P. 1



# OAR Box 1846 Prepped by Ollie Stewart



### Document

Number: 

# 8 ) ll-B- 5
Docket Number: 

# A-90-0 7

Printed 11/18/20087:30:14 AM Sheet 8of 93P. 2

^ £ 0 S, < * UNITE D STATE S ENVIRONMENTA L PROTECTIO N AGENC Y WASHINGTON , D.C
. 20460 

# { ] t g n »j. m FE B 2 5 199 2

MEMORANDUM 

## EP AAI R DOCKE T
SUBJECT : Cos t Estimate s fo r TSCATes t Guideline s FROM : Da n Axelradjl/ j

Regulator yc Tmpact s Branc h Economic s an

d Technolog

yDivisio n (TS-779 ) TO : Sher i Dunatchi k

Offic e o f Mobil e Source s OFFIC E O F PESTICIDE S AN D TOXI C SUBSTANCE S

Attached , a s requested , ar e cos t estimate s fo r th e TSC Ates t guideline s bein g use d b yyou r office
. Thi s packag e i s divide d int o thre e sections : 1
. Th e cos t estimates , summarize d i n a on e pag e table , followe d b ya se t detaile d estimate s fo r eac h tes t guideline ; 2
. An explanatio n o f th e methodolog yuse d t o develo p th e cos t estimates ; an

d 3
. Th e resum e o f Vincen t J . Piccirillo , th e consultan t wh o prepare s th e cos t estimates
. Pleas e cal l m e a t 382-371 3 i f yo u hav e an yquestion s concernin g thi s package
. Attachmen t

Printed on Recycled

PaperTSCA HEALT H EFFECT S GUIDELINE S P. 3

PROTOCO L / PROTOCO L NUM . SPECIE S ROUT E O F ADMIN .
Acut e Inhalatio nToxicit y 798.115 0 Rat s inhalatio n Subchroni c Inhalatio nToxicit y 798.245 0 Rat s inhalatio n Combine dChroni c Toxicity/Oncogenicit y 798.332 0 Rat s inhalatio n Inhalatio nDevelopmenta l Toxicit y (2 species ) 798.435 0 Mic e inhalatio n Rat s inhalatio n Developmenta l Toxicit y Preliminar y

Scree n 798.442 0 Mic e inhalatio n Rat s inhalatio n DAT E

06/24/9 1
07/19/9 0
08/15/9 0
07/19/9 0 07/19/9 0
07/19/9 0 07/19/9 0 Reproduction/fertilit y Effect s (2-Generatio nReproduction ) 798.470 0 Rat s inhalatio n07/19/9 0 Salmonell a typhimuriu m Revers e Mutatio nAssa y (Ame s Test ) 798.526 5 Salmonell a typhimuriu m 08/15/9 0 Detectio no f Gen e Mutation s i nSomati c Cell s i nCultur e 798.530 0 CHO/HGPR T I nVitr o Mammalia nCytogenetic s (Chrom o Aber ) 798.537 5 CH O Cell s 08/15/9 0
08/15/9 0 InViv o Mammalia nBon e Marrow Cytogenetic s Tests : Chromosoma l An a 798.538 5 Rat s inhalatio n07/20/9 0 BES T ESTIMAT E

1128 0
13971 0
126766 0
4860 0 4873 0
2381 0 2357 0
43965 0
384 0
924 0
866 0 lysi s (Chrom

o 2673 0 I nViv o Mammalia nBon e Marrow Cytogenetic s Tests : Micronucleu s Assa y 798.539 5 Mic e inhalatio n07/20/9 0 2462 0 Unschedule dDN

A Synthesi s inMammalia nCell s inCultur e 798.555 0 Ra t Hepatocyte s InVitr o Siste r Chromati dExchang e Assa y 798.590 0 CH O I nViv o Siste r Chromati dExchang e Assa y 798.591 5 Rat s inhalatio n Functiona l Observatio nBatter y 798.605 0 ACUT E Rat s inhalatio n SUBCHRONI C Rat s inhalatio n Genera l Metabolis m 798.710 0 Rat s inhalatio n 06/24/9 1
06/24/9 1
06/25/9 1

07/19/9 0
06/08/9 0
08/16/9 0 1050 0

1074 0
1587 0

1580 0
10433 0
16245 0 TES T COS T

940 0 -
10387 0 •
96815 0 •
4001 0 - 4019 0 -
1935 0 • 1921 0 •
32644 0 -
274 0 •
719 0 •
671 0 - Aber ) 2239 0 -
2052 0 •
872 0 -
892 0 •
1324 0 -

1320 0 -
7361 0 -
13543 0 - RANG E

1334 0
17751 0
158595 0
5803 0 5804 0
2866 0 2829 0
55939 0
497 0
1144 0
1076 0
3147 0
2914 0
1246 0
1276 0
1878 0

1866 0
13650 0
19225 0 BURDEN

15 8
204 6
2122 3
86 8 93 0
37 6 35 4
667 0
3 6
9 7
10 0
37 4
36 6
11 5
116
205

24 7
166 6
324 4

# T PROTOCO L # : 798.115 0

Acut e Inhalatio n Toxicit y P. 4

STUD Y DESIGN : Group l 2 3 Dos e Leve l

Lo wDos e Mi d Dos e High Dos e Numbe r o f Animal s 5 males , 5 female s 5 males , 5 female s 5 males , 5 female s TES T LIMIT :

I f a tes t a t a dos e concentratio n o f a t leas t 5 mg/lite r o r a t th e maxima l achievabl e concentration , wit h a n exposur e o f 4 hour s o r mor e produce s n o compound-relate d mortality , the n a thre e dos e leve l stud yi s no t necessary . ANIMA L SPECIE S O

R STRAIN : Laboratoryrats . AG E O F ANIMALS :

Youn g adul t animals
. DURATIO NAN D FREQUENC Y O F EXPOSURE :

Asingl e exposur e ove r a perio d o f a t leas t 4 hour s wit h anima l observation s fo r a t leas t 1 4 day s afte r exposure . ROUT E O F EXPOSURE :

Inhalation , fo r a t leas t 4 hours
. BODY WEIGHT :

Al l animal s prio r t o dosing , weekl ythereafte r and a t deat h o r sacrifice
. OBSERVATION S AN

D PHYSICA

L EXAMINATIONS : A t leas t cnc e daily
. Mor e frequen t observation s shoul d b e mad e during th e firs t fe wday s t o assur e th e observatio n o f transien t clinica l sign s an d precis e recordin g o f time o f death
. GROS S NECROPSY :

O n al l animal s including thos e foun d dead o r sacrifice d i n moribund condition
. Aful l serie s o f tissue s an

d organ s ar e take n an d preserved
.P. 5

PROTOCO L # : 798.115 0 Acut e Inhalatio n Toxicit y Rat/inhalatio n 06/24/9 1

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n Vet . Senio r tech
. set-u p compound prep . necrops y dat a recor d keepin g exposur e samplin g & grav.anal
. exposur e sys . developmen t Tech
. set-u p exposur e bod ywt./obs
. necrops y exposur e sys . developmen t Anima l car e set-u p beddin g chang e cag e cleanin g roommaintenanc e washin g feeders , etc . feedin g chambe r cleanin g Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (2 2 5 % o f D.L. ) TOTAL DIREC T LABO R No . o f Hour s 1 6 1 2 3 ( 3 ) 6 4 2 ( 5 ; ( 2 ; ( 3 ' ( 6 , ( 4 ( 8 t { 1 4 v 4 6 ( 5 ( 1 4 ( 9 ( 8 ( i o 1 7 ( 2 ( 1 ( 2 ( 2 ( 4 ( 1 \ *" * 1 2 8 4 15 4 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8
. 0 1

6.8 6

12.3 6 9.1 6 10.5 8 Tota l

Dollar s $55 2 $16 7 $9 8 $14 1 $41 5

$36 8

$11 7

$14 8 $7 3 $4 2 $2,12 3 $4,77 6 $6,89 9P. 6

PROTOCO L# : 798.115 0 Acut e Inhalatio nToxicit y Rat/inhalatio n 06/24/9 1

Othe r Direc t Cost s No . o f Unit s 8 4 5 4 8 0 1 4 Uni t

Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $5.0 0 Tota l

Dollar s Overtim e (anima l tech. ) Animal s Fee d Bedding Disposable s Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G§ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % c f Tota l Cost ) ESTIMATE D FINAL COST : $3 2 $38 3 $9 0 $1 6 $7 0 $21 2 $80 3 $7,70 2 $1,69 4 $9,39 6 $1,87 9 $11,27 5

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $11,28 0 $9,40 0 - $13,34 0

TOTAL : $11,28 0 $9,40 0 - $13,34 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , and fe e rate s assesse d withi n th e Unite d State s laboratories
.P. 7

PROTOCO L # : 798.115 0 06/24/9 1 Acut e Inhalatio n Toxicit y Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 5 mal e an

d 5 femal e rats .

- Stud yduratio n i s 2 weeks
.P. 8

798.245 0 INHALATIO N TOXICITY
STUD YDESIGN : Group Dose Level Number

of Animals 1 Contro l 1 0 males , 1 0 female s 2 Lo

w Dos e i o males , 1 0 female s 3 Mi dDos e 1 0 males , 1 0 female s 4 Hig

hDos e 1 0 males , 1 0 female s
ADDITIONA

L ANIMALS : I f interi

m sacrifice s ar e plannedthe numbe r o f animal s ineachgrou pshoul db e increase db y the numbe r o f animal s scheduledt o b e sacrificedduring th e cours e o f the study . A satellit e grou

po f 1 0 animal s pe r se xma y be treated wit hthe hig

hdos e leve l for 9 0 day s an dobserved for reversibility , persistenc e or delayedoccurrenc e o f toxi c effect s for a periodo f no t les s than2 8 days . ANIMA L SPECIE S O

RSTRAIN : Laborator

y rats . AG E O F ANIMALS :

Youn g adults . ROUT E O F EXPOSURE :

Inhalation . DURATIO N AN D FREQUENC

YOF EXPOSURE : Si xhour s pe r day , sevenday s pe r wee k for a period o f 9 0 days . BOD YWEIGHT :

Al l animal s ar e weigheda t initiatio no f the test , weekl y thereafte r an

da t deat ho r sacrifice . FOOD CONSUMPTION :

Measuredweekl y

i f abnorma l bod

y weigh t change s ar e observed . OBSERVATION S AN

D PHYSICA

L EXAMINATIONS : A t leas t onc e daily . HEMATOLOGY : Fiv

e animal s pe r se

xpe r grou

pprio r t o initiatio no f the test , afte r 3 0 day s o f exposur e an d jus t prio r t o termination . Evaluationinclude s hematocrit , hemoglobi nconcentration , tota l re dbloo d cel l count , tota l an

ddifferentia l whit e bloodcel l coun t an da measuremen t o f clottin g potential .P. 9

798.245 0 INHALATIONTOXICT Y (CONT. )
CLINICAL BIOCHEMISTRY :

Fiv e animal s pe r se x pe r group prio r t o initiatio n o f th e test , afte r 3 0 day s o f exposur e an d jus t prio r t o termination
. Th e parameter s t o b e measure d includ e test s fo r electrolyt e balance , carbohydrat e metabolis m an d live r and kidne yfunction
. URINALYSIS :

No t require d routinely
. OPHTHALMOLOGICAL EVALUATION :

Fiv e animal s pe r se x pe r group jus t prio r t o initiatio n an djus t prio r t o terminatio n o f th e test . GROS S NECROPSY :

O n al l animal s including thos e foun d dea d o r sacrifice d i n moribund condition
. Aful l serie s o f tissue s and organ s ar e take n and preserved
. ORGA NWEIGHTS : A t th e tim e o f termina l necropsy , th e liver , kidneys , adrenals , brai n an d gonad s ar e weighe d from al l animals
. HISTOPATHOLOGY :

Complet e histopathologyi s performe d o n th e respirator ytrac t an d o n al l organ s an d tissue s take n a t necrops yfro mal l animal s i n th e contro l an d hig h dos e groups . Al l gros s lesion s an d suspecte d targe t organ s ar e evaluate d fro mal l animals
.P.1 0

PROTOCO L # : 798.245 0 07/19/9 0 Subchroni c Exposur e Rat/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Bd
. Cert . Pathologis t slid e evaluatio n ( necrops ysupervisio n ( Q.A . Inspecto r Vet . Clinica l Chemis t Clinica l lab
. tech
. Histologis t Histolog

ytech
. Senio r tech
. set-u p i compoun d prep . i necrops y dat a recor d keepin g exposur e Tech
. set-u p exposur e bod ywt./obs
. necrops y bleeding Anima l car e set-u p bedding chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feeding chambe r cleanin g Hematologist ; Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTA L DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTA L DIREC T LABO R NO . o f Hour s 6 0 6 1 5 4 ) 7 ) 14 8 1 2 5 0 3 0 15 4 8 0 23 7 1 0 ] 1 5 ] 7 2 3 18 2 80 3 1 0 72 8 4 0 ( 1 5 

### [ 1 ° 20 0 [ 5 

### ( 1 ° ( 1 5 ( 8 ( 1 5 

### ( i o ( 13 7 6 0 8 4 1 1 2 1 5 197 1 Hourl y

Wag e 34.5 3 39.6 6
13.9 4 23.5 6 10.7 2 9.9 4 10.4 0 8.8 3 9.8 7

8.0 1

6.8 6

10.7 2 23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $2,07 2 $2,41 9 $2,06 3 $28 3 $53 6 $29 8 $1,60 2 $70 6 $2,33 9

$6,43 2

$1,37 2

$64 3 $18 5 $50 7 $11 0 $15 9 $21,72 6 $48,88 2 $70,60 8P.1 1

PROTOCO L # : 798.245 0 07/19/9 0 Subchroni c Exposur e Rat/inhalatio n

Othe r Direc t Cost s No . o f Unit s Uni t

Cos t Tota l

Dollar s Overtim e (anima l tech. ) 150. 0 $4.0 1 $60 1 Animal s 100. 0 $8.5 0 $85 0 Fee d 31. 0 $22.5 0 $69 8 Bedding 600. 0 $0.2 0 $12 0 Disposable s 91. 0 $5.0 0 $45 5 Histolog

ysupplie s 1. 0 $1,440.0 0 $1,44 0 Clinica l path , supplie s 1. 0 $1,080.0 0 $1,08 0 Vet . ophthalmologis t 2. 0 $1,000.0 0 $2,00 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) $2,17 3 TOTAL OTHE R DIREC T COSTS : $9,41 6 TOTAL BEFOR E G&A : $80,02 4 G@ A( 22 % o f Tota l Cost ) $17,60 5 TOTAL BEFOR E FEE : $97,62 9 FE E ( 20.0 % o f Tota l Cost ) $19,52 6 ESTIMATE D FINA

L COST : $117,15 5

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $117,15 0 $97,79 0 - $138,47 0 ANALYTICA L CHEM : $22,56 0 $6,08 0 - $39,04 0

TOTAL : $139,71 0 $103,87 0 - $177,51 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.1 2

PROTOCO L # : 798.245 0 07/19/9 0 Subchroni c Exposur e Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 1 0 mal e an

d 1 0 femal e rats .

