### Sanitized version
roved. O.M.B. No. 2070-0012. Approval Expires 10-31-96. 

### u. S. ENVIRONMENTAL PROTECTION AGENCY 

### AGENCY USE ONLY 

# &EPA PREMANUFACTURE NOTICE FOR NEW CHEMICAL SUBSTANCES 

### Whcit­ -: I completed 

### I sendthis : form to 

## I

,**DOCUMENTCONTROLO~CER O~CE**  OF POLLUTION PREVENTION AND TOXIC SUBSTANCES, 7407 U.S. E.P.A.401 M STREET, SW WASHINGTON D.C. 20460 Enter the total number ofpages in the Premanufacture Notice Date ofreceipt


# COMPANY**SA"rm?~n ~entconttolnuznber**  EPA case nuznber 

### 51010000 

## II GENERAL INSTRUCTIONS 

## TS­
•You must provide all information requested In this form to the extent that II Is known to orreasoubly ascertainable byyou. M.U:e rusonable estimates ifyou do

not have adual data.**~** •Beforeyou complete this form,you should rud the "InstructionsMmaal forPremanufadure Notificatioa" (the Instrudions M.anualls available from theToxic Substances Contral Ad (TSCA) Informatloll Sen>ice by calling**2D2-S~1404,** orfaxing**202-~S603).** ·Ifa userfee habeen remitted for this aotice (40 CFR 70(US), Indic.ale In theboxes abovethe TS-...erfee identification numberyoa have generated. Remember, your**u~r** fee ID numbermust also appeMonyourcorresponding fee remlltaAce, which 18 sentto: EPA, HQ Accounting Operations Branch <PM-266), P.O. 360399M, Pittsburgh,PA**1S2S1~99,** Athl. TSCAUserFee.**PmI-GE~L~ORMATION** You must provide the cuxrently coaed Chemical Abslrads (eA) N.une ofthe new chemical substance,even Ifyou dalm the identity as confidential. Yo'; may authorize ;motherperson to submitchemical Identityinformation foryou,but your submissionwill not be complete and the reviewwill nolbegin until EPA receives this Information. A letterin supportofyoursubmission should reference yourTS userfee identification number. You must submit m original and two copies ofthis notice including all test data. Ifyou claimed my information as confidential.a single**~tized** copy must also be submitted. Pmn - HUMAN EXPOSURE AND ENVIRONMENTALRELEASE Ifthere are several manufadure.processing. oruse OperatiolUl to bedescribed In Pm II. sections A and B ofthis notice, reproducethe sections
..needed. Pm ill- USTOFATTACHMENTS Attach additional sheets if there isnol enough sP'"""' to answera question fully. Label each continuation sheelwith thecorresponding section heading. InPm IlI,list these attachments, mytest data orotherdata ;md anyoptional Information included In thenotice. OPTIONALINFORMATION You may includeany**Info~ation** thatyou wantEPA to considerin evaluating the new substance. On page 11 ofthis form, space habeen provided foryou to describe pollution prevention and recycling informationyou may have regarding the new substance. So-called "binding" boxes areIncluded throughout this form foryou to indicate your willingness to bebound to certain sbtements you makein this notice, such as use, production volume, protectiveequipment ••. This option is intended to reduce delays that routinely accompany the development ofconsent orderS or Significant New Use Rules. Except in the aseofexemption applications (such a "Th1EA, LYE, LOREX) where certain infocnation provided in such nolifiation is bindingon the submitterwhen theAgency approves the exemption application, checldng a bindingbox In this notice.d.a.a..lul1 by itselfprohibitthe submitter from Ialerdevlatlng from theInformation (except chemical identity) reported in the form. CONFIDENTIALITYCLAIMS You may dalm any Infomution In thisnotice a confidential. To assert a claim on the form, marie (X) the confidentw box next to the information that you dalm as confidentw. To assert a dalm in an attachment, drcIe orbradtetthe information you claim a confidential. Ifyog dafm inhumation In thcPoticc as ronfidentla! yog mustalso providc j! unitized Ycajon of the notice lindudinc attachments). ForaddilionallnstructionsonclaimingInformation as confidential,read theInstrudionsManual.



# W MMk 00 If any Information In this notice is claimed as confldenllal. TESTDATA AND OlHERDATA You are required to submit all test data in yourpossession orconttol ;md to providea description of all otherdata nown to orreasonably ;ascertainablebyyou,ifthese dataarerelated to thehealth and envlranmental effects ofthe manufadure, pracessing,distribution in commerce, use, ordisposal ofthe new chemical substance. Standard Utenturedlations may besubmitted for data In the open scientific UtentuJ:e. Complete test dab (written In EncUshl. not summaries of data must be submitted Ittheydo Pot ilRxarIn tbe oxn literature. You should clearly Identifywhethertest data is on the substanceoron an analog. Also, the chemical compositloa of thetested material should be charaderized. Following are examplesof test data and oIherdata. Data should be submitted according to the requirements of §720.50 ofthePremanufadure NotificationRule (40 CFRPm720). TestData (CheckBelow any included in this notice)

Environmental fate data 

# D Yes • Otherdata 

# 0 Yes
• Health effects data 

## I]] Yes Risk assessments
• Environmental effectsdata 

# D Yes StrudureJadivity relationships

PhysiallChemical Properties'

# mYes Testdata not in the possession orconttol of the submitter "A physical md chemical properties worksheells located on the last page

of this form. TYPE OF NOTICE (Check Only One)



### [1] PMN (Premanufadure Notice)**'~::-:i** "C

## rl 

## D INTERMEDIATE PMN (submitted in sequence with flii;d**prod~~)** PMN> ...- ---; r'l 

## D SNUN (Significant New Use Notice) '"**C"~~** C:J !MEA (Test MarlcetingExemption Application) 

# D D LVE (Low VolumeExemption) @**~** CFR 723.50 (c)(1) c..!1 

# D 0) LOREX (Low ReleaselLowExposure Exemption) @ 723.SO(c)(2) 

# D LVE Modification 

# D LOREX Modification
IS nns A CONSOLIDATEDPMN7 

# D Yes

, ofchemicals <PreBotice Communication t required, enter t on page 3) EPA FORM mO-25 (Rev. 5-95) 

# / Replaces previous editions ofEPA Form 7710-25.Publicreportingburden for this collection ofinformation is estimated to average 110 hoursper response,including time for'reviewinginstructions, searchingexistingdata sources, gathering and maintainingthe dataneeded. andcompleting and reviewing the collection ofinformation. Send commentsregarding the burden estimate oranyother aspect of thiscoUection ofinformation, includingsuggestions for reducing this burden, to Oliei,Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W." Washington, D.C. 20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION Icertify that to thebeSt ofmyknowledgeand belief: 1. Thecompany named in Part I, section A, subsection 1aofthis notice form intends to manufactureorimportfor a commercial purpose, otherthan in small quantities solely for research and development, the substanceidentified in Part I, SectionB. 2. All information provided in this notice is complete and truthful as ofthedateofsubmission. 3. Iam submitting with this notice all test datain mypossession orcontrol and a descriptionofall otherdata knownto orreasonablyascertainable by me as required by§72050 ofthePremanufactureNotification Rule.
Additional Certification Statements: Ifyou aresubmittinga PMN, Intermediate PMN, Consolidated PMN, orSNUN, checkthe following userfee certification statementthat applies: 

### [K] The Companynamed in Part I,Section A has remitted the fee of$2500 specified in40 CFR 700.45 (b), or 

## D TheCompanynamed inPartI, Section A has remitted the fee of$1000 for an IntermediatePMN (defined @ 40 CFR 700.43) in accordance with40 CFR 7OO.45(b), or 

## D TheCompanynamed in PartI, Section A is a small business concern under40 CFR 700.43 and hasremitted a fee of$100 in accordance with 40 CFR700.45 (b). Ifyou are submittinga lowvolume exemption (LVE) application in accordance with40 CFR 723.50 (c) (1) ora Low release and low exposureexemption (LoI,UnO application in accordance with40 CFR 723.50 (c) (2), checkthe following certification statements: 

# D The manufacturer submittingthis noticeintends to manufacture orimportthenewchemical substancefor commercialpurposes,otherthan insmall quantities solelyfor research and development,under the termS of40 CFR 72350. 

# D Themanufactureris familiar with thetermsofthis sectionand willcomplywith thoseterms; and 

# D Thenew chemical substance for whichthe notice is submitted meets all applicableexemptionconditions. 

# D Ifthis application is for anL VE in accordance with 40 CFR 723.50 (c){1), the manufacturerintends to commence manufactureoftheexempted substancefor commercial purposes within 1 yearofthedateofthe expiration ofthe 30 dayreviewperiod. . ,

The accuracy ofthe statements you make in this notice should reflectyourbestprediction ofthe anticipated facts regarding the chemical

substance dESa'lDed herein. Any knowing and willfulmisinterpretation is subjectto criminal penalty pursuant to 18 USC 1001. Confidential Signatureand title of Authorized Official (Original Signature Required) Date

Date Signature ofagent. (ifapplicable)

FORM EPA7110-25 (Rev. 5-95) Page2

### PartI - GENERAL INFORMATION Conii-**f-...;S;..e;..;cti...;·..;.o.;.;n..;.A~-...;;S..;.UB~MI** __ I_I_ER_ID_ENTIFI

___ CA_TI_O_N

__

### ----------------------1 dentid
la. Person Submitting Notice (in US.)

b. Agent(lf applicable) Mailing address (number and Oty,State, ZIP Code

Nameofauthorized. oifid.al 

### Not applicable
Mailing address(numberandstreet) Oty, State, Code Telephone

Co Ifyou are submittingthisnotice as partofajoint submission,mark00 this box.**-:----------------~** Joint Nameofauthorizedoffidal TItle SWmtitter 

### Not applicable (Ifapplicable)
.**--;~~;;y----------------'-----------:----:--------:---** Mailingaddress (number andstreet)

2. Technical Contact 

## em U.s.) aty, State,ZIP Code

Name Company Mailingad.o:b:ess (numberandstreet) aty, State,ZIP Code Telephone TItle

Telephone I 

# x

.1 3. Ifyouhavehada prenotia!oommunication (PC) conalrningthisnotice andEPA assigned.a PCNumDe:r to thenotice. enter the number. Mark 00 Unone 4. Ifyouhave submitted an exemptionnotice/application {or thechemicalsubstana!covered.by thisnotice, entertheexemption Mark 00 numberassigned. byEPA. Ifyouhavewithdrawn a previously ifnone submitted. PMN enter the PMN number.· 

# >­ Ifyouhavesubmitted.a bona fiderequest (orthechemical substance Mark 00**-----:>~** 

## [XJ oovered. by this notice, enterthebonafide requestnumberassigned. ifnone**~~** 

## > Manufacture 2. Type ofNotice

- Mark 00 1. 

