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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 Project No. 29753

CMA Agreement No. EGE-74.o-00SE-BAT.NW -Amendment No.8

July 13, 1999

"Sbon-Term Studies tc Evaluate the Dosimetry aDd Modes ofActioa of 2-

ButozyetbaDol in B6C3Fl Mice"

R.A. Corleyl, K.K. WeitzI, TJ. ~, 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 lack of genotoxicity and the kDown

hemolytic mode of action of BE in rodents. it is likely that these potential treabnentrelated tumors occurred via indirect mechanisms involving chronic tissue injury. For the

liver tumors, the hemolytic. effects of BE and subsequ.em accumulation of degradation

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 of BE deposited on the fur or mucociliary clearance of

BE dissolved in the mucous layers of the 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 of spon13Dcously-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 of oral 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 hr to 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 inunediately post-exposme and in uriDe collected for

up to 18 br post-exposure. Small amounts of BE 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 of BE deposited on the fur ofmice represented 9-

13% ofthe total BAA eliminated in the 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 layer lining 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 caDnOt be ruled 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«t 29753

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 of neat BE is capable of causing 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 ofBE 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 of focal irritation in the forestomach similar in appearance to the lesioDS

observed in gavage and inhalation studies. One of three mice/group in each of the 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 playa role 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

may be dose-rate dependenl

In the liver evaluations. there was no evidence of degeneration and no corresponding

compensatory cellular regeneration 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 of these mice was evaluated and reported separately by the Indiana

University School ofMedicine (Kamendulis et aI.• 1999).
07

Project 29753

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INTRODUCTION

2-Butoxyetbanol (BE) was recendy evaluated in a series ofiIJhaIation toxicity studies by

the National Toxicology Program. While the technical report of this 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 the levels ofreactive oxygen 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 of BE dissolved in the mucous layers of the 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 of BE 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:

• Evaluate the potemial role oforal ingestion (via grooming) ofDeal 2-butox~1in

the production offorcstomach lesions ofmice exposed to BE vapor (Aim I)

• Evaluate the potential for increased oxidative SU'CSS associated with hemolysis iII the

production/progression ofliver lesions 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 of fur deposition on the internal dose of BE, samples of blood and urine were

analyzed for BE and its metabolite. butoxyacctic acid (BAA). ID addition to tne

dosimetry smdies, the dii'ect effect of BE on the forestomach ofmic:c was evaluated by

administering neat BE daily by oral gavage for up to five daYs. The forestomach of each

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 of hemolysis, 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 of BE on the foresromach of mice was evaluated histologically foUowing
08

Pro;ect 29753

Page 50f49

administration of saline solutions of BE by either intraperitoneal or subcutaneous

injection for J or 5 days.

METHODS

Test Materials. 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 test chemical 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 of mice for the NTP inhalation Sb.ldies. Animals

were ordered to arrive at approximately I0-11 ~eeks of age 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 of the

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 of 90 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 prior to placing on study.

Specific Aim·l: Deposition ofBE 0. tile fur or mice.

Study Design. Female B6C3FI were exposed by either whole-body (20 mice) or noseonly (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 their bodies washed two 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

Project 297S3

Pagc6of49

Inhalation Ex;)OSures.

Vapor Generation 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 of exposure 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 the exposure 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 of the 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 chamber positions.

Nose-Only Exposure Unit.

'The nose-only exposure uait was a flow-past type consisting of two 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 their expired 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 anicle concentration in

the exposure unit was determined prior to the study.

Chamber monitoring.

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 of the 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 of insaument 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

Project 297S3

Page 7 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 of 200°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.

GravimeaicalJy prepared 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 ofBE 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 of die sampling medium using

the same extraction procedure as the chamber grab samples. The quality CODuol check

standards were used to cbeck the 'preparation ofthe 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 of fur washes. blood and urine. At the end of each exposure (oose-onIy and

wbole·body). five mice were sacrifICed using CO: and transferred ~o individual beakers

and submerged in 50 ml of bot water. After rinsing in SO ml of water. the mice were

transfem:clto a second beaker containing an additional SO ml of hot 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 of ethylene glycol on the fur ofrats

and mice.

