Document Xzjnvv5dkoJMN2jJmJEpNZo7K

TOXICOLOGY AND APPLIED PHARMACOLOGY 98, 385-397 (1989) The Uptake and Elimination of 1,1,1-Trichloroethane during and following Inhalation Exposures in Rats12 Cham E. Dallas,13 2Raghupathy Ramanathan, Srinivasa Muralidhara, James M. Gallo,* and James V. Bruckner Department ofPharmacology and Toxicology and *Department ofPharmaceutics, College ofPharmacy, University ofGeorgia, Athens, Georgia 30602 ReceivedJuly 1,1988: acceptedJanuary 16,1989 The Uptake and Elimination of 1,1,1 -Trichloroethane during and following Inhalation Expo sures in Rats. Dallas, C. E., Ramanathan, R., Muralidhara, S., Gallo, J. M., and Bruckner, J. V. (1989). Toxicol. Appl. Pharmacol. 98, 385-397. The pharmacokinetics of 1,1,1-trichloroethane (TRI) was studied in male Sprague-Dawley rats in order to characterize and quantify TRI uptake and elimination by direct measurements of the inhaled and exhaled compound. Fifty or 500 ppm TRI was inhaled for 2 hr through a one-way breathing valve by unanesthetized rats of 325-375 g. Repetitive samples ofthe separate inhaled and exhaled breath streams, as well as arterial blood, were collected concurrently both during and following TRI inhalation and analyzed for TRI by gas chromatography. Respiratory rates and volumes were continuously monitored during and following exposure and were used in conjunction with the pharmacokinetic data to characterize profiles of uptake and elimination. TRI was very rapidly absorbed from the lung, in that substantial levels were present in arterial blood at the first sam pling time (i.e., 2 min). Blood and exhaled breath concentrations ofTRI increased rapidly after the initiation ofexposure, approaching but not reaching steady state during the 2-hr exposures. The blood and exhaled breath concentrations were directly proportional to the exposure concen tration during the exposures. Percentage uptake ofTRI decreased 30-35% during the first hour of inhalation, diminishing to approximately 45-50% by the end of the exposure. Total cumula tive uptake in the 50 and 500 ppm groups over the 2-hr inhalation exposures was determined to be 6 and 48 mg/kg body wt, respectively. By the end of the exposure period, 2.1 and 20.8 mg, respectively, of inhaled TRI was eliminated from rats inhaling 50 and 500 ppm TRI. A physio logical pharmacokinetic model for TRI inhalation was utilized to predict blood and exhaled breath concentrations for comparison with observed experimental values. Overall, values pre dicted by the physiological pharmacokinetic model for TRI levels in the blood and exhaled breath were in close agreement with measured values both during and following TRI inhalation. I9S9 Academic Press, Inc. 1 Research sponsored by U.S. EPA Cooperative Agree ment CR 812267 and the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under Grant Number AFOSR 87-0248. The U.S. Govern ment's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for govern mental purposes, is acknowledged. 2 Presented at the 26th Annual Meeting of the Society ofToxicology, Washington, D.C., February 1987. 3 To whom correspondence should be addressed. 1,1,1-Trichloroethane (TRI), also known as methyl chloroform, has been used in large quantities for decades in industry as a solvent and metal degreasing agent. Other applica tions include its use in adhesives, spot remov ers, aerosols, and water repellents. The toxic ity of TRI is considered to be of a relatively low order of magnitude, with depression of the central nervous system (CNS) (Torkelson 385 0041-008X/89 $3.00 Copyright 1989 by Academic Press, inc. All rights of reproduction in any form reserved. 386 DALLAS ET AL. and Rowe, 1981; KJeinfeld and Feiner, 1966; Stewart, 1968) and cardiac arrhythmias (Dornette and Jones, 1960; Reinhardt etal., 1973; Herd el al., 1974) the major effects seen after high doses in animals and humans. Hepatic and renal toxicity have been demonstrated only after very high acute doses in animals (Plaa and Larson, 1965; KJaassen and Plaa, 1966,1967;Gehring, 1968). Historically, hu man exposures to TRI have been of greatest significance in industry and other occupa tional settings, where exposures are primarily by inhalation. Workers are routinely exposed to TRI vapors in open or closed (i.e., recircu lating) work environments. Employees may be inadvertently exposed to high concentra tions when there has been a spill or equip ment malfunction. Studies of the pharmacokinetics of inhaled solvents such as TRI are playing an increas ingly important role in toxicology. Knowl edge of the uptake, disposition, and elimina tion of these chemicals is quite useful in health risk assessments. There is presently lit tle kinetic data available involving direct measurements of TRI in laboratory animals during inhalation exposures. The fate of 14CTRI has been investigated following the ter mination of single 6-hr 150 or 1500 ppm in halation exposures in rats and mice (Schu mann et al., 1982a). By 72 hr postexposure, 87-98% of the total recovered radioactivity was eliminated as unchanged TRI in the ex pired air. Respiratory elimination and me tabolism of TRI remained approximately the same after TRI inhalation exposures were re peated 5 days/week for 18 months (Schu mann et al., 1982b). The fraction of the total inhaled dose which is eliminated during on going inhalation exposures, however, has not been delineated in laboratory animals. Like wise, the rate and magnitude of uptake have not been quantified over time during the course of TRI inhalation exposures in ani mals. It was necessary, for example, for Schu mann et al. (1982a) to base estimates ofphar macokinetic parameters for rats on an as sumed constant uptake of 60% of inhaled TRI over 6 hr of exposure. Therefore, an objective of the current in vestigation was to provide accurate measure ments of the respiratory uptake and elimina tion of TRI during inhalation exposures. In haled and exhaled breath concentrations were monitored at frequent intervals in rats both during and following TRI inhalation, as were the minute volume and respiratory rate. Blood levels of TRI were monitored concur rently, so systemic uptake and elimination could be correlated with the respiratory mea surements. The exhaled breath and blood TRI concentrations were then utilized to assess the accuracy of values predicted by a physiologically based pharmacokinetic model for TRI inhalation. METHODS Animals. Adult, male Sprague-Dawley rats were ob tained from Charles River Breeding Laboratories (Ra leigh, NC). The animals were maintained on a constant light-dark cycle, with light from 0700 to 1900 hr and darkness from 1900 to 0700 hr. They were housed in stainless-steel cages in a ventilated animal rack. Tap wa ter and Ralston Purina Formulab Chow were provided ad libitum. The rats were used after at least a 14-day accli mation period, at which time they were approximately 12 weeks old and their body weight ranged from 32S to 375 g. Solvent exposures were initiated at approximately the same time each day (1000 to 1200 hr). Test material. 1,1,1-Trichloroethane, 98.3% mini mum purity, was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). The purity ofthe chemical during the conduct ofthe study was verified by gas chromatogra phy to be slightly less than 99%. Animal preparation. An indwelling carotid arterial cannula was surgically implanted into each animal. The rats were anesthetized for the surgical procedure by im injection of 0.8 ml/kg of a mixture consisting of keta mine HC1 (100 mg/ml):acepromazine maleate (10 mg/ ml):xylazine HC1 (20 mg/ml) in a proportion of 3:2:1 (v/ v/v). The cannulated animals were maintained in a har ness and pulley system that allowed relative freedom of movement in metabolism cages during a 24-hr recovery period. Inhalation exposures. Each cannulated rat was placed into a restraining tube ofthe type used in nose-only inha lation exposure chambers (Battelle-Geneve, Switzer- SL 300U KINETICS OF INHALED TRICHLOROETHANE influent mixture 1--| PNEUMOTACHOGRAPH | ^Sampling Ports ONE-WAY ____| EFFLUENT RESERVOIR 387 Strain Restraining Tube Blood Samples Jintegrator -- Transduce^mpl Transducer Amp. Respiratory Rate Signal Minute Volume Signal Blood Pressure Signal PHY5IOGRAPH Fig. 1. Schematic diagram of the inhalation exposure system. An unanesthetized rat in the restraining tube inhaled TRI through the one-way breathing valve attached to the face mask. TRI was inhaled from the influent mixture gas sampling bag and exhaled into the effluent reservoir bag. Inhaled and exhaled breath samples were taken from their respective sampling ports and arterial blood samples from an indwell ing cannula. The rate and volume of respiration were monitored on a physiograph. For sake ofclarity, the breathing valve, gas sampling bags, and other components are not drawn to scale. land). A face mask designed to fit the rat was held firmly in place on the animal's head by the use ofelastic straps, which were secured to the restraining tube. A miniatur ized one-way breathing valve (Hans Rudolph, Inc., St. Louis, MO) was attached to the face mask so that the valve entry port was directly adjacent to the nares of the test animal. The dead space of the valve was 0.5 ml. The valve was designed so that the negative pressure gener ated by the animal's inspiration pulled the inhalation dia phragm open and the exhalation diaphragm closed against its seat. Upon expiration, the positive pressure generated within the device pushed the exhalation dia phragm open and the inhalation diaphragm closed against its seat. This established separate and distinct air ways for the inhaled and exhaled breath streams with no significant mixing ofthe inhaled and exhaled air. The use of such a device for pharmacokinetic studies of inhaled halocarbons in small animals has been described in detail (Dallas el al., 1986). Inhalation and exhalation sampling ports were located immediately adjacent to the breathing valve. A known concentration of TRI was generated within a 70-liter gas sampling bag (Calibrated Instru ments, Ardsley, NY) by injecting the appropriate quan tity of the test chemical into the bag filled with air. Uni form dispersion of the vapor was ensured by a magnetic stirring bar within the bag. The bag was then connected in series by Teflon tubing with a pneumotachograph, a three-way connector, the breathing valve, and an empty 70-liter gas collection bag (Fig. 1). The latter bag served as a reservoir to collect exhaled gas. Thereby, a closed system was maintained to prevent release of the agent into the laboratory. TRI inhalation exposures were initi ated only after stable breathing patterns were established for the cannulated animals in the system. Just before the initiation of exposure, the solvent vapor was first drawn out of the gas sampling bag by an air pump attached to the three-way connector. In this manner, the animal was assured of being subjected at the very start of the expo sure to a TRI concentration equivalent to the target con centration in the bag, without significant dilution from dead space air in the system. The test animals then were subjected to 2-hr TRI inhalation exposures. During this period and for up to 4 hr afterward, inhaled and exhaled breath samples were taken from the sampling ports at approximately the same time as blood samples from the carotid artery cannula. Both air and blood samples were then analyzed for TRI content by gas chromatography. Respiratory measurements and calculations. The res piration of each animal was continuously monitored. The respiratory monitoring was conducted according to the methods previously used in solvent exposure studies by this laboratory (Dallas eld.. 1983, 1986). The airflow created by the animal's inspiration was detected by a pneumotachograph located in the inhaled airstream