Document 3kjpaNZR36qOL5z7wdywp5pn
IOFN 3VIHTRCOOPMAINCYROBCIOOMLPOOUGNIDCSALT-M2U5T4A0GECNoICCITaYndATS-S2A5Y4S1 CoC
oy tess 7
=
By: DKirriescttioern, E.MicMroorbcieallmanGse,netPihc.Ds. Deparement Nancy Marx,anMdicrobiologtst
a1
tom Prepared for:
GHeendeircaall DOefpfiacretsment S3t. CePanutle,r Minnesota 55101 Attention: WT.oxiCc.olMocgCyormSiecrkv,icePsh.D., Manager
(o SRI Project 1SC 4442-16
Approved: DDavitd C. CL. LJonFeb,oDierector Toxicology Laboratory
pn) L.ifAe. ScSikeinmcmeers,DiEvxiesciuotnive Director
FN +)
(431299)R3a2v6e-n5s2w0o0odCaAvbel.e:SMAeInIlNoTPUarMkP,KC.all+foTrniixa.4091205.373-1256 000080
statary
T-2341SRICoCInftoerrnmauttiaognealnicexaamcitnievdity34wiCtohmpasntyraicnosmpToAuLnSd3sS,T-T2A5L4S0I?C,oCTAaLnSd3S,
TASS, and mierosone
TAL00 of Salmonella typhimurium assay, in an assay conducted in
in the standard Ames Salmonella) desiccators, and with the yeast
aSnadccihnarotnhyeceasbsecnecreeviofsiaaeraD3t. livEearchmeatsasbaoyliwcasacpteirvfaotrimoend
in the system.
presence Neither
T-2540 assays
CoC nox T-2541 performed.
Coc
was
mutagenic
or
recombinogentc
in
any
of
the
1
000081
TNTRODUCTION
T-2561SRICoCInftoerrnmauttiaognaelniceixtaymibnyedin3MviCtornpoamniycrcoobmipooluongdiscaTl-2s5s4s0ayCsoCwiatnhd
strains TAIS3S, TAIS37, Salsenella trphimuriua
TALS3S, TASS, and TAL00 of the bacterium in the standard Anes Salmonella/microsone
assay,
cienreavnisaissseay DcJ.onduAcntAerdocilnorde1s2t5c4c-asttoirsm,ulaatnedd,witrhat tlheiveyreashtomoSgaecncahtaerommyecteas
bolic activation system provide metabolic steps
was included in the assay procedures to that the bacteria either are fncapable of
conducting or do not carry out under the assay conditions.
90%
reTlhieabalsesaiya pdreotceecdtuirnegwicatrhcSi.nogteynpshiamsurmiuutmagheanss,praonvdenitto habse
80 to about
the The
saasmseayreplrioacbediulrietywiicnhf5d.enciefryeivnigsiacehemisicaalbsouttha6t0%arreelnioatblecarfcnindoegteencitci.n'g
carcinogens because the
aasssaaygenstysstetmhsatdoinncorteasaelwamyistotpircovirdeeco1m0b0i%nactoirorne.lationHowwiecvher,
carcinogenicity investigations negative response conclusively
in animals, neither a positive nor a proves thata chemical is hazardous or
nonhazazdous to man.
2
600082
METHODS
TSAaIlSo3o7,nelTlAaISt3yS,phiTmAuSr,iumanSdtrTaAinIsGOTALS3S,
The Salmonella 2uxotrophs by virtue
typhimurium strains used at SRI are all of mutations fn the histidine operon.
histidine When these
choinsttaiidniinneg-deapetnrdaecnetofcehlilsstiadrieneg,roovnmlyoncemlilnsimatlhatmedrieuvmertagatro
plates histidine
independence (hts") are able to histidine allovs all the plated
form colonies. The bacteria to undergo
small a fev
smount of divisions;
in
many cases, this fants are easily
grovth scored
is as
essential for mutagenesis. colonies against the slight
The his" rever background groweh.
The but
spontaneous mutation frequency when a mutagen is added to the
of each strain is relatively agar, the mutation frequency
conscant, is
increased 2- to 100-fold, usually in a dose-related manner.
