BII-0908 

Table 14·4 Twenty-eight-day repeated-dose oral toxicity study in rats 

Summary of histopathological examinations 

Male Female 

Findings Grade 

Vehicle 

control 

Vehicle 

control 

(R ) ecove!l 

25 ISO 1,000 1,000 

(Recovery) 

Vehicle 

control 

Vehicle 

control 

(Recovery) 

25 150 1,000 1,000 (gfkgfd ) (Recovery) m . ay 

ta ta ta Ia fa fa fa ta Ia III III fa 

51' S 5 j 5 S 5 5 S S 5 S 

Axillar lymph node 

No abnormalities detected 515bl 515 515 515 

Mesenteric lymph node 

No abnonmalities detected 515 515 515 515 

Spleen 

No abnormalities detected 5/5 5/5 5/5 5/5 

Thymus 

No abnormalities detected 515 5f5 515 515 

<0 

I No abnormalities detected 515 SIS 5/5 515 -----------­

Thyroid 

No abnormalities detected 5f5 5f5 515 SIS 

PlInIlhyroid 

No abnonnalities detected 515 5/5 5/5 515 

Adrenal 

No abnonnalities detected 4/5 5/5 5/5 515 ................................................................... _ ....................................... _ ... __ .... __ ...... ___ ...................... #0 ...................................................... " ...... - ... _ ..... -- .......................... _ .... 


F()Cal hypertrophy ofcortical 

+ If5 015 0/5 0/5 cells 

Eyeball 

No abnormalities detected Sf' 515 !filS SIS 

Skin 

Scab [onmation + 1/1 

ta, tenmioal autopsy. 


a) Number ofanimals autopsied. 


b) Number of animals affected I Number of animals examined. 

A -, Not examined. W 

CJ1 

+, slight. 
B11-0908 

Addendum 1-1 	 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp. group Animal Administration Period (day) Recovery Period (day) 


(mglkgfday) No. Sings 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 22 23 24 25 26 27 28 I 2 3 4 5 6 7 8 9 10 II 12 13 14 


2 

Vehicle 3 control 

4 

, 

6 

Vehicle 7 

control 8 

(Recovery) 9 

I 

00 

o 

I 

10 

-: No abnormalities detected. 

w 

..j:lo. 

c::J) 
BII-0908 

Addcndum 1-2 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs ofindividual animals (males) 

Exp. group Animal Administration Period (day) 

(mglkglqay) No. Sings 2 34 S 6 1 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 


1l 

12 
 ---------~-----~-------.-"'-.--,,-.----------

2S 13 


14 


IS 

-: No abnormalities detected. 

I 


00 


I-' 


I 


W 

-1:>0 

--.J 
-----------------------------------------------

B11-0908 

Addendum 1·3 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp_ group Animal 	 Administration Period (day) 

(mglkglday) No. Sings I 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

16 


Salivation 15 17 20 21 


Decreased spontaneous 
 16 20 21 22 locomotion 

17 

150 Salivation 16 18 21 22 24 25 26 28 

18 

Salivation 	 10 13 14 15 16 17 19 20 21 22 23 24 25 27 

19 

Salivation 3 4 8 10 13 14 15 16 17 18 19 22 23 24 26 27 28 

1 

00 20 

N 

1 Salivation 13 15 16 17 26 27 28 

Decreased spontaneous 

locomotion 24 

.: No abnormalities detected. 

(..) 

~ 
B11-0908 

Addendum 1-4 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp. group Animal 	 Administration Period (day) 

(mglkgfday) No. Sings 2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

21 

Salivation 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Decreased spontaneous 

locomotion 6 7 8 9 12 13 14 15 16 17 21 22 24 25 26 27 28 

22 

Salivation 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Decreased spontaneous 

locomotion 7 9 13 14 16 17 18 19 21 22 23 24 27 28 

I 

00 

c.u 

I 

Moistening of mandible 

Moistening of mandible 

and neck 

1,000 

Loss of hair (mandible) 

Much food on tray 

Lacrimation 

23 

Salivation 2 3 4 :5 6 7 8 9 10 II 

13 15 17 18 21 

19 20 

19 20 21 

19 20 

12 13 14 15 16 17 18 19 20 21 

22 23 26 27 28 

25 

22 23 24 25 26 27 28 

22 

25 

22 23 24 25 26 27 28 

Decreased spontaneous 

locomotion 7 9 10 12 15 16 17 19 20 21 22 23 

Incomplete eyelid 

opening 7 

Moistening ofmandible 19 

C.:I 

..lIoo 

co 

Much food on tray 

-: No abnormalities detected. 

21 
BIl-0908 

Addendum 1-5 	Twenty-eigbt-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp. group Animal 	 Administration Period (day) 

(mglkg/day) No. Sings 2 3 4 5 6 7 8 9 IQ II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

I 

00 

If::o 

I 

24 

1,000 

25 

Salivation 

Decreased spontaneous 

locomotion 

Moistening ofmandible 

lollS ofhair (mandible) 

Much food on tray 

Salivation 

Decreased spontaneous 

locomotion 

Much food on tray 

-: No abnormalities detected. 

3 

3 

4 

4 

5 

5 

6 

6 

7 

7 

8 

8 

9 

9 

9 

10 Jl 

10 11 

12 13 

13 

12 13 

13 

14 15 

14 

15 

14 15 

14 15 

16 17 18 19 20 21 

20 21 

18 21 

19 20 21 

21 

16 17 18 19 20 21 

16 17 21 

22 23 24 25 26 27 28 

24 25 

22 

22 23 24 25 26 

22 23 24 25 26 27 28 

22 23 24 25 26 27 28 

22 23 24 26 

23 25 27 28 

(,.) 

t.n 

o 
BlI·0908 

Addendum 1-6 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp. group Animal Administration Period (day) Recovery Period (day) 

(llIJIk&Idlly) No. Sings 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2 3 4 5 6 7 8 9 10 II 12 13 14 

I 

00 

t;;"J 

I 

1,000 

(Recovery) 

26 

27 

Salivation 

Decreased spontaneous 

locomotion 

Loss afhair (mandible) 

Much food on tray 

Moistening of mandible 

Salivation 

Decreased spontaneous 

locomotion 

Loss of hair (mandible) 

Loss ofbair (mandible 

and neck) 

3 

3 

4 

4 

5 

5 

6 

6 

6 

7 

7 

7 

8 

8 

9 

9 

10 II 

11 

10 II 

11 

12 

12 

12 

12 

12 

13 14 15 16 17 18 

13 14 15 16 17 18 

13 14 15 16 17 18 

13 14 

13 14 15 16 17 18 

192021 22 23 

19 21 

19 20 21 22 23 

22 

23 

19 20 21 22 23 

19 20 21 22 23 

19 20 

20 21 22 23 

24 25 2627 28 

24 20 27 28 

24 25 26 27 28 

24 26 27 28 

24 25 26 27 28 I 2 3 4 5 6 7 8 9 10 11 

Moistening of mandible 15 17 18 26 

Moistening of mandible 

and neck 

-: No abnormalities detected. 

20 21 22 23 25 27 28 

w 

(J'I 
B1I-0908 

Addendum 1-7 	Twenty-eight-day repealed-dose oral toxicity study in rats 

Clinical signs of individual animals (males) 

Exp. group Animal 	 Administration Period (day) Recovery Period (day) 

(mg/kglday) No. Sings 2 3 4 .5 6 7 8 9 10 II 12 13 14 15 16 17 18 192021 22 23 24 25 2627 28 2 3 4 5 6 7 8 9 10 II 12 13 14 

21 

Salivation 

Decreased spontaneous 

locomotion 

2 3 4 5 6 7 8 9 to II U 

12 

13 14 15 

14 

16 17 18 19 20 21 

19 20 21 

22 23 24 25 26 27 28 

22 24 

Moistening ofmandible \3 15 17 18 21 22 23 

I 

00 

0':> 

I 

1,000 

(Recovery) 29 

Moistening ofmandible 

and neck 

Much food on tray 

Loss of hair (mandible) 

Salivation 

Decreased spontaneous 

locomotion 

3 4 5 6 7 8 9 10 

10 

11 

II 

12 

12 

13 

13 

14 

14 

15 

IS 

16 

16 

17 18 

17 

19 20 

21 

1920 21 

19 20 

25 

23 24 26 

22 23 

22 23 24 25 2627 28 

22 23 

Moistening of mandible 25 

30 

Salivation J 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 2223 24 25 26 27 28 

Decreased spontancous 

locomotion 10 13 14 15 16 17 18 23 24 25 26 27 28 

Much food on tray 20 21 22 23 25 27 3 

-; No abnormalities detected. 

(,..) 

<.n 

N 
811-0908 

Addendum 1-8 Twenty-eight-day repeated-dose oral toxicity study in rots 

Clinical signs of individual animals (females) 

Exp. group Animal Administration Period (day) Recovery Period (day) 

(mglkglday) . No. Sings 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 I 2 3 4 5 6 7 8 9 10 II 12 I3 14 

31 

32 

Vehicle 

--

33 control 

34 

-

35 

36 

-- Vehicle 37

control 38 

(Recovery) 39 

-

40 

I 

00 -: No abnormalities detected. -...l 


I 


w 

U1 

W 
Bll-0908 

Addendum 1-9 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual !IIlimals (females) 

Exp. group Animal Administration Period (day) 


(mglkglday) No. Sings 2 3 4 S 6 7 8 9 10 11 12 13 14 IS 16 17 18 19 20 21 22 23 24 2S 26 27 28 


41 


42 


25 43 


44 


45 


-: No abnormalities detected. 

I 


CO 


CO 


I 


w 

c.n 

-1:>0 
BII-0908 

Addendum 1-10 Twenty-eight-day repeated-do5e oral toxicity study in rats 

Clinical signs of individual anima.ls (females) 

Exp. group Animal Administration Period (day) 

(mglkgfday) No. Sings 2 3 4 S 6 1 8 9 10 11 11 \3 14 IS 16 17 1& 19 20 2J 22 23 2il 25 26 27 28 

46 

Salivation 24 

47 

150 

48 

Salivation 3 4 9 10 II 12 13 24 25 2627 28 

Salivation 3 13 

49 


so 

-: No abnorma.lities detected. 

I 

00 

to 


I 


W 

0'1 

0'1 
B11-0908 

Addendum I-II Twenty-eigbt-day repeated-dose oral toxicity study in rats 

Clinical signs ofindividualllIlimals (females) 

Exp. group Animal Administration Period (day) 

(mg/kg/day) No. Sings 1 2 3 4 .5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

51 

Salivation 1 2 3 4 5 6 7 8 9 }O II 12 13 14 }5 16 17 18 19 20 21 22 23 24 25 26 27 28 

Decreased spontaneous 

locomotion 10 11 14 16 17 28 

Incomplete eyelid 

opening 10 

Muc:h food on tray 24 

52 

Salivation 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

I 1,000 1:0 	 Decreased spontaneous o 	 6 8 II· 13 14 17 21 22 23 24 25 

I 	 locomotion 

Staining lower abdomen 26 27 28 

53 

Salivation 

Staining lower abdomen 

Decreased spontaneous 

locomotion 

3 4 

4 

5 

5 

5 

6 

6 

6 

7 

7 

7 

8 

8 

8 

9 

9 

10 

10 

11 12 13 

12 J3 

14 

14 

15 

15 

16 

16 

17 

17 

18 

18 

19 20 21 

20 

20 21 

22 

22 

24 25 

24 25 

26 27 28 

26 27 28 

27 

Much food on tray 21 22 23 25 26 27 

-: No abnormalities detected. 

(.) 

0) 

01 
Bll-0908 

Addendum 1-12 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (females) 

Exp. group Animal Administration Period (day) 

(mglkglday) No. Sings 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

54 

Salivation 2 3 4 :5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

Decreased spontaneous 

locomotion 5 7 9 10 II 12 J3 14 15 16 17 23 24 2:5 26 27 

Moistening of mandible 13 19 

1,000 

Much food on tray 

Loss ofhair (mandible) 

20 21 

20 21 

22 23 

22 23 

24 25 26 27 

, 

(.0 

...... 

I 

55 

Salivation 

Decreased spontaneous 

locomotion 

3 4 5 6 

6 

7 

7 

8 9 10 11 12 13 14 15 16 17 18 19 20 21 

14 15 16 17 

22 23 24 25 26 27 28 

27 28 

Staining lower abdomen HI 19 20 21 26 

Much food on tray 22 

-: No abnonnalities detected. 

W 

01 

....... 
BII"()908 

Addendum ]-13 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (females) 

Exp. group Animal Administration Period (day) Recovery Period (day) 

(mglkgfday) No. Sings 2 3 4 5 6 7 8 9 10 11 12 \3 14 15 16 17 18 1920 21 22 23 24 25 26 27 28 2 3 4 5 6 7 8 9 10 II 12 13 14 

56 

Salivation 

Staining lower abdomen 

Decreased spontaneous 

locomotion 

3 4 

4 

5 

5 

6 7 

7 

8 9 

9 

10 

10 

11 12 13 

13 

14 

14 

15 16 17 

16 17 

18 19 20 21 

19 

22 23 24 25 26 27 28 

26 

24 

Incomplete eyelid 

opening 

Loss of hair (mandible) 

9 10 

11 12 13 14 15 16 \7 18 1920 21 22 23 

I 

<.0 

~ 

I 

1,000 

(Recovery) 

Moistening of mandible 

Much food on tray 

13 18 19 20 21 

20 

23 24 25 26 

22 23 24 25 26 :I 

57 

SaJivation 

Staining lower abdomen 

Decreased spontaneous 

locomotion 

2 3 4 5 6 7 8 9 10 II 

11 

12 13 

12 

14 15 16 17 18 19 20 21 

20 

21 

22 23 

22 

22 23 

24 25 

24 25 

26 27 28 

26 27 28 

26 27 28 

Moistening of mandible 27 

Much food on tray 24 

-: No abnormalities detected, 

(.) 

CO 

01 
811-0908 

Addendum 1-14 Twenty-eight-day repeated-dose oral toxicity study in rus 

Clinical signs ofindividual animals (females) 

Exp. group Animal Administration Period (day) Recovery Period (day) 

(mglkgfday) No. Sings 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2 3 4 5 6 7 8 9 10 II 12 13 14 

58 

Salivation 

Decreased spontaneous 

locomoticm 

2 3 4 

4 

5 

5 

6 7 8 

6 7 

9 

9 

10 

10 

11 

11 

12 

12 

13 

13 

14 

14 

15 16 

16 

17 

17 

18 19 20 21 

20 21 

22 23 24 25 26 27 28 

22 23 24 26 27 

- - - . 

Incomplete eyelid 

opening 

Loss ofhair (mandible) 

6 

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

Moistening of mandible 17 

I 

~ 

<;=' 

Much food on tray 

1,000 Staining lower abdomen 

(Recovery) 59 

Salivation 

Decreased spontaneous 

locomotion 

Staining lower abdomen 

Incomplete eyelid 

opening 

2 3 4 

4 

4 

5 

5 

678 9 10 II 

II 

II 

12 13 

13 

14 15 16 17 18 

18 

20 

19 20 21 

20 

2J 28 

26 

22 23 24 25 26 27 28 

24 25 28 

. . . . - - -

Moistening of mandible 15 

Loss ofhair (mandible) 16 17 

Much food on tray 21 22 

w 

CJ1 

m 

-; No abnonnalities detected. 
BII-0908 

Addendum 1-1.5 Twenty-eight-day repeated-dose oral toxicity study in rats 

Clinical signs of individual animals (females) 

Exp. group Animal Administration Period (day) Recovery Period (day) 

(mglkglday) No. Sings 2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2 3 4 5 6 7 8 9 10 11 12 13 14 

60 

1,000 

(Recovery) 

Salivation 

Decreased spontaneous 

locomotion 

2 3 4 S 6 7 8 9 10 11 

10 

12 13 14 

14 

15 

1.5 

16 

16 

17 

17 

18 19 20 21 22 23 24 25 26 27 28 

24 26 27 

Staining lower abdomen 26 

-: No abnormalities detected. 

I 

<0 

~ 

I 

~ 

en 

o 
Addendum 2-\ Twenty-eight-day repeated-dose oral toxicity study in rats BIl'()908 


Detailed clinical observations ofindividual animals (Predosing) 


Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease of removal Vocalization 

0 0 

2 0 0 

3 0 0 

4 0 0 

Vehicle 5 0 0 

control 6 0 0 

7 0 0 

8 0 0 

9 0 0 

10 0 0 

11 0 0 

12 0 0 

25 13 0 0 

14 0 0 

Male 

15 0 0 

16 0 0 

17 0 0 

150 18 0 0 

19 0 0 

20 0 0 

2] ° 0 

22 0 0 

23 ° ° 24 0 0 

1,000 

25 

26 

0 

0 

0 

0 

27 0 0 

28 0 0 

29 0 0 

30 0 +1 

-95­ 361 
Addendum 2-2 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 


Detailed clinical observations of individual animals (predosing) 


Exp.group Removal from cage Sex Animal No. 

(mglkg/day) Ease of removal Vocalization 

31 0 0 

32 0 0 

33 0 0 

34 0 0 

Vehicle 35 0 0 

control 36 0 0 

37 0 0 

38 0 0 

39 0 0 

40 0 0 

41 0 +1 

42 0 0 

25 43 0 0 

44 0 0 

Female 45 0 0 

46 0 +1 

47 0 0 

150 48 0 0 

49 0 0 

50 0 0 

51 0 0 

52 0 0 

53 0 0 

54 0 0 

1,000 

55 0 0 

56 0 0 

57 0 0 

58 0 0 

59 0 0 

60 0 0 

-96­ 362 
Addendum 2·3 Twenty·eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (week I) 

Exp.group Removal from 

Sex Animal No. 

(mglkglday) Ease ofrernoval Vocalization 

1 0 0 

2 0 0 

3 0 0 

4 0 0 

Vehicle S 0 0 

control 6 0 0 

7 0 0 

8 0 0 

9 0 0 

10 0 0 

11 0 +1 

12 0 0 

25 13 0 0 

14 0 0 

Male 15 0 0 

16 0 0 

17 0 0 

150 18 0 0 

19 0 0 

20 0 0 

21 0 0 

22 0 +1 

23 0 0 

24 0 0 

1,000 

25 

26 

0 

0 

0 

0 

27 0 0 

28 0 0 

29 0 0 

30 0 0 

-97­ 363 
Addendum 2·4 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Detailed clinical observations of individual animals (week t) 

Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease ofremoval Vocalization 

31 0 0 

32 0 0 

33 0 0 

34 0 0 

Vehicle 35 0 0 

control 36 0 0 

37 0 0 

38 0 0 

39 0 0 

40 0 0 

41 0 0 

42 0 0 

25 43 0 0 

44 0 0 

Female 

45 0 0 

46 0 +1 

47 0 0 

150 48 0 0 

49 0 0 

50 0 0 

51 0 0 

52 0 0 

53 0 0 

54 0 0 

1,000 

55 

56 

0 

0 

0 

0 

57 0 0 

58 0 0 

59 0 0 

60 0 0 

-98­ 364 
Addendum 2-5 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Detailed clinical observations ofindividual animals (week 2) 

Exp.group Removal from cage Animal No. 

(mglkglday) Ease of removal Vocalization 

I 0 0 

2 0 0 

3 0 0 

4 0 ° Vehicle S 0 0 

control 6 0 0 

7 ° 0 

8 0 0 

9 0 0 

10 0 0 

II 0 0 

12 0 0 

25 13 0 0 

14 0 0 

IS 0 0 

Male 

16 0 ° 17 ° 0 

150 18 -I 0 

19 0 0 

20 ° 0 

21 0 0 

22 0 0 

23 0 0 

24 0 0 

],000 

25 

26 

0 

0 

0 

0 

27 0 0 

28 0 0 

29 0 0 

30 0 +1 

-99­ 365 
Addendum 2·6 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (week 2) 

Exp.group Removal from cage Animal No. 

(mglkglday) Ease of removal Vocalization 

31 0 0 

32 0 0 

33 0 0 

34 0 0 

Vehicle 35 0 0 

control 36 0 0 

37 0 0 

38 0 0 

39 0 0 

40 0 0 

41 0 0 

42 0 0 

25 43 0 0 

44 0 0 

Female 

45 0 0 

46 0 0 

47 0 0 

150 48 0 0 

49 0 0 

50 0 0 

51 0 0 

52 0 0 

53 0 0 

54 0 0 

55 0 0 

1,000 

56 0 0 

57 0 0 

58 0 0 

59 0 0 

60 0 +l 

-100­ 366 
Addendum 2·7 Twenty-eight-day repeated-dose oral toxicity study in rats BI1-0908 

Detailed clinical observations of individual animals (week 3) 

Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease of removal Vocalization 

0 0 

2 0 0 

3 0 0 

4 0 0 

Vehicle 5 0 0 

control 6 0 0 

7 0 0 

8 0 0 

9 0 0 

10 0 0 

11 0 0 

12 0 0 

25 13 0 0 

14 0 0 

Male IS 0 0 

16 0 0 

17 0 0 

150 18 0 0 

19 0 0 

20 0 0 

21 0 0 

22 0 0 

23 0 0 

24 0 0 

1,000 

25 

26 

0 

0 

0 

0 

27 0 0 

28 0 +1 

29 0 0 

30 0 0 

-101­ 367 
Addendum 2·8 Twenty·eight-day repeated-dose oral toxicity study in rats B1l..()9{)8 

Detailed clinical observations ofindividual animals (week 3) 

Exp.group Removal from cage Animal No. 

(mglkg/day) Ease of removal Vocalization 

31 0 0 

32 0 0 

33 0 0 

34 0 0 

Vehicle 35 0 0 

control 36 0 0 

37 0 0 

38 0 0 

39 0 0 

40 0 0 

41 -I 0 

42 0 0 

25 43 0 0 

44 0 0 

female 

45 0 +1 

46 0 +1 

47 0 0 

ISO 48 0 0 

49 0 0 

50 0 0 

51 0 0 

52 0 0 

53 0 0 

54 0 0 

1,000 

55 

56 

0 

0 

+l 

0 

57 0 0 

58 0 0 

59 0 0 

60 0 0 

-102­ 368 
Addendum 2-9 Twenty-eigbt-day repeated-dose oral toxicity study in rats Bll·0908 

Detailed cUnical observations of individual animals (week 4) 

Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease ofremoval Vocalization 

0 0 

2 0 0 

3 0 0 

4 0 0 

Vehicle 5 0 0 

control 6 0 0 

7 0 +1 

8 0 0 

9 0 0 

10 0 0 

11 0 +1 

12 ·1 0 

25 13 0 0 

14 0 0 

Male 15 0 0 

16 0 0 

17 0 0 

150 ]8 0 0 

19 0 +1 

20 0 0 

21 0 0 

22 0 0 

23 0 +1 

24 0 0 

1,000 

2S 

26 

0 

0 

0 

0 

27 0 0 

28 0 0 

29 0 0 

30 0 +] 

-103­ 369 
Addendum 2-10 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 

Detailed clinical observations of individual animals (week 4) 

Exp.group Removal from cage Sex Animal No. 

(mg/kgfday) Ease of removal Vocalization 

31 0 0 

32 0 0 

33 0 +) 

34 0 0 

Vehicle 35 0 0 

control 36 0 0 

37 0 0 

38 0 +1 

39 0 0 

40 0 +1 

41 0 +1 

42 0 +\ 

25 43 0 0 

44 0 0 

Female 

45 0 0 

46 0 0 

47 0 0 

150 48 0 0 

49 0 0 

50 0 0 

51 0 0 

52 0 0 

53 0 +1 

54 -\ 0 

1,000 

55 

56 

0 

0 

0 

+\ 

57 0 0 

58 0 0 

59 0 0 

60 0 0 

-104­ 370 
Addendum 2-11 Twenty-eight-day repeated-dose oral toxicity study in rats BIl-0908 

Detailed clinical observations of individual animals (Recovery week I) 

Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease of removal Vocalization 

6 0 0 

Vehicle 

control 

7 

8 

9 

0 

0 

0 

0 

0 

0 

Male 

10 

26 

0 

0 

0 

0 

27 0 +) 

1,000 28 0 +1 

29 0 0 

30 0 +) 

-105­ 311 
Addendum 2-12 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (Recovery week 1) 

Exp.group Removal from cage Selt Animal No. 

(mglkg/day) Ease ofremoval Vocaliwion 

36 0 0 

37 0 0 

Vehicle 

control 38 0 0 

39 0 0 

Female 40 0 0 

56 0 0 

57 0 0 

1,000 58 0 0 

.59 0 0 

60 0 0 

-106­ 372 
Addendum 2-J3 Twenty-eight-day repeated-dose oral toxicity study in rat~ BII-0908 

Detailed clinical observations of individual animals (Recovery week 2) 

Sex Exp.group 

(mglkg/day) 

Animal No. 

Removal 

Ease of removal 

from cage 

6 0 0 

Vehicle 

control 

7 

8 

9 

0 

0 

0 

+1 

0 

0 

Male 10 

26 

0 

0 

0 

0 

1,000 

27 

28 

29 

0 

0 

0 

0 

0 

0 

30 0 0 

VoealiZlltion 

-107­ 373 
Addendum 2-14 Twenty-eight-day repeated-dose oral toxicity study in rats B1I·0908 

Detailed clinical observations ofindividual animals (Recovery week 2) 

Exp.group Removal from cage Sex Animal No. 

(mglkglday) Ease of removal Vocalization 

36 0 0 

37 0 0 

Vehicle 

control 38 0 0 

39 0 0 

Female 40 0 +1 

56 0 0 

57 0 +1 

1,000 58 0 ° S9 ° 0 

60 ° 0 

-108­ 374 
Addendum 2·15 Twenty-eight-day repeated·dose oral toxicity study in rats Bll·0908 

Detailed clinical observations of individual animals (Predosing) 

Handling observations 

Sex I::':xp.group Animal No. Muscle Subnonnal Piloerection Staining Unkempt Paleness Reddening 

(mg/kglday) tone temperature hair hair 

0 

2 () 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

2S 13 0 

14 0 

Male 

lS 0 

16 0 

17 0 

150 18 0 

19 0 

20 0 

21 0 

22 0 

23 0 

24 0 

25 0 

1,000 

26 0 

27 0 

28 0 

29 0 

30 0 

-109­ 375 
Addendum 2-16 Twenty-eight-day repeated-dose oral toxicity study in rats 911-0908

Detailed clinical observations of individual animals (Predosing) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mglkgfday) tone temperature hair hair 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

Female 

45 0 

46 0 

47 0 

150 48 0 

49 0 

50 0 

51 0 

52 0 

53 0 

54 0 

1,000 

55 0 

56 0 

57 0 

58 0 

59 0 

60 0 

-110­ 376 
Addendum 2-17 Twenty-eight-day repeated·dose oral toxicity study in ra~ B11-0908 

Detailed clinical observations of individual animals (week 1) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mglkglday) tone temperature hair hair 

1 0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

25 

12 

13 

0 

0 

Male 

ISO 

14 

IS 

16 

17 

18 

0 

0 

0 

0 

0 

19 0 

20 0 

21 0 

22 0 

23 0 

24 0 

1,000 

25 

26 

0 

0 

27 0 

28 0 

29 0 

30 0 

-111­ 377 
Addendum 2-18 Twenty-eight-day repeated-dose oral toxicity study in rats BI1-0908 

Detailed clinical observations of individual animals (week 1) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mglkglday) tone temperature hair hair 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

Female 

45 0 

46 0 

47 0 

ISO 48 0 

49 0 

50 0 

51 0 

52 0 

53 0 +") 

54 0 

1,000 5S 

56 

0 

0 +1) 

57 0 

58 0 

59 0 + .) 

