Toxicology, 8 (1977) 231--249 © Elsevier/North-Holland Scientific Publishers. Ltd.

SHORT-TERM INHALATION TOXICITY STUDIES WITH P E R O X Y A C E T Y L N I T R A T E IN RATS*

A. KRUYSSE, V.J. FERON, H.R. IMMEL, B.J. SPIT and G.J. VAN ESCH** Central Institute for Nutrition and Food Research TNO, Zeist (The Netherlands)

(Received November 26th, 1976) (Revision received March 29th, 1977) (Accepted April 26th, 1977)

SUMMARY The inhalation t oxi c i t y of p e r o x y a c e t y l nitrate (PAN) was examined in acute (single exposure), sub-acute (4-week repeated exposure) and subchronic (13-week repeated exposure) studies in rats. The 4-h LCs0 was found to be 95 ppm. In the 4-week study rats were exposed to 0, 0.9, 4.1 or 11.8 ppm PAN vapour for 6 h/day, 5 days/week. Exposure to 11.8 ppm caused abnormal behaviour, growth retardation, mortality, elevated haemoglobin contents, haematocrit values and e r y t h r o c y t e counts, increased lung weights and severe in f lammato r y changes and epithelial hyper- and metaplasia in the respiratory tract. At 4.1 ppm minimal behavioral disturbance, transient growth depression, slightly increased lung weights and mild histopathological changes in the respiratory tract were found. At 0.9 ppm no treatment-related alterations were detected. In the 13-week study rats were exposed to 0, 0.2, 1.0 or 4.6 ppm PAN vapour for 6.5 h/day, 5 days/week. Exposure to 4.6 ppm resulted in changes similar to those f ound at 11.8 ppm in the 4-week experiment, b{lt no mortality occurred. At 1.0 ppm minimal irritation of the mucous membranes in the nasal cavity was the only PAN-related effect observed. No treatmentrelated changes were seen at 0.2 ppm. It was concluded that the no-toxicef f ect level is between 0.2 and 1.0 ppm, and very probably close to the upper value.

*The studies were carried out at the request and for account of the Dutch Ministry of Public Health and Environment. **National Institute of Public Health, Bilthoven, Netherlands. Abbreviations: FID, flame ionization detector; GLC, gas--liquid chromatography; PAN, peroxyacetyl nitrate; SA, serum albumin; SAP, serum alkaline phosphatase; SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase; TSP, total serum protein; UGOT, urine glutamic oxalacetic transaminase.

231

INTRODUCTION Peroxyacetyl nitrate (PAN) is one of the products that may be formed by photochemical processes in polluted air [29]. In the Netherlands varying amounts of PAN (never exceeding 16 ppb) have been measured in the Delft area [19]. This region is characterized by a concentration of chemical industries and a dense traffic. A few studies on the inhalation toxicity of PAN have been carried out in man and mice [5--7,27]. The 2-h LCs0 of PAN in mice was found to be 106 ppm [5], and mortality and severe damage to the trachea and lower respiratory tract were the major effects seen in mice following exposure to PAN at a level of 15 ppm for a period of 6 months [7]. However,no quantitative data seem to be available on the sub-acute and sub-chronic toxicities of PAN. This is unlike the situation with ozone having been known for many years as an important gaseous oxidant in photochemical smog, and of which many toxicological data are available [8,9,11,14,30]. In photochemical smog, ozone occurs in much higher concentrations than PAN, and in the Delft area ozone concentrations of more than 0.25 ppm have been found

