Acra pharmacol. et toxicol. 1979, 45, 352-378.

From the Research Laboratories, H. Lundbeck & Co. A h , Ottiliavej 7-9, DK-2500 Valby, Denmark

Studies on the Fate of Vegetable Oil after Intramuscular Injection into Experimental Animals BY Ove Svendsen and Tove Aaes-Jergensen (Received February 22, 1979 Accepted May 10, 1979)

Absfracr: The fate of sesame oil and Viscoleo was studied in dogs after single intramuscular injection into the thigh of 14C-labelledoil (0.5 ml/kg). Parts of Viscoleo was absorbed as liquid oil to the regional lymph nodes occasionally to such an extent that small oil droplets were drained from the iliac lymph nodes into the lymph of the thoracic duct causing pulmonary oil microembolisation. Sesame oil was also absorbed to the regional lymph nodes after single injection but in no case to such an extent that pulmonary oil microembolism occurred. In dogs which received unlabelled sesame oil (0.45 and 1.0 ml/kg) or Viscoleo (0.45 ml/kg) intramuscularly once a week for six months and finally an injection of 14C-labelledoil, pulmonary oil microembolism was seen in all three groups and most markedly after the higher sesame oil dose. The iliaclymph nodes in both sesame oil dose groups were highly enlarged and appeared cystic due to presence of oil. The lungs from the dogs receiving Viscoleo were seat of small mononuclear interstitial cell infiltrations sometimes present in the vicinity of oil microemboli. In the sesame oil groups oil was additionally found extravascularly in the lung interstities together with accumulation of macrophages and leucocytes and focal hemosiderosis in the lungs occurred on the highest dose level. In the sesame oil groups of dogs pulmonary oil microembolism was found microscopically in a higher number than that seen on autoradiograms. The embolisation obtained at one injection may thus not disappear before the next injection. Except for presence of very few oil microemboli and small focal areas with interstitial oil deposits the lungs were normal in dogs examined three months after six month chronic weekly injection of sesame oil. Small oil deposits were still present at the injection site and in the iliac lymph nodes ofthese dogs, but the lymphoid tissue of the nodes had partly recovered. Pulmonary oil microembolism was also seen microscopically in rabbits after intramuscular injection of sesame oil or Viscoleo three times a week for two weeks, and also in rats after injection of sesame oil three times a week for five weeks. The content ofradioactivity in the heart, kidneys, liver and lungs were generally low and the liver had the highest content (up to 1.32% of dose). The content in the lungs was not increased by presence of Viscoleo microemboli whereas sesame oil microembolisation significantly raised the amount of radioactivity in the lungs. The amounts of radioactivity at the injection site of dogs were reduced to half of the injected dose two days after injection of ''C-Viscoleo and five weeks after injection of "C-sesame oil. In rats receiving an intramuscular injection (0.5 ml) into the thigh of ''C-Viscoleo or l4Csesame oil the amounts of radioactivity at the injection site disappeared exponentially with time, and half of the dose had disappeared one week after injection of 14C-Viscoleoand9 weeks after injection of I4 C-sesame oil. The acute intravenous toxicity of sesame oil and Viscoleo was estimated in rabbits by determination of the LDSO dose which for sesame oil was 0.74 ml/kg and for Viscoleo 0.84 ml/kg. In the lungs of rabbits receiving 0.5 ml/kg of sesame oil or Viscoleo intravenously, numerous focal haemorrhages were present in the sesame oil group. Four weeks after the injection oil microembolisation was still pronounced in lungs from the sesame oil group while only few emboli were found in lungs from the Viscoleo group two weeks after the injection, In addition sesame oil was found extravascularly in the lung interstities with cellular infiltrate. Thus the pulmonary Viscoleo microemboli disappeared considerably faster than the sesame oil microemboli. In conclusion the present studies have revealed that chronic intramuscular injection of large volumes of two different oily drug vehicles may cause pulmonary oil microembolisation after lymphatic absorption as liquid oil. This occurred in spite of markedly different absorption rates of oil from the injection site. Toxicological implications of the present experiments with intramuscular injections ofoil were related to the injection site, the regional lymph nodes and the lungs. Consequently the present finding demonstrate that the regional lymph nodes and the lungs may be the organs of interest in future studies on the relations to the human clinic. If

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pulmonary oil microembolisation should occur in patients receiving large volumes of oil the pathological sequelae may according to the present findings in animals be without importance. Key-words: Sesame oil - Viscoleo

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injection, intramuscular - lymphogen absorption - dogs - rats - rabbits.

Different mineral and vegetable oils have been used extensively as vehicles for intramuscular drug preparations such as hormones, antibiotics, fat soluble vitamins, and neuroleptic and cytostatic depot preparations. Mineral oils cause intolerable local effects at the injection site (Symmers 1955; Anonymous 1965). They have consequently been abandoned as vehicles except for use as inflammatory adjuvant in preparations for experimental immunization programs. Vegetable oils also cause local effects at the injection site but to much less extent (Brown ef a/. 1944), and some are presently widely used as vehicles for a number of drug preparations. The fate of oil after intramuscular injection has not received much attention during the years since oil was introduced as vehicle for drug preparations. The metabolic fate of a mineral oil adjuvant has been studied by Bollinger (1970) after intramuscular injection of ''C-labelled mineral oil to rats and monkeys. It has been shown that some vegetable oils disappear slowly from the injection site in mice and rats (Deansly & Parkers 1935; Emery ef a/. 1941). That oil is subject to slow absorption from the injection site is generally accepted since the duration of pharmacological action of drugs dissolved or emulsified in oil is longer than that of a water solution. Slow release of drug has most convincingly been demonstrated for various intramuscular depot neuroleptic preparations (Dreyfuss efal. 1976;Pedersenetal. 1977;Aaes-Jsrgensen et al. 1977; Jergensen 1978). The oil is thought to disappear by local metabolic degradation, absorption to the blood and phagocytosis. Recent publications have demonstrated that oily drug preparations administered intramuscularly may be drained to the regional lymph nodes in man (Ahmed & Greenwood 1973) and animals (Behrens et a/. 1975). It has also been reported that sesame oil (Nakamoto ef a/. 1975; Hashida ef a/. 1977) and methyl oleate (Tanaka et a/. 1974) are subject to lymphatic absorption in rats after intramuscular

injection, and this absorption pathway is highly facilitated if the oil is administered as a water-in-oil emulsion. In this laboratory a high dose study in pigs has been carried out in order to elucidate if two widely used oily vehicles, sesame oil and Viscoleo, were subjected to lymphatic absorption after intramuscular administration. Viscoleo is a thin vegetable oil of triglycerides containing only short chain saturated fatty acids, caprylic acid ( 5 5 % ) , capric acid (40%) and lauric acid (5%). Sesame oil is a more viscous vegetable oil of triglycerides containing mainly long chain saturated and unsaturated fatty acids, palmitic acid (8.5%), stearic acid (4.5%), oleic acid (47.4%), linoleic acid (39%) and arachidic acid (0.6%). These two oils cover the broad spectrum of different vegetable oils. The iliac lymph nodes of the pigs were distended and black discoloured (the oil contained 0.5% Sudan black as a marker), and free oil droplets appeared in the lymph of the thoracic duct (personal observation, see appendix A). In addition black discolouration of the lungs was evident particularly with sesame oil. Microscopic examination revealed extensive oil microembolisation. Both oils were thus absorbed by the lymphatics, the oil retaining capacity of the regional lymph nodes being exceeded due to the high dose and pulmonary oil microembolisation developed subsequently . In a study on the pathomechanism of traumatic pulmonary fat embolisation Fuchsig (1966) gave an intramuscular injection of '3'J-labelled triolein into the lower limb of rabbits subjected to haemorrhagic shock and observed radioactivity in lymph from the thoracic duct and pulmonary oil microembolisation. Pulmonary oil microembolism after intramuscular administration of oil into normal animals has, however, never been described. The following studies which intended to elucidate elements of the fate of sesame oil and Viscoleo after intramuscular injection into experimental animals were therefore initiated.

