Six month inhalation studies of pirbuterol acetate aerosol in the beagle dog and squirrel monkey

FUNDAMENTAL A N D APPLIED T O X I C O L O G Y 1:426-431 (1981)

Six Month Inhalation Studies of Pirbuterol Acetate Aerosol in the Beagle Dog and Squirrel Monkey H.V. LEVINSKY,AC. BRECKENRIDGE, BR. LOUGH, BD.A. GONCI, AH.M. MclLHENNY Aand S. QURESHI B ADepartment of Drug Safety Evaluation, Pfizer Central Research, Eastern Point Road, Groton, Connecticut 06340; BDepartment of Inhalation Toxicology, Bio-Research Laboratories, Senneville, Quebec, Canada

ABSTRACT Six Month Inhalation Studies of Pirbuterol Acetate Aerosol in the Beagle Dog and Squirrel Monkey. Levinsky, H.V., Breckenridge, C., Lough, R., Gonci, D.A., McIIhenny, H.M. and Qureshi, S. (1981). Fundam. Appl. Toxicol. 1:426-431. Groups of Beagle dogs and Squirrel monkeys were exposed to ael:osols of pirbuterol acetate, a new bronchodilator at dose~ of 0, 200, 400 and 800 /~g of pirbuterol/kg body weight daily for 6 months. Each group consisted of 4 dogs or 6 imonkeys per sex. Dogs were exposed by face mask and monkeys were exposed by head only in a manifold. Plasma di'ug concentrations indicated that expected levels of drug exposure were achieved in both species. No significant alterations were revealed in physical appearance and body weights, hematological and blood biochemical analyses, urinalyses, ophthalmoscopy, assessment of cardiovascular status, pulmonary function or gross and histopathology. Examination of the respiratory tract showed no morphological changes that could be attributed to pirbuterol acetate inhalation. INTRODUCTION Pirbuterol (2 hydroxy-methyl-3-hydroxy-6 [ 1-hydroxy-2-tertbutylaminoethyl] pyridine) is a new bronchodilator, which is orally active in animals and effective in human patients with reversible chronic bronchospastic disease (Constantine eta/., 1979; Moore et al., 1978; Steen, 1974; Paterson et al., 1 977; Burki and Diamond, 1978). Because this selective beta-2 agonist bronchodilator will also be administered in aerosol form, animal studies using Beagle dogs and Squirrel monkeys were performed to establish the safety of this compound by the inhalation route. METHODS Materials Metered aerosol cannisters containing pirbuterol acetate or placebo aerosols were used with clinical mouthpieces attached in both studies. Each actuation of an active aerosol cannister supplied 200 micrograms of pirbuterol. The placebo aerosol contained equivalent amounts of excipients as the active aerosol but no pirbuterol. Animals

Dogs Purebred Beagle dogs were obtained from Ridglan Research Farms Inc., Mt. Horeb, Wisconsin. They were approximately 9 months of age and weighed 7.2 to 10.4 kg at initiation of treatment. Before receipt, all dogs had been immunized

against rabies, distemper and infectious canine hepatitis and had been subjected to an extensive regimen of anthelmintic therapy. During the five weeks prior to the first treatment they were acclimated to the laboratory setting, the face mask, and the restraining sling used during test substance administration. All dogs were housed individually in stainless steel cages equipped with an automatic watering system and a raised sleeping platform. The average room temperature and relative humidity during the study were 74 ~ F and 54% respectively. All dogs were fed 450 g of a 50:50 mixture of Romar 90 (J.E. Mondou, 90 Jean Talon East, Montreal, Quebec) and Purina Dog Chow~ once daily. Squirrel monkeys The Squirrel monkeys were obtained from Hazelton Prime Labs (Monmouth County Airport, Farmingdale, New Jersey). All animals had been tuberculin tested and treated for parasites and they were acclimated to the laboratory setting before the first inhalation exposure. At the initiation of treatment male monkeys weighed between 600 and 800 g and female weights ranged from 500 to 700 g. The animals were housed in pairs (1 female and 1 male together) in conventional stainless steel cages. They had ad libitum access to water and Purina Monkey Chow@ which was provided fresh daily. Experimental design Four groups of four dogs or six Squirrel monkeys per sex were assigned to treatments as follows: Group I served as controls and was exposed to placebo aerosols only. Groups II, III and IV were exposed to pirbuterol acetate aerosols at dose levels of 200, 400 and 800 micrograms of pirbuterol/kg body weight/day. Dogs received half of these doses twice daily (morning and afternoon), while Squirrel monkeys were exposed to the full dose in one single daily exposure period. Dose level selection The high dose of 800/~g pirbuterol/kg body weight is equivalent to 50-100 times the anticipated human therapeutic dose. This dose level was therefore judged to be sufficient to investigate effects associated with inhalation exposure to this compound. The total exposure time needed to achieve this dose in dogs (10-15 minutes twice daily) and Squirrel monkeys (30 minutes) is reasonable without causing undue stress in either species. Systemic effects of pirbuterol administration have been sufficiently investigated by other routes including intravenous administration. (Pfizer Inc., unpublished data).