60 0 

a) Lower abdomen 

-112­ 378 
Addendum 2·19 Twenty-eight-day repeated-dose oral toxicity study in rats B11·0908 

Detailed clinical observations ofindividual animals (week 2) 

Handling observations 

Sex Exp. group Animal No. Muscle SubnofTIUIl Piloerection Staining Unkempt Paleness Reddening 

(mglkglday) tone temperature hair hair 

1 0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

25 

II 

12 

13 

14 

0 

0 

0 

0 

Male IS 

16 

0 

0 

ISO 

17 

18 

19 

0 

0 

0 

20 0 

],000 

21 

22 

23 

24 

25 

26 

27 

28 

29 

30 

0 

0 

{) 

0 

0 

0 

0 

0 

0 

0 

-113­ 379 
Addendum 2·20 Twenty-eight-day repeated-dose oral toxicity study in rats BII·0908 

Detailed clinical observations of individual animals (week 2) 

Handling observations 

Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mg/kglday) tone temperature hair hair 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

Female 45 0 

46 0 

47 0 

150 48 0 

49 0 

50 0 

51 0 

52 0 

53 0 

54 0 

1,000 55 

56 

0 

0 

57 0 +") 

58 0 

59 0 

60 0 

II) Lower abdomen 

-114­

380 
Addendum 2-21 Twenty-eigbt-day repeated-dose oral toxicity study in rats 811-0908 

Detailed clinical observations of individual animals (week 3) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnonnal Piloerection Staining Unkempt Paleness Reddening 

(mglkgfday) tone temperature hair hair 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

1 0 

8 0 

9 0 

10 0 

11 0 

Male 

25 

150 

12 

13 

14 

15 

16 

11 

18 

0 

0 

0 

0 

0 

0 

0 

19 

20 

0 

0 

1,000 

21 

22 

23 

24 

2S 

26 

21 

28 

29 

30 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

-115­ 381 
Addendum 2-22 Twenty-eight-day·repeated-dose oral toxlcity study in rats 811-0908 

Detailed clinical observations of individual animals (week 3) 

Handling observations 

Sex Exp. group Animal No. Muscle SubnonnaJ Piloerection Staining Uulcempt Paleness ReddeI)ing 

(mg/kglday) tone temperature hair hair 

31 0 

32 0 

33 0 

34 0 

Vehicle 3S 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

2S 

42 

43 

0 

0 

44 0 

Female 4S 

46 

0 

0 

ISO 

47 

48 

0 

0 

49 0 

50 0 

51 0 

52 0 

53 0 

54 0 

1,000 SS 

56 

0 

0 

+a) 

57 0 

58 0 

59 0 

60 0 

a) Lower abdomen 

-116­

382 
Addendum 2-23 Twenty-eighl-day repealed.dose oral toxicity study in rats BJ 1·0908 

Detailed clinical observations of individual animals (week 4) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mglkglday) tone temperature hair hair 

I 0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

25 13 0 

14 0 

MaJe 

15 0 

16 0 

17 0 

150 18 0 

19 0 

20 0 

21 0 

22 0 +") 

23 0 

24 0 

25 0 

1,000 26 0 

27 0 +") 

28 0 +t) 

29 0 +h) 

30 0 

a) Moistening ofthe mandibule and neck: 

b) Moistening ofthe mandibule 

-117­

383 
Addendum 2·24 Twenty-eight-day repealed·dose oral toxicity study in rats BII-0908 

Detailed clinical observations ofindividual animals <week 4) 

Handling observations 

Sex Exp. group Animal No. Muscle Subnonnal Piloerection Staining Unkempt Paleness Reddening 

(mg/lcglday) tone temperature hair bair 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

3!) 0 

40 0 

25 

41 

42 

43 

0 

0 

0 

44 0 

Female 45 

46 

0 

0 

150 

47 

48 

4!) 

.50 

0 

0 

0 

0 

51 0 

52 0 + oj 

53 0 +a) 

54 0 

1,000 55 

56 

0 

0 

+a) 

+ &),0) 

57 0 + oj 

58 0 + 0) 

59 0 

60 0 +a) 

a) Lower abdomen 

b) Moistening oftbe mandibule 

-118­

384 
Addendum 2-25 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (Recovery week 1) 

Handling observations 

Sex Exp.group Animal No. Muscle Subnormal Piloerection Staining Unkempt Paleness Reddening 

(mglkglday) tone temperature hair hair 

6 0 

7 0 

Vehicle 

control 8 0 

9 0 

Male 10 0 

26 0 

27 0 

1,000 28 0 

29 0 

30 0 

-119­

385 
Addendum 2·26 Twenty-eight·day repeated·dose oral toxicity study in rats .B11-0908 

Detailed clinical observations of individual animals (Recovery week 1) 

Handling observations 

Exp. group Animal No. Muscle Subnormal Piloerection Staining Unkempt Palel'les5 Reddening 

(mglkglday) tone temperature hair hair 

36 0 

Vehicle 

control 

37 

38 

39 

0 

0 

I) 

Female 

40 

S6 

0 

0 

57 0 

1,000 58 0 

59 0 

60 0 

-120­

386 
Addendum 2·27 Twenty·eight-day repeated-dose oral toxicity study in rats BlI·Ol108 

Detailed clinical observations ofindividual animals (Recovery week 2) 

Handling observations 

Exp.group 

(mglkgfday) 

Animal No. Muscle 

tone 

Subnormal 

temperature 

Piloerection Staining 

hair 

Unkempt 

hair 

Paleness Reddening 

6 0 

Vehicle 

control 

7 

8 

9 

0 

0 

0 

Male 10 

26 

t} 

0 .~ 

27 0 

1,000 28 0 

29 0 

30 0 

-121­

387 
Addendum 2·28 Twenty-eight-day repeated·dose oral toxicity study in rats B11-0908 

Detailed clinical observations of individual animals (Rerovery week 2) 

Handling observations 

Exp. group Animal No. Muscle Subnonnal Piloerection Staining Unkempt Paleness Reddening 

(mglkJldal') tone temperature bair hair 

36 0 

37 0 

Vehicle 

control 38 0 

39 0 

Female 

40 0 

56 0 

57 0 

1,000 58 0 

59 0 

60 0 

-122­ 388 
Addendum 2-29 Twenty-eight-day repeated-dose orol toxicity study in rats 811-0908 

Detailed clinical observations of individual animals (predosing) 

Handling observations 

Sex Exp.group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkglday) sill: 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

25 13 0 

14 0 

15 0 

Male 

16 0 

17 0 

150 18 0 

19 a 

20 0 

21 0 

22 0 

23 0 

24 0 

1,000 

25 

26 

0 

0 

27 0 

28 0 

29 0 

30 0 

-123­ 389 
Addendum 2-30 Twenty-eight-day repeated-dose oral toxicity study in rats BIJ-0908 

Detailed clinical observations of indlvidual animals (Predosing) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkglday) size 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

Female 

25 

42 

43 

44 

45 

46 

0 

0 

0 

0 

0 

150 

47 

48 

49 

50 

0 

0 

0 

0 

51 0 

52 0 

53 

54 ° 

S5 ° 

1,000 

56 ° 0 

57 0 

58 0 

59 0 

60 0 

-124­ 390 
Addendum 2-31 Twenty-eight.day repeated.dose oral toxicity study in rats BlI-0908 

Detailed clinical observations of individual animals (week J) 

Handling observations 

Sex. Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkglday) size 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

II 0 

9 0 

10 0 

25 

II 

12 

13 

14 

0 

0 

0 

0 

Male 

150 

15 

16 

17 

18 

19 

20 

0 

0 

0 

0 

0 

0 

21 0 

22 0 

23 0 

24 0 

l,Ooo 

25 

26 

0 

0 

27 0 

28 0 

29 

30 ° 0 

-125­ 391 
Addendum 2-32 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (week I) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkglday) size 

31 0 

32 0 

33 0 

34 - 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

Female 45 0 

46 0 

47 0 

150 48 0 

49 0 

SO 0 

51 0 

52 0 

53 0 

54 0 

1,000 

55 

56 

0 

0 

57 0 

58 0 

59 0 

60 0 

-126­ 392 
Addendum 2·33 Twenty·eight-day repeated-dose oral toxicity study in ralS B11·0908 

Detailed clinical observations of individual animals (week 2) 

Handling observations 

Sex Exp.group AnImll.INo. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkglday) size 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

25 13 0 

14 0 

Male 

IS 0 

16 0 

17 0 

150 18 0 

19 0 

20 0 

21 0 

22 0 

23 0 

24 0 

1,000 

25 

26 

0 

0 

27 0 

23 0 

29 0 

30 0 

-127­ 393 
Addendum 2-34 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 

Detailed clinical observations ofindividual animals (week 2) 

Handling observations 

Sex Exp.group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkgfday) size 

31 0 

32 0 

33 0 

34 Q 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

Female 

45 0 

46 0 

47 0 

150 48 0 

49 0 

50 0 

51 0 

52 0 

53 0 

54 ·0 

1,000 

55 0 

56 0 

57 0 

58 0 

59 0 

60 0 

-128­

394 
Addendum 2-35 Twenty-eight-day repeated-dose oral toxicity study in rats BlI-0908 

Detailed clinical observations of individual animals (week 3) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkg/day) size 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

2.5 13 0 

14 0 

Male IS 0 

16 0 

17 0 

150 18 0 

19 0 

20 0 

21 0 

22 + 0 

23 0 

24 0 

1,000 

2.5 

26 

0 

0 

27 0 

28 0 

29 0 

30 0 

-129­

395 
Addendum 2-36 Twenty-eight-day repeated-dose oralloxicity study in rats 811-0908 

Detailed clinical observetions ofindividualanimais (week 3) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mgllcglday) size 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

2:5 43 0 

44 0 

Female 

45 

46 

0 

0 

150 

47 

48 

49 

0 

0 

0 

50 0 

51 0 

S2 0 

53 0 

54 0 

1,000 

55 

56 

0 

0 

57 0 

58 0 

59 0 

60 0 

-130­

396 
Addendum 2-37 TWCllty-cigbt-day repeated-dose oral tox,icity study in rats B11-0908 

Detailed clinical observations of individual animals (week 4) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Ex,ophthalmos Pupillary Salivation Secretion 

(m~day) size 

0 

2 0 

3 0 

4 0 

Vehicle 5 0 

control 6 0 

7 0 

8 0 

9 0 

10 0 

11 0 

12 0 

25 13 0 

14 0 

Male 15 0 

16 0 

17 0 

150 18 0 

19 0 

20 0 

21 0 

22 + 0 

23 0 

24 0 

25 0 

1,000 

26 0 

27 0 

28 0 

29 0 

30 0 

-131­

397 
Addendum 2-38 Twenty-eight-day repeated-dose oral toxicity study in rats BI1-0908 

Detailed clinical observations ofindividual animals (week 4) 

Handling observations 

Sex Exp. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillwy Salivation Secretion 

(mg/kg/day) size 

31 0 

32 0 

33 0 

34 0 

Vehicle 35 0 

control 36 0 

37 0 

38 0 

39 0 

40 0 

41 0 

42 0 

25 43 0 

44 0 

45 0 Female 

46 0 

47 0 

150 48 0 

49 0 

50 0 

51 0 

52 0 

53 0 

54 0 

1,000 

55 0 

56 0 

57 0 

58 0 

59 0 

60 0 

-132­ 398 
Addendum 2-39 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Detailed clinical observations ofindividualanimals (Recovery week I) 

Handling observations 

Sex Exp.group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mgfkglday) size 

6 0 

7 0 

Vehicle 

control 8 0 

9 Q 

10 0 

Male 

26 0 

27 0 

1,000 28 0 

29 0 

30 0 

-133­ 399 
Addendum 2-40 Twenty-eight-day repeated-dose oral toxicity study in rats BII.-0908 

Detailed clinical observations of individual animals (Recovery week: 1) 

Handling observations 

Sex Ex.p. group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkg/day) size 

36 o 

37 o 

Vehicle 

control 38 o 

39 o 

Female 40 o 

56 o 

57 o 

J,OOO 58 o 

59 o 

60 o 

134­

400 
Addendum 2-41 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 

Detailed clinical observations of individual animals (Recovery week 2) 

Handling observations 

Exp.group Animal No. Cyanosis Lacrimation Exophthalmos Pupillary Salivation Secretion 

(mglkgfday) size 

6 0 

7 0 

Vehicle 

control 8 0 

9 0 

Male 

10 0 

26 0 

27 0 

1,000 28 0 

29 0 

30 0 

-135­ 401 
Addendum 2-42 Twenty-eight-day repeatcd-dosc oral toxicity study in rats BIl-0908 

Detailed clinical ooservations ofindividual animals (Recovery week 2) 

Handling observations 

Sex Exp.group Animal No. Cyanosis Lacrimation Exophtbalmos Pupillary Salivation Secretion 

(rnglkglday) size 

36 0 

37 0 

Vehicle 

control 38 0 

39 0 

Female 

40 0 

56 0 

57 0 

1,000 58 0 

59 0 

60 0 

-136­

402 
Addendum 2-43 Twenty·eight-day repea1ecl-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (Preclosing) 

Observations in arena 

Sill( Exp.group Animal No. Posture Motor Respiration Lid Gail 

(mglkglday) activity closure 

1 0 0 0 

2 0 0 0 

3 0 0 0 

4 0 0 0 

Vehicle S 0 0 0 

control 6 0 0 0 

7 0 0 0 

8 0 0 0 

9 0 0 0 

10 0 +1 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 0 

14 0 0 0 

Male 

IS 0 0 0 

16 0 0 0 

17 0 0 0 

ISO 18 0 0 0 

J9 0 0 0 

20 0 0 0 

21 0 0 0 

22 0 0 0 

23 0 0 0 

24 0 0 0 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 0 0 0 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-137­

403 
Addendum 2-44 Twenty-eight·day repeated-dose oral toxicity study in rats Bll·0908 

Detailed clinical observations ofindividual animals (Preclosing) 

Observations in arena 

Sex Exp. group AnirnalNo. Posture Motor Respiration Lid Gait 

(mglkglday) activity closure 

31 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 Q 0 

control 36 0 0 0 

37 0 0 0 

38 0 0 0 

39 0 +1 0 

40 0 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 45 0 0 0 

46 0 0 0 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

50 0 ° 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 55 

56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

58 0 0 0 

59 ° 0 0 

60 ° 0 0 

-138-­

404 
Addendum 2-45 Twenty-eight-dIlY repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual Mimals (week 1) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mglkgfday) activity closure 

0 0 0 

2 0 -1 0 

3 0 0 0 

4 0 0 0 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 0 0 

8 0 +} 0 

9 0 0 0 

10 0 +1 0 

II 0 0 0 

12 0 0 0 

25 \3 0 0 0 

14 0 0 0 

Male 

15 0 +1 0 

16 0 0 0 

17 0 0 0 

150 18 0 +1 0 

19 0 0 0 

20 0 0 0 

21 0 0 0 

22 0 0 0 

23 0 0 0 

24 0 0 0 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 0 0 0 

28 0 0 0 

29 0 +1 0 

30 0 0 0 

-'-139­ 405 
Addendum 2-46 Twenty-eight-day repeated-dose oral toxicity study in rats 911-0908 

Detailed clinical observations of individual animals (week I) 

Observations in arena 

Sex. Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mglkgJday) aetlvity closure 

31 0 0 0 

32 0 +l 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 0 0 

control 36 0 +1 0 

37 0 0 0 

38 0 0 0 

39 0 0 0 

40 0 +1 0 

41 0 0 0 

2S 

42 

43 

0 

0 

0 

0 

0 

0 

Female 

150 

44 

45 

46 

47 

48 

49 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

50 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 

55 

56 

0 

0 

0 

+1 

0 

0 

57 0 0 0 

58 0 +1 0 

59 0 0 0 

60 0 0 0 

-140­ 406 
Addendum 2-47 Twenty-eight-da.y repeated-dose oral toxicity study in rats BII-0908 

Detailed clinical observations of individuaillnimals (week 2) 

Observations in arena 

Sex Exp.group Animal No. Posture Motor Respiration Lid Gait 

(mglkglday) activity closure 

0 Q 0 

2 0 0 0 

3 0 0 0 

4 0 0 0 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 0 0 

8 0 0 0 

9 0 Q 0 

10 Q +1 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 0 

14 0 0 0 

Male 

15 0 0 Q 

16 0 0 0 

17 0 0 0 

150 18 0 0 0 

19 0 0 0 

20 0 0 0 

21 0 0 0 

22 0 0 0 

23 0 0 0 

24 0 0 0 

1,000 

25 

26 

Q 

0 

0 

+1 

Q 

0 

27 0 0 0 

28 0 0 0 

29 0 +1 0 

30 0 0 0 

-141­ 407 
Addendum 2-48 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Detailed clinical observations ofindividual animals (week 2) 

Observations in lU'eTla 

Sex Ex.p. group Animal No. Posture Motor RespinUion Lid Gait 

(mgIkg{day) activity closure 

3] 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 .0 

Vehicle 35 0 0 0 

control 36 0 0 0 

37 0 0 0 

38 0 0 0 

39 0 0 0 

40 0 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 

45 0 +l 0 

46 0 0 0 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

SO 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 

55 

56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

58 0 0 0 

59 0 0 0 

60 0 +1 0 

-142­ 408 
Addendum 2-49 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical obse("\lations of individual animals (week 3) 

Ob5el"\iations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mglkglday) activity closure 

I 0 0 0 

2 0 0 0 

3 0 0 0 

4 0 0 0 :' 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 0 0 

8 0 0 0 

9 0 0 0 

10 0 0 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 0 

]4 0 0 0 

Male 

15 0 0 0 

16 0 0 0 

17 0 0 0 

150 18 0 0 0 

19 0 0 0 

20 0 0 0 

21 0 0 0 

22 0 0 0 

23 0 0 0 

24 0 0 0 

1,000 

2S 

26 

0 

0 

0 

0 

0 

0 

27 0 0 0 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-143­ 409 
Addendum 2·50 Twenty-eight-day repeated-dose oral toxicity study in rats BI1·0908 

Detailed clinical observations ofindividual animals (week: 3) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Galt 

(mglkglday) activity closure 

31 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 0 

Vehicle: 35 0 0 0 

control 36 0 0 0 

37 0 0 0 

38 0 0 0 

39 0 0 0 

40 0 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 

45 0 0 0 

46 0 0 0 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

50 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

.54 0 0 0 

1,000 55 

.56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

58 0 0 0 

59 0 0 0 

60 0 0 0 

-144­ 410 
Addendum 2-51 Twenty-eight-day repeated·dose oral toxicity study in rats 811...0908 

Detailed clinical observations of individual animals (week 4) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Oait 

(mgtkglday) activity closure 

0 0 0 

2 0 -1 0 

3 0 0 0 

4. 0 0 0 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 0 0 

8 0 0 0 

9 0 0 0 

10 0 0 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 0 

14 0 0 0 

Male 

15 0 -I 0 

16 0 0 0 

17 0 0 0 

150 18 0 0 0 

19 0 +1 0 

20 0 0 0 

21 0 0 0 

22 0 -I 0 

23 0 0 0 

24 0 0 0 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 0 -I 0 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-145­ 411 
Addendum 2·52 Twenty-eight-day repeated-dose oral toxieity study in rats Bll-0908 

Detailed clinical observations ofindividual animals (week 4) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mgllc&lday) activity closure 

31 0 0 0 

32 0 +1 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 0 0 

conlrol 36 0 0 0 

37 0 0 0 

38 0 0 0 

39 0 0 0 

40 0 -I 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 

45 0 0 0 

46 0 0 0 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

SO 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 -1 0 

1,000 

S5 

56 

0 

0 

+1 

+I 

0 

0 

57 0 +1 0 

58 0 0 0 

59 0 0 0 

60 0 0 0 

-146­ 412 
Addendum 2-53 Twcnty-eight-day repeated-dose oral toxicity study in rats Bl1-0908 

Detailed clinical observations of individual animals (Recovery week I) 

Observations in arena 

Sex Exp.group Animal No. Posture Motor Respiration Lid Galt 

(mglkg/day) activity closUre 

6 ° 0 0 

7 0 -1 0 

Vehicle 

control 8 0 0 0 

9 0 0 ° 

Male 10 0 0 ° 26 0 ° ° 27 0 0 0 

1,000 28 ° ° ° 29 0 ° 0 

30 0 0 0 

-147­ 413 
Addendum 2·54 Twenty-eight·day repeated.dose oral toxicity study in rats B11-0908 

Detailed clinical observations of individual animals (Recovery week 1) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mg1kglday) activity closure 

36 0 0 0 

37 0 0 0 

Vehicle 

control 38 0 0 0 

39 0 0 0 

Female 

40 0 0 0 

56 ° 0 

57 ° 0 0 0 

1,000 58 ° 0 0 

59 0 0 0 

60 0 0 0 

-148­ 414 
Addendum 2-55 Twenty-eight-day repeated-dose DraltDxicity study in rrlts B11-0908 

Detailed clinical observations of individual animals (Recovery week 2) 

Observations in arena 

Sex Exp.group Animal No. Posture Motor Respiration Lid Gail 

(mglkglday) activity closure 

6 0 0 0 

7 0 -1 0 

Vehicle 

cantrol 8 0 0 0 

9 0 0 0 

Male 

10 ° 0 0 

26 0 0 

27 0 0 ° 0 

1,000 28 0 0 0 

29 0 0 0 

30 0 0 0 

-149­ 415 
Addendum 2·56 Twenty-tight-day repeated·dose oral toxicity study in rats Bll·0908 

Detailed clinical observations ofindividual animals (Recovery week 2) 

Observations in arena 

Sex Exp. group Animal No. Posture Motor Respiration Lid Gait 

(mgfkglday) activity closure 

36 0 +1 0 

37 0 0 0 

Vehicle 

control 38 0 0 0 

3C;? 0 0 0 

Female 

40 0 0 0 

56 0 0 0 

57 0 

1,000 ° ° 58 0 ° 0 

59 0 0 0 

60 ° 0 ° 

-150­ 416 
Addendum 2-57 Twenty-eight-day repeated-dose oral toxicity study in rats BII·0908 

Detailed clinical observations ofindividual animals (Prcdosing) 

Observations in arena 

Sex Exp.group Animal No. Tremoritwitchl Defecation Urination Stereotypic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

0 0 3 

2 0 0 0 

3 0 1 2 

4 0 0 3 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 11 

8 0 0 

9 0 0 0 

10 0 0 I 

11 0 0 0 

25 

12 

13 

0 

0 

0 

0 

0 

21 

14 0 0 0 

Male 15 

16 

0 

0 

0 

0 J 

ISO 

17 

18 

0 

0 

0 

0 

16 

3 

J9 0 0 0 

20 0 2 

21 0 0 I 

22 0 0 0 

23 0 0 2 

24 0 0 S 

1,000 

25 0 0 0 

26 0 0 I 

27 0 0 2 

28 0 0 0 

29 0 1 3 

30 0 0 

-151­ 417 
Addendum 2-58 Twenty-eight-day repeated-dose oral toxicity study in rats Bl1-0908 

Detailed clinical observations of individual animals (Predosing) 

Observations in arena 

Sex Exp. group 

(mglkglday) 

Animal No. Tremor/twitch! 

convulsion 

Defecation 

(countlmin ) 

Urination 

(count/min) 

Stereotypic 

behavior 

Abnormal 

behavior 

31 0 0 0 

32 0 0 I 

33 0 0 0 

34 0 0, 4 

Vehicle 35 0 0 3 

control 36 0 0 5 

37 0 0 4 

38 0 0 

39 0 0 2 

40 0 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 7 

44 0 0 13 

Female 45 0 0 2 

46 0 0 2 

47 0 0 9 

150 48 0 0 0 

49 0 0 0 

SO 0 I 0 

51 0 0 4 

52 0 0 0 

53 0 0 0 

S4 0 0 0 

\,000 

55 0 0 3 

56 0 1 0 

57 0 0 3 

58 0 2 

59 0 0 2 

60 0 0 0 

-152­ 418 
Addendum 2·59 Twenty-eight·day repeated·dose oral toxicity study in rats BII·0908 

Detailed clinical observations of individual animals (week I) 

Observations in arena 

Sex Exp. group Animal No. Tremor/twitch! Defecation Urination Stcreotypic Abnormal 

(mglkg/day) convulsion (count/min) (count/min) behavior behavior 

I 0 0 0 

2 0 0 0 

3 0 0 I 

4 0 0 0, 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 0 4 

8 0 0 0 

9 0 0 0 

10 0 0 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 I 

14 0 0 0 

Male 

15 0 1 

16 0 0 0 

17 0 0 12 

150 18 0 0 2 

19 0 0 0 

20 0 0 0 

21 0 0 0 

22 0 0 0 

23 0 0 2 

24 0 0 I 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 0 0 2 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-153­ 419 
Addendum 2-60 Twenty-tight-day repeated-dose oral toxicity study in rats BII-0908 

Detailed clinical observations ofindividual animals (week 1) 

Observations in arena 

Exp. group Animal No. Tremorltwitcbl Defecation Urination Stereotypic Abnormal 

(mglkgldll)') convulsion (count/min) (countlmin) behavior behavior 

31 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 1 

Vehicle 35 0 0 0 

control 36 0 0 0 

37 0 0 2 

38 0 0 2 

39 0 0 0 

40 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 2 

44 0 0 7 

Female 

45 0 0 0 

46 0 0 8 

47 0 0 0 

ISO 48 0 0 0 

49 0 0 0 

50 0 0 0 

51 0 0 2 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 

55 

56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

58 0 0 

59 0 0 3 

60 0 0 

-154­ 420 
Addendum 2-61 Twenty-eight·day repeated-dose oral toxicity study in rats B11-0908 

Detailed clinical observations of individual animals (week 2) 

Observations in arena 

Sex Exp. group Animal No. Tremorltwitchl Defecation Urination Stereotypic Abnonnal 

(mglkglday) conVUlsion (count/min) (count/min) behavior behavior 

0 0 1 

2 0 0 0 

3 0 0 4 

4 0 0 0 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 2 2 

8 0 0 0 

9 0 0 0 

10 0 0 0 

II 0 0 0 

12 0 0 0 

25 13 0 0 0 

14 0 0 0 

Male 

15 0 3 

16 0 0 2 

]7 0 0 5 

1'50 18 0 0 0 

19 0 0 0 

20 0 0 

21 0 0 2 

22 0 0 0 

23 0 4 

24 0 1 

1.000 

25 0 0 1 

26 0 0 0 

27 0 2 1 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-155­ 421 
Addendum 2-62 Twenty-eight-day repeated-dose oral toxicity study in rats BlJ-0908 

Detailed clinical observations of individual animals (week 2) 

Observations in arena 

Sex Exp. group Animal No. Tremorltwitch/ Defecation Urination Stereotypic Abnormal 

(mglkglday) convulsion (countlmin) (counllmin) behavior behavior 

31 0 0 Q 

32 0 0 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 0 0 

control 36 0 0 0 

37 0 0 0 

38 0 0 2 

39 0 0 0 

40 0 0 I 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 

Female 

45 0 0 

46 0 0 2 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

50 () 0 0 

5] 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 

55 

56 

0 

0 

0 

0 0 

57 0 0 0 

58 0 0 4 

S9 0 0 0 

60 0 0 0 

-156­ 422 
Addendum 2-63 Twenty-eight-day repeated-dose oral toxicity study in rats Bl1-0908 

Detailed clinical observations of individual animals (week 3) 

Observations in arena 

Sex Exp. group Animal No. Tremorftwilcb/ Defecation Urination Stereotypic Abnonnal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

I 0 0 0 

2 0 0 0 

3 0 0 0 

4 0 0 I 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 3 I 

8 0 0 0 

9 0 0 0 

10 0 0 0 

11 0 0 0 

12 0 0 0 

25 13 0 0 0 

14 0 0 0 

Male 

15 0 0 0 

16 0 2 

17 0 0 2 

150 18 0 0 0 

19 0 0 0 

20 0 0 0 

21 0 0 1 

22 0 0 0 

23 0 0 0 

24 0 ) 0 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 () 0 4 

28 0 0 0 

29 0 0 0 

30 0 0 0 

-157­ 423 
Addendum 2·64 Twenty-eight-day repcated-dose oral toxicity study in rats BlJ-0908 

Detailed clinical observations of individual animals (week 3) 

Observations in arena 

Exp. group Animal No. Tremor/twitch! Defecation Urination Stcreotypic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior bellavior 

31 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 0 0 

control 36 0 0 0 

37 0 0 3 

38 0 0 0 

39 0 0 0 

40 0 0 2 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 

45 0 0 0 

46 0 0 13 

47 0 0 0 

ISO 48 0 0 0 

49 0 0 0 

50 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 I 

54 0 0 0 

1,000 55 

56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

S8 0 0 4 

59 0 0 0 

60 0 0 0 

-158­ 424 
Addendum 2-65 Twenty-eight-day repeated-dose oral toxicity study in rats BI1-0908 

Detailed clinical observations of individual animals (week 4) 

Observations in arena 

Sex Exp. group Animal No. Tremorltwitchl Defecation Urination Stercotypic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

I 0 0 0 

2 0 0 0 

3 0 0 0 

4 0 0 0 

Vehicle 5 0 0 0 

control 6 0 0 0 

7 0 I I 

8 0 0 0 

9 0 0 0 

10 0 0 0 

25 

11 

12 

13 

0 

0 

0 

0 

0 

0 

0 

0 

0 

14 0 0 0 

Male 15 

16 

17 

0 

0 

0 

I 

2 

0 

0 

1 

0 

150 18 0 0 0 

19 0 0 0 

20 0 0 0 

21 0 ] 0 

22 0 0 0 

23 0 0 0 

24 0 0 

1,000 

25 

26 

0 

0 

0 

0 

0 

0 

27 0 0 6 

28 0 0 0 

29 0 1 0 

30 0 0 0 

-159­ 425 
Addendum 2-66 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations ofindividual animals (week 4) 

Observations in arena 

Sex Exp. group Animal No. Tremorltwitchl Defecation Urination Stereotypic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

31 0 0 0 

32 0 0 0 

33 0 0 0 

34 0 0 0 

Vehicle 35 0 0 0 

control 36 0 0 0 

37 0 0 0 

38 0 0 0 

39 0 0 0 

40 0 0 0 

41 0 0 0 

42 0 0 0 

25 43 0 0 0 

44 0 0 0 

Female 

45 0 0 0 

46 0 0 6 

47 0 0 0 

150 48 0 0 0 

49 0 0 0 

50 0 0 0 

51 0 0 0 

52 0 0 0 

53 0 0 0 

54 0 0 0 

1,000 

55 

56 

0 

0 

0 

0 

0 

0 

57 0 0 0 

58 0 I 3 

59 0 0 0 

60 0 0 0 

-160­ 426 
Addendum 2-67 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Detailed clinical observations of individual animals (Recovery week I) 

Observations in arena 

Exp.group Animal No. Tremorltwitchl Defecation Urination Stereo typic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

6 0 0 0 

Vehicle 

control 

7 

8 

9 

0 

0 

Q 

2 

0 

0 

0 

0 

Male 

10 

26 

0 

0 

0 

0 

2 

0 

27 0 1 

1,000 28 0 0 0 

29 0 0 0 

30 0 0 0 

-161­ 427 
Addendum 2-68 Twenty-eight-day repeated-dosc oral toxicity study in rats BII-0908 

Detailed clinical observations ofindividual animals (Recovery week I) 

Observations in arena 

Exp. group Animal No. Tremor/twitch! Defecation Urination Stereotypic Abnormal 

(mglkgday) convulsion (count/min) (count/min) behavior behavior 

36 0 0 0 

37 0 0 3 

Vehicle 

control 38 0 0 0 

39 0 0 

Female 

40 0 0 0 

56 0 0 0 

57 0 0 0 

1,000 58 0 0 0 

59 0 0 0 

60 0 0 0 

-162­ 428 
Addendum 2-69 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Detailed clinical observations of individual animals (Recovery week 2) 

Observations in arena 

Sex Exp.group Animal No. Tremor/twitchl Defecation Urination Stereotypic Abnormal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

6 0 0 0 

Vehicle 

control 

7 

8 

9 

0 

0 

0 

2 

0 

0 

0 

0 

2 

Male 

10 

26 

0 

0 

0 

0 

0 

0 

27 0 0 I 

1,000 28 0 0 0 

29 0 0 0 

30 0 0 0 

-163­ 429 
Addendum 2·70 Twenty-eight-day repeated·dose oral toxicity study in rats B1I·0908 

Detailed clinical observations of individual animals (Recovery week 2) 

Observations in arena 

Sex Exp. group Animal No. Tremor/twitch! Defecation Urination Stereotypic Abnonnal 

(mglkglday) convulsion (count/min) (count/min) behavior behavior 

36 0 0 0 

37 0 0 0 

Vehicle 

control 38 0 0 0 

39 0 0 0 

Female 40 0 0 0 

56 0 0 0 

57 0 0 0 

1,000 58 0 0 0 

59 0 0 0 

60 0 0 0 

-164­ 430 
Addendum 3-1 Twenty-eight-day repeated·dose oral toxicity study in rats Bll·0908 

Reflex ofindividual animals (week 4) 

Sensorimotor function 

Exp. group Animal No. Approach contact! Pinna Pain response Pupillary Air righting 

(mglkglday) touch response response (tail pinch) reflex reflex. 