UniverWseitoybtoafinCeadliofuorrSn.ia eaytphBimeurrikuenlsetyr.ai'ns
from Dr. Bruce In addition to
Ames of the having muta-
tions (Efe)
in the histidine operon, all the indicator scrains char leads to a defective lipopolysaccharide coat;
have they
a mutation also have
baiodteilneti(obnio)thaantdcofnvertshegernepeasirinovfoluvletdraivniotlheet sy(nutvh)e-siinsducoefd tDhNeAvdiatmaamgien
(veh). aromatic
mThoelercuflaesm,utatthieroenbymakienscretahseinsgtratihnesmumtoargeenpiecrmeeaffbelcet toof matnhyeselarge
molecules. cheaically
The uvrb mutation causes or physically danaged DNA
decreased repair of some and thereby enhances che
types of strains'
Sensitivity to some mutagenic agents. by many mutagens that cause base-pair
Strain TALS3S substitutions.
is reverted to hist TALOO is derived
pflraosmaiTdALSp3R5UObLy. thTehiisntprloadsumcitdioinsofbeltiheeverdesitostacnacuesetarnansifnecrreafsaectoirn,error-
Prone DNA repair most mutagens."
Itnhataddlietaidosn,topmlaansymidmorPReMImOuLtatcioonfnesrsforresaisgtiavnecne dotsoe tohfe
3
00083
antibiotic ampicillin, which of the plasnid in the cell.
is a The
convenient marker to detect the presence presence of this plasmid also makes
strain TALOO sensitive to some frameshift mutagens [e.g., ICI-191, benzo(a)pyrene, aflatoxin B, and 7,12-dinethylbenz(a)anthracene].
Strains TALS] and TALS38 are reverted by many frameshift mutagens.
Strain TA9 which makes
is it
derived from TALS3S by more sensitive to some
the addition of the mutagenic agents.
plasaid
pRMIOL,
All indicator strains are kept at 4C on minimal agar plates
supplemented with an excess of biotin and histidine. The plates with
the plasaid-carrying strains ensure stable maintenance of
also contain ampicillin (25 ug/ml) to the plasmid pRMIOL. New stock culture
places are made every have been checked for
four to six weeks from single colony isolates that their genotypic characteristics (his, rfa, wr,
bio) and for the presence of the plasmid. For each experiment, am inoculua from the stock culture plates is grown overnight at 37C in
nutrient broth (Oxoid, CM67).
Aroclor 1254-Stimulated Metabolic Activation System
Some carcinogenic chemicals (e.g., of the aromatic amino type or the polycyclic hydrocarbon type) are inactive unless they are metabolized
to active forms. In animals and man, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large
number of these chemicals to carcinogens. *:*~'! Some of these inter mediate metabolites are very potent mutagens in the S. typhimurium test.
Anes has described the liver metabolic activation system that we use.' In brief, adult male rats (250 to 300 ) are given a single 500-ng/kg
intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated
biphenyls). This treatment enhances the synthesis of enzymes involved
in the metabolic conversion of chemicals. the animals' food is removed but drinking
Four water
days after the is provided ad
injection, libitum.
Oasn ftohlelofwisf.ch day, the rats are killed and the liver homogenate is prepared
" 000084
The livers are removed aseptically and placed in a preweighed
sterile glass beaker. The organ weight is determined, and all sub
sequent operations are conducted in an ice bath. The livers are vashed
with an equal volume of cold, sterile
minced with sterile surgical scissors
0.15 M KCL ( ml/g of wet organ),
in three voluaes of 0.15 M KCL,
~
and homogenized with a Pocter-Elvehjen apparatus. The homogenate ia
centrifuged for 10 minutes ac 9000 x g, and the supernatant, referred to
as the 5-9 fraction, is quickly frozen in dry ice and stored at 80C.
The metabolic activation mixture for each experiment consists of,
for 10 al:
1.00 al of 59 fraction
0.20 ml of MgCls (0.4 ) and KCI (1.65 )
+ 0.05 al of glucose-6-phosphate (1 )
:
0.40 al of NADP (0.1%)
+ 5.00 al of sodium phosphate buffer (0.2 , pH 7.4)
3.35 al of Hao.