be- SV 388 DALLAS ET AL. tween the influent bag and the breathing valve. The sig nal from the pneumotachograph and accompanying transducer was employed in recording the number ofres pirations per minute (/) in one channel of a physiograph. This signal was then intergrated over a 1-min in terval to yield the volume of respiration per minute, or minute volume (VE). A value for the average tidal vol ume (VT) during that 1-min interval was determined by dividing VE by /for that minute. An average value for these parameters for individual animals was obtained by averaging the measurements taken at 10-min intervals during the 2-hr exposure. The mean SE ofthese values for the 500 ppm exposure group (n = 6) were VE = 236.3 22.9 ml/min;/ = 135.3 6.6 breaths/min; and VT = 1.74 0.18 ml. The mean SE ofthese values for the 50 ppm exposure group (n = 6) were VE = 252 14.7 ml/min; /= 129.5 13.5 breaths/min; and VT = 1.96 0.1 ml. Since the VE and the TRI exhaled breath concentration at each sampling point were measured, subtraction ofthe quantity of TRI exhaled from the amount inhaled yielded an approximation of the quantity of TRI taken up each sampling period (cumulative uptake, or Gup.), Gup. = (Cinh vEi) - (C,* vEt), (1) where Ci,,h is the inhaled concentration; VE and Qih are the minute volume and exhaled breath measurements, respectively, made at each time point; and / is the interval oftime between sampling points (every 10 min for Qupl). The successive Gup. values are summed to determine cu mulative uptake over the 2-hr exposure. Determination of the cumulative elimination of TRI during inhalation exposure was made as a function of the GUp, and measurements of the inhaled dose. In their calculation of exhaled breath concentration, Ramsey and Andersen (1984) assumed that alveolar respiration accounts for 70% of total respiration, with 30% of total respiration delegated to the inhaled air that does not par ticipate in alveolar ventilation. By adding instrumental dead space of the breathing valve in the exposure system in the present study to this assumed physiological dead space, a value of 50% of total respiration was assigned to alveolar ventilation. Therefore, cumulative elimination (Gehm) ofTRI was estimated by Gehm = (Tmh Ta|v0 -- Gup<. (2) where the alveolar ventilation is = 0.5 VE and t is the time interval between sequential sampling ofthe exhaled breath. As for Gup., wit*1 sequential determination of Gehm it is possible to measure the cumulative elimination ofTRI during inhalation exposures. The successive elim ination ofTRI following exposure was calculated as Gehm = C,,hVEt. The percentage uptake (% Upt) of the total inhaled dose at each successive time point during the inhalation exposure period was calculated as Fig. 2. Diagram of the physiologically based pharma cokinetic model used to simulate the uptake and elimi nation ofinhaled TRI. The symbols and parameters used to describe the model are included in Table I and in the equations given under Methods. % Upt = ** ^v) --00 , t'inh (3) where the TRI alveolar concentration is Qjv = C^JN, in which Cln is the measured TRI arterial blood level and N is the blood;air partition coefficient for TRI. A physiologically based pharmacokinetic (PBPK) model was used to describe the disposition of TRI in the rat (Fig. 2). It was assumed that a blood flow-limited model was adequate to characterize the tissue distribu tion of TRI. Compartmental volumes and organ blood flows were obtained from the literature (Gerlowski and Jain, 1983; Ramsey and Andersen, 1984) and scaled to 340 g, the mean body weight of rats used in the present study. Partition coefficients and the metabolic rate con stant for TRI were taken from Gaigas et al. (1986, 1989), except for the richly perfused tis$ue:blood and lung: blood partition coefficients, which were assumed to be the same as the liverblood partition coefficient. The lung;air partition coefficient was then derived by multi plying the blood:air coefficient from Gargas et al. (1986) by the lung:blood coefficient. The alveolanlung mass transfer coefficient was estimated from the value used for methylene chloride (Angelo and Pritchard, 1984), Differential mass balance equations, incorporating the parameters listed in Table 1, that described the transport 30l3 KINETICS OF INHALED TRICHLOROETHANE 389 TABLE 1 Parameters for the Physiological Pharmacokinetic Model of TRI in the Rat (340 g) Parameter Value Alveolar ventilation rate (ml/min), VR. Inhaled gas concentration (/ig/ml), Cloh Blood flows (ml/min) Cardiac output, Qh Fat, Q, Liver, Qi, Muscle, Qw Richly perfused, Q, Tissue volumes (ml) Blood, Kb Fat, V, Liver, Vt Muscle, K,, Richly perfused, Vt Alveolar, V, Lung, V, Partition coefficients Lung.air, Fat:blood, Livenblood, Rh Lungiblood, R, Muscle:blood, Rm Richly perfused:blood, Rr Miscellaneous constants Lung:alveolar mass transfer coefficient, k Metabolic rate constant, Kf 126 (50 ppm exposure) 118 (500 ppm exposure) 0.279 (50 ppm exposure) 2.70 (500 ppm exposure) 106.4 9.4 39.8 12.8 44,4 25.4 30.5 13.6 248.0 17.0 2.0 3.97 8.6 47.7 1.49 1.49 0.55 1.49 500 ml/min 0.115 min 1 gas-tight, 1-ml syringe and injected directly onto an 8 ft x i in. stainless-steel column packed with 0,1 % AT 1000 on GraphPak. Standards were prepared in each of four 9-liter standard bottles with Teflon stoppers containing needles used for taking the air samples with the syringe. Operating temperatures were 150'C, injection port; 200'C, FID detector, 350'C, ECD detector; and 110'C, isothermal column operation. When using the ECD, gas flow rates of40 ml/min were employed for nitrogen (car rier gas), with an additional makeup gas flow rate of 30 ml/min to the detector. TRI levels in the blood were measured by GC headspace analysis. Blood samples were withdrawn from the arterial cannula via a stopcock by a 1-ml syringe. De pending on the anticipated blood concentration, be tween 25 and 200 gl of the blood was taken from the stopcock with an Eppendorf pipet and transferred to chilled headspace vials (Perkin-Elmer, Norwalk, CT). These vials were capped immediately with PTFE-lined butyl rubber septa and washers and tightly crimped. Each sample vial was then placed into the HS-6 autosampler unit ofa SIGMA 300 gas chromatograph (Perkin-Elmer, Norwalk, CT), where it was heated to 80C by a highprecision thermostat device. A predetermined volume of the vapor was then injected automatically into the col umn for analysis. Standard solutions were made and as sayed by diluting calculated amounts ofpure TRI in tolu ene, transferring to vials, and analyzing as previously de scribed. The concentration of TRI in the blood samples was then determined from a standard curve generated from blood that was spiked with these standard solutions. The column used was an 8 ft X J in. stainless-steel col umn packed with FFAP Chromasorb W-AW (80-100 mesh). Operating temperatures were 200*C, injection port; 350*C, ECD detector, and 85"C, column oven. The carrier gas was 5% argon-methane, at a flow rate of 40 ml/min with a makeup gas flow rate of 20 ml/min to the detector. of TRI in the rat were numerically integrated with the Advanced Continuous Simulation Language (ACSL) computer program (Mitchell and Gauthier, Concord, MA). The solution to the equations provided predicted TRI concentrations over time. The model-predicted cu mulative uptake values were the sum of the simulated amounts of TRI in each tissue compartment in the model. Analysis of TRI in air and blood. The concentration of TRI in the inhaled and exhaled air samples collected during and following the inhalation exposures were mea sured with a Tracor MT560 gas chromatograph (GC) (Tracor Instruments, Austin, TX). Analyses for the 500 ppm exposures were conducted using a flame ionization detector (FID), while the analyses for the 50 ppm expo sures were conducted using an electron capture detector (ECD). In either case, air samples were procured with a RESULTS The target concentrations for the TRI in halation exposures were 50 and 500 ppm. The starting concentration of TRI in the bag from which the test animal inhaled the test compound was measured just prior to the ini tiation of each exposure. TRI bag concentra tions were 515.8 20.6 and 53.6 2.2 ppm (x SE) for the 500 and 50 ppm groups, re spectively. The actual concentrations inhaled by the animals were determined by measure ments of air samples taken from the airway SL 030014 390 DALLAS ET AL. time{min) o 0 SO 100 150 200 250 300 350 lime(rnin) Fig. 3. Observed () and model-predicted (--) TR1 concentrations in the blood (top graph) and exhaled breath (bottom graph) of rats during and following a 2-hr 50 ppm inhalation exposure. Each point repre sents the mean value for six rats. immediately adjacent to the breathing valve. Inhaled TRI concentrations for the six rats in each group were 491.6 11 ppm for the 500 ppm exposures and 51.2 1.2 ppm (x SE) for the 50 ppm exposures. TRI concentrations in the blood and ex haled breath of rats during and following in halation of TRI are shown for 50 ppm expo sures in Fig. 3 and for 500 ppm exposures in Fig. 4. Concentrations of TRI in the exhaled breath generally paralleled concentrations in the arterial blood, though some differences were noted. TRI was rapidly absorbed from the lungs and readily available for distribu tion to tissues of the body, in that arterial blood concentrations of TRI were quite high at the first sampling time (i.e., 2 min). After an initial rapid rise, the blood levels increased steadily but did not reach steady state by the end ofthe 2-hr exposures. Exhaled breath lev els increased even more rapidly than blood levels after the initiation of exposures, attain ing near steady state within 10 to 15 min. The exhaled breath versus time curves were as ymptotic, in that they gradually increased throughout the remainder of the 2-hr inhala tion period. An increase in the inhaled con centration from 50 to 500 ppm produced an equivalent (i.e., 10-fold) increase in the ob served blood and exhaled breath concentra tions of TRI. Upon cessation of TRI inhala tion, the chemical was rapidly eliminated. As can be seen in Figs. 3 and 4, TRI concentra tions in the exhaled breath initially dimin ished more rapidly than did blood concentra tions. Disappearance of TRI from the blood paralleled that in the expired air during the latter part of the postexposure period. PBPK model-generated blood and exhaled breath concentrations of TRI are shown as solid lines in Figs. 2 and 3. Concentrations of TRI in the expired air were well simulated by the model during and following the 50 and 500 ppm exposures. Model predictions that TRI levels in the exhaled breath would quickly reach near steady state after the expo sures began were consistent with the observed data, with the observed levels slightly lower SL 030015 KINETICS OF INHALED TRICHLOROETHANE 391 Exhaled Breath 0 50 100 150 200 250 300 350 time(min) Fig. 4. Observed () and model-predicted (--) TRI concentrations in the blood (top graph) and exhaled breath (bottom graph) ofrats during and following a 2-hr 500 ppm inhalation exposure. Each point repre sents the mean value for six rats. than simulated levels over the course of the 50 and 500 ppm exposures. The model accu rately predicted both rapid and slow elimina tion phases of expiration of TRI postexpo sure. When the model was used to describe the time course of TRI in the arterial blood, a relatively good fit was obtained during the 500 ppm exposure (Fig. 4). Arterial blood concentrations were overpredicted by ap proximately 20% during the 50 ppm expo sure (Fig. 3). The model predicted a slightly more rapid decline in blood levels postexpo sure in both groups than was observed during the period of 130-200 min, but levels at sub sequent time points were accurately pre dicted. Percentage systemic uptake of TRI ap peared to be both concentration- and timedependent (Fig. 5). Although percentage up take was quite high during the initial minutes of inhalation of 50 and 500 ppm TRI, a de crease of 30-35% occurred during the first hour. Percentage uptake diminished more slowly during the remainder of the exposure period. As can be seen in Fig. 5, the mean val ues after 10 min are slightly but consistently lower in the 500 ppm group. Plots of cumulative uptake of TRI during the inhalation sessions, as calculated by Eq. TIME OF EXPOSURE (MIN) FIG. 5. Percentage uptake ofTRI during inhalation ex posures. Rats inhaled 50 or 500 ppm TRI for 2 hr. Each point represents the mean SE for six rats. The percent age uptake of the inhaled dose over time was determined after 1, 3, 5, 10, 15, and 20 min and at 10-min intervals thereafter. 