unpublished subchronic toxicity study: …6394 g-000-307 .i6 unpublished subchronic toxicity study:...
TRANSCRIPT
6394 G-000-307 . I 6
UNPUBLISHED SUBCHRONIC TOXICITY STUDY: NAPHTHALENE
O U I AND OU5 RI REPORTS) (C52904) FISCHER - 344 RATS - (USED AS A REFERENCE IN
03/04/80
C 5 2 9 0 4 NTB 75 REPORT
I
Bat te l le ' s Columbus L a b o r a t o r i e s Subcont rac t No. 76-34-106002
March 4 , 1980
Subchronic T o x i c i t y Study: f-Naphthalene3fC52904), It"r-=-=---- - F i s c h e r 344 R a t s _. - _ \ __,
I n t r o d u c t i o n
RECEIVE'
-
T h i s subchronic gavage t o x i c i t y s t u d y was conducted t o
de te rmine Maximum T o l e r a t e d Doses f o r t h e 104-week chronic t o x i c i t y
s tudy. Dose s e l e c t i o n s f o r t h i s subchronic s t u d y were based on c l i n i c a l
and p a t h o l o g i c a l r e s u l t s from a two-week r e p e a t e d dose s t u d y ,
confirmed by C h a r l e s Rose, Tracor J i t c o , I n c o r p o r a t e d , i n a l e t t e r d a t e d 1) . and were i
I
I A p r i l 20, 1979. I n t h i s r e p e a t e d dose s t u d y , t h e r e w a s no m o r t a l i t y a t
- 400 mg/kg, b u t c l i n i c a l s i g n s of l e t h a r g y and roughened h a i r c o a t s , and
weight g a i n d e p r e s s i o n s of 30-50 p e r c e n t were recorded. There was
marked l e t h a l i t y a t 800 mg/kg. The d o s e s e l e c t i o n s were 400, 200, 100,
50, and 25 mg/kg f o r b o t h sexes and were t o b e adminis te red a t t h e ra te
of 5 ml/kg of an imal body weight .
Materials and Methods
. Naphthalene, manufactured by F i s h e r S c i e n t i f i c Company, w a s
rece ived from Midwest Research I n s t i t u t e (MRI) on October 10, 1978. The
MRI A n a l y t i c a l Report i n d i c a t e d two i m p u r i t i e s (0.13 and 0.02 p e r c e n t o f
t h e major peak a r e a ) and a water c o n t e n t of 0 .23 - 0.04 p e r c e n t .
t h a l e n e i s a white c r y s t a l l i n e s o l i d which i s r e l a t i v e l y stable when s t x e d
a t room tempera ture (m.p. 80-81 C). A Copy of t h e Batch Chemical Data
Record i s a t t a c h e d as APpendix A.
+ Naph-
__ -
The s e l e c t e d d o s e s of 400, 200, 1'00, 50, and 25 mg/kg were
prepared by mixing a s t o c k s o l u t i o n of t h e h i g h e s t dose l eve l and sequen-
t i a l l y d i l u t i n g from t h i s s t o c k t o supply t h e remaining d o s e l e v e l s . The
*.
.. ! 639 4
2
d i l u e n t or v e h i c l e w a s co rn o i l , Mazola brand. The s t o c k s o l u t i o n w a s
p repared by p l ac ing a weighed p o r t i o n of naphtha lene i n t o a mixing
graduated c y l i n d e r and adding corn o i l t o make up t h e proper volume.
The mix tu re w a s mechanica l ly s t i r r e d f o r a p e r i o d of f i v e minu tes .
D i l u t i o n s f o r r a t s were prepared so t h a t t h e chemical would be d e l i v e r e d
t o t h e animal by gavage on t h e b a s i s of one m i l l i l i t e r of t h e m i x t u r e
p e r 200 grams body weight . Mixture q u a n t i t i e s were prepared weekly.
The a c t u a l q u a n t i t i e s mixed were changed once (doubled) d u r i n g t h e s t u d y
t o accommodate weight i n c r e a s e s i n t h e ra t s . The f i n a l m i x t u r e s were
composed of t h e fo l lowing :
Dose Level, Naphthalene, S e r i a l Corn O i l , T o t a l Volume,
mg/kg A S o l u t i o n , m l m l m l
400 21.6 -- Q.S. 270
200 135 135 270
100 135 135 270
50 135 13 5 270
25 135 135 27 0
For each dose l e v e l , t h e f i n a l m i x t u r e s were s t o r e d i n g l a s s
v i a l s a t room tempera ture .
days p r i o r t o use ; gavage t rea tment w a s f i v e consecu t ive dose days p e r
week. S t a i n l e s s steel gavage needles ’ and t u b e r c u l i n s y r i n g e s g radua ted
i n 0 .01 m l u n i t s were used t o a d m i n i s t e r t h e gavage doses . One a n a l y s i s
f o r dose c o n c e n t r a t i o n accuracy w a s performed; r e s u l t s were w i t h i n - 10
pe rcen t of t h e c o n c e n t r a t i o n s for a l l dose l e v e l s except t h e 200 mg/kg
which was 1 .76 p e r c e n t - o u t s i d e t h i s range.
Mixtures were prepared no more t h a n seven
+
Animals
The s tudy w a s performed wi th F i s c h e r 344 rats rece ived from
Char les R ive r , Po r t age , Michigan, on A p r i l 27, 1 9 7 9 , and he ld i n
q u a r a n t i n e 1 5 days. Dose a d m i n i s t r a t i o n began on May 1 4 , 1979, and
0800QZ
i
6394 - 3
cont inued f i v e days a week f o r 1 3 weeks. The s tudy concluded August 1 0 ,
1979. Copies of t h e Weekly Animal Report and BCL Misce l laneous Data
Sheet a re a t t a c h e d as Appendix B. Animals were examined d a i l y d u r i n g
q u a r a n t i n e ; no obse rvab le d i s e a s e w a s d e t e c t e d p r i o r t o randomiza t ion .
F ive males and f i v e females were t aken a t t h e t ime of randomiza t ion and
necrops ied ; no i n t e r n a l l e s i o n s o r d i s e a s e w e r e ev ident from t h i s g r o s s
examination. The rats were randomized i n t o test groups from t h e quar-
a n t i n e pool by t a b l e s of random numbers provided by D r . James J o i n e r ,
Tracor J i t c o , Inco rpora t ed . S i x t y rats of each sex were d i s t r i b u t e d
f i v e p e r cage i n t o 1 2 cages. The 11 cages w e r e t hen d i s t r i b u t e d from a
s e p a r a t e random numbers t a b l e t o f i v e dose l e v e l s and one c o n t r o l group
pe r sex.
r a t s ranged i n s t a r t i n g body weights from 170 t o 215 grams, while females
ranged from 135 t o 155 grams.