Assays tn agar To a sterile 13 x 100 mm test cube placed in a 43C heating block,
we add in the following order:
(1) 2.00 ml of 0.6% agar (2) 0.05 al of indicator orgentsas (3) 0.50 1 of metabolic activation mixture (optional) (4) 0.05 ml of a solution of the test chemical. This mixture is stirred gently and then poured onto minimal agar plates.' After the top agar has sec, the plates are incubated at 37C for 3 days. The number of his' revertant colonies 1s counted and recorded.
"The 0.67 agar contatas 0.05 uf histidine, 0.05 mf biotin, and 0.6% NaCl. "MOsfiluncKiainsHaePl,O.a,g0a.r2`angdploa3tf.e5sNgSc0oo,fn+s7iNHsa:tH0N,Ho,f,P3O,p+e4roRfsl0ic.tietrr,ic15acigdofmonaoghary,dra1t0e, 1o0f
5
000085
and
For 0.05
negative controls, ve use al of the solvent 1s used
steps (1), (2), and (3) for the test chemical.
(optional), Dimethyl
sulfoxide (DSO) was used For positive controls, we
as the solvent for T-2540 CoC test each culture by specific
and T-2541 CoC. mutagens known
fo revert each strain, using steps (1), (2), (3), (optional), and (4).
Assays in Desiceators for Volatile Compounds
highlyThveolsattainldeardcheAmmiescalpsl,atseotweesthaivsenmootdiefniteidreltyhesupirotcaebdluereforto tceosntdiuncgt
such testing. The Salmonella places assays in agar, but no test chemical
are prepared as described for the is added. The plates, with lids
removed, are placed side by side on a perforated shelf fn a desiccator (Figure 1). A known volume of the test chemical
9-liter is added
to
2 glass pecri bottom of the
plate shelf.
that is placed in the center of and attached to the A control chemical is tested simtlerly in each
Pelxapteerimiennta. rooTmhemadienstiacicnaetdoracis37seCa.ledA amnadgnpeltaiccedstoinrraermawgintehtivcansetsi,r
placed in the che cheadcal.
base of each desiccator, ensures adequate After incubation for hours, the places
dispersion of are removed from
the desiceators, their 37 for an additional
14ds are 64 hours.
replaced, and they are incubated at The number of his' revercants is
counted and recorded.
Saccharomyces cerevisiae D3
for
The yeast a mutation
S. cerevisise D3 1s a diploid leading to a defective enzyme
microorgants heterozygous in the adenine-etabolizing
pathway. When grow this mutation produce
on medium containing adenine, cells homozygous for a red pigment. These homozygous mutants can be
Ogfenethriastedrecformobmintahteiohneatlereovzeyngtotmeasy bybe miitncorteiacserdecboymbiinnactuiboant.ing Thtehe forregqauneinscmys
with various carcinogenic activicy of a compound or
or recosbinogenic its metabolite is
agents. The recombinogenic determined from the number
of red-piguenced colonies appearing on test plates.'
6
000086
DESICCATOR ASSAY
PORCELAIN
|c mm-- s a -- =
--
PETRI
WITH
PLATES
SALMONELLA
SHELF S -- EE
}
J fpGs LASS nPEnTRI
FAN ------)\ Ce J ------------------
TEST CHEMICAL
MAGNETIC
STIRRER
Figure 1
7
000087
A stock culture of S. cerevisize is stored at 4C. For each
experiment, broth containing 0.05% 0.35% peptone, 0.5% yeast extract,
MgSO. and 2%
0.15% KHaPO., 0.45% (NH.)aSO., dextrose is inoculated with a
sLhoaokpifnugl. of the stock culture and incubated overnight at 30C with
The in vitro yeast mitotic recombination assay in suspension 1s conducted as follows. The overnight culture is centrifuged and the cells are resuspended at a concentration of 10 cells/ml in 67 mi phosphate buffer (pH 7.4). To a sterile test cube are added:
* 1.00 al of the resuspended culture * 0.50 ml of either the metabolic activation mixture or buffer 0.20 ml of the test chemical 0.3m of buffer.
DSO was used as the solvent for T-2540 CoC and T-2541 CoC. Several dose levels of the test chemical (up to 5%, w/v or v/v) are tested in each experiment, and appropriate controls are included.
