P.1 

OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

66) ll-J-9 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:16 AM Sheet 66 of 93 
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PROTOCOL 

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EASL-M6215T 

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XBSTMfl FACILITY 

Toxicological Scleneta Laboratory 

Htalth and Environment Laboratoriea 

Baatman Kodak Conpany 

1100 Bldtevay Avenue 

Kodak Park (Bids. 920) 

locateter, IT 14631-3615 

LAMHTflOT WOJKCT IP 

Iff BS AMU 

BITOT sronsQi 

Ketoneo Panel 

Chemical Menufecturera Aaaoclatioa 

2501 M Street, BV 

Vaahlnfton, DC 20027 

December 7, 1990 
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HARL-MT621ST 

Page 2 of 11 

STUPV TITLE 

Meaityl Oxidei Combined Repeat Doee and Reproductive/Developmental Toxicity 

Screenins Test in the Rat 

____ 

The purpose of the screening test la 1) to determine the toxic effecta of 

meeltyl oxide in male and female rata, and 2) to detarmlne the potential for 

meaityl oxide to affect male and female reproductive performance following 

repeated expoeurca by inhalation. 

Tgsxins TMlhin 

_s__i 

Toxicological Sclencea Laboratory 

Health and Environment Laboratoriaa (BAIL) 

Eastman Kodak Company 

1100 Rldgevay Avenue 

Kodak Park (Bldg. 320) 

Rochaster, IT 14652-3615 

Ketones Panel 

Chemical Manufacturers Aeeociatlon 

2501 M Street, MW 

Washington, DC 20037 

Sponsor's Repreeentatlvet Barbare 0. Franela 

BTUPI PATES 

Initiation Data 

Experiment Start Data 

Treatment Period 

Experiment Completion Date 

Proposed Report Completion Data 

To Be Added 

To Be Added 

To Be Added 

To Be Added 

To Be Added 

BfiCULATOBY COMPLIAMCK 

Test Standard! 

Thia etudy vill be conducted according to the propoeed OBCD Guideline for a 

Combined Repeat Doee and Reproductive/Developmental Toxicity Screening Study. 
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HABL-+H6213T 

Page 3 of 11 

Thia etudy vill be conducted according to Good Laboratory Practice for 

Nonclinical Laboratory Studiee ae promulgated by the U. 8. Environmental 

Protection Agency Good Laboratory Practice Standard 40 CFR Part 792 and Annex 

2 of the OECD Guidellnea for Teetlng of Chemicala (C(81)30 (Final)) aa 

required by Council Directive 87/18/BBC of December IS, 1986. Inapectlona 

vill be performed by Quality Aeeurance Unite of Baatman Kodak Company to 

determine compliance of all phaaea of the etudy with good laboratory practice. 

TEST ARTICLE CHARACTERIEATIOH 

General Description 

Chemical lamet 

CAS lumbert 

HAELlo.l 

KAN lo. 

8RID Ko.t 

Experiment lo.t 

Physical Deecriptiont 

Source 

IBBXUAI 

Vepor Pressure (mm Rg) 

Flash Point (*C closed cup) 

Boiling Point <#C) 

Specific Gravity (20/20*C) 

Molecular Weight 

Meaityl Oxlds 

141-79-7 

To Bs Addsd 

To Bs Added 

To Ba Added 

To Be Added 

elasr liquid 

To Bt Added 

9.5 

n 129.0 

O.S57 

98.2 

[MO] 

Taat Artlcla Purity 

Purity analysis of the sample vill be conducted prior to the start of the 

study. The purity of tha tsst sample vill bs specified In tha Tasting Consent 

Order. The test saaple vill also ba snslytad for purity at tha completion of 

the etudy. 

fltorate Condition* 

The teet eample vill be kept in a chemical atorege area. 

vill be maintained in the BAIL Archive. 

Bafatv Precaution* 

A reaerve eample 

Preceutlona vill be taken to avoid contact betveea the teat article and 

clothing, akin, and eyes. Routine lndustrlsl hygiene practices vill ba 

employed ln handling of the teat article. 
— — l*U • I i W I *. i. ^ i* •-/ -? 