Copyright 1981,Societyof Toxicology

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PIRBUTEROL INHALATION IN DOGS AND MONKEYS

Description o f exposure

equipment

Dogs A special face mask has recently been developed which permits the administration of inhalant drugs as metered aerosol formulations to dogs per os (Krupa et al., 1977). This mask was used in the present study and consisted of a plastic cylinder fitted with one way valves in the closed end to allow entrance and exit of air. A receptacle for a metered aerosol container was mounted perpendicularly On the cylinder. The mask was fitted over the dog's muzzle in such a way that the nose was inside the cylinder. The aerosol administration was then timed to coincide with the opening and closing of the one-way valves and the respiratory excursions of the animal's body. Preliminary experimentation had shown that one actuation through the dog face mask delivered 100 micrograms pirbuterol to the face of a dog. Forty actuations would be required to obtain a 4,000 #g dose. Squirrel monkeys A head only exposure manifold was used which allowed simultaneous exposure of as many as 12 Squirrel monkeys. Two rows of six monkey boxes faced a common exposure manifold. The m'.onkeys were restrained by loose fitting neck yokes w h i c h allowed complete rotations of the animals with only the heads exposed to the aerosol in the manifold. A Roote's low pressure vacuum pump was used to exhaust air from the exposure manifold. This exhausted air was drawn through a Gelman flow meter and filter. At the intake side of the manifold a cannister rack accomodated five cannisters with clinical mouthpieces. The actuation of individual cannisters was accomplished by means of a series of five pneumatic cylinders which were mounted above the aerosol cannisters. The plate holding the cannister rack could be rotated through 180 ~ allowing inversion of the cartridges for agitation. On each treatment day, animals belonging to the same group were placed in the animal restraint exposure manifold. The position of each monkey w i t h i n the manifold was systematically changed over a 12 day period such that a given monkey occupied all 12 of the possible locations once during a 12 day rotation period. (Only eight locations were used after the three month interim sacrifice). After the animals were placed in the exposure manifold, they were taken to the inhalation room where a plexiglass top covering the manifold was taped in place and a 3 inch diameter exhaust line was connected to the exhaust port of the exposure manifold. Air flow through this line was .calibrated and maintained at a rate of 30 liters per minute. A series of 5 placebo control or pirbuterol acetate cannisters (depending on which group of animals was being housed in the exposure manifold) was selected and primed by shaking and actuation on five successive occasions. These cannisters were then individually weighed and inserted into the clinical mouthpieces. The cannister rack, mounted on the rotating platform above the pneumatic clyinders, was then rapidly rotated through 180 ~ repeatedly during a 20-second period. Preliminary validation experiments had shown that this procedure guaranteed a uniform suspension in the cannisters and thereby provided a uniform delivery of pirbuterol acetate during each actuation. Following the initial 20 seconds of shaking action the pneumatic cylinders were depressed sequentially by electromeFundamental and Applied Toxicology

