Fa= Approved. O.M..B. ~o. 2070-0012 Approval Expu-eo 10-31-96. 

U. S. ENVIRONMENTAL PROTECTION AGENCY 

A EAtA PREl\iA.NUFACTURE 0 1-\ NOTICE 

AGENCY USE ONLY 

Date of receipt 

Whl'n 

compktl'J 

~end t!ll' .. 

torm tll 

FOR NEW OIEMICAL SUBSTANCES 

DOCUMENT CONTROL OmCER 

OFFICE OF POLLUTION PREVENTION 

AND TOXIC SUBSTANCES, 7407 

U.S. E.P.A. 401 M STREET, SW 

. WASHINGTON. D.C. 20460 

Enter the total number of pages 

in the Premanufacture Notice 

'-· r 

Company Sanitized 

• You mut provide alllafoDIUitioa reqa..-..d Ia tlaia to-to the edeat tUt lt Ia kaoww. to or _.-,ly. ucertabaable by }'U'IL Make ~le estimata If you do 

not have actul. data. 

• Before yoa CXIIIlplete thia form. yoa llhoald lUd the "'utradiou Malul for Pftmauafac:tue Notificali011" (the lMtructiou M.u.ul ia n-ailable from the Toxic 

Substances Control Ad <TSCAl baformatioa Service 'by calling 2112~14CM,. or fllmlr; 202·554-5603l. 

• If a user fee haa been temitied for thia notice (40 CFR ?011..'-5), indiate iD the biiXIS ahem! the TS-uer fee ldmtifiati011 a'IIJII.ber you hne genaated. 

Remember, your uer fee ID number mut abo appear Oil your mnapoadillr; fee nmiltace, which Ia aeat to: EPA, HQ Aa::ouatiDg Operatiou Brmch (PM-266), 

P.O. 360399M,. Pitbb~ PA 15251-6399, Atm. TSCA UMrFPart I- GENERAL INFORMA noN 

You mut provide the CII.Il'elltly cum!d Chemical Ab.t:rada (CAl Name of the 

aew chemical•abiiQnce,. even If you c::J.al.Jil the ldeD.tlty • confldeatial. You.._,. 

authorlze aothu penon to nbmii chemka.lldatity iDfoDIUII:i011 foryoa. bat 

your •ubmiuioa wm-t be cumplete and the review will aot bepa .. w EPA 

receiva tAla illfo~oa. A 1etlez: ia ••ppod of yav .. .,__o• llhollld 

refereDCI! your TS uer fee ldeatifical:ioa aiUDbeL Yoa aut lllllnait • odp...l 

md two cupls of thia aotke illdadllls all tat data. If,_ dai.-1 aay 

IDformatioa • cunfid-tial. a .mgJ.e uaitlad mpy a..t ailo be nblllitt.d.. 

Part n- HUMAN EXPOSUltE AND ENVIKONMENTAL ~SE 

If there are ...vera! m.mafacture. piOCI!Uing, or ue operatiou to be dscrlbed Ia 

Part li, sediona A mel B of thia aotic:e,. repmclace the s.edla• • needed. 

Part ID- UST OF ATTACHMENTS 

Attach additional aheet8 If there ia not enoogh space to answer il question fully. 

Ubel each contlnaation sheet with the conapondillr; Ndioa headiJI.r;. In Part 

III. list thae attoK:hment., ;my tat d&a or other d&a ad ay optional 

information IJI.daded ill the notice. 

OPTIONAL INFORMATION 

You may illdade uy iDfol'JIUitioa tlUit yo• wul: EPA to CIIUici• b. enlutlaJ 

the aew •abstmce. 0. page 11 of thi8 fora. •!*Z U. b-. pzurided for yo• ID 

d-=ribe pollattoa pl'eYeDiioa md recydlaslafuz:aatiaR JV•..., li-. npaillls 

the new sabRance. 

So-c.alled "bindiJI.r;" box• are illdacloed tlmnaghollt tail &ma foryv• to ilacilca 

your willlnp- to be boaad to Cl!ltabl .. .._.....,. yo11allk ... b. thi8 aotlce, nell 

as uae,. produdlon volume, prota::tive ecrulpmeal ••• TliJ8 optioa. iate.ded to 

reduce dela}'8 th~ matinely aa::ompany the deorelopaamt .of CDII8at ordta or 

Significant New UIIC Rules. EJuzpt ill the CIM of eumptioa appUatia• lluch • 

TMEA, L VE, LOREX> where certala lnfouaatioa provided Ia nch •otlfiatioa ia 

binding oa the Rbmil:ter whea tile AlleDCY appiDV• lila DZ~Dptioa appllcatioa. 

checldll.& a biDdillr; box ill thia aotice ~by ll:8elf pmhlbtt the ll1lbmittllr 

from laer devtaill& &om the illfomutioa (except chaakalldatity) n:poned Ia 

the foiDI.. . -

CONFIDENnAUI'Y CLAIMS 

You may c:laia my l:a.fomudioa Ia tiLia Bot:ice • CIOIIfidestW. To -at a c:l.ta 

on the form, Ja.uk 00 tbe coafideatial box .at ID tile~ tlut yo• dai& 

• cunfideD.tial. To ..-a daia Ia - ettach•at, drde or bmck.et U.. 

IDfoanatioa yow. dalm • mafbieatial. If !pp da!m tvtnnnt!qn lp the pgtig: a· 

mnfldmtial YAP mplt abp mpyidc I vnHiv;d ycgiPP gf tllc pgtig; flpdgdt,nl 

i¢tlebmepgl. For addltio&al i..aadio .. - d.liJaia& IJabvwKI- • 

coa.fid-tial. rud the 1a.RradiDBa Manu.al. 

D Ma 00 If ~y infof'IIUitioa lD thia •oticz ia daimed • coafideatial. 

EPA FORM 7710-25 . (Rev. 5-95} 

. I 

TEST DATA AND 01liEll. DATA 

You aa required to nbmit all tat data iD your poueulon or 0011trol • 

and to proYiGe a cieKEipti- of all othB' de knowa 110 or -ably 

MC?Jta!nable by yoa.lf th- data ue re1Red to the ilealth ud 

m...UO...tal effec:D of the -afal:taa,. ~ diRdbatloa 

in CDIIIJIIell:a,.-.. or dlapoul of the •-c:beaic:alll1li:IRaD.CL Stadard 

lltentu. dtatloa..., 8e 1111bailted far- cia Ia the opa ldadflc 

iltantuJL CmgpJcte tat d;U !writtrp lp Ep&!bbl pgt pmmaria gf dW. 

m!!!!l be nP•Ittcd If tbct dp PPf-.JPllCII' tp the gpcp l!tmtpz. Youllhoukl 

de.ady idatlfy wiletller a.t daa 18 oa the nbRaDa: or 011 aa aalog. Al8o, 

the chaalcal mmpadloa of the le*d JUtaialllhollld be ciYracterizecL 

FollowiJlr; ilft -pi• of tat data ud other data. Data llhoald be 

•ubmitled ac=mil.Jlll to the nquiremma of §7211.50 of the Premaaafac:tue 

Noti6atioa 1tule (40 0'11. Part '720). 

Tat Data (Check Below my·indaded in thi8 aotice) 

• E.a'rimammtal f&e data [il Ys • Othudata 0 y.,. 

• Health effec:D data [if Yes IUI.k-en• 

• E.avirimmeatallffe:ta data D Ys Stmdareilldtrity rdationahip8 

• PhyU:al/C'aeakal Ptapedia • D Ys Tst data aot !.D. tlae ~-

or matrol of the .U.utter 

•A phymai and clte.ic:al piOpCHs _...... ia loc:aled oa the lat pap 

ofUU.fo-. 

~ TYPfi OP NOTICE (a.eck Oaly a. S 

~ PMN aor-..atadut ~ ~ fg ~ 0 IN'IEKMEDIATE PMN (aabaltteclla MCI"-~ ~ad PMN) . .._-

0 ~ n< 

SNUN (Sipifkaat Nellt U.e Notla=) :X - f"'1 

<JO 0 TMEA (fell MukeliaJEumptioa A~ 

D LVE CI.-Vol-Eze.ptioll) •• CFR~C)(]) 

0 LOREX a-~ ExJ>o-re Ex-p&.) e 723.!0(c)Q.) 

0 L VE Modl.fkatloa 0 LOREX Modifial:lc. 

IS nns A coNsoLIDATED PMN7 0 Y• 

lofc:haak::Ua 

!Preaotica Camm..ucatioa I ""'!aiftd, alar t on ~e 31 

Replaces previOWI editions of EPA Form 7710-25 . 

(oS ?,ll} 
..• 

Public reporting burden for this collection of inionnation is estimated to average 110 hours per response. including time for reviewing instructions, 

searching existing data sources, gathering and maintaining the data needed. and completing and reviewing the collection of information. Send 

comments regarding the burden estimate or any other aspect of this collection of information,. including suggestions for reducing this burden. 

to Otief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M St, S.W ... Washington. D.C. 20%0; and to the Office of 

Mana~ent and Bud~t. Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

I certify that to the best of my knowledge and belief: 

1. The company named in PaJ1 I, section A, subsection la of this notice form intends to manufacture or import for a 

commercial purpose, other than in small quantities solely fur research and development, the substance identified in 

Part I, Section B. 

2. All information provided in this notice is complete and truthful as of the date of submission. 

3. I am submitting with this notice all test data in my possession or control and a d~ption of all other data known to 

or reasonably ascertainable by me as required by §720.50 of the Premanufacture Notification Rule. 

Additional Certification Statements: 

If you are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNi.JN, check the following user fee certification 

statement that applies: 

0 The-Company named in Part I, Section A has remitted the fee of s2500 spe:ified in 40 CFR 700.45 (b), or -

0 The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined @ 40 CFR 700.43) in 

accordance with 40 CFR 700.45(b), or · 

0 The Company named in Part I, Section A is a small business concern under 40 CFR 700.43 and has remitted 

a fee of $100 in accordance with 40 CFR 700.45 (b). 

If you are submitting a low volume exemption (L VE) application in accordance with 40 CFR 723..50 (c) (1) or a Low 

release and low exposure exemption (LoREX) applicatiOn in accordance with 40 CFR 723..50 (c) (2), check the following 

certification statements: 

D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for 

commercial purposes, other than in small quantities solely for research and development, under the terms 

of 4D CFR 723.50. · . . , 

D The manufacturer is familiar with the terms of this section and will comply with those terms; and 

0 The new chemical substance for which the notice is submitted meets all applicable exemption conditions. 

0 If this application is for an L VEin accordance with 40 CFR 723.50 (c)(1), the manufacturer intends to commence 

manufacture of the exempted substance for commercial purposes within 1 year of the date of the expiration of the 

30 day review period. 

The accuracy ~f the statements you make in this notice should reflect yow- best predictial of the anticipated fads ieganfing the chemical 

substance described herein. Any knowing and wiJlful misinletpn!tation is subject to aiminal penalty pursuant to 18 USC 1001. r-C-o-nfi-.d-en-, -tW.--1 