0 0 0 + + 

2 0 0 0 + + 

3 0 0 0 + + 

4 0 0 0 + + 

Vehicle control 

5 0 0 0 + + 

6 0 0 0 + + 

7 0 0 0 + + 

8 0 0 0 + + 

9 0 0 0 + + 

10 0 0 0 + + 

11 0 0 0 + + 

12 0 0 0 + + 

25 13 0 0 0 + + 

14 0 0 0 + + 

Male 

IS 0 0 0 + + 

16 0 0 0 + + 

17 0 0 0 + + 

150 18 0 0 0 + + 

19 0 0 0 + + 

20 0 0 0 + + 

21 0 0 1'1 + + 

22 1'1 0 0 + + 

23 1'1 0 !'I + + 

24 0 0 0 + + 

1,000 

25 

26 

0 

0 

0 

0 

1'1 

1'1 

+ 

+ 

+ 

+ 

27 0 1'1 1'1 + + 

28 0 0 0 + + 

29 0 0 0 + + 

30 1'1 0 1'1 + + 

-165­ 431 
Addendum 3-2 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Reflex of individual animals (week 4) 

Sensorimotor function 

Sex Exp. group Animal No. Approach contact! PiMa Pain response Pupillary Air righting 

(mglkglday) touch response response (tail pinch) reflex reflex 

31 0 0 0 + + 

32 0 0 0 + + 

33 0 0 0 + + 

34 0 0 0 + + 

Vehicle control 35 0 0 0 + + 

36 0 0 0 + + 

37 0 0 0 + + 

38 0 0 0 + + 

39 0 0 0 + + 

40 0 0 0 + + 

41 0 0 0 + + 

42 0 0 0 + + 

25 43 0 0 0 + + 

44 0 0 0 + + 

Female 45 0 0 0 + + 

46 0 0 0 + + 

47 0 0 0 + + 

ISO 48 0 0 0 + + 

49 0 0 0 + + 

50 0 0 0 + + 

51 0 0 0 + + 

52 0 0 0 + + 

53 0 0 0 + + 

54 0 0 0 + + 

1.000 

55 0 0 0 + + 

56 0 0 0 + + 

57 0 0 0 + + 

58 0 0 0 + + 

59 0 0 0 + + 

6() 0 0 0 + + 

-166­ 432 
Addendum 4-1 Twenty·eight-day repeated-dose oral toxicity study in rats 811·0908 

Grip strength ofindividual animals (week 4) 

Sex Exp.group Animal No. Forelimb Hindlimb (g) 

(mglkg/day) Trial 1 

481 

Trial 2 

424 

Mean 

453 

Trial 1 

448 

Trial 2 

414 

Mean 

431 

2 413 397 405 458 407 433 

3 404 471 438 433 472 453 

4 382 472 427 414 435 425 

Vehicle 

control 

5 

6 

403 

493 

417 

388 

410 

441 

414 

371 

437 

379 

426 

375 

7 387 386 387 440 443 442 

8 455 424 440 412 385 399 

9 429 401 415 442 478 460 

10 345 381 363 381 459 420 

11 430 377 404 471 484 478 

12 375 384 380 465 431 448 

25 \3 449 428 439 483 426 455 

14 465 375 420 463 448 456 

Male 

15 457 423 440 455 488 472 

16 379 450 415 325 366 346 

17 368 449 409 464 429 447 

150 18 456 446 451 455 479 467 

19 340 424 382 433 418 426 

20 467 495 481 439 479 459 

21 353 434 394 345 363 354 

22 450 397 424 378 423 401 

23 459 498 479 420 417 419 

24 434 455 445 412 396 404 

1,000 25 

26 

414 

43] 

400 

484 

407 

458 

384 

383 

412 

416 

398 

400 

27 462 458 460 408 382 395 

28 326 385 356 433 344 389 

29 408 440 424 476 454 465 

30 398 412 405 400 454 427 

-167­ 433 
Addendum 4-2 Twenty-eigbt-day repeared-dose oral toxicity study in rats BII·090& 

Grip strength of individual animals (week 4) 

Sex Exp.group Animal No. Forelimb (g) Hindlimb (g) 

(mglkglday) 

31 

Trial 1 

425 

Tria! 2 

394 

Mean 

410 

Trial 1 

363 

Trial 2 

394 

Mean 

379 

32 369 327 348 375 345 360 

n 323 437 380 401 341 371 

34 340 352 346 407 386 397 

Vehicle 

control 

35 

36 

367 

373 

369 

397 

368 

385 

403 

350 

388 

375 

396 

363 

37 396 407 402 351 362 357 

38 406 382 394 439 417 428 

39 318 365 342 332 382 357 

40 322 371 347 363 386 375 

41 364 424 394 346 327 337 

42 321 376 349 393 388 391 

25 43 372 372 372 333 360 347 

44 335 399 367 367 356 362 

Female 45 377 326 352 356 411 384 

46 373 336 355 343 371 357 

47 374 376 375 357 331 344 

150 48 383 343 363 387 373 380 

49 369 421 395 404 358 381 

50 425 349 387 403 442 423 

51 332 327 330 411 374 393 

52 361 386 374 318 387 353 

53 340 339 340 421 402 412 

54 400 359 380 402 366 384 

1,000 55 

56 

312 

307 

336 

348 

324 

328 

324 

411 

362 

363 

343 

387 

57 376 398 387 381 424 403 

58 324 357 341 404 425 415 

59 397 422 410 392 377 385 

60 359 329 344 375 360 368 

-168­ 434 
Addendum 5-1 Twenty-eight-day repeated-dose oral toxicity study in rats BII-0908 

Motor activity of individual animals (week 4) 

Sex Exp. group Animal No. Interval (min) 

(mglkglday) 0-10 10-20 20-30 30·40 40-50 50-60 Total 

17 116 134 23 18 2 310 

2 166 .55 15 64 51 77 428 

3 206 172 138 22 4 0 542 

4 249 121 71 57 91 114 703 

Vehicle 5' 211 136 128 110 70 41 696 

control 6 177 71 4 74 3 70 399 

7 9 28 117 39 2 46 241 

8 302 140 92 74 118 173 899 

9 179 85 50 55 99 49 517 

10 227 148 45 50 4 38 512 

11 139 48 9 68 18 7 289 

12 283 136 107 15 72 46 659 

25 13 193 SI 73 29 8 106 490 

14 84 48 29 149 173 91 574 

Male 

15 239 116 80 42 55 48 580 

16 9 74 11 21 11 6 132 

17 207 135 51 63 67 100 623 

150 18 150 170 94 106. 122 52 694 

19 126 106 ISS 35 87 159 668 

20 140 30 30 4 46 39 289 

21 173 89 35 70 3 371 

22 371 157 197 102 232 154 1213 

23 166 54 65 88 59 124 556 

24 278 214 160 113 80 166 1011 

1,000 

25 

26 

251 

295 

176 

226 

166 

183 

154 

82 

57 

112 

40 

79 

844 

977 

27 197 78 88 36 116 65 580 

28 272 223 91 94 72 14 766 

29 265 157 127 139 107 9 804 

30 325 115 5 0 6 0 451 

-169­ 435 
Addendum 5-2 Twenty-eight-day repeated-dose oral toxicity study in rats B1I-0908 

Motor activity of individual animals (week 4) 

Sex Exp.group Animal No. Interval (min) 

(mglkglday) ()"10 10·20 20·30 30-40 4()"50 50-60 Total 

31 205 108 55 53 8 38 467 

32 317 123 106 36 0 15 597 

33 188 119 53 60 11 432 

34 261 261 152 83 35 0 792 

Vehicle 35 254· 56 59 0 17 33 419 

control 36 215 133 129 20 138 152 787 

37 180 97 102 25 39 74 517 

38 137 101 46 88 7 4 383 

39 307 129 203 131 20 122 912 

40 300 200 76 47 97 145 865 

41 234 149 104 13 72 20 592 

42 258 177 197 193 144 85 1054 

25 43 247 152 84 114 46 3 646 

44 250 134 110 0 119 135 748 

Female 

45 

46 

317 

142 

340 

84 

256 

4 

80 

7 

66 

0 

0 

]0 

1059 

247 

47 215 160 119 70 57 9 630 

150 48 198 89 7 18 75 2 389 

49 211 134 57 22 33 4 461 

50 208 136 46 84 88 26 588 

51 268 158 97 93 88 34 738 

52 353 268 69 204 221 48 1163 

53 294 135 93 92 31 108 753 

54 250 214 154 185 8 0 811 

1,000 

55 

56 

350 

324 

203 

190 

3] 

]57 

159 

15 

0 

114 

0 

155 

743 

955 

57 256 111 59 132 81 89 728 

58 204 155 206 118 [16 117 916 

59 327 211 223 227 157 174 1319 

60 278 148 62 0 15 163 666 

-170­ 436 
Addendum 5-3 Twcnly-eight-day repeated-dose oral toxicity study in rats 811-0908

Motor activity ofindividual animals (Recovery week 2) 

Sex Exp.group Animal No. Interval (min) 

(mglkglday) 0-10 10-20 20-30 30-40 40·50 50·60 Total 

36 157 127 91 7 116 72 570 

Vehicle 

control 

37 

38 

39 

128 

12 

235 

6 

5 

188 

25 

63 

33 

9 

40 

7 

2 

60 

35 

0 

45 

210 

53 

631 

Female 

40 

56 

180 

250 

51 

92 

21 

2 

0 

0 

8 

44 24 

261 

412 

57 204 87 43 34 91 32 491 

1,000 58 226 124 3 157 60 102 672 

59 282 268 237 8 58 0 853 

60 185 143 112 132 91 131 794 

-171­ 437 
Addendllll 6-1 Twenty-eight-day repeated-dose oral tOJicity study in rats 1111-0908 

Body weights of individual animals(g) 

Sex Exp.group Administration period 

(lig/kg/day) AninI No. -2 1 ' 3 8 12 17 21 26 28 (days) 

...... 

-.J 

t-:> 

I 

lIale 

Vebicle 

control 

25 

150 

1,000 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

27 

28 

29 

30 

140.7 

150.4 

155.3 

147.5 

149.1 

152.5 

149.8 

151.2 

145.4 

143.3 

149.3 

154.5 

141.7 

146.3 

151. 7 

155.2 

149.8 

151.4 

146.5 

140.7 

145.8 

149.3 

152.5 

150.8 

142.1 

152.6 

141. 6 

150.8 

143.4 

148.8 

155.2 

163.2 

169.2 

168.3 

169 1 

167.8 

160.5 

173.9 

163.3 

161.4 

169.4 

171. 2 

157.7 

184.3 

169.0 

171.2 

164.4 

169.1 

161.9 

155.2 

160.7 

167.5 

172.6 

167.5 

159.2 

171.5 

158.3 

167.9 

164.1 

16~L 5 

169.8 

182.8 

187.2 

183.6 

188.0 

182.8 

112.1 

193.3 

174.5 

174.4 

187.7 

185.7 

169.8 

180 .. 2 

181. 9 

185.4 

178.3 

188.3 

170.1 

167.3 

165.8 

169.0 

183.5 

180.3 

164.2 

180.4 

157.2 

168.3 

176.3 

114.~. 

204.4 232.3 

232.9 261,6 

231. 8 266.5 

226.2 257.5 

239.2 279.9 

228.3 258.4 

201.7 224.4 

238 1 273.8 

216.2 245.9 

208.1 232.7 

226.0 250.8 

221.4 253.4 

204.4 228.7 

235.3 266.7 

225.5 253.1 

225.2 249.5 

213.3 243.1 

240.8 280.5 

201.1 224.2 

201.9 229,2 

198.4 215.2 

201.6 330.7 

216.6 1148.9 

214.5 245.8 

201.7 221.6 

209.8 238.9 

186 3 208.5 

208.3 235.0 

227.0 262.3 

Z:()L.J._2~.4J__ 

266.7 

302.6 

305.8 

301.2 

336.2 

301.5 

264.0 

315.5 

287.9 

268.5 

283.5 

297.7 

.1 

17.8 

Z90.B 

277.3 

279.5 

341.3 

258.7 

31:1.1 

236.8 

268.6 

283.3 

277.4 

252.4 

280.6 

241.9 

268.7 

305.0 

~4_ 

295.9 

331.0 

335.5 

335.7 

359.6 

330.8 

295.3 

356.7 

321.7 

296.1 

310.4 

321.5 

290.4 

349.1 

319.0 

301.6 

306.0 

3U.4 

270.6 

~9i·6 

251.0 

292.8 

305.6 

300.9 

272.8 

306.9 

261.8 

288.9 

336.5 

~14,4. 

322,0 

359.5 

369 9 

374 9 

396.2 

356.3 

332.4 

381.9 

349.0 

319.9 

334.6 

351. 6 

312.6 

390.0 

340.3 

327.8 

336.1 

429.5 

Z89.S 

331.~ 

264.0 

315.5 

328.3 

322.3 

305.9 

334.0 

284.0 

314.2 

362.4 

301.0 

330.4 

368.2 

380.0 

386.2 

404.4 

368.2 

347 0 

392.3 

357 B 

330.9 

336.8 

361.7 

320.4 

403 0 

354.0 

336.3 

340.0 

445.0 

295.6 

337.8 

269.2 

318.4­

339.4­

330.4 

317.2 

342.4 

288.9 

320.7 

373.6 

305.1 

t; 

00 
AddendUll 6-2 	 TWenty-eight-dar repeated-dose oral toxicity study in rats Dll-0908 

Body weights of individual ani.als(g) 

Sex Exp.group 


(JIg/kg/day) Ani.al No. -2 1 3 8 12 17 21 26 28 


31 129.4 144.5 156.7 182.3 196.8 216.5 228.7 238.4 244.9 

32 113.9 125.4 129.9 147.6 159.5 174.3 185.9 199.6 203.4 

33 118.1 128.5 137.3 160.1 175.0 194.1 208.2 216.1 217.2 

34 133.0 144.8 155.2 177.2 184.7 201.5 216.9 224.1 236.6 

Vehicle 35 121.7 133.3 140.5 157.4 170.7 189.3 200.7 210.6 217.1 

control 36 124.3 133.0 142.7 163.2 170.3 190.4 198.6 211.1 209.1 

37 117.0 122.7 130.2 143.6 154.5 172 1 182.2 193.5 198.6 

38 131. 9 142.4 152.2 169.1 184 4 196.5 209.7 217.1 226.3 

39 119.0 132.2 138.0 158.2 168.7 185.8 193.2 195.9 204.5 

40 129.4 138.7 146.6 162.6 175.7 195.2 209.6 218.4 219.1 

41 129.5 139.2 144.4 171. 7 186.3 197.6 209.6 214 .4 223.8 

42 119.0 131. 7 142.6 156.4 162.4­ 176.5 189.0 200.5 206.2 

25 43 117.6 120.2 127.9 139.5 152.8 166.3 174.7 189.2 193.8 

44 127.3 135.1 144.2 166.1 180.3 202.1 213.4 223.3 235.7 

feJIale 45 122.3 135.3 144.3 153.5 164.7 173.0 183.8 192.1 198.6 

46 112.6 122.2 126.3 142.9 155.7 175.9 187.2 197.9 201. 3 

47 124.4 132.5 141.5 164.8 178.4 195.8 206.2 217.2 218.6 

150 48 128.6 143.7 153.6 191.6 193.0 202.1 212.5 226.0 238.4 

49 119.4 132.0 137.6 160.9 177. 2 200.6 209 3 218.9 228.3 

50 129.8 140.9 147.4 189.3 182.2 205.0 216.0 227.5 226.1 ....... 
 143:-6- ----­ -.:j 51 127.1 135.5 173.4 193.0 220.9 --Z35.4 241.3 244.4 

(jj 52 121. 0 130.2 135.1 160.5 185.5 202.1 216.3 225.0 226.9 

I 53 132.9 149.8 151.5 173.3 193.0 220.6 235.4 248.4 255.0 

54 117.9 125.3 132.3 150.1 161.2 179.7 191. 2 197.5 197.2 

1,000 	 55 122.4 129.9 136.7 158.8 174.0 197.1 206.0 214.2 225.6 

56 117.3 127.4 135.7 156.5 167.8 184.8 193 3 204.7 215.2 

57 123.7 136.8 148.8 176.4 192.8 211.1 22B.9 235.4 245.6 

58 129.6 140.7 154.9 169.5 186.8 210.7 218.5 228.0 232.3 

59 127.1 137.B 147.3 171.2 190.5 206.5 219.5 235.2 230.9 

60 U:l.2 132.0 138.!L l.61._L.__ 174.7 :t9~. 202.3 220.0 222.6 

.j::" 

W 

<0 
AddendUlD 6-3 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Body weights of individual aniaals(g) 

Sex Exp.group !tecover,. period 

(q/kg/day) Ani.al 110. 1 5 10 14 (days) 

6 

7 

372 .3 

351.8 

395.5 

375.3 

428.6 

403.1 

444.3 

421.6 

Vehicle 

control 

8 

9 

398.3 

361.3 

420.4 

380.6 

441. 4 

400.4 

453.7 

412 9 

lIale 10 334.8 359.2 381.2 401. 6 

26 340.3 366.1 399.4 424.0 

27 292.6 313.2 339.0 361.2 

1,000 28 

29 

323.0 

377.4 

346.4 

395.4 

377.7 

418.5 

399.2 

444.7 

30 304.8 311.6 329.7 350.8 

female 

Vehicle 

control 

36 

37 

38 

39 

40 

207.5 

202.3 

226.3 

207.5 

223.5 

218.3 

212.5 

232.6 

214.9 

235.1 

225.9 

215.8 

234.9 

215.8 

243.6 

228.7 

222.2 

243.2 

224.8 

249.2 

...... 

1.000 

56 

57 

58 

59 

60 

216.3 

240.5 

232.4 

233.8 

220.4 

211.3 

247.9 

240 4 

237.5 

232.8 

:laS.2 

247.0 

242.4 

244.5 

a.35.4­

229.7 

254.6 

246.0 

248.6 

240.2 

-.:J 

~ 

I 

~ 

~ 
Addendua 7-1 TweDty-eight-day repeated-dose oral toxicity study in rats 811-0908­

rood intakes of individual aniaa!s(g/rat/day) 

Sex Exp.group Administration period 

(llIg,/kg/day) 

-_._._- Anillal No. 1 3 8 15 2Z 28 (days) 

1 17.7 17.4 18.1 17.9 18.3 17.0 

2 19.0 22.1 23.1 21.2 21.3 19.2 

3 20.1 21.1 22.0 21.1 20.4 19.2 

4 20.0 19.6 21. 2 21.6 22.7 22.5 

Vehicle 5 22.1 21.1 24.9 25.3 23 9 21.7 

control 6 20.0 19.5 21.4 20.5 19.7 17.8 

7 19.1 17.9 18.2 18.0 20 0 20.9 

8 21.9 21.9 23.5 23.0 23.3 21.1 

9 19.8 18.1 20.1 21. 0 21. 5 20 2 

10 

11 

17.5 

21.1 

16.9 

20.7 

18.3 

20.2 

17.9 

18.2 

17,..L 

17.1 

lU 

16.5 

12 21. 2 20.7 21.1 21. 4 20.9 19.4 

25 13 18.2 17.0 19.4 18.4 18.6 17.3 

14 20.9 20.0 24.3 25.1 23.8 23.3 

Kale 15 20.7 18.9 20.6 20.2 19.3 17.4 

16 20.3 20.9 21.7 19.8 18.5 17.6 

17 20.5 19.7 20.7 21. 5 20.9 19.0 

150 18 22.0 21. 2 26.0 28.0 28.2 28.6 

19 21. 3 18.1 20.3 20.7 20.2 17.5 

...... 

~ 

20 

21 

18.6 

19.2 

17.5 

13.8 

18.8 

18.0 

20.5 

16.8 

21. 0 

17.1 

21.0 

16.5 

01 

I 

22 

23 

21.3 

20.9 

13.6 

16.9 

17.6 

20.2 

20.4 

22.S 

21.4 

22.0 

19.7 

22.6 

24 22.1 18.4 22.0 22 9 24.6 27.8 

1.000 25 19.6 14.4 18.7 19 2 20.7 24.S 

26 20.8 16.6 18.3 22.6 23.2 22.6 

27 19.4 13.4 15.2 17.8 17.9 18.2 

28 20.9 13.7 18.0 22.0 25.5 23.6 

29 20.8 18.5 22.8 23.7 24.4 22.1 

30 11L8 16.9 19.5 24.6 35.0 21.6 

~ 

~ 
AddenduDl 7-2 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Food intakes of individual aniaa)s(g/rat/day) 

Sex Exp.group Adllinistration period 

(lIIg!.kg/day) Aniad No. 1 3 8 15 22 28 (days) 

.... 

-.:J 

0:. 

( 

feaale 

Vehicle 

control 

25 

150 

1,000 

31 

32 

33 

34 

35 

36 

37 

38 

39 

40 

41 

42 

43 

44 

45 

46 

47 

48 

49 

50 

51 

52 

53 

54 

55 

56 

57 

58 

59 

tilL 

17.1 

13.8 

15.S 

17.2 

16.1 

16.0 

13.9 

16.7 

16.4 

17.5 

16.1 

15.4 

12.1 

16.1 

16.9 

14.6 

16.1 

17.2 

16.3 

16.6 

14.9 

15.8 

19.3 

13.6 

15.5 

15.6 

17.7 

17.2 

15.1 

U ..4 

17.1 

12.1 

15.1 

15.9 

13.6 

15.7 

12.3 

16.2 

14.3 

14.9 

14.4 

14.6 

12.2 

15.4 

16.4 

13.2 

14.5 

16.2 

13.7 

14.4 

12.9 

11.8 

12.9 

10.0 

12.0 

12.2 

14.8 

16.0 

13.1 

11.2 

16.3 

12.6 

15.7 

15.5 

12.9 

14 4 

12.0 

14.5 

14.6 

14.0 

16.4 

12.7 

11. 2 

15.5 

14.4 

13.3 

15.9 

18.8 

15.3 

16.1 

15.4 

14.6 

12.1 

11. 4 

14.3 

14.1 

17.1 

14.8 

14.0 

1::t.4 

14.1 

11.4 

15.3 

14.4 

13.1 

12.2 

11.1 

13.6 

12.3 

13.0 

14.8 

11.8 

10.4 

14.3 

12.9 

13.3 

13.8 

12.5 

15.4 

14.8 

16.1 

15.9 

16.7 

14.3 

16.1 

16.4 

15.5 

16.2 

15.9 

14.5 

13.7 

11.4 

14.2 

13.0 

13.8 

13.9 

11 4 

12.4 

12.2 

13.7 

13.1 

11.0 

11.4 

14.7 

11.6 

15.1 

14.3 

12.9 

14.8 

15.3 

16 7 

15.0 

18.4 

19.3 

17.5 

17.7 

16.6 

16.2 

16.2 

14 7 

12.2 

11.1 

12.4 

14.2 

13.5 

11.9 

11. 5 

12.3 

11. B 

12.3 

13.3 

11. 0 

11.2 

14.5 

12.5 

13.8 

13.2 

13.6 

12.4 

13.8 

14.5 

14.4 

19.0 

18.2 

17.9 

15.8 

16.0 

15.6 

15.0 

15.1 

:t 

N 
Addendll. 7-3 Tytnty-eight-day repeated-dose oral toxicity study in rats 811-0908 

lood intakes Qf indIvidual anilals(g/rat/day) 

Sex bp.group Recovery period 

(q/kg/day) Animal No. 8 14 (days) " 6 21.0 22.6 24.6 

7 23.3 26.3 26.3 

Vehicle 8 23.6 24.0 24.2 

control 9 24.1 24.5 25.6 

Kale 10 20.4 22.8 24.0 

26 25.7 26.3 28.8 

27 22.1 23.6 25.9 

1,000 28 22.9 25.0 25.7 

29 23.0 23.1 27.2 

30 19.9 22.4 25.8 

I-' 

-.;j 

-.;j 

1 

'e.ale 

Vehicle 

control 

1.000 

36 

37 

38 

39 

40 

58 

57 

58 

59 

*lQ 

17.7 

15.6 

16.1 

15.3 

17.3 

16.6 

16.8 

18.3 

18.1 n.! 