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chanical relays to actuate one cannister every 4 seconds. During each 4 second interval the cannister rack was again inverted through 180 ~ to ensure uniform mixing w i t h i n each cannister. After completion of a full cycle consisting of actuation of all 5 cannisters over a 20 second period the cycle began again starting with the first cannister. The 3 dose levels of pirbuterol acetate were achieved by varying the time that the 3 groups of monkeys were exposed to these conditions. During calibration work prior to the study, as well as at weekly intervals during the study, air samples were withdrawn from the exposure manifold and a nalyzed by a UV spectrophotometry method for the presence of pirbuterol. The air flow for these collections was 0,5 liters per minute simulating the average minute volume of a Squirrel monkey. Each air sample was collected for the entire exposure duration of the particular group of animals. The values obtained therefore approximated the amount of pirbuterol that could have been inhaled by a monkey located at the position of the sampling tube and breathing n o r m a l l y w i t h a minute volume of O.5 liters per minute. Based on actual mean minute volumes, body weights and amounts of pirbuterol recovered in the air samples, the exposure durations for the 3 treatment groups were adjusted periodically and ranged from 6 to 28 minutes. Particle size distribution o f aerosols Respirable drug delivery is defined here as that percentage of dose constituting particle sizes of less than 4 p. and was determined using a 6-stage cascade impactor (Model DCI-6, Delron Research Products Co., Powell, Ohio). Quantitation of particle size distribution was done chemically using spectrephotometric analysis for pirbuterol. Results indicated that about 54% of pirbuterol delivered through the dog face mask was less than 4/= in particle size as compared to 38% of the dose delivered through the clinical mouthpiece to be used in man and also used in the Squirrel monkey study. Clinical pathology and clinical evaluation Blood for hematology and serum chemistry analysis was collected from all dogs twice before to first exposure and again after two weeks, and one, two, four, and six months of compound administration. Blood was collected from Squirrel monkeys twice before first .exposure and at one, three and six months. For the hemogram standard methods were employed for determination of total and differential leukocyte count, red blood cell count, hemoglobin, packed cell volume, prothrombin time, clotting time, reticulocyte count, platelet count and erythrocyte sedimentation rate. Mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were calculated. Serum chemistry examinations included the following parameters: blood urea nitrogen, creatinine, glutamic pyruvic transaminase (SGPT), oxalacetic transaminase (SGOT), alkaline phosphatase, fasting glucose, total bilirubin, sodium, potassium, calcium, total protein, albumin and albumin/globulin ratio. In dogs direct bilirubin, lactic dehydrogenase (LDH) and uric acid values were also determined. Urine samples were obtained from all animals at the same frequency as the blood samples. Standard methods were used for assessment of volume, color, specific gravity, pH, ketones, bile pigments, protein, glucose, albumin and occult blood. In addition a microscopic examination of the urinary sediment was carried out. 427

LEVINSKY, BRECKENRIDGE, LOUGH, GONCI, MclLHENNY AND QURESHI

Ophthalmoscopic examinations with an indirect ophthalmoscope and a Haag-Streit slit lamp were conducted on all animals once before the onset of treatment and after 1,3 and 6 months of inhalation exposure. Electrocardiograms were obtained from all animals twice prior to first exposure and after 1, 3 and 6 months of daily exposure. The three standard leads (I, II and III) were used and the PR, QRS and QT intervals were measured on the lead II tracings. Indiredt measurements of systolic blood pressure (caudal artery) were conducted simultaneously. Pulmonary function studies were conducted on all animals twice before first exposure and after 1 (dogs only), 3 and 6 months of daily inhalation exposure. The assessment of several measurements relating to pulmonary function was achieved by the characterization of the distribution of inspired air in accordance w i t h the method described by Alarie et aL (1971) suitably modified for application to the dog or the Squirrel monkey. The respiratory rate (RR) and the tidal volume (Vt) were determined as part of this procedure. From these data t h e m i n u t e v o l u m e (V,~) was calculated (Vm = RR X Vt). In a d d i t i o n t h e w a s h i n g o u t of n i t r o g e n a s e a c h a n i m a l b r e a t h e d pure oxygen was studied and was expressed as the time taken to r e a c h 1% n i t r o g e n , ~ ( 1 % N2), a s t h e n u m b e r of b r e a t h s t a k e n d u r i n g t h a t t i m e (N i % N2) a n d a s c u m u l a t i v e tidal v o l u m e r e q u i r e d to r e a c h 1 % n i t r o g e n (CVt 1% N2).

