Document DDgqL79xm76DbyNKO9B9D6awM
ARRRE 3307
480
~~
CHROMOSOMAL ABERRATION TEST RESULTS USING MAMMALIAN CULTURE
CELLS
EE 1. General information
`Nomenclatureofnovel
{3
A
_
(according to [IUPAC nomenclature)
feompirricalmformula Structural formula and
I
ol
`method when unknown)
Purity of novel chemical
Bh
substance used in the
If [Lot No. of novel chemical
~
4
J
1
Hoke ofCiAmSpuNroi.ties
Melting point
DAVOT I S| [Vieponrpecsuee | =
=
Appearance at room Brown viscous
`Stability
Unknown
ete.
B
Sovak
`Water
om
Shand
solvents |
| socom |] 5<500 mmgg//mmLL ((mmeeaasusruerdoeondn ssittee)) |=*
1
H[yNdortoel]ysisproperty: unknown --_--
|
~ cn or s Bret TSCA h after preparation (confirmed on site at Hita Works)
IR
2
~
2. Cell ype and culture conditions
Cel name
cHLIU
Acquis.ition
SShiHinnakmkoaoFnoFuSoncudinadetanticioeonn,,
Bank
[Species | Chinese hamster | Acquisitiondate | April 17,2002__|
2st | serpin Serumtype and|
amountadded |
Newborn calf serum _(NBCS). 10%
Calyele About 15h
genNeroartoiofn ch(rmoomdoos)omes
Prodstar for We) Miko70J2un0y8a5k9u)KK.
196C
or | Plastic dish of SO am
Icnocnudbiatitoinosn "Temperature| concentration
37 + 05C 5%
3.59 mix
1) $9 acquisition, etc.
--~
Self-made or acquisition T-Self@maAccqeuisition (Oriental KoboKogyo KX.
[ Productiondate |
May9.2008
|
03050902
2) $9 preparation
[ Awmalwed | Denvaivemmera
[Spe|cRaiSDc|sorNamie g| n PBandsePR
|
[So 1mie| Dow |Abdommaicaviy |
Body wei:ght | 23352845| Do(sc@phearbiooddyawndeidgohs)age|| FpPPEByD30RmelmkegS/kgTohneesEitir ses
(3) $9mix composition
[Component _|'Amountin ImLof $9] Component | Amount in ImLof 9
SN
5 Glucos6e
wi
I~ mr Nephosphatebutfer| -- | HEPESQH72) |wml|
earn
C.ompany Saito, Does no conta V5 it
3
---
(4) $9 mix treatment conditions
Plate method 2. Flotation cell method 3. Other(
$9
SO (nal concentration) protein (final concentration)
[5%|
|
Ti mgml
|
[Rec | ovey tehame|
* 59 Assay Data (Oriental Kobo Kogyo KX.)
om sepokk[ram[PREF]| 4. Preparation of test substance solution Solventused[N| aDismtileledT Producer
|LotNo.|TGnjrecatdabele:| Purity) |
Reasons for The50t0estmsgu/bmstLaninceDwMaSsOnotanddiascseotolnvee,0dwh[isilce] 5500mmgg//mmLLiinn ddiissttiilllleedd wwaatteerr or
solvent selection | Sdhioscwoeldoraatgiooondatsrusopoemnstieomnpesrtaatteu;rfeuurtphet0rm2ohre,afttheerrperewpaasrantoiohne,attheumsidsissitiolnleodr
`water was chosen as the solvent.
