity shall 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., 

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 shall 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 shall be tested individually. The 

test session shall be long enough for motor activity to approach 

asymptotic levels by the last 20 percent of the session for nontreated control animals. All sessions shall have the same 

duration. Treatment groups shall be counter-balanced across test 

times. Animals' activity counts shall be collected in equal 

time periods of no greater than 10 minutes duration. Efforts 

shall 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, temperature, relative 

humidity, light conditions, odors, use of home cage or novel test 

cage, and environmental distractions. Additional information on 

35 
P.39 

Devel Neuro 

the conduct of a motor activity study may be obtained in the 

Office of Pesticides and Toxic Substances Neurotoxicity Screening 

Battery Guideline. 

(iv) Auditory startle test. An auditory startle 

habituation test shall be performed on the offspring on days 22 

and 60+2 . Details on the conduct of this testing may be 

obtained in Adams et al. (1985). 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) 

shall be made. While use of pre-pulse inhibition is not a 

requirement, it is highly recommended. Details on the conduct of 

this test may be obtained from Ison (1984). 

(v) Learning and memory tests. A test of 

associative learning and memory shall be conducted around the 

time of weaning (postnatal day 21-24) and at adulthood (postnatal 

day 60 + 2). The same or separate test(s) may be used at these 

two stages of development. Some flexibility is allowed in the 

choice of test(s) for learning and memory in weanling and adult 

rats. However, the test(s) must be designed so as to fulfill two 

criteria. First, learning must be assessed either as a change 

across several repeated learning trials or sessions, or, in tests 

involving a single trial, with reference to a condition that 

controls for non-associative effects of the training experience. 

Second, the test(s) shall include some measure of memory (shortterm or long-term) in addition to original learning 

(acquisition), but note that this measure of memory cannot be 

reported in the absence of a measure of acquisition obtained frora 

the same test. If the test(s) of learning and memory reveal(s) 

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 in 

Bushnell (1988) and for the infant rat in Green and Stanton 

(1988, Experiment 2); olfactory conditioning, as described in 

Kucharski and Spear (1984, Experiment 3); and acquisition and 

retention of schedule-controlled behavior, e.g., Cory-Slechta et 

al., 1983, and Campbell and Haroutunian, 1981. Additional tests 

for weanling rats are described in Spear and Campbell (1978) and 

Krasnegor et al. (1986), and for adult rats in Miller and 

Eckerman (1986). 

(iv) Neuropathology . Neuropathological 

evaluation shall be conducted on animals on postnatal day 11 and 

at the termination of the study. At 11 days of age, one male or 

female pup shall be removed from each litter such that equal 

36 
P.40 

Devel Neuro 

numbers of male and female offspring are removed from all litters 

combined. Of these, 6 male and 6 female pups will be sacrificed 

for neuropathological analysis. The pups will be killed by 

exposure to carbon dioxide and immediately thereafter the brains 

shall 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 shall be removed and 

weighed. In addition, 6 animals/sex/dose group (one male or 

female per litter) shall be sacrificed at the termination of the 

study for neuropathological evaluation. Neuropathological 

analysis of animals sacrificed at the termination of the study 

shall be performed in accordance with the Office of Pesticides 

and Toxic Substances Neurotoxicity Screening Battery. 

Neuropathological evaluation of animals sacrificed on postnatal 

day 11 and at termination of the study shall include a 

qualitative analysis and semi-quantitative analysis as well as 

simple morphometries. 

(A) Fixation and Processing of Tissue 

Samples for Postnatal Day 11 Animals. Immediately following 

removal, the brain shall be weighed and immersion fixed in an 

appropriate aldehyde fixative. The brains should be postfixed 

and processed according to standardized published histological 

protocols a such as the AFIP (1968), Spencer and Schaumburg 

(1980), Di Sant Agnese and De Mesy Jensen (1984), or Pender 

(1985). Paraffin embedding is acceptable but plastic embedding 

is preferred and recommended. Tissue blocks and slides shall be 

appropriately identified when stored. Histological sections 

shall be stained for hematoxylin and eosin, or a similar stain 

according to standard published protocols such as AFIP (1968), 

Ralis et al. (1973), or Bennet et al. (1976). For animals 

sacrificed at the termination of the study, methods for fixation 

and processing of tissue samples are provided in the section 

"Fixation and Processing of Tissue Samples" in the OPTS 

Neurotoxicity Screening Battery. 

(B) Qualitative Analysis. The purposes of 

the qualitative examination are: one, to identify regions within 

the nervous system exhibiting evidence of neuropathological 

alterations; two, to identify types of neuropathological 

alterations resulting from exposure to the test substance; and 

three, to determine the range of severity of the 

neuropathological alterations. Representative histological 

sections from the tissue samples shall be examined 

microscopically by an appropriately trained pathologist for 

evidence of neuropathological alterations. The following 

stepwise procedure is recommended for the qualitative analysis. 

First, sections from the high dose group are compared with those 

of the control group. If no evidence of neuropathological 

37 
P.41 

Devel Neuro 

alterations are 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 judgement and the kind of neuropathological 

alterations observed, it is recommended that additional methods 

such as Bodian's or Bielchowsky's silver methods and/or 

immunohistochemistry for glial fibrillary acid protein be used in 

conjunction with more standard stains to determine the lowest 

dose level at which neuropathological alterations are observed . 

Evaluation of postnatal day 11 pups is described in sections (l) 

and (2) below. For animals sacrificed at the termination of the 

study, the regions to be examined and the types of alterations 

that shall be assessed are identified in the section "Qualitative 

Examination" in the OPTS Neurotoxicity Screening Battery. 

(1) 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 cerebellum] to insure a thorough 

examination. 

