Neurotoxicologyand Teratology,Vol. 13, pp. 599-609. PergamonPress plc, 1991. Printed in the U.S.A.

0892-0362/91 $3.00 + .130

Interlaboratory Comparison of Motor Activity Experiments: Implications for Neurotoxicological Assessments 1 K. M . C R O F T O N , .2 J. L. H O W A R D , t V. C. M O S E R , § M. W . G I L L , ¶ L. W. R E I q ~ R , * H. A . TILSON~: 3 A N D R. C. M A c P H A I L *

*Neurotoxicology Division, Health Effects Research Laboratory U.S. Environmental Protection Agency, Research Triangle Park, NC tDivision of Pharmacology, Burroughs Wellcome Company, Research Triangle Park, NC SLaboratory of Molecular and Integrative Neuroscience National Institute of Environmental Health Sciences, Research Triangle Park, NC §NSI Technology Services Corp., Research Triangle Park, NC ¶Bushy Run Research Center, Union Carbide Corporation, Export, PA R e c e i v e d 15 January 1991 CROFTON, K. M., J. L. HOWARD, V. C. MOSER, M. W. GILL, L. W. REITER, H. A. TILSON AND R. C. MAcPHA/L. lnterlaboratory comparison of motor activity experiments: Implicationsfor neurotoxicological assessments. NEUROTOXICOL TERATOL 13(6) 599-609, 1991.--Motor activity is an important functional measure used in neurotoxicology. The effects of chemicals on motor activity, however, may depend on variables such as type of measurement apparatus, physical and environmental testing conditions, and many other experimental protocol and organismic variables. Due to the increasing use of motor activity in neurotoxicotogy, a major question concerns the potential for differences in experimental findings due to variations in sensitivity and reliability between different laboratories and devices used to measure motor activity. This study examined historical data from a number of laboratories that employed different devices and experimental protocols to measure motor activity. Four aspects of the motor activity data were compared: 1) within-laboratory control variability across time; 2) within-laboratory replicability of control data; 3) between-laboratory variability in the effects of chemicals; and 4) between-laboratory comparison of the control rates of habituation. The analyses indicated that there was a relatively restricted range of within-laboratory variability and reliability in control values, and that these ranges were comparable across laboratories. Similar profiles of habituation were also seen across the different laboratories. Moreover, in virtually every case, all laboratories were capable of detecting qualitatively similar changes in motor activity following acute exposure to a variety of chemicals. These data indicate a high degree of comparability in the data generated by the different devices and experimental protocols. Motor activity Interlaboratory comparison Within-laboratory reliability

Neurotoxicology

MOTOR activity is considered to be an " a p i c a l " test of nervous system function, in that it reflects the integrated output of the sensory, motor, and associative processes of the nervous system (22, 24, 44). Automated tests of motor activity provide objective and quantitative data on chemical-induced changes in nervous system function (24,44), and have been extensively used to examine the effects of chemicals in both pharmacology and toxicology (11, 12, 22, 44). Motor activity also has been used extensively in neuroscience to study the functional impact of di-

Within-laboratory variability

rect destruction of the nervous system, as well as subsequent alterations in the effects of centrally acting chemicals (11, 12, 19, 34, 46, 54, 55). Automated assessment of motor activity in humans also has proven to be an important variable in studying the effectiveness of drugs in human disease states such as psychiatric and arthritic disorders (56). Motor activity also has been shown to be sensitive, reliable, and efficient (3, 22, 41, 58, 59). Lastly, motor activity has been recommended by a number of national and international committees as an important component

1The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. eRequests for reprints should be addressed to Kevin M. Crofton, Ph.D., Neurotoxicology Division (MD-74B), Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. ~Present address: Neurotoxicotogy Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.

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CROFTON ET AL,

TABLE 1 PARTICIPATINGLABORATORIESAND TESTINGCONDITIONS Laboratory 1) Health Effects Research Laboratory, RTP, NC (K. M. Crofton and L. W. Reiter) 2) Burroughs Wellcome RTP, NC (J. L. Howard) 3) Health Effects Research Laboratory, RTP, NC (R. C. MacPhail) 4) NSI Technology Services RTP, NC (V. C. Moser) 5) Nat'l Inst. Environ. Hlth Sci., RTP, NC (H. A. Tilson) 6) Bushy Run Research Center, Export, PA (M. W. Gill)

System and Configuration

Strain and Sex

Age (days) or Weight (g)