Pathology After three months of pirbuterol acetate or placebo aerosol inhalation 2 monkeys/sex/group were killed by intravenous administration of pentobarbital sodium. The remaining 4 monkeys/sex/group and all dogs were similarly killed following completion of a six months exposure period. Gross examination of major organs and systems was performed at necropsy and heart, liver, lungs, kidneys, adrenals, gonads and spleen were weighed. A general histopathological evaluation of tissue sections stained with hematoxylin and eosin was performed. In the examination of the heart, sections were recovered from the walls of both ventricles and both atria, from the interventricular septum and from the papillary muscles. A portion of the formalin fixed liver was also deep frozen and stained with Oil Red O. Specialized histopathology of the respiratory tract was carried out on each animal in this study. During necropsy the mucosae of the nares and the paranasal sinuses with vestibulure, adjacent bone and nasal septum were processed according to the following special technique: Following the fixation of the base of the skull and maxilla and adjacent facial bones for one week in buffered formalin and decalcification of the tissues for five days in formic acid/sodium citrate, paraffin embedded sections 7 microns thick were cut from two blocks taken from the bridge of the nose. The resulting slides were stained with hematoxylin and eosin. In addition, portions of the larynx, trachea, major bronchi and of each lung lobe were placed in buffered 10% formalin for fixation and preservation. These sections were also stained with hematoxylin and eosin and subjected to histological examination. Bioavailability o f pirbuterol acetate Blood samples were obtained from all animals to determine the level of drug exposure. Sampling times were chosen based on extensive preliminary pharmacokinetic experimentation to coincide with maximal plasma levels in both species. Samples (2 mL, containing sodium heparin) were collected from all 428

TABLE 1 M e a n E s t i m a t e d E x p o s u r e to P i r b u t e r o l (/~glkglday) of S q u i r r e l M o n k e y s ^ Targeted Dose Levels 200 400 800

Mean Levels Achieved B Weeks I to 14 Weeks 15 to 26 Males 160 420 1130

Females 190 410 970

Males 220 580 890

Females 260 470 720

^There were 6 male and 6 female squirrel monkeys/dose level during weeks 1 to 14 and 4 monkeys/sex/dose thereafter. BChamber concentrations and group mean body weights determined weekly were used to calculate estimated exposure values based on a n average minute volume of 500 mL. Means for weeks 1 to 14 and 15 to 26 were then calculated and adjusted based on the actual group mean minute volumes determined 3 times d uring the study. Means were then rounded to the nearest 10,ug.

Squirrel monkeys 1-10 minutes following termination of exposure to the aerosol on four occasions: days 2 or 3, 25 or 26, 109 or 110 and 176 or 177. Blood samples(10 mL) were obtained from dogs before and 1 hr following termination of exposure to the morning dose on the first day and at 1 month and on the final day of the study. Plasma was maintained in a frozen condition until drug analysis with the standard gas chromatography spectrometry assay specific for pirbuterol (Falkner and Mcllhenny, 1976).

R ES UL TS Calculation of exposure

Dogs Preliminary experimentation had shown that one actuation through the dog face mask delivered 1(30 micrograms pirbuterol to the face of the dog. The number of actuations was adjusted daily to each dog's body weight. Squirrel monkeys During exposure validation experiments before the start of the monkey study average minute volumes of 500 mL and mean body weights of 800 g were used to select exposure durations necessary to achieve the targeted dose levels of pirbuterol. Table 1 summarizes the mean exposure data obtained during the study based on the amounts of pirbuterol recovered in the weekly manifold air samples, These means were corrected for the group mean body weights of the monkeys in the study and their actual minute volumes.

Clinical observations Both dogs and Squirrel monkeys quickly adapted to the manipulations associated w i t h the daily exposure routine. There was no indication of irritation to the respiratory tract during or immediately following inhalation exposure in dogs. In Squirrel monkeys pirbuterol acetate inhalation was associated with salivation and foaming at the mouth as well as occasional emesis during drug exposure. The incidence of both salivation and emesis was low. The intensity of the response was not related to dose and appeared to depend to some extent upon the disposition and perhaps excitability of the individual monkey. Male monkeys in all groups tended to develop open wounds or inflamed areas under one or both eyes, These sores are routinely observed in Squirrel monkeys and have been desFundam. AppL Toxlcol. (!)