[19]. To assess the acute, sub-acute and sub-chronic inhalation toxicities of PAN, a single exposure study as well as 4- and 13-week repeated exposure studies were carried o u t in rats. The results of these experiments are compared with data on the t oxi c i t y of ozone as found in the literature. EXPERIMENTAL Material and exposure system PAN was synthesized in the Central L a b o r a t o r y TNO, Delft, Netherlands, and stored, diluted in nitrogen (approx. 250 ppm ) in stainless steel bottles o f 50 1 capacity at a pressure of 15 kg/cm 2. Before use, gas samples of each bottle were analyzed by means of infrared spectroscopy.* From th e bottles the PAN-nitrogen m i xt ure was dosed at different concentrations to 15 1 glass exposure chambers [12] by means of a system consisting o f an o x i d a n t - p r o o f reducing valve, stainless steel needle valves, stainless steel/glass flowmeters and stainless steel and glass piping. For each c ha m ber the PAN-nitrogen m i x t u r e was diluted with measured flows o f filtered air and oxygen in the p r o p e r ratio in order to provide the test atmosphere. The total air flow rate per chamber was 5.5 l/min in single exposures and 6.5 1/min in repeated exposures. The chambers were kept at a temp er atu r e of 22--24°C. The relative hum i di t y of the at m osphere was 50 G0%. Each cham be r contained 10 rats. Animals and diet SPF-Wistar rats were obtained from The Central Institute for the Breeding o f L a b o r a t o r y Animals TNO, Zeist, Netherlands. After each exposure the *The synthesis and infrared analyses of PAN were performed by Dr. H. Nieboer, Central Laboratory TNO, Delft, Netherlands.

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surviving animals were provided with the Institute's stock diet and tap water ad libitum. The composition of the diet in percentages was: yellow maize, 29.7; soybean-oil meal, 11.0; whole meat, 36.0; fish meal, 7.0; meat scraps, 4.0; brewer's yeast, 3.0; grass meal, 3.0; dried whey, 2.0; steamed bone meal, 0.4; trace mineralized salt, 0.5; vitamin preparations, 0.4; soybean-oil, 3.0.

Experimental design and conduct Single exposure study. Seven groups of rats each consisting of 4 or 5 males and 4 or 5 females (9 weeks old, males weighing 205--255 g and females 161--202 g) were exposed once for a period of 4 h to atmospheres containing PAN vapour at levels varying from 78 to 151 ppm. The PAN concentrations were monitored by gas--liquid chromatography (GLC) using a flame ionization detector (FID). After an observation period of 14 days, the survivors were killed by intraperitoneal (i.p.) injection of a barbiturate and examined for gross pathological changes. Animals which died during the exposure or observation periods were also autopsied. The nasal cavity, trachea and lungs from all animals of a low, intermediate and high dose group were examined microscopically. In order to detect possible PAN-induced ultrastructural changes of the alveolar lung tissue, small pieces of peripheral pulmonary tissue of 2 male rats exposed to the sub-lethal concentration of 30 ppm for 4 h and killed after an observation period of 14 days, were fixed in 1% OsO4 (0.1 M sodium cacodylate buffer, pH 7.4, 2 h, 4°C), dehydrated in acetone and embedded in Epon 812. One micron sections were stained with toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 200 electron microscope at 60 kV. Four-week repeated exposure study. Four groups of 10 male and 10 female rats each (5 weeks old) were exposed to PAN vapour at concentrations of 0, 0.9 +- 0.3, 4.1 + 0.4 and 11.8 -+ 1.8 ppm {mean -+ S.D.), respectively for 6 h/day, 5 days/week during a 4-week period. Total oxidant determinations were made in air samples taken from the inhalation chambers using a Bergshoeff fourfold absorber [2] containing a 1% buffered potassium iodide solution. The iodine liberated was determined spectrophotometrically according to the method of Saltzman [24]. Individual body weights and food consumption per group were recorded weekly. Haematological data (haemoglobin content, haematocrit value and total counts of erythrocytes and leucocytes) were collected in week 4. In this week also urine examinations were made, including appearance, pH, glucose, protein, occult blood, ketones, using Labstix® test strips (Ames, Epernon, France) and microscopy of the sediment in pooled urine samples of each group. The day after the last exposure the rats were killed by exsanguination from the abdominal aorta after anaesthesia with barbiturate i.p. and examined for gross pathological changes. The heart, kidneys, liver, spleen and lungs with trachea and larynx were weighed. Tissue samples of these organs and of the head {after removal of the skin, brain and lower jaw) were fixed in a 4%