Duration of Test (min)

Epoch Length (min)

Housing Conditions

Figure-8 Maze (complex alleyways)

Long-Evans male

65-130 days

60

5

2-3/cage plastic w/bedding

Woodard Photoactometer (circular alleyway)

Long-Evans male

130--400 g

60

15

4-5/cage wire mesh

Motron (square field)

Long-Evans male

65-80 days

25-30

5-6

l/cage plastic w/bedding

Figure-8 Maze (complex alleyways)

Long-Evans male and female

70-90 days

60

5

1/cage wire mesh

Photocell (square field)

Fischer-344 male

80-140 days

60

10

4/cage plastic w/bedding

SDI Photocell (cage-rack mounted)

Fischer-344 male and female

70-84 days

90

10

1/cage wire mesh

of testing batteries to evaluate the neurotoxic potential of chemicals (13, 29-32, 37, 64). Because of its potential usefulness for assessing neurotoxic chemicals, the U.S. Environmental Protection Agency (EPA) has, under the auspices of the Toxic Substances Control Act (TSCA), issued a guideline for motor activity assessments (57). A revised motor activity guideline for testing toxic substances as well as pesticides regulated under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) will soon be published. A Developmental Neurotoxicity Guideline has recently been prepared that also requires motor activity testing (15). These guidelines do not specify any given apparatus or device for motor activity testing, but rather, identify certain generic requirements including monitoring by an automated apparatus, the capability to "measure increases and decreases in activity," "reliability of operations across devices and across days," and a duration of testing adequate to "allow motor activity to approach asymptotic levels by the last 20% of the session for nontreated control animals." There has been criticism of using motor activity in neurotoxicity testing (16, 24, 42). These criticisms assert that motor activity measures have inherently large variability (6, 23, 33, 48) and a lack of specificity for identifying neurotoxic agents (24). Concerns about reliability, sensitivity, efficiency, and specificity of motor activity measurements were recently discussed by MacPhail et al. (22). However, because of the likely increase in activity assessments in various neurotoxicology laboratories, there may be additional concern over potential differences in reliability across different devices and testing conditions. For the purposes of this exercise, reliability was defined as the reproducibility of test results in terms of: 1) the between-subject variability; 2) reproducibility of control values across time; and 3) the betweenlaboratory reproducibility of qualitative and quantitative effects of xenobiotics. The purpose of the present communication was to gather data sets, including both historical control values and data from common positive control chemicals, from a number of laboratories using different devices to monitor motor activity. The objective

was to determine the extent of differences between the various laboratories in terms of the following: 1) variability of control data, expressed as the distribution of coefficients of variation; 2) within-laboratory reliability, expressed as the reproducibility of control values across time; 3) between-laboratory variability in the effects of positive control compounds; and 4) comparability of the control rates of habituation observed in the different devices. Six laboratories representing a cross section of testing conditions and research environments were asked to participate. No attempt was made to standardize testing conditions, animals, age of testing, or dosing conditions. Much of the data had already been collected, but in some cases, new data were collected for this project. Positive control compounds that were selected had generally well-defined sites and/or mechanisms of action (e.g., carbaryl and inhibition of cholinesterase). Data also were obtained for a negative control compound, methylscopolamine, a peripherally acting analogue of scopolamine. METHOD

Apparatus and Procedures Testing conditions in the six laboratories are summarized in Table I. Details of the apparatus employed by each laboratory are described below. Laboratory 1. Motor activity was measured for one h in 16 figure-eight mazes, each consisting of a series of interconnected alleys (10 × 10 cm) converging on a central arena and covered with transparent acrylic plastic (8,35). Pine shavings were used beneath the wire bottom of the test chambers. The test chambers were located on a rack in a sound-attenuated room isolated by a door from the rest of the laboratory. Lighting was supplied by overhead fluorescent bulbs. Motor activity was detected by eight infrared phototransistor/photodiode (photobeam) pairs located 2.5-3.0 cm from the floor. Six of these were equally spaced around the figure-eight portion of the maze, and one pair was located in each of the blind alleys. Photodetectors were sampled at a 1-Hz rate by a microprocessor, and each time a photobeam