Signature and title of Authorized Official (Original Signature Required) Date 

Signature of agent- (if applicable) Date 

FORM EPA 771~25 (Rev. 5-95) Page2 
Part I- GENERAL INFORMATION 

Coofi· 

~~~~~~~~~~~----~~~~~~--------------------------------------------~dentiL 

la. Person 

Submitting 

Notice 

• (in U.S.) 

b. Agent (if 

applicable) 

and street) 

Cty, State, ZIP Code 

Name oi authorized. official 

Company 

Mailing address (number and street) 

Cty, State. ZIP Code 

Telepboae 

c. If you are summtting this notice. part of a joint submission. marie 00 ~box. ---------------~ 

Joint Name of autbCllized official 

Submitter 

(if applicable) 

2. Technical 

Contact 

(in U.S.} 

Company 

Mailing addrell (number mci street) 

Cty, State. ZIP Code 

Name 

Company 

Mailing address (number and street) 

Qty;State, ZIP Code 

3. If you: have had. a prenotice .communicatioa (PC) amCI!I'Ding this notice 

and EPA assigned a PC Number to the notice. enter the number. 

Poailica 

Telephone 

Telephcae 1 

I 

Mark (X) 

if none 

· 4. If you previously submitted an exemptKm applic::aticD foe the c:helnDi 

substaDce awered by tlUs-notioe. enter the exemptioa number l!!lllligned 

by EPA. If you previously submitted. PMN fot this .w..nce enter the L b 2 Y)? Made 00 

PMN number llllligried by EPA (i.e. witbdzawn ar inalmplele). . - 0 - 'J () If nooe · 

~ 

If you have submitted a notice of Bona fide intalt to manufacture ar 

import foe the chemical substance covered by this notice, entl!r the 

notice number asaigued by EPA. .... 

Type of Notice- Mark 00 1-D~-Optkm 

FORM EPA 7710-25 CRev. 5-95) Pap3 

MarkOO 

lineae 

----:)lo .. D 

3. DBoth 
Part I -GENERAL INFOR!viATION- Continued 

Section B - CHEMJCAL ID~VITTY INFORMATION: You. •ut provide • Clln'entiy comet O!cmaJ. Abatrac:t3 !CAl name Oi the ~b.ta:na bueoi em thnunth Collecuw lnoax (9(J) of CA nommcl..arun rules an~ <:oriYeZ~tioN. 

Uxltplete eitber item 1 (ClaA 1 or l~~abttancea) or l (Polymera) u appropriate. Complete all othEr itema. 

lf another ~ will submit chemic:al. identity informati011 for you (for either item 1 or 2), mark (X) the box at the risllt 0 

and addres of that in a sheet. )II. 

1. ClaN 1 or 2 c:bemicalmbiMa.Dces (for definiliona of cW8 1 and c:.la!e 2 substances, !lee the Iru~c:tioa• MamW) 

a. Cl.a!IS of substance- Mark (X) 1 0 ea.. 1 . or 2 D CJus 2 

b. O.emical name (Currently·c:on-ec:t O.emiaal. Abstract3 (CA) Name that is c:oosistent with T5CA Inventory listings for similar subetanc:es. 

For a- 1 substanc:e! a CA Index Name must be provided. Far au. 2 substaiia!s either a CA Index Name or CA Preferred Name 

be provided, whichever is appropriate bued on CA 9C nome:ndature rules and conventions.). · 

c. Please identify which method you used ID dewlap ar obtliD tbe specified c:bemic:al indentity i:nfcanati011 repcrte:i in this notice: 

(c:hec:k. one). . . 0 Method 1 (CAS Inventory Expert Service- a a:1py ri tbe Iden~tion QMethod 2 (Other Sourt:e) 

report obtained. from the CAS Inventory Expert Service must be 

submitted a an attachment to this notic:e) 

d. Molec:ular formula and CAS Registrjr Number Of a number already ex1sls for tbe subatance) 

CASt 

e. For a dua 1 subltanc:e. provide a cxmpj* mel cmra:t c:hemkal~inM:1:J.D'e dlagrmL Fer • d.. 2 IUJ:wannr - {1) IJit tbe immediate '\/ 

precunor Sl(u.taDces with their rapecttve CAS Repay Numbln. CZl Daaibe tbe Datare of the r-=tkla ar prcaa (3) lndicate the 11.. 

range oi cxmpoation md the typlc:al. cxmpaatlaa (where apprapdale). (4) Provide a curect repraatlatlve ar pctial c:bemiCII. 

structure diagrmn. as cxmplae • em be kDown, If aae c:. be ~y ...a:t.I:P&:l 

/ 

7 •. 

Mark (X) this box if you attach l ccnt!nuatioo sheet. 

FORM EPA m0-25 (Rev. 5-95) Page 4 
\ Part I -GENERAL INFORMATION- Continued 

~ Section B- CHEMICAL IDENTITY INFORMATION - Continued 

:z. Polymers (For a definition of polymer, see the lnatractiona ManuaL) Con: 

dent 

a. Indicate the number-average weight of the lowest molecular weight composition of the polymer you intend to-manufacture.. ~ 

Indicate maximum weight percent of low molecular weight speees (not inducting residual monomers, reactants, or solvents) below 

500 and below 1,000 absolute molecular weight of that composition. 

Describe the method• of meuun:ment or the bam for your estimatu: GPC 0 OtherO :<Specify> ~ 

il lowest number average maiecular weight: 

Ul maximum weight "' below 500 molecular weight: 

iiD maximum weight"' below 1000 molecular weight: 

D Mark (X) this box if yo~ attach a continuation sheet. 

b. You must make separate confidentiality claims for monomer or other reactant identity, a:nnposition information, and residual information. 

Mark (X) the "Confidential" box next to any item you claim as confidential. 

(1) - Provide the specific: c:hemic:al name and CAS Registry Number (if a number exists)· of eac:h monomer or other reactant used in the 

manufac:ture of the polymer. 

(2) - Mark (X) this column if entry in column (1) is alnfidentiaL 

(3) - Indicate the typical weight peram.t of eac:h monomer or other reactant in the polymer. . 

(4) - Mark 00 the identity column if yoa want a monomer or other reactant uaed at two weight percent or lea to be liated aa part of the 

polymer description on the TSCA Chemical Subatanc:e Inventory. · 

(5) - Mark (X) this column if entries in columns (3) and (4) are confidential. 

(6) - Indicate the maximum weight percent of eac:h mcn;tomer or other reac:tant that-may be present as a residual in the polymer as 

manufactured for c:cmmerdal purposes. 

(7) - Mark 00 this column if entry in column (6) is alnfidentiaL ' 

Monomer or other reactant and CAS Registry Number Confi- Typical Identity Con:.fi- Maximum Col 

dmtial c:ampositian MarkOO dmtial residual der 

(1) (2) (3) (4) (5) (6) . t 

.,;, r. 

·'" •% 

,. .,;, 

.,. ,. 

.,;, r. 

"· ,_ 

,. .,;, 

0 Mark (X) this box u you attach a alntinuatian sheet. 

c. Please identify whic:h method you U!ed to develop or obtain the sped.fied chemical identity informatiOn reported in this noti.ce. (c:hec:k one). D Method. 1 (CAS Inventory Expert Service- a copy d the identification report D Method. 2 (other source) · 

obtamed from CAS Invmtmy Expert Servia! must be submitted as 

an attac:hment to this notice) 

d. The omently correct <llemic:al Absttad:s (CA) lWl1e for the polymer that is amsistent with TSCA Inventory .l.istings far similar polymers. 

.. I e. Provide a correct representative or partial c:bemic:al structure diagram, as c:cmplete as c:an be known, u one c:an be reaaonably ucertained. 

...... 

. - -

. 

- I 

0 MarkOO this box u you attach a~~~ sheet. 

FORM EPA mG-25 (Rev. >95) PageS 
Part I -GENERAL INFORMATION- Continued 

~ Section B - CHEMICAL IDENTITY INFORMATION - Continued 

3. 1m purities 

(a.l- Identify each impu.Iity tha may be -!Ubly uticipaed to be prant In the chemic:.a.l nbtltaae • m.aaubaured far cnm.mad.a.l PurpoML 

Provide the CAS Regbtry NllDlber If available. U there 1ft IUiidmtified l.mp1Uitiea,. allu "'uidmtlfled. • 

(b) -&Um..te tM maximllDl weight'" of eaclllmpvity. U there are llllidaatified lmpuritiea, mtimllle their total weight'"· 

. Impurity and CAS Registry Number Maximum Confi- ~ 

(a) percent cient:ial jb) 

' 'l'o 'f.._ 

'l'o '/... 

'l'o 'f.._ 

cr. "~--

'!'0 

.,.. 

cr. 

0 Mark 00 this box if you attach a continuation sheet. 

4. Synonym•- Enter any chemical synonyms for the new chemical substance identified in subsedian 1 or 2. . ~~ 

X 

0 Mark 00 this box if you attach a continuation sheet. 

5. Trade idmt:ific:ati.OD - List trade names for the new chemical subl!ltance identified in subeed:i.oo 1 or 2. X 

0 Mark 00 this box if you attach a continuation sheet. 

6. Generic: c:hemic:al name - If you claim chemical identity as confidential. you·muat provide a generic chemical name far your substance 

that reveals the specific chemical identity of the new chemical substance to the maximum extent possible. 

Refer~ the TSCA Chemical Substance Inventory, 1985 Edidan, Appendix B for guidance on ~eloping 

genenc names. . 

l\ c y '/ \C\.\ ~ cf ~yo( 'rOK'j iW~',de_ 

D Mark 00 _this box if you attach a c:cntinuatian sheet. 

7. Byproduc:ts-...... my byprociucts resulting £ram the manufacture. processing. use, or dispoul. ci the new dlemical subetanc2. Provide the CAS 

R~~. ·Number if available. . . 

Byprociuc:t CAS Registry Number Confi-

(1) Q) dent:ial 

. -- x· 

-

D Mark 00 this box If you attach a mntinuaticn sheet. . -

FORM EPA 7710-25 (Rev. 5-95) Page6 
Part I -GENERAL INFORMATION- Contiriued 

~ Section C- PRODUCI'ION, IMPORT, AND USE INFORMATION: 

Mark 00 the "Confidential" box next to any item you claim as c:cmficlentiaL 

1. Production volume- Estimate the maximum production volume during the first 12 months of production. Also estimate the 

maximum production volume for any consecutive 12-month period during the first three years of production. Estimates should be 

100% new chemical substance basis. For a Low Volw:zte Exemption application, if you choose to have your notice reviewed at a lowE 

production volume than 10,000 kg/yr, specify the volume and mark (x) in the binding box. If granted, you are bound to this volum• 

Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confi.. Biadiq 

dential Optioa (100% new chemical substance basis) (100% new chemical substance basis) MukW 

X 

2. Use Information- You must make separate c:on&ientiality claims for the dei!laiption of the category of use, the percent of production volume 

devoted to each category, the formulation of the new substance, and other use information. Mark 00 the "Confidential" Box next to any item you 

claim as (X)nfidentiaL 

a. (1) -Describe each intended category of use of the new c:hemical substance by function and applicatiCll\. .. 

(2) - Mark 00 this column if entry in (X)Wmn (1) is amfidential busineas information (CBD. 

· (3) - Indicate your willingneas to have the information provided in calumn (1) biDding. 

(4) -Estimate the. percent of total production for the first three years devoted to each c:ategary of UBe. 

(5)- Mark (X) this calumn if entry 1n (X)lumn (4) is canfidmtial busineas information (CBD. - (6) - Estimate the percent of the new substance as formulated. in mixtures. suspealiaaa, emu1aions, solutions, or ~ 

as manufac:tured for commercial pmpoaes at sites under your control a-xi !!ted with each category of use. 

(7) - Mark (X) this calumn if entry in CDlumn (6) is c:cmfidential busineas infarmatiaa (CBD. 

(8) - Indicate " of prodw:t volume expected fa the listed "UIIe" sec:tan. Mark more than ane box if appropriate. Mark 

(X) to indicate your willingneas to have the Ulle type provided in (8) binding. 

(9) - Mark 00 this column if entry(ies) in a:ilimm (8) is (are) c:onfldential bwlinls inft;aw.aioa (CBD. 

. 

Category of Wll! (1) em 1liacUq Produc- CBI 'lit in pn " of subltance expected per Ule ~ (by function and application i.e. ·a dispersive dye for Optillla tioll" Foan- (8) Madt(x) ulation finishing polyester fibers) 15Ue- ICeD-• iJldul.. Cam- ~inciing (2) (3) (4) (5) (6) (7) limited ~er trial merdal Option I 

X " )< " ·'Y.. ·1 1\ 

% % 

% % 

% % . 

% % 

-

" " 

" % 

•If you have identified a "consumer" use, pleue provide on a continuation sheet a detailed description of the use(s) of this chemical 

substance in consumer products. In addition include estimates of the concentration of the new chemical substance as expected in 

consumer products and describe the chemical reactions by which this substance loees its identity in the consumer product. 

D Mark 00 this box if you attach a tDDtinuatian sheet. 

b. Generic If you claim any category of uae delaiptian iD IUbaec:tica 2a • amfidential. enter a geDeric dellaip&a of that 

use category. Read the IDM:rllctiaaa Mamaal fa examples of gmtric. uae dac:riptimiL • 

desaiption 

~ dk~~\U s:~ 

D Mark 00 ttUa boxtfyouattac:h a tDDtinuatiaa sheet. 

3. Hazard Infarmatioa - Include in the notice a copy of re.caatM facw!mile ol. any hazard warning~ label. material safety data llbeet. lliBdb 

or other information which will be provided liD any perBDD wbo Ia ~ly Watly liD be expcMCiiD thia suhMaDa! reprdiDg protec:tive OpbD 

lD (pment or practic:es for tbe sate handling, transport. uae.or disposal of tbe new 11dwtanc:e u.t tn put m ~ infmmatkm.yau mclude. ~ 

Mark (X) this box tf vou attach hazard information. . -

FORM EPA 7710-25 (Rev. 5-95) ,Pap7 
Part. II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

Complete section A for each type of manufacture, proceMing, IX' use operation involving the new chemical aubltaDCI! at inciuatriU sites you contrc 

Importen do not have to complete this 1ection for operation• outside the U.S. ; however, you may .till have reporting requirements if there are 

further induatrial"procesaing oru~e operation• after import. You mu1t deiCribe these operations. See.in1traction• manual. 

of the site at which the willoa:ur. 

Name 

address (number ~d street) 

City, County, State. ZIP Code 

If the same operation will oa:ur at more than one site. enter the number of sites. Identify the 

additional sites on a continuation sheet, and if any of the sites have significmtly different · ....., 

production rates or operations, include all the information requested in this sec:tion for thoee 

Mark 00 this box if 

b. TypeMark 00 

1. Batch 

attach a continuation sheet: 

. D Manufacturing 

2. Continuous 

D ProcEMng 

Hours/batch 

Hours/day 

D UseDays/yar 

.d. Process desc::ription Mark 00 to iDclic:Ue yaar williDp- to bave·yaar pmcaa clucriptiaa biDcliDsCon 

dent 

(1) Diagram the majarunit operation stepe and cbemical conversicna. Include interim storage and transport c:catainerl (specify-e.g. 5 gallon pails, 

gallon drum, rail car, tank truck,' etc.). . · 

(2) Provide the identity, the approximate weight (by kg/ day or kg/batch on a 1001fo new chemical substance bm), and entry point of all starting 

materials and feecistockB (including reactants, solvents, and catalysts, etc.), and oi all products, recycle streams, and wutes. lndude cleaning 

chemicals (note frequency if not used daily or per batch.). 

(3) Identify by number the points of releue, including small <r intermittant rel-. to the environment of the new chemical substance. 

-. 

FORM EPA 7710-25 (Rev. 5-95) PageS 
Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE- Continued 

~ Sedion A- INDUSTRIAL SITES CONTROLLED. BY THE SUBMIII EK- Continued 

2. Occupational E.xpo8ure -You must make separate mnfidentiality c:laims for the delaiption of worker activity, physical form of the new chemical 

substance, number of workers expoeed, and duration of activity. Mark (X) the "Confidential" box next to any item you claim u c:onfidential. 

(1) - Desc:ribe the activities (i.e. bag dumping, tote filling, unloading clrums, sampling, deaning, etc.) in which workers may be expoaeci to the 

substance. 

(2) -Mark 00 this column if entry in column (1) is ccnfidential business informatiOI\ (CBD. 

(3) - Desc:ribe an 'I protective equipment and engineering controls used to protect workers. 

(4) and (6) - Indicate you willingness to have the information provided in cclumn (3) or (5) binding. 

(5} - Indicate the physical form(s) and. 'ro new chemical substance (if in mixture) of the new c:hemical substance at the time of exposure (ie., solid: 

crystal, granule, powder, or dust) and ,. new chemical substance (if in mixture). 

(1)- Mark (X) this column if entry in cohmm·(S) is CXInfidential business information (CBD. 

(8) - Estimate the maximum number of worke:rs involved in each activity for all sites c:cmbined. 

(9) -Mark (X) this column if entry in column (8) is mnfidential business information (CBD. 

(10) and (11)- Estimate the maximum duration of the activity for any worker in hours per day and days per year. 

(12)- Mark (X) this column if entries in columns (10) and (11) are amfidential business information CCBD. 

Worker activity CBI Protective Equipment/ Bmdms Physical farm(s BIDdias CBI jof CBI Maxim_llll duration CB 

(i.e.. bag dumping, filling drums) Engineering Contzola OptiDa and,. new OptiCIIl Workers 

M.k(xl Hrs/day substance M.k(x) Exposed. Days/yr 

(1) (2) (3) (4) (5) (6) (1) (8) (9} (10} (11} (1~ 

.... 

. . 

D Mark (X) this box if you attach a amtinuatioll sheet. 

3. "Enviroamentallleleue ancl Diapoal- You m'Uit make separate amfidentiaUty claims for tbe releae nUIIlbK md. tbe amount of tbe new c:hemic 

substance rel.eued and other release ed disposal infarmatiOil. Mark (X) tbe "Confidential• box next to -=h item you claim • confidential. 

(1} - Enter the number of each releae point identified in the~ dieeaiptilm. part II. !edian A, subeectim 1d(3). 

(2} - Estimate the amount of tbe new substance rel..-d (a) directly to the eDYiramDIDt ar (b) into amtrol technology (in kg/ day or kg/batc:h). 

(3) - Mark 00 this c:alumn if entries in columns (1) and (2) are amfldi!Dtlal buslnees infarmatioll <CBD. 

(4) - Identify the media (stack air, .fugitive air (optional-see lnstructlon Manual), surface water, an-site ar off-site 1aNi or inc:ineratian, P01W, or 

other (specify}) to which the new substaDc:e will be released froiD that rel.eue point. 

(5) - a. Describe control technology, if any, ,and control efficiency that will be used ta limit the release of tbe new substance to the environment. 

For releases dlsposed of on land, c:harac:terlze the disposal method and state whether it is approved for disposal of RCRA hazardous waste. 

On a continuation sheet , for each site describe any additional disposal methods that will be used and whether tbe waste is subject to 

secondary or tertiisry on-site treatment. Jl. Estimate tbe amount releaaed to the environment after control technology (in kg/ day). 

(6) - Mark (X) this c:olumn if entries in columns (4) and (5) are amfidential business infarmatloD (CBD. 

m - Identify the destination(s) of releases to water. Please supply NPDES (Naticmal Pollutant Dlac:harge Elimination System) numbers for direc1 

. dischargers or NPDES numbers of tbePOI'W (Publldy Owned Treatment Works). Mark 00 If. tbe P01W DIIJDe or NPDES 41 i8 confidential 

business information (CBD. 

Release Amount of new subetanc:e em IM= CantroltecbnoJogy and effideDc:y (you may willh to optiooally attac:b effic:il!ncy data) c 

Number I Jli8dllls 

(1} (2a) (2b} ... --- (Sa)· : Wuk (X) (Sb} (3) (4) I 

I 

I 

I 

·I 

I 

I 

I· ' - I 

I ' I 

I ., I 

(1) Marx 00 the . D Po1w provide name
 below: CBI DNnipble 0 OtlMI'- Specify provide NPDES • 

destinatian(s) of - waterway 

releases to water. 

I 

0 Mark 00 this box if you attach a cxmt:inuaticm sbeet. 

FORM EPA 7710.25 (Rev. 5-95) 
______ ---u ... ~ ............ , • .a.AVL'ILYL.r:.J.'(J.AL RELEASE- Continued 

~.Section B-. INDUSTRIAL SITES CONTROLLED BY OTHERS· : · .·· , ·, ., · · · · · .· · , .. ~. . . 

Complete sedian B fer typ!.cal procemng or~ operatiaaa inval.vtng tbe new chemic:al Sllbstana It si.te5 you cio not aml:rtll.lm~ del net have to· 

eomplete this aed:i.on fa: ope:ralicm oul3icle tbe U.S.; howwK, you must npcrt my prcx:eSng ar \lie adivi.tles after iinpcrt. $ee the Instructicm 

ManuaL ~ • ,..,.a: JldXm B far Md!type af. ~g, ar IlK opntim bu:IOI'IIir&g Usc MID c1aUc&l ndls~. If the same <ipeiatian is pe:r:formecl 

at men thm ~e Ste delaibe the typicil operatlan ccmman to thale !i~ Identify add:itiCDal. lites an a cantiDuation INlet. · . - ..... · . .. : . . . · . . . 

Letter ' oi an Dllriltiat . ICBJ PJ...-.idi:N !quip./. . I,.. I CBi 11--. AmauDt ~ .CBI . Media of &e.. : CBl 

-d. · 0 Workm r;Jl · . · ltV ("I CoaaiaW rc..,. · · · . , .. _ N.w .' '.- &t Cmtzal . · · o 

Ad.- Expo.d -~. · Pb.,.alllaraadS 'lllllllaa . . 0 S.Jblaaal .· ·. Tedm...J......;. · 

ivity . .. . . !:1ft''.' 7 .. ~ . ..._,. 

n> CZl ·a> · c~ ·_. ~ .. ~- (5) . ,. ·: C6) : ·· · (i') (I) (9) n.W 1 n·llt). cit ... 02) 0 03) 

I 'A I 

X·-~- . ; ~ - ---- . r 'f·o r -~. 'f ' otx 

.. Y· _,;~ 

I ·· I l 

·:·'1' ·. . . I -. . · .. I ·. . I , I 

... ' ·~ ' I . , . I o • I ·. i,··. 

t :·-

. -I 

~ I 

I ·. ••• ·,~ :. I o ' 

;' ''! .. . . .. ... . ·-. . , '". '". ~ ', , ·:: • . • ·~' r. ' ·.·· . .-

(1~) :- Byproducts: _ . . .. • . . (15) 

FORMEPAi1l~~· (Rft . .5-95). ·. 0 ' ·-

. PaplO 

'o 
OPTIONAL POLLUTION PREVENTION INFORMATION 

To claim information in this section as confidential circle or bracket the specific information that you claim as confidential. 

In this section you may provide information not reported elsewhere in this form regarding your . 

efforts to reduce or minimize potential risks associated with activities surrounding 

manufacturing, processing, use and disposal of the PMN substance. Please include new 

information pertinent to pollution prevention, including source reduction, recycling activities 

and safer processes or products available due to the new chemical substance. Source reduction 

includes the reduction in the amount or toxicity of chemical wastes by technological 

modification, process and procedure modification, product reformulation, raw materials 

substitution, and/ or inventory control. Recycling refers·to the reclamation of useful chemical 

components from wastes that would otherwise be treated or released as air emissions or water 

discharges, or land disposal. DescriptionS of pollution prevention, source reduction and 

recycling should.emphasize potential risk reduction subsequent to compliance with existing 

regulatory requirements and can be either quantitative or qualitative. The EPA is interested in 

this information to assess overall net reductions in toxicity or environmental releases and 

exposures, not the shifting of risks to other environmental media or non-environmental areas 

(e~g., occupational or consumer ~sure) .. In addition,. information on the relative cost or 

performance characteristics of the PM:N substance to potential alternatives may be provided. 

All information provided in this section will be taken into consideration during the review 

of thiS substance. See Instructions Manual and Pollution Prevention GUidance manual for · 

guidance and examples. 

. Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in 

the volume manufactured; (3) a reduction in the generation of waste materials through recycling, source reduction or other 

means; (4) a reduction in potential toxicity or human exposure and/or environmental release; (5) an inaeue in product 

performance, a decrease in the cost of production ard./ or improved operatioll: efficiency of the new c:hemical substance in 

comparison to existing chemical substance used in similar applications; or (6) the extent to which the.new chemical substance 

may be a substitute for an existing substance that poses a greater overall risk to human health or the environment . 

. 

. 

-

0 Mark (X) this box if you attach a continuation sheet. .. 

fURM EPA 7710-25 <Rev. 5-95) Papll 
Part III-- LIST OF ATTACHMENTS 

Attach continuation sheets for sections of the form and test data and other data (including physical/chemical properties and 

structure/activity information), and optional information after this page. Oearly identify the attachment and the section of the form to 

which it relates, if appropriate. Number consecutively the pages of the attachments. In the column below, enter the inclusive page 

numbers of each attachment. 

Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the lnsbuctions Manual for 

guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy of the notice 

form a sanitized version of any attachment in which you claim information as confidential. 

Attachment name Attachment Confi· 

page number(s) dentia 

Material Safety Data Sheet (MSDS) 

'x 

X 

0 Mark (X) this box if you attach a continuation sheet. Enter the attachment name and number. 

FORM EPA 7710-25 (Rev. 5-95) Page 12 
I 

Material Safety Data Sheet 

. ~ Effective Date· '11-Mar . --'~000 

SECTION I MATERIAL IDENTIFIC ACTION - Trade name: . - ~ .... .... - - - - - -------- __ ..., -s:: 

- - Generic name : --, -- . ~ .-. - -- ~-·---- .. ----~ _;..;..:,;,;: -""'; ----~ - ~---" ::- -- -... ,.. 

- , .. _,.I-~-' --.:.;-=_-_:::__.:.~.:; ._:.._ --- ~:., ~ - --· - -· ··---· .J -,-.., ..... J ... ... ~u...;.i.. 

Chemical family : Acrylic monomer 

CAS No.: ~ .... ,..._- - . 

. - - -.: 

TSCA: - Manufacturer : ':;::.;:;.t;;:..~-" vu., .w~.u. 

address(Number, Street, City, State, and ZIP Code) : "I . .. ....... -- • , . ., • -:-: - - -- --- ·-- ·-- ·---~~- __ .:.J '--.. .,;_:. ----· ___ ... _ ----- . -· --- - __ ....... -...:.,:.-__ ---- - - Phone: ~ .. - _·.,;..·- '-'V-. ·-""'·"· 

Facsimile : r uJ.·u•viJv 1 I---

SECTION 11 HAZARDOUS INGREDIENTS 

pp.emic_al family : Acry_li_c monomer 

Molecular weight: Mw 249.29 

Content % : 88'"'-'98 wt% 

.. - -- -- ... 

'l- : ;I I 

;: -

Rational formula : -

Other components hazardous to health 

COMPONENTS CM:i No. % ACGIH : TLV TWA 

Toluene 108-88-3 < 1 50ppm 

impurities 1'""'-'11 

SECTION JI[ PHYSICAL/CHEMICAL DATA 

Appearance : solid 

Color : light yellow Specific gravity N.D. 

Odor: toluene's odor Solubility in water Insoluble 

Boiling point : 

SECTION IV STABILITY AND REACTMTY 

This product is considered to be stable under normal and anticipated storage and 

handling condition. A spontaneous polymerizing reaction may occur under the condition 

as follows-heat, shock, open flame, ultraviolet, radiation, direct sunlight radiation, 

oxidizing conditions, free radical initiators, inert gas atmosphere. 

SECTION v FIRE AND EXPLOSION 

Flash point : N.D. 'C Flammability limits in air : N.D. 

Ignition temperature : N.D. 

Extinguishing media 

Carbon Dioxide, Dry Chemical. Sand, Foam, Wate~· Fog. 

Hazardous decomposition products : (Products causing hazardous decomposition) 

Carbon Monoxide. Carbon Dioxide. Organic Acids. 

S12ecial fire-fighting j2rocecl ures : 

· Move container from fire areas if it can be done without risk. 

· Firefighters· should wear proper protective equipment. 

· Keep personnel removed from and upwind of fire. 

Unusual fire and exnlosion hazards : 

NIA 

13 
•, 

SECTION VT HEALTH HAZARD INFORMATION 

Acute toxicity 

Acute oral toxicity (LD50 in rat) : N.D. 

Skin irritation (in Rabbits) : N.D. 

Chronic toxicity 

.Al.VIES test negative 

Primary skin Irritation Index(PII) 0.0 (classified as non-irritant) 

First aid 

· Eye contact : 

Gently rinse the affected eyes with clean water for at least 15minutes. Arrange for 

transport to the nearest medical facility for examination and treatment by a physician 

as soon as possible. 

· Skin contact : 

Wash the affected area with water using a mild soap. 

· Inhalation : 

Rinse mouth with water. Give the person water. Do not induce vomiting as this may 

mcrease the risk of aspiration of the liquid into the lungs causing chemical 

pneumonia . 

Consumption of non-oily liquids(such as milk) may assist in delaying absorption of the 

liquid. 

Personal Erotection/E;sEosure control 

Respiratory protection : Chemical respirator with an organic absorbent in a cartridge. 

Hand, skin; and body protection : Gloves 

Eye protection : Safety goggles 

Other : Use with local exhaust ventilation. 

SECTION VJJ SPILLS, LEAKS, AND DISPOSAL PROCEDURES 

Accidental release measures 

Evacuate nonessential personnel. 

Shut off all sources of ignition; No flares, smoking or flames in area. 

Absorb spill with inert material (e.g., dry sand or earth), then place in a chemical waste 

container with covers for disposal, using nonsparking tools. 

Remove leaking containers to a safe place, iffeasible. 

DisQosal consideration 

This combustible material may be burned in a chemical incinerator equipped with an 

afterburner and scrubber. 

SECTION VI!/ STORAGE & HANDLING / TRANSPORT 

Storage & handling 

Keep tightly sealed and store indoors out of direct sunlight in a cool place. 

Keep away from sources of ignition - No smoking. 

TransQort 

IMDG code: N.D. 

SECTION !X GENERAL 

N.D. means No Data Available 

N/A means NOT Available 

CAS means Chemical Abstract Service(Registry number) 

To the best· of our knowledge, the information contained in this sheet lS 

correct. However, we also disclaim all responsibility for any consequences 

arising from its use. 

End ofPage 

1'+-· 
Ill and thier CAS Registry Nu1nber 

""'-

r 

Che111ical structures 

\ 

\ 

CAS No. 

II None 

None 

None 

i:' .......... 

,, 

Maxinu1n1 

Percent 

5.0 

1.0 

5.0 

Confidential 

X 

X 

X 

( 
PHYSIQ\,L AND CHEMICAL PROPERTIES WORKSHEET 

·To assist EPA's review of physical and chemical propertieS data, please complete the following worksheet for data you provide 

I 

and include it in the notice: Identify the property measured,, the page of the notice on which the property appears, the value 

of the property,. the units in which the property is measured (as necessary), and whether or not the property is claimed as 

confidential The physical state·of the neat substance should be provided. These measured properties should be for the neat (100% 

pure) chemical substance. Properties that are measured for mixtures or formulations should be so 11oted (% PMN substallce in_ ).You 

are .11ot required to stibmit this worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure 

· that amfidential information is properly protected. You shOuld submit this worksheet as a supplement to yoilr submission of test 

data. This worksheet is not a substitute for submission of test data. 

I Meumcl Confi- : 

Property Mark (X) if Page Value or dential 

provided number Eltilmle . 

(a) (b) (c) Mark (X) 

(MorE)· (d) 

. \ 

Physical state of neat substance _(s)_Q)_Ut) X 

Vapor pressure 

. @Temperature 

"C 

Torr 

Density/relative density g/an3 X 

SOlubility "C X @Temperature 

Solvent _g/L 

Solubility in water. @ Temperature · oc "C )( 

Meltjng temperature oc X 

Boiling/sublimation temperatUre @ torr pressure X . 

s~ )( 

Dissociation constant ' I 

Particle size distribution 

Octanol/water partition coefficient I 

I 

Henry;s Law constant ' 

Volitalization from water 

Volitalizallon from soil 

pH @ concenl:raticin 

Flammability 

Explodabllity 

Adsorption/ coefficient 

Other - Specify 

/b 
1'\ 
It<; 
•, 

FINAL REPORT 

M.S.I. REPORT No.9B374G 

Ready Biodegradability Test of . 

Subrni tted to 

Prepared by 

Mitsubishi Chemical Safety Institute Ltd. 

November 12, 1999 

t9 

7 
) 

COMPLIANCE WITH THE GLP STANDARDS 

Study No. 9B374G 

Study Title Ready Biodegradability Test of 

Sponsor a,~-.:..~-

The study described in this report was conducted in compliance with the Good 

Laboratory Practice {GLP) regulations recognized by the Basic Industries 

Bureau of the Japanese Ministry of International Trade and Industry. 

Facility Management 

7a,~~ a~E!EL Date 

Tadayo~hi Shigeoka, Ph~ 

Facility Management 

Head of Yokohama Laboratory 

Study Director 

Ju~:KOY:Su: B.Sc. 

Study Director 

Environmental Science Division 

::to 
Study No. 

Study Title 

Sponsor 

QUALITY ASSURANCE STATEMENT 

9B374G 

Ready Ei odegradabili ty Test of • - -~ ·---

-,, 

We hereby certify that the above study has been perfo~ed in accordance with 

the Good Laboratory Practice (GLP) regulations recognized by the Basic 

Industries Bureau of the Japanese Minis~ry of International Trade and 

Industry. 

Dates of inspections and reportings to the Study Director and the Facility 

Management are as follows : 

Date of Date of 

Inspection Reporting 

In-progress study September 28, 1999 September 28, 1999 

October 26, 1999 October 26, 1999 