The an imals were i n d i v i d u a l l y marked by t o e c l i p p i n g . Male
Animal Maintenance
R a t s were caged i n groups of f i v e an imals i n po lyca rbona te cages
suspended from s t a i n l e s s s t e e l r a c k s , bo th manufactured by Lab P roduc t s ,
Inco rpora t ed . Automatic wa te r ing dev ices manufactured by Edstrum Indus-
t r ies provided water ad l i b . Water w a s u n t r e a t e d a t t h e l a b o r a t o r y and
w a s supp l i ed by t h e C i t y of Columbus. Rats were housed i n Room 7C-207
c o n c u r r e n t l y w i t h mice a l s o on subchronic Naphthalene s tudy . Cages
bedded w i t h Absorb-Dri (Absorb-Dri, Inco rpora t ed ) were changed t w i c e
weekly; r a c k s h e l v e s were covered w i t h a spun-bonded p o l y e s t e r f i l t e r
(DuPont 2 0 2 4 ) . Racks were changed once every two weeks. Animal f e e d
w a s checked d a i l y i n cages and added as needed. Pur ina Lab Chow w a s
used; a Teed t a g i s appended t o t h i s r e p o r t . Fresh f eed and f e e d e r s
were provided weekly. Animal rooms were main ta ined a t 21-23 C and
40-60 p e r c e n t r e l a t i v e humidi ty , 1 2 hours of f l u o r e s c e n t l i g h t , and 15
a i r changes p e r hour. There were 1 2 r a t cages per 100 s q u a r e f e e t of
f l o o r space ; n e i t h e r cage p o s i t i o n s w i t h i n t h e r ack nor r a c k p o s i t i o n s
were r o t a t e d du r ing t h e s tudy .
000003
6394 4
Cages and f e e d e r s w e r e c leaned i n a cont inuous conveyor
washer manufactured by B lakes l ee , Chicago, I l l i n o i s . Cages r e c e i v e d a
one-wash and two-r inse sequence.
by Economics Labs, S t . P a u l , Minnesota, w a s used i n t h e wash sequence.
Temperature of t h e r i n s e water w a s 180 F or g r e a t e r .
i n a washer manufactured by Better B u i l t , Saddlebrook, New J e r s e y . The
r ack washer had a s i n g l e wash, double r i n s e sequence. The d e t e r g e n t and
r i n s e tempera ture were t h e same as t h a t used i n t h e t u n n e l washer. The
f l o o r i n Room 7C-207 w a s washed down d a i l y w i t h water and squeegeed d ry .
"Exceed" brand d e t e r g e n t , manufactured
Racks w e r e c l eaned
-
Design of t h e Study
F i v e dose l e v e l s of 400, 200, 100, 50 and 25 mg/kg w e r e admin-
i s t e r e d t o male and female rats by gavage a t a ra te of 0.5 m i l l i l i t e r s
p e r 100 grams of body weight .
Animals were gavaged i n a hood f i v e days a week f o r 13 weeks. Each
dose level and c o n t r o l group conta ined t e n r a t s of each sex .
Vehic l e c o n t r o l s r ece ived c o r n o i l . *
The subchronic s tudy began May 1 2 , 1979; an imals w e r e observed
twice each day f o r c l i n i c a l s i g n s of t o x i c i t y w i t h a t least s ix hour s
between o b s e r v a t i o n s . A l l c l i n i c a l s i g n s o r n e g a t i v e o b s e r v a t i o n s were
recorded d a i l y i n a bound o b s e r v a t i o n book. Weekly i n d i v i d u a l an ima l
weights and cage group food consumption were recorded . Day 9 1 o f t h e
2 s tudy w a s August 10, 1979; su rv iv ing an imals were te rmina ted w i t h CO
on August 1 4 and 15 w i t h f u l l necropsy and t i s s u e c o l l e c t i o n .
Resu 1 t s -
S u r v i v a l
During t h e 13 weeks of t h e subchronic s tudy a l l r a t s su rv ived
t o scheduled t e r m i n a t i o n except two 400 mg/kg males ( i n d i v i d u a l animal
numbers 4 and 9 ) .
s tudy . A s u r v i v a l t a b l e i s a t t a c h e d a s Appendix C .
These male r a t s d i ed du r ing t h e l a s t week of t h e
6394 5
Dai lv Observa t ions
Observa t ions were completed t w i c e d a i l y on a l l s t u d y an ima l s ,
and c l i n i c a l s i g n s o r n e g a t i v e o b s e r v a t i o n s were recorded d a i l y .
t h e c o u r s e of t h e 91 day s t u d y , c l i n i c a l s i g n s inc luded d i a r r h e a ,
l e t h a r g y , hunched p o s t u r e , and rough c o a t s . The h i g h e s t dosed r a t s
(400 mg/kg) had i n t e r m i t t e n t d i a r r h e a , males showing t h i s s i g n d u r i n g
weeks 3 , 4 , 10, and 13, and females d u r i n g weeks 4, 7 , 10, and 13.
During t h e f i n a l week of t h e s tudy t h e 400 mg/kg dosed an ima l s were
rough-coated; l e t h a r g i c , and females had a hunched p o s t u r e . Male ra t
number 9 showed a l l t h e s e c l i n i c a l s i g n s as w e l l as becoming p rone the
day p r i o r t o dea th . During week 1 0 a l l ra ts among t h e t h r e e h i g h e s t
dose groups showed rough h a i r c o a t s .
c l i n i c a l s i g n s were no ted d u r i n g t h e 13 weeks of t h i s s u b c h r o n i c s t u d y .
During
--
No o t h e r s i g n s of t o x i c i t y o r
D i e t Consumption and Body Weights
Cage group d i e t consumption w a s measured and r eco rded weekly.
T o t a l weekly group averages as w e l l as i n d i v i d u a l d a i l y consumption per
group i s inc luded i n t h e d i e t consumption t a b l e i n Appendix D. No d o s e
r e l a t e d t r e n d s are e v i d e n t among any of t h e dosed groups o r c o n t r o l s i n
e i t h e r male o r female rats.
Tables of body weight d a t a are inc luded i n Appendix E. Ind iv -
i d u a l animal weights w e r e t a k e n weekly d u r i n g t h e subchron ic s tudy . An
i n d i v i d u a l body weight summary t a b l e as w e l l as an a v e r a g e weekly i n d i v -
i d u a l body weight t a b l e w i t h pe rcen t d i f f e r e n t i a l weight changes compared
t o c o n t r o l s c a l c u l a t e d a re included;--Graphs are a l s o i n c l u d e d , p l o t t i n g
average .group body weight a g a i n s t t i m e . The p e r c e n t d i f f e r e n t i a l weight
changes show a d e f i n i t e dose r e l a t e d t r e n d among both male and f e n a l e
r a t s . The two h i g h e s t dose groups, 400 and 200 mg/kg, of male rats
have a g r e a t e r t h a n -10 p e r c e n t d i f f e r e n t i a l weight ; w h i l e t h e t h r e e
h i g h e s t d o s e groups ( i n c l u d i n g 100 mg/kg) of female rats have a g r e a t e r
than -10 pe rcen t d i f f e r e n t i a l weight change. The most marked d i f f e r e n c e s
6394 6
i n weight changes a r e i n t h e h i g h e s t male and female (4OOmg/kg) dosed
groups. Weight changes were watched c l o s e l y throughout t h i s s t u d y , and,
i n o r d e r t o gua ran tee a g a i n s t any dehydra t ion e f f e c t s , supplementa l water
d i s h e s were provided d a i l y t o t h o s e ra t groups ga in ing weight iess
r a p i d l y than o t h e r groups.
