Document mBYBmEG2yYb8eaGnd3LmgbG90
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VOLUME I
P. Box 178 Splnnetstown, PjA 18963
phone(21i5)536-4i10
fast(215) 536-1816
Study Tide
i Local Lymph Nodb Assay In Mice (LLjNA)
Test Article
- MTDID 6675.1
Positive Control
: alpha-hexyldnnamaldehyde (HCAj Technical Grade 85%, AkJnch Lot/Batcfi #131G2MO
Vehicle
: Dimethyl Sulfoxide (DMSO), Sigihe-Aldnch Batch/Lot #(34734ME
Author
: Melissa Kiifc. PhD .Study Director
Study Completed On : October 25,2007
Performing Laboratory :
MB BesearcbLaborartories
1785 Wentz Road P.O.Box 178 Spihfierstoyyn, PA 18988
MB Research Project # ; MB 0715961 26
MB Research Protocol # : 5650M
Sponsor
: 3M Corporate Toxicology & Regulatoiy Services 3M Center* Bldg 0220-06-E-03 oO*l roWaui.ri, AlyHlWt.i O p lw lv y v
Sponsor Study#
: 07-106
Citation
' Melissa Kirk, Ph.D (2007) Unpublished Report by MB Research Laboratories
P a g *1 o f9 2
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S tudrntte : LLNA
Project# : MB07-15961.26 Protocol 5656M ..........
T h )sstudyvrasoon durtedinxrtenQ e v^th jS o^
GFR 160.and 7 9 ^ R ^ 2 1 CFR 5$, and as soedfiad in Pifinriobs on t^ ^ h o ra fa rv Piartfe
publishedbjfthe Organization lo r BboooinicjS ia o g m a ^
189?, wfththo
following e(beptii3n;$:
T e s ta rtk te c iv a ra ^^
,wa.s:i
in addition, the testarfide
STUDY DIRECTOR
M elra^K ifk; P itD
_______ J O M M )
.... Date
phone; <215)53*4110
-f>9ea;PfaM^'-aMxiMKTai a p h n e w u ^ ro ltt bar* (215)53*1816
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PROJECT NUMBER :
TEST ARTICLE
:
SPONSOR
:
TITLE
:
MB 0715961.26
NTI1QID6675,
SM CCRP^f^TE TO)UCOL06Y & REGULATORY SERVICES
Local Lymph Nodo Assay in Mo (LLNA)
5650MI
fumale CBAfd
the (est arsele, MTDip.6675*
to be used in the definiti Local Lymph Node study. T be <rxntraeoos {25%, $0% and 100%)
o f the'test article were tested (2 animais per group T h ^ oorwentrations;cirespondd to
aoDroximately75%. 1 5 % a n d 2 9 ,9 % ^1 h e a g ^
Thieprelirninafyscreen
sfwwed t iniitBiton at any 100% BBs*di*^'tB;T*iti^rOapoaNntea#ana^
r
the definitive Local ! ..... ..................
---
.grtxjpw a&ieftunireatedto dse group, wfjW t dUtloiUTODMSO control ware admJitistered by topical
The mice vvergiven an intrapritonealirijBcflOB,ioifibe ttyroTdnpanlog 5 h b ro m a -2 t-d ^
five days ^lo w tfis th inifial dose, and fivehdurs pirtf t s a e tip i* A t sacrifice, the aurfeuter lymph nodes
werg isQ[Ari afrtgift-ceii suspensions of ivmDh rtodecelteqNCj) weregenetated, land the IN C
^ jpgpgfrw wasanaiyzed by flow cytom etryfor BitiU incorporation arid the total number o f LNC. The
amount o f prlifrting (#BrcHJ*) LtJG vras determlnea asa measure o f the proliferativeresponse of the
cat tyrnph ridde th Stimulation inde^ (SI) s calculated,by dlvklirig die proBfersrtive response <BrdU
incorporation) of each test srfictegrdup b the protlferatJvefespwifos f*h e ;V e h ^
or, for
the 100% test article group, by d ^ pm lif^e^jesponse of ttie naNe. control group. Test articles that
yielded a SI 3 vrrectfera<aert2Sdaspotentteitysensitizing substances
AHquets ofthe
e^
evaluation. CeBewere ashd arid resiiispenddn PhosphteSuffered Salh (PBS) containing the
viability dye 7AD.. Lh/e clle were analyzed by flow cytometry to determine %B cells, %T cells; and the
8:T cell ratio. Test articles iridoclrig; ari irwreas of:^5 % (1 i2 5 ift^ ) over toe vhide\(ornaive) central in
% B c^tlO rto e ^ B iT ra p iv ^ e ^
Summery. ABanimate suiyived tjhefn-life phase of the study and appeared normal. except for one
animal in the 16& dos group, vttWpti appeared emiacteted:arid srftM d ^a n o g e n ita l area oil Day 6
of Bte definitoroshtoi: Thte ariirriat Id be tejetded withBrdU, solyirjph riSdeswere not harvested
Thesigns observedlrithis aranfelare ndt`6efii|d te b festartMe-n^fed sfoee all ctoer ariimate in this
groupsppeared riorrftel Theraw eratw dlflleutiiesinadiiA^
lo thedosed ars Body-weight changes
iSte^swiiirriisi!fij the 100%dose group, vrtiiph
"' phono: (216)63S4110
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Summary (cont*d>:
ABSTRACT (contU)
. . fnclennallrritatlon
3 ^ S fc tih tw to M ^ ^
Th SI vale&for test articteMTDlD 6675,
at 25%i 50% SAd 100% were 0 8 ,2 ,4 and 2 8 ; respectively The SI value of
T'he test artcte elicite,d....a W,...d:Jiro e ams.e l,in % Bdve_lteofi1-A.0A5.fat_tJ2-5A%:4,AAl;A2//2': st5 03% a^n d .t a- l fiaSttV1S00G%Qf*lfrlOl Fofctincreeses in B:T rato were 1.04.af25%, 120 at50% , arid 2 21 at 10Q%, compared to vehicle, and
at 10C ^ ^ ^ d fe aie Naive control,
at 100%
artideisadw tT ^insiitjzw In theLtcalLynrij* NodeAssy. The test article has a calculated EC3 value
of 54:S% (16^4% as th ddive ingredient): Based oh ECETOC criteria, the active ingredient ih MTDIO
.... ' ...... .... ......
Cah te clasaiffed a weak sensitizer.
phone: (216) SS&4110
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--^
p-- - i '
: Study Title
Project # ' ................... Protocol
______
: LLNA - "
: M f 07-15961.26
: 5S60M
''
OBJECTIVE
To determine t
h
e
:^
. This s j^ y is d ^ tg n ^ bea
stand-alone a l^ a tfv ra s ^ y to T
S en ^ tetfo n A ss^ d efe^ inthefCCVAM
repc>(Ferfei3lReglster 63 C FE S /^p^,;July;i0, 1996), ^PiHMh E f l ^ Te^CukWinOj OPPTS
870:2000 final ^lclsline efliectareM^rch 2 0 0 ^ ^ 0 ^
e f f e c t April 2002.
Identity
T e stA itfe ie Characterization Provided by Stability Data Received Storage
Description Sample Preparation
: M TDID6675,'
V
,. ., ..... ....... . .... , . s ' ........... . ...., , : SeeAppendix K for TesfArticteChariecterization and C @ rdticaita.of/^iy^.
: 3M Corporate Toxicology BRegutatoryServices
: Not supplied by the sponsor;
: 07/06/07
,
: Roorntemperatureand humidity
: Clear liquid
'
...
: The tost article wassupplledasan aqueous solution containing 29.9% of
theactive ingredient It was used as received for,the.100% concentration,
and cfilutedwitataevahKte; DMSO. toappropriate concentrations,
,
identity
Provided by Diate Received Expiration Data Storage Description SarrtplePfeparation
POSITIVE CONTROL
ataita^exytcinnamsddehyde (HCA)Technical Grade 85%,
Aldrt<^Lot/Batd>il3102M O
.....
MB Research Laboratories
05/09/06
11700007
Room temperature and humidity:
Yellow liquid
204 pi of 85% HCA was added to 706 pi o f DMSO, to yield a 25% solution.
Identity ProvidedBy Received Date Expiration Date
Storage Description Sample Preparation
VEHICLE
Dimethyl Sulfpidde (DMSO)r S^hfia-AidUch Bafch/Lbt #04734ME MB Research itabqratdries 04/30/07 04/30/08 : Room temperature and humidity. : Clear liquid : Usedasreceived
phono: (215)636-4110
im wtantero^
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Project# : MB07<15961.2B Protocol : 5650M
Study Initiation ExperimentalStart Date Experimental Term Bat Draft ReportSigned 2 ^ DraftReportSubinitted Final ReportSigned
TEST PATES '
(date pra^pcol^nfd) (1st exposurBtdtestswbsta (last datedatecpliected} <j{ appfeable) {itappjKpabia} (study compietbn)
07/18/07 07/18/67 08/02/07 00/12/07 10/04/07 10/25/07
EXPERIMENTAL DESIGN
TestAnim als:
Female GBA/J mice ware re ^ e d fra m J a e k s p n ^ ^
06/26/07,07/10/07 & 07/17/071.
FoDawing an e g d lib ra fc n p a f^o T a t te a s ^ ^
non-pregnant female mice
were a3$ ig n e d to te
a c ^ p j^ r-b a s e d randomnumber program
7ne anlma)s were bom on 05/0i/07, Q5/15/07 & 05/22/07, 3 days. The Pay 1 bpdy weight range for the
defihithrestudyarom ^ 16T - Z? 2 gr?rns. The pay 1 body v ^ h t range fer die s t^n a h irn a ls w a s
1Qg:- g ra m s. T h e ; : v ^ c i s S f o n oifI^:^Hrmaiisat stoj>dy
did notexceed20% of the
m eanbodyw eight
The animals were identified bycage ootaHon and indelible tail marks, and housed 1/cagein suspended;
vdredagas. Beddngwas piaCed .b ^ ^
changed at fe a ^ thiee tirnes/iyeek. Fresh PMI
(Diet*5Q0l)ahdv*ate^
Theanim air^
oh abuts testa, iaa temperature-cpniroiledj had a l2 ^ o u r fight/dark c y d ft and was kept dean and
vermin free
1See Appendix 4 for AnimalHealth Report
phone: <215)53^4110
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Study TU> P ro je c t# Protocol
LLNA
MB 07-15961.26
56S0M
P o s in g :
EXPERIMENTAL DESIGN (confd)
...............
W P
consecwttveciiBiyi^TfiesiaBntrt(ro^'*iofra
t-
29^Sfe-_.^F.Wi0__
active inorecfieni ih tha test adl C fosriiiQ ite:^
eryftetna and m e ^ m m e n ts o f eai
20 hr after the Irsi dosa and:,72 Br alter wem ira;ao9c /Or* !.... . , .................. ....... , ,,, .........
asphyxjatksa 0 h a ^ P w 3 # J b s y G te i^ 0 DPY1
e xp re s a d a
pere# o f the Day 1 p r % w Valte, -Bar thickness Changs pf are man 25% were considered^ ...
bfe.|ngjraiiy i^nH icani(bad oo hitoHc^ Isterate^ OfeVliMiliiwfic$Bvaof a primarydannai irritation
response.
nofinw iw sttiav: The test arttete concentrations used fh the definitive LLNA sfody were chosen such that
me iTtaifrnum conohtraton tasted nia# t*" * fc*-" ksm^ ki
H t*} hwa a* simniiH hv
me sponsor
Six groups of CBA/J m ice. -------------- ,, ,,.,,,...
