A subacute toxicity study on 99m Tc stannous Glucoheptonate injection

A Subacute Toxicity Study on 99mTc Stannous Glucoheptonate Injection L. Belbeck, B.M. Bowen, J. Jeu and M. Richardson* ABSTRACT la dose humaine, sur la base du follow-up assessment in renal poids vif. Aucun des animaux transplant cases. Following intra- A subacute toxicity study on expe rimentaux ne manifesta de venous injection, a large propor- 99mTc stannous glucoheptonate signes cliniques. L'examen de tion of the dose is cleared from the was performed with rats, dogs l'urine et du sang des lapins ne plasma by glomerular filtration and rabbits, injected intraven- d6montra rien d'anormal. La and rapidly excreted. Partial tub- ously at ten to 100 times the necropsie, ainsi que l'examen ular reabsorption occurs, resulting human dose on a body weight microscopique des reins et des in retention in the cortex (1, 2, 18). basis. There were no abnormali- frottis de la moelle osseuse des The toxicity study presented in ties in the clinical status of any of rats et des lapins, ne revelerent this report forms part of the prec- the animals. No changes were rien d'anormal. L'histopatholo- linical drug submission for 9mTc found in urinalysis, blood chem- gie du foie d'un lapin qui avait stannous glucoheptonate. Radia- istry or hematology in the rab- reCu 100 fois la dose humaine de tion dosimetry for a recommended bits nor in gross examination, l'isotope experimental, revela la 10 mCi dose is presented in Table renal histology or bone marrow presence d'un foyer necrotique. I. smears in the rats and rabbits. Cette lesion incita les auteurs a It is expected that a patient Hepatic histology was also done. examiner le foie d'autres rats et would receive no more than a few A focal area of necrosis in a liver d'autres lapins experimentaux a doses in a lifetime for renal of one rabbit that had been la microscopie electronique. Ils assessment. Based on the small injected with 100 times the decelerent ainsi, dans le foie des amounts of drug likely to be given human dose was observed using animaux experimentaux qu'ils to a patient, the toxicity study was light microscopy. Examination examinerent, un reticulum undertaken on a subacute basis by electron microscopy in endoplasmique tantot lisse, using guidelines from the Food another group of rats and rab- vacuolaire et dilate, tantot and Drug Directorate of the bits was prompted by the obser- rugueux, d(pourvu de granules Department of National Health vation of that lesion. This et v6siculaire. La microanalyse and Welfare and from the Radia- revealed vacuolated and dilated aux rayons X d6montra une tion Protection Bureau of the smooth endoplasmic reticulum relation entre les changements Health Protection Branch. The and degranulated and vesicu- ultrastructuraux du foie et le guidelines for the toxicity study lated rough endoplasmic reticu- d6pdt d'Wtain dans cet organe. are for the nonradioactive form of lum in all the test livers. X-ray the drug, that is, Tc stannous microanalysis indicates that the glucoheptonate. ultrastructural changes are INTRODUCTION linked to the deposition of tin. A toxicity study was undertaken to evaluate a new formulation of R:SUMR 99mTc stannous glucoheptonate, a TABLE I. Radiation Dosimetry kidney scanning agent intended Radiation Dose Cette experience consistait a for use in the assessment of renal Organ (rads/10 mCi) etudier la toxicite subaigue! du perfusion, function and morphol- Kidney 2.0 glucoheptonate stanneux de ogy. Clinical indications for this Bladder wall 2.5 techn6tium99m, chez des rats, drug in nuclear medicine include Whole body 0.1 des chiens et des lapins auxquels investigation of space occupying Ovaries 0.19 on avait injecte, par la voie lesions, urinary retention states, Uterus 0.32 renovascular hypertension and Adrenals 0.18 intraveineuse, de dix a 100 fois *Central Animal Facilities (Belbeck), Department of Nuclear Medicine (Bowen and Jeu), Pathology Department (Richardson), McMaster University Medical Centre, Hamilton, Ontario. Submitted March 6, 1979. Can. J. comp. Med. 45: 299-303 (July 1981) 299 MATERIALS AND METHODS lb) two weeks after the last injec- EDAX was used here to determine tion. All organs were examined at the presence of technetium and tin Each millilitre of 99mTc stannous dissection. Kidney and liver spec- in control and test livers (6, 9). glucoheptonate contains: imens were examined using Samples for x-ray microanalysis Calcium glucoheptonate standard histological techniques were collected and prepared as for 14.28 mg (10% buffered formalin, 6 micron conventional transmission elec- Stannous chloride paraffin sections stained with tron microscopy with the omission 0.03 mg hematoxylin and eosin). The of postfixation with osmium and 99mTc sodium pertechnetate tissues were sliced in 5.0 mm sec- block staining with uranyl acetate. 