Arch. Toxicol. 34, 1 - - 8 (1975) (~ by Springer-Verlag t975
Original Investigations New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats*, * * G. M u a c e v i c C. H. Boehringer Sohn, Abt. Pharmakologie, Ingelheim/Rhein Received :February 7, 1975
Abstract. A new apparatus and method for the toxicological investigation of metered aerosols in rats, which is also suitable for tests in other small laboratory animals, is described. I t permits: 1. simultaneous treatment of 5 or more animals, 2. administration of metered aerosol doses to individual animals, 3. ventilation of the cages, 4. mechanical tilting of the metered aerosol packs to ensure thorough mixing of the content, and 5. continuous automatic tilting, administration and ventilation. Four metered aerosols, with and without ipratropium bromide, were investigated under different ventilation conditions. Blood gases and fluorinated chlorohydrocarbons (abbreviation: fluorocarbons) in the arterial blood were also determined. I n tests with spontaneous ventilation of the animal chambers without positive pressure, significant acidosis and hypoxia occurred after 40 puffs of metered aerosol. Where ventilation of the chambers was insufficient, the fluorocarbons led to dose-dependent toxic and lethal effects. The substance and the additives contained in the metered aerosol did not interfere with these effects. After active ventilation with 0.5 arm no symptoms of acidosis or hypoxia were observed. Up to 160 puffs of metered aerosol, no indications of toxic effects were established in the rats. Half-life of the fluorocarbons in the arterial blood after one puff of metered aerosol was 69 to S0 sec for fluorocarbon 11 and 57 to 67 see for fluorocarbon 12. Key word~: l~etered Aerosols - - Ipratropium Bromide - - New Apparatus - - Arterial Blood Gases - - l~luorocarbons in Arterial Blood. Zusammen/assung. Eine neue Apparatur und !~Iethode fiir die toxikologische Priifung yon Dosier-Aerosolen an Ratten, welehe im Prinzip auch fiir andere kleine Labortiere geeignet ist, wird beschrieben. Sie ermSglicht: 1. gleichzeitige Behandlung yon 5 oder mehr Tieren, 2. individuelle Verabreichung der Dosier-Aerosole, 3. Beliiftung der Tierk~fige, 4. Schiitteln tier Dosier-Aerosolbeh~ilter zur Durehmisehung des Inhalts, und 5. automatisehen Ablauf yon Mischen, Dosieren und Beliiftung. u Dosier-Aerosole mit und ohne Ipratropiumbromid wurden unter untersehiedlichen Belfiftungsbedingungen untersucht. Zus~tzlich wurden die Blutgase und die Treibgase im arteriellen Blur bestimmt. I n Vcrsuchen mit spontaner Beliiftung der Zwangsk~fige ohne positiven Druck ergab sich nach 40 Hfiben des Dosier-Aerosols eine signifikante Acidose und Hypoxie. Bei ungeniigender Beliiftung zeigten die Treibgase dosisabh~ngig eine toxische und letale Wirkung. Der Wirkstoff und die Zusatzstoffe des Dosier-Aerosols hatten darauf keinen EinflulL Bei aktiver Belfiftung mit 0,5 atii kam es nicht zu einer Acidose und Hypoxie, und die Ratten vertrugen bis t60 Hfibe des Dosier-Aerosols symptomfrei. Die Halbwertszeit der * Dedicated to Prof. Dr. Karl Zeile on the occasion of his 70th birthday. ** Presented in brief at the 33rd International Congress of the Pharmaceutical Sciences in 1973 in Stockholm.
2
G. Muacevic
Treibgase im arteriellen Blur nach einem Hub ])osier-Aerosol be~rug ffir Treibgas 7t/69 bis 80 sec, fiir Treibgas 12/57 bis 67 sec. Schli2sselw6rter: Dosier-Aerosole - - Ipratropiumbromid - - Neue ApparaSur- Arterielle Blutgase - - Fluorokohlenwasserstoffe im arteriellen Blur. The development of the metered aerosol has increased the importance of local aerosol administration of active substances in the upper and lower respiratory tracts accompanied in some cases by local absorption. Metered aerosols have been in use in medicine since 1956. E v e r y metered aerosol contains not only the active substance, but also a propellant, those most commonly used being compressed gases (nitrogen, carbon dioxide, nitrous oxide) or liquid gases (usually fluorinated ehlorohydrocarbons). Aerosol propellants have to be screened for their physiological effect and dermal and mucosal tolerance. Most of the fluorinated chlorohydrocarbon propellants (abbreviation: fluorocarbons) on the market have a maximal allowable concentration (MAC or TLV, in Germany MAK) of at least 500 p p m in air. Fluorocarbons l i (trichlorofluormethane), 12 (dichlorofiuoromethane) and l l 4 (i,2-dichlorotetrafluoroethane) have an MAC of 1000ppm, and are thus assigned to toxicity categories 5a or 6 (categories defined b y H. Kfibler, 1964). Categories I to 4 are not permitted for use in metered aerosols. Various apparatuses have been described for the laboratory investigation of atomized solutions and the investigation of cigarette smoke (Fultyn, 1961; H a n n a et al., 1970; Hinners, i966; HSbel, i972; Scholz, 1962), but a technique for testing regular metered aerosols on individual animals has hitherto not been published.
Description of the Apparatus Our apparatus consists of 5 glass immobilization tubes, tapered at one end to fit the nose and head of a rat. The size of the tubes is adequate for rats weighing approximately 200 g. The tail-end of the tube is closed with a sliding plastic bung which can be adjusted to the length of each individual rat. The metered aerosol test packs, of the type used in human medicine, are fixed at the end of each tube, 80 m m from the nose of the rat. A depressor bar activates all the packs simultaneously, and a tilting device ensures t h a t the contents of the aerosol packs are thoroughly mixed before each puff. Fresh air can be circulated at regular intervals to prevent hypoxia. This apparatus allows toxicological studies with metered aerosols to be carried out in groups of animals with a minimum of time and manpower. Each of the processes described: mixing, administration, and circulation of fresh air, can be programmed on the electronic control device to be carried out automatically in sequence. Air can be supplied as desired, either after each puff or after anything between 2 and 9 puffs, thus permitting the investigation of the toxicity of individual propellants under various ventilation conditions. The inhalation system is shown in diagrams I and 2. Five glass animal chambers (Figs. l . l and 2.2) are mounted on a plastic base (Fig. IA). Push-in sockets at the nose-end and clips at the taft-end of the base facilitate the removal, cleaning, and replacement of the tubes (1.3). A tumbler device (2.12), consisting of a base for the aerosol pack adapters and powered b y a pneumatic lifting cylinder (t.15),
New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats
3
--15
"=IP~ ~=,~,, -~..'.
