(a) Purpose. In the assessment and evaluation of the potential human health effects of substances, it may be necessary to test for neurotoxic effects. Substances that have been observed to cause neurotoxic signs (e.g., convulsions, tremors, ataxia) in other toxicity tests, as well as those having a structural similarity to known neurotoxicants, should be evaluated for neurotoxicity. The functional observational battery is a noninvasive procedure designed to detect gross functional deficits in young adults resulting from exposure to chemicals and to better quantify neurotoxic effects detected in other studies. This battery of tests is not intended to provide a detailed evaluation of neurotoxicity. It is designed to be used in conjunction with neuropathologic evaluation and/or general toxicity testing. Additional functional tests 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 central and/or peripheral 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.

(c) Principle of the test method. The material is administered by an appropriate route to laboratory rodents. The animals are observed under carefully standardized conditions with sufficient frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. Various functions that could be affected by neurotoxicants are assessed during each observation period.

(d) Test procedures—(1) Animal selection—(i) Species and strain. The laboratory rat or mouse is recommended. Although information will generally be lacking, whenever possible the choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies. The potential for combined studies should also be considered. Standard strains should be used.

(ii) Age. Young adult animals (at least 42 days old for the rat or mouse) shall be used.

(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level.

(B) The females shall be nulliparous and nonpregnant.

(2) Number of animals. At least eight animals of each sex should be used at each dose level and should be designated for behavioral testing. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the end of the study. Animals shall be randomly assigned to treatment and control groups.

(3) Control groups. (i) A concurrent (“sham” exposure or 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.

(ii) Concurrent or historic data from the laboratory performing the testing shall provide evidence of the ability of the procedures used to detect major neurotoxic endpoints such as limb weakness or paralysis (e.g., acrylamide), CNS stimulation (e.g., β, β′-iminodiproprionitrile) autonomatic signs (e.g., physostigmine).

(iii) A satellite group may be treated with the high dose level for the duration of exposure and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate duration, normally not less than 28 days.

(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.

(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.

(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.

(5) Duration and frequency of exposure. The duration and frequency of exposure will be specified in the test rule.

(6) Route of exposure. The test substance shall be administered by the route specified in the test rule. This route will usually be the one most closely approximating the expected route of human exposure. The exposure potocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline under subpart B or subpart C of this part.

(7) Combined protocol. Subjects used for other toxicity studies may be used if none of the requirements of either study are violated by the combination.

(8) Study conduct. (i) All animals in a given study should be observed carefully by trained technicians who are blind with respect to the animals' treatments. Standard procedures to minimize observer variability shall be followed. 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. All animals should be observed prior to initiation of exposure. Subsequent observations should be made with sufficent frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. At minimum, observations at 1 hour, 6 hours, 24 hours, 7 days, and 14 days and monthly thereafter are recommended. In a subchronic study, subsequent to the first exposure all observations should be made before the daily exposure. The animals should be removed from the home cage to a standard arena for observation. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables that can affect behavior are sound level, temperature, humidity, lighting, odors, time of day, and environmental distractions. Explicit, operationally defined scales for each function should be used. The development of objective quantitative measures of the observational endpoints specified is encouraged.

(ii) The following is a minimal list of observations that shall be noted:

(A) Any unusual responses with respect to body position, activity level, coordination of movement, and gait.

(B) Any unusual or bizarre behavior including, but not limited to, headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards.

(C) The presence of:

(1) Convulsions.

(2) Tremors.

(3) Increased levels of lacrimation and/or red-colored tears.

(4) Increased levels of salivation.

(5) Piloerection.

(6) Pupillary dilation or constriction.

(7) Unusual respiration (shallow, labored, dyspneic, gasping, and retching) and/or mouth breathing.

(8) Diarrhea.

(9) Excessive or diminished urination.

(10) Vocalization.

(D) Forelimb/hindlimb grip strength. The procedure described by Meyer et al. (1979), under paragraph (f)(9) of this section is recommended.