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Materials and Method A. Studies in dogs. I . Single intramuscular injection. i4C-labelled sesame oil or Viscoleo ( o h m vegetabile tenue, DAK 63) were given intramuscularly to dogs. Viscoleo was ''C-labelled by mixing sterile Viscoleo with small amounts ofglyceryl trioctanoate-'4C(NewEngland Nuclear) whereas sesame oil was 14C-labelledby mixing sterile sesame oil with small amounts of glyceryl trioleateI4C (Radiochemical Centre, Amersham). The specific activity of the sesame oil preparation was 37.0 pCi/ml while that of the Viscoleo preparations varied from 20.4 to 100.5 pCi/ml. Twenty-six pure-bred Beagle dogs of either sex (b.wt. 5.2-12.4 kg) were used. The labelled oil was given intramuscularly into the thigh (0.5 ml/kg) and the dogs were killed at different times after the injection. In the Viscoleo group two dogs were killed 1, 2, 3,4 and 6 days after the injection, while in the sesame oil group two dogs were killed 1, 2, 4, 6, 14, 21, 28 and 35 days after the injection. All dogs were autopsied after exsanguination in Narcodorma anaesthesia. During autopsy special interest was payed to the injection site, regional lymph nodes and lungs. The heart, kidneys, liver and lungs were dissected free and weighed. The inguinal superficial, popliteal and iliac lymph nodes from each side of the animal were also dissected free and weighed. The muscle tissue from the injected thigh was isolated from the bone and weighed. All organs and tissues were isolated carefully in order to avoid spreading of the radioactivity. Pieces of lung tissue were fixed in neutral phosphate buffered 10% formalin. Paraffin sections were stained with haematoxylin andeosin (H. +E.)and frozen sections (C02 freeze microtome) were stained with Sudan 111and hemalun for oil deposits. Preparation for liquid scintillation counting. Heart. kidneys. liver and lungs. Samples were homogenized with 4 volumes of water (Ultra Turrax homogenizer). To 200 p1 aliquots of the homogenates were added 400 p1 of Soluene 350s Solubilizer (Packard). After incubation for one hour at 50°, the samples were bleached by adding 200 p1 of isopropranol and 200 pl of 30% H~OZ-SOI. The samples were kept for 10 min. at room temperature, and then at 40" for 30 min. After cooling 10 ml of Insta Gel@(Packard) were added t o each sample. Lymph nodes. The samples were minced. One ml of Soluene 350s was added per 100 mg of lymph node. The samples 'were kept at 50" until the tissue was dissolved. To a 440 pI aliquot of the dissolved sample were added 200 pl of isopropanol and 200 pl of 30% H202-~01.,and the samples were treated as described above. Muscle tissue. The muscle tissue was homogenized in a MSE Atomix homogenizer. The homogenates were refluxed for one hour in two volumes of 0.5 M ethanolic KOH. After centrifugation thesupernatant was kept at 5"

overnight. The next day the supernatant was filtered to remove the stiffened fat. The fat was dissolved in 30 ml of chloroform. To 200 pl aliquots were added 10 ml of Insta [email protected] o 200 pl samples of the filtrate were added 200 p1 isopropranol and 200 pI 30% H~OZ-SOI.. and the samples were treated as described above. Autoradiography. Autoradiography was performed as described by Ullberg (1954). Pieces of lung were sliced into 40 p sections. After drying the sections were pressed against x-ray film (Structurix D 10, Agfa Gevaert). The time of exposure varied, depending on the amounts of radioactivity in the sections.

2. Chronic intramuscular injection. Three male and three female Beagle dogs received 0.45 ml/kg/week of Viscoleo and another three males and three females served as untreated control animals. For the sesame oil study eighteen Beagle dogs were divided into three groups each consisting of three males and three females. Group one and two received 0.45 and 1.0 ml/k$week of sesame oil, respectively, while the third group served as untreated control group. An additional group of four dogs (two males and two females) used for study of reversibility of treatment-induced changes received 0.45 ml/kg/week. Sterile Viscoleo and sesame oil in vials were used.The oil was injected intramuscularly into the thigh once a week for six months. The successive weekly injections were given alternately in the right and left thigh. The Viscoleo and sesame oil used for the last injection were 14 C-labelled as described above. Thegroup used for study of reversibility of treatment-induced changes received unlabelled sesame oil. The specific activity of the Viscoleo preparation was 50.0 pCi/ml while that of the sesame oil preparations were 88.6 and 43.2 pCi/ml for the group receiving 0.45 and 1.0 ml/kg, respectively. Haematological, clinical chemical and urinary examinations were performed once before dosing and in the 4th and 8th week and 4th and 6th month of the dosing period. The haematological tests comprised total red cell count, total white cell count, platelet count, haemoglobin concentration, packed cell volume, erythrocyte sedimentation rate, coagulation time, reticulocyte count and differential white cell count. The clinical chemical tests on blood comprised glucose, urea, creatinine, bilirubin, cholesterol, creatine phosphokinase, GOT, GPT, alkaline phosphatase, total serum protein, electrophoretic serum protein pattern, sodium and potassium. The urinalysis comprised colour, specific gravity, pH, spun sediment examination, protein, glucose, ketones, bile pigment and haemoglobin. Two dogs from each group were autopsied after exsanguination in NarcodormQ anaesthesia I , 2 and 6 days after the injection of ''C-labelled oil. The dogs used for study of reversibility of treatment-induced changes were autopsied 1 or 3 months (one male and one female at each time) after the last injection. The following organs were dissected free from fat and weighed: Kidneys, liver, ovaries, brain, pituitary, heart, pancreas, prostate, lungs,

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FATE O F INTRAMUSCULAR OIL thyroids, spleen, testes, uterus and adrenals. Small tissue samples from these organs and from the following organs and tissues were fixed in formalin except for the eyes which were fixed in Zenckers fluid: Lymph nodes (submandibular, prescapular and iliac or accessory iliac*, salivary glands (parotis and submaxillaris), thymus, oesophagus, stomach (fundus and pylorus), bone marrow urinary bladder, aorta, gall bladder, spinal cord with ganglions (cervical and lumbar intumescence), parathyroids, tonsils, duodenum, jejunum, terminal ileum, colon, injection site, skin, peripheral nerve (right radial), skeletal muscle and eyes. The earlier mentioned regional and corresponding contralateral lymph nodes and the muscle tissue from the thigh injected with ''C-labelled oil were isolated and weighed. Paraffin sections were stained with H.+E. whereas frozen sections of liver and lungs were stained with Sudan 111 and hemalun. Paraffin lung sections from the animals receiving sesame oil were also stained with Perk stain for demonstration of haemosiderin. The same organs and tissues as mentioned under single intramuscular injection were prepared for liquid scintillation counting and autoradiography. 0. Studies in rats.

I. Single intramuscular injecrion. A single intramuscular injection (0.5 ml) of ''C-labelled Viscoleo or sesame oil was given into the thigh of male rats (Wistar/Af/Han 67/Mol, SPF, 175-227 g b.wt.). The specific activity of the oil was 5.4 pCi/ml for Viscoleo and 5.5 pCi/mI for sesame oil. The rats were killed by chloroform inhalation at different times after the injection. From the Viscoleo group two rats were killed 7 and 16 hours, 1,10, 14and 21 days after the injection while four rats were killed 3,s and 7 days after the injection. From the sesame oil group two rats were killed 7 and 16 hours, I, 3,5,7, 10, 14.21.28.35 and 49 days after the injection. The injected hind leg was isolated by an incision through the hip joint, weighed and prepared for liquid scintillation counting. The leg was refluxed for one hour with 4 volumes of 0.5 M ethanolic (95%) KOH. After cooling 1 ml aliquots were counted in 10 ml of Insta Gel@. 2. Repeated intramuscular injection. Five groups of five male Wistar rats (125-185 g b.wt.) were used. One group served as control group and was not treated. Group two and three received intramuscularly 0.25 and 0.5 ml of Viscoleo, respectively, every Monday, Wednesday and Friday for five weeks. Group four and five received intramuscularly 0.25 and 0.5 ml of sesame oil, respectively, as mentioned for group two and three. In *The iliac lymph nodes from the dogs receiving I4Clabelled oil were used for scintillation counting while one or two small accessory iliac lymph nodes were used for microscopical examination.

all four groups the injections were given alternately in the right and left thigh. The rats were killed by chloroform inhalation three days after the last injection and autopsied. Tissue specimens from the injection site, the iliac lymph nodes and the lungs were fixed in formalin. Paraffin sections were stained with H.+E. and frozen lung sections with Sudan 111 and hemalun. C . Studies in rabbits. 1. Acute intravenous lethality.

Fourty-one New Zealand White rabbits 1.5-2.3 kg b.wt.) of either sex received different volumes of Viscoleo or sesame oil (table 1) intravenously into a marginal ear vein. The dose was administered during half a minute. The animals were observed regularly for 24 hours after the injection and clinical symptoms and the number of deaths were recorded. The LD5O dose was calculated from the death rats by probit analysis (Finney 1952). 2. Lung pathology after single intravenous injection. Thirthy rabbits (2.9-4.4 kg b.wt.) of either sex were used. Fourteen rabbits received an intravenous injection into a marginal ear vein of0.5ml/kgofViscoleo. Another fourteen rabbits received 0.5 ml/kg of sesame oil by the same route. The remaining two rabbits served as control animals and were not treated. Two rabbits from each group were killed by an intravenous overdose of Narcodorme 4 hours, 2 and 4 days, I , 2, 3 and 4 weeks after the injection. The control rabbits were killed 4 weeks after start of the experiment. The rabbits were autopsied and the lungs were fixed in formalin. Paraffin sections were stained with H. E. and frozen sections were stained with Sudan 111and hemalun. Seven rabbits receiving sesame oil died before planned day of autopsy. They were replaced by new rabbits receiving 0.5 or 0.25 ml/kg of sesame oil.