233

neutral formaldehyde solution. The lungs were fixed by intratracheal infusion with the formaldehyde solution at 10 cm water pressure. Suitable samples of the fixed material were embedded in paraffin wax (the head after decalcification in nitric acid). Sections of 5 ~m (four transverse sections of the nasal cavity and three longitudinal sections of the larynx and trachea with main bronchi) were stained with haematoxylin and eosin and examined microscopically. Special stains applied were periodic acid Schiff, Van Gieson and Kreyberg's m e t h o d for keratin and mucin. Thirteen-week repeated exposure study. Four groups of 10 male and 10 female rats each (4 weeks old) were exposed to 0, 0.20 -+ 0.04, 1.0 +- 0.1 and 4.6 -+ 0.5 ppm (mean + S.D.), respectively for 6.5 h/day, 5 days/week during a 13-week period. Air samples from each inhalation chamber were analyzed dally by means of both the method used in the 4-week study and GLC, using a 0.35 m × 4 mm stainless steel column packed with QF 14.8% diglycerol 0.13% on Chromosorb G-AW-DMCS and a FID. Individual body weights and food consumption per group were recorded weekly. Haematological data (haemoglobin content, haematocrit value, e r y t h r o c y t e and total and differential leucocyte counts) were collected in week 12. In this week the same kind of urine examinations as those in the 4-week study were made in pooled urine samples of each group, while specific gravity and urine glutamic oxalacetic transaminase (UGOT) activities were determined in urine samples of the individual rats. Determinations of serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and serum alkaline phosphatase (SAP) activities, and of total serum protein (TSP) and serum albumin (SA) were performed at the conclusion of the experiment. The day after the last exposure, the rats were killed in a way similar to t h a t used in the 4-week experiment. Heart, kidneys, liver, spleen, brain, testes, ovaries, thymus, thyroid, adrenals and lungs with trachea and larynx were weighed. In order to detect possible alterations of the alveolar surfactant in the lungs, alveolar bubble-stability tests were performed according to Pattie [20] and Slavkovik et al. [26] in air bubbles, isolated from pieces of fresh lung tissue of 3 males and 3 females from both the control and highest dose group. These rats were also used for determining the collagen and elastin content of the lungs. Samples of fresh lung tissue (stored at - 2 0 ° C ) were treated as described by Naum and Morgan [18]. Collagen was determined in the supernatant after autoclaving in water using the method of Stegemann and Stalder [28]. Elastin assays were carried out by the Lowry procedure [16] after solubilization of the residue by proteolysis with elastase. Tissue samples of the organs weighed and also of the salivary glands, head, stomach, pancreas, exorbital lachrymal glands, small and large intestines, aorta, mesenteric lymph nodes, urinary bladder, epididymis, prostate, uterus, skeletal muscle and skin were fixed and further processed according to the methods employed in the 4-week study. The head, larynx, trachea and pulmonary lobes of all animals were examined microscopically. Microscopy

234

of the other tissues was restricted to all males and females of the top dose and control groups. The following histochemical enzyme determinations were carried out on cryostat sections of specimens of gelatin-supported lungs (frozen in isopentane at - 7 0 ° C ) from 3 males and 3 females of each group: lactate dehydrogenase [21], glucose-6-phosphatase [10] and acid phosphatase [10]. For electron microscopical examinations samples of peripheral pulmonary tissue from one male and one female of each group were collected. The lungs of anaesthetized animals were fixed (with closed thoracic cavity) by intratracheal infusion of the fixative (3% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.05 M CaCl2 anhydride). The trachea was ligated and the expanded lungs with trachea were stored in the fixative (pH 7.4) at 4°C for 24 h. Small cubes were stored in the fixative for another 24 h. The material was washed in the cacodylate buffer without CaC12 during 2.5 h and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer with 0.05 M K3Fe(CN)6 (pH 7.4, 4°C). After dehydration and embedding in Epon 812, sections were stained with uranyl acetate and lead citrate and viewed with a Philips EM 200 electron microscope at 60 kV. Statistical analysis. Statistical analyses were carried out using the Studentt-test for the changes in body weights and organ-to-body weight ratios, whereas haematological and biochemical values were evaluated by means of the test of Wilcoxon. RESULTS