S p e c i a l Study - Hematology
The fo l lowing ana lyses were performed on venous blood c o l l e c t e d
r e t r o o r b i t a l l y t h e day of scheduled t e r m i n a t i o n ; Hemoglobin (HgB), hema-
t o c r i t (HCT) , t o t a l and d i f f e r e n t i a l w h i t e blood c e l l count (WBC), r e d
blood c e l l count (RBC), and mean c o r p u s c u l a r volume (MCV).- The r e s u l t s
a r e summarized i n Appendix F.
(SD) and sample s i z e ( N ) are p resen ted f o r each parameter and each
dosage group.
The mean v a l u e (f?), s t anda rd d e v i a t i o n
Among 400 mg/kg dosed male rats, a marg ina l d e c r e a s e i n the
hemoglobin and hematoc r i t i s appa ren t .
males are a moderate i n c r e a s e i n a b s o l u t e numbers of mature n e u t r o p h i l s
( s egs ) and a b s o l u t e dec rease i n lymphocytes w i t h a r e v e r s e i n t h e
neut rophi l / lymphocyte r a t i o , which r e s u l t s i n t h e t o t a l WBC count be ing
normal. Among female rats i n t h i s dose group, t h e r e i s a l s o a s l i g h t
i n c r e a s e i n t h e segmented n e u t r o p h i l s b u t t h e lymphocyte v a l u e a p p e a r s
normal. Females a l s o have a margina l dec rease i n t h e hemoglobin and
hematocr i t va lues . Although s t a t i s t i c a l a n a l y s e s were n o t performed
on t h i s d a t a , t h e most pronounced e f f e c t appea r s t o be i n t h e 400 mg/kg
male r a t s lymphocyte and n e u t r o p h i l va lues .
Also appa ren t i n t h i s group of
7
PATHOLOGY
Rats
6394
Methods :
D e t a i l e d necropsy examinat ions were performed on a l l ra ts i n
t h i s s t u d y . The fo l lowing t i s s u e s from a l l animals were p l a c e d i n
10 p e r c e n t n e u t r a l b u f f e r e d formal in f o r f i x a t i o n :
Mandibular lymph node Heart
S a l i v a r y g land Esophagus
Femur Stomach
Thyroid Uterus
._
P a r a t h y r o i d
Small i n t e s t i n e
Colon
Liver
Bra in (3 sections)
T.hymu S
Trachea
Pancreas
P r o s t a t e Spleen
T e s t e s Kidneys
Ovaries Adrenals
Lungs and mainstem bronchi Urinary b l a d d e r
Mammary gland
Eyes
P i t u i t a r y
T i s s u e s l i s t e d above from animals i n t h e c o n t r o l and h i g h e s t
dosage groups were processed i n t h e r o u t i n e manner, s e c t i o n e d a t 5 microns ,
s t a i n e d w i t h hematoxylin and e o s i n , and examined m i c r o s c o p i c a l l y . I n
a d d i t i o n , kidneys from males and thymus g l a n d s from females i n dosage
group D (100 mg/kg) were examined m i c r o s c o p i c a l l y s i n c e l e s i o n s which may
have been compound induced were observed i n t h e s e organs i n animals of
t h e r e s p e c t i v e s e x e s a t t h e h i g h e s t dosage group (400 mg/kg).
-
R e s u l t s :
6 3 9 4
8
Da'ryl Thake, D . V . M .
Necropsy o b s e r v a t i o n s f o r i n d i v i d u a l an imals are shown i n
Tables 1 2 through 1 7 w h i l e a summary of necropsy o b s e r v a t i o n s . i s shown
i n Table 18 . Les ions observed m i c r o s c o p i c a l l y a r e shown i n T a b l e s 1 9
through 2 1 . A summary of microscopic l e s i o n s by. s e x and 'dosage group i s
p resen ted i n Table 22 .
Necros is of r e n a l t u b u l a r e p i t h e l i u m was observed i n one male
r a t from t h e h i g h e s t dosage group (400 mg/kg) which d i e d spon taneous ly
l a t e i n t h e s tudy . There were no i n d i c a t i o n s of d e g e n e r a t i v e changes i n
kidneys from o t h e r an ima l s ; however, t h e p o s s i b i l i t y t h a t t h i s l e s i o n may
have been induced by t h e test compound cannot be r u l e d o u t . -
Deple t ion of thymic lymphocytes w a s observed i n f ema les from
t h e h i g h e s t dosage group (400 mg/kg). Th i s may have been due t o adminis-
t r a t i o n of t h e t es t compound s i n c e such changes are n o t common i n c o n t r o l
rats a t t h i s age . N e i t h e r of t h e changes d e s c r i b e d above occur red i n 1 - _.
xes a t t h e nex t lower dosage level (200 mg/kg).
anges were cons idered t o have been of spontaneous
o r i g i n w i t h no ev idence t h a t any r e s u l t e d from compound a d m i n i s t r a t i o n .
Necropsy o b s e r v a t i o n s g e n e r a l l y c o r r e l a t e d w e l l w i t h mic roscop ic changes
i n animals which were examined m i c r o s c o p i c a l l y .
Conclusions:
The on ly l e s i o n s which may have been induced by t h e t e s t compound
were r e n a l t u b u l a r n e c r o s i s i n one>e r a t a t t h e h i g h e s t dosage level
(400 mg/kg)-and d e p l e t i o n of thymic lymphocytes i n two f ema les from t h e
h i g h e s t dosage group (400 mg/kg).
a t t h e nex t lower dosage group (200 mg/kg).
- - .
These l e s i o n s were n o t p r e s e n t i n rats
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- .I+ Lulu l a , v) ar
C 0 L)
v)
a C (0 d cl
21
639 4
n
N cu
8
APPENDIX A
BATCH C H M I C A L DATA RECORD MRI ANALYTICAL REPORT
I
I
6394
..
6394
10. How was the physical propcrty obtained?
-
L'olumns *
cech core.. A t h i s page. hrough 15 &pply I O
DATARECORD
1 - Observed
2 - Literature 3 - Manufacturer's
Specifications
~~
ITEM C A R 0 C O L .
SECTION I
. . . . . :::<:. ,. ': ..fl :: ...__ . .,,_,. ::-.,.:,:;:.: .. . ,x::.: __._. ..,. :. .::::.
