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July 1999, Meeting Supplement 1

A Generic Protocol for Evaluating Chemical-Induced Neurotoxicity in Rats Following One-Time Dietary Administration 1

This document is supplement #1 for the session entitled "A Consultation on Protocol Design to Assess Acute Neurotoxicity Following Oral Administration of Pesticides", for the July 21, 1999 meeting of the FIFRA Scientific Advisory Panel.

June 30, 1999


This study protocol is for evaluating the potential for neurotoxicity of a cholinesterase-inhibiting chemical substance following one-time dietary administration.

Overview of Experimental Design

This study protocol consists of 4 treatment groups and 1 control group (24 animals per sex per group). Animals will be conditioned in a 2 week feeding adaptation phase prior to test article administration to eat their daily ration of food over a short time interval. During the conditioning phase, animals will receive two-thirds of their daily allotment of food for 1 hour from 6:00 to 7:00 am and one-third of their daily allotment of food for 1 hour from 5:00 to 6:00 pm. All feeding will be in the dark; red lights will be used when entering the animal room during the dark cycle. Animals will have free access to water throughout the study. Test article will be administered once on Study Day 1. Dosing will be staggered over 5 days. The test agent will be administered during the first daily feed. Eight animals per sex per group will be sacrificed at peak time of effect (as determined in a range finding study) for blood and brain cholinesterase inhibition. Functional Observational Battery (FOB) and locomotor activity (MA) determination will be conducted at the peak time of effect and on Study Day 15, for 16 animals per sex per group.

Following the day 15 FOB and MA determinations, 8 of the 16 animals will be sacrificied for blood and brain cholinesterase determinations. The remaining 8 animals per sex per group will be euthanized: no necropsy will be performed and carcasses will be discarded. The first daily feed will be shifted up to 1 hour earlier or later (one-fourth the animals per sex per group up 1 hour early, 30 minutes early, 30 minutes late, or 1 hour late) for those animals to be tested on that day. Observations for clinical signs, body weights, and food consumption will be measured during the course of the study.

Test Article Preparation

The test article will be used as received. A solution of the test article will be prepared in acetone or some other organic solvent. The solution is added to ground feed on a weight/weight basis and blended in a blender. The solvent will be allowed to evaporate from the resulting premix. The premix will then be thoroughly mixed with the appropriate amount of ground certified chow in a twin-shell blender to obtain the appropriate dietary concentrations. The blending time will be determined based on the results of the homogeneity analysis. Separate premixes will be prepared for each concentration of test diet. Solvent will also be added to the control diet and allowed to evaporate, in a similar fashion to that of the test article.

Homogeneity of Test Article in Diet

Prior to test article administration, test batches of the treated diets at the lowest and highest concentrations (ppm) will be prepared to assess the homogeneity of the test diets employing the same method and batch size to be used during the study. Test diets will be mixed in a twin-shell blender for 10 and/or 20 minutes. Following mixing, 10 samples (approximately 50 g each) from each batch will be collected and analyzed. The samples will be collected as the test diet is being dispensed at evenly distributed intervals. Samples from the 10-minute mixing interval will initially be analyzed. If the results of this analysis are unsatisfactory, based on precision and accuracy of the analytical method and other historical data, samples from the 20-minute mixing interval will be analyzed. Remixing may be necessary to obtain satisfactory homogeneity results.


Appropriately sized portions of the prepared diets mixed for homogeneity analysis will be stored under study room conditions for up to 14 days. Samples (duplicate 50 g samples.) of these stored diets will be collected after 7 and 14 days at room temperature and frozen. The 14-day samples will be analyzed to determine the stability of the test article in the diet. If the results of this analysis are unsatisfactory, the 7-day samples will be analyzed. Further testing may be necessary to obtain satisfactory stability results.

Concentration Analysis

Samples of test diet at each concentration will be collected for analysis of test article concentration. Concentration analysis results will be available prior to test article administration.

Test System

Species: Rat

Strain: Sprague-Dawley

Justification of Test System: The current state of scientific knowledge does not provide any acceptable alternatives, in vitro or otherwise, to the use of live animals to accomplish the purpose of this study. The rat is a universally used model for evaluating toxicity of various classes of chemicals and for which there is a large historical database. The Sponsor has asked that additional animals be ordered in case some of the animals do not adapt well to the timed feeding schedule.

