Subchronic Inhalation Neurotoxicity Studies of Ethyl Acetate in Rats

NeuroToxicology 24 (2003) 861–874

Subchronic Inhalation Neurotoxicity Studies of Ethyl Acetate in Rats Greg R. Christoph1, John F. Hansen1, Hon-Wing Leung2,* 1

Haskell Laboratory for Toxicology and Industrial Medicine, E.I. DuPont de Nemours and Company, Newark, DE 19714, USA 2 Consultant, 15 Deer Park Road, Danbury, CT 06811, USA Received 28 February 2003; accepted 5 May 2003

Abstract Rats were exposed to 0, 350, 750 or 1500 ppm of ethyl acetate by inhalation for 6 h per day, 5 days per week for 13 weeks. Functional observational battery (FOB) and motor activity tests occurred on non-exposure days during weeks 4, 8 and 13, after which tissues were microscopically examined for neuropathology. A subset of rats was monitored during a 4-week recovery period. Exposure to 750 and 1500 ppm, diminished behavioral responses to unexpected auditory stimuli during the exposure session and appeared to be an acute sedative effect. There were no signs of acute intoxication 30 min after exposure sessions ended. Rats exposed to 750 and 1500 ppm had reduced body weight, body weight gain, feed consumption, and feed efficiency, which fully or partially recovered within 4 weeks. Reductions in body weight gain and feed efficiency were observed in male rats exposed to 350 ppm. The principal behavioral effect of subchronic exposure was reduced motor activity in the 1500 ppm females, an effect that was not present after the 4-week recovery period. All other FOB and motor activity parameters were unaffected, and no pathology was observed in nervous system tissues. Operant sessions were conducted in another set of male rats preconditioned to a stable operant baseline under a multiple fixed ratio–fixed interval (FR–FI) schedule of food reinforcement. FR response rate, FR post-reinforcement pause duration, and the pattern of FI responding were not affected during or after the exposure series. In contrast, within-group FI rate for the treatment groups increased over time whereas those of the controls decreased. A historical control group, however, also showed a similar pattern of increase, indicating that these changes did not clearly represent a treatmentrelated effect. Results from these studies indicate a LOEL of 350 ppm for systemic toxicity based on the decreased body weight gain in male rats, and a LOEL of 1500 ppm for neurotoxicity based on the transient reduction in motor activity in female rats. In conclusion, there was no evidence that subchronic exposure up to 1500 ppm ethyl acetate produced any enduring neurotoxic effects in rats. # 2003 Elsevier Inc. All rights reserved.

Keywords: Ethyl acetate; Functional observational battery; Motor activity; Operant behavior; Solvents

INTRODUCTION Ethyl acetate, a volatile organic solvent, has substantial uses in industrial manufacturing processes and is contained in numerous consumer products, such as varnishes and lacquers. Worldwide production of ethyl acetate was 1,011,000 metric tons in 1998, and * Corresponding author. Tel.: þ1-203-790-4141; fax: þ1-203-790-4141. E-mail address: [email protected] (H.-W. Leung).

US production/sales was 117,727 metric tons in 1997. It has been estimated that 64–112 million consumers may encounter products containing ethyl acetate (USEPA, 1991). Ethyl acetate is regarded as having low toxicity (ACGIH, 1991), but acute exposure to high concentrations of ethyl acetate vapor can induce transient narcosis in laboratory animals (Proctor et al., 1988). This property is shared by many organic solvents and is presumably due to a depressant effect on CNS function (Andrews and Snyder, 1986). Disruption of schedule-controlled operant responding is the most

0161-813X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0161-813X(03)00074-3

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sensitive known measure for behavioral effects of acute ethyl acetate exposure. Glowa and Dews (1987) estimated that the acute inhalation concentration required to reduce fixed interval responding by 50% was about 600 ppm in mice. Although subchronic inhalation exposures to ethyl acetate have been examined with traditional toxicological measures in animals (Proctor et al., 1988; Smyth and Smyth, 1928), there have been no systematic studies of neurotoxicity. The present study focused on evaluating the potential for any behavioral and neuropathological effects following subchronic exposure to ethyl acetate.

MATERIALS AND METHODS Test Substance Monsanto Chemical Co. (Springfield, MA) supplied ethyl acetate in liquid form. Analysis by gas chromatography prior to and after completion of the subchronic exposure series indicated a purity of 99.92% relative to an analytical reference standard (Sigma–Aldrich). Known contaminants and their concentrations were water (0.029%), ethanol (0.018%), and acetic acid (0.0014%). Animals and Husbandry Crl:CD1BR (Sprague–Dawley) rats were organized into two sets, designated Neuro and Operant, corresponding to the different experimental procedures for each set. All rats were obtained from Charles River Laboratories (Raleigh, NC). Sixty male and 60 female rats in the Neuro set were 35 days old upon arrival, and 54 days old when the exposure series began. Females were nulliparous and non-pregnant. The Operant set consisted of 48 males that were 56 days old upon arrival. The subchronic exposure series began after they attained stable operant performance baselines when they were 146 days old. Only 40 males were used and evaluated in the definitive experiment; however, 48 male rats were trained to ensure that 40 rats with adequate baseline performance were available for testing. Tap water (United Water Delaware) was available ad libitum for all rats except during exposures and behavioral testing. Periodic water analyses confirmed that bacteria, coliforms, lead, and other contaminants had acceptably low levels. Feed consisted of Purina1 Certified Rodent Chow1 #5002 that met acceptable

