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Microfiche No. OTSoo01363



### FY1-0TS-I099-1363

07/13/99 

### Old Doc 10

10129/99 84000000oo3



### TSCA Section 

### FY1

Submitting Organization CHEM MFGS ASSN

FINAL REPORT. SHORT-TERM STIJDIES TO EVALUATE THE DOSIMETRY AND MODES OF ACTION OF 2-BUTOXYETHANOL IN B6C3Fl MlCE. WITH COVER LE1TER DATED 08/0611999 

### Chemical Category 2-BUTOXYE1liANOL.' 

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### FlNAL REPORT Battelle

### ProjectNo. 29753 CMA 

### AgreementNo. EGE-74.o-00SE-BAT.NW -AmendmentNo.8 July 13, 1999
"Sbon-Term Studies tc Evaluate the Dosimetry aDd Modes ofActioa of2- ButozyetbaDol in 

### B6C3Fl Mice"
R.A. Corleyl, K.K. WeitzI, 

### T

### J.**~,** 

### R.A.**MiIl~,** 

### and B.D. Thralll IBattelle Memorial Institute, Pacific 

### Nonhwest Division Chemical Dosimetry 

### PO 

### Box. 999. MSlN 

### P7-S9

Richland, WA 

### 99352 2Battelle

### Memorial Institute, 

### Columbus 

### Operations Toxicology PO 

### Box. 999, MSIN K4-IO

Richland, 

### WA 

### 99352 FOR The 

### Ethylene Glycol Ethers Panel Chemical Manufacturers Association 1300 Wilson 

### Blvd. Arlington, VA 22209 

### Corresponding Author: Richard 

### A. Corley ph: 

### 509-376-8462 fax: 509-376-9064 e-mail: ra corley@pnl.gov

# ~:05 Project 297S3 Page 2 of49 

### EXECUTIVE SUMMARY The 

### dr.lft NTP report on the chronic inhlation studies 

### with 2-butoxyethanol (BE) identified the forestomach and liver of B6C3FI mice as potential target organs for tumorigenicity (NTP, 1998). Based upon the general lackofgenotoxicity and the 

### kDown hemolytic mode ofaction ofBE in rodents. it is likely that 

### these potential treabnentrelated tumors occurred via indirect mechanisms involving chronic tissue injury. Forthe liver tumors, the hemolytic. effects ofBE and 

### subsequ.em accumulation ofdegradation byproduets may have increased the levels ofreactive oxygen 

### species. Mice 

### are peculiarly sensitive to oxidative stress, relative to rats and huawis. Furthermore, 

### bmnans are rdatively insensitive to BE-induced hemolYsis. For the forestomach tumors. chronic. contact irritation from grooming ofBE deposited on the 

### fur or mucociliary clearance of BE dissolved 

### in the mucous layers ofthe respiratory tract may have been a conaibuting factor. Alternatively, systemic delivery of BE or, more likely, 

### its major metabolite, butoxyacetic acid, at a high dose-rate may have caused chronic irritation in the mouse forestomach and subsequently increased the probability ofspon13Dcously-initiated cells progressing to 

### nunors. These potential 

### indirect mecbanisms were investigated in a 

### series of 

### studies conducted 

### at Battelle Northwest and at the Indiana University School of Medicine (Dr. 

### J. Klaunig, Principle Investigator). At Battelle Northwest, the potential role oforal ingestion via grooming in the production of forestomach lesio::s of female mice exposed to BE via whole body vs. nose-only inhalation exposures was investigated Small amounts ofBE were removed from the 

### fiB' offemale mice exposed for 

### 6 hrto target concentrations of250 ppm BE vapor. The total dose that was available for oral coasumption via grooming 

### was 9.2 :t: 2.9 mglkg for the whole-body exposure (229.2 ppm BE) and 7.5 :t: 2.3 mglkg for the nose-only exposure (241.8 ppm BE). Vcry little difference 

### was detected in the intema.I dose ofBE resulting from whole-body vs. nose-only exposure as 

### determined by analysis ofBE and its major metabolite. BAA, 

### in blood collected inunediatelypost-exposme and 

### in uriDe collected for up to 18 br post-exposure. Small amounts ofBE were 

### detected in the 

### uriDe which was atuibuted to residual BE on the fur and not from urinary elimination. Although this was not a mass-ba1ance study, the amounts ofBE deposited on the 

### fur ofmice represented 9- 13% ofthe total BAA eliminated inthe urine. BE deposited on the 

### fur and CODSWDed via grooming during the exposure 

### as well as orally consumed via mucociliary clearance of BE deposited 

### in the mucous layerlining the respiratory tract was not accounted for 

### in this study. Nevc:rtbeless, there is evidence that at least 

### a small amount of neat BE was available for oral consumption via grooming and that the poteDtial role for chronic. daily oral doses 

### in mouse forestomach toxicity caDnOtberuled O1.lL To evaluate whether 

### direct contact with BE can cause forestomach lesions and evaluate the potcnIiaJ role ofhemolysis-mediated oxidative stress 

### in the liver, neat BE (no vehicle) was 

### administered to male and female mice at 

### dose levels up to 1200 mg/lcgld for one week. Feed 

### was withdrawn 

### from each animal 4 

### hr prior to dosing and 

### returned 2-3 hr post-dosing 

### to mimic the conditions ofthe inhalation study. The 

### dose levels used 

### in this study resulted 

### in severe hemolysis 

### and monality that 

### was DOt 

### prediC1ed in a I-week

## dose range finding study and the two-week study 

### was tenninatecl 

### after only 4 doses. Neat BE

# 06 Proj«t29753 Page 3 of49
was cl:arly shown in this oral gavage 

### SlUdy to cause irritation to the mouse forestomach similar to that observed by the NTP following inhalation exposure. Forestomach lesioDS consisted of focal areas of irritation and epithelial hyperplasia. 

### The compensatory proliferative respoDSC was confumed by PCNA ill1I&':lDOhistoebcmistry. This clearly established thai direcl oral administration ofneat 

### BE is capable ofcausing lesions in the mouse forestomach. To investigate whether or nOI parecleral administration of 

### BE could 

### also produce forestOmach lesions in mice, additional mice were 

## admimstCred saline solutions of

### BE by either intraperitoneal (IP) or subcutaneous (SC) injection. Dose levels were 400 and 600 mglkgld for three consecutive days (probe SlUdy; 'n 

# =3/group) 

### or 0 (saline control) and 400 mglkgld for five coosecutive days (fuji study; n 

# = 6/group). AU three mice adminiSlel'ed 

### 600 mglkg 

### BE by IP injection for three days in the probe study showed clear evidence offocal irritation in the forestomach similar in appearance to the lesioDS observed in gavage and inhalation studies. One ofthree mice/group in each ofthe 400 mglkg IP and 400 and 600 mglkg SC groups in the 3-day probe study also had forestomach lesions. bUI these were considered minimal. Spomaneous lesions similar 10 these can 

### occ:ur in UDtreated mice. 

### In the 5-day study at 400 mg/kgld, 1/6 IP-dosed mice and 

### 216 SC-dosed mice also bad minimal lesions thai were polentially treatmenl-rewed; none were observed in me controls. Together. these data suggest thai forestomach lesions in lhe mouse may be induced by systemic admioistration and may DOl 

### require direct comact with 

### BE. It is further possible that the metabolite, 

### BAA. may playarole in producing the forestomach lesions since the 

### IP injection route 

### results in 

### fust-pass liver metabolism while SC injection 

### does DOl. InlC'eSlingly. these lesions bad not 

### heeD observed in previous drinking walei' studies conducted by

### NTP suggesting that the lesioDS maybe dose-rate dependenl In the liver evaluations. there was no evidence ofdegeneration and no corresponding compensatory cellularregeneration in mice 

### administered Deat 

### BE by gavage. 

### In addition. there was no remarkable accumulation of hemosiderin in Kuppfer cells (H&E stain) altbougb there was a clear dose-response increase in iron coDlent in Kuppfer cells (Perl stain) and to a much lesser degree in hepalOCytCS. The increase in iron may play a potentially significant role in**o~dative** 

## stress in these cells. 

### The potential for oxidative stress in the livers ofthese mice 

### was evaluated and reported separately by the Indiana University School ofMedicine (Kamendulis et

### aI.• 1999).

# 07 Project29753 Page 4 of49 INTRODUCTION 

### 2-Butoxyetbanol (BE) 

### was recendy evaluated in 

### a series ofiIJhaIation toxicity studies by the 

### National Toxicology 

### Program. While the 

### technical report 

### ofthis research 

### program bas 

### not been 

### reviewed and approved by the NTP Board 

### of Scientific Counselors, the 

### results have been prepared 

### for public review and comment (NTP, 1998). 

### Preliminary results from 

### those StUdies have identified the forestomach and liver of

### B6C3FI mic:c as potential target organs 

### for tumorigenicity. These 

### potential treatment-rewed tumors likely 

### oceurrc:d via indirect 

### mechanisms involving chronic tissue injury. 

### For the liver tumors, 

### the hemolytic effects of BE and 

### subsequent accumulation 

### of degradation byproduetS may have increased

### thelevels ofreactiveoxygen species. Mice arc 

### peculiarly sensitive to oxidative streSS. 

### relative to rats and 

### humans. Furthermore, hwnans 

### are relatively insensitive to BE-induced hemolysis which raises questions 

### regarding the ability to extrapolate the liver effects 

### observed 

### in mice to 

### human health 

### risk. For the forestomach tumors, 

### chronic. contact 

### irritation 

### ftom grooming 

### ofBE deposited on the fur 

### or mucociliary clearance ofBE dissolved in the 

### mucous layers ofthe 

### respiratory tract 

### may have been 

### a factor. The potential role for 

### direct 

### contact with Dc:31 

### BE 

### in forestomach toxicity 

### was also evaluated siDce exposure to 

### BE via 

### drinking water 

### did not cause these 

### effects (NTP, 1993). 

### Alternatively. systemic delivery ofBE 

### or, more ti.:l:cly. its major metabolite, butoxyacetic acid, at a bigh 

### dose-ratc may have ·caused**chrooi~** irritation 

### in 

### the mouse forestomach and subsequcndy increased the probability of spl.'Q!aDeouslyinitiated

### cells progressing to tumors. Therefore, 

### the specific ofmis study**W~** 

### to:

• Evaluatethe potemial role oforal 

### ingestion (via 

### grooming) ofDeal**2-butox~1** 

### in the 

### productionofforcstomach lesions ofmiceexposed to BE vapor(Aim I)



### • Evaluate the 

### potential for increased 

### oxidative SU'CSS 

### associated with hemolysis 

### iII the production/progressionofliverlesions in 

### mic:c (Aim 2)



### • Evaluate the potential role for systemic delivery of BE in producing forestomach lesions following routes of administration undergoing 

### either first-pass 

### metabolism (intraperitoneal injection) or systemic distribution prior to 

### metabolism (subcutaDeous 

### injection) (Aim 

### 3). To 

### accomplish these objectives, the amount ofBE deposited

### on the fur ofmice fonowing either

### a single**~hr.** 

### whole-body or

### nose-only exposure was determined. 

### To evallWe the impact offur 

### deposition on the internal dose 

### ofBE, samples ofblood and urine 

### were analyzed 

### for BE and 

### its metabolite. 

### butoxyacctic acid (BAA). ID addition 

### to tne dosimetry smdies, the 

### dii'ect effect ofBE on the forestomach ofmic:c 

### was evaluated 

### by administering neat 

### BE daily by 

### oral gavage for 

### up to five

# daYs. The forestomach ofeach mouse was evaluated histologically for evidcnc:c of 

### irritation and 

### inc:reased cell replication. 

### These same oraIly-dosed 

### animals 

### were also 

### used to 

### cvaIuase whether 

### the accumulation of

### iron in 

### Kupffer cells, as a secondary 

### event ofhemolysis, 

### is associated with 

### an increased 

### oxidative stress 

### iii the liver. 

### This latter 

### objective 

### was coaducted 

### in collaboration with 

### Dr. Jim KJaunig, Indiana 

### University Medical Center. FiDally, 

### the indirect effect 

### ofBE on the foresromach ofmice 

### was evaluated histologically foUowing

# 08 Pro;ect29753 Page 50f49 administration of saline solutions of BE by either intraperitoneal or subcutaneous injection for

### J or 

### 5 days.



### METHODS


### TestMaterials. The teSt chemical, 99+% spectI"OJ)botometric grade 2-butoxyethanol (BE, lot number 00437AS for Specific Aim 

### I and lot number 07847HN for Specific Aims 

### 2 and 3), 

### was purchased 

### &om Aldrich Chemical· Co. (Milwaukee, WI). Certificates of analysis received from Aldrich Chemical Co. indicated that the testchemical was 99.59010 

### and 99.87% pure by gas cluomatography for lot numbers 00437AS 

### and 07847HN, respectively. The teSt chemical was stored at**controll~** room temperature. 

### Test Animals. 

### A total of 

### SO young adult female 

### B6CJFI mice (including CXIraS for control blood and urine collection and matrix standards for analyses) were supplied by Taconic Labor:uory 

### Animals and Services (Germantown. NY), for 

### use Specific Aim I. Taconic Laboratory was the supplier ofmice for the NTP inhalation Sb.ldies. 

### Animals were ordered to arrive at approximately I0-11**~eeks** ofage so that the females were of sufficient size to fit in the nose-on.ly exposure system. Animals were fed Purina 5002 certified pelleted diet and fresh tap water was 

### provided QI/ libil1ml. Mice were housed individually in hanging wire stainless steel cages. Mice observed 

### for moribundity 

### and mOrtality twice daily. Body weights were determined 

### tbI= days after 

### arrival (for randomization and group assignment purposes) and prior to each exposure. 

### The mice were assigned to their respective exposure group (20 each) prior to the initiation ofthe first exposure. Each animal designated for nose-only exposure was acclimated to the nose-only restraiOlt device for four days prior to exposure. For Specific 