8.50 millicuries tions and random slices evaluated. The sections were collected on 200 Sodium chloride injection USP In the second phase of the study, mesh copper grids and carbon qs to 1.0 mL one rabbit was given a daily coated. Analysis was conducted in The recommended human dose intravenous injection, 50 times the a Philips 300 transmission electron of 99mTc stannous glucoheptonate is human dose, and a second rabbit, microscope fitted with a model 606 10.0 to 15.0 millicuries. The half- 100 times the human dose for five x-ray spectrometer, a model 707A life of technetium-99m is six hours. consecutive days. Two rats were energy dispersive analysis of x- All animals in this study were each given a daily intravenous rays system and an EDIT system intravenously injected with sam- injection, 100 times the human of EDAX International Inc. ples of this preparation that had dose for five consecutive days. A Femoral bone marrow aspirates decayed at least five days. Doses control rabbit and control rat were (0.2-0.5 mL) were performed were determined on a body weight given 0.9% sodium chloride injec- under anesthesia with a Rosenthal basis. tions. Three days following the last 16 gauge biopsy needle. Smears of In the first phase of the study ten injections, the controls, one test rat aspirated marrow were made and rats were each given a single and the rabbit that was adminis- stained with May Grunwald- intravenous injection of ten times tered 100 times the human dose Geimsa stain and cell morphology the human dose of Tc stannous glu- were stunned and exsanguinated as well as differential counts were coheptonate. Five dogs were each to remove the liver. The remaining performed. Normal stages of given a single intravenous injec- test rat and rabbit were sacri- hematopoietic cell growth and tion, ten times the human dose, and ficed in the same manner ten days normal cellularity were observed another five, 50 times the human after the last injection. The anim- in all cases. dose. One group of ten rabbits was als were not euthanized with given daily intravenous injections, sodium pentobarbital in this phase 50 times the human dose, and since the barbiturates can cause RESULTS another group of ten, 100 times the hypertrophy of the endoplasmic human dose for five consecutive reticulum in the liver (6). No changes were found in clini- days. Liver specimens for electron cal status, laboratory results, The clinical status of all animals microscopy were diced into 1.0 mm organ gross examination, renal was assessed over a two-week cubes and fixed in 2.5% glutaral- histology or assessment. period by performing physical dehyde in 0.2 M sodium cacodylate On light microscopy, a focal area examinations, measuring temper- pH 7.4 for six hours. The speci- of necrosis was found in the liver of atures and monitoring food and mens were then postfixed in 1% one rabbit that had been injected water intake. Blood samples were buffered osmium tetroxide for two with 100 times the human dose collected twice in the two-week hours, stained en bloc in aqueous (Fig. 1). This area appeared sim- period postinjection from the rab- uranyl acetate, dehydrated ilar to focal hepatic necrosis asso- bits via the central artery of the ear through graded ethanol, embedded ciated with Tyzzer's disease which for blood chemistry (BUN, creati- in Spurrs resin (17) and sectioned is caused by Bacillus piliformis nine, bilirubin T, bilirubin D, on a Reichert ultramicrome. Ultra (14). There appeared to be more SGOT, SGGT, alkaline phospha- thin sections were collected on 200 binucleated hepatocytes in the test tase) and hematology (RBC count, mesh copper grids stained with livers. These cells were counted WBC count, hemaglobin, hemato- lead citrate (19) and examined in a and found not to be significantly crit, mean corpuscular volume, Philips 301 electron microscope. different from control cells using mean corpuscular hemaglobin, Elemental microanalysis of the Student's t-test. mean corpuscular hemaglobin livers was carried out using an In transmission electron micros- concentration). The rabbits were energy dispersion x-ray analysis copy, it was observed that the maintained in metabolic cages and system (EDAX) which enables hepatocytes of the test animals had their urine collected for urinalysis chosen tissue regions to be exam- dilation and vesiculation of the (blood, ketones, glucose, protein). ined at the ultrastructural level smooth endoplasmic reticulum Three rats and three rabbits (4). All elements of atomic number and degranulation and vesicula- chosen randomly from each group 11 (sodium) and higher in the peri- tion of the rough endoplasmic were euthanized with euthanol odic table can be detected at a sen- reticulum. Free ribosomes were (sodium pentobarbital 240 mg/5 sitivity of at least 10-l' grams. The more abundant in the test livers. 