criteria for pesticides and other contaminants. Rats in the Neuro set were fed ad libitum, except during exposure sessions. Feed was available ad libitum for rats in the Operant set until they attained about 300 g. Thereafter, individual body weights were maintained between 280 and 313 g by presentation of about 60 performance-contingent grain-based chow pellets (45 mg, BioServe, F0165) during behavioral sessions and supplemental feeding after sessions as needed. The interval between supplemental feeding and the subsequent behavioral test session was typically 14 h. All rats were singly housed in wire-mesh, stainless steel cages. Male and female rats occupied separate cage racks in rooms maintained at 23  1 8C and 50  10% relative humidity. A timer controlled a 12 h light/12 h dark cycle, and all testing and handling of the rats occurred during the lighted portion of the cycle. Exposure Concentrations Selection of 1500 ppm as the highest concentration of ethyl acetate was based on a 10 h exposure, 2-week pilot experiment indicating that 1500 ppm decreased body weight and weight gain. The intermediate (750 ppm) and low (350 ppm) exposure concentrations were approximately equally spaced fractions of the highest concentration. Concurrent controls (0 ppm) were exposed to filtered, humidified air. General Experimental Design Neuro and Operant rats were concurrently exposed to ethyl acetate in the same suite of exposure chambers. Except as noted below for rats in the Neuro set, exposure sessions occurred 6 h per day, 5 days per week (Monday–Friday) for 13 weeks. The first week of exposure was defined as week 1 and the prior week was designated week 1. Neuro Set Rats were exposed to 0 ppm (18 of each sex), 350 ppm (12 of each sex), 750 ppm (12 of each sex), or 1500 ppm (18 of each sex) of ethyl acetate vapor. A computer randomization program sorted rats into control and treatment groups so that there were no significant differences (ANOVA, Dunnett’s P > 0:05) in mean body weights between groups for each sex. Neurobehavioral testing, consisting of a functional observational battery (FOB) and motor activity assessment, occurred during week 1 to establish baseline parameters. Four replicates, counterbalanced with

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respect to sex and treatment condition, each received neurobehavioral evaluations on a non-exposure day (i.e. one of the days between Tuesday and Friday, inclusive) during weeks 4, 8 and 13. This design ensured that an exposure day always preceded a neurobehavioral test day. Exposures continued through part of week 14 to ensure that each rat had at least 65 exposures. After the final exposure session, six randomly selected rats from each group were prepared for neuropathological evaluation. The remaining rats from the 350 and 750 ppm groups were euthanized by exposure to CO2. The remaining 12 rats of each sex in the control and 1500 ppm groups were monitored during a 4-week recovery period, and FOB and motor activity tests were conducted at the end of the recovery period (week 18). Operant Set Rats that received training on a multiple fixed ratio– fixed interval (FR–FI) schedule of reinforcement for 8 weeks (10 males per treatment condition) were exposed to ethyl acetate and evaluated. During the exposure series operant behavior sessions occurred in the morning 1–2 h prior to each exposure session. After the exposure series the rats had two additional weeks of daily operant sessions to assess any post-exposure changes. Inhalation Exposures Exposure Chambers Three 0.75 m3 stainless steel and glass chambers (350, 750 and 1500 ppm) and one 1.0 m3 (0 ppm) chamber were used. The chambers were operated in a one-pass, flow-through mode with airflow rates to achieve at least 12 air changes per hour, which provided sufficient oxygen and enabled adequate distribution of ethyl acetate in the chambers. During exposure sessions, rats occupied wire basket modules that provided sufficient space for whole-body movement (e.g. turning). Atmosphere Generation A displacement pump metered ethyl acetate into a heated glass flask where it evaporated under nitrogen to reduce the possibility of explosion. The nitrogen/ethyl acetate vapor was subsequently mixed with filtered, humidified air and ducted into an exposure chamber. Fine-tuning the flow rate of ethyl acetate into the flask produced the desired atmospheric concentrations. A baffle inside the chambers promoted uniform distribution. Filtered, humidified air, to which nitrogen was

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added in amounts comparable to that used for solvent vapor generation, flowed through the control chamber. Chamber exhaust passed through a water scrubber and discharged into a roof exhaust stack. Characterization of Test Atmospheres Ethyl acetate concentrations in the exposure chamber were determined within the breathing zone of the rats every 30 min by gas chromatography. Each chamber was fitted with non-reactive Teflon1 gas sampling lines, which were connected to a microprocessor-controlled, stream-selectable Hewlett-Packard 5890A gas chromatograph (GC) connected to a Hewlett-Packard 3396A integrator. The GC was fitted with a Restek RTX1-1 column and a flame ionization detector. Samples were chromatographed isothermally at 40 8C. Atmospheric concentrations of ethyl acetate in each chamber were determined by comparing the detector response of chamber samples to standard curves. Calibration standards consisted of three different concentrations spanning the range of targeted atmospheric concentrations. Standards were prepared prior to each exposure by the quantitative dilution of ethyl acetate in known amounts of filtered air at ambient laboratory temperature and pressure. Homogeneous distribution of ethyl acetate vapor within each chamber was verified by analysis of atmospheric concentrations drawn from sample ports at nine locations throughout the chamber. Chamber Environmental Monitoring Chamber airflow, temperature, and relative humidity were monitored continuously during exposure sessions. Anemometer probes located in the chamber inlet measured total chamber airflow. A thermistor and wet/ dry bulb hygrometer inside each chamber monitored temperature and relative humidity. Electrochemical sensors measured chamber oxygen concentration twice per exposure session. Exposure Sessions Exposure sessions typically started at 09:30 h and ended at 15:30 h. Rats remained in their respective exposure chambers for about 30 min after terminating the exposure, a period sufficient to allow ethyl acetate to be cleared from the chamber. Clinical Observations During each exposure session, startle responses to a standardized auditory stimulus were crudely and subjectively scored at 2 h intervals. The stimulus