# D Only

# 0~ 

# D 

# D 3. Both Bl.ndlng Option 

# D BlndingOplion Mark(x)
. Mark (x) FORM EPA7710-25 (Rev. 1-91) Page3

### Part I - GENERAL INFORMATION

­ Continued Section'B

- CHEl\1lCAL IDENTITYINFORMATION: You mustprovide a cu.rrently COlTectChemical Abstracts (CA) nameof the substance based on the ninthCollectiveIndex (9CI) ofCA nomenclaturerules and conventions. MarkCO the 'Confidential"box next to any item you claim as confidential. Complete eitheritem 1 (Cass 1 or2 substances) or 2 (Polymers) as appropriate. Complete all other items. Ifanother person will submit chemicalidentityinformation for you (for either item 1or Z), markCO the box at theright. 

# 0 the and address ofthat in a continuation sheet. ::;.... 1. Class 1 or 2 chemir::al substarttes (for definitions ofclass 1 and class Zsubstances, seethe Instructions Manual) a. Class ofsubstance

­ Mark 00 1 

# W Oass 1 or 2 

# 0 Class 2 b. o,emical name (Currently correct Chemical Abstracts (CA) Name thatis consistent with T5CA Inventorylistingsfor similar substances. For Qass 1 substances a CA Index Namemustbe provided. For Qass Zsubstances either a CA Index Nameor CA Preferred Name X be provided, whicheveris appropriatebasedon CA 90 nomenclaturerules andconventions.).

c. Pleaseidentifywhich methodyouused to develop or obtain thesped.fl.ed chemical indentity information reported in this notice:

(ched:: one). 

# D Method 1 (CASInventory ExpertService- a copy of the Identification WMethod2 (Other Source) report obtainedfrom theCAS InventoryExpert Servicemust be submittedas an attachment tothis notice) d. Molecularformula and CASRegistry Number (Ifa number already exists for the substance)

CAS'

e. For a cIass 1 substance, provide a complete andcorrectchemicalstructurediagram. For a class 2 substance

- (1) List theimmediate X

X

precursor substances with theirrespective CASRegistry Numbers. Q)Describe the natureof the reaction orprocess. (3) Indicate the X range ofcomposition and the typical composition (where appropriate). (4-) Provide a rorrectrepresentative orpartial chemical structure diagram, ascomplete as can beknown, ifonecan be reasonably ascertained.

MarkCO this box if attach a continuation sheet. FORM EPA 7710-25 (Rev. 5-95) Page 4­

### PartI

- GENERAL INFORMATION

- Continued**~** SectionB - CHEMICAL IDENTITYIN'FORMATION

- Continued 2. Polymen (For a definition ofpolymer, see the Instructions ManUllL) Confidential a. Indicate the number-average weight ofthe lowest mole<:Ul.ar weightcomposition ofthe polymeryou intend to manufacture. Indicate maximum weight percentof low molecular weight species (not indudingresidual monomers, reactants, or solvents) below SOO and below 1,000 absolute molecular weight ofthat composition. • Describe the methods ofmeasurementorthe basis foryow: estimates:GPC 

## 0 Other

# 0 :(Specify) i) lowest number avu:agemolecularweight: ii) maximum weight 'l'io belowsao molecularweight 

### Not applicable iii) maximumweight 'l'io below 1000 moleeular weight 

# o Mark00 this box ifyouattach a continuation sheet. b. You mustmakeseparate confidentiality claims for monomer or otherreactantidentity, composition information. andresidual information. Mark 00 the**~Confidentia1"** boxnext to anyitem youclai.m as con.6.dential.. (1)

- Provide the specific:chem.ic:a1 name and CAS Registry Number (ifaonumber exists) of each monomer orotherreactant used in the manufacture of thepolymer. (2)

- Mark (X) this column ifentry incolumn (1) is confidential.. (3)

- Indicate the typical weightpercentofeach monomer orotherreactant in the polymer. (4)

- Marle 00 the identity column ifyou want a monomerorotherreactantused at two weightpercent or**le~s** to be listed as partofthe polymerdescription on theTSCA ChemicalSublJlance Inventory. (5)

- Mark (X) this column ifentries in columns (3) and (4) arecon.6.dential.. (6)

- Indicate the maximum weight percentofeach monomer or other reactant that maybepresent as a residual in thepolymer as manufactured for**co=~** purposes. (7)

- Mark (X) this column ifentry in column (6) is confidential.. Monomerorother reactant andCASRegistry Number Confi- Typical Identity Confi- Maximum Confi· dential composition Mark (X) dential residual dential (1) (2) (3) (4) (5) (6) (7) 

### Not applicable 'l". 

# ,. 'r_ 

# ,. ,. 

# ,. 

# ,. ,. 

# ,. ,. ,. <V. 

# ,. 

# ,. 0 Mark (X) this box ifyouattach a continuation sheet. Co Please identify which method you usedto develop orobtain thespecified chem.ic:a1 identity informationreported in this notice. (check one).



# o Method 1 {CAS Inventory Expert Service- a copyoftheidentification report 

## 0 Method2 (other source) obtainedfrom CAS Inventory ExpertServicemustbesubmitted as an attachment to this notice) d.. Thecurrently correct Otem.ical Abstrad:9 (CA) name for the polymer that isconsistentwithTSCA Inventory listings for similarpolymers. 

### Not applicable

e. Provide a correct representativeorpartialchem.ic:a1 struc:tUIe diagram, as complete as can be known, ifonecan be reasonably ascertained.



### Not applicable



# o Mark (X) this box ifyou attach a continuation sheet. FORM EPA mo.25 (Rev. 5-9S) PageS

### Part I

- GENERAL INFORMATION

- Continued

## ~ Section B - CHEMICAL IDENTITY INFORMATION

- Continued 30 Impurities

Ca)

­ Identifyeach impurity th.at may be reason.ably antidp.ated to be pr_t In thechemical subsLana lIS m.anufadured for cnmmerd.a.l purposes. Provide theCAS Registry Number ifav.a.llable. Ifthere areunidentified Impurities,enter"unidentified." (b) -Estimatethemaximum weight 'I:. ofeach impurity. Ifthere are unidentified impurities, estim.atetheirtoW weight 'l'.. Impurity and CAS Registry Number Maximum Conti­ (a) percent dential '" (b) % X , % 

### X % % 

### r. % % 

# o Mark (X) this box ifyou attach a continuation sheet. 4. Synonyms

- Enter any chemical synonymsfor thenewchemical substanceidentified in subsection 1 or 2.

## ~::::1 X 

# o Mark (X) thisbox ifyou attach a continuation sheet. 5. Trade identification

- List tradenamesfor the newchemical substanceidentified in subsection 1 or2. 

### YDC-1312 

# 0 Mark (X) this box ifyou attach a continuation sheet. 6. Generic chemicalname­ [fyouclaim Ptemical identity asconfidential. youmustprovidea generic chemical namefor your**substan~** that reveals the specific chemical identity of the newchemical substanceto the maximumextentpossible. Refer to theTSCA OtemicalSubstanceInventory, 1985 Edition, AppendixBfor guidanceon developing genericnames. 

### Substituted phenylepoxide 

# D Mark (X) thisbox ifyou attach a continuation sheet. 7. Byproducts ' n.--,.;Jow, anybyproducts resulting from the manufacture,processing. use, or disposal of the new chemical substance. Provide theCAS RegIStry**Nu.in~** ifavailable. Byproduct CAS Registry Number Conti­ (1) (2) dential 

### None



# 0 Mark 0<) this box ifyouattach a continuation sheet. FORM EPA 77l0-2S (Rev. 5-95) Page 6

### Part I

-- GENERAL INFORMATION

­ Continued**~** Section C - PRODUCTION,IM:PORT, AND USE INFORMATION: Mark C<f'the -Confidential- box nextto any item you claim as confidential 1. Production volume

­ Estimate themaximum production volumeduring the first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during the first three years ofproduction. Estimates should be on 1000/0 new chemical substancebasis. Fora Low Volume Exemption applictztwn,

### ifyou cJwose to have your notice rf:Viewedat a lowerprodudwn volume than 10'{}(}o kg/yr, sper:ify the volume and mark (x) in the binding box. If granted, you are bound to this volume
.. Maximum first 12-month production (kg!yr) Maximum 12-month production (kg!yr) Confi- Binding dential Option (1000/0 new chemical substance basis) (1000/0 newchemical substancebasis) Mark (x)
X
2­ Use Information

- Youmustmakeseparateconfidentialityclaims for thedescription ofthecategory ofuse, thepercentofproductionvolume devoted to each category, theformulation ofthenewsubstance, and other useinformation. Mark (X) the**~~nfidential~** Box next to any itemyou claim as confidential. a. (1)

­ Describe each intendedcategory ofuseofthenewchemical substanceby function and application. (2)

- Mark (X) this column ifentry in column (1) is confidential businessinformation (CBO. (3) - Indicate your willillgness to have theinformationprovidedin column (1) binding. (4)

- EstiInate thepercentoftotal productionlor thefirst three yearsdevoted to each category ofuse. (5)

- Mark (X) this column ifentryincolumn (4) is confidential businessinformation (CBO. (6)

- EstiInate thepercentofthenewsubstanceas formulatedin mixtures. suspensions, emulsions, solutions, orgels as manufacturedfor commercialpurposes at sites under yourcontrol associated with each category ofuse. 

### m- Mark (X) this column ifentryincolumn (6) is confidential businessinformation (CBO. (8) -Indicate 'l'O ofproductvolume expected for thelisted**~use-** sectors. Mark morethan onebox ifappropriate. Mark (X) to indicate your willingness to have the use typeprovidedin (8) binding. (9)

­ Mark (X) this column ifentry(ies) in rolumn (8) is (are) confidential businessinformation (CBO. category of use (1) CBl Binding Produc- CBr 'l'Oin CBI

# f;.
0/

## ru"""'~ 

# """"'"" P'"

## ~ CDI (by function and applicationi.e. a dispersive dyefor OptiOD tion'l'O Form­ (8) MMIc(x) ulation finishing polyesterfibers) Con-' ICom- Binding (2) (3) (4) (5) (6) (7) ed sumer

# ~~erdal Option (9)

X**~** X**~** X X

• % 0/0 % 0/0 0/0 % 0/0 0/0 0/0 0/0 'j­ % % "Ifyou have identified a "consumer" use, please provideon a continuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumer products. In addition include estimates of the concentration ofthenew chemical substan<;eas expected in consumerproducts and descn'be the chemical reactions by which this substanceloses its identityin the consumerproduct. 