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uec iu.su S:D D NO.001 P.05 

HA2L-MT6215T 

Page 4 of 11 

lEfT SYSTEM 

Anlmala 

Sexually mature mele and female (nulliparoua and non-pregnant) Sprague-Devley 

rats (CD*(SD)BR) approximately 8-9 weeks of age vill bs obtalnsd from 

Charlee River Laboratories, Kingston, IT or other appropriate source. The 

anlmala vill be acclimated for at leaet five daye prior to study start. The 

health etetus of the animals vill be evaluated by a veterinarian prior to the 

stert of the study. After culling by ueuel procedures, forty-eight males and 

forty-eight/emalee vill be assigned to the study. Weight variation at etudy 

start ahould not exceed a 20Z of the mean velght for each aex. Animela vill 

be et leaat 10 veeka old at the beginning of the mating phase. The rat vaa 

ehoaen for this study becauee lt is a common representative apeelea for 

toxicity etudiea. Thia atrein haa a high fecundity end is routinely ussd in 

rodent reproduction end developmentel toxicity studies. The ret is one of the 

tvo primary epeclee recommended for use ln developmental toxicity atudlea by 

U.S. EPA and OECD guidellnea. 

Housing 

Anlmala vill be housed and exposed to the test material in appropriate arsas 

of the vivarium, HAEL. During tha acclimation period, anlmala vill be houeed 

by aex in groupa of five ln euapended atainleaa ateel vire mesh cages. 

During the exposure periods, anlmala vill ba housed singly in 

multicompartmented etelnless steel mesh cagea. 

Starting at etudy initiation and continuing through the prerneting period, 

animale vill be houeed oingly during nonexpoeure periods in suspended 

steinless steel mesh cegee. During tha mating period, tha animals vill be 

housed one male vith one female during nonexpoeure perloda in auapended 

etelnleee eteel meah cegea. Follovlng tha mating period, mele end female 

anlmala vill be houeed singly during nonexposure periods in suspended 

atainleaa steel meeh cages. On or about tha 19th day of gestation, solid 

bottom pans containing bedding materiel for nesting will bs put in ths 

nonexposure cagea of tha female rate. 

Target levela for expoaure and nonexpoeure environmenta are ebout 22 as 3*C 

end 30-70% relative humidity* Reletlva humidity levela may briefly be outaide 

of thie range during cleaning procaduree. Timere in the etudy rooma vill be 

programmed to provide a photoperlod of 12 hours from 6 em - 6 pm. Ro other 

animals vill be houeed in the ease room vith this study. Csges, recks, and 

feeders vill be veehed once per veek. Abaorbent paper under the cages vill be 

changed at least three times psr veek during the premating, mating, and 

geatetional periods. Bedding materiel ln the solid bottom pans vill ba 

changed only if neceaaary during ths post-partum period. 
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P««d and Watar 

Agvay* Prolab" Animal Diet (RMH 3200), certified ground chov vill be 

available ad libitum, during nonexpoeure perlode. Municipal tap vater (Monroe 

County Vater Authority) vill be supplied id libitum during nonexpoeure periode 

through an automatic vaterlng eyetem or in glees bottlee vhen the neeting pane 

ere ln the eagee. Ho knovn contaminants are expected to be preeent in the 

feed or vater vhich vould'interfere vith the outcome of the etudy. The veter 

le analysed for contaminants on a quarterly basis. 

Identification 

All adult rate vill bo identified by uniquely numbered motel eer tege end all 

adult male and female rata vill be aesigned a etudy number vithin doee groups. 

Randomization 

Culling and randomisation of the animele vill be done by computer-generated 

liate uelng the Automated Animal Toxicology System (AAT8). The meles and 

females vill be eeelgned to the etudy groups on ths basis of body vsighta ao 

that the mean body veighte ere almilar among the groupa at the etart of the 

etudy. Animele received vith the initial ehipment, but not used ln the study, 

vill be removed froa tbe etudy room:at the start of the treatment period. 

Records vill ba kept documenting the fsts of sll animals rscelved for the 

study. 

STUDY PESlfifl 

Iriitmtnt grawi 

The animals vill be assigned to three test groups and ens sir control group, 

each containing 12 male and 12 female rets. This group slse ves chosen ln 

order to yield approximately sight pregnant fsmslss for study in tho absenee 

of an adveree reproductive effect produced by the teat article. 

Exooaura Condition* 

The inhalation route vaa choeen alnce lt la a possible routs of expoeurs for 

man and ves recommended by the U.S. EPA. Exposure concentrations vill be 

selected besed on the results of a preliminary inhaletion study In pregnant 

female rata. The concentrationa vill ba included ln • protocol amendment. 

The higheet concentration ehould reeult in toxic effects but not produce 

mortality in the parental anlmala. The intermediete concentretion ahould 

produce minimel obeervable toxic effecta, and tha lovest concentration level 

ehould have a lov probability of producing toxic effsett. Except for 

treatment vith the teet substence, animals in the control group vill be 

handled ln a manner Identical to the teat anlmala. 
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Evpoaura Condltlona Cont. 