INTERLAB COMPARABILITY OF MOTOR ACTIVITY DATA

601

was interrupted, an activity count was registered. Photobeam calibration was checked by a program that alternately turned each photodiode on and off 1000 times. Data were collected from 1-h test sessions in 5-min bins. Male Long-Evans hooded rats were obtained at approximately 60 days of age, and were housed two per cage in standard plastic hanging cages (24 x 20 x 45 cm). All animals were given a ten-day acclimation period and were maintained on a 12:12-h photoperiod, L:D (0600:1800). Food (Purina Lab Chow) and tap water were provided ad lib in the home cage. Temperature was maintained at 21.0---2°C and relative humidity at 40---20%. All testing was done between 0900 and 1600 hours. Animals were transferred in their home cages from the animal colony (same building) to a holding room adjacent to the test room at least one hour prior to testing. Five minutes before testing, animals were transferred to smaller cages (21 x 15 x 27 cm) and placed on shelves in the test room, after which they were placed individually into test chambers. Maze assignments, order of testing, and time of day were counterbalanced across treatments. Laboratory 2. Motor activity was measured in 12 identical doughnut-shaped metal chambers with black walls, a removable metal top and a wire mesh floor (Woodard Photoactometers, Research Corporation, Herndon, VA). Each chamber was 31 cm in diameter and had six evenly spaced (60 ° intervals) infrared photobeams 1 cm from the floor; the circular alleyway was 18 cm high. The chambers were located on shelves in an isolated room, and white noise was used to mask ambient sounds. The chambers and room were dark during activity measurement. Each interruption of a photobeam was registered as an activity count on a Data General NOVA 3/12 microcomputer via an INTERACT interface and ACT software. Activity counts for each subject were collected in 15-min bins. Photobeam calibration was periodically checked by running a ruler around the inside of the cage and comparing the number of ruler circuits to the counts registered on the computer. Subjects were adult male Long-Evans rats weighing 130--400 grams. Animals were group housed 4-5/cage in a temperature (22---I°C) - and humidity-controlled (50-+ 3%) animal colony on a 12:12-h photoperiod, L:D (0600:1800). Food and water were available ad lib in the colony. Testing was conducted between 0800 and 1700 h. Animals were transported by cart in plastic cages from the animal colony to the test laboratory (same building, same floor) for later testing. Chamber bias was controlled for by counterbalancing. Laboratory 3. Motor activity was recorded in six commercial activity devices (Motron Electronic Motility Meters, Motron Produkter, Stockholm, Sweden). Each consisted of a platform containing a matrix of 40 photodetectors that were illuminated by a single overhead incandescent lamp (GE 30R20, 30 W). Each horizontal movement of a rat that interrupted a photobeam was recorded as an activity count. A Plexiglas chamber (33 x 21 × 26 cm) was placed on top of the platform, and had a removable lid with holes for ventilation. The apparatus was placed in a wooden enclosure lined with acoustically absorbent rubber ( F M l l , Southern Kinetics, Raleigh, NC) and containing a fan (Rotron No. WR2A1, Woodstock, NY) that provided continuous ventilation. The six chambers were located in a small test room that was lined with acoustical tile. Data collection was carried out with a microprocessor located in an adjacent room. Proper operation of the photodetectors and the data collection program were checked daily by turning the overhead lamp on and off. Motor activity of individual animals was measured during either 25- or 30-min sessions that were signalled by illumination of the overhead incandescent lamp. All testing was done between 0800 and 1300 hours. Groups of six adult male LongEvans rats, approximately 60-90 days of age, were transported