- Stud yduratio n i s 1 3 weeks
.P.1 3

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Mouse/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n ( Vet . Senio r tech
. set-u p ( compound prep . ( observation s 1 necrops yI dat a recor d keepin g i exposur e i c-sectio n i feta l evaluatio n Tech
. set-u p exposur e bod ywt./obs
. necrops y breedin g c-sectio n feta l evaluatio n Anima l car e set-u p beddin g chang e cag e cleanin g roommaintenanc e washin g feeders , etc . feeding chambe r cleanin g Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABO R No . o f Hour s 3 2 6 4 7 7 ) 8 21 3 2 5 ) 6 ) 5 6 ) 7 ) 1 2 ) 2 0 ) 2 0 ) 6 7 ' 34 6 2 5 8 0 8 6 2 0 2 8 ( 4 0 ( 6 7 6 8 ( 1 3 ( 8 ( 1 2 ( 4 ( 8 ( 8 ( 1 5 4 7 8 2 4 8 85 2 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $1,10 5 $89 2 $22 9 $18 8 $2,10 2

$2,77 1

$46 6

$9 2 $96 4 $22 0 $8 5 $9,11 6 $20,51 0 $29,62 6P.1 4

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Mouse/inhalatio n

Othe r Direc t Cost s Overtim e (anima l tech
. ) Animal s Fee d Beddin g Stain s Fixative s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTA L OTHE R DIREC T COSTS : TOTA L BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA L COST : No . o f Unit s 32. 0 250. 0 11. 0 480. 0 1. 0 1. 0 42. 0 D.L . ) Uni t Cos t $4.0 1 $2.8 0 $22.5 0 $0.2 0 $100.0 0 $600.0 0 $5.0 0 Tota l

Dollar s $12 8 $70 0 $24 8 $9 6 $10 0 $60 0 $21 0 $91 2 $2,99 3 $32,61 9 $7,17 6 $39,79 5 $7,95 9 $47,75 4

COS T COMPARISO N

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $47,75 0 $39,78 0 - $56,57 0 ANALYTICA L CHEM : $85 0 $23 0 - $1,46 0

TOTAL : $48,60 0 $40,01 0 - $58,03 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.1 5

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Mouse/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 2 0 femal e mice
.

- Stud yduratio n i s 6 weeks
.P.1 6

SYNOPSI S O F PROTOCO L

TYP E O F STUDY : Developmenta l Toxicit yStud yI n Rat s NUMBE R O F ANIMALS : 10 0 Femal e Rat s NUMBE R O F GROUPS : 3 Treate d An d 1 Contro l Grou

p (2 5 Female s Pe r Group )
METHO D O F COMPOUN

D ADMINISTRATION : Inhalation , 6 Hour s Pe r Da y DOSIN G PERIOD : Gestatio n Day s 6 Through 1 5 BOD Y WEIGHT/FEE D CONSUMPTION : Weekl y CESARIA NDELIVERY : Gestatio n Da y1 9
FETA L EVALUATION : 50 % O f Fetuse s Fo r Skeleta l Evaluation ; 50 % O f Fetuse s

Fo r Sof t Tissu e Evaluatio n GROS S NECROPSY : Al l Animal s

1 6P.1 7

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Rat/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n ( Vet . Senio r tech
. set-u p ( compound prep . . i observation s i necrops yi dat a recor d keepin g exposur e c-sectio n feta l evaluatio n Tech
. set-u p exposur e bod ywt./obs
. necrops y breedin g c-sectio n feta l evaluatio n Anima l car e set-u p beddin g chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feedin g chambe r cleaning Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABO R No . o f Hour s 3 2 6 2 7 7 ) 8 20 3 1 5 ) 6 ) 5 6 ) 7 ] 1 2 2 0 2 0 6 7 33 6 1 5 8 0 8 6 2 0 ( 2 8 : 4 0 ( 6 7 6 3 ( 8 [ 8 ( 1 2 ( 4 ( 8 ( 8 ( 1 5 4 7 8 2 4 8 82 5 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $1,10 5 $86 4 $22 9 $18 8 $2,00 4

$2,69 1

$43 2

$9 2 $96 4 $22 0 $8 5 $8,87 5 $19,96 8 $28,84 2P.1 8

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Rat/inhalatio n

Othe r Direc t Cost s Overtim e (anima l tech. ) Animal s Fee d Bedding Stain s Fixative s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTA L OTHE R DIREC T COSTS : TOTA L BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST : No . o f Unit s 32. 0 150. 0 25. 0 480. 0 1. 0 1. 0 42. 0 D.L . ) Uni t Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $100.0 0 $600.0 0 $5.0 0 Tota l

Dollar s $12 8 $1,27 5 $56 3 $9 6 $10 0 $60 0 $21 0 $88 7 $3,85 9 $32,70 2 $7,19 4 $39,89 6 $7,97 9 $47,87 5

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $47,88 0 $39,96 0 - $56,58 0 ANALYTICAL CHEM : $85 0 $23 0 - $1,46 0

TOTAL : $48,73 0 $40,19 0 - $58,04 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.1 9

PROTOCO L # : 798.435 0 07/19/9 0 Inhalatio n Developmenta l Toxicit y Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 2 0 femal e rats .

- Stud vduratio n i s 6 weeks
.P.2 0

798.332 0 COMBINED CHRONI C TOXICITY / ONCOGENICITY
STUD YDESIGN : Group 

### 1 2 3 4 Dose Level

Contro l Lo w Dos e Mi dDos e Hig hDos e Number c Oncogenicit y 5 0 pe r se x 5 0 pe r se x 5 0 pe r se x 5 0 pe r se x •)f

Animals Chroni c Tox j 2 0 pe r 2 0 pe r 2 0 pe r 2 0 pe r Lcit ; 

### se x se x se x se x ADDITIONA

L ANIMALS : The animal s i nthe chroni c toxicity group s ar e scheduledfor sacrific e afte r 1 2 month s o f treatment . I f additiona l interim sacrifice s ar e planned , the numbe r o f animal s i neachgrou pshoul dbe increasedb y the numbe r o f animal s scheduledt o be sacrificed durin g th e cours e o f the study . ANIMA L SPECIE S O

RSTRAIN : Laborator

y rat s or mice . AGE OF ANIMALS :

Approximatel y

4 2 day s a t the star t o f the study . DURATIO N AN D FREQUENC

YOF EXPOSURE : Sevenday s pe r wee k fo r a t leas t 2 4 month s for rat s an d1 8 month s for mice . ROUT E OF EXPOSURE :

Specifiedb

y tes t rule , ma

y be oral , derma l o r inhalation . BOD YWEIGHT :

Al l animals ; weekl y for the firs t 1 3 week s an dever y 4 week s thereafter . FOOD CONSUMPTION :

Al l animals ; weekl y for the firs t 1 3 week s an dever y 4 week s thereafte r i ntest s wher e the tes t chemica l i s administeredi nthe feed . Wate r consumptio ni s measuredunde r the sam e schedul e whenth e tes t chemica l i s administeredi nthe drinkin g water . OBSERVATION S AN

D PHYSICA

L EXAMINATIONS : Onc e dail y

wit hspecia l emphasi s o nth e appearanc e an

d progressiono f palpabl e tumors .P.2 1

798.332 0 COMBINED CHRONI C TOXICITY/ONCOGENICITY(CONT. )
HEMATOLOGY : Twent y

animal s pe r se

xpe r grou

pafte r 3 , 6 , 1 2 an d1 8 month s o f exposur e an djus t prio r t o stud y termination . Evaluatio ninclude s hematocrit , hemoglobi nconcentration , tota l re dbloo dcell , whit e bloodcel l an

dplatele t counts . Differentia l whit e bloodcel l count s wil l be performe do nal l contro l an

d highdos e animal s a t eachinterval . CLINICA L BIOCHEMISTRY : Te nanimal s pe r se xpe r grou

pprio r t o initiatio no f th e test , a t a midpoin t interva l an djus t prio r t o termination . The parameter s t o be measuredinclude test s for electrolyt e balance , carbohydrat e metabolism an dlive r an

dkidne y function . URINALYSIS :Te nanima l pe r se xpe r grou

pa t the sam

e interval s a s th e hematolog

y evaluations . Evaluation s t o include appearance , volume , specifi c gravit y , protein , glucose , ketones , bilirubin , occul t bloo dan d microscopi c evaluatio no f the sediment . OPHTHALMOLOGICA

L EVALUATION : Te nanimal s pe r se xpe r grou

pjus t prio r t o initiatio n an djus t prio r t o terminationo f the test . GROS S NECROPSY :

Onal l animal s includin g thos e foun

ddea dor sacrificed i nmoribun dcondition . A

ful l serie s o f tissue s an

dorgan s ar e takenan dpreserved . ORGA N WEIGHTS : A t the tim

e o f termina l necropsy , the liver , kidneys , adrenals , brai nan dgonad s ar e weighedfro m a t leas t 1 0 animal s pe r se

xpe r group . HISTOPATHOLOGY :

Complet e histopatholog y i s performedo

nal l organ s an

d tissue s takenat necrops y fro m al l animal s inthe contro l an

dhig hdos e grou

pa s wel l a s fro

m al l foun

d deadan dmoribun

dsacrificedanimals . Additionally , histopathologica l evaluatio ni s performedo

nal l gros s legions , targe t organ s an dthe lungs , live r an d kidney s fro

m al l animals .P.2 2

PROTOCO L # : 798.332 0 Combine d Chroni c Toxicity/Oncogenicit y Rat/inhalatio n 08/15/9 0

Direc t Labo r Stud yDirector-toxicologist(PhD ) Bd
. Cert . Pathologis t slid e evaluatio n ( necrops ysupervisio n i Q.A . Inspecto r Vet . Histologis t Histolog

ytech
. Senio r tech
. set-u p compound prep . necrops y dat a recor d keepin g exposur e Tech
. set-u p exposur e bod ywt./obs
. necrops y bleedin g Anima l car e set-u p bedding chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feeding chambe r cleanin g Hematologis t Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTA L DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABO R No . O f Hour s 42 4 45 8 41 1 ) 4 7 ) 153 9 5 7 107 5 56 0 331 2 6 0 12 2 4 7 , 15 9 292 4 977 2 [ 6 0 ' 877 2 81 2 10 5 2 3 277 0 3 0 42 4 63 6 5 3 10 6 [ 42 4 ' 109 7 9 3 1 2 25 0 9 0 10 6 2051 8 Hourl y

Wag e 34.5 3 39.6 6
13.9 4 23.5 6 10.4 0 8.8 3 9.8 7

8.0 1

6.8 6

10.7 2 23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $14,64 1 $18,16 4 $21,45 4 $1,34 3 $11,18 0 $4,94 5 $32,68 9

$78,27 4

$19,00 2

$99 7 $27 7 $3,09 0 $82 4 $1,12 1 $208,00 2 $468,00 3 $676,00 5P.2 3

- PROTOCO L # : 798.332 0

Combine d Chroni c Toxicity/Oncogenicit y Rat/inhalatio n

No . o f Uni t Othe r Direc t Cost s Unit s Cos t Overtim e (anima l tech. ) 141 0 $4.0 1 Animal s 60 0 $8.5 0 Fee d 127 3 $22.5 0 Bedding 2544 0 $0.2 0 Disposable s 73 1 $5.0 0 Histolog

ysupplie s 1 $10,080.0 0 Vet . ophthalmologis t 3 $1,000.0 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G§ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST :

COS T COMPARISON BES T ESTIMAT E PRIC E PROTOCO L ESTIMATE : $1,109,74 0 $925,59 0 ANALYTICAL CHEM : $157,92 0 $42,56 0

TOTAL : $1,267,66 0 $968,15 0 08/15/9 0

Tota l 

### Dollar s $5,64 7 $5,10 0 $28,64 3 $5,08 8 $3,65 5 $10,08 0 $3,00 0 $20,80 0 $82,01 3 $758,01 8 $166,76 4 $924,78 2 $184,95 6 $1,109,73 8

RANG E -$1,312,67 0

- $273,28 0 -$1,585,95 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.2 4

PROTOCO L # : 798.332 0 08/15/9 0 Combine d Chroni c Toxicity/Oncogenicit y Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 5 0 mal e an

d 5 0 femal e rat s a s wel l a s 3 dose d an d 1 contro l group o f 2 0 mal e an d 2 0 femal e rat s i n a satellit e group .

- Stud yduratio n i s 10 4 weeks
.P.2 5

PROTOCO L # : 798.442 0 Developmenta l Toxicit y(Preliminar y Mouse/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n ( Vet . Senio r tech
. set-u p ( compoun d prep . ( necrops y( dat a recor d keepin g i exposur e ( Tech
. set-u p exposur e bod ywt./obs
. necrops y breeding litte r bw./obs
. Anima l car e set-u p bedding chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feeding chambe r cleanin g Biostatisticia n Report/dat a coordinato r Repor t typing/processin g ComDute r code r SUBTOTA L DIREC T LABOR : Overhead.(225 % o f D.L. ) TOTA L DIREC T LABO R Screen )

No . o f Hour s 2 6 2 8 5 5 ) 7 4 7 2 0 ) 2 ) 5 ) 1 0 ] 1 0 14 9 2 0 4 0 1 6 1 5 2 8 ( 3 0 4 3 ( i o ( 4 ( 7 ( 3 ( 7 ( 4 ( 8 8 3 2 1 8 7 37 0 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 07/19/9 0

Tota l Dollar s $89 8 $39 0 $16 3 $16 5 $46 4

$1,19 3

$29 5

$18 5 $39 6 $16 5 $7 4 $4,38 8 $9,87 3 $14,26 1P.2 6

PROTOCO L # : 798.442 0 Developmenta l Toxicit y(PreliminaryScreen ) Mouse/inhalatio n

NO . O f Othe r Direc t Cost s Unit s Overtim e (anima l tech. ) 13. 0 Animal s 200. 0 Fee d 7. 0 Bedding 260. 0 Disposable s 32. 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST :

COS T COMPARISON BES T ESTIMAT E PROTOCO L ESTIMATE : $22,96 0 ANALYTICA L CHEM : $85 0

TOTAL : $23,81 0

Protoco l Estimate s ar e mad e b yvariance s i n 1 overhead , G&A , an

d fe e rate s assesse d withi n laboratories
. Uni t Cos t $4.0 1 $2.8 0 $22.5 0 $0.2 0 $5.0 0

PRIC E $19,12 0 $23 0 $19,35 0

th e fring e th e Unite d 07/19/9 0

Tota l Dollar s $5 2 $56 0 $15 8 $5 2 $16 0 $43 9 $1,42 0 $15,68 1 $3,45 0 $19,13 1 $3,82 6 $22,95 7

RANG E $27,20 0 $1,46 0 $28,66 0

benefits , 1 State sP.2 7

PROTOCO L # : 798.442 0 07/19/9 0 Developmenta l Toxicit y(PreliminaryScreen ) Mouse/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 1 dose d an d 1 contro l grou

p o f 3 0 femal e mice
.

- Stud yduratio n i s 4 1/ 2 weeks
.P.2 8

PROTOCO L # : 798.442 0 07/19/9 0 Developmenta l Toxicit y(PreliminaryScreen ) Rat/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n ( Vet . Senio r tech
. set-u p ( compoun d prep . ( necrops y( dat a recor d keepin g i exposur e ( Tech
. set-u p ( exposur e bod ywt./obs
. necrops y breeding litte r bw./obs
. Anima l car e set-u p bedding chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feeding chambe r cleaning Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABO R No . o f Hour s 2 6 2 6 5 5 ) ' 7 3 8 

### H ) 2 ) 5 ) 1 0 ) 1 0 ) 13 3 1 1 4 0 1 6 1 5 2 1 3 0 4 7 ( 5 ( 7 

### ( i o ( 3 ( 7 ( 7 ( 8 8 3 2 1 8 7 34 7 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $89 8 $36 2 $16 3 $16 5 $37 5

$1,06 5

$32 2

$18 5 $39 6 $16 5 $7 4 $4,17 1 $9,38 4 $13,55 4PROTOCO L # : 798.442 0

Developmenta l Toxicit y(PreliminaryScreen ) Rat/inhalatio n

NO . O f Othe r Direc t Cost s Unit s Overtim e (anima l tech. ) 13. 0 Animal s 108. 0 Fee d 15. 0 Beddin g 390. 0 Disposable s 32. 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST :

COS T COMPARISON BES T ESTIMAT E PROTOCO L ESTIMATE : $22,72 0 ANALYTICA L CHEM : $85 0
' TOTAL : $23,57 0

Protoco l Estimate s ar e mad e b yvariance s i n 1 overhead , G&A , an

d fe e rate s assesse d withi n laboratories
. Uni t Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $5.0 0

PRIC E $18,98 0 $23 0 $19,21 0

th e fring e th e Unite d P.2 9

07/19/9 0

Tota l Dollar s $5 2 $91 8 $33 8 $7 8 $16 0 $41 7 $1,96 3 $15,51 7 $3,41 4 $18,93 0 $3,78 6 $22,71 7

RANG E $26,83 0 $1,46 0 $28,29 0

benefits , 1 State sP.3 0

PROTOCO L # : 798.442 0 07/19/9 0 Developmenta l Toxicit y(PreliminaryScreen ) Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 1 dose d an d 1 contro l grou

p o f 3 0 femal e rats .