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was a sharp sound (112–117 dB, background noise level ¼ 73–75 dB) produced by striking a tethered steel nut against the metal wall of the chamber at a reproducible velocity. An observer made a visual judgment of the vigor of the response (excessive, normal, diminished, or no response). The chamber viewing windows permitted an unobstructed view of only about 50% of the rats in a given chamber, and the rapidity of the response precluded scoring responses for individual rats, so a group score was recorded. The response of rats in the control chamber was normal by definition. The observer had knowledge of the treatment condition of the rats. Standard clinical observation inventories were conducted immediately after each exposure session as the rats were removed from the chambers. Clinical observations were also conducted at weekly intervals prior to an exposure session to identify any enduring clinical signs that were not confounded by potential acute effects. Neuro Set Body Weight, Feed Consumption Body weights were recorded at least once per week throughout the exposure series. The amount of feed consumed was determined for the intervals between weighing, and feed efficiency for these intervals was computed as the ratio of body weight gained to the amount of feed consumed. Body weight, body weight gain, feed consumption, and feed efficiency data were first analyzed with Bartlett’s test for homogeneity of variance that enabled the use of parametric inferential statistics. Analysis of variance (ANOVA) followed by Dunnett’s identified treatment groups, if any, that significantly differed (P < 0:05) from control. Incidence data from clinical observations were analyzed with the Cochran–Armitage test for trend. The trend test first examined the data for all treatment groups within a sex, and if significant (P < 0:05), the test was repeated without the highest concentration group. The process of statistically deleting the highest concentration from the analysis continued until no significant differences were indicated. Functional Observational Battery The FOB evaluation was similar to previously published methods (Moser et al., 1988). Briefly, each rat was evaluated in three situations: (1) passive examination while the rat was inside a cage to which it had been acclimated for at least 10 min; (2) while handled

during removal from the cage; and (3) in an open field arena (85 cm  59 cm  20 cm). Parameters assessed in the acclimatization cage were posture, palpebral closure, writhing, circling and biting. Parameters assessed during removal from the cage and handling were ease of removal, ease of handling, muscle tone, vocalizations, piloerection, bite marks on tail and/or paws, palpebral closure, fur appearance, lacrimation, salivation and exophthalmus. Parameters assessed in the open field evaluation were righting reflex, breathing characteristics, convulsions/tremors, coordination, grooming, gait, locomotion, arousal, vocalizations, palpebral closure, defecation and urination. The open field evaluation also included assessments of responses to the experimenter’s finger approaching and touching the nose, a standardized auditory stimulus (‘‘finger cricket’’), and a mild pinch of the tail between the experimenter’s thumb and forefinger. Directing a beam of light into each eye enabled evaluation of pupillary constriction responses. A strain gauge device (Chatillon1) quantified forelimb and hindlimb grip strength. After gripping the horizontal wire grid, each subject was moved posteriorly until its paws released the grid. The force exerted on the grid at the moment of release was retained by the instrument. Grip strength measures were conducted three times for forelimbs and hindlimbs, and the results averaged. Landing foot splay was assessed by measuring the heel-to-heel distance from fresh ink impressions made when the rat was released from a height of about 30 cm onto a padded surface. Most of the categorical parameters in the FOB were subjectively scored according to simple rating scales. The rater had extensive experience in conducting FOB assessments and the same individual scored all rats for all time points. The rater was blind with respect to the treatment group of each animal. Testing order was counterbalanced with respect to exposure concentration and sex. The Cochran–Armitage test for trend was used for statistical analysis of all categorical observations in the FOB. Parametric analysis of continuous FOB variables (grip strength, foot splay) employed Bartlett’s test to verify acceptable homogeneity of variance. An ANOVA followed by Dunnett’s test identified significant differences (P < 0:05) between treatment and control groups. Separate analyses were conducted for each week and for each sex. Motor Activity After the FOB was complete for all rats within a replicate, the rats were individually tested in one of

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30 activity cages (41 cm  26 cm  18 cm). Groups and sexes were counterbalanced across the activity monitors to the fullest extent possible. The activity cages were located in an acoustically insulated room that had low-level ambient illumination. White noise masked any uncontrolled laboratory sounds. The activity monitoring devices (Coulbourn Instruments) enabled measurement of two dependent variables: time spent moving and number of movements. A continuous movement, regardless of its duration, was counted as one movement. Duration of movement and number of movements were recorded for each of six successive 10 min intervals and for the total 60 min session. Statistical analysis of motor activity data involved non-parametric methods because variance was not homogeneous (Bartlett’s test, P < 0:005). A Kruskal–Wallis test identified overall group differences, if any, and was followed by Dunn’s multiple comparisons that compared each treatment group to control. Separate analyses were conducted for each test week and for each sex. Neuropathology One day after the final exposure session, six randomly selected rats from each sex and treatment condition were anesthetized with pentobarbital followed by exsanguination and whole-body in situ perfusion with gluteraldehyde fixative. Selected tissues from the 0 and 1500 ppm groups were histologically processed and examined. Tissues from other groups were preserved for possible future evaluation. The tissues included: brain (forebrain, cerebrum, midbrain, pons, medulla and cerebellum), spinal cord (cervical and lumbar), sciatic nerve, tibial nerve, gasserian ganglia, cervical and lumbar dorsal root fibers and ganglia, cervical and lumbar ventral root fibers, and gastrocnemius muscle. The brain was cut into 2 mm coronal slabs throughout the anterior–posterior axis. Brain slabs and spinal cord segments (cervical: 5 and 6; lumbar: 1 and 2) were embedded in paraffin, sectioned (5 mm), and stained with hematoxylin and eosin. Additional sections of brain and spinal cord were stained with Luxol fast blue and periodic acid Schiff. Sciatic and tibial nerves and gasserian ganglia were embedded in glycol methacrylate, sectioned (3 mm), and stained with hematoxylin and eosin. Dorsal root ganglia, and dorsal and ventral root fibers were post-fixed in 1% osmium tetroxide, embedded in epoxy, sectioned (0.5 mm), and stained with toluidine blue. A board certified (A.C.V.P.) pathologist systematically examined 47 neuroanatomical loci by light microscopy.