Tesstosluubtsitonance
Dissolution _Suspension> Othe(r
)
Method for
--
nieisi," | timing alone gave a goo`dmaskuisnpgentshieons,ustpheunssithoen Labomixer was used for
soluble Storage time and temperatureofthe soplruetpiaornataifotner
[ram comenion |
Within 2 , room temperature
va Ge>
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1) Conditions for cell growth inhibition test
`Without metabolism| With metabolism
activation
activation
[S[ ee Sh 11RDoiuunma depilears:t6ic0dmismp h||DRioaumnetdepe rl:as6ti0cmdimsh|_|
Tneubato[r Noofimub|wo2n| prd5me|
gs
coe
|--No.ofin[oLcSuxl10a7mlt| edTc5xe10l7mlL s|
ho[AmountofS9mixadded| -- |Osmbdmn |
cTornedaittmi[ eonnts [[75599 proTtfeieinnaalfmicnoeanlcwceonintcmreaentwiroani| on|| _ o--n | _ 107o5m| n%gm|]|
|Recoverydme | igh | 18h |
~
Notes
a) Liquid quantity at treatment time. Recovery period [sic; level) after treatment: 5 mL/dish
b) Composition of medium used in the treatmentofthe test substance:
Without metabolism activation:
Medium 2.7 mL (= NBCS 0.3mL + 2-fold concentration MEM (x 2 MEM) 1.35mL + distilled water 1.05 mL) + test substance solution 0.3 mL. With metabolism activation:
Medium 2.2 mL (= NBCS 0.25mL + x2 MEM 1.125 mL+ distilled water 0.825 mL) + $9 mix 0.5 m+L test substance solution 0.3 mL
-
64747
Company Sanitized. Doss notcontain TSCA CBI
:
~~
(2) Cell growth andaberractelilon appearance frequenc:
doidt|r a alls SL =FEoeS)oT S eed | m kElLhE a=Ur --ey [C0 T7100 abundant| 0 1"210[100[abun | 2|0]
[30a [inion [Er[7882 1abundant |
50] no. |
139.0 T7905 Fr
hehe]
no. |781 |923 | abundant|mo | mo.
[156177051abundant |"no.| [3137176691 abundant| no. |
no. no.
| |
156 313
| |
864 #74
|abundant| |abundant|
no.| no. |
no. no.
| |
[res1750.0 T [1250 17530
abundant 1010 [625 abundant | 6 |0 |1250
| 79.1 | abundan | | 753 |abundam|
0 4
|0] |3|
[25007 | 642 | abundant
A S BL eECLm e Tamsmgsmmsn 000"|557| abundant
315 [os00 [726 | abundan [4 |0] 60 | 5000" | 660 |sbunaam | 4 |0]
~
~
Com St,sm 50
"
~~
Chromosome aberration test conditions
`Without metabolism| With metabolism
[ edmio | Anowr omm |Tpane] [Sh|a Roundplasticp dish|Founde pasicdish| [Si |Diametz er 60mm | Diae meter 60mm|
. ofNo imbm| onp2er5 do] e Cells |-- No.ofinoculatedcells |15x10%mL__| 15x107mL |
gg Amowtof| Som_ix|aOSd mie ashd|
S5BE3ST S [ ofemmS a acoil memc esi| oo nr | nce = en --n n w|a |ta 7si omano yn ]|
Recoveryme
| 18h | ish |
Notes
a) 'b)
hon mm soni Liquid quantity at treatment time. Recovery period [sic]aftertreatment:
Composition of medium used in the treatmentofthetest substance:
5 mL/dish
Medium 2.7 mL (= NBCS 0.3 mL + x2 MEM 1.35mL +distilled water 1.05 mL) +
~~
test substance solution 0.3 mL.
`With metabolism activation:
Medium 2.2 mL (= NBCS 0.25mL + x2 MEM 1.125mL + distilledwater 0.825 mL) + $9 mix 0.5mL + test substance solution 0.3 mL Cell growth was calculated from the numberofcells counted by electric cell counter (Microcell Counter CDA-500)
(4) Chromosome aberration test results (Table 1)
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`CompanySanhized. Do note conts ain TSCA Cay
,
stor| Ci ior Farias ~
6. Continuous treatment test
(1) Cell growih inhibitiontestconditions
Tetdwmion| onsen
[Spe|" Roundplasticdish
Se
I Diameter 60mm |
p-
NoN.oo.foifnicnuobcautloartsepdecrodlosse isi
memSCe |eATb OM Oe Ftest i substao nce son lutionda| ddeo d" ra03ai mmUsdo h n|
conditions
Recoveyime
|
on]
Cheibiltilon growth
Number (oMficcerlolcsecloluCnotuendtbeyreClDecAt-ri5c0c0e)ll counter
N4)otCeomposition of medium used in treatmentoftest substance
Mteesdtisuubmst4a.n5cmeLsol(u=tiNonB0.C50m.SL5mL + x2 MEM 2.25mL + distilled water 1.75 mL+)
--_
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CompanySanitzed.Doss not conta TSCA C1
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~~
(2) Cellgrowthandaberractelilon appearance frequency