I
n~ l_,_.'J_ _J-J_ . . . . . . . . I I
,_'.J_ _L.'-J
I
i I
!;! i ]
- I 1 I
j t
"1 ....
"
t I
~13
II ,j ,.~
,,, l l
I t
2
il]l~ I
' t
FI'I
3 l
10
2
7~
1
11
14
2
Figs. i and 2. 1 = base; 2 = glass immobilisation tube; 3 = raw of clips; d to 6 = solenoid valve; 7 = angle lever; 8 =adjusr ram; 9 = metered aerosol pack; 10 = lifting cylinder; 11 = metered aerosol adapter; 12 = tumbler device; 13 = tube for air supply; 14 = central valve; 15 = lifting cylinder t i l t s t h r o u g h 90 ~ a n d t h u s insures c o m p l e t e m i x i n g o f t h e c o n t e n t s of t h e aerosol packs. T h e lifting c y l i n d e r is r e g u l a t e d b y a solenoid v a l v e (1.4). M e t e r e d aerosol dosing is s y n c h r o n i z e d b y depression o f t h e aerosol p a c k s (2.9) in p o s i t i o n in t h e a d a p t e r s b y m e a n s of a n angle lever (2.7) f i t t e d w i t h a d j u s t a b l e r a m s (2.8). This causes a c t i v a t i o n o f t h e valves, a n d an aerosol dose is p r o j e c t e d t h r o u g h e a c h a d a p t e r ( 2 . i i ) a n d i n t o t h e n o s e - e n d o f t h e i m m o b i l i z a t i o n t u b e (2.2). This s y s t e m is also p o w e r e d b y a p n e u m a t i c lifting c y l i n d e r (2.10), r e g u l a t e d b y a
4
G. Muacevic
solenoid v a l v e (1.5 a n d 2.5). A c t i v e v e n t i l a t i o n b y circulation of fresh air t h r o u g h t h e a d a p t e r a n d nose-end of t h e tu b e s is again r e g u l a t e d by a solenoid v a l v e (1.6). T h e t i l t i n g d e v i c e a n d t h e a c t i v a t o r each r e q u ir e 4 a r m positive pressure, an d t h e v e n t i l a t i o n s y s t e m 0.5 a t m p o s i t i v e pressure. T h e s y s t e m ca n be controlled m a n u a l I y or a u t o m a t i c a l l y . Our electronic c ont ro l d e v i c e consists o f a series of t i m e - s e q u e n c e connectors. T h e d u r a t i o n of b o t h t h e i n t e r v a l b e t w e e n t h e puffs, a n d t h e a c t i v e v e n t i l a t i o n p r o c e d u r e can be r e g u l a t e d . T h e n u m b e r o f puffs to be a d m i n i s t e r e d prior to an a c t i v e v e n t i l a t i o n pha s e can be preselected. I f t h e preselector is n o t set, e v e r y puff of t h e m e t e r e d aerosol is followed b y v e n t i l a t i o n .
Methods Acute Inhalation Toxicity A series of metered aerosol preparations with and without ipratropium bromide (Sch 1000, Atrovent| (Bauer, 1974; Bauer et at., 1973) was tested for acute inhalation toxicity in rats, using the apparatus described here. Preparation No. I delivered per puff: 20 ~zg Sch 1000, 35 tzg soyabean lecithin, 17A65 mg fluorocarbon i t , 37.015 mg fluorocarbon 12, and 15.765 nag fluorocarbon l i 4 . Preparation No. 2 delivered per puff: 20 ~g Sch 1000, 70 ~g soyabean lecithin, i7A57 mg fluorocarbon t l , 36.996 mg fluorocarbon 12, and 15.757 mg fluorocarbon 114. Preparation No. 3 (placebo for No. 1) delivered per puff: 35 ~zg soyabean lecithin, i7A70 mg fluorocarbon l i , 37.025 mg fluorocarbon t2, and 15.770 mg fluorocarbon l l 4 . Preparation No. 4 (placebo for No. 2) delivered per puff: 70 ~g soyabean lecithin, 16.084 mg fluorocarbon t i , 37.762 mg fluorocarbon 12, and 16.084 mg fluorocarbon l l 4 . Each preparation was tested on t0 rats (strain Chbb: THOM (SPF), 5 ~, 5 9). 5, 10, 20, 80 or 160 puffs were offered to the individual groups on one day. Two administration and ventilation programs were employed: Program A. The rats received one puff of metered aerosol every 15 sec. After 5 puffs the apparatus was ventilated for t5 sec by circulation of fresh air. If more than 5 puffs were to be administered, the program was repeated as necessary. Program B. After each puff there was a t5-sec interval and then a 15-see active ventilation phase. This was repeated as necessary, up to as many as 160 puffs.
Determination o/Propellants in the Arterial Blood (Shargel et al., 1972) 5, 10, 15, 30 and 60 see after one puff of test preparation No. 3, 0.25 ml blood samples were taken from the carotid artery with a gas tight syringe. A carotid artery of the test animals had been cannulated with fine PVC catheters during brief ether anesthesia prior to commencement of the test. It was not necessary to remove the animals from the immobilization tubes for sampling, since the apparatus used here had been modified by a slit in the side of each tube, through which the catheter easily passed. The samples were injected directly into air-tight sample bottles containing 25 ml cooled hexane (Uvasol)/Ierek). The bottles were stored in ice until gas chromatography was carried out (HEWLETT-PACKAI~D, model 5750). An electron capture detector with Ni 6a was used. The pulse interval was 150 [zsec, and the detector temperature 250 ~ C. The glass column was 2 m long and had an internal diameter of 4 mm. PORAPACK Q, 80 to 100 mesh, was used as carrier for the stationary phase. The temperature of the column was 160 ~ C, and that of the injector 200 ~ C. Electrometer range 1, attenuation 32. Fluorocarbons i l and t2 were detected. :Fluorocarbon 114 could not be detected at the same time as its detection was much less sensitive (t:3:24 compared with fluorocarbons ~11 and 12).
Determination o/the Arterial Blood Gazes Two groups of 6 rats (body weight approximately 200 g) were used. 0.25 ml blood samples were taken from the carotid artery by the method described above, before and after treatment with preparation No. 3. The following programs were followed:
New Apparatus and l~ethod for the Toxicological Investigation of l~Ietered Aerosols in Rats
5
Group t received 40 puffs of the metered aerosol. 15 sec after each puff, the front end of the animal chambers was opened for t5 sec to allow passive ventilation. The second blood sample was taken after the final ventilation phase. Group 2 also received 40 puffs of this metered aerosol. 15 sec after each puff there was a 15-sec active ventilation phase at 0.5 arm positive pressure (program B, above). The second blood sample was taken after the last ventilation phase. The blood gas values were obtained by the Micro-Astrup method (BMS 2 MK 2, Radiometer). The acid-base equilibrium and the partial pressures PO 2 and PCO2 were determined (Siggard-Andersen, 1960).