. . . . . . . ...... 1. ~ r m i c a l (hnmnn N J m r
. . : .... .: , . .. : .. . . . . .. ... . . . . . . . . . .. , , . Naphthalene
13. Methods of Prepatation or Synthesis* .
I
2. N a m e o f LlJnufaciurrr ' FOR OFFICE USE Fisher Scientific Company I fDo Nor C o d e )
43
Naphthalene Scintanalyzed
5. Is batch . l a b e l e d ? (C.8. isotope) I 2 - N o
1 - Yes'
6. I s there a 1 - Y e s reserve supply of batch? 1 2 - N o
.I 'm l I
I 7. I s b a t c h 1 - Y e s (Results o/ analysis)* I
<. . I ' 1 2 - N o purity t e s t e d ?
8. mysical State of Chemical Batch
I - Solid (Indicate melting range in I t o n 9) 2 - Liquid (lndicaie boiling range in It - 9 ) 3 - Gel (Indicde liqui/icaiia range in Item 9) 4 - Gas (Skip I t n No. 9)
COMMENTS: Refer t o Item No. when making comments.
11. 3/28/78
9. Ctitical Trmprraturc Ronpe (Dcgrrcs Crnti~udel
I 3 2 - 2
7 m l 4 0
I ... SECTION II
. :.
The following information should be provided only if available. Enter "1" in appropriate boxes and report da ta in Comment area.
. .
11. Date Received from Supplier*
12. Date of Manufacture and Original Storage by Manufacturer*
14. Special Treatment Applied After Receipt ( c g . pwi/icmion methods)*
1
CARDNUMBER
Bioassayer: Battelle Research Institute
I. BULK CHEMICAL STUDIES
was slightly high. major component. tive area of 0.10% of the major peak area. cated two impurities (0.13 and 0.02% of the major peak area). 0.23 2 0 .04(6 )% water by titration with Karl Fischer reagent. nuclear magnetic resonance spectra were consistent with literature spectra.
of 2 weeks at temperatures of up to 60°C.
B. ANALYSIS: The elemental analysis for carbon was slightly low while that f o r hydrogen Thin layer chromatography using two developing systems indicated only the
Vapor phase chromatography by one system indicated one impurity with a rela- A second vapor phase chromatographic system indi--
Naphthalene was found to contai: The ultraviolet, infrared and
C. HEAT STABILITY: Naphthalene is stable when stored as the bulk chemical for a period
11. CHEElICAL/VEHICLE STUDIES A. ACUTE TOXICITY (LD50) DOSAGE MXXING PRQTOCOL: Naphthalene can be suspended in 0.5%
aqueous methyl cellulose, and the suspension remains visually homogeneous for a maximum of 1 min, if the dry chemical is first ground in a mortar and the naphthalene/methyl cellulose mixture is sonicated f o r 30 min. treatment with the vortex mixer for 1 mln.
When the mixture separates, it cap be rehomogenized by
' COS! M EN T H EQU I H ED
6394 , . ...
11.
corn
111.
CHEMICAL/VEHICLE STUDIES (Concluded) B. CHRONIC LEVEL SThBILITY STUDY: Naphthalene is stable when dissolved in o i l at a dose level of 4% and stored at room temperature for 7 days.
ANALYSIS PROTOCOL FOR USE BY BIOASSAYERS A. BULK CHEMICAL
1. GENERAL PROTOCOL: The objective is to determine whether a given compound as received and stored is, and remains, identical to that received and analyzed by MRI.
stable standard to be used for comparison with the stored bulk chemical. There- fore, when you receive the bulk shipment, remove standard samples of 1 g for 60lids or 2 ml for liquids for each testing time.
closed and, if possible, sealed. Place samples in a freezer at -2OOC for stor- age prior to analysis.
t o analysis. Perform analyses in tandem so that t e s t sample results can be com- pared to the standard. reports. Slight differences may occur between the MRI report and your analyses. However, your standard and sample analyses should be identical to each other and identical to MRI data within expected variation limits.
2. PURITY: Vapor-phase chromatography (this report, Section I-B-l-d- System 2; 10% Carbowax 20 MTPA).
3. IDENTITY: Infrared spectroscopy (this report, Section I-B-2-b-(1)).
The most efficient method for determining stability is to maintain a
Place each sample in a glass vial with a Teflon-lined top tightly
Remove a single standard vial from the freezer approximately 4 hr prior
Also compare the results obtained in the MRI analytical
*
B. CHEMICAL/VEHICLE: Follow the analysis and extraction procedures described above in Sections 11-B-2 and 11-B-3 for the chronic level 7-day stability study.
IV. STORAGE CONDITIONS
Naphthalene should be stored in sealed containers at room temperature away from oxidizing materials and sources of ignition. stable for 2 weeks at temperatures of up t o 6 O o C , indicating a stability of at least 24 weeks at room temperature (25OC). stable for 7 days at 25OC when dissolved in corn oil at a dose level of 4%. Naphthalene dust forms an explosive mixture in air at high concentrations; therefore, care should be exercised when preparing to open storage vessels containing naphthalene.
The bulk material is
Naphthalene was also found to be '
V. HANDLING PROTOCOL
Naphthalene is irritating to the eyes and skin, and is toxic if in- gested. Acute overexposure may cause dermatitis, headache, and nausea. Chronic overexposure causes renal shutdown and eye cataracts. This material is also sus- pected of causing neoplasms in rats. tected when working with naphthalene. Coveralls, gloves, eye and foot protection are necessary. Respiratory protection should be provided in areas where naphtha- lene dust may form. Dust type cannister or air-supplied masks are suitable. AN work should be conducted in well-ventilated areas. Although we did not encounter this problem, it is very possible to create a dust when handling solid naphthalene. Care should be taken to prevent this since high concentrations of naphthalene dust are explosive in air.
Thus, all personnel should be fully pro-
noon22
.MIDWEST RESEARCH INSTITUTE TRACOR J I T C O , I N C . SUBCONTRACT NO. 74-24-106002
September 1 9 , 1978
COMPOUND: Naphthalene
'10H8
SUBMITTED B Y : . Tracor J i t c o CBDS NO.: C52904 MRI REQUEST N O . : 335 PROJECTED USE: Bioassay s t u d i e s a t
TOXICITY :L/ orl-chd LDL, : 100 mg/kg orl-hum LDLo: 50 mg/kg or l - ra t LD50:1,780 mg/kg scu - ra t TDL, : 3,500 mg/kg/98DI
AQTX: Aquat ic T o x i c i t y Rat ing:
Bat te l le Research I n s t i t u t e
T€X : NE0
TLm96:lO-1 ppm
TRACOR J I T C O P.O. NO.: 3274 .
P.O. DATE: 3-17-78 RECEIPT DATE: 3-28-78
MANUFACTURER DATA COMPANY: F i s h e r S c i e n t i f i c
SIZE: 48 x 1 l b LOT NO.: 775379 BATCH NO.: 01' PURITY: S c i n t i l l a t i o n g rade ANALYTICAL DATA: None enc losed
Company -
I. BULK CHEMICAL STUDIES
A. HONOGEMIZATION
The c o n t e n t s of t h e 48 b o t t l e s were combined i n a f i b e r b o a r d drum and t h e drum shaken and r o l l e d f o r 5 min. Samples were t a k e n and t h e re- mainder of t h e naph tha lene r e t u r n e d to t h e o r i g i n a l c o n t a i n e r s . .
