**~** Ryuich Arita with collaboration of Katsumi Miyazaki and Shutoko Mure *: Metabolic test ofhexabromocyclododecane. Test on chemical substances used in household items. Studies on pharmacodynamics of hexabromocyclodoecane_-_'_

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### * Department of Pharmacy, Hokkaido University Hospital

Translated from Japanese by the Ralph McElroy Co., Custom Division P.O. Box 4828, Austin, Texas 78765 USA1 Code: 237-58354
METABOLIC TEST OF HEXABROMOCYCLODODECANE Hexabromocyclododecane, commonlyused as a flame retardant in household items, containsmany bromine atoms in themolecule and is extremely soluble in lipids; there is the possibility that undesirable phenomena such as its accumulation in tissue might result from its ingestion. However, at the present time, there has been almost no report on the pharmacodynamics of this compound. In this study, we examined its excretionin urine and feces as well as its distribution into organs.

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Hexabromododecane Experimental Samples and reagents: We used Pyroguard SR-103 (SR-103, hereafter) from Daiichi Kogyo SeiyakuK.K. as hexabromocyclododecane. We have confirmed that there was a single peak in gas chromatography of SR-103.As reagents, we used reagents of special class fromWako Junyaku K.K. Experimental animals: We usedmale Wistar strain rats at a body weight of 260-300 g as the experimental animal. Rats were maintained on a constant composition diet(Oriental Kobo Kogyo K.K.). We prepared a fine suspension of SR-103 in olive oil to be orally administered under ether anesthesia using a stomach tube at a dosage of 500 mg/kg. In this procedure, the fine suspension was mixed well to homogeneity in a mortar before administration (100mg/mL)

. 2

Oral administrationwas performed for 5 days consecutively and urine and feces were dailycollectedby separatelyhousing rats in a glass cage (Sugiyama Gen, type 5T). 24 h after the final administration, autopsywas performed and organs such as the spleen, pancreas, kidneys, liver, heart and fatty tissue were collected for examination. Gas chromatography (GC): Hitachi 663 type was used as the instrument. Measurement was performedunder the following conditions: detector: FID, column: 3% silicone OV-1 on chromosorb WAW DMCS, 60-80 mesh, 2.0 mx 

### 3 mm 

### id, column temperature: 240° for 4 min to attain a constanttemperature followed by temperature rise at the rate of 2°/min to reach 265°, temperature at the injection and detector: 300°, carrier gas: nitrogen (40 mL/min). Usually 2

**~L** of a sample were injected and the concentration in the sample was calculated fromthepeak height ratiowith respect to the internal standard (n-octacosane)

. Quantification of SR-103 in feces: Distilledwater was added to the total amount of feces collected at 4 mL/g, and the mixturewas mixed well in a mortar to obtain a suspension of the particles. 2 mL of distilled water were added to 1 mL of the suspension followed by addition of 4 mL of a mixture of chloroformand acetone at a ratio of 4:1 for extraction with shaking for 10min. The mixture was centrifuged at 1000 g for 5 min and the solvent was removedunder reduced pressure from 1 mL of the organic layer. The residue was dissolved in 100**~L** of the extraction solvent described above containing the internal standard {n-octacosane, 200**~g/mL)** to prepare a sample for GC measurement. The calibration curve was prepared as follows: 2 mL of distilled water were added to 1 mL of a fine suspension of blank feces followed by the addition of 4 mL of extraction solvent described above containing SR-103 at known concentrations and shaking the mixture. The extract was treated similar to the sample described above to prepare the calibration line. 3

Detection of SR-103 in urine: After measurement of the volume, 1 mL of a urine sample was added to 2 rnL of distilled water followed by procedures similarto those for fecal samples to obtain a specimen for GC measurement. The calibration curve was prepared to the calibration curve for the fecal samples after addition of 1 mL of blank urine to 2 mL of distilledwater. Absorption experiment using a loop of small intestine: A loop of small intestine was prepared according to themethod of Levy, et al. fromjustbelow the pylorus {closedbile duct) with a length of about 20 em. A sample was preparedin the form of a fine suspension inphysiological saline {10 mg/mL) and 2 mL of the suspension was introduced into the loop. 1 h after the introduction, the loop was isolatedand the content of the loop was poured into a 10-mLmeasuring cylinder. About 7 mL of