Results 1. Acute Inhalation Toxicity The various inhalation toxieities of the different m e t h o d s are shown in Tables l and 2. Only with p r o g r a m A (minimal ventilation of the immobilization tubes) was a dose-dependent lethal effect observed (Table 2). The inclusion or absence of the anticholinergie substance ipratropium bromide (Atrovent| in the aerosol a n d the v a r y i n g a m o u n t s of s o y a b e a n lecithin m a d e no difference to the effect (Table 1). After high doses the animals lost consciousness and died of acute respiratory failure, which shows t h a t the toxic effect is caused b y the propellant, and only occurs w h e n ventilation is poor. I n addition to the tests with active substance, a group of rats was tested u n d e r simulated conditions with e m p t y aerosol packs a n d no active ventilation. These animals survived the test and did n o t suffer lack of o x y g e n (Table t). Aerosol application b y p r o g r a m B did n o t result in a toxic effect (Table 2). This m e t h o d is comparable with conditions in h u m a n aerosol therapy. Table 1. Acute toxicity with metered aerosols in rats. Mortalities per group of t0 animals. Chamber ventilation after every 5th puff (program A) 5 puffs 10 puffs 20 puffs 40 puffs 80 puffs 160 puffs
Atrovent| 20 ~g/puff, No. 1 Atrovent| 20 ~g/puff, No. 2 placebo No. 3 placebo No. 4
0/10
0/10
7/10
5It0
8/10
7/10
0/10 0/10 0/10
0/10 1/10 0/10
3/10 5/t0 4/10
1/t0 3/10 3/10
3/10 6/10 5/I0
7/10 8/t0 7/t0
t 60 puffs from empty packs, without ventilation 0/t0
Table 2. Acute toxicity with metered aerosols in rats. Mortalities per group of 10 animals. Chamber ventilation after every puff (program B)
Atrovent| 20 btg/puff, No. 1 Atrovent| 20 ~g/puff, iN'o. 2 placebo No. 3 placebo No. 4
5 puffs
20 puffs
40 puffs
80 puffs
160 puffs
0/10
0/10
0/10
0/10
0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/t0
6
G. Muacevie
2. Propellants in the Arterial Blood o/the Rat Gas c h r o m a t o g r a p h y of blood samples t a k e n 5 to 60 sec after a d m i n i s t r a t i o n of the placebo (preparation No. 3) showed t h a t the m a x i m u m c o n c e n t r a t i o n of fluorocarbons i i a n d i 2 is to be f o u n d 5 sec after a d m i n i s t r a t i o n . T a b l e 3 shows t h e results of these tests i n groups of 6 rats. E x c e p t at the time of m a x i m u m , t h e blood level of fluorocarbon i i was significantly higher t h a n t h a t of fluorocarbon i2. Table 3. Fluorocarbons in the arterial blood of the rat after I puff of placebo aerosol pack No. 3. Sampling times 1 to 60 sec after administration; n = 6/sampling time substance
fluorocarbon l i fluorocarbon 12 significance t-test for paired comparisons
time after administration immediately 5 sec 75 sec ~g/ml ~zg/ml ~g/ml ~+s ~+s ~+_s
30 see ~g/ml ~_+s
60 sec ~g/ml ~+s
24.0 _+6.4 16.3 _+5.2
29.1 _+ 9.5 28.4 _+10.5
27.7 _+5.6 21.0 _+5.6
24.2 + 5.5 18.5 _+6.2
t8.3 _+2.7 i5.2 _+2.2
P < 0.001
P > 0.05
P < 0.001
P < 0.01
P < 0.001
The blood levels a n d t h e levels of significance of the i n d i v i d u a l times are given i n T a b l e 3. T h e m e a n half-lives, e v a l u a t e d graphically o n a semi-logarithmic network, were f o u n d to be 80 sec for fluorocarbon l i a n d 57 see for fluorocarbon 12. The m e a n difference b e t w e e n the half-lives of F l i a n d F i 2 has a significance of P < 0.05. A f u r t h e r test i n 2 animals, where the propellant levels were measured b e t w e e n 15 a n d 120 see after a d m i n i s t r a t i o n , showed again t h a t fluorocarbon 12 is e l i m i n a t e d from the blood more r a p i d l y t h a n fluorocarbon i i . The half-lives were 68 a n d 77 see for fluorocarbon l i , a n d 51 a n d 66.5 see for fluorocarbon 12.
3. Arterial Blood Gases T a b l e 4 shows the values for the arterial blood gases. The i m p o r t a n c e of a d e q u a t e v e n t i l a t i o n of the aerosol c h a m b e r becomes e v i d e n t from these data. After 40 puffs of placebo m e t e r e d aerosol No. 3 with only passive v e n t i l a t i o n phases, h y p o x i a a n d acidosis were found. The base excess increased a n d the CO S p a r t i a l pressure t e n d e d to rise (Table 4 A). W h e n the c h a m b e r was actively v e n t i l a t e d , h y p o x i a a n d acidosis did n o t occur, the base excess increased b y less a n d t h e PCO 2 r e m a i n e d c o n s t a n t (Table 4 B). The slight decrease i n b i c a r b o n a t e was the same in b o t h groups. Tables 4 A and B. Arterial blood gas values for the rat after 40 puffs of placebo aerosol No. 3. A: passive ventilation (front of test chamber open) after each puff. B: automatic active ventilation (circulation of air at 0.5 arm) after each puff A pO~ t s
pC02 2 t
1
pH 2
t
1
2 t
Base excess 1 2
10t.9 50.5" 38.9 53.7 N.S. 7.35 7.18" - 3.8 +47 _+21.0 +_4.2 _+12.6 _+0.02 +_0.11 +1.5
t
Bicarbonate 1 2
t
- 9.2 * 20.9 t9.6 •.S. +_5.t _+1.8 _+5.2
New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats
7
Table. 4B pO~ 1 8
pCO~ 2 t
i
pH 2
t
l
2 t
Base excess 1 2 t
Bicarbonate 1 2 t
86.3 91.2N.S. 35.9 38.9 N.S. 7.36 7.29* - 4 . 9 - 7 . 4 ** 19.6 18.3 * +10.4 _+11.9 +2.6 +3.0 +_0.03+0.03 _ + t . 8 _+2.5 +i.8 _+2.1
Blood samples taken 15 sec after the last puff. I = original value; 2 = value after treatment; t = t-test for pairs; 9 = mean value for 6 rats; s = standard deviation; N.S. = not significant; * = significant (P < 0.05), ** highly significant (B < 0.01).