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Operant Set Apparatus Behavioral training and testing were conducted in 20– 24 nominally identical operant chambers (Coulbourn, EC10-10) enclosed in sound-attenuating cubicles. White noise (76  2 dB) from a speaker in each chamber masked extraneous sounds. Illumination of an overhead lamp (house light) in each chamber designated an active operant session. One wall of the experimental chamber was equipped with cue lamps, a speaker for presentation of a tone (2.9 kHz, 75  2 dB), a dispensing trough for delivering reinforcement pellets, and a response manipulandum (lever). All experimental events were controlled and recorded by computer systems with Coulbourn L2T2 software packages. Body Weight Body weights were controlled within a target range of 280–313 g throughout training and testing. Daily body weight data were used to individually tailor the amount of feed provided to each rat each day. The minimum daily ration of standard chow was about 10 g in addition to the reinforcement pellets in the operant task. Training After learning to press the lever under a schedule of continuous reinforcement, the number of responses required for reinforcement was progressively increased until the rats attained a fixed ratio 20 (FR 20) schedule. After behavior appropriate to the FR 20 schedule was established, the fixed interval 120 s (FI 120 s) schedule component was introduced. The cue lamps were continuously illuminated during the FR schedule, whereas the continuous presence of a tone (and non-illumination of the cue lamps) signaled the FI component. The two schedule components, FR 20 (four consecutive sub-components) and FI 120 s (two consecutive subcomponents), comprised one cycle of the multiple schedule. Sessions terminated after 47 min and typically included 10 full cycles of the multiple schedule. Eight weeks of operant training (5 days per week) on the multiple schedule were provided prior to beginning the exposure series. Dependent Variables The principal dependent variables for the FR component were running rate of response and postreinforcement pause duration. Experience indicated that even well-trained rats occasionally failed to terminate a high-rate run of responses immediately upon delivery of reinforcement, and the responses that

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overflowed from one schedule sub-component to another interfered with estimates of FR running rate and pause duration. A 500 ms overflow window at the beginning of each FR sub-component was established to capture the overflow responses and exclude their influence on running rate and pause measures. FR running rate was defined as the number of responses after the overflow window divided by the time between the first response after the overflow window and the reinforced response. FR post-reinforcement pause duration was defined as the time between the previous reinforcement and the first FR response that occurred after the overflow window. FR pause duration was computed only for the second, third and fourth FR sub-components within the four repetitions of each FR component. The pause at the beginning of the first FR 20 requirement in each set of four was excluded because it did not follow FR reinforcement as in the three subsequent FR sub-components. The principal dependent variables for the FI component were overall rate of response and index of curvature. FI rate of response was defined as the number of FI responses divided by the duration of the FI component. FI index of curvature is a metric of the extent to which a rat responds with an accelerating or decelerating rate during the FI. The index was computed as described by Fry et al. (1960), except that each 120 s interval was divided into ten 12 s segments and the equation was modified accordingly. Under these conditions the index has a range of 0.9 and þ0.9. A positive index indicates an accelerating rate during the interval, and its value increases in proportion to the extent of acceleration. A negative index indicates a decelerating rate. The index of curvature is an unreliable measure if the response rate is extremely low (Allen and McPhail, 1991), so at least 10 responses during a given FI 120 s sub-component were required to include the index for a given FI subcomponent in the session mean. Retrospectively, index values were very rarely excluded due to low response rate. Group Assignment After 8 weeks of training, eight rats with the highest or lowest values for the dependent variables were excluded from the study. The remaining 40 rats were assigned to four groups of 10 rats each by stratified randomization such that the mean values for the dependent variables were not significantly different between groups. Data Analysis Within-subject coefficients of variation (COVwithin) for each rat were computed for each of the performance

measures over the five sessions during baseline (week 1), and these coefficients served as a metric of session-to-session stability. Analysis of treatment-related effects focused on data collected during the same weeks that neurobehavioral procedures occurred for rats in the Neuro set, i.e. weeks 1, 4, 8 and 13. Operant data for the final week of the post-exposure period (week 15) were also included in the analysis. For a given dependent variable, the values during sessions on Tuesday through Friday were averaged for each rat, and this weekly mean represented the entire week’s performance for the subject. Data from Mondays were not included because it followed a 2day period during which exposures did not occur, whereas sessions on Tuesday through Friday were always preceded by an exposure day. There were rare occasions of equipment failure (e.g. pellet dispenser malfunction) when a particular session for a given rat was excluded from the weekly means. Statistical analysis began with the Shapiro–Wilks test and Levene test to detect deviations from a normal distribution and homogeneity of variance, respectively. The data were then subjected to univariate ANOVAwith group (0, 350, 750 and 1500 ppm) as a between-subject factor and week (1, 4, 8, 13 and 15) as a repeated measures factor. The ANOVA also examined the interaction of the main factors. The analysis plan specified that a significant group main effect would trigger contrasts to test for differences between control and each treatment group for each week in the analysis. A significant group  week interaction triggered contrasts that compared changes in performance of a treated group between week 1 and subsequent weeks relative to changes in performance of the control group over the same period. The week main effect and associated contrasts for specific groups could only identify a change over weeks without specifying whether the change was different from that of the control group. If deviations from homogeneity of variance or normality were indicated, then non-parametric analysis consisted of the Kruskal–Wallis test to detect significant differences among groups. Dunn’s multiple comparisons were then used to compare each treatment group to control during each week. RESULTS Exposures Chamber Concentrations The ranges of chamber concentrations were 340–380, 660–780 and 1400–1600 ppm, respectively.