Exposurea vill be for eix hours per dey, seven daye per veek. Bxpoeuree vill 

be conducted ln 420 L etalnleee eteel and glaee inhalation chambers. Chambers 

vill be maintained under negative pressure (0.5 Inch voter) vith 12 to 13 sir 

changea per hour. 

Vepors vill be generated by metering the test material dropvlse Into a glass 

bead-packed column eupplled vith metered, dried, oil-free compreessd air. 

Chamber vapor concentrations vill be determined at leeet once per hour using a 

MIRAN* IA infrared analyser equipped for automated sampling and analysis. 

In addition, temperature, humidity, and sirflov in chambers will bs monitored 

and recorded at least once every half hour. Nominal chamber vapor 

concentration (ratio of volume or mass of teet materiel ussd and total 

airflow) vill be calculated dally. Animal cage eeelgnments vill be roteted 

dally vithin their respective chambers to minimiss potential positional 

effecte. Distribution of the teet material vill be determined initially by 

measurement from numerous positions throughout each chamber and vill ba 

compared to that of a fixed reference peel tion vithin eech chamber. 

8ubeequently, ell test data vill bs rscordod from ths reference poaltlon. 

Duration of Treatment 

The etudy ia composed of four phaeees premetlng (14 daye)j mating (up to 14 

days); pregnancy (21-22 days)* and sarly lactation (4 days). Females will be 

exposed only until Dey 20 of geetation (approximately 55-48 expoeuree). All 

mele rata vill be expoeed throughout the entire study, a period of 

epproximately 40-53 daye. Males vill be exposed until the dey prior to 

euthanaeie. Males vill bs euthanatized after last littsr has bean 

euthanatlsed. Exposuree vill be conducted on e ssven day/veek beela. 

Mating 

Melee end femelea vill be meted ltl vithin the ease dose group. Femalea not 

lneemlnated during the first weak of meting will ba placed during a second 

week of meting vith a mala vhich had successfully mated with a female during 

the flrat week. The ear tag numbera of the mated paira -will be recorded. 

Copulation vill be verified through Identification of sperm ln vaginal smears 

or by appearance of a copulation plug. The morning of tha day a plug or 

vaginal sperm la obeerved vill be eonsidsred Day 0 of gestation. Following 

copulation, the melea and females will bs seperstsd and housed individually 

until study termlnetlon. 

Body Walahe hataflnatlona 

The female animele will be weighed on Daye 0, 7, and 14 of ths prsmstlng 

period, Deye 0, 7, 14, and 20 of gsststion, and Days 0 and 4 post-parturn. The 

male anlmala vill be velghed on Daye 0, 7, and 14 of the premetlng period, end 

veekly thereafter. All anlmala vill be velghed at the beginning of the mating 

period and veekly during the cohabitation period. All adult anlmala vill be 

velghed end faeted the dey before euthanaeie. 
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BAEL-MT6215T 

Page 7 of 11 

read Consumption 

Feed consumption vill be determined et leaet once e veek for all animele 

during the premetlng period. Feed consumption vill not be determined during 

the mating period. Feed consumption for mated females vill be determined at 

leaet once a week during geatatlon and from Day 0 to Day 4 poetparturn. 

Resumption of weekly feed consumption determinetions will bsgln for msle 

animals after the 14-day mating period (efter the end of the longeet poeeible 

mating period). 

Clinical ObaarTatlona 

Clinical observations vill be made et leaet once deily. Obaervetlona vill 

include, but not be limited to, examination of the heir, akin, eyee, mueoua 

membranea, reeplratory eyetem, circulatory system, snd sutonomlc and central 

nervoue systems, fecss, urins, snd general activity. Poot-perturn, neonatal 

observations vill bs conducted simllsrly. Ths dams will bs observed for ths 

beginning of parturition and this dsts will bs ussd to ealeulats tha gaatatlon 

period. All pupa will be examined, without hendling, aa aoon aa poeeible 

efter birth to determine the number of vieble sAd stillborn pupa. Tha day on 

which the dam has completely finishsd delivering will ba coneidered 

post-pertum Day o. Llttere will be examined for ebnormalltiss on Day o and 

Day 4 poat-partum. The number of pups (sliva and dead) will ba counted and 

eexed. Live pupa will be velghed ee a litter and mean pup velghta vill be 

calculated. 