- Stud yduratio n i s 4 1/ 2 weeks
.P.3 1

SYNOPSI S O F PROTOCO L

TYP E O F STUDY : Reproductio n And Fertilit yEffect s NUMBE R O F ANIMALS : 20 0 Tota l (10 0 Males , 10 0 Females )
NUMBE R O F GROUPS : 3 Treate d And 1 Contro l (2 5 Males , 2 5 Females/Group )
NUMBE R O F GENERATIONS : Tw o NUMBE R O F LITTER S PE R GENERATION : On e METHO D O F COMPOUN

D ADMINISTRATION : Inhalatio n DOSIN G REGIMEN : Seve n Day s Pe r Wee k Throughou t Study ; P Male s And Female s

Dose d Fo r 1 0 Week s Prio r T o An d Throug h Breeding , F . Parenta l Animal s Fo r 8 Week s Prio r T o An d Throug h Breeding ; Parenta l Female s Dose d Throug h Gestatio n An d Lactatio n IN-LIF E OBSERVATIONS : Twic e Dail yFo r Obviou s Sign s O f Toxicity ; Physica l Examination s Weekl y BODY WEIGHT/FEE D CONSUMPTION : Weekl yFo r Parenta l Animal s PU P BODY WEIGHTS : A t Birth , And O n Day s 4 , 7 , 14 , And 21 ^ Individua l Pup s LITTE R CULLING : Day4 T o 4 Male s An d 4 Female s Pe r Litte r GROS S NECROPSY : Al l Parenta l Animal s An d Al l F

_Animal s A t Day2 1 O f Lactatio n And F . Animal s No t Selecte d A s Parenta l TISSU E PRESERVATION : Reproductiv e An d Targe t Organ s O n Al l P j An d F { Animal s Selecte d Fo r Matin g HISTOPATHOLOGY : Reproductiv e And Targe t Organ s O n Al l Hig h Dos e An d Contro l P . And F . Animal s Selecte d Fo r Matin g

1 8P.3 2

PROTOCO L # : 798.470 0 07/19/9 0 Reproductio n an

d Fertilit yEffect s Rat/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Bd
. Cert . Pathologis t slid e evaluatio n ( necrops ysupervisio n ( Q.A . Inspecto r Vet . Histologis t Histologytech
. Senio r tech
. set-u p i compound prep . i observation s ( necrops y dat a recor d keepin g exposur e Tech
. set-u p exposur e bod ywt./ob s . necrops y breedin g Anima l car e set-u p beddin g chang e cag e cleaning roommaintenanc e washin g feeders , etc . feeding chambe r cleaning Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABOR NO . O f Hour s 14 8 24 0 21 1 ) 2 9 ) 49 5 2 3 33 8 17 6 141 4 3 6 ) 3 5 ) 76 0 ; 2 9 t 5 6 49 8 299 5 3 6 199 2 88 1 4 4 ( 4 2 62 0 ( 1 8 ( 4 9 ( 7 4 ( I 9 ( 3 7 ( 4 9 ( 37 4 8 7 8 2 4 3 7 659 6 Hourl y

Wag e 34.5 3 39.6 6
13.9 4 23.5 6 10.4 0 8.8 3 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $5,11 0 $9,51 8 $6,90 0 $54 2 $3,51 5 $1,55 4 $13,95 6

$23,99 0

$4,25 3

$18 5 $96 4 $22 0 $39 1 $71,10 0 $159,97 4 $231,07 4P.3 3

PROTOCO L # : 798.470 0 Reproductio n an

d Fertilit yEffect s Rat/inhalatio n

No . o f Othe r Direc t Cost s Unit s Overtim e (anima l tech. ) 148. 0 Animal s 180. 0 Fee d 134. 0 Beddin g 2960. 0 Stain s 1. 0 Fixative s 1. 0 Disposable s 259. 0 Histolog ysupplie s 1
. 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST :

COS T COMPARISON BES T ESTIMAT E PROTOCO L ESTIMATE : $369,43 0 ';

- ANALYTICAL CHEM : $70,22 0

TOTAL : $439,65 0

Protoco l Estimate s ar e mad e b yvariance s i n 1 overhead , G&A , an

d fe e rate s assesse d withi n laboratories
. Uni t Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $200.0 0 $600.0 0 $5.0 0 $6,336.0 0

PRIC E $307,52 0 $18,92 0 $326,44 0

th e fring e the Unite d 07/19/9 0

Tota l Dollar s $59 3 $1,53 0 $3,01 5 $59 2 $20 0 $60 0 $1,29 5 $6,33 6 $7,11 0 $21,27 1 $252,34 5 $55,51 6 $307,86 0 $61,57 2 $369,43 2

RANG E $437,88 0 $121,51 0 $559,39 0

benefits , 1 State sP.3 4

PROTOCO L # : 798.470 0 07/19/9 0 Reproductio n and Fertilit yEffect s Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e findin g studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 2 0 mal e an

d 2 4 femal e rats .

- Stud yduratio n i s 3 5 weeks
.P.3 5

798.526 5 SALMONELL A TYPHIMURIU

M REVERS E MUTATIO

N ASSA Y
STUD Y DESIGNPERSTRAIN : Group 

### 1 2 3 4 5 6 7 Dose Level

Vehicl e Contro l Positiv e Contro l Concentratio n 1 Concentratio n 2 Concentratio n 3 Concentratio n 4 Concentratio n 5 

### Conditions With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n NUMBE R O F BACTERIA L STRAINS : Salmonell a typhimuriu mstrain s TA1535 , T A1537 , TA9 8 and TA100
. POSITIVE CONTROLS :

Th e positiv e contro l shoul d b e strai n specific
. NUMBE R O F PLATE S PE R CONCENTRATION :

Platin g shoul d b e don e i n triplicate
. CONDITIONS :

Wit h and withou t metaboli c activation
. Activatio n i s achieve d byth e additio n o f th e S- 9 fractio n fromth e live r o f rodent s treate d wit h a metaboli c activato r suc h a s Arochlo r 1254
. EVALUATION : Individua l plate s ar e counte d fo r th e numbe r o f

revertan t colonies
.P.3 6

PROTOCO L # : 798.526 5 Th e Salmonell a Typhimuriu m Revers e Mutatio n Assay Direc t plat e incorporatio n metho d 08/15/9 0

Direc t Labo r No . o f Hour s 5 3 8 8 ) 1 6 6 ) 1 0 ) 3 1 3 6 Hourl y

Wag e 26.4 4 13.9 4 17.3 0 9.9 4
9.1 6 10.5 8 Tota l

Dollar s $13 2 $4 2 $13 8 $15 9 $2 7 $1 1 $51 0 $1,14 6 $1,65 6 Stud yDirector-geneticist/microbio
. Q.A . Inspecto r Geneticis t plat e countin g Laboratorytechnicia n set-u p aga r overlay/platin g Repor t typing/processin g Compute r code r SUBTOTA L DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABORP.3 7

PROTOCO L #. : 798.526 5 Th e Salmonell a Typhimuriu mRevers e Mutatio n Assay Direc t plat e incorporatio n metho d 08/15/9 0

Othe r Direc t Cost s No . O f Unit s 1. 0 1. 0 2. 0 Uni t

Cos t $25.0 0 $300.0 0 $5.0 0 Tota l

Dollar s $2 5 $30 0 $1 0 $5 1 $38 6 $2,04 2 $44 9 $2,49 1 $49 8 $2,98 9 Bacteri a Plates/medi a supplie s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST : D.L .

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $2,99 0 $2,51 0 - $3,51 0 ANALYTICAL CHEM : $85 0 $23 0 - $1,46 0

TOTAL : $3,84 0 $2,74 0 - $4,97 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.3 8

PROTOCO L # : 798.526 5 08/15/9 0 Th e Salmonell a Typhimuriu mRevers e Mutatio n Assay Direc t plat e incorporatio n metho d

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Base d o n 5 strains , 5 cone , 2 positiv e an d 1 negativ e contro l group s wit h confirmin g assay .P.3 9

Group 

### 1 2 3 4 5 6 Dose Level

Vehicl e Contro l Positiv e Contro l Concentratio n 1 Concentratio n 2 Concentratio n 3 Concentratio n 4 798.530 0 DETECTIO N O F GEN E MUTATION S I N SOMATI C CELL S IN

CULTUR E
STUD Y DESIG

NPE R STRAIN : 

### Conditions With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n CEL L LINES :

Avariet yo f cel l line s ar e use d i n thi s assa y includin g subclone s o f th e Chines e hamste r ovary cells , th e L5178 Y (mous e lymphoma ) cell s an d V-7 9 cells
. NUMBE R O F CULTURE S PE R CONCENTRATION :

A t leas t duplicat e culture s shoul d b e ru n fo r eac h concentratio n an

d condition
. CONDITIONS :

Wit h and withou t metaboli c activation
. Activatio n i s • achieve d byth e additio n a chemica l known t o stimulat e th e metaboli c activit yo f th e tes t syste mt o th e cultur e media
. TES T PROCEDURES/EVALUATION :

Th e cell s ar e expose d t o th e tes t substanc e i n cultur e medi a wit h an d withou t metaboli c activatio n fo r a perio d o f 1 t o 5 hours
. A t th e en d o f th e exposur e period , th e cell s ar e washe d an d culture d t o determin e viabilit yan d th e expressio n o f mutate d cells
. Th e cell s ar e the n grow n i n selectiv e medi a fo r th e determinatio n o f th e number s o f mutant s an d th e efficienc yo f th e cell s fo r cloning
.P.4 0

PROTOCO L # : 798.530 0 Detectio n o f Gen e Mutation s i n Somati c Cell s i n Cultur e CHO/HGPR T cell s 08/15/9 0

Direc t Labo r No . o f Hour s 2 0 8 4 8 7 4 8 6 1 6 6 2 0 1 2 6 6 2 8 9 7 ) ) )
) ) ) ) ) ) Hourl y Wag e 26.4 4

13.9 4 17.3 0

9.9 4
23.0 8 9.1 6 Tota l

Dollar s $52 9
$9 8 $83 0

$11 9 $4 6 $7 3 $1,69 6 $3,81 5 $5,51 0 Stud yDirector-geneticist/microbio
. stud ysupervisio n ( dat a evaluatio n ( repor t preparatio n ( Q.A . Inspecto r Geneticis t set-u p ( cel l preparatio n ( exposur e ( cel l count s ( Laborator ytechnicia n glasswar e prep . ( medi a pre p ( Biostatisticia n Repor t typing/processin g SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTA L DIREC T LABOR

# k PROTOCO L # : 798.530 0

Detectio n o f Gen e Mutation s i n Somati c Cell s i n Cultur e CHO/HGPR T cell s

No . o f Uni t Othe r Direc t Cost s Unit s Cos t CH O cell s 1. 0 $40.0 0 Disposable s 2. 0 $5.0 0 Lab . supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST :

COS T COMPARISON BES T ESTIMAT E PRIC E PROTOCO L ESTIMATE : $8,39 0 $6,96 0 ANALYTICA

L CHEM : $85 0 $23 0 TOTAL : $9,24 0 $7,19 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d laboratories
. P.4 1

08/15/9 0

Tota l Dollar s $4 0 $1 0 $17 0 $22 0 $5,73 0 $1,26 1 $6,99 1 $1,39 8 $8,38 9

RANG E $9,98 0 $1,46 0 $11,44 0

benefits , State sP.4 2

PROTOCO L # : 798.530 0 08/15/9 0 Detectio n o f Gen e Mutation s in Somati c Cell s i n Cultur e CHO/HGPR T cell s

ASSUMPTIONS :

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Base d o n singl e culture s a t eac h level , triplicat e plate s pe r dos e fo r cytotoxicity
.

- Fiv e plate s pe r dos e evaluate d fo r mutation
.

- Fiv e dos e level s plu s positiv e an d negativ e controls
.

- Al l group s teste d wit h an d withou t metaboli c activation
.P.4 3

Group 

### 1 2 3 4 5 6 Dose Level

Vehicl e Contro l Positiv e Contro l Concentratio n 1 Concentratio n 2 Concentratio n 3 Concentratio n 4 798.537 5 I NVITR O MAMMALIA NCYTOGENETIC S
STUD Y DESIG

NPE R STRAIN : 

### Conditions With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n With/Withou t Activatio n CEL L LINES :

Avariet yo f cel l line s and primar ycel l culture s ca n b e use d i n thi s assay . Chines e hamste r ovaryand huma n lymphocyte s ar e frequentl yused
.
NUMBE R O F CULTURE S PE R CONCENTRATION :

A t leas t tw o fo r eac h experimenta l harves t tim e poin t wit h fou r tim e points
. CONDITIONS :

Wit h and withou t metaboli c activation
. Activatio n i s achieve d b

yth e additio n a chemica l known t o stimulat e th e metaboli c activit yo f th e tes t systemt o th e cultur e media
. EVALUATION : Prio r t o harvest , a mitoti c spindl e inhibito r i s adde d

t o th e cel l cultur e t o sto p th e cell s i n metaphase
. Chromosoma l preparation s ar e mad e an d th e slide s ar e evaluated
. Th e differen t type s o f chromosoma l aberration s ar e enumerate d fo r th e contro l an d treate d groups .



# IP.4 4

PROTOCO

L# : 798.537 5 08/15/9 0 I n Vitr o Mammalia n cytogenetic s Chromoson e Aberrations/CH O cell s

Direc t Labo r Stud yDirector-geneticist/microbi o stud ysupervisio n dat a evaluatio n repor t preparatio n Q.A . Inspecto r Geneticis t set-u p cel l preparation/maintenanc e staining scoring Laboratorytechnicia n set-u p cel l preparation/maintenanc e staining scoring Biostatisticia n Repor t typing/processin g SUBTOTA L DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTA L DIREC T LABO R No . O f Hour s 1 6 ( 4 ( 4 ( 8 8 3 6 ( 6 ( i o ( 6 ( 1 4 3 4 ( 4 ( 2 0 ( 2 ( 8 2 4 10 0 ) ) )
) ) ) ) ) ) ) ) Hourl y Wag e 26.4 4

13.9 4 17.3 0

9.9 4

23.0 8 9.1 6 Tota l

Dollar s $42 3
$11 2 $62 3

$33 8

$4 6 $3 7 $1,57 8 $3,55 1 $5,12 9PROTOCO L # : 798.537 5

I n Vitr o Mammalia n Cytogenetic s Chromoson e Aberrations/CH O cell s

No . o f Uni t Othe r Direc t Cost s Unit s Cos t CH O cell s 1. 0 $40.0 0 Disposable s 2. 0 $5.0 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST :

COS T COMPARISO N BES T ESTIMAT E PRIC E PROTOCO L ESTIMATE : $7,81 0 $6,48 0 ANALYTICA L CHEM : $85 0 $ 2 3 0

TOTAL : $8,66 0 $6,71 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d laboratories
. P.4 5

08/15/9 0

Tota l Dollar s $4 0 $1 0 $15 8 $20 8 $5,33 7 $1,17 4 $6,51 1 $1,30 2 $7,81 3

RANG E $9,30 0 $1,46 0 $10,76 0

benefits , State sP.4 6

PROTOCO L # : 798.537 5 I n Vitr o Mammalia n Cytogenetic s Chromoson e Aberrations/CH O cell s 08/15/9 0

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Base d o n tw o culture s a t eac h level , 2 4 hr
. screening
.

- Fou r dos e level s plu s positiv e an d negativ e controls
.