Discussion The apparatus described here permits differentiation between the toxicity of propellants and of the test substance in toxicological studies with metered aerosols in small laboratory animals. The ventilation of the testchamber is variable, so that the effects of propellants in combination with hypoxia can be investigated. Our toxicity studies with this apparatus found a lethal effect of the propellants, independent of the active substance and its additives, only when the animal chamber was poorly ventilated (Tables I and 2). The analysis of the blood gases showed the importance of good ventilation (Table 4). When the animal chamber was ventilated b y active circulation of air, hypoxia and acidosis did not occur. Our apparatus differs from other systems designed for the screening of aerosols in laboratory animals in that individual doses can be administered, and the dilution of the aerosol with the air in a large common chamber is avoided. We consider this to be of advantage as we reduce the dead space to a minimum and thus more closely parallel the therapeutic conditions, since such aerosols are usually administered directly into the oral cavity. The importance of active circulation of air after each puff, ensuring removal of any uninhaled aerosol residue, and sufficient ventilation, has already been emphasized. The tilting device ensures that the metered aerosol suspension is evenly mixed at every puff. I t is also possible to study the effect of the propellants, since a ventilation phase need not follow every puff, but can be set to occur after every 2 to 9 puffs. A slight modification to the animal chamber permits arterial blood samples to be taken from the immobilized rats directly after inhalation of a metered dose. Analysis of the arterial blood showed that a proportion of the aerosol propellant gas phase is absorbed. The concentrations of fluorocarbons l i and i2 found b y us in the arterial blood of the rat (Table 3) compare well with the findings of Shargel et al. (i972) in the dog, and the half-lives calculated by us also agree with those given by these authors. Paterson et al. (i972) measured a concentration of 0.1 to 3.0~g/ml fluorocarbon l i in human arterial blood after inhalation of 2 puffs. The considerably lower levels in human blood are due to the fact that the air space in the studies in man was far greater than in the animal studies. Although the air spaces in the studies with the rat and the dog also differed, this was compensated for by the far higher respiration frequency in the rat, and the values obtained for these two species were thus comparable. I n our investigations the maximum concentrations of fluorocarbons i i and 12 in the arterial blood were practically the same, but fluorocarbon 12 was eliminated much more rapidly, possibly due to differences in the solubility and volatility of
8
G. Muacevic
t h e two. A f t e r i n h a l a t i o n of m e t e r e d aerosols w i t h p r o p e l l a n t m i x t u r e s consisting o f fluorocarbons l i , i2, a n d i l 3 or i i , 12, a n d i t 4 , t h e a r t e r i a l b l o o d of m a n was f o u n d to c o n t a i n o n l y fluorocarbon l i (Dollery et al., t970). P a t e r s o n et al. (197~) f o u n d t r a c e s o f fluorocarbon t2. I t is possible t h a t t h e d i s c r e p a n c y in t h e v a r i o u s findings is d u e to t h e different m e t h o d s of blood-gas analysis used. I n t h e two t e s t s cited, gas c h r o m a t o g r a p h y was carried o u t with direct injection of t h e blood s a m p l e into t h e gas c h r o m a t o g r a p h , m e a n i n g t h a t a n y of t h e h i g h l y v o l a t i l e fluorocarbon 12 (Graf et al., 2969) in t h e s a m p l e m a y h a v e escaped before c h r o m a t o g r a p h i c analysis, a p o s s i b i l i t y which was e x c l u d e d b y us, since we p u t t h e blood s a m p l e s d i r e c t l y into p r e c o o l e d h e x a n e u n t i l c h r o m a t o g r a p h y was begun.
Acknowledgements. I am most indebted to Dr. K. Brandt and Mr. K. Ramthun for their gas chromatographic analysis of the propellants, to Mr. K. H. Bozung for his measurement of the arterial blood gases, to Mr. L. Nikolaidis for his practical assistance in the inhalation studies, to Mr. G. Goetze and Mr. F. Pabst for their technical assistance with the inhalation equipment, )fir. F. Otterbach and Mr. K. Hemmkeppler for designing the electronic control system, and Dr. F. Knappon for his consultation and help with the biometric calculations. References Bauer, R.: Pharmakologie und Pharmakokinetic yon Atrovent. Wien. med. Wschr. 124 (Suppl. 21), 12--15 (1974) Bauer, R., Wick, H. : Ipratropiumbromid (Sch 1000) als Aerosol - - ein Anticholincrgicum zur Behandlung obstruktiver Atemwegserkrankungen. I. Int. Kongr. ffir Aerosole in der Medizin, t9.--21.9. 1973, Baden bei Wien Dollery, C. T., Davies, D. S., Draffan, G. H., Williams Faith, M.: Blood concentrations in man of fluorinated hydrocarbons after inhalation of pressurized aerosols. Lancet 1970 H, l i 6 4 Fultyn, R. V. : Contaminant generators for continuous exposure inhalation chambers. Amer. industr. Hyg. Ass. J. 22, 49--53 (1961) Graf, E., Graser, M. : Pharmazeutische Aerosole. Mitt. dtsch, pharm. Ges. 39, 97 (1969) Hanna, M. G., 2qettesheim, P., Gilbert, J. R. : Inhalation carcinogenesis. U.S. Atomic energy commission, Oak Ridge, ~970, AEC Symposium Series 18, pp. 55--72, 393, 394, 416, 4t7 Hinners, R. G. : Animal exposure chambers in air pollution studies. Arch. environm. Hlth 13, 609--6i5 (1966) HSbel, M. : Resorption yon Wirkungssubstanzen aus Aerosolen in Abhiingigkcit vom Atmungstyp. Arch. int. Pharmacodyn. 198, 76--84 (1972) Kfibler, H. : The physiological properties of aerosol propellants. Aerosol Age 9, 44--50 and 90---91 (1964) Paterson, J. W., Sudlow, M. F., Walker, S. R. : Blood levels of fluorinated hydrocarbons in asthmatic patients after inhalation of pressurized aerosols. Lancet 1971 H, 565 Scholz, J. : Neue texikologische Untersuchungen einiger als Treibgas verwendeter FrigenTypen. Fortschr. d. biol. Aerosolf. i957--1961, Ber. fiber den 4. Aerosol-Kongrel3, S. 420 bis 429. Stuttgart: Schattauer t962 Shargel, L., Koss, R. : Determination of fluorinated hydrocarbon propellants in blood of dogs after aerosol administration. J. pharm. Sci. 61, 1445--1449 (1972) Siggaard-Andersen, 0. : A new acid-base nomogram. An improved method for the calculation of the relevant blood acid-base data. Scand. J. clin. Lab. Invest. 12, 177 (1960) Dr. G. )fuacevic C. H. Boehringer Sohn Abteilung Pharmakologie D-6507 Ingelheim am Rhein Federal Republic of Germany