(E) Sensory function. A simple assessment of sensory function (vision, audition, pain perception) shall be made. Marshall et al. (1971) under paragraph (f)(8) of this section have described a neurologic exam for this purpose; these procedures are also discussed by Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under paragraph (f)(7) of this section described a number of reflex tests intended to detect gross sensory deficits, including the visual placing response, Preyer reflex, and tail pinch. Many procedures have been developed for assessing pain perception (e.g., Ankier, 1974 under paragraph (f)(1) of this section; D'Amour and Smith 1941 under paragraph (f)(3) of this section; Evans 1971 under paragraph (f)(6) of this section).

(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792 subpart J the final test report must include the following information.

(1) Description of system and test methods. (i) A detailed description of the procedures used to standardize observation, including the arena and operational definitions for scoring observations.

(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historic data may be used if all aspects of the experimental protocol are the same, including personnel.

(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).

(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 percentage of animals showing each abnormal sign at each observation time.

(D) The mean and standard deviation for each continuous endpoint at each observation time.

(3) Evaluation of data. The findings of a functional observational battery should be evaluated in the context of preceding and/or concurrent toxicity studies and any correlative 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 statistical analyses. Choice of analyses should consider tests appropriate to the experimental design and needed adjustments for multiple comparisons.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Ankier, S.I. “New hot plate tests to quantify antinociceptic and narcotic antagonist activities,” European Journal of Pharmacology, 27: 1-4 (1974).

(2) Coughenour, L.L., McLean, J.R. and Parker, R.B. “A new device for the rapid measurement of impaired motor function in mice,” Pharmacology, Biochemistry and Behavior, 6: 351-353 (1977).

(3) D'Amour, F.E., Smith, D.L. “A method for determining loss of pain sensation,” Journal of Pharmacology and Experimental Therapeutics, 72: 74-79 (1941).

(4) Deuel, R.K. “Determining sensory deficits in animals,” Methods in Psychobiology Ed. Myers R.D. (New York: Academic Press, 1977) pp. 99-125.

(5) 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).

(6) Evans, W.O. “A new technique for the investigation of some analgesic drugs on reflexive behavior in the rat,” Psychopharmacologia, 2: 318-325 (1961).

(7) Irwin, S. “Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse,” Psychopharmacologia, 13: 222-257 (1968).

(8) Marshall, J.F., Turner, B.H., Teitlbaum, P. “Sensory neglect produced by lateral hypothalamic damage,” Science, 174: 523-525 (1971).

(9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. “A method for the routine assessment of fore- and hindlimb grip strength of rats and mice,” Neurobehavioral Toxicology, 1: 233-236 (1979).

(a) Purpose—(1) General. In the assessment and evaluation of the toxic characteristics of a substance, determination of the effects of administration of the substance on motor activity is useful when neurotoxicity is suspected.

(2) Acute Motor Activity Test. The purpose of the acute motor activity test is to examine changes in motor activity occurring over a range of acute exposure levels. These changes may then be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce acute neurotoxicity and may be used to screen members of a class of substances for known neurotoxicity, and/or to establish a dosage regimen prior to the initiation of subchronic neurotoxicity testing.

(3) Subchronic Motor Activity Test. The purpose of the subchronic motor activity test is to determine whether the repeated administration of a suspected neurotoxicant results in changes in motor activity. These changes may be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce subchronic neurotoxicity.

(b) Definitions. (1) Neurotoxicity is the adverse effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.

(2) Motor activity is any movement of the experimental animal.

(3) 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.

(c) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. Measurements of motor activity are made. The exposure levels at which significant changes in motor activity are produced are compared to those levels which produce toxic effects not originating in the central and/or peripheral nervous system.

(d) Test procedures—(1) Animal selection—(i) Species and strain. Testing shall be performed in a laboratory rat or mouse. The choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.

(ii) Age. Young adult animals (at least 42 days old for rat or mouse) should be used.