### 4 physiological saline were used to wash inside the loop and the solution was added to the cylinder. The total volume in the cylinder was brought up to 10 mL. To 5 mL of this solution, 5 mL of the mixture of chloroform-acetone (4:1) were added followed by extraction of the mixture with shaking for 10min. The mixture was centrifuged (1000 g for 5 min) and the organic layer was separated. The solvent was removed under reducedpressure from 3 mL of the organic layer, and the residue thus obtained was treated similarly to the case of fecal samples to be used as a specimen for GC measurement. The measurement of SR-103 in the intestinal tissue was carried out as follows: the loop was cut intopieces about 1 em long and the pieces were homogenized (Waring [transliteration] blender, Nissei AM4 type, 6000 rpm for 15min) after addition of 20 mL physiologcal saline. After addition of 2 mL of physiological saline used to wash inside the blender and 20 mL of chloroformacetone mixture (4:1) to the homogenate, the mixture was shaken for 10 in for extraction, followedby centrifugation at 1000 g for 5 min. 

### 4mL of the organic layerwere exposed to reduced pressure to remove the solvent and the residue was treated similarly to the above to obtain a sample for GC measurement. The calibration line was prepared as follows: extraction solvent containing SR-103 at a known concentration was added at the same volume employed above to the fluid in the loop obtained after introduction of 2 mL ofphysiological saline into a loop of another rat and homogenate of intestinal tissue followed by the same procedures. Distribution in various organs: the organ weight was measured for extirpated spleen, pancreas, liver, kidneys and5 heart and each organ was chopped into fine pieces. After addition of distilled water at 4 times the volume of each organ sample, a homogenate was obtained with a Waringblender. 4 mL of extraction solvent (chloroform-acetone, 4:1) were added to 3 mL of the homogenate to obtain a sample for GC measurement by the procedures similar to those for fecal samples. Fatty tissue was collected form the periphery of a kidney and 10 mL of the extraction solvent were directly added to 0.5 g of this fatty tissue followed by stirring (6000 rpm, 5 min) in a Waring blender for the extractionprocedure. The calibration curve for each organ was preparedby extraction of eachblank organ homogenate with the extraction solvent containing SR-103 at known concentrations followed by extraction and others procedures.
Results and discussion
Evaluation of quantificationmethod of SR-103 inbiological samples
In quantification of SR-103 inbiological samples, we performed examination of the extraction solvent for SR-103. We tried chloroform, chloroform-acetonemixture and n-pentane as the solvent and we observed that chloroformandpentane were markedly inferior to the chloroform-acetonemix:.ure in solubility of SR103 (less than 2 mg/mL), although theywere excellent in preventing extraction ofbiological blanks. Consequently, these two solvents were found inadequate for recovery of SR-103 from fecal samples after administration of SR-103 at a dosage of6 500 mg/kg/. Then we examined chloroform-acetonemixture and we decided to use the chloroform-acetonemixture at a 4:1 ratio as the extraction solvent, which is satisfactory inprevention of interference of biological blanks and in the solubility of SR-103 (more than 100 mg/mL). We examined the quantification method by means of GC after separation frombiological components following the conditions of quantitative analysis and experimental procedures described in the Experimental section. As shown in the gas chromatogramof a fecal sample, it was possible to detect SR103 well separated from biological components (Figure 1) and an excellent linearity was obtained in the calibration wave (Figure 2). Similarlyexcellent results were obtainedin urine samples and homogenate samples of various organs. Fromthese results, we concluded that SR-103 inbiological samples canbe quantified accurately and simplyby this method of quantification. The limit of quantification in this method was about 5 ug/mL in urine and about 20 ug/mL inhomogenates of biological samples (feces and various organs with addedwater 4 times in volume for homogenate preparation)