Original Investigations New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats*, * * G. M u a c e v i c C. H. Boehringer Sohn, Abt. Pharmakologie, Ingelheim/Rhein Received :February 7, 1975
Abstract. A new apparatus and method for the toxicological investigation of metered aerosols in rats, which is also suitable for tests in other small laboratory animals, is described. I t permits: 1. simultaneous treatment of 5 or more animals, 2. administration of metered aerosol doses to individual animals, 3. ventilation of the cages, 4. mechanical tilting of the metered aerosol packs to ensure thorough mixing of the content, and 5. continuous automatic tilting, administration and ventilation. Four metered aerosols, with and without ipratropium bromide, were investigated under different ventilation conditions. Blood gases and fluorinated chlorohydrocarbons (abbreviation: fluorocarbons) in the arterial blood were also determined. I n tests with spontaneous ventilation of the animal chambers without positive pressure, significant acidosis and hypoxia occurred after 40 puffs of metered aerosol. Where ventilation of the chambers was insufficient, the fluorocarbons led to dose-dependent toxic and lethal effects. The substance and the additives contained in the metered aerosol did not interfere with these effects. After active ventilation with 0.5 arm no symptoms of acidosis or hypoxia were observed. Up to 160 puffs of metered aerosol, no indications of toxic effects were established in the rats. Half-life of the fluorocarbons in the arterial blood after one puff of metered aerosol was 69 to S0 sec for fluorocarbon 11 and 57 to 67 see for fluorocarbon 12. Key word~: l~etered Aerosols - - Ipratropium Bromide - - New Apparatus - - Arterial Blood Gases - - l~luorocarbons in Arterial Blood. Zusammen/assung. Eine neue Apparatur und !~Iethode fiir die toxikologische Priifung yon Dosier-Aerosolen an Ratten, welehe im Prinzip auch fiir andere kleine Labortiere geeignet ist, wird beschrieben. Sie ermSglicht: 1. gleichzeitige Behandlung yon 5 oder mehr Tieren, 2. individuelle Verabreichung der Dosier-Aerosole, 3. Beliiftung der Tierk~fige, 4. Schiitteln tier Dosier-Aerosolbeh~ilter zur Durehmisehung des Inhalts, und 5. automatisehen Ablauf yon Mischen, Dosieren und Beliiftung. u Dosier-Aerosole mit und ohne Ipratropiumbromid wurden unter untersehiedlichen Belfiftungsbedingungen untersucht. Zus~tzlich wurden die Blutgase und die Treibgase im arteriellen Blur bestimmt. I n Vcrsuchen mit spontaner Beliiftung der Zwangsk~fige ohne positiven Druck ergab sich nach 40 Hfiben des Dosier-Aerosols eine signifikante Acidose und Hypoxie. Bei ungeniigender Beliiftung zeigten die Treibgase dosisabh~ngig eine toxische und letale Wirkung. Der Wirkstoff und die Zusatzstoffe des Dosier-Aerosols hatten darauf keinen EinflulL Bei aktiver Belfiftung mit 0,5 atii kam es nicht zu einer Acidose und Hypoxie, und die Ratten vertrugen bis t60 Hfibe des Dosier-Aerosols symptomfrei. Die Halbwertszeit der * Dedicated to Prof. Dr. Karl Zeile on the occasion of his 70th birthday. ** Presented in brief at the 33rd International Congress of the Pharmaceutical Sciences in 1973 in Stockholm.
2
G. Muacevic
Treibgase im arteriellen Blur nach einem Hub ])osier-Aerosol be~rug ffir Treibgas 7t/69 bis 80 sec, fiir Treibgas 12/57 bis 67 sec. Schli2sselw6rter: Dosier-Aerosole - - Ipratropiumbromid - - Neue ApparaSur- Arterielle Blutgase - - Fluorokohlenwasserstoffe im arteriellen Blur. The development of the metered aerosol has increased the importance of local aerosol administration of active substances in the upper and lower respiratory tracts accompanied in some cases by local absorption. Metered aerosols have been in use in medicine since 1956. E v e r y metered aerosol contains not only the active substance, but also a propellant, those most commonly used being compressed gases (nitrogen, carbon dioxide, nitrous oxide) or liquid gases (usually fluorinated ehlorohydrocarbons). Aerosol propellants have to be screened for their physiological effect and dermal and mucosal tolerance. Most of the fluorinated chlorohydrocarbon propellants (abbreviation: fluorocarbons) on the market have a maximal allowable concentration (MAC or TLV, in Germany MAK) of at least 500 p p m in air. Fluorocarbons l i (trichlorofluormethane), 12 (dichlorofiuoromethane) and l l 4 (i,2-dichlorotetrafluoroethane) have an MAC of 1000ppm, and are thus assigned to toxicity categories 5a or 6 (categories defined b y H. Kfibler, 1964). Categories I to 4 are not permitted for use in metered aerosols. Various apparatuses have been described for the laboratory investigation of atomized solutions and the investigation of cigarette smoke (Fultyn, 1961; H a n n a et al., 1970; Hinners, i966; HSbel, i972; Scholz, 1962), but a technique for testing regular metered aerosols on individual animals has hitherto not been published.
Description of the Apparatus Our apparatus consists of 5 glass immobilization tubes, tapered at one end to fit the nose and head of a rat. The size of the tubes is adequate for rats weighing approximately 200 g. The tail-end of the tube is closed with a sliding plastic bung which can be adjusted to the length of each individual rat. The metered aerosol test packs, of the type used in human medicine, are fixed at the end of each tube, 80 m m from the nose of the rat. A depressor bar activates all the packs simultaneously, and a tilting device ensures t h a t the contents of the aerosol packs are thoroughly mixed before each puff. Fresh air can be circulated at regular intervals to prevent hypoxia. This apparatus allows toxicological studies with metered aerosols to be carried out in groups of animals with a minimum of time and manpower. Each of the processes described: mixing, administration, and circulation of fresh air, can be programmed on the electronic control device to be carried out automatically in sequence. Air can be supplied as desired, either after each puff or after anything between 2 and 9 puffs, thus permitting the investigation of the toxicity of individual propellants under various ventilation conditions. The inhalation system is shown in diagrams I and 2. Five glass animal chambers (Figs. l . l and 2.2) are mounted on a plastic base (Fig. IA). Push-in sockets at the nose-end and clips at the taft-end of the base facilitate the removal, cleaning, and replacement of the tubes (1.3). A tumbler device (2.12), consisting of a base for the aerosol pack adapters and powered b y a pneumatic lifting cylinder (t.15),
New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats
3
--15
"=IP~ ~=,~,, -~..'.
I
n~ l_,_.'J_ _J-J_ . . . . . . . . I I
,_'.J_ _L.'-J
I
i I
!;! i ]
- I 1 I
j t
"1 ....