(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level for the motor activity test.

(B) The females shall be nulliparous and nonpregnant.

(2) Number of animals. Animals shall be randomly assigned to test and control groups. Each test or control group must be designed to contain a sufficient number of animals at the completion of the study to detect a 40 percent change in activity of the test groups relative to the control group with 90 percent power at the 5 percent level. For most designs, calculations can be made according to Dixon and Massey (1957) under paragraph (f)(1) of this section, Neter and Wasserman (1974) under paragraph (f)(5) of this section, Sokal and Rohlf (1969) under paragraph (f)(9) of this section, or Jensen (1972) under paragraph (f)(3) of this section.

(3) Control groups. (i) A concurrent control group is required. This group must be an untreated group, or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control group are required.

(ii) Positive control data are required to demonstrate the sensitivity and reliability of the activity measuring device and testing procedure. These data should demonstrate the ability to detect increases or decreases in activity and to generate a dose-effect curve or its equivalent using three values of the dose or equivalent independent variable. A single administration of the dose (or equivalent) is sufficient. It is recommended that chemical exposure be used to collect positive control data. 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.

(iii) A satellite group may be treated with the high dose level for 90 days and observed for reversibility, persistence or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.

(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.

(i) The highest dose shall produce (A) clear effects on motor activity or (B) life-threatening toxicity.

(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.

(5) Duration of testing. The duration of exposure will be specified in the test rule.

(6) Route of administration. The test substance shall be administered by the method specified in the test rule. This will usually be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline.

(7) Combined protocol. The tests described herein 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) General. Motor activity must be monitored by an automated activity recording apparatus. The device used must be capable of detecting both increases and decreases in activity, i.e. baseline activity as measured by the device must not be so low as to preclude decreases nor so high as to preclude increases. Each device shall be tested by standard procedure 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 most treatments and animals. All sessions should have the same duration. Treatment groups shall be counter-balanced across test times. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage and environmental distractions. Tests shall be executed by an appropriately trained individual.

(ii) Acute. Testing shall be timed to include the time of peak signs.

(iii) Subchronic. All animals shall be tested prior to initiation of exposure and at 30 ±2, 60 ±2 and 90 ±2 days during the exposure period. Testing shall occur prior to the daily exposure. Animals shall be weighed on each test day and at least once weekly during the exposure period.

(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report must include the following information:

(1) Description of system and test methods. (i) Positive control data from the laboratory performing the test which demonstrate the sensitivity of the procedure being used.

(ii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups.

(2) Results. The following information must be arranged by test group (dose level).

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

(A) Its identification number.

(B) Body weight, total session activity counts, and intrasession subtotals for each date measured.

(ii) Group summary data should also be reported.

(3) Evaluation of data. An evaluation of the test results (including statistical analysis comparing total activity counts at the end of exposure of treatment vs control animals must be made and supplied. This submission must include dose-effect curves for motor activity expressed as activity counts.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis 2nd Ed. (New York: McGraw-Hill, 1957).

(2) Finger, F.W. “Measuring behavioral activity,” Methods in Psychobiology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-19.

(3) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T 2 Statistics,” Biometrics, 28:39-53 (1972).

(4) 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).

(5) Neter, J. and Wasserman, W. Applied Linear Statistical Models. Homewood, Richard D. Irwin, Inc., 1974.

(6) Reiter, L.E. “Use of activity measures in behavioral toxicology,” Environmental Health Perspectives, 26:9-20 (1978).

(7) Reiter, L.W. and MacPhail, R.C. “Motor Activity: A survey of methods with potential use in toxicity testing,” Neurobehavioral Toxicology, 1: Suppl. 1, 53-66 (1979).

(8) Robbins, T.W. “A critique of the methods available for the measurement of spontaneous motor activity,” Handbook of Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder, S.H. (New York: Plenum, 1977) pp. 37-82.

(9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H. Freeman and Co., 1969).