- Al l group s teste d wit h an d withou t metaboli c activation
.P.4 7

798.538 5 IN

VIV O MAMMALIA

N BON E MARRO W CYTOGENETIC S TESTS : CHROMOSOMA

L ANALYSI S
STUD YDESIGN : Group 1 2 3 4 5 Dose Level

Negativ e Contro l Positiv e Contro l Lo w Dos e Mi dDos e Hig hDos e Number of Animals 1 5 males , 1 5 female s 1 5 males , 1 5 female s 1 5 males , 1 5 female s 1 5 males , 1 5 female s 1 5 males , 1 5 female s
ANIMA L SPECIE S O

RSTRAIN : Laborator

y rats , mic e or hamster s AG E O F ANIMALS :

Sexuall y

mature , youn

g adults . FREQUENCYO F EXPOSURE :

A singl e exposur e i s mos t widel y usedbu t othe r treatmen t schedule s ma y b e used . ROUT E O F EXPOSURE :

Oral , vi a gavage , o r intraperitonea l injection . Othe r route s ca

nbe selected , i f appropriate . SACRIFIC E SCHEDULE :

Fiv e male s an

dfiv e female s fro

m eachgrou

par e sacrificeda 6 , 2 4 an d4 8 hour s afte r dosing . EVALUATION : Bone marro w sample s ar e takenfro m eachanima l an

d slide s ar e preparedfor evaluation . Chromosoma l change s i nthe cell s i nmetaphas e ar e evaluatedfro m eachanimal .P.4 8

PROTOCO L # : 798.538 5 07/20/9 0 I n Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Chromosoma l Analysi s Rat/inhalatio n

Direc t Labo r Stud yDirector-geneticist/microbio
. Q.A . Inspecto r Vet . Clinica l lab
. tech
. Geneticis t Senio r tech
. set-u p ( compound prep . necrops y dat a recor d keepin g exposur e Tech
. set-u p exposur e bod ywt./obs
. necrops y Anima l car e set-u p beddin g chang e cag e cleanin g roommaintenanc e washin g feeders , etc . feeding chambe r cleanin g Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTA L DIREC T LABO R No . O f Hour s 3 6 2 8 6 7 5 5 0 3 5 

### i s ; 3 ' 1 3 2 2 6 6 1 5 8 5 ( 3 8 2 9 8 ( 4 ( 6 ( 2 ( 3 ( 4 ( 2 2 3 0 1 2 2 37 1 Hourl y

Wag e 26.4 4 13.9 4 23.5 6 9.9 4 17.3 0 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $95 2 $39 0 $14 1 $74 6 $86 5 $34 5

$52 9

$19 9

$4 6 $37 1 $11 0 $2 1 $4,71 5 $10,60 9 $15,32 4P.4 9

PROTOCO L # : 798.538 5 07/20/9 0 In Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Chromosoma l Analysi s Rat/inhalatio n

Othe r Direc t Cost s No . o f Unit s 6. 0 180. 0 11. 0 240. 0 300. 0 2. 0 Uni t

Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $2.0 0 $5.0 0 Tota l

Dollar s $2 4 $1,53 0 $24 8 $4 8 $60 0 $1 0 $47 2 $2,93 1 $18,25 5 $4,01 6 $22,27 1 $4,45 4 $26,72 6 Overtim e (anima l tech. ) Animal s Fee d Beddin g Stains/slide s Disposable s Lab
. supplie s (10 % o f SubTot , TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST : D.L . )

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $26,73 0 $22,39 0 - $31,47 0

TOTAL : $26,73 0 $22,39 0 - $31,47 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.5 0

PROTOCO L # : 798.538 5 07/20/9 0 I n Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Chromosoma l Analysi s Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e findin g studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dosed , 1 positiv e and 1 negativ e contro l group s o f 5 mal e an d 5 femal e rat s fo r thre e sampling interval s a t 6 , 2 4 an d 4 8 hour s afte r treatment .P.5 1

PROTOCO L 798.539 5 07/20/9 0 I n Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Micronucleu s Assa y Mouse/inhalatio n

Direc t Labo r Stud yDirector-geneticist/microbio
. Q.A . Inspecto r Vet . Clinica l lab
. tech
. Geneticis t Senio r tech
. set-u p compoun d prep . necrops y dat a recor d keepin g exposur e Tech
. set-u p exposur e bod ywt./obs
. necrops y Anima l car e set-u p beddin g chang e cag e cleanin g roo m maintenanc e washin g feeders , feedin g chambe r cleanin g Biostatisticia n etc .
Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTA L DIREC T LABOR : Overhea d (225 % TOTA L DIREC T LABO R o f D.L. ) No . o f Hour s 3 6 2 7 6 7 5 5 0 3 5 1 5 ) 3 ) 1 3 2 ' 2 6 6 1 5 8 5 3 8 2 2 ( 8 ( 2 ( 3 ( 2 ( 3 ( 2 ( 2 2 3 0 1 2 2 36 3 Hourl y

Wag e 26.4 4 13.9 4 23.5 6 9 .9 4 17.3 0 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $95 2 $37 6 $14 1 $74 6 $86 5 $34 5

$52 9

$15 1

$4 6 $37 1 $11 0 $2 1 $4,65 3 $10,47 0 $15,12 3P.5 2

PROTOCO L # : 798.539 5 I n Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Micronucleu s Assay Mouse/inhalatio n

No . o f Othe r Direc t Cost s Unit s Overtim e (anima l tech. ) 6. 0 Animal s 180. 0 Fee d 3. 0 Bedding 120. 0 Stains/slide s 300. 0 Disposable s 2. 0 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST :

COS T COMPARISON BES T ESTIMAT E PROTOCO L ESTIMATE : $24,62 0

TOTAL : $24,62 0

Protoco l Estimate s ar e mad e b yvariance s i n 1 overhead , G&A , an

d fe e rate s assesse d withi n laboratories
. Uni t Cos t $4.0 1 $2.8 0 $22.5 0 $0.2 0 $2.0 0 $5.0 0

PRIC E $20,52 0 $20,52 0

th e fring e th e Unite d 07/20/9 0

Tota l Dollar s $2 4 $50 4 $6 8 $2 4 $60 0 $1 0 $46 5 $1,69 5 $16,81 8 $3,70 0 $20,51 7 $4,10 3 $24,62 1

RANG E $29,14 0 $29,14 0

benefits , 1 State sP.5 3

PROTOCO L # : 798.539 5 07/20/9 0 I n Viv o Mammalia n Bon e Marro wCytogenetic s Tests : Micronucleu s Assay Mouse/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dosed , 1 positiv e an d 1 negativ e contro l group s o f 5 mal e an d 5 femal e mic e fo r thre e samplin g interval s a t 6 , 2 4 an d 4 8 hour s afte r treatment .P.5 4

PROTOCO L # : 798.555 0 Unschedule d DN

ASynthesi s i n Mammalia n Cell s i n Cultur e

CEL L TYPE :

Ra t Hepatocyte s TES T CONCENTRATIONS :

Fiv e tes t articl e concentrations , positiv e an d negativ e controls
. RADIOTRACE R DETERMINATION :

Autoradiography
. NUMBE R O F CULTURES :

Si x pe r concentration
. CEL L COUNTING :

Fift y(50 ) cell s pe r culture
.J&J55
_

PROTOCO L # : 798.555 0 Unschedule d DN

ASynthesi s i n Mammalia n Cell s i n Cultur e Primar yculture s o f roden t hepatocyte s 06/24/9 1

Direc t Labo r No . o f Hour s Hourl y

Wag e Tota l

Dollar s Stud yDirector-geneticist/raicrobio
. stud ysupervisio n dat a evaluatio n repor t preparatio n Q.A . Inspecto r Geneticis t set-up cel l preparation/maintenanc e stainin g scorin g Laborator ytechnicia n set-u p cel l preparation/maintenanc e stainin g scorin g Anima l car e set-up beddin g chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feedin g Biostatisticia n Repor t typing/processin g SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABOR 3 2 1 6 8 8 9 3 6 6 1 0 6 1 4 3 4 4 2 0 8 n l 1 1 1 

# i

<z 1 2 4 11 5 26.4 4

13.9 4 17.3 0

9.9 4

6.8 6

23.0 8 9.1 6 $84 6

$12 5 $62 3

$33 8

$4 8

$4 6 $3 7 $2,06 3 $4,64 2 $6,70 5P.5 6

PROTOCO L # : 798.535 0 06/24/9 1 Unschedule d DN

ASyntnesi s in Mammalia n Cell s i n Cultur e Primaryculture s o f roden t hepatocyte s

Othe r Direc t Cost s Animal s Fee d Beddin g Medi a & Stain s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) No . o f Unit s 2. 0 1. 0 40. 0 1. 0 2. 0 D.L. ) 

### uni t Cos t $8.5 0 $22.5 0 $0.2 0 $200.0 0 $5.0 0 Tota l

Dollar s $1 7 $2 3 $ 8 $20 0 $1 0 $20 6 $46 4 $7,16 9 $1,57 7 $8,74 6 $1,74 9 ESTIMATE D FINA L COST : $10,49 5

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $10,50 0 $8,72 0 - $12,46 0

TOTAL : $10,50 0 $8,72 0 - $12,46 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.5 7

PROTOCO L # : 798.555 0 06/24/9 1 Unschedule d DN

ASynthesi s i n Mammalia n Cell s i n Cultur e Primaryculture s o f roden t hepatocyte s

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e findin g studies
.

- Doe s no t includ e cos t fo r analytica l chemistry
.

- Base d o n si x culture s a t eac h level , 2 4 hr
. screening
.

- Fiv e dos e level s plu s positiv e an d negativ e controls
.

- Al l group s teste d withou t metaboli c activation
.

- UD S i s measure d b yautoradiograph

ytechnigue
.P.5 8



### PROTOCO L# t 798.590 0 I n Vitr o Siste r

Chromati dExchang e Assa y

CEL L TYPE :

Chines e Hamste r Ovar y TES T CONCENTRATIONS :

Fiv e tes t articl e concentrations , positiv e and negativ e controls
. SC E DETERMINATION :

Fluorescenc e plu s Giems a Metho d NUMBE R O F CULTURES :

A t leas t tw o independen t culture s pe r dat a point
. CEL L EXPOSURE :

Cell s shoul d b e expose d t o th e tes t chemica l durin g a t leas t tw o time intervals , e.g. , G O an d S phases
.P.5 9

PROTOCO L # : 798.590 0 06/24/9 1 I n Vitr o Siste r Chromati d Exchang e Assa y Chines e Hamste r OvaryCell s

Direc t Labo r Stud yDirector-geneticist/microbio
. stud ysupervisio n dat a evaluatio n repor t preparatio n Q.A . Inspecto r Geneticis t set-up cel l preparation/maintenanc e stainin g scorin g Laborator ytechnicia n set-u p cel l preparation/maintenanc e stainin g scorin g Biostatisticia n Repor t typing/processin g SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIREC T LABO R No . o f Hour s 3 4 ( 1 6 ( 6 ( 1 2 9 3 6 ( 6 ( 1 2 ( 6 ( 1 2 4 0 ( 6 2 4 ( 2 ( 8 2 4 11 6 ) ) )
) ) ) ) ) ) ) ) Hourl y Wag e 26.4 4

13.9 4 17.3 0

9.9 4

23.0 8 9.1 6 Tota l

Dollar s $89 9
$12 5 $62 3

$39 8

$4 6 $3 7 $2,12 8 $4,78 7 $6,91 5P.6 0

PROTOCO L # : 798.590 0 I n Vitr o Siste r Chromati d Exchang e Assa y Chines e Hamste r Ovar yCell s 06/24/9 1

Othe r Direc t Cost s No . o f Unit s Uni t

Cos t Tota l

Dollar s $20 0 $1 0 $21 3 $42 3 $7,33 8 $1,61 4 $8,95 2 $1,79 0 $10,74 2 Medi a & stain s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTA L OTHE R DIREC T COSTS : TOTA L BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % O f Tota l Cost ) ESTIMATE D FINA L COST : 1. 0 2. 0 D.L . ; $200.0 0 $5.0 0

COS T COMPARISO N

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $10,74 0 $8,92 0 - $12,76 0

TOTAL : $10,74 0 $8,92 0 - $12,76 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.6 1

PROTOCO L # : 798.590 0 I n Vitr o Siste r Chromati d Exchang e Assay Chines e Hamste r Ovar yCell s 06/24/9 1

ASSUMPTIONS :

- Doe s no t includ e cos t

- Doe s no t includ e cos t

- Base d o n tw o culture s

- Fiv e dos e level s plu s 

### fo r dos e rang e findin g studies , fo r analytica l chemistry , fo r eac hexperimenta l point , positiv e an

dnegativ e controls

.

- Al l group s teste dwit handv/ithou t metaboli c activation

.PROTOCO L # : 798.591 5

I n Viv o Siste r Chromati d Exchang e Assay

STUDY DESIGN : Grou p l 2 3 Dos e Leve l

Lo wDos e Mi d Dos e Hig h Dos e 06/25/9 1

vssa y

Numbe r o f Animal s 5 males , 5 female s 5 males , 5 female s 5 males , 5 female s P.6 2

ANIMA L SPECIE S OR STRAIN :

Laborator yrats . AG E O F ANIMALS :

Youn g adul t animals
. DURATIONAND FREQUENC Y O F EXPOSURE :

Asingl e exposur e ove r a perio d o f a t leas t 4 hour s followe d b

yBdr U injection s an d treatmen t wit h a spindl e inhibito r 2 hour s prio r t o sacrifice
. ROUT E O F EXPOSURE :

Inhalation , fo r a t leas t 4 hours
. CEL L TYPE :

Bon e marro w TES T CONCENTRATIONS :

Thre e tes t articl e concentrations , positiv e an d negativ e controls
.P.6 3

PROTOCO L 0i798.591 5 I n Viv o Siste r Chromati d Exchang e Assa y Rat/inhalatio n 06/25/9 1

Direc t Labo r Stud yDirector-geneticist/microbio , Q.A . Inspecto r Vet . Clinica l lab
. tech
. Geneticis t Senio r tech
. set-u p compound prep . necrops y dat a recor d keepin g exposur e Tech '

. set-u p exposur e bod ywt./obs
. necrops y Anima l car e set-up beddin g chang e cag e cleanin g roommaintenanc e washin g feeders , etc . feedin g chambe r cleanin g Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTA L DIREC T LABO R No . o f Hour s 3 6 1 5 6 2 5 1 7 1 8 ( 5 ) ( 3 ) ( 4 ) ( 2 ( 4 ) 3 4 ( 5 ] ( 1 4 ( 2 ( 1 3 ' 2 0 ( 3 ( 2 ( 3 ( 2 ( 3 ( 2 (5 2 3 0 1 2 1 20 2 Hourl y

Wag e 26.4 4 13.9 4 23.5 6 9.9 4 17.3 0 9.8 7

8.0 1

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 Tota l

Dollar s $95 2 $20 9 $14 1 $24 9 $29 4 $17 8

$27 2

$13 7

$4 6 $37 1 $11 0 $2 1 $2,98 0 $6,70 5 $9,68 5P.6 4

PROTOCO L # : 798.591 5 I n Viv o Siste r Chromati d Exchang e Assay Rat/inhalatio n 06/25/9 1

Othe r Direc t Cost s Overtim e (anima l tech. ) Animal s Fee d Beddin g Stains/slide s Disposable s Lab
. supplie s (10 % o f SubTot
. TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST : No . o f Unit s 6. 0 60. 0 4. 0 120. 0 100, 0 2. 0 D.L. ) Uni t Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $2.0 0 $5.0 0 Tota l

Dollar s $2 4 $51 0 $9 0 $2 4 $20 0 $1 0 $29 8 $1,15 6 $10,84 2 $2,38 5 $13,22 7 $2,64 5 $15,87 2

COS T COMPARISON

BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $15,87 0 $13,24 0 - $18,78 0

TOTAL : $15,87 0 $13,24 0 - $18,78 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d state s laboratories
.P.6 5

PROTOCO L # : 798.591 5 06/25/9 1 I n Viv o Siste r Chromati d Exchang e Assay Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e findin g studies
.

- Doe s no t includ e cos t fo r analytica l chemistry
.

- Stud ycompose d o f 3 dosed , 1 positiv e an d l negativ e contro l group s o f 5 mal e an d 5 femal e rats .

- Animal s administere d Brd U afte r exposur e t o tes t chemica l an d a spindl e inhibito r 2 hour s prio r t o sacrifice
.