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PIRBUTEROL INHALATION IN DOGS AND MONKEYS

cribed as indicating abscesses of canine teeth with involvement of the maxillary sinus (Ulrich et aL, 1977). There was no effect of treatment on body weights, food consumption or ophthalmological findings in either species. Some elevations in serum transaminase levels were linked to parasite infestation which is a common finding in Squirrel monkeys (Duncan and Prasse, 1977). There were, however, no changes in any serum chemistry, hematology or urinalysis para meters in either the monkey or the dog that could be linked to the inhalation treatment. Examination of bone marrow obtained terminally from dogs (sternum) and monkeys (femur) revealed no evidence of treatment related changes in the cell-fat or myeloiderythroid ratios.

Cardiovascular and pulmonary studies There was no deleterious effect of pirbuterol acetate or placebo aerosol inhalation on systolic blood pressure, ECG tracings or pulmonary function in either species. Gross Pathology : The most consistent gross observation in dogs was related to lung worm infestation w h i c h was diagnosed equally in treatment and control animals. In monkeys round or tape worms were identified in several animals and a few males presented canine tooth abscesses. None of these or other incidental findings was apparently associated with pirbuterol acetate exposure and there was no effect on organ weights.

Dogs Pulmonary alterations attributed to lung worm infestation (Filaroides hirth/~ were frequently noted substantiating the gross pathological observations. The specialized histopathological examination of other components of the respiratory system (nasal cavities, larynx, trachea, and major bronchi) did not reveal pathological findings with the exception of a mild chronic tracheitis with focal collections of lymphocytes and plasma cells in three placebo control and 4 pirbuterol acetate dogs. All of these changes were mild in nature and never extensive enough to have marked a possible effect of pirbuterol inhalation on the respiratory tract. Squirrel monkeys Chronic mild focal to multifocal hepatitis was observed in 13 of 16 animals killed after three months of treatment and in 22 TABLE 2 P l a s m a C o n c e n t r a t i o n s (Means +S.D.) of P i r b u t e r o l in D o g s F o l l o w i n g I n h a l a t i o n of the Pirbuterol Acetate Aerosol Formulation ng Pirbuterol/mL plasma 1 hr post first divided dose

Dose

Day 1

Day 31

Day 180

400

12.3(8) A +5.8

16.6(8) +6.8

17.0(7) +4.9

200

6.9(8) •

8.6(8) +4.2

6.4(8) +1.2

100

Detailed histopathological examination of the respiratory tract revealed minor changes attributable to intercurrent bacterial a n d / o r parasitic infections. Pulmonary perivasculitis and peribronchiolitis were revealed in 13 of 16 animals killed after three months of treatment and in 17 of 32 monkeys killed at the end of six months of treatment. In all cases only small portions of the pulmonary tissue were involved with the remaining parenchyma showing no evidence of the pathological alterations. Other respiratory tract lesions included a mild chronic laryngitis, mild rhinitis, and small abscesses of the nasal bones of some animals w i t h o u t any correlations w i t h treatment. Mild chronic interstitial myocarditis with random focal collections of lymphocytes was occasionally seen. This Condition has been described in nonhuman primates and its origin is unclear (McNulty and Malinow, 1972).

Bioavailability o f pirbuterol acetate

Dogs

Histopathology

/~glkg b.i.d.

of 32 monkeys spcrificed at the end of six months of treatment. Biliary hyperplasia, periportal fibrosis and pericholangitis were also observed in several animals, especially those,that had trematodes. These inflammatory processes involved only small focal areas in the sections examined and were never extensive enough to have caused functional disturbances. Two experimental animals revealed hepatic alterations characterized by hyalin and hydropic (vacuolar) degeneration. In the absence of a regular pattern and dose response the significance of these two observations could not be determined.

3.1(S)

4.O(S)

S.6(7)

•

•

•

Aparentheses contain numbers of animals used in calculation of means. Fundamental and Applied Toxicology

(1) 11-12/81

Pirbuterol plasma levels were detected in all animals on the first day of the study and concentrations exhibited a near proportional relationship with the dose (Table 2). No sex related differences were apparent. At the one-month interval and at termination of the study, similar mean drug concentrations were maintained at each dose level and in both sexes, although the variability w i t h i n groups sometimes exceeded 5 fold. There was no evidence for a decline or a n accumulation of plasma pirbuterol concentrations during the course of the study.