## D Mark (X) this box ifyouattach a continuation sheet. b. Generic Ifyouclaim anycategory ofusedescription in subsection 2a as confidential, enter a genericdescription of that use category. Read theInstructionsManualfor examplesof genericusedescriptions. description 

### Electric device


# o Mark (X) thisbox ifyou attach a continuation sheet. 3. Hazard**~nfonna~on** -Includein thenotice a copyofreasonablefacsimile ofanyhazard warningstatement,label. materialsafety datasheet, Blacilns

or**o~er** information which will be provided to anyperson who isreasonably likely tobeexposed to this substance regardingprotective Opdoa 

### LX] eqwpment**p~ces** for the safe handling, transport,use,ordisposal ofthenew substance. Ustin part

## m hazardinformationyouinclude. M&d<(;jd XMark (X) thisbox if YOU attach hazardinformation. FORMEPA771O-25 {Rev.5-95} Page7

### Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE SectionA - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER M":I'k (X) the**'Co~dentialff** box next to anyitem you claim as confidential. Complel;j!.section A for each type of maJ.'tUiacture, processing, oruse operation involving the,lJew chemical substance at industrial sites you controL Importers do not have to complete this section for operations outside the U.S. ;however,you may stillhave reporting requirements ifthere are further industrial pmcessingoruse operations alter import. You must desaibe these operations. See instructionsmanuaL 1. Operation desaiption a. - Enter the identityofthe site at which the oOE!ration will occur. Name

### Not applicable address (number andstreet) aty, County,State,ZIP Code
Ifthe sameoperation will occur at more than one site, enter the numberofsites. Identify the additional sites on a continuation sheet, and if any ofthe sites havesignificantlydifferent**~** i'roduction rates oroperations,include all the information requested in this section for those**~** Con.6 dential.



# o Manufacturing 

# P Processing
Hours/batch b. TypeMark CO 

# o Use Batches/year 1. Batch Maximumkg/day (100"
_ c.hemlc.al Hours/day Days/year mbotanc:d 2. Continuous d. Processdesaiption 

## 0 Mark00 to indicateyourwillingness to have yourprocess description binding. (1) Diagram themajorurdtoperation stepsand chemical conversions. Include interim storage and transport containers (sped..fy-e.g. 5 gallon pails,55 gallondrum, rail car, tank truck. etc.). (2) Provide theidentity, theappmximate weight (by kg!dayorkg/batch on an 100% newchemicalsubstance basis), andentry pointof all starting materials andfeedstocks (includingreactants, solvents, andcatalysts, etc.), andofall products, recycle streams, and wastes. Includecleaning chemicals (notefrequency ifnot used daily orper batch.). (3) Identify by number thepoints ofrelease, including small orintermittantreleases, to theenvironmentof thenewchemical substance.


### Not applicable



# o Mark CO this box if you attach a continuation sheet. FORM EPA7710-25 (Rev. 5-95) PageS

## I Part II**~-** 

### HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

­ Continued 

## I**~** SectionA - INDUSTRlAL SITES CONTROLLED BY THE SUBMIII t.K

­ Continued 2. O<:rupational Exposure

­ You must make separate confidentiality claims for the description of worker activity, physicalform ofthe newchemical

substance, number of workers exposed. and duration of activity. Mark (X) the -Confidential- box next to anyitem you claim as confidentiaL (1)

­ Describe the activities (e.g. bag dumping. tote filling. unloading drums, sampling. clea.ni..ng. etc.) in which workers may be exposed to the substance. (2)

- Mark (X) this colw::n:n ifentryin column (1) is confidential business information (CEl). (3) ­ Describe anyprotectiveequipment and engineeringcontrolsused to protect workers. (4) and (6)

­ Indicate you will1r].gness to have theinformation providedin column (3) or (5) binding. (5) ­ Indicate the physicalformes) of the newchemical substance (e.g. solid: crystal, granule, powder, or dust) and % newchemical substance (ifpartof a mixture) at the time of exposure.**~** . (7)

­**~~** (X) this colw::n:n ifentry in column (5) isconfidential businessinformation (CBl). (8) ­ . -atethe maximum number ofworkers involved in each activity for all sitescombined. (9)

­ Mark (X) this colw::n:n ifentryin colw::n:n (8) is confidential businessinformation (CBl). (10) and en)

­ Estimate themaximumduration of the activity for any workerin hours per day and days peryear. (12)

- Mark (X) this colw::n:n ifentries in colw::n:ns (10) and (11) areconfidential businessinformation (CEl)• Worker activity CBI ProtectiveEqllipmenll Binding .t'llYSlcal tormls) tBinding CEI II of CBr Maximum duration CBI (e.g. bagdumping. fiJ..J.i.ng drums) Engineering; ControLs Option (e.g.soUd:powder) pption Workers Mark (x) and \ID new**~lc.W** Exposed HIs/day Days/yr (1) m m (4)**subs~.gce ~(61**  m {al 121 (10) (11) (12) 

### Not appUcab e

I 

# :0 Mark (X) this box ifyouattach a continuation sheet.

3. EnvironmentalRelease and Disposal- ¥oumustmakeseparateCOnfidentiality claimsfor the release number and theamountofthe newchemical substancereleased and otherreleaseanddisposal. information. Mark (Xl theRConfidential" boxnext to each item youclaim as confidential.
. (1)

- Enter thenumberofeach releasepointidentified in theprocess description, partIl, sectionA, subsection ld(3). (2)

- Estimate the amount ofthenewsubstancereleased (a) directly to theenvironment or (b) into control technology (in kg/day orkg/batch). (3)

- Mark (X) this colw::n:n ifentries incolumns (1) and (2) are confidentialbusinessinformation (CEl). (4)

- Identify themedia ofrelease Le. stack air, fugitive air (optional-see InstructionManual), surface water, on-site oroff-siteland orindneration, P01W, or other(please specify) to which thenewsubstance will bereleased from thatrelease point. (S)

- a. Desaibecontrol technology, ifany,,andcontrol efficiencythatwill beused to limit therelease of the new substance to the environment. rorreleasesdisposed. ofon land.characterize the disposal method andstate whether itis approvedfor d.i.spo5al ofRCRAhazardous waste. On a continuationsheet, for each sitedescribeany additionaldisposal methods that will used and whether the wasteis subject to secondaryor tertiaryon-sitetreatment. b. Estimate the amountreleased to theenvironment after control technology (in kg/day). (6)

- Mark (Xl this colw::n:n ifentries in columns (4) and (5) are confidentialbusinessinformation (CEI).
. (7)

- Identify the destination(s) ofreleases to water. PleasesupplyNPDES (NationalPoUutant Discharge Elimination System) numbersfor direct dischargersorNPDES numbers oftheP01W(Publidy OwnedTreatmentWorks). Mark (Xl if thePOTW name orNPDES.# is confidential businessinformation (CBO. Release Amountof-new substance 

### em Mediaof Control technology and efficiency (youmay wish to optionallyattach effidencydata) CBl Number reJea5ed release e.f;.lItxXUr : Binding , (1) (23) ab) (3) (4) (Sa) Mulr.: ():) (5b) (6)

. " I !**I~Ul-** cttJtJ.l..l.'-o.u.J..V I I I : I I I I I I I I I I (7) Mark (Xl the 

# o P01Wprovidename(s) below: CEI 

# o Navigable 

## 0 Other

- Specify provide NPDES # CBI destination(s) of f- waterway releases to water. 

# D Mark (Xl this box ifyou atbch a continll3tion sheet. FORM EPA 7710-25 (Rev. 5-95) Page9

# I Part II

­ HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued**~** Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS Completesection Bfor typical processingoruse operationsinvolving the new chemical substance at sites you do not rontrol.Importers do nothave to complete this section for operationsoutside the US.; however. youmust report any processingor use activities after import See the Instructions Manual. Compli:lI: aseparausection B for t!ach I:ypt!of prrx:.essing, oruse opt!TlI.tion inrolmng the nt!Wchem:iaz1 subst.o.nCl!. Ifthe sameoperation isperformed at morethan one site describe the typical operation common to thesesites. IdentUy additionalsites on a continuation sheet. 1. Operation Description· To claim information in this section as confidential. circleor bracket the specific information that you claim as confidential. (1) ­ Diagram the majorunitoperation steps.and chemical conversions, includinginterim storage and transport containers (spediy- e.g. 5 gallon pails, 55gallon drums, rail cars, tank"trucks, etc). On the d.ia.gram, identify byletter and brieflydescribe each worker activity. (2)

­ Provide theidentity. the approximate weight (by kg/day orkg/batch, on an 100% new chemical substance basis), and entry pointofall feedstocks (includingreactants, solvents andcatalysts, etc) and of all products, recycle streams, and wastes. Includecleaning chemicals (note frequency ifnot used daily orper batch). (3) - Identify by number thepoints ofrelease, including smallorintermittentreieases, to the environmentof the newchemical substance. (4) Please enter the # ofsites (remember.toidentUy tlIe locations ofthese sites on a continuation sheet); # of Sites

< 

### See the attachment at Page 15.



# D Mark (X) this box ifyouattach a

### contin~tion sheet. 2. WorkerExposure/EnvironmentalRelease (1)

­ From the diagram above, provide theletterfor each worker activity. Complete 2-8 for each worker activitydescribed. (2)

­ Estimate the numberof workersexposedforall sites combined. (4) ­ Estimate the typicalduration ofexposureperworkerin (a) hoursper day and (b) daysper year. (6) ­ Describe physicalform ofexposureand % newchemical substance (ifin mixture), and anyprotective equipmentand engineering controls used to protect workers. (1)

­ Estimate thepercentofthe newsubstance as formulated when packagedorused as a final product. (9) From theprocess diagramabove, enterthe number ofeach releasepoint. Complete 9-13 for each release point identified. (10) Estimatethe amount ofthenewsUbstancereleased (a) directly to theenvironment or (b) intocontrol technology to the environment (in kg! dayorkg/batch). (12) ­ Describemedia ofreleasei.e. stackair, fugitive air (optional-seeInstructionsManual), surfacewater, on-siteoroff-sitelandorincineration, PO'IW, orother (specify) and control technology, ifany, that willbe used to limit therelease ofthe new substance to theenvironment. (14) - IdentUy byproductswhich mayresultfrom the operation. (3), (5), (8), (11), (13) and (15).Mark 00 this column if any oftheproceedingentriesareconfidentialbusiness information (CBD. Letter # of CBI Duration CBI Protective Equip. / 'fGin CBI Reiease Amount of CBI Media ofRelease CBI

of Workers of EngineeringControls/ Form- Number New &reControl Act· Exposed Exposure PhysicalForm and 'fG ulation Substance Technology ivity new substance Released I (1) (2) (3) (4a) I (4b) (5) (6) (1) (8) (9) (10a) 

# : nab) (11 (12) (13) I I I X I X I 

### X I X X I I I I I I I I I I I I I I I I I I I (14) - Byproducts: None (15) 

# D Mark (X) this box ifyou attach a rontinuation sheet. FORM EPA 7710-25 (Rev. 5-95) Page 10

### OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle orbracketthe specific information that you claim as confidential. 

