~ Ryuich Arita with collaboration of Katsumi Miyazaki and Shutoko 

Mure *: Metabolic test of hexabromocyclododecane. Test on 

chemical substances used in household items. Studies on 

pharmacodynamics of hexabromocyclodoecane_-_'_ -(-198-3)_-~-·- :-;-:-:;-----J_· 

[no source given) ' - - ., :' · ' ·'· 1 

:_ . . .:l i :, ···--,...,_.. ____ ····-·---~~--~- ' 

('. -. -....., .. --

'• ( -· - ; ; _; " 

* Department of Pharmacy, Hokkaido University Hospital 

Translated from Japanese by the Ralph McElroy Co., Custom Division 

P.O. Box 4828, Austin, Texas 78765 USA 
1 

Code: 237-58354 

METABOLIC TEST OF HEXABROMOCYCLODODECANE 

Hexabromocyclododecane, commonly used as a flame retardant 

in household items, contains many bromine atoms in the molecule 

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 excretion in urine and feces 

as well as its distribution into organs. 

'--~-.. ••••••• v • ' vv ' " , .... ·~· •••• 

l 

Hexabromododecane 

Experimental 

Samples and reagents: We used Pyroguard SR-103 (SR-103, 

hereafter) from Daiichi Kogyo Seiyaku K.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 from Wako 

Junyaku K.K. 

Experimental animals: We used male 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 

( 100 mg/mL) . 

2 

Oral administration was performed for 5 days consecutively 

and urine and feces were daily collected by separately housing 

rats in a glass cage (Sugiyama Gen, type 5T). 24 h after the 

final administration, autopsy was 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 performed under the following 

conditions: detector: FID, column: 3% silicone OV-1 on chromosorb 

W AW DMCS, 60-80 mesh, 2.0 m x 3 mm id, column temperature: 240° 

for 4 min to attain a constant temperature 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 from the peak height ratio with 

respect to the internal standard (n-octacosane) . 

Quantification of SR-103 in feces: Distilled water was added 

to the total amount of feces collected at 4 mL/g, and the mixture 
was 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 

chloroform and acetone at a ratio of 4:1 for extraction with 

shaking for 10 min. The mixture was centrifuged at 1000 g for 5 

min and the solvent was removed under 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 similar to 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 distilled water. 

Absorption experiment using a loop of small intestine: A 

loop of small intestine was prepared according to the method of 

Levy, et al. from just below the pylorus {closed bile duct) with 

a length of about 20 em. A sample was prepared in the form of a 

fine suspension in physiological saline {10 mg/mL) and 2 mL of 

the suspension was introduced into the loop. 1 h after the 

introduction, the loop was isolated and the content of the loop 

was poured into a 10-mL measuring 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 10 min. The mixture 

was centrifuged (1000 g for 5 min) and the organic layer was 

separated. The solvent was removed under reduced pressure 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 into pieces about 1 em 

long and the pieces were homogenized (Waring [transliteration] 

blender, Nissei AM4 type, 6000 rpm for 15 min) 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, followed by centrifugation 

at 1000 g for 5 min. 4 mL of the organic layer were 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 of physiological 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 and 
5 

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 Waring blender. 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 extraction procedure. 

The calibration curve for each organ was prepared by 

extraction of each blank organ homogenate with the extraction 

solvent containing SR-103 at known concentrations followed by 

extraction and others procedures. 

Results and discussion 

Evaluation of quantification method of SR-103 in biological 

samples 

In quantification of SR-103 in biological samples, we 

performed examination of the extraction solvent for SR-103. We 

tried chloroform, chloroform-acetone mixture and n-pentane as the 

solvent and we observed that chloroform and pentane were markedly 

inferior to the chloroform-acetone mix :.ure in solubility of SR103 (less than 2 mg/mL), although they were excellent in 

preventing extraction of biological blanks. Consequently, these 

two solvents were found inadequate for recovery of SR-103 from 

fecal samples after administration of SR-103 at a dosage of 
6 

500 mg/kg/. Then we examined chloroform-acetone mixture and we 

decided to use the chloroform-acetone mixture at a 4:1 ratio as 

the extraction solvent, which is satisfactory in prevention 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 from biological components following 

the conditions of quantitative analysis and experimental 

procedures described in the Experimental section. As shown in the 

gas chromatogram of 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). Similarly excellent results were obtained in urine samples 

and homogenate samples of various organs. From these results, we 

concluded that SR-103 in biological samples can be quantified 

accurately and simply by this method of quantification. 