in individual plastic cages from the colony room to the laboratory (same building, same floor) where they were treated and held for testing. All rats were otherwise individually housed in the colony room maintained at 21.0-+2.0°C, 55% humidity, with a 12:12-h light:dark cycle (0600:1800). Food (Purina Lab Chow) and water were available ad lib in the home cages. Laboratory 4. Motor activity was measured in figure-eight mazes of similar size and photobeam placement to those used in Laboratory 01. Plastic-backed absorbent paper was used beneath the wire bottom of the test chambers, which were located on racks in an isolated room. Adult Long-Evans hooded rats of both sexes (Charles River Laboratories, Portage, ME or Raleigh, NC) were obtained at 60 days of age and quarantined for at least one week. Rats were singly housed in wire mesh cages ( 1 5 x 2 2 x 17.5 cm). Food (Purina Lab Chow) and water were available ad lib in the colony. The animal colony was maintained at 21.0-+2.0°C, 55% humidity, with a 12:12-h light:dark cycle (0600:1800). Testing took place between 0800 and 01600 h. At the specified postdosing time, rats were placed in small transport cages and taken to the test room. The session was initiated as soon as all rats were placed in the mazes. Treatment conditions were counterbalanced across mazes and time of day. Laboratory 5. Motor activity was measured in four black Plexiglas rectangular boxes (21 x 18 x 43 cm) contained within Coulbourn (Model El0-20) sound- and light-attenuating cubicles. The cubicles were located on a rack in a room isolated from the laboratory. There was no light in the testing boxes. Within each activity box, two parallel rows of infrared photobeams, 20 per side, were positioned along the long axis of the chamber, 5.5 cm and 13 cm from the wire floor insert in the bottom of the activity box. Adjacent photobeams were separated by a space of 3.9 cm. Each interruption of a photobeam was registered as an activity count. The lower photobeams assessed horizontal activity, while the upper detectors measured vertical activity (i.e., rearing). Output from the photobeams was transmitted to a PDP 11 computer and stored for later analysis. Litter or shavings were not used in the box. Calibration of photobeams was routinely checked by running a ruler perpendicular to the plane of the photobeams and counting the number of LED emissions in a test circuit located externally to the test chambers. Male Fischer-344 rats were obtained from Charles River (Raleigh, NC) and housed 4/cage in plastic cages with corncob bedding. Lab chow (NIH diet 31) and tap water were available ad lib in the colony room. The animal colony was maintained at 2 1 . 0 - 2 . 0 ° C , and 55% relative humidity with a 12:12-h light: dark cycle (0600:1800). All testing was done between 0900 and 1600 hours. Animals were brought in their home cages on carts to the testing area, which was located in the same building and floor as the colony room. The animals were usually housed singly and were removed one at a time and placed individually into the activity testing chambers. Animals not being tested sat in their home cages on the cart in a waiting area. Box assignments, order of running and time of day were counterbalanced across treatment groups. Laboratory 6. Motor activity was measured in 30 identical rectangular cages (24 x 18 z 41 cm) constructed of stainless steel (back and side walls) and wire mesh (floor and front wall) mounted on a double-sided rack (15 enclosures/side). Enclosures were stacked in 6 rows with 2 or 3 enclosures/row/rack side. The long dimension of each enclosure faced outward to the room. Each enclosure was equipped with two sets of infrared photobeams. One set of four photobeams projected from the front to the back of the enclosure 2.5 cm from the floor and was spaced 7.5 cm apart. A second set of eight photobeams projected side to side 12.5 cm from the floor and was spaced 2.5 cm apart. Sequential interruption of 3 photobeams in the

602

CROFTON ET AL.

TABLE 2 CHEMICALS TESTED, ROUTE OF ADMINISTRATION, PRETEST DOSING TIMES, VEHICLE, AND NUMBER OF ANIMALS PER TREATMENT GROUP Compound Stimulants d-Amphetamine Scopolamine Triadimefon Depressants Carbaryl Chlorpromazine Endosulfan Physostigmine Miscellaneous Cypermethrin Methylscopolamine

LAB 1

LAB 2

LAB 3

LAB 4

SC, 20 min, S, 10 IP, 0 min, S, 6 IP, 30 min, S, 8 SC, 20 min, S, 10 IP, 0 min, S, 20 IP, 30 min, S, 6 nt PO, 60 min, CO, 8 PO, 60 min, CO, 12

IP, 30 rain, S, 9 IP, 30 min, S, 8 IP, 20 min, E, 8

nt IP, 30 min, CO, 10 IP, 0min, S, 10 IP, 60 min, S, 9 nt PO, 60 rain, CO, 10 IP, 0 min, S, 36 SC, 15 min, S, 8

IP, 30 min, E, 8 IP, 30 min, CO, 6 IP, 30 min, S, 9 IP, 30 min, S, 6 PO, 120 min, CO, 12 PO, 90 min, CO, 8 nt IP, 20 min, S, 6

PO, 90 min, CO, 10 nt SC, 20 min, S, 9 IP, 0 min, S, 6

PO, 120 min, CO, 6 IP, 30 min, S, 6

LAB 5

LAB 6

SC 15 min, S, 8 PO, 120 min, W, 28 SC, 15 min, S, 8 nt* PO, 60 min, CO, 8 PO, 60 min, CO, 10 nt IP, 60 rain, S, 8 nt SC, 15 min, S, 8