### In this section you mayprovideinformation notreported elsewherein this form regarding your efforts to reduce orminimize potential risks associated with activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new informationpertinenuopollutionprevention, including source reduction, recycling activities

.and safer processes orproducts available due to the new chemical substance. Source reduction includes thereduction in the amountor toxicity ofchemical wastes by technological modification, process and procedure modification, productreformulation, rawmaterials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes thatwould otherwise be 

### treated or released as air 

### emissions or water discharges, or land disposal. Descriptions ofpollution prevention, source reduction and recycling should emphasizepotential risk reduction subsequent to compl.!-ance with existing regulatory requirements and canbeeither quantitativeor qualitative. The EPA is interested in 

### this information to assess overall netreductionsin toxicity or environmentalreleases and exposures, notthe shiftingofrisks to other environmental mediaornon-environmental areas (e.g., occupational orconsumer exposure). In 

### addition, information on therelative cost or performance characteristics ofthe P:M::N" substance to potential alternatives may be 

### provided. All informationprovided inthis section will be taken into considerationduring the review ofthis substance. See the revised Instructions Manual thatincludes a Pollution Prevention manual for guidance and examples. Describe the expected net benefits, such as (1) an overall reduction in risk tohuman health orthe environment; (2) a reduction in the volume manufactured;(3) a reduction in the generation ofwaste materials throughrecycling, source reduction or other means; (4) a reduction in potential toxicity orhuman exposure and/or environmental release; (5) anincrease in product performance, a decrease in thecost ofproductionand/or improvedoperation efficiencyofthenew chemical substancein comparisonto existing chemical substancesused in similarapplications; or (6) the extentto which thenewchemical substance maybea substitutefor an existing substancethat poses a greateroverallriskto human health ortheenvironment.


### Not applicable



# o Mart< (X) this box if you attach a continuation sheet.

FORM EPA 7710-25 <Rev. 5-95) Page 11

### PartIII

- LIST OFATTACHMENTS
Attach continuation sheets for sections of the form and test data andotherdata (including physical/chemicalpropertiesand structure/activityinformation), and optional information after this page. Qearlyidentify the attachment and the section of the form to which itrelates, ifappropriate. NUIllber consecutively the pagesof the attachments. In thecolumn below, enter the inclusive page nUIllbers ofeach attachment. Mark (X) the ·Confidential"box next to any attachment name you claim as confidential. Read the Instructions Manualfor guidanceonhowtoclaim any informationin an attachment as confidential. You mustinclude with the sanitized copy of thenotice form a sanitized versionofanyattachmentin which youclaim information asconfidential. Attachment name Attachment ConfipagenUIllber(s) dential 

### Material SafetyData Sheet (MSDS) 1 6 1 

### 7 

### Physical and Chemical Properties Worksheet 1 

### 3 

### IR Chart 1 4 

### Continuation sheet of Part 11, 

### Section B.l 1 5 

### Bacterial Reverse Mutation Study of YDC-1312 1 8 

### - 3 6 

### Chromosome Aberration Study of YDC-1312 in Cultured Mammalian Cells 3 7 5 5



# 0 Mark (X) thisbox ifyou attach a continuation sheet. Enter theattachment name and nUIllber. FORM EPA mO-25 (Rev. 5-95) Page 12PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review of physical and chemical propertiesdata, pleasecomplete the following worksheet for data you provide and include it in the notice. Identify the property measured, the pageofthenoticeon which the property appears, thevalue of the property, the units in which the property is measured (as necessary), and whether ornot thepropertyisclaimedas confidential. Thephysical stateof theneat substanceshould be provided.. These measured properties should be for theneat (100'l'. pure) chemical substance. Properties that aremeasured for mixtures or formulations should be sonoted (0/., PMN substance in ).You are not required to submit this worksheet; however, EPA stronglyrecommendsthat you do so, asit will simplify reviewand ensure that confidential information is properly protected.. You should submit this worksheet as a supplement to yoursubmission oftest data. This worksheet is not a substitutefor submissionof test data. Property Page Value M
.....I'I!<! ContiMark00 if or dential provided number Eotimat. (a) (b) (c) Mark 00 (M or E) (d) Physical stateofneat substance X X (s)
__ (l)
__ (sV Vaporpressure ·C @Temperature Torr 20°C X 1. 12 M Density/relative density g/cm3 Solubility "C

@Temperature

Solvent g/L



## . "C X 

### Insoluble E Solubility in water @Temperature "C X 141°C M Melting temperature 

## ·c @ 2 torr pressure Boi!ing/sublimationtemperature X 184°C M Spectra I R 

### spectra X 14 M
Dissociation constant Particlesizedistribution Octanol/waterpartition coefficient Henry's Lawconstant Volitalization from water Volilalization from soil pH @concentration 

### Flash point X 206°C Flammability Explodability Adsorption/ coefficient Other

­ Specify

PAGE 13

## o u

a:l ..I-l a:l 'tJ ..I-l C Q) S Q) s... :::l til a:l Q) S 0:: H I t-< U:.. .::t Q) flO a:l 0....Continuation Sheet of Section B. 1 of Part

### II 

## CONFIDENTIAL

Page 15U.S. Department of Labor Occupational Safety and Health Administration (Non-Mandatory Form) Form Approved OMB No. 1218-007 Note: Blank spaces are not pennitted. Ifany item is not applicable, or infonnation is available the s ace must be marked to indicate th, Section I Manufacturer's Name 

### TOHTO KASEl CO. LTD. Emergency Telephone Number Address (Number, Street, City, State, and Zip Code) NihonbashiBakurocho l-chome, Chuo-ku, Telephone Number for Information +81-3-3665-1701

__**........:T::..;o:.:k::li~yo~"**.=J..=a:.A;:.:;Plan==-

___________--I Date Prepared October 2000 Signature of Preparer (Optional) Section 

# n - Hazardous Ingredients/Identity Information Hazardous Components (Specific Chemicalldentiryj Common Name(s) OSHA PEL ACGIH TLV Other Limits % 

### None Non-Hazardous Components YDC-1312 None None None 99%

S f 

# m Ph
. I/Ch

ec Ion 

# - wSlca emlcaI Characterls 'f ICS Boiling Point Specific Gravity (H2O::1) 

### 184't at 2 mmHg 1.12 Vapor Pressure (mmHg) Melting Point 

### 141't Not determined Vapor Density (Air - 1) Evaporation Rate 

### Notdetermined (Butyl Acetate:: 1) 

### Notdetermined

., SolubIlity In Water 

### Insoluble Appearance and Oder 

### Transparentlightyellow solid losion Hazard Data UEL Flammable Limits 

### 206't 

### N.A. Extinguishing Media 

### Water spray, dry

### chemical, carbon dioxide, or chemicalfoam Special Fire Fighting Procedures 

### Wear self-containedbreathingapparatus in

### the case offire atthe close area. Unusual Fire and Explosion Hazards 

### Not in

### particular. (Reproduce locally) OSHA 174, Sept 1985 Page 16

### Section 

# v
.. Reactivitv Data 

### ­ Stability Unstable Conditions to Avoid 

### Strongoxidizer, strongacids andbases Stable X Incompatibility Materials to Avoid) 

### None Hazardous Decomposition or Byproduc

### Carbonmono-oxide Hazardous Polymerization May Occur Will Not Occur 

### X Conditions to Avoid 

### Curingagents andhightemperature C>



### Section VI
.. Health Hazard Dau Route{s) ofEntry: Inhalation? Skin? Ingestion? 

### No No No Health Hazards (Acute and Chronic) 

### Positive atAmes testbut negative at chromosomal aberration test. Notknown about other chronic effects. Carcinogenicity: 

### NTP? IARC Monograph? 

### No OSHA Regulated? 

### No No Signs and Symptoms ofExposure 

### Notknown
Medical Conditions Generally Aggravated by Exposure 

### Not known Emergency and First Aid Procedures 

### Eye contact: Immediatelyflush withplentyofwater for atleast 15 minutes. Get medical aid. Skin contact: Washwithplentyofsoap andwaterfor atleast 15 minutes. Get medical aid, ifirritation develops orpersists. Ingestion: Rinse outthe
Sweep and scoop up for disposal, avoiding dustgenaration. Washtrace residueswithwater. Do not allow residues to enterwaterways. Waste Disposal Method 

### Waste material may be dumped orincinerated underconditions which meet all 

### federal, state and local environmentalregulations. Precautions to Be Taken in Handling and Storing 

### Precautions mustbetakento avoid contact withthe mouse withplenty ofwater and getmedical attentionimmediately.

skin and eyes. Store in a cool, dry, 

### ventilatedplace andina tightlyclosed container. Other Precautions 

### Not in

### particular Section V1II
.. Control Measures Respiratory Protection (Specify Type) 

### Seekprofessional advice priorto respirator selection and use. Ventilation Local Exhaust 

### Recommended Mechanical (GeneraC

### Recommended Special 

### Notrequired Other 

### Notrequired Protective Gloves 

### Rubber gloves Eye Protection 

### Safety chemical goggles Other Protective Clothing or Equipment 

### Recommended 

### to wear chemical apron. Work/Hygienic Practices Wash thoroughly afterhandling, and avoid contactwith chemical. Page 2 USGPO: 1966-491-529/45775 

### Page 17

## 4L134 

# CONFIRMATORY STATEMENT OF THE STUDY REPORT
General Testing Research Center, Japan Oilstuff Inspectors' Corporation

Sponsor : TOHOTO Kasel CO., LTD. Title : Bacterial Reverse Mutation Study of YDC-1312 Study No. : 4L134

The said study has been conducted in General Testing Research Center, Japan Oilstuff Inspectors' Corporation. The original study report was written in Japanese language and translated into English language. I hereby declare that this report reflects faithfully the original study report as much as possible to my knowledge.

## /}~} 

# .1/ i

- .. Translator

# ~.**~~** Date: 

# 1£1,.1

# Z IffY V E ii

## chi Mach i

## gaki, 

### M. 

## S. Scientist of Mutagenicity Testing Group

2

### 4L134


## Submitted to: TOHOTO Kasei CO., LTD.


# (REPORT)
Bacterial Reverse Mutation Study of YDC-1312 (Study No. : 4L134)

June 15. 

## 1994
GENERAL TESTING RESEARCH CENTER, JAPAN OILSTUFF INSPECTORS' CORPORATION

i !' " 

### 3 .A ..J

## 41134 

# STAT EMEN T
General Testing Research Center. Japan Oi lstuff Inspectors' Corporation

Sponsor : TOHOTOKasei CO.• LTD. Titie Bacterial Reverse Mutation Study of YDC-1312 Study No. 4L134

The study in this report was conducted in compliance with the following Good Laboratory Practice Standards. rJapanese GLP Standerds Applied to Industrial Chemicals CKanpogyo No. 39, Yakuhatsu No. 229. 59 Kikyoku No. 85, 1984) J



## rGLP Standards under the Japanese Industrial Saftey and Health Law (Notification No. 76. Ministry of Labor, 1988) J



## Management Osamu Ueno sealed Date: June 15. 1994 Director of Microbiological Laboratory

4 

# 20

### 4L134



# .. 

# QUALITY ASSURANCE STATEMENT
General Testing Research Center. Japan Oi lstuff Inspectors' Corporation

Sponsor TOHOTO Kasei CO
.. LTD. Title Bacterial Reverse Mutation Study of YDC-1312 Study No. 4L134

By the inspections and audits listed below. we hereby certify that this study was carried out in accordance with its protocol and with our Standard Operating Procedures. and that this report faithfully describes the methods and procedures employed, and that the reported results reflect accurately the raw data of the study. Inspection Date of inspection Date of reporting to or audit or audit the study director and to the management Study protocol Apri 1 27, 1994 April 27. 1994 Study procedure June 1. 