"
t I
~13
II ,j ,.~
,,, l l
I t
2
il]l~ I
' t
FI'I
3 l
10
2
7~
1
11
14
2
Figs. i and 2. 1 = base; 2 = glass immobilisation tube; 3 = raw of clips; d to 6 = solenoid valve; 7 = angle lever; 8 =adjusr ram; 9 = metered aerosol pack; 10 = lifting cylinder; 11 = metered aerosol adapter; 12 = tumbler device; 13 = tube for air supply; 14 = central valve; 15 = lifting cylinder t i l t s t h r o u g h 90 ~ a n d t h u s insures c o m p l e t e m i x i n g o f t h e c o n t e n t s of t h e aerosol packs. T h e lifting c y l i n d e r is r e g u l a t e d b y a solenoid v a l v e (1.4). M e t e r e d aerosol dosing is s y n c h r o n i z e d b y depression o f t h e aerosol p a c k s (2.9) in p o s i t i o n in t h e a d a p t e r s b y m e a n s of a n angle lever (2.7) f i t t e d w i t h a d j u s t a b l e r a m s (2.8). This causes a c t i v a t i o n o f t h e valves, a n d an aerosol dose is p r o j e c t e d t h r o u g h e a c h a d a p t e r ( 2 . i i ) a n d i n t o t h e n o s e - e n d o f t h e i m m o b i l i z a t i o n t u b e (2.2). This s y s t e m is also p o w e r e d b y a p n e u m a t i c lifting c y l i n d e r (2.10), r e g u l a t e d b y a
4
G. Muacevic
solenoid v a l v e (1.5 a n d 2.5). A c t i v e v e n t i l a t i o n b y circulation of fresh air t h r o u g h t h e a d a p t e r a n d nose-end of t h e tu b e s is again r e g u l a t e d by a solenoid v a l v e (1.6). T h e t i l t i n g d e v i c e a n d t h e a c t i v a t o r each r e q u ir e 4 a r m positive pressure, an d t h e v e n t i l a t i o n s y s t e m 0.5 a t m p o s i t i v e pressure. T h e s y s t e m ca n be controlled m a n u a l I y or a u t o m a t i c a l l y . Our electronic c ont ro l d e v i c e consists o f a series of t i m e - s e q u e n c e connectors. T h e d u r a t i o n of b o t h t h e i n t e r v a l b e t w e e n t h e puffs, a n d t h e a c t i v e v e n t i l a t i o n p r o c e d u r e can be r e g u l a t e d . T h e n u m b e r o f puffs to be a d m i n i s t e r e d prior to an a c t i v e v e n t i l a t i o n pha s e can be preselected. I f t h e preselector is n o t set, e v e r y puff of t h e m e t e r e d aerosol is followed b y v e n t i l a t i o n .
Methods Acute Inhalation Toxicity A series of metered aerosol preparations with and without ipratropium bromide (Sch 1000, Atrovent| (Bauer, 1974; Bauer et at., 1973) was tested for acute inhalation toxicity in rats, using the apparatus described here. Preparation No. I delivered per puff: 20 ~zg Sch 1000, 35 tzg soyabean lecithin, 17A65 mg fluorocarbon i t , 37.015 mg fluorocarbon 12, and 15.765 nag fluorocarbon l i 4 . Preparation No. 2 delivered per puff: 20 ~g Sch 1000, 70 ~g soyabean lecithin, i7A57 mg fluorocarbon t l , 36.996 mg fluorocarbon 12, and 15.757 mg fluorocarbon 114. Preparation No. 3 (placebo for No. 1) delivered per puff: 35 ~zg soyabean lecithin, i7A70 mg fluorocarbon l i , 37.025 mg fluorocarbon t2, and 15.770 mg fluorocarbon l l 4 . Preparation No. 4 (placebo for No. 2) delivered per puff: 70 ~g soyabean lecithin, 16.084 mg fluorocarbon t i , 37.762 mg fluorocarbon 12, and 16.084 mg fluorocarbon l l 4 . Each preparation was tested on t0 rats (strain Chbb: THOM (SPF), 5 ~, 5 9). 5, 10, 20, 80 or 160 puffs were offered to the individual groups on one day. Two administration and ventilation programs were employed: Program A. The rats received one puff of metered aerosol every 15 sec. After 5 puffs the apparatus was ventilated for t5 sec by circulation of fresh air. If more than 5 puffs were to be administered, the program was repeated as necessary. Program B. After each puff there was a t5-sec interval and then a 15-see active ventilation phase. This was repeated as necessary, up to as many as 160 puffs.
Determination o/Propellants in the Arterial Blood (Shargel et al., 1972) 5, 10, 15, 30 and 60 see after one puff of test preparation No. 3, 0.25 ml blood samples were taken from the carotid artery with a gas tight syringe. A carotid artery of the test animals had been cannulated with fine PVC catheters during brief ether anesthesia prior to commencement of the test. It was not necessary to remove the animals from the immobilization tubes for sampling, since the apparatus used here had been modified by a slit in the side of each tube, through which the catheter easily passed. The samples were injected directly into air-tight sample bottles containing 25 ml cooled hexane (Uvasol)/Ierek). The bottles were stored in ice until gas chromatography was carried out (HEWLETT-PACKAI~D, model 5750). An electron capture detector with Ni 6a was used. The pulse interval was 150 [zsec, and the detector temperature 250 ~ C. The glass column was 2 m long and had an internal diameter of 4 mm. PORAPACK Q, 80 to 100 mesh, was used as carrier for the stationary phase. The temperature of the column was 160 ~ C, and that of the injector 200 ~ C. Electrometer range 1, attenuation 32. Fluorocarbons i l and t2 were detected. :Fluorocarbon 114 could not be detected at the same time as its detection was much less sensitive (t:3:24 compared with fluorocarbons ~11 and 12).
Determination o/the Arterial Blood Gazes Two groups of 6 rats (body weight approximately 200 g) were used. 0.25 ml blood samples were taken from the carotid artery by the method described above, before and after treatment with preparation No. 3. The following programs were followed:
New Apparatus and l~ethod for the Toxicological Investigation of l~Ietered Aerosols in Rats
5
Group t received 40 puffs of the metered aerosol. 15 sec after each puff, the front end of the animal chambers was opened for t5 sec to allow passive ventilation. The second blood sample was taken after the final ventilation phase. Group 2 also received 40 puffs of this metered aerosol. 15 sec after each puff there was a 15-sec active ventilation phase at 0.5 arm positive pressure (program B, above). The second blood sample was taken after the last ventilation phase. The blood gas values were obtained by the Micro-Astrup method (BMS 2 MK 2, Radiometer). The acid-base equilibrium and the partial pressures PO 2 and PCO2 were determined (Siggard-Andersen, 1960).