Final report November 12, 1999 November 12, 1999 

Quality Assurance Personnel 

Date 

Yoshihiro Miura, M.Sc. 

Date 

Tsuneki Susuki, B.Sc. 
) 

I, the undersigned, declare that the work was performed according to the 

procedures herein described, and that this report provides a true and correct 

record of the results obtained. 

Date 

Rie Yamaguchi 

Environmental Science Division 
1 

2 

3 

4 

5 

6 

7 

8 

9 

SUMMARY 

STUDY TITLE 

PURPOSE 

TEST GUIDELINES 

GLP COMPLIANCE 

STUDY PERIOD 

SPONSOR 

TESTING FACILITY 

CONTENTS 

STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS 

TEST SUBSTANCE 

10 CONFIRMATION OF IDENTITY AND STABILITY 

10 .1 IDENTITY 

10.2 STABILITY 

11 TEST METHODS 

11.1 ACTIVATED SLUDGE 

11.2 EXPOSURE CONDITIONS 

11.3 

11.4 

11.5 

11. 6 

11.7 

BOD MEASUREMENT 

pH MEASUREMENT 

DOC MEASUREMENT 

MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION 

EQUATIONS FOR CALCULATION OF DEGRADABILITY 

12 RESULTS 

12.1 OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD 

12.2 

12.3 

12.4 

12.5 

12.6 

13 

pH MEASUREMENT 

ACTIVITY OF SLUDGE 

DEGRADABILITY BASED ON BOD 

DEGRADABILITY BASED ON DOC 

DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE 

CONCENTRATION 

CONCLUSIONS 

TABLES AND FIGURES 

Page 

6 

7 

7 

7 

7 

7 

7 

7 

B 

9 

10 

10 

10 

11 

11 

11 

12 

12 

12 

13 

14 

15 

15 

15 

15 

15 

15 

16 

16 

17-34 
'. 

···;~~ 

SUMMARY. 

STUDY TITLE 

Ready Biodegradability Test of 

METHOD 

The method described in the Order, which prescribes tne procedure of testing 

new chemical substances as required by the Chemical Substances Control L·aw 

{Japanese Law 117, 1973). ("Ready biodegradability, Modified MITI test" in 

OECD Guidelines for Testing of Chemicals). 

Test bottle Bottle 1 aniline + sludge + basal medium 

Bottle 2 sludge + basal medium 

Bottles 3 ....... 5 test substance (ARONIX T0-1534)+ sludge 

+ basal medium 

Bottle 6 : test substance (ARON!X T0-15 3 4 ) + water 

Measurements Biochemical oxygen demand (BOD) (for 28 days) 

Dissolved organic carbon (DOC) (at day 28) 

Residual test substance concentration (at day 28) 

[measured by high performance ~iquid chromatography} 

RESULTS 

Measured values (day 28) 

BOD,· mg 

DOC, mg/1 

14.1 

43.5 

8.7 14.1 

43.6 44.5 

Theoretical 

6 value 

0. 0 60.7 

62.7 62. 6 

Test substance, mg/1 <1 <1 <1 107.5 100.0 

*1 value corrected with BOD or DOC value of Bottle 2. 

Degradabilities (%) 

Bottle No. 

3 4 5 average 

BOD 23 14 23 20 

DOC 31 30 29 30 

Test substance >99 >99 >99 >99 

CONCLUSION 

From the degradability results based on the BOD and DOC measurements, it can 

be concluded that the test substance is not read.i,ly biodegradable under the 

conditions of the Modified MITI test. 

Although the degree of degradability based on the residual test substance 

concentration was calculated to be >99, the degree of degradability based on 

the BOD was 20~ in average, DOC was 30!t. in average. Th.:=.refore, it was 

suggested that the test substance was transformed, and transformed product(s) 

was remained in the test solution. 
l STUDY TITLE 

Ready Biodegradability Test of _- • 

2 PURPOSE 

This study was conducted to evaluate the ready biodegradability of the test 

substance (ARONIX T0-1534) for notification under the Chemical Substances 

Control Law of Japan. 

3 TEST GUIDELINES 

The test was conducted according to the method described in the o=der, which 

prescribes the procedure of testing new chemical substances as required by 

the Chemical Substances Control Law (Japanese Law 117, 1973) . This method .is 

also known as the "Ready biodegradability, Modified MITI test" in the OECD 

Guidelines for the Testing .of Chemicals No. 301C. 

4 GLP COMPLIANCE 

The test was conducted according to the Good Laboratory Practice (GLP) 

regulations recognized by the Basic 'Industries Bureau of the Japanese 

Ministry of International Trade and Industry. 

5 STUDY PERIOD 

September 24, 1999 to November 12, 1999 

(BOD measurement : September 28, 1999 to October 26, 1999) 

6 SPONSOR 

:-.. ' -~!'""-

7 TESTING FACILITY 

Yokohama Laboratory 

Mitsubishi Chemical Safety Institute Ltd. (M.S.I.) 

1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan 

Head Office: 

Mitsubishi Chemical Safety Institute Ltd. (M.S. I .. ) 

1-30 Shiba 2-chome, Minato-ku, Tokyo 105-0014, Japan 
-

·.~~;~~; 

) 

B STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS 

The following records, the raw data, and a small sample of the test 

substance will be stored. in the archives of M.S.I. for 10 years after the 

submission of the final report. After this period, the sponsor will be 

contacted for the approval of the disposal of data. Data can be stored in the 

archives for a further specified period at the sponsor's request with an 

additional fee. 

1) Protocol and its amendment. 

2) Final report. 

3) Raw data. 

4) Quality assurance reports. 

5) Test substance (approx. 2 g). 

6) Other documents required under the MITI GLP regulations. 
) 

9 TEST SUBS'r.ANCE 

l)Name* 

2)Chemical name* 

3)Structural formula* 

4)Molecular formula 

5)Molecular weight* 

6)Elemental composition 

J 

e 

_, 

. 249.29 

C 62.6%, H 6.1%, 0 25.7%, N 5.6% 

(Calculated by M.S.I.) 

7) Physico-chemical properti·es* 

I 

Solubility 

Melting point 

B) Batch* 

9) Purity* 

Water insoluble 

DMSD, Acetone, DMF, toluene, MIBK, 

Acetonitrile oo 

61 oc 

Lot No. NH-9301-01 

. 94.1% 

Component of impurities 

(Dimpuri ty 1 

@impurity 2 

\ 

....) 

2. 7% (Q)+@) 
10)Appearance 

11)Supplied quantity* 

12) Supplier 

@impurity · 3 

/• 

.. 

', 

) 

white powder 

0.1 kg (net). 

10 CONFr.RMATION OF IDENTITY AND STABILITY· 

10 . 1 IDENTITY 

3.2% 

I 

\-,.. 

* provided by the sponsor. 

For the identification of the test substance, the infrared absorption 

spectrum of the test substance was measured. The measured spectrum was 

consistent with.that supplied by the sponsor. 

1)Apparatus Perkin-ELmer model 1640 infrared spectrophotometer. 

(Figure 1 (P-21)) 

1 0 . 2 STABILITY 

After the exposure period, the infrared absorption spectrum of the test 

substance that had been stored in a desiccator during the course of the 

) study was measured. The spectrum was consistent with that shown in Fig. 1, 

indicating that the test substance was stable under the storage condition. 

1) Apparatus Perkin-Elmer model 1640 infrared spectrophotometer. 

[Figure 2 (P-22)) 
ll TEST METHODS 

The test was performed according to the method described in the Order, which 

prescribes the procedure of testing new chemical substances as required by 

the Chemical Substances Control Law (Japanese Law 117, 1973}. This method is 

also known as the '!Ready biodegradability, Modified MITI test" in OECD 

Guidelines for Testing of Chemicals No.301C (1992}. 

The test substance was exposed to the activated sludge in a closed-syst;em 

oxygen consuntption measuring apparatus. The_ biochemical oxygen demand (BOD l 

was measured over a 2 8 day period. After this period, the concentrations of 

the dissolved organic carbon (DOC.) and the r'esidual test substance in the 

test bottles were measured. The biodegradability of the test substance was 

evaluated from these results. 

11.1 ACTIVATED SLUDGE 

1)Mixed liquor suspended solid (MLSS) : 3700 mg/1 

2}Source 

3)Date of receipt 

Chemical Biotesting Center, Chemical 

Inspection and Testing Institute, Japan 

September 10, 1999 

11.2 EXPOSURE CONDITIONS 

1) Temperature 25 ± 1 "C 

2)Exposure period 

3)Test volume 

28 days 

300 ml 

4)Concentration test substance (bottles 3-6) 

aniline*l(bottle 1) 

100 mg/1 

100 mg/1 

activated sludge (bottles 1-5) 30 mg/1 

*l Reference substance : KANTO CHEMICAL Co., INC. Lot No.103Gl134 

5)Test bottle contents 

Bottle 1 

Bottle 2 

Bottles 3-5 

Bottle 6 

Aniline + activated sludge + basal medium 

29~(30 mg) of aniline was added to the basal 

medium*2, then activated sludge was added. 

Activated sludge + basal medium 

Activated sludge was added to the basal medium*=. 

Test substance + activated sludge + basal medium 

30 mg of the test substance· was added to the basal 

medium*2 , then activated sludge was added. 

Test substance + ultra-pure water 

30 mg of the test substance was added to 300 ml of 

ultra-pure water (purified by Milli-Q, >lOMohm-cm). 

*2 The total volume of the basal medium and the sludge was held fi:,ed at 300 ml. 
~.·. 

11 . 3 BOD MEP.SUREMENT 

The BOD was measured for 28 days. 

1)Apparatus closed system oxygen consumption measuring apparatus 

Ohkura Electric Co., Model OM-2001 (M.S.I. ID No.F). 

11.4 pH MEASUREMENT 

After the exposure period, 20 ml of the test solution in each test bottle 

was transferred into a 20-ml glass beaker for pH mea~urement. 

After pH measurement, the test solution was returned to each bottle for 

measurement of residual test substance concentration. 

1)Apparatus : ·pH meter, ORION Research, Model 720A 

11 . 5 DOC MEASUREMENT 

,~:::~ The concentration of the DOC was measured as follows. 

1)Apparatus 

2)Conditions 

TOC analyzer, Shimadzu Co., model TOC-5000 

Furnace temperature 

Air flow rate 

Sensi ti vi.ty 

Injection volume 

3)Calibration curve 

'680" C (TC) 

150. ml/min. 

X 5 

50~ 

The following standard solutions were injected into the TOC analyzer. 

The calibration curve was prepared by the data processor ·of the analyzer. 

standard solutions 

TC (total carbon) 20 and 50 mg C/1 aqueous solutions of potassium 

biphthalate. 

IC(inorganic carbon) 0 mg C/1 Ultra-pure water (purified by Milli-Q) 

and 10 mg C/1 aqueous solution of sodium hydrogen 

carbonate and sodium carbonate. 

· .) 4) Measurement of DOC in the bottles·. 

Ten milliliter of the test solution in each bottle were transferred into 

a 10-ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes. Five 

milliliter of· supernatant were used for the measurement of DOC. 

The remaining supernatant and the precipitate were returned to each 

bottle for the measurement of the residual test substance concentration. 
11.6 MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION 

. The concentration of the residual test substance was measured by high 

performance liquid chromatography (HPLC) as follows: 

1) Appara t\ls 

HPLC Shimadzu Co., Model LC-10AWS-3 

Pump Shimadzu Co. 1 Model LC-10AD 

Auto Injecor Shimadzu Co., Model SIL-l OA.'<L 

uv-vrs Detector: Shimadzu Co. 1 Model SPD-M10AVP 

Col~ Oven Shimadzu Co., Model CTO-lOAC 

Work Station Shimadzu Co. 1 Model CLASS-LC10 

Degasser Shimadzu Co. 1 Model DGU"-14A 

Communication bus module : Shimadzu Co., Model CBM-lOA 

2)Conditions 

Column 

Mobile phase 

Flow rate 

Wavelength 

GL sciences Inc. Inertsil ODS-3V, 4. 6 rnrn i. d. X 150 mm 

Acetonitrile : Water = 60 : 40 

1 ml/rnin. 

230 nm 

Attenuation [Detector] -10 "' BOO AU/FS 

Injection volume: 20 ~ 

Oven Temperature :. 25 OC · 

3)Calibration curve 

The standard solutions of the test· substance with 0, 25, 50, and 100 

mg/1 in 40% acetonitrile I water. were prepared. and analyzed. The total 

peak area of the test .substance was calculated by adding the area of the 

peaks with retention time of about 3 .1, 3. 7, 4. 4 and 6. 9 minutes. The 

total peak area of the test substance was plotted against the 

concentration. The correlation coef.ficient was calculated to be 1. 00 by 

the method described in Japanese Industrial Standards Z9041-1968. 

Concentration of the test substance for each bottles was calculated from 

ratio of total peak area for the sample to that for the standard solution 

with 50 mg/1 in 40% acetonitrile I water. 

[Figure.3 (P-23) and Figure 4 (P-24...,..·25)] 

4)Recovery test 

Duplicate t~st bottles (identical to the test bottles 3-5 described in § 

11.2) were kept in a closed-system oxygen consumption measuring apparatus 

at 25±1t for 30 minutes. 

Each test solution was treated by the procedures described in § 11.6. 6) . 

The concentration of the test substance was measured by HPLC. The average 

recovery of the test substance was 98 %. 

[Table 3 (P-19) and Figure 5 (P-26...,..,27)] 

The concentration of the residual test substance measured for bottles 3 .......... 

6 were corrected with the average recovery value. 

·3\ 
,· 

5)Detection limit 

The detection limit of the test substance was calculated to be 1 mg/1 

based on the minimum detectable peak area of 1000 rnAbs • sec for the 

largest peak (retention time of about 4.4 minutes) in the HPLC 

chromatogram. (Table 3 {P-19) and Figure 5 (P-26 ....... ·27) J 

6)Measurement of the test substance in test bottles 

The content in each bottle was separately transferred into a 500-ml 

glass volumetric flask, which was the brought up to 500 rnl with 

acetonitrile. Ten milliliter of the test solution was centrifuged at 3000 

rpm for 10 minutes. The supernatant was analyzed by HPLC. The test 

substance concentration was analyzed by HPLC under the conditions 

described in § 11. 6. 2) . 

11.7 EQUATIONS FOR CALCULATION OF DEGRADABILITY 

Equations for calculation of degradability based on BOD, DOC, and residual 

te~t substance concentration are as follows: 

1)Degradability based on BOD 

Degradability {%) = {BODs- BODb)/ThOD X 100 

where BODs 

BODb 

ThOD 

ThOD 

aniline 

Oxygen consumption {mg) in bottles 1, 3, 4 and 5. 

Oxygen consumption (rng) in bottle 2. 

Theoretical oxygen demand (mg) of aniline and the 

test substance. 

C6H1N + 35/ 40z -+ 6COz + 7 /2Hz0. + NOz 

ThOD 90.2 mg Oz/30 rng-aniline 

test substance C13H1sO,N + 63/ 40z - 13COz + 15/2Hz0 + NO: 

ThOD : 60.7 rng Oz/30 rng-test substance 

) 2)Degradability based on DOC 

Degradability (%) = {1- (DOCs-DOCb)/DOCc}X100 

where DOCs 

DOCb 

DOCc 

DOC (mg/1) in bottles 3, 4 and 5. 

DOC (mg/1) in bottle 2. 

DOC (mg/1) in bottle 6. 

3)Degradability based on residual test substance concentration 

Degradability (%) = (1 - Cs/Cc) X 100 

where Cs 

Cc 

concentration (mg/1) in bottles 3, 4 and 5. 

concentration (mg/1) in bottle 6. 
·~. 

j -·· 

12 RESULTS 

There was no specific circumstances which might have affected the 

reliability of the test results. 

12.1 OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD 

The solution in bottle 1 was·cloudy yellow. The solutions in bottles 2, 3, 

4, 5 and 6 were colorless. Growth of the sludg.e was observed in bottle 1 in 

contrast with control (bottle 2). No growth was observed in bottles 3, 4 and 

5. 

12 . 2 pH MEASUREMENT 

After 28 days of exposure, the pH was determined to be 6.6, 6.6, 6.5 and 

5.6 for bottles 3, 4, 5 and 6, respectively. (Table 1 (P-17)) 

12. 3 ACTIVITY OF SLUDGE 

The degradabili ty of aniline, based on the ·BOD measurements was 55 % after 

7 days. The activity of the sludge was thus shown to be satisfactory. 

[Table 1 (P-17) and Figure 6 (P-28 ........ 31)) 

12 . 4 DEGRADABILITY BASED ON BOD 

BOD*1 in bottles 3, 4, and 5 (as corrected with the value in bottle 2) were 

14.1, 8.7 and 14.1 mg, respectively. BOD in bottle 6 was 0.0 mg. 

(* 1 Maximum theoretical value= 60.7 mg) 

The degree of degradability based on. the BOD measurements were 23, 14 and 

23 % for bottles 3, 4, and 5. 

[Table 1 (P-17} and Figure 6 (P-28----31)] 

12.5 DEGRADABILITY BASED ON DOC 

DOC*2 in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 

43.5, 43.6, and 44.5 mg/1, respectively. DOC in b~ttle 6 was 62.7 mg/1. 

(*2 Maximum theoretical value= 62.6 rng/1) 

The degree of degradability based on the DOC measurements were 31, 30 and 

29 % for bottles 3, 4, and 5. 

(Table 1 (P-17) and Table 2 (P-18)] 
•, 

_ .. -

) 

12.6 DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE CONCENTRATION 

The test substance*J was not detected in bottles 3, 4 and 5, and detected 

107.5 mg/1 in bottle 6. 

(*3 Initial concentration= 100 mg/l} 

The deg~ee of degradability based on the residual test substance 

concentration were >99, >99 and >99 % for bottles 3, 4 and 5, respectively. 

[Table 1 (P-17), Table 4 (P-20), and Figure 7 (P-32~34)) 

13 CONCLUSIONS 

From the degradability results based on the BOD and DOC measurements, it can 

be concluded that the test substance is not readily biodegradable under the 

conditions of the Modified MIT! test. 

Although the degree of degradabili ty based on the residual test substance 

concentration was calculated to be >99, the degree of degradability based on 

the BOD was 20% in average, DOC was 30% in average. Therefore, it was 

suggested that the test substance was transformed, and transformed product(s) 

was remained in the test solution. 
' 

Table 1 Summary of the test results 

Degradabili ties were calculated by the equations shown in Sec. 11. 7. 

) D eg ;ra dabili tv based BOD 

Bottle Test ThOD day 7 day 14 

No. substance mg BOD % Degra- BOD % Degraill I! dai.Jilitv rug dabilitv 

1 aniline 90.2 50.6 55 63.2 67 

2 ----- --- 1.3 --- 2.9 ---

3 ,. -- "' . - 60. 7 7. 3 10 14.6 19 

4 1_ ·- & - . 60. 7 5. 8 7 11. 6 14 

5 - - 60. 7 6. 6 9 14.6 19 

----

--- ---

6 • r ' 60. 7 0.0 ---

___ 

---

-· 

-

. L__ __ ___:_ __ ._ 

) D -· ra dabili tv based .. DOC 

Bottle Organic carbon TC IC DOC % DegraNo. added wg/l wg/l wg/1 wg/1 dab iii ty 

2 ----- 1.6 0.0 1.6 ----

3 62.6 45. 1 0.0 45. 1 31 

4 62.6 45.2 0.0 45. 2 30 

5 62.6 46. 1 0.0 46. 1 29 

6 62.6 62J - ___ _Q. Q - -·--- 62.7 --·---- ----J 

da 21 da' 28 

BOD % Degra- BOD % Degra- 

llj:! dabilitv mg dabilitv 

65.4 69 65.4 6

3.2 --- 3.2 --- 

17.3 23 17.3 211.9 14 11. 9 116.0 21 11.3 2

0.0 --- 0.0 --- 

d) D - --dabilitv based .. 

Bottle Concentration 

No. wg/l 

2 <1 

3 <1 

4 <1 

5 <1 

6 1Q7. 5 
J _. 

Table 2 Re.aulta of DOC mea~urement 

Standard 

TC: &o; o •elL 

vIa. I : al 

MN 

f area 

1 

2 

3 

24849 

24809. 

24829 

24829 

Bottl.e 2 

TC: 

f area 

TC: 20 .o •elL 

vla.l :a2 

liN 

RMK 

f· area 

1 10174 

2 10149 

3 10110 . 

10144 

B o t t I e S 

TC 

f area 

IC: 10.0 ar/L 

vial :a3 • 

liN 

• a.rea 

1 

2 

3 

4973 

6062 

6111 

&048 

RIIK 

-----------------~---------- -------------------~--------

loiN 

1 

2 

3 

827 

782 

72.4 

771 

1. 89 

1. SB 

1.48 

1. 58 liN 

1 c rc 

# area lle/1." .RMK 

1 0 0.00 

2 0 o.oo 

3 0 o.oo 

1 22188 

2 :noos 3 22054 

22075 

# area 

1 0 

2 0 

3 0 

46.21 

44.95 

45 .os· 

45.09 

aefl. 

o.oo 

o.oo 

0.00 

RIIK 

---------------------------- ---------------------------- MN 0 

Bottle 6 

TC: 

0.00 MN 0 

B o t t l e B 

TC: 

:o.oo 

---------------------------- ----------------------------

MN 

IC 

loiN 

# area 

1 

2 

3 

22451 

22682 

22624 

22552 

# area 

1 

2 

3 

0 

0 

0 

0 

TOC-5000 DATA 

mc/1. 

45.88 

46.13 

46.21 

46.07 

me/L 

0.00 

0.00 

0.00 

0.00 

RE.PORT 

RMK 

KN 

IC: 

RMK 

MN 

# area 

1 30478 

2 30708 

3 30838 

306U 

# area 

1 

2 

3 

0 

.0 

0 

0 

me/1. 

62.28 

62.73 

82.99 

62.66 

me/1. 

o.oo 

0.00 

0.00 

0.0~ 

---------------------------------------------------- SPL # TC,u/L RWK IC.me/L RWK TOC,mr/L 

---------------------------------------------------- 2 1. 58 0.00 1. 68 

3 46.09 0.00 46.09 

4 45 .1 e o.oo 46.16 

5 46.07 0.00 46.07 

6 62.66 0.00 82.66 

RMK 

RMK 

<----

<----

<----

<---~ 

<----

B 

B 

B 

B 

B 

IC: 0.0 ae/L 

vial :a4 

# area 

1 

2 

3 

0 

0 

0 

------:--------

B o t t l e 4 

TC 

MN 

IC 

# area 

"1 22012 

2 21993 

3 22317 

22107 

# area 

1 0 

2 0 

3 0 

ae/L 

44.98 

44.93 

45.59 

45.18 

1u/L 

0.00 

0.00 

0.00 

0 

RMK 

RMK 

---------------------------- liN 0 0. QQ. 

0 t t l e 2 0 t t I e 8 0 t t l e 4 

0 t t I e 5 0 t t I e 8 
w 

_...) 

Table 3 Calculation of recovery and detection limit 

Concentration in 

Peak solution mdl Recovered Added Recovery Detection 

area for HPLC in bottle mass mass limit 

mAbs·sec analysis mg mg % mg/l 

(A) (B) (C) (D) (E) (F) (G) 

Recover}': test 1 4096927 58.8 98.1 . 29.4 30.0 98 ----Recovery test 2 4078677 58.6 97.6 .' 29.3 30.0 98 ----

Blank <2000 <O. 1 <O. 1 ------- Q.O ---- 

----------

Average recovery: 

50 mg/l 

98% 

Standard solution Concentration 

Peak area 

Volume of solution for HPLC analysis· Volume of test solution for HPLC analysis Volume of test solution in bottle 

(H): 

(I): 

(J) : (K): 

(L): 

3481229 mAbs ·sec 

0. 500 1 o, 300 1 

0. 300 1 

Equation : B=H X A+ I C=HXA+IXJ+K D=CXL F=D+EX 100 

Calculation of detection limit 

50 mg/1 std. Blank 

Retention Peak area Rate of peak Peak area 

time min mAbs·sec area % mAbs·sec · 

3. 1 14652 0.4 ----

3. 7 . 41184 1.2 ----

4.4 3334330 95.8 <1000 

6. 9 91063 2. 6 ----

_ 

3481229 100.0 <2000 

- -----

The minimum detectable peak. area of the test substance was calculated by 

the following equation based on the mi.nimum detectable peak .area of 1000 

UL'\bs· sec for the largest peak (retention time 4. 4 min.) in HPLC chromatogram. 

Minimum detectable peak area = 

(mAbs· sec) 

3481229 

(mAbs·sec) 

1000 (mAbs·sec) 

X ----------

3334330 (mAbs·sec) 

G: raise decimal fractions 

1044 (mAbs·sec) 

< 2000 (IDA.bs ·sec) 
w 

0\J 

·· . 

. .. ·:. ! 

'--

Table 4 Residual .test substance concentration in test bottles 

Bottle 

No. 

2 

3 

4 

5 

6 

Concentration in 

Peak solution mg/l 

for HPLC in bottle 

·mAbs·sec analysis 

(A) (8) (C) 

<2000 (0. 1 <1 

(2000 . (0.1 <1 

(2000 <O. 1 · <1 

<2000 <O. 1 <1 

3447013 62.2 107.5 

Standard solution Concentration (D): 

Peak area (E): 

Volume of solution for HPLC analysis (F): 

Volume of solution for.HPLC analysis (G): 

Average recovery (H): 

Equation :B=DXA+E 100. 

50 mg/1 

2772249 mAbs ·sec 

0. 500 1 

0."295 1 

98 96 
Figure 1 Infrared absorption spectra oftJ:te test substance 

Measured by the. !!J)onsor 

100 

10 

70 

eo 

ISO 

3D 

2000 • 1511D' 1000 500 

w.......oor. 1cm-1J 

Measured.by M.S.I 

X: ~000.0. ~00.00 cm-1; 0.2~.~5.39 ~T 99/09/03 10:18 

~ scans; mace ratlo; r-esol 4.00 cm-1: apoc str-ong 

smooth 32.0 cm-1; flat; 

9837 .o!G {lfll'/vr 1 ot. no . NH-930 1-01 R. Y lllllaguch 1 

) thr-eshold 3.00X; bane · 

cm-1 X cm-1 X cm-1 X cm-1 X 

3451.4 30.28 2942.8 16.89 1708.2 0.23 1633.5 16.75 

1401.5 3.68 1368.2 1:5.19 1:3:30.1 17.2:3 1298.9 15.20 

1187.5 2.25 1073A z2.'os 1018.2 15:76 965.5 11.27 

945.3 27.50 900.7 30.39 B11.B 22.54 744.5 28.78 

716.7 26.22 669.4 35.40 548.3 .34.98 462.6 32.99 

409.4 33.19 

21 paeks 1ound 

<16 .<17 

IT 

_.I 

C-H stretch 

·o.&:-f00-0 ---J-,5U-0---11--rO-o-, ~~-~-0 --.--\ !SUD 

C=O stretch 

\ ~---~;-

C-N stretch C-0-C stretch 
) 

Figure 2 Infrared absorption spectrum of the test substance stored in desiccator 

x: ~ooo.o, 40o.oo cm-1; 0.10,63.53 XT 

4 scans; 110de r-atio: r-esol 4 .co cm-1: 

smooth 11.:3 cm-1: flat: · 

96374G : : ~ ." _ ,'1ot..no .HH-9301-01 

thr-eehold e.oox: band. 

cm-i X cm-1 l cm-1 

3449.2 '49.65 2943.7 21.46 1711.6 

1-437,0 10.22 1398 ,B 1.:35 1370.1 

1:301.9 19.17 1187.2 0.21 1077.1 

986.5 6.81 946.0 36.56 902.4 

832.6 40.22 812.3, 23.42 748.0 

670.5 50.37 550.5 52.29 48:3.5 

24 peak= fo!Jnd 

73.RS 

IT 

~ C-H stretch 

99/10/28 09:53 

IPOC Str-ong . 

l 

0.09 

12.37 

:33.99 

42.36 

37.45 

47.59 .. 

cm-1 

1634.1 

1331.5 

1017.7 

861.7 

713.4 

H:3.9 

X 

21.56 

16.75 

14.60 

44.50 

34.76 

49.8:3 

··l.ID.f---.,---.,---.,....--,.---+-'--,--==f-.....;...---r""c-----,- 4DDD 3SOO 31100 Z:SDD 20110 . co"' 5DD 

C=O stretch C-N stretch C-0-C stretch 
) 

Figure 3 Calibration Curve of the Test Substance 

Curve Fitting [Least Square Method] 

J!!P..Y.:LDa_g;_ ----- Concentration Peak Area 

--1'1..9.~.---- ... X. ~II!&LJ.L Y .~!!'Ab~..§~c;:l__ · 1 0 0 

2 25 . 1781328 

3 50 3580618 

-~-~------- 100 70825§3.· 

Y = 1. 2138E+04 · + 7. 0834E+04 X X 

r = 0. 99998 

8000000 

"' u 

Q) 

7000000 

6000000 

~ 5000000 ..c 

< 

e 

'-" 4000000 

C"d 

~ 

::: 3000000 

~ 0.... 

2000000 

1000000 

Calibration Curve· 

0 ~--~----~--~----~--~--~ 

0 20 40 60 80 -100 120 

Concentration (mg/1) 

'I-I 
\ 

. 

Figure 4 HPLC chromatograms of the test substance ---Calibration curve 

1 .\SS-LC!O Yer. =I. 60 11:' r'-I=STDO. KOl 15:68:54 

!:·J" : . . .. . 

;!J : !18374G . 

ii:Jdi: : 20 

i.i/11:111 : SI'IHI!OAYJ> 

I ,·1-14>: R. YAIIAGUCJU 

• hf' :f, ·: ICIIRIIl.IIOl 

·•• tni1'7.J.-

I 

BOO I 0. 2 230nll 

600 

-(:: 

("400 

200 

2 4' 0 6 . 0 

..• . 

1 ! t'-tlllh!Jll:ith- !I 

Std. 0 mg/1 in 40 % Ac.etoni tri 1e / Water. 

8 

aln 

a..ASS-ti:lO Ver. =1. 60 "*' -··-IJ=2 r' -I=ST025. KOl 99/

: ... 

1D : 118374G 

. lt.AJI: : 20 . 

!*Hill .I SPIHIJOAYJ> 

H;f; I R. JAJIAGIJOII: 

.. n,•· : IO!RIIt.IIOt 

IIAh• . 

2:loq11 

600 

400 

2QO 

n 

0 

-- t'-?"*'-· -

:e !1ll 

l ... ID __rl_______ci_ 

2 4 ·6 

PKNO O.Ml Till& AREA Jllf«) ax«: III!IGIIT NAII

I 2 3.116 

2 2 3.698 

3. 2 

4 2 6.939 

7432 

18701 

1713878 

41317 

Tc!.al.Atea 1781328 

1114 

2590 

201954 

3365 

I 00. 0000 209023 

Std. 25 mg/1 in 40 I I Water 
'--· 

Figure 4 (Continued) 

l"USS-LCLO Ver. =1. 60 $-.Xj'.Lfi.JJ-=1 99/08/'1:1 16:21:58 tn·" : .-. ·-

JD : 98374G 

ilo)..JI : 20 

fiilfl It · : SPIHII OA TJ1 

R. YAXAGUCHI 

11,1' 1. : IO!RJII.IIOI 

.... ?117},.7,1. ... 

I 

600 

... 

11; t 

. 200 

ol I \ . ! I . ' ' ·8 ' 0 

.... ... 

0!1/0 TillE 

I 2 3.120 

2 2 . 3. 702 

3 2 4.396 

t 2 6.946' 

2' 

AREA IDNO 

14197 

38764 

3«1049 

86608 

Total Area 3580618 

4 

cooc lEIGHT NAliE 

2l78 

6258 

4011W5 

6891 

100. ooOo 4111172 

Std. 50 mg/1 in 40 'Acetonitrile I Water 

8 

lllil 

a.ASS-LC10 Yer.=l.60 $-.X;'.t..+:l .l-t"-11flt=4 f'-1"5111100.K01 99

r.n·., ·: < ..• 

IP : 9B374G 

: :.10 .. 

II : · SI'IHII DA YJ1 

H:fi 1 II. YAIIAGVOII 

111r :fi ! IOIRII1.11Df 

... ... 

a\bs 800_,.., ""a.:':2:"·-::o230n=-..... ·

1100 

400 

200 

.I 

ol 

0 

... t' ... 

I 

2 

PK1«J 01.'11 TIIIB AIII!A 

I 2 3.128 28!1115 

z 2 3. 709 78343 

3 2 4.402 8799328 

4 2 8.955 .176019 

Total . 7082683 

!§ ! 

!2 . 2 1 \ 

I 

4 8 

IDI«J OK: IIEIGI!f NA11B 

. 4390 

10113& 

787214 . 

13873 

100.0000 816113 

Std. 100 mgll in 40 I Acetonitrile I later 
., . 

Figure 5 HPLC chromatograms of the test substance ---Recovery and detection limit 

The recoveries and detection limit are shown in Table 3. 

Yor. =1. 6o I'J.fJ.Jfft-:1 K01 99/09/28 11:48:1Z 

, : r • : .fWUJr• · 

. II : 98374G 

i'.ldi : 20 

: · 

: R: YAIIAGUOIJ 

·1,1' <S ·: ·ICJ!RIIt.IID1 

.• J•T•r1.1.-

. 

BOO I Ch2 230o• I 

hOO 

-F 

200 

CD 

l!l 

OJ I § . ! I 1 \ I ; I I 

o ;! • . . a 8 

,,.. t' -JU' -

l'liNO OtNO TillE 

I 2 .3. Ill 

2 2 3. 696 

3 2. 4. 31!8 

4 2 6.9-14 

ARPA IDNO 

14652 . 

. 41184 

3334330·. 

91063 

Total Area 3481229 

CONC HE Ictn' NAlll! 

21)8 

li284 

:161708 

6851 

100. OQOO 381959. 

Std. 50 mg/l in: Acetonitrile I Water· 

llf.n . 

<USS-lC10 Yer. =i. 60 vJ.f!JI'-Jt=l -tJUt=2· f'-I=R_BL 11:01 ·I)

t:rr •· : 

ID . : 98374G . 

· !tAll: : 20 

. l*f:lUI·· : 

f" R. YAIIAGIJOII· 

. ·. n,r :·IOIRJit.Jllll ..;.,..,.,., ... _ 

iiAbs' .. 

. 80010i'2 230o•· 

1100 

-400 

. 200 

n•· 

0 2 

. 

-- t' -JW -· - . II !!· 

Blank 

4 fi .. 
Figure 5 (Continued) 

i.AS5-LCIO Ver, =1. 60 #-.J=X 1«!1. Jl01 99/09/28 12: ll:te 

,: 7·., : {Wrm--f -

IU : 9B3HG 

.tAli ,: 20·· 

;;uua. ·: SPIHIIO.Aop 

r·,· It YAIIAGUOir 

: IOIRIII.IIOt 

..• '"•r71·-

""b• BOO I 0!2 .· .. . •. . I 

600 

\ 

I 

0 1 o • o .} \ 

1 

!· , I 

0 

... 

. o.NO Till!! 

I 2 .!1.118 

2 2 :i.'702 

3 2 .f. 397 

2 6.965 

2 

AREA JDNO 

'16587 

46498 

3935782 

119060 

Total Area 4096927 

Recovery test l 

.. 

aN; 

6 

IIEIGifl' NAliE 

2361' 

6816 

.437600 

7547. 

100.-0000· 453323· 

8 

•in 

. 

-: 

lUSS-t.ClO Ver.=-LeO i'$'-f.lJ=oi f'-l=ft H02.Jt:01

t!lr • : -

:JD . : '98374G. 

U:J..8: ·: 20: . 

lllfltf i SI'IHW1Aop f'-. It-I f. : R. WIAGUCIIl n,•· <15.: 10111111.1101 

.... , .... 

10\bs 

soot 012 230n• 

1100 

.4110 

0»1 

Ol " ci I o· 2 

- t' -11-$' ... - . a.NO ·rnm ARI!A Tim 

. '1 .2 3. 123. 

2 2 3. 709' 

. 3 2 (. 40lt 

4 2 6.975 

"15707 .. 

43990 

3922745. 

96235 

Total Area 4078677 

Recovery test 2 

... 

0 

.... 

!;i 

0 

4 

rnNC 

• 6 

JIBIGHT N

2293 

6674· 

436688 .. 

7264 

·100,0000 451898 
·Figure 6 BOD chart 

1\) 1\) 1\) 1\) 

Ill I) 0 _. N c.l .eL - N t

-·---. -.- 75- i . ! ----------- !" • • 

--·-

.. ! ... 

so+-· 

---,-.--

.... 6 ---+--+-

·- :----

. .!! 

· lleasuremen t of BOD •· ···· ·- ·· ·• ···· ! ··· ··· 

:-: ..• 

llodel Olof-2001 (I. D,. II:F) -: . 

Cfuiit' speed · · ' ··!' 

.. . . · 

_ ,;; ___ .£;:}it 

-'--: -· -· -3 ··--AROIIIX·T1>71634· .. · -.;. , . 30-. 

-yJ ----:: : · _.-. !: .:.:_: __ 

6 . J.ROHIX TO-II'i34 ... 'iOO.:.L <.::+-:- . ' _,__;_ _____ _____ .. j 

. 

.:. -· i .• -- 

. . . ; -: : : ; ' : r;cc l 

: , 0 f day , . . 1 : , : ; . I 

;: .. ! .... L' .. ... : ;·_·_; __ l_:_i __ ; .: .. :. ! ...... ........... ! .. l ..... ........ .! 

Ll C!; f!:CII.\"1:-,;-1·1 tiD. F'teAC1l.'5 k'O!I.SS..\5HI 
Figure 6 (Continued) 

;. 01 (I -l CD U) 

... 

6 N Col 

I s:J i ;---1-:t=i"·l-....... r ···!·+! ............ , ! • i :-: '·t--r-·1-f ·-··:. 1--!· :··: ·-;···1 I :I·:-- f---.H

J_iBott e 5 .. ·· , ... i .. -; .. !:.:.1 .. .. : .. . J i: i: ) · i. : .. ;._ .:-...::.-:.:...;:·-L.!. ·::.1 ... :·:: .. : : 

-; 'ottle'i! .. J .; .••. :.-!: ... +-t··l; _;_:..! ( 1·'-··' i ·: ... :.. • ! ', .... -;--:--7-H-+i-' · · i; : ..... .. ·.j .. -.l·t:! · .. 1 .. :

cJ· ,Botti• < . -•·-· .. ,_. j .... I. ,_ , __ -' r:, +···J __ , ., .. •-- . '-i -' 1- . _, .. ' , ' ----- - ., .. •--• .. , -•· ' +· ,_ ' ' ·' -·-

•j 

. ... 

7f 

(168) 

! . :-; I . : . .. 1 • j ··•••• ·-'- , • I • I , • CHART tt0.P"1!'RCfOC: . !· • KO<i-\YA7':1 • .. 1t.Ot.'!:-i-r . . ... 
2 

23 

22 

I 

J •• 

i 

i 

I 

I 

I 

~ .. 

I. 

I· 

i 

i . 1·:' 

21 I, .... · 

!·' 

L ' .. 

I 

17 

.. , 
:.!$..! .. 
Figure 7 HPLC chromatograms of the test substance 

--- Measurement of residual test substance concentration 

The concentration of residual test substance in bottle is shown in Table 4. 

CLASS-l.CIO Vor. =1. 60 1-J:f.lJI'-ij-=j "'*" -t!l"Jt=l 7"-J:f_STD.I(OJ 99/10/26 16:07:55 t:.-7·, : 

ID : 98374G. 

!V-A: : 20 

. : SPIHIIOAyp 

t..,· R. YMIAGUOU 

n, 1-.1; : IOIR.IIt. v01 

..-. 1•1H"'.J.-

IIAbs I 

aoo I a.z 230o• 

600 

200 

ol 

0 2 

- t" -· - PKNO O.NO . TillE AREA 

I z 3. 225 11316. 

2 2 3.880 28250 

3 z 4.654 2672910 

{ 2 7.519 69173 

Total Area 2772249 

In 

"" "" ci 

IDNO 

··f8 

ci 

' .f 

.... 

. ... 

"' 

1 

100.0000 

I 

6 

HEIGHT NAVE 

1713 

3978 

328783 

6070• 

339'!4 I 

Std. 50 mg/1 in Acetonitrile / Water 

"' ti 

8 

aln 

a..ASS-l.CJO· Yer. "'1. 60 1-J:f.l.Jf=l f"-J=T_N02 •. KOI 99

· t/t, : 

ID : 98374G 

U:.A:R :20 

tlii:JS .: SPIHilOAYp t": .R. YAIIAGIJOII 

l:l7t" 4'. : IOlRIIl.IIOl 

-

soo I ChZ 230n• 

600 

. 400 

. 201! 

Ol I'-. 

0 2 l 6 

- -

!I II 

Bottle 2 (blank test of sludge) 
Figure 7 (Continued) 

CLASS-LCIO Ver.•'i. 60 t"-1=1_1103. KOt 99/10/26 ·15:31;00 t;,:r t. : W"nJv-F . · 

I D : llll37 4G 

li:J-Q i20 

: SPIHIIDAYp t'' l--145 :· R.: Y ,a.JIACUOll 

J:l,f"4', : DOlRIIL.IIOl 

... ,,.,.,., ... 

-

m.\bs 

800 I Ot2 230n• I 

600 

200 

0 2 4 . 6 8 

•In 

""* 

!! II 

Bottle 3 (sludge + test substance) 

. :_' .. '!·f 

CUSS-LCIO Ver."'L.60' U'-).JJ-=13 r·-t=T_IIm.KO

lr : Hf"Tifl-·-'f 

ID : 98374G 

fV..fi : 20 

I SPIHIIOAvp f-" R. Y AllAGUOU. · 

J;f,r if. : IOIRIII.IIOt 

- I•T)I"'-' .... 

.ilba . . . . . . . . , . . · .. 

aoo I a.z 23011• 

61)0 •. 

400 

ill 

...l 

. 0. 2 '-4 6 

. - t"-1V*"-) - ·Pml a.NO TI!IF. AREA tom . aJNC. IEIGHI' 

1 2 1. 352 13H419 J49994 

131Hl9 '100. 0000 14!1994 