### Aims 2 and 3, a loral of90 male and 90 female B6C3FI mice (including extras) and 36 female B6C3FI mice, respect:ively, were 

### also supplied by Taconic. All animals 

### were acclimated to the laboratory environment for at**I~** one week priorto placing on study. 

### Specific Aim·l: Deposition ofBE 0. 

### tile fur ormice. Study Design. Female B6C3FI 

### were exposed by either whole-body (20 mice) or nose

### only (20 mice) inhalation, for a single 6-br exposure period to a larget concentration of 250 ppm BE. This concentration was the highest concenuation used in the NTP inbaJation bioassay

## <NTP, 1998). Immediately following each exposure (whole-body and nose-only), five mice were quickly sacrificed 

### aDd theirbodies washedtwo times each with bot tap water. 

### Each water wash was analyzed for BE. Another 10 mice were 

### transferred to metabolism cages for an 18-hr**post-ex~** urine collection. The urine and cage washes were analyzed for BE and its major metabolite, bUlOll:yaCeOC acid (BAA). The remaining 5 mice were kiUed immediately post-exposure and their blood collected into 

### heparinized tubes for analysis ofBE and BAA.

# 09 Project297S3 Pagc6of49 Inhalation Ex;)OSures. 

### VaporGeneration s.vstem. The test article was pumped by a precision metering pump (Model 302; Gilson IDe, MiddlelOD, WI) intO a 25 mm x 300 mm tbRaded-glass air sampling manifold (Ace Glass 

### lnc:., Vineland NJ) (Figure I). BE was 

### vaporized on the beated (-IM°C) 

### glass surface of the generator while a metered air stream flowed through the 

### generator (-20 

### LJmin) and 

### then into the 

### exposure chamber (whole body or nose-only). The buildup of vapor concentration in the exposure chambers at the 

### beginning ofexposure to 9()D/o of its stable fmal concentration (T90) was cstimaIcd to be 10 min for the whole body 

### chamber (-85 L mixing volume) and 

### 3 min for the 

### nose-oDly system. 

### Exposure duration 

### was 6 hours 

### + T90 from the time theexposure was initiated. 

### Whole Body

### Ezposure Chamber. lbe whole body exposure chamber 

### was an 85 L polycarbonate box. Mouse cages 

### were suspeDded 

### in the center of the box above a stainless steel catch 

### pan. BE vapor 

### was introduced into the chamber from 4 

### pons along the top ofthe chainber and was exhausted from 4 

### pons below the catcb pan. Eight sample 

### pons were distributed across one face of the c:bamber, 4 ofthe ports 

### were adjacent to the cage units and 4 ports 

### were located above the 

### cages. The 

### generator airflow (-20 Umin) resulted in -14 air 

### changes per hour. The distribution of

### BE vapor in the chamber was 

### determined prior to the stan ofthe 5tUdy by comparing: the concentration from 

### 3 different chamberpositions. 

### Nose-Only Exposure Unit. 

### 'The nose-only exposure uait was 

### a flow-past type 

### consisting oftwo 

### conceoaic 

### stainlesssteel manifolds; one supplied fresh vapor to the breathing zone of each animal and one 

### removed the 

### expired and excess 

### vapor. Airflow through the unit was -20 Umin from the generator (inlet) with 

### an exhaust flow of 

### -18 Umio to prevent the animals from 

### rcbreathing theirexpired

### air. The exposure unit consisted of) tiers of8 exposure ports. For concentration determination, three of the 

### pons (one from each tier) were used to collect samples ofthe breathing atmosphere; samples 

### were also collected from the inlet manifold using an additional pon from the lowest tier. Uniformity ofthe test anicleconcentration in the exposure

### unit was determined prior to the study. 

### Chambermonitoring. The exposure concentrations ofBE were detennined by 

### gas chromatographic analysis of atmospbere 

### Mgrab samples" taken directly from the exposure chamber during animal 

### exposure. Grab 

### samples were collected on cbarcoa1 

### sampling tubes (ORBO-JOl). 

### Known volwnes ofthe chamber atmosphere wete collected using a 

### calibrated criticalorifice-controlled sampler at a constant flow rate of -0.2 

### LJminute for 3 minutes 

### each.. An extraction solution was added to the primary charcoal bed after sample coUection to 

### desorb the test chemical 

### from the charcoal. 

### The extraction solution (tOluene) contained an intemal standard (l-pbenylbexane) to correct for variations in injection volume and drift ofinsaument sensitivity. 

## The sample extracts were analyzed using a HP-5890 gas chromatograph (Ge) equipped with a flame ionization detector operated at 300°C and a

# 10 Project297S3 Page7 of49



# . . 30-m x 0.53-mm 

### 10. Rtx-S. 1.5-1UJ'1 film thickness capillary column 

### (Rc:stek Corp.• Bellefonte, 

### PAl. The carrier 

### gas 

### was helium at a head pressure of

### -6 psi. 

### The column was maintained at 60°C for 

### 1.0 minute 

### and then heated at 16°Clminute to 

### a tiDal temperature of200°C. Under these conditions. BE had 

### a retention time of-4.4 minutes (figure

### 2). 'Data were obtained

### using HP ChemStation software. version 

### A04.02. GravimeaicalJyprepared standards made 

### from high purity Aldrich Chemical Co. BE 

### (loc number 07847HN) and the intemal standard in toluene were used to calibrate the GC for quantitation of

### BE in the grab samples. Two independently weighed stock solutions were used each time the standard series was prepared. One 

### stock solution 

### was used to prepare the calibration standards. The quality conuol check 

### standards were prepared with the second 

### stock solution and were analyzed in the presence ofdie sampling medium using the same extraction procedure as the chamber grab samples. The 

### quality CODuol check 

### standards were used to cbeck 

### the'preparation of

### the standards. sample exttaction from 

### me sampling media.. and any drift of instrument calibration throughout the analysis. 

### The analyzed concentrations ofthe quality control check staDdards were required to be within ±10%oftheir prepared 

### concentrations for a valid analysis. Nominal exposure concentrations 

### were calculated using the 

### mass ofleSt article pumped. the total time during whicb 

## rpe metering 

### punip was naming and the dilution 

### airflow into the chamber. This value was compared to the average exposure concentrations obtained by 

### grab samples. Collection offur washes. blood 

### and urine. At the end ofeach exposure (oose-onIy and wbole·body). five mice were sacrifICed using CO: and transferred**~o** individual 

### beakers and submerged in 50 ml ofbot water. After rinsing in SO ml ofwater. the mice were transfem:clto a second beakercontaining an additional SO ml ofhot water. Each fur rinse 

### was stored frozen (-70°C) until analyzed. This fur washing procedure was similar to that used by Tyl et aI. (1995) to 

### determine the deposition ofethylene glycol on the fur ofrats and mice. At the end ofeach exposure (nose-only and whole-body). five mice were immediately anesthetized using 70010 C02 and samples ofblood were collected via the retro-orbital sinus. 

### The time ofeach blood sample 

### was accurately recorded. Following coUection. each blood sample

### was maintained on ice until placed in freezer 

### (.700q. At· the end, of each exposure (oose-only and whole-body). 10 mice were placed in individual metabolism cages (Lab Products.' Seaford., Delaware) for an IS-hour urine collection. 

### The urine samples were collected over dry ice. Mice were provided with access to water but not 

### food. Following urine sample collection. each metabolism cage was 

### rinsed with -25mL ofdistilled water. Each urine sample and cage rinse 

### was stored 

### frozen (-700C) until analyzed. Analyses of fur. Samples of each fur wash were analyzed directly using a Hewlett Packard (HP) 6890 GC equiped wi'th a flame ionization detector (FlO) (Hewlett Packard; Palo Alto. CAl. Cbromatograpy was achieved using a 

### nB.Wax capillary column (IS

# 11 Project 297S3 Page 8 of49 meter x 0.53 mmid x 1.0 film thickness. 

### J &W Scientific, Folsom. CA.) 

### in the spiitiess injectioa mode. 

### Hdium was used as the carrier 

### gas at a const3Dt 

### coItImD bead 

### pressure of4.0 psi. One ,.d ofneat (water) sample 

### was injected onto a large 4 mmid injection liner to facilitate the water vapor expansion generated at the constant injection 

### temperatUreof2050<:. The oven tempetature 

### was initially maintained at SQllC for 2.0 

### min then ramping at 

### 3()iC1miD to

### 24()11C. FlOtemperat!Jre 

### was 27fPC. 

### Undertheseconditions, BE bad 

### a retention time of-3.2 minutes. Analysis ofblood 

### and urine. A GC method developed previously to analyze for related glycol ethers 

### and their aIkoxyacetic acid metabolites was modified to simultaneously analyze for 

### BE and the metabolite. 

### BAA in blood (Corley et al., 1998). 

### Urine samples 

### and cage washes were also aDaIyzed by 

### this method for BE and BAA. The method involved the acidification ofa weigbed quamity (-02.5 g) ofblood, urine or cage 

### wash. with 

### an equal volume of0.9 M H2SO
.. addition of02.5 g 

### Na2S04 (to improve extraction efficiencies) and extracted with 0.25 

### ml ethyl acetate 

### comaiDing known CODCeDtratiODS of internal standards (etboxyetbanol [EE] 

### and ethoxyacetic acid 

### [EAA]) to correct for possible extrae:tioalinsuument variations during analysis. Separation and quantitatioo 

### was achieved using a 30 m x 

### 032 mmid x 

### O.S J.UD 

### film thickness Stabilwax-DA capillary column (Restec:, Bellefonte, PAl 

### and a Hewlett 

### Packard 6890 GC with flame ionization detection. Splidess injections of-1.5 

## f.lL ofextract were 

### made at an injector IempCr'aIUr'e of2()()l'C. The initial ovc:o temperature 

### was 7fPC for 2.5 min 

### then increased at 20"C1min to 24()l1C and held for one 

### min. Hydrogen was, used as 

### the carrier gas at IS psi constant 

### pressure. The 

### FlO temperature 

### was 27fPC. For the preccdiDg 

### conditions 

### EE. BE, EAA 

### and BAA bad retention times of 

### -3. -4. -7 and 

### -8 minutes. respeaively (only approximate retentiOTl times are**~"ided** 

### due to slight variations 

### in retention times observed 

### for eacb matrix). Specific Aim 2: EffectoforaUy-lldmiDistered ae.t BE 'JD the**~orestom8C"** aDd

## mer. Probe Study. 

### Male and 

### female B6C3Fl 

### mice, 5lsa1dose,**~c:re** 

### administered 

### neat BE by oral gavage at 

### 100,400 or 

### 800 mglkglday for 

### 5**~e** days. After 

### two days of dosing at 800 mglkg, DO clinical signs oftoxicity were observed. Therefore, 

### animals assigned 

### to the 

### 100 mg/kglday group were 

### administered 

### 1200 mglkglday for 

### the remaining 3 days ofthe probe study. Feed 

### and waterwere removed at 

### -7:00 

### a.m. and the dosing 

### was initiated at approximately -11:00 

### a.m. to mimic 

### the fasting state 

### associated 

### with inbalatioa stUdies. Feed aDd water

### were retumed to the 

### 3nimals by 2:00 p.m. Each 

### animal was weigbed on the morning ofthe 

### first dose and the volume ofBE delivered 

### adjusted 10 

### deliverIbe appropriaIe mglkg 

### dose level for 

### each animal. The animals were observed for overt signs oftoxicity; animals DOt expeaed to survive Ibe next 

### dose were humanely sacrificed. The 

### purpose of

### this probe study was 

### to 

### detctmine ifthe 

### animals would survive 

### the trcaanent regimc:o and select dose levels for the definitive study descnbed below. 

### Each surviving animal 

### was weighed 

### and sacrificed by cervical dislocation four 

### hours after the final dose. Stotnaeb (empty), liver 

### and spiceD 

### weigbls were 

### recorded and blood 

### was dr.lwn by eardiac puDeturC for detertnilWion of the hema1OC1'it. Slices ofesophagus, 'forestomach and glandular 

### stomach. as well as liver

# 12 Project 29753 Page 9 of49
were fixed 

### in neuttal buffered formalin. Selected samples were evaluated as 

### dc:scribed below 

### for the Full Study. Full Studv Design. 

### Based on me results 

### &om the 

### probe study, male and female B6C3F1 mice. 16/scxldosc. were initially treated by oral gavage with saline. 400, 800 or 1200 mgllcg ofneal 2-butoxyetbanol. Each animal was 

### weighed at the beginning ofthe week to adjust the dosing volume to the 

### appropriate mg/kg 

### body weigbt. Priorto the first week ofdosing a group of8lsex/dose 

### was implanted with 

### 5-day osmotic minipumps delivering bromodeoxyuridine for determination ofcdls in S-pbase 

### in the liver. The dosing regimen

. was to have been 5 days/week according 10 

### the following schedule:

0.400. 800. 1200mgIkg (D 

### =8) 

# + SaaifICC SSuM!WTbFSSuMTWTbF 

# + Pump Implam Group I (I wit)
Group 0 (2 wit) 

### ----t
.. 0,400,800, 1200m.gJkg (II = 8)



# + Sac:rif"1CC 

### During the first two days ofdosing. excessive mortality 

### was observed 

### across tt'eatmeD1 groups thai was not anticipated based on the 

### results from the probe study. The mortality was 

### observed in all 

### groups, except 

### comrols, with and 

### without implanted 

### osmotic: minipumps. 

### Therefore, each dose level 

### was reduced in balfresulting in the following dose levels: saline CODtrol, 200, 400 or 600 mgllcglday. During the next two days of 