Monkeys Pirbuterol plasma concentrations were detected in all monkeys of the study (Table 3). In terms of actual dose levels delivered during the first month of the study, mean plasma concentrations exhibited a near proportional relationship with the dose. The determination of individual plasma drug concentrations during the fourth and final months indicated that bioavailability of pirbuterol from the aerosol was maintained in all monkeys studied for the complete six month period. The apparent decrease in mean plasma levels during the fourth and sixth months was consistent with somewhat lower chamber air concentrations during those two particular weeks. In general, plasma concentrations were relatively uniformly achieved with an inter-animal variability of no more than 3fold w i t h i n each dose level, although levels occasionally varied more than 5-fold. The variation was apparently not related to the several-minute differences between animals in blood sampling times after removal from the chamber, or to the chamber position of the monkeys; it is more likely a reflection of individual differences in kinetics of drug disposition, as observed in other species with pirbuterol (Mcllhenny, unpublished data). 429

LEVINSKY, BRECKENRIDGE, LOUGH, GONCI, MclLHENNY AND QURESHI TABLE 3 Plasma Concentrations (Means +_S.D.)'of Pirbuterol in Squirrel Monkeys Following Inhalatiofi of the Pirbuterol Acetate Aerosol Formulation Males Dose

ng Pirbuterol/mL plasma, post-exposure period a

/~g/kg

Day 2

Day 25

Day 110

Day 176

800

350(6)8 ___208

373(6) -t-114

197(4) ___62

129(4) -t-45

400

133(6) •

155(6) __.61

115(3) +85

200

54.5(5) •

58.0(6) ___6

44.7(4) •

61.5(4) -t-18 23.0(4) •

Females ng Pirbuterol/mL plasma, post-exposure period Dose

Day 3

Day 26

Day 109

Day 177

800

434(6) • .

477(6) +179

134(4) +39

110(4) +14

400

136(6) i -1-45 ~

127(6) -I-35

200

72.6(5) ___46 9

63.5(5) +4

80.7(4) •

53.5(4) •

71.8(3) ___46

46.6(3) ___14

^Samples were obtained within 10 minutes after removal of monkeys from the exposure manifold. BParentheses contain numbers of animals used to calculate means. DISCUSSION The inhalation exposure systems for dogs and monkeys were carefully validated prior to the beginning of these studies and were thoroughly monitored during the experimental period. These studies were designed to simulate human exposure conditions and did so very well. Aerosol generation occurred through the clinical mouthpiece that will be used in man and was therefore equivalent. Aerosols generated for either species contained at least the same percentage of respirable particles of drug as obtained in clinical use. Previous animal studies have shown that pirbuterol is deposited at high concentrations in the larynx, trachea and in all lobes of the lung following inhalation in a chamber (rats) or with the face mask device (dogs). In dogs, respiratory tissue concentrations exceeded by at least 100-fold concentrations of pirbuterol in the plasma. Separate plasma pharmacokinetic studies in the anesthetized cynomolgus monkey indicated that pirbuterol is rapidly absorbed from the lung following administration of the aerosol through an endotracheal tube (Pfizer, unpublished data). Based on this extensive pretrial experimentation, it was concluded that expression of the dose as the amount presented to the face of the dog (nasal and buccal cavities combined) was a realistic measure of exposure when compared to the human situation and that monkeys were also adequately exposed. Data obtained during the study indicated that all animals were actually exposed to pirbuterol acetate at the three intended dose levels of 200, 400 and 800/zg/kg/day. These levels correspond to 50-100 times the anticipated human therapeutic dose. Exposure and absorption was also confirmed by plasma levels of pirbuterol at several time points during the studies. Although plasma samples were never intended to 430