16.5 

14.6 

16.2 

14.8 

17.7 

18.4 

17.1 

18.1 

17.5 

18.5 

16.7 

16.5 

16.0 

15.9 

18.7 

16.8 

17.2 

18.0 

ULI 

18,2 

~ 

~ 

w 
Addendull B-1 	 Twenty-eight-day repeated-dose oral toxicity study in fats Bll-0908 

Hematological data of individual animals 

Exp.group RBC tlBC lib Ht tlCV MeR MCRe Platelet Reticulo P T APTT 


Sex (llg/tg/day) Animal No. (xl04 / ,uL) (xlOa/ ,uL) (g/dL) (%) (fL) (pg) (g/dL) (xl04 / ,uL) (%) (sec) (sec) 


1 800 150 15.9 47.9 59.9 19.9 33.2 108.3 2.5 18.1 30.9 

2 755 121 15.2 45.5 60.2 20.2 33.5 102.8 2.7 17.3 28.0 

3 742 109 15.1 44.1 59.4 20.3 34.2 105.8 2.4 18.3 27.4 

4 683 118 13.8 40.8 59.8 20.2 33.8 96.6 3.0 16.7 26.1 

Vehicle 	 5 763 _!~,!__________l,.§_.:...L____4 5 _·j,_________?.~_~_~ _______J_U ________Jl_~_c.!3 ____.J!.9 . 7 2.3 _Ul_~_'! _________~L·_?_______ control recov;r-;-­

6 802 128 15.1 44.5 55.4 18.8 33.9 114.3 2.2 24.6 35.3 

7 753 92 14.7 43.3 57.5 19.6 34.0 120.0 2.6 24.4 35.3 

8 746 127 15.2 44.4 59.5 20.4 34.2 103.2 2.1 18.2 36.9 

9 861 125 15.8 46.3 53.7 18.3 34.1 109.2 2.1 18.0 37.9 

10 791 104 15.3 44.8 56.6 19.4 34.2 102.8 2.8 15.9 32.1 

11 751 103 15.0 44.0 58.5 20.0 34.1 95.4 2.0 14.7 25.9 

12 749 136 14.9 43.7 58.4 19 9 34.l 99.4 2.8 15.4 31.3 

25 13 724 126 14.4 43.5 60.1 19.9 33.1 99.8 2.4 17.5 32.3 

14 742 145 14.3 42.8 57.7 19.3 33.4 103.2 2.5 15.9 27.5 

Kale 15 742 91 15.0 44.9 60.5 20.3 33.5 102.9 2.2 16.6 26.8 

16 711 154 14.8 44.4 62.5 20.9 33.4 99.9 2.7 16.4 25.7 

17 781 78 14.9 45.2 57.9 19.1 32.9 108.1 2.1 14.8 27.1 

150 18 730 150 14.7 43.7 59.9 20.1 33.6 107.5 2.8 18.9 28.5 

I 19 781 111 15.3 46.0 58.9 19.6 33.3 94.5 1.6 24.3 34.1 
 f-' 20 741 77 15.0 45.2 61.1 20.2 33.1 87.0 2.5 18.2 28.0
 ....;j 

21 842 134 16.0 47.9 56.9 19.0 33.3 111.6 ·Z.4 17.0 36.2 

I 	 22 791 142 14.4 43.1 54.5 18.2 33.5 117.7 2.2 16.2 30.5 

23 738 83 14.9 44.8 60.6 20.2 33.3 92.5 2.6 18.3 30.3 

24 817 142 16.1 48.7 59.6 19.7 33.1 93.6 3.7 18.2 30.3 

00 

1,000 __~?______I§.~_______!.QQ_________l,..?__'..Q_______H..:..~___~?_:_1!.________!_~_L_____1L.!!._______1 0l_!_L___ ?..:....~___________l,.~--'_!.._______~_~.:_?_______ Recovery 26 796 141 15.0 44.8 56.3 18.9 33.5 115.2 2.6 28.2 39.1 

27 742 126 14.5 43.6 58.7 19.5 33.3 117.2 2.9 14.5 32.7 

28 744 146 14.4 41. 6 55.9 19.3 34.5 109.7 2.8 19.4 36.8 

29 731 127 14.6 43.5 59.4 20.0 33.7 103.1 . 3.3 17.7 38.1 

30 789 116 15.3 45.6 57.8 19.5 33.7 99.1 2.0 17.4 31. 7 

~ 

~ 

~ 
Addendum 8-2 Twenty-eigbt-day repeated-dose oral toxicity study in rats 811-0908 

Heaatoiogical data of individual aniaals 

Exp.group RBC WIIC Hb Ht KCV MeR IICHC Platelet Reticulo l' T APTT 

Sex (I,/ltg/day) Anillal No. (xlO'/tlL) (xl0z/,tlL) (g/dL) (%) (fL) (pg) (g/dL) (xl04 /,tlL) (%) (sec) (sec) 

31 743 103 14.8 43.2 58.2 19.9 34.1 106.4 2.0 14.0 27.1 

32 774 102 15.1 45.0 58.1 19.5 33.5 99.6 1.7 14.3 21.4 

33 771 113 15.1 44.2 57.3 19.6 34.2 100.8 1.9 15.8 22.6 

Vehicle 

34 741 105 14.7 42.9 57.9 19.9 34.3 104.5 1.6 16.1 28.2 ___~1_____ 7E!.?.___..___ ___I.1..J!.._____.1..!..·..L______J?.9_:...?.____.__!_!1.c.2________~l..2________!.?_e.~1_____J_:_l:!.__________l:.!L~_____~_~_._~_______ control Recovery 

36 803 81 15.8 46.1 57.4 19.7 34.3 114.3 2.5 14.5 27.7 

37 787 98 14.8 42.3 53.7 18.8 35.0 140.2 '1.8 14.8 20.7 

38 754 84 14.6 41.7 55.3 19.' 35.1 122.4 1.S 14.4 25.7 

39 791 90 14.6 41.7 52.7 18.4 35.0 116.2 1.9 14.0 22.8 

40 750 108 14.9 41.6 55.5 19.9 35.& 1~9.9 2.~. 14.6 24.2 

41 774 87 15.4 4S.4 58.7 19.9 33.991.7 1.6 13.6 26.5 

42 767 57 15.1 44.8 58.5 19.7 33~8 107.1 1.5 13.7 24.1 

25 43 760 112 14.9 44.1 58.1 19.7 33.8 112.2 1.7 11.2 25.9 

44 742 74 14.1 41.9 56.5 19.0 33.7 115.0 2.1 15.3 28.0 

Feaale 45 

46 

47 

770 

788 

760 

101 

80 

92 

15.3 

15.5 

14.9 

44.9 

45.1 

44.3 

§8.~ 

57.S 

58.3 

19.8 

19.7 

19.7 

34.0 

.4.4 

33.7 

130.3 

110;4 

111.9 

1.6 

1.6 

1.9 

Ui..3 

14.8 

14.2 

26.0 

23.9 

24.6 

150 48 750 92 14.4 42.9 57.2 19.1 33.6 94.' 2.1 15.1 24.7 

I-' 

-l 

~ 

49 

50 

51 

740 

739 

737 

77 

143 

103 

14.4 

15.4 

14.5 

42.4 

44.8 

42.9 

57.3 

60.7 

58.2 

19.4 

2Q.8 

19.7 

33.9 

34.3 

33.8 

102.2 

119.9 

117.6 

1.7 

1 ..6 

1.9 

15.7 

15.0 

15.0 

22.6 

19.8 

22.7 

I 52 759 127 15.0 44.5 5&.7 19.8 33.8 110.9 2.0 14.5 24.8 

53 716 134 14.2 41.7 58.2 19.8 .4.1 119.3 2.2 14.3 23.4 

54 727 134 14.0 41.1 56.6 19.3 34.1 108.9 1.8 13.5 19.7 

1,000 55 749 93 14.9 43.5 58.1 .iiieoverY'-----------------------------------­ 19.9

- 94.2 99.5 2.1 16.7 27.7 -----. 56 807 71 14.9 43.1 53.4 18.4 34 5 130.5 1.8 14.7 26.0 

57 760 89 14.9 43.0 56.5 19.6 34.7 121.8 1.8 13.9 21.2 

58 805 85 15.0 42.6 52.8 18.6 35.2 119.7 1.9 14.7 24.4 

59 783 57 14.6 41.9 53.5 18.7 34.9 113.1 1.9 14.4 22.1 

60 766 78 14.9 42.7 55.7 19.5 34.9 11~.4 J.9 15.1 25.8 

~ 

~ 

()1 
5 

10 

15 

20 

25 

30 

AddendUJI 8-3 Twenty-eight-day repeated-dose oral toxicity study in rats B11-0908 

Heaatological data of individual ani.als 

I!xp.grollP 	 Differentiation of leukocyte (") 

Sex (ag/kg/day) Animal No. Meutro £osino Baso LYlIPh 110110 LIlC 

1 12.7 0.6 0.2 83.4 1.9 1.3 

2 11.1 0.4 0.2 84.7 2.5 1.1 

3 20.6 0.5 0.2 73.3 4.1 1.4 

Vehicle 

cOlltrol 

4 

ie"COVeri15 4 

16.4 

0.4 0.2 80.3 2.9 O.B 

Q..:_!L____O_~____.1.1_~_____4.2 ___-L..1 

6 12.0 0.5 0.2 82.B 3.5 1.1 

7 26.7 0.8 0.2 68.7 2.3 1.2 

8 18.0 0.4 0.3 74.9 4.0 2.4 

9 10.7 0.5 0.2 84.7 3.0 1.0 

10.3 0.7 0.1 85.8 2.0 1.1 

11 20.1 0.7 0.2 75.4 3.1 0.4 

13 15.2 0.3 0.1 81.4 2.1 0.9 

25 13 15.0 0.5 0.1 79.7 2.B 1.9 

14 14.4 0.4 0.2 79.6 3.5 1.9 

Hale 23.5 0.5 0.1 73.6 1.0 1.2 

16 17.9 0.3 0.2 76.7 2.7 2.2 

17 25.1 1.3 0.0 69.5 2.7 1.3 

150 18 15.6 0.5 0.2 79.7 3.3 0.6 

I 19 21. 6 0.6 0.1 72.6 4.1 0.9 
 I-' 19.9 0.7 0.1 76.0 2.5 0.8
 00 21 17.3 0.5 0.2 78.2 2.3 1.4 

I 	 22 28.6 0.9 0.1 65 6 4.0 0.7 


23 21.4 0.5 0.2 73.3 3.9 0.6 


24 21. 2 0.6 0.2 70.9 5.5 1.6 


0 

1,000 24.2 0.6 _______q..:..t__-1Q_~L_.__ 4.2 ••___...2...:..!.______ 

llecovery 26 14.4 0.4 0.1 2.1 

27 11.4 0.7 o 1 2.5 

28 10.2 0.8 0.1 1.5 

29 15.5 0.5 0.1 7 

11 ..5 1.1 0 

t 

C) 
Addendull 8-4 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Hematological data of individual ani.als 

Exp.group 	 Differentiation of leukocyte (") 

In (llg/kg/day) Anilal Ho. Neutro Eosino IbO L)"Iph Hono LOC 

31 15.0 1.2 0.1 80.6 2.6 0,6 

32 14.3 0.7 0.1 82.4 1.5 1.1 

33 10.5 1.1 0.1 84.2 3.2 1.0 

34 8.5 1.0 0.2 88.0 1.2 1.1 

Vehicle 35 12.3 0.6 0.1 85.3 0:8 0.9 RecoV;;-ry----------------------------------------------------------,---­ control 

36 9.8 1.4 0.1 85.4 2.1 1.1 

37 16.0 0.6 0.2 78.8 2.9 1.4 

38 18.1 1.2 0.2 77.3 :.L 1 1.1 

39 10.1 2.3 0.1 83.1 3.6 0.8 

40 20.0 1.3 0.3 73.7 3.1 1.6 

41 20.4 1.3 0.1 74.8 2.6 0.9 

42 14.8 1.0 0.1 82.2 1.3 0.7 

25 43 13.9 0.9 0.2 82.4 1.6 1.0 

44 20.3 0.7 0.1 75.3 2.6 0.9 

Pelale 45 13.6 1.8 0.1 81.0 2.0 1.5 

46 16.9 0.7 0.1 79.2 1.9 1.2 

47 15.2 0.8 0.1 81.2 1.7 0.9 

150 48 17.2 0.4 0.1 80.2 1.3 0.9 

..... 49 27.6 0.8 0.1 68.9 1.8 0.7 

00 50 6.1 0.7 0.1 90.2 1.3 1.6 ..... 51 18.8 l.Q 0.1 76.6 2.7 0.8 

I 	 52 15.2 0.5 0.2 78.7 4.5 0.8 


53 23.0 1.7 0.2 69.8 4.4 1.0 


54 11.9 3.2 0.1 80.4 3.7 0.7 


1.000 55 18.6 0.7 0.1 74.0 3.6 3.0 iiicov'{;j:i-------------------------------------------------------------------­

56 11.8 1.5 0.1 82.6 2.7 1.3 

57 18.3 1.0 0.1 75.3 4.0 1.3 

58 9.0 1.8 0.1 83.9 3.6 1.6 

59 9.6 1.0 0.1 84 4 3.4 1.4 

60 9.6 1.0 0.1 85.9 2.. a 1.3 

t 


-....I 
Addendum 9-1 Twenty-eight-day repeated-dose oral toxicity study in rats 8l1-0908 

Blood cbs.ical data of individual animals 

Ixp.group AST ALI ALP ChE y -GTP T-Cho TO Glucose T-Protein Al bUllin A/G ratio 

s~ (lig/kg/day) AninI No. (1l1/L) (lUlL) (lUlL) (lOlL) (IlllL) (llg/dL) (lig/dL) (mg/dL) (g/dL) (g/dL) 

1 73 Z1 709 47 0.6 49 10 155 5.7 2.7 0.90 

3 63 18 518 75 0.8 54 47 158 5.8 2.8 0.93 

3 63 16 665 46 0.9 52 133 178 ~.1 2.9 0.91 

4 55 15 366 71 0.9 48 76 176 6.0 2.7 0.82 

Vehide control 5 59 20 537 83 0,7 58 131 169 6.0 2.8 0.88 hcovery------------------------------------------------------------------------.-----------------------------------­

6 

7 

57 

86 

19 

26 

273 

387 

55 

44 

0.8 

0.5 

46 

55 

87 

74 

161 

145 

6.0 

6.1 

2,8 

2.6 

0.88 

0.74 

8 69 26 333 61 0.8 48 67 138 6.0 2.7 0.82 

9 58 22 327 47 1.0 43 39 160 6.2 2.7 0.77 

10 58 lit _..3~ .~_6 0.9 6,5 63 1$7 5.6 2.7 0.93 

11 58 15 677 67 0.7 58 69 193 5.7 2.7 0.90 

12 88 18 495 37 0.8 50 71 151 6.0 2.7 0.82 

85 13 63 22 576 55 1.0 50 36 143 6.3 2.8 0.80 

14 73 26 568 43 0.7 49 84 143 5.6 2.8 1.00 

Hale 15 .lt316 555 68 0.9 43 50 169 5.4 a.6 0.93 

16 72 27 550 43 0.9 46 69 148 5T 2.8 1.00 

17 54 19 477 49 0.6 53 69 160 5.6 2.8 1.00 

150 18 57 22 350 85 0.6 58 179 154 5.7 2.8 0~97 

....... 

00 

t-..:l 

19 

10 

21 

58 

59 

72 

24 

20 

43 

632 

647 

590 

42 

5Z 

56 

0.9 

1.0 

1.0 

54 

51 

71 

60 

36 

29 

158 

190 

165 

5.9 

5.9 

6 3 

2.S 

3.0 

3.1 

0.90 

1.03 

0.97 

I 22 77 49 509 67 0 8 48 29 125 5.9 2.9 0.97 

23 62 37 452 79 0.8 57 19 142 5.8 3.0 1.07 

24 66 39 382 61 0.8 52 107 145 5.8 3 1 1.15 

1.000 25 80 51 640 42 0.8 51 34 123 5.8 3.1 1.15 Recover,.-----~-----------------------------------------------------------------.. -----------------------~

26 63 21 338 52 0.8 52 53 142 6.0 2.8 0.88 

27 71 24 311 44 0.6 49 54 109 5.6 2.6 0.87 

28 61 22 255 64 0.6 62 44 128 5.8 2.6 0.81 

29 66 23 321 45 0.5 49 81 142 5 8 2.5 0.76 

30 85 34 332 42 0.8 46 47 136 5.7 2.7 0.90 

~ 

~ 

00 
Addendum 9-3 Tventy-eight-day repeated-dose oral toxicity study in rats BJI-090B 

Blood chemical data of individual animals 

Exp.group AS! ALT ALP ehB l' -GTI' T-Cho TG Glucose T-Protein AlbU1llin A/G ratio 

Su (lIg/kg/day) Auiaal No. (IU/L) (IlJ/L) (Io/L) (lUlL) (Io/L) (llg/dL) (mg/dL) (~/dL) (g/dL) (g/dL) 

31 66 16 273 275 0.9 60 40 119 6.0 3.1 1.07 

32 76 23 779 212 0.9 63 24 143 5.9 2.9 0.97 

33 75 21 351 300 0.9 73 22 119 5.8 3.0 1.07 

Vehicle 

control 

34 61 29 374 304 0.6 54 31 133 6.1 3.1 ],03

35 68 17 197 183 1.0 63 19 128 6.3 3.1 0.97 fecovery------------------------------------------------------------.--------------------------------------------------------­

36 52 15 211 277 0.6 66 20 114 6.3 2.9 0.85 

37 66 20 334 600 1.0 76 78 118 8.1 3.0 0.97 

38 77 21 173 484 1.0 49 13 116 6.6 3.3 1.00 

39 67 18 161 488 0.8 85 26 112 6.1 3.0 0.97 

40 58 18 217 449 0.8 65 44 151 6.5 2.9 0.81 

41 59 17 259 166 0.7 45 16 106 ~:6 2.9 1.07 

42 62 16 242 224 0.7 48 16 105 5.9 2.8 0.90 

25 43 59 21 312 281 0.9 59 20 138 6.2 3.1 1.00 

44 57 18 250 328 0.9 43 35 121 5.9 3.0 1.03 

felale 45 73 22 297 339 1.0 52 21 116 5.8 3.1 1.15 

4'6' 50 26 451 162 0.7 75 28 141 5.9 3.1 1.11 

47 57 15 303 370 O.H 45 26 128 5.9 3.1 1.11 

150 48 47 12 271 267 1.5 54 22 153 5.9 2.9 0.97 

....... 

00 

c.o 

49 

50 

51 

53 

73 

67 

16 

21 

39 

456 

273 

473 

266 

228 

143 

0.8 

1.1 

1.2 

83 

61 

71 

25 

28 

32 

143 

128 

132 

5.7 

6.3 

-~.9 

2.8 

3.2 

3.0 

0.97 

1.03 

1.03 

I 52 92 68 260 119 0.8 13 23 132 6.6 3.2 0.94 

53 61 31 344 145 0.8 84 23 125 6.3 3.2 1.03 

54 55 38 210 86 1.0 64 28 131 5.8 3.2 1.23 

1,000 55 54 23 362 132 0.8 84 23 150 6.3 3.2 1.03 Rec-o;;ry--------------------------------------------------------------------------------------...-----------------------------------------------­ sa 76 16 212 167 1.4 77 44 146 5.8 2.8 0.93 

57 60 21 194 212 1.4 98 lOB 139 6.3 3.0 0.91 

58 67 20 203 342 0.8 86 34 115 6.4 3 0 0.88 

59 69 19 242 470 o.B 78 14 140 6.2 3.0 0.94 

60 80 23 Z32 286 0.9 68 28 115 5.9 3.0 1.03 

..1:10 

..1:10 

to 
Addendua 9-3 	 Twenty-eight-day repeated-dose oral toxicity study in rats 1111-0908 

Blood chemical data of individual animals 

I!xp.group BUN Creatinine T-Bil Ca IP Ha K Cl 


&ex hlg/kg/day) Anilal Ko. (ag/dL) (mg/dL) (ag/dL) (ag/dL) (lig/dL) (aEq/L) (lIt:q/L) (mEq/L) 


1 7.5 0.17 0.06 9.9 7.4 143 4.3 105.8 

2 7.6 0.19 0.06 10.1 9.0 143 4.1 104.6 

3 8.4 O. :n 0.04 10.3 7.8 143 4.2 107.2 

4 11.2 0.27 0.04 10.3 7.6 144 4.0 105.7 

VehIcle 5 8.1 0.19 0.10 10.3 8.1 145 3.8 106.3 Riicoveri----------------------------------------------------------------------------­ control 

6 12.2 0.21 0.06 10.0 7.1 142 3.7 103.8 

7 13.5 0.23 0.07 10.2 7.4 143 3.6 104.0 

8 14.3 0.22 0.06 9.7 6.6 142 4.0 104.5 

9 13.7 0.22 0.06 9.9 7.3 144 4.0 106.5 

10 16.9 0.23 0.07 9.7 6_.8 141 4.1 103.7 

11 10.7 0.22 0.08 9.9 7.6 143 4.3 104.8 

12 7.9 0.24 0.04 9.9 7.3 144 4.4 107.8 

25 13 8.9 O. Z1 0.07 10.2 7.1 145 4.1 107.6 

14 6.7 0.15 0.05 10.2 7.7 144 4.2 106.2 

Kale 15 10.7 0.24 0.06 10.3 9.2 145 4.8 107.8 

16 8.0 0.17 0.09 9.8 6.8 142 4.3 105. i-­

17 9.6 0.22 0.06 9.8 7.3 143 4.2 106.7 

150 18 9.5 0.21 0.05 10.4 9.1 143 4.3 104.5 

I 19 6.8 0.21 0.06 10.3 8.4 144 3.9 106.0 ...... 20 11.0 0.20 0.07 10.3 9.2 144 4.2 105.0 00 

!I>- 21 17.7 0.29 0.14 10.1 9.4 141 4.3 105.2 


I 22 19.2 0.31 0.13 9.9 9.3 142 4.3 106.1 


23 9.0 0.20 0.07 9.9 9.2 144 4.5 107.9 

24 12 .0 0.24 0.08 9.9 9.4 144 4.3 107.4 

1,000 25 10.6 0.~4 0.08 10.3 8.6 144 3.9 108.8 Recoveri---------------------------------------------------------------------------------------­

26 13.9 0.19 0.05 9.7 7.3 144 4.3 105.9 

27 17.5 0.21 0.09 9.7 6.8 140 4.4 104.2 

28 12.6 0.19 0.08 9.6 6.8 143 4.3 105.7 

29 14.3 0.20 0.08 9.7 8.2 143 4.3 103.2 

3.0 13.1 ILJ.J3 0.06 S.l 6 •.6 144 3.9 105.3 

~ 

OJ 

o 
Addendull. 9-4 Twenty-tight-day repeated-dose oral toxicity study in rats B11-0908 

Blood cheaical data of individual anilals 

l!xp.group BUN Creatinine T-Bil Ca JP lIa K Cl 

S.I (lIIg/kg/day) Anilal No. (mg/dL) (mg/dL) (llg/dL) (ag/dL) (lIg/dL) (IIEq/L) (IliEq/L) (dq/L) 

31 10.2 0.22 0,07 9.9 6.7 144 3.6 10B.6 

32 9.1 0.22 0.08 9.9 B.9 142 3.9 107.3 

33 11.8 0.24 0.04 9.7 6.7 143 3.9 107.3 

34 10.6 0.23 0.04 9.8 6.6 144 4.0 111.4 

Vehicle 35 9.2 0.23 0.07 10.1 7.8 142 _____ ________l.Q]...:..!L____ control Recovery 36 19.0 0.27 0.07 9.8 4.8 141 3.8 108.0 

37 13.2 0.26 0.09 9.9 5.0 141 3.4 103.9 

38 21. 2 0.37 0.06 9.8 5.2 142 37 106.4 

39 16.3 0.26 0.13 9.6 5.5 142 3.8 107.0 

40 15.4 0.26 0.09 9.9 5.5 142 3.7 106.7 

41 8.7 0.19 0.03 9.5 6.4 143 3.6 108.6 

42 11. 0 0.28 0.05 9.2 6.6 142 4.1 110.0 

25 43 13.5 0.25 0.04 10.0 7.5 143 3.9 109.4 

44 9.4 0.20 0.04 9.9 6.4 144 3.6 108.6 

lemale 45 14.4 0.27 0.05 9.9 7.3 144 3.,7 110.3 

46 14.2 0.19 0.09 10.8 9.1 142 4.4 ---'103.7 

47 9.3 0.18 0.05 10.1 7.3 142 4.0 106.7 

150 48 9.8 0.21 0.06 9.8 7.9 143 3.8 108.3 

49 7.3 0.21 0.04 9.7 6.4 142 4.3 10B.9 I-' 50 10.5 0.25 0.09 9.9 6.3 143 3.8 108.4 00 

01 51 8.0 0.28 0.08 10.1 8.9 143 4.3 106.5 

I 52 11.3 0.26 0.08 10.3 8.8 145 3.7 107.0 

53 8.5 0.22 0.08 10.1 9.0 144 3.9 107.3 

54 15.9 0.30 0.10 9.5 8.3 131 4.3 98.6 

1,000 55 11.6 0.26 0.10 10.3 B.8 140 3.8 103.1 Il.ecOvery----------------·---·------------------------------------------------------.------------------­

56 14.6 0.24 0.07 9.5 6.4 143 4.3 109.7 

57 21.6 0.26 0.13 10.2 6.8 140 4.7 104.6 

58 14.4 0.22 0.08 10.0 5.9 141 3.9 104.6 

59 13.6 0.24 0.07 9.5 5.7 144 3.3 107.0 

60 18.8 0.30 0.08 9.5 5.9 142 4.0 IJ~.9. 6 

~ 

01 
Addendum 10-1 TWentY-eight-day repeated-dose oral toxicity study in rats Bll-0908 

Urinalytic data of indiTidual aniaa18 

Exp.group Urine volUie Uosa 


Sex (a,!kg/day) Ani_I 110. (1111) (lIllsa/L) 


1 9 536 


2 20 250 


3 10 613 


4 4 1914 


Vehicle 5 23 276 


control Recovery 


6 16 569 


7 11 637 


8 6 1193 


9 9 852 


10 	 5 17~2 

11 3 2040 


12 5 1211 


25 13 3 2138 


14 7 876 


!lale 15 3 2276 


---------_....­

16 2 2032 


17 2 3034 


150 	 18 8 1411 


19 9 532 
 ...... 20 6 1048 


00 21 6 970
 Q':J 22 8 615
 I 
 23 12 726 


24 20 308 


1.000 25 29 217 
 ----------------_...... _.....­ Recovery 


26 15 633 


27 16 656 


28 13 653 


29 6 1437 


30 18 378 


~ 

CJ1 

I'.) 
AddendWi 10-2 	 Twenty-eicht-day repeated-dose oral toxicity study in rats Bl1-0908 

Urinalytic data of individual animals 

Rxp.group Urine volua8 Uosm 


Sex (Ig/kg/da,) Anilal 110. (IlL) (IOsa/L) 


Vehide 

control 

31 

32 

33 

34 

35 

Recovery 

36 

37 

38 

39 

40 

11 

2 

2 

7 

5 

8 

7 

5 

2 

6 

563 

2204 

2566 

694 

1017 

779 

928 

1069 

2472 

la63 

....... 

Pellie 

25 

150 

41 

43 

43 

44 

4§ 

46 

47 

48 

49 

50 

18 

2 

1 

10 

zg 

'1 

4 

3 

8 

5 

263 

1796 

1461 

474 

U3 

692 

1244 

1225 

626 

1037 

00 51 38 85 

-l 52 32 168 I 53 14 545 

54 13 555 

1,000 55 9 886 -------_..._-­ Recover, 

56 7 862 

57 4 1556 

58 5 1356 

59 2 2302 

6Q 3 1490 

~ 

CJ'1 

W 
Addendum 10-3 Twenty-eight-day repeated-dose oral toxicity study in rab 611-0908 

Urinalytic data of individual animals 

Exp.group Color Turbidity pH Protein Glucose Occult blood 

Sex (mg/kg/day) Animal No. 