### administc:riDg the lowerdose levels (total offour days ofdosing) 

### the mortality continued and the study 

### was terminated on the 

### fifth day. Each surviving animal 

### was weighed and sacrificed by cervical dislocation the day 

### after Ilhe 

### fiDaI dose. Stomach (empty). liver aDd spleen 

### weights were recorded and blood 

### was drawn bycardiac puncture for dclennination ofthe hcnwoc:riL Slices of

### esophagus. foresromach and glandular stomach, as well as liver were fixed in 

### oeutral buffered formalin for histopaUwlogy. The remaining liver tissues (-0.5 g) was 

### snap frozen in liquid nitnlgeD for aDalysis ofmarkers for oxidative SU'CSS byJim KJaunig, 

### lDdiaDaUniversity School ofMediciDc. Afterdisc:ussioas 

### with the 

### sponsor. the tissues taken from the tenninal sacrifice were aDalyzed as 

### described below although 

### the group sizes were significantly affected by the mortality. This action was considered nea:ssary to complete the 

### smdy prior to 

### the meeting ofthe NTP BoanI of Scientific CouDc::ilors to review 

### the NTP report 

### aDd precluded repeating the study 

### iD the 3\ailable time.**Fores~omach** 

### Analysis. Paraffin-embedded tissues were 

### serially secticmed and stained withhematoxylin and eosin (H&E) oramibodics against proliferating cell nuclearantigen (PCNA). HiSlologic:al aDalysis (H&.E) 

### wc:rc CODducted 10 detennine evidence ofnecrosis and/or irritation of

### the 

### gastric: mucosa.. The primary determinat.ioa of

### cells 

### in S-pbasc was accomplished by 

### aDa1yscs of PCNA inc:orporatioo according to Eldridge aDd Goldsworthy (1996). Unit 

### length labeling indices (ULLI) 

### were determined by counting

# tA: 13 Project29753 Page 100f49 the number of

### cells in S-pbase per millimeterofthe mucosa within the forestomach. 

### The number oflabeled epithelial cells were scored 

### in at least four 0.25 mm random sections 

### peraDimal. Liver Analysis. 

### Serial scctious from liver slices were sWned with either H&E., Perl's stain 

### (ferroeyanide) or antibodies against bromodeoxyuridine (BrdU). Histological analysis (H&E) were 

### conducted to determine 

### if there 

### was evidence of necrosis and whether the necrosis was diffuse or localized near iron-loaded Kupfer cells. Perl's slain 

### was 

### used to determine the extent ofiron deposition 

### in Kupffer 

### cells and/or bepatoc:ytes. Analysis of BrdU incorporation into S-pbase nuclei was conducted by 

### immunohistochemistry, using the procedure described by 

### Stauber and 

### Bull (1997). Visualization ofBrdU-reaetivc: cells 

### was accomplished with the chromagcn 3-amiDo-9- etbylcarbazole with 

### a bematoxyliD 

### counterstain. A 

### miDimum of 2000 bepatocellular nuclei was scoredby lightmicroscopy in atleast 10randomlyselected fields to

## determine the labeling index for cells 

### in S-pbase. Specific Aim 3: Effect

### 01 IP vs SC-llClmhaistered BE OD 

### the 10restomllCb. 

## Probe Study. Female B6C3FI mice, 3/group were administered saline solutions ofBE by either intraperitoneal (IP) or subcutaneous (SC) injection at 400 or 600 mgllcgld for 3 days. Aftertwo days of

### dosing. one 600 mglkg IP mouse 

### was sacrificed moribund. Ful! Study. 

### Groups of6 female B6C3Fl mice 

### werc: administered 0 or 400 mglkgld BE by either IP or SC injection for 

### S days 

### and necropsied on the afternoon ofthe Slh day of 

### dosing. Terminal body weights 

### and liver weights were measured and livers were flasb

### frozeD and stored at -8O"C for potential aaalysis by Indiana University. The stomach from each 

### animal 

### was dissected. inflated with formalin and 

### fixed in neutral buffered fonnalin for histopathology. 

### F2!W0Jn3ch Analysis. Paraffin-embedded 

### tissues 

### were serially scetioned and stained**~th** hematoxylin and eosin (H&E). Histological analysis (H&E) 

### werc: conducted to determine evidence ofnecrosis and/orirritationofthe gastric mucosa.



### RESULTS AND DISCUSSION Specific 

### AiIII 1: DepositiOD 01 BE 08 tile far

### 01 mice, 

### Wbole-bodY CbaJpber 

## ooc:ration. The 

### distribution 

### of BE vapor CODCCDtr3Iions in 

### the 

### exposurechamber

### was 

### determined prior to 

### the study. Theconce:nnations were measured at 

### 3 chamberpositions during the 

### first hour ofa test generation

### aDd again daring

### the last hour of

### the rest generation. The concemrations were 238.1 

### ± 15 ppm (0.6% relative staDdard deviation) for the 6 samples. 

### The analytical concentration averaged 95% of

### the nominal cODCCDtraricm (250.6ppm).

# ~: 14 Project 29753 Page 11 of49 During the animal 

### exposure. the 

### conc:cntrarionofBE was measured at 3 chamber

### positions during the first 

### hour of

### the 

### exposure study aDd again during the last hourofthe study. 

### The anaiytical 

### c:bamber concentration was 

### 229.2 

### ± 1.0 ppm 

### (92";" ofthe target 

### cooccntration with 0.4% relative 

### staDdard deviation) for the 

### 6 samples. The average analytical concentrariOD was 92.4% ofthe 

### nomiDal coocentratiOD (247.9 ppm). Nose-only chamber operation. 

### The distribution of BE vapor concentratiODS 

### in the exposure chamber was determined priorto the study. The concentrations were measured at a port on each tier and aD 

### additional 

### pan on the lower tier (sampling from the inlet manifold) during the 

# rust hour ofa test generation and again during the 

### last bour ofthe test**generation~** The concentration was 241.8 ± 4.7 ppm (1.9% relative standard deviation) for the 8 samples. A nominal 

### CODCectI'ation was DOt determined 

### due to a stoppage ofthe metering pump for 3D**lDld~** 

### time in the period berwecn the time 

### the two setS ofgrab samples were taken. The measured meaD amcentratiOD was 96.7% ofthe target 

### CODCCDtrationof250 ppm. During the animal 

### exposure, the concentration ofBE vapor was measured at a port on each tier and aD 

### additional port on the lower tier (sampling 

### &om the inlet manifold) during the first hoarofa test generation and again 

### duriDg the last hour of

### the study. 

### The average 

### concentration was 245.7 ± 1.8 ppm (0.7% relative standard 

### deviation) for the 8 samples. 

### The analytical 

### CODCentration averaged 99.4% of the 

### DOrDina1 concentration (247.0

### ppm). Qinical observations. 

### There were no early deaths from either exposure. There were no visible signs of overt toxicity during either the nose-only or whole-body exposure. However, a large number (7/10) of

# me urine samples collected 

### &om the mice following the oose-onlyexposure comained a red colored

### material. possibly blood. 

### In 

### addition 1/10 of

## mecage rinse samples were significantlytinted red. Qeoosition ofBE on the fur. An average of205 J.&g ofBE was detected 

### on the fur offive female mice exposed whole-body to 229 ppm BE vapor (Table I). Sligbdy less BE. 170 j.lglmouse, 

### was detected 

### on the fur offemale mice 

### exposed nose-only to 242 

### ppm BE. Residual BE 

### on the fur ofthe 

### nosc-<lnly exposed mice was attributed to"the use ofa positive-pressure. flow-past exposure system. 

### Most ofthe resiciuaJ 

### BE was detected 

### in the first 

### SO

### m1 wash (see Appendix A-I for individual animal and fur wasb data) thus the method 

### used to wash the fur

### was considered to be effective. An example 

### c:bromatogram 

### from the fur wasb is shown in Figure 3. However, evaporative losses ofBE from the fur probablyocc:um:d during the time for the chamber tobe shut down aDd evacuated aDd the 

### animals 

### sacrificed via**C~ pri~**  to inunersion 

### in 50 

### ml of

### hatwater. When corrccted for the actual BE exposure concentration, whole body exposures averaged 25% more 

### BE.on the fur than the cort':SpOnding nose-only exposures.**Whil~** there 

### was clear evidence that residual BE adsorbs to the fur of mice, the total dose measured post-c:xposure that could be available to 

### grooming and ultimately come into comact with the forestomacb was less dlan 10 mglkg.

# 01 Project 29753**Pa~** 120(49
BE and BAA in blood and urine. The concenuation ofBE 

### in the blood offive female mice at the end ofeach exposure averaged 3.0 and 3.9 mgll for the whole-body and noseonly 

### exposures, respectively (fable 2; Appendix A-2). The concentrations ofthe major metabolite, BAA, 

### in blood were 235 

### and 390 mgII for the whole-body and nose-only exposures respectively (Table 2; Appendix A-2). These concentrations ofBE and BAA were similar to the levels reponed in the NTP chronic toxicokinetic study (Dill et aI., 1998). An examplechromatogramfrom the blood analyses is shown 

### in Figure4. Low levels ofunmetabolized BE «100 JL8) were found in the urine ofmice collected 0- 

### J8 hr after both whole-bodyand nose-only 

### exposures (Table 3; Appendix A-3), The BE in urine was preswned to have come 

### from the 

### fur ofthe mice since no 

### BE is expected to ha'le been excreted umnetabolized 

### in urine based upon data obtained. fu;m '13!S (Corley et 

### aI., 1994). The total amounts offree 

### BAA in me urine indicate little difference between the two methods of exposure 

### and were similar to levels reponed in the 

### NTP chronic toxicokinetic study 

### (Dill et 

### aI., 1998). 

### AD example chromatogram from the urine analyses is shown in Figure 5. Specific Aim 

### 2: Effect oforaDy-administered neat BE DD the forestomach aDd liver. 

### Probe Study. 

### After two consecutive days of dosing. no oven signs of toxicity were observed in male or female mice dosed as high as 800 mgIlcgld Therefore, after consultation with the sponsor, the mice 

### from me lower dose group (100 mglkgld) were administered 1200 mglkgld for the remaining three days of dosing. One mortality occurred (high dose male) that 

### was due to gavage error. All mice were necropsied approximately 4 hr after the 

### fiDaJ dose. Body weigbts. organ weights (stomach, liver and spleen) and hematocrits from each ofthe mice are suounarized 

### in Table 

### 4. Individual animal data are presented in Appendix B-1. Both absolute and relative organ weights were increasedovercontrols for both sexes. Hematoerits were 

### also significantly affected with the top 

### dose group (1200 mg/kgld) decreased 15-300/0 

### from contrbllevels. Based on these data, the dose levels for,the full study were selected. to be 400, 800 and 1200 mglkgld Full Study. Aftertwo consecutive days ofdosing, mortality

### was observed 

### inall treatment 

### groups except controls. Therefore, dose levels for each group were reduced in 

### half resulting in dose levels of200, 400 and 600 mglkgld Overthe next two days ofdosing. the mortality 

### continued to be excessive and the study

### was terminated on the 

### fifth day with no further dosing. Terminal body weights were significantly 

### decreased from pre-study and a dose-response mortality 

### was observed (Table 5). Hematocrits were 

### also decreased by as much as 23-29% from controls (Table 5). Relative liver weights 

### and bom absolute and relative stOmach 

### and spleen weights were**in~** over controls (Table 6). Individual animal body weights, organ weights 

### and hematoerits are summarized 

### in 

### AppendixB-2.

# 02 Project29753 Page :3 of49 

### Forestomach Histology. Evaluation ofH&E-stained slides from mice 

### exposed to neat BE by gavage for four days resulted 

### in lesions related to BE exposure in the forestomachs ofmale 

### and female mice (see Appendix B-3). These lesions included epithelial hyperplasia and inflammation of the forestomach. In the low dose group, minimal to mild forestomaCh epithelial hyperplasia was obsezved in both male and female mice. The change was cbaIacterized by focal areas ofincreased layers of epithelial cells. In mice from the mid exposure group, minimal to moderate forestomach epithelial hyperplasia 

### was obserlled 

### in both sexes. The epithelial hyperplasia, although appearing somewhat focal, 

### was more expansive than observed 

### in low dose mice and the epithelial thickening due to increased epithelial cell layers was more prominenL 

### There were down-gi-owths ofproliferating epithelial cells 

### into the lamina propria. 

### In 

### this 

### exposure group several mice bad significant infLltraces of newropbils and mOl!Otluclear inflammatory cells in the submucosa and muscularis. Both sexes of mice from the high exposure group had minimal to marked forestomach epithelial hype;plasia. Inflammation ofthe submucosa and muscularis 

### was, again. commonly associated with the hyperplasia 

### and 

### in one 

### high dose male appeared to have extended to the periumea.l surface. A very tiny ulcer with

. miniIDaI forestomach epithelial hyperplasia at ilS border was observed in one female control and minimal focal necrosis and forestomach epithelial hypetplasia at the limiting ridge (junction offorestOmach and glandular sromach) 

### in another female control. These were considered spontaneous changes thatare notuncommon 

### in mice. Initial eff'orlS at calculating a unit length labeling index using PCNA immunohistochemistry in random sections of forestomach as origiDally planned wa.<; found to be an inappropriate technique due to the focal nature ofthe'lesions. Therefore.. analysis of labeling indices by PCNA was limited to a qualitative assessment of the relative amount ofS-phase cells 

### in hislologically-nonnal forestomach epithelium VemJS the epithelium surrounding histological lesions. There was evidence ofan increase 

### in PCNA staining intensity in regions smrounding focal areas of hyperplasia and inflammation, as compared to either the forestomach epithelium ofcontrol mice or to histologically-normal regions within the forestomach ofBE-exposed mice. The intensity ofPCNA staining within each forestomach section was more closely associated with the apparent severity ofthe lesion rather than BE dose although the incidei1ce and degree of lesions was associated with BE dose. Lesions with focal regions ofinflammation and thickening ofthe epithelium showed greater overall PCNA·staining as well as clearer evidence ofS-phase cells. 