+

3. Repeated intramuscular injection. Ten rabbits of either sex (3.1-4.2 kg b.wt.)were divided into two groups of five animals. One group received an intramuscular injection of 1 .O ml/kg of Viscoleo every Monday, Wednesday and Friday for two weeks alternately into the right and left thigh. The other group received sesame oil by an identical schedule. Both oils were stained by addition of 0.5% Sudan black. The rabbits were killed by an intravenous overdose of Narcodorme three days after the last injection and autopsied. The lungs, iliac lymph nodes and muscle tissue from the injection sites were fixed in formalin. Paraffin sections were stained with H.+E. and frozen sections from each lobe of the lungs were stained with Sudan I11 and hemalun.

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Fig. 1. Iliac lymph nodes (R and C) in dogs autopsied 2 (a) and 3 (b) days after single injection of ''C-Viscoleo (0.5 ml/kg). The regional iliac lymph nodes (R) are enlarged and small oil cysts (arrows) are visible through the capsule.

Results A. Studies in dogs. 1. Single intramuscular injection.

Gross pathological findings. The iliac lymph node regional to the injection site was enlarged in all dogs receiving Viscoleo and transparent subcapsular oil droplets were present in the node from one or both dogs examined 2, 3 and 4 days after the injection (fig. I). In the muscle tissue at the injection site from most of the dogs and in the subcutaneous tissue covering the injection site varying degrees of oedema, hyperaemia and heamorrhage were observed. In the sesame oil group pin-point subcapsular oil droplets in the regional iliac lymph node were only seen in two dogs examined 21 and 28 days after the injection, respectively. The size of the contralateral iliac lymph node tended in nine dogs to be greater than the regional node. Large oil deposits were found in the thigh from all dogs. The depot was mainly located to subcutaneous tissue and inter-

muscular connective tissue around the sciatic nerve. The oil was situated freely in the tissue in dogs examined within the first week after the injection. In dogs examined two weeks or later after the injection the oil was encapsulated in a thin fibrous membrane (fig. 2). Microscopic findings. Small mononuclear interstitial cell infiltrations and perivascular cuffings were found in lungs from most of the dogs, and focal accumulations of alveolar foam cells were present in three dogs. These changes are normal occasional findings in Beagle dogs from our colony. Interstitial accumulations of lipid loaded cells were general findings in frozen lung sections from all dogs. The accumulations were most frequently located to the subpleural lung tissue. This is also a well known finding in dogs from our colony. Few pulmonary fat microemboli were seen in frozen sections from one dog autopsied 6 days after the injection of Viscoleo. The emboli were located

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Fig. 2. The thigh of a dog au topsied 21 days after single injection of ''C-labelled sesame oil (0.5 ml/kg). 1The superficial muscles have been removed. Interstitial oil cysts envz:loped in transparent membrane are indicated by arrow heads.

in a small area with mononuclear interstitial cell accumulation. Similar accumulations appeared also without fat embolisation. In addition, free fat droplets appeared localized to empty spaces in the lung tissue from one dog autopsied 1 day and one dog autopsied 3 days after the injection and from both dogs autopsied 2 days after the injection. Several pulmonary fat emboli were found in one dog receiving sesame oil (autopsied 6 days after the injection) but the regional iliac lymph node was not enlarged and oil droplets did not appear through the capsule. One fat embolus appeared in another dog autopsied 28 days after the injection. Besides, free fat droplets in the lung tissue were seen in one dog autopsied 1 day after the injection. Distribution of radioactivity.

The content of radioactivity in organs and lymph nodes is shown in figs. 3 and 4. The contents are expressed as percentage of dose. The individual values of the two dogs examined at each time varied considerably in several cases. Six days after the injection of 14C-Viscoleothe mean content

of radioactivity in the kidneys, liver and heart had declined compared to the content 3 or 4 days after the injection. The mean content in lungs was highest 6 days after the injection, in mean 0.34%of the dose. Peak content in regional iliac lymph node was seen 2 days after injection of ''C-Viscoleo (fig. 4). The activity in one of these nodes corresponds to the activity in about 100 p1 Viscoleo. The activity was low in the contralateral iliac lymph nodes. The activity in the regional popliteal and inguinal superficial lymph nodes was also considerably higher than that in corresponding contralateral nodes. The percentage of dose values from the thigh receiving 14C-Viscoleoare shown in fig. 5 . The content of radioactivity seems to decline exponentially and the half-life for the disappearance of radioactivity has been estimated to about 2 days. In the I4C-sesame oil group the amount of radioactivity in heart, liver and kidneys (fig. 3) increased during the entire observation period. The content in liver was the highest obtained (mean 0.15% of dose). The highest content obtained in

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OVE SVENDSEN AND TOVE AAES-JBRGENSEN

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FATE OF INTRAMUSCULAR OIL

lungs and heart was lower, 0.042% and 0.055% of dose, respectively. In the regional iliac lymph nodes the highest amount of radioactivity was obtained 28 days after the injection of I4C-sesame oil (fig. 4). These lymph nodes contained the highest amounts obtained in any of the lymph nodes and the content was up to 425 times higher than that of the contralateral iliac lymph node. In the regional popliteal and inguinal superficial lymph nodes the content was also considerably higher than that in the corresponding contralateral nodes. About 70% of the injected dose of I4C-sesame oil was found in the thigh 1 and 2 days after the injection (fig. 5). A part of the remaining 30% may have disappeared through needle canal in the skin and during processing of the tissue. After 35 days about 50% of the initial dose was still present at the injection site. Autoradiography The autoradiograms revealed radioactive spots (fig. 6) in lung tissue from two dogs in the I4CViscoleo group (2 and 6 days after the injection) and from one dog in the I4C-sesame oil group (6 days after the injection). The remaining autoradiograms showed radioactivity equally distributed throughout the lung tissue representing metabolized oil. 2. Chronic intramuscular injection. Except for slight lameness in a few dogs after the first injections of Viscoleo no clinical signs related to the medication were observed throughout the study.

Gross pathological findings. Subcutaneous and intramuscular yellow discolouration and slight fibrosis were found in both thighs from most of the dogs from the Viscoleo group. Oedema and slight hyperaemia or haemorrhage were also observed in the thigh. Small oil cysts up to 4 mm in diameter were present in the intermuscular connective tissue. The iliac lymph nodes were small in four of these dogs but in two dogs the size of the nodes was increased and small oil cysts appeared beneath the capsule. Blood deposits were present in the nodes from three dogs. In the sesame oil group large oil deposits were

363

found at the injection site, and slight yellow discolouration and fibrosis were seen. The lymph nodes regional to the last injection were enlarged in all dogs. The regional iliac lymph nodes were extremely enlarged and contained numerous large oil cysts in the high dose group (fig. 7), and to a lesser extent in the lower dose group. Enlargement of the regional iliac lymph nodes was most pronounced in dogs autopsied I and 2 days after the injection. The contralateral iliac lymph nodes were also enlarged but only slightly. The iliac lymph nodes were still enlarged in dogs killed 1 o r 3 months after the last injection of sesame oil and small subcapsular oil droplets were also present. Microscopic findings. Changes related to treatment with Viscoleo appeared at the injection sites, in the iliac lymph nodes and the lungs. At the injection sites and in the accessory iliac lymph nodes oil cysts surrounded by a thin reactive granulomateous zone were general findings. In addition, haemosiderosis appeared in some nodes. In the lungs oil microembolism was seen in all treated dogs and the interstitial mononuclear cell infiltrations occasionally found in the control dogs were more frequent in the treated group and sometimes associated with the emboli. Oil microembolism was also demonstrated in the lungs from all dogs autopsied I , 2 and 6 days after the last injection of sesame oil but most pronounced in the group receiving 1.0 ml/k$week (fig. 8). In all dogs from the high sesame oil dose group and in some of the dogs from the low dose group focal interstitial oil accumulations were also present. In these foci interstitial infiltration with polymorphonuclear leucocytes and macrophages were seen. Finally, focal haemosiderosis appeared in Per1 stained lung sections from dogs in the high dose group. Oil microemboli were also found in lungs from both dogs autopsied 1 month after the last injection of sesame oil and in one dog autopsied 3 months after the last injection. Focal areas with interstitial oil deposits were occasionally found in frozen lung sections from these dogs. In the iliac lymph nodes oil cysts were present both one and three months after the last injection togetherwith haemosiderosis. The cysts were lined by a single layer of flat cells

364

OVE SVENDSEN AND TOVE AAES-JPIRGENSEN

and occasionally by a thin reactive granulomateous zone. Solitary foreign body giant cells were now and then seen close to the cysts. The size and number of oil cysts had decreased and the lymphoid tissue partly recovered three months after the last injection. Small oil cysts appeared also at the injection site three months after the last injection.