392 DALLAS ET AL. Fig. 6. Cumulative uptake of TRI during inhalation exposures. Rats inhaled 50 or 500 ppm TRI for 2 hr. The quantity of inhaled TRI retained during successive 10min intervals was calculated on the basis ofthe measured minute volume and difference between inhaled and ex haled TRI concentrations. Each point represents the mean SE for six rats. The diminutive SE bars are omit ted from the 50 ppm values for sake ofclarity. nation, as determined by Eq. (2), was propor tional to the inhaled concentration. By the end of the 2-hr exposure to 50 and 500 ppm TRI, 2.1 0.2 and 20.8 3.0 mg (* SE), respectively, were eliminated from the rats in the exhaled breath. Model-predicted elimina tion at the end of 2 hr in the 50 and 500 ppm groups was approximately 40 and 50% greater, respectively, than these measured values. Following the termination of expo sure, TRI was eliminated in the exhaled breath in progressively smaller quantities, as reflected by postexposure plateaus in the elimination curves. During the 2-hr postex posure period, an additional 0.3 and 3.3 mg of TRI were eliminated from the animals in the 50 and 500 ppm exposure groups, respec tively. DISCUSSION (1), are shown in Fig. 6. Cumulative uptake, as determined from direct measurements of the minute volume and TRI concentrations in the inhaled and exhaled breath, was not linear in either the 50 or 500 ppm animals. The departure from linearity was more ap parent at the higher exposure level. Total cu mulative uptake during the 2-hr exposures, as ascertained from the direct measurement data, was 2.2 0.2 and 16.7 0.9 mg (Jc SE) in the 50 and 500 ppm groups, respec tively. Predicted values for uptake, derived by summating the predicted levels of TRI in the model compartments, were significantly less than these measured uptake values (i.e., after 2 hr exposure to 500 ppm TRI, predicted up take was 50% of measured uptake). Cumulative elimination of TRI in the ex haled breath during and following inhalation exposure is shown in Fig. 7. During TRI ex posure, TRI in the pulmonary blood and TRI not absorbed from the alveolar space each contribute to the TRI eliminated in the ex haled breath. Cumulative pulmonary elimi Pharmacokinetic studies are playing an in creasingly important role in toxicology and TIME (MIN) Fig. 7. Cumulative elimination ofTRI during and fol lowing inhalation exposures. Rats inhaled 50 or 500 ppm TRI for 2 hr. The quantity of inhaled TRI eliminated in the breath over time was calculated using direct measure ments of the minute volume and TRI concentrations in the inhaled and exhaled breath. Each point is the mean SE for six rats. Cumulative elimination was deter mined for successive 10-min intervals during the 2-hr ex posure, and for successive 15-min intervals postexpo sure. SL 030017 KINETICS OF INHALED TRICHLOROETHANE 393 in health risk assessments (Clark and Smith, 1984; Clewell and Andersen, 1985; NRC, 1987). Unfortunately, there is relatively little information available on the uptake and dis position of TRI and many other VOCs dur ing ongoing inhalation exposures. Most phar macokinetic studies of TRI have focused on the elimination of the chemical and its me tabolites following the cessation of exposures (Stewart et al., 1969; Ikeda and Ohtsuji, 1972; Eben and Kimmerle, 1974; Seki et al., 1975; Holmberg et al., 1977; Caperos et al., 1982; Schumann et al., 1982a,b). Although Schumann et al. (1982a) utilized rats with an indwelling jugular cannula, just three blood samples were taken and analyzed for TRI content during a 6-hr inhalation session. Similarly, blood levels appear to have been taken for TRI analysis only three times from human volunteers during 6 hr of TRI inhala tion (Nolan et al., 1984). These few time points are not sufficient to accurately define blood concentration versus time profiles, or to recognize changes which may occur in ki netics during the course of exposures. The monitoring of blood and breath levels of VOCs in animals has been primarily re stricted to times after termination of expo sures, due to problems involving restricted access to subjects in inhalation chambers and metabolism cages and technical difficulties in working with small animals. In the present study we have utilized a technique which al lowed direct, simultaneous measurements of respiratory parameters, inhaled and exhaled breath concentrations ofTRI, and TRI blood levels in unanesthetized rats during expo sures. The separation of the inhaled and ex haled breath streams, by use of a miniatur ized one-way breathing valve, facilitated ac curate serial determinations of airflow and TRI concentrations in the inhaled and ex haled breath. A similar non rebreathing valve has been used previously for assessing respi ratory volumes and gas exchange (Mauderly et al., 1979), though it has apparently not been used in pharmacokinetic studies. This approach allowed us to directly monitor res piratory uptake and elimination of TRI, a VOC for which pulmonary clearance is the major route ofelimination. Indeed by the end of the 2-hr 50 and 500 ppm inhalation expo sures, we determined that 52.5 and 56.3%, re spectively, of the inhaled dose had been ex haled. Percentage uptake of inhaled TRI was highly time-dependent. The percentage up take of inhaled TRI has apparently not been previously determined in laboratory animals. Schumann et al. (1982a) assumed that 60% of inspired TRI was absorbed by rats throughout a 6-hr exposure. The rate of transfer of TRI from alveoli to blood should initially be very rapid, but become progres sively slower as the chemical accumulates in the blood and tissues. This pattern was re flected by the time course of systemic uptake ofTRI in the current study, where percentage uptake decreased from more than 80% at the beginning to less than 50% at the end of the 2-hr exposure. Initial uptake of inhaled TRI is governed by tissue loading and metabo lism. Once the tissues have reached steady state, continued uptake will be dependent upon the rate of metabolism ofthe chemicals. Since TRI is very poorly metabolized by the rat and by humans (Ikeda and Ohtsuji, 1972; Schumann et al., 1982a; Nolan et al., 1984), percentage uptake would be expected to be very low once steady state was reached. Steady state was not reached in our study, as percentage uptake progressively decreased over the 2 hr of exposure. Monster et al. (1979) found that percentage uptake of in haled TRI by humans decreased rapidly from approximately 95% at the onset to 30% at the end of 4-hr exposures. Nolan et al. (1984) re ported that human volunteers exposed for 6 hr to 35 or 350 ppm TRI retained about 25% of the chemical to which they were exposed. The greater percentage uptake in rats than in humans is consistent with a higher TRI blood:air partition coefficient and greater car diac output/pulmonary blood flow in rats SL 030018 394 DALLAS ET AL. than in humans (Reitz et ai, 1988). Since sys temic uptake ofTRI is time-dependent, aver age values of percentage uptake for short in tervals may be misleading and have little rele vance for health risk assessments. TRI exhibits linear kinetics over a wide dosage range. Exhaled breath levels and blood levels of TRI were directly propor tional to the inhaled concentration (i.e., 50 and 500 ppm) of TRI throughout the 2-hr ex posures in the current study. Similar findings were reported in humans exposed for 6 hr to 35 and 350 ppm TRI (Nolan et ai., 1984). Schumann et ai (1982a) found that the amount of TRI exhaled by rats and mice in creased eight- to ninefold when the inhaled concentration ofTRI was increased from 150 to 1500 ppm. These investigators also ob served that blood levels, tissue levels, and body burden of 14C-TRI were each propor tional to exposure level in both species. Al though TRI was poorly metabolized, Schu mann et al. (1982a) demonstrated that its biotransformation by mice and rats was a dose-dependent, saturable process. Meta bolic saturation in rats was believed to occur between 500 and 1500 ppm, if not near 500 ppm. Metabolic saturation, however, had lit tle apparent effect on the overall pharmacoki netics of TRI, since biotransformation was a minor route of elimination. The kinetics of TRI is governed largely by its partition co efficients (e.g., bloodiair, tissue:blood) and the physiology (e.g., respiratory rate and vol ume, cardiac output, tissue volumes, and blood flow rates) of the animal. The major route of elimination of TRI in laboratory animals and in man is exhalation of the parent compound (Schumann et al., 1982a; Nolan et ai, 1984). Nolan and his col leagues measured TRI in the exhaled breath of male human volunteers during and after 6-hr inhalation sessions. The exhaled breath levels after 1.5 hr of exposure to 35 and 350 ppm TRI were 0.14 and 1.28 ug/ml, respec tively. Assuming a linear scaleup to a 50 and 500 ppm exposure, the exhaled breath levels in humans would be 0.2 and 1.83 /xg/ml. These values are quite comparable to exhaled breath levels measured in the present study after 1.5 hr of exposure of rats to 50 and 500 ppm TRI (i.e., 0.21 and 2.16 respec tively). The similarity in magnitude in ex haled breath levels of TRI between rats and man is an unexpected finding. It would be an ticipated that alveolar and presumably ex haled breath concentrations ofTRI would be lower in rats than in man, due to the rat's higher bloodiair partition coefficient and greater percentage uptake of inhaled TRI, The aforementioned physiological param eters and biochemical constants were used to input into a PBPK model for inhalation of TRI. Our model accurately predicted the time courses of TRI in the blood and exhaled breath of rats both during and following ex posure to 50 and 500 ppm TRI. Cumulative uptake over the 2-hr exposure, however, was underpredicted by our model. The source of the discrepancy between the predicted and the measured uptake value is unclear. Revi sions of the model may be warranted by find ings in ongoing studies ofTRI concentrations in tissues of exposed animals. These data should be useful in verifying tissueiblood par tition coefficients, tissue compartments, and tissue volumes. Reitz et ai (1988), for exam ple, found that most of the changes in the pharmacokinetics of TRI in older rats could be accounted for by increasing the size of the fat compartment in their PBPK model. Reitz et ai (1988) used their model to accurately predict blood and exhaled breath concentra tions measured in humans subjected to TRI inhalation exposures. The investigators also utilized the PBPK model to predict TRI blood levels and amounts metabolized post- exposure in mice, rats, and humans, as well as to describe the kinetics of TRI in rats after iv injection, bolus gavage, and drinking water administration. Thus, it appears that PBPK models can be quite useful in predicting the time course of TRI concentrations in the SL 300l9 KINETICS OF INHALED TRICHLOROETHANE 395 body of different species under different ex posure conditions. Major