Bottle 4 (sludge + test substance) 
Figure 7 (Continued) 

Cl.ASS-L.CIO Vor.=l:60 J-"-I=T_N05.JI:Ol 99/10/28 16:54:0• 

t!l7"-" : 

ID . : 91l374G · 

ltJ,.Jl· : 20. 

l;t . : SPIHIIOA ..-p.. t"" lr-1-1,: L YAliAGIJOU n1r ·: IOIRIII.IIOl 

- 1•'1',.'-" -

II>Ab.s . • ' •• • • 1 

BOO I 012 "230iuo 

600 

if) 400 

f" 

200 ,._ ., 

n 

0 ' 

0 2 4 6 8 

•In 

- -

PliNO CbNO TTII!l Am Illli"O lEIGHT twiE 

I 2 I. 337 . I 207!>90 160131 

1207090 100.0000. 150131 

Bottle 5 (sludge + test substance) 

/J:ftl; 

•.• . 

tUSS-I.CIO Yer. =t. 60 5-A'I'J..JJ%1 t:l;" f" NOll; KOl 

f"/J" ft : (WT!JI-t -

ID · . : 98374G · 

tJ:J,.t : 20. 

lltlflll I SPIHIIOAwp 

I. YIJIAGUOII 

JM • : IOIRIII. 1101 

- ,..,.,.,1-

I!Abs , 

8001 Cb2 

' 

600_, 

.00· 

200 

J ./\ 

0 .. 

- t" -11-*" -· -· PKNO O.NO TillE 

I 2 3.225 

2 2 .3.881 

3 2 4.656 

4 2 7.62:1 

d. 

I 

2 

AIIEA . ID.'II 

15797 

28958 

3322859 

79399 

Total Area 3447013 

·!§ 

i 

d 

I 

4 

eo«: 

100.0000 

Bottle 6 (water + test substance) 

6 

lElGHT 

2166" 

3990 

416658 

6762 

42911fi8 
! 

' 

CONFIDENTIAL 

Sponsor 

' . I• 

J .. :: .. T 

TSI 173/992799/SE 

T0-1534 

SKIN IRRITATION TO THE RABBIT 

Research Laboratory 

Huntingdon Life Sciences Ltd 

P.O. Box2 

Huntingdon 

Cambridgeshire 

PE18 6ES 

ENGLAND 

Report issued 1 June 1999 
l 

l TSI 173/992799/SE 

CONTENTS 

Page 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS..................... 3 

QUALITY ASSURANCE STAT'EMENT ......................................................................... 4 

RESPONSIBLE PERSONNEL .................... ...... ....................................................... .. ...... 5 

SUlvll\1AR Y . . . . .. . . . .. . . . . . . . . . . .. . . .. . . . . . ... ..... .. .. . ... . . .. . . . . ... . .. .... .. ...... .. . . . .. .. .. . .. . .. . . ........ .. . ............. 6 

INTRODUCTION ............................................................................................................ 7 

TEST SUBSTANCE......................................................................................................... 8 

EXPER.llv1ENT AL PROCEDURE..................................................................................... 9 

RESULTS......................................................................................................................... 12 

CONCLUSION................................................................................................................. 12 

TABLE 

1. Dermal reactions . . . . . . . . . . . . . . . . . . . . . ... . . ... ... . . .. . ... . . . . . . .. . .. ..... .. . . . . . . . . . ... .. . . . . .. . . .. .. . .. . .. ....... .. . . . . . . . 13 
TSI 173/992799/SE 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS 

The study described in this report was conducted in compliance with the following Good Laboratory Practice 

standards and I consider the data generated to be valid. 

The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). 

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV /MC/CHEM(98) 17. 

EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15129), and from 

1 May 1999 EC Commission Directive 1999/11/EC of8 March 1999 (Official Journal No L 77/8) . 

...... ~0.~ ................ . Stephen J. Mason, B.Sc. (Hons.), 

Study Director, 

Huntingdon Life Sciences Ltd. 

. .. l .. J:~.I~lf.'········ Date 
r 

I 

TSI 173/992799/SE 

QUALITY ASSURANCE STATEMENT 

The following have been inspected or audited in relation to this study 

Study Phases Inspected Date of Inspection Date ofReporting 

Protocol Audit 10 March 1999 10 March 1999 

Process Based Inspections 

Husbandry 19 March 1999 19 March 1999 

Housing/Environment 19 March 1999 19 March 1999 

Test Material:-

Control 19 March 1999 19 March 1999 

Treatment Procedure 19 March 1999 19 March 1999 

Scoring 19 March 1999 19 March 1999 

Records Audit 19 March 1999 19 March 1999 

Report Audit 18 April 1999 20 April 1999 

Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director 

and Company Management as indicated above. 

Process based inspections: At or about the time this study was in progress inspections and audits of 

routine and repetitive procedures employed on this type of study were carried out. These were conducted 

and reported to appropriate Company Management as indicated above. 

Report Audit: This report has been audited by the Quality Assurance Department. This audit was 

conducted and reported to the Study Director and Company Management as indicated above. 

The methods, procedures and observations were found to be accurately described and the reported results 

to reflect the raw data . 

. ... t.t~~~:-. ...... !3.~:::.~ ................... .. Margaret Blows, 

Quality Assurance Group Leader, 

Department of Quality Assurance, 

Huntingdon Life Sciences Ltd. 

. .. ~.J .... M~ .. i.':l.:i:;. 

Date 
Stephen J. Mason, B.Sc. (Hons.), 

Study Director, 

Huntingdon Life Sciences Ltd. 

TSI 173/992799/SE 

RESPONSffiLE PERSONNEL 
f. 

f 

l 

' 

TSI 173/992799/SE 

SUMMARY 

A study was performed to assess the skin irritation potential ofT -· _._;,t to the rabbit. The method followed 

was that described in the Code of Federal Regulations, Consumer Product Safety Commission, Subchapter C 

- Federal Hazardous Substances Act Regulations, Title 16, Section 1500.41. Method of testing primary 

irritant substances, 1 January 1994. 

Six rabbits were each administered a dermal dose of0.5 ml ofthe test substance to one intact and one abraded 

skin site and observed for three days. 

A single occlusive application of' · - ( to intact and abraded rabbit skin for twenty-four hours elicited no 

dermal irritation. 

The Primary Irritation Index (PII) was calculated to be 0.0 and· ->-, ·is classified as non-irritant. 
TSI 173/992799/SE 

INTRODUCTION 

The study was designed to assess skin irritation potential of': __ :....... following a single dermal application to 

rabbits. The test substance may come into contact with skin during handling or use. 

The study was conducted in compliance with the Code of Federal Regulations, Consumer Product Safety 

Commission, Subcbapt~r C - Federal Hazardous Substances Act Regulations, Title 16, Section 1500.41. 

Method of testing primary irritant substances, 1 January 1994. 

The albino rabbit was chosen as it has been shown to be a suitable model for skin irritation studies and is the 

animal recommended in the test guideline. 

The amount oftest substance administered was chosen in compliance with the guideline. 

The protocol was approved by Huntingdon Life Sciences Management on 25 February 1999, by the Sponsor 

on 3 March 1999 and by the Study Director on 4 March 1999. 

The experimental phase ofthe study was undertaken between 9 and 12 March 1999. 
.. 

· TSI 173/992799/SE 

TEST SUBSTANCE 

Identity: __ _,.,. 

Chemical name: -.i ---' , 

Intended use: Coating 

Appearance: Clear liquid 

Storage conditions: Room temperature in the dark 

Lot number: 80622 

Expiry: 22 February 2000 

Purity: >95% 

Sample received: 25 February 1999 
TSI 173/992799/SE 

EXPE~NTALPROCEDURE 

ANIMAL MANAGEMENT 

Animals for this study were selected from a stock supply ofhealthy adult rabbits of the New Zealand White 

Strain, obtained from Charles River UK Ltd, Margate, Kent, England. 

They were in the weight range of2.1 to 3.3 kg and at least 10 weeks of age, prior to treatment (Day 1). All 

rabbits were acclimatised to the experimental environment for a period of six days prior to the start of the 

study. 

The rabbits were selected without conscious bias for the study. They were housed individually in metal cages 

with perforated floors in Building Rl4 Room 5. 

A standard laboratory rabbit diet (Special Diet Services STANRAB (P) SQC pellet) and drinking water were 

provided ad libitum. The batch of diet used for the study was analysed by the supplier for nutrients, possible 

contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified 

amounts, might have an undesirable effect on the test system. The animals were fed hay on arrival and 

subsequently three times a week. 

The water supplied to Huntingdon Life Sciences by Anglian Water was potable water for human 

consumption. Anglian Water takes its guidelines on water quality from the EEC directive relating to water 

for human consumption (80/778/EEC) and conforms to the United Kingdom Water Act 1989 and subsequent 

amendments. Results of routine physical and chemical examination of drinking water at Consumers· taps. as 

conducted by the supplier, are made available to Huntingdon Life Sciences Ltd as quarterly summaries. 

Animal room temperature was maintained at 19 to 20°C and relative humidity at 35 to 52%. These 

environmental parameters were recorded daily. Lighting was controlled by means of a time switch to give 

12 hours of artificial light (0700- 1900 hours) in each 24 hours period. 

Each animal was identified by a numbered aluminium tag placed through the edge of one ear. This 

identification was unique within the Department throughout the duration of the study. Each cage was 

identified by a coloured label displaying the study number, animal number and initials of the Study Director 

and Home Office licensee. 

TEST SUBSTANCE PREPARATION 

T0-1534 was administered as supplied by the Sponsor. 

The absorption of the test substance was not determined. 

Characterisation of the homogeneity, stability and purity ofthe test substance was not undertaken as part of 

this study and remains the responsibility of the Sponsor. 

&r 
TSI 173/992799/SE 

TREATMENT PROCEDURE 

Approximately 24 hours prior to application of the test substance, hair was removed with electric clippers 

from the dorso-lumbar region of each rabbit exposing an area of skin approximately 100 mm x 100 mm. 

Immediately prior to application of the test substance two areas of skin each approximately 25 mm x 25 mm 

were identified in the clipped dorso-lumbar region, one to the left and one to the right of the spine. The area 

on the right was abraded using the tip of a scalpel blade to make minor incisions through the stratum 

corneum. These were not deep enough to reach the dennis or produce bleeding. The area on the left 

remained intact. 

Approximately 0.5 mi of the test substance was applied under 25 mm x 25 mm porous gauze pads to one 

intact and one abraded skin site. 

The treatment sites were covered with "Elastoplast" elastic adhesive dressing backed with "Sleek" waterproof 

strapping for twenty-four hours. The animals were not restrained during the exposure period and were 

returned to their cages immediately after treatment. 

At the end of the 24-hour exposure period, the occlusive dressing and gauze pads were removed and the 

treatment sites were washed with warm water (38°C) to remove any residual test substance. The treated areas 

were blotted dry with absorbent paper. 

OBSERVATIONS 

Clinical signs 

All animals were observed daily for signs of ill health or toxicity. 

Dermal responses 

Examination of the treated skin was made on Day 2 (ie approximately 60 minutes after removal of the 

dressings) and on Day 4 (equivalent to 72 hours after exposure). 

Local dermal irritation was assessed using the prescribed numerical system: 

Erythema and eschar formation: 

No erythema 

Very slight erythema (barely perceptible) 

Well-defined erythema 

Moderate to severe erythema 

Severe erythema (beet redness) or eschar fonnation (injuries in depth) 

preventing erythema reading 

0 

1 

2· 

.., 

.) 

4 
TSI 173/992799/SE 

Oedema fonnation: 

No oedema 

Very slight oedema (barely perceptible) 

Slight oedema (edges of area well-defined by definite raising) 

Moderate oedema (edges raised approximately 1 millimetre) 

Severe oedema (raised more than 1 millimetre and extending 

beyond the area of exposure) 

CALCULATION OF PRIMARY IRRITATION INDEX 

0 

1 

2 

., 

,;) 

4 

The numerical scores for erythema and oedema at both the 24 and 72 hour readings for both intact and 

abraded skin sites were added together and divided by 24 to give the Primary Irritation Index (PII). 

The PIT was used to classify the test substance as follows: 

PII 

0 

>0- 2.0 

2.1-5.0 

5.1-6.0 

>6.0 

Classification 

Non-irritant 

Mildly irritating 

Moderate irritant 

Moderate to severe irritant 

Severe irritant 

Based on Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and 

Drug Officials ofthe United States. 1959, p 47. 

ARCHIVES 

All raw data and study related documents generated during the course of the study at Huntingdon, together 

with a copy of the final report will be lodged in the Huntingdon Life Sciences Ltd Archive, Huntingdon. 

Such records will be retained for a minimum period offive years from the date of issue ofthe final report. At 

the end of the five year retention period the client will be contacted and advice sought on the future 

requirements. Under no circumstances will any item be discarded without the client's lmowledge. 

DEVIATIONS FROM PROTOCOL 

There were no deviations from the protocol. 
TSI 173/992799/SE 

RESULTS 

CLINICAL SIGNS 

There were no signs of toxicity or ill health in any rabbit during the observation period. 

DERMAL RESPONSES 

The numerical values given to the dermal reactions elicited b} : ,. are shown in Table l. 

No dermal irritation was observed in any a.rllmal throughout the study. 

The Primary Irritation Index (PII) was calculated to be 0.0. 

CONCLUSION 

A single occlusive application of to intact and abraded rabbit skin for twenty-four hours elicited no 

dermal irritation and is classified as non-irritant. 

12 : 
TSI 173/992799/SE 

TABLE I 

Dermal reactions 

Rabbit no. & E=Erythema 24 hours 72 hours 

sex 0= Oedema 

Intact Abraded Intact Abraded 

2098Male E 0 0 0 0 

0 0 0 0 0 

2099Male E 0 0 0 0 

0 0 0 0 0 

2100Male E 0 0 0 0 

0 0 0 0 0 

2101 Male E 0 0 0 0 

0 0 0 0 0 

2102Male E 0 0 0 0 

0 0 0 0 0 

2103 Male E 0 0 0 0 

0 0 0 0 0 

Primary Irritation Index= 0.0 

13 : 
F i n. a 1 Report 

Mutagenic ty Study of 

in Bacterial Reverse Mutation Assay 

(StudyNumber 5739) 

BML. INC. 
AUTHENTICATION 

I, the undersigned, hereby dedare that this report is an accurate translation of the original report 

written in Japanese, No. 5739, issued on Msuch 20, 1998, by BltfL, Inc. 

Title of Report: A mutagenicity study of.'- : .:-.- --'- \"' .... ·· .. , 

ethyl ester in Bacterial Reverse Mutation Assay 

Report Number: 5739 

Signature: 

T Yoshihiro Oguma 

Study Director, 

Division of Safety Evaluation 8c Reagent 

BML, Inc. 

Date: f-'1 C( r. 3 I 
STATEMENT OF STUDY DIRECTOR 

Title of Study. A mutRgenicity study ~ __ .• ' -- -, . ~ .. _., 

ethyl ester in Bacterial Reverse MutRtion Assay 

Study Number: 5739 

This is to certify that the this study was conducted in accordance with "Standards to be observed by 

Mutagenicity Testing Institution~' {Notification No. 76, Ministry of Labor, Japan, September 1, 1988). 

sprure: 1~ UJ- tft 'l4L-"'"-

Yoshihiro Ogui: 

Study Director, 

Division of Safety Evaluation & Reagent 

BML, Inc. 
QUALITY ASSURANCE STATEMENT 

Tide of Study: A mutagenicity study of ' ""' • 

ethyl ester in Bacterial Reverse Mutation Assay 

Study Number: 5739 

This study was conducted in accorda.nce with "Standards to be observed by Mutagenicity Testing 

Institutions" (Notiiication No. 76,; Ministry of Labour, japan, September 1, 1988). 

The circumstances of audits a.nd inspections are as follows. 

Date of inspection 

February 12, 1998 

February 23, 1998 

March 19, 1998 

March 12, 1998 

March 18, 1998 

March 23, 1998 

March 31, 2000 

Phases 

Review of draft protocol 

and SOP 

Review of protocol 

and SOP 

Review of amendment 

to the protocol 

General inspection 

Review of draft report 

and audit of mw data 

Review of fina.l report 

(in japanese) 

Review of .5na.I report 

(in English) 

Date of Report to Management 

February 12, 1998 

February 24, 1998 

March 20, 1998 

March 12, 1998 

March 19, 1998 

March 23, 1998 

March 31, 2000 

I certify that the methods and procedures used in the test are described precisely in the final report, . 

and that the reported results re.Dect the mw data accurately. 

Signature: 

Masahiro Kurar.now 

Manager, Qulliity Assumnce 

Quality Management Division 

B.M.L, Inc. 

Date: 
~tudy number : 5739 

Title : A mutagenic! ty study of 

. -~· ~- k in bacterial reverse mutation assay 

Purpose of the test: To assess the effect of the test substance in bacterial reverse 

mutation assay. 

This study was performed 

for Mutagenicity Tests using 

in accordance with the guidelines set forth in "Standards 

Microorganisms" (Notification No. 67, June 2, 1997,. Ministry 

o f L abo u r , J ap an) . 

And it was conducted in compliance with "Standards to be Observed by Mutagenicity 

Testing Institutions" (Notification No. 76, Ministry of Labour, Japan, September 1, 1988). 

Sponsor 

Name 

Address 

Testing facility 

Name 

Address 

Date of study 

Initiation 

Completion 

Persons in charge 

Study Director 

·-..:;:;.- _. ~ - '' . . . ' 

Section of Safety Evaluation, 

1361-1, Matoba, Kawagoe-shi, 

February 10, 1998 

March 20, 1998 

.. 

:":.::.::::--·:: --·. . -.r- ... - ,. 

Division of Biocellology, 

Saitama 350-1101 Japan 

Yoshihiro Oguma 

BML, INC. 

Persons in charge of study Miko Sarada, Hiroshi Seki, Fumio Yokota 

Name of the person who prepared the final report, and date of preparation 

Yoshihiro Oguma 

Title : Study Director 

Date : March 20 1998 ----~~~~--~~---

certify that this study was conducted under the specified protocoL 

K iyosh i Och i 

Title :Administrator 

'70 Date : ----~~~~~~;_-- March 20 1998 
I. ' Summary 

Mutagenicity potential of - - L --

l' was assessed by using Salmonella typhimurium strains TAlOO, TA1535, TA98, 

TA1537 and Escherichia coli WP2 uvrA. 

In this study, neither increase in the number of revertant 

as many as that of the negative control in any strains of 

colonies more than twice 

base-pair substitution 

type or frameshift type, with or without metabolic activation, nor dose-related response 

was observed. 

Thus, it is considered· that 

has no mutagenicity to bacteria under the conditions of this 

study. 

?! 
2. Date of test 

Investigation of the solubility February 6, 1998 

Initiation of the dose-finding test February 25, 1998 

Initiation of the main test March 11, 1998 

Completion of the main test March 14, 1998 

3. Test substance 

(1) Name{Another name} 

(2) BML-CODE 

(3) Lot No. 

(4) Structural formula 

(5) Purity 

n ""' -- . --

" 

~' - -

BML-3845 

71107 

Over 99wt% 

(6) Name and concentration of impurities Toluene:under lwt% 

(7) CAS No. 

(8) Molecular weight 249. 27 

(9) Melting point 50"C 

(10) Boiling point 

(11) Vapor pressure 

(12) Partition coefficient 

(13) Appearance at room temperature Wb it e so I i d 

(14) Stability 

(15) Degree of solubility 

(16) Stability in solvent 

(17) Condition of storage 

(18) Notice in handling 

Stable at room temperature 

Water: insoluble 

Dimethyl sulfoxide(DMSO): over 50mg/ml 

Acetone: soluble 

Toluene: soluble 

Stable in DMSO, acetone and toluene 

Room temperature in shade 

Wear the gloves in order to prevent the rash 

The test substance was analysed and all the data are retained by the sponsor. 

The solubility in DMSO was tested by BML, INC. 

_1( 2 
~ ~ester strains 

(1) Strains used 1

' · 2' · 3' 

The following 5 strains were used. 

Base-pair substitution type : 

Salmonella typhimurium TA!OO, TA!535 

Escherichia coli WP2 uvrA 

F r arne s h i f t type : 

Salmonella typhimurium TA98, TAI537 

(2) Supplier and date obtained 

S. typhimurium TA100 

The Division of Mutagenesis, National Institute of 

Japan (The Division of Genetics and Mutagenesis, 

Research Center National Institute of Hygienic Science, 

; April 15, 1982 

Other S. typhimurium TA strains 

Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 

E. coli WP2 uvrA 

Hygienic 

Biological 

Sciences, 

Safety 

Japan at present) 

Department of Molecular Oncology, Institute of Medical Science, University of 

Tokyo, Japan; November 24, 1987 

(3) Characterization date 

S. typhimurium TA100 December 25, 1997 

S. typhimurium TA1535 February 16, 1998 

E. coli WP2 uvrA December 2, 1997 

S. typhimurium TA98 December 25, 1997 

~ typhimurium TA1537 February 16, 1998 

(4) Reason for choice 

These strains are very sensitive to mutagens and used most commonly in mutagenicity 

study in bacterial reverse mutation assay. 

(5) Condition of storage 

The cell suspension and DMSO (spectrophotometric grade) were mixed 

0. 8 ml to 0. 07 ml. The mixture was subdivided into 0. 3 ml aliquots, 

were frozen and stored at -80l:. 

in a 

and 

ratio of 

then they 
5. Con t ro I compounds 

(1) Negative control 

Negative control plates were treated with only DMSO. 

(2) Positive controls 

Strain Without S9 (/.L g/plate) With S9(/.Lg/plate) 

TA100 AF-2 (0. 0 1) B [a] P ( 5. 0) 

TA1535 NaN3 (0. 5 ) 2AA ( 2. 0) 

WP2 uvrA AF-2 (0. 0 1) 2AA (1 0. 0) 

TA98 AF-2 (0. 1 ) B [a] P ( 5. 0) 

TA1537 ICR-191 (1. 0 ) B [a] P ( 5. 0) 

AF-2 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 

(Wako Pure Chemical Industries, Ltd.; Lot No. SAJ0748; Special grade of JIS; 

Purity, more than 95%) 

NaN3 Sodium azide 

(Wako Pure Chemical Industries, Ltd.; Lot No. DCJ6398; Special grade of JIS; 

Purity, more than 95%) 

ICR-191 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine ·2HCI 

(Polysc'iences, Inc. ; Lot No. 52300; Pur.i ty, more than 95%) 

2AA 2-Aminoanthracene 

(Wako Pure Chemical Industries, Ltd.; Lot No. KPQ0892; Special grade of JIS; 

Purity, more than 95%) 

B [a] P Benzo [a] pyrene 

(Wako Pure Chemical Industries, Ltd.; Lot No. KCF4188; Special grade of J!S; 

Purity, more than 95%) 

1) Solvents 

AF-2, ICR-191, 2AA and B[a]P were dissolved in DMSO (Wako · Pure Chemical Industries, 

Ltd.; Lot No. TPK5055). NaN3 was dissolved in distilled water for injection (Hikari 

Pharmacy Co., Ltd.; Lot No. 9708ST). These solutions were subdivided, stored at -20't: 

and thawed just prior to use. 

2) Reason for choice of the positive control compounds 

These compounds were chosen in accordance with the guideline of the Industrial 

Safety and Health La~ 
6. Materials 

(1) S9 mix 

1) S9 

Name 

Manufacturer 

Lot No. 

Manufacture date 

Purchase date 

Storage 

Materials for S9 

S9 Fraction 

Kikkoman Corporation 

RAA-375 

January 9, 1998 

January 20, 1998 

Stored at below -80 \: in freezer 

Fraction 

Species and strain Sprague-Dawley rat 

Sex Male 

Age 

Weight 

7 weeks 

183- 245g 

Inducing substance Phenobar bit a I (PB) and 5, 6-benzo flavone (BF) 

Administration i.p. injection 

Dose 

2) Cofactor 

Name 

Manufacturer 

Lot No. 

Storage 

3) Composition of 

Water 

S9 Fraction 

MgCI2 

KCl 

G-6-P 

NADPH 

NADH 

PB=30t60t60+60 mg/kg (4 days); BF=80 mg/kg(at the third day) 

Cofactor 

Boehringer Mannheim L L 

725 

Stored at below 5 'C in refrigerator 

S9 mix (per 1ml) 

0. 9 ml 

0. 1 ml 

8. 0 tL mo I 

33. 0 tL mo I 

5. 0 tL mo I 

4. 0 tL mol 

4. 0 tL mo I 

Na-Phosphate buffer (pH 7. 4) 100. 0 tL mol 

(2) Top agar 

Used agar 

Name 

Manufacturer 

Lot No. 

Bacto-Agar 

Difco Laboratories 

99703JA 
· (3) Minimal glucose agar plate 

Name 

Manufacturer 

Lot No. 

Manufacture date 

Purchase date 

Storage 

7. Method 

(1) Solvent 

Name 

Manufacturer 

Lot No. 

Purity 

Minimal glucose agar medium M 

ORIENTAL YEAST CO. , LTD. 

BM010AN 

January 28, 1998 

February 10, 1998 

Stored at room temperature 

Dimethyl sulfoxide (DMSO) 

Wako Pure Chemical Industries, Ltd. 

TPK5055 

More than 99% (Special grade of JIS) 

(2) Preparation of the test substance solution 

Based on the information from the sponsor that the test substance was insoluble 

in water, the solubility test the test 

substance dissolved at 50 mg/ml 

was performed in 

in DMSO. DMSO was 

DMSO. From the result, 

solvent for preparation 

of the test substance solutioa The preparation was 

used as 

carried out just prior to use. 

(3) Condition of pre-culture 

I) Nutrient Broth 

Name Nutrient Broth No. 2 

Manufacturer Oxoid Limited 

Lot No. 105 59702 

2) Incubator 

Model Cool Bath Shaker ML-10 

Manufacturer Taitech Co., Ltd. 

3) Pre-culture procedure 

Bacterial cells of each strain (20 111 of S. typhimurium TA strains, 5 111 of E. coli 

WP2 uvrA) were inoculated into a L-form cui ture tube (capacity of 35 ml) containing 

10 ml of Nutrient Broth. This culture tube was left at 4 'C for 7 hours and 45 

minutes and incubated shaking (IOO rpm) at 37 'C for 8 hours. After incubation, optical 

density was measurel The bacterial cultures were stored at room temperature and 

used within 2 hours after incubation. 
4) Number of bacteria at the end of pre-culture 

Number of bacteria in cui ture was estimated from optical density at the end of 

pre-culture. 

S. typhimurium TA100 

S. typhimurium TA1535 

E. coli WP2 uvrA 

~ typhimurium TA98 

S. typhimuri um TA1537 

(4) Dose selection 

dose-finding test 

1. 48X 10 9/ml 

1.88Xl0 9/ml 

4.59Xl0 9/ml 

2.14X10 9/ml 

1.59X10 9/ml 

main test 

1. 46X 10 9/ml 

1. 80Xl0 9/ml 

4. 57Xl0 9/ml 

2. lOX 10 9/ml 

1. 49 X 10 9/ml 

The dose range was determined from the dose-finding test using the following 

seven dose levels: 1. 2, 4. 9, 20, 78, 313, 1250 and 5000 ug/plate. 

In the dose-finding test, growth inhibition was observed at 1250 ug/plate and more 

in S.typhimurium TAstrains without metabolic activation and S.typhimurium TA1537 with 

metabolic activation, and 5000 ug/plate in E.coli WP2 uvrA with or without metabolic 

activation and in S.typhimurium TAIOO, TA1535 and TA98 with metabolic activation. 

Precipitate by the test substance on the plate was not observed with or without 

metabolic activatio~ 

Therefore, as the highest dose level of the test substance in the main test, 1250 

ug/plate was selected for s. typfdmurium TA strains without metabolic activation and 

s. typhimurium TA1537 with metabolic activation, 5000 ug/plate was selected for E. coli 

WP2 uvrA with or without metabolic activation and S. typhimurium TAlOO, TA1535 and 

TA98 with metabolic activation. These highest doses were diluted 5 times (using a 

common ratio of 2) to provide a total of 6 dose levels for all strains with or 

without metabolic activatio~· 

(5) Test procedures (the preincubation method) 41 · 51 · 61 · 71 

For the tests wi thou! metabolic activation, 0. 5 ml of 0. 1M sodium-phosphate buffer 

(pH7. 4) and 0. 1 ml of each bacterial cul lure were added to a tube containing 0. 1 ml 

of the test substance solution. For the tests with metabolic activation, 0. 5 ml of 

S9 mix instead of 0. 1M sodium-phosphate buffer was added to each tube. The mixture 

was preincubated at 37 t: for 20 min shaking horizontally. Then 2. 0 ml of top agar 

was added to the mixture, and the contents in each tube were poured over ·the 

surface of the minimal glucose agar plate. For the sterility test, 0.1 ml of the 

test solution at the highest dose and 0.1 ml of S9 mix were put into each tube, 

2. 0 ml of top agar was then added, and the contents in each tube were poured 

over the surface of each minimal glucose agar plate. 

After incubation at 37 t: for 48 hours, the revertant colonies on the plate were 

counted and the bacterial cells were observed by a stereoscopic microscope in order 

to check whether or not growth of the tester strains was inhibited. As top agar, 

soft agar solution (0.6% Bacto-agar and 0.5% NaCl) with 1/10 volume of 0.5 mM biotin 

- 0. 5 mM L-histidine for the S. typhimurium TA strains or with 1/10 volume of 0. 5 mM 

tryptophan for the E coli strain was used. Two minimal glucose agar plates were used 

for each dose I eve I. 

7'l 
(6)' Counting procedure 

The number of revertant colonies was counted by colony counter. 

1) Colony counter 

Model 

Manufacturer 

2) Correct ions 

Correction with 

3) Coefficient 

data 

- I 00 

101 - 400 

401 -

8. Judging Criteria s> 

the 

Colony Analyzer 

System Science 

coefficient 

coefficient 

I. II 

I. I6 

I. 25 

CA-7TI 

Co., Ltd. 

In the dose-finding test and the main 

the test plates increased significantly 

test, if the number of revertant colonies on 

in comparison with that on the control plates 

(more than twice as many as that of the negative control), and dose-response and 

reproducibility were observed, the test substance was judged positive. Statistical 

analysis was not done. 

9. Results and discussion 

The results were shown in the Table 

In both the dose-finding rest and 

of revertant colonies more than twice 

I, 

the 

2 and the Figure I, 2. 

main test, nei !her increase 

as many as that of the 

in any strains of base-pair substitution type or frameshift type, 

metabolic activation, nor dose-related response was observed. 

in the number 

negative control 

with or without 

The revertants of the positive controls showed increase more than twice as many 

as that of the negative controls, indicating that this study was performed properly. 

From these results, mutagenicity of the test substance was judged negative. 

The growth inhibition was observed at doses marked (*) in the Table 1 and 2, but 

the precipitate by the test substance was not observed on the plates with or without 

metabolic activation. 

In the sterility test on test solution and S9 mix, no growth of bacteria was 

observed. 

10. Deviation from protocol, etc. 

During the study period, there was no environmental factor that was thought to 

have affected the credibility of the test, or there was no deviation from the 

protocol. 
11. Cone l us ion 

From the results above, it was concluded that~~ . ------::· "• 

.. -- --- .. was non-mutagenic in bacterial reverse mutation 

assay under the experimental condi lions. 

12. Storage of records and test substance 

All records and test substance generated during this study (listed below) will be 

stored in the GLP archival area of BML, INC. for 10 years after the application of 

permission. At the end of the period, the sponsor wi 11 be consul ted regarding the 

handling of them. 

Stored records and test substance 

1) Protocol and Amendments 

Z) Records concerning test substances 

3) Raw data 

4) Test substance 

5) Records on activities of Quality Assurance Unit 

6) Draft of final report 

7) Final report 

13. References 

1) B. N. Ames, F. D. Lee, and W. E. Durston: An Improved Bacterial Test System for the 

Detection and Classification of Mutagens and Carcinogens, Proc. Nat!. Acad. Sci. 

USA Vol. l.Q., No.3, pp. 782-786, March 1973 

2) J. McCann, N. E. Spingarn, J. Kobor i, and B. N. 

Mutagens: Bacterial Tester Strains with R Factor 

USA Vol. 11. No.3, pp. 979-983, March 1975 

Ames: Detection of 

Plasmids, Proc. 

Carcinogens 

Natl. Acad. 

as 

sci. 

3) M.H.L. Green and W. J. Muriel: Mutagen Testing using Trpt Reversion in Escherichia coli, 

Mutation Res. Vol. 38, pp. 3-32, 1976 

4) T. Yahagi, M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada 

of N-nitrosamines on Salmonella, Mutation Res. Vol. 48, pp. 121-130, 

Mutagenic i t,i e s 

1977 

5) D. M. Maron and B. N. Ames: Revised Methods 

1983 

for the Salmonella Mutagenic! ty Test, 

Mutation Res. Vol.ffi, pp. 173-215, 

6) A guidebook for Mutagenicity Studies using Bacteria, New Edition, Ed. by Chemical 

Substances 

of Labor, 

Investigation Division, Industrial Safety and Health Department, Ministry 