### LiverH"ulOlogy. 

## Based on H&E-staiIJed sections, liver mOtphology 

### appeared normal in 

### both male and female mice and at all BE dose groups. In addition, no**treatmeDt-re~** accumulation of hemosiderin in Kupff'ercells was observed in anyexposure group. Highly g1ycogenatedappearing liver cells were observed with low occurrence, lack ofa dose response, and variable group distribution and WCJe, therefore, notconsidered exposure-related..

# :03 Project 29753 Page 140f49 There was a clear treatment-rela.ted increase in hepatic iron accumulation based on analysis ofsections stained with Perl's stain. Enumeration ofpositive staining cells 

### in random liver sCctions demonstrated 

### that the iron accwnulation 

### in both male and female mice exposed to BE 

### was dose-related. Although some minimal iron accumuI8tion was occasionally observed in parenchymal cells. the predominant cells affected were Kupffer·s cells. There 

### was a trend for greater iron accumulation 

### in male mice as compared to females 

### (Figure 6). Although this sex-dependcnt trend was observed in both the probe and full study, it 

### was not statistically significant based on the scoring methods used. Analysis ofproliferation indices by 

### PCNA staining revealed no significant differences 

### in the fraction ofS-phase cells between control animals and BE-treated animals 

### in either male or female mice (Table 7). The mean fraction ofcells in S-pbase in liver ranged

. between 0.14 and 0.16 % for males and between 0.16 and 0.19% for females. No treannenl-related differences were observed in BE-exposed animals ofeither 

### sex. Sexdependent differences in PCNA labeling indices have been previously reported (Ellridge and Goldsworthy. 1996). 

### Specific Aim 3: EffectofIP vs 

### SC-admiaistered BE on the 

### forestomach. The dose level of600 mg'kg IP resulted in the moribund sacrifice ofone mouse aftertwo doses 

### in the probe study. Therefore., a dose of400 mg'kgld for five 

### days 

### was used in the 5-day study. All animals survived the 5-day dosing regimen with no clinical observations of treatment-related effects. Terminal body weights were similar to pre-;study body weights in all dose groups. including conlrOls (Appendix C-I). Liver weights were slightly elevated in both the IP and SC treatment groups (Appendix C-I). 

### Forestomach Histology 

### In the probe study, IP injection of 600 mglkg for three days 

### was associated with forestomach epithelial hyperplasia and inflammation (Appendix C-2). There were forestomach lesions 

### in one mouse per group in the 400 mg!kg IP and the 400 and 600 mglkg SC injection groups. Although suggestive ofa treabDent-related response., similar

. lesions occur spontaneously 

### in mice. 

### In the 5-day study. 1/6 mice in the 400 mglkg IP group bad forestomach epithelial hyperplasia 

### and 216 mice in the 400 mglkg SC group 

### bad forestomach epithelialhyperplasia and inflammation (Appendix C-3). The lesion prcsem 

### in

# the IP-dosed mouse 

### was similar to spontaDeous lesions but those in the two SC-dosed mice appeared subjectively different from spontaneous lesions. Regardless, all 1hree mice (IP 

### and SC) had minimal lesions thaI could be identified when evaluatedblindly; association 

### with BE treatment 

### was consideredpossible.

# :04 Project 29753 Page ISof49

CONCLUSIONS Small amounts of

### BE (134

- 318 f.lg) were removed from the fur offemale mice exposed either whole-body or nose-ooly for 

### 6 br to target concentrations of

### 250 ppm 

### BE vapor. The total dose that was available for oral conswnption via grooming was 9.2 ± 2.9 mglkg for the whole-body exposure and 7.5 ± 2.3 mglkg for the oose-only exposure. Few differences were detected in the internal 

### dose of

### BE resulting from whole-body 

### vs. noseonly exposure as determined by analysis ofBE and its major metabolite BAA 

### in blood collected immediatelypost-exposure and in urine collected for

### up to 18 brpost-exposure. 

### Although this 

### was not 

### a mass-balance 

### stUdy for the total fate of

### BE. the amounts of

### BE deposited 00 the furofmice represented 

### 9-13% oftbc total 

### BAA eliminalcd in the urioe. This calculation is, however, speculative since the amount ofBAA found in the urine collected 18 br post-exposure is likely an underestimate ofthe total absorbed dose. 

### In addition, the amounts of

### BE deposited on the fur and coDSlDlled via grooming 

### during the

. exposure as well as orally consumed via mucociliary clearance of

### BE deposited 

### in the

mucous layer lining the 

### respiratory tract was not accounted for 

### in this study. Nevertheless, there is evidence that at least 

### a small amount ofneat 

### BE was available for oral consumption via grooming and Chat the potential role for chronic, daily oral 

### doses 

### in mouse forestomach toxicity**~t** be ruled ouL Oral dosing ofneat 

### BE to male and female mice 

### 4 hr after feed was withheld resulted in clear dose"response irritation to the forestomach. No overt toxicity 

### was observed by H&E 

### stain in the liver. The dose levels used 

### in this study were clearly excessive with 

### severe hemolysis and mortality observed at all dose levels in the 

### full study that were not
. predicted from the 

### probe study. Dose levels 

### used in this study were initially 0, 400, 800 and 1200 mglkgld for two days. After two days, each dose group reduced in halfto 0, 200, 400 and 600 mglkgld for two additional days of dosing before the study 

# was tenninated on the 

### fifth day. Overall mortality was 0, 6, 8 and 11 ofsixteen male mice and 0, 6, 12 and 10 ofsixteen female mice at the control, low, middle 

### and high dose levels, respectively. Forestomach lesions consisted of focal areas of irritation and epithelial hyperplasia. The prOliferative response was confirmed by PCNA immunohistochemistry ofepithelial cells surrounding the lesions vs. normal cells 

### in the forestomach. Similar forestomach lesioDS were observed following IP injection of600 mglkg for 3 days 

### in 313 female mice. Minimal forestomach lesions were observed 

### in 1/6 and 216 female mice following IP and SC injections of 400 mglkgtd for 5 days, respectively. These data suggest that syst!=Mic admimstration of BE can result 

### in forestomach lesions 

### and the direct contact with BE may not 

### be a 

### requiremenL It is funber possible that the metabolite, BAA, may playa role io producing the forestomach lesions since the IP injection route results in 

### first-pass liver metabolism while SC injection docs DOt. Interestingly, these lesions had DOt been obseJved in previous drinking water studies conducted by NTP (NTP, 

### 1993) suggesting that the lesions may

### be dose-rate

### depeDdenL

# :05 Project 29753 Page 160f49 ID me livers ofmice orally-dosed with neat BE, there was no evidence ofdegeneration and nO corresponding compensatory cellular regeneration. 

### In addition, there ""'as no remarkable accwnuJation ofhemosiderin 

### in Kuppfer cells although there was a clear dose-response increase 

### in iron content in Kuppfer cells and to a much lesser degree 

### iD bepatocytes. The significaDt increase 

### in iron may playa potel'ltially significant role 

### iD oxidative stress in these 

### cells. The potential for oxidative StresS in the livers ofJ1ese mice were evaluated 

### and reponed separately 

### by the Indiana University Medical Center (Kamendulis et al., 1999). 

### In**~clusion,** these dara coupled with the evaluations ofKemendulis et aI. (1999) are consistent with the hypothesis that the turnors observed 

### in the foreslOmacbs 

### and livers of B6C3FI mice following chronic inhalation exposure are me result of indirect mechanisms associated with chronic tissue injury. The lesiODS 

### in the forestOmach appear to

## be dose-rate dependent and may not require direct contact with the 

### test material. The lesions in the liver appear to be secondary to bemolysis and may result from increased oxidative

### mess associated with iron deposition.



### ACKNOWLEDGMENTS The authors greatly appreciate me technical assistance of T. Luders. 

### B. Myers. K. Studniski, and Dr. L. Sasser 

### in the conduct ofthe oral gavage studies and J. Blessing, M. Vucelick, B. 

### MacisalK:, E. Rossignol. B. 

### lhomas, G. Ell and C. Simmons 

### in the conduct ofthe inhalation studies.

# :06 PIojec129753 Page 17,,[';9 

### REFERENCES Corley. R.A.• 

### Bormen, GoA. 

### and Gbanayem. B.L (1994). Physiologically-Based Pharmacokinetics 

### of 2-Butoxyethanol and its Major Metabolite, Butoxyacetic Acid, in Rats and Hmnans. 

### ToDcol. Appl. PharmDcol. 129,61-79. Dill J.A., 

### Lee. K.M., Bates,**DJ~** Anderson, OJ., Johnson, R.E., 

### Cbou. BJ.• Burka, LT. and Roycroft, J.H. (1998). Toxicokinetics of inhaled 2-bulOxyethanol and its major metabolite, 2-butoxyacetic acid, in F344 rats and B6C3FI mice. 

### To:xicol. Appl. PhaTmilcol. 

### 153, 227-242. Eldridge, S.R. and Goldswonh. S.M. (1996). CeU 

### proliferation rau:s in 

### common cancer larget tissues of B6C3FI mice and F344 rats: Effects of age, gender and choice of marlcer. 

### Fund. Appl. Toxicol. 32, 159-167. Kamendulis,**LM~** Park, 

### JJ. and Klatmig. J.E. (1999). Potential mechanisms for rodent liver toxicity by 2-butoxyetbanol: Oxidative stress "studies. 

### Draft Progress Report. February 8, 1999. National Toxicology Program (NTP). (1998). 

### DtaftTechnical Report 

### on the Toxicology and Carcinogenesis Studiesof2-Butoxycthanol (CAS No. 111-76-2) in F344/N 

### Rats and B6C3FI Mice 

### (Inhalation Studies). NIH Publication No. 98-3974. National Toxicology 

### Program (NTP). (1993). Ethylene Glycol Ethers 2- Methoxyetbanol. 2-Ethoxyethanol and 2-Butoxyethanol Administered in Drinking Water to F344/N Rats and B6C3Fl Mice. NIH Publication No. 93-3349. Stauber. AJ. and Bull, 

### RJ. 1997. Differences in phenotypeand cell replicative behavior ofhepatic tumors induced bydicbloroacetate (DCA) and tricbJoroacewe (TCA).
. 

### ToricoL Appl. Pharmacal. 144,235-246. Tyl. R.

### W., Ba11antYne. 

### B.• Fisber. 

## L.e.. Fait, D.L, Savine, T.A., Dodd, D.E
.. Klonne, D.R. and Prins, I.M. (1995). Evaluation ofthe developmentallOxicity ofethylene glycol aerosol 

### in the CD rat and CD-I mouse by whole-body exposun:. 

### Fund. Appl. Toricol. 24,57-75.

# :07 Project297S3 Page 180f49 

### Fagan 1. Vaporgcncating system 

### used for 

### wbole-bodyand 

### nose-only

### exposures to target 

### concenr.ratioos

### 0(250 ppm 

### BE. To Building Exhaust 

# +

Stainless Steel Hood

Power Supply 

# , Vapor L..acIen Gas To Exposure Unit Tape



# 1

I.!::==:!.J Secondary Container Bubble Removing

Syringe Compressed Air

# 08 Proje<:l:297S3 Page 190f49 

### Fagare 2. Example cbrornaIDgramofsampleextIaCt 

### from nose-only exposure 

### amaosphere. 

### Peaks at 

### -4.4 minutes 

### and -7.7 minuteS are BE aDd 

### I-pbeoylhexane internal standard, respectively.

. .**--·~A:~_m_.i..-rtIt'o.**



# -.**I~.~**



# ..**~.** 

# _. !

# , , • i

# 09 Project297S3 Page 20 of49 

### Fagare 3. Example chromatograms from (a) control wasb water 

### and (t,) 

### fur wash 

### from a 

### mouseexposed whole body

### to 

### 229 

### ppmBE for 

### 6hr. (a) Control wasb waIer

Pet" ro.-.aaT

....**~J** ... IlDii.DI 

# ...



# .**~'**
II·

11-



# ..

--------------



# .. 11-

II··

11-

.



# .... 

# --.-----

# .._---

(b) 

### Furwasb 

# T

I, .' 

## -._----- •



# _._---------------"--- .:._._--- "

### ..**~-'----'-"'--'--­** 

### .,.

### ;-'-------.

### --_._---:: ..

## _-------- 

## ----------_._-.

# __._. 

# . 

# . .

# 10 Project 29753 Page 21 of49 FJgare 4. Example cbromatograms 

### fiom (a) control mouse blood 

### and (b) blood 

### from a 

### mouseexposed whole-bodyto 229 ppm 

### BE for 6 hr. (a) Comrol mouseblood

1I'",,,,,lO.lICl!'T. 

### _'Da 

# .. : 

# _.



# --

....

.....**12-~_Acid** 

# I '::::::;: ".**'~----~-------..;..;..> --~--** 
(b) 

### Blood sample 

### from exposed mouse

""

__ '''''

__ ''11I01'-


# ...

_

.



# .....

# 11 Proje.."t 29753 Pagellof49 

### FagareS. Examplechrorrwograms from (a)control mouse urineand (b) 

### urine from a mouse exposedwhole-bodyto 

### 229 ppm 

### BE for6 hr. (a) Control urine



# .. , 

# --

'

# ...



# -- •.

..

....**----~~~._-'-~"---_.** 

### ---

(b)

## uriDefrom exposedmouse
..,.'" lG.....r.

_ ... 

# .. 

# _. _. ,,..- 

# .•.



# . : .: 

# I 2.£_,...oc AcId 

# I

. "'&
. . 1 

# •.'**12~~.1::**

. 