PO, 240 min, CO, 8 nt IP, 30 min, S, 8 SC, 15 min, S, 8

nt PO, 120 rain, W, 28 nt nt nt nt

*Not tested. Route of administration: SC = subcutaneous; PO = per os; IP = intraperitoneal. Vehicles: S = saline; CO = corn oil; W = distilled water; E = emulphor (5%)/ethanol (5%)/water (90%) suspension.

lower set was recorded as one ambulatory activity count. Interruption of one or two adjacent photobeams was recorded as a fine activity count. Horizontal activity was defined as the sum of fine and ambulatory activity counts. Interruption of any of the upper set of photobeams was recorded as a vertical activity count. Simultaneous interruption of adjacent photobeams in the upper set also was recorded as one vertical activity count. Photobeam calibration was routinely checked by manually interrupting photobeams in predetermined sequences and by an integral diagnostics program. The testing rack and data collection software were manufactured by SDI Inc. (San Diego, CA). The testing rack was located in a sound-attenuated room maintained at 21.0°C, 50% humidity, 60 dB of white noise, with a 12:12-h light:dark cycle (0500:1700). Testing took place between 0830 and 01600 h. The animals were housed individually in stainless steel cages with wire mesh floors (23 x 18 × 15 cm) in a room located in the same building as the testing room. Animals were transferred to the testing room and immediately placed in the test chambers. Test sessions were started simultaneously for all animals.

Data Collection and Analyses Historical control data. Data sets were assembled from historical records of each laboratory. Inclusion of data was restricted to nontreated and nonaffected vehicle-treated (e.g., saline, corn oil) groups of rats, horizontally directed activity, and total counts per test session. For each data set, the following information was requested: 1) date of collection; 2) treatment (nontreated or type of vehicle); 3) number of animals/group; 4) group mean and standard deviation; and 5) sex, age and strain. Habituation data. Each laboratory also submitted data from one or more test sessions expressed as the mean counts per epoch within a session. The test session length and duration of the epochs varied between laboratories depending on historical precedent. Chemical data. All chemicals tested are listed by laboratory in Table 2. For each compound, the following-information was requested: 1) time between dosing and testing; 2) sex of subjects; 3) number of animals per group; 4) dosages of test mate-

rial; 5) mean and standard error of the total counts per test session (nontransformed raw scores); 6) route of administration; and 7) vehicle. For ease of comparison between laboratories, all group mean treatment data were expressed as percentages of the appropriate vehicle control means. Data analysis. All data sets were entered into computer files and summary data were compiled using SAS (47). For the historical control data, coefficients of variation [CV = (standard deviation/mean) × 100] were calculated to represent between-subject variability in nontreated and/or vehicle-treated control groups. The distribution of CVs was plotted as a percent of the total number of observations. Distributions were tested for normality using the Kolmogorov D statistic or Shapiro-Wilk statistic (47). The stability of control means over time was analyzed by plotting the control means vs. time and computing a grand mean and grand CV for each laboratory. Due to the limited number of data sets submitted from Lab 6, no historical data descriptors were calculated. For the chemical data, EDsos were calculated using a modification of a probit program (47). An EDso was defined as the dosage that produced a 50% increase or decrease in motor activity relative to the vehicle-control mean and regardless of whether asymptotic levels were reached. Statistical analyses of the chemical data were performed to determine significant differences between treated and the nontreated vehicle groups. The LOEL (lowest observed effects level) was defined as the lowest dose that produced statistically significant changes in motor activity as compared to the control groups. Specifics of the statistical evaluations were left to the discretion of each investigator. RESULTS

Historical Control Data Figure 1 shows the relative-frequency distribution of between-subject control CVs for each laboratory. These data indicate a remarkably good consistency between Laboratories 1 through 4, with the mean CVs ranging from 18.9 to 24.6%. The overall mean between-subject control CV for the five laboratories was 23.2%. Lab 5 had a slightly higher mean CV of 30.7%.