_.
concentrations: vehicle t pooRiva oontrol to me dorstim of each earonce dafly for three oonsecutiye days
(atat&rwtibrateiym e santetim eeaehday) ek>n<^trations cif 2l5%,O0% and 100% ofthetestartrcte.
con^poriding toapproidmatoly 1 & % 1 5 % a n d 2 9 .2 % o ^
were tested. Thetst
a ^ w ^ p ^ ^ Z S p l/M a r .' AaTVe group of 5 a n in w ls i^ le ftO n tre a T ^d e fa u ltye h id e ,
Atone:01l>^-<^'^:'ri? G Q ) vifisiit-Kit:Jws^-tfci$r;.toFjtai^ vehicl has pirx^upW'Soiffeii^ineaailW-'^^:*w^poil!fsees:a*0!iilrbl;.S ^
-jqut as w tl aa with other
sensitizers and nortaehsltizers The testarticle dose conrtentraBorte w et* proposed by MB and approved ........ - i ifritatkmmreshola Thedoslng schedule lOltowsi
Dose Groups:
TestSubstance N a iv e ............. V e h ic le -<MSQ) , Positive;q4ntrd 25^M T O lb^e75; 50% MTDID8675, 100% MTDID 6675,
Anim ates . 1- , 6-10 ; : 11-15 " ' 31-38
36-40
.. 41-45
Type and Frequency o f Observationa:
All animals in the study were observed one daily m rougho^ the stedy for cjinica! s^ns, either o f tocal irritation at the appRcation sits p r of systemic foxioity, and.fbr mdftaflty. B r^y v y ^ h te were re c o rd ^ cm Day l . immedetely priori? do^ng^ahcl on Day6, prior to BrtU fnjecIBqn Bar nfreasurements were tike n
from 1animate and recorded on Days t, 3, and 6
___________________ ______________
phone: <21 8364110
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EXPERIMENTALDESGN (cont'd)
Nodo Isolation and Processing:
Oq[tay$ japgn^ateilyp to
were a dose
D j$ J *? | ^ :P !!f^ 1 m g /f| th BirdUwas adnifnfefiwW by intraperitenOal injection using a 27-
g g tijo pftaiHto T$is fty im i^ ^
inq^rppralbdtiiib the PNA of (onfi^apiig ciieiis. Including
prolffisratihg n o d a lly m p h ^e s r^ M
nddesWre iM la^(se>^>jM ndi)(D
feriyijiph in d e location) and single-cell suspensBrts were generated in Fetal Bovine Serum (FBS) arid
fixed with 8594 ethanol The cell suspensions were Used to determine BrdU incorporation into the
Flow Cytometry:
Flow cytometrtarmlys were cofKlucted ustoga FACScaoflow cytomet8r (Becton Dickinson, San Jose,
CA) equipped with ah O m nK ftrpiraargt^
at 488 nm with 15 mWo f power For DNA and/or
6MU dttontfn&tiorisi d m p s o fh u d e iw e ro e ^
gates seton integrated red
fludresceiw signals The histogramsgeo^ated iith e s e e x p s d n ^ ^
usingCea Quest
Steare<Bton Dickinson IrnrttdrtocySM pet^^Systems^^
DeterminationofStimulatlon index:
PrnUferaBon ofl;vmohocvtes:i# o f BrciU * cells); Measured aliquots o f fixed ceHs were washed and
V i m HCI Mrttainlng 0.5% Trfton X-100 This acid denaturation step allows the BrdU
antibody to quantitatively interact with BrdU that has been incorporated into the cellularJDNA Following a
on*fcour denaturadon pqdpdv & te s ^ j)je s w80?neutralized by washing:^ ^iu m te tra b b ra te (pH 8(5).
. a b u f f e r (1.6%'Bovine
^cj0^rrt:,iirjkd.iei.
(flgorasceSn^tiugteayahtbay'^
USA), The nucleiwere again
^^
eefeih^ phase}wasd^tto!^
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SOM ' ' ......
CellNum ber Determination: Force number dafenm taatidhlbn& ^
offoced
cate transferred tB a tube c ^ t^ jiir ^ propkfiu^ focfide. TW enurW ^ crfc^ls in each aliquot
(representing a 1/500 dBiiiipn o ftlfe lN C s u s ^ ^
The total number o f celis in the lymph node virasdetermined b smplini art afiquot that represented 1/500 (dilution fa c to r* 500) of the tots number of odist the node (Equation 1) To determine the number o f BrdU+ cefe in th lyrtiph hode^the tote number o L^Is counted in art aliquoto f the cell suspension w asm uddied b th e p e rto r^ ^
Equation it (Mean # ofC ete in ABquot) X (p a u p i Factor) Total # of Celts in Node
Equation 2: 0dtal # ofCells in Node) X (Bmu* CeteVK = # Proliferating Lymphocytes
The means and sfandaitfdeviations ^ Analysis ofStim ulatlonlndex Data:
dosegroup
calculated fm each group- Them ean number d pidiw rating ,l^
then
divided | y the mean h u trite f of PWifafating lymphocytes I the vehicle cgntrtf group. ((Forfae1o0%
test articfagroup, the mean number o f P ro B fe ra ^ tmphoytes iMas dhddeft by h mean number o f
BrdU* JG Wthe naive controlgroupSincebo' DMSO wasb s e d in ^ d c s e ip r p a ra ^ ^
This "Tes# Control Raffe'is th^Stimuiatioin Index!' (SI) andvv:<racUltedfef^
Equation i befew: ' '
. , ,,
Mean * of Proliferating Lymphocytes Per anted______ vehlde control group
Stim ulation Index |$ t) per animal
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Protocol
$65081
EXPERIMENTAL DESIGN (eonfd)
EG3 calcu1ation: rv*irt ^ h <rfgca; 7toecon<ntrat^
` ., SI 3 (EC3)*as calculated according to thelbllowing :
Equation.^ E3 = v ^ r e a, are the efcncentrations ofthe TestArticle and b,dareS I valuesateachrespective concentration.
The skin sensitization potencycanbe dassifted aceordingto thefoltovnngtable2^
Category Extreme
Strong; : Moderate
Weak
EC3TCU <0.1
i0.1r<1 a t-< io
fc10-Sl00
3 p ri^^C o n ta d S e n sh fe a B b n :
-
^ toxIrtiio^G feffllcate CETOf*Technical Report 87.20
'"^.**rite5dv postofficebox178, pkmriiowri, pa1M68 Page1CoT62
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Aliquots o f LNC {approximately 5 x 10ta*fls)4wereaterfirkdwith t^;;flOYsoen^
arrthmouse
3220 antibocty (B ceBs) and 8riti-mouseCD3 ahtibodyTell*) in PBSrbaseif buffer usitigi aoappropri
6ter (BD Bfosdehces) (kllsw ene then V i^hed ancl FesusperKI^I in PBS containing
7AD LiyeHs^reari^edby fldwcvtoi^^
t cefej andtheBiT celi
ratiq. The mean yatues fbr each parameter were calculated fcr-a^scti te ^ ^draMp'aiKl
to the
vehicle control grp Values for the 100% test article igrp were also comparec to tile naTvecontrof
group
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EXPERIMENTALDESIGN (contd)
Interpretatioh dfthe Data:
A test article ts considered to hwre annuitizin g pptent!ai rf t^ im e .n t results ip a 3-ioM or greater
increase in the-,Lhi<SproNfereiton-p fepwr
control. Tlierefcye. fest a r^ e s that^feld a Sl s Sare charaderized as p p te n tia rs e n ^
If atest article hasan & > 3 :sm d lsi# ffte a n ^
m m U hdphertypingdate^^
Ytfietherthatfest arficteisa sensitizeror irritant. For
:knliiitaunQ^eiib!|)DP^^
re ^ o if> ,l 26-fold i>25% over vehidex^ntroi) are
considered predict!ve o f a to e sensitizer
Retention o f tire Date:
Upon signing; the final reporValirew data, supporting documentation and reports aresubmitted to the
AixdiMst by tite Study
The raw data fe fflediat MB Research by project number The final report
9filed eiM B ReaeaTOhi^ sponsor narne and MB project n u iitf r
The test article will be returned to the submitter following submission of the report.
Amendment of the Protocol:
wasEC3 <alculated for the,:te^ a ^ e and lrK:lyded in thfr report The EG3 value is a representative of
potency SeeAppendbr L for protocol in ftsentlrety
Deviations to the Good Laboratory Practices
Although complete,test articte cn ara^ tnlttetibn, information 6ntodidetotity,sfo
hot provided to thestudy director prior to study purity, oompdsition and uniform i^ was provided In
addition the te ^ artide :chairacteflzatiim, p ro ^
was not
conducted uncteir ihe G c ^ . U bora^#PrdC dcas, Becaused fth e short duration, the information provided
phone: {215) 536-1110
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StudyTftle Prefect# Protocol
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'
: N&97459B1.26
: 5S50M
RESULTSAND DISCUSSION
Test A rtide Formulationand Dosing: The test article formed a.homogenous mixture Itifo e vehicle, DMSO No cHffiwlQgshirers rppdenced with the application o f the test artices to ie ears or wh foe retenifoh o fte d a H ^ ^
Body Weights (Appendix A):
Body weight changes were normal, extopt for the animal in the 100% dose group, which lost 1.4 grams
and appeared ^ a c ia t d " ' '
"'
M o rte titya n d S y^rn lcO b se w a tlo n 8 (A p p e n d b t B):
A il animals survNed foe viite phase of the study and appeared normal, except forone animal in die
100% d c^g ro u p .V ^ich a p p a ila em aciated,ai^
D ayfloifthe
deffoltive study. This enirrai could not be injected vfifoBrdU. so lymph nodes were not harvested- The
signsobaervd fa fob'animaLare not believed fo W te s t a rtN ^^
group appeared normal
'`
Ear swelling (Table 1, Appendices C & D):
A preliminary dermal irritation screen was performefoand ear s w e iili^ measurementsw ^rp used to
assess irritation potential (See Appendix C for data and Appendix OA x digram ). Ear thidcnsss
measurements were performed on Day 1 prior todpsing; On.bay 3 before the third test article application,
and on Day Sfceforesaoffica None ofth e concentrations tested resuilsd. ^ increases fo ear thickness
greater than 26%, andtherefore was rrotc o n sid e i^ irRtetfng Based wd ^ ^p re lim in a ry dermal
irritation screen results and test artfctesolubjTitydata* concentrations o f 25%* 50% .and 100% of foe to s t'
a rtfcie w ife used for foe definitive local Lymph Node Assay., These cancentnatipns corresponded to
approodmately T S ^ 1 .5 % -s n trii& d % c f * e - ii # ig r^ ^
\"
inthedefinfovte stody.none of foeM tD ib ^ S s , , :
' ooi^entrations tested resulted
in dermal irritation. The positivecontrol25% MCA, had agreaterthan2556;increase in ear swelling on
Day 6, which is aaisistent with the literature hteioricaJ results and based on otar general experience
occurs in about 90% of studies When using 25% HGA In the vahScle OMSO This resuttls considered
miadiy to moderately irritating
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Table 1: Ear Swelling
Treatm ent Naive DMSO (Vehicle) 25% HCA Positive Gcntrol)* ....... 25% MTOiD 66:75. 5d% M TB iD 6675. 100% WTDID 6675V
M ean!EarThiekneeiilmnri) Pre<tos(ng 48 H r
iD ey-l); (I* * ? )... (0 **6 1
0.18 0,18 0.18
0.19 0.20 0.20
0.19
0:24
0.26
0.19 0.20 0.20
0.18 0.20 0.20
0.19 0; 10 0.20
Chan!re/% ) D ayS -
o iy 1 Day 1 0:0% 0.0%
5.3% 5.3% 26.3% 36.8%*
5.3% 5.3% 11,1% 111%
0.0% 5.3%
a s a lest substance (eadKng to increases in ear thickness of greater than 25% Is considered irritating considered niifdiy to moderately M ating
phonw
1765wentiroad, p^1orn<s*b6x178, spTmMnrtMm, palSsSS .Pag14ofS2' '
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;
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RESULTS AND DISCUSSION (eontti)
Mean Number and Percent of ProUferallnfl Lymph Node Cells(Tabta2):
The total n i^ lw o f proliferattog lyropho^tes v ^ calculatod t^ m uitif^biatotai cell nurnber by tho
--
. , The mean total lymphocyte. prolifwatfon for each of
Table 2: Mean N um berofProG feratingC ells In the Lymph Nodes o feach Treatm entG roup
Treatm ent Naive DMSO P/ehfcleV 25% HGA iPosftive Control) 25% MTDID 66751 50% MTDID6675. 100% MTDID 6675.