(a) Purpose. The techniques in this guideline are designed to develop data on morphologic changes in the nervous system for chemical substances and mixtures subject to such testing under the Toxic Substances Control Act. The data will detect and characterize morphologic changes, if and when they occur, and determine a no-effect level for such changes. Neuropathological evaluation should be complemented by other neurotoxicity studies, e.g. behavioral and neurophysiological studies. Neuropathological evaluation may be done following acute, subchronic or chronic exposure.

(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent.

(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 sacrificed and tissues in the nervous system are examined grossly and prepared for microscopic examination. Starting with the highest dosage level, tissues are examined under the light microscope for morphologic changes, until a no effect level is determined. In cases where light microscopy has revealed neuropathology, the no effect level may be confirmed by electron microscopy.

(d) Test procedure—(1) Animal selection—(i) Species and strain. Testing shall be performed in the species being used in other tests for neurotoxicity. This will generally be the laboratory rat. The choice of species shall take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.

(ii) Age. Animals shall be young adults (150-200 gm for rats) at the start of exposure.

(iii) Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects.

(2) Number of animals. A minimum of six animals per group shall be used. The tissues from each animal shall be examined separately. It is recomse (iv)mended that ten animals per group be used.

(3) Control groups. (i) A concurrent control group(s) is (are) required. This group must be an untreated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the vehicle used has a known or potential toxic property, both untreated and vehicle control groups are required.

(ii) A satellite group of animals may be treated with the high level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length; normally not less than 28 days.

(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.

(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.

(ii) The data from the lower doses must show either (A) graded dose-dependent effects at two dose levels or (B) no effects at two dose levels, respectively.

(5) Duration of testing. The exposure duration will be specified in the test rule. This will generally be 90 days exposure.

(6) Route of administration. The test substance shall be administered by a route specified in the test rule. This will generally be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity guideline.

(7) Combined protocol. The tests described herein 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) Observation of animals. All toxicological (e.g., weight loss) and neurological signs (e.g., motor disturbance) shall be recorded frequently enough to observe any abnormality, and not less than weekly.

(ii) Sacrifice of animals—(A) General. The goal of the techniques outlined for sacrifice of animals and preparation of tissues is preservation of tissues morphology to simulate the living state of the cell.

(B) Perfusion technique. Animals shall be perfused in situ by a generally recognized technique. For fixation suitable for light or electronic microscopy, saline solution followed by buffered 2.5 percent glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended. While some minor modifications or variations in procedures are used in different laboratories, a detailed and standard procedure for vascular perfusion may be found in the text by Zeman and Innes (1963) under paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of this section, and by Spencer and Schaumburg (1980) under paragraph (f)(6) of this section. A more sophisticated technique is described by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.

(C) Removal of brain and cord. After perfusion, the bonystructure (cranium and vertebral column) shall be exposed. Animals shall then be stored in fixative-filled bags at 4 °C for 8-12 hours. The cranium and vertebral column shall be removed carefully by trained technicians without physical damage of the brain and cord. Detailed dissection procedures may be found in the text by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section. After removal, simple measurement of the size (length and width) and weight of the whole brain (cerebrum, cerebellum, pons-medulla) shall be made. Any abnormal coloration or discoloration of the brain and cord shall also be noted and recorded.

(D) Sampling. Unless a given test rule specifies otherwise, cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum, the midbrain, the cerebellum and pons, and the medulla oblongata; the spinal cord at cervical and lumbar swelling (C3-C6 and L1-L4); Gasserian ganglia, dorsal root ganglia (C3-C6, L1-L4), dorsal and ventral root fibers (C3-C6, L 1-L4), proximal sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at knee). Other sites and tissue elements (e.g., gastrocnemius muscle) should be examined if deemed necessary. Any observable gross changes shall be recorded.