The suspension mixture is incubated at 30C for 4 hours on a roller drum. The sample is then diluted serially in sterile physiologic saline, and a volume of 0.2 ml of the 107 and 10 dilutions is spread on plates containing the same ingredients as the broth plus 2.0% agar; 4ve places are spread with the 10~* dilution and three plates are spread wich che 10 dilution. The plates are incubated for 2 days at 30 C, folloved by 2 days at 4 C to enhance the development of the red pigment indicative of adenine-deficient homozygosity. Plates containing the 10=* dilution are scanned with a dissecting microscope at 10 X magnification, and the number of mitotic recombinants (red colonies or red sectors) is recorded. The surviving fraction of organisms is determined from the total number of colonies appearing on the plates of the 10~* dilution.
The number of mitotic recombinants is calculated per 10 survivors. A positive response in this assay is indicated by a dose-related increase of more than 3-fold in the absolute number of mitotic recombinants per m10illisluirvtievrorass.well as in the relative number of mitotic recombinants per
8
000088
RESULTS AND DISCUSSION
and 7T-a2b5l1esCo1Canidn 2thepreAsmeesntSatlhemonreelslual/tmsicorfosooumre taessstasy.of TTh-e25d4a0taCoiCn
each table show the results of a duplicate assay performed on separate
days. 10 to
Both 5,000
compounds ug/plate,
were both
tested over a wide range of dose levels, with and without metabolic activation.
from
Compound T-2540 CoC was toxic to the bacteria at 5,000 ug/plate. No
bdaocskeg-rroeulnadtedcouinntcrweaasseobinsertvheednuimnbeeirthoefrhiassstaiyd.ineThreerveefrotraen,tsweovecronctlhuede
scthaatndacrodmpSoaulnmdosneTl-l2a540plaCtoeC ianndcorTp-o2r54a1tioCnoCaswsearye. not mutagenic in the
Table 3 conducted in
presents the results of an assay of T-2540 CoC and T-2541 desiccators with strains TA98 and TAL00. The compounds
CoC
were tested over a range Toxicity was observed at
of dose levels from 0.1 to 1.0 ml per desiccator with
5.0 al T-2540
per CoC
desiccator. and at
5-0 vas
ml per desiccator with T-2541 CoC. observed with or without metabolic
Because no activation,
mutagenic response no further testing
was performed in desiccators.
72540TheCoCresaureltspreosfeamtiecdrobiinolTaobgliecsal4 astsharyosugwhit6.h
S. cerevisiae D3 on In a preliminary
experiment (Table 4),
conducted organisus
over a range of concentrations from 0.1 to exposed to T-2540 CoC showed less than 50%
5.0% survival
at concentrations of 0.5 and higher. Therefore, the retested over a range of concentrations from 0.025 to
compound vas 0.25% (Table
5).
There was a slight increase in the number of mitotic recombinants both
w0.i2t5h%,andandwitthhoisut imnectraebaoseliwcasacntoitvadtoisoen-raetlactoendc.entrCaotmipoonusnd fTr-o2m5400.07C5oC to
was tested activation
once more within and from 0.09 to
a narrower range, from 0.07 0.4% with activation (Table
to 0.2% 6). In
without this
assay, there was an increase in the number of mitotic recombinants only
9
00089
aocbse0r.v3e%dwiitnhboatchtivaastsiaoyns. werBeecnaeuistehetrhedoisnec-rreealseastedinnomritorteipcrodruecciobmlbei,nants we do not believe that T-2540 CoC vas recombinogenic with S. cerevisiae D3. with ST.ablceesrev7istihareougD3h. 9 AprepsreenltinitnhaeryreseuxlptesrimofentassdaeytserwmiitnhedT-th2a5t41 CoC Twi-t2h541actCiovCatviasonto(xTiacbleto7)t.he Tyheeastcomatpou0n.d5%wawsithtoeusttedacttiwviacteimoonreandat5.0% concentrations from 0.025 to 0.25% without activation and 0.25 to 2.5% with activation (Table 8), and at concentrations from 0.07 to 0.2% without activation and 0.2 to 1.0% with activation (Table 9). T-2541 Coc showed several increases in the number of mitotic recombinants per 10 survivors. However, as with T-2540 CoC, these increases were neither dose-related nor repeatable; therefore, we conclude that T-2541 CoC was not recombinogenic in this assay. not muItnagseunmiacryw,itwheS.contcylpuhdiemurtihuamt, coormporuencdombTi-n2o5g4e0niCcoCwaintdh ST.-25c4e1revCiosCiaweerDe3.