. Excretion during repeated administration and absorption from the small intestine Figure 3 shows the cumulative fecal excretion (mg/g body weight) in each of 4 rats administeredwith SR-103 repeatedly for 5 days. Table I shows the average daily excretion with· respect to the administered amount andthe average cumulative excretion with respect to the cumulative average administered as the mean among the rats.7 As the result, it is clear that the cumulative fecal excretion increased with time, even if there were variations among the rate (Figure 3 ) . Furthermore, the average daily rate of excretion was 29-37% of the administered amount in 4 rats (Table I, left) and the cumulative excretion was roughly constant at 32-35% with respect to the cumulative amount of administration in the test period (Table I, right)

. Fromthese observations, a strongpossibility is suggested that an approximately constant fraction of SR-103 is taken up in the body from the administered amount at each ad!ninistration. Table II shows the result of experiments for absorption using the loop of upper jejunum. About 12% of the administered amount was found to be accumulated in the intestinal tissue, and the amount remaining in the lumen loop was small. Fromthese results, it canbe suggested that SR-103 canbe absorbed from the intestine. However, urinary excretion of SR-103 was not observed under the conditions of this experiment.
Distribution in tissues Among tissues of various organs tested in this experiment, SR-103 was detected only in fatty tissue. Table II shows the result ofmeasurements of SR-103 fat 24 h after repeated administration of SR-103 for 5 days. SR-103 was shown to have been accumulated at 0.3-0.7 mg in 1 g of fat. In this laboratory, we made a similartest on 2,4,6- tribromophenyl-2-methyl-2,3-dibromopropyl ether (TBPPE), a bromine flame retardant similar to SR-103.8 

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Under roughly identical conditions of repeated administration as in this test, we found TBPPE was detected in fatty tissue (0.21, 0.80, 1.2 mg/g of fat, n 

### = 3) and none was detected in other organs. In the experiment with a single administration at the same dosage, TBPPE was detected at a concentration of 0.9 mg/g in fat while the amount therein was below the detection limit for SR-103. With regard to transfer into fatty tissue, TBPPE was suggested to be more superior than SR-103. Difference in excretion fromTBPPE
After oral administration ofTBPPE, we detected various peaks in GC attributable tometabolites including 2,4,6-tribromophenol (TBP) in the fecal sample. Also, TBP was detected in urine. In comparison, we did not detect peaks different from those of the blank and theoretically derived from metabolites in fecal and urine samples after administration of SR-103. This observation seems to be an important difference between the two compounds. As the results of experiments for pharmacodynamics of SR-103 using rats, we observed that this substance without alteration was excreted at about 35% of the dosage administered and therefore there 

### is a possibility that the rest couldbe taken up in the body, including the intestinal tissue. In this experiment,9

we did not detect urinary excretion and we believe that, while SR-103 is partially accumulated in fatty tissues, there is a high possibility that it is widely distributedin the body at a concentrationbelow the detection limit.
Conclusion
1. Hexabromocyclododecane was absorbed in the intestines following oral administration and about 35% of the amount administeredwas excreted in feces. 2. No urinary excretionwas observedunder the experimental conditions of this study. 3. No peak attributable to themetabolites of SR-103 was observed in GC, except those peaks due to the blank. 4. After repeated administration, we observed accumulation of SR-103 in fatty tissue. 5. We did not detect distribution of SR-103 to other organs.0 

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### Table 11. PercentofResiduorSR-103 inthe IntestinalLoopond TissueoftileUpperSmall IntestineofRots, Loop Tissue Rat 1 16.3 11.6 Rat 2 4.8 12.3 

### 14

## Table III. ConcentrationofSR-103InFatTissueafter MultiPleOralAdministrationofSR-103to Rotsfor5Days,

Rat A Rat B Rat C Rat D 

# .• ConcentrationofSR-103 ( mg/g tissue J
0.67 0.48 0.47 0.33 

### 15