MeanLNC %BrdU+
. Vehicle (xIO*}
LNC
. - * , 1463
036
Lymphocyte Proliferafloh
<#BIU+V
5100
2560
0.61 " 41929 `
DMSO 12462
1.48
183010
DMSO
798
1 2 7 ' : 9824
DMSO ...1193 2 3 9 28365
DMSO
1281
3.80
45277
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RESULTS AND DISCUSSION (conftI|
Stimulation Indices werecaicuiawa ay wviuinyic
iw S
animal By the mean nomber o pmHfefaSng ly m p lfo cy te $ in tb e ^
test
ade atuP the total lym]iocyte proUSNrstteowas dWded fc^thato
9 toum
the SI forthe10Q %
based on the vehK^ control ^ u p
As theiKlTvegroupis the control fortho 100% test arttctegroup, ttwcajculsted SI forthtegrpup wasl^
The mean Si ndstandard
for each
calculated (Table 3); An SI a 3
indicatesapotenttal sensitizing response
graph)
Table 3: Stimulation Index
Treatm ent
.... ................,--
Nah/A
__ ________ _
OMSOfVehlcle) 25% HCA (Positive Control)
.. .= .......... ......... . . _
25% MTD1D6675.
....... - - ........
50% MTDID 6675, J 100% MTID 6675.
(fold to naive)
100% MTDID 6675..
. .. (fold to vehicle)
SI 1.0 1,0 15,3* 0.8 2.4 8.9* 3.8*
S.D. W-0-6) (+/-0.5)
(t/r.0.1) </-14) (+1-5.6) W-24) '
a SI 3 indicates a potential sensitizing response
1000Statisticalevaluation ofthe calculated Slvalueswerftm^
SraphPad jnStat^,
c1990-1I D99S4'! v''2*.05a+(SeeAAMpApaenn^dtvixnl) However, <<faformination oftestarticle denial sensitizationwas
tb^assBedd oonn the criterion that test rtidesthatyeld a Si k 3 are characterized as potential sensitizing
Substances
EC3 calculation: The EC3 was calculated to be 54 .8% This corresponds to a concentration of 16 4% of the active
phorrt.: (Zjl 6564)10
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... Prolbcct
it p
.
RESULTS AND DISCUSSION (confd)
Immunophenotyplng ofLym ph N odeC eite-B and T cells (TaWes 4 ..Appendices G & H ):
TTw %B cellar %T cells, and B:T oeU were detemiRed by flew cytometry ae described*! the Experimental Design section of Hib report. Gowp means and standard deviations are tabulated below. (See Appendix fof indlvWuafdata and Appendtic H for g r^ rte )' ,
T a b le s
% CD3+ (T CSelb)
T reatm ent Naive DMSO (Vehicle)
Fold % B220IB Celle) compared % CD3(T Cells)
Mean &D. to vehicle Mean S.O.
13J i*M ,6)
f--i,
70JS (A 57)
_ TT.8 <*/.2.7> ' mm*' , ' 73.0 (**4.0)
25% HCA (Positive Control)
27.1 (/2i8) 2.304 5 8 3 W- 1.5)
25% MTDID 6875. .
123 M m , 135
73^2 (+A3.8)
50% MTDID 6675. . 100% MTDID 6675,
100% MTDID 5675.
... 143
. 1,22
ifold to Naive) : 22.4 (+/-3.0) ...........
(foWtoVefitele) J & L *.M m l
1.62 1.91#
72.9 <+/28) 6 2 3 (*#-43) 6 23 M- 4.3)
# sgreaterthan 125<qW(a25%incre3se) ow\mhiclecpnSols pp#iensliiz&^ response
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M B Research Laboratories
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r e s u l t s a n d fts e u s s io N (cont'd)
T ab le 5: B:T Lym phocyte Cell Ratio
Treatm ent Naive
___ .
FokP com pared -... Mean s.D. to Vehicle
0 20 (+A0.O9)
0.T8 W-0.06)
S8 ' i: I1
25% HGA (Positive Control)
............ ........ 0.46 (+/-0.0 2.84#
25% M TD ID 6675.
........
.. 0.17 f+A0.03) L04
IU1% MTDId 6675:
.
,, ... ... 020 ui-or n 1.20
100% MlTDlO 6675.
{fold to Naive) 0.36 (+/-o.on 1.79
100% MTDlD6675,-1 .................
(fold to Vehicle: 0.36 (4-A0.07) 2.21#
#m than 1.264btd (*26% Increaee) owvehicle control = positivo sensitizing response
The test article etidteda fold iocreaseln %8 oeRso f 105 at 25%, 1.22 at50% ^nd. 191 at 100%, oomoared to 8ie v e h k ^ coritroi^ and an increase o f 1.82*foid at 100% compared to the naive c ^ tro l I S S ^ S i f S r S i d v v e w p la t , 120 a t $0%, and 2 21 a t 100%, compared to vehicle, and
179 a t 100% compared to the NaiVe control
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CONCLUSION
Topical application o f the test article MTOID 6675,
. . , at 100% (2^8% as the
'aceyarlnoinwMBne-
Iw -^StCS-s^S as weD s a >25% increase in %B
celteand In the B:T ag a ^ ^ r e d ip both vehiaeahd naive controls i^a re fp ie . ^ is t^ a rifo i is a
derma) sensitizer in Locai! L y rn ^ Ndde A^say The test rticfe has a calculated EC3 value of S i 8%
(16 4% as. the activ indredieht) Based n ECETOC criteria, the activ ingredient in MTOID 6675,
/cahbedassifidasaw ek sensitizer
FINAL REPORT
Approved by;
:PlsJj'QO`r.
MelissaKirfc, Ph D Study Director
- .....istiiaitiwps j p!ea^fiiiB#aeat'^T.^astowwwdhaws^ we*
Phon8: f2is^$3fr4110
'
c ' r <>
' . "'
PagelSottZ
.........
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f f ^ y ^ noiaJ9uu|ds
^
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ss
so m
t i _____m ___
' 20- "
tr`QZ,
S I 22
S i 8 02
s i 622
e i 2
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6'ABQ
s 01 02
902 602 SOI S i2 re i
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#
OS NV3W
a ' Si " :d S d Si d 21 .
-Amm. SL----xs # V
jofluoo OABieod -VOH %92
s 0 si or 02 si 61
i >0 seti* m a
s
ro 122 SftZ 2E2 S i? 612
*22 0*90
S i S02 581 212
oe 002 fTZ
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as
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ir 21 01 Si JH. so fi
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s i 112 SZ2 822 112 81 .Sftfe .
9*8fl '
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9A16N
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W0S99 : |ooo*aid 9ri96S^2Q8A : #Wf<Mdl _______ wn : sMiApms_______________________^ ________
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Appendix A (confd): Body Weight in grant*
25% MTD1D6675,
50% M TDID 675,
An.# Sett D ay!
11 36. p 4 M
37 ; F
186
38 F 167
38 F 176
40 P 21.5
" ME3JT"I B
-S0., 1.8
V .5
0 ay6-..;. '
4.5 21.1 ' 190 19-6. ,
22i4.42- '
21 ; 5
B.w. Change
2 2 23 20 2.9 24 oi
5
100% MTD ID 6675,
A lt#
42 43 44 45
:Sac:'
F F
F F F MEAN SO. #
DW1 281 210
200 199 20:8 0.4 OS
5
**. 2JL6 1 198 2L7 208 22.1 2t;o ' U'
8
&W-Gtiahge
34
-.4.4:
17 10
13
' ' 12
if
"
5
phone: (216)$384110
'' "
`
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'; Pag21 of52 .
-*
....... ftod (215)53$-
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StudyTiUe : LLNA
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Protocol : 56S0 M
AppendixB: Systemic Observations
TIME p e r io d s ; UP
1 2
Da3
Naive
T N I M Ai 1 ... # / S E T
2IP m
m
5IP
uD..a.y...6... ...
TIME
1
PERIODS; 18/F
Da1 Day;2
Day-3
Day 4
DayS
Day
ITIME-
Oy 1 Day 2 1Day 3 Day 4 Day5 Day 6
I
/F
DM SO^Vhide
A N I M A L M I S. E X
7/F.. - BIP
9/T
25% H6A Positive Control
A N I M A L #/ SEX
.. 12/F
13/F
14/F
'IIF 1F
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Studytitfe r LLNA g n M i f : MB 07-15961.26
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Appendix B fconfd): Systemic Observations
100% MTDID 6675, MTDID6575,
Noentry [ndicatesarumal jsppearednormal etthatoteonrafion period
R =v^bgenHalarea wet
W=
1 = Red ^bstanee^atihjectionaita
__________________________________
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Appendix C: W itiary Deirrial liirfjEatiort Dose Ra^g FfihO^ (E^r Sh(yMlwa Soreer*
Treatment 25% M TD ID 6675, S0%WrrblD.B675, 100% MTDIO 6875..
....Mean BarThickness(mm)
Chant\* m ...
Pre-dosing [ 48 Hr End In-Life D * y 3 - Day 6 -
(P a y ti (Pay 3).
day t Day i
............. 0.19 1 0.19
0.19 0.0% 0.0%
............. .