At the end of each exposure (nose-only and whole-body). five mice were immediately

anesthetized using 70010 C02 and samples of blood were collected via the retro-orbital

sinus. The time of each 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 of distilled 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 of neat (water) sample was injected onto a large 4 mmid

injection liner to facilitate the water vapor expansion generated at the constant injection

temperatUre of2050<:. The oven tempetature was initially maintained at SQllC for 2.0 min

then ramping at 3()iC1miD to 24()11C. FlO temperat!Jre was 27fPC. Under these conditions,

BE bad a retention time of-3.2 minutes.

Analysis of blood 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 of a weigbed quamity (-02.5 g) of blood, 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: Effect of oraUy-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 ~edays. After two days of

dosing at 800 mglkg, DO clinical signs of toxicity 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 water were 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 of the first dose and the volume of BE delivered

adjusted 10 deliver Ibe 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 if the 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 of esophagus, '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 of neal 2-butoxyetbanol. Each animal was weighed at the beginning of the week

to adjust the dosing volume to the appropriate mg/kg body weigbt. Prior to 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. 1200 mgIkg (D =8)

+

SaaifICC

SSuM!WTbFSSuMTWTbF

+

Pump

Implam

Group I (I wit)

Group 0 (2 wit) ----t.. 0,400,800, 1200 m.gJkg (II = 8)

+

Sac:rif"1CC

During the first two days of dosing. 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 balf resulting in the following

dose levels: saline CODtrol, 200, 400 or 600 mgllcglday. During the next two days of

administc:riDg the lower dose levels (total offour days of dosing) 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 by cardiac puncture for dclennination

of the 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 of markers for oxidative

SU'CSS by Jim KJaunig, lDdiaDa University School ofMediciDc. After disc: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 of the 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

with hematoxylin and eosin (H&E) or amibodics against proliferating cell nuclear antigen

(PCNA). HiSlologic:al aDalysis (H&.E) wc:rc CODducted 10 detennine evidence of necrosis

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

Project 29753

Page 100f49

the number ofcells in S-pbase per millimeter ofthe 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 of iron 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 of BrdU-reaetivc: cells was accomplished with the chromagcn 3-amiDo-9-

etbylcarbazole with a bematoxyliD counterstain. A miDimum of 2000 bepatocellular

nuclei was scored by light microscopy in at least 10 randomly selected 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. After two days ofdosing. one 600 mglkg IP mouse was sacrificed moribund.

Ful! Study. Groups of 6 female B6C3Fl mice werc: administered 0 or 400 mglkgld BE

by either IP or SC injection for S days and necropsied on the afternoon of the Slh day of

dosing. Terminal body weights and liver weights were measured and livers were flasbfrozeD 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/or irritation ofthe gastric mucosa.

RESULTS AND DISCUSSION

Specific AiIII 1: DepositiOD 01BE 08 tile far 01 mice,

Wbole-bodY CbaJpber ooc:ration. The distribution of BE vapor CODCCDtr3Iions in the

exposure chamber was determined prior to the study. The conce:nnations were measured at

3 chamber positions during the first hour of a test generation aDd again daring the last hour

ofthe rest generation. The concemrations were 238.1 ± 15 ppm (0.6% relative staDdard

deviation) for the 6 samples. The analytical concentration averaged 95% ofthe nominal

cODCCDtraricm (250.6 ppm).
~: 14

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During the animal exposure. the conc:cntrarion ofBE was measured at 3 chamber positions

during the first hour ofthe exposure study aDd again during the last hour ofthe study. The

anaiytical c:bamber concentration was 229.2 ± 1.0 ppm (92";" of the 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 prior to 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 of a 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 of grab samples were taken. The measured meaD amcentratiOD was 96.7%

ofthe target CODCCDtration of250 ppm.

During the animal exposure, the concentration of BE 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 hoar ofa test generation and again duriDg the last hour ofthe 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) ofme urine samples collected &om the mice following

the oose-only exposure comained a red colored material. possibly blood. In addition 1/10

ofme cage rinse samples were significantly tinted red.

Qeoosition ofBE on the fur. An average of 205 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 of female mice exposed nose-only to 242 ppm BE.

Residual BE on the fur of the nosc-<lnly exposed mice was attributed to"the use of a

positive-pressure. flow-past exposure system. Most of the 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 to be shut down aDd evacuated aDd the

animals sacrificed via C~ pri~ to inunersion in 50 ml ofhat water.