Single exposure study In the single exposure test, the rats kept their eyes closed when exposed and eye irritation was evident. At the beginning of an exposure, a respiration frequency of approx. 60 per min and frequent coughing were observed. This mode of respiration was followed by a dyspnoeic pattern of several rapid breathing movements with apnoeic pauses. After 2 h the respiration frequency was approx. 90 per min and m o u t h breathing occurred. In some animals at each exposure level the respiration became laboured at last. All deaths among males and most deaths among females occurred within 24 h after the start of an exposure. The 4-h LCs0 was calculated according to the m e t h o d of Litchfield and Wflcoxon [15], and was found to be 95 ppm with 116 and 78 as the 95% confidence limits. Gross examination at autopsy revealed focal congestion, severe oedema and focal emphysema in the lungs of rats that died during exposure or within 24 h after exposure. Animals which survived the 14-day observation period did not show PAN-related gross changes. However, when the thoracic cavity was opened, the lungs did not collapse well, indicating reduced elasticity of the pulmonary tissue. Microscopic examination of high dose rats showed focal desquamation of the epithelium lining the trachea and nasal cavity. The main findings in the

235

lungs i n c l u d e d focal c o n g e s t i o n and e m p h y s e m a , extensive alveolar and perivascular o e d e m a , d e s t r u c t i o n o f b r o n c h i a l and b r o n c h i o l a r e p i t h e l i u m and, occasionally, slightly t h i c k e n e d interalveolar septa. T h e histological changes in intermediate dose animals t h a t died were very similar to those f o u n d in the high dose group. H o w e v e r , the nasal cavity was n o t visibly a f f e c t e d . T h e animals killed at the end o f the o b s e r v a t i o n p e r i o d s h o w e d an increased n u m b e r o f m u c o u s cells in the trachea. In the lungs, alveolar o e d e m a , focal a c c u m u l a t i o n s o f cellular debris and p o l y m o r p h nuclear l e u c o c y t e s , increased n u m b e r s o f alveolar m a c r o p h a g e s , h y p e r p l a s t i c (regenerating) bronchial and b r o n c h i o l a r e p i t h e l i u m and t h i c k e n e d interalveolar septa we.re observed. In a d d i t i o n , p r o l i f e r a t i o n o f fibroblast-like cells o c c u r r e d in the p e r i b r o n c h i a l and p e r i b r o n c h i o l a r areas. T h e nasal cavity and t r a c h e a o f the low dose animals did n o t s h o w t r e a t m e n t - r e l a t e d changes. T h e interalveolar septa o f the lungs were t h i c k e n e d and the n u m b e r o f alveolar m a c r o p h a g e s was increased. In a d d i t i o n , perivascular infiltrates o f eosinophils, hyperplasia o f b r o n c h i o l a r e p i t h e l i u m and p e r i b r o n c h i o l a r p r o l i f e r a t i o n o f fibroblast-like cells were f r e q u e n t l y seen. T h i c k e n e d interalveolar septa with m a n y cells and w i d e n e d capillaries

Fig. 1. Blood-air barrier in alveolar septum. Swollen epithelial cells, severely damaged endothelial cells and focal thickening of basal membranes. As, alveolar space; Er, erythrocyte. Male rat exposed to 30 ppm PAN for 4 h and killed after a recovery period of 14 days. Uranyl acetate and lead citrate, x 24 000.

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[zig. 2. Swolle~l b l o o d capillary m alveolar s e p t u m . Only remua~lt~ o1' Lype I e p i t h e l i u m are left. D e n u d e d basal m e m b r a n e is visible (arrows). As, alveolar space; Er, e r y t h r o c y t e . Male rat e x p o s e d t o 30 p p m P A N for 4 h a n d killed a f t e r a r e c o v e r y p e r i o d o f 14 days. U r a n y l a c e t a t e a n d lead citrate. × 8 800.

containing ballooned erythrocytes were found at microscopic examination of one micron sections of alveolar tissue of rats exposed to 30 ppm PAN for 4 h and killed after a recovery period of 14 days. Ultrastructurally, the blood-'air barrier of these rats appeared to be 3--4 times thicker than normal {Fig. 1), which was mainly due to swollen cell processes of type I pneumocytes. Focal changes included severely damaged endothelial cells, denuded basal membranes and widened capillaries often containing electron-lucid erythrocytes with interrupted cell membranes (Fig. 2). The interstitium of the interalveolar septa often contained an increased amount of collagen, which could not always be distinguished from fibrin. Considerable amounts of cellular d~bris were seen in the alveolar spaces.