1 Y NT 6.5 1+ 

2 SY NT 7.0 ± 

3 Sy NT 6.5 1+ 

4 Y NT 6.0 z+ 

Vehicle 5 SY NT 7.0 ± 

control RecoverY 

6 SY NT 7.0 1+ 

7 SY NT 7.0 1+ ± 

8 Y NT 6.5 1+ 

9 SY NT 6.5 ± 

10 Y NT 6.5 1+ 

n12 y 

Y 

JIlT 

NT 

'6.0 

6.5 

2+ 

1+ 

25 13 Y NT 6.0 2+ 

14 Y NT 6.5 1+ 

Male 15 

16 

Y 

Y 

NT 

NT 

6.0 

6.0 

z+ 

2+ 

17 Y NT 5.0 2+ 

150 18 Y NT 6.0 2 

19 SY NT 6.5 1 

I-' 20 Y NT 6.5 1+ 

00 

00 

I 

1,000 

SY, Slightly yellow. 

Y. Yellow. 

NT. No turbidity. 

21 

22 

23 

24 

25 

Recovery 

26 

27 

28 

29 

30 

Y 

SY 

SY 

SY 

SY 

SY 

SY 

SY 

Y 

SY 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

11<1 

NT 

NT 

6.0 1+ 

6.0 1+ 

6.0 1+ 

6.5 ± 

6.;:,.5____= 

7.0 

7.0 

7.0 

6.5 

7.0 

± 

± 

1+ 

1+ 

± 

~ 

(J'I 

~ 
Addendum 1(}-4 Twenty-eight-day repeated-dose oral 611-0908 toxicity study in rats 

Urinalytic data of individual animals 

Sex 

Exp.group 

(mg/kg!day) Animal No. 

Color Turbidity pH Protein Glucose Occult blood 

Female 

Vlit,lic\tt 

control 

25 

u 

32 

33 

3435 

&iCOV81j'. 

36 

37 

38 

39 

40 

41 

42 

43 

44 

45 

w 

Y 

Y 

SYY 

SY 

Y 

Y 

Y 

Y 

$V 

Y 

Y 

SY 

SY 

~ 

NI' 

NT 

NTNT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

u ± 

5.0 2+ 

5.0 2+ 

6.5 1+ 6.5 __..!.l+.!-_____________ 

,._­

6.5 ± 

6.5 1+ 

6.5 1+ 

6.0 2+ 

6.5 1+ 

7.0 .::t 

6.0 2+ 

6.0 Z+ 

6.5 1+ 

7.0 

........ 

00 

<.0 

I 

150 

1.000 

46 

47 

48 

49 

50 

n 

~ 

~ 

~ 

H 

SY 

Y 

Y 

Y 

Y 

SY 

SY 

SY 

SY 

SY 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

NT 

6.5 

6.0 

6.5 

6.5 

6.0 

7~ 

U 

U 

U 

U 

1+ 

2+ 

1+ 

1+ 

1+ 

± 

1+ 

Recovery 

56 

57 

58 

59 

60 

SY 

Y 

Y 

Y 

'Y 

NT 

NT 

NT 

NT 

NT 

7.0 

6.5 

6.5 

6.5 

6.5 

± 

1+ 

1+ 

2+ 

1+ 

SY. Slightly yeUow. 


Y, YliUow. 


NT. NQ turbidity. 


oj:>. 

CJ'l 

CJ'l 
Addendum 10-5 Twenty-eight-day repeated-dose oral toxicity study in rats 811-0908 

Urinaiytic data. of individual anima.Js (Urinary sediment) 

...... 

(0 

o 

I 

Exp.group 

Sex (mg/kg/day)

Mala 

Animal No. 

-I-­

Z 

3 

4 

Vehicle 5 

control Recovery 

6 

7 

8 

9 

to 

If 

12 

25 13 

14 

15 

16 

17 

150 18 

1.000 

19 

20 

21 

22 

23 

24 

Red blood ceUsal 

a 

0 

0 

0 

0 

0 

0 

0 

0 

Q 

0 

0 

0 

0 

0 

U 

0 

0 

0 

0 

0 

0 

0 

0 

White blood cells') Epithelial cells,l 

0 0 

0 2 

0 3 

0 0 

0 

-----------_. 

0 0 

0 2 

1 0 

0 2 

00--­

0 0 

0 1 

0 0 

0 1 

0 0 

0 I 

0 0 

0 0 

0 3 

0 0 

14 18 

5 33 

11 84 

6 11 

CMtS') 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Crystals') 

+ 

+ 

± 

+ 

+ 

± 

j:: 

27 

28 

29 

30 

o 

o 

o 

o 

o 

o 

o 

o 

o 

o 

2 

o 

2 

1 

o 

o 

o 

o 

o 

o 

± 

a) Number of cells/l0 views (x 400). 

b) Number ofcasts/18X 18 mm2

• 

c) Incidence of crystals/I8 X 18 mm z 

. 

.". 

c.n 

C) 
Addendum 10-6 Twenty-eight-day repeated-dose oral toxicity study in rats Bll-090S 

Urinalytic data of individual animals (Urinary sediment) 

Exp.group Red blood cells·) White blood cells') Epithelial cells.,) Castsbi CrystalsC

) 

Sex (mg/kg/day) Animal No. 

-31-­

32 

33 

34 

Vehicle 35 

control 

37 

38 

39 

40 

4I 

42 

25 43 

44 

Female 45 

45 

47 

ISO 48 

...... 49 

~ 50 ..... 

I -61 

52 

53 

54 

1,000 

Recovery 

56 

57 

58 

59 

60 

a) Number of cells/lO views (X 400>' 

b) Number of cuts/IS X 18 mm 2

• 

cllncidence of crystals/IS X 18 mm 2

• 

~ 

U1 

..... 

0 0 

0 0 

0 0 

0 0 

0 0 

o 0 

0 2 

0 I 

0 0 

0 I 

0 0­

() 1 

0 0 

0 1 

() () 

0 0 

0 0 

0 1 

0 0 

0 0 

·00 

0 0 

0 0 

0 

0 0 

0 2 

0 0 

0 0 

0 0 

2 

1 

3 

1 

3 

0 

1 

1 

Z 

1 

O~~ 0 

2 0 

1 0 

1 0 

0 () 

~ 0 

0 () 

! () ++ 

0 () 

4 () 

9 

0 

4 

12 

1 

4 

3 

2 

2 

o 

o 

o 

o 

0 

0 

0 

0 :t 

0 

0 

0 

() 

() 

0 

0 + 

0 

0 :t 

0 + 
Addendum 11-1 Twenty-eigbt-day repeated-dose oral toxicity study in rats Bll-0908 

Absolute organ weights of individual anilals 

Sex Exp.group 

(llg/kg/day) Animal No. 

Liver 

(g) 

Heart 

(g) 

Iidncy 

(g) 

Testis 

(g) 

Epididymis 

(g) 

Ovary 

(lIIg) 

Brain 

(g) 

Spleen 

(g) 

Thymus 

(lIg) 

Adrenal 

(mg) 

Body lIeight 

(g) 

. .. . -----.-...--. 1.85 0.63 466.2 41.3 313.2 

2 11.21 1.15 2.32 2.63 0.76 1.82 0.60 802.4 44.2 352.1 

3 11.60 1.20 2.48 3.19 0.77 2.08 0.55 540.4 53.3 356.9 

4 12.75 1.15 2.31 3.42 0.76 2.04 0.81 640.0 58.0 363.2 hhlc~ control 5 12.28 1.19 2.54 3.24 0.74 2.07 0.67 594.9 53.7 383.4 RecoVilj.·i--·---------------------------------·-·---.. ------.--------------------.----.---------------------------------------.-------------------­ 6 11.48 1 33 2.69 3.03 1.02 2.14 0.67 663.2 50.7 423.8 

7 10.64 1.25 2.73 3.36 1.18 2.27 0.63 513.3 48.3 393.4 

8 11.44 1.41 3.02 3.08 1.12 2.10 0.79 557.1 58.0 427.5 

9 11.23 1.44 2.89 3.61 1.17 2.13 0.70 414.7 56.1 380.1 

10 10.00 1.26 2.75 3.25 1.01 2.02 0.59 499.5 54.9 374.2 

11 10.96 1.03 2.343.37 0.82 1.98 0.54 6306' 52.1 323.7 

12 11.99 1.25 2.56 3.21 0.83 2.00 0.70 533.2 47.3 344.0 

25 13 9.54 1.09 2.17 2.93 0.71 1.95 0.66 508.7 50.0 302 5 

14 10.76 1.15 3.00 3.02 0.84 1.95 0.64 733.0 44.2 383.8 

Kale 15 lO.19 L15 Z,S7 .a.21 Q.S.1. 1.94 0.58 621..._; 45.8 337.1 

16 10.0Z-----1.15 2.72 2.92 0.66 1.90 0.67 570.5 38.0 318.4 

17 10.98 1.01 2.24 2.95 0.73 2.00 0 43 282.4 39 6 324.1 

150 18 13.84 1.38 2.58 3.04 0.73 2.03 0.67 668.2 52.7 420 7 ~ 19 9.24 1.02 1.95 3.03 0.78 1.96 0.48 380.9 42.6 280.4 

>.0 20 10.52 1.23 2.30 3.06 0.85 Z.09 0.60 636.5 39.4 322.7 

tv 

I 21 

22 

10.02 

10.82 

0.98 

1.14 

2.28 

2.85 

2.93 

3.13 

0.66 

0.75 

1.80 

1.BO 

0.38 

0.44 

300.6 

362.5 

33.4 

44.5 24~ 290.7 

23 11.73 1.15 2.95 3.33 0.87 1.82 0.S2 369.4 47.1 310.3 

24 12.57 1.13 2.69 2.77 0.65 1.92 0.66 420.4 41.7 301.9 

1.000 25 10.58 0.97 2.79 3.38 0.78 1.88 0.39 413.2 42.8 282.3 RecOvery----------------------------------------------------.-------------------------..-------------------------.--------------------------------­ 26 11.74 1.37 3.14 3.06 0.86 2.04 0.70 442.6 46.1 386.3 

27 9.33 1.13 2.91 3.08 0.91 2.01 0.54 372.7 40.4 333.7 

28 10.63 1.17 2.86 3.42 0.94 1.96 0.56 601.0 54.9 371.5 

29 12.51 1.50 3.31 3.04 1.01 1.97 0.77 539.2 57.2 414.9 

30 8.48. 1..:12 2.69 2.92 0.91 1.99Q.SL 305.9 37.1 3aa.6 

.j». 

CJ'1 

CO 
Addendum 11-& Twentr-eight-day repeated-dose oral toxicity study in rats 811-0908 

Absolute organ weights of individual animals 

Sex Exp.group Liver Heart Kidney Testis EpididYliis Ovary Brain Spleen Thymus Adrenal Body weight (mg/kg/day) Animal No. (g) (g) (g) (g) (g) (Ilg) (g) (g) (lIg) (mg) (g) 

31 7.20 0.96 1. 69 57.1 1. 86 0.37 535.1 54.8 234.1 

32 5.61 0.68 1. 58 76.4 1. 82 o 39 425.6 61. 9 190.1 

33 6.52 0.83 1.48 81.1 1. 89 0.53 .537.2 58.8 201.7 

34 6.22 0.80 1 64 83.4 1. 90 0.41 555.5 64.0 220.6 

Vehicle 35 6.50 0.79 1.43 82.5 1.62 0.40 547.5 54.3 208.8 iii-covery----------------------------------------------------------------------------------------------------------------------------------­ control 

36 6.02 0.82 1. 43 74.1 1.83 0.49 488.4 57.2 217.3 

37 5.67 0.74 1. 48 66.7 1. 93 0.45 403.3 57.7 209.9 

38 6.02 0.77 1. 56 88.7 1.84 0.44 330.4 66.5 232.6 

39 5.71 o 84 1. 65 82.3 1. 96 0.48 408.0 65.6 212.9 

40 6.20 0.83 1. 61 72.6 1.85 0.45 436.2 52.0 230.9 

41 

42 

6.42 

5.17 

0.86 

0.65 

i-.69 

1. 39 

83.7 

66.3 

1. 95 

1. 86 

0.46 

0.33 

655.8 

453.3 

65.8 

64.0 

214.1 

197.0 

135 43 5.85 0.75 1. 35 69.6 1. 75 0.32 504.8 59.0 183.2 

44 6.79 0.72 1. 65 60.1 1 .97 0.35 431.9 72.9 224.7 

Pellale 45 5.40 0.65 1.17 74.6 1. 71 0.38 379.8 53.8 189.0 

..... 

<:0 

150 

46 

47 

48 

49 

50 

6.07 

8.02 

7.43 

7.33 

6.92 

0.72 

0.90 

0.83 

0.86 

0:.81 

1. 53 

1. 74 

1.68 

l..67 

1. 45 

65.2 

78.7 

76.6 

80.5 

83.4 

- 1~78 

1.86 

1.88 

1. 93 

1. 87 

0.37 

0.51 

0.39 

0.48 

0.41 

414.3 

345.9 

433.0 

408.9 

556.1 

50.7 

88.5 

64.7 

64.7 

74.6 

190.2 

ZOB.5 

219.4 

214.3 

218.1 

CO 

I 51 

52 

9.67 

9.11 

0.89 

0.83 

2.13 

1. 81 

83.2 

65.4 

1. 82 

1. 82 

0.48 

0.47 

410.3 

373.9 

48.8 

70.2 

230.7 

210.4 

53 10.63 0.93 2.49 71.7 1.88 0.38 371.7 72.7 230.3 

54 7.57 0.75 1. 77 98.8 1. 82 0.41 366.9 55.3 189.6 

1,000 55 9.52 0.85 2.04 - 78.1 1.86 0.46 343.0 57.1 203.9 ie.;-ov8r;-------------------·------------------------------------------------------------------------------------------------------------------------­

56 6.18 0.81 1. 63 83.4 1. 87 0.44 398 4 61.7 210.6 

57 7.47 1. 05 1. 68 73.3 1. 76 0.55 443.6 61.0 241.0 

58 6.88 1. 03 2.04 84.4 1.85 0.51 482.3 60.1 233.1 

59 6.84 0.90 1.88 94.5 1.91 0.60 455.1 73.5 234.0 

60 6 ..14 .0.88 1.56 -----­ 81.2 l.U 0.5a 345.0 75.5 224.4 

.j:I. 

CJ1 

co 
Addendum 12-1 Twenty-eight-day repeated-dose oral toxicity study In rats 811-0908 

Relative organ weights of iodividual animals 

Sex Exp.group 

(Ig/kg/day) Animal No. 

Liver 

(g/IOOg) 

Heart 

(g/100r;) 

Kidner 

(g/lOOg) 

Testis 

(g/IOOg) 

Epididyllis 

(g/IOOg) 

Ovary 

(lg/l00g) 

Brain 

(g/100g) 

Spleen 

(g/100g) 

ThYIUS 

(lg/100g) 

Adrenal 

(mg/IOOg) 

Body weight 

(g) 

Vellicle 

cootrol 

1 

2 

3 

4 

5 Rec'overy 

6 

7 

8 

9 

10 

3.07 

3 18 

3.25 

3.51 

3.20 

2.71 

2.70 

2.68 

2.95 

2.67 

0.38 

0.33 

0.34 

0.32 

0.31 

0.31 

0.32 

0.33 

0.38 

0.34 

0.75 0.83 0.22 0.59 0.20 148.9 13-:2 313.2 

0.66 0.75 0.22 0.53 0 17 227.9 12.6 352.1 

0.69 0.89 0.22 0.58 0.15 151.4 14.9 356.9 

0.64 0.94 0.21 0.56 0.22 176.2 16.0 363.2 

0.66 0.85 0.19 0.54 0.17 155.2 14.0 383.4 --------------------------------------------------------------------------------------------------------­

0.63 0.71 0.24 0.50 0.16 156.5 12.0 423.8 

0 69 0.85 0.30 0.58 0.16 130.5 12.3 393.4 

0.71 0.72 0.26 0.49 0 18 130.3 13.6 427.5 

0.76 0.95 0.31 0.56 0.18 109.1 14.8 380.1 

0.73 0.87 0.27 0.54 0.16 133.5 14.7 374 2 

...... 

~ 

Kale 

25 

150 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

3.39 

3.49 

3.15 

2.80 

3.02 

3.15 

3.39 

3.29 

3.30 

3.26 

0.32 

0.36 

0.36 

0.30 

0.34 

0.36 

0.31 

0.33 

0.36 

0.38 

0.72 

0.74 

0.72 

0.78 

0.70 

0.85 

0.69 

0.61 

0.70 

0.71 

1.04 

0.93 

0.97 

0.79 

0.95 

0.92 

0.91 

0.72 

1.08 

0.95 

0.25 

0.24 

0.23 

0.22 

0.24 

0.21 

0.23 

0.17 

0.28 

0.26 

0.61 

0.58 

0.64 

0.51 

0.58 

0.60 

0.62 

0.48 

0.70 

0.65 

0.17 

0.20 

0.22 

0.17 

0.17 

0.21 

0.13 

0.16 

0.17 

0.19 

194.8 

. 155.0 

168.2 

191.0 

184.4 

179.2 

87.1 

158.8 

135 B 

197.2 

16.1 

13.7 

16.5 

11.5 

13.6 

11.9 

12.2 

12.5 

15.2 

12.2 

323 7 

344.0 

302.5 

383.8 

337.1 

318.4 

324.1 

420.7 

280.4 

322.7 

,p. 

I 

1,000 

21 4.08 0.40 0.93 1.19 0.27 0.73 0.15 122.4 13.6 245.5 

22 3.72 0.39 0.98 1.08 0.26 0.62 0.15 121.7 15.3 290.7 

23 3.78 0.37 0.95 1.07 0.28 0.59 0.17 119.0 15.2 310.3 

24 4.16 0.37 0.89 0.92 0.22 0.64 0.22 139.3 13.8 301.9 

25 3.75 0.34 0.99 1.20 0,28 0.67 0.14 146.4 15.2 282.3 iie-cover-i----------------------------------------------.. -----------------------------------------------------------------------------.-------­

26 

27 

28 

29 

30 

3.04 

2.80 

2.86 

3.02 

2.58 

0.35 

0.34 

0.31 

0.36 

0.37 

0.81 

0.87 

0.77 

0.80 

0.82 

0.79 

0.92 

0,92 

0.73 

0.89 

0.22 

0.27 

0.25 

0.24 

0.2.S 

0.53 

0.60 

0.53 

0.47 

0.61 

0.18 

0 16 

0.15 

0.19 

0.16 

114.6 

111.7 

161.8 

130.0 

93.1 

11,9 386 3 

12.1 333.7 

14.8 371,5 

13.8 414.9 

11.3328.6 

""­ 0) 

o 
Addendull 12-2 Twenty-ei,ht-day repeated-dose oral toxicity study in rats 811-0908 

Relative organ weights of individual ani.als 

Sex Exp.group Liver Heart Kidner Testis Epidid)ll!is Ovary Brain Spleen ThYlius Adrenal Body weight (q/kg/day) MiDI 110. (,/IOOg) (g/IOO,) (g/IOOg) (g/IOOg) (g/100g) (llg/IOO,) (g/100g) (g/IOOg) (llg/100g) (llg/100g) (g) 

31 3.08 0.41 0.72 24 4 0.79 0.16 228.6 23 4 234.1 

Vehicle 

control 

32 2.95 0.36 0.83 40.2 0.96 0.21 223.9 32.6 190.1 

33 3.14 0.40 0.71 39.0 0.91 0.26 258.6 28.3 207.7 

34 2.82 0.36 0.74 37.8 0.86 0.19 251.8 29.0 220.6 

35 3.11 0.38 0.68 39.5 0.78 0.19 262.2 26.0 208.8 iiicovii-ry------------------------------------------------------------------------------..-.------- ...-------------------------------------­ 36 

37 

2.77 

2.70 

0.38 

0.35 

0.66 

0.71 

34.1 

31.8 

o 84 

0.92 

0.23 

0.21 

224.8 

192.1 

26.3 

27.5 

217.3 

209.9 

38 2.59 0.33 0.67 38.1 0.79 0.19 142.0 28 6 232.6 

..... 

<0 

Fellale 

25 

150 

39 

40 

41 

42 

43 

44 

45 

46 

47 

48 

49 

50 

2.68 

2.69 

3.00 

2.62 

3.19 

3.02 

2.86 

3.19 

3.85 

3.39 

3 42 

3.17 

0.39 

0.36 

0.40 

0.33 

0.41 

0.32 

0.34 

0.38 

0.43 

0.38 

0.40 

0.37 

0.78 

0.70 

0.79 

0.71 

0.74 

0.73 

0.62 

0.80 

0.83 

0.77 

o 78 

0.66 

38.7 

31.4 

39.1 

33.7 

38.0 

26.7 

39.5 

34.3 

37.7 

34.9 

37.6 

38.2 

0.92 

0.80 

0.91 

0.94 

0.96 

0.88 

0.90 

0.94 

0.89 

0.86 

0.90 

0.86 

0.23 

0.19 

0.21 

0.17 

0.17 

o 16 

0.20 

0.19 

0.24 

0.18 

0.22 

0.19 

191.6 

188.9 

306.3 

230 1 

275.5 

192.2 

201·.9 

217.8 

165.9 

197.4 

190.8 

255.0 

30.8 

22.5 

30.7 

32.5 

32.2 

32.4 

28.5 

26.7 

42.4 

29.5 

30.2 

34.2 

212.9 

230.9 ·ZT4-.-i-­

197.0 

183.2 

224.7 

189.0 

190.2 

208.5 

219. '1 

214.3 

218.1 

CJl 

J 

1,000 

51 4.19 0.39 0.92 36.1 0.79 0.21 177.9 21. 2 230.7 

52 4.33 0.39 0.86 31. 1 0.87 o 22 177.7 33.4 210.4­

53 4.62 0.40 1. 08 31. 1 0.82 0.17 161.4 31. 6 230.3 

54 3.99 o 40 0.93 52.1 o 96 0.22 193.5 29.2 189.6 

55 4.67 0.42 1.00 - 38.3 0.91 0.23 168.2 28.0 203.9 Reco;e-r-i------------------------------------------··------------------------------------------------------------------------------------------------------------------­

56 

57 

58 

59 

60 

2.93 

3.10 

2.95 

2.92 

2.74 

0.38 

0.44 

0.44 

0.38 

0.39 

0.77 

0.70 

0.88 

0.80 

0.70 

39.6 

30.4 

36.2 

40.4 

36.2 

0.89 

0.73 

0.79 

0.82 

0.B2 

0.21 

0.23 

0.22 

0.26 

0.23 

189.2 

184.1 

206.9 

194.5 

153.7 

29.3 

25.3 

25.8 

31. 4 

33.6 

210.6 

241.0 

233.1 

234.0 

224.4 

~ 

(l) 
BII-0908 

Addendum 

Sex 

13-1 Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings of individual animals 

Animal Exp.group N Fate Macroscopic findings o. Histopathological findings') 

1 ta No abnonnalities detected No abnormalities detected ................... a9 ................ "' .......... 00_ .. ____ ....................................................... ___ .... '" ............. "' .............. __ ......... "' ......... ___ .' 


2 ta No abnonnalities detected No abnonnalities detected ~ .. _ ........................... _ ........................................................................... __ ......................... _ .... e ........................................................ , 


3 ta No abnonnalities detected Adrenal 

Male Vehicle control 

.•.•..•. _......................._•.. _..••.•.....•.•...•..•••X?c.~l.~~~~l?~y. .':~ P~r:!~~!.~:!~.:.... 4 ta No abnonnalities detected No abnormalities detected ,. ................................................... _ ........................... _ .... _ ......................... _a. .... _ ........ "' ................... _ .................. ___ .... __ .......... ~ 

5 ta No abnonnalities detected No abnormalities detected 

a) Organs/tissues examined as follows: trachea, lungs. forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, 

colon, rectum, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, cerebrum, cerebellum, 

pons, spinal cord, sciatic nerve, bone marrow, axillar lymph node, mesenteric lymph node, spleen, thymus, pituitary gland, 

thyroid, parathyroid, adrenals and eye ball. 

ta, terminal autopsy. 

+,slight. 

-196­ 462 
B11-0908 

Addendum 13-2 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings of individual animals 

Animal Sex Exp.group N Fate Macroscopic findings Histopathological findings') o. 

6 ta No abnormalities detected No abnormalities detected ....................................................................................................-............................................................... ., .. , 

7 ta No abnormalities detected No abnormalities detected Vehicle control ...................................................................................................... Male 8 ta No abnormalities detected No abnormalities detected (Recovery) 

9 ta No abnormalities detected No abnormalities detected ........... .... _... ............................................ "" ............................................... "' ..................-.................................................................. 
 10 ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver, kidneys, epididymides, cerebrum, cerebellum and pons, 

ta, terminal autopsy. 

-197­ 463 
Bll-0908 

Addendum 13-3 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings of individual animals 

Exp.group Animal F Sex 	 ate Macroscopic fIDdings Histopathological findings') (mglkgldax) No. 

11 ta No abnormalities detected No abnormalities detected 

12 ta No abnolmalities detected No abnormalities detected 

13 ta No abnormalities detected Liver 

MaJe 	 25 ................._._...._...._......._...._..............._. !.?!:~l. !'~~~~~~~ ~.~~:p.~!,?!.?'!~.;. _. __ ._.' 

14 ta No abnormalities detected No abnormalities detected .....................__ .. -_ ............... _- ... '" ........................................................... ~ ...... "' ........... -.......... ,. ... ,.. ................................ . 


15 ta No abnormalities detected Liver 

Microgranuloma + 

a) Organs/tissues examined as follows: liver. 


ta, terminal autopsy. 


+, slight. 


-198­ 464 
BII-0908 

Addendum 13-4 Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings ofindividual animals 

Exp.group Animal Fate Macroscopic fmdings 


(mgfkglday) No. 


Sex Histopathological findings&) 

16 ta Jejunum Jejunum 

Diverticuluin Diverticulum 

Liver 

Male • _ •••••••••••• __ •• _ ••••••••__•• _ ••••• _ ••••••••••••••••• 0.0 •••~!~~<?~':!!~!,l!~:!'.•••....••..__ '_0'.__ • 150 

17 ta No abnormalities detected No abnormalities detected 

18 ta No abnormalities detected No abnormalities detected ................. "' ........................................................... .... "' .. .................................................................. _-............................ _... . 


19 ta No abnormalities detected No abnormalities detected 

20 ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver and macroscopic lesion. 


ta, terminal autopsy. 


+, slight. 


-199­ 465 
B11-0908 

Addendum 13-5 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological fIndings of individual animals 

Sex Exp.group Animal Fate Macroscopic fmdings Histopathological findings·) {mg/l<Jyday) No. 

21 ta Liver No abnonnalities detected 

Enlargement Liver 

Kidney No abnonnalities detected 

Enlargement (bilateral) Kidney 

No abnonnalities detected 

22 ta Kidney . Liver 

Enlargement (bilateral) Microgranuloma + 

Skin Kidney 

Loss ofhair (neck) No abnonnalities detected 

Skin 

Male 1,000 Scab fonnation + ..................... ,. .. "' .... -.... ,. ............. _.. _. '"'., .... - ........................ -­ _.............................. -_ ............................... _...... ,. ............................ 23 ta Kidney Kidney 

Enlargement (bilateral) Pelvic dilatation, unilateral + 

.................. ~:~~j.S .~i}~!~!!1?!.l.(~i~~9••..•••.•......•••..••.•••••.••.•.••••••••••.•.••• ___ .•••.•. 

24 ta Liver Liver 

Enlargement Diffuse fatty cbange ofhapatocytes + 

Kidney Kidney 

25 ta Kidney No abnormalities detected 

Enlargement (bilateral) Kidney 

No abnonnalities detected 

a) Organs/tissues examined as follows: trachea, lungs, forestomach, glandular stomach, duodenum,jejunum, ileum, cecum, 

colon, rectum, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, cerebrum, cerebellum, 

pons, spinal cord, sciatic nerve, bone marrow, axillar lymph node, mesenteric lymph node, spleen, thymus, pituitary gland, 

thyroid, parathyroid, adrenals, eye ball and macroscopic lesion. 

ta, tenninaJ autopsy. 

+, slight. 

-200­ 466 
l 

BII-0908 

Addendum 13·6 Twenty-eight-day repeated-dose oral toxicity study ill rats 

Pathological fmdings of individual animals 

Exp.group Animal Sex (!P8!kglday) No. Fate Macroscopic fmdings Histopatbological findings' 

1,000 Male (Recovery) 

26 ta No abnormalities detected No abnormalities detected ....................................................... "' ...... '" "' ........ 4> ..... _ ...... __ ..........................__ ....... ................'..........,. ...". ...................... . 