- 1/ F a i r c h i l d , Edward J . , Ph.D. ( e d . ) , "Regis t ry o f Toxic E f f e c t s of Chemical Subs tances , " U.S. Depar tnent of H e a l t h , Education and Wel fa re , C i n c i n n a t i , Ohio, p . 566 (1977).
B. ANALYSIS
1. ' ASSAY
a. CHROMATOGRAPHY-TLC
Plates: Silica gel F-254, 0.25 nun layer, precoated
Amount spotted: 100 and 300 vg (10 and 30 u1 of i 10 p g / p 1 solution in acetone)
System 1: Hexanes (100%)
Rf: 0.62 Rst: 1.29
Ref. Standard: Pyrene (1 ~1 of a 10 P g l p 1 solution 'in acetone)
Visualization: UV (254 nm) and iodine vapor
System 2: Pentane:carbon tetrachloride (50:50) Rf: 0.75 RSt: 1.08
b. ELEMENTAL ANALYSIS
Element C H Theory 93.70 6.30 Determined 93.06 6.59
92.87 6.70 x D/T 99.22 105.48
c. WATER ANALYSIS (Karl Fischer)
0.23 - + 0.04(6)%
d. VAPOR PHASE CHROMATOGWIiY
Instrument: Varian 3740 Detector: Flame ionization Inlet temperature: 200°C Detector temperature: 25OOC Carrier gas: Nitrogen Carrier flow rate: 70 cc/min
(1) System 1
Column: 3% SP2100 on 100/120 Supelcoport, 1.8 m x
Oven temperature program: 5 min at 50°C, then
Sample injected:
4 mm ID; glass
50 to 25OoC at 1O0C/min 5 l.11 of a 10 mg/ml solution in
methylene chloride to quantitate impurities and 5 l.11 of a 5 mg/ml solution in methylene chloride to check for detector overloading
Results: Major peak and one impurity after the ma- jor peak which had an area of O . l X , relative to the area of the major peak.
Retention Time Retention Time Area 4, - Peak (mid (Relative to Naphthalene) (Relative to Naphthalene)
1 , 12.0 2 13.9
1.00 1.16
100 0.10
(2) System 2
Colunm: 1 O X Carbowax 20M-TPA on 80/100 Chromasorb
&en temperature program:
Sample injected:
W (AW); 1.8 m x 4 mm ID; glass 5 min at 5OoC, then 50 to 200OC at 10°C/min
5 l.11 of a 10 mg/ml solution in methylene chloride to quantitate impurities and 5 l.11 of a 5 mg/ml solution in methylene chloride to check for detector overloading.
Results: Major peak and two impurities, one before and one after the major peak. The impurities had a combined area of 0.15%, relative to the area of the major peak.
RetentiTn Time Retention Tine Area % Peak ' (Relative to Naphthalene) (Relative to Naphthalene)
1 12.3 2 * 12.8 3 14.2
0.96 1.00 1.10
0.02
0.13 100
- . - . - - . . . . - . - . . . .
I 394
2. IDENTITY (major component only unless (a) mixture specified or (b) major component not nominal ingredient)
a. PHYSICAL PROPERTIES
De t ernine d Literature Values
(1) Appearance: White crystalline solid
(2) m.p.: 80-81°C (visual, capil- 80.2"&/
b. SPECTRAL DATA
(1) Infrared
Instrument: Beckman IR-12 Consistent with lit- Cell: salt melt Results: See attached spec-
erature spectrum, 31
trum
(2) Ultraviolet/Visible
Instrument: Cary 118 - 41 No absorbance between 350 and
800 nm (visible region) at a concentration of 9.3 mg/ml
E x 10-2
312 1.779 5 0.018(6) 310 2.922 286 36.32 + 0.16(6) 285 42.20 275 54.51 0.21(6) 273 65.64 265 48.86 5 0.16(6) 265 57.83
-258 (shoulder) 34.02 + 0 . 1 2 ( 6 ) 257 38.55 221 1,022.63 6.82(6) 221 1,417 16 -
Solvent: Hexane Solvent: Cyclohexane
- 21
- 3 /
- 4/
Windholz, Martha, et al. , eds. , The Merck Index, Merck and Company, Inc. ,
"Sadtler Standard Spectra," Sadtler Research Laboratories, PhiladelFhia,
"Sadtler Standard. Spectra," Sadtler Res"earch Laboratories, Philadelphia,
Rahway, New Jersey, Ninth Edition, p. 828 (1976).
Pennsylvania, IR No. 865.
Pennsylvania, W No. 265.
I -
Instrument: Varian 3740 (autosampler) Detector: Flame ionization Inlet temperature: 2 0 0 ° C Detector temperature: 3 0 0 ° C Carrier gas: Nitrogen Carrier flow rate: 70 cc/min. Column: 3% SP2100 on 100/120 Supelcoport; 1.8 m x 4 nun ID; glass Oven temperature program: 8 O o C , isothermal Retention times: Naphthalene 6.3 min
Decane (internal standard) 9.7 min
3. RESULTS
Recovery ( X of -20°C Sample) Storage Temperature
- 2 0 ° C soc
2 S ° C 6 O o C
100.0 + 0.4(6) 100.0 0.4(6) 100.4 + 0.4(6) 100.2 - 0.4(6)
4. CONCLUSIONS a
Naphthalene is stable when stored as the bulk chemical for a period of 2 weeks at temperatures of up to 6 O O C .
11. CHPiICAL/VEHICLE STUDIES
A. ACUTE TOXICITY (LD50) DOSAGE NIXING PROTOCOL
1. TYPICAL DOSAGE LEVEL
The dosage concentration was calculated to be 240 mg/ml using the following data:
Weight of test animal: 100-g .LD50: 2,400 mg/kg , orl-rat/ Volume to be administered: 1 m l Solvent: 0.5% methyl cellulose in water
- 61 Safety and Toxicity Data for naphthalene prepared by Tracor Jitco, Inc., dated March 3, 1978.
2. MXING
Naphthalene would not dissolve or suspend in water or corn oil at the above concentration. methyl cellulose.
However, the compound would suspend in 0 . 5 % aqueous
Naphthalene was ground in a mortar before weighing 7 2 0 mg into a 5 ml vial. volume to 3 m l . The mixture was sonicated 15 min, placed on a vortex mixer for 2 min, and sonicated again for 15 min. pension which remained visually uniform f o r at least 1 min and could be drawn through an 18-gauge gavage needle.
Aqueous methyl cellulose (0.5%) was added to bring the total
This procedure produced a sus-
When the uniform mixture began to separate, it could be rehomogenized by treatment with the vortex mixer for 1 min.