(2) Types of Alterations. Guidance for 

neuropathological examination for indications of developmental 

insult to the brain can be found in Friede (1975) and Suzuki 

(1980). 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: 

a) gross changes in the size or 

shape of brain regions such as alterations in the size of the 

cerebral hemispheres or the normal pattern of foliation of the 

cerebellum; 

b) the death of neuronal 

precursors, abnormal proliferation, or abnormal 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 Miale and 

Sidman (1961) for discussion]; 

c) abnormal differentiation, while 

more apparent with special stains, may also be indicated by 

shrunken and malformed cell bodies; 

d) evidence of hydrocephalus, in 

particular enlargement of the ventricles, stenosis of the 

cerebral aqueduct and general thinning of the cerebral 

hemispheres. 

38 
Devel Neuro 

(C) Subjective Diagnosis. If any evidence 

of neuropathological alterations is found in the qualitative 

examination, then a subjective diagnosis will be performed for 

the purpose of'evaluating dose-response relationships. All 

regions of the brain exhibiting any evidence of neuropathological 

changes shall 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 

severity 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 statistical analyses performed to 

evaluate dose-response relationships. For each type of doserelated lesion observed, examples of different ranges of severity 

shall 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 very extensive. 

(D) Simple Morphometric Analysis. Since the 

disruption of developmental processes is sometimes more clearly 

reflected in the rate or extent of growth of particular brain 

regions, some form of morphometric analysis shall be performed on 

postnatal day 11 and at the termination of the study to assess 

the structural development of the brain. At a minimum, 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 (1971). 

(e) Data collection, reporting, and evaluation. The 

following specific information shall be reported: 

(1) Description of test system and test methods. A 

description of the general design of the experiment should be 

provided. This shall include: 

(i) A detailed description of the procedures used 

to standardize observations and procedures as well as operational 

definitions for scoring observations. 

(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 laboratory must 

demonstrate competence in evaluating effects in neonatal animals 

perinatally exposed to chemicals and establish test norms for the 

appropriate age group. 

(iii) Procedures for calibrating and ensuring the 

equivalence of devices and the balancing of treatment groups in 

testing procedures. 

(iv) A short justification explaining any 

decisions involving professional judgment. 

(2) Results. The following information must be 

arranged by each treatment and control group: 

(i) In tabular form, data for each animal must be 

39 
P.43 

Devel Neuro 

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; total session 

activity counts and intrasession subtotals on each day measured; 

auditory startle response amplitude per session and intrasession 

amplitudes on each day measured; appropriate data for each 

repeated trial (or session) showing acquisition and retention 

scores on the test(s) of learning and memory on each day 

measured; time and cause of death (if appropriate), any 

neurological signs observed, a list of structures examined as 

well as the location(s), nature, frequency, and extent of 

lesion(s); and brain weights. Inclusion of photomicrographs 

demonstrating typical examples of the type and extent of the 

neuropathological alterations observed is recommended. Any 

diagnoses derived from neurological signs and lesions, including 

naturally-occurring diseases or conditions, should also be 

recorded. 

(ii) Summary data for each treatment and control 

group must include: 

(A) The number of animals at the start of the 

test. 

(B) The body weights 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 observation time. 

(E) The percentage of animals showing each 

abnormal sign at each observation time. 

(F) The mean and standard deviation for each 

continuous end point at each observation time. These will 

include body weight, motor activity counts, auditory startle 

responses, performance in learning and memory test(s), 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 for each animal. 

(I) The values of all morphometric 

measurements made for each animal listed by treatment group. 

(3) Evaluation of data. An evaluation of test results 

must be made. The evaluation shall include the relationship 

between the doses of the test substance and the presence or 

absence, incidence, and extent of any neurotoxic effect. The 

evaluation shall include appropriate statistical analyses. The 

choice of analyses shall consider tests appropriate to the 

experimental design and needed adjustments for multiple 

40 
P.44 

Devel Neuro 

comparisons. The evaluation shall include the relationship, if 

any, between observed neuropathological and behavioral 

alterations. 

(e) References. For additional background information on 

this test guideline the following references should be consulted: 

Adams, J., Buelke-Sam, J., Kimmel, CA., Nelson, C.J., Reiter, 

L.W., Sobotka, T.J., Tilson, H.A., Nelson, B.K. Collaborative 

behavioral teratology study: Protocol design and testing 

procedure. Neurobehavioral Toxicology and Teratology. 7:579-586 

(1985) . 

Bennet, H. S., Wyrick, A. D., Lee, S.W., McNeil, J.H. Science 

and art in the 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). 

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). 

Campbell, B.A., Harouturiian, V. Effects of age on long-term 

memory: Retention of fixed interval responding. Journal of 

Gerontology 36:338-341 (1981). 

Cory-Slechta, D.A., Weiss, B., Cox, C. Delayed behavioral 

toxicity of lead with increasing exposure concentration. 

Toxicology and Applied Pharmacology 71:342-352 (1983). 

Di Sant Agnese, P. A., De Mesy Jensen, K. Dibasic staining of 

large epoxy sections and application to surgical pathology. 

American Journal of Clinical Pathology 81:25-29 (1984). 

Friede, R. L. Developmental Neuropathology. New York: Springer 

Verlag (1975). 

Green, R.J., Stanton, M.E. Differential ontogeny of working 

memory and reference memory in the rat. Behavioral Neuroscience 

103:98-105 (1989). 

Ison, J.R. Reflex modification as an objective test for sensory 

processing following toxicant exposure. Neurobehavioral 

Toxicology and Teratology 6:437-445 (1984). 