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

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BE and BAA in blood and urine. The concenuation of BE in the blood of five 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 example chromatogram from the blood analyses is shown in Figure 4.

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

J8 hr after both whole-body and 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 of oraDy-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 of the mice are suounarized in Table 4. Individual

animal data are presented in Appendix B-1. Both absolute and relative organ weights

were increased over controls 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. After two consecutive days of dosing, mortality was observed in all treatment

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

resulting in dose levels of 200, 400 and 600 mglkgld Over the next two days of dosing.

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

Appendix B-2.
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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 of male 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 of increased 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 of proliferating

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 of the 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 of forestOmach and glandular sromach) in another female control. These

were considered spontaneous changes that are not uncommon 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 of S-phase cells in hislologically-nonnal forestomach epithelium VemJS

the epithelium surrounding histological lesions. There was evidence of an increase in

PCNA staining intensity in regions smrounding focal areas of hyperplasia and

inflammation, as compared to either the forestomach epithelium of control mice or to

histologically-normal regions within the forestomach ofBE-exposed mice. The intensity

of PCNA 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 of inflammation and

thickening of the epithelium showed greater overall PCNA·staining as well as clearer

evidence ofS-phase cells.

Liver H"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'er cells was observed in any exposure group. Highly g1ycogenatedappearing liver cells were observed with low occurrence, lack of a dose response, and

variable group distribution and WCJe, therefore, not considered exposure-related..
:03

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There was a clear treatment-rela.ted increase in hepatic iron accumulation based on

analysis of sections stained with Perl's stain. Enumeration of positive 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 of S-phase cells between control animals and BE-treated animals in either

male or female mice (Table 7). The mean fraction of cells 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 of either sex. Sexdependent differences in PCNA labeling indices have been previously reported (Ellridge

and Goldsworthy. 1996).

Specific Aim 3: Effect of IP vs SC-admiaistered BE on the forestomach.

The dose level of600 mg'kg IP resulted in the moribund sacrifice of one mouse after two

doses in the probe study. Therefore., a dose of 400 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 of a 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 evaluated blindly; association with BE

treatment was considered possible.
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CONCLUSIONS

Small amounts ofBE (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 ofBE resulting from whole-body vs. noseonly exposure as determined by analysis of BE and its major metabolite BAA in blood

collected immediately post-exposure and in urine collected for up to 18 br post-exposure.

Although this was not a mass-balance stUdy for the total fate of BE. the amounts of BE

deposited 00 the fur of mice represented 9-13% oftbc total BAA eliminalcd in the urioe.

This calculation is, however, speculative since the amount of BAA found in the urine

collected 18 br post-exposure is likely an underestimate of the total absorbed dose. In

addition, the amounts ofBE 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 of neat 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 half to 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 of sixteen male mice

and 0, 6, 12 and 10 of sixteen 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 of epithelial cells surrounding the lesions vs. normal cells in the

forestomach. Similar forestomach lesioDS were observed following IP injection of 600

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
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ID me livers of mice orally-dosed with neat BE, there was no evidence of degeneration

and nO corresponding compensatory cellular regeneration. In addition, there ""'as no

remarkable accwnuJation of hemosiderin 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 of J1ese

mice were evaluated and reponed separately by the Indiana University Medical Center

(Kamendulis et al., 1999).

In ~clusion, these dara coupled with the evaluations of Kemendulis 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.
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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). Dtaft Technical Report on the Toxicology

and Carcinogenesis Studies of2-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 phenotype and cell replicative behavior

ofhepatic tumors induced by dicbloroacetate (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 of ethylene glycol

aerosol in the CD rat and CD-I mouse by whole-body exposun:. Fund. Appl. Toricol.

24,57-75.
:07

Project 297S3

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Fagan 1. Vapor gcncating system used for wbole-body and 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 cbrornaIDgram ofsample extIaCt 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

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Page 20 of 49

Fagare 3. Example chromatograms from (a) control wasb water and (t,) fur wash from a

mouse exposed whole body to 229 ppm BE for 6 hr.

(a) Control wasb waIer

Pet" ro.-.aaT....~J...IlDii.DI

...

.~'

II·

11-

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

..

11-

II··

11-.

....

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

(b) Furwasb

.'