The limit of quantification in this method was about 5 ug/mL 

in urine and about 20 ug/mL in homogenates of biological samples 

(feces and various organs with added water 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 administered with SR-103 repeatedly 

for 5 days. Table I shows the average daily excretion with· 

respect to the administered amount and the 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) . From these observations, a 

strong possibility 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. From these 

results, it can be suggested that SR-103 can be 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 of measurements 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 similar test on 2,4,6-

tribromophenyl-2-methyl-2,3-dibromopropyl ether (TBPPE), a 

bromine flame retardant similar to SR-103. 
8 

'OBr .. CHJ 

B 0-CHz- C -CHz r I I 

Br Br 

TB Pf> E 

Br · 

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 from TBPPE 

After oral administration of TBPPE, we detected various 

peaks in GC attributable to metabolites 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 could be 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 distributed in the body at a 

concentration below the detection limit. 

Conclusion 

1. Hexabromocyclododecane was absorbed in the intestines 

following oral administration and about 35% of the amount 

administered was excreted in feces. 

2. No urinary excretion was observed under the experimental 

conditions of this study. 

3. No peak attributable to the metabolites 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 

. 

• 

'. 

' 

:' . 

:' 

. ' :. 

'I 

:' 

. 

' . • 

\ . 

'\I, 

' 

' 

Internal standard 

SR-103 

/ 

4 8 12' 14 

Time ( min ) 

Fig. 1 Gas Chromatograms of SR-103 Extracted from 

Rat Feces:, 

after oral administration of SR-103 

----- : fecal blank 

10 
11 

2.0 

0 

>.-1 

"' 

H "' 

..c:: "' 

"' >.-1 

QJ 

..c:: 

~ l.O "' QJ 

"' 

.• 

20 80 160 240 320 400 

Con en. ( )lg/ml homogena :te ) 

Fig, 2 Standard Curve of SR-103 Obtained from Rat Feces. 
1.4 

1.2 

"' ., 

"' E 

1.0 ..., 

"" "' . .; 

I!) 

:> Rat A 

,., 0.8 

'0 

0 

.0 

... 

I!) 0.6 Rat c Cl. 

'0 

I!) 

'"' I!) 

... 

C) 0.4 X 

I!) Rat D ..., 

:;l " 

0 

~ 0. 2 

Time{ day ) after administration 

Fig, 3 Cumulative Fecal Excretion after Multiple Oral Administration 

of SR-103 to Rots for 5 Days, 

Dose : SOOmg/kg 

12 
13 

Table I. Fecal Excretion after Oral Administration of SR-103 to Rats. 

Time{day) 

n 

v - 1 

1 - 2 

2 - 3 

3 - 4 

4 - 5 

Percent 

per day 

3 ~ ·• 7 + 

28.8 + 

- 37.4 + 

36.2 + 

32.5 + 

excreted 

9.43 

14.33 

12.98 

9.85 

7.47 

Time{day) 

0 - 1 

0 - 2 

0 - .3 

0 - 4 

0 - 5 

Each data represents ~ean + S.E.M. { n = 4 ) . 

Percent of cumulative 

excretion 

34 .. 7 + 9.43 

31.7 + 11.07 -

33.6 + 10.04 

34.4 + 8.68 - 34.0 + 7.28 
Table 11. Percent of Residuor SR-103 in the Intestinal Loop ond 

Tissue of tile Upper Small Intestine of Rots, 

Loop Tissue 

Rat 1 16.3 11.6 

Rat 2 4.8 12.3 

14 
Table III. Concentration of SR-103 In Fat Tissue after 

MultiPle Oral Administration of SR-103 to 

Rots for 5 Days, 

Rat A 

Rat B 

Rat C 

Rat D .• 

Concentration of SR-103 

( mg/g tissue J 

0.67 

0.48 

0.47 

0.33 

15