CllnlCBl EltlWlQBY IlifllnitlflDJ on HSlBS 

At nscropsy, blood vill bs collected from tha poeterior vena cava of all male 

animele While the reta are under CO2 aneatheaia. All aaeaya will ba 

conducted by the Anlmel Clinical Analyaie Croup, Toxicological Scieneea 

Laboratory, RABL* 

Hematology teeta will include1 hematocrit, hemoglobin concentration, red 

blood cell count, and white blood eell count. Fixed blood ameara will be 

prepered and etored. 

Clinical chemlotry teete will includes aapertate smlnotrsnofsrase (AST), 

alanine aalnotraneferaee (ALT), glucoss, ursa nitrogen (UN), gamma glutamyl 

transpeptidase (GGTP), totel bilirubin (direct bilirubin may be measured if 

totsl bilirubin ia elevated), creatinine, total protein, albumin, 

albumin/globulin ratio (A/0 ratio), potaaaium, chlorlda, calcium, and 

phoaphorua. 

Urinalysis 

urlnalyals ia not recommended on a routine basis in thia typs of screening 

teet, but haa bean included ln thia protocol baaed upon tha known propertlea 

of tha teat materiel. During the week prior to necropey, male rata will be 

placed ln meteboliem cegea for approximately 16 houra for urine collection. 

Feed end vater vill be availeble gd libitum during the collection period. The 

osmolality end volume of the urlnee vill be measured. Microscopic examination 

vill be conducted on urinary sediment for the preoenee of essta, red blood 

cells, white blood cells, crystals, snd bactsrla. 
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BABL-MI6215T 

Page 8 of 11 

WicropiY 

Animals that die or srs euthanatized beeauee of morbidity will bo nscropslsd 

ae aoon aa possible. Surviving male and female enimele will be aneethetlsed 

with CO2, and exsanguinated by covering the posterior vsns cava. All 

animals vill bs fsstsd overnight prior to scheduled nseropsy, and blood vill 

be collected from melee for anelyale prior to exeangulnatlon. All adult 

animele vill be aubjected to a full necropay vhich Includes examination of the 

external surface of the body, all oriflcea and the cranial, thoracic and 

abdominal cavities end their contante. Special attention will be paid to tha 

organe of the reproductive system. Femalaa that fall to litter will be 

euthanatized approximately five days after their expeeted delivery date and 

the uteri vill be examined for implantation aitea and corpora lutaa. Dame 

thet litter vill be examined for implantation sitae and corpora lutaa at the 

time of necropsy. 

Dead pupa end pups euthanatized et termination of the study vill be examined 

maeroeeopieally for externel ebnormslltlee, but not nscropslsd-. Tissusa from 

the offspring vill not bs collected. 

Wet weights of ths livsr, kidneys, thymus, lungs, testes, and epididymides 

vill be collected et necropsy. Palrsd organs vill bs velghed togethsr. 

Organ/body weight ratios will bs calculated. 

Hlatopatholoav 

The tiesuss listsd bslov snd tissues from terget organs identified during 

necropey exeminetion vill be collected from all adult animals and rateined in 

epproprlate fixetivea. The tiaauea to be collected in buffered neutral 10X 

formalin includes liver (2 sections), kidneys (1 section/kidney), adrenal 

glande, brain (3 aectiona Including msdulls oblongata, pons, cerebellar » 

cortex, end cerebral cortex), heart, epleen, thymus, lungs, naaal paaaegea,

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xxa^' 

overiea, and target organs. Ths tsstss snd spidldymidsa will bs preeerved in 

Bouln's fixative. 

Histological evaluation will ba conducted oa tha tieauee Hated above from 

control and high doaa sals and female animals. If ebnormalitiee or equivocal 

results are eeen ln aay of these tissues, tha asms tissusa froa lowsr doss 

groups will bs examined. Rletopathology vill alao be'performed oa the organe 

and tiaauea Hated above for all adult animele vhich die or ere euthanatized 

prior to study termination. Histopathology vill ba performed on significant 

gross lesions ia all adult animals. Ovaries froa say salaal vhich fella to 

complete its pregnancy vill be examined. 
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HAEL-MT6215T 

Page 9 of 11 

fiTATTSTTCAL PBOCRDUBEg 

Mean valuee vill be calculated for chamber coneentretion, chamber temperature, 

chamber relative humidity, edult body veight, feed consumption, cllnicel 

laboratory determlnatione, organ weights, geetetlon period, litter else, pup 

body veight, pup body veight gala, and pup % survival. All mean data, vhere 

Applicable, vill be evaluated using the following computer-generated 

etatietical teste» Bartlett's teet (p < 0.01), one-way analysis of variance 

(ANOVA) (p < 0.05), and Duncan's multiple range test (p < 0.05) to 

indicate etetistical significsncs. Batlos such as X mortality and X pregnancy 

vill be compared uelng Chl Square Tebles. Transformations to normalise 

dietribution of the dots vill be performed vhere appropriate. A t-teet may bs 

performed vhere the Bartlett's test lndlcetee e significant difference in 

variancee vhich might Interfere vith interpretation of analytical reeults. 