Korenbrot, C.C, Huhtaniemi, I.T., Weiner, R.W. Preputial 

separation as an external sign of pubertal development in the 

male rat. Biology of Reproduction 17:298-303 (1977). 

Krasnegor, N. A., Blass, E. M., Hofer, M. A., Smotherman, w. P. 

(eds.) Perinatal Development: A Psychobiological Perspective. 

41 
P.45 

Devel Neuro 

Orlando: Academic Press (1986). 

Kucharski, D., Spear, N. E. Conditioning of aversion to an odor 

paired with peripheral shock in the developing rat. 

Developmental Psychobiology 17:465-479 (1984). 

Luna, L. G. Manual of Histologic Staining Methods of the Armed 

Forces Institute of Pathology. (Third Edition) New York: McGraw 

Hill (1968). 

Miale, I. E., Sidman, R. An autoradiographic analysis of 

histogenesis in the mouse cerebellum. Experimental Neurology 

4:277-296 (1961). 

Miller, D. B., Eckerman, D. A. Learning and memory measures. In: 

Neurobehavioral Toxicology. Z. Annau (ed). Baltimore: Johns 

Hopkins University Press, pp. 94-149 (1986). 

Pender, M. P. A simple method for high resolution light 

microscopy of nervous tissue. Journal of Neuroscience Methods 

15:213-218 (1985) 

Ralis, H. M., Beesley, R. A., Ralis, Z. A. Techniques in 

Neurohistology. London: Butterworths (1973) 

Rodier, P. M., Gramann, W. J. Morphologic effects of 

interference with cell proliferation in the early fetal period. 

Neurobehavioral Toxicology 1:128-135 (1971). 

Spear, N. E., Campbell, B. A. (eds.) Ontogeny of Learning and 

Memory. New Jersey: Erlbaum (1979). 

Spencer, P. S., Schaumburg, H. H. (eds.) Experimental and 

Clinical Neurotoxicology. Baltimore: Williams and Wilkins (1980) 

Suzuki, K. Special vulnerabilities of the developing nervous 

system. In: Experimental and Clinical Neurotoxicology. P. S. 

Spencer and H. H. Schaumburg (eds.) Baltimore: Williams and 

Wilkins, pp. 48-61 (1980). 

US Environmental Protection Agency. Office of Pesticides and 

Toxic Substances Neurotoxicity Screening Battery (1990). 

42 
P.46 

Devel Neuro 

NOTES 

Response to Public and SAP Comment 

on the 

Developmental Neurotoxicity Study 

EPA's Work Group on Developmental Neurotoxicology would like 

to acknowledge and thank the individuals and organizations that 

provided comment on the "Developmental Neurotoxicity Study" 

protocol. The Work Group has reviewed all of the comments and 

taken them into consideration in revising the protocol. 

Responding to every individual comment is beyond the scope of 

this effort. Therefore, response will be limited to those 

comments that were raised by more than one individual or 

organization and which significantly impact the design of the 

study. 

1. COMMENT: The developmental neurotoxicity protocol is too 

complex and should be restricted to those agents for which there 

is sufficient justification to undergo such testing. A simpler, 

"tier 1" test should be developed that could be used more 

routinely. 

EPA RESPONSE: The Agency had a number of discussions on this 

issue. At first, the Agency considered development of a two-tier 

approach within the confines of a single study design. That is, 

measurements would have been carried out periodically during 

postnatal development. The "tier 1" component would have been 

the study carried until postnatal day 24 and the "tier 2" 

component would have been an extension of "tier 1" into 

adulthood. Whether or not the "tier 2" component would have been 

conducted would have depended on the analysis of the data up to 

postnatal day 24. This proposal was presented at several 

scientific meetings. However, it was criticized by the public 

for several reasons. First, concerns were raised that 

assessments only through the time of weaning may not be 

sufficiently sensitive to detect all potential developmental 

neurotoxicants. That is, unless assessments were carried out 

into adulthood, there would be a possibility that some potential 

developmental neurotoxicants would not be identified. Second, 

there was a strong sentiment among scientists from industry and 

contract laboratories that the assessments from tier 1 would not 

be completed in time for a decision to be made as to whether or 

not to proceed with the tier 2. In light of these concerns, the 

Agency has decided to publish the protocol as a single test to be 

conducted in its entirety, as it had been proposed. In the 

meantime, the Agency will be considering more feasible approaches 

43 
P.47 

Devel Neuro 

for development as a "screen" or "tier 1" type of protocol and 

encourages the development of screening methodologies by 

scientists working in this area. 

2.COMMENT: Reference to the highest dose level as inducing 

"some overt maternal toxicity" was considered to be excessive. 

Some suggested that the highest dose level should be below the 

"threshold" for "minimal maternal toxicity." 

EPA RESPONSE: The Agency disagrees. The protocol further 

qualifies that the highest dose "shall not result in a reduction 

in weight gain exceeding 20% during gestation and lactation" and 

that it "will not induce in utero or neonatal death or 

malformations sufficient to preclude a meaningful evaluation of 

neurotoxicity." This represents as minimal a toxic level as one 

could require in order to ensure that the agent has been 

adequately tested across an appropriate range of dose levels. The 

Agency is satisfied with this requirement in the guideline and 

does not believe a change is necessary. 

3.COMMENT: The specified duration of dosing, that is day 6 of 

gestation through day 21 postnatally, is excessive. Dosing of 

the dams postnatally should not be required because of potential 

effects on maternal behavior, milk production, or milk let-down, 

or sequestration of the agent in the milk with transfer to the 

pup, any of which may alter maternal-neonatal interactions. 