# - ,-



# ..**·~12.~** 

# J**.~** 

# ;



# .... ....

.,**.....::...~** .....**'~'.** _ · ;_...**"'--~-.-;.~l-:-.---.'~~** '--"



## "---"..----------------------:,-._-----



## --

"

# .12 Project29753 Page23of49 Figure 

### 6. The relative intensity ofPerl 

### staining as a function ofBE 

### dose 

### in male and female mice 

### deIcnnined from the fuJI 

### study. Each value is the mean 

## <:t s.d) nwnber of positive cells per field (0.25 x 0.25 mm) from a1 least 4 mice, 

### determined by counting 

### S random fields 

### persection.

25

5 

### .c 20 E on 

# ... e :e 15 

# .. U 

# .. > 

## ! 10 o a. r:

200 300 Final 2·BE Dose (mglkg)

# 13 Project29753 Page 24 of49 Tab-'. 1. Residual BE (mean ± s.d) removed 

### from the fur offive female mice exposed 

### eitherwhole-bo:dY or

### nose-oDlyfor

### 6 br

### to a target

### concenaationof250ppmBE.



### Exposure

Group 

### ExposureCone. BodyWI. (ppm) (g) 

### Total BE (118) Total BE Total BE (mgIkg BW) (mg/\cgfppm) Nose.()nJy Whole-Body 241.8 

### 2292 22.7 ± 0.4 170±52 7.5 ± 23 0.031 ±0.009 22.0:t: 

### 1.3 205 :t: 

### 69 92

# = 2.9 0.040

# = 0.013

Table 

### 2.. ConcentJitiOD ofBE 

### and BAA 

### in the

### blood offive female 

### mice atIbe 

### eDd of

### a 6- hI' whole-body ornose-oolyexposure to a

### targetc:cmccuaationof

### 2SOppmBE. Exposure Exposure 

### Cone. BodyWI. BE 

## BAA

Group 

### (ppm) (g) (mgIL) (mgIL) Nose-Only 

### 241.8 21.9: 1.1 

### 3.94: 1.70 389.5 :t:33.8 Whole-Body 

### 229.2 21.6:1.2 3.02: 1.2S 235.4 ± 

### 52.3
.

Table3. Amounts offree BE

### and BAA excreted 

### in the urine often female mice 

### 18 hrafter the end 

### ofa 6-br 

### whole-body or**~y** exposure to a target 

### concentration of250 ppm BE. 

### Exposure

Group Nose-oaIy 

### \'-'bole-Body**~CODC.** (ppm) 

### 241.8 229.2 Total BE (J1g) 

### 67.6:23.3 68.7±39.6 Total BAA (JIg) 

### 2021 ±537 1783 :t:645

# 14 Project297S3 PagelS of49 

### Table 4. 

### Body weights. organ weights (absolute 

### and relative) and bcmatoerits from 

### five 

### micelscxldose gavaged with neat BE 

### for five consecutive days (Probe Study). 

### Da%a are expressed

### as the 

### mean with the standard 

### deviation aD the 

### nextline. (a) Males 

### Dose BW SlOmaCh Liver Spleen HCT (mgIkg) (g) (g) (%BW) (g) (o/oBW) (g) (%BW) (%) 

### 0 2636 0.182 0.685 1.099 4.158 0.066 0248 47.8
. 1.74 0.045 0.140 0.142 0283 0.018 0.053 2.1 400 . 26.76 0258 0.963 1.178 4.389 0.156 0.582 48.0 1.91 0.058 0.196 0.149 0.305 0.051 0.181 4.1 800 2820 0262 9·931 1220 4320 0.170 0.606 N/A 152 0.028 '0.105 0.125 0.283 0.014 0.083 N/A 1200- 23.18 0228 0.978 0.925 3.988 0.075 0325 40.8 1.62 0.036 0.102 0.084 0.140 0.006 0.037 2.2

. • Mice were 

### dosed with 100mglkgtd BE for two days followed by 3 days at 1200 mglkgld

(see teXt). (b)Fcmales



### Dose BW Stomach Liver Spleen HCT (mglkg) (g) 

### (g) (%BW) (g) (%BW) (g) (%BW) (%) 0 20.4 0.174 0.858 0.810 3.979 0.074 0363 503 1.0 0.015 0.105 0.038 0.057 0.011 0.045 2.6 

### 400 20.7 0276 1327 0.782 3.782 0.092 0.451 46.0 1.6 0.052 0.193 0.061 0353 0.019 0.129 5.4 800 19.8. 0248 1250 0.818 4.124 0.158 .0.797 N/A 0.8 0.034 0.163 0.054 0243 

### 0.OS7 0296 N/A 1200· 19.0 0302 1.619 0.766 4.061 0.092 0.491 35.8 1.7 0.111 0.687 0.009 0292 0.022 0.134 83

• Mice were 

### dosed with 100mglkgld BE for two days foUowed 

### by 3 days at 1200 mgIkgId (sec text).Project 29753 Page 26 of49 

### Table 

### 5. Body weights, monality and hematocrits from 

### 16 micelsexJdose administered neat BE by gavage for foW" consecutive 

### days (Full Study). Terminal body weights and hematocrits determined only 

### in animals surviving until terminal sacrifice. (a) Males 

### Pre-Study Tetminal Dose BodyWt. BodyWt. HCf (mglkw

### d) (g) (g) Mortality (%) 

### 0 26.8: 1.1 

### 21.2 ± 1.0 0/16 

### 52.1 ±3.3 4001200" 26.3: 1.2 26.9: 0.1 6/16 51.0±2.6 8001400" 27.1 ± I.l 

### 25.5: 2.S 8/16 46.6±3..2 1200/600" 26.9: 1.7 23.2±2.9 11/16 4O.0±2.6

• After two days ofdosing the mortality was excessive: tbcR:fc=. surviving mice from each dose group were**admiI1ist~** half of Ibeir correspoDdiDg ori(tiDal target dose for two additional days prior to SDIdy termiDation. (b) 

### Females Pre-Study Tennina1 Dose BodyWt. BodyWt HCf (mglkw d) (g) (g) Mortality (%) 

### 0 20.5 ± 1.8 20.8 ±0.7 0/16 

### 50.9±2.5 4001200" 19.5 ± 

### 2.5 17.5 ±4.3 6/16 4'P 8001400" 20.4±2.0 19.7 ±2.2 12116 37.5 ± 1.3 1200/600" 

### 20.8±1.6 19.4±2.1 10/16 

### 363±1.5
1 Aftertwo days ofdosing !hemortality was excessive: therefore. surviving mice from each dose group were adlDillisU:rcd IIIlf of Ibeir corresponding original target dose for twO additional days prior to SDIdy tcrmiIIatioII.

. . b UDable to oblaiDblood samples via orbilal sinus punC1UJ'C from all but ODe mouse.

# C:02 Project 29753 Page 27 of49 Table 6. Temtinal body weights and organ weights (absolute and relative) from 

### up to 16 micelsexldose gavaged with neat BE for four consecutive days (Full Study). TermiJJal body weights and organ weights determined only in animals surviving 

### until temUDal sacrifice (see Table 5 for monalitydata). 

### Dataare expressed as the mean with the standard deviation on the next line. (a) Males

Dose BW Stomach Liver Spleen Kidney (mglkg) (g) (g) (%BW) (g) (%BW) (g) (%BW) (g) (o/oBW) 0 27.2 0.1723 0.6353 1.2285 4.5197 0.072 0.267 0.1926 0.7091 1.0 0.0127 0.0548 0.1466 0.4528 0.012 0.050 0.0155 0.0432 4001200· 26.9 0.1785 0.6666 1.4485 5.3887 0.0985 0.3680 0.1895 0.7067 0.7 0.0213 0.0955 0.1893 0.6224 0.0231. 0.0917 0.0105 0.0542 800/400· 

### 25.5 0.2075 0.8166 1.2956 5.0576 0.1158 0.4500 0.1895 0.7440 2.5 0.0261 0.1014 0.2201 0.5120 0.0385 0.1262 0.0221 0.0657 12oo/6O(f 23.2 0.2036 0.8713 1.2168 5.1115 0.1154 0.4917 0.1712 0.7407 2.9 0.0507 0.1568 ·0.2335 0.4185 0.0514 0.1770 0.0189 0.0462
(b) 

### Females 

### Dose BW Stomach Liver Spleen Kidney (mglk:g) (g) (g) (o/oBW) (g) (%BW) (g) (%BW) (g) (o/oBW) 0 20.8 0.1568 0.7562 0.9485 45588 0.075 0.3623 0.1254 0.6036 0.7 0.0324 0.1707 0.0589 0.2056 0.009 0.0415 0.0127 0.0671 4001200a 175 0.1443 0.8255 0.6350 3.9379 0.0665 0.3496 0.1165 0.6712 4.3 0.0358 0.0734 0.2299 0.8666 0.0449 0.1655 0.0251 0.0820 

### 800/400- 19.7 0.1803 0.9176 1.0933 55288 0.1680 0.8723 0.1388 0.7016
. 2.20 0.0175 0.0599 0.1869 0.4706 0.0493 0.3184 0.0243 0.0560 1200/600" 19.4 0.1970 1.0184 1.0093 5.2250 0.1860 0.9549 0.1310 0.6795 2.1 0.0213 0.0865 0.1655 0.2000 0.0400 0.1598 0.0062 0.0563

• After twO days ofdosing the monality was excessive: lhen:fore, survivingnUee tiom cadi dose group were admiDistered balf of Ibeir eorrespondjng origiDal large! dose for twO additiooal days prior to study rermiDation.

# C:03 Project 29753 Page 28of49 

### Table 

### 7. Percentage of

### S-phase cells 

### in liver ofmice treated with 

### BE. Values (mean 

## ± s.d.) were determined 

### by analysis of 

### PCNA 

### staining by 

### scoring a minimwn 

### of 

### 2000 hepatocytes from 

### randomly selected fields, with 

### a minimwn of 4 animals scored 

### per treatment

### group.

% 

### S-Pbase 

### DoseGrou 

### Males Females ContrOl 0.15:0.06 0.18 :0.05 

### 4001200**mgtk~** 

### 0.15 :0.06 0.19:0.08 800/400**mglk~** 

### 0.16:0.06 0.17:0.06 1200/600mglkt 

### 0.16:0.09 0.18 :0.09

• After lWO days ofdosilIg !be

### monalny w:IS excessive: lberdore. surviviDgmice from each dose group were admiDistercdba1foflbeircom:spoading originall3rgetdose fortwoadditional dayspriorto studyrcmirwion. S-phase nuclei were scoredbasedontbc: mcdIodtqlOned byEldridge ami GoIdswonby (1996).

# 0 Appendix A-I 

### Projecl29753 Illdividual Animal Dala

- 

### Fur Dcposilion SlUdy Page 29

### or49 Appendix A·I Individual Anil1)al Data

- FurDeposilion Study Residual2.Buloxyethanol on the FurofMice- NoseOnlyExposure (241.8 ppm) Residual BE Residual BE 

### Body WI. Amount of 

### BE in Wash Total BE in Wash (mg BE/kg '. (mg 

### BElkg BW AnimallD (g) 

### Wash No. Wash Waler(g) (Jlg1ml) (Jig) BW) Ippm Exposure) 306 22.5 I 

### 48.982 4.63 226.92 2 49.118 0.426
. 20.94 247.9 11.02 0.0456 307 23.1 

### I 

### 48.479 3.61 174.89 2 48.721 0.49 23.79 198.68 8.60 0.0356 308 22.1 I 

### 48.988 2.24 109.91 2 48.562 0.51 24.67 134.58 6.09 0.0252 309 22.5 ,I 

### 48.772 2.29 111.86


# --- --- 2 49.026 0.47 ll,ll 

### 134.71 5.99 0.0248 310 23.1 1 49.114 2.29 112.3 2 49.196 0.44 133.27 5.80 0.0240**----~----** 

### Mean: 22.66 169.97 7.50 0.0310



### SD: 0.43 51.69 2.27 0.0094

### AppendiKA-I Projeel 29753 

# 0 Individual Animal Data

- 

### FurDCllosilion

### Study Page 30or49 

# Ul
Appendix A-I Individual Animal Dala

- FurDeposilion Siudy Residual 2·Buloxyelhanol on the FurofMice

- Whole Body Exposure (229.2 ppm) Residual BE
. Residual BE Body WI. Amounlof BE in Wash Tolal BE in Wash (mg BE!kg (mc OE!kg BW Animal 10 

### (g) Wash No.. Wash Weier (g) ().lglml) ().Ig) OW) !ppm Exposure) 106 2J.8 I 

### 48.741 J.88 189.08 2 48.633 0.68 33.18 222.26 9.34 0.0408 101 21.1 I 

### 49.259 2.12 134.15 2 49.403 0.59 29.29 163.44 7.53 0.0329 108 22.6 I 

### 49.163 5.72. 281.00 . 2 48.666 0.16 36.15 J17.75 14.06 0.0613 109 20.4 I 

### 49.291 3.10 153.01 2 49.206 0.43 

## ill 174.30 8.54 0.0373 110 21.6 I**49.1~9** 

### 2.66 130.93 2 49.050 O.JO 

### IDQ 145.53 6.14 0.0294 Mean: 22.02 204.66 9.24 0.0403

SD: 1.21 69.31 2.87 0.0125Appendix A-2 Individual Animal Dala

- Fur DcrosilionSrudy 

### Appendix A-2 

### Individual Animal Oala

- FurDeposilion Study Concenlralionof2·Buto"yelhanol (BE)/Buto"yacelic Acid (BAA) in Blood Project 29753 Page: 31 of49 

# n o 0'\

Animal No. Exposure Body

### WI. (g) 

## DE (mgtl) BAA (mgll) 

### 101 102 

### 103 104 IllS 

### Mean:

SO: 