7) Testing 

Tamura, 

Japan Industrial Safety and Health Association, 1986 

Methods for Environmental Mutagens : Ed. by Taj ima, 

Koudan-sha Co., pp. 56-68, 1980 

Kada, Kondo and 
Table 1 

Table of Dose-Finding Test Results 

Name of test substanct.: ·· --·" ac --- - .... : :.;':..1;.;. -.. ; :. ' : ~;:.-J. 

.No. 5 7 3 9 

A term of test February 25. !998 - February 28, 1998 

Number of revertants (number of colonies/plate) 

With(+) or Test substance 

without(-) concentration Base-pair substitution type Frameshi f t type 

S9mix ( t.L g/pl at e) 

TAlOO TA1535 WP2uvrA 

Solvent . 109 23 21 

control 142 ( 12 6) 22 ( 23) 20 ( 

143 25 18 

l. 2 124 ( !34) 20 ( 23) 27 ( 

145 18 19 

4. 9 141 ( 143) 20 ( 19) 23 ( 

159 22 19 

2 0 122 ( 141) 22 ( 22) 19 ( 

-89 mix 

124 27 19 

7 8 156 ( 140) 20 ( 24) 18 ( 

146 20 19 

3 1 3 134 ( 140) 17 ( 19) 17 ( 

118* 11* 22 

1 2 5 0 102* ( 11 O) 1* ( 9) 17 ( 

0* 0* 13* 

50 0 0 0* ( O) 0* ( 0) 20* ( 

Solvent 146 17 38 

control 141 ( 144) 20 ( 19) 33 ( 

146 22 29 

1. 2 !50 ( 148) 17 '• ( 20) 24 ( 

147 13 28 

4. 9 143 ( 145) 14 ( 14) 29 ( 

!58 25 26 

2 0 118 ( 138) 25 ( 25) 27 ( 

+S9 mix 

152 25 28 

7 8 147 ( 150) 24 ( 25) 27 ( 

141 22 28 

3 1 3 156 ( 149) 20 ( 21) 20 ( 

133 26 20 

1 2 5 0 !51 ( 142) 21 ( 24) 19 ( 

70* 2* 17* 

5 0 0 0 61* ( 66) 6* ( 4) 9* ( 

Name AF-2 'I NaN, •2 AF-2 

Positive 

control Concentration 

not (ug/plate) 0. 0 1 0. 5 0. 0 1 

requiring 

S9mix Number of 514 412 136 

colonies/plate 555 ( 535) 400 ( 406) !55 ( 

Name B [a] P •• 2AA •• 2AA 

Positive 

control Concentration 

requiring (t.Lg/plate) 5. 0 2. 0 1 0- 0 

S9mix 

Number of 870 310 407 

colonies/plate 841 ( 856) 306 ( 308) 454 ( 

#1 :2-(2-Furyl)-3-(5-ni tro-2-funl)acryl amide #2:Sodium azide 

f3:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropy!amino)acridine·2HC1 

#4:Benzo[a)pyrene #5:2-Aminoanthracene 

*:Growth inhibition of tester strains was observed. 

21) 

23) 

21) 

19) 

19) 

!8) 

20) 

17) 

36) 

27) 

29) 

27) 

28) 

24) 

20) 

13) 

146) 

431) 

TA98 TA1537 

27 13 

21 ( 24) 12 ( 13) 

17 9 

23 ( 20) 14 ( 12) 

23 11 

22 ( 23) 13 ( 12) 

17 12 

23 ( 20) 8 ( 1 O) 

22 8 

26 ( 24) 7 ( 8) 

24 7 

20 ( 22) 7 ( 7) 

8* 6* 

13* ( 11) 2* ( 4) 

0* 0* 

0* ( O) 0* ( O) 

30 22 

30 ( 30) 17 ( 20) 

28 14 

28 ( 28) 20 ( 17) 

31 19 

23 ( 27) 17 ( 18) 

24 17 

29 ( 27) 18 ( 18) 

28 19 

29 ( 29) 17 ( 18) 

30 17 

30 ( 30) 19 ( 18) 

31 6* 

28 ( 30) 12* ( 9) 

11* 3* 

!H ( 11) 8* ( 6) 

AF-2 ICR-191"' 

0. 1 1- 0 

613 1482 

646 ( 630) 1551 ( 1517) 

B [a] p B [a J P 

5. 0 5. 0 

210 61 

205 ( 208) 69 ( 65) 
T-a b 1 e 2 

Table of Test Results 

Name of test substance:~ . 

No. 5 7 3 9 

A term of test March II. 1998 - March 14, 1998 

Number of revertants (number of colonies/plate) With(+) or Test substance 

without(-) concentration Base-pair substitution type 

S9mix (.ug/p!ate) TA100 TA1535 WP2uvrA 

Solvent !51 17 20 

control 147 ( 149) 14 ( 16) 21 ( 

154 . 15 

3 9 142 ( 148) 17 ( 16) 

152 13 

7 8 !56 13) \ ( !54) 12 ( 

146 II 22 

1 5 6 !50 ( 148) I2 ( 12) 23 ( 

-S9 mix !54 I3 26 

3 1 3 154 ( I 54) !0 ( 12) 22 ( 

163 7 22 

6 2 5 148 ( 156) !3 ( I O) 26 ( 

131 * H 16 

1 2 5 0 123* ( 127) 10* ( 9) 22 ( 

19* 

2 5 0 0 16* ( 

D* 

5 0 0 0 D* ( 

Solvent 160 13 

13) 1 

36 

control 163 ( 162) 13 ( 37 ( 

3 9 

7 8 

176 15 29 

1 5 6 !59 ( I68) 16 ( !6) 36 ( 

168 18 28 

3 1 3 !51 ( 160) 12 ( !5) 3! ( 

+S9 mix 

!55 21 34 

6 2 5 172 ( 164) 14 ( 18) 38 ( 

175 12 36 

1 2 5 0 171 ( 173) 16 ( 14) 26 ( 

69* 16* 21* 

2 5 0 0 65* ( 67) 10* ( !3) 21* ( 

56* 8* 

9) I 16* 

5 0 0 0 65* ( 6!) 10* ( 17* ( 

Name AF-2 ., NaN, ., AF-2 

Positive 

control Concentration 

not ( .u g/p I at e) 0. 0 1 0. 5 0. 0 1 

requiring 

S9mix Number of 558 421 166 

colonies/plate 516 ( 537) 410 ( 416) 134 ( 

Name B (a] P . '2AA . ' 2AA 

Positive 

control Cone en t ration 

requiring (.LL g/plate) 5. 0 2. 0 1 0. 0 

S9mix 

Number of 949 322 428 

colonies/plate 966 ( 958) 289 ( 306) 437 ( 

#!:2-(2-Fury1)-3-(5-nitro-2-fury!)acry1amide #2:Sodium azide 

#3:2-Methoxy-6-chloro-9-[3-(2-chloroethy!)aminopropy!amino)acrldine·2HCI 

#4:Benzo [a]pyrene #5:2-Amlnoanthracene 

*:Growth inhibition of tester strains was observed. ~~ 

21) 

23) 

24) 

24) 

!9) 

!8) 

0) 

37) 

33) 

30) 

36) 

3!) 

24) 

17) 

!50) 

433) 

Frameshift type 

TA98 TA1537 

27 11 

20 ( 24) 11 ( II) 

20 8 

20 ( 20) 11 ( 1 0) 

19 9 

2I ( 20) 6 ( 8) 

27 8 

18 ( 23) 10 ( 9) 

2I 7 

19 ( 20) 8 ( 8) 

22 8 

25 ( 24) 8 ( 8) 

16* D* 

6* ( II) 0* ( O) 

41 21 

32 ( 37) 16 ( 19) 

19 

15 ( 17) 

15 

19 ( 17) 

36 15 

31 ( 34) I6 ( I 6) 

28 II 

31 ( 30) 18 ( 15) 

34 14 

33 ( 34) 9 ( 12) 

31 8* 

31 ( 31) 9* ( g) 

18* 

18* ( 18) 

12* 

II* ( 12) 

AF-2 ICR-191"' 

0. 1 1 . 0 

627 1505 

588 ( 608) 1574 ( 1540) 

B [a J p B [a] P 

5. 0 5. 0 

258 61 

265 ( 262) 68 ( 65) 
.a. ..._0 • ..L 

(Colonies/Plate) D o s e - r e s p o n s e 

,,, '20'0 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 39 78 156 313 625 

Fig. 2 

(Colonies/Plate) D o s e - r e s p o n s e 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 39 78 156 313 625 

.::O'Jc u r v e 

1250 2500 

c u r v e 

1250 2500 

No.5739 

( s 9 - ) 

oTA100 

eTA1535 

OWP2uvrA 

*TA98 

+TA1537 

5000 ( 11 g/plate) 

No.5739 

( s 9 + ) 

oTAlOO 

eTA1535 

OWP2uvrA 

*TA98 

+TA1537 

5000 ( 11 g/plate) 
VERIFICATION OF TRANSLATION 

This is to certify that English translation listed below conforms to the Japanese 

original report. 

Title : Acute Oral Toxicity Study of lmidoacrylate in Rats 

Study No. : 10101 

Ut dwk i, t}JJ r;ti~k~ 

Hideaki Hiratsuka, Ph.D. 

Study Director 

Panapharm Laboratories Co., Ltd. 

-83-

Date l 
PPL .t<'J.N.P..L !lliPUl{l': lUlUl 

Acute Oral Toxicity Study of Imidoacrylate in Rats 

- Final Report -

Aprill2, 2001 

Sponsor : ·' 

-· :::: ::· .. 

Organization under contract 

: Mitsubisbi Chemical Safety Institute Ltd. 

1-30, Shiba 2-cbome, Minato-ku, Tokyo 105-0014, Japan 

Test Facility : Panapbarm Laboratories Co., Ltd. 

1285, Kurisaki-machi, Uta, Kumamoto 869-0425, Japan 

-84--
QUALITY ASSURANCE STATEMENT 

Study No. : 10101 

Title :Acute Oral Toxicity Study of Imidoacrylate in Rats 

Personnel of the Quality Assurance Unit inspected each phase of this study. The 

phases, dates of inspection and dates of the QA report to the study director and the 

management are listed below. 

Phase 

·Protocol 

· Animal receipt and body weight measurement 

· Information, receipt, storage, and preparation 

of test substance mixture 

· Administration and clinical observation 

·Necropsy 

·Raw data (Draft report) and draft report 

·ditto (reinspection) 

· Return document oftest substance 

· Final report and all records 

Date of 

inspection 

Jan.24,2001 

Jan.30,2001 

Feb.6,2001 

Feb.6,2001 

Feb.20,2001 

Mar. 21, 2001 -

Mar. 22, 2001 

Mar. 23, 2001 

Mar. 24, 2001 

Apr. 12, 2001 

Date of 

QAreport 

Jan.24,2001 

Jan.30,2001 

Feb.6,2001 

Feb.6,2001 

Feb.20,2001 

Mar. 22, 2001 

Mar. 23,2001 

Mar. 24, 2001 · 

Apr.12, 2001 

I assure that this study was conducted in compliance with OECD Principles of Good 

Laboratory Practice (recommended by OECD, 1997) and this report accurately reflects 

the methods and raw data of this study. 

Manager of Quality Assurance Unit 

April 12, 2001 Hi detosbj Ogata sealed 

Panapharm Laboratories Co., Ltd. 

-85-.-
J:>J:>L .l:<lNAL .liliJ:>U.lU: lUlUl 

Signature 

Study Director 

April 12, 2001 Hjdeaki Hiratsuka sealed 

Panapharm Laboratories Co., Ltd. 

-86--

- i -
Study Schedule 

1. Initiation of Study : 

2. Animal Receipt : 

3. Initiation of Experiment : 

4. Administration: 

5. Necropsy: 

6. End of Experiment : 

7. Ena of Study : 

Conduct of Study 

1. Guideline 

rrLrll'V~LKCrU.tU: .LU.LUl 

January 24,2001 

January 30, 2001 

February 6, 2001 

February 6, 2001 

February 20, 2001 

February 20, 2001 

April12, 2001 

OECD Guideline for Testing of Chemicals " Acute Oral Toxicity " ( 401, Adopted by the Council 

on 24th February 1987) 

2.GlP 

OBCD Principles of Good Laboratory Practice (recommended by OBCD, 1997) 

Unforeseeable Circumstances that may have Adverse Effects on the Reliability of the Study and Deviations 

from the Protocol 

This study was carried out in accordance with its protocol, and there were no unforeseeable 

circumstances that might have adverse effects on the reliability of the study. 

Kinds of Records and Location of Storage 

1. Protocol 

2. Records of animals 

3. Records of animal room condition 

4. Records of test substance 

5. Records of study 

6. CD-R 

7. Reports 

-87--

- ll -
.I:".I:"L .I:'J.NAL .K.J:;.I:"UK!': lUlUl 

8. Other data concerning with study 

All records described above will be retained for ten years after the submission of the final report 

(Aprill3, 2001 - Apri112, 2011) in archive of PPL. After ten years, transaction of records will be 

discussed with the sponsor. 

Responsible Personnel 

1. Study Director : Hideaki Hiratsuka 

2. Technician in Charge : Kumiko Honda 

3. Pathologist : Masao Hamamura 

4. Veterinarian (Quarantine) : Emiko Kuwasaki 

5. Test Substance Management 

: Hiroyuki Izumi 

6. Preparation of Test Substance 

7. Animal Receipt 

8. Animal Husbandry 

9. Grouping 

10. Administration 

11. Clinical Observation 

: Mikako Sukita 

: Ryuji Yoshida 

: Hideaki Hirll.tsuka, Eiji Murata, Yasuo Nakaoka, Ryuji Yoshida, 

Hidehiro Ogata, Kumiko Honda, Kenji Ichimura, Takeshi Higuchi, 

Terohiko Enoshima 

: Kumiko Honda 

: Hideaki Hiratsuka 

: Hideaki Hiratsuka, Eiji Murata, Yasuo Nakaoka, Ryuji Yoshida, 

Hidehiro Ogata, Kumiko Honda, Kenji Ichimura, Takeshi Higuchi, 

Teruhiko Enoshima 

12. Measurement of Body Weight 

13. Necropsy 

: Hideaki Hiratsuka, Ryuji Yoshida, Hidehiro Ogata, Kumiko Honda, 

K.enji Ichimura 

: Hideaki Hiratsuka 

14. Pathological Examination : Masao Hamamura 

15. Management of Computer System 

: Ryoichi Tagami, Miho Iwanaga 

-88-_ 

-iii-
J.J.L....l'.L.L"'ru....~J.Vl'\..1, J..VJ.\!J. 

Signature 

Study Schedule 

Conduct of Study 

Contents 

e I. 0 0 0 ° 0 I I 0 I 0 I 0 I 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 0 I 

Unforeseeable Circumstances that may have Adverse Effects 

Page 

ii 

ii 

on the Reliability of the Study and Deviations from the Protocol · · · · · · · · · · · · · · · · · · · · · · · · · ii 

Kinds of Records and Location of Storage 

Responsible Personnel 

Summary 

Objective 

Materials and Methods 

Results 

Discussion 

ii 

iii 

1 

2 

2 

4 

5 

Fig. 1 Body weight changes in rats after a single oral administration of Imidoacrylate · · · · · · · 6 

Table 1 Mortality and approximate lethal dose in rats after a single oral administration of 

Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · · · · 7 

Table 2 Clinical signs in rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · 8 

Table 3 Body weights in rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · 9 

Table 4 Necropsy findings in rats after a single oral administration of Imidoacrylate · · · · · · · · · · 10 

Appendix (Individual data) 

-89--

-iv-
L L J.., .L'.Ll. 'lru..,. J.'\o..lj.L VJ.\.1. J.VJ.VJ. 

Summary 

lmidoacrylate was administered once orally to Crj: CD (SD) IGS rats at the dose levels of 1000 and 2000 

mglkg. Five males and five females were used in each group. Obtained results are shown below. 

No males and females died in any groups. 

Hypoactivity, salivation, loose stool, and lacrimation were observed on the day of administration, 

however, these signs disappeared by 6 hour after administration. No abnormal signs were observed up to 

the end of observation. 

Body weights in all animals increased normally. 

Necropsy revealed no abnormal findings in any animals. 

These results suggested that approximate lethal dose of Imidoacrylate was greater than 2000 mg!kg for 

male and female in the present study condition. 

-90--

- l -
PPL .t<'lNAL .t<.bPU.I:U: lUlUl 

Objective 

Imidoacrylate was administered orally to rats, and its acute toxicity and approximate lethal dose were 

investigated. 

Materials and Methods 

1. Test substance and Vehicle 

Imidoacrylate (Lot No. NL-9301-02) supplied from Mitsubishi Chemical Safety Institute Ltd. was 

used for this study. This test substance was a yellowish solid, and its purity was 95.4%. The 

remaining test substance was returned to the sponsor from Panapharm Laboratories Co., Ltd. for 

chemical analysis, and the stability of the test substance during the period of use in the study was 

confirmed (The analytical data were :filed in the raw data). Corn oil (Nacalai Tesque, Inc., Lot No. 

VOR1096) was used as vehicle. The test substance was stored in a locker in Test Substance Room at 

room temperature shielded from light and the vehicle was stored in a locker in Test Substance Room at 

room temperature. 

2. Animal and Animal Husbandry 

Twenty-one male and 21 female Crj: CD (SD) IGS rats aged 5 weeks were purchased from Charles 

River Japan, Inc. The animals of both sexes were quarantined and acclimatized for 6 days. In this 

period, all animals were examined for clinical signs and weighed in order to confinn the health 

conditions. One day before administration, 10 male and 10 female rats were selected by the stratified 

sequential randomization method and used for this study at age of 6 weeks. Body weights on the day 

of administration were 161.5 - 172.1 g for males and 111.2 - 125.8 g for females. Individual body 

weight was within ± 20% of the mean body weight. Animal room No. 03 in banier system A Area 

was used for this study. The animal room was controlled at temperature 24 ± 2°C (permissible range: 

21-27°C), humidity 55 ± 10% (permissible range: 35-75%), 12-hour light/dark cycle (lighting from 

7:00A.M. to 7:00P.M.) and ventilation 13-15 changes/hour. The animals were housed in stainless 

steel cage (W260xH200xD380 mm) at one animal per cage. Actual ranges of temperature and 

humidity in the animal room during the study were 21 - 26°C and 47 - 54%, respectively. The 

animals were allowed to receive an autoclaved pellet diet (CRF-1, Oriental Yeast Co., Ltd.) and to 

drink well water supplemented sodium hypochlorite (approximately 2 ppm) ad libitum. The diet 

containing impurities below the permissible limit, which was verified by Japan Food Research 

-91--

- 2 -
t't'L .l"lNAL Kbt'U!U: !U!Ul 

Laboratories (a juridical foundation) at the request of Oriental Yeast Co., Ltd. Water was analyzed at 

K.akujo Nankyukaken Center Co., Ltd., and it was verified that impurities were below the permissible 

limit. All housing equipments were sterilized by autoclaving before use. The animal cage and rack 

were changed once at group assignment, and the sanitary tray was changed twice a week. The floor of 

the animal room was cleaned and wiped every day with a disinfectant-soaked mop. 

3. Group Constitution, Reason for Determination of Dose Levels, and Group Assignment 

Group constitution was shown below. 

Dose level Concentration Dose volume Number of Animal 

Group Sex 

(mgfkg) (w/v %) (mlJkg) animal Number 

cf' 5 101-105 

Low-dose 1000 5 20 ~ 5 201-205 

r:J 5 106-110 

High-dose 2000 10 20 ~ 5 206-210 

Because the toxicity of the test substance was thought to be weak, 2000 mg!kg, which is the 

maximum dose described in OECD Guideline for Testing of Chemicals 401, was set as the high dose. 

In ad,dition, 1000 mgfkg was set according to a common ratio of 2. 

The number of animals per group was set at 5 each for males and females. The animals were 

allocated to each group one-day before administration by the stratified sequential randomization 

method on the basis of their body weights. After group allocation, the animals were identified 

individually by tail number with oily felt pen and ear punch. Cage card listing the study number, 

animal number, dose level, and sex were attached to each cage. Finally, the remaining animals were 

excluded from the study. 

4. Route and Method of Administration 

Oral route was chosen to evaluate the safety of the test substance when exposed by oral route. All 

animals were fasted for about 17 hours, and the test substance was orally administered with a stomach 

tube. Dose volume was 20 ml)kg. Individual dose volume was calculated from the body weight on 

the day of administration. The animals were given diet 3 hours after administration. 

5. Method of Preparation of Test Substance Mixture 

On the day of administration, Imidoacrylate was suspended in corn oil at the concentrations of 5 and 

10 (w/v) %. 

-92--

- 3 -
6. Method and Frequency of Qinical Observation, Examination, and Measurement 

1) Clinical Observation 

Observation period was for 14 days after administration. Animals were observed frequently 

for clinical signs and mortality until 6 hours after administration and twice daily (AM and PM) up 

to 13 days after administration. Animals were also observed once in the morning on Day 14. 

2) Body Weight 

Body weights were measured before the administration (Day 0), and Days 1, 3, 7 and 14. 

3) Necropsy 

All animals were eutbanized by bloodletting under ether anesthesia and then subjected to 

necropsy. 

7. Statistical Analysis 

Mean and standard deviation of body weights were calculated in each group. 

Results 

1. Mortality 

Mortality and approximate lethal dose are shown in Table 1. 

No males and females died in any groups. Therefore, approximate lethal dose was greater than 

2000 mglkg for male and female. 

2. Clinical Observation 

Results of the clinical observation are shown in Table 2 and Appendix 1, 2. 

Hypoactivity, salivation, and loose stool were observed from 15 min after administration in males 

and females in the 1000 mg!kg group. In addition to these signs, lacrimation was observed in males 

and females in the 2000 mg!kg group. These signs disappeared by 6 hours after administration, and 

no abnormal signs were observed up to the end of observation. 

3. Body Weight 

Body weight changes are shown in Fig.l, Table 3, and Appendix 3, 4. 

Body weights in all animals increased normally. 

-93-

- 4 -
4. Necropsy 

Necropsy findings are shown in Table 4. 

No abnonnal findings were observed in any animals. 

Discussion 

Imidoacrylate was administered orally to male and female rats with the dose levels of 1000 and 2000 

mglkg, and its acute toxicity and approximate lethal dose were investigated. 

No males and females died in the 1000 and 2000 mg!kg groups. 

Hypoactivity, salivation, loose stool, and lacrimation were observed on the day of administration. 

However, these signs disappeared by 6 hours after administration, and no abnormal signs were observed up 

to the end of observation. 

Body weights in all animals increased normally. 

Necropsy revealed no abnormal findings in any animals. 

These results suggested that approximate lethal dose of Imidoacrylate was greater than 2000 mglkg for 

male and female in the present study condition. 

-94-

- :::> -
0\ 

I 

\0 

Ul 

I 

I 

,..--., 

b.O 

'---' 

.... 

.c 

bJJ 

·v 

;>-, 

-o 

0 

0) 

350-, 

300 

250 

200 

150 

Male 

1000 mg/kg 

--<>-- 2000 mg/kg 

.--T I I I I I I I I I T--T --I . I 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Days after administration 

250 

200 

..... 

..c:: 

a.o 

...... 150 

"'0 

0 

co 

100 

50 

Fema

--o--- 1000 mg/kg 

---<>-- 2000 mg/kg 

I I I I I I I I 1 

0 I 2 3 4 5 6 7 8 

Days after adm

Fig.l Body weight changes in rats after a single oral administration of Imidoacrylate 
' \0 -._] 0\ 

I ' 

Table 1 

Sex 

Male 

Female 

Mortality and aEEroximate lethal dose in rats after a single oral administration of Imidoacrllate 

Group Number Time after administration 

and of Minute Hour Day 

dose animals 0 15 30 45 1 3 6 1 2 3 4 5 6 7"-'14 

1000 mg/kg 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2000 mg/kg 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

1000 mglkg 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2000 mg/kg 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Total n

o

dead an

0 0 

0 

0 
I 

Xl \0 'I 

I 

I 

Table 2 Clinical signs in rats after a single oral_administration of Imidoacrvlate 

Sex Group 

Male 

Female 

and 

dose 

1000 mglk:g 

2000 mg/kg 

1000 mglk:g 

2000 mg/kg 

Clinical sign 

Number of animals 

No abnormality 

Hypoactivity 

Salivation 

Lacrimation 

Loose stool 

Number of animals 

No abnormality 

Hypoactivity 

Salivation 

Lacrimation 

Loose stool 

Number of animals 

No abnormality 

Hypoactivity 

Salivation 

Lacrimation 

Loose stool 

Number of animals 

No abnormality 

Hypo activity 

Salivation 

Lacrimation 

Loose stool r 

Minute 

0 15 30 45. 

5 5 5 5 

5 3 0 0 

0 2 5 5 

0 1 2 3 

0 0 0 .o 

0 0 0 0 

5 5 5 5 

5 4 0 0 0 0 5 5 0 1 2 3 

0 0 2 1 

0 0 0 0 

5 5 5 5 

5 4 3 0 

0 0 0 5 

0 1 2 2 

0 0 0 0 

0 0 0 0 

5 5 5 5 5 2 0 0 

0 1 5 5 

0 2 4 4 

0 0 1 1 

0 0 0 0 

Time after administration 

Hour 

1 3 6 

5 5 5 

0 3 5 

5 0 0 

2 0 0 

0 0 0 0 2 0 

5 5 5 

0 5 5 

5 0 0 

3 0 0 

1 0 0 

0 0 0 

5 5 5 

0 4 5 

5 0 0 

2 0 0 

0 0 0 

0 1 0 

5 5 5 

0 5 5 

5 0 0 

1 0 0 

0 0 0 

0 0 0 

Da

1 2 3 4 

5 

5 

0 

0 0 

0 

5 

5 

0 0 

0 

0 

5 5 

5 5 

0 0 

0 0 

0 0 

0 0 

5 5 5 5 

5 5 5 0 0 0 0 0 0 

0 0 0 

5 0 

0 

0 

0 0 0 0 

5 5 5 5 

5 5 5 5 

0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 

5 5 5 5 5 5 5 5 0 0 0 0 

0 0 0 0 

0 0 0 0 

0 0 0 0 
Table 3 Body weights in rats after a single oral administration of Imidoacrylate 

Group Body weight (g) on day Sex and 

dose 0 I 3 7 14 

Male 1000 mg/kg N 5 5 5 5 5 Mean I69.0 182. I 206.6 256.4 326.8 S.D. ±4. 3 ±4.9 ±4. 6 ±1. 2 ±1. 5 

2000 mg/kg N 5 5 5 5 5 Mean 167. 9 179. 8 198.5 245.0 315. 1 S.D. ±3.1 ±6. 6 ±4. 9 ±7. 2 ± 12.1 

Female 1000 mg/kg N 5 5 5 5 5 Mean 117.0 127. 1 142.9 163. 1 189.4 S.D. ±5. 8 ±6. 8 ±5.3 ±6. 7 ±10.1 

2000 mg/kg N 5 . 5 5 5 5 Mean 1I8. 3 128. 1 141. 9 161. 5 189. 3 S.D. ±5. 8 ±7. 3 ±12. 3 ±13. 9 ±15.1 

I 

\() 

00 
Table 4 Necropsy findings in rats after a single oral administration of Imidoacrylate 

Organs and 

All organs and tissues 

NR: No remarkable changes. 

I 

\0 

( \0 

( I 

I 

Sex 

Group and dose 

Number of animals 

Male 

1000 mg/kg 2000 mg/kg 1000 mg/kg 

5 5 5 

NR NR NR 

Female 

2000 mg/kg 

5 

NR 
.t'.t'L .t'.U"'AL lU:J:"U.K.J.: lUJ.Ul 

Acute Oral Toxicity Study of Imidoacrylate in Rats 

Appendix 

(Individual data) 

-100-
rrL r.u'l.M.L .1.\..CrU.l\.J.: ~u~uJ. 

Content 

Page 

Appendix 1 Individual clinical signs in male rats after a single oral administration of 

lmidoacrylate · · · · · · · · · · · · · · · · · · · · · · · 1 

Appendix 2 Individual clinical signs in female rats after a single oral administration of 

Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · 2 

Appendix 3 Individual body weights in male rats after a single oral administration of 

lmidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · 3 

Appendix 4 Individual body weights in female rats after a single oral administration of 

Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · 4 

-101--
I 

...... 

C) 

N 

I 

Appendix 1 Individual clinical signs in male rats after a single_QraJadministration of Imidoacrylate 

Group Animal Time after administration 

and No. Minute Hour 

dose 0 15 30 45 1 3 6 

1000 mglkg 101 - a a,b a,b a d 

102 - a,b a,b 

103 

104 

105 

2000 mglkg 106 

107 

108 

b 

a 

a a 

a,b,c a,b,c 

a 

a,b a,b 

a,b a,b 

a a 

a a 

a,b,c a,b,c 

a,b a,b 

a a 

109 - - a a a,b 

d 

110 - - a a,b a 

Abbreviations : -, no abnormality; a, bypoactivity; b, salivation; c, lacrimation; d, loose stool 

D

1 2 3 4 
I 

...... 

N o 

(.)) 

I 

Appendix 2 Individual clinical signs in female rats after a single or-al administration of Imido acrylate 

Group Animal Time after administration 

and No. Minute Hour 

dose 0 15 30 45 1 3 6 1 

1000 mg/kg 201 

2000 mg/kg 

202 203 

204 205 206 

207 

208 

209 

210 

b 

b 

b 

a 

b 

b 

a,b,c 

a,b 

a 

a,b 

a,b 

a a 

a,b a,b 

a a 

a,b a,b 

a a d 

a,b,c a 

a,b a 

a a 

a,b a 

a,b a,b 

Abbreviations : -, no abnormality; a, hypoactivity; b, salivation; c, lacrimation; d, loose stool 

2 3 
Appendix 3 

Group and 

dose 

1000 mg/kg 

ZOOO mg/kg 

I 

1-' 

0 

""' I 

Individual body weights in male rats after a single oral administration of Imidoacrylate 

Animal Body weight(g) on day 

No. 0 1 3 7 14 

l 01 161. 5 174.0 200.4 245.8 313. 9 102 171. 3 186.5 211. 0 264.0 328.0 103 170. 5 182.8 208.6 259. 7 328.4 104 169. 4 181. 9 209.8 259. 7 330.5 I05 I 72. l I85.4 203. I 252. 6 333.0 

106 I 63. I I73. 7 192.9 238. 7 302.8 107 170. 7 182. 7 203. 1 249. 5 324 .. 3 108 167.2 171. 7 193. 5 236. 9 301. 3 109 170.3 184.4 200.9 246.0 326.4 110 168.3 186.4 202. 2 254.0 320.8 
Appendix 4 

Group and dose 

1000 mg/kg 

2000 mg/kg 

I 

f-' 

0 

Ul 

I 

Individual body wefghls in female rats after a single oral administration of Imidoacrylate 

Animal Body weight(g) on day 

No. 0 1 3 1 14 

201 125. 8 138. 2 152. 3 173. 1 204. 6 202 119. 0 129.0 141. 5 164. 5 188. 2 203 Ill. 4 I 21. 9 140. 6 160.7 187. 5 204 116. 3 123. 1 140. 9 162. 8 190.4 205 I I 2. 5 123. 2 139. 3 154. 6 116. 2 

206 l 23. 7 137.0 158.3 177. 7 204.3 207 124. 9 134.8 151. 4 174. 1 205. 2 208 115. 9 121. 8 132. 0 146. 2 17Z.O 209 115. 8 125.4 137. 2 156.0 187.3 210 111. 2 121. 7 130.6 153. 1 177.7 
. :,•· 

.LGa 

FINAL REPORT 

... - -::.::...-:':"-.. - -=--- fl 

,, 

Induction of micronuclei in the bone marrow of treated mice 

Study Number: S277 

Sponsor 

~ _, .. . . -~. -· -::-: ·- ... ~ --

'II 

- .....,...,. . -

-106-. 

Test Facility 

Toxicology Center 

LGCI, Life Sciences R&D 

1 04-1 Munji-dong 

Yuseong-gu 

Daejeon 

305-380 

KOREA 
lo 

study Number. S277 . Report 

CONTENTS 

Page 

CONTENTS ................................................................................................... 1 

STUDY DIRECTOR AUTHENTICATION AND 

GLP COMPLIANCE STATEMENT ................................................................. .4 

QUALITY ASSURANCE STATEMENT ................................................... ~ ...... 6 

RESPONSIBLE PERSONNEL ....................................................................... 6 

ARCHIVE STATEMENT ................................................................................ 6 

1. SUMMARY .............................................................................. ; ..................... 7 

2. INTRODUCTION ........................................................................................... 8 

2. 1 Study title ....................................................................................................... 8 

2.2 Study Objective .............................................................................................. 8 

3. STUDY DATES .............................................................................................. 8 

4. TEST ARTICLE AND FORMULATION ............................................................ 8 

4.1 Test article ........................................................................................ , ............ 8 

4.2 Formulation ..................................................................................................... 8 

5. CONTROL ARTICLES ................................................................................... 9 

5.1 Negative control article .................................................................................... 9 

5.2 Positive control article .................................................................................... 9 

6. TEST SYSTEM .............................................................................................. 9 

6.1 Animals: supply, acclimatization and allocation .............................................. 9 

6.2 Animals: housing, diet and water supply ....................... : .............................. 1 0 

7. TREATMENT ............................................................................................... 10 

7.1 Preliminary dose finding experiment ............................................................ 10 

7.2 Justification of dose levels ............................................................................. 11 

7.3 Dose groups and animal numbers of micronucleus test. ................................ 11 

7.4 Route and duration of administration ........................................................... 11 

8. OBSERVATIONS ........................................................................................ 11 

8.1 Mortality ....................................................................................................... 11 

8.2 Clinical signs ................................................................................................ 11 

8.3 Body weight .. · ............................................................................................... 12 

9. NECROPSY AND SLIDE PREPARATION ................................................. 12 
study Number: S277 Report 

10. SCORING MI.CRONUCLEI ........................................................................... 12 

11. ANALYSIS OF RESULTS ............................................................................ 12 

11 .1 Treatment of data ........................................................................................ 12 

11 .2 Acceptance criteria ...................................................................................... 13 

11.3 Evaluation criteria ........................................................................................ 13 

12. RESULTS .................................................................................................... 13 

12.1 Mortalities .................................................................................................... 13 

12.2 Clinical findings ............................................................................................ 13 

12.3 Body weights ............ : ............................................................ : ..................... 13 

12.4 Micronuclei findings ........................................ ~.......................................... 13 

13. CONCLUSION .............................................................................................. 13 

14. REFERENCES ............................................................................................ 14 

15. PROTOCOL DEV.IATIONS .......................................................................... 14. 

16. TABLES ....................................................................................................... 15 

17. APPENDICES ............................................................................................. 20 
study Number: s2n Report 

LISt OFT ABLES 

Table 1. - - __ . - . ~ _ :; Summary of MNPCE frequency and PCEI(PCE+NCE) 

ratio ............................................................................................................... 15 

Table 2. / .. ~ ~,. - = :- • - /: Summary of group mean body weight changes ............... 16 

Table 3. ·- _. ::· · -::. :Summary of Mortality ..................................................... 17 

Table 4. :-r--~· ···--:-:: · -~ .: Summary of group mean body weight changes 

(Preliminary range f1nding) .......................................................................................... 18 