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The mean values, expressed to two significant digits, for the entire exposure series were equal to the targeted values of 350, 750 and 1500 ppm. Chamber Environmental Conditions Mean values for temperature, relative humidity, and airflow were similar between the four chambers. Mean daily chamber temperatures ranged between 22 and 24 8C; mean daily humidity ranged between 32 and 56%, and oxygen concentration ranged between 20 and 21%. Airflow rates were targeted at 200 l/min and ranged from 190 to 220 l/min over the course of the study. Clinical Observations Crude observations of the startle response during exposure indicated that the 1500 and 750 ppm groups displayed a diminished response one or more times during most of the exposure sessions (78 and 85%, respectively) in the series. The assessment did not suggest systematic changes in the likelihood or degree of effect between sessions. The 350 ppm rats had only one notation of a diminished response during a single session. Clinical observations conducted immediately after exposure sessions revealed no overt signs that would suggest adverse effects on nervous system function, such as lethargy or altered gait. The only treatment-related sign was stained chin that had significantly greater incidence in the 1500 ppm male group (Neuro set) than control (3/18 versus 0/18) during the course of the exposure series. Other occasional signs such as stained perineum were not systematically related to treatment. The clinical signs inventory conducted at weekly intervals prior to exposure sessions included no statistically significant or otherwise remarkable observations. Neuro Set Body Weight and Body Weight Gain Exposure to 750 and 1500 ppm significantly reduced body weight for both males and females (Fig. 1). Body weights for the 350 ppm rats, although slightly less than control, were not significantly affected. Over the entire exposure series, statistically significant (P < 0:05) reductions in body weight gain for the 1500, 750 and 350 ppm groups were 28, 17 and 12% less than control (males) and 30, 25 and 11% less than control (females). The body weight parameters for male and female rats exposed to 1500 ppm showed some evidence of recovery during the 4-week postexposure period.

Fig. 1. Mean body weights for ad libitum fed rats during and after subchronic exposure to ethyl acetate.

Feed Consumption and Feed Efficiency Over the 13-week exposure series, daily feed consumption decreased 13 and 9% (both P < 0:05) for the 1500 and 750 ppm male groups, respectively (control mean  S:D: ¼ 27:8  2:6 g). Feed consumption by 1500 ppm females decreased 8% (P < 0:05) relative to control (control mean  S:D: ¼ 18:2  1:3 g), whereas that for 350 and 750 ppm females was unaffected. The mean efficiency offeed consumed, that is grams of body weight gained per day divided by grams of feed consumed, was significantly reduced during the course of the study for all male treatment groups and for the 1500 and 750 ppm females. Over the entire exposure period, mean feed efficiency for the 1500, 750 and 350 ppm males was 17, 8 and 9%, respectively, less than control (all P < 0:05, control mean  S:D: ¼ 0:113  0:007). Feed efficiency for the 1500 and 750 ppm females during this period was 25 and 20% less than control (P < 0:05, control mean  S:D: ¼ 0:064  0:008). Feed efficiency was greater than control for the male and female 1500 ppm groups during the 4-week recovery period, suggesting a compensatory rebound for this parameter. FOB: Forelimb Grip Strength There were no treatment-related effects on forelimb grip strength in male or female rats. Table 1 shows that forelimb grip strength increased between baseline and week 8 for all groups as the rats matured, but there were no remarkable or significant differences between groups at any time point.

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Table 1 Mean forelimb and hindlimb grip strength in rats Group (ppm)

N

Week 1

Week 4

Week 8

Week 13

Week 18a

Forelimb grip strengthb Male 0 350 750 1500

18 12 12 18

0.44 0.42 0.45 0.44

(0.08) (0.12) (0.07) (0.06)

0.76 0.76 0.76 0.73

(0.17) (0.14) (0.20) (0.19)

1.12 1.23 1.18 1.20

(0.29) (0.26) (0.18) (0.24)

1.11 1.05 1.18 1.14

(0.28) (0.17) (0.19) (0.21)

1.20 (0.27)

18 12 12 18

0.41 0.44 0.43 0.43

(0.07) (0.07) (0.05) (0.08)

0.58 0.62 0.59 0.62

(0.15) (0.13) (0.10) (0.12)

0.79 0.87 0.78 0.87

(0.19) (0.17) (0.24) (0.16)

0.80 0.77 0.75 0.79

(0.23) (0.22) (0.16) (0.26)

0.71 (0.15)

18 12 12 18

0.41 0.41 0.44 0.41

(0.08) (0.09) (0.08) (0.04)

0.75 0.68 0.77 0.68

(0.15) (0.13) (0.15) (0.14)

1.03 1.05 1.05 1.02

(0.20) (0.23) (0.14) (0.25)

1.05 1.14 1.14 1.18

(0.23) (0.27) (0.17) (0.25)

1.34 (0.16)

18 12 12 18

0.41 0.40 0.42 0.43

(0.08) (0.07) (0.06) (0.09)

0.60 0.61 0.61 0.65

(0.14) (0.13) (0.10) (0.14)

0.80 0.83 0.86 0.89

(0.16) (0.16) (0.12) (0.12)

0.79 0.98 0.87 0.92

(0.14) (0.18)* (0.19) (0.15)*

0.90 (0.19)

Female 0 350 750 1500 Hindlimb grip strengthb Male 0 350 750 1500 Female 0 350 750 1500

1.15 (0.16)

0.81 (0.22)

1.18 (0.25)

0.90 (0.23)

Data are presented for test weeks before (week 1), during (weeks 4, 8 and 13), and after (week 18) subchronic exposure to designated atmospheric concentrations of ethyl acetate. a N ¼ 12 during week 18. b Grip strength in grams (S.D.). * Significantly different relative to 0 ppm group of same sex (P < 0:05).

effects on hindlimb grip strength for females during weeks 1, 4 and 8, the 350 and 1500 ppm groups had significantly greater hindlimb grip strength than control during week 13. At the end of the recovery period this effect was not present.