27 ta No abnormalities detected No abnormall~BS detected .................................. "" .............................................-......................................................... ,..............--_....... 
 28 ta No abn~lities detected No abnormalities de~ted .......................................................""'.............................................-..............-._-................................,.....,. ............ 


3.0 ta No abnormalities detected No abnormalities detected 

a) Organs/tiSSUes examined as follows: liver, kidneys, epididymides. cerebrum. cerebellum and pons. 

ta, terminal autopsy. 

-201­ 467 
1311-0908 

Addendum 13-7 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological ti.ndings of individual animals 

Animal F Sex Exp.group ate Macroscopic findings Histopathological findings') No. 

31 ta No abnonnafnie[l detected No abne>rmalities detected .................. "" ...... to ................................... __ ............................ __ .......................-....'!"..................."' ......... - ............. '"',.~ 

32 ta No abnMnalines detected No abnonnalities derected ..............................................-................................................._._.................-.......................... -_ .............. 
 Female Yehiclecontrol 33 ta No abnormalities detected No abnormalities detected 

~ 

"........................... -......................................................................-....-........,. ...............-........................-_......... .
 34 fa No abnormalities detected No abnormalities detected ........... "............. "'..................." 	 ........-..................-.........-_ ..-................ " .............................................
~ 

3S ta No~bnormaJities detected No abnormalities detected 

a) Organs/tissues examined as follows: trachea, lungs, forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, 

colon, rectum, liver, heart, kidneys, urinary bladder, ovaries, uterus, vagina, cerebrum, cerebellum, pons, spinal cord, 

sciatic nerve, bone marrow, axillar Jymph node, mesenteric lymph node, spleen, thymus, pituitary gland, thyroid, parathyroid, 

adrenals and eye ball. 

ta, terminal autopsy. 

-202­ 468 
811-0908 

Addendum 13-8 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological fmdings ofindividual animals 

Animal F Sex Exp.group N ate Macroscopic fmdings Histopathological findings') o. 

36 ta No abnormalities detected No abnormalities detected ... __ ................................................................................................ __ ........... OJ ......... _ ............ OM _ ............................ 10' .... _ .......... . 

37 ta No abnormalities detected No abnormalities detected 

Female Vehicle control 

(Recovery) 38 ta No abnormalities detected No abnormalities detected ................... _.............................................­.............................................................."" ................................................................. 39 ta No abnormalities detected No abnormalities detected .......................................... "'_ ..................... --........................................ _....... "'­ _........................................................................ 40 ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver, heart and kidneys. 

ta, terminal autopsy. 

-203­ 469 
Bll-0908 

Addendum 13-9 	Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological fmdings ofindividual animals 

Sex Exp.group Animal Fate Macroscopic fmdings Histopathological findingsa) (mg/kglday) No. 

41 ta No abnormalities detected No abnormalities detected ........................................................................... _........................................................................_.................... "". 

42 ta No abnormalities detected Kidney 

Female ..............................................•.•............~)!t:!:?:.~r-:~ ~.~!?:!.l!~."t.............. . 25 

43 ta No abnormalities detected No abnormalities detected 

44 ta No abnormalities detected No abnormalities detected 

4S ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver and kidneys. 


ta. terminal autopsy. 


+, slight. 


-204­ 470 
811-0908 

Addendum 13·10 Twenty-eight-day repeated-dose oral toxicity study in rat~ 

Pathological findings of individual animals 

Sex Exp.group Animal Fate Macroscopic findings Histopathological findings') 
 (mli!<liday) No. 


46 ta No abnormalities detected No abnormalities detected .. -. "' ....... - .......... - _.. -- - ...... _... ----.................................................................................................................... __ .- .............. -_ ... . 


47 ta Liver No abnormalities detected 

Enlargement Liver 

No abnormalities detected _............................- .... _................................................................................................._.......'............................... ,., .. _......
~ 

Female 150 48 ta Glandular stomach Glandular stomach 

Blackish region ofrnucosa Necrosis offundic mucosa + 

..................t~.! .~).........___..................._._............... _ ...................... . 
 49 ta No abnormalities detected No abnormalities detected 

50 ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver, kidneys and macroscopic lesion. 


ta, terminal autopsy. 


+, slight. 


-205­ 471 
B 11-0908 

Addendum 13-11 	 Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings of individual animals 

Exp.group Animal Sex 	 Fate Macroscopic findings Histopathological fmdings') (mg!Jcslday) 	 No. 

51 ta Liver Liver 

Enlargement No abnonnalities detected 

Kidney Kidney 

Enlargement (bilateral) Degeneration and necrosis of 

tubular epithelium with regenerative 

. epithelium + . ... -~ .................................... _.. -...................................... -_ .............................................. - .... - .. -_ ............................. 


52 ta Liver Liver 

Enlargement Fatty change of periportal 

hepatocytes + ............. ----_._ .................-..... -_ .......... -.. _............................................................. _... __ ........ -_ ........ -_ .. _.... -.. -............ . 


53 ta Liver Liver 

Enlargement Fatty change of periportal 

Kidney hepatocytes + 

Enlargement (bilateral) Microgranuloma + 

Female 1,000 Kidney 

No abnormalities detected .......................... -_ ............................................. _.. ---_. -........................ -........ -............ -_ .... _.. -_ .... -_ ...... - ...... _.. _........ -_ .... -_ .. , 


54 ta Liver Lung 

Enlargement Focal inflanunation + 

Kidney Liver 

Enlargement (bilateral) No abnormalities detected 

Kidney 

No abnormalities detected 

55 ta Liver Liver 

Enlargement No abnormalities detected 

Kidney Kidney 

Enlargement (bilateral) 	 Degeneration and necrosis of 

tubular epithelium with regenerative 

epithelium + 

a) Organs/tissues examined as follows: trachea, lungs, forestomach, glandular stomach, duodenum,jejunum, ileum, cecum, 

colon, rectum, liver, heart, kidneys, urinary bladder, ovaries, uterus, vagina, cerebrum, 'cerebellum, pons, spinal cord, 

sciatic nerve, bone marrow, axillar lymph node, mesenteric lymph node, spleen, thymus, pituitary gland, thyroid, parathyroid, 

adrenals and eye ball. 

ta, tenninal autopsy. 

+, slight. 

-206-	 472 

BII-0908 

Addendum 13-12 Twenty-eight-day repeated-dose oral toxicity study in rats 

Pathological findings of individual animals 

Sex Exp.group 

(rng/kglday) 

Animal 

No. 

Fate Macroscopic fmdings Histopathological findings') 

Female 1,000 

(Recovery) 

56 ta No abnonnalities detected No abnormalities detected ........................................ -_ ... ---_ .... --- --_ .. --_ .......................... -_ .. _..... _.......... _.................. _.. --_ ..... -_ .. --_ ...... -_ .... -_ ...... . 

57 ta No abnonnalities detected No abnormalities detected 

58 ta No abnormalities detected No abnormalities detected .......... _.. ------_ .... _................_................... -_ ...................... _...................... --_ ...... -­....----_ .... _...... ---_ .. _.. ---­ _._-. 

59 ta No abnormalities detected No abnormalities detected 

60 ta No abnormalities detected No abnormalities detected 

a) Organs/tissues examined as follows: liver, heart and kidneys. 

ta, terminal autopsy. 

-207­ 473 
811-0908 


HISTOPATHOLOGICAL PHOTOS 


474 
BII-0908 

Photo. ) 	 Liver of a female rat from vehicle control group. 

NonnaJ. 

No. 33 animal. HE. )( 180. 

Photo. 2 	 Liver ofa female rat from 1,000 mglkglday group. 

Fatty change ofperiportaJ bepatocytes. 

No. 53 animal. HE. )( )80. 

-1­

475 
BII..(l908 

Photo. 3 	 Kidney ofa female rat trom vehicle control group. 

Normal. 

No. 33 animal. HE. x180. 

Photo. 4 	 Kidney of. female rat trom J,000 mglkglday group. 

Degeneration and necrosis oftubular epithelium with regenerative epithelium. 

No. 51 animal. HE. x180. 

-2­

476 
Bll-0908 

PHYSICOCHEMICAL REPORT 


477 
cERJ 


Receipt No. 827-08-D-3387 

STUDY CODE: XIS-0908 

FINAL REPORT 

STABILITY ANALYSIS OF KarenzMT (Mixture of PEMB and PE3MB), 


HOMOGENEITY, STABILITY AND CONCENTRATION ANALYSES OF THE 


TEST SUBSTANCE FORMULATION 


March 2009 

Hila Laboratory 


Chemicals Evaluation and Research Institute, Japan 


-1­

478 
XlS-0908 

STATEMENT 


TITLE OF STADY 

Stability Analysis of KareniMT (Mixture of PEMB and PE3MB), Homogeneity, 

Stability and Concentration Analyses of the Test Substance Formulation (Study Code: 

X1S-090S) 

I, the undersigned, hereby declare that this report provides a correct English translation of the 

final report (Study Code: X18-0908, issued on March 16, 2009). 

Yuji Kusune, M.S. 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

-ii479 
XlS-0908 

GLP STATEMENT 

Hita Laboratory 


Chemicals Evaluation and Research Institute, Japan 


~Q.,-,r:__-",S=h","ow.:..:.=a""D=e=n=ko",-,,-,K=.K=. 

Title: Stability Analysis of KarenzMT (Mixture of PEMB and_ PE3~B), Homogeneity, 

Stability and Concentration Analyses ofthe Test Substance Formulation 

Study Code: X 18-0908 

I, the undersigned, hereby declare that this study was conducted in compliance with "Concerning 

Standard of the Testing Facilities Conducting the Test Relating to the New Chemical 

Substances" on Japanese GLP (Notification No. 1121003 of the Phannaceutical and Food Safety 

Bureau, MHL W, No.3 (November J7, 2003) of the Manufacturing Industries Bureau, MET! & 

No. 031121004 ofthe Environmental Health Department, MOE (November 21, 2003)). 

I also con finned that this report accurately reflected the raw data and the test data were valid. 

Study Director: Sisned in original March 16,2009 

Yuji Kusune, M.S. 

111­

480 
XIS-0908 

QUALITY ASSl.JRA.~CE STATEMENT 

Hita Laboratory 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Shows Denko K.K. 

Title: Stability Analysis of KarenzMT (Mixture of PEMB and PE3fvtID,-Ho!Il~!tce.ity"" 

Stability and Concentration Analyses of the Test Substance Formulation 

Study Code: X 18-0908 

This study was inspected by Quality Assurance Unit of Hita Laboratory, Chemicals Evaluation 

and Research Institute, Japan. The dates inspected and the dates reported these results to the 

study director and management are as follows 

Phasc Date of lnspection 

Date Reported to Study 

Director and Management 

Protocol October 10, 2008 October 10, 2008 

IR Spectrum of Test Substance October] 4, 2008 October 14,2008 

Homogeneity and Stability 

Analyses ofTest Substance 

Formulation 

October 14, 2008 October 14, 2008 

Confirmation of the Answer from 

Study Director (Protocol) October 17,2008 October 17,2008 

Amendment to Protocol October 27, 2008 October 27, 2008 

Concentration Analysis of Test 

Substance Formulation November 6, 2008 November 6, 2008 

Amendment to Protocol (No.2) November 10, 2008 November 10, 2008 

Raw Data and Draft Final Report February 24, 2009 February 24, 2009 

Reinspection ofRaw Data and 

Draft Final Report February 27, 2009 February 27, 2009 

Amendment to Protocol (No.3) March 12, 2009 March 12, 2009 

Draft Final Report (Second Time) March 12, 2009 March 12, 2009 

Final Report March 16, 2009 March 16, 2009 

I, the undersigned, hereby declare that this report provides an accurate description ofthe methods 

and procedures used in this study, and that the reported results accurately reflect obtained raw 

data. 

Head, Quality Assurance Unit: Signed in original March 16, 2009 

Ryuichiro Mizuguchi, D.S. 

-iv481 
X18-0908 


TABLE 01" CONTENTS 


Page 


TITLE ... ,............................................................................................................................ 3 


SPONSOR ......................................................................................................................... 3 


TESTING F ACILITY ....................................................................................................... 3 


PURPOSE OF STUDy ....·..····....·.. ............ ....................................... ............... ................. 3 


GLP COMPLIANCE ........................................................................................................ 3 


PERIOD OF STUDY ........................................................................................................ 3 


STORAGE AND RETENTION PERIOD OF DATA ........................................................ 4 


RETENTION OF ORIGINAL DOCUMENTS ................................................................ 4 


STUDY DIRECTOR AND PERSONS CONCERNED WITH THE STUDY AND THE 


OPERATION .................................................................................................................... 4 


APPROVAL BY AUTHOR .............................................................................................. 4 


SUMMARY ...................................................................................................................... 5 


MATERIALS ......... .... ..... ..... ............ ................. ..... ......... ................. ............ ........... .......... 6 


J. 	 TEST SUBSTANCE ................................................................................................... 6 


METHODS ........................................................................................................................ 7 


1. 	 SUBJECT STUDY ..................................................................................................... 7 


2. 	 STABILITY ANALYSIS OF THE TEST SUBSTANCE .................. ;...................... 7 


3. 	 HOMOGENEITY, STABILITY AND CONCENTRATION ANALYSES OF THE 

TEST SUBSTANCE FORMULA TION ....................................................... ,............. 8 

THE ENVIRONMENTAL FACTORS THAT MIGHT HAVE AFFECTED THE 

RELIABILITY OF THE STUDY RESULTS ................................................................. 12 

RESULTS AND DISCUSSION ...................................................................................... 12 


I. 	 RESULTS ................................................................................................................. 12 


2. 	 DISCUSSION ........................................................................................................... 13 


-1­

482 
Xl 8-0908 

TABLES 

Homogeneity and stability analyses ofthe test substance formulation 14 

2 Concentration analysis ofthe dose formulation ........................................................ 14 

FIGURES 

IR spectrum measured prior to the administration period ............................ .,........... 15 

2 IR spectrum measured after the end of the administration period ............................ 15 

REFERENCE 

1 IR spectrum provided by the sponsor ....................................................................'" 15 

-2­

483 
X18-0908 

Study Code: X 18-0908 

Test Substance Code: HR7645 

Sponsor Code: s-o 170 

TITLE 

Stability Analysis of KarenzMT (Mixture of PEMB and PE3MB), Homogeneity, Stability and 

Concentration Analyses ofthe Test Substance Formulation 

SPONSOR 

Showa Denko K.K. 

13-9, Shiba Daimon l-chome, Minato-ku, Tokyo 105-8518, Japan 

TESTING FACILITY 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan 

PURPOSE OF STUDY 

The purpose of this study is to determine the stability of the test substance during the dosing 

period, and homogeneity, stability and concentration of the test substance in formulation in 

"Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of KarenzMT (Mixture of PEMB and 

PE3MB) in Rats" (Study Code: B 11-0908). 

GLP COMPLIANCE 

This study was conducted in compliance with "Concerning Standard of the Testing Facilities 

Conducting the Test Relating to the New Chemical Substances" on Japanese GLP (Notification 

No. 1121003 of the Pharmaceutical and Food Safety Bureau, MHLW, No.3 (November 17, 

2003) of the Manufacturing Industries Bureau, MET! & No. 031121004 of the Environmental 

Health Department, MOE (November 21,2003). 

PERIOD OF STUDY 

Commencement of Study: October 9,2008 

Initiation of Examination (Initiation of Analysis); October 14, 2008 

Termination ofExamination (Termination of Analysis); December 26, 2008 

Completion of Study: March 16, 2009 

-3­

484 
X18-0908 

STORAGE AND RETENTION PERIOD OF DATA 

The raw data, protocol, study contract documents, test substance information, final report and 

other record documents will be retained in the archive of the Hita Laboratory of our organization 

for the same period of BII-0908 paper data. After termination of the retention period, any 

measures taken win be done so with the approval ofthe sponsor. 

RETENTION OF ORIGINAL DOCUMENTS 

An original protocol and an original final report will be retained at Hita Laboratory. The copies 

of their original that the study director will be recognized to be accurate copy will be sent to the 

sponsor. 

STUDY DIRECTOR AND PERSONS CONCERNED WITH THE STUDY AND THE 

OPERATION 

Study director: Yuji Kusune, M.S. 

Study staff: 	 Masaru Suzuki, B.S. 

(Analysis ofthe test substance) 

Saari Takakura, J.L.A.T., JAC.L.C.T. 

(Preparation of the test substance formulation) 

APPROVAL BY AUTHOR 

Study director: Signed in oriiinal March 16, 2009 

Yuji Kusune, M.S. 

Analytical Chemistry Section 

Hita Laboratory 

-4­

485 
XlS-0908 

SUMMARY 

The test substance (KarenzMT (Mixture of PEMB and PE3MB)) was stable during the dosing 


period ofsubject study (Study Code: B 11-0908). 


The test substance in 10.0 and 0.25 w/v% formulations was stable for 8 days after preparation at 


cold and dark place and showed good homogeneity. The concentration oftest substance in 10.0, 


1.5 and 0.25 w/v% dose formulations for subject study was acceptable level. 

-5 

486 
X18-0908 


MATERIALS 

1. TEST SUBSTA.NCE (INFORMATION PROVIDED BY THE SPONSOR) 

-6­

487 
XlS-0908 

1.8 	 Storage Conditions 

Stored at room temperature (cabinet No. 1 in test substance storage room, tolerance 

temperature: 10-30°C). Actual temperature between received of test substance to 

termination of examination was 19.0-22.4°C. It satisfied tolerance. Avoid oxidizing 

agent. 

1.9 	 Handling Precaution 

Glove, mask, cap, goggles and lab coat were worn. The test substance was treated in the 

draft since the test substance has characteristic odor. 

METHODS 

1. 	 SUBJECT STUDY 

Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of KarenzMT (Mixture of PEMB 

and PE3MB) in Rats (Study Code: B11-0908) 

2. 	 STABILITY ANALYSIS OF mE TEST SUBSTANCE 

The infrared (IR) spectrum was measured by IR spectrophotometer before and after the 

dosing period of subject study. 

2.1 	 Measurement of IR 

1) Instrument 

IR spectrophotometer: FT-720 (HORIBA, Ltd.) 

2) Condition 

Wave number: 4600 em'] - 400 cm'l 

3) 	 Pre-Treatment 


Potassium bromide neat 


2.2 	 Criteria for Judgment 

IR spectrum ofthe test substance that measured prior to dosing in our laboratory should be 

identical with provided from the sponsor. The test substance was judged to be stable 

when there are no differences in the IR spectrums at before and after dosing period. 

-7 

488 
XlS-090S 

3. 	 HOMOGENEITY, STABILITY AND CONCENTRATION ANALYSES OF THE 

TEST SUBSTANCE FORMULATION 

In the homogeneity analysis, the samples were taken (n=l) from the upper, middle and 

lower layers of formulations immediately after preparation, respectively. These samples 

were pretreated and measured (n=l) with high performance liquid chromatography 

(HPLC). 

In the stability analysis, the formulations were stored at cold and dark place for 8 days, and 

the sample was taken (n=l) from the middle layer of the formulations at point of 

measurement (3 days and 9days after preparation). These samples were pretreated and 

measured (n=l) with HPLC. 

In the concentration analysis, the samples were taken (n=1) from the middle layer of dose 

formulations immediately after preparation for subject study. These samples were 

pretreated and measured (n=1) with HPLC. 

3.1 	 The Test Substance Formulation 

1) Homogeneity and Stability Analyses 

(1) Concentration 

10.0 and 0.25 w/v% 

(2) Preparation Method 

Weighed 5.00 g ofthe test substance in a beaker, and mixed with olive oil to make 

50 mL, and used as 10.0 w/v% formulation. Accurate 2.5 mL of 10.0 w/v% 

formulation was diluted with olive oil to make 100 mL, and used as 0.25 w/v% 

formulation. 

Vehicle: olive oil (Lot No. 0460YS, Fujimi Pharmaceutical) 

2) 	 Concentration Analysis 

The 10.0, 1.5 and 0.25 w/v% dose formulations at first preparation for subject study 

were used. 

3.2 	 Outline of Analytical Method 

The analytical method was decided, according to results of validation of the analytical 

method on non-GLP at the test facility. 

1) Validation ofthe Analytical Method 

(1) Preparation for Measurement Sample 

a) 	 Standard Stock Solution for Validation ofthe Anal)'1ical Method 

Weighed 0.10187 g of the test substance, dissolved in 2-propanol to make 

100 mL, and used this solution as 1

°18.7 J.lg/mL standard stock solution for 

validation ofthe analytical method. 

b) Sample for Specificity 

The standard stock solution for validation of the analytical method was diluted 

with 2-propanol to make 50,9 llg/mL standard solution and 50.9 ~lg/mL 

-8 

489 
Xl 8-0908 

vehicle-containing standard solution (containing 2 v/v% vehicle (olive oil)). 

2-Propanol was used as solvent blank, and vehicle blank (containing 2 v/v% 

vehicle) was prepared. 

c) 	 Sample for Linearity 

The standard stock solution for validation of the analytical method was diluted 

with 2-propanol to make 25.5, 50.9') and 102 IlglmL standard solutions. 

*I: It was llsed 50.9 J1g/mL standard solution of sample for specificity. 

d) Sample for Accuracy and Repeatability 

Standard solutions (concentration: 25.5, 50.9 and 102 llg/rnL, each 

concentration: n=3) were prepared in the same way of sample for linearity. 

(2) Specificity 

Samples for specificity were measured with HPLC. The variation of detection 

value (total peak area) of the test substance between standard solution with and 

without vehicle was -2.0%. Therefore, it was confirmed that the result of 

variation satisfied criteria for judgment (within ±5%). In the results of HPLC 

analysis of solvent blank and vehicle blank, it was confirmed there were no 

background and interfering peaks at the elution peak position of test substance. 

(3) Linearity 

Samples for linearity were measured with HPLC. The calibration curve was 

made by the concentration of the test substance in the horizontal line and the 

detection value of test substance in the vertical line. The coefficient of 

correlation of calibration curve which was obtained from least square was R=0.999. 

Therefore, it was continued that the result of linearity satisfied criteria for 

judgment (more than 0.999). 

(4) Accuracy and Repeatability 

Samples for accuracy and repeatability were measured with HPLC. The 

concentrations of the test substance were calculated with regression formula that 

was obtained at linearity. Accuracy and repeatability were calculated with these 

values. 

Accuracy of25.5 llg/mL standard solution was 0.4, -3.1 and -0.4%. 

Accuracy of 50.9 llg/mL standard solution was -0.4, -1.2 and -1.0%. 

Accuracy of 102 llg/mL standard solution was 0.0, 0.0 and 1.0%. 

Repeatability of 25.5, 50.9 and 102 IlglmL standard solution was 1.9, 0.4 and 0.6%, 

respectively. 

It was confirmed that the result of accuracy and repeatability satisfied criteria for 

judgment (accuracy: within ±J 0%, repeatability: less than 5%). 

9 

490 
XJ8-0908 

2) 	 Preparation for Standard Solution 

Weighed 0.10113 g of the test substance dissolved in 2-propanol to make 100 mL, and 

used this solution as J 0 11.3 J.l.g/mL standard stock solution. Accurate 1 mL of 

standard stock solution diluted with 2-propanol to make 20 mL, and 50.6 JlglmL 

standard solution was prepared. 

3) 	 Pre-Treatment 


Formulations were mixed well using a magnetic stirrer. 


(1) 10.0 w/v% Formulation (Homogeneity, Stability and Concentration Analyses) 

Accurate 0.5 mL of fonnulation was dissolved in 2-propano! to make 50 mL. 

Accurate I mL of this solution was diluted with 2-propanol to make 20 mL, and 

served as an analytical sample (dilution rate: 2000). 

(2) 1.5 w/v% Formulation (Concentration Analysis) 

Accurate 0.5 mL of formulation was dissolved in 2-propanol to make 30 mL. 

Accurate 2 mL of this solution was diluted with 2-propano! to make 10 mL, and 

served as an analytical sample (dilution rate: 300). 

(3) 0.25 w/v% Formulation (Homogeneity, Stability and Concentration Analyses) 

Accurate 0.5 mL of formulation was dissolved in 2-propanol to make 25 mL, and 

served as an analytica.1 sample (dilution rate: 50). 

4) 	 Conditions of HPLC Analysis 

(1) Instruments (HPLC7) 

Interface: D-7000IF (Hitachi, Ltd.) 

Pump: L-71 00 (Hitachi, Ltd.) 

Auto sampler: L-noo (Hitachi, Ltd.) 

LN-VIS detector: L-7400 (Hitachi, Ltd.) 

Column oven: L-7300 (Hitachi, Ltd.) 

Data processor: D-7000 HPLC System manager (Hitachi, Ltd.) 

(2) Conditions 

Column: L-column ODS (4.6 mm LD. x 50 mm, CERl) 

Column temperature: 40°C 

Mobile phase: 0.5 v/v% phosphoric acid solution/acetonitrile == 45/55 (v/v) 

Flow rate: 1 mLimin 

Wavelength: 210nm 

Injection volume: 10 J.l.L 

3.3 	 Data Processing 

1) Detection Value 

A total peak area was used as the detection value. 

2) 	 Quantitative Analytical Method 

In validation of the analytical method, the result of linearity was a straight line range 

-10­

491 
X18-0908 

of25.5, 50.9 and J02 iJ.gltnL standard solutions, and it passed through the origin ofthe 

coordinates. Therefore, the concentrations of analytical samples were detel1l1ined by 

single level calibration. 

3) 	 Calculation of the Test Substance Concentration in FOl1l1ulation 

Concentration of test substance in each sample (C: w/v%) was calculated with the 

equation shown below and rounded off to three significant figures. 

CsxA x D C 

Asx 10000 

Cs: Test substance concentration in standard solution (!lg/mL) 


As: Detection value of test substance in standard solution 


A: Detection value oftest substance in each analytical sample 

D: 	 Dilution rate in each analytical sample 

3.4 Criteria for Judgment 

1) 	 Homogeneity Analysis 

The test substance was judged as homogeneous dispersion in vehicle if a coefficient of 

variation (CV) was within 5%. The CV was calculated using the following equation: 

CV(%) =:; Standard deviation for concentration of test substance in each x 100 

Mean concentration of test substance in each layer 

2) 	 Stability Analysis 

The test substance was judged as stable state in vehicle if a rate to the nominal 

concentration for the actual concentration (R.N.) and a rate to the mean concentration 

immediately after preparation for the actual concentration (R.P.) were within the range 

of ]OO±1 0%. The R.N. and R.P. were calculated using the following equation: 

R.N.(%) = Actual concentration x 100 


Nominal concentration 


R.P.(%) = Actual concentration x 100 

Mean concentration immediately after preparation 

3) 	 Concentration Analysis 

It was confirmed that R.N. was within the range of 100±1O%. The R.N. was 

calculated using the following equation: 

-11 

492 
X18-0908 

R.N.(%) = concentration x 100 


Nominal concentration 


ENVIRONMENTAL FACTORS THAT MIGHT HAVE AFFECTED RELIABILITY OF 

STUDY RESULTS 

There were no factors that might have affected the reliability ofthe study data. 

RESULTS AND DISCUSSION 

1. 	 RESULTS 

1.1 	 Stability Analysis of the Test Substance 

IR spectrum of test substance provided by the sponsor (Reference 1) was identical with 

that measured before dosing period for subject study (Figure 1). 

There were no differences in the IR spectra between before and after dosing period 

(Figures 1,2). 

1.2 	 Homogeneity, Stability and Concentration Analyses of the Test Substance 

Fonnulation 

1) Homogeneity and Stability Analyses 

The results of homogeneity and stability analyses of the test substance formulation are 

shown in Table 1. 

(1) Homogeneity Analysis 

CV of 10.0 and 0.25 w/v% formulations were 0.6 and 1.0%, respectively. The 

results satisfied criteria for judgment. 

(2) Stability Analysis 

a) 	 10.0 w/v% Formulation 

At immediately after preparation, R.N. were 101 to 102%. 

At 3 days after preparation, R.N. was 100%, and R.P. was 98.0%. 

At 9 days after preparation, R.N. was 101%, and R.P. was 99.0%. 

All the results of R.N. and R.P. satisfied criteria for judgment. 

b) 0.25 w/v% Formulation 

At immediately after preparation, R.N. were 102 to 104%. 

At 3 days after preparation, R.N. was 102%, and R.P. was 99.6%. 

At 9 days after preparation, R.N. was 102%, and R.P. was 100%. 

AU the results of R.N. and R.P. satisfied criteria for judgment. 