Without metabolism activation (24-0)
(Dgomse)|| groCSewl|tlh
| Relative
| splitPienxdex
| Incidence|
|| of split
Aberration cll pearance
conToqge|ncy? N(um5e)rical
[3oI 00o oimwe|]igJ wos]F7 amaann||e B 0 s 0 e 0
[T(E1o5E6t8s2|2n0o8 |= amfemw 0 56 0 Ts] e]
e 260 Toonee ] ----
[250077165| no. |nome | mo. | no |
[5E 0067T | 11T7 | mo. | nome | no. | mo |
or no dani miccscope )b) aCbaulncdualnatt:edsubfyfiscpileinttceslpllisticnel5ls0.0 cells afew: few split cells
d) 50 cells were observed
rare: very few split cells none: no split cells at all
~
F: Test substance precipitation occurred at the beginning and end of the treatment,
Note) Upper limit of dosage was set at 5000 g/mL
--_ coe
Company Sess Doss etcontan750Aot
,
~~
(3) Chromosome aberration test conditions
CL amin wom Swe
| Rowdpwuda
[Sk |Damce tr60mm |
5 miJdish
Roofesto prde Cll
No. of inoculated cells'
: Amount of test substance solution addedTM |0_. __5_ ml/dish|
TTrarme amentime| | ondtion
Recoveytime
|
on
|
Note
2) Composition of mediumusedin treatment of test substance
Medium 4.5 mL (= NBCS 0.5 m+L x2 MEM 2.25mL + distilled water 1.75 mL) + test substance solution 0.5 mL
Number of cells counted by electric cell counter (Microcell Counter CDA-500) for cell growth
(4) Chromosome aberration test results (Table 2)
--_
(5) Split index in chromosome aberration test (Table 3)
7| Rresetortattionn snd Erm oniey ae [ren Game e Reasons for evaluation i `The evaluation is given because of no chromosomal aberration increase in both the
~
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`CompanySenlized.DossnotcontainTSCA C1
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--_
) Notes
1. Reasons for setting dosageoftest substance,
Cell growth inhibition test results
Seem[orm | ston |sompgnnin
In short-term treatment, the maximum dosage for observing mid-term split images needed for analysisofchromosomal aberrations had an upper limit of 000 g/mL in the presence or absence of the S9 mix. Bothinthe presence and absenceofthe S9 mix, with a `maximum dosage at 5000 ug/ml, and with the appearance of light cell toxicity, 1580 and 500
ng/mL diluted at a nominal ratio [sic]of V10 were set.
In the continuous treatment, the maximum dosage for the observationofthe mid-term
split image needed for analysisof chromosomal aberrations was 78.1 pg/mL. At 78.1 pg/mL,
cell growth was 91.5%, and the relative split index was inhibited to 20.4% of the negative
control, thus a distinct cell toxicity was judged to occur. Thus, the concentration for inhibiting
50% of spit cells in the cell growth inhibition test was about 52 pg/mL; with a maximum dosage
~~
of78.1 pg/mL, 39.1 and 19.5 pg/mL diluted by a nominal ratioof 2 were set.
2. Toxicity evaluation in continuous treatment
AU78.1 pg/mLsetas the maximum dosage used inthecontinuous treatment, the cell
growth in the chromosome aberration test is 86.9%; cell growth inhibition exceeding 50% is not
observed, while a relative split index compared with the negative control is decreased to 38.3%;
thus, itijudged that a distinct cell toxicity needed for evaluating chromosomal aberrations
occurred.
3. Chromosome aberration evaluation standard
A positivejudgment is given when the appearance frequencyofcells having structural
`aberrations and numerical" aberrations increased more than 10% with a dependence on the
dosage or when an increase exceeding 5% is regenerated in the chromosome aberration test and
confirmation test; all others arejudged as negative.
4. Figures
Figure 1: Cell growth inhibition test results of short-term treatment usin
3
Figure 2: Chromosome aberration test results of short-term treatment usin
Figure 3: Cell growth inhibition test resulsofcontinuous treatment using
Figure 4; Chromosome aberration test result of continuous treatment usin
~~
*Not certainif"numerical" inthis case indicate the "numofbsbeerrartio"ns.)