Furthermore, it was noted that pups would be undergoing 

observations and testing while potentially being exposed to the 

agent via the milk; thus, alterations in these measurements may 

be due to pharmacologic action of the agent rather than to a 

neurotoxic effect. It would not be possible to distinguish these 

effects. 

EPA RESPONSE: After careful consideration of all of the issues, 

the Agency has revised the protocol so that the period of 

exposure is now from day 6 of gestation through day 10 

postnatally. The rationale behind this revision was as follows: 

First, the Agency strongly believes that dosing should 

continue into the postnatal period for several reasons. These 

include: 1) several major events that occur prenatally in the 

nervous system of the human are still going through critical 

stages postnatally in the rat, and 2) exposure to the still 

developing organism may occur when agents are transferred from 

the mother to the offspring via the milk. An alternaltive 

postnatal exposure route that has been suggested is direct dosing 

of neonates, but the Agency believes that more work is needed to 

develop better methods before this should be adopted. 

Second, although the Agency feels strongly that dosing 

44 
P.48 

Devel Neuro 

should continue into the postnatal period for the reasons 

identified above, it recognizes the potential confounding factors 

with postnatal dosing that were raised in the public comments and 

has, therefore, adopted a compromise position. The Agency has 

modified the duration of exposure so that it ceases on postnatal 

day 10. The Agency chose this time for cessation of exposure for 

the following reasons: 1) while the revised period of exposure 

does not cover the entire period of lactation, it should still be 

sufficient to detect any potential effect that may be caused 

through exposure via breast milk, 2) any agent with a reasonable 

half-life would, theoretically, be eliminated sufficiently by the 

time testing begins (day 13 for motor activity) and would not 

significantly influence the results, and 3) while the nervous 

system is still undergoing some development beyond this time and, 

thus, effects on these events may be missed, the majority of the 

critical periods for CNS development have occurred by this time, 

most notably, proliferation of neuronal precursors in the 

cerebellum and hippocampus. Thus, it was felt that dosing through 

postnatal day 10 would maximize the detection of most 

developmental neurotoxic effects while minimizing the potential 

for pharmacologic influences of the agent on the outcome of 

functional evaluations; 

4.COMMENT: The requirement for oral dosing by intubation is too 

rigid. Greater flexibility should be allowed for exposure via 

other routes. 

EPA RESPONSE: The protocol has been revised to read: "The test 

substance or vehicle shall be administered orally. Other routes 

of administration may be acceptable, on a case-by-case basis, 

with ample justification/reasoning for this selection." The 

Agency, however, recognizes that conduct via other routes of 

exposure may necessitate modifications of the protocol because of 

potential problems with postnatal (lactational) exposure, that 

may be considered too drastic, and, thus, unacceptable. EPA is 

conducting research in this area and encourages others to do so, 

as well, in order to address the complications that may arise in 

studies conducted via routes other than oral. 

5.COMMENT: The frequency of monitoring motor activity is 

excessive; furthermore, inclusion of two preweaning evaluations 

will separate the pups from the mother for lengths of time that 

may be detrimental to the pups. 

EPA RESPONSE: In the proposal, EPA specified monitoring of motor 

activity on days 13, 17, 21, and day 60 (±2). These days were 

selected because they represent critical periods of motor 

development. Testing over a number of days provides the assessor 

45 
P.49 

Devel Neuro 

with information regarding the developmental pattern of motor 

activity. Interpretation of data from just a single day of 

testing would be much more difficult to interpret and less 

meaningful than more complete data on the ontogeny of motor 

activity and within-session habituation. Furthermore, the Agency 

does not have any reason to believe that handling of the pups, or 

separation from the mother for the length of time needed to carry 

out the motor activity testing, will result in any adverse 

effects on the pups; treated and control pups will be handled in 

the same manner to avoid any bias in the data. 

6.COMMENT: A number of comments were made regarding the 

section on neuropathology. A suggestion was made to include 

simple morphometries. In some cases, certain aspects of the 

Agency's procedures related to neuropathological evaluation were 

questioned. These included: 1) conducting neuropathology on 

animals sacrificed on postnatal day 4, 2) the number of animals 

included for neuropathologic evaluation, 3) plastic and paraffin 

embedding of tissue samples, 4) the qualitative examination, 5) 

the list of specified regions of the nervous system to be 

examined, 6) the semi-quantitative analysis (subjective 

diagnosis), and 7) the reporting of results. 

EPA RESPONSE: The Agency has agreed to adopt the suggestion of 

including simple morphometries. Detection of disruption of the 

development of the nervous system is the major purpose of this 

test. Compounds may alter nervous system development in a 

variety of ways including altering the rate and extent of growth 

of the nervous system. Alterations of this type are not always 

accompanied by gross neuropathological alterations. Thus, 

assessment of the extent of potential change in the normal state 

of development should be included. The approach will be to make 

simple measurements in regions known to be undergoing extensive 

growth at the time of sacrifice. 

The Agency has formulated the following response to the 

questions raised regarding the aforementioned procedures: 

1) The protocol has been revised to include 

neuropathological examination of animals sacrificed on postnatal 

day 11, the day after dosing ends, rather than postnatal day 4. 

This revision is based on several advantages to assessing the 

later time point. First, cumulative injury should be more 

apparent if exposure continues for an additional 6 days 

postnatally. Second, by postnatal day 11, several brain regions 

are approaching maximal proliferative activity (e.g., 

cerebellum). Third, a greater number of brain regions undergo 

further development during the additional 6 days, thus, the 

likelihood of revealing potential vulnerability is increased. 

Furthermore, brains of postnatal day 11 pups are more amenable to 

routine neuropathological analysis. 