- Afte r sacrifice , bon e marro wi s extracte d an d slide s mad e an

d prepare d fo r SC E evaluation
.P.6 6

PROTOCO L # : 798.605 0 07/19/9 0 Functiona l Observationa l Battery(Acute ) Rat/inhalatio n

Direc t Labo r Stud yDirector-toxicologist(PhD ) Q.A . Inspecto r Vet . Pathologis t necrops ysupervisio n ( Vet . Senio r tech
. set-u p i compound prep . I necrops yi dat a recor d keepin g i exposur e i FO B testin g i Tech
. set-u p exposur e bod ywt./obs
. necrops y FO B testin g Anima l car e set-u p beddin g chang e cag e cleanin g roommaintenanc e washin g feeders , etc . feeding chambe r cleanin g Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTAL DIREC T LABOR : Overhea d (225 % o f D.L. ) TOTAL DIRECT,LABO R NO . O f Hour s 

### 1 6 1 8 

### 7 7 ) 

### 6 4 2 9 ) 2 ) 7 ; 6 ) 2 ) 1 6 ] 

### 9 8 9 1 

### 8 1 8 I 1 5 ( 4 8 

### 2 3 [ 5 ( 3 

### 4 ( 2 ( 4 ( 3 : 2 

### 2 1 

### 8 4 24 3 Hourl y

Wag e 34.5 3 13.9 4 32.6 9 23.5 6 9.8 7

8.0 1

6.8 6

12.3 6 9.1 6 10.5 8 Tota l

Dollar s $55 2 $25 1 $22 9 $14 1 $41 5

$78 5

$15 8

$26 0 

### $7 3 $4 2 $2,90 6 $6,53 9 $9,44 5

•T-.KS>--v-

-PROTOCO L # : 798.605 0

Functiona l Observationa l Battery(Acute ) Rat/inhalatio n

No . o f Othe r Direc t Cost s Unit s Overtim e (anima l tech. ) 8 Animal s 9 0 Fee d 7 Beddin g 16 0 Disposable s 1 4 Lab
. supplie s (10 % o f SubTot
. D.L. ) TOTAL OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@A( 22 % o f Tota l Cost ) TOTAL BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINAL COST :

COS T COMPARISON BES T ESTIMAT E PROTOCO L ESTIMATE : $15,80 0

TOTAL : $15,80 0 Uni t Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $5.0 0

PRIC E $13,20 0 - $13,20 0 -

Protoco l Estimate s ar e mad e b yvariances , i n th e fring e overhead , G&A , and fe e rate s assesse d withi n laboratories
.

•••'•.•v-'-A . •- ' .•-£*>•"--.•-:..•--s-rf ^ .^•-••;-»':<i A .-..-A'MifjijV.rViis-:-' : P.6 7

07/19/9 0

Tota l Dollar s 

### $3 2 $76 5 $15 8 

### $3 2 $7 0 $29 1 $1,34 7 $10,79 2 $2,37 4 $13,16 6 $2,63 3 $15,79 9

RANG E $18,66 0 $18,66 0

benefits , th e Unite d State*

-

r :•• - . :..- ~ .

'^^izZyiyy*•?'•*. . . ^i-

- ... .

- «H* A•:*,.«*-
.P.6 8

PROTOCO L # : 798.605 0 07/19/9 0 Functiona l Observationa l Battery(Acute ) Rat/inhalatio n

ASSUMPTIONS :

- Doe s no t includ e cos t fo r dos e rang e finding studies
.

- Doe s no t includ e cos t fo r analytica l metho d developmen t o r validation
.

- Stud ycompose d o f 3 dose d an d 1 contro l grou

p o f 1 0 mal e an

d 1 0 femal e rats .

- Stud yduratio n i s 2 weeks
.P.6 9

798.605 0 FUNCTIONA L OBSERVATIONA

L BATTER

Y
STUD YDESIGN : Grou p 1 2 3 4 Dos e Leve l

Contro l Lo w dos e Middos e Hig hdos e Number o f Animal s

1 0 males , 1 0 female s 1 0 males , 1 0 female s 1 0 males , 1 0 female s 1 0 males , 1 0 female s SATELLIT E GROUPS :

A satellit e grou

pma y b e treate

dwit hth e hig hdos e leve l fo

r th e duratio

no f exposur e an

dobserve

dfo r reversibility , persistenc e or delaye doccurrenc e o f toxi c effect s for a post-treatmen t perio dof appropriat e duration , normall y not les s tha

n2 8 days (no t include

di nth e cos t estimate) . ANIMA L SPECIE S O

RSTRAIN : Laborator

y rats . AG E O F ANIMALS : A t leas t 4 2 day s DURATIO N AN D FREQUENC

YO F EXPOSURE : Dail y dosing , seve

nday s pe r wee

k fo r 13 weeks . ROUT E O F EXPOSURE : Dermal , non-occluded . OTHER : Concurren t o r historica l dat a fro m th e laborator

y performin

g th e tes t t o provide evidenc e tha t th e observatio

nprocedure s detec t neurotoxi c changes . Th e test s ma

y b e performedo

nsubject s fro

m othe r toxicity

test s providedtha t th e requirement s o f neithe r stud

y ar e violatedb

y combination . BOD YWEIGHTS :

Weekly , throughou t th e study . OBSERVATIO

N INTERVALS : Prio r t o stud

y initiation , 1 , 6 an d2 4 hour s afte r initia l treatment , day s 7 an d14 of stud

y an dmonthl y thereafter . OBSERVATIONS : A . An

y unusua l response s withrespec t t o bod y position , activit y

level , coordinationan dgait . B . Unusua l o

r bizzar e behavio r suc ha s headflicking , hea d searching , compulsiv e bitin

g or licking , sel f mutilation , circlin g an dwalkin

g backwards .P.7 0



### c .

D . toiicit v

ll'D? SUC h" th ° S e t 3nPie«U y observe din toxicit y

studie s an

d considere drelate dt o slllvatfnn 3 1 Chan ? e s ' e ' 8 - increase dlacrimatio no r const*!.?? ' con ^^i°n s o r tremors , pupi l dilatio no r diarrhl ! , ' chan « e s i nrespiratio no r urination , diarrhea , piloerectio nan d vocalization . Forelim

ban d hindlia bgri pstrength .

E " ^uictii n S llX* ° f te ; t £ t 0 evaluat e •"•«* « onsenso r

i»ef„ i ; r f fl8Xe s an dP a i nPerception . Thes e ma y includ e test s suc ha s auditor y startl e respons e Tickin g refl6X ' ^ i n " y "«« . ho t plaE"; * balanc f MnS?? 8 ! ' b f lanC B bea m •1« i "bri»« . rotoro d r!.«« n h f ndllB bs Pi* y measurements , visua l placin g response , to e and/o r tai l pinc hresponse , visua l <no-i?fuI MPO ?" # , ' n dreactio nt o Poaitiona l change s (positiv e geotropis m an d awkwar dpositions) . ASSUMPTIONS :

hlstorirl f h " Hi l ° f th e re « uir * dequipmen t an d o f thii"estr PerienC e a " d COntro i dat a fo r th e conduc t yP.7 1

CF R 79B.605 0 0 3 JU N199 0

PRGTOCD L LSTIMAT E FUNCTIONA L OBSERVATIONA L DATTER Y

Ra t . ' Inhalatio n Subcroni c

Direc t Labo r Num

. O f Hour s Hou r1 y

Waa e Tota l Dollar s
Btuu vDi rToxicoloqist(PhD )

. Ar-t

. ;,i=aecta r ete n nar yPathologis t Necrops ysupervisio n _a a A n i rn a 1 Ve t et " inar i a n Senio ri-irtirna l Technicia n Se cu p Compoun d preparatio n Dat a recor d keepin g Necrops v Exposur e FO B testin g r-.i \ i ina i Tecn n i ci a n

. OI J - . .vrf i ul'its /

O b se rva t i ori s

. ) f i L rG i j 3 V :..Gu:ui' e

•

_o
_. = •

= C i ("iC j ni'lliiia l Jai'Stdlket " ;":ocii , -se t

u p Deddin a chang e !"•>ooi" n iiia i n t e n anc e Cau e cleanin g Feed i n g hashin g feeders , etc . ChaitiDe r cleanin g Diostat i st i cia n f-.eoor t Writer/Dat a Coordinato r Wor d Processor/Typis t Compute r Code r SUBTOTA L DIREC T LABOR : \
( ( ( ( ( ( \ (

I . (. ( ( ( s i ( ( 60
. 0 0 ;31.0 0 7
. 0 0 7.00 ) 12.0 0 262.0 0 

### a . 

### oo ) 15.00 ) 23.00 ) 7
. 00 ) 182.00 ) 30.00 ) 394.00
. 3.00 ) o3.00 ) 15.00 ) 72B.OO ) i-30.00 ) 199.0 0 4
. 00 ) 10.00 ) 3
. 00 ) 15.00 ) 10.00 ) 15.00 ) 137.00 ) 8
. 0 0 33.0 0 20
. 0 0 15.0 0 34.5 3

13.9 4 32.6 9 23.5 6 9.3 7

Q . 0 1

5.8 6

23.0 3 9
. 1 6 10.5 3 ,071.3 0

,12?.1 4 228.8 3 2B2.7 2 :.535.? 4

." , 160.9 4

J . . JUwJ , 1 4

184.6 4 407.3 8 183.2 0 .158.7 0 $15,758.93
Overhea d (225.0'/

. o f D.L. ) TOTA L DIREC TLABOR : $51,216.5 2P.7 2

PROTOCO L # : 4 0 CF R 798.605 0 0 8 JUN199 0

FUNCTIONA L OBSERVATIONA L DATTER Y

Ra t / Inhalatio n Subcroni c

Othe r Direc t Cost s

Over t ijie : rimma l Technicia n Rat s Fee d Eieddin g Disposable s La b Supplie s ( 10.07
. o f SubTot
. D.L. ) TOTA L OTHE R DIREC T COSTS :

TOTAL COO T DEFOR E GS<A : G& A( 22.07
. o f Tota l Cost ) "OTAL COO T BEFOR E FEE : FE E ; ' 20.07
. o f Tota l Cost ) Num . O f Unit s

150.0 0 90.0 0 24
. 0 0 600.0 0 91
. 0 0 .L. ) Uni t

Cos t

4.0 1 8.5 0 22.5 0 0
. 2 0 5.0 0 1575.8 9 1

$4 600.7 5 765.0 0 540.0 0 120.0 0 455.0 0 ,575.8 9 .056.6 4

ISTITIATED FINA L COST : -••• O O a-^

. > •- > • X > 12.160.1 0 •-E67

. 433

. 2 7 13.486.6 5
£80.919.9 2

PROTOCO L ESTIMATE : ANALYITCA L CHEM. :

TOTAL : COS T COMPARISO N

BES T ESTIMAT E $80,92 0 $23,41 0 $104,33 0 PRIC E RANG E $6 : SO O 96,00 0 $6,31 0 - 40,50 0 $73,61 0 - 136,50 0

;:,sjj"ft>*
.P.7 3

PROTOCO L it s 4 0 CF R 798.605 0 0 8 JU N 179 0

FUNCTIONA L OBSERVATIONA L BATTER Y

R a t / Inhalatio n Subcroni c

Protoco l Estimate s are mad e b

yvariance s i n th e fring e benefit : overhead , G&A , and fe e rate s assese d withi n th e Unite d State s 1aborstarles
.

ASSUMPTIONS :

- 1 contro l & 3 treate d group s ot 1 0 male s and 1 0 female s each
.

- N o analytica l chemistr yvalidatio n o f stabilit yo r dosag e verification
.
ANALYTICA L CHEMISTR Y COSTS : Analvtica l Chemistr ycost s ar e estimate d t o rang e fro m: £ 6,31 0 t o $ 40,500
.P.7 4

798.710 0 METABOLIS M
STUD YDESIGN : Group 1 2 3 4 Dose Level

Lo w Dos e Lo w Dos e Lo w Dos e Hig hDos e itout

e I V x l * O Rx l * * O Rxl 4 ** * O Rx l Number

of Animals 4 males , 4 female s 4 males , 4 female s 4 males , 4 female s 4 males , 4 female s singl e intravenou s dos e singl e ora l dos e ** * dail y

ora l dos e for 1 4 day s * * *

ANIMA L 8PECIE S O

RSTRAIN : Laborator

y rat s (dog s sometime s used) . AG E OF ANIMALS :

Youn g adults . NATURE OF TH E CHEMICAL :

Analyticall y

pure , radiolabelledtes t chemica l shoul d be used . DURATIO N OF TEST : A t leas t 7 day s afte r administratio no f the radiolabelled tes t chemica l or unti l 95 % recover y o f the administereddos e i s excreted . ROUT E OF EXPOSURE :

Ora l dosin

g ma y be achievedvi a gavag e o r capsul e (generall y

i ndo g studies) . The intravenou s dos e grou pi s used for compariso nt o the ora l dos e group s t o determine o f the rat e an ddegre e o f absorptiono f the tes t chemical . DOSIN GREGIMEN : Al l animal s i ngroup s 1, 2 an d4 receiv

e a singl e dos e o f the radiolabelledtes t chemical . The grou p3 animal s receive singl e ora l dose s o f nonradiolabelled materia l dail y

for 1 4 consecutiv e days . Twenty-four hour s afte r the las t dose , th e grou p3 animal s receiv e a singl e ora l dos e o f the radiolabelledtes t chemical .P.7 5

798.710 0 METABOLIS M (CONT. )

SAMPL E COLLECTION/ANALYSIS :

Quantitie s o f radiolabe l inthe urine , fece s an d expiredai r ar e determinedi nsample s collecteda t 4,8,12,2 4 an

d3 6 hours , 2,3,4,5, 6 an d7 day s pos t dosin g wit hthe radiolabelledtes t chemical . A t sacrifice , the brain , fat , gonads , bone , heart , kidneys , liver , lungs , muscle , spleen , tissue s showin g gros s patholog

y an dcarcas s ar e retainedan dthe quantity

o f radiolabe l determined . Appropriat e analytica l procedure s ar e followedt o determine t o the exten t o f metabolis m o f the tes t chemica l inurin e an dfece s sample s fro m al l groups . The identificatio

no f the majo r metabolite s i s no t requiredbu t ma

y be requested .PROTOCO L # : 798.710 0

Metabolis m Rat/inhalatio n

Direc t Labo r Stud yDirector-anal
. chem
. Q.A . Inspecto r Chemis t extraction s ( GC/TLC , GC/M S ( dat a collection/analysi s analytica l monitorin g Chemistr ytech
. set-u p radiocarbo n analysi s homogenization/combustio n TLC/extraction s dat a reductio n dat a entr y Senio r tech
. set-u p compoun d prep . dat a recor d keepin g sampl e collectio n exposur e Tech
. set-u p exposur e anima l roo mactivitie s bodywt./obs
. sampl e collectio n Chem
. lab
. assistan t homogenization/combustio n glasswar e prep./maintenanc e radiocarbo n analysi s Anima l car e set-u p metab
. cage s beddin g chang e cag e cleanin g roo mmaintenanc e washin g feeders , etc . feedin g chambe r cleanin g Biostatisticia n Report/dat a coordinato r Repor t typing/processin g Compute r code r SUBTOTA L DIREC T LABOR : No . o f Hour s 6 0 24 3 29 5 4 8 , 19 2 4 8 7 121 9 1 6 50 0 17 3 12 8 13 4 26 8 4 8 8 1 8 1 3 1 8 13 5 1 0 7 2 1 5 1 2 2 6 110 5 17 3 3 2 90 0 4 3 5 2 3 3 5 2 2 3 8 4 0 4 0 3 323 9 Hourl y

Wag e 21.6 3 13.9 4 12.7 4

9.3 8

9.8 7

8.0 1

7.0 9

6.8 6

23.0 8 12.3 6 9.1 6 10.5 8 08/16/9 0

Tota l Dollar s $1,29 8 $3,38 7 $3,75 8

$11,43 4

$47 4

$1,08 1

$7,83 4
$29 5

$18 5 $49 4 $36 6 $3 2 $30,63 9 P.7 6P.7 7

PROTOCO L # : 798.710 0 Metabolis m Rat/inhalatio n 08/16/9 0

Othe r Direc t Cost s No . O f Unit s 10. 0 40. 0 5. 0 100. 0 1. 0 1. 0 22. 0 1. 0 Uni t