1w

Distribution of radioactivity. The content of radioactivity in organs and lymph nodes from dogs receiving ''C-Viscoleo is shown in figs. 3 and 4. The mean content in heart, liver, lungs and kidneys was highest 6 days after the injection. The largest amounts were found in

1M:

t

50

40 ' 30

w

B

0

20

8

E7 1 0 '

B 1

2

3

4 5 6 DAYS AFTER INECIIM(

1

2

3

4

5

6

DAYS MIER INXCIION

0

8

0

: 1.0 W K G

0

0.45 M/KG

D 1

2

3

4 5 . 6 DAYS d T C R INJCCllON

365

FATE O F INTRAMUSCULAR OIL

100

1:. b

I.

0

40 '

30

'

20

'

0

'

50 ( 0 )

0

w

ff

25 c w

10 w

E 2

6

4

8

10

12

14

18

16

DAYS

20

22

AFTER INJECTION

a 0

a

so..

8

40 '

30 .. %

s

20

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= 10" a. w

F 4

8

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20

24

28

32

, 36

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DAYS

44

48

AFTER INJECTION

Fig. 5 . Disappearance of radioactivity from the injection site after intramuscular injection of 14C-labelledsesame oil or Viscoleo into dogs and rats. Results are expressed as % of dose. A-Dogs, single injection of Viscoleo (0.5 ml/kg) B -Dogs, chronic injection of Viscoleo (0.45 rnl/kg/week) C -Dogs, single injection of sesame oil (0.5 ml/kg) D-Dogs, chronic injection of sesame oil (0.45 and 1.0 ml/kg/week) E -Rats, single injection of Viscoleo (0.5 ml) F-Rats, single injection of sesame oil (0.5 ml) ( 0 ) Oil was missed during excision.

366

OVE SVENDSEN AND TOVE AAES-JBRGENSEN

the liver (0.56% of dose) andlungs (0.28% ofdose). Among the lymph nodes the highest amounts of radioactivity were obtained in the regional iliac lymph nodes. The remaining lymph nodes contained very small amounts of radioactivity but the content in the regional nodes was higher than that in the corresponding contralateral nodes. The content of radioactivity at the injection site is shown in fig. 5. The content was reduced to half of the initial level about 2 days after the injection of 14 C-Viscoleo. Generally, the amounts of radioactivity in organs and lymph nodes (figs. 4 and 5) from the dogs receiving 0.45 ml/kg/week of sesame oil were similar to those found after single administration but the regional iliac lymph nodes contained higher amounts. The content of radioactivity in the regional iliac lymph nodes was high (up to 4.4% of the dose) in the group receiving I .O ml/k$week. The amounts of radioactivity at the injection sites were relatively low one day after the injection of I4C-sesame oil and did not decrease during the following 5 days. A utoradiography.

Several radioactive spots were found in the lung tissue from all dogs in the Viscoleo group (fig. 6). Radioactive spots were also found in one dog from the group receiving 0.45 ml/kg of I4C-sesame oil and in four dogs from the group receiving 1.0 ml/kg of I4C-sesame oil. Three of the latter four dogs had relatively high amounts of radioactivity (up to 0.24% of the dose) in the lungs.

Haematology, clinical chemistry and urinalysis. No changes related to the treatments were seen. Organ weights. No changes related to the treatments were seen. B. Studies in rats. I . Single intramuscular injection - radioactivity at injecrion site. The results obtained are presented in fig. 5. In the 14 C-Viscoleo group the amount of radioactivity in the thigh disappeared exponentially with time and the half-life for the disappearance has been estimated to about 7 days. The amount of radioactivity in the thigh from the I4C-sesame oil group also

declined exponentially with time and the half-life for the disappearance has been estimated to about 9 weeks.

2. Repeated intramuscular injection. Macroscopic findings. A large oil depot was found at the injection site. The size of the depot was dose dependent. Most of the oil was present in few large cysts located to the connective tissue between the muscle groups. In the Viscoleo group no changes appeared in the iliac lymph nodes o r in the lungs. In the iliac lymph nodes from two rats in the low and four rats in the high sesame oil dose group pin-point subcapsular oil droplets were observed. The lungs from these groups were normal. Microscopic jindings. Few large irregular and a number of small cysts appeared at the injection site. The cyst membranes were mostly composed of two layers; an inner layer of macrophages and an outer thin layer of fibrous tissue. Granulation tissue was sometimes present in the outer layer of the Viscoleo cycts. In the Viscoleo group few small cycts appeared between the muscle fibres in the muscle bundles. The membrane of these cysts was composed as described above for the large cysts. A small number of muscle fibres adjacent to these cysts were atrophic, and a necrotic muscle fibre infiltrated with macrophages was sometimes seen. In the sesame oil groups a higher number of small oil cysts were seen between the muscle fibres in the muscle bundles. In most cases the cyst membrane was a single layer of flat cells. The iliac lymph nodes and the lungs from the Viscoleo groups did not reveal changes related to the treatment, i.e. no cysts were found in the lymph nodes and no oil microemboli in the lungs. Few and small oil cysts appeared in the iliac lymph nodes from four rats in the group receiving 0.25 ml of sesame oil. More numerous and larger oil cysts were present in the nodes from all rats in the group receiving 0.5 ml of sesame oil. A number of oil microemboli was observed in the frozen lung sections from two rats in the group receiving0.5 ml of sesame oil (fig. 9). Some of the emboli were surrounded by a mononuclear cell accumulation. Furthermore, these lungs were seat of peribronchi-

FATE OF INTRAMUSCULAR OIL

Fig. 6 . Autoradiograms of lungs from dogs 6 days after intramuscular injection of ''C-labelled Viscoleo or sesame oil. Several spots are localized to the lung parenchyma. A-Single injection of Viscoleo, 0.5 ml/kg B - Chronic injection of Viscoleo, 0.45 ml/kg/week C-Chronic injection of sesame oil, 1.0 ml/kg/week.

368

OVE SVENDSEN AND TOVE AAES-JBRGENSEN

a1 and septa1 infiltration with histiocytes and eosinophilic granulocytes. No changes were seen in the lungs from the group receiving 0.25 ml of sesame oil. C . Studies in rabbits. 1. Acute intravenous lethality.

The results obtained are shown in table 1. The rabbits that died showed respiratory distress few minutes after the injection and most of them had

convulsive attacks shortly before death. They died within 30 min. after the injection except for 3 rabbits receiving 0.75 ml/kg of sesame oil. One of these died about four hours after the injection and two died during the night.

2. Lung pathology after single intravenous injection. Numerous focal pulmonary haemorrhages were present in the lungs from most rabbits treated with

Fig. 7. Iliac lymph nodes (Rand C) in dogs autopsied 1 (a) and 6 (b) days after weekly intramuscular injection of sesame oil (1.0 ml/kg/week alternately into the right and left thigh) for six months. Both iliac lymph nodes are enlarged, most pronounced of the node regional (R) to the last injection and numerous oil cysts (arrows) are visible through the capsule.

FATE OF INTRAMUSCULAR OIL

369

Fig. 8. Microscopical appearance of lung from a dog treated as mentioned in fig. 7. Oil microembolisation is evident. The arrow heads indicate interstitial sudanophilic material, probably representing extravasated oil. Note the associated cellular infiltrate (Sudan 111, magnification X 170).

Fig. 9. Microscopical appearance of lung from a rat given repeated intramuscular injections of sesame oil for 5 weeks. Few oil microemboli are present. Note the associated cellular infiltrate (Sudan 111, MagnificationX 130).

370

OVE SVENDSEN AND TOVE AAES-JBRGENSEN Table 1.