46 
P.50 

Devel Neuro 

2) The requirement of 6 male and 6 female animals per dose 

group is to ensure an adequate sample size for statistical 

analysis of incidence and severity of neuropathological 

alterations as well as the morphometric analysis. 

3) The goal of the procedures outlined for the preparation 

and processing of tissue samples is to optimally preserve tissue 

morphology for microscopic examination. The higher resolution 

obtainable in plastic embedded tissue is considered to optimize 

the detection of a number of types of lesions, particularly in 

the peripheral nervous system. In contrast, paraffin-embedded 

material is more amenable to sampling large regions of the 

nervous system and is considered optimal for a variety of special 

stains that may be useful in characterizing neuropathological 

alterations. Several organizations felt the requirement for 

separate animals for plastic and paraffin-embedding of tissue 

samples was excessive. Furthermore, commentors presented views 

that differences in plastic and paraffin techniques did not 

require the use of separate animals and that in an appropriate . 

tiered evaluation scheme both plastic and paraffin-embedding 

could be used with tissue samples from the same animals. The 

requirement for separate animals is now eliminated, with paraffin 

being acceptable and plastic being recommended. 

4) Many regions of the developing brain have been 

demonstrated to be sensitive to neurotoxic insult. The purpose 

of the qualitative examination is to identify regions within the 

developing brain that exhibit evidence of neuropathological 

alterations and to identify types of neuropathological 

alterations that result from exposure to the test substance. A 

stepwise evaluation is recommended since, if evidence of 

neuropathological alterations is not observed at the high dose 

level, additional processing of tissue samples is not required. 

5) The Agency received some comments that the list of 

specified regions of the nervous system to be examined was 

inadequate, while others felt it was too detailed. Moreover, 

comments were received that argued that the list of potential 

types of neuropathological alterations also was too restricted. 

These lists were intended to serve as guidance. Since they 

appear to be subject to misinterpretation, the requirement for a 

thorough examination of the nervous system for any evidence of 

neuropathological alteration is now explicitly stated. However, 

it is noted that examination of the developing brain is 

particularly difficult and not usually the subject of routine 

neuropathological analysis. Therefore, a list of regions and the 

types of major alterations to be evaluated are included. In 

addition, a list of all structures examined is required in the 

final report. The requirement for examination of more than one 

section per region, however, has now been deleted. 

6) The purpose of the semi-quantitative analysis is to 

evaluate the relationship between the incidence and severity of 

the neuropathological alterations and the exposure. Since the 

47 
P.51 

Devel Neuro 

rating scale is by necessity subjective, it is necessary to 

ensure that any bias resulting from the previous qualitative 

examination of the tissue samples is minimized. Several 

organizations commented that blind evaluation of tissue sections 

should not be required. In the initial qualitative analysis, in 

which the types of lesions and regions affected are first 

identified, blind evaluation is not required. However, in the 

semi-quantitative analysis in which the dose-response 

relationship is evaluated, it is imperative that the evaluation 

be as objective as possible. Moreover, since the semiquantitative analysis focuses on a limited number of regions for 

lesions previously described in the qualitative analysis, blind 

reading is required to ensure objectivity. Thus, it is required 

that the subjective rating of the severity and incidence be 

performed without knowledge of treatment. 

7) The data and analyses supplied in the report must be 

evaluated by Agency risk assessors. Thus, the report must be 

sufficiently detailed for the Agency to evaluate the quality of 

the study. Since no list of regions to be examined is outlined -

in the guideline, a list of regions examined must be supplied 

with the report. Similarly, an adequate description of lesions 

observed must be supplied also. The Agency received comments 

that the requirement of photomicrographs to document 

neuropathological alterations was extremely costly. The Agency 

has decided to recommend, rather than require, the use of 

photomicrographs to aid in the description of typical examples of 

treatment-related lesions. 

7. COMMENT: Additional validation is needed before the glial 

fibrillary acidic protein (GFAP) radioimmunoassay should be 

included as part of the battery of tests in the developmental 

neurotoxicity study. Use of special stains should be at the 

discretion of the pathologist conducting the study. 

EPA RESPONSE: Although, EPA believes that the GFAP assay has 

been shown to be sensitive to the neurotoxic effects of agents in 

both the adult and developing nervous systems, this assay has 

been deleted at this time. However, the Agency will continue 

using this assay experimentally and encourages others to do so, 

as well, in an effort to obtain additional validation of its use 

as a means to assess the neurotoxic potential of agents. In 

addition, if GFAP immunohistochemistry is used as a special stain 

in the neuropathology segment of the testing protocol and 

evidence of a glial response to toxicant injury is observed, 

application of the radioimmunoassay is encouraged in order to 

provide objective, quantitative dose-response data. 

48 
P.52 

SCHEDULE-CONTROLLED OPERANT BEHAVIOR

 1 

(a) Purpose. In the assessment and evaluation of the 

potential human health effects of substances, it may be necessary 

to test for functional neurotoxic effects. Substances that have 

been observed to produce neurotoxic signs in other toxicity 

studies (e.g., CNS depression or stimulation), as well as 

substances with a structural similarity to neurotoxicants 

affecting performance, learning, or memory may be appropriate to 

evaluate with this test. This guideline defines procedures for 

conducting studies of schedule-controlled operant behavior, 

one way of evaluating the rate and pattern of a class of learned 

behavior (Dews, 1972; NAS 1975, 1977, 1982). Our purpose 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 toxicological effects seen. 

Operant behavior tests may be also used to evaluate many other 

aspects of behavior (Laties, 1978). Additional tests may be 

necessary to completely assess the effects of any substance on 

learning, memory, or behavioral performance. 

(b) Definitions.' 

(1) Neurotoxicity. Neurotoxicity is any adverse effect 

on the structure or function of the nervous system related to 

exposure to a chemical substance. 