INTERLAB COMPARABILITY OF MOTOR ACTIVITY DATA

il ~2 =77 i t Lab I 4~ l ~ Total#CV =22.1%t

== .%

Mean

i : ta~

q

Coefficent of Variation

ii ~ Toatl #1=51 ~

Coefficent of Variation

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The distribution of the CVs was not normal for Labs 1-4 [p distribution was positively skewed (the skewness statistic ranged from 0.27 to 0.81)]. In contrast, the data from Lab 5 were normally distributed (p>0.15). Figure 2 shows that control mean values were relatively stable across time. This is especially apparent for Laboratories 1 and 5. There was a slight drift upwards in time for Labs 2 and 3. The upward trend for Lab 3 resulted mainly from a shift in total test time from 25 to 30 minutes during 1987. Data for Lab 4 also were stable over time, except for a few relatively low values from vehicle-treated groups during the fall of 1988. The mean and CV for the control values combined across time for each individual laboratory are seen in the insets for Fig. 2. The overall mean CV for replication over time was 15.9%.

603

doses of d-amphetamine, some devices (Labs 1, 3, and 4) registered fewer counts than at lower doses, resulting in an "inverted U"-shaped dose-response function. Other devices (Labs 2, 5, and 6) obtained monotonically increasing activity with increasing dose. Scopolamine. The results with scopolamine were also in good agreement between laboratories (Fig. 5) in that all laboratories reported increases in activity following administration of scopolamine. Labs 2 and 5 demonstrated the largest increases of 260% and 120% above control levels, respectively. Labs 1 and 4 had peak increases of 83 and 40%, respectively. Note that the increase in activity for the 0.3-mg/kg dose for Lab 3 was not significant so no LOEL could be calculated, and that the peak increase for Lab 4 was only 40% so no EDso could be calculated. There was general agreement in that the LOELs ranged from 0.19 to 1.00 mg/kg and EDso values ranged from 0.19 to 0.58 mg/kg (Table 3). Triadimefon. All five laboratories reported hyperactivity following exposure to triadimefon (Fig. 5). LOELs ranged from 50 to 100 mg/kg. All labs reported "inverted U"-shaped functions, in that higher doses produced less effects than did intermediate doses. The EDsos ranged from 28.9 to 120.7 mg/kg.

Chemical Data--Agents That Decreased Motor Activity Carbaryl. Three out of three labs reported dose-dependent decreases in activity following exposure to carbaryl (Fig. 6). There was a remarkable similarity in the dose-effect curves in that the EDsos for the three laboratories were 13.3, 13.6, and 17.6 mg/kg. LOELs ranged from 8.0 to 10.0 mg/kg. Chlorpromazine. All six laboratories reported that chlorpromazine decreased motor activity (Fig. 6). Labs 1-5 reported EDsos between 2.3 and 4.7 mg/kg, whereas Lab 6 had a slightly higher EDso of 7.9 mg/kg (Table 3). LOELs ranged from 1.0 to 5.6 mg/kg (Table 3). Endosulfan. There was good agreement between laboratories for the effects of endosulfan (Fig. 6). All labs reported decreased activity, and the dose-effect curves revealed a LOEL of 5 mg/ kg in all cases. The EDsos ranged from 8.5 to 16.2 mg/kg (Table 3). Physostigmine. Four out of four labs reported dose-dependent decreases in activity following administration of physostigmine (Fig. 6). The dose-effect curves were similar in that the LOELs ranged from 0.1 to 0.56 mg/kg and ED~os ranged from 0.21 to 0.40 mg/kg (Table 3). Chemical Data--MisceUaneous

Habituation Data Figure 3 illustrates the habituation data for each laboratory. Although the data were collected in different interval lengths (epochs) across laboratories and the total counts per test session were different, a decrement in the number of counts per interval occurred in all cases within the session. The habituation data for all laboratories, expressed as a percent of the initial interval's value, are presented in Fig. 4.

Chemical Data--Agents That Increase Motor Activity d-Amphetamine. All laboratories reported increased activity following dosing with d-amphetanaine (Fig. 5). Regardless of the measurement device, LOELs ranging from 0.38 to 1.25 mg/kg represent increased activity relative to vehicle control levels. EDsos ranged from 0.54 to 2.67 mg/kg (Table 3). At higher

Methylscopolamine. There was some inconsistency in the resuits of testing methylscopolamine (Fig. 7). Labs 1, 3 and 5 reported no significant effects at doses up to 3 mg/kg. Lab 4 demonstrated a "U"-shaped function, a slight decrease at an intermediate dose (1.0 mg/kg) and no effect at higher or lower doses. Lab 2 reported a slight decrease only at the highest dose tested (0.5 mg/kg). These decreases in activity produced by methylscopolamine were no greater than 29 and 24%, for Lab 2 and Lab 4, respectively. No EDsos could be calculated for methylscopolamine. Cypermethrin. The effects of cypermethrin are presented for three laboratories in Fig. 7. Labs 1 and 4 reported decreases in activity over a wide range of doses. Lab 3 reported a decrease (--25%) at the intermediate dose, followed by a substantial increase at the highest dose of cypermethrin. In Lab 3, this dose was reported to produce clear signs of the Type II pyrethroid

604

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5 10 15

20

25

30

35

40

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606

CROFTON ET AL.