species differences have been ob served in the pharmacokinetics of inhaled TRI. After 1.5 hr of exposure to 35 or 350 ppm TRI, humans had mean blood levels of 0.14 and 1.62 ^ig/ml, respectively (Nolan et ah, 1984). After being normalized for differ ences in inhaled concentrations, mean blood levels in rats in the present investigation were approximately 3.6-fold higher than the levels measured in humans. Schumann et al. (1982a) reported blood levels of TRI in rats similar to those observed in the present study (when normalized for inhaled concentra tion). The lower blood levels in humans are consistent with a lower TRI blood:air parti tion coefficient for man (2.53 versus 5.76 for rats) and the greater adipose tissue volume in man (23.1% versus 11% in rats). In a compar ison with the normalized data of Schumann et ah (1982a), Nolan et ah (1984) noted that blood levels in mice and rats inhaling TRI were 17.3 and 3.5 times higher, respectively, than those measured in humans. When deter mining the actual inhaled dose at equivalent inhaled concentrations, one must consider the wide variation in volume of respiration and body weight between species. Assuming a 4.2 liters/min alveolar ventilation and 70 kg body wt for man (Ganong, 1979), the rats in the present study received an inhaled dose ap proximately six times greater than that of the humans in the study by Nolan et ah (1984). Nolan and his colleagues determined follow ing a 6-hr exposure to 150 ppm TRI that mice, rats, and humans metabolized 0.16, 0.06, and 0.014 ^mol/kg/ppm, respectively. Thus, mice and rats should be more suscepti ble than humans to TRI toxicity at equiva lent inhaled concentrations, due to signifi cantly greater systemic absorption and me tabolism of the chemical. Meaningful health risk assessments require a careful selection of the measure of dose. In the present study systemic uptake of TRI is measured directly during the initial phase of inhalation exposure, when significant loading of tissues is occurring. Once tissue loading is completed (i.e., steady state is reached), very little uptake of TRI should occur because of the poor metabolism of the chemical. Thus, the common practice of assessing dose by multiplying ventilation rate by inhaled con centration would be very misleading during prolonged exposures. A more logical measure of target organ dose or tissue exposure would be the area under the tissue concentration versus time curve. The concept and rationale for selection of appropriate target organ dose measures (i.e., tissue dosimetry) are discussed in detail by Andersen (1987), It is important that target organs and mechanisms oftoxicity be elucidated, so that the agent(s) responsible for toxic effects are identified and can subse quently be quantified and correlated with the magnitude of toxicity in the target tissue(s). It is not clear for TRI whether the parent com pound or its metabolites should serve as the dose measure, or surrogate. Near-lethal expo sures are required for effects on most target organs. Carcinogenicity bioassays have been negative, or inconclusive. Reitz et ah (1988) decided to use the average concentration of TRI in the liver over a lifetime (ACL) as a dose surrogate. These investigators used a PBPK model to calculate ACLs for compari son of internal doses received by mice and rats in long-term toxicity studies versus hu mans drinking TRI-contaminated water. Unfortunately, there is a paucity of data on actual concentrations of TRI in the liver and other organs. Direct measurement studies are needed to generate tissue concentration ver sus time data sets for rigorous validation of PBPK model predictions of dose surrogates. APPENDIX Mass Balance Differential Equationsfor Physiological Pharmacokinetic Model* Arterial Blood dQ, Vb dt SL 030020 396 DALLAS ET AL. Venous Blood dCv _ _ Cy C,,, Vb dt ~Quru + qTMX + Qt~+Qr%-QtCv K( Kr Alveolar Space the other symbols. *V(0 = 1 for / < 120 min and0forr> 120 min. ACKNOWLEDGMENTS The authors are grateful to Ms. Linda Quigley, Miss Elizabeth Lehman, and Mrs. Judy Bates for their exper tise in preparation ofthis manuscript. a dt = VR.Cinh ,(/) -- VRaCa + h Liver r"f=a(c`-S Muscle dCm dt Qm Fat dQr Vt dt Richly Perfused Note. *Ci - concentration of TRI in com partment i. See Table 1 for the definition of REFERENCES Andersen, M. E. (1987). Tissue dosimetry in risk assess ment, or what's the problem here anyway? In Pharma cokinetics in Risk Assessment, Vol. 8, Drinking Water andHealth, pp. 8-26. National Academy Press, Wash ington, DC. Angelo, M. J., and Pritchard, A. B. (1984). Simula tions of methylene chloride pharmacokinetics using a physiologically based model. Regul. Toxicol. Pharma col. 4,329-339. Caperos, J. R., Droz, P. O., Hake, C. L., Humbert, B. E., and Jacot-Guillarmod, A. (1982). 1,1,1-Trichloroethane exposure, biological monitoring by breath and urine analysis. Int. Arch. Occup. Environ. Health 49,293-303. Clark, B., and Smith, D. A. (1984). Pharmacokinetics and toxicity testing. CRCCrit. Rev. Toxicol 12, 343385. Clewell, H. J., and Andersen, M. E. (1985). Risk as sessment extrapolations and physiological modeling. Toxicol. Ind. Health 1,111-131. Dallas, C. E., Bruckner, J. V., Maedgen, J. L., and Weir, F. W. (1986). A method for direct measurement ofsystemic uptake and elimination ofvolatile organics in small animals. J. Pharmacol. Methods 16,239-250. Dallas, C. E,, Weir, F. W., Feldman, S,, Putcha, L,, and Bruckner, J. V. (1983). The uptake and disposi tion of 1,1-dichloroethylene in rats during inhalation exposure. Toxicol. Appl. Pharmacol. 68, 140-151. Dornette, W. H. L., and Jones, J. P. (1960). Clinical experiences with 1,1,1-trichloroethane. A preliminary report of50 anesthetic administrations. Anesth. Analg. 39,249-253. Eben, A., and Kjmmerle, G. (1974), Metabolism, ex cretion and toxicology of methyl chloroform in acute and subacute exposed rats. Arch. Toxikol. 31, 233242. Ganong, W. F. (1979). Review ofMedical Physiology, p. 500. Lange Medical Publications, Los Altos, CA. Gargas, M. L., Andersen, M. E., and Clewell, H. J., Ill (1986). A physiologically based simulation ap proach for determining metabolic constants from gas uptake data. Toxicol. Appl. Pharmacol. 86,341-352. SL 030021 KINETICS OF INHALED TRICHLOROETHANE 397 Gargas, M. L., Burgess, R. J., Voisard, D. E,, Ca son, G. H., and Andersen, M, E. (1989). Partition coefficients of low molecular weight volatile chemicals in various liquids and tissues. Toxicol Appl, Pharma col. 98,87-99. Gehring, P. J. (1968). Hepatoxic potency of various chlorinated hydrocarbon vapors relative to their nar cotic and lethal potencies in mice. Toxicol. Appl Phar macol 13,287-293. Gerlowski, L. E., and Jain, R. K. (1983). Physiologi cally-based pharmacokinetic modeling: Principles and applications. J. Pham. Sci. 72, 1103-1127. Herd, P. a., Lipsky, M., and Martin, H. F, (1974). Cardiovascular effects of 1,1,1-trichloroethane. Arch. Environ. Health 28,227-233. Holmberg, B., Jakobson, I., and Sigvardsson, K. (1977). A study on the distribution of methyl chloro form and n-octane in the mouse during and after inha lation. Scand. J. Work Environ. Health 3,43-52. Ikeda, M., and Ohtsuji, H. (1972). A comparative study of the excretion of Fujiwara reaction-positive substances in urine of humans and rodents given trichloro- or tetrachloro-derivatives ofethane and eth ylene. Brit. J. Ind. Med. 29,99-104. Klaassen, C. D., and Plaa, G. L. (1966). Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice. Toxicol. Appl. Pharmacol 9,139-151. Klaassen, C. D., and Plaa, G. L. (1967). Relative effects of various chlorinated hydrocarbons on liver and kidney function in dogs. Toxicol. Appl. Pharma col. 10,119-131. Kleinfeld, M., and Feiner, B. (1966). Health hazards associated with work in confined spaces. J. Occup. Med. 8,358-364. Mauderly, J. L., Tesarek, J. E., Sifford, L. J., and SlFFORD, L. J. (1979). Respiratory measurements of unsedated small laboratory mammals using nonre breathing valves. LabAnim. Sci. 29,323-329. Monster, a. C., Boersma, G., and Steenweg, H. (1979). Kinetics of 1,1,1-trichloroethane in volun teers: Influence of exposure concentration and work load. Int Arch. Occup. Environ Health 42,293-302. Morgan, A,, Black, A., and Belcher, D. R. (1972). Studies on the absorption of halogenated hydrocar bons and their excretion in breath using 38C1 tracer techniques. Ann. Occup. Hyg. 15,273-282. Nolan, R. J., Freshour, N. L., Rick, D. L., Mc Carty, L. P., and Saunders, J. H. (1984). Kinetics and metabolism of inhaled methyl chloroform (1,1,1trichloroethane) in male volunteers. Fundam. Appl. Toxicol. 4,654-662. National Research Council (NRC) (1987). Pharmacoki netics in Risk Assessment, Vol. 8, Drinking Water and Health. National Academy Press, Washington, DC. Plaa, G. L., and Larson, R. E. (1965). Relative neph rotoxic properties ofchlorinated methane, ethane, and ethylene derivatives in mice. Toxicol Appl Phamacol. 7,37-44. Ramsey, J. C., and Andersen, M. E. (1984). A physio logically based description of the inhalation pharma cokinetics of styrene in rats and humans. Toxicol. Appl Pharmacol 73, 159-175. Reinhardt, C. F., Mullin, L. S., and Maxfiled, M. E. (1973). Epinephrine-induced cardiac arrhyth mic potential of some common industrial solvents. J. Occup. Med. 15,953-955. Reitz, R. H., McDougal, J. N., Himmelstein, M. W,, Nolan, R. J., and Schumann, A. M. (1988). Phys iologically based pharmacokinetic modeling with methylchloroform: Implications for interspecies, high dose/low dose, and dose route extrapolations. Toxicol Appl Pharmacol. 95,185-199. Schumann, A. M., Fox, T. R., and Watanabe, P. G. (1982a). [14C]Methyl chloroform (1,1,1-trichloroeth ane): Pharmacokinetics in rats and mice following in halation exposure. Toxicol. Appl Pharmacol. 62,390401. Schumann, A. M., Fox, T. R., and Watanabe, P. G. (1982b). A comparison of the fate of inhaled methyl chloroform (1,1,1-trichloroethane) following single or repeated exposure in rats and mice. Fundam. Appl. Toxicol 2,27-32. Seki, Y., Urashima, Y., Aikawa, H., Matsumura, H., Ichikawa, Y., Hiratsuka, F., Yoshioka, Y., Shimbo, S., and Ikeda, M. (1975). Trichloro-compounds in the urine of humans exposed to methyl chloroform at subthreshold levels. Int. Arch. Arbeitsmed. 34,39-49. Stewart, R. D. (1968). The toxicology of 1,1,1-trichlo roethane. Ann. Occup. Hyg. 11,71-79. Stewart, R. D,, Gay, H. H., Schaffer, A. W., Erley, D. S., and Rowe, V. K. (1969). Experimental human exposure to methyl chloroform vapor. Arch. Environ. Health 19,467-472. Torkelson, T. R., and Rowe, V. K. (1981). Haloge nated aliphatic hydrocarbons containing chlorine, bromine and iodine. In Patty's Industrial Hygiene and Toxicology (G. D. Clayton and E. Clayton, Eds.), 3rd ed., pp. 3502-3510. Wiley, New York. Veng-Pedersen, P. (1984). Pulmonary absorption and excretion of compounds in the gas phase: Theoretical pharmacokinetic and toxicokinetic analysis. J. Pharm. Sci 73, 1136-1141. SL 030022 TOXICOLOGY AND APPLIED PHARMACOLOGY 98, 398-412 (1989) Type I and Type II Pyrethroids Increase Inhibition in the Hippocampal Dentate Gyrus of the Rat1 Robert M. Joy,* Timothy E. Albertson,f and David E. Ray$ *Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine; fDepartment ofInternal Medicine, School ofMedicine. University ofCalifornia, Davis, California 95616; and %MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SMS 4EF, United Kingdom Received July 22, 1988; acceptedJanuary 3,1989 Type 1 and Type II Pyrethroids Increase Inhibition in the Hippocampal Dentate Gyrus ofthe Rat. Joy, R. M., Albertson, T. E., and Ray, D. E. (1989) Toxicol. Appl. Pharmacol, 98,398412. Urethane-anesthetized rats were prepared for stimulation of the perforant path and for recording from the granule cell region ofthe hippocampal dentate gyrus. Subjects were adminis tered varying doses of allethrin (a prototype type I pyrethroid) or deltametbrin (a prototype type II pyrethroid), and the excitability of the perforant path and granule cells was tested. Both pyrethroids produced a dose-dependent decrease in the responsiveness ofgranule cells, following stimulation of the perforant path, that lasted up to 100 msec. Analysis suggested that the pyrethroid-induced effects were associated with an increase in intemeuronally mediated inhibition. Neither the perforant path axon or terminal nor the granule cell was affected by doses which appeared to affect intemeurons. Basal excitability of the granule cells was also decreased by deltamethrin. This effect may have been secondary to an increase in tonic inhibition evoked by the same mechanisms responsible for the increase in phasic inhibition. 