Results 1. Acute Inhalation Toxicity The various inhalation toxieities of the different m e t h o d s are shown in Tables l and 2. Only with p r o g r a m A (minimal ventilation of the immobilization tubes) was a dose-dependent lethal effect observed (Table 2). The inclusion or absence of the anticholinergie substance ipratropium bromide (Atrovent| in the aerosol a n d the v a r y i n g a m o u n t s of s o y a b e a n lecithin m a d e no difference to the effect (Table 1). After high doses the animals lost consciousness and died of acute respiratory failure, which shows t h a t the toxic effect is caused b y the propellant, and only occurs w h e n ventilation is poor. I n addition to the tests with active substance, a group of rats was tested u n d e r simulated conditions with e m p t y aerosol packs a n d no active ventilation. These animals survived the test and did n o t suffer lack of o x y g e n (Table t). Aerosol application b y p r o g r a m B did n o t result in a toxic effect (Table 2). This m e t h o d is comparable with conditions in h u m a n aerosol therapy. Table 1. Acute toxicity with metered aerosols in rats. Mortalities per group of t0 animals. Chamber ventilation after every 5th puff (program A) 5 puffs 10 puffs 20 puffs 40 puffs 80 puffs 160 puffs
Atrovent| 20 ~g/puff, No. 1 Atrovent| 20 ~g/puff, No. 2 placebo No. 3 placebo No. 4
0/10
0/10
7/10
5It0
8/10
7/10
0/10 0/10 0/10
0/10 1/10 0/10
3/10 5/t0 4/10
1/t0 3/10 3/10
3/10 6/10 5/I0
7/10 8/t0 7/t0
t 60 puffs from empty packs, without ventilation 0/t0
Table 2. Acute toxicity with metered aerosols in rats. Mortalities per group of 10 animals. Chamber ventilation after every puff (program B)
Atrovent| 20 btg/puff, No. 1 Atrovent| 20 ~g/puff, iN'o. 2 placebo No. 3 placebo No. 4
5 puffs
20 puffs
40 puffs
80 puffs
160 puffs
0/10
0/10
0/10
0/10
0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/10
0/10 0/10 0/t0
6
G. Muacevie
2. Propellants in the Arterial Blood o/the Rat Gas c h r o m a t o g r a p h y of blood samples t a k e n 5 to 60 sec after a d m i n i s t r a t i o n of the placebo (preparation No. 3) showed t h a t the m a x i m u m c o n c e n t r a t i o n of fluorocarbons i i a n d i 2 is to be f o u n d 5 sec after a d m i n i s t r a t i o n . T a b l e 3 shows t h e results of these tests i n groups of 6 rats. E x c e p t at the time of m a x i m u m , t h e blood level of fluorocarbon i i was significantly higher t h a n t h a t of fluorocarbon i2. Table 3. Fluorocarbons in the arterial blood of the rat after I puff of placebo aerosol pack No. 3. Sampling times 1 to 60 sec after administration; n = 6/sampling time substance
fluorocarbon l i fluorocarbon 12 significance t-test for paired comparisons
time after administration immediately 5 sec 75 sec ~g/ml ~zg/ml ~g/ml ~+s ~+s ~+_s
30 see ~g/ml ~_+s
60 sec ~g/ml ~+s
24.0 _+6.4 16.3 _+5.2
29.1 _+ 9.5 28.4 _+10.5
27.7 _+5.6 21.0 _+5.6
24.2 + 5.5 18.5 _+6.2
t8.3 _+2.7 i5.2 _+2.2
P < 0.001
P > 0.05
P < 0.001
P < 0.01
P < 0.001
The blood levels a n d t h e levels of significance of the i n d i v i d u a l times are given i n T a b l e 3. T h e m e a n half-lives, e v a l u a t e d graphically o n a semi-logarithmic network, were f o u n d to be 80 sec for fluorocarbon l i a n d 57 see for fluorocarbon 12. The m e a n difference b e t w e e n the half-lives of F l i a n d F i 2 has a significance of P < 0.05. A f u r t h e r test i n 2 animals, where the propellant levels were measured b e t w e e n 15 a n d 120 see after a d m i n i s t r a t i o n , showed again t h a t fluorocarbon 12 is e l i m i n a t e d from the blood more r a p i d l y t h a n fluorocarbon i i . The half-lives were 68 a n d 77 see for fluorocarbon l i , a n d 51 a n d 66.5 see for fluorocarbon 12.
3. Arterial Blood Gases T a b l e 4 shows the values for the arterial blood gases. The i m p o r t a n c e of a d e q u a t e v e n t i l a t i o n of the aerosol c h a m b e r becomes e v i d e n t from these data. After 40 puffs of placebo m e t e r e d aerosol No. 3 with only passive v e n t i l a t i o n phases, h y p o x i a a n d acidosis were found. The base excess increased a n d the CO S p a r t i a l pressure t e n d e d to rise (Table 4 A). W h e n the c h a m b e r was actively v e n t i l a t e d , h y p o x i a a n d acidosis did n o t occur, the base excess increased b y less a n d t h e PCO 2 r e m a i n e d c o n s t a n t (Table 4 B). The slight decrease i n b i c a r b o n a t e was the same in b o t h groups. Tables 4 A and B. Arterial blood gas values for the rat after 40 puffs of placebo aerosol No. 3. A: passive ventilation (front of test chamber open) after each puff. B: automatic active ventilation (circulation of air at 0.5 arm) after each puff A pO~ t s
pC02 2 t
1
pH 2
t
1
2 t
Base excess 1 2
10t.9 50.5" 38.9 53.7 N.S. 7.35 7.18" - 3.8 +47 _+21.0 +_4.2 _+12.6 _+0.02 +_0.11 +1.5
t
Bicarbonate 1 2
t
- 9.2 * 20.9 t9.6 •.S. +_5.t _+1.8 _+5.2
New Apparatus and Method for the Toxicological Investigation of Metered Aerosols in Rats
7
Table. 4B pO~ 1 8
pCO~ 2 t
i
pH 2
t
l
2 t
Base excess 1 2 t
Bicarbonate 1 2 t
86.3 91.2N.S. 35.9 38.9 N.S. 7.36 7.29* - 4 . 9 - 7 . 4 ** 19.6 18.3 * +10.4 _+11.9 +2.6 +3.0 +_0.03+0.03 _ + t . 8 _+2.5 +i.8 _+2.1
Blood samples taken 15 sec after the last puff. I = original value; 2 = value after treatment; t = t-test for pairs; 9 = mean value for 6 rats; s = standard deviation; N.S. = not significant; * = significant (P < 0.05), ** highly significant (B < 0.01).