0,20 | 0:21
0.21 5.0% 5.0%
0.20 I 0.20
0.19 0:10% -5.0%
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Appendix D: Location o f ^Di^nlhg l ^ p h Nodte
Ear thickness
measurements
B*s*ofwr
Jvgoter bttiircotfoo
Flginrc J. la te ra l ylew
General location
ofnode
From6. Saftrtadln TheMurihelotali^T^ Cftem<i^a<Comp<K^,NlSilS*ryilCC^^<994 "
M e^farM sassirigm A lbr^
phone: {216)5364110
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1*1*86
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..................
____________ ;_____
Study THte : LLNA Project # : MB07-15961.26 Protocol : S6S0M
Appendix E; Stimulation Index
Treatm ent Naive
Total # Cells
l Anim al# IriN odejrtO * % Brdtl+ ProfttBratlon
1 1486 021 3120
' 2 ' 463 0 48 2220
3 2658 0.39 10366
4
1435
031
4449
. 5 ... . 1273 . .. .042 .
5346
Mean
U&
J5W
5100
StDev
785
010
3179
SI
0,6 04 20
09 1.0 1.0 0.6
D M 80 (Vehicle)
6 2608
7 2368 8 3829
9 641
10 3352
Mean ..... 2560
StDev
1220
031 052' 056 079 0.38
051 019
8086
12315 21440
5066 12739
... 11929.... 6187
07 10 18 0.4 .11 1.0.. 0.5
25%HCA {Positive Control)
11 12 13 14 15
Mean StDev
12012 15215 12142 11614 11325; 12462 1673
138 1 37 180 140. 1.41 1.48
0 18
165766 208446 213559 162598 159683 183010
28146
139 17:5 183
136 13.4
15.3a 24
25%MTDID 6676,
31 32 33
34 35
Mean
S tD e v
771
665 711 1076 766 798 162
t.46 1.51 1.64
086 1.44
127 030
11407 10034 7364
9254 110.30
9824 1600
10 08 06 08
0,9
0.8 0.1
50%MTDID 6675.
36 37
38 39 40
Mean StDev
2298 601
1228 469 1371
1193 729
237286 226 211
2,33 239 029
54451 17309 27742 9.885 31938 28265 17010
46 15 23 0.8 2.7 2.4 14
a=Sfe3
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Appendix & Stimulation W doxiconfd)
Treatment 100% MTDID 6675,
TbtaJlfCeile
Animal# lnNodex10'
41 990
42 ' :2
43 1962
44 1911
45 242
Mean
1291,
StQev
827
%BrdU+ : 8.19 2
3.38 372 4.91 30 078
.
Lymphocyte Preliferatioii
31689 2
66578: 71071
11870 45277 28416
SI 82
t 131 13.9
2.3 8.9a 56
a 81*3
Z /tfilmal#42was <Dosicktobe injected withBfdU. KwaesaccifioedM^ token. ' .
group, butnodestwerenot
phono: (215) 636-4110
1765wantzracd, postofficobox178; spto^Mtowi^*18*58
Paso^<itS2
fax: (215)5361816
9ttt*9es(SK)
noni ed `UMOfrXHflKtem k **t P l ^ ^ 'pewfruaMS9Xt
0U?-9S (SIZ)
Occupo aAgisod)
vbH%c (w <w \) o rn a
Stlmulatan Index
* \z
siinssij AwsvepoNqdiuXi (iKxri
usw eaJi 'SA xpui uoije|nui!is y xipuaddv
WOSSS : iooiaid 9?l96$l~0Sl i P^OJd
w i i : eniiL Ap b is ___________________ ________ __ ________ --
-- -- --------- ---------------------- s e jJ O iB iO q B i i|O je s s y g w
aazu iN vs v o s i
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Appendix G: Immunophenotyping --B am i T celts
Traabnent N a fre
.
1 Animal
1 2 3 4 5
Mean
1243.. 21M 13.38
9.26 1258
1357 4'ST
%T 7443 6154 6812 74.56; 73.79 70.49 567
B & fta U a
0.17 035 020 Q12 0.17
020 0109
DMSO (Vehicle)
6 7
8 9
18 Mean St Dev
1021 925 1144 1626 11.71 11.77 270
78.95 74.91
68.12 6750
75;58:
7257 5 05
013 612 0.17 0,24
0.15 0.16 005
26% HCA (Positive Control)
11 12 18 14 15
Mean St Dev
29 72 2646 2340 3010 25.59 2705 2 84
58 78 6619 6041 58 32, 57:65 5857 165
051 047 039 052 044 046 0,05
25% MTDID6675,
31 32 33 34
35 Mean S t Dev
1248 11.17 1484 1240 10.84 12.35 1:57
7015 7283 6887 7598 77.92
73.15 3,81
0.18 0.15 022 016 014 0.17 003
50% MTOID 6675,
36 37 38 39 40
Mean St Dev
12:47 : 1365 1532 1359
16.54 1451 1 61
76 42 72:02 6883 7398 73.30 72.81 2.79
016 019 022 0.18
023 0.20 6 03
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Appendix G: Immunophenotyping - B andT cells (confd)
100% MTDID 9675,
41 42 43
44 45 Moan S t Dev
25.87 . :k
2054 23 93 1&4422.46 298
61.83 2
0 85
5695 .'m M 62.67
427
042 Z
031 0.42 030
0.36 0 07
nodeswere nottaken
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Appendixl: Statistical Analysis
Study Title : ULNA Project# : MB 07-15961.28
Protocol : 5660M
C om D arisonO fS lvaluesbvS tudent-s t-Test
DMSO(Vehicte)vs; 25% HGAiPositive Control)
DMSO.{Vahfcte):vs: 25%MT01D6675;
DM80 (Vehicle) vs. 0% ^
Mean .... Difference P Value
.... -14.340
..................... -0.1800
ns
................,, -1380
ns
Com oaroon o f S lva tu e sb v S tudent's t-Test
Naive vs; ld b A MTDJD6675. .. ......................................................
Mean: Difference P Value
-7.895 * * *
* * * = statistically sighi#icant(999% ajnfidence) os = not statistically significant (p>0.05)
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fax: (216)536-1816
CCOO Q.
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S: $
Foradditionaldetaitsregardirtgour health
$9> !
25 | oa0*C*:
G :S
25 1
p it
r-
o3'
1
C
Ift
8
I
a
0
1
*!*
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p
CO 4^
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Appendix J
MB 07 1598125
P a ra s ite s N Q P Q S ftW E R E ,l.T S ;
Aftho^shmostofUfesorgan^ in inostfacSities Ifany of
in lo rfo re v^r sa ^^ from mercmraresuspervdedan^
unacceptable notified
Organism
..........
Fleas. furmite*, lce
.
FoIScferites
......
Pinwomis
Roundwormsand other helminths,
Tapeworms.
.........
Gncophatiozoon cunicuf
.
Sample fsted
-Test; MerpoO
..
;
--'M r.H iir
Fur. ' ' Visual: . ............ M S
s i c u a s .y is i^
.... m s
Cdcum Visual
ms
tnteStihe ., Visual
MS
Intestine Visual
was?
Serum CLISA
Dec` a i epJjDk
0/06 m e m $ om o/os. .....caw < m fl VOS MS G f- , - i.
Jan`06
0/06 ww MW? M>6 vos 0/21
.......
O pportunistic organism s --M O PO SfTIVE RESULTS
These organismsarago<ally rcgfiie<} as p p d u h i s t f c ^ ^ con^iate dlsast oriotertena wm.re^afch under certaKtdreumstan.cas,g-, rnmurwdftfdrr*. thty nottoleratedin;^
mfce Shippingffmtheanoai$ii<rtstoeoed.
'
O rganism ,,
.......... . ' Sample -Test ' ' ' ' "
.......
,7 ^ ::. ..'M iigd'.
'
M * r `07 e c W
BafdoteHabfonc/saptica
TreCheOCdlture . m s
Cotya&bacSriom.kutscheiT .. .. Trachee, Culture ... .
HefKobacterspp
intastin nrtfy " '
or feces
'
~ ms
<M06
0S 0/06
Klebsiellapneumona ,
" " - Trampa, ...... intestino. Culturo:
. ' orfecs .
, wo?
W06
Murine norovirus(MdV) . , : ....... sw tim ...... e l s a ............ ,
Pa$te?lfprttmofrOp
. ... Trachea Outturn . ..
<W20t <wo?
<W2 0/06
PreumocysOscrini/
Lung
PCB .
......... > . .
'
Pseodon/onpsspp..
Infestine r feoes
<wo$
0/6
Slapitpococeviautans
Trdfm Outturn ......
m s W0
Simptooocousspp.
^ Trachea Cultore.
M s 0/06
Oppctnlsiicprotozoa:
. Spttondevs)
(e g
, $$nf*>
tntestjo Mere
m s <V06
Sep`06
om
0/6
0/6
Jan` 06
0 /0 6 :.......
0/06
0/6 ..
M>6
(W0S
. ;-t:
fl/06... (W06. .. ` . . ..
<vw M i
M
<m
0/06
0/06: .
0/06 ' ; 0 0 6 .
-
O th e r (R o n p ath o g en ie ) o rg a n is m s P o s itiv e r e s u lfc fo r lc r g n is m
Tfies orgniscnsaregnerai^.Vteaptfs nomralflora fhifeof asexportons
disease Or intocfote yrtth rosit^ Ifariy of these organismsare ^^hl'lih
shipments am noilsuspended:
' . ---
niatki ni ih(|Nrt;bat .....
O rganism
'' Sample Test
-
Tested Method
' .Mar *07' Dec <06
Klebsiellaspp..otherthan K. pneumoniae Trachea.
.
. .. '
intasine;. Culture ' . o r foce ': . ..
,
3 /06 .
Nonpathogentcprotozoa (e g ., trichonwnadst
intestine ttere- .. . . ' . _ , M S . / /$
S ^ `06 4/06 0/6
i
/ l D vi
-
..... __ '
.J . yr "
Jun *66 2/06 (W06
Peggyi. Oannemao. VMO, MS. DACLAM
TO e Jackson tb o r a to iy
AREA: MP-15
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Appendix <J
MS 07-15961 26
Animat Health Report
R eport #0507MP15
OVERALL HEALTH STATUS
(rEdxpcoeslilteivnetresults)
{SKujppeatr^tteM.m s, parasites oroppcrtunwto)
O(nGo opaotdhogens or parasites-)
MP-tS IS OPERATE) AS A:
Maximum barrier 0 High Barrier * Standard barrier Q Research Standard barrier t.oVr bamV
MP IS IS IN: Production Facility
Q Research facility
Q Other Area*
Organiste EGVcIPRtrVUImIS(EeTlSihVlteirraSs) ytms
Sample.
Tnated .