### 1994 June 1. 

### 1994 Study June 15, 1994 June 15, 1994 report

Quality Assurance Unit =Ma::..:sc.;;;:.a.::....;yo:...;:.s..:.::..h=--i..::..::K.:...;id:.::a'----=...:se::.:;a:...:.l.:::.;ed:.... Date: June 15, 1994

5

### 4L134 

# OUTLINE OF THE STUDY
1. Title : Bacterial Reverse Mutation Study of YDC-1312 (Study No. : 4Ll34) 2. Purpose : To assess the mutagenicity of the test substance in bacterial mutation assays. using 

### Salmonella tyPhimurium and 

### Escherichia coli strains. 3. Guideline : Japanese Guidelines on Industrial Chemicals(Kanpogyo No.70Q. Yakuhatsu No. 1039. 61 Kikypku No. 1014. 1986) and guidelines under the Japanese Industrial Safety and Health Law (Notification No. 77. Ministry of Labor. 1988) 4. G L P 

### : Japanese GLP Standerds Applied to Industrial Chemicals (Kanpogyo. No. 39, kuhatsu No. 229. 59 Kikyoku No. 85. 1984) and GLP Standards**unde~** 

### the Japanese Industrial Saftey and Health Law (Notification No. 76, Ministry of Labor. 1988) 5. Sponsor : TOHOTO Kasei CO.. LTD. 07-14 Higashikasai. 3-chome. Edogawa-ku, Tokyo) (Responsible person) Yoshiaki Nakamura 6. Contractor : Mitubichi Chemical Safety Institute Ltd. (1-30, 2-Chome, Shiba, Minato-ku, Tokyo) 7. Testing Facility: General Testing Research Center. Japan Oi lstuff Inspectors' Corporation (10-4, l-Chome, Mikagetsukamachi, Higashinada-ku, Kobe-shi, Hyogo) 8. Management : Osamu Ueno 9. Study Director : Elichi Machigakl 10. Other Contributors :Masato Nakamura, Manabu Kajihara. Mikiko Tatsumi 9. Study Date : (Initiation of the Study) April 27, 1994 (Submission of the Final Report) June 15, 1994 10. No statistical methods were used for data analysis. 11. There was no unforeseeable circumstances that may have ill effects on test results. 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. Further retention wi II be discussed with the sponsor.

6

### 4Ll34 

# Con ten t s Page Summary
........................."............................................... 

### 8
Materials and Methods 1. 

### T est rnat eria 1

### s .. . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . ... ... . . . . . ... 9 

### 2. Bacterial tester strains
....................................... 10 3. Media
..................................................................... 11 4. S9 mix
.................................................................. 11 5. Test method
............................................................ 12
Results and Conclusion
................................................ 14
References
.................................................................. 14
Tables
.................................................................. 15 Figures
.................................................................. 18

7

### 4L134 

# Summary
YDC-1312 was examined for the mutagenicity in bacterial assays using five different strains, SaLmoneLla tyPhimurium 

## TA9S, TAI00, TA1535, TA1537 and Escherichia coLi 

## WP2 uvrA-. 

## The numbers of revertants induced by the test substance were more than double of the solvent control value in S. typhimurium 

## TAI00 and TA1535 with S9 mix. These findings led to the conclusion that YDC-1312 is mutagenic under the test conditions employed.

S

## 4L134 

# Materials and Methods 1. Test materials Confidential


### , 1. 

## 1 Test substance

1.2 Negative and positive controls The negative controls and the positive controls were as follows. Supplier Name Lot Purity 

## Abbreviation 

## I number (%)

i 

## Negative controls dimethylsulfoxide DMSO Wako Pure Chemical APE5938 min.99.5 Industries. Ltd. I 

## Positive controls 

# I 2-(2-furyl)-3-(5-nitro­ AF-2 Wa k0 

## Pure C hem i c a I . PDF0626 98"'-'102 2-furyl)acrylamide Industries. Ltd. sodium azide NaNs 

## Wako Pure Chemical DSG1561 Industries. Ltd. 92. 2 9-:-aminoacridine 9-AA SIGMA Chemical Company 80F-0186**~** 

## 98 

## N- ethyl-N' -nitro-N­ ENNG SIGMA Chemical Company 56F-3651**~** 

## 97 nitrosoguanidine 2-aminoanthracene 2-AA Wako Pure Chemical TSE4201 92.6 Industries. Ltd.

9

### --



### --

-- 

### 4L134 

## 2. Bacterial tester strains 2.1 Strains I} 

## Genetically different five strains of Salmonella typhimurium TA98. TA100, TA1535. TA1537 and Escherichia coli WP2 uvrA- were employed. These strains were received from Mistubishi Chemical Safety Institute Ltd. on July 8, 1988. The genetic properties of the strains are listed in the following table. The genetic properties of the strains are listed in the following table. Strain Gene Additional mutations Affected. Repair LPS R-factor Mutation type 

## S. tyPhimurium

TA98 his D uvr B ria pKM 101 Frameshift his G 

## uvr B ria pKM 101 Base-pair change TA1535 TA100 his G 

## uvr B Base-pair change TA1537
. ria his 

## C uvr B ria Frameshift E. coli

WP2 uvrA

­ trP E uvr A**Base~pair** 

## change 

## + ! I 

## 2.2 Check on genotypic characteristics The tester strains were checked on their genotypic characteristics such as amino acid requirement, UV sensitivity. membrane mutation and R-factor before the study. 2.3 Storage Eight ml of fresh bacterial suspension (cultured with shaking at 37°C for about 7 hours in a liquid growth medium) was**mi~ed** 

## with 0.7 ml of DMSO and quickly frozen in a dry ice-acetone mixture. The stocks of the tester strains were stored below -80°C. 2.4 Bacterial suspension Twenty /11 

## of stock cultures of tester strains were inoculated against the 10ml liquid growth medium and grown with shaking at 37°C for about 7 hours. Densities of the fresh bacterial cultures were measured with spectrometer.

1 0

### 4L134 3 Media 3.1 Liquid growth medium Oxoid Nutrient Broth No.2 (Unipath Ltd. Lot NQ 19947080) was dissolved in distilled water (25g/1!.) and autoclaved at 121°C for 15 minutes. 3.2 Minimal glucose agar plate The agar plate ( CLIMEDIA®AM-N MEDIUM) was purchased from Nisshln Flour Milling Co
.. Ltd
.. The dose finding tests were used lot No. AN090BJ prepared on February 21. 1994. and the main test were used lot No. ANlS0CJ prepared on March 28, 1994. 

### 3.3 Top agar Aqueous amino acid solution for S. 

### tyPhimurium strains. which contained Lhistidine (Wako Pure Chemical Industries. Ltd.) and D-biotin (Wako Pure Chemical Industries, Ltd.) in 0.5 ruM 

### respectively was prepared, sterilized by filtration (0.45tim 

### pore size) and stored in a refrigerator. Aqueous amino acid solution for E. 

### coli. which contained 0.5 ruM 

### L-tryptophane (Wako Pure Chemical Industries. Ltd.) was prepared. sterilized by filtration (0.45 tim 

### pore size) and stored in a refrigerator. O.S g of powdered agar ( Bact-Agar; Difco Laboratories) and 0.5 g of sodium chloride were dissolved in 100 ml of distilled water and autoclaved. The above mentioned amino acid solution was added to this agar solution in the ratio of 1 to 10 and kept at about 45°C until use.
4 S9 mix 4. 1 S9 S9 from Kikkoman Corporation was the 9000Xg supernatant fraction of liver homoganate prepared from Sprague Dawley rats which were given i.p. injection of phenobarbital and 5.S-benzoflavone. The dose finding tests were used lot No. RAA-305 prepared on February 18. 1994. and the main test were used lot No. RAA-309 prepared on May 13. 1994. The S9 was stored below -80°C.
1 1

### 4L134 4.2 S9 mix The composition of S9 mix is listed in the following table. S9 mix was freshly "Prepared for each assay. and was stored in the ice. Components S9 MgCh • 6H 2

### O KCI D-glucose 6-phosphate /3 

### -NADPH i 

### /3 

### -NADH Na-phosphate buffer (pH 7.4) Distilled water Amount in 1ml 0.1 ml 8 /l

### moI 33 /lmo 

### I

5 /l

### moI 4 /l

### moI 4 /l

### moI 100 /lmol remainder

5 Test method 2) 

### 5.1 Preparation of test substance solutions and positive controls The test substance was dissolved in DMSO. and diluted into each concentration. The test substance solution was prepared just before use. As for**p~sitive** 

### controls. NaN 3 

### was dissolved in DW. the others were dissolved in DMSO. 5.2 Test substance concentrations In the dose finding test and the main test. five dose levels was graded at nominal ratio of 2 (313 to 5000/lg 

### per plate). 5.3 Reverse mutation test The reverse mutation test was done by preincubation method. Point one ml of a test solution. a 0.1 ml aliquot of one of the bacterial suspensions and 0.5 ml of either Q 

### 1 Msodium phosphate buffer (pH 7.4) for assay without metabolic activation or S9 mix for assay with metabolic activation were mixed in a sterilized test tube and incubated with gentle shaking at 37°C for 20 

### min. After preincubation, 2 

### ml of mol ten top agar was added to this mixture and poured onto a minimal glucose agar plate. When the agar overlay solidified. these plates were incubated at 37°C for 48 hours.

1 2 r. ,.... ;;..()

## 4L134 The presence of the background lawn was observed using a stereoscopic microscope and microbial growth was confirmed. Revertant colonies were counted with eye or an automatic colony counter. Two plates per one dose level were used for each assay. Negative and positive control tests were also done in each assay. The positive controls and the concentrations were as follows. Strain I 

## -S9 Mix Cttg/plate) tS9 Mix Cttg/plate) 

## S. tyPhimllriWlt 

### I



## TA98 TA100. TA1535 TA1537 E. coLi



## WP2 llvrA

- 

## AF-2 AF-2 NaNs 

## 9-AA ENNG 0.1 0.01 0.5 80

2 2-AA 2-AA 2-AA 2-AA 2-AA 0.5 1 2 2 10 5.4 Contamination test The highest dose suspensionon and S9 mix were tested for the bacterial contamination. 5.5 Criteria for judging the results The test substance is judged to be positive in this assay when the mean number of revertants is more than double of the negative control value and when it increases significantly dose-dependent. When the test substance is judged to be positive. specific activity was calculated as a induced revertants per Img test substance.