Lethality after intravenous injection of Viscoleo or sesame oil in various doses into rabbits. Viscoleo

Dose (ml/kg) 1.25

0.5

1 .o

5

5

Number of animals tested

LD50 (95% confidence limits)

1.5

5

5 0.84 ml/kg (0.58-1.21)

Number of animals died

0.50

Sesame oil Number of animals tested

2

1

5

5

3

Dose (ml/kg) 0.65 0.75 1.0 5

5

5

1.25

LD50 (95%confidence limits)

3

0.74 ml/kg (0.64-0.85) Number of animals died

0

0

sesame oil. Focal pulmonary haemorrhages were also present in rabbits examined up to 2 weeks after the injection of Viscoleo but the number and size declined during that period and they were rare and small in animals examined 3 or 4 weeks after the injection. Pronounced oil microembolism occurred in all rabbits examined 4 hours, 2 and 4 days and 1 week after the injection. Two weeks after the injection very few emboli were found in lungs from rabbits treated with Viscoleo but the embolisation was still pronounced in lungs from rabbits examined4 weeks after injection ofsesame oil. In these rabbits sesame oil was additionally present in the lung interstities accompanied by cellular infiltration. 3 . Repeated intramuscular injection.

Macroscopic findings. The main part of the plantar muscle tissue of both thighs from the group receiving Viscoleo containing 0.5% Sudan black was markedly black discoloured and after incision oil fled from cysts of different size. Round black areas from pin-point up to 2 mm in diameter were seen subcapsularly in the iliac lymph nodes. Few small round haemorrhages appeared subpleurally in the lungs from two animals. The lungs from the remaining animals were

5

4

3

seat of more pronounced haemorrhage and hyperaemia. On the dorsal surface of the diaphragm lung lobe from one animal few pin-point black spots were observed. The findings in the thigh from the group receiving sesame oil were essentially like those described for the Viscoleo group. Pin-point or larger black spots in varying numbers appeared subcapsularly in the iliac lymph nodes. The lungs were seat of focal hyperaemia, and few focal haemorrhages were seen. Small focal infiltrations were found in the lungs from one animal.

Miscroscopic findings. In the Viscoleo group numerous small round o r oval cysts were found between the muscle fibres in the muscle bundles and in granulation tissue of different thickness surrounding the muscles. The cyst membranes were composed of one to several layers of macrophages. Some of the membranes surrounding larger cysts were also composed of an outer layer of fibrous tissue. The granulation tissue in the interstities was slightly infiltrated with eosinophilic granulocytes and occasionally with multinucleated foreign body giant cells. Besides, focal haemorrhage was seen. Few degenerated muscles fibres appeared occasionally adjacent to

FATE OF INTRAMUSCULAR OIL

37 1

Viscoleo or sesame oil was roughly similar to that in dogs receiving chronic injections. All six dogs in the chronic Viscoleo study had, however, radioactive spots in the lungs while this was only found in two out of ten dogs in the single injection study. The total amount of radioactivity in lungs from dogs in the chronic Viscoleo study were grossly similar to that of dogs in the single injection study. The numerous radioactive oil microemboli in lungs from the chronic Viscoleo study may therefore not contribute significantly to the total radioactivity in the lungs. In the chronic sesame oil study, on the other hand, the radioactive oil microemboli elevated significantly the total radioactivity in the lungs. The content of radioactivity i n regional iliac lymph nodes from dogs in the chronic Viscoleo study was low. The highest amount obtained was about 0.5% of the dose, while the highest amount obtained in the single injection study was 2.8%. This finding and the fact that all dogs in the chronic study had pulmonary oil microembolism and that the disappearance rate of radioactivity from the injection site was similar to that in the single injection study suggest that the oil retaining capaDiscussion city of the iliac lymph nodes has been reduced by The present studies have demonstrated that Visco- prolonged exposure to the oil. Consequently, the leo and sesame oil administered intramuscularly to least dose of oil which can give rise to pulmonary experimental animals are subject to lymphogenic oil microembolisation may be less after chronic absorption to the regional lymph nodes as liquid administration than after single administration. In oil, and that oil absorbed by this pathway may give the sesame oil studies the content of radioactivity in rise to pulmonary oil microembolism if the oil re- the regional iliac lymph nodes was several fold taining capacity of the lymph nodes draining into higher in the chronic injection study (up to 4.4% of the thoracic duct is exceeded. Pulmonary oil micro- the dose) compared to the single injection study, embolism accompanied by presence of subcapsular and pulmonary oil microembolisation appeared microscopically in all dogs in the chronic study. oil droplets in enlarged regional iliac lymph node was seen in some dogs receiving a single intra- This also indicates that the least dose of sesame oil which can give rise to pulmonary oil micromuscular injection of 0.5 ml/kg of Viscoleo but not embolisation is lowered after chronic administraafter a single intramuscular injection of 0.5 ml/kg of sesame oil. After chronic intramuscular injec- tion. The fact that all dogs receiving chronic Viscoleo injections had radioactive spots in the tions pulmonary oil microembolism was seen in all lungs while only one out of six dogs receiving dogs receiving Viscoleo (0.45 ml/k$week) and in chronic injections of the low dose of sesame oil had all dogs receiving sesame oil (0.45 or 1.0 ml/k$ week). Pulmonary oil microembolism was also radioactive spots in the lungs suggests, however, observed in rabbits after chronic intramuscular that Viscoleo also after chronic administration injection of Viscoleo or sesame oil but in rats only passes the lymph nodes more quickly than sesame after chronic intramuscular injection of sesame oil. oil. After chronic injection considerable lymphatic The distribution pattern of radioactivity in dogs absorption of sesame oil may take place since the after single intramuscular injection of L4C-labelled regional iliac lymph nodes contained high amounts the cysts. Small oil cysts were seen in the iliac lymph nodes from three rabbits. They were surrounded by a thin reactive granulomateous zone. Oil microembolism was demonstrated in the lungs from the three animals having oil cysts in the iliac lymph nodes. Several small mononuclear cell accumulations and small perivascular cuffings were found in the lungs from all animals. These infiltrations are well known findings in the lungs of rabbits. Intermuscular granulation tissue containing small and thin walled cysts were consistent findings at the injection site from rabbits receiving sesame oil. The cysts were normally lined by a single layer of flat cells. Haemorrhages and foreign body giant cells were occasional findings. The iliac lymph nodes from one rabbit contained small oil cysts. The cysts were surrounded by a thin reactive zone. The lungs from this rabbit contained numerous oil microemboli. The lymph nodes and the lungs from the remaining rabbits were without remarks except for the occurrence of small mononuclear cell accumulations and perivascular cuffings in the lungs.

372

OVE SVENDSEN AND TOVE AAES-JBRGENSEN

of radioactivity already one day after the injection. Autoradiographically pulmonary oil ,microembolism was only found in one dog given 0.45 ml/kg/ week of sesame oil and in four dogs given 1.0 ml/kg/week. Since all six dogs in each group had pulmonary oil microembolisation microscopically this difference indicates that the oil microemboli obtained at one injection did not disappear from the lungs before the next injection. In support of this, it took more than four weeks for the rabbit lungs to be cleared of oil after intravenous injection of sesame oil while Viscoleo microemboli were only present in very low numbers two weeks after intravenous injection. Slow clearance of sesame oil from the lungs is consistent with findings in rats after intravenous injection of 13'J-labelled triolein (Paredes et af. 1965) and in dogs after intralymphatic or intravenous injection of "'J-labelled iodized puppyseed oil (Lipiodol) (Koehler et al. 1964; Threefoot 1967). Oleic acid is a major component of sesame oil, and the chemical composition of puppyseed oil is close to that of sesame oil since about 85% of the fatty acids are oleic acid and linolic acid. The major portion of neutral fat emboli in the lungs is thought to be removed by lipolytic activity of lipase, the level of which is elevated in blood and lung tissue after intravenous injection of neutral fat (Peltier & Scott 1957; Armstrong ez al. 1967). Viscoleo is less viscous and less lipophilic than sesame oil. These differences favour the biological degradation of Viscoleo. One dog killed 6 days after single injection of ''C-labelled sesame oil had radioactive spots in the lung tissue. Several fat microemboli were demonstrated microscopically in frozen lung sections from this dog and the lungs contained relatively high amounts of radioactivity. This finding would suggest lymphohaematogenic absorption of liquid sesame oil if the regional iliac lymph node contained macroscopically visible oil droplets or the size of the node was increased. But the size of this node was not increased and it did not contain visible oil droplets. Furthermore, the concentration of radioactivity in the node was lower than that of the other dog killed 6 days after the injection, and also lower than that of the two dogs having macroscopically visible subcapsular oil droplets in the iliac lymph nodes. This indicates that the