(2) Behavioral toxicity is any adverse change in the 

functioning of the organism with respect to its environment in 

relation to exposure to a chemical substance. 

(3) Operant, operant behavior, operant conditioning. 

An operant is a class of behavioral responses which changes or 

operates on the environment in the same way. Operant behavior is 

further distinguished as behavior which is modified by its 

consequences. Operant conditioning is the experimental procedure 

used to modify some class of behavior by reinforcement or 

punishment. 

(4) Schedule of reinforcement. A schedule of 

reinforcement specifies the relation between behavioral responses 

and the delivery of reinforcers, such as food or water (Ferster 

and Skinner, 1957). For example, a fixed ratio (FR) schedule 

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 

minutes). 

(c) 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 then administered according to the 

experimental design (between groups or within subjects) and the 

duration of exposure (acute or repeated). Measurements of the 

49 
P.53 

Neuro Behav 

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. Comparisons are 

made between any exposures that influence the behavior and 

exposures that have neuropathological effects or effects on other 

targets of the chemical. 

(d) Test Procedures. 

(1) Experimental design. These test procedures may be 

used to evaluate the behavior of experimental animals receiving 

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 (pre-exposure and 

post-exposure) are recommended 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. Approximately equal numbers of male 

and female animals are required for each dose level and control 

group. Virgin females should be used. 

(iv) Experimental history. Animals should be 

experimentally and chemically naive. 

(3) Number of animals. Six to twelve animals should be 

exposed to each level of the test substance and/or control 

procedure. 

(4) Control groups. 

(i) A concurrent control group or control 

session(s) (according to the design of the study) are required. 

For control groups, subjects shall 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 shall 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. Data shall be collected according to 

the same experimental design as that proposed for the test 

substance. Positive control data shall be collected at the time 

of the test study unless the laboratory can demonstrate the 

adequacy of historical data for this purpose, i.e., by the 

approach outlined in this guideline . 

(5) Dose levels and dose selection. At least 3 doses 

50 
P.54 

Neuro Behav 

shall 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. We strongly 

encourage the use of equally spaced doses and a rationale for 

dose selection that will maximally support detection of doseeffect relations. 

(i) Acute studies. The high dose need not be 

greater than 2 g/Kg. Otherwise, the high dose shall 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 shall 

produce minimal effects, e.g., an ED10, or alternatively, no 

effects. 

(ii) Subchronic (and Chronic) Studies. The high 

dose need not be greater than lg/Kg. Otherwise, the high dose 

shall result in significant neurotoxic effects or other clearly 

toxic effects, but not produce an incidence of fatalities that . 

would prevent a meaningful evaluation of the data. The middle and 

low doses should be fractions of the high dose. The loweset dose 

shall 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. For many materials, it should be recognized 

that more than one route of exposure may be important and that 

these criteria may conflict with one another. The route that 

best meets these criteria should be selected. Dietary feeding 

will be generally be acceptable for repeated exposure studies. 

(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 delivery of reinforcers shall 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 periodically be 

calibrated. 

(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 

51 
P.55 

Neuro Behav 

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 criterion 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. 

(B) Freguency of testing. The maintenance of 

stable operant behavior normally will require regular and 

frequent (e.g., 5 days a week) testing sessions. Animals shouldbe 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 minutes should be 

adequate. 

(v) Schedule selection. The schedule of 

reinforcement chosen should generate response rates that may 

increase or decrease as a function of exposure. Many schedules 

of reinforcement can do this: a single schedule maintaining a 

moderate response rate; fixed-interval schedules, which engender 

a variety of response rates in each interval; or multiple 

schedules, where different components may maintain high and low 

response rates. 

(e) Data reporting and evaluation. The final test report 

must include the following information. 

(1) Description of equipment and test methods, (i) A 

description of the experimental chambers, programming equipment, 

data collection devices, 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. 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. We encourage submission of such data to 

52 
P.56 

Neuro Behav 

facilitate the rapid and complete review of the significance of 

effects seen. 

(2) Results, (i) Data for each animal should be 

arranged by test group in tabular form including the animal 

identification number, body weight, pre-exposure 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 shall include the relationship between 

the doses of the test substance and the incidence and magnitude 

of any observed effects, i.e. dose-effect curves for any effects 

seen. 

(ii) The evaluation should include appropriate 

statistical analyses. Choice of analyses should consider tests 

appropriate to the experimental design, including repeated 

measures. There may be many acceptable ways to analyze data. 

(iii) Any known citations from the open literature 

related to the interpretation of the neurotoxicity of the test 

material shall also be included. 

(f) References. For additional background information on 

this test guideline the following references should be consulted. 

Dews, P.B. "Assessing the Effects of Drugs", In Methods in 

Psychobiology. Vol. 2, Ed., R.D. Myers (New York: Academic Press, 

1972) 83-124. 

Ferster, CB . Skinner, B.F. Schedules of Reinforcement. 

(New York: Appleton-Century-Crofts, 1957). 

Laties, V.G. "How Operant Conditioning can Contribute to 

Behavioral Toxicology". Environmental Health Perspectives. 28:29-

35 (1978). 

National Academy of Science. Principles for Evaluating 

Chemicals in the Environment. (Washington, DC: National Academy 

of Sciences, 1975). 

National Academy of Science. Principles and Procedures for 

Evaluating the Toxicity of Household Substances. (Washington, 

DC: National Academy of Sciences, 1977). 

National Academy of Science. "Strategies to determine needs 

and priorities for toxicity testing." Appendix 3B. Reference 

Protocol Guidelines For Neurobehavioral Toxicity Tests. 2:123-129 

(1982) . 