1 3

### 4Ll34 

# Results and Conclusion The results are shown in Tables 1**~** 

### 2 and Figures 1**~** 

### 6. In the main test. the numbers of revertants induced by the test substance were more than double of the solvent control value in S. tyPhimurium 

### TAI00 and TA1535 with S9 mix. The reproducibility of the these results was confirmed in the dose finding test.

Maximum specific activity was 496 colonies/mg in TAI00 with 89 mix at 625u 

### g/plate.

Microbial growth inhibition was not observed. Precipitated test substance was observed on the agar plate. It was shown as 

### " t" in the tables. In the positive control tests useing specific mutagBns. they induced significant revertants exceeding those of the negative control.

No statistical methods were used for data analysis. The sponser confirmed that the lot of the test substance used in this study was stable during testing period. These results led to the conclusion that YDC-1312 is mutagenic under the test conditions employed.


# References 1) 

### Maron. D. M. and Ames. B. 

### N. (1983) Revised methods for the Salmonella

mutagenici ty test, Mut. Res
.. 113. 173-215. 2) Mutagenicity test in Industrial Safety and Health Law (1991) The test guideline and the GLP. edited by the Chemical Substance Investigation Division, Ministry of Labor of Japan. Japan Industrial Safety and Health Associat ion, Tokyo.

1 4 r. r' . ,**L~** UI

### 4L134 Table 1 Results of the Dose-finding Test Test substance
. YDC-1312 Testing periods From May 10. Number of revertants 1994 to May 13. 1994 Cnumberof colonies/plate) With (+) 

### or 

## I Test material without( ) concentration Base-pair change type Frameshift type S9 Mix C/.lg /plate ) TA 100 TA1535 WP2 lLvrA- 

### TA 98 TA1537 Solvent 101 16 19 15 8. control 124 C113) 15 ( 16) 16 ( 18) 14 ( 15) 5 ( 7) t 

### 105 15 29 11 10 156 108 ( 107) 12 ( 14) 19 ( 24) 13 C 12) 9 ( 10) t 

### 117 10 21 13 3 313 98 ( 108) 9 ( 10) 19 C 20) 16 ( 15) 11 ( 7) - S 

### 9mix t 

### 107 16 23 12 7 625 111 ( 109) 7 ( 12) 12 ( 18) 12 ( 12) 3 ( 5) t 

### 138 15 11 11 6 1250 112 ( 125) 11 ( 13) 10 ( 11) 

### 12 ( 12) 8 ( 7) t 

### 118 5 17 16 4 2500 111 ( 115) 8 ( 

### 7) 15 ( 16) 13 ( 15) 6 ( 5) t 

### 117 5 15 10 6 5000 123 C120) 6 ( 6) 14 ( 15) 15 C 13) 8 ( 7) I 

### Solvent 98 10 18 25 14 control 100 ( 99) 10 ( 10) 18 ( 18) 13 ( 19) 10 ( 12) 140 10 19 24 6 156 133 ( 137) 8 ( 9) 13 ( 16) 22 ( 23) 10 ( 8) 192 76 20 I 

### 19 12 313 238 ( 215) 71 ( 74) 24 ( 22) 39 ( 29) 13 ( 13)
+ S 

### 9mix 373 211 22 24 13 625 406 ( 390) 203 ( 207) 20 ( 21) 31 ( 28) 12 ( 13) t 

### 451 248 22 29 10 1250 452 ( 452) 266 ( 257) 24 ( 23) 25 ( 27) 7 ( 9) t I 

### 465 237 23 25 10 2500 439 ( 452) 241 ( 239) 27 ( 25) 25 ( 25) 7 ( 9) t

# ~ro 194 10 16 12 5000 343 ( 361) 230 ( 212) 22 C 16) 31 ( 24) 7 ( 10) Name. AF 2 NaNs ENNG 

### AF-2 9-AA Posi ti 

### ve control Concentration 0.01 0.5 2 0.1 80 not (p.g /plate ) requiring S9 Mix Number of 433 394 831 443 458 colonies/plate 431 ( 432) 382 ( 388) 843 ( 837) 441 ( 442) 498 ( 478) Name 

### BP 2-AA 2-AA 

### BP BP Positive control Concentration 5 2 10 5 5 requiring (p.g /plate ) I 

### S9 Mix Number of 1096 263
I 

### 924 283 190 colonies/plate 1043 (070) 264 ( 264) 946 ( 935) 287 ( 285) 194 ( 192) I

•



### Af-2 :**2-(2:f~rYl)-3-(5-nitro~2-fur¥l~acrylamide.** 

### NaN 3 : 

### sodium azide, ENNG : N-ethyl-N' nitro-NnltroSOguanldlne, 9-AA : 9-amlnoacrldlne. BP : benzo[a]pyran. 2-AA : 2-aminoanthracene. Notes () : Mean t : 

### Precipitated test substance was observed on the ager plate.

1 5

### Table 2 Results of the Main Test 8L134 Test substance
. YDC-1312 I 

### Testing periods From May 31. 1994 to June 3, 1994 Number of revertants (number of**colonies~plate** 

### ) With (+) 

### or Test material Frameshift type without( ) concentration Base-pair change type S9 Mix (ttg**~plate** 

### ) TA 100 TA1535 WP2 uvrA- 

### TA 98 TA1537 Solvent 123 8 11 

### 18 9 control 100 ( 112) 7 ( 8) 10 ( 11) 20 ( 19) 8 ( 

### 9) 

### t 136 C 10 C 

### 17 C 

### 28 C 7 C 156 94 ( 115) 10 ( 10) 9 ( 13) 24 ( 26) 5 ( 6) 

### t 106 C 12 C 

### 18 C 24 C 4 C 313 126 ( 116) 9 ( 11) 15 ( 17) 19 ( 22) 6 ( 5) 

### t 116 C 8 C 15 C 23 C 3 C S 

### 9mix 625 122 ( 119) 11 ( 10) 22 ( 19) 22 ( 23) 8 ( 6) 

### t 97 C 13 C 11 

### C 17 C 4 C 1250 128 ( 113) 14 ( 14) 14 ( 13) 19 ( 18) 9 ( 7) 

### t 111 C 11 

### C 14 C 23 C 5 C 2500 119 ( 115) 11 ( 11) 

### 14 ( 14) 19 ( 21) 13 ( 9) 

### t 103 C 11 

### C 15 C 10 C 5 C 5000 106 ( 105) 9 ( 10) 18 ( 17) 21 ( 16) 4 ( 5) Solvent 101 4 21 29 9 control 101 ( 101) 8 ( 6) 17 ( 19) 28 ( 29) 7 ( 8) 90 17 17 31 9 156 128 ( 109) 20 ( 19) 12 ( 15) 31 ( 31) 7 ( 8) 248 110 29

# 28)~ 10 313 230 ( 239) 99 ( 105) 27 ( 7 ( 9) + S 

### 9mix 452 247 21 25 625 369 ( 411) 272 ( 260) 21 ( 21) 37 ( 31) ) 

### t 465 C 375 C 28 C 30 C 17 C 1250 484 ( 475) 369 ( 372) 19 ( 24) 30 ( 30) 10 ( 14) 

### t 474 C 315 C 27 C 26 C 8 C 2500 452 ( 463) 315 ( 315) 21 ( 24) 24 ( 25) 14 ( 11) t 

### 390 C 292 C 16 C 22 

### C 5 C 5000 381 ( 386) 273 ( 283) 18 ( 17) 39 ( 3D 7 ( 6) Name AF 2 

### Positive NaN! ENNG 

### AF-2 9-AA control Concentration 0.01 O. 

### 5 2 0.1 80 not (ttg**~plate** 

### ) requiring S9 Mix Number of 469 290 1094 517 782**colonies~plate** 

### 500 ( 485) 281 ( 286) 1148 (121) 519 ( 518) 483 ( 633) Name 

### BP 2-AA 2-AA 

### BP BP Posi ti

### ve control Concentration 5 2 10 5 5 requiring (ttg**~plate** 

### ) S9 Mix Number of 951 218 870 268 159**colonles~plate** 

### 953 ( 952) 216 ( 217) 745 ( 808) 308 ( 288) 142 ( 151) Af-2 :**2-(2~f~ryl)-3-(5-ni**

### tro-;-2-fur¥i!acry)amide. NaNs : 

### sodium azide, ENNG : N-ethy)-N'

### ni tro-NnltroSOguanldlne. 9-AA : 9-amlnoacrldlne. BP : benzo[a]pyran. 2-AA : 2-aminoanthracene. Notes (): Mean 

### t : Precipitated test substance was observed on the ager plate.

1 6

### Results of Specific Activity 

## Test substance YDC-1312 

# / Bacterial WitholJt S9 mix With S9 mix Strains Specific IConcentration** Specific Concentration** Activity* (,ug/plate) Activity * 

## (,ug/plate) TAlOO 466 625 TA1535 315 625 Dose finding WP2 uvrA

## test TA98 TA1537

I 

## TAlOO 496 625

TA1535 406 625 Main test WP2 uvrA

## TA98 TA1537 

# * : The number of induced revertants per 1 mg test substance. 

# ** : Maximum value of specific activity was mentioned at in the dose finding test and the main test.

1 7600**,-----~------,------,~-----,------~----_,** 

# ; (TroD)

Revertants per plate 4 50-----­

# 1 .. ",:"",
....................................: .......................· ....·

..·

....f
..··.. ............, ..................................................................... ! 

# i

3 0 0
....................................+ ....................................j
..................................

### 1..
..................................:................................... ....................................



## iii 

## i i**OL-----~------~----~~----~------~-----** 

# o 156 313 625 1250 2500 5000 Concentration (.ug/plate) Fig. 1 Dose response curve of YDC-1312 (Main Test) 

## (0···0 : -S9mix. 

### 0-0: +S9mix)

40**~----~------~----~,------~,------~-----,** (W P 

# 2u v r A 

### -,J

3 0
....................................+ ....................................j
....................................+ ....................................j
...................................

# + ................................... Revertants per plate 2

# O------l.::.~-::~~t~~::::T:::-:::.r

# ...______9 _. _._.'

1 0 ·
..

# ·~·~ .. ..

# ~:. .. .. .. .. .. ..

# T
..............................· ..·!·
..··..·......· ..·..·

....·

........

# ·r ..................· ..·..· ..·

..·:....· ..................· ....·

..·

..

# + .................................

o L-

____ ______ ____ ______ ______ ____**~**



# o 156 313 625 1250 2500 5000 Concentration (.ug/plate) Fig. 2 Dose response curve of YDC-1312 (Main Test) 

## (0···0 : -S9mix. 

## 0-0: +S9mix)

1 8

## Revertants per plate

Revertants per plate 40

30

20

10



### o

400

300

200

100



### o 

### (TA 1

# 53 5 

# S9Mix 

## J



## .

...····0·····
.. ,

# ... '" .()

## ....... 

## ....



# o 156 313 625 1250 2500 5000 Concentration (.u g/p

## late) Fig. 3 

## Dose response curve of YDC-1312 (Main Test)

. (0···0 : 

## -S9mix)

(TA 1

## ~ 3 5**+~**

# '9M~>--. .n.·..·...····· 

# ;V 

# ,/

r 

# V

o 156 313 625 1250 2500 5000 Concentration (.ug/plate) Fig. 