3 . CONCLUSIONS
Naphthalene can be suspended in 0 . 5 % aqueous methyl cellulose, and the suspension remains visually homogeneous for a maximum of 1 min, if the dry chemical is first ground in a mortar and the naphthalene/methyl cel- lulose mixture is sonicated for 30 min. When the mixture separates, it can * be rehomogenized by treatment with the vortex mixer for 1 min.
B. CHRONIC LEVEL STUDIES - SFVEN-DAY, ROOM TEIQERATURE STABILITY IN CORN OIL AT A 4% DOSE LEVEL
1. SAMPLE PREPARATION AND STORAGE
Naphthalene ( 2 . 1 3 9 + 0.001 g) was placed in a 50-ml volumetric flask and diluted to the volume mark with corn oil (44 .357 - + 0.001 g). The chemical dissolved upon sonicating for 5 min to give a solution of 46 .00 - + 0 . 0 2 mg/g or 42 .78 - + 0 . 0 2 mg/d ( 4 . 2 9 % w/v or 4 . 6 0 % w/w).
As soon as the solution had been.prepared, eight accurately weighed 1 . 6 0 g aliquots were removed and sealed in separate 60-ml septum vials (Microsep F-138 gas chromatography septa with TeflonB film facing, from Canton Biomedical Products, Inc.; aluminum crimp seals from Wheaton Scientific Company, Inc.) . zero-time, samples and for storage at 1, 5 and 7 days, respectively.
Duplicate aliquots were used as initial, or
2 . SAMPLE EXTRACTION ANXI ANALYSIS
Extracting solvent containing an internal reference standard was prepared by weighing 4 . 5 7 1 - + 0.0001 g of 1,2-bis(2-methoxyetho?cy)ethane,
. ”. 63-9 4 transferring to a l-liter volumetric flask, and diluting to the mark with ab- solute methanol. Concentration of reference standard: 4.571 + 0.0002 mg/ml. -
To extract each sample aliquot, the septum vial was opened, 50 m l of the extracting solvent was added by volumetric pipette, and the vial im- mediately resealed. The corn oil/methanol-mixture was manually shaken for . 15 sec, sonicated 30 sec, and 10 m l of the resulting suspension was decanted into a 12-ml centrifuge tube and centrifuged for 5 min. A portion of the clear, methanolic supernatant solution ( 5 m l ) was then transferred to an 8 .5 ml septum vial for subsequent analysis by the gas chromatographic system outlined below:
Instrument: Bendix 2500 with Hewlett-Packard 3380A Automatic Integrator
Column: 2 mu ID, glass, silanized
Detection: Flame ionization Temperatures: Inlet, 18OOC
10% SP-2330 on 100/120 mesh Chromosorb W(AW), 1.8 m x
Oven, 125*C, isothermal Detector: 220°C
Carrier gas: Nitrogen *
Flow rate: 30 cc/min Volume of solution injected: 4 ~1 Retention times: Test chemical, 6.0 min
Reference standard, 8.3
3. QUALITY COhTROL PROTOCOLS
Analyses were performed in duplicate using 1,2-bis(2-methoxyethoxy)- ethane as an internal reference standard. Recovery studies were performed in duplicate at the same concentration level as the test samples. Gas chromato- graphic linearity was determined with standard solutions in methanol at 1.84, 1.38 and 0.72 mg/ml concentrations f o r the naphthalene and 5.06, 3.79, and 2 . 5 3 mg/ml for the internal reference. The least squares plot correlation coefficients were > 0.999 for both the test chemical and the internal refer- ence (effectively 1.0, linear). -
c 4. RESULTS
6394
Storage Time Average % (w/w) Chemical Found in (days) Chemical/Vehicle Mixturedl
1 5 7
4.57 + 0.072/ 4.53 5: 0.07 4.58 - 0.07
- a/ - b/
- c /
Corrected for zero-time recovery yield of 94.4 - + 0.8%. Theoretical concentration-of chemical in corn oil,
The error values in this table are average deviations . obtained in the analytical measurements of the test
4.601 - + 0.002% (w/w).
solutions.
5 . CONCLUSION
Naphthalene is stable when dissolved in corn oil at a dose level of 4% and stored at room temperature for 7 days.
111. ANALYSIS PROTOCOL FOR USE BY BIOASSAYERS
A. BULK CHEMICAL
1. GENERAL PROTOCOL
The objective is to determine whether a given compound as received and stored is, and remains, identical to that received a n d analyzed by MRI.
The most efficient method for determining stability is to maintain a stable standard to be used for comparison with the stored bulk chemical. Therefore, when you receive the bulk shipment, remove standard samples of 1 g for solids or 2 m l for liquids for each testing tine.
Place each sample in a glass vial with a Teflon-lined top tightly closed and, if possible, sealed. Place samples in a freezer at -2OOC for- storage prior to analysis.
Remove a single stand2rd vial from the freezer approximately 4 hr prior to analysis. Perform analyses in tandem so that test sample results can be compared to the standard. Also compare the results obtained i n the MRI analytical reports. and your analyses. However, your standard and sample analyses should be identical to each other and identical to IRI data within expected variation limits.
Slight differences may occur between the MRI report
689 4 2 . PURITY
Vapor-phase chromatography (this report, Section I-B-1-d-System 2 ; 10% Carbowax 20 MTPA).
3. IDENTITY
Infrared spectroscopy (this report, Section I-B-2-b-(1)).
B. CHEMICAL/VEHICLE
Follow the analysis and extraction procedures described above in Sections 11-B-2 and 11-B-3 for the chronic level 7-day stability study.
IV. STORAGE CONDITIONS
Naphthalene should be stored in sealed containers at room tem- perature away from oxidizing materials and sources of ignition. The bulk material is stable for 2 weeks at temperatures of up to 60°C, indicating a stability of at least 24 weeks at room temperature (25OC). Naphthalene was also found to be stable for 7 days at 25OC when dissolved in corn oil at a dose level of 4%. Naphthalene dust forms an explosive mixture in air at high concentrations; therefore, care should be exercised when preparing to open storage vessels containing naphthalene.
*
V. HANDLING PROTOCOL
Naphthalene is irritating to the eyes and skin, and is toxic if ingested. Acute overexposure may cause dermatitis, headache, and nausea. Chronic overexposure causes renal shutdown and eye cataracts. This mate- rial is also suspected of causing neoplasms in rats. Thus, all personnel should be fully protected when working with naphthalene. Coveralls, gloves, eye and foot protection are necessary. provided in areas where naphthalene dust may form. air-supplied masks are suitable. ventilated areas. Although we did not encounter this problem, it is very possible to create a dust when handling solid naphthalene. be taken to prevent this since high concentrations of naphthalene dust are explosive in air.
Respiratory protection should be Dust type cannister or
AN work should be conducted in well-
Care should
689 4 MIDhTST RESEARCH INSTITUTE
Bulk Chemical Studies
Mike Cannon Junior Chemist
&>,LA Linda Siemann Junior Chemist
Dave Steele Assistant Chemist
Richard Brown Associate Chemist
Approved :
Evelyn aurrill, Ph.D. Senkor Advisor for Chemistry
$!*--* E J Woodhouse, Ph.D.