FOB: Hindlimb Grip Strength Hindlimb grip strength appeared to increase for all groups over time as the rats matured (Table 1). For male rats there were no meaningful differences from control at any time point. Although there were no

Table 2 Mean foot splay (heel-to-heel distance) after landing from a release height of 30 cm Group (ppm)

N

Week 1

Week 4

Week 8

Week 13

Week 18a

Landing foot sprayb Male 0 350 750 1500

18 12 12 18

6.3 6.3 6.3 6.3

(2.0) (1.9) (1.3) (1.8)

7.6 7.1 7.8 7.3

(2.5) (2.3) (1.2) (2.2)

8.3 7.8 7.7 8.6

(2.1) (2.7) (1.9) (1.8)

7.9 7.1 7.6 7.8

(2.3) (2.6) (1.8) (2.4)

8.5 (2.6)

18 12 12 18

5.4 6.9 5.5 5.8

(2.0) (2.0) (1.5) (2.2)

6.7 7.7 6.4 7.2

(1.7) (1.6) (2.0) (1.9)

6.8 7.2 7.1 6.9

(1.9) (1.7) (1.9) (2.1)

6.5 7.7 6.5 7.1

(1.8) (2.4) (2.3) (1.9)

6.3 (2.2)

Female 0 350 750 1500

8.0 (2.5)

6.8 (1.7)

Data are presented for test weeks before (week 1), during (weeks 4, 8 and 13), and after (week 18) subchronic exposure to designated atmospheric concentrations of ethyl acetate. a N ¼ 12 during week 18. b Mean foot splay in cm (S.D.).

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FOB: Footsplay Landing foot splay values increased as the rats matured, but there were no differences between treated and control rats of either sex (Table 2).

These differences did not subsequently recur. All other FOB parameters were not significantly affected in males, and none of the dependent variables were altered in female rats at any time point.

Other FOB Parameters Male rats exposed to 1500 ppm had a reduced incidence of defecation (2/18 versus 8/18, P < 0:05) and urination (2/18 versus 9/18, P < 0:05) in the open field arena relative to the control group during week 4.

Motor Activity: Duration of Movements Total duration of movement by male rats was unaffected by exposure to ethyl acetate during all test weeks (Fig. 2A). The pattern of declining duration of movement within the 60 min session is shown in Fig. 2B for

Fig. 2. Duration of movements during 60 min motor activity assessments. (A) Mean total duration of movement during 60 min assessments for male rats. (B) Mean duration of movement for each 10 min interval during the week 4 assessment for males. (C) Mean total duration of movement in female rats. (B) Mean duration of movement for each 10 min interval within the week 13 test session for females. Error bars (1 S.D.) are shown only for the control group to avoid error bar clutter. Significantly less than control (P < 0:05) by non-parametric analysis.

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week 4, which included the largest difference (not significant) in total session activity between control and 1500 ppm males. The pattern of declining activity for male rats was not significantly affected during any session. Fig. 2C and D show similar results for female rats, but there were two statistically significant differences between treatment groups and control. During week 13, the mean total duration of movements for the 1500 ppm group was reduced (22%) relative to control, and one 10 min epoch within the 60 min session was also reduced (Fig. 2D). Otherwise, all female exposure groups were similar to control at all time points. At the end of the 4-week post-exposure evaluation period, no activity differences between control and 1500 ppm female rats were detected.

were not significantly altered by exposure for both male and female rats (data not shown). Like the results for duration of movement, the total number of movements for the 1500 ppm females during test week 13 was lower than control, but not significantly so. Neuropathological Evaluation There were no treatment-related, abnormal microscopic observations in the central or peripheral nervous system in males or females. There were, however, one or two examples of neuron/myelin degeneration within most neuroanatomical sites or individual nerve fibers examined. The frequency of these observations was the same regardless of treatment condition. Tissues from the 350 and 750 ppm groups were not processed to slides for examination because there were no treatment-related neuropathological effects in the 1500 ppm rats.

Motor Activity: Number of Movements The total number of movements and number of movements within each 10 min interval of the sessions

Table 3 Metrics of operant performance subjected to inferential statistical analysis 350 ppmb

750 ppma

1500 ppmb

Operant performance measures Mean fixed ratio running response rate 1 199 (57) 4 231 (74) 8 235 (88) 13 231 (92) 15 235 (77)

183 194 207 204 213

(43) (41) (46) (45) (45)

219 237 251 247 252

(69) (85) (92) (95) (94)

212 232 251 247 248

(68) (67) (68) (62) (56)

Mean fixed ratio pause duration 1 4 8 13 15

7.0 7.3 7.3 8.8 7.7

(2.8) (2.5) (2.6) (3.1) (3.1)

6.8 7.0 6.6 7.4 7.4

(3.1) (3.6) (2.9) (3.1) (3.5)

6.0 5.8 5.9 6.2 6.4

(1.9) (1.5) (1.7) (1.8) (2.5)

6.9 7.8 8.3 8.5 8.3

(2.4) (3.5) (4.0) (4.9) (5.1)

Mean fixed interval response rate 1 4 8 13 15

56 43 40 32 34

(18) (18) (18) (17) (18)

62 72 75 76 75

(21) (23)*,# (23)*,# (19)**,# (18)**,#

68 66 73 79 83

(22) (27) (27)*,# (33)**,# (35)**,#

58 65 74 69 70

(23) (30)# (37)*,# (42)**,# (44)**,#

Week

0 ppma

Mean fixed interval index of curvature 1 0.47 4 0.50 8 0.50 13 0.53 15 0.50

(0.06) (0.19) (0.18) (0.10) (0.14)

0.46 0.46 0.46 0.44 0.43

(0.07) (0.10) (0.09) (0.11) (0.12)

0.45 0.49 0.49 0.48 0.44

(0.09) (0.10) (0.08) (0.12) (0.11)

0.49 (0.07) 0.49 (0.08) 0.47 (0.10) 0.49 (0.14) 0.47 (0.13)

Week 1 serves as pre-exposure baseline. Weeks 4, 8 and 13 occurred during the exposure series, and week 15 was the second week after termination of exposures. a N ¼ 10. b N ¼ 10, except FI Rate for which N ¼ 9. * Treatment mean is significantly different relative to control (P < 0:02). ** Treatment mean is significantly different relative to control (P < 0:008). # Change between week 1 and test week for treatment group is significantly different relative to change over same period for control (P < 0:005).