quantitate exposure or absorption the levels obtained were well w i t h i n the ranges expected based on pretrial experimentation and therefore, validated the experimental design and justified our dosage calculations. Pirbuterol acetate inhalation in the monkey was associated with some salivation and foaming at the mouth as well as occasional emesis during drug exposure. The incidence was not dose related and the intensity of the response appeared to depend on the individual disposition of the animals. Otherwise exposure to pirbuterol acetate or placebo aerosols for 6 months was well tolerated by Beagle dogs and Squirrel monkeys. None of the conventional parameters used to assess toxicity revealed any treatment related changes. The cardiovascular system and pulmonary function were not impaired. Hepatic lesions observed in Squirrel monkeys were considered to represent residual alterations produced as a result of spontaneous disease or their sequelae and not by administration of the test or placebo aerosols. (McClure et aL, 1978; Jubb and Kennedy, 1970). Complete gross and histopathological investigation of all animals which included a special examination of the respiratory tract revealed no morphological changes that could be attributed to the inhalation of pirbuterol acetate. It was thus Concluded that daily inhalation exposure of Beagle dogs and Squirrel monkeys to aerosols of pirbuterol acetate at dose levels of 200, 400 and 800/~g pirbuterol/kg was well tolerated and produced no signs of toxicity. A CKNO WL ED G E M E N TS Mr. S.A. DiRoma's contribution to the monkey validation study is greately appreciated. The technical assistance of Ms. Vivian Vaupshas and Mr. Bruce Hollomby is gratefully acknowledged. REFERENCES Alarie, Y., Krumm, H., Jennings, H. and Haddock, R. (1971). Distribution of Ventilation in Cynomolgus Monkeys (Measurement with real-timed digital computerization). Arch. Environ. Health 22:633-642. Burki, N.K. and Diamond, L. (1978). Long-term Oral Bronchodilator Therapy of Asthma With Pirbuterol. Clin. PharmacoL Ther. 24(1):84-89. Constantine, J.W., MclIhenny, H.M. and Moore, P.F. (1979). Pharmacokinetics and Cardiopulmonary Effects in Dogs of Sublingual Pirbuterol, A New Bronchodilator. J. PharmacoL Exp. Ther. 208(3):371-376. Duncan, J.R. and Prasse, K.W. (1971). Veterinary Laboratory Medicine, Clinical Pathology, p. 77, Iowa State University Press. Falkner, F.F. and Mcllhenny, H.M. (1976). Selected Ion Monitoring Assay for the Bronchodilator Pirbuterol. Biomedical3fass Spectrometry 3:207-211. Jubb, K.V.F. and Kennedy, P.C. (1970). Pathology of Domestic Animals, Volume 2, p. 25, Academic Press, New York. Krupa, V., Owston, E. and lndacochea-Redmond, N. (1977). The Design and Application of a Mask for Oral Inhalation of Aerosols to Conscious Beagles. First Int. Congress on Toxicology, Toronto. McClure, H.M., Chapman, W.L., Hooper, B.E., Smith, F.G. and Fletcher, O.I. (1978). The Digestive System in Pathology of Laboratory Anhnals, Vol. 1, ed. Benirschke, Garner and lones, pp. 243-244, Springer-Verlag, New York. Fundam. Appl. ToxicoL (1)

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PIRBUTEROLINHALATION IN DOGS AND MONKEYS McNulty, W.P. and Malinow, M.R. (1972). The Cardiovascular System. In: Pathology of Simian Primates, PArt: General Pathology ed. Fiennes, pp. 756-808, Karger, New York. Moore, P.F., Constantine, J.W. and Barth, W.E. (1978). Pirbuterol, A Selective Beta2 Adrenergic Bronchodilator. J. PharmacoL Exp. Ther. 207(2):410-418. Paterson, I.C., Willey, R.F. and Grant, I.W.B. (1977). Effects of

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Prolonged Administration of Pirbuterol by Mouth in Chronic Asthma. Br. J. Clin. PharmacoL 4(3):376-377. Steen, S.N. (1974). Effect of Pirbuterol on Specific Airway Conductance in Patients with Chronic Bronchospastic Disease; IRCS Libr. Compend. 2(2):1088. Ulrich, C.E., Monaco, R.A. and Messer, S.M. (1977). Baseline Pulmonary Physiologic Values for the Squirrel Monkey (Saimiri sciureus). Lab. Anim. Sci. 27:1024-1027.

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