Table 5. , .. _ =-~ ·· --:- = : _ :.: Summary of Mortality (Preliminary range finding) ............ 19 

LIST OF APPENDICES 

Appendix 1. . _ , __ ,,-- , _ ·: PCE, NCE, and MNPCE on each animal .................. 20 

Appendix 2 .. - __ ...... _ : .: Individual Body weights ............................................ 23 

Appendix 3. ~- _. • · : Individual clinical signs ............................................... 24 

Appendix 4. _ ~:":'~:.-_ . _. : Individual Body weights (Preliminary range finding) .. 25 

Appendix 5 ... >.-:..: ::·:-:-__ , _ ~: Individual clinical signs (Preliminary range finding) ... 26 

Appendix 6. Certificate of Analysis .......................................................................... 27 
Study Number: S277 

STUDY DIRECTOR AUTHENTICATION 

AND GLP COMPLIANCE STATEMENT 

Report 

Induction of micronuclei in the bone marrow of treated mice 

Study Number: S277 

I, the undersigned, hereb~ declare that the work described in this report was performed under my 

supervision, as Study Director, and that the report provides a true and accurate record of the results 

obtained. 

The study was performed in accordance with the agreed protocol, unless otherwise stated, and the 

study objectives were achieved. The study was also performed in accordance with LCCI Standard 

Operating Procedures and the principles of the following codes of Good Laboratory Practice: 

Korea National Institute of Environmental Research 

Good Laboratory Practice Regulations for the Testing of Chemicals 

KNIER Notification No.1998-41, 23 December 1998 

Organization for Economic Co-operation and Development 

Principles. on Good Laboratory Practices (as revised in 1997) 

Paris 1998 

D'- d~ P~.JL ).O 0 I ' f-l. t.u;:;y. ' '.L... 2..-

l 

Du Hee Park, DVM, MSc. 

Study Director 

Toxicology Center 

LGCI/Llfe Sciences R&D 

Confirmed by: 

Sangk n Lee, Ph.D. 

Management 

Toxicology Center 

LGCI/Life Sciences R&D 

Date 1; 

Date • 

Pa.-110-:n 
Study Number; Sf:!J Report 

QUALITY ASSURANCE STATEMENT 

Study Number: S277 

Study Title:~--- ·... ;:Induction of micronuclei in the bone marrow of 

treated mice 

The study described in this report was subject to audit by the independent Quality Assurance 

Unit as indicated below. 

The findings of each audit were reported to the Study Director and Management as prescribed 

by Standard Operating Procedures . 

.. The report audit was designed to confirm that as far as can be reasonably establi-shed the 

methods described and results incorporated in the report accurately reflect the raw data 

produced during the study. 

Audit Type 

Study-based 

Ha-Kang Kim 

Section of GLP 

Quality Assurance 

LGCI, Life Sciences R&D 

Audit Phase 

Study protocol 

In process 

Raw data 

Study report 

Audit Date 

27 Jun 2001 

11 Jul 2001 

13 Jul 2001 

26 Jul2001 

20 Aug 2001 

20 Aug 2001 

Page 5 of27 

-111-

Report to Study Report to 

Director Management 

28 Jun 2001 29 Jun ·2001 

11 JLil 2001 12 Jul2001 

13 Jul 2001 14 Jul2001 

26 Jul 2001 30 Jul2001 

22 Aug 2001 23 Aug 2001 

22 Aug 2001 23 Aug 2001 

Date 
Study Number: S277 Report 

·RESPONSIBLE PERSONNEL 

Induction of micronuclei in the bone marrow of treated mice 

The following staffs were responsible for key elements of the study. 

Director of Toxicology: SangKyun Lee, PhD 

Study Director: Du Hee Park, DVM MSc 

Quality Assurance Unit Ha-kang Kim, MSc 

'The following staffs were responsible for 'key elements of the study: 

Responsible Scientist: 

Formulation: 

Yun Wha Jung, MSc 

Ok Youn Kim, BSc 

Eun-Kyeu Son, MSc 

ARCHIVE STATEMENT · 

All primary data or authenticated copies thereof, specimens and the final report will be retained in 

the Archives and the Document Control Center (DCC) of LGCI/Life Sciences R&D for five years 

after submission of the final report. At this time the Sponsor will be contacted to determine whether 

data should be returned, retained or destroyed on their behalf. 
.. ,·· . ~ . . 

Study Number: S277 Report 

1. SUMMARY 

The potential of . ~ 1 to induce micronuclei was determined in vivo in bone 

marrow cells of mice. 

For the micronucleus test, preliminary dose range finding experiment was done. In the 

preliminary experiment, 2000 mg/kg dosing resulted in all animal death and two consecutive 

1000 mg/kg dosing was thought to be around LD50• 

In the micronucleus experiment, test article groups of six male of ICR mice were dosed once 

daily on two consecutive days by intraperitoneal route with · ·- :=::-:L~. · ~ ~ _ • at dose levels of 

125, 250 and 500 mglkg/day. 

Animals in the negative control groups were dosed with vehicle via the same route following 

the same treatment schedule as that in the test article groups. Animals in the positive control 

group were dosed once by intraperitoneal injection with Mitomycin C at 2 mg/kg. . 

Bone marrow samples were collected from animals in the test article and negative control 

groups at 24 hours after the second dosing, while they were collected from positive control 

animals at 24 hours after the first dosing. 

'. In the micronucleus test, mortality case was not observed. After administration, hypoactivity, 

rough fur, ataxia, abdominal distension were·observed in the highest test article groups (500 

mg/kg). Mean body weights of all groups were not decreased by the administrations. 

Bone marrow smear slides from all groups were analyzed. The ratio of polychromatic 

erythrocytes (PCE), normochromatic erythrocytes (NCE) and the total number of micronucleated 

polychromatic erythrocytes (MNPCE) were counted for each animal. 

All animals dosed with ; .. :·_ ·-.:::- - ~ ;. - ' exhibited similar PCEJ(PCE+NCE) ratios and 

MNPCE frequencies to those of historical negative control animals. 

MNPCE numbers of negative control group fell. within historical range, but in the statistical 

analysis, it was different with test article groups. It was not considered meaningful value of the 

statistical difference (p=D.02). 

It is concluded that :: ~';-,;> ;_· . - , did not induce micronuclei in the mouse bone marrow 

cells under the conditions employed for this test. 

P -113- '7 ag --. 
study Number: S277 Report 

2. INTRODUCTION 

2.1 Study title 

;· ·--" --- . Induction of micronuclei in the bone marrow of treated mice 

2.2 Study objective 

3. 

4. 

The objective of this study was to assess the ability of the test article, .'. ~ =-- _ , to 

induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of treated mice. 

STUDY DATES 

Study initiation: 26 June 2001 

Experimental phase initiation: 28 June 2001 

Animal arrival: 28 June and 6 July 2001 

Treatment: 11 July and 12 July 2001 

Slide preparation: 13 July 2001 

Experimental phase complete: 07 August 2001 

Study c9mpletion: 22 August. 2001 

TEST ARTICLE AND FORMULATION 

The Sponsor provided the test article and a Certificate of Analysis (COA). The COA was 

added in this report (Appendix 6). 

4.1 Test article 

Test article identity: 

Chemical name: 

Supplied by: 

Lot number: 

Purity: 

Test article received and quantity: 

Storage conditions: 

Appearance: 

Stability of test article: 

Safety precautions: 

Current expiry date: 

4.2 Formulation 

- ' 

r 

not applicable 

NE-1301-07 

above 95% 

15 June 2001, 50 g 

under 25'C 

White solid 

stable in air and water, unstable to light and heat 

This article can be polymerized by UV light, heat and 

oxidation 

Not provided 

Not provided 

Preliminary solubility assessment was made. Test article was insoluble in water and corn oil and 

suspension status was not a satisfying one to injection. 5% Pluronic F-68 in saline was chosen 

as a solvent to improve suspension status. Formulations were prepared in solution with a solvent 
::·.:·. 

·,(. 

·~· 

study Number: S277 Report 

on the day of dosing. The formulations were handled at the room temperature during the 

dosing period. All treatment solutions were prepared on the day of administration and dosed 

within approximately 1 hour of the initial formulation of the test article. Details on treatment 

solutions are shown below: 

Concentration of Dose levels of 

~periment treatment solution Administered 

Dose range finding 

Micronucleus 

experiment 

5. CONTROL ARTICLES 

5.1 Negative control article 

(mg/ml) 

100 

200 

12.5 

25.0 

50.0 

(mg/kg) 

1000 

2000 

125 

250 

500 

Negative (5% Pluronic F-68 in saline) control treatments were administered the vehicle alone 

with the same dosing volume (1 0 ml!kg), once daily for two consecutive days. 

5.2 Positive control article 

Positive control treatment was administered the Mitomycin c· (MMC, Sigma Chemical Co.). It· 

was freshly prepared in water for injection at concentration of 0.2 mg/ml and dosed via 

intraperitoneal route at a dose level of 2 mg/kg. MMC was administered once, 24 hours prior to 

necropsy. 

6. TEST SYSTEM 

6.1 Animals: supply, acclimatization and allocation 

6.1.1 

6.1.2 

6.1.3 

Animals 

Species I Strain: 

Age ordered: 

Number of animals ordered: 

Weight range ordered: 

Supplier: 

Justification of test system: 

Acclimatization 

Duration: 

Husbandry conditions: 

Allocation to treatment groups 

Allocation: 

Method: 

Mouse /ICR(SPF) 

7 weeks. 

20 males for preliminary toxicity study 

40 males for the micronucleus study 

within an 20% of the overall mean 

Daehan Biolink Co. Ltd., Korea. 

The mouse was selected because it was a rodent 

species recommended for the micronucleus test by 

other various regulatory authorities and background 

data were available. 

5 days prior to dosing to allow for acclimatization. 

Refer to Section 6.2. 

One day prior to treatment. 

Random. 

Pa -115- _27 ,_ -
Study Number: S277 

6.1.4 Identification 

Numbering: 

Method: 

Cage labels: 

Unique for each animal within study. 

Tail marking method. 

Report 

Colored label displaying the dose level, study number, 

animal number 

6.2 Animals: housing, diet and water supply 

6.2.1 Environmental control 

Rodent facility: 

Air supply: 

Temperature: 

Relative humidity: 

Lighting: 

6.2.2 Animal accommodation 

Animals per cage: 

Cage material and flooring: 

6.2.3 Diet and water supply 

Diet supply 

Diet name: 

Diet type:. 

Availability: 

Certification: ·'": .. ·· 

Limited access (Room no. 1 PB71) 

Hepa-filtered, not recirculated 

Was maintained within the range of 19.47 to 23.22"C. 

Was maintained within the range of 57.34 to 69.88 %. 

12 hours light : 12 hours dark. 

Five or Six of the same sex, unless reduced by 

mortality or isolation. 

Polycarbonate cages {W260 x 0280 x H180 mm) with 

bedding. 

Rodent feed (Purina, Korea) 

Pellet. 

Non-restricted. 

Before delivery, each batch .of diet was analyzed by the 

supplier for various nutritional components and 

chemical and microbiological contaminants. Supplier's 

analytical certificates were scrutinized and approved 

before any batch of diet was released for use. 

This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. 

Water supply 

Supply: 

Availability: 

Certification: 

7. TREATMENT 

Filtered and UV -treated tap water. 

Non-restricted via polycarbonate bottles with sipper 

tubes. 

The water was routinely analyzed for the specific 

contaminants at Daejeon Regional Institute Health and 

Environment. Certificates of analysis were received 

and archived. 

7.1 Preliminary dose finding experiment 

Test article was administered by intraperitoneal route for two consecutive: days with 6 males in 

each dose group. At 2000 mg/kg, all animals were dead in 30 minutes after test article 

administrations. At the dose level of 1000 mg/kg, three of six animals were dead in 3 hours 

after the second test article administration( day 2). 

Clinical signs of dead animals were severe in the both dose groups(see appendix 5). From the 

results of this experiment, the highest dose level of micronucleus study was determined as 500 

mg/kg/day. 

Pag-n~=~-?7 
' . 

. ,. 

..... 

.;_. 

study Number: S277 Report 

7.2 Justification of dose levels 

Preliminary dose finding experiment was performed. Regarding the results of preliminary dose 

range finding study, high dose level of micronucleus test was determined. 

7.3 Dose groups and animal numbers of micronucleus test 

After randomization, 6 mouse were assigned a cage and were grouped to 3 treatment, 1 vehicle 

control and 1 positive control groups. A color-coded card that had labeled study number, group 

assignment, and animal numbers was placed on the front of each cage. Control animals were 

received vehicle only. Individual animals doses was calculated using a individual body weight. A 

constant dose volume of 1 0 ml/kg body weight was used. 

Dose 24 hour Sampling_iMale) 

Group Test article (mg/kg) Total Number of Number of 

treated animals Treated animals 

N~ative Vehicle 0 6 M01 ~ M06 

Test Low (125) 6 M07- M12 -- Middle(250) 6 M13- M18 Article -. - High (500) 6 M19- M24 

Positive MMC 2 6 M25- M30 

7.4 Route and duration of administration 

7.4.1 

7.4.2 

Route: 

Justification: 

7.4.3 

7.4.4 

Administration: 

Frequency: 

8. OBSERVATIONS 

8.1 Mortality 

Debilitated animals: 

Premature sacrifice: 

8.2 Clinical signs 

Animals: 

Deviations from normal: 

recorded at the time in respect 

of · 

Additional observations: 

Intraperitoneal route 

The Intraperitoneal route was chosen for this study 

because it was recommended ·exposure route by the 

various authorities for micronucleus test.· 

Was administered by a syringe with 22 - 26G needle. 

Dosed once daily two consecutive days 

Observed carefully, may be isolated to prevent 

cannibalism. 

Animals may be killed on humane grounds or if 

considered in extremis. 

Individually observed after dosing hourly during the first 

4 hours with special attention, and daily for a total of 3 

days. 

Nature and severity. 

Date and time of onset. 

Duration and progress of the observed condition. 

Changes in skin and fur, eyes and mucous membrane, 

and also respiratory, circulatory, autonomic and central 

nervous systems, and somatomotor activity and 
study Number: 8277 

8.3 Body weight 

Individual bodyweight recording: 

behavior pattem. 

At the first dosing (Day 1 ). 

At the second dosing (Day 2). 

At necropsy (Day 3). 

The day of first dosing was designated as Day 1. 

9. NECROPSY AND SLIDE PREPARATION 

Report 

Test article-and vehicle-treated mice were killed in groups, 24 hours after the second 

administration. MMC-treated mice were killed 24 hours after the single dose administration. 

Mice were killed by cervical dislocation in the same sequence used for the dosing. · 

Both femurs from each animal was exposed, removed, cleaned of adherent tissue and the ends 

removed from the shanks, Using a syringe and needle, bone marrow was flushed from the 

marrow cavity with 0.5 ml fetal bovine serum into appropriately labeled centrifuge tubes (1 per 

animal). 

The tubes were centrifuged at 1250 x 'g' for 2-3 minutes; the serum was aspirated to leave 1 or 

2 drops and the cell pellet. The pellet was mixe'd into this small volume of serum in each tube, 

and from each tube 1 drop(about 6 pl) of suspension was place on the end of each of 2 slides 

labeled with the appropriate study number, sampling time, date of preparation. A smear was 

made from the drop by drawing the end of a clean slide along the labeled slide. Two slides were 

prepared .each animal. 

Slides were allowed to .~ir-dry and then fix~d for 5 minutes in absolute methanol before being 

stained. After ·air-drying, slides were stained for 30 minutes in '5% Giemsa stain in Sorensen's 

buffer,· pH6.8. Stained slides were rinsed for 4- 5 seconds in 0.004% citric acid solution and 

then were rinsed 3 times in distilled water. After air-drying, randomized numeric numbers was 

labeled which serve as the code so analysis can be conducted "blind". Slide was examined 

under microscope (x 1 000). · 

1 D. SCORING MICRONUCLEI 

The slides from all groups were arranged by randomized numeric number order. Initially the 

relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or purple stained 

enucleate cells, and normochromatic erythrocytes (NCE), seen as pink-stained enucleate cells, 

was determined until a total of at least 500 cells (PCE plus NCE) has been analyzed and 

counting will continue until at least 1000 PCE per slide have been examined. All 

micronucleated polychromatic erythrocytes (MNPCE) observed during counting were recorded. 

Total2000 PCE(1000 per each slide) per animal was examined. 

11. ANALYSIS OF RESULTS 

11.1 Treatment of data 

After completion of microscopic analysis and decoding of the data, the ratio of PCE/NCE for 

each animal and the mean for each group was calculated, and the frequency of MNPCE for 

each animal and the mean for each group will also be determined. The PCE/NCE ratio was 

examined to see if there was any decrease in groups of treated animals that could be taken as 

evidence of bone marrow toxicity. 

The number of MNPCE in each treated group will then be compared with the numbers in 

vehicle control groups by using a 2 x 2 contingency table to determine chi-square. 

Probability values of p :5 0.05 was accepted as significant. 

P -118-:27 ag_ ~· 
::':' 

Study Number: S277 Report 

11 ;2 Acceptance criteria 

This micronucleus test was considered valid because the following criteria were met: 

1) The frequencies of MNPCE in vehicle control groups fell within or close to the historical 

vehicle control range, and 

2) The positive control chemical (MMC) induced a statistically significant increase in the 

frequency of MNPCE, and 

3) At least 5 animals out of each group were available for analysis. 

11.3 Evaluation criteria 

The test article was considered as positive in this assay if: 

1) The assay was valid (see acceptance criteria). 

2) Statistically significant increase in the frequency of MNPCE at one or more concentrations, 

and 

3) The frequency of MNPCE at such a point exceeded the historical control range. 

12. RESULTS 

12.1 Mortalities (Table 3, Appendix 3) 

Any treat-related death was not observed in test article groups. 

12.2 Clinical findings (Table 3, Appendix 3} 

At the highest dose level (500 mg/kg}, animals showed hypoactivity, rough fur, ataxia, 

abdominal distension. Abdominal distension seemed to be caused by intestinal distension 

(enlarged and full of food) 

12.3 Body weights (Table 2, Appendix 2) 

Mean body weights of all groups were not decreased by the administrations. In the highest 

dose, body weight was increased after one day after test article administration (day 2) due to 

abdominal distension. 

12.4 Micronuclei findings {Table 1, Appendix 1) 

13. 

MNPCE numbers of negative control group fell within historical range, but in the statistical 

analysis, it was different with test article groups (p=0.02). It was considered due to 

unexpected low frequency numbers of MNPCE in the negative control group that could often 

affect the statistical results. It was not considered meaningful value of the statistical 

difference, because MNPCE numbers of test article groups were all similar with historical 

negative control (saline). All animals dosed with .· -- · · - exhibited similar 

PCE/(PCE+NCE) ratios to those of negative control groups. MNPCE frequencies of all 

animals dosed with were also considered similar to those of historical 

negative control groups. 

The positive control group exhibited clear increases in the frequencies of MNPCE (p:;; 0.001 ). 

CONCLUSION 

It is concluded· that did not exhibit clastogenic activity in the 

polychromatic erythrocytes in the bone marrow of mice when tested under the conditions 

employed for this study. 
study Number: S277 Report 

14. REFERENCES 

Wei L Y, Chao J S and Hong C C (1997) Assessment of the ability of propoxur, methomyl and 

aldicarb, three carbamate insecticides to induce micronuclei in vitro in cultured chinese 

hamster ovary veils and in vivo in BALB/c mice. Environmental and Molecular Mutagenesis 29, 

386-393. 

Crebelli R, Carere A, Leopardi P, Conti L, Fassio F, Raiteri F, Barone D, Ciliutti P, Cinelli Sand 

Vericat J A (1999) Evaluation of 1 0 aliphatic halogenated hydrocarbons in the mouse bone 

marrow micronudeus test. Mutagenesis 14, 207-215. 

Higashikumi N and Sutou S (1995) An optimal, generalized sampling time 30± 6 h after double 

dosing in the mouse peripheral blood micronucleus test. Mutagenesis 10, 313-319 

Proudlock R J, Statham J and Howard W (1997) Evaluation of the rat bone marrow and 

peripheral blood micronucleus test using monocrotaline. Mutation Res 392, 243-249. 

Holden H E, Majeske J B and Studwell D (1997) A direct comparison of mouse and rat bone 

marrow and blood as target tissues in the micronucleus assays. Mutation Res 391, 87-89. 

Chao J S, Wei L Y •. Huang M C, Liang S C and Chen H H C (1999) Genotoxic effects of 

triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo. Mutation 

Res 444, 167-174. 

Asano N, Katsuma Y, Tamura H, Higashikuni N and Hayashi M (1998) An automated new 

technique for scoring the rodent micronucleus assay: computerized image analysis of acridine 

orange. supravitally stained periph~ral blood cells. Mutation Res 404 149-154. 

Lovell D P, Anderson D, Albanese R, Amphlett G E, Clare G, Ferguson R, Richold M, 

Papworth D G, and Savage J R K (1989) Statistical analysis of in vivo cytogenetic assays. In 

"Statistical evaluation of mutagenicity test data" Report of the UKEMS Sub-committee on 

Guidelines for Mutagenicity Testing. Part Ill. Ed D J Kirkland. Cambridge University Press, pp 

184-232. 

15. PROTOCOL DEVIATIONS 

1. Acclimatization period was stated 6 days in the protocol, but in this study, acclimatization 

period was 5 days which was marginal period of regulations. 

-120-. Pag~ -- · 27 
study Number: S277 Report 

16. Tables 

Table 1. - - · - ::. -:.: Summary of MNPCE frequency and PCE/(PCE+NCE) ratio 

Group mean 

Group mean frequency of MNPCE 

Dose group PCE 

PCE+NCE 

(per 1000) 

Vehicle 0.58 0.33* 

10 ml/kg 

-~ ·- ----· . ,, . - - ' ,. 0.66 0.83 

125 mg!kg 

- '-.. ~ ,., . 

0.60 1.08 

250 mg/kg 

j -. ~ ... 0.42 1.25 

500 mg/kg 

Mitomycin C 0.33 81.5** 

2 mg/kg 

*: Statistical significance was observed (P=0.02). 

MNPCE numbers of negative control group fell within historical range, but in the statistical 

analysis, it was different with test article groups. It was not considered meaningful value of 

the statistical difference. 

**:Statistical significance was observed (P<0.01). 

P -12l-_r27 a~:,. --
Study Number: S277 Report 

Table 2. "'~ ·~: Summary of group mean body weight changes 

(unit: Q) 

Dose group Gro~ mean body weight 

Day 1 Day2 Day3 

Vehicle 34.13 33.95 34.70 

10 ml/kg 

.' .. 33.68 34.33 34.22 

125 mg/kg 

- . . - 34.13 33.88 34.17 

250 mg/kQ 

- . - ·.-~ . '\-. 34.37 36.10* 34.17 

500 mg/kp 

Mitomycin C - 33.78 34.68 

2 mg/kg 

l j -: Not applicable 

1 "': Statistical significance was observed (P<0.05). 

\: Pa;_ 22-~27 

jL 
..... 

:•·. 

study Number: S277 

Table 3. .. : Summary of Mortality 

Dose group Day 1 

Vehicle 0 

10 ml/kg 

I .. 0 

125 mg/kg 

t .. .. - 0 

250 mg/kg 

.. .. 0 

500 mg/kg 

Mitomycin C - 2 mg/kg 

Sa: Sacrifice for bone marrow sampling 

- : Not applicable 

Mortality 

Day2 

-123-

Parre 1 / -;r'27 

0 

0 

0 

0 

0 

Report 

Day3 

Sa 

Sa 

Sa 

Sa 

Sa 
Study Number: s2n Report 

Table 4 .. ' ~: Summary of group mean body weight changes {Preliminary 

range finding) 

(unit:_g_l 

Dose group Day 1 Day2 

1 •••• ..._- ___ ... _ 

. - 32.7 33.1 

1000 mg/kg 

. ' 

. ~ . - '· . ' 33.7 -

2000 mg/kg 

-:Not applicable 

Pag-124<27 
!·+ t study Number: S277 Report '\,' 

t 

>: 

f 6

,, Table 5.. ._;.:Summary of Mortallty{Preliminary range finding) 

f;: 

I 

~: 

~· w 

~: ~-

~-€· 

~'· g, 

F; 

m' t '" (' i:". 

~1: 1': 

'-

fi: f -: Not applicable 

f. 

~ li 

i.'· 

},t 

)j 

Dose group 

1000 mg/kg 

-

2000 mg/kg 

-125-

Page . ::_, "27 

Number of dead animal/Total animal 

Day 1 Day2 Day3 

0/6 3/6 -

6/6 - -
i 

:~' 

~·. 

'·• 

study Number: S277 

17. Appendices 

Appendix 1. 1- . •·'· 

Dose group 

Animal 

Number 

M1 

d' 

M2 

Vehicle d' 

10 ml/kg M3 

d' 

M4 

d' 

M5 

d' 

M6 

d' 

M7 

d' 

MB 

: . ' d' .,.., . .. 

125 mg/kg M9 

d' 

M10 

d' 

M11 

d' 

M12 

d' 

Report 

-. ·; =:-= , PCE, NCE, and MNPCE on each animal 

Slide PCE:NCE PCE PCE MNPCE 

Counted PCE+NCE Counted counted .per1000 

A 230:270 0.46 1000 0 0 

B 241 :259 0.48 1000 1 1 

Total 471:529 0.47 2000 1 0.5 

A 290:219 0.57 1000 0 0 

B 283:217 0.57 1000 0 0 

Total 573:436 0.57 2000 0 0 

A 395: 105 0.79 1000 1 1 

B 354:146 0.71 1000 0 o· 

Total 749:251 0.75 2000 1 0.5 

A 259:242 0.52 1000 0 0 

B 275:226 0.55 1000 0 0 

Total 534:468 0.53 2000 0 0 

A 302: 199 0.60 1000 0 0 

B 309: 193 0.62 1000 0 0 

Total 611 : 392 0.61 2000 0 0 

A 264:239 0.52 1000 0 0 

B 264:236 .. 0.53 1000 2 2 .. 

Total 528:475 '· > 0.53 2000' 2 1 

A 302:202 0.60 1000 0 0 

B 307: 193 0.61 1000 3 3 

Total 609:395 0.61 2000 3 1.5 

A 294:206 0.59 1000 1 1 

B 344: 156 0.69 1000 1 1 

Total 638:362 0.64 2000 2 1 

A 326: 174 0.65 1000 0 0 

B 270: 130 0.68 1000 0 0 

Total 596:304 0.66 2000 0 0 

A 285:239 0.54 1000 0 0 

B 301: 199 0.60 1000 0 0 

Total 586:438 0.57 2000 0 0 

A 470: 139 0.77 1000 1 1 

B 564: 173 0.77 1000 0 0 

Total 1034: 312 0.77 2000 1 0.5 

A 358: 157 0.70 1000 2 2' 

B 360: 175 0.67 1000 2 2 

Total 718:332 0.68 2000 4 2 

Pa:_126-.tj27 
r .. 1;,• 

:.: 

!': 

i.' 

study Number: S277 Report 

Appendix 1. ,: PCE, NCE, and MNPCE on each animal each animal 

(continued) 

Dose group 

Animal Slide 

Number 

M13 A 

~ 8 

Total 

M14 A 

~ 8 .. ~ 

Total 

250 mg/kg M15 A 

~ 8 

Total 

M16 A 

~ 8 

Total 

M17 A 

~ 8 

Total 

M18 A 

~ 8 

Total 

M19 A 

~ 8 

Total 

M20 A 

~ B ~ 

' ~ Total 

500 mg/kg M21 A 

~ 8 

Total 

M22 A 

~ 8 

Total 

M23 A 

~ B 

Total 

M24 A 

6' 8 

Total 

PCE:NCE PCE 

Counted PCE+NCE 

330:275 0.55 

249:280 0.47 

579:555 0.51 

514: 302 0.63 

464:291 0.61 

978:593 0.62 

281 : 223 0.56 

362:272 0.57 

643:495 0.57 

457:229 0.67 

339:214 0.61 

796:443 0.64 

364: 177 0.67 

362: 174 . 0.68 

726: 351 0.67 

305: 198 0.61 

309:229. 0.57 

614·: 427 0.59 

272:275 0.50 

275:322 0.46 

547:597 0.48 

531 : 555 0.49 

302:270 0.53 

833:825 0.50 

184: 375 0.33 

208:431 0.33 

392: 806 0.33 

250:273 0.48 

375:538 0.41 

625: 811 0.44 

316: 668 0.32 

344:596 0.37 

660 : 1264 0.34 

380: 260 0.59 

223:277 0.45 

603: 537 0.53 

-127-

Page ~J- of27 

PCE MNPCE 

Counted counted per1000 

1000 0 0 

1000 0 0 

2000 0 0 

1000 1 . 1 

1000 3 3 

2000 4 2 

1000 0 0 

1000 2 2 

2000 2 1 

1000 3 3 

1000 0 0 

2000 3 1.5 

1000 2 2 

1000 2 2 

2000 4 2 

1000 0 0 

1000 0 0 

2000 0 .. ., 0 

1000 0 0 

1000 2 2 

2000 2 1 

1000 2 2 

1000 1 1 

2000 3 1.5 

1000 0 0 

1000 1 1 

2000 1 0.5 

1000 3 3 

1000 1 1 

2000 4 2 

1000 3 3 

1000 0 0 

2000 3 1.5 

1000 2 2 

1000 0 0 

2000 2 1 
i:. 

:'i' 

i., 

,',.·· 

~·. 

Study Number: S277 

Appendix 1 

continued) 

Dose group 

Animal Slide 

Number 

M25 A 

~ 8 

Total 

M26 A 

MMC ~ 8 

Total 

2 mg/kg M27 A 

~ 8 

Total 

M28 A 

~ 8 

Total 

M29 A 

~ 8 

Total 

M30 A 

6' 8 

Total 

Report 

:: PCE, NCE, and MNPCE on each animal 

PCE:NCE EQ£ PCE MNPCE 

Counted PCE+NCE Counted counted per 1000 

219:291 0.43 1000 94 94 

198:305 0.39 1000 83 83 

417: 596 0.41 2000 177 88.5 

243:376 0.39 1000 93 93 

138:364 0.27 1000 74 74 

381 :740 0.34 2000 167 83.5 

166: 334 0.33 1000 77 77 

156:344 0.31 1000 75 75 

322:678 0.32 2000 152 76 

125: 385 0.25 1000 71 71 

127:379 0.25 1000 76 76 

252:764 0.25 2000 147 73.5 

271 :330 0.45 1000 100 100 

182: 360 0.34 1000 85 85 

453:690 0.40 2000 185 92.5 

148: 361 0.29 1000 78 78 

135: 369 0.27 1000 72 72 

283:730 0.28 2000· 150 75. ..... ·:·.··' 
Study Number: S2n 

Appendix2. 

Dose group 

Vehicle 

10 ml/kg 

I - r 

" 

125 mg/kg 

~:. ~-~. 

·. -· ~· ,._ 

250 mg/kg 

- ··-· 

500 mg/kg 

Mitomycin C 

2 mg/kg 

t 

!:• 

- : Not applicable 

: Individual Body weights 

Animal 

Number 

M1 

M2 

M3 

M4 

M5 

M6 

M7 

MS 

M9 

M10 

M11 

M12 

M13 

M14 

M15 

M16 

M17 

M18 

M19 

M20 

M21 

M22 

M23 

M24 

M25 

M26 

M27 

M28 

M29 

M30 

Day 1 

32.7 

32.7 

33.4 

35.0 

35.7 

35.3 

32.6 

32.4 

35.2 

32.8 

34.7 

34.4 

33.6 

33.3 

33.0 

35.0 

33.5 

36.4 

33.5 

33.8 

33.9 

34.5 

34:3 

36.2 

-

-

-

-

-

-

-129- Pagc -- (27 

Report 

1unlt: fl) 

Day2 Day3 

32.7 33.8 

31.9 32.1 

33.2 34.1 

34.2 34.4 

35.5 36.3 

36.2 37.5 

32.8 32.6 

33.0 33.4 

34.6 33.6 

34.7 34.6 

35.2 35.5 

35.7 35.6 

33.3 33.7 

33.6 33.9 

33.5 33.7 

34.3 34.7 

32.6 33.3 

36.0 35.7 

35.2 32.6 

34.9 35.0 

36.1 33.1 

34.8 34.0 

38.0 33.0 

37.6 37.3 

31.5 32.6 

33.6 34.3 

33.4 33.7 

34.4 34.5 

33.9 35.7 

35.9 37.3 
,f 

;, 

~·. 

: ~:= 

( 

~··· 

o' 

~ ·. :. 

: .. 

'l_'· 

Study Number: S277 

Appendix 3. .-- --A: Individual clinical si_gns 

Dose group 

Animal Day 1 

Number 

M1 N 

M2 N 

Vehicle M3 N 

10 ml/kg M4 N 

M5 N 

M6 N 

M7 N 

M8 N . -· -- . 

-·. M9 N 

125 mg/kg M10 N 

M11 N 

M12 N 

M13 N 

M14 Hypoactivity ..... --- p 

---· M15 N 

250 mg/kg M16 N 

M17 Hypoactivity 

M18 N 

M19 Ataxia; hyPoac~ivity 

M20 Ataxia, hypoactivity -- -- M21 N 

500 mg/kg M22 Ataxia, hypoactivity 

M23 Hypoactivity 

M24 Ataxia, Hypoactivity 

M25 - M26 - Mitomycin C M27 -

2 mg/kg M28 - M29 - M30 -

N: Normal 

A* : Common clinical signs (Abdominal distension, rough fur) 

-:Not applicable 

Report 

Day2 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

N 

A*,.ataxiq, hypoactivity, dyspnea 

A* 

A* 

A*, hypoactivity 

A* 

A*, h_ypoactiv!!Y 

N 

N 

N 

N 

N 

N 
Study Number: S277 Report 

Appendix4. .: Individual Body weights (Preliminary range finding} 

(unit: Q\ 

Dose group 

Animal Day 1 Day2 Number 

M13 32.7 33.3 

M14 32.6 32.5 .. :..·-- : ._. . ~ M15 33.9 33.8 

1000 mg/kg M16 33.3 33.9 

M3 32.6 33.4 

M4 31.1 31.4 

M9 32.1 - M10 32.5 - : .. . ; ... M11 34.3 -. 

2000 mg/kg M12 34.3 - M2 34.1 - M1 35.0 -

-:Not applicable 
i. 

I. 

:~ 

~· 

,, 

I' c 

.,, 

,, 

f· 

i 

t 

~~· 

study Number: S277 

Appendix 5. 

Dose group 

__ ..j. 

1000 mg/kg 

2000 mg/kg 

N: Normal 

D: dead 

Report 

: Individual clinical signs (Preliminary range finding) 

Animal Day 1 Day2 Number 

M9 N D 

M10 N D 

M11 N Hypoactivity, tremor 

M12 N Hypoactivlty, tremor, dyspnea, ataxia 

M2 N Hypoactivity, ataxia, tremor 

M1 Hyt:mactivity D 

M13 D - M14 D - M15 D - M16 D - M3 D - M4 D -

(showed severe clinical signs and dead 10 to 120 minutes after administration) 

(Major clinical signs of dead animals were dyspnea, convulsion, ataxia, and lateral position) 

- : Not applicable 
Study Numper: S277 

18. Certificate of Analysis 

Al~ ~i~Ai 

CERTIFICATE OF ANALYSIS 

Report 

g: 

f< 

'I v ... -· .. t ... 

I () l I 

t ~ I 

·r - -::-. ·== ··=- ·=~---..... --~-.7~TE \f~'r\~=- ~~--, 1~ li "C -~, ~ -=· - ' . . l\ 

t II ·,i:\'ESCTH?T!O)( ljJ: ,Yl.\, tF.RL-\l) '--... ----- -~~~ (: if - .. I 

r II CAS ~ro. I :=.,____ -------··-.. -----·-'1 \:' f --~·-o-~ ~4 :i I ---· . .:~. 1::." l'i t '. I 

!; l\ 'Ct;~['O~iFH. N.~:vrEl ·- · .::~~-:~ (LO'T. 0;_-~. T~E --:;,c~}l}_£ -130 1- D1__;: 

f, ll ~ t~ ~ ,~ ... ("::''Ell T • h\ "' - 7.} %~ ·t!, -er I •. 

' ' -'~.L :I. r..J-.' ·-.)U b I•.'.CA'::l Q:::> __ T?.r_l(ir -~·o.'.\ ~- 1\ (A\10l'NT~ ______ ___c_:.:_.· __ ,\ ~~------~----;, 