Cos t $4.0 1 $8.5 0 $22.5 0 $0.2 0 $1,500.0 0 $200.0 0 $5.0 0 $6,000.0 0 Tota l

Dollar s $4 0 $34 0 $11 3 $2 0 $1,50 0 $20 0 $11 0 $6,00 0 $3,06 4 $11,38 7 $110,96 5 $24,41 2 $135,37 7 $27,07 5 $162,45 2 Overtim e (anima l tech. ) Animal s Fee d Beddin g Solvent s TL C plate s Disposable s Countin g accessories/autoradiogr a Lab
. supplie s (10 % o f SubTot
. D.L . TOTA L OTHE R DIREC T COSTS : TOTAL BEFOR E G&A : G@ A( 22 % o f Tota l Cost ) TOTA L BEFOR E FEE : FE E ( 20.0 % o f Tota l Cost ) ESTIMATE D FINA

L COST :

COS T COMPARISO N BES T ESTIMAT E PRIC E RANG E PROTOCO L ESTIMATE : $162,45 0

TOTAL : $162,45 0 $135,43 0 - $135,43 0 - $192,25 0

$192,25 0

Protoco l Estimate s ar e mad e b yvariance s i n th e fring e benefits , overhead , G&A , an

d fe e rate s assesse d withi n th e Unite d State s laboratories
.P.7S
_

PROTOCO L # : 798.710 0 Metabolis m Rat/inhalatio n 08/16/9 0

ASSUMPTIONS : TL C fo r quantificatio n utilize s poole d sample s fro msam e sexe d animal s fro

meac h grou

p an d a t eac h interval
. GC/M S metabolit e identificatio n assume d o n spot s fro m thre e intervals
. Exposur e vi a th e a s a comparativ e Doe s no t includ e Doe s no t includ e validation
. Doe s no t includ e o r fo r synthesi s gavag e rout e wit h a n intravenou s group

contro l dos e rang e finding studies
.

analytica l metho d developmen t cos t cos t fo r fo r o r cos t fo r radiolabelle d material($10,000-20,000 ) o f metabolite s standards
.P.7 9

I . COS T ESTIMATIO

N PROCEDURE S

A . OVERVIE

W
The methodolog

y discussedbelo

w describe s the step s taken i nth e preparationo f cos t estimate s for chemica l fate , environ menta l effect s an

d healt heffect s test s a s requiredb y the Offic e
o f Toxi c Substances , U.S . Environmenta l Protectio nAgency . Thi s methodolog

y ha s beendevelopedfro m contracto r experienc e i n servin g the testin

g need s o f variou s governmenta l agencie s an d industry . The principl e component s o f the cos t estimate s ar e tota l direc t labo r (TDL) , Overhead(OH) , Othe r Direc t Cost s (ODC) , Genera l an

dAdministrativ

e Cos t (G&A ) an

d Fee . Eacho f thes e component s ar e discussedi ndetai l below .

B . TOTA

L DIREC T LABO

R
Uponreceip t o f a tas k assignment , th e progra

m manage r reviews the scope o f wor k t o determin e the requirement s t o b e met . I f th e assignmen t require s th e costin g o f a standardtes t guideline , a cop

y o f tha t guidelin e i s secured , the tes t procedure s withi nthe guidelin e ar e reviewedan da noutline o f the stud

y protoco l i s prepared . Occasionally , a tes t rul e require s th e combinatio no f testing procedure s fro m severa l test s
o r evaluationo f journa l article s for cos t considerations . I n thes e cases , a stud y design/protoco l i s preparedt o compl y wit h
curren t regulator

y requirement s an

dreviewedfor technica l an d logistica l feasibility . An y question s tha t aris e ar e passedP.8 0

throug hth e EPA/RI B contac t t o the appropriat e technica l personne l withi nthe Agency . Uponcompletio no f th e stud y design , th e outlin e o f the test , a s indicatedabove , i s prepared .
The outlin e i s a

nitemize

dbreakdow

no f the stud

y desig n int o the variou s component s required b y th e protocol , e.g. , the numbe r o f animals , duratio no f the test , dosin g regime , special ize dtests , etc . The outline i s furthe r subdividedint o tas k component s accordin g t o technica l areas , e.g . anima l care , his tology , analytica l chemistry , etc . The requiredtask s ar e reviewedan

d the usua l tim

e requiredfor completio ni s estimated . Afte r completiono f thi s proces s for al l task s containe di nthe outline , a labo r category/tas k performanc e shee t i s completed . Thi s shee t list s variou s jo bcategorie s requiredb y the test , th e tota l numbe r o f labo r hour s for tha t categor y an da breakdow

no f th e hour s int o the variou s task s performed .
The tota l labo r hour s for eachjo bcategor

y ar e thenmulti pliedb y th e hourl y

rat e assignedt o tha t labo r categor y t o determine th e tota l labo r dollars . Th e tota l direc t labo r (TDL ) i s the su

m o f the labo r dollar s fo r eachjobcategory . The salar y rate s currentl y

i nus e ar e estimate s o f industr y average s obtaine dfro m testin g laboratories , governmen t contractors , placemen t service s an dtechnica l jo

bplacemen t advertisement s i n November , 1985 .P.8 1

C . OVERHEA

D
The overhead rat e i s applie da s a percentag e o f TDL . Thi s percentag e represent s thos e cost s incurredb y a laborator

y i nth e conduc t o f it s business ; tha t i s facility operation , fringe benefit s an

dindirec t labor . The cost s associatedwit hthe operationo f a testin g labor ator y facility

include factor s suc ha s mortgag e o r rent payments , utilities , depreciationan damortizatio

no f non-expendabl e equip ment , insurance , propert y taxes , repair s an dmaintenanc e o f the facilit y an dequipment . Indirec t suppor t cost s ar e als o include d i noverhead . Thes e cost s include item s suc ha s fee s an

d licenses , publication , postage , compute r expenses , travel , offic e supplie s an

dmaterials , recruitmen t an drelocationo f personnel . Indirec t labo r costs , i.e. , thos e associatedwit hpersonne l wh o are requiredfo r the functionin g o f a laborator

y bu t ar e no t directl y

allottedt o the study , ar e als o included i noverhead . Indirec t labo r include s security , maintenanc e an dsupervisor

y personnel .
Becaus e o f the confidentia l natur e o f suchfinancia l infor mation , privat e laboratories , a s a practice , d o no t include over hea drate s i nthei r bids . Thes e rate s are , however , supplie dt o th e governmen t b

y contractor s inthe bidding process . Overhea d rate s i ncurren t us e ar e thos e acceptedb y testing laboratorie s inbiddin

g governmen t contract s a s wel l a s rate s calculatedb y Mathtechfro

m laborator

y competitiv

e bid s fo r variou s programs .P.8 2

D . OTHE

RDIREC T COST S

Othe r direc t cost s (ODC ) consis t o f laborator y an d expendabl e supplies , subcontractedservice s an dovertim

e premium .
1 ) Laborator

y an dExpendabl e Supplie s Thes e supplie s ar e thos e item s requiredspecificall y fo r the test . They

include animal s an dassociated product s (feed , bedding , anima l roo m supplies) , chemical s an

dreagents , an

ddisposabl e supplie s for specializedtest s (histology , analytica l chemistry) . The cost s fo r equipmen t t o conduc t the test s ar e no t included . I t i s assumedtha t a laborator y conductin

g a specifi c tes t woul dhav e the majo r equipmen t require d for tha t test . The allocatio no f cost s associated wit h majo r equipmen t ar e include dinthe overheadcalcula tio ndiscussedabove . 2 ) SubcontractedService s Sinc e i t i s no t cos t effectiv e fo r a laborator

y t o hav e al l o f the requiredequipmen t or specifi c expertis e t o conduc t a broadvariet y o f tests , som

e servic e ma

y b e subcontracted . A s anexample , ophthalmologica l examinationmay

be require

don e o r tw o time s pe r yea r b

y a specifi c laborator y requirin

g the service s o f a consultin

g ophthalmologist . Thes e fee s ar e include d a s a nODC . 3 ) Overtim

e Premiu

m Certai nemployee s may be requiredt o perfor m function s o ntest s tha t requir e mor e tha neigh t hour s pe r da y or 4 0 hour s pe r week . The functionin g o f a nanima l testin

g laborator

y require s seve

nda y pe r wee k coverage . Overtim e i s includeda s a nODC .

E . GENERA

L AN D ADMINISTRATIV

E COST S
Thes e cost s ar e the salarie s o f the suppor t service s require di nthe administrativ e functio

no f the compan

y or corporatio

nrathe r thanthe laboratory . Thes e service s includ e the accounting , personnel , administrative , purchasing , payroll ,PJ33

_

lega l an

dmarketin

g departments . The G& A rate s ar e les s variabl e thanthos e for overhea d an danaverag e o f 15 % cover s a broa d rang e o f variou s businesses .

F . FE E
The fe e or profi t assessedb y a testin

g laborator

y ca n
var y markedly . Factor s involvedi ndeterminin g a fe e rat e include the availabl e capacit y o f the laboratory , specifi c capabilit y

o f the laboratory , duratio no f the test , th e typ e o f tes t an d curren t marke t conditions .

G . FINA

L COS T DETERMINATIO N
The initia l ste

pi nthe preparatio no f the cos t estimat e i s the completio

no f the labo r hou r estimate s an d determination
o f the othe r direc t cost s a s requiredb y th e protoco l for the study . Wit hthi s information , the cos t estimatio nproceed s a s follows :
Tota l hour s xhourl y rat e Overhea d% xTD L

% G& A xTota l 1 above =

% Fe e xTota l 2 above = Tota l Direc t Labo r (TDL ) + Tota l Overhead(OH ) + Othe r Direc t Cost s



### +

+ Tota l Tota l Tota l Tota l 

### 1 G& A 2 Fe e
Estimatedcost s Su m o f component s abov eP.8 4

Dependin

g o nthe duratio

no f the test , salar y adjustment s
an dinflationfactor s ma y b e includedfor the TDL an dthe ODC , respectively , basedo ncurren t governmen t standard s an dthe
consumer s pric e index .
A nestimated cos t rang e i s the ncalculatedb y substitutin

g the lowes t an

dhighes t expectedoverhead rate s int o the genera l calculation . Overhea di s useda s the mai nvariabl e becaus e i t i s th e mos t significan t cos t componen t i nthe overal l tes t cos t an d
ca nvary greatl y

fro m compan y t o compan

y an deve nwithi nspeci fi c department s withi na company . G& A rate s ar e generall y con sisten t fro

m the businesse s surveyed . N o consideratio

nfor marke t condition s ar e mad e s o a reasonabl e fe e rat e i s maintained i nthe estimation . Unde r slo w marke t conditions , thi s componen t would , i nfact , decreas e i nthe contrac t laborator y busines s i n orde r t o increas e the competitivenes s o f the bi dt o potentia l sponsors . Industria l laboratorie s sho w highe r overhea

drate s than thos e usedfo r the abov e estimationprocedures . The estimated tes t cost s betwee nthe industria l an dcontrac t laboratories , however , woul dbe similar . The highe r overhea drate s o f the industria l laboratorie s woul db e balancedb y the fe e chargedb

y th e contrac t laboratories,i.e. , the industria l laboratorie s do
no t charg e themselve s a fe e for doing studie s i nhouse .P.8 5

II . ANALYTICA

L CHEMISTR

YREQUIREMENT S

A nintegra l par t o f the conduc t o f a nacceptabl e tes t i s the
assuranc e tha t th e tes t organism s ar e exposed t o the appropriat e dos e or concentrationo f the tes t chemical . The dos e or concen tratio nlevel s canb e directl y calculated(nomina l concentratio n
or dose ) base donth e mixin g procedur e o r the generatio nproce dures . A s anexample , th e amoun t o f tes t chemica l (mg ) adde dt o
a measuredamoun t o f dosin g vehicl e o r wate r (ml ) calculate s t o a specifi c concentration(mg/ml) . Becaus e o f the chemica l or phys ica l natur e o f the tes t chemical , the calculatednomina l concen tratio nma y b e severa l fol ddifferen t fro m the actua l concentra tio nt o whic hthe organis m i s exposed . The tes t chemica l ma y als o rapidl y

degrad e unde r specifi c circumstance s s o tha t the concentratio

nma y be correc t at the star t o f the test , bu t signi ficantl y

lowe r a fe

w hour s t o severa l day s later .
I norde r t o assur e tha t the correc t exposur e occur s durin g specifi c tests , certai no f th e Toxi c Substance s Contro l Ac t test in g guideline s specif y the analysi s o f dosing sample s t o deter mine actua l concentration s o f exposur e a t severa l interval s durin g the tes t period . Thes e requirement s ar e specificall y foun di nthe tes t guideline s for the environmenta l effect s test s
an dfor the healt heffect s test s b y the inhalatio

nroute . Althoug hno t directl y

a par t o f the individua l TSC A testin g guideline s fo r othe r healt heffect s tests , the requiremen t for analytica l verificationo f tes t chemica l purity , stability an d appropriat e concentrationi ndosin g solution s ar e mandatedb y the TSC A Goo dLaborator

y Practic e Standard s (4 0 CF RPar t 792 ,P.8 6

Novembe r 29 , 1983) . O f course , analytica l determination s are th e ke y aspect s o f bioconcentrationan dmetabolism

studies . Analytica l chemistr y determination s ar e comprisedo f tw o distinc t phases : methodvalidation / verificationan dsampl e analysis . Eachphas e i s ar e discussedbelow . The metho

dvalidation / verificatio nphas e involve s the setu p
an dtria l analysi s o f know nstandard s t o assur e tha t th e pres cribe dmethod s wor k fo r bot hthe testin g laborator

y an dthe specifi c matri xt o b e analyzedfor tes t chemica l content . Becaus e o f variation s i ntechnique s an dequipmen t fro

m on e labor ator y t o another , i t i s essentia l tha t the laborator y analyzin

g the tes t sample s demonstrate s consistenc y o f result s wit hthe laborator

y tha t developedthe methods . Differen t procedure s ma y be require

di nthe preparationo f sample s basedo nthe matri xi

n whichth

e tes t chemica l i s mixed . Sample s i nwate r ma y be directl y

analyzedwit hminima l preparation ; sample s fro m tes t chemical / die t admixture s ma y requir e extensive extractio nan d cleanu pprio r t o analysis . Metho d validation / verificationmus t b e completedprio r t o th e conduc t o f al l tests . Onc e a laborator y ha s complete

d the metho dvalidationassociatedwit ha particula r chemica l ina specifi c matrix , the methodvalidatio ndoe s no t needt o be repeate dfor subsequen t test s o f a relatednature . Thus , aP.8 7

laborator

y conductin

g severa l aquati c test s wit ha specifi c tes t chemica l woul db e requiredt o validat e the methodonc e an dappl y thi s methodt o the sample s fro m severa l simila r tests . Sampl e analysi s schedule s ar e state di nthe environmenta l effect s guideline s a s wel l a s the inhalatio nhealt heffec t guideline s s o tha t a naccurat e numbe r o f requiredsample s ca nbe determinedfor eachtest . Fo r the remainin g healt heffect s tests , certai nassumption s mus t b e made indeterminin g the frequenc

y for samplin

g an danalysi s a s wel l a s stability testin g
an dhomogeneit y

o f mixing evaluation s inorde r t o compl y wit hth

e GLPs . Thes e area s o f the GLP s ar e currentl y unde r revisio

nan d the number s o f sample s requiredfor analysi s ar e expectedt o change .
The cost s for methodvalidation / verificationan dfo r sampl e analysi s var

y due t o the degree s o f complexity requiredinth

e preparationo f the tes t sampl e t o a nanalyzabl e form , th e type o f equipmen t necessary , the specification s relate dt o the chemica l unde r analysi s an

d the suitabilit y

o f th e metho dt o automation . The numbe r o f sample s analyzeda t eac hinterva l als o i s important t o the cost . Th

e instrumen t setu

ptime , a s a

nexample , i s th

e
sam e whethe r one sampl e o r multipl e sample s ar e analyzed . Standard s ar e als o analyzedregardles s o f the numbe r o f sample s analyzed a t a particula r interval . I f sample s fro m severa l interval s canbe save dan danalyzeda t the sam e time , som

e cos t efficiencie s woul dbe realized .P.8 8

Th e metho d validatio n cos t range s fo r variou s analytica l method s ar e a s follows :
Ga s chromatography / Liqui d chromatograph y$220 0 t o 700 0 Ultraviolet/Visibl e Absorbanc e Spectroscopy$ 55 0 t o 135 0 Atomi c Absorptio n Spectrophotometry$27 5 t o 55 0
Th e analytica l cos t pe r sampl e fo r th e abov e method s ar e estimate d a s follows :
Ga s Chromatography / Liqui d Chromatograph y$ 5 7 t o 12 2 Ultraviolet/Visibl e Absorbanc e Spectroscop y$ 2 5 t o 4 2 Atomi c Absorptio n Spectrophotometr y$ 1 9 t o 3 0
Expecte d analytica l costs , wher e available , ar e liste d fo r individua l guideline s i n Attachmen t I . Thes e cost s als o appea r i n th e detai l cos t breakdown s o f th e guideline s whic h ar e containe d i n Appendice s 5 and 6 (Volume I ) an d Appendi x 7 (Volum e II)
. Abrei f descriptio n o f th e thre e analytica l method s liste d abov e i s als o include d a s Attachmen t II .