### WB WB WD 

### WB WB 22.9 4.68 22.2 2.21 21.4 2.84 19.6 3.82 21.7 1.57 21.6 3.02 1.23 1.25 195.0 206.9 

### 215.5 234.2 325.5 235.4 52.3 301 302 303 304 305 

### Mean:



# so: NO 20.8 5.95 409.7 

### NO 21.0 5.49 364.2 

### NO 21.8 3.18 401.1 

### NO 22.1 3.12 345.1 

### NO 23.6 1.99 427.2 21.9 3.94 389.5 1.1 I.

### 70 33.8 

### WB

# = 229.2 

### ppm Whole 

### Bodyexposure. 

### NO

# = 241.8 ppm NoseOnlyexposure.Appendix A·J Individual Animal Dala

- FurDeposilion SlUdy 

### Appendix A-J Individual Animal Data

- Fur Deposition Study 2-Buloxyethanol (BE) and BUloxyacclic Acid (BAA) Levels in Urinecollecled 18 hr POSI-Exposure Prajeel29153 Page nof49 

# o Q

Animal No. Exposure Total BE in Urine (j.1g) Total BAA in Urine (J.lg) III WB 104.4 2]] 1.8 112 WB 80.1 2641.4 113 WB 130.7 2678.1 114 WB 74.6 1527.8 115 WB 43.8 1546.2 116 WB 5.9 813.6 117 WB 90.6 I 

### 1395.5 118 WB 81.1 2165.7 119 

# wn 8.5 1051.0 120 WB 68.1 1674.8 Mean: 68.7 1782.6

SD: 39.6 645.0 311 NO 67.5 312 NO' 94.6 313 NO 112.9 314 NO 6].0 315 NO 80.0 316 NO 32.0 317 NO 55.8 318 NO 47.6

. 319 NO 57.1



### 120_
. NO

__ _ __ .. 65.3**M~n: ~J**  

### SD: 23.3 2695.0 1709.7 1143.5 1583.8 210),5 1948.6 1949.5 1674.8 2825.3 2571.6 2020.5

537.4**Appendi~** "-, Individual Animal Dala

- Gavage Sludy. Probe 

### Appendix 8-1 

### Individual Animal Dala

- Gavage Study, Probe Body Weighls, Organ Weighls and Hemalocrils for Anir:nals al Tenninal Sacrifice Saline Control- Males (Probe) Projeci 29753 Page)) of49

# .~ Q 

# QO

Relative Relative Tenninal Relalive Liver Liver Spleen Spleen BodyWeighl Siomach WI. Stomach WI. WI. Wt. WI. WI. HCT Animal II (8) (g) (% 

### BW) (g) (% 

### BW) (g) (% 

### BW) (%) 

### 1 24.4 0.140 0.574 0.987 4.045 0.050 0.205 48 2 27.9 0.190 0.681 1.200 4.301 0.080 0.287 

### NS 3 28.1 0,220 0.783 1.280 4.555 0.090 0.320 45 4 26.7 0.230 0.861 1.090 4.082 0.060 0.225 48 5 24.7 0.130 0.526 0.940 3.806 0.050 0.202 SO



### Mean: 26.36 0.182 0.685 1.099 4.158 0.066 0.248 47.8



### SO: 1.74 0.045 0.140 0.142 0.283 0.018 0.053 2.1

### NS"~N-:-o-S=-am-'-p-:-Ie; NW"Nol Weighed; N/A=Not

### Applicable. 400 mglkg/d

- Males (Probe) 

### Relalive Relalive Terminal Relative Liver Liver Spleen Spleen Body Weighl SiomachWI. Siomach WI. WI. WI. WI. WI. HCT Animal II 

### {gL___ (g) 

### (% 

### BW) (g) 

### (% 

### OW) (g) 

### (% 

### DW) (%) 

### II 23.4 0.220 0.940 0.920 3.932 0.120 0.513 48 12 28.1 0.230 0.819 1.200 4.270 0.110 0.391 53 IJ 

### 27.3 0.310 1.136 1.290 4.725 0.240 0.879 47 14 27.8 0.330 1.187 1.270 4.568 0.160 0.576 42 

### IS 27.2 0.200 0.735 1.210 4.449 0.1 SO 

### 0.55I 

### SO'

Mean: 26.76 0.258 0.963 1.178 4.389 0.156 0.582 48.0



### SD: 1.91 0.058 0.196 0.149 

### 0.305 0.051 0.181 4.1 

### NS=No Sample; NW=Nol Weighed; N/A=Nol

### Applicable.Appendix 8·1 Individual Animal Dala -Gavage Sludy, Probe



### 800 mglkgld .:- Males (Probe) ProjecI29753 Page 34 or49 

# c o

Tenninal Relative Body Weight Stomach Wt. Stomach WI. Animal II 

### (g) (g) (% 

### BW) 16 29.1 0.290 0.997 17 28.8 0.230 0.799 18 25.5 0.260 1.020 19 28.7 , 0.240 0.836 20 28.9 0.290 1.003



### Mean: 28.20 0.262 0.931

SD: 1.52 0.028 0.105 

### NS=No Sample; NW=Not Weighed; N/A=Not Applicable. Relative Relative Liver Liver Spleen Spleen WI. Wt, Wt. Wt, lIeT (g) 

### (% 

### OW) (gL(% 

### OW) 1.370 4.708 0.160 0.550 

### NS 1.280 4.444 0.160 0.556 

### NS 1.040 4.078 0.190 0.745 

### NS 1.250 4.355 0.160 0.557 

### NS 1.160 4.014 0.180 0.623 

### NS 1.220 4.320 0.170 0.606 N/A 0.125 0.283 0.014 0.083 

### NIA

IZOO mglkg/d

- Males (Probe) 

### Relative Relalive Tenninal Relative Liver Liver Spleen Spleen Body Weight Stomoch Wt. Stomach WI. Wt, WI. WI. WI. IICT Animal 1# 

### (g) (g) (% 

### BW) (g) (% 

### BW) (g) (% 

### OW) (%) 

### 6 22 0.220 1.000 0.880 4.000 0.070 0.318 42 7 24.2 0.260 1.074
. 1.010 4.174 0.080 0.331 43 8 21.6 0.180 0.833 0.830 3.843**~l.O80** 

### 0.370 38 9 24.9 0.250 1.004 0.980 3.936 0.070 0.281 40



### Mean: 23.18 0.228 0.978 0.925 3.988 0,075 0.325 40.8

SD: 1.62 0.036 0.102 0.084 0.140 0.006 0.037 2.2 

### NS=No Sample; NW=NotWeighed; N/A=Not Applicable.AJlpendix D·) Individual Animal Daln

- Gavage Siudy, rrobe



### Saline Conlrol- Females (Probe) Projecl 29753 Page 35 of49 

# () 1-6 

# o

Relative Relative' Tenninal Relative Liver Liver Spleen Spleen Body Weight Stomach Wt. Stomach WI. WI. WI. WI. WI. HCT Animal" {&)**(~~uu-<o/~B_W)** 

### (g) (% 

### OW) (g) (% 

### BW) (%) 

### 102 21.6 0.170 0.787 0.860 3.981 0.090 0.417 54 101 19.8 0.160 0.808 0.790 3.990 0.080 0.404

-48 103 21.1 0.160 0.758 0.840 3.981 0.070 0.332 

### NS 104 20.3 0.190 0.936 0.790 3.892 0.070 0.345 49 105 19.0 0.190 1.000 0.770 4.053 0.060 0.316 50



### Mean: 20.4 0.174
. 0.858 0.810 3.979 0.074 0.363 50.3

SD: 1.0 0.015 0.105 O.oJ8 0.057 0.011 0.045 2.6 

### NS=No Sample; NW=Nol Weighed; N/A=Not Applicable. 400 mglkgld

- Females (Probe) 

### Relative Relative Tenninal Relative Liver Liver Spleen Spleen Body Weight Siomach WI. Stomach WI. WI. Wt. WI. WI. IICT Animal" (8) (8) (% 

### BW) (8) (% 

### BW) (8) (% 

### OW) (%) 

### III 22.2 0.360 1.622 0.820 3.694 0.080 0.360 50 112 18.6 0.220 1.183 0.700 3.763 0.120 0.645 38 113 21.6 0.260 1.204 0.160 3.519 0.070 0.324 48 114 19.6 0.280 1.429 0.860 4.388 0.100 0.510 48 115 21.7 0.260 1.198 0.770 3.548 0.090 0.415 

### NS

Mean: 20.7 0.276 1.327 0.782 3.782 0.092 0.451 46.0



### SO: 1.6 0.052 0.193 

### 0.061 0.353 0.019 0.129 5.4



### _

NS=No Sample; NW=Nol Weighed; N/A=Nol

### Applicable.Appendix (]·I Individual Animal Dolo

- Gavage Study. Probe



### 800 mglkgld

- Females (Probe) Project 29753 Page 36 of49 

### I-l I-l

Relative Relative Tenninal Relalive Liver Liver Spleen Spleen BodyWeight StomachWt, Siomach WI. WI. WI. WI. WI. HCT Animal ft 

### (8) (g)**~ro** 

### BW) (g) 

### (% OW) (g) 

### (% OW) (%) 

### 116 19.1 0.220 1.152 0.790 4.136 0,230 1.204 NS 117 21.2 0.280 1.321 0.880 4, I5I 0,180 0,849 NS 118 19.7 0.230 1.168 0.840 4.264 0,180 0.914 NS 119 19.9 0.220 1.106 '0.740 3.719 0.110 0.553 NS 120 19.3 0.290 1.503 0.840 4.352 0.090 0.466 NS

Mean: 19.8 0.248 1.250 0.818 4.124 0.158 0,797 N/A



### SD: (l,8 0.034 0.163 0.054 0.243 0.057 0.296 N/A 

### NS=No Sample; NW=Nol Weighed; N/A:;;Nol Applicable.
1200 mglkgld

- Females (Probe) Relative Relalive Tenninal Relative Liver Liver Spleen Spleen Body Weight Stomach WI. Stomach WI. WI. Wt. WI. Wt, HCT Animal tI 

### (gl (g) 

### (%HWL- (g) 

### (%BW) (g) 

### (%BW) (%) 106 18.0 0.220 1.222 0.760 4.222 0.120 0.667 35 107 18.8 0.310 1.649 0.770 4.096 0.100 0.532 25 108 .17.5 0.490 2.800 0.760 4.343 0.070 0.400 38 109 21.8 0.260 1.193 0.780 3.578 0.070 0.321 45 110 18.7 0.230 1.230 0.760 4.064 0.100 0.535 NS



### Mean: 19.0 0.302 1.619 0.766 4.061 0.092 0.491 35.8

SD: 1.7 0.111 0.687 0.009 0.292 0.022 0.134 8.3 NS=No Sample; NW=Not Weighed; N/A=Nol Applicable. ..Appendill B·2 Individual Animal Dala

- Oavage Sl\Idy. Full
Appendix 8·Z 

### Individual Animal Data-Gavage Study, Full Dosing Regimen, Body Weights, Organ Weighls and Hemalocrils for Animals atTerminal Sacrifice Plojeel 29753 Page)7 of49 

# N

Relative Relalive Spleen Kidney Kidney

WI. WI. WI. IleT (% 

### OW) (8) (% 

### OW) (%)



### 0.374 0.198 0.770 55 0.205 0.173 0.668 54 0.247' 0.182 0.682 54 0.243 0.187 0.688 47 0.273 0.200 0.719 48 0.287 0.183 0.665 

### NS 0.269 0.194 0.705 52 0.235 0.224 0.775 55 

# ____-=- .**.;:S=-=~lIn;.;.:e'_"C;.;:oc::n=tr.::.:ol'_-....:.M:.:;I=..:le=_=sc..>.(F::...;u::..:;II:..:S::.;tu::.;d::.ly}** _ 

### Pre· exposure Terminal Days Days. Relalive Relalive

Body Body Dosed Dosed Stomach Stomach Liver Liver Spleen Animal Weighl Weight 1200 600 

### WI. WI. WI. WI. WI. 1# 

### (g} (g) mglk8 mglkg (8) (% 

### OW) (8) (% 

### BW) 

# --.W I 26.1 25.7 2 2 0.165 0.642 1.071 4.167 0.096 3 24.9 25.9 2 2 0.196 0.757 1.105 4.266 0,053 5 26.1 26.7 .2 2 0.164 0.614 1.252 4.689 0.066 7 26.8 27.2 2 2 0.166 0.610 1.272 4.676 0.066 9 26.6 27.8 2 2 0.164 0,590 1.520 5.468 0.076 II 27.8 27.5 2 2 0.179 0.651 1.098 3.993 0.079 13 27.4 27.5 2 2 0.160 0.582 1.207 4.389 0.074 15 28.4 28.9 2 2 0.184 0.637 1.303 4.509 0.068 

### Mean: 26.76 2V 0.1723 0.6353 1.2285 4.5197 0.072



### SD: 1.10 1.0 0.0127 0.0548 0.1466 0.4528 0.012 

### NS=No Sample; NW=Not Weighed; N/A=Nol Applicable. 0.267 0.1926 0.7091 52.1 0.050 0.0155 0.0432 3.3Appcndilt B·2 Individual Animal Dala

- Gavage Study, Full



### 400/200 mg/kg/d

- Males (Full Study) Projecl 297S) Pagel8or49

## .~) l-'

Preexposure Terminal Days Days Relalive**Rclaliv~** 

### Relalive Relalivc

Body Oody Dosed Dosed Siomac Siomac Liver Liver Spleen Spleen Kidney Kidney Animal Weighl Weight 400 200 hWt. hWI. WI. WI. WI. WI. WI. WI. HeT #I (8) (8) 