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Operant Set Body Weight Body weights maintained a mean of 301 g for all groups throughout the study. Individual rats were occasionally outside the target range of 280–313 g, but these excursions were rapidly corrected, and did not appear to adversely affect the behavioral results. Despite attempts to maintain a constant weight by controlled feeding, test weeks 1–3, 5 and 6 included significantly lower body weights for the 1500 ppm group relative to control. The reduced weights, however, were all within 2.5% of the mean body weight of the control group and did not appear to affect the rats’ operant performance. Baseline Operant Performance Performance on the multiple schedule of reinforcement during week 1 was adequately under control of the multiple schedule of reinforcement as judged by quantitative performance measures (Table 3) and subjective evaluation of cumulative response records (not shown). FR performance during baseline was characterized by a brief pause after each reinforcement followed by a high-rate run of responding. Of about 40 overflow windows per session, 32% included an overflow response. Overflow windows rarely contained more than one response. Overall response rates for the FI component were moderate, and the accelerating rate during the FI was quantitatively characterized by a positive index of curvature. In all respects, baseline performance was qualitatively and quantitatively very similar to that of three independent sets of 40 rats that were trained in a nominally identical fashion. Session-to-session stability for all dependent variables except overflow responses was generally acceptable. FR rate and FR pause duration had the greatest session-tosession consistency during baseline (mean COVwithin ¼ 6:7 and 7.9, respectively). FI rate and FI index of curvature were more variable during baseline (COVwithin ¼ 14:1 and 9.9, respectively). The number of overflow responses varied erratically within subjects during baseline (mean COVwithin ¼ 30:7), and this measure was also characterized by a high degree of between-subject variation (mean  S:D: ¼ 13  8). During the baseline period, only 2 of the 200 individual COVwithin values (i.e. 40 rats  5 variables) exceeded the respective variable’s mean COVwithin by more than 3 S.D. These two outliers were FI rate metrics for one rat each in the 350 and 750 ppm groups. According to the baseline stability criteria established prior to the study, their FI rate data for the two outliers, but not the data for the other measures, were excluded from subsequent analysis.

Fig. 3. Operant performance during the fixed ratio component of a multiple FR 20–FI 120 s schedule of reinforcement. Rats were exposed to various concentration of ethyl acetate between weeks 1 and 13, inclusive. (A) Mean running rate of response (N ¼ 10 per group). (B) Mean post-reinforcement pause duration (N ¼ 10 per group). Dependent variables are defined in text. Error bars (1 S.D.) are shown only for the control group.

FR Response Rate Ethyl acetate exposure did not affect FR rate of response (Table 3). All groups drifted toward increasing response rates over weeks, especially during the first few weeks of exposure, but the increase was apparent in all groups (Fig. 3A). There were no statistically significant changes in performance during the post-exposure period (test weeks 14 and 15) relative to the immediately preceding weeks. FR Pause Duration FR pause duration did not exhibit time-dependent trends that were systematically related to treatment (Table 3 and Fig. 3B). Overflow Responses The number of overflow responses continued to be highly variable both between and within subjects during the exposure series. In the context of this extensive variability, there was no suggestive or statistical evidence that overflow responses changed in a treatment-dependent fashion (data not shown).

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sessions whereas that for the 350 ppm and control group generally remained constant. FI Index of Curvature The index of curvature for the control group appeared to increase slightly over time, whereas that of the treatment groups remained generally constant with some idiosyncratic variation (Fig. 4B). None of these differences, however, indicated any particular treatment-related trend (Table 3).

DISCUSSION

Fig. 4. Operant performance during the fixed interval component of a multiple FR 20–FI 120 s schedule of reinforcement. Rats were exposed to various concentration of ethyl acetate between weeks 1 and 13, inclusive. (A) Mean rate of response (N ¼ 10 for 0 and 1500 ppm groups and N ¼ 9 for the other groups). (B) Mean index of curvature (N ¼ 10). Dependent variables are defined in text. Error bars (1 S.D.) are shown only for the control group.

FI Response Rate Response rate for the control group steadily drifted toward decreasing values after baseline (test week 1), eventually attaining a mean value during week 13 that was only 57% of baseline performance (Table 3 and Fig. 4A). This decrease over time was not characteristic of any of the treatment groups. On the contrary, the mean FI response rates for the groups exposed to ethyl acetate appeared to uniformly drift toward increasing values over time. All treatment groups had significantly greater FI rate than control during weeks 8, 13 and 15. Statistical analysis also confirmed that the decreases in mean FI rate between week 1 and subsequent weeks for the control group were significantly different from the increase over the same period for the treatment groups. Contrasts examining these time and treatment interactions were significant for the 350 and 1500 ppm groups during week 4 and were also significant for all treatment groups during weeks 8, 13 and 15. Table 3 shows that between-subject variation appeared to increase for the 750 and 1500 ppm groups over

Subchronic inhalation exposure to 750 ppm ethyl acetate caused clear evidence of systemic toxicity. In rats fed ad libitum, exposure to ethyl acetate caused reductions in body weight, body weight gain, feed consumption, and feed efficiency. These effects began to recover or recovered fully during the 4-week postexposure period. Neuropathological evaluation revealed only minor examples of neuronal/myelin damage that occurred equally in treated and control groups. Minor lesions of this type and frequency occur spontaneously in rats (Eisenbrandt et al., 1990). Ethyl acetate exposure (750 and 1500 ppm) appeared to have transient acute effects on nervous system function. The data suggest that exposure to 750 and 1500 ppm ethyl acetate reduced the vigor of the startle response and probably represents an initial stage in the concentration-dependent ability of many organic solvents to induce acute CNS depression, sedation, and narcosis (Andrews and Snyder, 1986). There was not a progression toward more pronounced reductions in startle behavior over successive exposures, suggesting that acute and mild CNS depression was reinstated on a repeated basis. Since clinical observations conducted immediately after daily exposures revealed no signs of nervous system dysfunction, the acute sedative effects were either so mild that they could not be detected by a standard clinical evaluation or the sedative effects wane quite rapidly after termination of exposure. The principal question addressed by the experimental design was whether subchronic exposure would promote the emergence of enduring behavioral effects. Among the 37 FOB parameters, 2 motor activity measures, and 5 operant dependent variables, only a few findings merit discussion. Indeed, a remarkable outcome is how normal the behavior of the rats was after months of exposure with concentrations high enough to presumably induce transient signs of CNS depression on an almost daily basis.