10
000090
|
Compound NegTaotnisv)econtrol PosiSSodtdmiiivunemocsaocsneitsdrdetonle
NZ-iatnochfrlaunoinreens
S.k asic R2
MAecttaibvoaltiicon +-
+: i: -::: : ++ i+ ++
Table 1 IN VITRO ATS2S5A4Y0S CWIoTCHaSndaiTo2N5E4L1LAGTC.yuRING
HoficrCoogmpeoaumnsd Added per Plate
TAISES THiAsLtiSdine ReTveArLtaSnts per PTlAace THI BZo2u8 491us smoowa1 3wo 4 0so1m6
0L0o0 5.0
369 531 76 261 wr osy mow a 859
r1al0os
a2 a 7 3v13o 6 w28 239 31ma7
25
wos moe
s100 100
1030 wou
17183 87
aw6 ow1s Wom
27wonoyn mammmoa
1050000 51000
26,002178 Brae
1489 a1s 1um w2% 43% er stu ur ler 30
aai 0 est ser
510 100
uBBo oosy;dwoom3 o %wom ow33
asoe4 w10s u9s 3 4 1% 1
151050000000
20a32%8
aw1mm omaow
T2owos310t
a3 o2a 44
awi13s ok sar
(conttaued)
--_compouns 2s coc
gSSs. 82g
fNeetcbaibaotlitoen ::-: 3++ :: +
Table 1 (Concluded) I UITRDATSSAITS WCoIdTHaSnadnTsOIuLSAAGTGYPE
hadoMefiscCrpooegremapnFsaote 105000 1S.l35o00o00t1 251000
p1sr.0e0n0d!
TT MIaAciSdine Resvesteancs B per piaceor
BDoZoooommmoowemo4eso6 umo3mmoewnos oo3ow% oumoon nowaenodyees
3Zo5n B
nmoomno hoa onnonh AW B uw
iipsamn wom
B0Boomouoommow ooon%x Bom momo
wnBooomnm ow ow
hu4s oBnow wabaeen wow on
Bodom ouoxm own 4 dem
hin.eocnoesp.ount forsed a prectpicate at chia concentratson.
compound NeBgiatoive control PoSsoideituvme aCzoindeerol Simcirnoofslcroirdeinnse 2-tochzanine
SSEp Tawa 38
Mfeettsibuoalttieon -:
: ++ -z -: -: ++: +1+
IN VITRO TAS2S5A4Y0S AdoWdfiecdCepooengrpoomuPsnldate
soLoo 5100 2Tso 25 510
51000 L500000
5100 150000 31.000000
Tavte 2 ICoTCHanSdAL1HO-N2E5L1LAGToYPING
TRIE TAHtSsc)idine ReTveIrtaSnts aTwmomswos wwoosw
505 380 wr 390
8a0es
2v%ousnls6 wo 38
BW ome7 ou8 Buu
w9 ouow wom
BBo 20 TTM oer
112s3 Toa
wesmr sso7r
oVBoo noommoonwoamosuwowsou
BW Bosuoumw : oa
w7 omow wow
pes PTlAaStSe wow omow
33m am3s ue wl
amon doms 1o0w Qe
Sue64a0o
3om o2m wow wous
oBw owkawno x
lour w ar agr oesm
xoBn o6 ow soaww
0osm 9
omw ow2 won
0ga wm
(continue)
conpound asi cae
@gg x g2
Hfoetctaubaotlitoen ::::
+ii ::i
Table 2 (concluded)
In yo Tas2s5a4v0s Gwiomansd a25v41eCoTG PR
AddoHefidcCroopmgeprroaunHnsadce ERIS MaisStiaine tevertancsper piace
10050 sm
Wmooaoommosmars7as2ws5 aw sos sod
mBwoooaAu how
doanwe ao%nw Gola
p1.e00l01
Bosox 3 uww onRoo7 nonnoa wsioem
0500 5,
B ZTooo nsoe omom ooos eommmouooAn se Mov Don
manoomnowo5s
os ow
mom dn a
5L.000000!