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8. Others Test facilites
a H3i-t8a22WoIsrhkiisT ,;HitaK,agOakiutaBusshitsu Heikakenkjukiku
Fax 0973 (23) 9800
Principal tester Name. `Test Department 3
Yoshikazu Anjimi
[Byerene| 623/03 wo 11/13/y03es
|
LLTetNo
777
Reed
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[TFaebsletm3:eSnptliit minede)x in|c5h9rmoimoxs|omDeoasbaegrerat(ioungt/esnt)(c|ontRienuloautsivtereastpmietnitn)dex
Negative control
(distilled water)
100
#0
CoerrTwo----|
Relative split index is calculated based on 100% as the split index of the negative control. a) Calculated with the observation of 1000 cells per dose.
--~
64747
CompanySanltizod. DossnotcontainTSCA CBI
5
--
Cell growth (%)
T w| o e
.
PY
or
a
Dose (pg/mL)
- 89 mix
~
Cell growth (%)
S " e
.
Py
wo
Po
es Dose (g/mL)
Figure 1: Cel growth inhibition test els of shotfem reatnent wo --)
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CompanySalized. Dos ot ont TSA cn
--
Appeara%nce frequency of chromosome aberration cells (5%)
`Onumerical aberration
structural aberration
6
| |
'
|
||
: | To
0-
Soo
Dose (ug/mL)
- $9 mix
Appearance frequency of chromosome aberration cells (%)
~~
*
sOtnruumcetruircaallaabbeerrrraattiioonn
||
|
wo.
I) 10 DoSseo(umgi/xml) on)|
~
Figure 2 Chromosome aberration test results ofshot erm treatment wr f--)
64747
Company SanftizedD,oes notcontalnTSCA cay
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--_
Cel groTwoth (%)
oN
o0
10\ 0 L L000 10000
2D4oshet(reuagt/mmeln)t
Figure 3 Cell growth inhibition test resultsofcontinuous treatment wn j--)
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1 ~ Appeara3n0ce frequeOncnyuomfercihcralomaobserormaetiaobnerration cells (%)
structural sberration
'
|
10
oo
155
391
pi
Dose (ug/ml)
24h treatment
~~
Figure 4: Chromosome aberration test results of continuous treatment in --
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Company Sanlized. Doosnotcontain TSCA CBI
1
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Responsibility Chart
`Title: Chromosomal aberration test ---
`mammalian cultured cells
Principal tester Yoshikazu Ajimi
`Test planning, management of overall test, specimen observation,resultsanalysis, evaluation, report preparation
g
Preparation and treatmentof test substance, specimen observation
[ShinyaWakaborasi |specmen isrvaion
--~
-- 6a47
`CompanySanltzed.Doesnotcontain TSCA CBI
RELIABILITY WARRANTY
Kagakubusshitsu Heikakenkyukiko Hita Works
Test Requester: Dupont K.K.
Test
Test
Title:
Code:
Chromosomal
K06-1022
aberration
test
--...
`mammalian
cultured
cells
KagakubTuhsisshitetsstuhHaesibkeaeknenakuyduitkeidkoor. iSnusppeercvtiesdibonyotrheiQnsApecDtiivoinsidoantosfHaintdadaWtoerskrse,ported o the principal tester and managemenrte given below.
`Auditing or inspection tems
inAsupdecittiionngdoart| e | inspAeucdtiitoinngreosurlts
lest plan documented
Fe"Asmtesnudbmsetnantocefprteesptarpaltainodnoacnudmetnettsare
report date
T7505 gangs] | 7772003| 77005]
~~
RAemcoerdnindg
manoedf
neresptotrt
pldarnadftocument
(2)
[722005[7730005]
|| 110052417722000053|TTo0r201172300050--|]
[D[T EFoAimnmnaaaetllnrrrdeeempppeooornrttddroy rafafttteesrtvevipileaewnwdocs ument (No.3) o |1--j1001r70s37002r7n 72a000000330--| o1] 0307p2003
accurate"lTyhirsefrleepcotrtthaeccruarwatdealtyaodefstchreibeesstst.h testing method and procedure and the reported results
November 13, 2003 Keiji Shiraishi
Reliability Warranty Officer
Language Services Unit Phoenix Translations January 14,2004 --~ cara
`Company Sanitizod. Dossnotcontain TSCA CBI