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FIG. 6. Effects of carbaryl, chlorpromazine, endosulfan and physostigmine on motor activity. Data are plotted as the percentage of the vehicle control mean ( -+SE) for each laboratory. Control means, respectively, are as follows: (A) carbaryl--330, 3418 and 354, for Labs 1, 3, and 5; (B) chlorpromazine--311, 897, 3372, 293, 1762 and 857 for Labs 1--6; (C) endosulfan--226, 2856 and 323, for Labs 1, 3 and 4; (D) physostigmine--296, 897, 3918 and 1486, for Labs 1, 2, 3 and 5. Key to symbols is the same as Fig. 5.

nonstandardized conditions, yielded lower ratios. The mean for the behavioral EDsos from the present study was 2.81. The mean for the behavioral LOELs was 3.55. It remains to be determined to what extent these ratios would be decreased further by standardizing several of the key variables. The comparison of the habituation data yielded small interlaboratory differences. Regardless of the configuration of the device, the rate of habituation was similar (Figs. 3 and 4). These results suggest that a time period of approximately 50 to 60 minutes may be necessary to adequately study toxicant effects on the habituation of motor activity. One exception to this may be the device used in Lab 6 which appears to require a slightly longer period of time (e.g., 70-80 min) (note that Lab 6 sub-

ables (e.g., age and strain of rat) that existed between laboratories. Variables such as these have been shown to be important in the effects of chemicals on motor activity (7, 12, 19, 44, 60). These differences in EDsos between laboratories are lower than the variability between laboratories for LDso estimates. In a study authorized by the Commission of the European Communities (under nonstandardized test conditions), ratios of maximal to minimal LD~o values for 5 chemicals ranged from 3.6 to 11.9 with a mean of 6.96 (18,65). In a follow-up study that employed more standardized test conditions, the range of ratios for these same 5 chemicals was from 2.4 to 8.4 with a mean of 4.4 [EPA/CEC, 1979 as cited in (65)]. The present interlaboratory comparison of behavioral data for 9 compounds, tested under

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Dosage (mg/kg) FIG. 7. Effects of methylscopolamine and cypermethrin on motor activity. Data are plotted as the percentage of the vehicle control mean ( _ SE) for each laboratory. Control means, respectively, are as follows: (A) methylscopolamine--275, 897, 4357, 335 and 1788, for Labs 1-5; and (B) cypermethrin--321, 3572 and 342, for Labs I, 3 and 4. Key to symbols is the same as Fig. 5.

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mitted data from 90-rain test sessions). These results are consistent with a previous report that habituation rates were similar between four different types of activity devices, jiggle cages, photocell cages, a circular open field, and running wheels (53). Taken together, these data suggest that these diverse devices measure the same process or construct (i.e., habituation). In summary, this study demonstrates that motor activity measurements taken in different laboratories under disparate testing conditions are reliable, reproducible, and sensitive to a variety of chemical agents. Regardless of geometric and mechanical differences that result in different baselines of activity, these devices yield generally similar levels of both within- and betweenexperiment control variability. The relatively low levels of withinand between-experiment variability as well as the good comparability between laboratories argue that the belief of inherently large variability in motor activity measurements (6, 23, 33, 48)

is wrong. Moreover, the results of testing a variety of pharmacological and pesticidal chemicals indicate that these devices are capable of detecting chemical-induced alterations in motor function that in almost all cases are qualitatively and quantitatively similar. Since all the chemicals tested here affected motor activity due to neuroactive properties, future studies should also compare the effects of neuropathic chemicals on motor activity. Further work should also compare the results of repeated dosing and testing studies. The data presented here suggest that the specifics of device design may not be the most important variable in motor activity testing. Rather, well-defined standardized testing conditions within a laboratory appear to be of paramount importance in reducing within and between experimental error. Finally, we thoroughly encourage similar retrospective analyses for other measures of nervous system function likely to be routinely used to assess the neurotoxic potential of chemicals.

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