~~~~~- I· 

;11. II' I .)-I )' ..,L .;;, c.\· cl I ..: --- ,..: .,t.. -6/. :~ -:-:1. I -~~ .1.4 i(-. ~.:~.'1· iJ:: -: "':, - ,__ ..,., ' I· ~ I~ ~ ....., \:.., ..., .. :.1 I ' I 'T;..·-T :::::·,.:JECrFic~'"'ri·J · -Es---r R-=·s'-L1' 1't:"c·:· ·..,rE.,."iOL>. It · ~--7'2S1._JI.S:'I-·! ~,_:,l 1 -- =-=---·····--~-L--l-_. __ ..._ '-- . _..._1. ·....:..___:_r. 1 

ij ~- .-'..PPEA.c\.-1..:-ICE 1 ; .:Vfh ;{e. ~· "f'id .. .. ~) ----------r---·---.-- ------~-------- I , DFSS!J''I' ~t (.01: ; : . . . j 

- . - ...:..,_ - ) ... ·-- ...;.___ .. -----__;..-.,-

I 

Par?o ?7 n( 2 7 

--133-
I o ~ 

. B001286 

Submitted to: 

( R e p o· r t J 

Chromosomal Aberration Study of~. ''"~ in Cultured Mammalian Cells 

(Study No. : B001286) 

May 22, 2001 

GENERAL TESTING RESEARCH CENTER. JAPAN DILSTUFF INSPECTORS' CORPORATION 

-134-. ,-
B001286 

S t a t e m e n t 

General Testing Research Center. 

Japan Oilstuff Inspectors' Corporation 

Sponsor 

Title Chromosomal Aberration Study of • in Cultured Mammalian Cells. 

Study No. B001286 

The study for the report has been conducted in compliance with the following 

Good Laboratory Practice Standard. 

rJapanese GLP standards Applied to Industrial Chemicals (Kanpogyo No.39, 

Yakuhatsu No.229. 59 Kikyoku No. 85. 1984; revised in 1988 and 2000 )J 

Management 

Masakatsu Usami 

Study Director 

Masato Nakamura 

-135-
B001286 

Quality Assurance Statement 

General Testing Research Center, 

Japan Oils tuff Inspectors' Corporation 

Sponsor 

Title Chromosomal Aberration Study of in Cultured Mammal·ian Cells 

.Study No. B001286 

.By the inspections and audits listed below, we hereby certify that the study 

was carried out in accordance with its protocol and with our Standard Operating 

Procedures, and that the report faithfully describes the methods and procedures 

applied, and that the reported results reflect the raw data of the study 

accurately. 