I,T

-._-----

•

.,._._---------------"--- .:._._---" ..~-'----'-"'--'--­

;-'-------.--_._---:: .._------------------_._-.__._. . . .
10

Project 29753

Page 21 of49

FJgare 4. Example cbromatograms fiom (a) control mouse blood and (b) blood from a

mouse exposed whole-body to 229 ppm BE for 6 hr.

(a) Comrol mouse blood

1I'",,,,,lO.lICl!'T. _'Da .. :

_.

--

....

..... 12-~_Acid I

'::::::;: ".

'~----~-------..;..;..> --~--

(b) Blood sample from exposed mouse

""__'''''__''11I01'-

...

_.

.....
11

Proje.."t 29753

Pagellof49

Fagare S. Example chrorrwograms from (a) control mouse urine and (b) urine from a

mouse exposed whole-body to 229 ppm BE for 6 hr.

(a) Control urine

.. ,

--

'...

--•.

..

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

(b) uriDe from exposed mouse

..,.'" lG.....r. _ ... _...

_.

,,..-

.•.

.:

.:

I2.£_,...oc AcId I . "'& .

. 1

•..' 12~~.1:: - ,-

..·~12.~ J .~ ; .... .... ., .....::...~ ..... '~'._ · ;_..."'--~-.-;.~l-:-.---.'~~ '--"

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

"
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Project 29753

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Figure 6. The relative intensity of Perl staining as a function of BE 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

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Tab-'. 1. Residual BE (mean ± s.d) removed from the fur of five female mice exposed

either whole-bo:dY or nose-oDly for 6 br to a target concenaation of250 ppm BE.

Exposure

Group

Exposure Cone. Body WI.

(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 at Ibe eDd ofa 6-

hI' whole-body or nose-ooly exposure to a target c:cmccuaation of2SO ppm BE.

Exposure Exposure Cone. Body WI. 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 .

Table 3. Amounts offree BE and BAA excreted in the urine often female mice 18 hr after

the end of a 6-br whole-body or ~yexposure to a target concentration of 250 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
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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 next line.

(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 100 mglkgtd 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 100 mglkgld BE for two days foUowed by 3 days at 1200 mgIkgId

(sec text).
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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

(mglkwd) (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

(mglkwd) (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 !he mortality 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 oblaiD blood 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 monality data). Data are 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, surviving nUee 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 of mice 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

Dose Grou 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

1200/600 mglkt 0.16:0.09 0.18 :0.09

• After lWO days ofdosilIg !be monalny w:IS excessive: lberdore. surviviDg mice from each dose group were

admiDistercd ba1foflbeir com:spoading originall3rget dose for two additional days prior to study rcmirwion.

S-phase nuclei were scored based on tbc: mcdIod tqlOned by Eldridge ami GoIdswonby (1996).
0 Appendix A-I Projecl29753 Illdividual Animal Dala - Fur Dcposilion SlUdy Page 29 or 49

Appendix A·I

Individual Anil1)al Data - Fur Deposilion Study

Residual2.Buloxyethanol on the FurofMice- Nose Only Exposure (241.8 ppm)

Residual BE Residual BE

Body WI. Amount of BE in Wash Total BE in Wash (mg BE/kg '. (mg BEl kg 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
AppendiK A-I Projeel 29753 0

Individual Animal Data - Fur DCllosilion Study Page 30 or49 Ul

Appendix A-I

Individual Animal Dala - Fur Deposilion Siudy

Residual 2·Buloxyelhanol on the Fur of Mice - 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 0.0125
Appendix A-2

Individual Animal Dala - Fur Dcrosilion Srudy

Appendix A-2

Individual Animal Oala - Fur Deposilion Study

Concenlralion of2·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 Body exposure.