Mr eest<hod
Serum
Swum
EBUUSSA*
Haotaaovms:
Srum 'k i m '
Kvirus
.Serem ELISA
Snrtn ; Engyroe
~Serum EUSA.
t^feehmnuteyire$(MMV)
Srum ELISA
Mouse L>tO n^alow i^(M C ^
Serum EUSA Srum ELISA'
......r Serum , ELISA
" MMbuu#s;&patfrVtCn>fevvtKtriSttS1M(MPTVV)
' ' ...Serum ELISA
Serum 1FA
flf^riyeourtariafav^irus ~Reovims3IR3K>3)
. . Sttim Srum
. .Swrn'r
ELiSA EUSA EUSA
Relaying IBPIM)
Serum , ELISA
Settclalvims
' ' Swum EUSA
SACTERIA ft MYCOPLASMA
C/Uttw ife " ' '
Si
Myscpfasma pu/mows _
Salmonspp aosttviim pfornf
. TSrearuchmea
'
"
Serum
dInetfeasctiense
llrtstiiie
orfeoes
Serum
ELISA
Culture EUSA
Culture
Culture
EUSA
.
6pT10ah80re0.HJM0a4aadr2icn2soSMor,ffriMIeGlagEbt0o(4r66a4t02o93r)y
Page :3Wof 52
May W Fb `07 N ov`0 Aug *06
0/21
0/21
0m/21i
m i dm
0/10
mi 0/21 ' m i mi mi < m 0/21 m i G1 0/18 m i om
,-e
- , 021 XW21 0/21 . 0/21 ' 0/21
onx mi om
'm i
mi
mi mi
0/21
0/21 mi
mi
mi
0/21 . oa-i
0/2-1 ' 0/21 ,,
0/21 .
0/21 0/20
0121 ' '
... 0/21
021 021
0/21 0/21
0o2m1
0/21 oo/mii
0/06
m\
m001/02i16.
0/06 0/06 0/06 0/06
am 0/06 am 0/06
om "
0/21 *
rFnoo>n-aitdodriitniognparol dgeratamilasnrdesghairpdpinbgToguprohliecaylth
eeh itp;//fom ^^
TSCA SANITIZED
Appendix J
MB 07-15961 26
Parasites - NO PO SITIVE R ESU LTS Although most ofthese organisms have KattedpOfenSatto caus disease o r interfere witH research^ theyareurracceptable la m c^fa cititl s;. if onyiof trese o 9 S *^ re fo h ia li^ p W ^
Organism
.
Sarhpfe T*st fest! ... Method
Fleas, ter mits,iic&
Fur Visual
FptMernites .
. . ' 5- " ' ' * 5d#sufe ' Visual j :. ,
Pinworma
.... ................ ' ' Cecum ' Visual
Roundworms and other hetmaithsi
Intestine' Visual
Tapeworms ' ' ' ' . . ....
lritesfirie Visual
EncopeStozoncuniouli
__
Swum;...... E LIS A __
M ay` 07 0706 <WK M W6
'
Fcb` 07
<V06 om am am 0/6 0/21
Nov `0$ ug.*.:
0/06 0/0$ om
m 0706.
0/0$ 0706 0706
OWW ..076
0/21
O pportunistic organism s - NO POSITIVE
Theseorganism sare generally recognized as opportunistic pathogeny that may com plicate disease o r interfere w ith research under
certain circumstances, e g , h m w n o ^ R ^ t ntc, T h y ra itp t;t ^ ^ investigation dsignedto Bmlnate a ll infected m it ; SWppirigfrom tha area is notstopped
- p o d ^ --ra * a .* e rs . 6 ^ ...............
Organism
Bardatone brondiisoGce............. CdfynBbacterium kutsctir
Samplo 'Tsti'- : " '
. .Tested . Method
'
Trachea Culture . Trachea Culture ...
May*?
0706 ~0f6
He/icobacterspp
.S S
' .."
. s/06 ;
Tracha, ... '
WeOsMapneumoniae
intestine Culture or feces * . . '
0706
Murine narovirus(MW) Pastoureilo pfaumoimpkxt
Sertmt . ELISIA . . ........ M i " Trachea Culture . .. 0706
PnanimocvsSscarin
' Urna - ' PCR . : ..
' - . '
Pseudomonas spp
Intestino or feces Cu,turB
[..f 6/0$
Trachea Cuttur:
0706
Sbeptccusspp.
. Trachea Culture .. '
0706.
Opportunistic protozoa (eg,. Giardia, SpirotctaoS)
Intestete Mero
0/66
Feb *07
o m 0*06 o m 0/06
.0/66 0/06
Aag^` 06
0/06 0/06 0/06
o m 0/06 0/0$ o m 0/21 , o m 0/06 o m . et
0/06 /P6 0/06
o m 0/06 0/0$ 0/06 0/06 o m
0/06 ; 0/06 r . 0/06
*......... 1
- . ........
Other (nonpathogentc) organism s P ositive re su lts fo r 1 organism sOOUoilJUCKHHytnismswiUrttle or no jjpteritial to cause
cfiseaSer InterferewHIrresearch Ifany iof thesoorgonisms re foundiri;any IAX mouseh ft findirrg Isnotedfrithis repori, but shipments ararne suspended:
Organism
Sample Test Tasted . Method
Trachea,
Kibsiita spp. otherthan K pneumoniae Ihtesiihei Culture
orfeces
i.. -
Npi^thogenic pmtoza^eg:, Wchomohadsi
intestine Micro.
`.May.*07 Feb` 07 Noy*06 AM)6
" ..
...
-2/0$; V WDS
/06 om
2706 0/06
1/06 0/06
,
-- :--------PeggyJ- Oannman, V)MD, MS, DACLM Sr. Director, Laboratory Animat Health Services
n. ,
' , '
` .. . ..
^
Th Jackson Laboratory
600 Malo re e ! B arH artorj ME0460& 1-800 422-MICE(642)
Y` ;
Pape 3T o f 52
For additional details regarding o u r health iiyiiHuiUKj lA y y id iii o|w ontppKi(|policy sebnpWiaxrnkMaxx>n^ftaOfi
TSCA SANITIZED
Appendix J
M & Q M & e i 2$
Animal Health Report
AREA: AX-9
Report # 0607AX9
Check out web site for recent updates to tbis report http://iaxmice jax .0r9/health/axd pdf
OVERAU:HEALTH STATUS'
--------------------------------------- ------ -----------------------------
E x ce lle n t (no;positive issuits)
AX-9 IS OPERATED AS A:
i S u p e rio r
'
, parasites cxoppCrtunists)
Q:(nGoopoadthogens or parasites)
Maximum batrler High Barrier * Standard barrier Cl Resear ch Standard barrier Low barrier
AX-SMS IN:
Production fa c ility
Q Research Facility
Q Other Area*
Othara ^ fioi^sfflail pr^u<^c^-bi^ i^ordieslrrtaimaihed as in-houseresatifcesorsources (Swordemand micefordstnoutloa
Mouse pathogens - NO POSITIVE RESULTS
u
------------ s j r -
tef taletfnce wfth research
Ifany oftheAeorgapems arefaund,foanyiAX mouse, attshipments iipm me rppmaresuspertO^eodiktstometamarie:
Ot0` " em
.....
.. VIRUSES ' .... ' '
' .S S S "T S S I T
' > * M a r`07 M e w ^
* ---------------- ----------------------- -------- ---------------
6ctrorneavirus
Gpvii (Thaller's) \Aus Hantaanvirus ....................... Kvirus Lartlnd^ydrbflenaseelavallnii virus Lymphocyticchoriomeningitis (LGMV) M6usominutftvitus(MMV) Mouseadrmovirus (MAV)
MousecytqmegabViri(MCMV) Molise hepatiUsvirus (MHV)........ Moui&barvovirtfS/MPV) . Moosethymb WiU5S.(MTV)
Serum: aiS A ... Semm ELISA ' ' ...Serum ELISA ' Serum: EUSA TMScrum ' " ErttymO '
' SeUwii ELISA " ' Setups. . EUSA.........
_Sefurh ELISA Serum ELtSA ' Serum EUSA ....
Sbrum EUSA serum FA
.
: " "
"
c m """ cm cm ' ~
--'
~~cm ~ cm
'- '
c m ... m i"
`'
,
m b ' 0/21 "" ~ 0/2T ...
0/20 0/21 Q/2J
one ~ 0/21
cm
0/21____
b/Ib~
-ITT"
~~m C
......
c m " cm
Mi
one mo
"
__ j m ~ Mi "
m o. 121 M l
m o __0/21
0/2)
0/20 "
cm
Eneumari&Vlo^ Polyema.viru.s
Serum ELISA Tsarum ` eusa7 .
cm " om ~"' '"~ ir
Ml " Ml " "
Raovirus3iRE03)
Serum ! ELISA
c m .j. .fctl
RotavihisfiEDlM)
Serum EUSA'' " ......
cm m e m i
:Seridaitfrus'.................. .
..., S6rum' ~ eusa;
c m .... a m
M l 0/21
1
1
i
I?3 i
""
"" '
BACTERIA &MYCOPLASMA
car baefflus
.
SlreplcfcadUusmonilifomls
h4ycoplastjnapulmonis
Serum EUSA Trachea Culture ... Serum EUSA
0/22 ' ,, 0/21 n`""" ........ -- 0/06 0/05 0/06 0/06 0/22 0/20 0/21 0/21
Gitobaiiey rodentium
22
...... o/os 0/05 0/06 0/06
Salmonellasw Clostridium pH/brme
S S e Serum: ELISA
c m 0/05 0/06 0/06
'
0/20 . .
Ml
6t0h0e MJaaciknsSotnifeleatboratory
BarHarbor. PEQ46Q9 i-0OO4^lifGE(M23)
Page & Of62
rFtorraitdddriirtiisojnparlodgeratamilsanredgsahrdipinpgin,gouprohliecaylth
see:htto:/fiaxmioe;iaXdWhbalthi
TSCA SANITIZED
Appendix J
MB 07 1596126
Parasites - NO PO SITIVE RESULTS
AKhough most ofthese organisms have limited pdtenlial ftycause disease or Interferewith research, they are unacoeptatye
Irt mostfacHliit. ({any ofthe oi^irtiisirfi are;
th Ibom S^'fBtH^peittSeKltWMIx^ie^nKnsare notified
Organism
_ IS L t
* * * `07 * * m
Flaasrfurrntte8,llce ... , .. _
^
. sfarei , .........0/05 . 0/P5: 0/05
Follicle mites
" >..? ' '
Stibotifls ' Visual _ : 0/06 l '~ Q/05 . 0/06 .'.
~~Pn^rve .. " T I "
Ccuffi Visual ~ ~ ... ~ 0/061 0/P5 0/06:
Rooridwonrtsahd otherhatmtnthS ......... Intestine1 Visual___________0/06
6 /0 5 __ 0/06
"Yapewftts
"***. ........ *
Irrfestbe V is u a l_______ 0/06
Q/PS _ 0/06
" EnoeohelHozooncurcult
_;Sotwn gU S A ........ ... O fli. .....______ 0/21
0/06 P/05 0/06 0/06
0/06 ~___
O pportunistic organism s-- NO PO SITIVE RESULTS . .. i:
ThSaorganismsare generally rac^te^ M io ^
mayxnBlcafe d&esasedr Inhere vhresirch Under
certain cireumstances, , lrwTHjnod^pienl mice. Th^are rK>ttoteratod In any Production aroa - pttltlve reslts triggeradncat
investigation designed t^raiinetealj trided fiifc:. Stppirig from the area is ndtstopped
1 i 1
I
Organism
BordeM lebroricflseptica
H elicobattefsQ p
KlebsieBa pneum oniae
Murine riorovlrusiMNV)
Pastaum ltapneum otiotiica Pneumocystis carino Pseudomona? spp S tao h ilo co co u sau m is S lre p to c o c o J s s o o .