Chemical Vehicle Studies
&-w* Lorren D. Kurtz
Junior Chemist
Bruce -&/& Schworer
Thornton C. Carpenter, Ph.D. Associate Chemist
L l Principal Chemist
APPENDIX B
-
WEEaY Ah’IMAL REPORT BCL MISCELLANEOUS DATA SHEET
n
;I) m
3 v, x
!
3 u
) EM-360 60 MHz NMR SPEC 'OMETER ~ ~ ~ 0 3 3
-._._._...._............-- 6394
A N I M A L S
T H I S LYEEK
A N llLl A LS 0 I SC A R D E i FFIOP.1 H O L D I N G CAGES D U R IN C Q U A ;1 A N T I rJ E
1
T H I S WEEK
A N I rrlA L D E N T I F I C A T I O N
L A 8 0 R A T C R Y D E N T I F I C A T I O N
\ .-. - 1
u
I . BCL MISCELLWEOUS DATA SHEET
NA?E OF CHEMICAL: Naphthalene C 52904 BCL 28
CHMICAL PURITY: 99.6 %
OBSERVATIOB DATES: August 11, 1979 to August 14, 1979 ; 2-4 days
!
MDOMIZED BODS WEIGHT RANGES USED IN TEST:
a c e $ 18-21 g Piice 3 22-28 g Age 8 weeks
---kts $ 135-155 g Rat- 170-215 g Age 6 weeks
mws :
Date of Birth:
a c e March 13, 1979
--Rats March 27, 1979
1
1
639 4
A P P r n I X c
SURVIVAL TABLE
6694 i
0 3 0 0 0 0 0 rlrlclrlrl
o o o o c o rlrlrlrlrlrl
ooococ r l r l r l r l r - ! r l
0 0 0 0 0 0 rlrlrlrlrlrl
ooooco rlrlrlrlrirl
0 0 0 0 0 0 rlrlrlrlrirl
oocooc rlrlrirlrlrl
m
3 0 0 0 0 0 0 rlrlrlrlrlrl
c o o o o o rlrlrlrlrirl 31 ni
0 0 0 0 0 0 rlrlrlrlrlrl
.. x '0 1 U
4
0 0 0 0 0 0 rlrll-lrlrld
0 0 0 0 0 0 rlrlrlrlrlrl
0.c c 0 c c, rldr-lrlrl-
occooo ririrldrlrl
ooocco ' rlrlrlrirlrl
occccc rlrlrldrirl
rl 0 Li
c c o o v r u CCOvrN t: u w l - l 0 u
rl 0
I
!
APPENDIX D
LTEKLS DIET C O H S W T I O N
639 4
6 3 9 4 m b u u u m u u u u u u r l r l r l r l r l "
. . . . . .
0 0 d c i c o h l . . . . . .
I - l v l h l b d d . . . . . .
0 m 4 m o a r l c o u o u m o b c o c o m c o ci
. . . . . .
c o c i 0 ~ O U
a c o c o u m a a m m o l n m . . . . . .
I-l
3 a 1 U VI
w 0 p:
01
o m m u m c o N b U U c i c o r l o b m c m r l r l rl
. . . . . .
c o N a m N O O
O m a b N m o o o o c i o 4 r l r l I - l c i I - l
. . . . . . m o m m r n a . . . . . . r i h o b m m a 0 b . O \r) a
ci 0
o o o o m u U N " ' 0
V
0 0 0 m N L ) C o o o o m o o o o m h l u 21' N d u
C C V
. . . . . . * . .
A P P r n I X E
GROUP AVERAGE BODY LTIGHTS, WEEKLY IhXIVIDUAL STARTING AKD FINAL BODY WEIGHTS
GRAPHS
! 6 3 9 4 1
6 3 9 4
\ 0 h U N U Q \ . . . . . .
h m u b m m . . . . . .
. . . . . .
. . . . . . m o u c o m h U h h c o 0 1 C n N N N N N N
r l 0 U N U h
N m m c o r l m u m u h o 3 c o N N N N C V N
. . . . . .
m . c o 0 LW 0 03
O u 3 m h C c m N m u m ' rn L 1 N N N N N N
. . . . . .
m h m C J 0 r - I
h O m m d N O N - J m U w - N N N N N N
. . . . . .
\ 0 0 r A 4 O h . . . . . .
V
'.J
m N h w h m \ o c o m o m o \ 0 c o m o m o d 4 4 N d N
. . . . . .
d U d d N * . . . . . .
m m u m u m . . . . . .
m m m N o m d d r l
. . .
0 1 \ 0 0
h U 0 m \ 0 h 4 4 4
. . .
. . . m m u \ o h m 4 d l - l
U o o o o m ~ O o O m N u U N 4 G , o
U
. . .
..-- I..% .,..._ - (
INDIVIDUAL STARTING AND FINAL BODY WEIGHTS (GRAMS), j?!D ?%CENT INCREASE, OF FISCHER 344 RATS DURING
THE SUBCHRONIC TOXICITY STUDY OF NAPHTHALENE
6394
Dose Group, Animal P e r c e n t Number Day 0 Day 91 I n c r e a s e . m g k
400
200
100
50
I
1 2 3 4 5 6 7 8 9
1 0
1 2 3 4 5 6 7 8 9
10 1 2 3 4 5 6 7 8 9
1 0
1 2 3
' 4 5 6 7 8 9
10
Males
204 197 190 166 187 202 205 185 188 179
18 9 173 178 210 193 ' 172 188 182 197 209
177 196 210 183 18 5 172 201 1 7 9 18 5 197
180 207 200 213 167
. 209 202 200 186 168
255 289 228
-
-- 236 266 292 27 2 162 255
309 28 5 284 333 337 28 7 3 28 291 289 324
308 343 .