1989 Academic Pies, Inc. The synthetic pyrethroids constitute a diverse group of chemicals with important insecti cidal actions. They are highly toxic to a wide spectrum of insects but possess relatively low oral toxicity to mammals. Their intravenous toxicity is much greater, however, suggesting that this selectivity is at least partly based on pharmacokinetic and metabolic factors (Vijverberg and van den Bercken, 1982). The symptoms produced in mammals vary de pending upon the specific pyrethroid in volved. Some produce primarily tremor asso ciated with increased sensitivity to external stimuli (type I pyrethroids). Others produce a state more characterized by salivation and a wobbling motor dysfunction that progresses 1 This research was supported in part by the Thomas Henry Curtis Endowment Fund of the University ofCal ifornia and in part by USPHS Grant 2S07RR05457. to choreoathetoid movements and convul sions (type II pyrethroids) (Verschoyle and Aldridge, 1980). Most pyrethroids can be placed into one or the other of these two cate gories, although examples intermediate in symptomatology are known to exist. All of the pyrethroids that produce mam malian toxicity are known to interact with so dium channels in nervous tissue. They all prolong the sodium current evoked by depo larization of the membrane (Narahashi, 1971, 1985, 1986; Vijverberg and van den Bercken, 1982; Vijverberg and de Weille, 1985; Vijverberg el al,, 1986). At least part of the distinction between type I and type II pyrethroids appears to be related to the time during which the sodium channel remains in an open state. Type I pyrethroids hold the so dium channel open for relatively short peri ods of time (milliseconds). This effect pro- 0041-008X/89 $3.00 Copyright 1989 by Academic Press, Inc All rights of reproduction in any form reserved 398 St I UNDAMENTAL AND APPLIED TOXICOLOGY I 1,61 1-625 (1988) 1,1,1-Trichloroethane Formulation: A Chronic Inhalation Toxicity and Oncogenicity Study in Fischer 344 Rats and B6C3F1 Mice J. F. Quast, L. L. Calhoun, and L. E. Frauson Mammalian and Environmental Toxicology Research Laboratory, The Dow Chemical Company, ISOS Building, Midland, Michigan 48674 Received October 19, 1987; accepted May 25, 1988 1,1,1-Trichloroethane Formulation: A Chronic Inhalation Toxicity and Oncogenicity Study in Fischer 344 Rats and B6C3F1 Mice. Quast, J. F., Calhoun, L. L., and Frauson, L. E. (1988). Fundam. Appl. Toxicol, 11, 611-625. Groups of male and female Fischer 344 rats and B6C3F1 mice (80/sex/group) were exposed to vapor concentrations of0, 150, 500, or 1500 ppm 1,1,1-trichloroethane formulation 6 hr/day, 5 days/week, for 2 years. Ten rats and mice/ sex from each group were predesignated for interim sacrifices after 6, 12, and 18 months of exposure. Fifty rats and mice/sex/group were assigned to the study to be terminated after 24 months. Parameters measured during the study included mortality, in-life clinical signs oftoxic ity, hematology, urinalysis (rats only), clinical chemistry, body weight, organ weights (liver, kid neys, brain, heart, testes), gross pathology, and histopathology. Inhalation exposure of male and female Fischer 344 rats to 1500 ppm vapor of the 1,1,1-trichloroethane formulation for 2 years resulted in a significant decrease in body weights offemales. In addition, very slight microscopic hepatic effects were seen in the liver of 1500 ppm-exposed male and female rats necropsied at 6, 12, and 18 months. The hepatic effects could not be discerned at 24 months due to confounding geriatric changes. In the rats exposed to 150 and 500 ppm there were no changes that were considered due to exposure to the 1,1,1 -trichloroethane formulation. There were no toxic effects noted in male or female mice at any exposure concentration tested. There were no indications ofan oncogenic effect in rats or mice following 2 years ofexposure to this 1,1,1 -trichloroethane formulation. 1988 Society ofToxicology. 1,1,1 -Trichloroethane (methyl chloroform) is used in formulations for cold cleaning and in dustrial degreasing processes as well as in aerosol applications. It is the major ingredi ent of CHLOROTHENE1 solvents. Com mercial formulations commonly contain as much as 5% (total volume) chemical stabiliz ers and traces of chlorinated aliphatic hydro carbons. A review of the scientific literature on the toxicity of 1,1,1-trichloroethane in rats and mice indicates that the two species respond to the solvent in comparable fashion over a similar range of exposure concentrations, 1 Registered trademark of The Dow Chemical Com pany. The LC50 for a single 7-hr exposure of rats to 1,1,1-trichloroethane was reported to be 14,250 ppm (Adams el al,, 1950). Gehring (1968) found that the time to 50% mortality (LT50) for mice exposed to 13,500 ppm was approximately 6 hr. One study compared the response of rats and mice to 1,1,1-trichloro ethane inhalation exposure (rats and mice, published in two parts, MacEwen and Vernot, 1974; McNutt et al, 1975). Continuous exposure, 24 hr/day, of rats and mice to 0, 250, or 1000 ppm 1,1,1-trichloroethane for 14 weeks resulted in the following findings in 1000 ppm-exposed animals: increased rela tive liver weights in both rats and mice, in creased liver triglycerides in mice, and micro scopic liver changes detectable in rats and 0272-0590/88 S3.00 Copyright C 1988 by the Society ofToxfcotogy. All rights of reproduction in iny form reserved. SL 030024 612 QUAST, CALHOUN. AND FRAUSON mice. At 250 ppm the findings were increased relative liver weight and minimal micro scopic liver changes in the rats and minimal cytoplasmic alterations ofcentrilobular hepatocytes observed only by electron microscopy in the mice. The National Cancer Institute (NCI) has reported the results of a carcinogenesis bioas say of a 1,1,1-trichloroethane formulation (containing 3% 1,4-dioxane and 2% minor impurities) in Osborne-Mendel rats and B6C3F1 mice (NCI, 1977). The test material was administered by gavage in com oil to 50 animals/sex/species, 5 days/week for 78 weeks. Rats received doses of 750 or 1500 mg/kg/day. Dose levels for mice were initially 0, 2000, or 4000 mg/kg/day but were in creased twice during the study, resulting in time-weighted average doses of 2807 and 5615 mg/kg/day. There was a moderate de crease in body weight gain observed in the high-dose male rats during the first year and in both sexes of mice throughout the study. Early mortality attributed to intercurrent murine pneumonia was observed in both sexes of rats and in female mice. All surviving rats were killed at 117 weeks of age and mice at 96 weeks of age. No neoplastic or nonneo plastic pathological findings were observed that were attributable to treatment. However, the study was ruled inadequate by NCI for as sessment of carcinogenicity due to a short ened life span in rats and mice. A chronic inhalation study of a 1,1,1-trichloroethane formulation in Sprague-Dawley rats has been conducted in this laboratory (Quast et al,, 1978; Rampy et ai, 1977). The rats were exposed to 0, 875, or 1750 ppm of the test material 6 hr/day, 5 days/week, for 12 months. The animals were then allowed to survive until 31 months. No treatment-re lated changes were detected except that fe male rats exposed to 1750 ppm showed an in creased incidence of focal hepatocellular al terations. No evidence for a carcinogenic response was detected; the tumor incidence of 1,1,1-trichloroethane-exposed rats was comparable to that of controls. A report of a long-term ingestion study of 1,1,1-trichloroethane in Sprague-Dawiey rats has recently been published (Maltoni et ai. 1986). Forty male and forty female rats were dosed by gavage with 500 mg/kg body wt/day 1,1,1-trichloroethane in olive oil, while 50 rats of each sex served as controls and were dosed with olive oil alone. Animals were treated once daily, 4-5 days/week, for 104 weeks. After this time the surviving ani mals were held without treatment until death (up to 141 weeks). The authors reported an increase in the incidence of rats with leuke mia, particularly immunoblastic lymphosar comas. The authors stated that "The design and size of our experiment do not allow us to draw definite conclusions" but recom mended additional testing to assess the carci nogenic potential of 1,1,1 -trichloroethane. In preparation for selecting appropriate ex posure concentrations for the 2-year inhala tion study herein reported, a 90-day sub chronic inhalation study of the 1,1,1-trichlo roethane formulation was conducted in this laboratory (Calhoun et ai, 1981). Groups of rats and mice exposed to 2000 ppm, 6 hr/day, 5 days/week, showed statistically significant changes in body and liver weights and mini mal microscopic changes in the olfactory epi thelium. Based on these findings in the subchronic inhalation study, the maximum ex posure level selected for the 2-year study was 1500 ppm. METHODS Test Material A production-grade 1,1,1-trichloroethane formula tion, approximately 94% (by volume) 1,1,1 -trichloroeth ane, 5% stabilizers (butylene oxide, t-amyl alcohol, methyl butynol, nitroethane, and nitromethane), and <1% minor impurities, was used for the study. Each drum of material was analyzed by gas-liquid chromatog raphy when opened for use. Analyses ofsamples from all drums oftest material used in the study were comparable and showed no sample degradation during the test in terval. SL 030025 E\pernncntL, Groups ot multancousl 500, or 1500 non. these ct s 14 mg/htei .bvs/week Itiexposun Male and and male an were purcha tage, Michi) designated h of 10 predesi ducted after tional 10 am test approxit .ssment; re repeatedly e' Schumann e I The test at a statistical r ] identified by singly; rats i Food (Putin, | Louis, MO)' during expo times from a | . .posed to their respect tion chantbt plied during Exposures Exposure1 x 8 ft (14.5 shape of | :..'sin-coated j ated under < tamed at a s I thesurroum lar 12-hr ph [ controlled b at approxim | mately 50% corded daily I Vapors ol | tcrated by .;^iassJ-tub. I The air supr | minimum e tion of the t I pors from th | her inlet du 1.1.1 -TRICHLOROETHANE ONCOGENICITY STUDY 613 Experimental Design Groups of Fischer 344 rals and B6C3F1 mice were si multaneously exposed to target concentrations ofO, 1 SO, 500, or 1500 ppm of the 1.1,1-trichloroethane formula tion; these concentrations correspond to 0,0.82. 