### 1 0ATTACHMEN T I ENVIRONMENTA

L EFFECT S TEST S P.8 9

797.105 0

797.116 0

797.130 0

797.133 0

797.135 0

797.140 0

797.152 0 Alga l acut e toxicity tes t GC/L C $171 0 t o $366 0 UV/VI S $ 75 0 t o $126 0 A A $ 57 0 t o $ 90 0 Lemna actur e toxicity tes t GC/L C $205 2 t o $439 2 UV/VI S $ 90 0 t o $151 2 A A $ 68 4 t o $108 0 Daphni dacut e toxicicty tes t Stati c (ST ) GC/L C $102 6 t o $219 6 UV/VI S $ 45 0 t o $ 76 5 A A $ 34 2 t o $ 54 0 Flowthroug

h(FT ) GC/L C $228 0 t o $488 0 UV/VI S $100 0 t o $168 0 A A $ 76 0 t o $120 0 Daphni dchroni c toxicity tes t Flowthroug

h(FT ) GC/L C $592 8 t o $1268 8 UV/VI S $260 0 t o $436 8 A A $197 6 t o $312 0 Daphni dchroni c toxicity tes t GC/L C $467 4 t o $1000 4 UV/VI S $205 0 t o $344 4 A A $155 8 t o $246 0 Fis hacut e toxicity Stati c (ST ) GC/L C $102 6 t o $219 6 UV/VI S $ 45 0 t o $ 75 6 A A $ 34 2 t o $ 54 0 Flowthroug

h(FT ) GC/L C $228 0 t o $488 0 UV/VI S $100 0 t o $168 0 A A $ 76 0 t o $120 0 Fis hbioconcentratio ntes t GC/L C $1140 0 t o $2240 0 UV/VI S $ 500 0 t o $ 840 0 A A $ 380 0 t o $ 600 0 3 0 sample s

3 6 sample s

1 8 sample s

4 0 sample s

10 4 sample s

8 2 sample s

1 8 sample s

4 0 sample s

20 0 sample s

1 1P.9 0

797.160 0 ATTACHMENT I (Continued ) ENVIRONMENTA

L EFFECT S TEST S Fis hearl y

lif e stage toxicity tes t Minnow s GC/L C UV/VI S A A Trou t GC/L C UV/VI S A A $490 2 t o $1049 2

$215 0 t o $361 2 $163 4 t o $258 0 $832 2 t o $1781 2 $365 0 t o 613 2 $277 4 t o 438 0 797.180 0

797.183 0

797.193 0

797.195 0 Oyste r acut e toxicity tes t GC/L C $159 6 t o $341 6 UV/VI S $ 70 0 t o $117 6 A A $ 53 2 t o $ 84 0 Oyste r bioconcentrationtes t

GC/L C $912 0 t o $1952 0 UV/VI S $400 0 t o $672 0 A A $304 0 t o $480 0 Mysi dshrim

pacut e toxicity

tes t Stati c (ST ) GC/L C $102 6 t o $219 6 UV/VI S $ 45 0 t o $ 75 6 A A $ 34 2 t o $ 54 0 Flowthroug

h(FT ) GC/L C $307 8 t o $658 8 UV/VI S $135 0 t o $226 8 A A $102 6 t o $162 0 Mysi dshrim

pchroni c toxicity tes t GC/L C $615 6 t o $1317 6 UV/VI S $270 0 t o $453 6 A A $205 2 t o $324 0 8 6 sample s

14 6 sample s

2 8 sample s

16 0 sample s

1 8 sample s

5 4 sample s

10 8 sample s

797.275 0 See

dgermination/roo t elongatio ntoxicity

tes t 1 2 sample s GC/L C $68 4 t o $146 4 UV/VI S $30 0 t o $ 50 4 A A $22 8 t o $ 36 0 797.280 0 Earl y

seedlin

g growthtoxicity

tes t GC/L C $57 0 t o $122 0 UV/VI S $25 0 t o $ 42 0 A A $19 0 t o $ 30 0 Marine sedimen t toxicit y tes t - Schwart z GC/L C $136 8 t o $292 8 UV/VI S $ 60 0 t o $100 8 A A $ 45 6 t o $ 72 0 1 0 sample s

2 4 sample s

1 2 

# JP.9 1

ATTACHMENT I (CONTINUED ) HEALT H EFFECT S TEST S 798.110 0 N/ A 798.115 0 Acut e inhalationtoxicity

4 intervale s x3 concentration s + contro l - 1 6 sample s GC/L C $91 2 t o $195 2 UV/VI S $40 0 t o $ 67 2 A A $30 4 t o $ 48 0 798.117 5 N/ A 798.225 0 N/ A 798.245 0 Inhalatio ntoxicit y Initia l an

dweekl y

monitorin

g o n3 concentration s an d contrao l a t 3 tim

e period s for eachinterva l - 16 8 sample s GC/L C $957 6 t o $2049 6 UV/VI S $420 0 t o $ 705 6 A A $319 2 t o $ 504 0 798.265 0 ora l toxicity

Gavag e an

ddrinking wate r Stability

assay s 2 4 sample s Weekl y concentrationassays 5 2 sample s

Tota l 7 6 sample s GC/L C $432 2 t o $927 2 UV/VI S $190 0 t o $319 2 A A $144 4 t o $228 0 Dietar y Sam e a s above + homogeneity o f mixin

g 2 4 sample s Tota l 10 0 sample s GC/L C $570 0 t o $1220 0 UV/VI S $250 0 t o $ 420 0 A A $190 0 t o $ 300 0

1 3ATTACHMENT I (CONTINUED ) HEALT H EFFECT S TEST S P.9 2

798.330 0

798.410 0 798.470 0 Oncogenicity

Gavag e an

ddrinkin

g wate r Stability

assay s Concentratio

nassas y Weekl y for 1 3 week s followe d b y monthl y

fo r 2 1 month s Tota l 2 4 sample s

28 0 30 4 GC/L C UV/VI S A A Dietar y Sam e a s abov

e + $1732 8 t o $3708 8 $ 760 0 t o $1276 8 $ 577 6 t o $ 912 0 homogeneit y

o f mixin

g 2 4 Tota l 32 8 $1869 6 t o $4001 6 $ 820 0 t o $1377 6 $ 623 2 t o $ 984 0 sample s sample s

GC/L C UV/VI S A A Inhalatio

n Equilibriu

m assay s Weekl y chambe r monitoring a t 3 interval s pe r chambe r 124 8

Tota l 129 6 GC/L C $7387 2 t o $25811 2 UV/VI S $3240 0 t o $ 5443 2 A A $2462 4 t o $ 3888 0 sample s sample s

4 8 sample s sample s sample s

N/ A Reproductionan

dfertility

effect s Stability

assay s Concentratio

nassay s Weekl y for 4 week s followedb y monthl y for 1 0 month s

Tota l GC/L C UV/VI S A A Dietar y Sam e a s abov

e 2 4 sample s
5 6 8 0

GC/L C UV/VI S A A $456 0 t o $976 0

$200 0 t o $336 0 $152 0 t o $240 0 homogeneit y

o f mixin

g 2 4 Tota l 10 4 $592 8 t o $1268 8 $260 0 t o $ 436 8 $197 6 t o $ 312 0 sample s sample s

sample s sample s

1 4P.9 3

ATTACHMEN

T I (CONTINUED ) HEALT H EFFECT S TEST S
798.470 0 Reproductionan dfertility

effect s (continued ) Inhalation Equilibrationassays 4 8 sample s Weekl y chambe r monitorin g a t 3 interval s pe r chambe r 48 0 sample s

Tota l 52 8 sample s GC/L C $3009 6 t o $6441 6 UV/VI S $1320 0 t o $2217 6 A A $1003 2 t o $1584 0 798.490 0 N/ A SUBPART F - GENETI C TOXICITY Becaus e o f the natur e o f thes e tests , no analytica l determinatio

nar e generall y

required . Exception s ma y include thos e mammaliantest s wit hmultipl e dos e regime s or vi a the inhalatio

nroutes . SUBPART G- NEUROTOXICITY 798.605 0 Functiona l observatio nbattery 798.620 0 Moto r activity 798.640 0 Neuropatholog y 798.650 0 Schedule-controlledoperan t behavio r

Analytica l cost s ar e dependen t o nth e exposur e protoco l for the animal s o nthes e tests .



### 1 5ATTACHMEN T I I DESCRIPTIO

N OF ANALYTICA

L CHEMISTR

YTECHNIQUE S
UV/VIS : ULTRAVIOLET/VISIBL E ABSORBANC E SPECTROSCOP Y Instrumenta l methodfor measurin g th e ocncentrationo f a compoun d i nsolutio

nbase do

nit' s ability

t o absorbligh t a t a particular wavelength . Thi s techniqu e i s generall y applicabl e t o ver y simpl e solution s o f a fe w components . I t i s usuall y applie dt o th e measuremen t o f organi c compounds . The tes t solution s mus t b e clea r (fre

e o f particulat e matter) , wit honl y the component o f interes t absorbin

g ligh t a t a particula r wavelength . Tes t solution s ar e usuall y pouredint o transparen t vessel s tha t are inserte d one a t a tim

e int o th

e instrumen t an

dthe absorbanc e valu e (amoun

t o f ligh t absorbed ) i s rea d of f a gaug e o r digita l display . The majo r complication s i nthi s typ e o f analysi s are dissolutio

no f the compoun

di na nappropriat e solven t and/or clarificatio

no f the resultin

g solution . Thi s i s one o f th e simple r instrumenta l method s o f analysis . Interference s fro m variou s component s o f a comple xsampl e (on

e wit hman

y substance s othe r tha

nthe compoun

do f interes t present ) limi t usefulnes s o f i t i nman

y circumstances . Som e sample s canb e analyzeddirectl y whil e other s may

requir e som

e pretreatment .

LC/GC : LIQUI D CHROMATOGRAPHY/GA S CHROMATOGRAPH Y Instrumenta l methodfor measurin g the concentrationo f a compoun d or grou po f compound s i na solutio nb y separatin

g the m alon g a strea m flowin g liqui d o r ga s whichpasse s continuousl y throug ha detector . The strea m i s passedthroug

ha colum

npackedwith materia l tha t create s the separatio no f component s befor e reachin g the detector . The detecto r respond s t o specifi c physica l or chemica l characteristic s o f the compound s includin g bu t b y n o mean s limitedt o light absorption . Separatio nan d detectio

no f the compound s usuall y occur s ove r a tim e spa nfro m a fe w minute s t o severa l hour s dependin g upo nthe analysi s conditions . The choic e o f suc hparameter s a s the type o f column , detector , liqui do r ga s movin g throug

hthe system , temperatur e an dman y othe r factor s candetermine the tim e for the analysi s o f a solutio

na s wel l a s the amoun t o f sampl e preparationneede d befor e analysis . The benefit s o f LC/G C analysi s ove r simpl e absorbanc e spectroscop y ar e adde

dsensitivit y

for mos t compounds , th e ability

t o analyz e very

comple xmixture s an

dmor e tha

non e component i na solution . Solution s mus t b e fre e o f particulates , an dcompound s tha t ar e no t compatibl e wit hth e flowin g liqui dor gas . Sampl e preparationcanofte ninvolv

e severa l step s designe d t o dissolv

e a compoun

di na particula r solvent , pu t i t int o a for m tha t give s a respons e i na detecto r and/o r remov e interferin

g substances . The majo r complication s o f LC/G C



### 1 6P.9 5

ATTACHMENT I I (CONTINUED ) DESCRIPTIO

N OF ANALYTICA

L CHEMISTR

YTECHNIQUE S (CONTINUED ) analysi s ar e instability o f instruments , interference s fro m simila r compound s an dinteraction s betweenthe flowin g stream

s an dth e column s tha t produc e the separatio no f components . Thes e technique s ar e usuall y appliedt o organi c compounds . Mos t sample s requir e som e pretreatment .

AA : ATOMI C ABSORPTIO N SPECTROPHOTOMETR

Y Instrumenta l metho

d for measuring the concentratio no f inorgani c (elemental ) component s o f a solutio nbasedo

nthei r ability

t o absor blight a t particula r wavelength s whe nheatedb y a flam e or electri c current . Whe nelement s ar e heatedenoug ht o vaporiz e an dfor m atoms , the

y wil l absor

blight a t wavelength s characteristi c o f eachelement . I nthi s type o f analysi s o f a solutio ncontainin

g anelemen t i s burne da s par t o f a ful e mixtur e o r heated b y anelectrica l current . A t highenoug h temperature s atom

s ar e formedi nth e exhaus t gases . A bea m o f light fro

m a lam ptha t emit s ligh t a t the sam e wavelength s a s thos e absorbedb

y the elemen t i s passedthroug hthe vapor . The amoun t o f light tha t i s absorbedi nthe vapo r i s proportiona l t o the concentratio

no f the elemen t i nth e solution . The elemen t bein g analyzedfor determine s the type o f lamp , wavelengt hbein g monitored b

y the instrument , type (temperature ) o f the flam e an d sensitivity

o f the assay . Ther e ar e a numbe r o f know n interference s i nA

A analysis . Man

y o f the m canb e eliminated or a t leas t minimized b y modification s o f the technique . The majo r complication s o f the methodar e removin g organi c component s fro m a sampl e withou t los s o f the elemen t o f interest , dissolutiono f th e elemen t an

dremova l o f inter-elemen t interferences . Almos t al l sample s analyzedb y thi s methodrequir e som e for m o f pretreatmen t ranin g fro m simpl e additio

no f a naci dt o extensiv e ashin g an ddissolutionprocedure s fro ver y comple xsamples .