300 The mitochondria in the test anim- Using x-ray microanalysis, the als showed no abnormalities (Figs. K and L peaks of tin were observed 2 and 3). in the livers from the treated U-A ,' TO4' .;4 e 4 -l..: 'i! #AW 1 .>... ,k,e. a Fig. 4. Spectrum of x-ray microanalysis W of control rat liver. Fig. 1. Focal area of necrosis in one area of the liver of a rabbit that had been injected intravenously with 100 times the human dose on a body weight basis of Tc stannous glucoheptonate for five consecutive days. The rabbit was sacrificed two weeks after the last injection. (a) xl00 (b) x500 Fig. 5. Spectrum of x-ray microanalysis of test rat liver with characteristic energy peaks of tin. animals, but not from the controls (Figs. 4 and 5). Technetium was not found because the amount Fig. 2. Livers of control animals. No abnormalities were observed. injected was below detectable (a) Rat liver - X7800 limits. (b) Rabbit liver - X7800 -4. i .14F .' -. ..x DISCUSSION : ;e-F :; A,- .W.. . .. 1%b .t XI. Focal hepatic necrosis was iv T.- .. ;a observed by light microscopy in a s-,. ht:i} .e I.: rabbit that had received five intravenous injections, 100 times the human dose of 99mTc stannous glucoheptonate. To eliminate the possibility of concurrent disease and to further define the lesion, other test livers were examined by electron microscopy. These showed vesiculation and dilatation of smooth endoplasmic reticulum Fig. 3. Livers of test animals that had been injected intravenously with 100 times the and degranulation of rough endo- human dose on a body weight basis of Tc stannous glucoheptonate for five consecu- plasmic reticulum. X-ray microa- tive days. The animals were sacrificed three days after the last injection. The smooth nalysis revealed the deposition of endoplasmic reticulum is vacuolated and dilated. The rough endoplasmic reticulum tin in the hepatic lesions. One can- is degranulated and vasiculated in comparison to control specimens. (a) Rat liver -x7800 not conclude the precise chemical (b) Rabbit liver -x7800 nature of the toxic component, 301  except that tin is somehow after examining the same speci- To further put the observations involved. mens with light microscopy it was from this study into perspective Toxicity due to tin itself has been noted that there was moderate to one must consider that a clinical studied elsewhere. The toxic mani- marked fatty change without an dose of 99mTc stannous glucohepto- festations of tin include pathologi- increase in fibrous tissue or glyco- nate would be administered no cal changes in the liver and kidney gen impairment (10). Further stu- more than a few times in a patient's and brain edema. Rats adminis- dies with high levels of ingestion lifetime. In addition, a commercial tered an LD50 dose equivalent to over a prolonged period in both formulation of this drug, Frosst 32.0 mg of stannous chloride man (13) and baboon (12) includes Image Glucoheptonate Kit' has intraperitoneally die as a result of this proliferation of smooth ER but been in clinical use for about three lower nephron necrosis (15). In in addition is characterized by a years with apparent safety. rabbits that were given one or decrease in microtubules, an In conclusion, five daily intrav- more grams of stannous chloride accumulation of protein and fat, enous injections, 50 to 100 times every six to ten days, the animals swelling, and abnormal cristae of the human dose of 99rTn stannous died after one to two months with the mitochondria, and ultimately glucoheptonate, produces ultra- gastritis, degeneration of the liver necrosis (11). Furthermore, on structural changes in rat and rab- and kidney and paralysis of the light microscopy lesions are char- bit livers. However, the use of this hind legs (3). In a subacute study acterized by necrosis, fibrosis and drug for the approved indications where rats ingested 5 ppm stan- inflammation. The interpretation and in the recommended dose is nous chloride in drinking water by others are that changes in the not unwarranted. (36.68 ,g/100 g body weight/day) smooth ER are adaptive following from the time of weaning to the induction of detoxifying enzymes. time of natural death there was an The changes in other organelles REFERENCES increased incidence of fatty are the result of a toxic metabolite, change in the liver and vacuolar acetaldehyde (11). Thus, although 1. BOYD, R.E., J. ROBSON and F.C. HUNT. QQmTc gluconate complexes for changes in the proximal convo- Tc stannous glucoheptonate may renal scintigraphy. Br. J. Radiol. 46: luted tubules (16). induce certain enzyme systems 604-612. 1973. To put the observation of focal with concomitant anatomical 2. BOYD, R.E., J. ROBSON and W.J. hepatic necrosis into perspective, a change, these changes are tran- SORBY. Studies with a technetium comparison is made with a drug in sient and do not cause serious renal scanning agent. I. Chemical and experimental 'aspects. Aust. N. Z. J. clinical use. 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