Four-week repeated exposure study Controls and low dose animals (0.9 ppm) in the 4-week experiment were sleeping during the exposures. Animals of the mid dose group (4.1 ppm) were more or less restless throughout the exposures, and those of the top dose group (11.8 ppm) kept their eyes closed, did not sleep and coughed frequently. Deaths (4 males and 5 femal,es) occurred only at the highest exposure level. A severe growth depres§ion was observed at the highest exposure level in both sexes, whereas in the mid dose group only males showed a slight and transient reduction of growth (Table I). Food consumption and food 237

TABLE I M E A N B O D Y W E I G H T S (g) O F R A T S E X P O S E D PAN (ppm)

TO PAN FOR 4 WEEKS

Mean body weight at end of week 0

1

2

3

4

73 73 73 73

113 111 108 a 69 c

150 149 142 a 75 c

186 191 182 82 c

225 231 221 91 c

69 69 69 69

98 99 97 64 c

118 123 117 69 c

134 142 a 136 75 c

152 159 152 82 c

Males 0.9 4.1 11.8

Females 0 0.9 4.1 11.8 ap < 0.05. c p < 0.001.

T A B L E II MEAN HAEMATOLOGICAL PAN (ppm)

VALUES

OF RATS EXPOSED TO PAN FOR 4 WEEKS

Number of rats

Hb (g/lO0 ml)

Packed RBC a cell vol. (%) (× 1 0 6 / m m 3)

WBC b (X 1 0 3 / m m 3)

10 10 10 6

14.9 14.4 14.9 16.8 c

45.1 43.5 44.7 53.8 c

6.6 6.7 6.7 8.0 c

13.8 13.9 15.8 12.4

10 10 10 5

16.2 16.8 16.4 18.0

47.4 49.1 47.1 57.2 c

7.3 7.6 7.2 8.2

17,4 14.7 16.2 13.7

Males 0 0.9 4.1 11.8

Females 0 0.9 4.1 11.8

a R e d b l o o d cells. b W h i t e b l o o d ceils. e p < 0.05.

efficiency were considerably diminished only in the top dose group. Haematocrit values, haemoglobin contents and erythrocytes counts were clearly elevated at the highest exposure level in both sexes (Table II). At this level the urine of males contained a fairly high a m o u n t of crystals, that of females a relatively high number of bacteria. In both sexes of the highest dose group the relative heart and kidney weights were significantly elevated, which may at least partly be due to the

238

severe growth depression, because the absolute weights were much lower than in controls. Relative spleen weights were significantly decreased in males and females of the top dose group, whereas lung-to-body weight ratios were increased in the two highest dose groups, the response being positively related to the dose. Only the lungs of top dose rats showed gross changes attributable to treatment. The lungs of these rats did not collapse well and looked slightly too spongy. Several of the animals that died before the end of the test period showed confluent pneumonic areas. In one case adhesions had developed between the lungs and the diaphragm. Histopathological changes that could be ascribed to the inhalation of PAN were observed in the respiratory tract only. In the nasal cavity, trachea, bronchi and lungs these alterations were found at the mid and high exposure levels, whereas laryngeal changes were noticed only in rats of the top dose group. The injuries observed in "the nasal cavity of animals exposed to 11.8 ppm PAN mainly consisted of rhinitis, squamous metaplasia of the lining e p i t h e l i u m and slight ulceration in the vestibular area. An exudative rhinitis was often visible in both the naso-maxillary and ethmoid regions. Non-keratinized stratified squamous epithelium was found to cover fairly large areas of the naso-maxillary turbinates and the nasal septum. At the 4.1 ppm level nearly all animals showed a slight to severe rhinitis characterized by neutrophilic exudation in the lumen and occasionally metaplastic squamous epithelium. Areas in the larynx which are normally lined by stratified non-keratinizing squamous epithelium were covered with a thick layer of keratin in several rats of the highest dose group. Focal necrosis of the laryngeal epithelium and severe laryngitis were observed in a few top dose animals. Hyper- and metaplasia of the tracheal epithelium were c o m m o n findings at the highest exposure level. Various degrees of stratification and flattening of the epithelial cells were observed. Keratinization did n o t occur. The epithelial alterations were generally more marked in the distal than in the proximal part of the trachea. In a few animals of the top dose group plugs of mucus and cellular d~bris occurred in the tracheal lumen. Occasionally a slight tracheitis or peritracheitis was encountered. At the intermediate exposure level minimal changes of the tracheal epithelium (hyper/metaplasia) were found in a few rats only. At 11.8 ppm the bronchial tree was severely damaged. The epithelial lining of bronchi and bronchioli varied from simple epithelium consisting of irregularly-shaped low columnar, cuboidal or flattened cells to well,differentiated keratinized stratified squamous epithelium (Fig. 3). In general, the number of mucus-producing cells in the bronchial epithelium was clearly reduced, but in some animals there seemed to be locally an increased number of these cells. Conspicuous and consistent features were peribronchial and peribronchiolar fibrosis accompanied by slight infiltrates of chronic inflammatory cells (Fig. 4). Slight hypertrophy of the muscular layer was visible in some instances.