pulmonary oil microembolisation in this particular dog may have arisen from an accidental intravenous injection of some parts of the oil or that lymphogenic absorbed oil may have been drained into the blood by lympho-venous anastomoses which have been shown to be present close to the popliteal lymph node (Bron et al. 1963). Pulmonary fat microemboli were, also demonstrated microscopically in one dog killed 28 days after single injection of 14C-sesame oil but the autoradiograms failed to show radioactive spots in the lung tissue. The reason for this discrepancy may be that the lung sections used for autoradiography have not contained oil microemboli or that the emboli may represent endogenous fat as discussed later. Few reports dealing with the fate of intramuscularly administered fat or oil have been found in the literature (Bisgard & Baker 1940; Szabo et al. 1963; Fuchsig 1966; Bollinger 1970; Hashida et al. 1977), and mosts are reports on experimental studies of the pathomechanism of pulmonary fat embolisation after trauma. Bisgard & Baker (1940) found fat loaded phagocytes in lung capillaries and alveoli fourty-eight hours after intramuscular injection of bone marrow fat suspension into the thigh of rabbits and suggested that the fat had been transported to the lungs by means of phagocytes. Szabo et al. (1963) studied the appearance of radioactivity in blood and thoracic duct lymph during six hours after intramuscular injection of "'J-labelled tnolein into the thigh of dogs and found only insignificant indications of lymphogenic absorption of the oil. On the other hand, Fuchsig (1966) observed radioactivity in lungs and thoracic duct lymph after intramuscular injection of 13'J-labelled triolein into the lower limb of rabbits with haemorrhagic shock, and pulmonary oil microembolisation was found at histopathological examination (Brucke et al. 1965). The present studies have demonstrated that pulmonary oil microembolisation may arise in normal animals after single intramuscular injection of Viscoleo and after chronic intramuscular injections of Viscoleo or sesame oil. Hashida et al. (1977) found relatively high concentrations of radioactivity in the regional lymph nodes after single intramuscular injection of sesame oil or sesame oil emulsions (mixed with 14C-Iabelledtripalmitin) into the thigh of rats, and

FATE OF INTRAMUSCULAR OIL

oil droplets were observed microscopically in the node fifteen minutes after injection of a water-in-oil emulsion containing gelatine. Pulmonary fat embolism has been described to arise under three clearly different circumstances: First of all it is a well known, although rare clinical complication following bone fracture or soft tissue injury (for review see Peltier 1957 and Sevitt 1962) and a common histological but clinically insignificant sequelae to trauma. Pulmonary fat embolism has also been observed histologically in a variety of conditions such as burns, diabetes mellitus, infections and prolonged high dose treatment with steroids (vide Mahley et a/. 1972). Secondly, pulmonary oil embolism is a very common and accepted entity after lymphography by use of oily contrast media (Bron et al. 1963; Richardson et a/. 1966). Thirdly, pulmonary fat embolisation may be a normally occurring phenomenon since chylomicrons in the thoracic duct lymph may coalesce under certain conditions (Volkheimer et al. 1965). Intravenous injection of neutral fat and fatty acids have been widely used for the study of the structural and functional consequences of pulmonary fat embolism (Jefferson & Necheles 1948; Armin & Grant 1951; Peltier 1956; Sevitt 1962; Baker et al. 1969; Jacobs er al. 1973; Parker et al. 1974; Reidbord 1974; Derks & Jacobovitz-Derks 1977). Neutral fat embolisation after intravenous administration does not reproduce the haemorrhagic pneumonia found in the fatal traumatic fat embolisation syndrome (Schulz 1963; Baker et a/. 1969; Jacobs et al. 1973; Reidbord 1974). It appears that fatty acids and not neutral fat cause the destructive pulmonary changes. In neutral fat embolisation an initial stage of mechanicalvascular obstruction is followed by a chemical stage related to the toxicity of free fatty acids liberated by the action of blood and pulmonary lipase (Peltier 1956; Peltier & Scott 1957). Death from intravenously administered neutral fat is strictly caused by vascular obstruction (Peltier 1956). Neutral fat embolisation depresses the lung surfactant activity mainly during the hydrolysis stage while fatty acid embolisation promtly depresses the activity of lung surfactant (Hamilton et a\. 1964). Intravenous injections of oleic acid have been used by morphologists and physiologists to reproduce the chemical stage of fat embolism (Jefferson

373

& Necheles 1948; Peltier 1956; Ashbaugh & Uzawa 1968; Parker e f a/. 1974). Capillary congestion,

alveolar haemorrhage and septa1 necrosis are consistent microscopic findings after intravenous injection of oleic acid to dogs and rabbits (Schulz 1963; Derks & Jacobovitz-Derks 1977). Endothelial cell and type I cell necrosis were among the most severe acute damages seen in the electron microscope and pulmonary fibrosis developed in a late stage. In neutral fat embolism endothelial swelling is the most characteristic ultrastructural finding (Schulz 1963). The acute toxicity of Viscoleo administered intravenously to rabbits was similar to that of sesame oil. The lethal effect was most likely caused by fatal circulatory disturbances due to massive pulmonary oil embolisation. The acute intravenous lethality of neutral fats and fatty acids has been extensively investigated (vide Peltier 1957) and the results show that fatty acids are far more toxic than neutral fats. The present results on the acute toxicity of Viscoleo and sesame oil are in agreement with those reported with neutral fats. By complicated histological procedures and fat extractions Armin & Grant (1951) obtained evidence, that intravenous injection of 0.15 ml/kg of perirenal fat into rabbits caused pulmonary embolisation more pronounced than in cases of gross fat embolism in man. Pulmonary oil embolisation is a normal entity after lymphography with the oily contrast medium Lipiodolo (Bron et a/. 1963; Goldberg & Feinberg 1963; Gough et af. 1964, Guiney el al. 1964)and the embolisation is so marked that chest X-rays show presence of fine pulmonary stippling. No changes in the ventilatory ability but a fall in the diffusion capacity have been observed when the normally recommended dose, 6-10 ml, was administered into the lymphatics of one lower extremity of adults (Fraimow et af. 1965). No pulmonary symptoms may develop even after higher doses (Gough et al. 1964) but pulmonary complications occurred in patients with underlying pulmonary disease (Bron et a/. 1963; Weg & Harkleroad 1968). The acute toxic effect of LipiodoP injected into the lymphatics of anaesthetized rabbits has been studied by Guiney et al. (1964). The average dose in fatal cases was 0.97 ml/kg and the cause of death was massive embolisation of the contrast medium in the lungs.

3 74

OVE SVENDSEN A N D TOVE AAES-JPIRGENSEN

This dose is close to the LD5O doses obtained in the present study after intravenous injection of sesame oil or Viscoleo. Finally pulmonary fat embolisation may be a physiological phenomenon. Volkheimer et af. (1965) observed pulmonary fat embolisation in dogs given a fatty meal 1 hour and 40 min. before they were anaesthetizedfor 50min. andsubjectedto passive abdominal compression. The authors explain their finding in the following way. During the anaesthetic period the normal chyle discharge from the thoracic duct decreased and large quantities of chyle accumulated in the thoracic duct. The chylomicrons in the stagnated lymph may confluence to microemboli which were discharged to the venous blood after compression of the abdomen. Intravascular small fat droplets in lung parenchyma have been described to develop postmortem in traumatized rabbits (Allardyce 1971). Small fat emboli have also been described in pig lungs obtained from slaughterhouse (Brunner 1972a 8t b) and they are now and then found in lungs from untreated rabbits fixed in formalin immediately after exsanguination (personal observation). Since the lungs from the animals in the present study were fixed in formalin immediately after death, post mortem development of fat droplets was prevented but contamination with endogenous fat during processing of the tissue cannot be excluded. This may account for the presence of free fat droplets localized to embty spaces in frozen lung sections from dogs in the present study since radioactive spots in the lung tissue were only found in autoradiograms from one of the four dogs having free fat droplets in the lung tissue. Interstitial accumulations of lipid loaded cells in the lungs have been described in human autopsy material (Bonfiglio & Schenk 1974) and in rabbit lungs (Galindo & Imaedi 1966). Similar accumulations have also been seen in lungs from untreated rats, rabbits, dogs and pigs in this laboratory (personal observation, not published). It is well known that the lungs clear and metabolize circulating lipids and also synthetize lipids (Nasr & Heinemann 1965; Morgan 1971). No increase in the number of lipid loaded cells or in the lipid load itself were seen in the present study. However,