BWoowwo:aomomoBmoounboBoaob a odag
1 texte "he compound foreed a precipitate at this concentration.
[SdI --ompownd Rietagsdoegsse rast coc
ggox g&
ras coo
*1, torte.
nies " p TesS tap mRoR00 --
wosotte H-- UI gain peta pr pace
: xorow Activation _Deisn tcostor TT T W oomw va womu
:. 1Lo mwooww own omous
:::-
830te
xFI i
om ow wm oko
::3: b50it1e B 22To0oh%row 0ohBwoorymooau
:::: 30ot1l8a Byow omo08s oo0oow%om =oowm
:::: p5iota @B5 x ooooB%now omoN m oeimom
bie 4 I VITI0 ASSAYS 2WIT5H0SaGcactumRomces cermvIsiag 03
compound
AYcatcisvbaotliiocn C(oJnPuceernoctrervnaittvi)on CIT(T1s0puec7re)tauTosspercent T M(is1o0r%te )e tecor nbgSaearna vtisses
NTegsatoive Control
:-
es oww as3%
isas
[ 1.2.3.4-- Dleporybucane :-
0x.i0
s Samw asem
121600
250 coc es =
::-
001310
:
6
ins, oeow%on i3so
@867s
7T
[3
gg 8sg
::+
o0i1so
:
50
w mssoownls SeWeoo
na8
TH T
1 tose.
| ma
I Hs [ i-- rast cc 8$oggyu
notes ume sss wrTsscousonces coms >
tesotte gCraRecUep :: .1 oasm :::- ooooFisn ::Tomboeh ::: Soosih
TSTiEoRErsee SSionres siecpnts,i
A oma 2i0
anoonon ooam 2i0
Bo moooownnoR
we ow
i5i5s
wHuooownn oeoen won
u51s 3
HHoooosn a 3i
Negastoive contro Fos1i1c2,i3v,e4 cDoinscpeoanlybusane 2540 coc 2S$. &@Ss
Tanke 6 =IN VITRO ASSAYS WITT H skH ccuRoeces crmvist 13
Metabolic -+ :< -:: :++: ::
Concpeenrtcernattion GeITeSuperrviavorrs TMitoe rte tr aconba gaantos
8e 3 wa0 aieo
3563
0o.0o25
52 8PE aw
117%00
00o0.ss0 or
[LrE ew7 5e50o0 a 50
a5737 53
0:2 0o0.2r09
a8 PeEaR wo
2) 35.00
3 a42
sow
004
50 2 auSieo
ia)is
Compound Nbegsatoive Control Pos1t,c2,i3v,e4cDolnetprooxlyhutane T2541 Coc
S. S2 2
Table 7 IN VITRO ASSAYS WITTH23S4A1CCHCAoRCORYCES CEREVISIAE D3
AMcettiavbaotliiocn ++-z :++ ++
ConPceerncternattion (wlvor vie)
0ola.05 00.31 slloo 0o1s BLog
CellSsurpvorivaolrs 107) Pereemt [6s EI]0
2s.s5 3wm
52.30 3"3 TMT TH
75.60 97 s2n1 2n
EMCicorte (16) 33ss eoomn
w1o0 TT
2.400 w0o
Reconbnants, Survivors "5i.s4 12640000 uw20 T 537s 2i
"1, conte.
compouns Negeatoive Conceal Fos1t1e2t,v3e.4cloenproorlybutane 2s cor ggSs. 28S
ae 8 IN VITRO ASSAYS UITTH SihccuGuoRtowtcss cesEvISIAE 53
MAcettiavbaotliiocn -: -: ::: ::+ :i
CWornoceacrnceernav/ttai)on 00.002s xooaursss a02s 000.752s5 2350
GOTTsSurp1ve0it7v)oortspexcene TMi(t1o5e t%t)e Rr ecombm tSeaeavnite vsers
1r2e ooww 3280
5238
sEAeI mm
1250%0
Bsssosownom ei3n0o
n35.55
$mom sh
35s
PS5E e0ba TWeo
1x5.6
sCEoa 0%H
2
ore.