Op|x>^i$Ua irotzoa (eg.,
S rik sn tB s
.
Sandia Test.......
Testad Method
.
J n n *67 IVar *07 Dee.*06 iSep*06
Trachea Cultura
.... 0/06
0/05 0/06 0 /0 6 ................
Trechea Culture
0/06
0 /0 5
0 /0 6
0/0 6
Intt^rer o ro orTetes . Trache,- . ...
intestine. Culture
0 /0
o/o
0/0 5 0 /0 5
jtt/06 0 /0 6
0 /0 6
0/06 r
orfeces SeriaTrachea
lung
ELISA Culture
PGR:
mi . me
.' -r '
0 /2 0
0/21
-
me 0/06
0/06 ;
. . -. . ' . t . V
_ '
a s ? '"
Tracha Cultur
Trachea Culture
0 /0 6 0 /0 6 0 /0 6
0 /0 5
ms
0 /0 5
. 0/06
0 /0 6 0 /0 6
0/06
0/06 0/06
intestine Micro
Q/06
0 /0 5
0 /0 6
0/06
Cither (nonpathogenlc) o rg an ism s-- Positive resulte fo r 1 organism
Thesepraanisms are generally viewed as rKtrmal flore dti^ce oras exbgriosmteroiaarSsmswith titaor no potential to cause
disease 6r lnte^ere wi(h rM.srch tf^ y othe^ e^artisms arefound l/reny.jAlXmcuse;the finding Is noted in this mjoort, but
stiiprnentsarenOtsuSpridc
1
Organism
S2? *L
* `7
Klebsiella spp other than K.. pneumona Tinratecshanae. Cult.ure ' " . . . .3./6. 6. . 3/05 3/06 4/06
_____ ' "
___ or feces
...... ...... ' ' ' '_______________ ____ i __
' Nonpathogenicfwotozoa Cegr,
intestine Micro
Q/06 0/05
0106. 0/06;
trichomonadsl - _ _------------------------ ----------------------------------____________________ '----------.------
PSregDgiyreJc, tDora, olanbeomraatno,rVyMAOn,imMaSl ,HDeAalQthLASMervices TC0h0eJMaaciknsSotnreleatborat,ory B1a8r0Ht*a4r2b2o-rM,MICE04(664023f) .
For additional datsBIs regardingour health momferingprgnamand shipping policy seahtto://iaXflifca.tax.ora/health
Paga
.... -
''
TSCA SANITIZED
Appendix K
MB 07-15961 26
. p. 40
M i R e s e a r c h L a jb e m to r ie s
.... 17SSW *ntt Road
P.& Box 178 Splnnoratown.PA 1886$ ' phone ( ^ 5) 536-41TO
fisx (215) 6^6-1816
inAomoilaricewttt Good LabCfatoryPiaoticetOLP) regulations, a cbaracterizatw oftfie test
srtiete te nMuiredand SfioUtd mOude entity, sbwgthv pufi^v compoSffien. stabnrty and nntomTrtv This data must be reyievwd'by Study Dimtetorprfbr to"ittsdy InftiASon aa^i vwll bo
(be Anil refWfe <EPA 40 CFR 160,.105a M 792 105<PDA 21 CFR 58,105. OECD
62). " ..... . ''" '
'
-
-
in atM ^ . ff) f adf^diaradofteah'dn sBchjld be performed ih eompirahr;With the Good
( nharatory Practices
,.
Any exceptions to the G IP requirements witt be intficated in the CompHahte Statement ofihe
fihaJrefJort
Accbrffingly; pi ^ -arppty H iefM ow rfhginforrodonte
TsArtlcifltdentity
Strength
Purity
Composition
Stability
Jt/a
S* t
Uniformity
te a
if; This tAaracterization w as conducted under GLPs (or)
|g [ tV^s -chaae*iBrfccati&W"
BY (sigpatute)
*%
u f / s '/ m v ___ (tuiV
fOR:
undef S ti> s -tebmfiany)
T:\WRKETINGiteinpiaies^TA CHAR INFOfOKM aoc
P ag40ofii
TSCA SANITIZED
Appendix K
MB 07-15961 26
p. 41
Aa ISO90Q1/ 200
tibo
W titifkB8B:
-
m t o s t & te
\MTDmi6675
FnaetNm ts
' M att* 22.200:7
TheMunjfcof .... ...MiHWpiSTS, ................ , ... Wftss&^ectedtLC/MSand
rtandtodfoton fnefrtoxtotoniine! theabsoluteslight percent
ptd^ aadto .......
chaiftctecizeu maiiy hnpuritycoi^^
T lie q a a ita i^ ^
coo^ositoiutltwute th #
wereerivedfi^t&eccmbinedlJStSand^
Sample:
,M H fflO # 667S,
TABLE-I
'-qoeom
Coa>>odtiBdResulte identified Componente ^
W
lAyMs
uitt
-
e-w'^-MNft A^niriswseslute\Vt.%
Coocenftetious
PgvTffgr i.frrniruvn^.-t>i.n-,>atolriliiii^mwiiMittV
0^
0 745* 0007%
00354* 00006% 0 (12*00002%
"" 2 Iue kveb ofot&ruawWpi^ oompowafow^i^
.......' " **-nMR speetr*
Page )ol
WeJtefeeiK
3M MRD Analytical Laboiatoiy Request <
P a o s e to fs j
TSCA SANITIZED
______
Appendix L
I MB 07-15961,28
......
M l RESEARCH LABORATORIES
STANDARD PROTOCOL __________0 & S M __________
1.0 TtTLEQP STUDY; LOCAL LYMPH NODE ASSAY IN MICE (LLNA)
2.0 OBJECTIVE: f o determine the sensitizing potential o f topically applied fast substances, th is lX N A protai, modified using floyy< ^ m e try analysis, is designed to be an altematt/e assay for the Bushier Guinea Pig Sensitization Assay defined in die ICCVAMreport{83 CFR 37405-6; July
10,1998) and fee L I^ A as defined In EpA OPPTS 870 2800, Final Guideline (March 200$), and OECD TestGufeeiine 428, effedke April 2002
3.0 TEST ARTICLE;
3:1? Source: Air test artides w ifi be supplied bv the sponsor Priorto initiation o f the study, the sponscrsheuld provtdetestarticle characterisation to fee Study Director that should include, if technically feasible, fee name and quantfees o f unknown contaminants arid impurities. Refer to section I3 4 l;3ofth isprotoopifor additlonalinformation.
3 2: i ahfti- Eachtestarfesfewin be identified by source, name, and/orcoda number, date o freceipt at MB Research, and MB Praject Nirmfcer
3 3: Storage: The test article will be stored at room temperature and humidity unless otherwise specified by the Sponsor
34: Hazards: Basedon the infonnatfon provided by the Sponsor, appropriate routine safety
piieeatiHbns vdfc
in fee hanging of fee test a r t ^
3 5: vehicle: AS necessary, a suitable vehicle will be added to the test articte lo generate dilutions of the test article Thevrehicle w ill be AOO (acetone:oBve oil. 4:1) unless otherwise directed by the Sporisdf When a vehicle or diluent other than AOO will be used, it must be one that does not elicitanyslgnificanftoxifieffectsanddoesrtot substantiallyalterthechem icaiortoxicolt^ical properties Of the test article. Solubility testing and the use of vehicles other than AOOwill be documented in the raw data*6
4 1 : Animal Requirements: 4 1 1 : Total Number o f Animals 4 1 2 : NumberofGrotips
4,1 3: No. Anlmals/GrOuo 4.14; Soecies/Strain 4.15: Age
IRRITATION
PRESCREEN 6
QUANTITATIVE
IRRITATION
TEST (if needed) atleast 12
MAiN at least25
at least 6, including one (1) control group receiving vehicle Slone and (1) positive control group,; plus at least three (3) test groups receding consecutive concentrations
at least 5 (all female)
CBA/J or CBA/JHsd
8-12 weeks old at study initiation (age matched +/- one week)
ms.wentz mad, p,o<box 178
phOtM^(nS)S3S4 l 10
splnwnrtawiiw 1$
faxr(215) 53S-1816
TSCA SANITIZED
Appendix L
MB 07-1596126 STUDY TITLE: Local Lym ph Node Assay In Mice (LLNA)4
p. 43
42: 4 2 1 : Soedas/Sex: L L N A u s e s fe m a te m fc e ,^ ^ therdte (hernostdetaited information avanabraaboutthe Indocttonsnd regulationjjf
4.2 2 :
Strain; m p ra to c o l utflfess young adult ( fe ttw e e W fo m ^ w3j,CBA/JW sd. o r CBAiCa strain mice ara apceptebteias oeOTand.EPA t^st guidelines) for use in the assay sin?, in several fofec-teboratory validation studies, they displayed comparable response. The source and strain used he indicated in the study
repent
4.2 3: Number of Animals:
:
\.
the4 2:31 irritation Prescreen: The 6 mouse pratraraenis mlnimumnumberneededto
datermtnetf the testerticte has demnat irritation properties at the highest attainable
uraentraifcw ^
42 32 w henir bedetermlned.
The optional QwanB&tiveirritation Test (12 mice) is used need to
4 2 3 3. Main T est The minimum number of animals in the definitivelest is25Jin 5 groups of 5
mice each. OccsdonaBy, especially when dehnal iiritaH&n fe present an a d d itio n a l!^ treatmerttgroups (5-10 mk);may Be necessary. For ^e c i^ .^ e W c fe s or fomwIatiQns, a itelVe group o r e Second vehidei group, may Ira needed _p e U.NA permits tt>e
reductfonofahlm tfsrequir^ suitance8<m paredto3tbdies1ny?^ngtheusei0fgulneairfg9.Them fntm um nurTiber
per group recommended by IGGVAM and the EPArOPPTS 8702600 and OECD #429
test guidelines is five mice.
,.
,
4.3: Husbandry.
4 .3 1 :
&)
4.3.2: Housing: Animals w ill be housed individually in rBawmendafions in We Guide for BraCare and Use of Laboratory Animals DHEW(NIH)
and is equipped with a 12-hour ligM/daric cycle Temperature and humidity \Mlt be
ro o n ^ using
devices..
4 3.3: Food: Fresh PM1 (Diet #5001) will be available at all times
1765w ilts rowt p o l>ost178 ' phon {215)63*4*10
........................
Pape43<452
' ' ' ^ i i 5 ) SS-ieis
TSCA SANITIZED
Appendix L
MB07-15881.26
STUDY TITLE: Local Lymph Node Assay in M ice (LIN A )
........................