(iii) Specimen storage. Tissue samples from both the central and peripheral nervous system shall be further immersion fixed and stored in appropriate fixative (e.g., 10 percent buffered formalin for light microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered paraformaldehyde for electron microscopy) for future examination. The volume of fixative versus the volume of tissues in a specimen jar shall be no less than 25:1. All stored tissues shall be washed with buffer for at least 2 hours prior to further tissue processing.

(iv) Histopathology examination. (A) Fixation. Tissue specimens stored in 10 percent buffered formalin may be used for this purpose. All tissues must be immersion fixed in fixative for at least 48 hours prior to further tissue processing.

(B) Dehydration. All tissue specimens shall be washed for at least 1 hour with water or buffer, prior to dehydration. (A longer washing time is needed if the specimens have been stored in fixative for a prolonged period of time.) Dehydration can be performed with increasing concentration of graded ethanols up to absolute alcohol.

(C) Clearing and embedding. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast. Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded together in one single block for sectioning. All tissue blocks shall be labelled showing at least the experiment number, animal number, and specimens embedded.

(D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall be prepared from the tissue blocks and mounted on standard glass slides. It is recommended that several additional sections be made from each block at this time for possible future needs for special stainings. All tissue blocks and slides shall be filed and stored in properly labeled files or boxes.

(E) Histopathological techniques. Although the information available for a given chemical substance may dictate test-rule specific changes, the following general testing sequence is proposed for gathering histopathological data:

(1) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background.

(2) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by the use of specific techniques. If H&E screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: neuronal body (e.g., Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g., Kluver's Luxol Fast Blue) and neurofibrils (e.g., Bielchosky). In addition, peripheral nerve fiber teasing shall be used. Detailed staining methodology is available in standard histotechnological manuals such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al. (1973) under paragraph (f)(5) of this section, and Chang (1979) under paragraph (f)(2) of this section. The nerve fiber teasing technique is discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If a lesion(s) is observed, the special techniques shall be repeated in the next lower treatment group until no further lesion is detectable.

(3) Alternative technique. If the anatomical locus of expected neuro-pathology is well-defined, epoxy-embedded sections stained with toluidine blue may be used for small sized tissue samples. This technique obviates the need for special stains for cellular components. Detailed methodology is available in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section.

(4) Electron microscopy. Based on the results of light microscopic evaluation, specific tissue sites which reveal a lesion(s) shall be further evaluated by electron microscopy in the highest treatment group which does not reveal any light microscopic lesion. If a lesion is observed, the next lower treatment group shall be evaluated until no significant lesion is found. Detailed methodology is available in Hayat (1970) under paragraph (f)(3) of this section.

(F) Examination—(1) General. All stained microscopic slides shall be examined with a standard research microscope. Examples of cellular alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and tissue changes (e.g., gliosis, leukocytic infiltration, and cystic formation) shall be recorded and photographed.

(2) Electron microscopy. Since the size of the tissue samples that can be examined is very small, at least 3 to 4 tissue blocks from each sampling site must be examined. Tissue sections must be examined with a transmission electron microscope. Three main categories of structural changes must be considered:

(i) Neuronal body. The shape and position of the nucleus and nucleolus as well as any change in the chromatin patterns shall be noted. Within the neuronal cytoplasm, cytoplasmic organelles such as mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments, endoplasmic reticulum and polyribosomes (Nissl substance), Golgi complex, and secretory granules shall be examined.

(ii) Neuronal processes. The structural integrity or alterations of dendrites, axons (myelinated and unmyelinated), myelin sheaths, and synapses shall be noted.

(iii) Supporting cells. Attention must also be paid to the number and structural integrity of the neuroglial elements (oligodendrocytes, astrocytes, and microglia) of the central nervous system, and the Schwann cells, satellite cells, and capsule cells of the peripheral nervous system. Any changes in the endothelial cells and ependymal lining cells shall also be noted whenever possible. The nature, severity, and frequency of each type of lesion in each specimen must be recorded. Representative lesions must be photographed and labeled appropriately.