### 1 7P.9 6

III . RANGE-FINDIN

GTEST S
A majo r objectiv

e i nthe toxicologica l assessmen t o f an y tes t chemica l i s the determinationo f bot hclearcu t effec t an dn o effec t levels . Thes e level s ar e equall y important for bot h healt heffect s assessment s an denvironmenta l impac t evaluations . I norde r t o selec t th e appropriat e dos e o r concentratio nrange s for the definitiv

e tests , range-findin g test s ar e frequentl y conducted . Th e Toxi c Substance s Contro l Ac t (TSCA ) testin g guideline s for man y o f the environmenta l effect s test s recogniz e the importanc e o f range-findin g evaluations . Althoug hno t a requiremen t o f an

y o f the tes t guidelines , statement s sucha s " a range-findin

g tes t shoul db e conducted " o r "i t ma y be necessar

y t o perfor

m a range-findin

g test " ar e foun

dinal l o f the aquati c tes t guideline s excep t for the bioconcentratio ntests . The bioconcentrationtest s requir e informatio nfro m acut e toxicit y test s t o establis hthe appropriat e concentratio nfor evaluation . I nthes e cases , the acut e test s canb e considere da s range findin g tests .
I nthe example s above , the mai npurpos e o f th e range-findin

g test s i s t o establis hthe tes t chemica l concentration s t o be used i nth e definitive tests . I nspecifi c cases , suc ha s the Alga l Acut e Toxicit y

Tes t (797.1050 ) an dthe Lemn

a Acut e Toxicit y

Tes t (797.1160) , the result s fro m the range-findin

g ma y determine tha t
a definitive tes t i s no t necessary . The range-findin g tes t for



### 1 8P.9 7

th e Mysi dShrim

pAcut e Toxicity

Tes t (797.1930 ) i s als o necessar y t o detex-min

e whic hlif e stag e (juvenil e o r young adult ) shoul dbe utilize di nthe definitiv e test . The Healt hEffect s Testin g Guideline s do no t us e th e term inolog y "range-findin

g tests " withi nthe guidelines , however , a fe w studie s discus s range-findin g test s using othe r terminology . Th e InhalationDevelopmenta l Toxicit y (798.4350) , the Preliminar y Developmenta l Toxicity Screen(798.4420 ) an d Developmenta l Toxicity

(798.4900 ) guideline s stat e " a pilo t or tria l stud y ma y be advisable " for the selectio no f th e appro-priat e dos e levels . Som e o f the Geneti c Toxicity guideline s for variou s plat e assay s discus s initiall y

performin

g th e tes t wit ha broa drange o f con centrations . Othe r test s d o no t include suchstatements . The majorit y

o f the Healt hEffec t test s ar e specifi c i nthe number o f dos e group s tha t mus t be usedan dth e criteri a for acceptability

o f the stud

y wit ha clea r cu t dos e respons e relationshi pan

da no-observed-toxic-effec t level . The highes t dos e leve l i nrodent s shoul dresul t i ntoxi c effects , bu t no t produc e a

nincidenc e o f fatalitie s whic hwoul dpreven t a meaningfu l evaluation . The lowes t dos e leve l shoul dno t produc e
an y evidenc e o f toxicity . I norde r t o mee t thes e criteria , i t i s important t o conside r wel l designed , tes t chemica l specifi c range-findin

g tests .



### 1 9P.9 8

The progressioni nthe developmen t o f toxicolog y program

s include s the completiono f th e acut e test s an

drepeateddos e range-findin

g test s t o selec t dose s for the subchroni c (SC) , developmenta l (DEV ) an dreproductiv

e (REP ) tests , the conduc t o f th e SC , DE

V an dREP , a s required , and , i f necessary , th e conduc t
o f the chroni c an

d oncogenicity

tests . Th e result s o f al l pre viou s test s ar e use d i nthe selectio no f th e dos e t o b e use di n th e chroni c tests . I ncase s wher e n o previou s dat a exis t i na particula r anima l specie s b y a specifi c dosin

g regimen , furthe r range-findin

g test s ma

y b e conducted . A s anexample , th e major it y o f th e acute , subchroni c an dreproductiv

e test s ar e conducte d inth e rat . Thi s dat a woul dno t be meaningfu l i nthe selectio n
o f dose s t o b e use di na mous e chroni c o r oncogenicity test . Therefore , additiona l "range-finding " studie s appropriat e t o tha t specie s ma

y b e necessary .
The stud

y designfor range-findin g shoul dfollo

w th e sam e principle s an

dpractice s o f the definitiv e study . I nthe range findin g tes t a limitednumbe r o f th e specie s unde r tes t ar e expose dt o a wid e rang e tes t chemica l concentration s or dose s fo r
a duratio

nconsideredt o be acceptabl e t o ascertai nresult s tha t
ar e meaningfu l t o the definitiv e study . Fo r example , a 4- 6 wee k range-findin

g stud y wit hfiv

e mic e pe r sexpe r grou pdose dvi a th e die t a t 5 o r 6 broadl y space dconcentration s ma y be sufficient t o selec t th e level s t o b e testedi na 2-year mous e oncogenicity

b y the dietar

y route .



### 2 0P.9 9

The range-findin

g test s recommende

dfor the Environmenta l Effect s guideline s ar e outlinedi nsufficien t detai l s o tha t on e
ca neffectivel y

planth e test . The Healt hEffect s tests , however , requir e the skill s o f the toxicologis t i nreviewin g th e purpos e o f the definitive tes t an dascertainin

g the potentia l toxicitie s o f the tes t chemica l s o tha t the range-findin g ca nbe properl y

designed . A s anexample , th e acut e ora l LD5 0 o f th e chemica l ha s bee ndeterminedan di t i s know

ntha t singl e ora l dose s caus e moderat e t o sever e irritatio no f the stomach . Th e rang e findin

g tes t for a chroni c ora l gavag e stud y woul dbe designedt o fin

ddos e range s tha t woul dneithe r b e toxi c no r produc e stomachirritationuponrepeatedadministration . A s a genera l rule , range-findin g dat a shoul db e generatedprio r t o th e initiatio

no f an y definitive tes t i nwhic hthe endpoint s ar e the determinatio

no f clearcu t effec t an dn o effec t level s o r wher e additiona l informatio ni s necessar

y befor e the definitiv e tes t
ca ninitiate , e.g. , a non-cytotoxi c leve l for a mammaliancel l cultur e test .
Becaus e o f the lac k o f definitio nfor range-findin g test s in Healt hEffect s an

dthe uncertaint y

o f conductin

g range-findin

g test s i nthe Environmenta l Effect s guidelines , the cost s associatedwit hthes e test s ar e no t include di nthe tes t cos t evaluations . Som

e considerationshoul dbe givent o includin g thes e cost s i nthe future .



### 2 1P. 1

VINCEN T J . PICCIRILL O
TECHNICA L QUALIFICATION S

o
o Board-certifie d toxicologis t wit h te n year s experienc e i n chemica l ris k assesssment . Leadin g exper t i n TSC Atestin g requirements
. Seve n year s experienc e wit h OT S regulatoryanalysis
.
EDUCATION ; 197 6 197 4 197 0 Diplomate , America n Boar d o f Toxicology

Ph.D. , Medica l Colleg e o f Virginia , Pharmacology B.A. , Pennsylvani a Stat e University , Biologica l Scienc e

1967-7 8 1974-7 6 Consultant , Mathtech , Inc .

Directo r o f Toxicology , Tegeri s Laboratories , Inc . (formerl yBorristo n Laboratories , Inc. ) Senio r Staf f Scientist , Hazelto n Laboratorie s Senio r Toxicologist , Roh man d Haa s
EXPERIENCE : A s an independen t consultant , Dr . Piccirill o assist s Mathtech , Inc . i n developin g protocol s fo r toxicologica l an d environmenta l test s require d b

yU.S.EP Aunde r Section s 4 an d 5 o f th e Toxi c Substance s Contro l Act . H e ha s als o assiste d i n th e developmen t o f a n automate d mode l t o determin e th e cost s o f suc h test s an d i n th e preparatio n o f th e TSC ATes t Cos t Manual
. A s toxicologydivisio n directo r fo r Tegeri s Laboratories , Dr . Piccirill o wa s responsibl e fo r financial , technica l an d manageria l aspect s o f al l studie s conducte d b yth e division
. Th e divisio n wa s comprise d o f approximatel y2 0 senio r an d junio r scientists , technicians , repor t writers/dat a coordinator s an d anima l caretaker s wit h a n annua l budge t i n exces s o f $ 2 million
. H e worke d closel ywit h industria l sponsor s i n assurin g tha t stud y protocol s complie d wit h intende d registratio n guideline s o r othe r tes t standard s (FIFRA , TSCA , OECD , FDA , OSHA)
. Technica l
Pag e 1 o f 4P. 2

expertis e coveredth e design , implementation , evaluationan d repor t preparatio

no f acute , subchroni c an dchronic/oncogeni c studie s inrodents , rabbits , guine a pig s an dcanine s b

y al l classica l route s o f administratio n(intubation , feeding , dermal , ocular , intravenous , wate r dosing , etc.) . H e serveda s principa l investigato r fo

r tw o majo r NIOS H contract s t o evaluat e the Chernof f technique s a s a neffectiv

e scree nfo r reproductiv

e hazar dpotentia l o f priorit y chemicals . Als o wit hTegeris , Dr . Piccarill o serve da s th e progra

m manage r for a subcontrac t t o perfor m cos t an

deconomi c analysi s o f TSCA , FIFRA , an

dOECD tes t standard s an dtes t rule s for EPA . Thi s progra m va s expandedt o include evaluatio no f tes t standard s unde r th

e Premanufactur e NotificationProgra m (PMN ) o f TSCA . H e wa s laborator

y representativ

e t o th e Regulator

y Affair s Committe e o f th e Nationa l Associationfor Lif e Scienc e Industrie s (NALSI) , wher e h e wa s involve

di nreview , comment an ddisseminationo f informatio

nregardin

g regulator

y matter s affectin

g membe r companies . Wit hHazeltonLaboratories , Dr . Piccarill o conductedresearch studie s t o evaluat e the safety o f variou s tes t material s includin

g drugs , cosmetics , foo dan dcolor additives , an d industrial , agricultura l an dhousehol dchemical s inexperimenta l animals . He wa s responsibl e for th e developmen t o f experimenta l stud y designs , coordinationo f th e laborator

y work , an

d interpretatio

no f th e dat a resultin

g fro m acute , subacut e an d chroni c toxicity

studie s a s wel l a s teratology , carcinogencit y an dreproductio

nstudies . Othe r activitie s includedsupervisio n o f the canine toxicolog y laboratory . Whil e wit hRoh

m an d Haas , Dr . Piccarill o wa s responsibl e for establishin

g in-hous e subchroni c an dspecializedtoxicit y testin g capabilities . H

e wa s als o responsibl e fo r designin

g protocols , monitorin

g projects , supervisin g personnel , an

dwriting reports . I naddition , he monitoredtoxicit y studie s awarde

dt o extramura l researchtestin

g laboratorie s an

dpreparedSectio

nC submission s t o the Environmenta l ProtectionAgency coverin

g herbicid e an

d plan t fungicide areas .
AFFILIATIONS ; Society o f Toxicolog

y AmericanColleg e o f Toxicolog y AmericanAssociatio nfo r Laborator

y Anima l Scienc e (AALAS ) Nationa l Associationo f Lif e Scienc e Industrie s (NALSI) , Regulator

y Affair s Committe e

Pag e 2 o f 4P. 3

PUBLICATION S AND PRESENTATIONS :
Chambers , J.W . an

d Piccirillo , V.J. , Th e effect s o f ethano l o n amin o aci d uptak e an d utilizatio n b

yth e live r an

d othe r organs , Quart . J . stud
. Ale
. 14 : (3) , 707-719
. (1973 ) Piccirillo , V.J . (Doctora l Thesis)
. Th e effect s o f ethano l o n amin o aci d metabolis mi n th e live r an d othe r organ s i n th e rat . Medica l Colleg e o f Virginia
. (1974 ) Piccirillo , V.J . an d Chambers , J.W. , Th e effect s o f ethano l metabolite s o n ure a productio n b yth e isolate d perfuse d ra t liver
. IRC S Med
. Sci
. 3: 186
. (1975 ) Piccirillo , V.J . an d Chambers , J.W. , Inhibitio n o f hepati c uptak e o f alpha aminoisobutryi c aci d b yethanol : Effec t o f o f pyrazol e an

d metabolite s o f ethanol
. Res . Commun . Chem
. Path
. Pharmcaol
. 12 : (2 ) 297
. (1976)
. Piccirillo , V . J . an d Quesenberry , R.P. , Reproductiv e capacitie s o f contro l mallar d duck s (Ana s Platyrhynchos ) durin g a on e generatio n reproductio n study , J . Env . Path
. Toxicol
. 4 , Part s 5
. 6 : 133-140
. (1980)
. Piccirillo , V.J . an d Owen , T.B. , Inhalatio n Bioassayo f Cigarett e Smok e i n Dogs . Director yo f on-goin g researc h i n Smokin g an dHealth
. (1980 ) Piccirillo , V.J. , Orlando , D.A. , an d Quesenberry , R.P. , Reproductiv e capacitie s o f contro l bobwhit e quai l (Colinu s virginianus ) durin g a on e generatio n reproductio n study
. J Env . Path
. Toxicol
. (Accepte d January , 1981 ) Parker , CM. , Piccirillo , V.J. , Kurtz , S.L. , Garner , F.M. , Gardiner , T.H . an

d Va n Gelder , G.A . Si x mont h feedin g stud y o f Fenvalerat e i n Dogs . Fundam
. Appl
. Toxicol
. 4(4) : 577-58 6 (1984)
. Schuler , R.L. , Hardin , B.D. , Neimeier , R.W. , Boot h G. , Hazleden , K. , Piccirillo , V.J . an d Smith , K . Result s o f testin g fiftee n glyco l ether s i n a shorttermi n viv o reproductiv e toxicit yassay . Environ
. Healt h Perspect . 57:141-14 6 (1984)
. Brown , J.H. , Piccirillo , V.J . and Hartman , W.C , Jojobutter-51 , A primar yderma l irritatio n stud yi n rabbits
. Cosmetic s an d toiletries , 99(51:5 7 (1984)
. Haider , C.A. , Holdsworth , C.E. , Cockrell , B.Y. , an d Piccirillo , V.J. , Hydrocarbo n nephropath yi n mal e rats : Identificatio n o f th e nphrotoxi c component s o f unleade d gasoline
. Tox . an d Industr
. Health
. (Accepte d September , 1985)
.

Pag e 3 o f 4P. 4

Piccirillo , V.J. , an dChambers , J.W. , Th e effec t o f pyrazol e o n th e inhibitio n o f AI B uptak e b yethano l i n th e isolate d perfuse d ra t liver
. Fe d Proc . 32 : 698
. (1973)
. Presente d a t th e FASE B meeting , Atlanti c City , Ne wJersey , 1973
. Chambers , J . W . an

d Piccirillo , V.J. , Increas e i n amin o aci d incorporatio n int o protei n an d decreas e in ' hepati c ure a synthesi s produce d b yethanol
. Fed
. Proc . 3_3_ : 554 , (1974)
. Presente d a t th e FASE B meeting , Atlanti c City , Ne w Jersey , 1974
. Piccirillo , V.J . and Chambers , J.W . Ethano l effec t o n incorporatio n o f alanin e int o protei n i n variou s organ s o f th e rat . Th e Pharmacologi s 16. : (2)
. 303 , (1974)
. Presente d a t th e ASPE T meetin g Montreal , Canada , 1974
. Piccirillo , J.V . an d Quesenberry , R.P. , Biologica l variatio n i n th e reproductiv e capacitie s o f contro l mallar d duck s (Ana s platyrhynchos ) durin g on e generatio n reproductio n studies
. Toxicol
. Appl
. Pharmacol
. 4J3. : Par t 2 : 1979
. Presente d a t th e SO T meeting , Ne wOrleans , Louisiana , 1979
. Parker , CM. , Piccirillo , V.J. , Kurtz , S.L. , an d Garner , F.M. , Si x Mont h Feedin g Stud yo f Fenvalerat e i n Dogs . Th e Toxicologist , Vo. , No . 1 , March , 1983
. Presente d a t th e SO T meeting , La s Vegas , Nevada , 1983
. Neal , B.H. , Piccirillo , V.J. , Rowland , J. C an d Kastello , M.D. , Preclinica l Toxicit yo f Vehicl e 1 2 i n Beagl e Dogs . Th e Toxicologist , Vo . 3 , No . 1 , March , 1983
. Presente d a t th e SO T meeting , La s Veags , Nevada , 1983
. Lunchick , C , Hartman , W.C . an d Piccirillo , V.J . Variabilit yo f th e dominan t letha l respons e i n th e ra t t o orall y administere d triethylenemelamine
. Presente d a t th e NCAB chapte r o f SO T meeting
. Spring , 1983
. Piccirillo , V.J. , Healt h Effect s an d Environmen t Effect s Guidelines : Evaluatio n an d Cos t Estimation
. Presente d t o th e RegulatoryImpact s Branch , U.S . Environmenta l Protectio n Agency , Washington , D.C . o n Augus t 6 an d 13 , 1985
. Piccirillo , V.J. , J.K . Orrell , an d P.M . Godfrey , TSC ATes t Guidelin e Cos t Estimate s prepare d fo r th e Economic s an d Technolog

yDivision , Offic e o f Toxi c Substances , U.S . Environmenta l Protectio n Agency , Washington , D.C .

Pag e 4 o f 4