ftATA ZTOfUeE 

The final report, data eheets, snd ell non-perlehabls rev date vill bs storsd 

in ths HAEL srchlvss for at leest tsa years, 

REPORT 

The final tsst report vill include a deecriptlon of the materials and methode, 

the reeults, end a discussion sad interpretetion of the reeults. The report 

vill elao include the epeelee/atraln of rati abnormal clinical eigne (day of 

observation and clinical couree)} day of deeth of ell animals including those 

surviving to study terminations, a summary of ths numbsr of implentetlone, 

litter size snd aean pup weights} toxic offsets on reproduction, offeprlng, 

and poetnetel grovth} body end organ veight data on all parental animele} feed 

coneumption data} hematology end clinical ehemietry reeulte} necropey 

findinge} hlatopathology reeults) and etatietical treetneat of tha atudy 

reeulte. 

Summery tablea included la the report vill preoaat tha following dates 

Exposure condltlona 

Number of anlmala at tha start of ths tsst 

Fertility indices (mele end female) 

Maternal mortality 

Numbsr of viable littera (Geetetioa Index) 

Mean geetetioa period a BB 

lumber of totally reaorbad litters 

Mean maternal body velghta « BD 

Mean female body weights s 80 (prior to pregnancy) 

Msan male body weights * 80 

Mean maternal body weight change during geetatloa a 80 
— . . P.11 

KODAK 320 ItL NO . ab-/22-u^ y Dec lU.yo 9 = 55 No.001 P. n 

BAEL-MT621ST 

Page 10 of ll 

Summary tablee cent. 

Mean feed consumption for male sad female enimele during the premetlng 

period s SD 

Mean maternal feed consumption during gestation a SD 

Mean feed consumption for male enimele during the poetmating 

period t SD 

Mean number of live pupe on Daye 0 ead 4 poet-partum * 8D 

Mesa litter else (live + deed pups) oa Day 0 poat-pertua ft 80 

Mean number of dead pupa on Day 0 poet-partua a 8D 

Mssa y pup survivsl on Day 0 poet-partua ft 80 

Mean X pup eurvival from Daye 0-4 poet-pertum a SD (Viability Index) 

Mean pup body vaighte on Days 0 snd 4 post-partum a SD 

Heaa pup body veight gala Days 0-4 post-partum « 80 

Sex ratio for pupe on days 0 sad 4 

Mean male hematology valuee at nseropsy a SD 

Mean male clinical cheaietrles at necropsy a 80 

Individusl dsts vill bs preeented in the appendlcee end vill includes 

Individual female body veight - pregnant females 

Individusl female body veight - non-pregnant feaalsa 

Individual male body veight 

Individusl fssd consumption - prsgnant feaalsa 

Individual feed consumption - non-pregnant fsaalas 

Individual feed consumption • males 

Individuel reproduction and litter data 

Individual aale hematology data 

Individual mele clinical ehemlatry data 

Individual groae pathology data for adult animals 

Individusl histopathology data for adult anlmala 

Individual groea abnormality data for offeprlng 

Chemical purity, etebllity, and concentration analyaee report 

PaftJKCT FAtTTCIPAHTg 

Study Director Walter J. Kraeavage, U.S. 

Study Technicians Llee G. Bernard, A.A.S. 

Beede A. Moultoa 

Heaatologlat/Cllalcal Chemlet R. B. Emmons, E.g. 

Psthologiat Milan 8. Vlaovle, D.V.M., Ph.D. 

Laboratory Animal Medicine Gordon J. Hankinaon, O.V.M., M.S. 

Analytical Chemist Kenneth A. Roblllard, Ph.D. 

RIOBBJUI 

Propossd OECD Guideline for Testing of Chemicels (1990). Combined 

Repeet Doee end Reproductive/Developmental Toxicity Screening Teet. 
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SIgBATURE PAflB 

HABL-MT6215T 

Pege 11 of 11 

Study Director Date 

Director, Memmelien Toxicology Section, BAEL Date 

Director, Toxleologicel Sciencee Laboratory, BAEL Date 

HABL Quellty Aeeurence Unit Data 

Sponeor'e Repreeentetlve Dete