2.73, or 8 19 mg/liter in air. Exposures were conducted 6 hr/day, days/week (except holidays), for 24 months (total of 516 exposure days). Male and female Fischer 344 rats, 4-6 weeks of age, and male and female B6C3F1 mice, 5-6 weeks of age, were purchased from Charles River Laboratories, Por tage, Michigan. Fifty animals/sex/species/group were designated for 24 months of exposure. Interim sacrifices of 10 predesignated animals/sex/species/group were con ducted after 6, 12, and 18 months ofexposure. An addi tional 10 animals/sex/species/group were maintained on test approximately 16 months for pharmacokinetic as sessment; results of the pharmacokinetic studies in the repeatedly exposed rats and mice have been reported by Schumann el al. (1982). The test animals were assigned to treatment groups by a statistical randomization procedure. The animals were identified by a numbered metal eartag. Mice were housed singly; rats of the same sex were housed two per cage. Food (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO) was available to all animals adlibitum except during exposure. Water was available ad libitum at all times from an automatic watering system. Rats and mice exposed to the 1,1,1-trichloroethane formulation and their respective controls were maintained in the inhala tion chambers throughout the study with clean air sup plied during nonexposure periods. Exposures Exposures were conducted in walk-in chambers 8X8 X 8 ft (14.5 m5), equipped with stainless-steel ceilings in the shape of a regular quadrangular pyramid and epoxy resin-coated walls and floors. The chambers were oper ated under dynamic airflow conditions and were main tained at a slight negative pressure relative to the air in the surrounding area. The chambers were kept on a regu lar 12-hr photocycle. Air supplied to the chambers was controlled by a system designed to maintain temperature at approximately 70*F and relative humidity at approxi mately 50%. The temperature and humidity were re corded daily during exposure. Vapors of the 1,1,1-trichloroethane formulation were generated by metering the liquid at a calculated rate into a glass J-tube vaporization apparatus (Miller et al.. 1980). The air supplied to the J-tube assembly was heated to the minimum extent necessary to ensure complete vaporiza tion of the test material (approximately 90-100C). Va pors from the J-tube were then introduced into the cham ber inlet ducts where there was further mixing and dilu tion to the intended target concentrations. Total airflow through the chambers was maintained at approximately 2000 liters/min. The daily nominal concentration of the 1,1,1-trichloroethane formulation vapor in each chamber was calcu lated from the total amount of liquid vaporized and the total chamber airflow. The analytical concentration of 1,1,1-trichloroethane vapor in the chamber was deter mined by infrared spectrophotometry (Miran 1A, Foxboro Analytical, Norwalk, CT) at a wavelength of 9.2 pm. Each exposure chamber was analyzed at least once per hour during the 6-hr exposure period. The output of the infrared analyzer was fed to a microprocessor which calculated the time-weighted average (TWA) exposure concentration for each chamber. There was close agreement between mean daily TWA analytical concentration and mean daily nominal con centration for each exposure chamber. The TWA analyt ical values were 151 2, 502 5, and 1505 11 ppm for the 516 exposure days. The distribution of the 1,1,1trichloroethane vapor within each chamber was deter mined to be uniform (within 10% of the chamber TWA) prior to initiation ofthe study. Clinical Determinations Animals were observed daily for changes in appear ance and demeanor. Each animal on test was checked for palpable masses prior to initiation of the study and monthly from the 12th month ofexposure until termina tion. In-life palpable "masses" included not only possible neoplasms, but any swelling regardless oflocation or neo plastic character. Animals were weighed at approxi mately monthly intervals throughout the study. Clinical laboratory studies were conducted for all sur viving animals that were designated for the 6-, 12-, and 18-month interim sacrifices. Ten rats/sex/exposure group were used for all clinical laboratory studies at 24 months. Total red blood cells, hemoglobin concentra tion, packed cell volume, total white cells, and platelets were evaluated (Ortho ELT-8, Ortho Instruments, Bos ton, MA) for all samples except those of mice at the 6month interval. Differential white blood cell counts (Honeywell ACS-1000, Honeywell, Inc., Denver, CO) were determined for all samples from the 12-month in terval, and for control and 1500-ppm samples at the re maining intervals (except mice at 6 months). Urine sam ples were obtained from the same rats (not from mice) 1-2 weeks prior to each scheduled necropsy for analysis of specific gravity (T. S. Meter, American Optical Co., Keene, NH), pH, protein, glucose, ketones, bilirubin, blood, and urobilinogen (Multistix, Ames Co., Elkhart, IN). Clinical chemistry determinations (CentrifiChem System, Methods File, Union Carbide Corp., Rye, NY) on all samples included serum levels of urea nitrogen, alkaline phosphatase activity, glutamic-pyruvic trans- SL 030026 614 QUAST. CALHOUN. AND FRAUSON TABLE 1 Tissues Collected and Preserved at Necropsy Adrenals Aorta Bone Bone marrow Brain Cecum Cervix Coagulating glands Epididymides Esophagus Eyes Gallbladder (mice) Heart Kidneys Lacrimal/ Harderian glands Large intestine (colon, rectum) Larynx Liver Lungs Mammary gland Mediastinal lymph node Mediastinal tissues Mesenteric lymph node Mesenteric tissues Nasal tissues Oral tissues Ovaries Oviducts Pancreas Parathyroid glands Peripheral nerve Pituitary Prostate Salivary glands Seminal vesicles Skeletal muscle Skin and subcutis Small intestine (duodenum. jejunum. ileum) Spinal cord Spleen Stomach Testes Thymus Thyroid gland Tongue Trachea Urinary bladder Uterus Vagina aminase activity, glucose, cholesterol, triglycerides, total protein, albumin, and globulin. Blood samples were col lected from the orbital sinus of rats 1-2 weeks prior to necropsy for hematology and from severed cervical ves sels at necropsy for clinical chemistry. Blood samples were collected from the orbital sinus of the mice at the time of necropsy for all determinations. Pathology Rats and mice submitted for necropsy were weighed, anesthetized with methoxyflurane, and bled for clinical hematology and chemistry determinations. A complete gross necropsy was performed by a veterinary patholo gist, and tissues listed in Table 1 were preserved in neu tral, phosphate-buffered 10% formalin. Examination of the eyes by a moist microscope slide technique under fluorescent illumination was performed. Liver, kidneys, brain, heart, and testes (males) were weighed and the or gan weight'.final body weight ratios were calculated. Ani mals that died or were submitted moribund were exam ined in a similar manner, except organs were not weighed and the detailed eye examinations were not conducted. The tissues (Table 1) were processed in the standard manner, embedded in paraffin, sectioned at 5-6 tttn, stained with hematoxylin and eosin, and examined mi croscopically by a veterinary pathologist. All tissues from control and 1500 ppm-exposed groups of rats and mice designated for 6-, 12-, 18-, and 24-month (terminal) sac rifices were examined. At the 6-, 12-, and 18-monih in terim sacrifices, only selected tissues from mice and livers from rats were generally examined from animals in the groups exposed to 150 and 500 ppm. For male and fe male rats exposed to 150 and 500 ppm and assigned to the 24-month portion of the study, histopathologic eval uation of the following tissues was performed: liver, spleen, kidneys, pituitary, adrenals, thyroid, testes, mes enteric lymph node, lung, trachea, larynx, and nasal tur binates. All tissues of male and female mice exposed to 150 or 500 ppm and assigned to the 24-month portion of the study were examined. In addition, any grossly ob served lesions suggestive of a tumor were examined mi croscopically from all animals. Statistics Cumulative mortality data were tabulated by daily in tervals and analyzed for overall differences by the Gehan-Wilcoxon test (Breslow, 1970), a - 0,05. Body weight data were evaluated by Dunnett's test, a = 0.05, two-sided (Steel and Torrie, 1960); statistical outliers were identified by the sequential procedure described by Grubbs (1969) and were excluded from further calcula tions. Interim data collected through 18 months ofthe study, including absolute and relative organ weights, urinary specific gravity, hematology (except differential leuko cyte counts), and clinical chemistry data, were statisti cally evaluated as follows. Statistical outliers were deter mined by the procedure described by Grubbs (1969) but were not excluded from calculations. The data were eval uated by Bartlett's test for equality of variances (Winer, 1971), a = 0.01. Based on the outcome of Bartlett's test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was signifi cant, it was followed by Dunnett's test (Winer, 1971) or the Wilcoxon rank-sum test (Hollander and Wolfe, 1973), a = 0.05, two-sided, with Bonferroni's correction (Miller, 1966). Clinical laboratory data (hematology, urine specific gravity, and clinical chemistry) and organ/body weight data obtained from the 24-month sacrifice were not sta tistically analyzed for differences from control because by 24 months these parameters generally reflect a variety ofgeriatric changes, spontaneous neoplasms, and agonal changes. Statistical evaluation ofthese data is ofdoubtful scientific merit. Descriptive statistics were determined for differential leukocyte counts at all intervals; however, no statistical analysis ofthese data was conducted. For the results of the histopathologic examination of the tissues of mice scheduled for 24 months ofexposure, Fisher's exact probability test (Siegel, 1956) was used as the main interpretive statistical comparison. To ensure that pairwise comparisons had not missed any observa- 0300^7 sl* 1,1,1-TRICHLOROETHANE ONCOGENICITY STUDY SURVIVAL 615 Fig. 1. Survival of male and female rats and mice exposed to vapors of a 1,1,1 -trichloroethane formula tion for 2 years. tions, the Cochran-Armitage trend test (Armitage, 1971) was also applied ifFisher's test was negative. The Bonferroni correction procedure (Miller, 1966) was used when the control incidence of the lesion was at least 6%. The nominal a level in all cases was 0.05, one-sided. For tissues intended for histopathologic examination from all rats at all dose levels scheduled for 2 years of exposure, the incidences of specific observations were first tested for deviation from linearity using ordinal spacings ofthe doses. Iflinearity was not rejected the data were then tested for a dose-response relationship using the Cochran-Armitage trend test (Armitage, 1971). Ifthe trend was statistically significant, or if significant devia tion from linearity was found, incidences for each dose were compared with that of the control using a pairwise x2 test with Yate's correction (Fleiss, 1981). For tissues which were evaluated from all control and high-doselevel rats, but only from selected rats from the middleand low-dose groups, statistical analysis was limited to the pairwise comparison of control and top dose. The nominal o levels used were a = 0.01 (x2 test for linearity); a - 0.02, two-sided (trend test); and a - 0.05, one-sided (pairwise comparison Yates x2 test). The trend test, rather than Fisher's method, was used as the primary sta tistical tool for the evaluation of histopathologic data on rats because it is a global test utilizing all the data and because of its superior operating characteristics (Haseman, 1983, 1985). The trend test is more sensitive than Fisher's test and achieves its nominal a level more uni formly (Park, 1985; Park and Kociba, 1985). Since multiple, interrelated parameters were statisti cally compared in the same group of animals, the fre quency offalse-positive errors may be much greater than the nominal a level. Thus, in addition to statistical analy ses, the final toxicologic interpretation of the data also considered factors such as dose-response relationships and whether or not the findings appeared to be plausible and consistent in light of other biologic findings. RESULTS AND DISCUSSION Rats There were no statistically significant