(e) Data collection, reporting, and evaluation. In addition to information meeting the requirements stated under 40 CFR part 792 subpart J, the following specific information shall be reported:

(1) Description of test system and test methods. A description of the general design of the experiment shall be provided. This shall include a short justification explaining any decisions where professional judgment is involved such as fixation technique and choice of stains.

(2) Results. All observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:

(i) Description of signs and lesions for each animal. For each animal, data must be submitted showing its identification (animal number, treatment, dose, duration), neurologic signs, location(s) nature of, frequency, and severity of lesion(s). A commonly-used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity ranging from very slight to extensive may be used. Any diagnoses derived from neurologic signs and lesions including naturally occurring diseases or conditions, should also be recorded.

(ii) Counts and incidence of lesions, by test group. Data shall be tabulated to show:

(A) The number of animals used in each group, the number of animals displaying specific neurologic signs, and the number of animals in which any lesion was found;

(B) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesion.

(iii) Evaluation of data. (A) An evaluation of the data based on gross necropsy findings and microscopic pathology observations shall be made and supplied. The evaluation shall include the relationship, if any, between the animal's exposure to the test substance and the frequency and severity of the lesions observed.

(B) The evaluation of dose-response, if existent, for various groups shall be given, and a description of statistical method must be presented. The evaluation of neuropathology data should include, where applicable, an assessment in conjunction with other neurotoxicity studies performed (eg. electrophysiological, behavioral, neurochemical).

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-Hill (1968).

(2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (Springfield, IL: Charles C. Thomas, 1979).

(3) Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (New York: Van Nostrand Reinhold, 1970)

(4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (New York: Springer-Verlag, 1974).

(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (London: Butterworths, 1973).

(6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).

(7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat. (New York: Academic, 1963).

(a) Purpose. (1) 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 known neurotoxicants should be evaluated for these effects.

(2) This guideline defines procedures for conducting studies of schedule-controlled operant behavior, one way of evaluating functional neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this section). Our purpose is to evaluate the effects of acute and repeated exposures on the rate and pattern of responding under schedules of reinforcement. Operant behavior tests may be used to evaluate many other aspects of behavior (Laties, 1978 under paragraph (f)(3) of this section). Additional tests may be necessary to completely assess the behavioral effects of any substance. Behavioral evaluation should be used in conjunction with neuropathologic evaluation and the evaluation of other toxic effects.

(b) Definitions—(1) Neurotoxicity. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent. Behavioral toxicity is an adverse change in the functioning of the organism with respect to its environment following exposure to a chemical agent.

(2) Operant, operant behavior, operant conditioning. An operant is a class of behavioral responses which change 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.

(3) 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 under paragraph (f)(2) of this section). For example, a fixed ratio (FR) schedule requires a fixed number of responses to produce a reinforcer (e.g. FR 30). On 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 operant behavior are repeated. 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. (A) For most studies, the laboratory mouse or rat is recommended. Standard strains should be used.

(B) 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. (A) Approximately equal numbers of male and female animals are required for each dose level and control group.

(B) 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. If post exposure effects are examined, a separate group, 6 to 12 additional animals not sacrificed for pathology, will required in subchronic studies.

(4) Control groups—(i) Untreated controls. A concurrent “sham” exposure or vehicle control group or session (according to the design of the study) is required. The subjects should be treated similarly except that administration of the test substance is omitted.

(ii) Positive controls. Positive control data is required to demonstrate that the experimental procedures, under the specific conditions in the testing laboratory, 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 should be collected according to the same experimental design as that proposed for the test substance. Historical data on the procedure collected in the same species and under the same conditions in the testing laboratory may be acceptable, but the presentation of concurrent control data is strongly encouraged since it provides evidence that the test has remained sensitive.

(5) Dose levels and dose selection. At least 3 doses, equally spaced over a log scale (e.g., 10, 30, 100), over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.

(i) The highest dose shall produce: (A) Clear behavioral effects; or (B) life-threatening toxicity.