Inspection 

or audit 

St.udy 

protocol 

Study 

procedure 

Draft 

report 

Study 

report 

Quality Assurance Unit 

Date of inspection 

or audit 

March 21. 2001 

April 6, 2001 

May 18, 2001 

·May 22, 2001 

Sadayoshi Ohtubo 

Date of reporting to 

the study director 

and to the management 

March 21. 2001 

Ap·r i 1 6, 2001 

May 18, 2001 

May 22, 2001 

Date: M/l.Jf. ))., :zoo/ 

(! 
1. Title 

2. Purpose 

3. Guideline 

4. G LP 

5. Sponsor 

6. Organization 

in Contractor 

B001286 

Outline of the Study 

:Chromosomal Aberr.a:t ion Stu.dy of -- _ · in Gu 1 tured Mammal ian 

Cells. (Study No.: B001286) 

:To assess the clastogenicity of the test substance by the 

chromosomal aberration tests using cultured mammalian cells. 

:Guidelines under the Japanese Industrial Safety and Health Law 

(1997) 

: GLP under the Japanese Industrial Safety and Health Law (1984; 

revised 1988 and 2000) 

' 1 

(RespGnsible person) 

:Mitsubishi Chemical Safety Institute Ltd. 

(1-30, 2-Chome, Shiba, Minato-ku, Tokyo) 

7. Testing Facility:Gene-ral Testing Research Center, 

Japan Oilstuff Inspectors' Corporation 

(10-4. 1-Chome. Mikagetsukamachi. Higashinada-ku, Kobe-shi, Hyogo) 

8. Study Contributors 

Management :Masakatsu Usami 

Responsibility person of data keepi.ng:Etsurou Fujiwara 

Study Director Date: %.y l21 lw/ 

Masato Nakamura 

Other Contributors :Osamu Iwaihara. Sumiyo Mitsui 

9. Study Date :(Initiation of the Study) March 21, 2001 

(Initiation of the Test) March 23. 2001 

(Submission of the Test) May 10, 2001 

(Submission of the Final Report) May 22. 2001 

10. Data analysis :No statistical methods were used. 

11. There was no unforeseeable circumstances that had affected test results undesirably. 

12. Retention :All records will be retained in the safekeeping facility of General 

Testing Research Center for 10 years after the submission of the 

final report. Furt-137-._:ention will be discussed with the sponsor. 

4 
B001286 

Contents 

Page 

Summary ........................................................................ 6 

Materials and Methods ................................................... · 7 

1. Test materials ...................................................... 7 

2. Indicator cell line ................................................. 9 

3. Culture media .......................................................... 9 

4. 89 mix .................................................................. 9 

5. Test method .................. ,......................................... 10 

Resu Its and Dis cuss ion ·················· ··· ··· ························ 16 

References · · .. ·· · .. · ·· ··· ... · · · · .. · · · · · · · ·· · · · · · · .. · .. · · .............. ·· · ·.. 17 

Tables .................................................................. 18 

Figures .................................................................. 19 

Photograph .................................................................. 22 

The attached sheet l(Structural formula) ........................ 23 

The attached sheet 2 (Mitotic Index) ........................... 24 

-138-

5 
B001286 

Summary 

A chromosomal aberration study of · in vitro was made with an 

established cell line (CHL/IU) from the lungs of a female Chinese hamster. 

The pulse treatment test was made and the incidences of structural chromosomal 

aberration was 65.5% at 12.5 t.tg/ml without metabolic activation and· the 

incidences of structural chromosomal aberration was 10.5% at 150 fl. g/ml and 

31.5% at 200 {.l.g/ml with metabolic activation. The incidences of numerical 

aberration were less than 5% in all treatment groups both of without and with 

metabolic activation. 

From these findings, it has come to conclusion that 

potential in the CHL/IU ce 11 1 in e. 

-139-;-

6 

-- " has a clas togenic 
B001286 

Materials·and Methods 

1. Test materials 

1. 1 Substance to be tested 

Name of the new 

chemical substance - - ,"J ··-. - - ... 

-. - ~ - . - - (IUPAC nomenclature) 

Trade name 

Structural formula 

or rational formula 

(or outline of [Refer-to the sheet attached 1) 

manufacturing 

method, in case both 

are unknown) 

Purity of the new Lot No. of the new 

chemical substance 95.4% chemical substance to NL-9301-02 

tested [GPC area] be tested 

Name and concentration of impurities 

CAS. No. Vapor pressure 

Molecular weight 249.29 Partition coefficient 

'" Melting point 61 "C Appearance at ambient ligbtyellow· 

temperatures so 1 i d 

Boi 1 ing point 

Stab i·l i ty 

Solvent Solubilities Stability 

in Solvent 

Water insoluble 

So 1 ubi 1 i ties in DMSO soluble 

several solvents 

Acetone soluble at 500 mg/ml 1> stable 2) 

others( ) 

DMSO : dimethylsulfoxide 

Storage condition : kept at a room temperature, darkplace, s·hield the 1 ight 

1) : According to examination of vehicle. 

2) : Heat of dissolution, discoloring and foam formation were not observed during preparation for dissolving the test substance to be tested. 

-140- 7 
B001286 

1.2 Preparation of test 

1) Negative control mater1al .(solvent) 

Ace tone ( Lot No. KSH4520, Wa.ko Pure Chemica 1 Labor a tory Co. , Ltd. ) 

2) Solvent selection and preparation for making solution of test substance 

Based on the information in which the test substance was not dissolved in 

water and was dissolved in DMSO and acetone, the examination of vehicle was 

per'formed at sal ine(50 mg/ml), DMSO(lOOO mg/ml), l%CMC-:Na solution(50 mg/ml) 

and acetone(500 mg/ml). The test substance was insoluble in saline, l%CMCNa solution and DMSO. However, the test substance was dissolved in acetone. 

Therefore, acetone was used as solvent for the test substance. 

The test substance solution was diluted with acetone to the required 

concentrations. 

1.3 Positive material 

1) Positive controls 

Mi Urni,ycin C CMMC, Lot No. KSM4520, purity 99. 8%, Wak6 Pure Chemical 

Laboratory Co., Ltd.) 

Benzo[a]pyrene CBP, Lot No. GGOL purity 95. 6%, Tokyo Kasei Kogyo Go., 

Ltd.) 

2) Positive control solutions 

MMC was dissolved in saline (Otsuka Pharmaceutical Factory, Inc.) to a 

concentration of 1.5 ,ug/ml before use. BP was dissolved in DMSO (Dojin 

Chemical Laboratory Co., Ltd.) to 4 mg/ml, frozen, and thawed at the room 

temperature before use. 

-141- --.1-

8 
B001286 

2. Indicator cell line 

The CHL/IU cell line, originally derived from the lung of a female Chinese 

hamster, was used. The cells were purchased f.rom Dainippon Pharmaceutical Co., 

Ltd. A cell suspension was mixed with one tenth volume of DMSO, and then 

frozen in 1 ml and stored in liquid nitrogen. The suspended cells were used . . 

after thawing and cultivation through up to 5 passages. The cel'ls were 

cultured in plastic dishes (Becton Dickinson and Company; diameter of 6 em or 

10 em) in an automated C02 incubator (Tabai Espec Corp. model BNA-111A, 121DA; 

C02 5%, temperature 37°C, humid condition). 

The final doubling time of the cell used for the test was about 16.7 hours. 

3. Culture media 

3.1 Eagle's minimum essential medium 

Eagle's minimum essential medium CMEM; Eagle's MEM "Nissui(i)", Nissui 

Pharmaceutical Co., Ltd·.) of 8. 3g ·was dissolved i·n 880 ml.of distilled water 

and autoclaved at 121 oc for 15 min. The solution was supplemented with'· 

8. 8ml of sterilized 2. 92% L-glutamine solution and 11.2 ml of sterilized 10% 

sodium bicarbonate solution.· 

3.2 Growth medium 

Nine hundred ml of the above MEM solution was supplemented with 100 ml of 

heat-inactivated (56 °C, 30 min.) calf serum (GIBCO BRL; Lot No. 296130). 

4. S9 mix 

4. 1 89 

89 from Kikkoman Corporation was a supernatant fraction of liver homogenate 

prepared from rats which were ,given i.p. injection of phenobarbital at 30, 60, 

60 and 60 mg/kg every day and 5, 6-benzoflavone at 80 mg/kg at the third PB 

injection. The S9 was stored below -BO"C. 

-142-_ 

9 
Source of 89 

lot No. : RAA-440 

Prepared on : February 16. 2001. 

Using animal 

Species, Strain : Sprague-Dawley Rat 

Sex, Age (in weeks) : Male. 7 weeks old 

Weight : 211 - 245 g 

Final concentration of the S9 was 5%. protein of 89 was 1. 341 mg/ml. 

4.2 S9 mix 

B001286 

The following table shows the composition of 1 ml of 89 mix. Each of 89 mix 

was prepared freshly for each assay and wa~ stored in the ice. 

S9 

MgCl2 C6H2D) 

KCl 

D-glQcose-6-phosphate 

/3 -NADP+ 

HEPES buffer (pH 7.2) 

Sterilized distilled water 

5. Test Method 

5.1 Pulse treatment test 

1) Cell growth inhibition test 

(1) Test substance concentration 

0.3 ml 

5 11- mo 1 

33 11- mo 1 

5 11- mo 1 

.4 11- mo 1 

4 11- mo 1 

remainder 

The highest concentration was set at 5000 ,ug/ml. Then, following 

concentrations were graded at a common ratio of 4. 

(2) Treatment of cells 

In a 6 em plastic plate, 5 ml of cell suspension (4X108/ml) were incubated 

for 3 days. After removal of the cui ture medium from each plate, the 

following table shows the composition,· treatment of cells was performed for 6 

hours and the tests were made using two plates for each concentration. After 6 

hr. incubation, the cells were washed three times with MEM. and incubated in 

5 ml of a fresh growth medium for a further 18 hr. 

-143-_ 

1 0 
B001286 

In the negative control group, cells were treated with vehicle. 

test substance solutions S9 mix the growth and negative control medium 

without metabolic 0.03 ml 3. 0 ml - activation 

with metabo 1 i c 0.03 ml 0. 5 ml 2. 5 ml 

activation 

(3) Measurement of surviving cells 

Cells were washed with Dulbecco's phosphate buff~red saline·CPBS(-); 

Dulbecco' s PBS uNissui", Nissui Pharmaceutical Co., Ltd.), fixed with 

methanol for 15 min., stained with 0.1% crystal violet solution for 10 min,, 

and washed with water and dried. Cell density was measured with a cell density 

counter (Monocellator, Olympus Co., qd.). 

(4) Measurement of 50% inhibition concentration of cell growth 

The ratio of surviving cells in treatment groups were calculated in 

comparison with negative control. Then, a survival curve was drewed, and 50% 

inhibition concentration of cell growth (IC5o) was calculated. 

2) Chromosomal aberration test 

(1) Test substance concentration 

Fifty percent inhibition concentration of cell growth was calculated to be a 

13.9 pg/ml without metabolic activation and 136 ,ug/ml with metabolic 

act iva t i on. 

Without metabolic activation 

concentration negative 0. 08 0.3 1. 25 5 2"0 78 313 1250 5000 

(,ug/ml) control 

cell growth 100 96 85 77 73 38 0 0 0 0 

-144-_ 

1 1 
B001286 

With metabolic activation 

concentration negative 0.08 0 .. 3 1. 25 5 20 78 313 1250 5000 (~/ml) control 

cell growth 

100 95 88 87 93 88 81 . 12 0 0 

The concentration was set at 50, 25, 12.5, 6.3 and 3.1 f.lg/ml without 

metabolic activation and 300, 200, 150, 100, 50 and 25 f.lg/ml with metabolic 

activation on the result of cell growth inhibition tests. 

In positive controls, final concentrations. of MMC and BP were set at 0.15 

and 20 ,ug/ml. respectively, which are the concentration levels as· known to 

induce chromosomal aberration. 

(2) Treatment of cells 

The cultured cells were treated with the test substance using the same 

procedure as described above. 

For positive controls, the cells were treffted with a mixture of 2. 7 ml of 

the growth medium an.d 0.3 ml MMC solution in the treatment without metabolic 

activation and treated with a mixture of 2. 5 ml of the growth medium and 0.5 

ml of 89 mix and 15 f..ll BP solution in the treatment with metabolic activation. 

(3) Preparation of specimens and measurement of surviving cells 

Cell density was measured in the preparation of specimens. The cultured 

cells were treated with the test substance using the same procedure as 

described above. 

Before 2 hours from the end of the treatment, colcemid was added to the 

medium in each plate to a final concentration of 0.1/.lg/ml to arrest- cells in 

metaphase during preparing specimen. 

The cells were then washed with PBS(-), dissociated with 0.25% trypsin 

solution, and centrifuged (1000 rpm, 5 min.; same below) for collecting them. 

-145--

1 2 
B001286 

After removal of the supernatant, 4 ml of 0. 075 M potassium chloride 

solution was added for hypotonic treatment (37 QC, 15 min.). The cells were 

then fixed in 0. 5 ml of cold methanol/acetic acid mixture (3/1. v/v). After 

their centrifugation, the supernatant was removed and 4 ml of a fresh 

fixative added. The fixation procedure was repeated 3 to 4 times. Thereafter. 

the cells were susp.ended in a small amount of the fixative, and drops of the 

cell suspension were placed in two positions on a slide glass and dried. The 

ce·lls were stained for 20 min. with 3% Giemsa' s solution and then diluted 

with 1/15 M phosphate buffer (pH 6.8). The slides were then washed with water, 

dried, and coated with a mounting medium. Three slides were prepared for each 

plate. 

(4) Observation 

Q) PreliminarY. observation 

Each specimen was observed first to confirm that it has an adequate (50 

cells for one plate) number of mitotic ·cells~ F-or po·sitive. and nega:tive 

controls, the incidence of structural aberrations was confirmed to be 

appropriate. 

aD Structural aberration and numerical aberration 

Each specimen was coded, 100 metaphase cells for each plate (i.e. 200 cells 

for each concentration) were observed in a blind manner. 

Metaphase cells of which chromQsomes are spread well were selected and 

observed for structural aberrations. Cells without aberration but not having 

25+2 chromosomes were omitted from the survey. 

Structural aberrations were classified as follows 1l 

J

chromatid breaks (ctb) 

chromatid exchanges (cte) 

1 chromosome breaks (csb) 

chromosome exchanges Ccse, 

other aberrations (frg) 

dicentric, ring, etc.) 

-146-;_ 

1 3 
B001286 

A ~gap" was distinguished from a ~break" as follows. The ~gap" is an 

achromatic region in a single chromatid which is narrower than the width of 

the chromatid. Gaps are not included to the chromosomal aberration. The 

recorded gaps were distinguished from that of the other structural aberration. 

Numerical aberration was observed and polyploid cells, which include 

endoreduplicated cells, were scored. 

@ Mitotic index 

One thousand cells from each plate (i. ~ 2000 cells for Bach concentration) 

were observed to calculate mitotic index. 

(5)J udgemen t 

A cell with at least one structural chromosomal aberration was classified as 

aberrant cell.. The number pf .. aberra.n.t cells was counted in different kinds of 

two ways, one includes cells with no aberration other than ga~s (tgap), and 

another excludes such above cells (-gap). 

The chromosomal aberrati6ri potenti~l of the test substance was judged as 

follows. When both of incidence of -gap aberrant cells and that of numerical 

aberrations are less than.5%. the substance is negative (-). When either of 

incidences is 5% or more and less t~an 10%. the test substance is inconclusive 

(+). When either of incidences is 10% or more, the test substance is 

positive (+). 

When the chromosomal aberration test is inconclusive or positive, the 

reproducibility of these results or dose-dependent confirms. 

Treatment concentration of confirmation examination is decided according to 

the incidence of the chromosome aberration. According to case, examine by the 

equal ratio. It is judged positive in the case where reproducibility is 

admitted. 

-147-·- 1 4 
B001286 

5.2 Taking data 

Each of the sums and the incidence (%) of the chro~osome aberration and 

numerical aberrations was shown. The chromosomal aberration was shown dividing 

the number of cells in the type. Each of Cell growth index in cerl growth 

inhibition tests and chromosome aberration .tests were shown. 

When the test wa·s positive, the "D2o" values were calculated from the·test 

results. The "D~o" value is the concentration (mg/ml) required to induce any 

aberration in 20~ of metaphases. The microscopic photograph of a typical 

chromosomal aberration is appended. 

-148-_ 
B001286 

Results and Discussion 

Jn the cell growth inhibition tests of pulse treatment test, fifty percent 

inhibition concentration of cell growth was calculated to be 13.9 ~/ml 

without metabolic activation and 136 ~/ml with metabolic activation. 

On the basis of the cell growth inhibition tests, the concentration was set 

at 50, 25, 12. 5, 6. 3 and 3.1 ,Llg/ml without metabolic activation and ·300, 200, 

150. 100, 50 and 25 ,Llg/ml with metabolic activation(Figure 1. 2). 

In the cell growth inhibition test of pulse treatment more than 1250 ,Ltg/ml, 

precipitated test·substance was observed in the medium before incubation, but 

was not observed after incubation. 

In the chromosomal aberration test of pulse treatment test, the incidences 

of structural chromosomal.aberration was 65.5% at 12.5 11 g/ml without 

metabolic activation and the incidences of structural chromosomal aberration 

was 10.5% at 150 11 g/ml and 31.5% at 200 11 g/ml with metabolic activation. 

The incidences of numerical aberration were less than 5% in all treatment 

groups both of without and with metabolic activation. The incidences of 

structural aberrations in the positive control groups were significantly 

higher than ones in negative control groups (Table 1 and Figure 3, 4, 5 and 

6). 

D2o value was calculated from the treatment group with the increase in 

structural chromosomal aberration cell. and was 0. 005 mg/ml without metabolic 

activation and 0.17 mg/ml with metabolic activation. 

Microscopic photographs of normal cell for the negative control and 

structural cell in w"ithout metabolic activation are shown in photo. 1, 2. 

No statistical method was used for the analysis of data. 

From these findings, it has come to conclusion that 

potential in this CHL/IU cell line system. 

-149-_ 

1 f) 

~- have clastogenic 
B001286 

Reference 

1) Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of 

Japan : Atlas of chromosomal aberrations induced by chemicals. Asakura 

Press. Tokyo, 1988. 

-150--

1 7 
,_:. 

co 

I 

...... 

o:.n 

,__. 

I 

I 

hat aubrtence : T0-1534 

Tobie 1 Rl!sult of the chrorrosorral aberration test ( pulse treat11ent test ) 

... 

Test subslanee 

Ho. of calla with chro1110ao•l structunl aberrations 

S9•ix dose levels Ho. No. of cell• tat•l of tiae (h) cells Chra ... t ld breaks ChroMilfd Chro1110some break• Chromosollll! Other axchanrea aberration • aberration• lpc/al) analvzed (H) Heeative 100 0 1 1 0 0 2 6-JB - contra I 100 1 0 0 0 0 1 (Ace lone) zoo 1 co. 5) 1 co. 5) 1 co. 5} 0 co.o 0 (0. Ol 3 (1. 5) 

100 0 7 0 0 0 2 6-18 - 3. 1 100 0 0 0 0 0 0 

200 0 co.m 2 Ct. 0) 0 co. 0) 0 co. 0) 0 (O.Ol 2 ct. 0) 

100 3 4 0 0 0 7 6-18 - 6. 3 100 1 . 5 0 0 0 t2 

200 10 15. Ol 9 (4. 5) 0 CO. Ol 0 ·co. 01 0 CO.Ol 19 (9. 5l 

100 45 42 0 0 5 67 6-18 - 12.5 100 d7 49 0 0 4 69 

200 92 (46. {)) 91 145.51 0 (0. 0) 0 f0.01 g (4. 5) 131 (65. 5) 

6-18 - 25 r T 0 X J 

6-18 - 50 r T 0 X J 

Pas ltive 

6-18 100 27 37 1 0 0 59 - contra I 100 79 43 0 0 0 59 (IIC) D. 15 200 56 68. iii 80 (40 Ol 1 (0 5\ 0 co 0\ 0 (0 0) ·lis (59 0) Hera the 

6-18 

100 0 0 0 0 0 0 control 100 0 0 0 0 0 0 (Ace lone) 200 0 CO.Ol 0 (0. Ol 0 co OJ 0 co. Ol . 0 (0. 0) 0 (0.0 

6-18 100 1 1 0 0 0 2 25 100 2 0 0 0 0 2 

200 3 Cl. 5 1 co. 51 0 co. 0 0 10. OJ 0 CO.Ol 4 (2 Ol 

6-18 

100 0 1 0 1 0 2 50 100 2 1 0 0 0 3 

200 2 11. 01 2 Ct. 01 0 co. OJ 1 CO. 51 0 IO. 0) 5 (2. 5) 

6-18 

100 0 3 0 1 0 4 100 100 2 1 0 0 0 3 

200 2 ll. 01 4 (1.01 0 10. 0) 1 co. 51 0 CO. 0) 7 (3. 5) 

100 .ol 5 0 0 0 g 6-18 • 150 too 8 5 1 0 0 12 

100 12 (6. OJ 10 f5. 0} 1 co.61 0 ro.m ·0 CO. OJ 21 (10.51 

6-18 100 70 25 0 0 0 28 + 200 100 22 27 0 0 2 35 

100 42 (21. 0} 51 116. 0) 0 CO. OJ 0 CO.Ol 2 Cl. Ol 63 (31. 51 

6-18 • 300 r T 0 X J 

Positive 

6-18 

100 10 39 0 ol ol 46 • contra I 100 20 62 0 3 ol 67 

I BP l ZO 100 I 3o £15. mltot (50. 511 0 co. m I 3 fl. 511 0 COOJittJ <56.51 I 

le.st substance treabumt tine ( 6 hours ) - reeaver thre ( 18 hours ) 

• :Others in structural aberration : fracnentation (ercept pulverization). •• Othus in nu11erlcal aberration : endoreduplicated cells 

MlfC : llitonycin C. BP : Banzo (a) pyrene 

fTOXJ : lfetahue ce I I had not obh fned nore then 50 ce f Is 

Ho. of cells with 

IIP!I 

1 

0 

1 (0. 51 

1 

0 

1 -co. 5> 

0 

1 

1 10.51 

0 0 

0 IO.Ol 

-

0 

0 

0 co 0\ 

0 

0 

0 IO.m 

0 

0 

0 fO 01 

1 

0 

1 10.51 

1 

0 

1 co. 51 

0 

0 

0 ro 0\ 

1 

0 

1 10.51 

·o 

0 

0 -fO. 01 

Cell •ro•lh Ho. 

index (JC) of cells 

analyz.ed 

100 

100 100 

2.00 

100 

84 100 

200 

100 

57 100 

zoo 

100 

39 100 

zoo 

10 

0 

100 

I 100 

700 

100 

100 100 

200 

100 

91 100 

200 

100 

78 100 

200 

100 

65 100 

200 

100 

60 100 

100 

100 

24 100 

200 

11 

too 

I too 

200 I 
8001286 

Pulse treatment test 

100 

• Cell growth inhibition test 

. o- Chromosomal aberration test 

,-..... 

?!2- .._.... 

P< 

CD 

"'0 

•1"'"1 = 

" ..t:: 50 '\ ..., 

13: '\ 

0 

~ D 

t:lLJ ' r--1 ' r--1 

CD ' 

u ' 

' 

' 

b .. .. .. .. .. 0 

0 25 50 75 100 

Concentration ( Jl g/ml) 

Fig. 1 Cell· growth inhbition treated with 

without metabolic activation 

100 

• Cell growth inhibition test 

- o- Chromosomal aberration test 

----. 

?!2- ... 

" ........... " ><: 

CD 

"'0 

•.-I = 

..c: 50 ..., 

13:: '\ 0 

/-; '\ 

bD \. 

r--1 '\ r--1 

CD 

u o .. .. .. .. .. .. 

0 

0 100 200 300 400 500 

Concentration ( J.L g/ml) 

Fig.2 Cell growth inhibition treated with 

with metabolic activation 

-152-_ 

1~ 
100.0 

I 

80. 0 

,.-..., 

'?fi. 

'-' 60. 0 

(].) 

0 

.t:: 

(].) 

"'CC 

•r-1 

0 40.0 .t:: 

H 

20. 0 

0.0 

0 

10.0 

8.0 

,....._ 

cf2- 6. 0 "-" 

(].) 

0 

.t:: 

(].) 

"'CC 

•r-1 4. 0 

o· 

.t:: 

H 

2. 0 

0.0 

0 

B001286 

Pulse treatment test 

_.,_Without metabolic act1 vat ion I 

) 

7 

/_ 

_/ 

- -

3. 1 6. 3 12. 5 

Concentratin ( JJ. g/ml) 

F.ig. 3 Incidence of structural aberrent cell treated with 

I ~Without metabolic activation I 

~ 

.. 

-

3. 1 6. 3 

Concentration ( J.l. g/ml) 

Fig.4 Incidence of numerical aberrant cell treated 

with _ 

-153-. 

12. 5 
40.0 

30.0 

,-._ 

?Je. 

......, 

Cll 

c.J 

§ 20.0 

""0 

•.-I 

c.J 

c:: 

~ 

,-._ 

~ 0 

........ 

Cll 

c.J 

c:: 

Cll 

'1j 

•.-I 

c.J 

c:: 

1-1 

10.0 

0. 0 

10.0 

8. 0 

6. 0 

4.0 

2. 0 

0. 0 

I 

0 

I 

0 

B001286 

Pulse treatment test 

- -o- With metabolic activation I 

I 

I 

, 

I 

, 

I 

, 

I 

, 

I 

n ,. .. .. .. ,. .. - 0 - - ---0 0 -- -- - - - -- 25 50 100 150 200 

Concentratin ( /.1 g/ml) 

Fig. ·s Incidence of structural aberrent cell treated with 

- o- With metabolic activation I 

,.0- .. .. ---- 0- .. .. - .. 

25 50 1 DO 150 

Concentration ( /.1 g/ml) 

Fig. 6 Incidence of numerical aberrant cell treated 

with · 

-154--

---

200 
Photo. 1 Normal cell for the negative control 

(Without metabolic activation) 

Photo. 2 Structural aberration cell for the treated group 

(Without metabolic activation: 12.5 ,ug/ml) 

-155-. 

? ? 

B001286 

·r· 

t 

I, 

1! 

j 

'J 

. 

1 

i 

1 i

":.: 

~ 11 

I . 

. 

. 

' 

I 

2: 

'.i 

:.: 

I· 
Stractural fomula of To-1534 

CD Main chemical substance (Lot. No. NL-9301-02) 

® Impurity 1 

® Impurity 2 

/ 

/ 

@ Impurity 3 

\ ' 

-156--

Purity 

CD 95.4% 

B001286 ~ 

(not separate GD and QD) 

Purity 

l. 6%(®+@) 

Purity 

@3.0% 
/-() 

B001286 

(The attached sheet 2) 

Mitotic Index 

(1) Pulse treatment tests 

Without metabolic activation With metabolic activation 

( 6-18hr.) ( 6-18hr.) 

Treatment Number Mitotic Treatment Number Mitotic 

Concentration of cells index Concentration of cells index 

(,Lt&/ml) (%) (.u&/ml) (%) 

Negative Negative 

control 2000 9. 3 control 2000 14.3 

(Acetone) (Acetone) 

3. 1 2000 9. 8 25 2000 I 15. 7 .. 

6.3 2000 8.2 50 2000 15.2 

12.5 2000 1.6 100 2000 8. 7 I 

25 r TOX J r TOX J 150 2000 8.4 

50 r TOX J r TOX J 200 2000 1.2 

300 r TOX J r TOX J 

Positive 2000 5.4 

con t ro-1 (MMC) Positive. 2000 6. 0 

con t ro 1 (BP) 

MMC : Mitomycin C, BP : Benzo (a) pyrene 

r TOX J : Metahase cell had not obtained more than 50 cells 

-157-. 

2 4