differences.in survival rates of male or female exposed rats when compared with their re spective controls (Fig. 1). The body weights of female rats of both 500 ppm- and 1500 ppm-exposed groups SL 030028 616 Quast, calhoun, and frauson BODY WEIGHTS FIG. 2, Body weights of male and female rats and mice exposed to vapors of a 1,1,1-trichlotoethane formulation for 2 years. were decreased compared with the controls through much of the study (Fig. 2). The 500 ppm-exposed female rats had statistically de creased body weights from approximately the 7th month to the 18th month, and the 1500 ppm-exposed group of female rats showed a statistical decrease in weight from approxi mately 11 through 24 months. It should be noted that these groups weighed slightly less than their respective control groups prior to initiation of exposure. Nonetheless, the greater and more consistent decreased body weight in the 1500 ppm-exposed females ap pears to have been due to exposure to the test material. Other statistically significant devia tions in body weight were observed, but these were interpreted to be a reflection of normal biological variability since they did not follow a pattern that suggested an exposure-related effect. There were no unusual in-life clinical ob servations during the study that were consid ered to be exposure related. The hematology, urinalysis, and clinical chemistry values, absolute organ weights, and relative organ weights (g/100 g body wt) for male and female rats sacrificed at the various time intervals throughout the study were un affected by treatment. The gross necropsy observations for all groups of rats from the predesignated interim sacrifices or from the 2-year portion of the study did not reveal an exposure-related effect. There were no early deaths in groups ofrats sacrificed at 6 and 12 months. Several rats predesignated for the 18-month sacrifice died prior to the scheduled sacrifice. Review of the gross and microscopic findings in the animals that died revealed a spectrum of normal agerelated changes not considered related to ex posure. The only apparent exposure-related effect observed in rats from the 6-, 12-, and 18month interim sacrifices was found micro- 030029 SL 1,1,1-TRtCHLOROETHANE ONCOGENICITY STUDY 617 TABLE 2 Selected Neoplasms: Rats" Males Females Exposure concentration (ppm) 0 150 500 Number of rats examined 50 50 50 1500 50 0 150 500 1500 50 50 50 50 Subcutaneous tissue (number oftissues examined) 50 50 50 Fibroma, benign, primary 11 6 2b Testes (number of tissues examined) 50 50 50 Interstitial cell tumor, unilateral, benign, primary 7 11 3 Interstitial cell tumor, bilateral, benign, primary 36 30 38 Interstitial cell tumor, unilateral or bilateral 43 41 41 Multiple organs (number oftissues examined) Mononuclear cell leukemia, malignant 50 50 50 24 29 23 50 0bc 50 4 50 50 01 50 _ d -- 4 -- ---- 45' -- -- -- 49 -- -- -- 50 50 50 50 20 17 25 18 50 0 _ -- -- -- 50 11 Data are the number ofanimals with the specified observation noted on microscopic examination oftissues. * Statistically identified difference from control group, Yates x2 test, a = 0.05. c Linear trend by Cochran-Armitage Test, a = 0.02 (two-sided). d Not applicable. scopically in the liver. An accentuation of the normal hepatic lobular pattern was observed in males and females in the 1500 ppm-exposed group. The effect on the hepatocytes consisted of altered cytoplasmic staining in the cells surrounding the central vein. Gener ally, the hepatocytes in the portal region ap peared smaller in the exposed rats when com pared with their respective controls. The very slight response in the hepatocytes surround ing the central vein was interpreted to have resulted from exposure to the test material and/or its metabolites. The minimal expo sure-related effects in the liver observed at earlier necropsy times were no longer dis cernible after 2 years of exposure due to con founding geriatric changes. The only neoplastic lesions that were statis tically identified as different from controls were subcutaneous fibromas and bilateral testicular interstitial cell neoplasms in male rats (Table 2). The incidence ofsubcutaneous fibromas in male rats was statistically de creased compared with controls at the 1500ppm exposure level. The total incidence of unilateral or bilateral benign testicular inter stitial cell tumors was not statistically differ ent from that of controls at any exposure level; however, the number of rats with bilat eral tumors was statistically identified as in creased from controls. Since the total number of male rats with interstitial cell tumors was comparable in all groups and this tumor type has such a high incidence in the Fischer 344 rat, this finding was not considered to have any toxicological significance. There was no indication of an increased incidence of mononuclear cell leukemia, or any lymphoreticular proliferative process, in rats ofeither sex (Tables 2 and 3). Other statistically identi fied observations were noted and interpreted to be reflective of normal variability in aged rats. Due to normal age-related geriatric changes there were no discernible exposurerelated microscopic hepatic effects noted in the rats at the end of the 2-year study. Mice There were no statistically significant differences in survival rates of male or female exposed groups of mice when compared with their respective controls (Fig. 1). There was 030030 SV> 618 QUAST, CALHOUN, AND FRAUSON TABLE 3 Tumor Incidence: Rats" Males Females Exposure concentration (ppm) 0 150 500 1500 0 150 500 1500 Number of rats examined 50 50 50 50 50 50 50 50 Adrenals (number of tissues examined) 50 50 50 50 49 50 50 50 Adenoma, cortex, benign 1 00 00 0 1 1 Carcinoma, cortex, malignant 1 1 0 00 0 0 0 Ganglioneuroma, benign 0 1 0 00 0 0 0 Pheochromocytoma, benign 525 41 0 1 0 Pheochromocytoma, malignant 1I1 11 10 0 Brain (number oftissues examined) Astrocytoma, malignant 50 0 1 50 50 2 0 50 1 __* 0 10 0-- l Cervix (number oftissues examined) __ __ __ __ 50 3 1 50 Fibroma, benign -- ---- -- --w 1 0 0 1 Adenocarcinoma, malignant ---- -- -- 0 0 1 0 Stromal sarcoma, malignant ---- -- 0 20 0 Epididymides (number oftissues examined) Sarcoma, undifferentiated, malignant 50 0 0-- 3 49 __ -- __ 0 1-- -- -- -- Jejunum (number oftissues examined) 39 2 0 41 41 0 0 37 Adenocarcinoma, malignant 10 0 0-- -- 0 Leiomyosarcoma, malignant 0 l-- 0 0-- -- 0 Kidneys (number oftissues examined) 50 50 50 50 50 50 50 50 Adenoma, tubule cell, benign 000 10 0 0 0 Mixed mesenchymal tumor, malignant 00 1 10 0 0 0 Liver (number oftissues examined) 50 50 50 50 50 50 50 50 Neoplastic nodule, benign 1 1 I 41 10 0 Carcinoma, hepatocellular, malignant 003 01 00 0 Lung (number oftissues examined) 50 49 50 50 50 50 50 50 Adenoma, alveolar/bronchiolar, benign 00 1 10 0 0 0 Mammary gland (number oftissues examined) 49 1 1 50 50 3 8 49 Adenoma, benign 1 00 00 1 1 I Fibroadenoma, benign 10I 36 0 7 4 Adenocarcinoma, malignant 000 01 2 1 1 Multiple organs (number oftissues examined) 50 50 50 50 50 50 50 50 Mesothelioma, malignant 213 30 0 0 0 Mononuclear cell leukemia, malignant 24 29 23 20 17 25 18 11 Ovaries (number oftissues examined) __ __ __ __ 47 1 2 50 Granulosa/thecal cell tumor, benign Granulosa/thecal cell tumor, malignant ---- -- -- 0 0 0 ---- -- -- 0 1 0 2 0 Pancreas (number oftissues examined) 50 5 6 50 48 0 0 49 Adenoma, acinar cell, benign 1 00 1 0-- -- 0 Adenoma, islet cell, benign 7 14 2 0-- -- 1 Carcinoma, islet cell, malignant 331 1 0-- -- 0 Pituitary (number oftissues examined) 47 48 50 50 49 49 48 47 Adenoma, benign 13 18 13 10 22 20 19 16 Carcinoma, malignant 232 22 2 0 0 Preputial/clitoral gland (number oftissues examined) 3 1 0 43 10 1 Carcinoma, malignant 1 1-- 21 0 __ 0 Squamous cell carcinoma, malignant 0 0-- 10 0-- 0 " Data are the number of animals with the specified observation. Only tissues with a primary tumor are tabulated; other examined tissues are not shown. b --, Not applicable. 030031 SL 1.1,1-TRICHLOR.OETHANE ONCOGENICITY STUDY 619 TABLE 3--Continued Males Females Exposure concentration (ppm) Number of rats examined 0 50 150 500 1500 0 150 500 1500 50 50 50 50 50 50 50 Prostate (n umber of tissues exami ned) Adenoma, benign Salivary gland (number of tissues examined) Fibrosarcoma, malignant Spleen (number of tissues examined) Histiocytic sarcoma, malignant Subcutaneous tissue (number of tissues examined) Fibroma, benign Neurofibroma, benign Trichoepithelioma, benign Fibrosarcoma, malignant Myxosarcoma, malignant Osteosarcoma, malignant Testes (number oftissues examined) Interstitial cell tumor, benign, unilateral Interstitial cell tumor, benign, bilateral Thyroid (number of tissues examined) Adenoma, "C" cell, benign Adenoma, follicular cell, benign Carcinoma, "C" cell, malignant Carcinoma, follicular cell, malignant Thymus (number of tissues examined) Thymoma, malignant Tongue (number of tissues examined) Squamous cell papilloma, benign . Urinary bladder (number oftissues examined) Transitional cell papilloma, benign Uterus (number of tissues examined) Adenoma, benign Endometrial stromal polyp, benign Leiomyoma, benign Adenocarcinoma, malignant Squamous cell carcinoma, malignant 50 0 0 50 1-- -- 0-- -- -- -- 50 0 0 50 50 0 1 50 0-- -- 0 0-- 1 0 50 48 50 50 49 50 50 50 1 00 00 0 0 0 12 8 4 67 0 2 3 11 6 2 0 4-- 1 0 00 1 1 0-- 0 0 000 1 0-- 0 0 000 0 1-- 0 0 000 0 1-- 0 0 1 20 0 0-- 0 0 50 50 50 50 -- -- -- -- 7 11 3 4-- -- -- -- 36 30 38 45 -- -- -- -- 47 44 49 45 45 49 45 48 222 22 0 2 0 1 10 11 00 1 212 11 13 1 000 00 0 0 1 38 0 1 43 43 0 0 42 0 -- 1 0 0-- -- 0 50 0 1 50 50 1 0 ?0 0-- 1 00 I-- 0 47 2 0 48 46 0 0 49 0 2-- 0 0-- -- 0 __ __ __ __ 50 12 11 50 ---- -- -- 0 1 0 0 -- -- -- -- 14 8 9 7 ---- -- -- 0 0 1 ---- -- -- 0 1 0 ---- -- -- 0 0 1 0 2 0 a slightly greater, not statistically identified, frequency of dead or moribund male mice submitted for necropsy from the 500-ppm group during the last several months of the study. The body weights of male and female mice of all exposed groups were comparable to those of the controls throughout the study (Pig. 2). There were no unusual in-life clinical ob servations during the study that were consid ered to be exposure related. In all groups of male mice the most common in-life palpable "mass" noted was in the preputial area. These were not neoplasms but rather were the result of an inflammatory process involving the penile and preputial region. Some mice recovered from the inflammatory process and no gross or microscopic lesion was found. However, swelling, ulceration, para phimosis, and urinary tract obstruction which resulted in death or a moribund condi tion were observed in some mice from all ex posure groups. The 500-ppm group of male 621 620 QUAST. CALHOUN, AND FRAUSON TABLE 4 Selected Neoplasms: Mice" Males Females Exposure concentration (ppm) 0 150 500 1500 0 150 500 1500 Number of mice examined 50 50 50 50 so 50 50 50 Lacrimal/Harderian gland(s) (number oftissues examined) Adenoma, benign, primary Cystadenoma, acini, benign, primary Cystadenoma, acini, benign, primary (two) Total number of mice with adenoma or cystadenoma Multiple organs (number of tissues examined) Leukemia--myeloid cell--primary in bone marrow Lymphosarcoma, localized or disseminated Primary in Peyer's patch Primary in kidneys Primary in liver Primary in mediastinal lymph node Primary in mesenteric lymph node Primary in subcutaneous lymph node Primary in miscellaneous lymph nodes Primary in spleen Total number of mice with lymphosarcoma ofany site Liver (number oftissues examined) Benign, primary hepatocellular neoplasm (adenoma) Malignant, primary hepatocellular neoplasm (carcinoma) Total with one or more primary hepatocellular neoplasms, benign or malignant 50 0 8 0 8 50 0 l 0 0 0 7 0 0 0 8 50 26 12 29 49 50 00 75 10 85 50 50 00 10 00 00 01 33 10 00 00 54 50 50 13c 19 