53 
P.57 

Neuro Behav 

NOTES 

1. This guideline has only been modified for clarity of prose and 

to make generic changes to conform to other guideline revisions 

in sections such as dose selection and route of administration. 

Otherwise, it is essentially identical to the guideline 

previously published by OTS in the Federal Register and codified 

in 40 CFR 798.6500 

2. Positive control data need only be generated roughly once 

every year as long as most conditions remain the same. 

54 
P.58 

PERIPHERAL NERVE FUNCTION1 

(a) Purpose. In the assessment and evaluation of the potential 

human health effects of substances, it may be necessary to test for 

neurophysiological effects. Substances that have been shown to 

produce peripheral neuropathy in other neurotoxicity studies (or 

other neuropathological changes in peripheral nerves), as well as 

substances with a structural similarity to those causing such 

effects, may be appropriate to evaluate with this test. This 

guideline defines procedures for evaluating certain aspects of the 

neurophysiological functioning of peripheral nerves. Our purpose is 

to evaluate the effects of exposures on the velocity and amplitude 

of conduction of peripheral nerves. Any observed effects should be 

evaluated in the context of both the concordance between functional 

neurological and neuropathological effects and with respect to any 

other toxicological effects seen. Additional tests may be 

necessary to completely assess the neurophysiological effects of 

any substance. 

(b) Definitions. 

(1) Neurotoxicity. Neurotoxicity is any adverse effect 

on the structure or function of the nervous system related to 

exposure to a chemical substance. 

(2) Conduction velocity is the speed at which the 

compound nerve action potential traverses a nerve. 

(3) Amplitude is the voltage excursion recorded during 

the process of recording the compound nerve action potential. It 

is an indirect measure of the number of axons firing. 

(c) 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. 

(d) 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 

shall be balanced to ensure that differences are not systemically 

related to treatment. 

(ii) Age and weight. Young adult animals (at least 

60 days for rats) must be used. Age (± 15 days for rats must not 

55 
P.59 

Periph Neuro 

vary across groups. Weights should be within ± io percent of the 

mean. 

(iii) Sex. Either (or both) sex(es) may be used. 

Sex must not vary across groups. 

(2) Number of animals. 20 animals should be exposed to 

each level of the test substance and/or control procedure. The goal 

is to be able to detect a 10 percent change from normal conduction 

velocity at the 5 percent level with 90 percent power. 

(3) Control groups, (i) A concurrent control group is 

required. For control groups, subjects shall 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 shall provide evidence that the experimental 

procedures are sensitive to substances known to affect peripheral 

nerve function. Positive control data shall be collected at the 

time of the test study unless the laboratory can demonstrate the 

adequacy of historical data for this purpose, i.e., by the approach 

outlined in this guideline . 

(4) Dose levels and dose selection. At least 3 doses 

shall be used in addition to the vehicle control group. Ideally, 

the data should be sufficient to produce a dose-effect curve. We 

strongly encourage the use of equally spaced doses and a rationale 

for dose selection that will maximally support detection of doseeffect relations. 

(i) Acute studies. The high dose need not be greater 

than 2 g/Kg. Otherwise, the high dose shall 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 shall produce minimal 

effects, e.g., an ED10, or alternatively, no effects. 

(ii) Subchronic (and Chronic) Studies. The high dose 

need not be greater than lg/Kg. Otherwise, the high dose shall 

result in significant neurotoxic effects or other clearly toxic 

effects, but not produce an incidence of fatalities that would 

prevent a meaningful evaluation of the data. The middle and low 

doses should be fractions of the high dose. The loweset dose shall 

produce minimal effects, e.g an ED10, or alternatively, no effects. 

(5) Route of administration. Selection of route may be 

based on several criteria including, the most likely route of human 

exposure, bioavailability, the likelihood of observing effects, 

practical difficulties, and the likelihood of producing nonspecific effects. For many materials, it should be recognized that 

more than one route of exposure may be important and that these 

criteria may conflict with one another. The route that best meets 

these criteria should be selected. Dietary feeding will be 

generally be acceptable for repeated exposure studies. 

(6) Combined protocol. The test described in this 

guideline may be combined with any other toxicity study, as long as 

56 
P.60 

Periph Neuro 

none of the requirements of either are violated by the combination. 

(7) Study conduct, (i) Choice of nervefs). The nerve 

conduction velocity test must separately asses the properties of 

both sensory and motor nerve axons. Either a hind limb (e.g., 

tibial) or tail (e.g., ventral caudal) nerve must be chosen. 

Response amplitude may be measured in a mixed nerve. 

(ii) Preparation. (A) In vivo testing of 

anesthetized animals is required. A barbiturate anesthetic is 

appropriate. Care should be taken to ensure that all animals are 

administered an equivalent dosage and that the dosage is not 

excessive. If dissection is used, extreme caution must be observed 

to avoid damage to either the nerve or the immediate vascular 

supply. 

(B) Both core and nerve temperature must be 

monitored and kept constant (±0.5 °C) during the study. Monitoring 

of skin temperature is adequate if it can be demonstrated that the 

skin temperature reflects the nerve temperature in the preparation 

under use. Skin temperature should be monitored with a needle 

thermistor at a constant site, the midpoint of the nerve segment to 

be tested. 

(CJ Electrodes. (1) Choice of Electrodes. 

Electrodes for stimulation and recording may be made of any 

conventional electrode material, such as stainless steel, although 

electrodes made of non-polarizing materials are preferable. If 

surface electrodes are used, care must be taken to ensure that good 

electrical contact is achieved between the electrode and the tissue 

surface. Following each application, all electrodes must be 

thoroughly cleaned. 