## 4 Dose response curve of YDC-1312 (Main Test) (0-0 : 

## +S9mix)

1 9**40~----~------~----~------~----~-----,** 

# (T A 9 8)

j


## Revertants per plate

10i···_·····_·
..·······..··.. ·..

### ·+....·..· ..··_--_·_..··.. ··I·
..·.... ·....·..·............···+..· ....·

..··..........· ..······i
...... _·-_..·..·.............

### +............._ ....................**O~----~------~----~------~----~-----** 

# o 156 313 625 1250 2500 5000 Concentration (tLg/plate) Fig. 

## 5 Dose response curve of YDC-1312 (Main 

## Test) (0···0 : -S9mix; 

## 0-0: +S9mix)



## Revertants per plate**40,-----~------~----~------~----~-----,** 

### (T ! 

### 1 5 3 7 .'

301
......·_..·..·....···.._·..··...."
..·......_ ..·····..··....·......·i
......· ..··..· ..· ........··..·f·
....··....·······..·..........·j
....·....·..........· ........·..·..

### ·+........· ..··......·_........· ....1

201
..·....·....·................·· ..·t....·····....·......·.. ··....··....·I·
......·..........··....·..·......·,..............·..........··..·..···..1
..·..·....··..·..· ............··..

### +..· ............· ..·

.............. 1 

## lOR:._

156 313 625 1250 2500 5000 Concentration (tLg/plate) Fig. 6 

## Dose response curve of YDC-1312 (Main Test) (0"'0 : -S9mix, 

## 0-0: +S9mix)



### 2 0

## 4L435 

# CONFIRMATORY STATEMENT OF THE STUDY REPORT

, General Testing Research Center, Japan Oi lstuff Inspectors' Corporation

Sponsor : TOHOTO 

## Kasei Co., Ltd. Title Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells Study No. 4L435

The said study has been conducted in General Testing Research Center. Japan Oilstuff Inspectors' Corporation. The original study report was written in Japanese language and translated into English language. I hereby declare that this report reflects faithfully the original study report as much as possible to my knowledge.

Date:

# fiJ", 

# Il, 11Yt

r 

### I 

## Masato Nakamura, M.S. Scientist of Mutagenicity Testing Group

2 

## Translator

## 4L435
Submitted to: TOHOTO Kasei Co., Ltd.


# (REPORT]
Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells
(Study**N~** 

## : 4L435)

March 16, 1995
GENERAL TESTING RESEARCH CENTER, JAPAN OILSTUFF INSPECTORS' CORPORATION

3

### 4L435 

# STAT EMEN T
General Testing Research Center. Japan Oi lstuff Inspectors' Corporation

Sponsor TOHOTO Kasei Co
.. Ltd. Title Chromosoma1 Aberration Study of YDC-1312 in Cultured Mammalian Cells Study No. 4L435

The study in this report was conducted in compliance with the following Good Laboratory Practice Standards. rJapanese GLP standards Applied to Industrial Chemicals CKanpogyo No. 39. Yakuhatsu No. 229. 59 Kikyoku No. 85. 1984) J



### rOECD Principles of Good Laboratory Practice (1981)J

Management Osamu Ueno sealed Date: March 16. 1995**--~==~~------~~~** 

### Director of Microbiological Laboratory

4

## 4L435 

# QUALITY ASSURANCE STATEMENT
General Testing Research Center. Japan Oi lstuff Inspectors' Corporation

Sponsor : TOHOTO Kasei Co
.. Ltd. Title Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells Study No. 4L435

By the inspections and audits listed below. we hereby certify that this study was carried out in accordance with its protocol and with our Standard Operating Procedures, and that this report faithfully describes the methods and procedures employed. and that the reported results reflect accurately the raw data of the study.

Inspection Date of inspection Date of reporting to or audit or audit the study director and to the management Study protocol October 25, 1994 October 25. 1994 Study procedure November 7. 1994 November 7. 1994 Study March 16. 1995 March 16. 1995 report Quality Assurance Unit Masayoshi Kida sealed Date: March 16. 1995

5

## 4L435 

# OUTLINE OF THE STUDY
1. 

## Title : Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells (Study No. : 4L435) 2. Purpose :To assess the clastogenicity of the test substance by the chromosomal aberration tests in cultured mammalian cells. 3. Guideline : Japanese Guidelines on Industrial Chemicals(Kanpogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014, 1986) and EEC Guidelines (1992)
. 4. G L P 

## : Japanese GLP Standerds Applied to Industrial Chemicals (Kanpogyo No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, 1984) and OECD GLP (1981) 5. Sponsor :TOHOTO Kasei Co., Ltd. (17-14 Higashikasai 3-chome. Edogawa-ku. Tokyo) (Responsible person) Yoshiaki Nakamura 6. Contractor :Mitsubishi Chemical Safety Institute Ltd. (1-30, Shiba 2-Chome. Minato-ku. Tokyo) 7. Testing Facility : General Testing Research Center. Japan Oilstuff Inspectors' Corporation (10-4. Mikagetsukamachi 1-Chome, Higashinada-ku. Kobe-shi, Hyogo) 8. Management :Osamu Deno 9. Study Director :Eiichi Machigaki 10. Other Contributors :Masato Nakamura 11. Study Date : Onitiation of the Study) October 16. 1994 (Initiation of Cell Growth Inhibition Test) October 28. 1994 (Submission of Chromosomal Aberration Test) February 14. 1995 (Submission of the Final Report) March 16. 1995 12. There was no unforeseeable circumstances that may have ill effects on test results. 13. 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. Further retention will be discussed with the sponsor. 6 

# 4.1.

### 4L435 

# Contents Page Summary
.. ····· .. ··· .. ···· ...................................................... 8
Materials and Methods 1. 

### Test materials
...................................................... 9 

### 2. Indicator cellIine
................................................ 10 3. 

### Cu1ture.media
......................................................... 10 4. S9 

### Mix
................................................................... 10 

### 5. Preparation of test material solutions
..................... 11 6. Cell growth inhibition test .................................... 11 7. Chromosomal aberration test .................................... 13
Resu 1

### ts and Canclusion
................................................ 16
References
................................................................... 16
Tables
.................................................................. 17 Figures
.................................................................. 19



### 7

### 4L435 

# Summary

An 

### in vitro chromosomal aberration study of YDC-1312 was conducted with an establ ished cell line (CHL!IU) from the lungs of a female Chinese hams ter. On the basis of the cell growth inhibition test. the chromosomal aberration test was set at 5000. 2500 and 1200.ug/ml in 24 and 48 hours treatment without metabolic activation and the metabolic activation. In the chromosomal aberration test. test substance did not significantly increaced cells with structural chromosomal and numerical aberration (polyploid cell) wi thand without metabalic activati

### on. From these findings. it was concluded that YDC-1312 has no clastogenic potential in the CHL/IU ce 11 line.

8

## 4L435 

# Materials and Methods 1. 

## Test materials Confidential 

## 1. 1 Test substance

1.2 Preparation of test substance Negative control material (solvent) 1% sodium carboxymethylcellulose solution (l%-CMC-Na. Wako Pure Chemical Laboratory Co
.. Ltd. Lot No. LKE1873) 1.3 Positive material Positive controls Mitomycin C (MMC, Kyowa Hakko Kogyo Co
.. Ltd., Lot No.770AA!' purity 100%) Benzo[a]pyrene (BP. Tokyo Kasei Kogyo Co., Ltd, Lot No. AXOL purity 99.5%)

9

## 4L435 2. Indicator cell line The CHL/IU cell line. originally derived from the lung of a female Chinese**hamste~** 

## was used. The cells were purchased from Dainippon Pharmaceutical Co.• Ltd( prepared on January 21, 1992). Acell suspension was mixed wi th one tenth volume of dimethylsulfoxide (DMSO). then frozen in 1 ml and stored in liquid nitrogen. The cells were used after thawing and cultivation through up to 5 passages. Cells were cultured in plastic dishes (Becton Dickinson and Company; diameter of 6 cm or 10 cm) in an automated CO 2 

## incubator CTabai Espec Corp. model BNA-llIA. 121DA; CO 2 

## 5%. temperature 37°C. humid condition).
3. Culture media 

# a 1 Eagle's minimum essential medium Eagle's minimum essential medium (MEM; Eagle's MEM "NissuiCD", Nissui Pharmaceutical Co., Ltd.) 8.3gwere dissolved in 880 ml of distilled water and autoc1aved at 121°C for 15 min. This solution was supplemented with 8.8ml of sterilized 2.92% L-glutamine solution and 11.4 ml of sterilized 10% sodium bicarbonate**solutio~** 

## 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 (GIBCD BRL; Lot No. 38N3025).
4. S9 mix 4. 1 

## S9 The S9 used. which was a preparation from the liver of male Sprague-Dawley rats treated with phenobarbital (PB; 30, 60 and 60 mg/kg respectively on three successive days, i.

## p.) and 5,6-benzoflavone (80 mg/kg v. p. at the third PB injection), was purchased from Kikkoman Corporation (Lot No. RAA-310. prepared on June 3, 1994). The S9 fraction was stored at below -80°C.