increased amounts of fat have been described in the cytoplasm of pulmonary histiocytes after intravenous injection of autogenous fat into rats (Reidbord 1974) and oleic acid into dogs (Derks & Jacobovitz-Derks 1977). This suggests that embolic fat may be transferred to histiocytes. The pathogenesis of histiocytic cell infiltrations in the lung interstities is not clear but they may represent foreign body reactions to the oil. Histiocytic cell accumulations were also found around oil droplets in the muscle tissue at the injection site and in the iliac lymph nodes. The histiocytic cell accumulations usually disappear without trace when the accumulating stimulus subsides and if necrosis of the parenchymal elements does not take place. The lungs from dogs in the chronic toxicity studies were not seat of irreversible changes. The cellular infiltration had disappeared three months after the last injection of sesame oil. A morphological study of the lungsover a two week period after intravenous administration of autogenous fat to rats has been reported by Reidbord (1974). Next to fat embolisation, infiltration with histiocytes and polymorphonuclear leucocytes were noted. The histiocytic cell infiltration was most prominent three days after the injection. Ten days after the injection no fat emboli were found but fat containing histiocytes were numerous. After two weeks the lungs were normal. This finding supports the suggestion that the histiocytic cell accumulations found in the present study may be reversible. The reactive changes observed in the regional accessory iliac lymph nodes from dogs in the chronic injection studies were close to those described in lymph nodes after Lipiodolo lymphography in dogs (Kraus 1967). Lipiodol deposits in the lymph nodes disappear slowly. Koehler et al. (1964) found 13.7% of the dose seventeen days after lymphography in dogs and deposits were present microscopically fifteen months after the injection (Kraus 1967). In the present chronic injection study sesame oil deposits were observed macroscopically three months after the last injection. In a chronic toxicity study of flupentixol palmitate in Viscoleo only few and very small oil droplets were found microscopically in the iliac lymph nodes three months after the last injection (persona1 observation). These findings indicate

FATE OF INTRAMUSCULAR OIL

that Viscoleo disappears faster from the lymph nodes than sesame oil and Lipiodol. It has been demonstrated by the present studies that sesame oil was absorbed from the intramuscular injection site in rats considerably slower (50% in 9 weeks) than Viscoleo (50% in 1 week). The disappearance rate of Viscoleo from the intramuscular injection site in dogs was fast (50% in 2 days) compared to that of sesame oil (50% in 5 weeks). Both oils disappeared faster from the injection site in dogs than in rats. One of the reasons for this species difference may be the different distribution patterns of the oils at the injection site. In dogs the oil depot was distributed as a number of small droplets over a large area while in rats the depot was localized to few large droplets. Hashida et a/. (1977) estimated the halflife for the disappearance of ''C-labelled sesame oil from the injection site of rats to approximately 10 days. This is considerably faster than seen in the present study but they followed the contents of radioactivity for three days only and the injected volumes were considerably lower. The unexpectedly low concentrations of radioactivity found at the injection site of rats killed during the first day after injection of sesame oil seem difficult to explain. However, parts of the oil may have disappeared through the needle canal and small amounts of oil may also have disappeared during processing of the isolated thigh, since encapsulation had not taken place at that time. The present study has demonstrated that lymphatic absorption is one of the pathways for disappearance of oil from the injection site after intramuscular injection. However, the studies have only given vague indications as to what extent lymphatic absorption of liquid oil takes place. The amonts of radioactivity in the regional lymph nodes and in the lungs suggest, however, that lymphatic absorption may be a minor pathway. Preliminary results from a study o n the amounts of radioactivity in lymph collected from dogs with chronic thoracic duct cannula support this suggestion since only about 2% of the injected dose (0.5 ml/kg of ''C-labelled Viscoleo) was demonstrated in the lymph collected continuously during 6 days after the injection (Svendsen, Boeck and Aaes-Jnrgensen, unpublished results). Negligible

375

amounts of radioactivity were found in the thoracic duct lymph from rats after intramuscular injection of '"J-labelled triolein in sesame oil (Nakamoto et al. 1975) or ''C-labelled methyl oleate (Tanaka et al. 1974). Aaes-Jnrgensen et al. (1977) demonstrated relatively high levels of clopenthixol decanoate in lung tissue from dogs given 100 o r 30 mg/kg/week (20% in Viscoleo) in a six month chronic toxicity study. This finding was unexpected since clopenthixol decanoate is hydrolysed very fast in vitro in presence of various kinds of tissue homogenates o r serum (loc. cit. 1977). The finding of pulmonaryoil microemboli in dog lungs from the present study suggests that clopenthixol decanoate may have been brought to the lungs via the thoracic duct protected against hydrolytic degradation in absorbed oil droplets. The serum levels of radioactivity in rats after intramuscular administration of 'H-labelled flupentixol decanoate or clopenthixol decanoate dissolved in Viscoleo differ clearly from that in dogs (Aaes-Jsrgensen et a/. 1977; Jmgensen 1978). I n rats the highest serum levels are seen 2 to 6 hours after the injection. Thereafter the concentration decline3 rather fast until 2-4 days after the injection when the decline slows down. In dogs low initial serum levels were seen on the first day after the injection. The peak level appeared four days after the injection and then the decline was similar to the slow phase of the decline in rats. This difference in the shape of the serum level curves can partly be explained from the different disappearance rates of the oily vehicle from the injection site of the two animal species. The fact that the serum level of flupentixol in man closely follows that in dogs (Jsrgensen 1978) suggests that the fate of oil in man may be more like that in dogs than in rats. General considerations. Before employing the findings of the present study to considerations upon toxicological consequences of the clinical use of intramuscular depot preparations containing Viscoleo or sesame oil, it has to be emphasized that most depot preparations are recommended in the human clinic in doses of 2-4 ml with an interval of 2 to 4 weeks. This corresponds to about 0.05 ml/kg of the oily vehicle. The dogs in the present study received 0.45-1.0

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ml/kg which corresponds to about 30 ml or more in humans. However, it seems not quite reasonable t o compare the dose in ml/kg body weight used in humans with that used in dogs of the present study. Actually, the total dose given to the dogs was about 5-10 ml at each injection and this is higher than the total dose recommended in humans. Furthermore, the injections were given once a week in dogs. Follow-up chest X-rays of patients treated for a long period (years) with oily depot preparations have not revealed pulmonary abnormalities (Dencker, Gravem and Fog, personal communication). A histological study of human lung tissue and iliac lymph nodes obtained at autopsy from schizophrenic patients treated for a prolonged period with oily depot preparations is in progress, and until now no indications (4 patients) of lymphogenic absorption of liquid oil or pulmonary oil microembolisation have been seen. Thus, it is unknown if lymphogenic absorption of liquid oil from oily depot preparations may give rise to pulmonary oil microembolisation in patients. It does seem, however, reasonable to expect this to occur to some extent after long term and frequent administration of large volumes. Pulmonary injury induced by the embolisation is o n the other hand not to be expected unless extremely high volumes are administered frequently. Acknowledgements The technical assistance of Mrs. Yvonne Bresson, Anna Grethe Eriksen, Maria Galambosi, Bente Hansen, Elna Jacobsen, Inger Jensen, Kirsten Kjaergaard, Birgitte Nielsen and Mr. Allan Jsrgensen is highly acknowledged. References Aaes-Jergensen, T., K. Fredricson Overe, K.P. Begese & A. Jergensen: Pharmacokineticstudies on clopenthixol decanoate: a comparison with clopenthixol in dogs and rats. Act4 pharmacol. et toxicol. 1977, 41, 103-120. Ahmed, A. & N. Greenwood: Lymphadenopathy following repeated oil-based injections. J. Path. 1973, 111, 207-209. Allardyce, D. B.: The postmortem interval as a factor in fat embolism. Arch. Path. 1971, 92, 248-253. Armin, J. & R. T. Grant: Observations on gross pulmonary fat metabolism in man and the rabbit. Clin.Sci. 1951,10,441-469. Armstrong, H. J., M. C. Kuenzig & L. F. Peltier: Lung