MB RESEARCH LABS
PROTOCOL NO: 56S0M
_________________;..,...... .............________- _______ ;__________ ;___ -EAgNQ;,?gf.H
4 3 4: Water viiMl bB available a t all times
4 3 4 .1 : Analysis of W ater and AccePtabte Levels of Contamlnants: nalyste o f water is
peHOnried apptxto ^^
per p a re a d results are ta m p a n
o f acceptable l v k o f contaminante as provided by the water testing laboratory
list
4.4: eoritrol of Blas:
od non-
pregrianft mice of fe e s a m e a g e s p a c lf^
beaseigned tagroups u|ng standard
acceptedmethods Of teOdomfitetife The method for attalnina the random numbers v* be
reobrdd in the studfiie.
4 4.1: PragstudV Body Weights; At the iititiatton o f the study, the weight variation of test animals Will not exceed 2Q percent o f the mean body weight
4 5: Identification:
4 .51: Cace: Each oage w ill be identified by a cage tag indicatkig the date of dosing, test article .........Wnffficatioo, M Bprojectnurnber,dcelevel, numberand sexofanimals
4,5.2: Animaf: JEach animalw iil be identtfiecl by an indelible tail mark coorespondlng to the numbers ddamnted on the dataeollection forms
S.OEXPERIMENTALPESIGN:
5 1 : Intrduetiri: The LIN A determines the sensitization potential o fa test substance by measuring
thepflBteatib:M lpiftc ytsin th auricular lriipbn^
(ears).
Lynoi'cxife p>tr|Teratlpn WHI be:measured by determining the incorporation ofbmmodeoxyurldlhe
(B fdU )iisingafl
m thodshwntobe e q u iv ^ n tto 'T-thym ldinebased
measiilritiiete ^ ^ h o ^ p i^ f r a t lo r i-
52: Summary o f Experimental Design:
LLNA PROTOCOL P A V i
DAY 2 DAY 3
DAY 4
DAY 5
DAVO
DAY 7+
7* DAYS
T BW .ET
T
T ET
T * Topical appBcatibnpftestsubstance, yehkdeorcontrol
BrdU,
P
BW .ET
6 2 H,.Si ......... .. .
BjcdO ...
3W ET
P
- At 5 hrs pn^sacrifice ( t* 8^ his), systemic administration o f BrdU in PB8 (200 fit per moU$e;i4>.); at sacrifice, excision and processing o f each mouse lymph node set (on an individual animal baste); preparation o f a slngle-oell suspension o f Lymph Node
Cells'(LNG)
- BOdy Weight * EarThickness measured (digital micrometer c r Peaoock Dial thtcknees gauge) within
24 hrs pre test, on Day 3 prior to dosing and pre-sacrifice on Day 6
Post In-life phase, ex-vivo flow r^tometry procedures performed; measurement of Brd incxtfixjratfen irto jypph node celle (LNC) analyzed, and Stimulation Index (SI) and other parameters calculated
17$ S \w to 4road,p. box 178 phbm* (216}5164110
Mg:44d f 6Z':
plnnerstown, p al8968 fax:{215>536-ie i#
TSCA SANITIZED
Appendix L
MB 0715661.26 STUDY TITLE: Local Lym ph Node Assay In Mica (ULNA)
MB RESEARCH LABS P ^ T d C il^ M: 5650M
PAE NO: 4 o f 11
Will be ^ l t d th e lb llc H ^
o fp re fe re ^ J a ra c c c ^ ^
Sponsor, th e dfeuft yhfclls /t Q ;4 :l fee Si^ton 3|.5) ,lf 0 :1 S n o t ueUI asthe
vehicle. Ihesohdwy vehteles ih sctfen & 3 1 vrtll^IrtvssU gatd frsofpW Iitydfthe test
art|c|e(8jItmti\roi|^^
based h th SporisoCs filstoirteat ififfcatfion and solubility data.
. ;, r
5.3.1: Optional LLNA Vahldes:
.
4:1 v/v Actone^OBve PHiA) Dmthyf:suiftdde (DMSO) ;
.
4:3:3 OMSOActnetQhnPl (D$A 433) '
Acetone
.......
N,N43imettylformarnide (t)MF) Dinwtlylacefemld (D M A ).
4:3:3 i& h ie th y te c e t^
(D ^E 433),
Ethanol (50%,$%, or 1$} Methyl ethyl ketone (MEK), aka 2-8tanone UH pare Ptroiaturo
Propylene gycol(PQ)
' ' ...
The preferred vehicleAOQfe preparedbyadding4 parts (ml) piacetene forever 1 pati (nil)
oliveoil. Whollyaqueousvehicle are to be avplded as pertestguidelines The vehiclewSl
hL^>edtdiwth
^cOFUsaTti^tlpn;
Sateii^F....
appncabte)tStofiage/haftdlirtgand,t^
5.3.2:
Vehicle Preference: W h e re p c s s to le th e fo i^
be used for the iL N A (In
wper ^ p r ^ r o n ^ AOO > Dlmthylsulftdd > D*AE-433 ( * t)* E )> Acetne. If O is
not to l^ s ^ <cm:ff ia
r s l e w p i | he ipiPhRaH^
rif whlc^)
the test artcle teadde distobe used), th S p o ra ro rv
tt vehicle in secdbn 1 3 1 3
o f the SPONSOR REQUEST section o f this protocol: .......... .
........................ :......
54: Positive Control: The mc^arote sm sflizef alpha-hepcyl cinnamic a W e l^
MB)
at 25% or 50% In AQO (or suitable vehlde) wfll e
a-ndicatd"jby
Sponsor In section 13, AddiBna postive mtrotesuch s th e
dinjtrocfttorobenzene (DNGB. dippBed byMB) at01% iniA O O (or suitable vehicle) my be
added at the Spcnsor's.opponi,
^ 1hmi^nopttnotplrig:ndp^rits:m be
added to the study, These hmteplS have produced consistent responses ip the Lljs vtdth the
solvents AOO or DiAE 433 Other positive, negative, naive, or irritant controls ma be added by
the Sponsor, in consultation with th Study Director (se section 13:1 2)
5 5: Negative Control: There, is no suggested negative control for theLLNA Anegativecontrol substance or a.naye control group may ba added as an iaddifbhai group a tg ie ppion Lthe
Sponsor and at ^d itfo n a l oerpense (see sector 13.1.2) All test Prde grobps wSl be compared to ieir respectiv&vehicle control group
1768wentz roatl,p.A box 178 pHowis {215) 6864110
- plnnerztown, pa 18968.
fw :(2l 5) 838481
. p. 46
TSCA SANITIZED
Appendix L
MB 07.1596.1.26 STUDY TITLE: Local Lym ph Nda Assay in Mlce(LLNA)
MB RESEARCH LABS
PROTOCLNO:56BOM P A a N :B C f 11
5 6: Test Solution Preparation
5 6.1: Safety:; SUbSiBRO#'
m ubecan^
(to w o m d u h n g so lu i^ Z-
if the fest
56 2:
Tftst Article Pmoration: Tli8 8iinpl8 PWP3?^o^y/ill be d o & in i^te d In the: f^iydsjta Pwsh test substance sQltions/suepenslons will be prepared on each tree&nent day. Substances of
low solubility oan be mixed using a mechanical agitator or usinga magnetic stirrer, Heat
aboye 38*0 vvhi not be feed tm te ^flfe sU b sta ^^
to be batstable,
5 6 3: T<stArticle Conranhations: Ttoefost article is normay assayed a t three to five consecutive concentrations from within the following range:
100%. 50%, 25%, 10%. 5%. 25%-, 1.0%, 0 5%. 0 25% , efe. (WfVfor solids, v/yfor liquids)
The Sponsor will Endicate concentrations to be used in Section 134 2 based upon previous
experience orstudies ( if available). structure activity analyst, dermal M fefen and solubility Optimai test concentrattons will be prepared based u|XOTfee ma^mum solublllty o f the te ^
a rtd e in toe vehictor whife avoiding overt or severe systemte toxjcHy: or fecal irrifetion, Intoe
eyentof no tobcdfe.Bi4Pjpptfen&.^r
*n
p iy o f or in addition to an Irritation Prescreen (see 5.6 3 1 ) to determine irritation and
56.3 t: 5 6 .3 2 :
irritation prascreen: /\n ioiuoi imiauun usyoii ;*<*...g: edema)will beperfermod using
i
aiitifesforitoe3
obtainableIn chosen
vehicle). Six mice (twopar concentration)will be ipedend thepresoeen wwbe
conducted under tdntiifei ctmdlUonsas top mainstudy, e x c i terthe assessment of
lymphradeproiifora^
lfnoinitafiontaobseryed;(ear swelling <25%). then
RHSi^iBianfei^issiiaiMrii^ utloid:lr*.i^.|Ttaia-srtSil|)i., ^significant irflfionfe25% increase inear swelling) observed, a Quantitative lrritetion Test (see 5 63 2) should be
performed.
lrritatlon;Pratacraen(5631). an
StS^^^Scmsseeif
Irrfathfij,
a eii^irattons of
in toe be
ar^ Kn = 3mfoe, or 6
article sfiouks lae
5 .7: Topical Application: 5 71: Safety: Gloves must be worn during this operation.
5 *2
^ n b ^ ^ o n oflhe t^substanee to toe dorsum <rf both ears one fim^per day for three
consecutive days Control mice will be treatediMto the vehWealone Theopptication volume (25 ul per ear) will be administered using a positive defacementpipetto ana win pe spread over theientirC domal surface of the ear: The time of dosing will be recorded
1765wahiz-rol,P&. bo* 17 phot: (215) 636-4110
Page48of 52
eplnnwatown, pa 18888 fax:(216)836-1816
p. 47
TSCA SANITIZED
Appendix L
807-15981.25
STUDY TTTLE: LocalLym phN ode A asaylhM IcefLLN A)
..................
MB RESEARCH LABS
PROTOCOL NO; 58S0M
________________ ;___________________________________________________ PAGE NOi 6 Of 11
5 8: Administration and incorporation o f StdUfnvivQ: Five d a p after the first topical applicatimi o f
test article, a irm k ^ M ll be lhJe^ed in^perHoiieally w ^ 200 pi of a & B ro n ^2 ' <)(y-Uridihe
solution (m a . m gtf fri P B ).....
...................
5,9; Observations:
-..-'V . . . .
5 91: Dermal Reactions: AS mioe wiK
application site
. -. ,;
'
5 9 2: Systemic: A i a m W i^tim , tntee w8l b observedricectail^r f o r d i r i f c a l signs, either Of
loca) irntatiqnat thapplfcaffon site orofsysteirilc to io c ty M b ^
bi
systematically recorded, with records malritatnedfor ech irid ^ u a i rrtose..
5.9,3: BodvW ldhts: BQdywteightSwfll be fea^edprartastam Jprior to BrdU infection; Significant weight toss' (2gr Or more) will be noted and addressed,
5 94: Ear S te llin a : Both ears o f each animal Wffl be observed for edema and/or erythema, and ear . thickness measurements uriti be taken on Day 1 (pre-dose), Day 3 (atapproxlmateiy 48 hours
D te ith^nassgauge)'
. .? '
,.
510: Post Mortem LvmPh Wode Extiactlon:
.