(2) Electrode placement. Electrode 

placement must be constant with respect to anatomical landmarks 

across animals (e.g., a fixed number of millimeters (mm) 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. 

(3) Recording conditions. The animal 

should be grounded at about the 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. 

(D) The electrical stimulator must be isolated from 

ground. Biphasic or balanced pair stimuli to reduce polarization 

effects are acceptable. A constant current stimulator is preferred 

(and required for polarized electrodes) and should operate from 

about 10 uA to about 10 mA. If a constant voltage stimulator is 

used, it should operate to 250V. All equipment shall be calibrated 

with respect to time, voltage, and temperature. 

57 
P.61 

Periph Neuro 

(E) The recording environment should be enclosed in 

a Faraday cage unless electromagnetic field pickup can be shown to 

be more than 1.5 times the amplifier baseline noise, under 

recording conditions. The recording output should be amplified 

sufficiently to render the compound action potentially easily 

measurable with an oscilloscope. The amplifier should pass signals 

between 2.0 Hz and 4 kHz without more than a 3dB decrement. The 

preamplifier must be capacitatively coupled or, if direct coupled 

to the first stages, must be able to tolerate any DC potentials 

which the electrode-preparation interface produces, and operate 

without significant current leakage through the recording 

electrodes. 

(F) A hard copy must be available for all waveforms 

or averaged waveforms from which measurements are derived, and for 

all control recording required by this standard. Hard copies must 

include a time and voltage calibration signal. 

(iii) 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. Response 

peak latency may be read off the oscilloscope following single 

sweeps or determined by an average of a fired number of responsors. 

The baseline-to-peak height technique (Daube, 1980) 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 supra-maximal. 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 cathodeanode distance, and with the cathode located toward the recording 

electrode. The cathode to cathode distance between the two sets of 

stimulating electrodes should be divided by the difference between 

the two latencies of muscle action potential in order to obtain 

conduction velocity. Placement of electrodes shall 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 

58 
P.62 

Periph Neuro 

conduction velocity is calculated by dividing the distance between 

the two stimulating cathodes by the difference between the two 

latencies of the largest primary peak of the somatosensory evoked 

potential. Between 64 and 123 responses should be averaged. The 

stimulation frequency should be about 0.5 Hz. Stimulus intensity 

should be the same as that used for determining the motor 

conduction velocity. Should the peak of the somatosensory response 

be so broad that it cannot be replicated with an accuracy of less 

than 5 percent of the latency difference observed, then a point on 

the rising phase of the potential should be chosen, e.g., at a 

voltage that is 50 percent of the peak voltage. Alternatively, the 

sensory nerve conduction velocity can be obtained from a purely 

sensory nerve or from stimulation of the dorsal rootlets of a mixed 

nerve, using two recording electrode pairs. 

(e) Data collection, reporting and evaluation. The final test 

report must include the following information. 

(1) Description of equipment and test methods. A 

description of the experimental chambers, programming equipment, 

data collection devices, and environmental test conditions should 

be provided. 

(i) A "description of the experimental design 

including procedures for balancing treatment groups should be 

provided. 

(ii) 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 experimental protocol are the 

same. Historical control data can be critical in the interpretation 

of study findings. We encourage submission of such data to 

facilitate the rapid and complete review of the significance of 

effects seen. 

(iii) Hard copies of waveforms from which 

measurements were made as well as control recordings should be 

included. 

(iv) Voltage and time calibration referable to the 

standards of the Bureau of Standards or to other standards of 

accuracy sufficient for the measurements used should be included. 

(v) Data demonstrating that nerve temperature was 

maintained constant throughout the recording period should also be 

included. 

(2) Results. Data for each animal should be arranged by 

test group in tabular form including the animal identification 

number, body weight, nerve conduction velocity, and amplitude. 

Group summary data should also be reported, including standard 

measures of central tendency and variability, e.g., means and 

standard deviations, and results of statistical analyses. 

(3) Evaluation of data. (i) The findings should be 

evaluated in the context of preceding and/or concurrent toxicity 

studies and any correlated functional and histopathological 

59 
P.63 

Periph Neuro 

findings. The evaluation shall include the relationship between the 

doses of the test substance and the incidence and magnitude of any 

observed effects, i.e. dose-effect curves for any effects seen.. 

(ii) The evaluation should include appropriate 

statistical analyses. Choice of analyses should consider tests 

appropriate to the experimental design, including repeated 

measures. There may be many acceptable ways to analyze data. 

(iii) Any known citations from the open literature 

related to the interpretation of the neurotoxicity of the test 

material shall also be included. 

(f) References. For additional background information on this 

test guideline the following references should be consulted: 

Aminoff, M.J. (Ed.). Electrodiagnosis in Clinical Neurology. 

(New York: Churchill Livingstone, 1980). 

Daube, J. "Nerve Conduction Studies," Electrodiagnosis in 

Clinical Neurology. Ed. M.J. Aminoff (New York: Churchill 

Livingstone, 1980). pp. 229-264. 

Glatt, A.F., H.-N. Talaat and W.P. Koella "Testing of 

peripheral nerve function in chronic experiments in rats," 

Pharmacology and Therapeutics. 5:539-534 (1979). 

Johnson, E.W. Practical Electromyography. (Baltimore: 

Williams and Wilkins, 1980). 

NOTES 

1. This guideline has only been modified for clarity of prose and 

to make generic changes to conform to other guideline revisions in 

sections such as dose selection and route of administration. 

Otherwise, it is essentially identical to the guideline previously 

published by OTS in the Federal Register and codified in 40 CFR 

798.6850. 

2. Positive control data need only be generated roughly once every 

year as long as most conditions remain the same. 

60