NO =241.8 ppm Nose Only exposure.
Appendix A·J

Individual Animal Dala - Fur Deposilion SlUdy

Appendix A-J

Individual Animal Data - Fur Deposition Study

2-Buloxyethanol (BE) and BUloxyacclic Acid (BAA) Levels in Urine collecled 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

Body Weighl 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 Siomach WI. 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 0.55 I 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=Not Weighed; 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

Body Weight Stomach Wt, 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 at Terminal 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 3.3
Appcndilt 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 h Wt. h WI. 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 h WI. . 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 (Full Study)

Project 29753

rage 40 or 49

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 h WI. h WI. 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

Saline Control- 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 2i.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=Not Weighed; 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 - Gavage Study, Full

Appendix D·]

Individual Animal Data·· Gavage Sludy, Full

Histopolhological (H&E) Observalions of Forestomach and Liver

Group Animal" Observalions

Projeci 29753

Page 43 of 49

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, liver nonnal

-- ._- -... '-'..~~~.'~~~-= ~_-.~~:~: ~:~_~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, liver cells seem highly

I~'-::~--:-"-:.-.:.:--.:: .-..J. _: _-~ ItJ:~ij_~o~~~:t~!Ch~p.i~h~Uo.!.~_.pl~!i~!!~·~i.!l~~L-.~_ .. _..

.M!c1 D()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 glandular stomach 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 inflammatory cells in

.. . __.... .... __I .._.. __ . _~~~_sl.!~J!I.u~l)~a andm1.!~.~!~!!!!J.i.Y~~_!I()m,lll'_ .... _. __ ... ..._._ ..... _. _
Appendix 8·2

Individ.ual Animal Para - Gavage Study, full

Projcct 29753

Page 44 of49

o

til

Appendix B-3 (conUnued)

Individual Animal Data - Gavage Sludy, Full

Histopothological (H&E) Observalions of Foreslomach 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

12001600 mglkg I 603

---_ ...._---------1·----· .... --.. 608

minimal ulcer of forestomach, 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!1B p~opria, liver nO.rnlllL __ ._ .. __ ........ __ .... mild forestomach cpilhelial hyperplasia wilh inflammation in Ihe

muscularis. There is a small island ofepilhelium in the innamed area and

.evidence of eXlension 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 of Foreslomach 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 ulcer and minimal epilhelial hyperplasia,

liver normal

_::=-::'_=~_-=-_~.-_-~.=:~~~~: 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, liver 110 rJl111L. .. . __

III foreslomach normal,liver normal ------- -..-....- -..--... '-'1'13 .--. 'mirilmlilrociii necrosis and ep'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·celis-in· -. Ihe musculll;!s, liver normal . - ----..-_.. _---- -,--_.__ .. --.-------- ---- -- --_._-_._-------------- - --.----_...- ..- ------- .. ---- -- ------- ---- _..

5I3 mild forestoma~h epithelial hyperplasia, liver appears 10 be highly

I . _ 1-.. .._. 8!yc~8~l'!.a!~. __ ._ __
Appendix D·2

Individual Animal Data - Oavage Study. Full

ProJeCI297S3

rage 46 of49

o

-....)

Appendll B-3 (conCinued)

Individual Animal Dolo - Gavage Study. Full

Hislopalhological (H&E) Observalions ofForeslomach and (.jvcr

Animal #I Observations

High Dose Female

Group

701 I mild foreslomach epilheliat hypeijiliis18 with some: IrtnDmmation In the

lamina propria. Liver normal. . . .-----.----•.-'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 Innammalion in Ihe muscuillris,

liver not presenl; spleen is within normal limits -•. ···r···" ..... -..... ·'1--'·'109"-· ""giiiiiifiifar stomach iiorrnai,niIlifforeslom8Cii epilheliaI hyperplasiaand _. minimallnnammalory cell infillrate In lamina propria. liver normal .-- .....-..-.- ----1"-- 7is' I(si<i"iniich noi preseiiij: IIver·normiir-..··---··---·--· _. -.-----.-.. -.. _ •• ••••• __ • •••• _._. __ • __ •• __•••• _ •• _ ........ _ ••• _. • ~_. • 0_0 __..__ •• •• _a • • •• ~._
08

Appendix e-I • Project 297S3

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 and Terminal Body aDd Liver Weights

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 oftbe Forestomach

Project 29753

Page4lof49

GToup

IP 400 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 - IP vs, 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 blinded evaluation)

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

HypeIplasia, epithelium. forestomach +1;

inflammation +1; very minimal diffuse epithelial

thickening with submucosal mononuclear inflammatory

cells (also identified by blinded evaluation)

Normal

Hyperplasia, epithelium. forestomach +1;

inflammation +1; very minimal diffuse epithelial

thickening with submucosal mononuclear inflammatory

'cells (also identified by blinded evaluation)