### mglks mglk8 (8) (% OW) (8) (% OW) (8) 

### (0/0 OW) (g) (%"BW) 

### (0/0) 

### 203 25.8 26.2 2 2 0.191 0.729 1.392 5.3 13 0.125 0.477 0.205 0.782 53 205 25.2 26.3 2 2 0.199 0.757 1.255 4.772 0.097 0.369 0.186 0.707 52 207 26.4 27.3 2 2 0.173 0.634 1.707 6.253 0.069 0.253 0.185 0.678 48 211 27.9 27.6 2 2 0.151 0,547 1.440 S,217 0.103 0,373 0.182 0,659 NS Mean: 26.33 26.85 0.1785 0.6666 1.4485 5.3887 0.0985 0.3680 0.1895 0.7067 Sl.0

SO: 1.16 0.70 0.0213 0.0955 0.1893 0.6224 0.0231 0.0917 0.0105 0.0542 2.6 NS=No Sample; NW=Not Weighed; N/A=Not Applicable.Appendix B·2 Individual Animal Dala Gavage Study. Full



### 800/400 mg/kg/d

- Miles (Full Study) Projeel 297Sl Page 390f49 

### I-l

Preexposure Tenninal Days Days Relalive Relalive Relalive Relalive

Body Body Dosed Dosed Siomac Stomac Liver Liver Spleen Spleen Kidney Kidney Animal Weighl Weight 800 400 

### hWI.
. h

### WI. WI. WI. WI. WI. WI. WI. Her II (8) (8) 

### mglkg mglkg (8) (% OW) (8) (% OW) (8) (% OW) (8) (% OW) (%) 

### 402 25.2 22.9 2 2 0.209 0.913 1.126 4.917 0.096 0.419 0.162 0.707 46 404 26.4 22.2 2 2' 0.176 0.793 1.030 4.640 0.091 0.410 0.181 0.815 42 408 26.8 25.9 2 2 0.223 0.861 1.340 5.174 0.156 0.602 0.210 0.811 48 410 26.9 27.0 2 2 0.219 0.811 1.547 5.730 0.094' 0.348 0.182 0.674 52 411 27.4 25.1 2 2 0.183 0.729 1.068 4.255 0.088 0.351 0.164 0.653 48 412' .27.5 27.5 2 2 0.176 0.640 .1.589 5.778 0.188 0.684 0.222 0.807 44 414 28.3 24.0 2 2 0.231 0.963· 1.202 5.008 0.083 0.346 0.186 0.775 

### NS 41S 

### 28.S 29.S 2 2 0.243 0.824 1.463 4.959 0.130 0.441 0.209 0.708 46 

### Mean: 27.13 25.51 0.2075 0.8166 1.2956 5.0576 0.1158 0.4500 0.1895 0.7440 46,6



### SD: 1.06 2.46 0.0261 0.1014 0.2201 0.5120 0.0385 0.1262 0.0221 0.0657 3.2 

### NS=No Sample; NW=Nol Weighed; N/A=Nol

### Applicable.AJlpcndill B·2 Individual Animal Dala

- Gavage Study, full



### 1200/600 mg/ka/d- Males (FullStudy) Project 29753 rage 40

## or49 

# o

"""""

Preexposure Tcnninal Days Days Relative

. Relalive Relalive Rclalive

Body' Body Dosed Dosed Siomac Siomac Liver Liver Splcen Spleen Kidney Kidney Animal Weighl Weighl 1200 600 

### hWI. hWI. WI. WI. WI. WI. WI. WI. HeT**_#~_(s.L-** 

### (g) mglkg mglkg --.J1l) (% 

### OW) (Il) (% 

### BW) (g) (% 

### OW) (g). (% 

### BW) (%)



### 602 24.8 20.0 2 2 0.142 0.710 1.027 5.135 0.125 0.625 0.160 0.800 38 603 26.1 21.1 2 2 0.155 0.735 

### NW NW

_0.077 0.365 0.148 0.701 

### NS 608 29.1 22.7 2 2 0.243 1.070 1.029 4.533 0.082 0.361 0.167 0.736 

### NS 610 28.1 27.3 2 2 0.233 0.853 1.508 5.524 0.201 0.7]6 0.189 0.692 43 616 26.6 24.8 2 2 0.245 0.988 1.30] 5.254 0.092 0,371 0.192 0.774 ]9 

### Mcan: 26.94 -23.18 0.20360.8713 1.21685.11150.11540.49170.17120.7407 40.0



### SD: 1.69 2.93 0.0507 0.1568 0.2335 0.4185 0.0514 0.1770 0.0189 0.0462 2.6 

### NS=No Samplc; NW=Nol Weighed; N/A=Nol Applicable.Appendix 8·2 Individual Animal Dala

- Gavage SlUdy. Full Projecl 29753 Page 41 of49 

# o N

SalineControl- Females (Full Study) Pre- Relative exposure Tenninal Days Days Relative Liver Relalive Relative

Body Body

. Dosed Dosed Stomach Stomach Livcr WI. Spleen Spleen Kidney Kidney Animal Weight Weight 1200 600 

### Wt. WI. WI. (% WI. WI. WI. WI. HCT 1# 

### (S) (g) 

### mglkg mglkg (g) (%OW) (g) 

### OW) (g) 

### (%BW) (g) (%BW) (%) 

### 101 21.7 20.3 2 2 0.115 0.567 0.951 4.685 0.063 0.310 0.124 0.611 53 103 20.4 20.3 2 2 0.175 0.862 0.930, 4.581 0.086 0.424 0.116 0.571 48 105 21.2 20.2 2 2 0.170 0.842 0.836 4.139 0.074 0.366 0.114 0.564 53 107 20.8 20.7 2 2 0.138 0.667 0.949 4.585 0.075 0.362 0.129 0.623 49 109 21.1 21.0 2 2 0.149 0.710 0.935 4.452 0.088 0.419 0.136 0.648 52 II 

### f

. 21.1 22.1 2 2 

### 0.145 0.656 1.050 4.751 0.078 0.353 0.129 0.584 53 113 2

### i.7 21.6 2 2 

### 0.140 .0.648 0.971 4.495 0.075 0.347 0.108 0.500 52 115 16.2 20.2 2 2 0.222 1.099 0.966 4.782 ·0.064 0.317
. 0.147 0.728 47 

### MC[lIl: 20.53 20.8 0.1568 0.7562 0.9485 4.5588 0.075 0.3623 0.1254 0.6036 50.9

SO: 1.80 0.7 0.0324 0.1707 0.0589 0.2056 0.009 0.0415 0.0127 0.0671 2.5

___ 400/200 mglkgld

- 

### Females (Full Study) Pre- Relative exposure Tenninal Days Days
. Relative Liver Relative Relative

Body Body Dosed Dosed Stomach Stomach Liver WI. Spleen Spleen Kidney Kidney Animal Weight Weight 400 200 

### WI. WI. WI. (% 

### WI. WI. WI. WI. HCT II 

### (g) .JgL. 

### mglkg mglkg (g) (% 

### BW) (gL 

## llWl_ (g) 

## (% BW) (g) (%_BW) (%) 

### 301 18.2 12.8 2 2 0.103 0.805 0.378 2.953 0.021 0.164 0.08 0.625 

### NS 305 2\.6 19.2 2 2 0.178 0.927 0.821 4.276 0.078 0.406 0.132 0.688 

### NS 311 16.7 15.4 2 2 0.126 0.818 0.706 4.584 0.043 0.279 0.12 0.779 

### NS 313 21.6 22.6 2 2 0.170 0.752 

### NW NW 0.124 0.549 0.134 0.593 47 

### Mean: 19.53 17.51) 0.1443 0.8255 0.6350 3.9379 0.0665 0.3496 0.1165 0.6712

SD: 2.47 4.30 0.0358 0.0734 0.2299 0.8666 0.0449 0.1655 0.0251 0.0820 NIA. 

### NS=No Sample; NW=NotWeighed; N/A=Not Applicable.AppcndiK B·2 Individual Animal Dala

- Gavage ShJdy. Full



### 800/400 mwkwd

- Females (Full Study) Project 29753 Page 42 of49 

## Q 

# W

.Animal /I 

### 502 505 510 513 

### Meim:

SD: Preexposure Tenninal Days Days Relative

Body Body Dosed Doscd Stomach Stomach Weight Weight 800 400 

### WI. WI
.

(g) (g) 

### mglkg**n~_lg)** 

### _(% 

### BW) 17.9 17.5 2 2 0.162 0.926 21.7 22.1 2 2 0.204 0.923 19.7 18.2 2 2 0.179 0.984 22.3 21;0 2 2 0.176' 0.8]8 20.40 19.70 0.1803 0.9176"

2.0 2.20 0.0175 0.0599 

### \ Relative Relative Rclative Liver Liver Splecn Splccn Kidney Kidney WI. WI. WI. WI. Wt. WI. 

## lIer (8) (% BW) (8) (% UW) (8) (% BW) (%) 

### 0.996 5.691 0.151 0.863 0.118 0.674 39 1.270 5.747 0.125 0.566 0.173 0.783 ]8 0.879 4.830 0.239 1.313 0.126 0.692 36 1.228 5.848 0.157 0.748 0.138 0.657 37 1.09]] 5.52880.16800.87230.13880.7016 37.5 0.1869 0.4706 0.0493 0.3184 0.0243 0.0560 1.29 11001600 mK!kgfd

- Females (Full Study) 

### Pre· exposure Tenninal Days Days
. Relative Relative Relative Relative

Body Body Dosed Dosed Stomach Stomach Liver Liver' Spleen Spleen Kidney Kidney Animal Weight Weighl 1200 600 

### WI. WI. WI. WI. WI. WI. WI. WI. 

## lIer # (g)**(g~glkg m~~(g)**  

### (%BW)Jg) (%BWl (g_)_(%BW) 

### (g) (%BW)

__

## 00 701 21.2 14.8 2 2 0.131 0.885 0.686 4.635 0.081 0.547 0.106 0.716 35 702 17.8 17.4 2 2 0.214 1.230 1.096 6.299 0.194 1.115 0.122 0.701 40 703 21.1 19.8 2 2 0.208 1.05 I 

### NW 0.227 1.146 0.128 0.646 36 707 21.0 19.8 2 2 0.218 1.101 1.013 5.116 0.203 1.025 0.130 0.657 38 709 21.1 16.5 2 2 0.169 1.024 0.842 5.103 0.133 0.806 0.126 0.764 35 715 22.5 21.S 2 2 0.193 0.898 1.173 5.456 0.181 0.842 0.14 0.651 

### NS Mean: 20.7833 19.4000 0.1970 1.0184 1.0093 5.2250 0.1860 0.9549 0.1310 0.6795 36.3

SD: 

### 1.5664 2.0928 0.0213 0.0865

- 0.1655 0.2000 0.0400 0.1598 0.0062 0.0563 1.5 

### NS=No Sample; NW=Not Weighed; NIA=Not Applicable.Arrendix 0·2 Individual Animal Dala - GavageStudy, Full 

### Appendix D·] Individual Animal Data··Gavage Sludy, Full Histopolhological (H&E) 

### Observalions ofForestomach and Liver Group Animal" Observalions Projeci 29753 Page 43 of49 

# o**_~l?!!~r~I.~.II.I~** 

### ... ___._ _ ._...1 .'**fo~c.~~oinach.a.'!~ gl~.n~l!!ar_~I()J!la~hq.'!C?~~I!.liv~r**  

### nonnal

_ 

## ._ _ .. ..__ _ .._.._ .. .3...**.(!:I~_~~~~.o.~h. p~e~e~.lpivernQnn.o.l.**  

### _

_ _ ,_,_",
.. . _____ S**.fore~I()ll1ach** 

### "()'!na!!**H~~r** 

### nonnal

...._ _ 

### 7 forestomach normal, livernonnal 

# --._-- ...'-'

..**~~~.'~~~-= ~_-.~~:~:** **~:~_~f~.·. ::·_to!~~~iri~~h:n~iOl~~;)i~ei** **~n~JiTI~C:-_~:·.:.:·._.· ~::~**  

# ..- :-

__:-_: :

__::::**~:: '.~'~'-** 

# ..'_':.

..... __.""'

.._ .1.

## L.**_.(()r~_~~ol11o.~h~~~.g.ta,!~~lo.!_s'~!!1.o.~~ Ilo~o.!,.** **!!.~~f..'!.()t.p~~.~e_nL** 

### __ _ .



## .. _ _..._ .__ !**~.. fl!r~~lolTlach** **.al1~.g!all~_~I.o.~~~()~.!~~~~()f!.ll_al!.!!~e~** 

### .1l2.nna.I

.._. __._._ ..