239

Fig. 3. B r o n c h u s lined with m e t a p l a s t i c sLratil+ied s q u a m o u s e p i t h e l i u m . F e m a l e r a t r e p e a t e d l y e x p o s e d t o 11.8 p p m PAN for 4 weeks. H a n d E. X 165.

D

Fig. 4. P e r i b r o n c h i a l fibrosis. B r o n c h u s is lined by m e t a p l a s t i e e p i t h e l i u m . F e m a l e r a t r e p e a t e d l y e x p o s e d to 11.8 p p m PAN for 4 weeks. H a n d E. X 40.

240

Fig. 5. Bronciaiole containing plug of mucinous material interspersed with desquamated epithelial cells and macrophages. Peribronchio[ar fibrosis. Female rat repeatedly exposed to 1 1 . 8 p p m P A N f o r 4 weeks. H a n d E. x 165.

Fig. 6. Peripheral bronchiole and proximal alveoli containing eosinophilic material, cellular d~bris and macrophages. Male rat repeatedly exposed to 11.8 ppm PAN for 4 weeks. H and E. × 160.

241

Fig. 7. G r o u p of severely d a m a g e d alveoli. Note e o s i n o p h i l i c " m e m b r a n e s " cellular d~bris a n d alveolar m a e r o p h a g e s . Male rat r e p e a t e d l y e x p o s e d to 11.8 p p m P A N for 4 weeks. H a n d E. × 450.

Bronchioles often contained plugs of mucus interspersed with desquamated epithelial cells and macrophages (Fig. 5). Occasionally, a suppurative bronchopneumonia had developed in some severely affected areas. The pulmonary tissue exhibited a scala of pathological changes (Figs. 6 and 7) such as focal alveolar oedema often accompanied by eosinophilic "membranes", increased numbers of alveolar macrophages, disruption of alveolar walls associated with a c c u m u l a t i o n s o f cellular d6bris, thickened alveolar septa containing increased numbers of cells (viz. inflammatory cells, type II pneumocytes, fibroblasts). Occasionally, groups of alveoli were completely consolidated. At the 4.1 ppm level only a few animals showed some of the above mentioned broncho-alveolar lesions to a minimal degree. The most constant features were focal increases in the number of intra-alveolar cells (macrophages, desquamated type II pneumocytes), increased numbers of mucusproducing cells in the bronchial epithelium and slightly thickened alveolar septa. However, the lungs were histologically usually indistinguishable from those of the controls.

Thirteen-week repeated exposure study In the 13-week study the behaviour of the low (0.2 ppm) and mid (1.0 ppm) dose animals did not differ from that of the controls. In the first week the animals of the highest dose group (4.6 ppm) kept their eyes closed, 242

T A B L E III M E A N B O D Y W E I G H T S (g) O F G R O U P S O F 10 M A L E A N D 10 F E M A L E R A T S E X P O S E D T O P A N F O R 13 W E E K S PAN (ppm)

M e a n b o d y w e i g h t at e n d o f w e e k 0

1

4

6

8

10

13

Males 0 0.2 1.0 4.6

61 61 61 61

93 98 93 82 c

206 216 216 180 b

262 273 274 239 a

301 317 313 286

328 340 340 304 a

362 368 366 331 a

Females 0 0.2 1.0 4.6

53 53 53 53

79 82 81 73 a

132 144 a 137 131

155 168 a 161 158

173 185 a 176 178

183 195 185 185

198 211 197 197

a p < 0.05. b p < 0.01. c p < 0.001.