Discussion The apparatus described here permits differentiation between the toxicity of propellants and of the test substance in toxicological studies with metered aerosols in small laboratory animals. The ventilation of the testchamber is variable, so that the effects of propellants in combination with hypoxia can be investigated. Our toxicity studies with this apparatus found a lethal effect of the propellants, independent of the active substance and its additives, only when the animal chamber was poorly ventilated (Tables I and 2). The analysis of the blood gases showed the importance of good ventilation (Table 4). When the animal chamber was ventilated b y active circulation of air, hypoxia and acidosis did not occur. Our apparatus differs from other systems designed for the screening of aerosols in laboratory animals in that individual doses can be administered, and the dilution of the aerosol with the air in a large common chamber is avoided. We consider this to be of advantage as we reduce the dead space to a minimum and thus more closely parallel the therapeutic conditions, since such aerosols are usually administered directly into the oral cavity. The importance of active circulation of air after each puff, ensuring removal of any uninhaled aerosol residue, and sufficient ventilation, has already been emphasized. The tilting device ensures that the metered aerosol suspension is evenly mixed at every puff. I t is also possible to study the effect of the propellants, since a ventilation phase need not follow every puff, but can be set to occur after every 2 to 9 puffs. A slight modification to the animal chamber permits arterial blood samples to be taken from the immobilized rats directly after inhalation of a metered dose. Analysis of the arterial blood showed that a proportion of the aerosol propellant gas phase is absorbed. The concentrations of fluorocarbons l i and i2 found b y us in the arterial blood of the rat (Table 3) compare well with the findings of Shargel et al. (i972) in the dog, and the half-lives calculated by us also agree with those given by these authors. Paterson et al. (i972) measured a concentration of 0.1 to 3.0~g/ml fluorocarbon l i in human arterial blood after inhalation of 2 puffs. The considerably lower levels in human blood are due to the fact that the air space in the studies in man was far greater than in the animal studies. Although the air spaces in the studies with the rat and the dog also differed, this was compensated for by the far higher respiration frequency in the rat, and the values obtained for these two species were thus comparable. I n our investigations the maximum concentrations of fluorocarbons i i and 12 in the arterial blood were practically the same, but fluorocarbon 12 was eliminated much more rapidly, possibly due to differences in the solubility and volatility of
8
G. Muacevic
t h e two. A f t e r i n h a l a t i o n of m e t e r e d aerosols w i t h p r o p e l l a n t m i x t u r e s consisting o f fluorocarbons l i , i2, a n d i l 3 or i i , 12, a n d i t 4 , t h e a r t e r i a l b l o o d of m a n was f o u n d to c o n t a i n o n l y fluorocarbon l i (Dollery et al., t970). P a t e r s o n et al. (197~) f o u n d t r a c e s o f fluorocarbon t2. I t is possible t h a t t h e d i s c r e p a n c y in t h e v a r i o u s findings is d u e to t h e different m e t h o d s of blood-gas analysis used. I n t h e two t e s t s cited, gas c h r o m a t o g r a p h y was carried o u t with direct injection of t h e blood s a m p l e into t h e gas c h r o m a t o g r a p h , m e a n i n g t h a t a n y of t h e h i g h l y v o l a t i l e fluorocarbon 12 (Graf et al., 2969) in t h e s a m p l e m a y h a v e escaped before c h r o m a t o g r a p h i c analysis, a p o s s i b i l i t y which was e x c l u d e d b y us, since we p u t t h e blood s a m p l e s d i r e c t l y into p r e c o o l e d h e x a n e u n t i l c h r o m a t o g r a p h y was begun.
Acknowledgements. I am most indebted to Dr. K. Brandt and Mr. K. Ramthun for their gas chromatographic analysis of the propellants, to Mr. K. H. Bozung for his measurement of the arterial blood gases, to Mr. L. Nikolaidis for his practical assistance in the inhalation studies, to Mr. G. Goetze and Mr. F. Pabst for their technical assistance with the inhalation equipment, )fir. F. Otterbach and Mr. K. Hemmkeppler for designing the electronic control system, and Dr. F. Knappon for his consultation and help with the biometric calculations. References Bauer, R.: Pharmakologie und Pharmakokinetic yon Atrovent. Wien. med. Wschr. 124 (Suppl. 21), 12--15 (1974) Bauer, R., Wick, H. : Ipratropiumbromid (Sch 1000) als Aerosol - - ein Anticholincrgicum zur Behandlung obstruktiver Atemwegserkrankungen. I. Int. Kongr. ffir Aerosole in der Medizin, t9.--21.9. 1973, Baden bei Wien Dollery, C. T., Davies, D. S., Draffan, G. H., Williams Faith, M.: Blood concentrations in man of fluorinated hydrocarbons after inhalation of pressurized aerosols. Lancet 1970 H, l i 6 4 Fultyn, R. V. : Contaminant generators for continuous exposure inhalation chambers. Amer. industr. Hyg. Ass. J. 22, 49--53 (1961) Graf, E., Graser, M. : Pharmazeutische Aerosole. Mitt. dtsch, pharm. Ges. 39, 97 (1969) Hanna, M. G., 2qettesheim, P., Gilbert, J. R. : Inhalation carcinogenesis. U.S. Atomic energy commission, Oak Ridge, ~970, AEC Symposium Series 18, pp. 55--72, 393, 394, 416, 4t7 Hinners, R. G. : Animal exposure chambers in air pollution studies. Arch. environm. Hlth 13, 609--6i5 (1966) HSbel, M. : Resorption yon Wirkungssubstanzen aus Aerosolen in Abhiingigkcit vom Atmungstyp. Arch. int. Pharmacodyn. 198, 76--84 (1972) Kfibler, H. : The physiological properties of aerosol propellants. Aerosol Age 9, 44--50 and 90---91 (1964) Paterson, J. W., Sudlow, M. F., Walker, S. R. : Blood levels of fluorinated hydrocarbons in asthmatic patients after inhalation of pressurized aerosols. Lancet 1971 H, 565 Scholz, J. : Neue texikologische Untersuchungen einiger als Treibgas verwendeter FrigenTypen. Fortschr. d. biol. Aerosolf. i957--1961, Ber. fiber den 4. Aerosol-Kongrel3, S. 420 bis 429. Stuttgart: Schattauer t962 Shargel, L., Koss, R. : Determination of fluorinated hydrocarbon propellants in blood of dogs after aerosol administration. J. pharm. Sci. 61, 1445--1449 (1972) Siggaard-Andersen, 0. : A new acid-base nomogram. An improved method for the calculation of the relevant blood acid-base data. Scand. J. clin. Lab. Invest. 12, 177 (1960) Dr. G. )fuacevic C. H. Boehringer Sohn Abteilung Pharmakologie D-6507 Ingelheim am Rhein Federal Republic of Germany