## . .

__.. _. _ ..!**~.** 

## _**(f~.r.~~t()I1!~~~** 

### ,!ot**pr_e~~n.Q, gtandul~!.~t~m.a~~**  

### _normo..I.. liver**~o~a.1**

# --t~£J~~~~t-=~~~-~I~~:~-~~f~~**_~~ --to{~~~£~£l~~:~j~~~!~~~tf:~~~~~~~~~~·i.i~i-~:~~='** **:~~.'.~---'-**

# --- 207 minimal forestomach epithelial hyperplasia, livercells seem highly
I

# ~'-::~--:-"-:.-.:.:--.:: .- ..

# J. _:

# _-~ ItJ

# :~ij_~o~~~:t~!Ch~p.i~h~Uo.!.~_.pl~!i~!**!~·~i.!l~~L-.~_** .. _ .. .M!c1D()se Male**.~.** 

# 191**.!!!i!~ {l?.!~!~.!!!.~~h...~p!!!l~!!.~!J1~~I~!!.a.!.!i~~~~m!o.L_._**  

## . __. . ...._ ...

.**~OOL4QC:l.~~L** 

### ...**~Q_~_.** 

### _**8!~!1.~ula_~~!~!l:'_~~~ nl)~~!,-f~!~~tC!.~o.~.!!.!!~~~h** **!iY~~.I1l?_~~L** 

### __ _. ... _. _

._ .••

__ ••

__• _. ••••_. _00 

# 108._**!11.i!lJ!!1_a!ro!~~~o.n:t~~.~.~P!t~~!i!!Lhyp.~~L~sj~l!i,!,er_nl).~!-1I** 

## ...._. .._ .. 1- ._ ------.-

__. .-.-

# _1**!~.____!!1iW fq~~~I()!!1.a..c~** **~p.i~~.I~!.h~!'P!o.!i!'!i~~r 11t?!!J1~I**  

### ..._.. _. . _ .



## ____ . .....__ ... _.1!_!**J~~Q!!lM_h_P!~~~!!~k!!Y~r no_~_!L**  

### . ._. . . .._.._ __.. __ 

### 412 glandularstomach normal, moderate forestomach epithelial hyperplasia

__ 00_. _. ... __ •• .,. .._ _ .__ .••**~ilh _i.!!O.!!.!!'.'!!~~~ry** **~~!!~i.!I_~!J_~'!l~~()~!.!l.I!~_'!.uscuJ.~!!~!. ~i_Y.!l~.~t?~~!**  00..

_ 1 .
.. . . ..__. 

### 4)4
.**l11oderatef()re~I()I11~~~ ep!lh~.Ii~!** **~yp~~lo.s!~,.!.i.Y~!.!!~~~1** 

## ..__ .... _ 415 moderate forestomach epithelial hyperplasia with inflammatorycells in

.. . __.... .... __I

.. _.. __ .**_~~~_sl.!~J!I.u~l)~a andm1.!~.~!~!!!!J.i.Y~~_!I()m,lll'_**  

### .... _.__... ..._._..... _. _Appendix 8·2 Individ.ual Animal Para

- GavageStudy, full Projcct 29753 Page 44 of49 

# o til
Appendix B-3 (conUnued) 

### Individual Animal Data

- Gavage Sludy, Full Histopothological (H&E) 

### Observalions ofForeslomach and Liver Group Animal II 

### Observalions**J:!!8~ J?'!s~.~-!!~_._._**  

# L.

__**.~QL** 

## ..**..I.~! L~**  

## ..**r()r~!to_R)_~~h._~p.i !h..~li~!!ayp~_rp'!!s_iD.!-'J.y.~.r..!!S![I!'!~**  

# t_

_ __.._ _ _

610

l .•.••.• . __._ ••

__._._.. .,1

__ ._ ... , __ . 0



### 12001600mglkg 

### I 603

---_...._---------1·----·
.... --.. 

### 608 minimal ulcerofforestomach, minimal foreslomach epilhelial**h.yp~~!~~~!.I.iv_e~_~~~!.** 

### __ _ .. _ _ .._

.. . _ _ .. . glandular slomach nonnal, mild foreslomach epithelial hyperplasio with a

_**s.'!1_~!L ~~~ces.~.i!1.th~**  

### la.".!i!1 B**p~opria,** 

### liver

### nO.rnlllL

__ ._ .. __ ........__.... mild forestomach cpilhelial hyperplasia wilh inflammation in Ihe muscularis. There is asmall islandofepilhelium in the innamed area and

.evidence ofeXlension ofthe innammalion into the peritoneal cavity. Liver nonnal

_ .. -----. 

### ----- 

# ...._----_.......

# , ...- 

### ..

### - ..

### _-. -- .

### __..

### --

### .. "'." - 

### ------------. --._.. 

### _-- .....

### _ ...__.....

# __.-....

### ----_._- 

### 616 marked forestomach epithelial hyperplasia with epithelial downgroWlh through lhe lamina propria, muscularis mucosa and inlo lhe outer muscular layers. This is associate.d wilh prominenl inflammalion of(he**.m_u~~ll!~i~ _~i.v_e.!.~Q~.!lL**  

### . _ ._ _.'--
.. _ .. _. ."
..Ap(lCndix 8·2 Individual Animal Data - Gavagc Study. Full Projcct 29753 Page 45 of49 

## Q 

# 0\
Appendix B-3 (continued) 

### Individual Animal Pollia - 

### Gavage Siudy, Full Hislopolhological (H&E) Observations ofForeslomach and Liver Group Animal #I 

### Observalions .

.**5;~~~~I~ma~~_....:1.-** 

# _J**~L** 

# .._**.f.~r~~Q.~~~Jtn~!'!1!~11Ji.Y!~L** 

## =:**mi.,!i!l:1_~I..~~!!~~!lobuh1!-!}~~~o.~.i~** 

### ....___._._.. . 103 forestomach = 

### minimal focal ulcerand minimal epilhelial hyperplasia, livernormal



# _**::=-::'_=~_-=-_~.-_-~.=:~~~~: t~** 

# __**:J~S·-·.·.~ :f~res-~~~~.n_o~~i:Jiit:~~~~~L**  

# _._-..**-_~:.** 

# :-

__**::-.~~:_.=.'.':.'**

# __**:~:=._-~_~~~=-'.:-**



## __..**.I~! Jf~r~s!l?_I!l-a~~!I_l?~** **pr~~.~l). 81~!!.du!ar~Il?~~~h_'!<:J".!!~ll** **It~~~~g~_al** 

### .._._. _. _..LO.9
..**!o.r~~!~I!1~~~** 

### n_ormal,liver110

### rJl111L.

.. .__

III foreslomach normal,livernormal



## ------- -..-....- - ..--...'-'1'13 .--. 

# 'mirilmlilrociiinecrosisandep'iiheiiBi"hype;j)iasia-ai 1_

.....__._.__ ._•.•

__._ __,__ ..__ -. .**g!~!IAl!!lI!.'f~r~~~~~~~hj!!~~lio.!!,J.i~~!.!!!>..,:!!!~.1..** 

### .__... -,,_ ...----- .



# . .... ..**---'_!~ for~~IC?.'!'~~Jt** **1l()~lI.!!.IJ~~!.!'~.~lI!.** 

### :_ ... _ .. .. .__ .... . .. ,



### _**J,.o~ _~~~.** **!:~!Jl_~le** 

## .. _,,1_
....

### ~Ol_ 

### ..__**.rc~~~st~_m..~~h._!I~".!1~!!.~~~r!l~t:!!1~I_** 

### -- ...-.-- ..-... ---..-'-

---.... --.- ..



## ___________.._ .....**~o~. mR~** **!.l?!~S!~~~~~. ep.il~_e_Ii!'Lh~rpJ.~.i~,** **!!Y~Ln.~r.!'!1_~!..** 

# __ .. .... _ 311 glandular stomach normal, minimal forestomach epilhelial hyperplasia, minimal focal foreslomach innammslion, liver normal 

# , ..__**~_~::~-~.~~:-.:~-_·~.-=:I:~~-_H~.** 

# ==:**"8!!~~~~i.~!Q'!1.a~~ ~"-!1i~_r~i~~!~~iCE~o~~~~-r-n~i!ii~',=~~_~-** **::'~::·.** Mid Dose Female

_. .. 1._.'

# ~_~~.**1_~~;~':~r;~f::r!~~:~p~~~~~~~fu~:!r~~~~;~4~~~t:~ .~_~.I_I~_i~.** _ .. 

### 505 minimal foreslomach ulcer, moderate forestomach epilhelial hyperplasia



# ---------.---. -.-----SI·i.--.-,

# ~~:~1;~!sT~~~ce~I:~f.~~TI~~~!;;~~·/f;~~1~;~maIOry·ce**lis-in·** -. Ihe musculll;!s, livernormal
.
- 

### ---- 

# ..

# -_
.._----

# -,--_.

__.. --.-------- ---- -- --_._-_._-------------- - --. 

### ---

# -_
...-

### ..-

### ------- .. ---- -- ------- ----

_.. 

### 5I

### 3 mild**forestoma~h** 

### epithelial hyperplasia, liverappears 10

### be highly 1-..
.._.**8!yc~8~l'!.a!~.** 

## __._ __ I .

_Appendix D·2 Individual Animal Data

- Oavage Study. Full 

### ProJeCI297S3 rage 46 of49 

# o -....) Appendll B-3 (conCinued) 

### Individual Animal Dolo

- GavageStudy. Full Hislopalhological (H&E) ObservalionsofForeslomachand (.jvcr

Animal #I 

### Observations 

### HighDose Female Group 701 

### I mild foreslomach epilheliat hypeijiliis18with some: IrtnDmmationIn the laminapropria. Livernormal.

. .

.-----.---- •. -'702 

# ---I inild-roresiomach'ePJiiieii81 hYPerpiftslii.-liverappellrslo

## hehlgfiry·-._- glycogenaled.

# :==~~=-~~:~=~~~I=~~~~r:-'I~i~d~t?~i:i~~~-t~:~l~~~;:ht:~~~~~~~~~~~~dera,e~'=~:~ forestomach epllhelial hyperplasia with Innammalionin Ihe muscuillris, livernot presenl; spleenis within normal limits

-•. ···r···"

..... -.....

## ·'1--'·'109"-· ""giiiiiifiifarstomachiiorrnai,niIlifforeslom8Cii epilheliaIhyperplasia

# and_. minimallnnammalorycell infillrate In laminapropria. livernormal



### .--.....- ..-.- ----1"-- 

# 7i

# s' I(si<i"iniich

# noipreseiiij:IIver·normiir-

..··---··---·--·_.-.-----.-.. - ..

_ •• •••••

__ • •••• _._. __ •

__ ••

__••••

_ ••

_ ........ _ ••• _. •**~_.** • 0_0

__ ..__ •• •• _a • • ••**~._**

# 08 Appendix e-I • Project297S3 IndMduaI Animal Daz:a -IP vs. SC IDjcaiou Page47of49 

### Appeadix

### C-l Individual Animal Data
- 

### IP

### vs. SC Injection, Full Study

.....Pre-Exposure Body 

### Weights 

### andTerminal Body

### aDd 

### LiverWeights Group Animal Prc-exp. 

### TenniDal LiverWt 

### Ret Liver 

### # BW(g) BW(g} (g) Wt.(%) 

### SQ SaliDe 101 24.6 24.9 1.08 4.34 102 22.5 21.6 0.89 4.12 103 21.7 20.6 0.79 3.84 104 22.4 21.5 0.92 4.28 105 23.2 

### 23.4 0.91 3.89 106 22.1 0.84 Mean: 228 22.4 . 0.905 4.09

SD: 1.0 1.7 0.099 0.23 

### IP Saline 

## III 22.0 21.4 0.90 4.21 112 23.4 24.1 1.02 4.23 113 226 21.8 0.18 4.04 114 21.7 21.4 0.87 4.07 

### I1S 22.0 21.4 0.94 4.40 116 22.4 22.1 0.77 3.48 

### Mean: 22.4 22.0 0.897 4.07

SO: 0.6 1.1 0.083 0.31 SQ400mgIkg 201 22.1 22.4 1.03 4.59 202 

### 22.2 21.7 0.92 424 203 23.4 23.5 1.10 4.68 204 

## 22S 22.6 1.02 4.51 205 23.2 23.7 120 

### 5.06 206 21.4 22.6 J.14 5.04 Mean: 22.5 228 1.068 4.69



### SD: 0.7 0.7 0.099 0.32 1P400mglkg 211 23.7 23.9 1.10 4.60 212 24.6 24.1 1.02 4.23 213 .21.6 23.4 0.88 3.76 214 22.1 21.8 1.00 4.59 215 

### 22.2 21.7 0.96 4.42 216 24.3 24.5 

### 0.97 3.96 

### Mean: 23.1 23.2 0.988 426

SO: 1.3 1.2 0.073 034

# 09 Appcadix C-J IndividUa1 Animal 

### om-IP "S. SC Injection Appeudix C-2 Individual 

### Animal 

### DaIa

- IP vs. SC Injection, 

### Probe Study Histopathological Observations oftbeForestomach Project29753 Page4lof49



### GToup IP400 mglkg



### IP 600.mglkg

SC

### 400 

### mgllcg

SC 

### 600 mglkg Animal # 

### 24 16 

### 7 

### 2

10


### 23
18 11 g 31 

### 20 

### 21 Observations hyperplasia. epithelium, forestomach +1 normal normal 

### hyperplasia. epithelium, forestomach 

### +2; inflammation

### +2; necrosis +1 

### hyperplasia, epithelium. forestomach +2; inflammation +1; 

### ulcer+1 hyperplasia, epithelium,**f~mach** +2; inflammation

### +1 hyperplasia. 

### epithelium. forestomach DOnna) normal hype:plasia, epithelium, forestomach normal normal" •.J 

# 10 Appendix C-I Individual Animal Data

- IPvs, SC IDjec:Qon AppeDdiJ: 

### C-3 Individual 

### Animal Data

- IP vs. SC Injection. Full Study Histopathological Observations ofthe Forestomach Projea 29753 Page490f49

Group IP Control



### IP400 mglkg

SCContrOl

SC400mglkg Animal # 

### III 112 113 

### 114 

### lIS 1I6 

### 211 212 213 

### 214 215 216 101 102 103 104 

### 105 106 

### 201 202 203 204



### 205 206 Observations 

### Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Hypc:zplasia, epithelium.,foresoomacb +1 (focal); (also identified by blindedevaluation) 

### Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal HypeIplasia, epithelium. forestomach 

### +1; inflammation + 1; very minimal diffuseepithelial thickening 

### with submucosal mononuclearinflammatory cells (also identified by blinded evaluation) 

### Normal Hyperplasia, epithelium. forestomach 

### +1; inflammation +1; very minimal diffuseepithelial thickening 

### with submucosal mononuclearinflammatory 'cells (also identified by blindedevaluation)