Expected Age and Weight: Animals will be approximately 6 to 8 weeks of age at arrival, and approximately 8 to 10 weeks at initiation of dosing. Animals will be greater than 175 g by initiation of dosing.


All animals will be permitted an acclimation period of approximately 2 weeks. Animals will be acclimated for 2 days using standard acclimation procedures: ad libitum food and water, and a light/dark cycle of 12/12 hours to allow animals to familiarize themselves with their new surroundings. On or after acclimation day 3 animals will begin a feeding acclimation, conditioning them to eat two-thirds of their daily allotment of food (approximately 16.5 grams) for 1 hour at 6:00 to 7:00 am and one-third of their daily allotment from 5:00 to 6:00 pm.

Environmental Conditions

Upon arrival and for the first 2 days fluorescent lighting will be provided on a 12/12 hour light/dark cycle. Starting on acclimation day 3 fluorescent lighting will be provided for approximately 10 hours per day, from 7: 00 am to 5: 00 pm to coincide with the feeding cycle, so that all feeding is in the dark. The rat is a nocturnal animal. By providing food in the dark it is more likely to eat its allotment of food. Red lights will be used by technicians when entering the animal room during the dark cycle. Temperature and humidity will be monitored and recorded daily and maintained to the maximum extent possible between 64 to 79o F and 30 to 70%, respectively. Animal rooms will have at least 10 air exchanges per hour.

Diet and Drinking Water

The basal diet will be certified (with organophosphate analysis) meal rodent chow. Tap water will be supplied ad libitum to all animals via an automatic water system unless otherwise indicated.

Test Article Administration

The test article will be administered orally only once, via dietary admixture. The test article will be available in the diet for 1 hour on Study Day 1 (toward the end of the dark cycle) during the first daily feed. Dosing will be staggered over 3 or 4 days. On the first day of staggered treatment, 12 animals per sex per group will receive test article, and 4 per sex per group on all other days.

Dose Levels

Dose levels will be based on the results from a dose-range finding study, mg per kg body weight.

Ante-mortum Study Evaluations

Cageside Observations: All animals will be observed at least twice a day for morbidity, mortality, injury, and availability of water. Any animals in poor health will be identified for further monitoring and possible euthanasia.

Detailed Clinical Examinations: A detailed clinical examination of each animal will be performed once prior to study initiation, each study day for each animal not receiving FOB and locomotor activity assessments on that day, and at termination. The examination will include, but will not be limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration and palpation of tissue masses.

Body Weights: Body weights will be measured and recorded several times: within 3 days of arrival, at least once prior to randomization, on the day prior to test article administration (Study Day - 1), the day of test article administration (Study Day 1), 7 days post dose (Study Day 8) and at termination.

Food Consumption: Animal will be given a measured amount of food at each feeding (16.5 grams for the am feed and 8.5 grams for the pm feed). Food consumption will be measured for each feeding, each day throughout the study. At least 2 days of food consumption collected prior to test article administration will be used for calculation of the required concentration of test agent for delivery of the target dose. Food consumption from the test article administration feeding will be used to calculated compound consumption.

Morbidity: Any animal showing signs of severe debility or toxicity, particularly if death appears imminent, will be euthanized for humane reasons. All animals euthanized in extremis or found dead will be subjected to a routine necropsy and carcasses will be discarded.

Plasma and Red Blood Cell (RBC) Cholinesterase Evaluations: Plasma and RBC (erythrocyte) cholinesterase evaluations will be performed on the same animals predose (16/sex/group), and at Study Day 1 at the peak time of effect (8/sex/group) and also at termination Study Day 15 (8/sex/group). Approximately 1 mL of blood will be collected for cholinesterase evaluation in plasma and erythrocytes (RBCs) via the jugular vein from unanesthetized animals for predose evaluation and via cardiac puncture at termination. Animals will be euthanized by carbon dioxide inhalation prior to cardiac puncture. The order of bleeding and analysis will be alternating (one animal from each dose group, then repeating) to reduce handling and time biases.

Neurobehavioral Evaluations: Behavioral tests will be conducted on animals without knowledge on the part of the testers of the treatment groups. The animals will be evaluated using the following neurobehavioral evaluations.