10 12 22 28 50 50 50 50 00 0 0 43 1 2 00 0 0 43 1 2 50 50 50 50 10 0 0 00 0 0 00 0 0 00 0 0 02 0 1 4 12 13 12 00 0 0 01 0 1 12 1 1 5 17 14 15 50 50 50 50 16 10 9 5 12 4 1 5 24 13 10 10 50 1 6 0 7* 50 0 1 1 1 1 11 0 0 2 17 50 5 2 7 * Data are the number ofanimals with the specified observation noted on microscopic examination oftissues. b Statistically identified linear trend by Cochran-Armitage test, a = 0.05 (one-sided). c Statistically identified difference from control group by Fisher's exact probability test, a = 0.05. mice was more frequently affected than the other groups, and this was the primary reason for the slightly higher mortality in this group during the last few months of the study. The hematology and clinical chemistry values for male and female mice sacrificed at the various time intervals throughout the study were unaffected by treatment. The final body weights, absolute organ weights, and relative organ weights (g/100 g body wt) for male and female mice sacrificed at the vari ous time intervals were considered to be un affected by exposure. The gross necropsy observations for all groups of mice from the predesignated interim sacrifices or from the 2-year portion of the study did not reveal a significant adverse effect that was considered due to exposure. Al though the livers of some male mice in the 1500-ppm group at 6 months appeared grossly to have an accentuated lobular pattern, there were no findings on microscopic examination of the liver or evaluation of liver weights and clinical chemistry values that suggested an ex posure-related effect. Eight male mice and three female mice from various exposure groups died prior to scheduled sacrifices at 6, 12, and 18 months. For two of the mice an apparent cause of death was not determined. The remaining mice died with inflammation and obstruction of the genitourinary tract (4), trauma (2), liver tumor (one control and one 1500 ppm male), and bilateral adrenal gland tumor (one 150 ppm male). Histopatb rificed after to vapors ol lation did changes. N differences logically ether a dc cal plausit experience In gene statistical! vations w mice. An dilatation group. T1 sistent wi tions wh obstructi nary tra< most prt conditio exposur tion; sp urinary logical Proteus r servatic a statistic y were c< aviously volvinj tumor male i crease rnic oflive, -vice of mal p to norma formouse a the indica :ovaphore ion at sex C dsub* Ev et ai, data tistic exac mor rim; 0033 rs^ 03 622 QUAST, CALHOUN, AND FRAUSON TABLE 5 Tumor Incidence: Mice" Males Females Exposure concentration (ppm) Number of mice examined 0 50 150 500 1500 0 150 500 1500 50 50 50 50 50 50 50 Adrenals (number of tissues examined) Adenoma, cortex, benign (two) Pheochromocytoma, benign Pheochromocytoma, malignant, no metastasis Pheochromocytoma, malignant, metastasis Bone (number oftissues examined) Osteogenic sarcoma, pelvis, malignant, metastasis Bone marrow (number of tissues examined) Leukemia--myeloid cell, multiple organs, malignant Mast cell tumor, multiple organs, malignant, metastasis Brain (number oftissues examined) Granular cell tumor, meninges, benign Cecum (number of tissues examined) Lymphosarcoma, Peyer's patch, malignant, metastasis Cervix (number of tissues examined) Leiomyoma, muscularis, benign Stromal cell sarcoma, malignant, no metastasis Epididymides (number oftissues examined) Adenoma, head, benign Fibrosarcoma, tail, malignant, no metastasis Kidneys (number of tissues examined) Adenoma, cortex, benign Adenocarcinoma, cortex, malignant, no metastasis Lymphosarcoma, malignant, no metastasis Lacrimal/Harderian gland(s) (number of tissues examined) Adenoma, benign Cystadenoma, acini, benign Cystadenoma, acini, benign (two) Large intestine (number oftissues examined) Squamous cell carcinoma, anus-rectum, malignant, no metastasis Liver (number oftissues examined) Adenoma, hepatocellular, benign Adenoma, hepatocellular, benign (two) Adenoma, hepatocellular, benign (three) Hemangioma, benign Carcinoma, hepatocellular, malignant, no metastasis Carcinoma, hepatocellular, malignant no metastasis (two) Carcinoma, hepatocellular, malignant metastasis Lymphosarcoma, malignant, no metastasis Lungs (number oftissues examined) Adenoma, alveoli, benign Adenoma, alveoli, benign (two) Adenocarcinoma, alveoli, malignant, no metastasis 50 48 48 0 01 0 00 0 00 0 00 50 49 50 0 10 50 49 50 0 00 0 10 50 50 50 1 00 50 50 50 0 10 _b -- -- -- -- -- -- ____ 50 50 49 1 00 2 01 50 50 50 0 01 1 00 0 00 50 49 50 0 00 8 75 0 10 50 50 50 0 00 50 50 50 20 11 16 4 23 2 00 0 01 8 55 0 00 4 67 0 00 50 50 50 10 7 4 0 0 -0 2 20 49 0 0 0 0 50 0 50 1 0 50 0 50 0 ____ ____ 50 0 1 50 0 1 0 50 0 4 0 50 49 0 1 1 0 50 0 50 0 0 50 0 50 0 50 0 0 ____ -- _ 50 0 0 0 50 0 3 0 50 48 50 00 01 00 11 50 50 00 50 50 00 00 50 50 00 50 50 00 50 49 10 00 -- ------. -- -- ____ 50 -SO 00 00 00 50 so 00 I2 00 50 50 50 0 0 0 0 50 0 50 0 0 50 0 50 0 50 0 1 -- -- ____ 50 1 0 1 50 1 6 0 50 00 0 0 1 50 50 50 50 _50 12 8 8 4 3 41 1 1 2 01 0 0 0 00 0 0 1 73 1 1 2 0I 00 50 0 4 00 0 0 50 50 50 50 32 3 4 01 0 1 01 00 0 0 1 50 6 0 0 " Data are the number of animals with the specified observation. Only tissues with a primary tumor are tabulated; other examined tissues are not shown. * --, Not applicable. 030035 Females 150 500 1500 50 50 50 48 50 00 01 00 1 50 ,, 50 00 50 50 00 00 50 50 00 50 50 00 50 49 10 00 -- --. ---- ---- 50 50 00 00 00 _S0 0 12 00 0 50 4 5 5i 1 ( 5( ( Sf 0 50 0 1 _ -- ____ 50 I 0 1 50 1 6 0 50 00 0 50 i4 11 )0 0 I 1 50 3 2 0 1 2 00 40 01 50 50 46 I0 00 are tabulated; 1.1,1 -TR1CHLOROETHANE ONCOGENICITY STUDY TABLE 5--Continued 623 Exposure concentration (ppm) Number of mice examined 0 50 immary gland (number of tissues examined) Fibroadenoma, benign Carcinoma, malignant, no metastasis Carcinoma, malignant, no metastasis (two) Carcinoma, malignant, metastasis Mediastinal lymph node (number of tissues examined) Lymphosarcoma, multiple organs, malignant, metastasis Mesenteric lymph node (number oftissues examined) Lymphosarcoma, malignant, no metastasis Lymphosarcoma, multiple organs, malignant, metastasis Multiple organs (number of tissues examined) Lymphosarcoma, subcutaneous, malignant, no metastasis Lymphosarcoma, multiple organs, malignant, metastasis Reticulum celt sarcoma, lumbar, malignant, metastasis Oral tissues (number oftissues examined) Odontoma, benign Squamous papilloma, hard palate, benign Ovaries (number oftissues examined) Granulosa--thecal cell tumor, benign Teratoma, benign Granulosa--thecal cell tumor, malignant, metastasis Oviducts (number oftissues examined) Papillary adenoma, benign Pancreas (number oftissues examined) Adenoma, islets, benign Adenocarcinoma, ducts, malignant, metastasis Pituitary (number of tissues examined) Adenoma, anterior (pars distalis), benign Adenoma, pars intermedia, benign Carcinoma, anterior (pars distalis), malignant, no metastasis Carcinoma anterior (pars distalis), malignant, metastasis Skin and subcutis (number oftissues examined) Hemangioma, subcutaneous, benign Squamous papilloma, benign Basal cell carcinoma, malignant, no metastasis Fibrosarcoma, head, malignant, no metastasis Fibrosarcoma, subcutaneous, malignant, no metastasis Fibrosarcoma, pinna, malignant, no metastasis Fibrosarcoma, pinna, malignant, metastsis Undifferentiated sarcoma, subcutaneous, malignant, no metastasis Small intestine (number oftissues examined) Polyp, mucosa, benign Adenocarcinoma, mucosa, malignant, no metastasis Lymphosarcoma, Peyer's patch, malignant, no metastasis Spleen (number of tissues examined) Hemangioma, benign Lymphosarcoma, malignant, no metastasis 0 -- -- -- 43 0 49 0 7 50 0 0 0 14 0 0 -- -- -- -- __ -- 50 0 0 49 0 1 0 0 48 0 0 0 0 0 0 0 0 50 0 0 1 50 0 0 Males Females 150 500 1500 0 150 500 1500 50 50 50 50 50 50 50 00 0 45 44 48 ---- -- 2 3 1 ---- -- 3 3 2 ---- -- I 0 0 39 33 0I 50 45 00 33 50 50 41 35 35 33 02 0 1 49 50 49 49 00 0 0 4 12 13 12 50 50 50 50 48 2 2 0 1 40 i 50 1 10 50 10 00 0 0 00 01 0 1 00 00 1 0 16 8 8 4 11 11 00 00 0 0 20 00 0 0 -- -- 43 47 45 ---- -- 1 1 0 ---- -- 0 0 1 -- -- ---- 0 0 0 __ __ 50 50 49 ---- 000 50 48 50 50 50 50 00 02 0 0 00 00 0 0 46 47 44 43 45 48 01 1 10 5 11 00 03 0 1 0 0 0 9 1 0 49 0 0 1 50 1 50 0 1 48 5 0 00 00 49 48 00 00 00 01 00 00 00 01 00 01 0 1 49 50 49 50 10 0 1 01 00 01 0 0 00 0 0 00 0 0 00 I 0 00 1 0 0 1 49 2 0 0 0 1 0 0 00 50 50 0 0 00 00 1 0 50 50 50 50 00 2 0 00 1 0 0 50 0 0 00 50 49 01 00 00 0 0 50 50 50 50 01 0 0 00 1 1 1 50 0 1 003& 03 624 QUAST, CALHOUN, AND FRAUSON TABLE 5--Continued Males Females Exposure concentration (ppm) Number of mice examined 0 50 150 500 1500 0 150 500 1500 50 50 50 50 50 50 50 Lymphosarcoma, multiple organs, malignant, metastasis Reticulum cell sarcoma, multiple organs, malignant, metastasis Stomach (number oftissues examined) Papilloma, nonglandular mucosa, benign Polypoid adenoma, glandular mucosa, benign Squamous cell carcinoma, nonglandular mucosa, malignant, no metastasis Thyroid gland (number oftissues examined) Adenoma, follicle(s), benign Tongue (number of tissues examined) Squamous papilloma, benign Squamous cell carcinoma, malignant, no metastasis Uterus (number of tissues examined) Endometrial stromal polyp, benign Hemangioma, benign Leiomyoma, muscularis, benign Adenocarcinoma, malignant, no metastasis Adenocarcinoma, malignant, metastasis Leiomyosarcoma, muscularis, malignant, metastasis Reticulum cell sarcoma, malignant, metastasis Stromal cell sarcoma, malignant, no metastasis Stromal cell sarcoma, malignant, metastasis Vagina (number oftissues examined) Fibrosarcoma, malignant, no metastasis 0 0 50 0 0 0 48 1 50 0 0 -- -- -- -- -- -- -- -- -- -- -- -- 00 00 50 50 01 10 0 47 0 50 0 1 -- -- -- -- -- -- -- -- -- -- -- 0 49 1 50 0 0 -- -- -- -- -- -- -- -- -- -- -- 12 10 49 50 01 00 0 48 0 50 0 0 -- -- -- -- -- -- -- -- -- -- -- -- 1 49 1 50 1 0 50 2 1 2 0 0 1 0 0 I 50 1 00 00 50 50 10 00 00 50 50 10 50 50 00 01 50 49 02 10 02 10 00 00 0I 10 00 50 49 00 1 0 50 1 0 0 50 1 50 0 0 50 2 0 1 0 1 0 0 0 0 50 0 1978). There was no indication of an in* creased incidence of Iymphoreticular prolif erative processes in either Fischer 344 rats or B6C3F1 mice in this 2-year inhalation study. A previous gavage bioassay study conducted in rats also did not demonstrate an oncogenic effect with a 1,1,1-trichloroethane formula tion (NCI, 1977). The present study in rats, as well as several other chronic bioassays, does not indicate an increased incidence of immunoblastic lymphosarcoma as suggested by the inconclusive bioassay conducted by Maltoni et ah (1986). In conclusion, the repeated exposure of male and female Fischer 344 rats and B6C3F1 mice to 0, 150, 500, or 1500 ppm of a 1,1,1 -trichloroethane formulation 5 days per week for 2 years resulted in very slight mi croscopic hepatic effects in rats. The hepatic effect was discernible in the rats from the 1500-ppm group sacrificed at 6, 12, and 18 months. Due to confounding geriatric changes in aged rats, the liver effects were no longer detected at the end of the 2-year study. A slight exposure-related decrease in body weight was observed in the 1500-ppm female rats. There were no changes in the rats ex posed to 150 and 500 ppm, or in the mice at any exposure level, that were considered due to the 1,1,1 -trichloroethane formulation. Ex posure to vapors of the 1,1,1-trichloroethane formulation for 2 years did not result in an oncogenic effect in either the Fischer 344 rat or the B6C3F1 mouse. ACKNOWLEDGMENTS The authors express their appreciation to Fran Stafford for her contribution in the preparation ofthis report and to Jessie Norris for her editorial assistance. 0I 00 50 50 01 00 00 50 50 01 SO 50 00 10 49 50 22 00 2I 00 0] 00 10 00 00 49 50 0 from the !, and 18 geriatric were no ^ar study, in body m female - rats exe mice at lered due ition. 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