(ii) The data from the lower doses must show either: (A) Graded dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose levels, respectively.

(6) Duration of exposure. The duration and frequency of exposure will be specified in the test rule.

(7) Route of Administration. The route of administration will also be specified in the test rule and will usually be identical to one of the anticipated or actual routes of human exposure. For some chemicals, another route (e.g. parenteral) may be justified. The exposure protocol should conform to that outlined in the appropriate acute or subchronic toxicity study guideline under subpart B or subpart C of this part.

(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) Deprivation and training. (A) If a nonpreferred positive reinforcer is used, all subjects should be deprived of food until they reach a fixed percentage (e.g. 80 to 90 percent, commonly) of their ad libitum body weight or for a fixed period (e.g., 18 hours) prior to training. Deprivation should be kept constant throughout the study.

(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.

(C) Cumulative records of cumulative responding over time for each animal should be presented to demonstrate that the pattern of responding is representative of that generated by the schedule of reinforcement.

(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, usually shortly after exposure. For subchronic studies, subjects should be tested prior to daily exposure in order to assess cumulative effects.

(B) Frequency of testing. The maintenance of stable operant behavior normally will require regular and frequent (e.g., 5 days a week) testing sessions. Animals should be weighed on each test day.

(C) Duration of testing. (1) 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.

(2) If the nature or duration of effects following cessation of repeated exposure are a concern, animals from the high dose group should be tested following exposure for a suitable period of time.

(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. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report should contain the following information:

(1) Description of system, test methods, experimental design, and control data. (i) A description of the experimental chamber, programming equipment, data collection devices, and environmental conditions.

(ii) A description of the experimental design including counterbalancing procedures, and the stability criterion.

(iii) A description and statistical evaluation of positive control and other control data, including standard measures of central tendency, variability, coefficient of variation of response rates, and the slope of the dose-effect curve.

(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 of responding, changes in response rate produced by the chemical, and group data for the same variables, including standard measures of central tendency, variability and coefficient of variation.

(ii) A description and statistical evaluation of the test results: With particular reference to the overall statistical procedures (e.g., parametric or nonparametric) dose-effect curve, and calculation of slope. Presentation of calculations is encouraged.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Dews, P.B. “Assessing the Effects of Drugs,” Methods in Psychobiology, Vol. 2, Ed., R.D. Myers (New York: Academic Press, 1972) 83-124.

(2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New York: Appleton-Century-Crofts, 1957).

(3) Laties, V.G. “How Operant Conditioning can Contribute to Behavioral Toxicology,” Environmental Health Perspectives, 28: 29-35 (1978).

(4) National Academy of Science. Principles for Evaluating Chemicals in the Environment. (Washington, DC: National Academy of Sciences, 1975).

(5) National Academy of Science. Principles and Procedures for Evaluating the Toxicity of Household Substances. (Washington, DC: National Academy of Sciences, 1977).

(6) 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).

(a) Purpose. In the assessment and evaluation of the toxic characteristics of organophosphorus substances the determination of subchronic delayed neurotoxicity may be carried out, usually after initial information on delayed neurotoxicity has been obtained by acute testing or by the demonstration of inhibition and aging of neurotoxic esterase in hen neural tissue. The subchronic delayed neurotoxicity test provides information on possible health hazards likely to arise from repeated exposures over a limited period of time. It will provide information on dose response and can provide an estimate of a non-effect level which can be of use for establishing safety criteria for exposure.

(b) Definitions. Subchronic delayed neurotoxicity is a prolonged, delayed-onset locomoter ataxia resulting from repeated daily administration of the test substance.

(c) Principle of the test method. Multiple dose levels of the test substance are administered orally to domestic hens (Gallus gallus domesticus) for 90 days. The animals are observed at least daily for behavioral abnormalities, locomotor ataxia and paralysis. Histopathological examination of selected neural tissues is undertaken at the termination of the test period.