were restless and occasionally coughed frequently when exposed. In the second week these rats began to show adaptation to the treatment and were sleeping for long periods, but they remained restless while sleeping. Up to week 3 slight respiration irregularities were observed. No deaths occurred. At the highest exposure level, growth was retarded in males throughout the experimental period and in females during the first week only (Table III). This phenomenon was accompanied by decreased food intake. The blood of males and females of the high dose group contained a decreased number of lymphocytes and an increased number of neutrophilic leucocytes (Table IV). The males of this group showed slightly elevated haemoglobin contents. The SAP activity was slightly higher (statistically significantly) in males of the top dose group than in controls. Neither the composition nor the specific gravity and UGOT activity of the urine were adversely affected by PAN. Increased organ-to-body weight ratios were noticed for the testes, thyroid and lungs of top dose rats (Table V). In males of the three test groups the relative liver weights were significantly lower than in controls, b u t there was no dose-response relationship. The alveolar surfactant of the lungs was not noticeably affected by PAN as appeared from negative results of the bubble-stability test. The amounts of collagen and elastin in lungs of top dose rats did not differ significantly from those of lungs of controls. At autopsy, the lungs of 2 males and 1 female of the highest concentration group looked spongy and collapsed insufficiently. No other gross changes were seen that could be ascribed to PAN exposure. Histopathological changes

243

t~

15.4 15.2 15.0 15.4

15.3 15.4 15.4 15.9 c

a Red blood cells. b White blood cells. c p < 0.05.

0 0.2 1.0 4.6

Females

0 0.2 1.0 4.6

Males

47.0 46.9 47.0 47.4

47.1 47.4 47.6 49.0

Packed cell volume (%)

Hb (g/100 ml)

PAN (ppm)

7.8 7.7 7.8 8.0

8.1 8.0 8.2 8.0

AND

13.4 11.1 13.6 13.6

14.9 14.5 14.7 13.4

RATS EXPOSED

90.5 89.8 85.5 83.6 c

86.6 86.1 80.7 78.0 c

Lymph

7.8 8.5 13.0 15.3 c

12.1 12.7 17.8 21.5 c

Neutr

Differential c o u n t

10 F E M A L E

WBC b (X 1 0 3 / m m 3)

Leucocytes

O F 10 M A L E

RBC a (x 1 0 ' / m m 3)

OF GROUPS

VALUES

IV

MEAN HAEMATOLOGICAL F O R 13 W E E K S

TABLE

0.7 1.7 1.5 1.1

1.4 1.2 1.5 0.5

Eos

TO PAN

0 0 0 0

0 0 0 0

Mono

TABLE V MEAN BODY WEIGHTS (g) AND MEAN RELATIVE ORGAN WEIGHTS (g/100 g body weight) OF RATS EXPOSED TO PAN FOR 13 WEEKS Organ

PAN (ppm) 0

0.2

1.0

4.6

362 0.318 0.62 3.59 0.182 0.53 0.89 0.104 0.0036 0.0107 0.46

368 0.313 0.60 3.29 c 0.172 0.53 0.92 0.099 0.0047 0.0103 0.42

366 0.311 0.61 3.35 a 0.166 a 0.52 0.92 0.088 0.0033 0.0095 0.46

331 a 0.318 0.63 3.34 b 0.179 0.57 1.03 a 0.084 0.0054 a 0.0123 0.56

198 0.352 0.66 3.42 0.211 0.84 0.0343 0.188 0.0067 0.0204 0.57

211 0.346 0.64 3.25 0.215 0.83 0.0291 0.145 0.0081 0.0227 0.54

197 0.357 0.65 3.48 0.199 0.89 0.0302 0.149 0.0053 0.0226 0.53

197 0.368 0.62 3.35 0.223 0.86 0.0279 0.140 0.0064 0.0220 0.73 a

Males

Body weight Heart Kidneys Liver Spleen Brain Testes Thymus Thyroid Adrenals Lungs Females

Body weight Heart Kidneys Liver Spleen Brain Ovaries Thymus Thyroid Adrenals Lungs a p < 0.05. bp