Table 5 IN VITRO ASSAYS WTI2T5H41SACCCaIcARONICES CEREVISIAE D3
Compound
MAcettiavbaotliiocn
Concpeenrtcernattion (uly or y/o
ollaSuprevrivao1rs 2107) percent
T M(ic1o0tse )e Recr osRbSneuarnvm ci2e0vors
Negastoive Control
-:
8T3e 1000 p3e.}0
3563
Pos1.t2.e5.t4vDeleCopnoerroslbutane -+
00.0022s8
5&.2 B ow w
110000
< T2541 coc 2
--
d0.o0s7
:
os
8227 99% 53.00
36s1
0 7 so
0
s3g
-z paotz
epit 7 00
11
8
++
0ol2i
+ o's
687s aa 5w5o0 62 a eo
25 57
i+
Toos
pwnet n5 oon5.0
2n
REFERENCES
1. Jc3.a0r0cMiccnChaoengmnie,cnaslEs.a.sCmhouPirt,oacg.eE.nNsaYtai.mnasAtachakedi.,SaSlacnuido.nBe.UlSlAaN./1m2iA,cnerso5.s1o3m5e-D5e1tt3ee9sctt:i(o1n9A79so)sf.ay of 2. waF.liktyKh.lantiZitinrmgomuesarmgaeanncnitds,anidnl-RSm.aectcShhcyahlur-ao3im-eynrci.etsroc-Ielnr-denuvicittsriiooanseo:goufaMnmoiildt.iontGeiecma.ngdeGneeontehcreo.rnversion
100, 63-69 (1967).
3+
Dg.oreJ.eniBnrgusitcekchannidqueV.s 83-96 (1973).
Wv.ithMayyeera.st.New
Ednevvierloonp.menHtesaltihn
mPeurtsapgeecntiicvietsy
6,
4: 2Ba5nNinf.orfaAlmuueeossrh,einfetE.maGni.dtagoGetunhmsee:ry,arMoeJ.mtaatbA.iocliMtiaelmslienrea,ndcaarndcdeirnHio.vgaetBniasvr.etsschPo.rfos2.-CaacNreectci.ynro:gens
Acad. Sci. USA 69, 3128-3132 (1972).
5. cBt.aersctNi.nsoAygmseetnses,m. Ff.oPrrDo.tch.eLeedN,aett.eacnAdtciaoWd.n. Ea.nSdciD.ucrlsaUtsSoAsni.7f0i,caAtn7i8o2in-m7p8or6fovem(du1c9a7b3ga)ec.ntserainadl
6:
g3.reN.mutAamgese,ns:W. for activation
Ea.nAdDsubtraascpttloeenr,itaesE.tforYsaymdsaetsteaemkcitc,ioomnba.indninFP.grocDl..iveLNaerte..hoAmcCoaagdre.cniaSnteoieg.ens
USA 70, 2281-2285 (1973).
7. JcP.laarscMmicinCdaosng.ne,nsNP.raoscE..muNtSaaptgi.enngsaAm:cna,d.BaJ.cSctieK.roibaoUlrSiA,t7e2sa,tnder97B.0s-t9Nr.8a3inAsm(e1sw9.i75t)h.DRetefcatcitoonr of
8. MKp.orloepEc.e.rMtoGyernto.felaGpaelnnaessta.iadn1d6M7B6,.A.f3D1r.7o-m3S2ti7otcsk(eu1rl9.7t9r)a.vSieoglreetg-aptrioontecotfintghepmruotpearteoyr.
9+ mc3.uatracN.giennoAimgecesin,scyaJ.ntedsMtcm.CuatnanMg,uetnasatniwdointE.hRecYsha.ema3sS1aa,klim.o3n47e-lM3le6at4/hmoad(ns1m9af7lo5ir)snd-emtieccrteisnigne
10.
aL.cetDv.itKyieri,n Ec.igYaarmeacscaekis,aokaendco5.ndeN.nsaAtneess.. 11, 4159-4163 (1974).
DPreotce.ctNiaotn. ofAcamdu.tagseern.icUS
13.
Lh.ipAs Clin.
PaTnoodxiircitoehlre. ar9no,dle7V.6o1f-F.7m7u1tSatga(em1on9ni7.6c).scMrueteangienngic-tceasrtcsinfoogrenciacrcirneolgaetniiocnity.
2
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