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Female rats exposed to 350 ppm had significantly greater hindlimb grip strength near the end of the exposure series. Since the magnitude of change was not concentration-dependent, the most plausible explanation is that the difference was spurious. During week 4 assessments, but not thereafter, the incidence of 1500 ppm male rats that defecated and urinated in the open field was less than that of the control group. The toxicological relevance of these findings is unclear. These effects can be triggered by some agents that affect autonomic function, such as cholinesterase inhibitors (Moser, 1995), but they are usually accompanied by a constellation of other autonomic and neurological signs, none of which were detected in the present study. Therefore, these transient findings, which did not persist after the fourth week, are probably artifacts, and are unlikely to be treatment-related. The reduction in motor activity for the 1500 ppm female group is regarded as a treatment-related effect because it occurred in the high concentration group during the final week of exposure. Like body weight and feeding parameters, the motor activity measure recovered after the exposure series. The non-specific nature of the motor activity test does not permit determination of whether the change was due to an effect on the nervous system or was secondary to another cause, such as poor health status as reflected by reduced body weight and feeding parameters. Up to 1500 ppm of ethyl acetate did not affect either the rate or pattern of FR performance as indicated by the absence of demonstrable effects on FR running rate and FR pause duration. The overflow window technique, while useful for preventing contamination of FR rate and pause measures by accidental overflow responses, had too much variation to be a useful variable, so the absence of effects on overflow responding is not especially meaningful. The fundamental scallop pattern of responding under control of an FI schedule was not altered by exposure to ethyl acetate, as indicated by the absence of effects on FI index of curvature. In contrast to these unaffected measures, significant differences between FI response rate for control and all treatment groups developed during the exposure series and persisted during the 2-week postexposure period. The principal reason the treatment groups differed from the control group is that control FI rate declined about 40% relative to baseline during the exposure series whereas FI rates for the treatment groups all increased over time. Some investigators (e.g. Glowa and McPhail, 1995) have suggested that increases in behavioral variation can be associated with treatment effects, and it is

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noteworthy that between-subject variation for FI rate did not change substantially for control or 350 ppm groups during the exposure series, whereas variability for 750 and 1500 ppm groups increased substantially. There is, however, reason to doubt that FI rate or between-subject variation was genuinely affected by exposure to ethyl acetate. Determination of whether a 40% decline in control FI rate over months is typical for sham-exposed rats is critical for interpretation of the differences between treated and control subjects. Data collected for an independent set of control rats in the same laboratory can help clarify the ways in which FI response rates can spontaneously change over time. These historical control rats were the same strain and age and were obtained from the same supplier. Furthermore, all procedural details, including details of the sham exposure, were nominally identical to those used for the concurrent control group in the present investigation. Fig. 5 shows that FI rate for the historical control was very similar to that of the 1500 ppm ethyl acetate group

Fig. 5. Top: mean fixed interval rate of response for a shamexposed historical control, the concurrent 0 ppm ethyl acetate control group, and the 1500 ppm ethyl acetate group. Procedures for the historical and concurrent controls were nominally identical (N ¼ 10 per group). Bottom: standard deviations for the data summarized in the top panel are presented to illustrate betweensubject variation during the course of sham exposure.

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and that both differed substantially from the concurrent control. Fig. 5 also shows that variability as measured by the standard deviation for FI rate also changed similarly for the historical control and 1500 ppm groups. These comparisons indicate that a wide range of spontaneous changes in FI rate over time are possible in untreated rats. Presumably, the absence of contingency pressure on slower or faster FI response rates can permit the FI rate values to drift idiosyncratically. This drift is a property of the schedule of reinforcement and not a property of ethyl acetate treatment. Although the performance of a concurrent control group surely merits emphasis in determination of treatment-related effects, the performance of the historical control should not be ignored. It is plausible that ethyl acetate had no genuine effect on FI rate, just as it was without effect on FI index of curvature, FR rate and FR pause duration. At best, the FI rate data are equivocal, and it is premature to conclude that FI rate was affected by treatment. In contrast to the lack of clear evidence for treatment-related effects of subchronic ethyl acetate exposure on operant behavior, Glowa and Dews (1987) found that acute exposure markedly reduced the rate of FI responding in mice in a concentration-dependent fashion. The different results stem from the different experimental questions underlying the experimental designs. Glowa and Dews (1987) conducted the operant sessions while the subjects were being exposed and assessed how exposure affected operant performance at the time of peak acute action. The present investigation inquired instead whether repeated exposure caused enduring changes in performance that were not confounded by the immediate consequences of acute exposure. The present work does not provide clear evidence that subchronic exposure up to 1500 ppm has enduring effects on operant performance. Integration of the results reported here indicate that transient, acute behavioral effects of ethyl acetate are detectable with lower exposure concentrations than those affecting a more enduring, yet recoverable, reduction in motor activity. The results additionally suggest that exposure levels that provide protection from acute sedative effects and subchronic exposure effects on measures such as body weight should also adequately protect individuals from enduring changes in nervous system structure and function.

ACKNOWLEDGEMENTS The authors thank Dr. John Green for expert statistical advice. The technical assistance of Nancy Betts, Thomas Kegelman, Carolyn Lloyd, Craig Marshall, Janet Maslanka, Kathleen Mikles, Ahmad Pulliam, and Jim Riggin is appreciated. The Oxo Process Panel of the American Chemistry Council (1300 Wilson Blvd., Arlington, VA 22209, USA) supported this work.

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