lipase levels in normal rats and rats with experimentally produced fat embolism. Proc. SOC.Exp. Biol. Med. 1967,124,959-961. Anonymous: A letter regarding mineral oil adjuvant preparations from the Department of Health, Education, and Welfare, Food and Drug administration. Ann. Allergy 1965, 23, 558-562. Ashbaugh, D. G. & T. Uzawa: Respiratory and hemodynamic changes after injection of free fatty acids. J. Surg. Res. 1968, 8, 417-423. Baker, P. L., M. C. Kuenzig& L. F. Peltier: Experimental fat embolism in dogs. J. Trauma 1969, 9, 577-586. Behrens, H., G. Matschullat & K. Tuch: uber die Vertraglichkeit bliger Vitaminlosungen beim Schaf nach intramuskullrer Applikation. Duch. Tierarztl. Wschr. 1975, 82, 27-31. Bisgard, J. D. & C. Baker: Experimental fat embolism. Am. J. Surg. 1940, 47, 466-478. Bollinger, J. N.: Metabolic fate of mineral oil adjuvants using “C-labeled tracers. I: Mineral oil. J. Pharm. Sci. 1970, 59, 1084-1088. Bonfiglio, T. A. & E. A. Schenk: Lipid deposits in pulmonary connective tissue. Arch. Parhol. 1974, 97, 48-50. Bron, K. M., S. Baum & H. L. Abrams: Oil embolism in lymphangiography. Radiology 1963, 80, 194-202. Brown, W. E., V. M.Wilder & P. Schwartz: A study of oils used for intramuscular injections. J. Lab. Clin. Med. 1944, 29, 259-264. Brunner, P.: Haufigkeit und Intensitat von Lungenfettembolien bei Schlachtschweinen. Zbl. ANg. Path., 1972a, 115,391-394. Brunner, P.: Erganzende Untersuchungen zum Vorkommen von Lungenfettembolien bei Schlachttieren. Zbl. Allg. Path. 1972b, 116, 295-299. Briicke, P., G. Bliimel & R. Gottlob: fiber die Fettresorption bei Volumenmangel: Ein experimenteller Beitrag zur Pathogenese der Fettembolie. Ach. Klin. Chir. 1965,313, 1049-1053. Deansly, R. & A. S. Parkes: Note on the subcutaneous absorption of oils by rats and mice, with special reference t o the assay of oestrin. J. Physiol. 1933, 78. 155-160. Derks, C. M. & D. Jacobovitz-Derks: Embolic pneumopathy induced by oleic acid. A systematic morphologic study. Am. J. Path. 1977, 87, 143-158. Dreyfuss, J., J. M. Shaw & J. J. Ross: Fluphenazine enanthate and fluphenazine decanoate: Intramuscular injection and esterification as requirements for slowrelease characteristics in dogs. J. Pharm. Sci. 1976,65, 1310-1315. Emery, F. E., C. S. Matthews & E. L. Schwabe: The absorption of stilbestrol and theelin from cysts of sesame and peanut oils. J. Lab. Clin.Med. 1941, 27, 622-627. Finney, D. J.: Probit analysis. Cambridge University Press 1952,2nd ed., pp. 65-72. Fraimow, W., S. Wallace, P. Lewis, R. R. Greening & R. T. Cathcart: Changes in pulmonary function due to lymphangiography. Radiology 1965, 85.23 1-241. Fuchsig, P.: Neue Erkenntnisse in Pathogenese und

FATE OF INTRAMUSCULAR OIL Therapie der Fettembolie. Arch. Klin. Chir. 1966, 316,243-252. Galindo, B. & T. Imaeda: Cellular response to Freund's adjuvant in the rabbit lung. An electron microscope study. Lab. Invest. 1966, 15, 1659-1681. Goldberg, M. E. & S. B. Feinberg: Pulmonary infarction following lymphangiography in dogs: Its implications in human studies. Radiology 1963, 81, 479-483. Gough, J. H., M. H. Gough & M. L. Thomas: Pulmonary complications following lymphography. With a note on technique. Brit. J. Radiol. 1964, 37, 416-421. Guiney, E. J., M. H. Gough & J. B. Kinmonth: Lymphography with fat-soluble contrast media. Studies of their effect in lepus cuniculus. J. Cardiovasc. Surg. 1964, 5, 346-354. Hamilton, R. W., R. F. Hustead & L. F. Peltier: Fat embolism: The effect of particulate embolism on lung surfactant. Surgery 1964, 56, 53-56. Hashida, M., M. Egawa, S. Muranishi & H. Sezaki: Role of intramuscular administration of water-in-oil emulsions as a method for increasing the delivery of anticancer agents to regional lymphatics. .I. Pharmacokin. Biopharm. 1977, 5, 225-239. Jacobs, R. R., E. J. Wheeler, C. Jelenko 111, T. F. McDonald & F. E. Bliven: Fat embolism: A microscopic and ultrastructure evaluation of two animal models. J. Trauma 1973, 13, 980-993. Jefferson, N. C. & H. Necheles: Oleic acid toxicity and fat embolism. Proc. Soc. Exp. Biol. Med 1948, 68, 248-250. Jmgensen, A,: Pharmacokinetic studies on flupenthixol decanoate, a depot neuroleptic of the thioxanthene group. Drug Metabolism Reviews 1978. 8, 235-249. Koehler, P. R., W. A. Meyers, J. F. Skelley & B. Schaffer: Body distribution of ethiodol following lymphangiography. Radiology 1964, 82, 866-871. Kraus, R.: The histologic picture of the lymph node up to 15 months after lymphography with lipiodol UF. In: Progress of lymphology. Ed.: A . Riittimann. Georg Thieme Verlag, Stuttgart, 1967, pp. 329-330. Mahley, R. W., M. E. Gray & V. S. LeQuire: Role of plasma lipoproteins in cortisone-induced fat embolism. Am. J. Parhol. 1972, 66, 43-64. Morgan, T. E.: Pulmonary surfactant. New Ilngl. J. Med. 1971, 284, 1185-1193. Nakamoto, Y.,M. Fujiwara, T. Noguchi. T. Kimura. S. Muranishi & H. Sezaki: Studies on pharmaceutical modification of anticancer agents. I. Enhancement of lymphatic transport of mitomycin C by parenteral emulsions. Chem. Pharm. Bull. (Tokyo) 1975. 23, 2232-2238. Nasr. K. & H. 0. Heinemann: Lipid synthesis by rabbit lung tissue in v i m . Am. J. Physiol. 1965,208, 118-121.

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Paredes, S . , F. Comer, S. Rubin, F. Adler & L. F. Peltier: Fat embolism. Distribution of fat tagged with 131 J within the body of the rat at various times following intravenous injection. J. Bone Joint Surg. 1965,47, 1216-1220. Parker, F. B., S. D. Wax, K. Kusajima & W.R. Webb: Hemodynamic and pathological findings in experimental fat embolism. Arch. Surg. 1974, 108, 70-74. Pedersen, V., 0. Svendsen, P. Danneskiold-Samsee, V. Boeck t I. Molter Nielsen: Pharmacology of cis (Z)clopenthixol decanoate, a depot neuroleptic. Acra pharmacol. et toxicol. 1977, 40, 482-490. Peltier, L. F.: Fat embolism. 111. The toxic properties of neutral fat and free fatty acids. Surgery 1956, 40, 665-670. Peltier, L. F.: An appraisal of the problem of fat embolism. In(. Abstr. Surg. 1957, 104, 313-324. Peltier, L. F. &.I. R. Scott: Fat embolism. Changes in the level of the blood lipase following the intravenous injection of neutral fat, fatty acids, and other substances into dogs. Surgery 1957, 42, 541-547. Reidbord, H. E.: Pulmonary fat embolism. An ultrastructural study. Arch. Parhol. 1974, 98, 122-125. Richardson, P., E. H. Crosby, N. A. Bean & D. Dexter: Pulmonary oil deposition in patients subject to lymphography. Detection by chronic photoscan and sputum examination. Canad. Med. Ass. J. 1966,94,1086-1091. Schulz, H.: Some new observations on the submicroscopic pathology of the lung. Pulmonary adenomatosis and fat embolism. Lab. Invest. 1963, 12, 616-627. Sevitt, S.: Fur embolism. Butterworth Ltd., London, 1962. Symmers, W. St. C.: Simulation of cancer by oil granulomas of therapeutic origin. Brit. Med. J. 1955,2, 15361539. Szabo, G . , P. SerCnyi & L. Kocshr: Fat embolism: Fat absorption from the site of injury. Surgery 1963, 54, 756-760. Tanaka, T., H. Kobayashi, K. Okumura, S. Muranishi & H. Sezaki: Intramuscular absorption of drugs from oily solutions in the rat. Chem. Pharm. Bull. (Tokyo) 1974, 22, 1275-1284. Threefoot, S . A,: Fate of oil in the lungs after lymphography. In: Progress in lymphology. Ed.: A. Riittimann. Georg Thieme Verlag, Stuttgart, 1967, pp, 321-322. Ullberg, S.: Studies on the distribution and fate of "Slabeled benzylpenicillin in the body. Acta Radiol. 1954, 188, SUPPI.,22-31. Volkheimer, G., H. Wendland, M. Walkenback & J . Unger: Untersuchungen zum Chyle Jet Effect. Das Deutsche Grsimdheits~,esen1965, 20, 66-67. Weg, J. G. & L. E. Harkleroad: Aberrations in pulmonary function due to lymphangiography. Dis. Chest. 1968. 53, 534-540.

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Appendix A Black stained oil droplets (arrows) in branches of the thoracic duct (small arrow heads) from pigs. Black discoloured iliac lymph nodes (large arrow heads). Ten ml of Viscoleo (a)or sesame oil (b)containingO.S% Sudan black as a marker were given intramuscularly into each thigh five times with two to three days intervals (total dose 100 nil). The pigs were killed three days after the last injection and autopsied.