(d) Test procedures—(1) Animal selection. The adult domestic laying hen, aged 8 to 14 months, is recommended. Standard size breeds and strains should be employed.

(2) Number of animals. Ten hens should be used for each treatment and control group.

(3) Control group—(i) General. A concurrent control group should be used. This group should be treated in a manner identical to the treated group, except that administration of the test substance is omitted.

(ii) Reference substances. If a positive control is used, a substance which is known to produce delayed neurotoxicity should be employed. Examples of such substances are triorthocresyl phosphate (TOCP) and leptophos.

(4) 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.

(5) Dose levels. At least three dose levels should be used in addition to the control group(s). The highest dose level should result in toxic effects, preferably delayed neurotoxicity, but not produce an incidence of fatalities which would prevent a meaningful evaluation. The lowest dose level should not produce any evidence of toxicity.

(6) Route of administration. Oral dosing each day for at least 5 days per week should be carried out, preferably by gavage or administration of gelatine capsules.

(7) Study conduct—(i) General. 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. The test or control substance should be administered and observations begun. All hens should be carefully observed at least once daily throughout the test period. Signs of toxicity should be 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 once 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. The hens should be weighed weekly. Any moribund hens should be removed and sacrificed.

(ii) Pathology—(A) Gross necropsy. In the presence of clinical signs of delayed neurotoxicity useful information may be provided by gross necropsy.

(B) Histopathology. Tissues from all animals should be fixed in situ, using perfusion techniques. Sections should include medulla oblongata, spinal cord and peripheral nerves. The spinal cord sections should be taken from the upper cervical bulb, the mid-thoracic and lumbosacral regions. Sections of the proximal region of the tibial nerve and its branches and of the sciatic nerve should be taken. Sections should be stained with appropriate myelin and axon-specific stains. Microscopic examination should be carried out on all hens in the control and high-dose groups. Microscopic examination should also be carried out on hens in the low and intermediate dose groups when there is evidence of effects in the high-dose group.

(e) Data reporting and evaluation—(1) Test report. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report must include the following information:

(i) Toxic response data by group with a description of clinical manifestations of nervous system damage; where a grading system is used the criteria should be defined.

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

(iii) The day of observation of each abnormal sign and its subsequent course.

(iv) Body weight data.

(v) Necropsy findings for each animal, when performed.

(vi) A detailed description of all histopathological findings.

(vii) Statistical treatment of results, where appropriate.

(2) Treatment of results. (i) 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.

(ii) All observed results should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.

(3) Evaluation of results. The findings of a subchronic delayed neurotoxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the incidence and severity of observed neurotoxic effects and any other observed effects and histopathological findings in the treated and control groups. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level based on lack of clinical signs and histopathological changes.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Abou-Donia, M.B. “Organophosphorus ester-induced delayed neurotoxicity” Annual Review of Pharmacology and Toxicology, 21:511-548 (1981).

(2) Abou-Donia, M.B., Pressing, S.H. “Delayed neurotoxicity from continuous low-dose oral administration of leptophos to hens.” Toxicology and Applied Pharmacology, 38:595-608 (1976).

(3) Baron, R.L. (ed). “Pesticide Induced Delayed Neurotoxicity,” Proceedings of a Conference, February 19-20, 1976, Washington, DC. U.S. Environmental Protection Agency. EPA Report No. 600/1-76-025, Washington, DC (1976).

(4) Cavanaugh, J.B. “Peripheral neuropathy caused by chemical agents” Critical Reviews of Toxicity, 2:365-417 CRC Press, Inc. (1973).

(5) Johannsen, F.R., Wright, P.L., Gordon, D.E., Levinskas, G.L., Radue, R.W., Graham, P.R. “Evaluation of delayed neurotoxicity and dose-response relationship of phosphate esters in the adult hen,” Toxicology and Applied Pharmacology, 41:291-304 (1977).

(6) Johnson, M.K. “Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure/activity studies,” Archives of Toxicology, 34:259-288 (1975).