-12 

493 
Xl 8-0908 

2) 	 Concentration Analysis 

The results of concentration analysis of the test substance formulation are shown in 

Table 2. 

R.N. of 10.0, 1.5 and 0.25 w/v% dose formulations were 99.1 to 101%. All the 

results satisfied criteria for judgment. 

2. 	 DISCUSSION 

The test substance was stable during the dosing period of subject study. 

The test substance in 10.0 and 0.25 w/v% formulations was stable for 8 days after 

preparation at cold and dark place and showed good homogeneity. The concentration of 

test substance in 10.0, 1.5 and 0.25 wlv'% dose formulations for subject study was 

acceptable leveL 

-13­

494 
XIS-0908 

Table 1 Homo eneity and stabIlity analyses ofthe test substance £ ormu l' atlOn 

Nominal Time point Layer of Actual R.N. Mean cone. cone. of 

measurement cone. (%) (w/v%) (w/v%) measurement (w/v%) 

Immediately Upper lO.2 102 

after Middle 10.2 102 10.2 

preparation Lower 10.1 101 

10.0 

3 days 

after Middle 10.0 100 - preparation 

9 days 

after Middle 10.1 101 - preparation 

Immediately Upper 0.259 104 

after Middle 0.254 102 0.256 

preparation Lower 0.256 102 

0.25 

3 days 

after Middle 0.255 102 . preparation 

9 days 

after Middle 0.256 102 - preparation 

R.P. CV 

(%) (%) 

- 0.6 

98.0 -

99.0 -

- 1.0 

99.6 -

100 -

I 

I 

R.N.: Rate to the nominal concentration 

R.P.: Rate to the concentration measured immediately after preparation 

cv: Coefficient of variation 

C I . Table 2 oncen trf a Ion analYSIS I . 0 f th e d ose fiormu atlon

Date of analysis 

Nominal cone. 

(w/v%) 

Actual cone. 

(w/v%) 

R.N. 

(%) 

10.0 9.91 99.1 

November 6, 2008 1.5 1.51 101 

0.25 0.250 100 

R.N.: Rate to the nominal concentration 

-14­

495 
X18-0908 


ns.2F__­

..; 

Figure 1 IR spectrum measured prior to the administration period 

111.t r-------------------~ 

0.1001"" to ••• ,., • Ii • Q •• 1 • •• , I 4600 40lI0 181. tOOO 10lI0 coo. 0 

Figure 2 IR spectrum measured after the end ofthe administration period 

. ' •• L-'_----' 

1000 400 

Reference 1 IR spectrum provided by the sponsor 

-15­

496 
CERi 


Receipt number 662-08-E-4746 

Study number 94746 

FINAL REPORT 


A 96-hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and PE3MB) 

with Medaka 

April 15, 2009 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

497 
94746 


STATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 	 Showa Denko KK 

Title 	 A 96-hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and 

PE3MB) with Medaka 

Study number 	 94746 

I, the undersigned, hereby declare that this report provides a correct English 

translation of the Final Report (Study No. 94746, issued on April 15, 2009) 

Date f<..c I 7, 	~o r

Study Director :lalcJ.l 1ltri1:uuI.U4 

Takeshi Matsuura 

498 
94746 


GLPSTATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 	 Showa Denko KK 

Title 	 A 96·hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and 

PE3MB) with Medaka 

Study number 	 94746 

The study described in this report was conducted in compliance with the following GLP 

principles: 

(1) 	 "Standard Concerning Testing Facility Relating to New Chemical Substances" 

(November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of 

Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries 

Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental 

Policy Bureau, Ministry of the Environment) 

(2) 	 "OECD Principles of Good Laboratory Practice (November 26, 1997, ENV/MC/CHEM 

(98)17)" 

This final report reflects the raw data accurately and it has been confirmed that the test data 

are valid. 

Date 	 April 15, 2009 

Study Director 	 Signed in Qri&inal 

Takeshi Matsuura 

499 
94746 

QUALITY ASSURANCE STATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 	 Showa Denko KK 

Title 	 A 96-hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and 

PE3MB) with Medaka 

Study number 	 94746 

I assure that the final report accurately describes the test methods and procedures, and 

that the reported results accurately reflect the raw data of the study. 

The inspections and audit of this study were carried out and the results were reported to 

the Study Director and the Test Facility Management by Quality Assurance Unit as foHows. 

Date of report to 


Item of inspection Jaudit 
 Date of inspection Jaudit Study Director and 

Test Facility Management 

Study plan draft February 26, 2009 February 26, 2009 

Study plan February 26, 2009 February 26, 2009 "---_._--­ Amendment to study plan March 6, 2009 March 6, 2009 

,-­

March 2, 2009 March 6, 2009 

after the exposure 

Start of the exposure and 

March 6, 2009 March 6, 2009 .---. i--'-­ -,~.--,...

Raw data and final report draft April 14, 2009 April 15, 2009 -. -_... Final report April 15, 2009 April 15, 2009 

Date April 15, 2009 

Head of Quality Assurance Unit Signed in original 

Keiji Shiraishi 

500 
94746 


CONTENTS 


Page 

Title ................................................................... ·..········..·····..··· ...... ····· ..·..,······..·1 

Sponsor ...... · .. ·..· .. · ..······..·· .. ····· ...... ··.... ··· .... ·..· .... ·· ...... ·· .. · ..·..· ...... ···· .. ······· ..···· .. ···1 

Test facil ity ......................................................................................................... 1 

Objective···..············..·······..··· .............................................................................. 1 

Test method ........................................................................................................ 1 

Applied GLp· .. ···· ..···········..·..···············..· ...... ······· ..·········..·······..·········..·· .... · ..··..·1 

Dates·· .... ······· ..·· .. ············· ..·..······.. ·· ....· .... ··· ........................................................ 2 

Storage of test item, raw data, etc. ····· .. ······ .... · .... ·· .... ·· .. ·· .. ··.... ·· ..·..· .... · .... · .... · .... ·2 

Personnel .... · .. ··................................................................................................... 3 

Approval of final report .... ··....·..···...................................................................... 3 

SUMMARY ............................................................................ · ........ · .... ·..·..·..····4 

1. Test item ....... , ..................................................................... · .... ···· ..·..··..··.... · .... ·6 


2. Test sample ..................................................................................... ···· .... ···· .. · .... ·7 


3. Test materials and methods .............................................................................. 8 


4. Results and discussion ...................................................................................... 13 


5. Factors that affected reliability of test results .. · .... ··· .... · .. · .......................... · ..··.. 14 


Tables and Figure 

Table 1 Cumulative mortality 

Table 2 Observed abnormal response 

Table 3-1 Dissolved oxygen concentration of test solutions 

Table 3-2 pH of test solutions 

Table 3-3 Temperature of test solutions 

Table 4 LCso and LLso to Medaka 

Figure 1 Concentration-cumulative mortality curve 

Appendix 1 Chemical characteristics of dilution water 

Appendix 2 Analytical method and measured concentration of test item 

Appendix 3 Calibration curve and chromatogram 

Additional data 1 Results of preliminary studies 

Additional data 2 Results of measured concentration of each component of test item 

Additional data 3 Qualitative analysis of transformed substance 

501 

Title 

Sponsor 

Test facility 

Objective 

Test method 

Applied GLP 

94746 

A 96-hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and 

PE3MB) with Medaka 

Showa Denko KK 

13-9, Shiba Daimon l-Chome, Minato-ku, Tokyo 105-8518, Japan 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

3-2-7 Miyanojin, Kurume-shi, Fukuoka 839-0801, Japan 

The objective of this study is to determine the acute effects of the 

Karenz MT (Mixture of PEMB and .PE3MB) on fish. 

This study was performed according to the following test methods and 

guidance document. 

(1) Fish, Acute Toxicity Test stipulated in the "Testing Methods for 

New Chemical Substances" (November 21, 2003; No. 1121002, 

Pharmaceutical and Food Safety Bureau, Ministry of Health, 

Labour and Welfare; November 13, 2003, No.2, Manufacturing 

Industries Bureau, Ministry of Economy, Trade and Industry; No. 

031121002, Environmental Policy Bureau, Ministry of the 

Environment) 

(2) 	OECD Guidelines for Testing of Chemicals, Section 2 : Effects on 

Biotic Systems, 203 Fish, Acute Toxicity Tcst (Guideline 203, 

1992) 

(3) 	OECD Guidance Document 23 "Guidance Document on Aquatic 

Toxicity Testing of Difficult Substance and Mixtures" (September 

2000) 

This study was conducted in compliance with the following GLP 

principles: 

(1) 	 "Standard Concerning Testing Facility Relating to New Chemical 

Substances" (November 21, 2003; No. 1121003, Pharmaccutical 

and Food Safety Bureau, Ministry of Health, Labour and Welfare; 

November 17, 2003, No.3, Manufacturing Industries Bureau, 

Ministry of Economy, Trade and Industry; No. 031121004, 

Environmental Policy Bureau, Ministry of the Environment) 

(2) 	 "OECD Principles of Good Laboratory Practice (November 26, 

1997, ENV/MC/CHEM(98)17)" 

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Dates 

Study initiation date February 26, 2009 

Experimental starting date March 2, 2009 

Experimental completion date March 6, 2009 

Study completion date April 15, 2009 

Storage of test item, raw data, etc. 

(1) Test item 

The test sample'l will be sealed in a storage vessel and stored in archives in this 

laboratory for ten years after the receipt of notice specified under Clause 1 or Clause 

2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 

5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning 

Examination and Regulation of Manufacture, etc. of Chemical Substances". If it is 

not stable for the storage period, it will be stored as long while it is kept stable. 

Treatment of the item supplied by the sponsor after the storage period will be 

discussed with sponsor. 

*1 It will be stored as the common sample for storage of these studies (Study Nos. 

94744,94745 and 94746). 

(2) Raw data and materials 

Raw data, the study plan, documents concerning the study presented by the 

sponsor, the final report and necessary materials will be stored in archives in this 

laboratory for ten years after the receipt of the notice specified under Clause 1 or 

CJause 2 in Article 4, CJause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in 

Article 5-4 Or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning 

Examination and Regulation of Manufacture, etc. of Chemical Substances". 

Treatment of raw data and materials, etc. after the storage period wiU be discussed 

with sponsor. 

503 ·2· 
Personnel 

Study Director 

Stud y personal 

Biology 

Analytical chemistry 

Approval of final report 

Study Director 

94746 

Takeshi Matsuura 

Section 4 (Eco-toxicity test area) 

Jun Sugimura, Miyuki Narasaki, 

Manko Tanaka, Hiroyuki Togawa 

Takeshi Matsuura, Makiko Anai 

Hiroyuki Togawa, Fumiko Kamiaka, 

Tomoyo Kagamiyama, Mariko Tanaka 

Hitomi Fukui, Hiromi Andoh 

Date 	 April 15, 2009 

Signature 	 Signed in original 

Takeshi Matsuura 

504 -3­
94746 

SUMMARY 

Title 

A 96-hour Acute Toxicity Study of Karenz MT (Mixture of PEMB and PE3MB) 

with Medaka 

Test conditions 

(1) Test item 

(2) Test organism 

(3) Exposure duration 

(4) Test concentration 

(5) Replicate 

(6) The number of organisms 

(7) Dilution water 

(8) Type of test 

(9) Preparation of test solution 

(10) Volume of test solution 

(11) Temperature of test solutions 

(12) Irradiation condition 

(13) Feeding 

(14) Aeration 

Karenz MT (Mixture of PEMB and PE3MB) 

Medaka (Oryzias lalipes) 

96 hours 

Five exposure levels of 5.0, 3.1, 2.0, 1.2 and 0.8 

mglL (a geometric series with a factor of 1.6) as 

loading rate [respectively 0.51, 0.65, 0.25,0.14 and 

0.12 mgIL as measured concentration] and control 

One replicate/test level 

Seven fish/test level 

Dechlorinated tap water 

Semi-static (renewal at every 24 hours) 

Test sample and dilution water were mixed to 

produce each nominal lording rate, and then they 

were stirred for 48 hours. They were filtrated to 

prepare the test solution. 

About 4.9 Utest level 

23.9 to 24.2°C 

Artificial light of white fluorescent lamp, 

16-hour light/8-hour dark 

No feeding 

No aeration 

(15)Analysis of concentration of test item in test solution 

HPLC analysis 

(At the start of the exposure, before and after the 

renewal and the end of the exposure) 

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Results 

(1) Solubility in dilution water (24±1°C) 

10 mgfL (measured value in preliminary study) 

(2) Concentration of test item in test solution (Percentage of the initial concentration) 

At the start of the exposure and after the renewal 

Below the lower detection limit (0.019 mg/L) to 0.98 mgfL 

At the before the renewal and the end of the exposure 

Below the lower detection limit (0.019 mg/L) to 0.92 mglL (50 to 97%) 

(3) 96-hour LC50 (Median Lethal Concentration) 0.16mgfL 

(4) The lowest concentration causing 100% mortality at 96 hours 0.25mgfL 

(5) The highest concentration causing 0% mortality at 96 hours 0.12 mgfL 

[Reference value: 96-hour U50 (Median Lethal Loading); 1.4 mgIL (nominal loading rate)] 

[The values of (3), (4), (5) are based on geometric mean of the measured concentrations.] 

506 -5­
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1. Test item 

.

-------..... - .....-_ .....-._._--------_ ....----------... ~--- ..-----..----.. -----~-.--- ....-----.-------.----.. -----.~---------. 

. 

507 -6­
94746 

2. Test sample :-.-... ~.------- .. ---.-.--.-----.--.----...-....-----...._------.......------......_------_ .._-------......-_._-_._._.---._._---;
 . .
 . . 


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2.5 	 Storage condition and confirmation of stability at storage condition 

Storage condition Dark storage place at room temperature and avoided 

contacting with oxidizing agent. 

Confirmation of stability The stability of the test item during the test period was 

confirmed by no alteration in the IR spectra of the test 

item before the experimental start and after the 

experimental completion. 

3. Test materials and methods 

3.1 Test organism 

(1) 	 Species 


Medaka (Oryzias latipes) 


(2) 	 Reason for selection of species 


This species is recommended in the test guidelines. 


(3) 	 Size 

Totallength 2.3 ± 1.2 cm 

Size of test organism was applied the regulated size set to test method (1). 

(4) 	 Supplier 

Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan 

3-2-7 Miyanojin, Kurume-shi, Fukuoka 839-0801, Japan 

(5) Acclimation 

Medaka hatched out on November 26, 2008 were acclimated for 24 days by 

flow-through condition under the same water quality (dechlorinated tap water), 

temperature (24±1 QC), light and dark period (16-hour light/8-hour dark) as test 

condition (on February 6 - March 2, 2009). The mortality was 0% during the 7 

days before the start of the exposure. The test organism at the start of the 

exposure was 3-month-old fish. The test organisms were not treated with a 

medicament for external disinfection. The test organisms were fed the feed 

mixture for carp (2C), and not fed for 24 hours before the start of the exposure. 

Dissolved oxygen concentration in breeding water during acclimation was kept not 

less than 80% of air saturation value. A 96-hour acute toxicity test of CUS04 

5HzO (Reagent chemical, Wako Pure Chemical Industries, Ltd.) to confirm 

reproducibility of the test system was carried out on October 20 - October 24, 2008, 

and the 96-hour LCso was 0.34 mglL. This value was within the stipulated range 

(mean ± 2S.D. : 0.14 - 0.97 mgIL) [mean ± S.D. : 0.56 ± 0.21 mgIL (n=42)J to 

background data in this laboratory. AU of the values shown above for the 

reference substance were converted into CUS04 value, 

509 -8­
94746 


(6) 	 Allocation to the test groups 

Medaka were allocated at random to each test group. 

3.2 	 Dilution water 

Dechlorinated tap water, aerated sufficiently and controlled temperature, was used. 

Some chemical characteristics of the dilution water measured regularly are listed in 

Appendix 1. 

3.3 Test apparatus and equipment 

(1) 	 Test apparatus 

Test vessel : 7.8 L Glass tank (diameter: 24 ern, depth: 28 em) 

The test vessels were covered with lid in order to prevent dust, and 

volatilization of the test solution. 

(2) Test equipment 

Water bath Plastic tank 

Warming/cooling unit (Type HCA 250, Sato craft) 

3.4 Test conditions 

(1) Conditions of exposure 

(a) Type of test 

The test organisms were exposed to the test solution containing the test item. 

The test solutions were renewed at every 24 hours as semi-static regime. 

(b) Exposure duration 

96 hours 

(c) Test concentration 

Five exposure levels of 5.0, 3.1, 2.0, 1.2 and 0.8 mg/L (a geometric series 

with a factor of 1.6) as loading rate were used in the test. The exposure levels 

and the factor were decided based on the results of preliminary studies. The 

results of the preliminary studies are shown in Additional data 1. 

(d) Control 

The dilution water without the test item, which was treated in the same 

stirring manner as the test solution, was used as the control. 

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(e) 	 Replicates 


One replicate/test level 


(f) 	 The number of organisms 


Seven fish/test level 


(g) 	 Volume of test solution 


About 4.9 Utest level 


(2) Conditions of test environment 

(a) 	 Water temperature 


24±1°C 


(b) Dissolved oxygen concentration 

The test was performed in the condition where dissolved oxygen 

concentration was at least 60% or more of the saturated concentration at the 

water temperature. Aeration was not used during the exposure. 

(c) 	 pH 


The test was performed without adjusting pH. 


(d) 	 Irradiation condition 

Artificial light of white fluorescent lamp, 16-hour light/8-hour dark 

(e) 	 Feeding 


Test organisms were not fed during the exposure. 


3.5 Preparation of test solution 

The test sample and dilution water were mixed to produce each nominal lording rate 

and stirred by magnetic stirrer for about 48 hours. The solution was filtrated with a 

glass fiber filter (ADVANTEC, GB-l40, 0.4 /1-m filter unit) by suction. Mter the 

filtration, the filtrate was aerated for about one hour under about 24°C in order to 

recover decreased dissolved oxygen concentration by suction and then used for test 

solution. 

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3.6 Observation and measurements 

(1) Observation of test organisms 

Mortality and visible abnormality were observed at 3, 24, 48, 72 and 96 hours 

after the start of the exposure. A fish was considered as dead if the observable 

motion (motion of mouth and opercula etc.) were not observed and touching of the 

caudal peduncle with glass rod produced no reaction. 

(2) Total length and body weight of test organism 

The test organisms in the control group were used for measuring total length 

and body weight after the end of the exposure. 

(3) Appearance of test solution 

Appearance of the test solutions was observed at the start and before renewal 

of test solutions (24 hours after). 

(4) Quality of test solutions 

Dissolved oxygen concentration, pH and water temperature of the test 

solutions were measured at the start of the exposure, before and after the renewal, 

and the end of the exposure. They were measured also at the time that confirmed 

all test organisms dead at the exposure level, where all test organisms died at the 

exposure level. The dissolved oxygen concentration measurements were carried 

out with an oxygen meter (YSI Incorporated, YSI MODEL 58). The pH 

measurements were carried out with a pH meter (DKK-TOA, Model HM-21P). 

The temperature measurements wer:e carried out with a calibrated red alcohol 

thermometer of glass stick type. 

(5) Concentration of test item in test solution 

The concentration of the test item in the test solutions was measured at the 

start of the exposure, before and after the renewal, and at the end of the exposure. 

The measurement was conducted also at the time that confirmed all test organisms 

dead at the exposure level, where all test organisms died at the exposure level. 

The test solution was taken out from the middle layer of the test vessel and was 

used for the measurement. The concentration of the test item was analyzed by 

high-performance liquid chromatography (HPLC). Analytical method and 

measured concentration of test item are shown in Appendix 2, and analytical 

calibration curve and chromatograms are shown in Appendix 3. 

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3.7 Calculating method of LCso'3 and LLso'4 

Results of this study were estimated using Computer Program (running on 

Microsoft software "Excel") developed by our laboratory. 

(1) 	 LCso 

The results of this study were estimated by geometric mean of the measured 

concentrations For 48 and 72 hours, the LCso was calculated by Probit analysis, 

and the 95% confidence interval and the slope of the dose-response curve were also 

calculated. The 96-hour LCso was calculated by Binomial test. For 24 hours, the 

LCso was estimated as "> the highest test concentration (0.65 mg/L)" since the 

mortality over 50% was not observed. 

(2) 	 LLso (reference) 

The results of this study were estimated by the nominal loading rate For 48, 

72 and 96 hours, the LCso was calculated by Binomial test. For 24 hours, the 

LLso was estimated as "> the highest test concentration (5.0 mglL)" since the 

mortality over 50% was not observed. 

*3 	 LCso (Median Lethal Concentration) : The test item concentration at which 50% of 

the test organisms causes mortality during the exposure. 

*4 	 LLso (Median Lethal Loading) : The test item nominal concentration at which 50% 

of the test organisms causes mortality during the exposure. 

3.8 	 Validity of the test 

(1) 	 The mortality in the control should not exceed one fish. 

(2) 	 Dissolved oxygen concentration must be at least 60% of the air saturation value at 

the water temperature in the test during exposure. 

3.9 	 Treatment of numerical values 

Values were rounded off in accordance with JIS Z 8401 rule B, 1999. 

(JIS ; Japanese Industrial Standards) 

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4. Results and discussion 

The test concentrations in the following sentence is expressed using geometric mean 

of the measured concentrations which are 0.51,0.65,0.25,0.14 and 0.12 mg/L, which are 

respectivel y 5.0, 3.1, 2.0, 1.2 and 0.8 mg/L as the loading rate. 

4.1 	 Mortality 

Cumulative mortality of each observation time was shown in Table 1. 

Concentration-cumulative mortality curve is shown in Figure 1. 

The lowest concentration causing 100% mortality within 96 hours was 0.25 mg/L, 

and the highest concentration causing no mortality within 96 hours was 0.12 mg/L. 

Number of dead fish in the control at the end of the exposure was 0, which meets the 

criterion for the validity of the test (Le. not exceed one fish). 

4.2 	 Observed abnonnal response 

The abnormal responses observed during the exposure are shown in Table 2. 

No abnonnal responses were obtained in the controL 

The following results of observation were based on the comparison with the control 

organisms. Observed abnormal responses during the exposure were swimming at the 

water surface, light body color and reduced activity. 

4.3 	 Size of test organism [Mean ± Standard deviation (n=7)] 

Total length 2.5 ± 0.076 em 

Body weight 0.13 ± 0.011 g 

4.4 	 Observation and measurement of test solution 

(1) 	 Appearance oftest solution 

The test solutions were clear and colorless at the start of the exposure. The 

appearance kept until before the renewal. 

(2) 	 Quality of test solutions 

Qualities of the test solutions are shown in Tables 3-1, 3-2 and 3-3. 

The measured values of dissolved oxygen concentration, pH and temperature 

during the exposure ranged from 7.2 to 8.4 mglL, 7.5 to 7.9 and 23.9 to 24.2°C, 

respectively. The measured values of dissolved oxygen concentration met the 

criterion for the study validity (at least 60% or more of saturate concentration'5 at 

the water temperature). 

*5 	 Saturated dissolved oxygen concentration (23 to 25°C): 8.39 to 8.11 mg/L 

(JIS K 0102, 2008) 

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(3) Concentration of test item in test solution 

The results of the measured concentrations of the test item are shown in 

Appendix 2. 

The measured concentrations of the test item in the test solution were from 

below the lower detection limit (0.019 mglL) to 0.98 mglL at the start of the 

exposure and after the renewal, and from below the lower detection limit (0.019 

mglL) to 0.92 mglL (50 to 97% of the initial measured concentration) before the 

renewal and the end of the exposure containing with the time all test organisms 

died. 

4.5 	 LC50 and LLso 

The LCsos and LLsos at each observation time are shown in Table 4. 

The 48-hour and 96-hour LCso were respectively 0.49 mgIL (95% confidence 

interval: 0.012 to 0.60 mglL) and 0.16 mglL. 

As reference value, the 48-hour and 96-hour LLso estimated based on the loading 

rate were respectively 2.6 mgIL and 1.4 mgIL. 

4.6 Discussion 

This study was conducted as a test in order to estimate LC50 of the test item for the 

test organisms below the solubility of the test item in dilution water. As a result, the 

96-hour LCso based on the measured concentration was 0.16 mglL, the 96-hour LCso 

based on the nominal loading rate was 1.4 mglL. 

The concentration-response of cumulative mortality was not linear between 3.1 and 

5.0 mglL. However, it was confirmed that the measured concentration in the test 

solution during the exposure was also not linear at the same exposure levels. 

Therefore, the concentration-response of mortality was linear. Since the cause of the 

reversal of the test item concentration was the reversal of the PEMB concentration, it 

was highly likely that PEMB concentration was involved in the effect on organism 

greatly (reference: Additional data 2). It was considered that expected causes of 

reversal of the concentration were to be poorly water-soluble for the test item, and to 

adopt the method to prepare the test solutions each exposure level. In addition, 

because it was confirmed that the converted products derived from the test item 

produced in the test solution during exposure, there was a possibility that the test item 

concentration was affected by productive condition of the converted products 

(reference: Additional data 3). 

Since it was confirmed that the maintenance ratio of the test item concentration in 

the test solution varied, the definitive study was conducted the renewal as much as 

possible. The environmental conditions were within the suitable range, therefore, it is 

concluded that this study complied with the applied test guidelines. 

5. Factors that affected reliability of test results 

There were no factors which might have affected the reliability of the test. 

515 -14­
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Table 1 Cumulative mortality 

Measured Cumulative mortality (%) 

concentration*6 

(mglL) 3 hours 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 0 

0.12 0 0 0 0 0 

0.14 0 0 0 0 29 

0.25 0 0 0 86 100 

0.65 0 14 86 100 100 

0.51 0 0 57 86 100 

*6 	 Geometric mean of measured concentration (also expressed as measured 

concentration in the following tables and figure) 

Table 2 Observed abnormal response 

Measured 

concentration 

(mglL) 

Result of observation 

(Left column: Number of affected fish I Total survival number, 

Right column: Symptom detail) 

3 hours 24 hours 48 hours 72 hours 96 hours 

Control on 1 N 

I 017 [ N on 1 N j on I N on I N 

0.12 

I 

on N on! N on N I 

on I N on N 

0.14 on N on N on ::::Ei N 

N 0/1 : N ! 

0/5 N 

0.25 on N on, N on - -

0.65 on ' N 0/6 N III AS(l) - 1 

! - - -

0.51 0/7 N 0/7 N 113 

A1S(1} 

LT(I) 

RA(l) 

on N - -

N: Normal (No abnormal response) 


Value in parentheses expresses the number of individuals shown the symptom. 


- : All test organisms died. 


Abbreviation of symptoms 


AS : Swimming at the surface 


LT: Light body color 


RA : Reduced activity 


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Table 3-1 Dissolved oxygen concentration of test solutions 

Measured 24 hours 48 hours 72 hours I 

concentration At the At the 

slart Before the After the Before the After the Defore the After the end (mglL) renewal renewal renewal renewal renewal renewal 

Control 8.3 7.7 8.4 7.8 8.2 7.8 8.4 7.9 

0.12 7.8 7.6 8.4 7.7 8.1 7.8 8.3 7.8 

0.14 8.1 7.6 8.4 7.6 8.1 7.6 8.2 7.7 

0.25 8.2 7.5 8.3 7.6 8.1 7.7 8.1 8.1 * 

0.65 8.1 7.4 8.3 7.2 8.2 8.1* - -

0.51 8.2 7.6 8.3 7.5 8.2 8.0 8.2 8.2* 

Unit: mgIL 


- : All test organisms died. 


* :It indicates the measured value at the time that confirmed all test organisms dead. 

Table 3-2 pH of test solutions 

Measured 

concentration 

(mglL) 

At the 

start 

24 hours 48 hours 72 hours 

At the 

end Before the 

renewal 

After the 

renewal 

Before the 

renewal 

After the 

renewal 

Before the 

renewal 

After the 

renewal 

Control 7.7 7.5 7.7 7.6 7.7 7.5 7.7 7.5 

0.12 7.9 7.5 I 7.9 7.5 7.9 7.5 7.9 7.5 

0.14 7.9 7.5 7.9 7.5 7.9 7.5 7.9 7.5 

0.25 7.9 7.5 7.9 7.5 7.9 7.5 7.9 7.8* 

0.65 7.9 7.5 7.9 7.5 7.9 7.7* - - 0.51 7.9 7.5 7.9 7.5 7.9 7.7 7.9 7.8* 

- : All test organisms died. 