5..10.1: Sacrifice: AnN afe ^p v^ng s e V ^ arid enduring sigr o f diSteess and pdlh, or amtmads in a moribund condition and not expected to survive unf$ the next observation interval unti be humanely sacrificed using CO* On the day of study termination, five (5) hours after the injection ofBrdO; th e S u rtriv ln g m k ^
5.10 Z Exclston and Preparation of Lvmph Nde Celb-. F o ik n ^ sacrifice, all Of the drertnjng auricular lymph nodes fro m e a ^ mouse will be excised arid epmnd. On an individuai
ftomthelNC suspension
511: Optfonai lnvnunophenotvping Cell Treatment An.aliquotofthe cellswill bestoredat 4*C in
storaganiedia for up to 72 hoursforoptional ftowcytometiyanalysis Qfimmunophewtw)ew
surfaoemarker expression,e.gr%B220+. %CD3+, %CD69+, and/ori|l^,`+<lls, as per MB
SOP voi VII D i.
........... ...........
512; Fixation of Cells: An aliquotoftoaUslCwill betwes^rvadaritii asufebtebufieror atcohorand will be stored at ~18 to*3QC untilfarther processed. Storageehoura not exceed twomonths
S .13: Determination of Cell Proliferation:
5 13,1: Enumeration ofCete: After propidium iodide stpinto&asingle-itel^
be
analyzed iiSrn$a BedonrDickfospnflowcytQineters^ficalfyprogrammedfor propidium
kxfidestaining ^ enumerate nucleatedcess inthalymr^ ncde ceUsu^nsion The total
numberof cells permousewiil be calcufatedfromtoe vaiuesobtalhed by muWplying bythe
appropriate difutlbnfactors ;t
-,
1785w?ntt wd, pA b<Bt178 ................. .......
phon (5)838-4110
..................................
: p189S9
fax: (218)635.4818
p. 48
TSCA SANITIZED
Appendix L
MB 07-1596126
STUDY TITLE: Local Lym ph Node Assay in Mice (LLNA)
DABRESEARCH LABS PROTOCOL NO: 5650M
514: ilu 'iu iiila atfon of BrdU.tncorooraflon:
5.14.1: Acid Denaturationof ONA: DNAof LNCwill be add denatured so that theBrdlJ antibody can acoess and quantitetivelytnterartv^th the BtoU that has been incorporated into thecellular
0NA
5.142: NeutraftzatlOnofthe Cellular Adaterlali Samplea will be neutralized bywashingthe oeflswith boratebuffer(pH8:5)/orotheroomparableneijtrai^ionbyffer
5:14 3: BrdU-soecrflc Statnlnadf Celte: Nudd wiH be washed with astalnlng biitfer and meubated
with BfdUpeoificfluore8oent ahtib<rfy opnjusate ThenucieiwBi bewashedagain with
stafaingbufferandresuspendedin.PBSdontelningR^
propidiumtodide, Foliowlng at least a3CHn4nutelncubatidn atrpom temperature, the total
DfoAcontentofftenucfieii Is:Vyjsilias thep^rtoentitetgieof
BrdU e
percentage of proliferating iymphocytesj.wfll tedeterminedusing ftoweytometry
515: Test Duration: The test durationofthe study is at least eleven (11) days
61: FlowCytometer FlowCytometry and all cell processing will be conducted aecording to MB
ReseaK^ laltoratodes SfendardOperating Procedures. Lymphnode ceii analyseswUI be
performed using a Beeton-Dickinson FACScan flowcytometerusing Ilm W ofpowerat488 nm
esteitationwavefengtb. BD CeilQuestver 3,3 acquisition software ona fttacintosh 04 acquisition
systemwillheused tocaptureandstoredale (Ust
as LMD or ,FCS)on a
dedidatedsecuier^tworkdrive. DatefflesvillbeanalyzedusingftowJoforPG orCellQuest to
determine sppraprtate analysisgate and % positiveLNC populations.
7..0: DATA ANALYSISAND CALCULATTOMOFSTIMULATION INDEXfSH:
71: oata Analvste: Foranalysls o f IrKfivtoual animal lymph node sets (right and left side draining local
hO desJ.thaproliferativeraspanseafiym ^ number d f Brdli-posItiye lymphocytes per (individual ahimal} lymph node sets, The mean value f th tQtolnumbrfBrtiOHtositVecellsand.its associated standard deviation (S.D.) w ill be calculated for eachg^oup, The S lt i.e. the natio of th mean BidUInctoqxeration into LNC o f each fest arflde grodp divided by tltot o f tha vehicle groupV wiif be calculated for eaci1) tost group
accordingto Equation 1 below:
Equation 1: SI *
Ateah#BrdU +cells`mTreatm6nt Group ------ -- - - -- ------ . .
Mean #BrdU+cells inVehicle Group
7.2: EQuivocal Results: Intoeeaeowheredose-reiated increases nceirproJifration {f e., BrdlJ posityeclte)reslt1ri a SI thatappToachesOutdoes not reach 3, theregulatoryguidelines may toquir thatdditinat tssCsbe performed using higherconrantratidnsofthe test substance (or in
andtofvehicle) if possible in such cases, theffectofthe vehicle on the outcomeshouldalso be examined
1765wtz wa, p d box178 flru>rw :(2lS )M (H l10
P a g *4 8 o f8 2
sptmwi>town, pa i8958 fx; <2t5) 83S-1S1#
p. 49
TSCA SANITIZED
Appendix L
MB 07^1$961.26
STUDY TITLE: Local Lym ph Node Assay In Mice (LLNA) __________ . . ._____________ ____________
........... ...... .............. IMS RESEARCH LABS PROTOCOL NO: 5850M , PAGE NO: 8 o f 11
7 3: fitnUKtfcfll Analysis: For each testgraqp, the indhrfdua! Sl valuesatongjvilh thfi mean Sl and
standatodeviatton w illb e calculated irfurthersfafisScal analysis i# re q g i^ . ^ analysis w iil be
. pettomied only upon request by the sponsor
..,
,
S-A* DATAfNTERFRETATiON; r V " ' ' ` " ' 'V ' ' ;
"" " ' '
81: interpretation: A substancewiUberegarded as a sensitizerinthe IlN A Katleast pne ,
concentration pfd t^aiik;le results In a S-toJdor greater In e ^ fe In
relative
tothat ofControl Chicle) lymph nodes, as indicated by an SI *3 .0. TTtodatashoBld also he
c o m p a tiT & w ith a ^
aW w ughjin;altow aitoetto
hightopical application concentiations, tor toca) irritation; systemictotfcStyorimmunological
suppression. '
"' . "
9.0: P I^ E IW ld D F T tS S iiE ;
' `J .
V
v
91: Tissue Otherthan the lymph nodesprocessedas above, noothertissueswill be token
9 2: optional tissues; At the Optionof the Sponsor, the dosagetete(earn)yW! be excised and
preserved in 10% neutrat formalin tor HASstaining,and;nfs$o^eAhqfc0C9 evyailuisdfQCi atan
adcifttonalcoBt Ottwrite6uef or organsmay bespeciSed bythe'Sp<msorto beisotetedand
preserved. HistopathologywWbe performed byM Ray Brown, D.V.M., PhD., DACVP,
Research Pathology Services, In c , New Britain;, PA.
..
10.0: ftEVESiQNOF THE PROTOCOL: Any amendment to or devtetionfrtm ttaV
b e fu lly
in fl^ s m d y tir^ u d ^
ibr tbe
f<i^ safd
the date thereof
....
.
--
110: RECORDS TO BE MAINTAINED:
1 1 1 ; Collection of Data: All data generated during the conduct of the study w ill be recorded in ink on worksheets. All entries Will be dated, initialed and verified by another person. Flow cytometry
data files wiB be wrfte-piptocted, becked-up and a copy stored olfrSlte TTie crigtoai computeracquired data wi|l be analyzed and histograms, dot.plots and % positive LNC will be printed out for each anlntol arid endpoint, and included w itotoeraw itote
11 2: Reports:
1 1 2 1 : Draft Report A draft report w ill be submitted to the sponsor prior to submission of the final report
176SwantsiaA rM> box ITS phon*;(2i#)e*1<0
Pag4Sof52
................
, splmwnrtown, pa18968
TSCA SANITIZED
Appendix L
MB07-1g961,26
STUDY TITLE: Local Lym ph Mode Assay in Mice (LLNA)
....................................
MB RESEARCH LABS
PROTQCDLNO: 5650M
...-----------------------------*-------- -- ----------:---------------------------- -- ..................... ....P X C E N fra d fT *
tt2 2 : 3Baa|,^Rort; Pd!dMn0 approval by the sponsorofthe draft report, toefinal reportwill be
submittedandwillihctude, isotnbt belfmftiedto:
..................
Species, strain, sex, number, age and sourceof test animate
Equilibration, housing conditionsduringexposure and post-exposure; bedding maMal; roomtemperature arid humidity, fight/darkcycle, dietandwater
Method of randomassignment
Physical nature, purity, stabitlty. and lotnumber of testartfcie Justification % c h ( ^ ofebMentAmhicle
lndMd.ua! andtestgroupdata (I e, meanandstef dev.) presented In tabutefonn
Description o fadverse effects: of treatment onthe mice
L is td re f^ n o a s r^ jn to e r^
indevelopteg the protocol, perinhing the
mathg' and in te w p ^
andcompiBhg arai e\mlua|hg fa u lts .................. ........................" ..........................
* The;w m bef pf re s p e g h m V rp jc je ^
M jP fcg#toe^i ilJ + clte ft>r each animal will 'aBtM'siTq^^ (orinip(f^;:;i ib .....................
11 S: Retention o f Data:
i 1.31: Raw Data: The original raw data w ill be filed at MB Research by project number.
11 3 2 : FlnalR eport The original final reportw ill be submitted to the sponsor and a true and certified copy o f toe original report wl be Wedat MB Research by sponsor name and MB project number.
113 3: TestArticte: Anyrernainingtest article w ill be returned to the sponsor upon: submission o f the study report.
1 1 3 4 : Tissues, cells, blocks & slides will be .stored at MB Research arid indexed by sponsor name
and MB projed number; The sponsor M il be contacted to determlriefinal disposifidn upon
submission of the report
...... ...........
12 ^ Thfe stodyiW! be conducted naccordance wito the Good Laboratory Practices o f the EPA, 40
CFR160 and 702, FDA21QFR58, and as spedfid in the Prindptes oh Good Laboratory
Practice, published by the Organization for Economic Cooperation & Development (OECD),
1007
............
.............
12 2:. Protocol: MB Research w ijl hve on file a oopy of this protocol, signed and dated by both the resporistofe MB Study Directorand the Sponsors autoorized reprsentative.
123: Quality Assurance: The Quality Assurance Unit will inspectt least onecritical phase of this study,audit Mae raw data and audit the report in accordance wito toe Standard
17M ii^eebA :lM >i.bm t.1?S: phona:(21S)SS<W110
.......................
nag so of 52
*Mre**Wvyn,pi89.88 fax: <21S) 836-181
6 I d.
TSCA SANITIZED
Appendix L
MB 07-15961 26
V
Paged of S2
--I
3 CoO
8 w J>z > o 2 CO 2$ >
0M*>1Xti
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f03o r N m
D
p Orol