Functional Observational Battery (FOB) Evaluations: FOB evaluations will be conducted at peak time of effect and 14 days following test article administration (Study Day 15) for 16 animals per sex per group. Each animal will be observed for a minimum of 3 minutes in a black plexiglass, open-field observation box measuring 20 in. by 20 in. by 8 in. Parameters evaluated will be based on those outlined in Moser et al.(1988). The following evaluations will be conducted:

  1. Assessment of signs of autonomic function: Ranking of the degree of lacrimation and salivation, with a range of severity scores from none to severe. Presence or absence of piloerection and exophthalmus. Measurement of urination and defecation including polyunia. and diarrhea. Pupillary function as indicated by constriction of the pupil in response to light. Degree of palpebral closure, e.g. ptosis.

  2. Description, incidence and severity of convulsions, tremors, abnormal motor movements, both in 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 general level of activity during observations of the unperturbed subject in the open field, with a range of severity scores from unresponsive to hyperreactivity.

  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 the procedure described by Meyer et al. (1979).

  8. Quantitative measure of landing foot (hindfoot) splay as described by Edwards and Parker (1977).

  9. Sensorimotor responses to stimuli of different modalities will be used to detect gross sensory deficits. Pain perception will be assessed by a ranking or measure of the reaction to a tail pinch and measuring the latency to a nociceptive (thermal) response when the subject is placed on a heated (52 oC) surface, as described by Ankier (1974). The response to a mechanically produced "click" will be ranked 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.

  12. Other measures to be recorded are counts of rearing activity in the open field, ranking of air righting. body temperature measured rectally, and spontaneous or excessive vocalizations. Alterations in rate and ease of respiration (e.g. rales or dyspnea), and sensorimotor responses to visual (approaching blunt object) and proprioceptive (touch on rump with blunt object) stimuli.

Motor Activity: Two similar methods were described for motor activity (MA) testing. The first method involves testing of each animal once before initiation of treatment and on days 1 and 15 at approximately the same time each day after the FOB. The animals will be placed in an automated photocell activity recording device for 40 minutes. Activity counts will be recorded at 2 minute intervals.

The second method for motor activity evaluation is conducted at peak time of effect and 14 days following test article administration (Study Day 15) for 16 animals per sex per group. Animals will be monitored (recorded) for 6 consecutive 10 minute intervals allowing for examination of both exploratory and acclimation activity levels. Movement is recorded by 16 photocells each in two horizontal and one vertical phase. Two of these planes are used to record horizontal activity and intersect at right angles to form a grid pattern. The third plane is located above the first two and records vertical activity. A range of different activities will be recorded but only the following will be used in comparisons between treated and control animals as the most representative activity parameters: horizontal activity, vertical activity, total distance (cm.) and stereotypic activity.

Brain Cholinesterase

Brain cholinesterase evaluations will be performed on the same animals for which plasma and RBC cholinesterase determinations will be conducted. Eight animals per sex per group on Study Day 1 at peak time of effect and at termination Study Day 15 (8/sex/group) will be used. Following blood collection, animals will be exsanguinated. The skull will be opened and brain removed and weighed. The cortex, hippocampus, and striatum. will be dissected and weighed separately. Brain sections will be stored frozen prior to assay for brain cholinesterase. No necropsy will be performed. Carcasses will be discarded. All other animals will be euthanized and no necropsy will be performed (8 animals/sex/group) following the Study Day 15 FOB and locomotor activity assessment. Carcasses will be discarded.


Ankier, S. 1. (1974). New hot plate tests to quantify anti-nociceptive and narcotic antagonist activities. Europ. J Pharmacol. 27, 1-4.

Edwards, P. M. and Parker. V. H. (1977). A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats. Toxicology and Applied Pharmacology 40, 589-591.

Meyer, 0. A.. Tilson, H. A., Byrd, W. C., and Riley, M. T. (1979). A method for the routine assessment of fore- and hindlimb -rip strength of rats and mice. Neurobehav. Toxicol. 1, 233-236.

Moser, V. C., McCormick, J. P., Creason, J. P., and MacPhail, R. C. (1988). Comparison of chlordimeform and carbarvi usin- a functional observational battery. Fund. Appl. Toxicol. 11. 189-206.

1 This document was prepared by EPA, and is a composite of two similar test protocols recently submitted to the Agency. Not all details of the submitted protocols are described here: only those details deemed relevant by the Agency for purposes of the FIFRA Scientific Advisory Panel session are provided in this document. The text in this document is essentially identical to that used in the submitted protocols. Back to Top Scientific Advisory Panel (SAP): July 1999 Meeting Supplement #1

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