* : It indicates the measured value at the time that confirmed all test organisms dead. 

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Table 3-3 Temperature of test solutions 

Measured 'A h . I tt e concentratIOn 

(mglL) : start 

24 hQurs 48 hours 72 hours 

At the 

end Before the 

renewal 

After tbe 

renewal 

Defore the 

renewal 

Afterlhe 

renewal 

Before the 

renewal 

Afterlbe 

renewal 

Control 23.9 24.0 24.0 24.0 24.1 24.0 24.1 24.0 

0.12 23.9 24.0 24.0 24.1 24.2 24.0 24.1 24.0 

0.14 24.0 24.0 24.0 24.2 24.2 24.1 24.1 24.0 

0.25 24.0 24.0 24.0 24.1 24.2 24.0 24.1 24.0* 

0.65 24.0 24.0 24.0 24.2 24.2 24.0· - - 0.51 24.0 24.0 24.0 24.1 24.1 24.0 24.1 24.1* 

Unit: °C 

- : All test organisms died. 

'" : It indicates the measured value at the time that confirmed all test organisms dead. 

Table 4 LCso and LLso to Medaka 

Exposure 

duration 

LCso 

(mgIL) 

95% confidence 

intervals 

(mgIL) 

(Slope of the 

dose-response 

curve) 

Statistical 

procedure ! 

used for 

determination 

of LCso 

Reference values 

Statistical 

LLso'7 procedure 

used for (mglL) detennination 

ofLLso 

24 hours >0.65 -

( -) - >5.0 -

48 hours 0.49 0.012 to 0.60 

(9.4) 

Probit 

analysis 2.6 Binomial 

test 

72 hours 0.23 0.16 to 0.32 

(5.1) 

Probit 

analysis 1.7 Binomial 

test 

96 hours 0.16 Binomial test 1.4 Binomial 

test 

-: Not obtained 

*7 Based on loading rate 

-17­ 518 
94746 


100 

80 

~ 

~ '-' 

>. 60 .,... 

-

"3.... 

0 

e 

~ 

·5 

..!:l 

:::I 40 e 

:::I 

U 

20 

o 

. 

. 

. 

; ....................• 

. 

.. 

.

. 

-G--24hour 

~48hour 

...•... 72 hour 

---.- 96 hour 

0.1 1 

Measured concentration (mgfL) 

Figure 1 Concentration-cumulative mortality curve 

519 -18­
94746 


Appendix 1 


Chemical characteristics of dilution water 

520 

94746 

Chemical characteristics of dilution water (Sampling on January 14,2009) 

Parameter Unit ResulCs Lower limil or determinalion 

Total hardness (as CaC03) mg/L 42 1

Suspended solid mg/L < ] 1

pH - 7.9 (22"C) - Total organic carbon mg/L 0.4 0.1

Chemical oxygen demand mg/L 0.6 0.5 

Residual chlorine mg/L < 0.02 0.02 

Ammonium ion mg/L < 0.01 0.01 

Total cyan mg/L < 0.01 0.01 

Alkalinity mg/L 29 1

Electric conductivity mS/m 17.1 - Organicphospborous mg/L < 0.1 0.1 

Alkylmercury mg/L < 0.0005 0.0005 

Mercury mg/L < 0.0005 0.0005 

Cadmium mg/L < 0.001 0.001 

Chromium (VI) mg/L <0.01 0.01 

Lead mg/L < 0.005 0.005 

Arsenic mg/L < 0.001 0.001 

BOTOn mg/L 0.08 0.01 

Fluorine mg/L < 0.1 0.1 

Iron mg/L < 0.01 om 

Copper mglL < 0.005 0.005 

Cobalt mg/L < 0.001 0.001 

Manganese mg/L < 0.01 0.01 

Zinc mg/L < 0.005 0.005 

Aluminum mglL OJ)42 0.001 

Nickel mg/L < 0.001 0.001 

Silver mg/L < 0.0001 0.0001 

Sulfate ion mg/L 20.1 0.1 

Chloride ion mg/L 12 1 

Sodium mg/L 14.9 0.01 

Potassium mg/L 1.8 0.01 

Calcium mg/L 11.5 0.01 

Magnesium mg/L 3.3 0.01 

1,2-dichloropropane mg/L <0.0001 O.[)OOl 

Chlol'Othalonil mg/L < 0.0001 0.0001 

Propyzamide mglL <0.0001 0.0001

Chlornitrofen mg/L < 0.0001 0.0001 

Simazine mglL < 0.0001 0.0001 

Tbiobencarb mg/L < 0.0001 0.0001

Diazinon mg/L < 0.0001 0.0001 

Isoxathion mg/L < 0.0001 0.0001 

Fenitrolhioll mg/L < 0.0001 0.0001 

EPN mg/L < 0.0001 0.0001 

Dichlorvos mg/L < 0.0001 0.0001 

Iprobenfos mg/L < 0.0001 0.0001 

PCB mg/L < 0.0005 0.0005 

521 -1 Appendix )­
94746 


Appendix 2 


Analytical method and measured concentration of test item 

522 

94746 


1. Pretreatment of test solution 

The test solutions sampled were pretreated according to the following flow scheme 

to prepare the sample for high-performance liquid chromatography (HPLC) analysis. 

Flow scheme 

I Test solution 100 mL I 

+-Chloroform 30 mL (graduated cylinder) 

• Shaking (10 min) 

I IWater layer I IChloroform layer I 

• Transferred chloroform layer to the eggplant flask with 

a beaker 

• Dryness (rotary evaporator, about 40°C, nitrogen stream) 

+-Acetonitrile / water"l (111 v/v) about 1 mL (pasteur pipette) 

• Ultrasonic irradiation (ultrasonic cleaner, about 30 sec) 

• Filling up to 2 mL [acetonitrile / water"l (1/1 v/v), 

volumetric flask] 

I Sample for HPLC analysis I 

*1 Tap water was treated with ultrapure water system. 

523 -1 Appendix 2­
94746 


2. Method of analysis 

The pretreated samples for HPLC analysis were quantitatively analyzed by HPLC 

under the following conditions to determine the concentration of the test item. The 

test item was detected as two peaks on the chromatogram. Therefore, these were 

analyzed as the test item. The concentration of the test item in each sample for HPLC 

analysis was determined on the basis of a comparison of the total peak area on the 

chromatogram of the sample solution with that of a standard solution. Some 

chromatograms obtained are shown in Appendix 3. 

Analytical conditions 

Instrument 	 High-performance liquid chromatograph 

SHIMADZU LC-2010A 

(Equipped with UV -VIS detector) 

Column 	 L-column ODS 

(15 cm x 2.1 mm I.D., Chemicals Evaluation and 

Research Institute) 

Column temp. 40°C 

Eluent A: Acetonitrile I phosphoric acid (1000/1 v/v) 

B: Water"l I phosphoric acid (1000/1 v/v) 

Gradient condition 

Time (min) A(%) B(%) 

0.00 50 	 50 

20.00 100 o 

40.00 100 o 

Flow rate 0.2 mIJmin 

Wave length 210nm 

Injection volume lO.uL 


Detector output 2V/AU 


3. Preparation of standard solution 

The standard solution to determine the concentration of the test item in the sample 

for HPLC analysis was prepared as follows. 

Test sample of 100 mg was precisely weighed with an electronic balance and 

dissolved in acetonitrile to obtain 1000 mgIL solution of the test item. The solution 

was diluted with acetonitrile / water"1 (111 v/v) to prepare 50 mglL of standard solution. 

524 -2 Appendix 2­
94746 


4. Calibration curve 

The standard solutions of 5.0, 25, 50 and 100 mgIL were prepared for HPLC 

analysis by the same procedure as described in section 3. These soiutions were 

analyzed according to the quantitative analytical conditions described in section 2. A 

calibration curve was drawn from the relationship between the concentrations of 

standard solution and the total peak area on the chromatogram, and the determination 

was confirmed. The calibration curve is shown in Appendix 3. The determination 

limit of the test item was the lowest concentration of the standard solution (5.0 mglL) 

within the calibration range. Therefore, the determination limit of the test item in the 

test solution was 0.11 mglL in consideration of pretreatment. 

5. Recovery test and blank test 

5.1 Method 

The recovery test was conducted by adding the test item (prepared with 

acetonitrile) to dechlorinated tap water according to pretreatment of test solution 

described in section 1. The blank test was also conducted using dechlorinated tap 

water (added acetonitrile) without the test item in the same way as the recovery test. 

The recovery test and the blank test were performed in duplicate. 

Amount of the test item added 100 Itg 

5.2 Result 

As a result of analysis by the method of section 5.1, no peak of the test item 

appeared in the chromatogram of the blank test. Two individual recovery rates and 

their average on the analytical procedure are shown below. The average of recovery 

rate was used as correction factor for the determination of the test item concentrations 

in the test solutions. 

Recovery rate for pretreatment 


87%, 89% average 88% 


525 -3 Appendix 2­
94746 


6. Results of measurement 

The results of the measured concentrations of the test item in the test solution are 

shown below. 

Appendix table 2-1 Measured concentrations of test item in test solutions 

Measured concentration (mg/L) 

Nominal (Percentage of measured concentration versus that at each preparation %) 

concentration 

(mglL) At the 

start 

Control n.d. 

0.8 0.16 

1.2 n.d:3 

2.0 n.d:4 

3.1 0.69 

5.0 0.51 

24 hours 

old new 

n.d. n.d. 

n.d:4 0.17 

n.d."3 0.25 

n.d."3 0.61 

0.59 0.74 (86) 

0.39 0.92 (75) 

G 48 hours hOUrs

old new new 

n.d. n.d. n.d. n.d. 

'4 0.22 0.11 n.d. 0.19 (53) 

0.22 0.41 0.25 0.35 (85) (61) 

0.39 0.65 0.37 0.63 (64) (57) 

0.54 0.72 

0.65'2 - (73) (91) 

0.89 0.26 0.13 0.98 (97) (50) 

At the Geometric 

end mean 

n.d. /

0.12 0.12 (65) 

0.21 0.14 (61) 

0.46"2 

0.25 (74) 

- 0.65 

0.92*2 

0.51 (94) 

old : before the renewal 

new : after the renewal 

n.d. ; < 0.11 mgIL 


- : It shows no measurement because all test organisms died. 


*2 It indicates the measured value at the time that confirmed all test organisms dead. 


*3 In accordance with DECO Guidance Document No.23, the detection limit (0.019 mgIL) 


was adopted for the calculation of the geometric mean, because the test item was not 

detected. 

*4 	 In accordance with DECD Guidance Document No.23, half the determination limit of the 

test item (0.055 mgIL) was adopted for the calculation of geometric mean, because the test 

item was detected but not quantified. 

526 -4 Appendix 2­
94746 


Appendix 3 


Calibration curve and chromatogram 

527 

94746 


900000 

~ C\1 450000 

~ 

~ 

~ 

o 

o 	 50 100 

Concentration (mgIL) 

y = 8519x 


r =1.00 


Concentration Peak area 

(mgIL) (}.tV' sec) 

5.0 41878 

25 212165 

50 429373 

100 850423 

Appendix figure 3-1 Calibration curve of test item for analysis by HPLC 

528 -1 Appendix 3­
---

94746 

Study No. 94746 

l!:tt;LILmcLL___ . __._____.. Operating Date: Mar. 02, 2009 

PlCNO Time Area lie I cht Cone. 

Cain) ().IV·seo) ().I V) (~) 

I 8.68 61153 5309 14.60 

2 	 15.22 361357 39470 85.4Q - Total 423110 100.00 

I 

_L_.__._JJ .­

. I • 0.0 10.0 20. 0 30.0 Operator: Hiroyuki Togawa 

94746LOh/Control -.. Operat i nit Date : liar. 02. 2009 

PKNO Time Area Height Cone. 

(min) 	 (p V ·sec) ().IV) ~) 

n.d. n.d. 

~- ~ i --"~--.. ,.---_. 

0.0 10'.0 20.0 30. Operator: Hlroy"'l Togawa 

94746/0hLO.8 mg/l 	 Operat i nr; Date : liar. 02. 2009 ..-~"-~-- -",'~~"-<..----.

PKNO 	 Time Area Height CoIm. 

(pain) (ltY·sec) ().IV) ~) 

1 8.68 800B 799 13.51 

2 	 Iii. 23 51846 4344 86.49 

Total 59943 100.00 

!. I .1 

. 	 , . 110 10.0 20.0 30.. Operator : HI royuki TOlal!a 

Date: Uar.4.2009 

Appendix figure 3-2-1 HPLC chromatograms at start of exposure 

529 -2 Appendix 3­
94746 

Study No 94746 

Oparatinr Date . ller. 02.2009 94746iOhLl.2 mgiL 

PKNO Tim. Area fie leht Cone. 

jill n) (,U'l'680) (pV) (\) 

n.d. n.d. 

-"----"'_~

~ __.. .J.. ~ -~

0.'0 ;0:0 20.0 

___~'__0 __••_ ..... 

30.0 Operator : Hi royuki TOlaR 

94146/9.bfl.<LM__________. ----. ..­

PKNO 

Operatiol Date: Mar. 02, 2009 

Time Area Heicht Cone. 

(min) (J.l V·sec) (IlV) (t.) 

n.d. n.d. 

---1~__

~ .__....,A. ~ . 

0.0 10.0

, ,~-" 

20.0 

---_. 

,

311.(1 Operato r ; Hi royuk i Togawa 

94146/0hLlilJ!&lb...~ ______ ..___.__.__ Operating !late: Iler.02.2009 

PKNO 

1 

2 

Time 

(min) 

8.10 

15.25 

Area 

(jl V'sec) 

93175 

162954 

Height

(/IV) 

8671 

15911 

Cone. 

<") 

36.38 

63.62 

Total 256129 100.00 

1.. 1 1 -.... -~----~"-. 

. , . C1.0 10.' ao :!fl. Operator : Hi royuk i Toga"a 

FIg 

Appendix figure 3-2-2 HPLC chromatograms at start of exposure 

530 -3 Appendix)­
94746 

Study No. 94746 

94746/0h/5.0 mgjL Ope rat ing Date : liar. 02, 2009 

/'KilO Ti.& Ar ea He i IIht Cone, em i n) (1/ Nee) (I/ V) (I) 

8.68 104447 9705 54.88 .1.~. 23 85859 7627 45. 12 

Total 1911306 100.00 

Operator : Hi royuk I 10ga1lll 

Fig 

Date: Mar. 4, 2009 N8IIIe: H~rQluh I~Gwg 

,~ ~a..!; ~.<,/ 

Appendix figure 3-2-3 HPLC chromatogram at start of exposure 

531 -4 Appendix 3­
94746 

Study No 94746 

std. 50 m&Ll 	 __.._ .·4. ___ ~~· ___ Oparatinll Data: Mar.03.2009

PKNO Time Area Heiiht Cone. 

Ullin) (SlY,sIIe) (St V) 0') 

1 8.72 62228 5300 14.57 

2 	 1!.f8 364889 39552 85.43 

Total 427117 100.00 

I ........... A 

. . 0.0 10.0 

94146[Z4hLControllMO 

I

20.0 3110 

---- ­ ~- ---.¥.---­

PKNO 

Operator: HiIOyuki Togawa 

Operating Date : liar. 03. 2009 

Til1lll Ana Height Cone. 

(mi n) (I! V'sec) (SI V) (Il 

n.d. n.d. 

i ~ ~ 

. 

0.0 10.0 20.0 

94146/24h/O. 8 m,!.vMllI~__.... _. 

---~ 30.0 

- I'lOO 

Operator: HiroYllki Togawa 

Operating Dab : Mar. 03.2009 

Time Area Height Conc. 

(min) (II Y·sec) (fl V) (S) 

n.d. n.d. 

~ ~ --~--------- ",. .----~.---

0.'0 Hi. a to. 0 JOo Operator : HI royuk i Togalla 

Fig 

Appendix figure 3-3-1 HPLC chromatograms before renewal at 24 hours 

532 -5 Appendix 3­
94746 

Study No. 94746 

94746/24hLLUgf~*1l\L Operating Date: Mar. 04.2009 ..m ~"""~"..--... 

PKNO 	 Time Area Height Cone. 

(min) 	 (Il V·sec) (pV) (I) 

n.d. n.d. 

~ ~ ......A.---.____•.___.-A--...________..... 

.. I 0:0 	 Hi. 0 20.0 300 Operator : Hi royuki Togalla 

Operating Date: Mar. 04. 2009 94H~Lf5bLLQ..lJlW.jt!*liI ..---.--­ ~--

Pl(NO 	 Time Area He i,tit Corn;. 

(min) (IlV·seo) (/I V) (I) 

n.d. n.d. 

~ w._______ ~.---""'--.----"--

Co ,0. 0 

94746124bn.L!!l&LLj~ 

2!UI 

.-~.. 

31). 

PKNO 

1 

2 

Operator : 111 royuki TogaWi 

Operating Date: Mar. 04. 2009 

tillie Area Heieht Cone. 

(min) (/I Y·aee) (Il V) <l) 

8.72 87316 7650 39.33 

HU8 134617 12606 _ 60.61 

Total 221993 100.00 

~_~ 2 __ ....L<­ __. I 

'11.11 U).O 2O~0 - 30.0 Operator: Hiroyukl Togawa 

FIg 

Appendix figure 3-3-2 HPLC chromatograms before renewal at 24 hours 

533 -6 Appendix 3­
94746 

Study No. 94746 

Operatinl Date : Mar. ()4, 2009 

PKNO 	 Tillie Arell Height Cone. 

(ml rll (p V-sec) (p V) (" 

1 8,73 818.41 7395 56.50 

2 	 15.25 63010 5063 43.50 

Tota I '44951 100.00 

30.0 Operator: Hiroyukl Togawa 

Fig 

Date Mar. 4. 2009 Name :H\Yop,l~; TOdAWo. 

/g..~ 

~-1,) ...... !.\,..' 

Appendix figure 3-3-3 HPLC chromatogram before renewal at 24 hours 

534 -7 Appendix]­
94746 


Additional data 1 


Results of preliminary studies 

535 

94746 


1. Solubility of test item in dilution water 

It was expected that the solubility of the test item in dilution water was below 100 

mg/L, therefore, the preliminary study for measurement of the solubility of the test item in 

dilution water was conducted. 

1.1 Measurement of water solubility of test item 

(1) Method 

The test sample and dilution water were mixed to prepare about 100 mgIL and 

they were stirred for 24 and 48 hours under the test condition (24±1 QC). After that 

the mixture was left at rest for about one hour, the middle layer was collected and 

filtrated" with a glass fiber filter (ADV ANTEC, GB-140, 0.4 11m filter unit). The 

filtrate was analyzed with high-performance liquid chromatography (HPLC) as 

described in Appendix 2. 

* 	 Test solution was filtered after the filter was washed with about 100 mL of the test 

solution. 

(2) Result 

Sample name 

Measured concentration (mglL) Arithmetic 

mean (mglL) Stirring for 24 hours Stirring for 48 hours 

Sample-l 10 -----­

10 

Sample-2 10 

Sample-3 10 

-------­

Sample-4 

-------­

----­ 10 

Sample-5 

-------­

11 

Sample-6 -----­ 9.2 

1.2 Summary 

It was decided that the solubility of the test item in dilution water was 10 mg/L, 

which was the average of measured concentration stirred for 24 hours and 48 hours. 

536 -1 Additional data 1­
94746 


2. Effect on test organism 

2.1 Preliminary study 

(1) Method 

The test sample and dilution water were mixed to produce each nominal loading 

rate and it was stirred by magnetic stirrer for about 48 hours. After the filtration, the 

filtrate was aerated for about one hour under about 242C in order to recover decreased 

dissol ved oxygen concentration by suction filtration, and the filtrate was used for the 

test solution. The test organisms were exposed to the test solution in order to 

confirm effect of test item. The concentration of test item in the test solutions were 

measured at the same time. 

(2) Result 

Nominal 

concentration 

(mglL) 

Left column: Cumulative mortality (%) 

Right column: Existence of abnormal response 

abnormalities: * no abnormalities: N 

3 hours 24 hours 48 hours 72 hours 96 hours 

1.2 0 , N ·· 

0 · · N

· 0 ·· , N

· 

0 · , ·• N 0 · · N

3.6 0 , 

· N · · 

25 · N ··

· 

50 : 

· 

* 100 ! - 100 i . 

7.3 0 · N 25 · · N · · 

100 · - ·· 

100 1 

, - 100 : - · 12 0 ·

· N

· · 1001 - 100 ·: - · 

100 : - · 100 , : - Exposure system: semi-static (renewal at 48 hours after) 


Number of organisms/volume of test solution: 4 fish/About 2.5 L 


Aeration: conducted 


- All test organisms died. 


Nominal 

concentration 

Measured concentration 

(m ;/L) 

(mglL) At the start After 48 hours 

1.2 0.30 n.d. 

12 1.7 1.0 

(60) 

n.d.: <0.11 mgIL 

2.2 Summary 

It was confirmed that the measured concentration of test item decreased at 48 

hours after. Therefore, the definitive study would be conducted as semi-static regime 

(renewal at every 24 hours) in consideration of maintenance of the concentration. 

537 -2 Additional data 1­
94746 


3. Operation of the definitive study 

Based on the results of the preliminary study, the definitive study was conducted 

with five concentrations of the stock solution content 5.0, 3.1, 2.0, 1.2 and 0.8% (a 

geometric series with a factor of 1.6) and the control by semi-static regime (renewal at 

every 24 hours). Aeration was not conducted during the exposure. The preparation 

method of the test solution was the same as the preHminary study. The concentrations of 

test item in the test solutions were measured at the start of the exposure, before and after 

the renewal and the end of the exposure. 

538 -3 Additional data 1­
94746 


Additional data 2 


Calculation results of concentrations of each component 

539 

94746 

1. Calculation results of concentrations of each component 

The test item was detected as two peaks in chromatogram. Calling Peak 1 and 2 

in order of elution, each peak corresponded to PE3MB and PEMB (Information supplied 

by the sponsor). Based on the resu1ts of HPLC analysis in definitive study in each 

exposure level, the concentrations of each component were calculated. Results of 

calculation are shown below. The concentrations of each component in each sample 

for HPLC analysis were determined on the basis of a comparison of the each peak area 

on the chromatogram of the sample solution with that of a standard solution. In 

addition. concentrations of each component in standard solution was calculated based on 

content percentage (PE3MB: 14.l%, PEMB: 81.7%). 

Additional figure 2-1 Concentration of PE3MB (Peak 1) in test solutions 

Nominal 

concentration 

(mgIL) 

Measured concentration (mglL) 

(Percentage of measured concentration versus that at each preparation %) 

At the 

start 

24 hours 48 hours 72 hours At the 

end 

Geometric 

mean old new old new old new 

Control n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. V 

0.8 0.023 n.d. '3 0.026 n.d:3 0.044 0.029 

(65) 0.031 0.025 

(81) 0.022 

1.2 n.d:3 n.d.'3 0.059 0.057 

(96) 0.084 0.085 

(101) 0.058 0.068 (118) 0.041 

2.0 0.022 n.d. "3 0.16 0.099 

(63) 0.15 0.14 (92) 0.15 

0.14*2 

(89) 0.078 

3.1 0.27 0.25 

(93) 0.31 0.25 (80) 0.23 

0.24*2 

(109) - - 0.26 

5.0 0.30 0.23 

(78) 0.44 0040 

(91) 0.15 0.095 

(65) 0.50 

0047*2 

(94) 0.28 

old: before the renewal 

new : after the renewal 

n.d. : < 0.018 mglL 


- : It shows no measurement because all test organisms died. 


*2 It indicates the measured value at the time that confirmed all test organisms dead. 


*3 In accordance with OECD Guidance Document No.23, half the determination limit of the 


test item (0.0090 mgIL = 0.018 mglU2) was adopted for the calculation of geometric 

mean, because the test item was not detected and the detection Hmit (0.022 mgIL) was 

higher than half the determination limit of the test item. 

540 -1 Additional data 2­
94746 


Additional figure 2-2 Concentration of PEMB (Peak 2) in test solutions 

Measured concentration (mglL) 

Nominal (Percentage of measured concentration versus that at each preparation %) 

concentration 

(mg/L) At the 24 hours 

start old 

Control n.d. n.d. 

0.8 0.13 0.043 

(33) 

1.2 n.d.'3 n.d.'3 

2.0 n.d. '4 n.d. '3 

3.1 0.41 0.34 

(82) 

5.0 0.22 0.16 

(73) 

new 

n.d. 

0.14 

0.19 

0.44 

0.42 

0.47 

48 hours 

old new 

n.d. n.d. 

0.071 0.16 (52) 

0.15 0.31 (81) 

0.28 0.47 (64) 

0.28 0.46 (67) 

0.47 0.099 (101) 

72 hours At the 

old new end 

n.d. n.d. n.d. 

0.082 0.15 0.094 

(51) (62) 

0.16 0.28 0.14 

(52) (51) 

0.22 0.45 

0.31'2 

(47) (69) 

0.40'2 - . (86) 

0.037 0.48 

0.45"2 

(38) (94) 

Geometric 

mean 

/ 

0.10 

0.083 

0.13 

0.38 

0.22 

old: before the renewal 

new: after the renewal 

n.d. : < 0.013 mg/L 


- : It shows no measurement because all test organisms died. 


*2 It indicates the measured value at the time that confinned all test organisms dead. 


*3 In accordance with OEeD Guidance Document No.23, half the determination limit of the 


test item (0.0065 mglL = 0.013 mg/U2) was adopted for the calculation of geometric 

mean, because the test item was not detected and the detection limit (0.022 mglL) was 

higher than half the determination limit of the test item. 

*4 	 In accordance with OECD Guidance Document No.23, half the detennination limit of the 

test item (0.0065 mgIL = 0.013 mg/U2) was adopted for the calculation of geometric 

mean, because the test item was detected but not quantified. 

541 -2 Additional data 2­
94746 


Additional data 3 


Qualitative analysis of converted products 

542 

94746 

1. Qualitative analysis of converted products 

The peaks which were likely converted pr09ucts except the test item were 

detected on the HPLC chromatogram of test solution in the definitive study. 

Therefore, the sample was qualitatively analyzed by liquid chJomatograph~mass 

spectrometry (LC~MS) and estimated structures. 

1.1 Method of analysis 

Standard solution and the pretreated sample of 5.0 mgIL at the time that all test 

organisms died for HPLC analysis described in appendix 2 were qualitatively analyzed 

by LC~MS under the following conditions. 

Analytical conditions 

Instrument Liquid chromatograph ~ mass spectrometer 

HPLC system SIDMADZU LC~20A Series 

Mass spectrometer Thermo Electron TSQ Quantum Discovery 

Pump (Post~column) HITACHI ~62oo 

Conditions of liquid chromatograph 

Column L-column ODS 

(15 cm x 2.1 mm I.D., Chemicals Evaluation and 

Research Institute) 

Column temp. 40llC 

Eluent A: Acetonitrile I formic acid (1000/1 v/v) 

B: Water'! I fonnic acid (1000/1 v/v) 

Gradient condition 

Time (min) A(%) B(%) 

0.00 50 50 

20.00 100 o 

40.00 100 o 

Flow rate 0.2mllmin 

Injection volume 5pL 

Post-column 

Solution Acetonitrile I water'} (1/1 v/v) "2 

Flow ratc 0.1 mUmin 

*1 Tap water was treated with ultrapure water system. 

*2 Containing 25 mmol/L ammonium acetate. 

543 ·1 Additional dala 3· 
94746 


Conditions of mass spectrometer 


Ionization mode Electrospray ionization (ESI) 


Detection ion Positive 


Measure mode Scan 


Scan range (mlz) 150-600 


Capillary temp. 3()()2C 


1.2 Results 

Converted products, Peak A, B, C, D, E and F detected on total ion current (TIC) 

chromatogram (Additional figure 3-4) were qualitatively analyzed by LC-MS and 

estimated as foUowing structures. Also, it was recognized that Peak 1 and 2 were test 

item. 

Peak 

No. 

Retention time 

on TIC 

chromatogram 

(min) 

Estimated structure 

and molecular formula 

Estimated 

molecular 

weight 

Additional 

figure No. 

A 7.4 

7.6 

· · · · · · · ·

· · · · · 

· · 

· · · · · ·· · · · 

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Figure 3-5 

Figure 3-13 

Figure 3-6 

Figure 3-14 B 

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1 8.1 Figure 3-7 

Figure 3-15 

544 -2 Additional data :;­