368 341 334 319 341 327. 315 338
331 354 34 1 402 315 365 349 350 377 3 28
25 47 20
26 32 42 47
-14 42
63 65 60 59 75 67 74 60 47 55
74 75 75 8 6 81 8 5 70 83 70 72
84 71 7 1 89 89 75 73 75
103 95
--
689 4 (
'INDIVIDUAL STAXTING AND FINAL BODY \EIGHTS (Continued)
Dose Group, Animal Percent Increase Day 0 Day 91 Number mg 1 kg
Males (Continued)
25
Control
-400
200
1 2 3 4 5 6 7 8 9
10
1 2 3
o 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9
10
1
3 4 5 6 7 8 9
10
2
204 181 188 188 188 19 7 1 8 2 205 185 210
199 186 18 7 196 194 208 183 1 9 1 198 208
Females
1 3 1 134 139 140 1 4 1 138 148 134
- , 132 139
1 4 4 145 136 140 149 131 134 131 135 140
364 365 350 327 327 344 345 380 35 3 379
355 32 3 34 6 366 36 7 360 351 336 336 349
128 162 167 184 133
1 7 7 165 163 1 2 1
198 207 193 185 1 9 9 1 7 4 182 1 8 7 1 9 1 189
1 6 7-
78 102
86 74 74 75 90 85 9 1 80
78 74 85 87 89 73 92 76 70 68
. - 2 20 20 31
- 6 21 20 23 23
-13
38 43 4 2 32 34 33 36 43 4 1 35
I N D I V I D U A L S T A R T I S G Ah9 FINAL BODY WEIGHTS (Continued)
Dose Group, A n i m a l P e r c e n t m d k e Number Day Q Day 9 1 Increase
Females (Continued)
100 1 2 3 4 5 6 7 8 9
10
5 0 . . 1 2 3 4
= 5 . .6
7 8 9 10
25 1 2 3 4 5 6 7 8 9
1 0
Con t ro l 1 2 3 4
' 5 ' 6
7 8 9
10
1 5 4 1 5 0 1 5 1 1 3 3 1 3 5 1 4 4 1 4 5 1 4 2 1 4 4 1 4 1
1 4 0 1 3 2 15 3 1 3 8 1 4 6 1 5 2 1 4 8 1 4 6 1 4 5 1 3 5
1 4 1 1 4 0 1 3 7 1 3 8 1 4 3 1 4 4 1 4 0 1 4 2 1 3 2 1 4 7
1 5 3 . 1 4 4
1 4 7 1 4 3 1 3 9 1 4 5 1 4 2 1 3 7 1 3 5 1 3 7
201 205 203 .
1 8 3 1 8 9 205 1 8 8 205 203 1 9 0
2 0 1 1 8 4 1 9 9 208 1 9 7 210 208 226 203 1 9 9
1 8 8 1 9 8 195 1 9 5 203 203 202 205 1 8 8 201'
2 1 7 1 9 1 210 1 9 1 1 9 6 219 218 204 1 9 1 197
3 1 37 34 38 4 0 42 30 44 4 1 35
4 4 39 30 5 1 35 38 4 1 5 5 4 0 47
L
33 4 1 42 4 1 42 4 1 4 4 44 42 3-7
4 2 33 4 3 34 4 1 5 1 54 4 9 4 1 44
d U U
350
340
330
32 0
31 Q
300
. .
-.BO. -. .
280
. _.
21 0
. .
.260
. . .
250
. . . .
2 4 0
. . . .
230
220
210
200
. (
190
165
....
. . . .
. . . . . . . . .
.. .- ... - ... - . . . . . .
- . . . . . . . . . . . . . . .
....... - ... - . . . . . .
. . . . . _- - - . . . -. .
6394
I I I I I I I
6 3 9 4
. . - -.
-.
. . . . . .
.
-.
.......... -. -
- 210
200.
c
7% 180, 4 CJ
.. - 3 .. . . . .
' - ? . - ,170 C n
. . . . a, OD
u CJ 3 150; > .. <
140
-130
0
. . . . . . . . . . . . . .
- KAPTHALENE. .
.. -. .. .... - _ _ . . . . . . . . . - .. . . . . SUBCHRONIC STUDY _. _ _ -_ -
WEIGHT CURVE FOR FEMALE F344 RATS
...
.... - . . .
. . - ..
.... -. . . . . . . . . . . . . . . . . - -. . .
. . . . . .
. .
. . . - . . . . .
- . . . . . . .
. . . . . . . . . . .
....... -. ..... ........ \ . . . .
. - -. ..... - - - . . .- ...... _. . - . . . . . . . . .
. . -
. . legend: -- ..
. . . - . ..
A .- 400. mg/kg --
. . . . . . . _ .. t 200 mg/kg
6 10.0 =g/kg
A 50 mg/kg
0 25 mg/kg
0 v e h i c l e c o n t r o l
2 4 1 0 12
I
APPENDIX F
HEMATOLOGY TABLE
6594
6394 0 4
ro 0 4
Y
n m
- 4 r l<
N 4
u
. . h
Y 9
r.4 d U
0 0- . . ' 0
n 0 4
0 . W
1
m G oru or- l n l n o m r - r . 0 0 ' 9 4 - u 4 coo m a m o i . . . . . . dr l :dd N C V 0 4 00 A,/ ,I
a m 4
n n n n 0 0 h O n m 4 4 l n 4 I - W Y Y W Y W
a m o m or. d m o m psm C V N 94 or- 4 l n o o m a N m r . m c o I - 0 3 m m m I - C o r - I - I - l n
. . . . . . . . . . . .
A n n n O n n O z z s z z s
00 00 00 00 00 00
n n n n O n o n
Y w w u v Y m 2 4 C n 4 r -
00 00 00 00 0 0 0 0
I
n 0 4
04 m m a m rl
W
. .
n 0 rl
OI- 0- m u 4
W
. . n
m
0- N
m W
9":
n 0 rl Y
c m ? 9 O m N
n m N o 3 N u l
c o u rl
W
. . n m 0 0
a
W
n 0 rl
04 r l m a u rl
Y
. . n 0, Y
0 0 3
m m rl
9 9
n 0 rl
a m m
W
h a 3 &I v)
I-
m e 4
r . m m
Y
? ? C
In N
9 I- a
rl
n n n n O n n O s z e c ! s 5
00 00 00 00 00 00
n n n n O O n O n Y V o ' d ~ ~ s ~
00 0 0 0 0 00 P O 0 0
n n n n n O n O O O ' O
rl m m o oco 0- 0.u 00
r n r l m o u o u o r 4 o u r l ~ r n r n m r n m r n . . . . . . . . . .
n n n n n 0 0 0 0 0 . - = ! E ! E ! s s 9
n n n n n O n 0 0 0 0
r n m o m r l N rnm 0 0 - m m s z s e s s
r n a d r 4 m m m u . f.?: I-!?
Q)os3ocooo jocoocoo . . . . . .
- m o N l n m m u m r l m OI-
m*u. TU. N N N m N C V a m m o m o g o & d m o o 3 0
. . . . n n n n n O n 0 0 0 0 5 2 s s s c
m m u u m c D r l o 4 m I- u o w I - h l a 9I- m r - m o r-rl coo000 I-0 coo C O N . . . . . . . . . . . .
n n n n n e n 0 0 0 0 1 -
W Y Y W Y Y . . . . s z s s s z 0 ad o u o m o m 00
r n r l l n m m a c o m 0 0 mco o m o m o w o m u m r l I-0 a m I-N m403r4 9rl.Irl url u u u u u u u u u u u u
. . . . . . . . . . . . a m , 4 4 4 m r l mr4 m N ?". ""9 '4? ""
n n n n n O n 0 0 0 0
m 4 a o m m 9 m o . f I-m ~ m u u r n m 1 - c o u m r 4 m
4 4 r l r l r l 4
W 4 c s s s s
d d d d a o -0 .Io m o . . . . . . . .
. .
rl a rl
0 . u m o o o o ii a m m o u 0 5060.49
r u In 0 . 3 0 0 c m m o o o 0 9 4 c Y . f V
APPENDIX G .
PURINA LAB CHOW FEED TAG
639 4 1
.
.