1 0

## 4L435 4.2 S9 mix The following table shows the composition of 1 ml of S9 mix. The S9 mix was freshly prepared for each test and kept on ice until use. S9 0.3 ml MgCl 2 

## C6H2

## 0) 5 fl

## mo 1 

## KCI 33 flmo 

### I 

## D-glucose 6-phosphate 5 fl

## moI {3 

## -NADP+ 4 flmo 

### 1 

## HEPES buffer (pH 7.2) 4 fl

## moI distilled water remaining


## & Preparation of test material solutions 5.1 Solvent selection and preparation of test material solutions The test substance was insoluble in water. soluble more than 5% in DMSO. The test substance was most uniformalY suspended in l%-CMC-Na. Therefore 1%­ CMC-Na was used as the solvent for the test substance. The test substance suspension was diluted with 1%-CMC-Na to the required concentrations. 5.2 Positive control solutions MMC was dissolved in saline (Otsuka Pharmaceutical) to a concentration of 3 flg/ml 

## before use. BP was dissolved in DMSO (Dojin Chemical Laboratory Co
.. Ltd.) to 4 mglm1. frozen. and thawed at the room temperature before use.
6 Cell growth inhibition test A cell growth inhibition test was conducted to determine appropriate concentrations for the chromosomal aberration tests. 6.1 Test substance concentration Seven concentrations of 5000, 2500. 1250. 625. 313, 156 and 78fl 

## g/ml solution were used in the tests.

1 1

### 4L435 6.2 Treatment of cells In a 6 em plastic plate. 5 ml of cell suspension C4x10 3

### /ml) were incubated for 3 days. After removal of the culture medium from each plate. wi thout metabolic activation treatment of cells was performed for 24 or 48 hours using 0.5 ml of each test substance suspension mixed separately with 4.5 ml of a fresh .growth medium. With metabolic activation. treatment of cells with 89 Mix was performed for 6 h using 0.3 ml of each test substance suspension mixed with 0.5 ml of S9 Mix and 2.2 ml of the growth medium. With metabolic -activation. treatment of cells without S9 Mix was performed for 6 h using 0.3 ml of each test substance solution mixed with 2.7 ml of a growth medium. After 6 h incubation, the cells were washed three times wi th MEM. and incubated in 5 ml or a fresh growth medium for a further 18 h. In the negative control group. cells were treated wi th vehicle Cl%-CMC-Na). The test was performed using two plates for each concentration. 6.3 Measurement of surviving cells Cells were washed with Dulbecco' s phosphate buffer solution CPB8(-); Dulbecco's PBS "Nissui", Nissui Pharmaceutical Co., Ltd.), fixed wi th methanol for 15 min., stained wi th 0.1 % 

### crystal violet solution for 10 min., washed with water and dried. Cell density was measured with a cell density counter CMonocellator, Olympus Co., Ltd.). 6.4 Measurement of 50% inhibition concentration of cell growth The ratio of the number of surviving cells in each treatment to the number surviving in its corresponding negative control was calculated, a survival curve drawn and 50% inhibition concentration of cell growth (IC 50) 

### calculated.

1 2

### 4L435 7. Chromosomal aberration test 7.1 Test substance concentration In the cell growth inhibition tests, the test substance did not inhibit cell growth more than 50% at 24 hours without metabolic activation and with metabolic activation, the test substance concentrations resulting in 50% inhibition were 3154t1g/ml at 48 

### hours without metabolic activation. On the basis of the cell growth inhibition tests. the highest concentration in the chromosomal aberration test was set at 5000,ug/ml 24 and 48 

### hours

, .' 

### without metabolic activation. and with metabolic activation. In positive controls. final concentrations of MMC and BP were set at 0.03 and 20,lig/ml. respectively. which are the concentration levels known to induce chromosomal aberration. 7.2Treatment of cells The cultured cells were treated with the test substance using the same procedure as described in section 6.2. As positive controls. cells were treated with a mixture of 0.05 ml of MMC solution and 5 ml of**th~** 

### growth medium without metabolic activation. with a mixture of 2.5 ml of the growth medium. 0.5 ml 89 Mix and 0.015 ml of BP solution wi th metabol ic activation with S9 Mix. and with a mixture of 3 ml of the growth medium and 15,li1 of BP solution with metabolic activation without S9 Mix. 7.3 Preparation of specimens In preparation of specimens. at 2 h before the end of treatment, colcemid was added to the medium in each plate to a final concentration of 0.1 ,lig/ml to arrest cells in metaphase. The cells were then washed with PBS(-). dissociated with 0.25% trypsin solution. and centrifuged ClOOO rpm, 5 min.; same below) for collection. After removal of the supernatant. 4 ml of 0.075 M potassium chloride solution was added for hYPotonic treatment (37°C. 15 min.).

1 3

### 4L435 

## The cells were then fixed in 4 

## ml of cold methanol/acetic acid mixture (3/1. v/v). AfteFcentrifugation, the supernatant was removed and 4 ml of a fresh fixative added. This fixation procedure was repeated 3 to 4 times. Thereafter, the cells were suspended 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 cells were stained for 20 min. with 3% Giemsa solution diluted with 1/15 Mphosphate 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. 7.4 Observation (1) 

## Structural aberration and numerical aberration Each specimen was coded, 100 metaphase cells for each plate (Le. 200 cells for each concentration) were observed in a blind manner. Metaphase cells with well-spread chromosomes were selected and**obs~rved** 

## for structural aberrations. Cells without aberration but not having 25 2 chromosomes were omitted from the survey. Structural aberrations were classified as follows ll : 

## gaps (g. including chromatid and chromosome type lesions) chromatid breaks (ctb) 

# f

chromatid exchanges (cte) chromosome breaks (csb) chromosome exchanges (cse, dicentric. ring, etc.) 

# 1

other aberrations (frg) A "gap" was distinguished from a "break" as follows. The "gap" is an achromatic region in a single chromatid which is equal to, or slightly larger than, the width of the chromatid, whose shape is clearly defined. (2) 

## Numerical aberration (polyploid cell) Numerical aberration were observed and polyploid cells, including

.endoredupl icated cells, were scored.

1 4

### 4L435 

## 7.5 Judgement " Acell having at least one structural chromosomal aberration was classified as an aberrant cell. The number of aberrant cells was counted in two ways. that is incru ding eel Is wi t h no aberration other than gaps (+

## gap) , and excluding such cells (-gap). The chromosomal aberration potential of the test substance was judged as follows.

The number of aberrant cells was statistically compared with that of the control by the 

# x 2

## -test. The number of polyploid cells was also analyzed in the same manner.

The test substance was judged to induce chromosomal aberration when the number of aberrant cells is significantly different from tllat of "the solvent control and any of the following is observed: 

# CD Significance at P 

## <0.001 

## GD Obvious dose-dependent increase @ 

## Reproducible increase at the significant concentration 7.6 Collect of data Each of the sums and the incidence (I) 

## of the chromosome aberration and numerical aberrations was shown. The chromosomeal aberration was shown dividing the number of cells in the type. When the test was positive. the 

## "D 20 " 

## values were calculated from result. The "D 20 " 

## value is the concentration (mg/mD required to induce any aberration in 20% of metaphases. The microscopic photograph of a typical chromosomal aberration is appended.

1:"":0 

### 1 5 

## v

## 4L435 

# Results and Conclusion In the cell growth inhibition tests, the test substance did not inhibit cell growth more than 50% at 24 hours without metabolic activation and with metabolic activation. the test substance concentrations resulting in 50% inhibition were 3154,ug/ml at 48 hours without metabolic activation (Figure 1. 2).



## On the basis of the cell growth inhibition tests. the chromosomal aberration test was set at 5000. 2500 and 1250 ,ug/ml 24 and 48 hours without metabolic aritivation and with metabolic**activatio~** 

## In the chromosomal aberration test. the test substance did not significantly increased cells with structural chromosomal aberrations and numerical aberration (polyploid cell) with and without metabolic activation. In the negative control. the incidences of aberrant cells were low. and in the positive controls, aberrant cells were increased significantly (Table L 

## 2 and Figure 3, 4). The sponser confirmed that the lot of the test substance used in this study was stable during testing period. From these findings, it was concluded that YDC-1312 does not have clastogenic potential in this CHL/IU cell Iine system.


# Reference 1) Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of

Japan: Atlas of chromosomal aberrations induced by chemicals, Asakura Press, TokYO, 1988. 2) OECD guideline for testing of chemicals, Section 4 Health effects» GENETIC TOXICICOLOG" 473, (997).

1 6 r' t...' 

### 1TABLE 1 RESULTS OF THE CHROMOSOMAL ABERRATION TEST (WITHOUT METABOUC ACnVATION) Test substance: YDC-1312

I--' --J



# ("" C.,) Treatment Exposure Dose No. of Polyploids No. of cells with chromosomal structural aberrations time levels cells Judge- Gaps Chromatid type* Chromosomal type* Others* Total group (h) (J.l. g/mO al1alysed (%) ment** g ctb ' cte csb 

# loo 0 0 0 0 Negative 24 0 100 0 / 0 0 0 control 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) (l%-CMC-Na) 100 0 0 0 0 48 0 100 0 / 

### 0 0 0 200 0(0.0) 0(0.0) OCO.O) 0(0.0) 0(0.0) 100 0 0 0 0 24 1250 100 0 

# - 0 0 0 

## t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 100 0 0 0 0 24 2500 100 0 

# - 0 1 0 

## t 200 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 100 0 0 1 0 24 5000 100 0 

# - 0 0 0 Test 

## t 200 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) substance 100 0 0 0 O. 48 1250 100 0 

# - 0 0 0 

## t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) HO.5) 100 0 0 0 0 48 2500 100 0 

# - 0 0 0 

## t 200 O{O.O) 0(0.0) 0(0.0) 0(0.0) 0<0.0) toO 0 1 1 0 48 5000 100 0 

## - 0 0 0 

## t 200 0(0.0) 1(0.5) 1(0.5) 0(0.0) O(O.O} 100 0 0 9 19 Positive 24 0.03 100 0 

# - 2 10 15 control 200 0(0.0) 2(1.0) 19(9.5) 34(17.0) 0(0.0) 100 0 2 10 26 (MMC) 48 0.03 100 0 

# - 1 12 26 200 0(0.0)

. 3(1.5)_**~2(t** 1.0)**~(~6.0)** 3(1.5)
. * ctb: chromatid break. cte : chromatid exchange. csb : chromosome break. cse : chromosome exchange.

others: fragmentation(except pulverization) ** X2-test : significantly different from negative control: +++(p(O.OOl) l%-CMC-Na: 1% sodium carboxymethylcellulose solution. MMC : mitomycin C 

### t : undissolved test substance was observed in medium at cell tratment time. cse -g +g 0 0 0 0 0 0 0 0 0 0

0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 0 0 0 0 0 0 0 0 0

0(0.0) 0(0.0) 0(0.0) 0(0.0) () 0 0 0 0 0 0 0 0 0

0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 0 0 0 0 0 0 0 1 1

0(0.0) 0(0.0) 1(0.5) 1(0.5) 0 0 0 1 1 0 0 0 0 0

0(0.0)
. 0(0.0) 1(0.5) 1(0.5)**.~** 0 .1

0 0 0 0 1 0 0 1 1

0(0.0) 0(0.0) 1(0.5) 1(0.5) 0 0 0 0 0 0 0 0 0 0

0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 0 0 1 2 0 0 0 0 0

0(0.0) 0(0.0) 1(0.5) 2(1.0) 0 0 0 27 27 0 0 0 25 27

0(0.0) 0(0.0) 52(26.0) 54(27.0) 0 1 1 34 

### 36 3 0 0 37 38 1(0.5)_**~(0.5)** 71(35.§)_]1(37.0) Judgement**J I /
/



# -



# -

-



# -
i



## -



# -
+++

+++

,.r.:.. r:­ ,.r.:.. c...:> c..nTABLE 2 RESULTS OF TH IHROMOSOMAL ABERRATION TEST (WITH ME". IOUC ACTIVATION) Test substance: YDC-1312

I-' co



## rn Treatment With or Dose No. of Polyploids No. of cells with chromosomal structural aberrations without levels cells Judge- Gaps Chromatid tvpe* Chromosomal type* group S9 Mix (11 g/mD analysed (%) ment** g ctb cte csb 100 0 0 

### 0 0 Negative 

### - 0 100 0 / 1 0 1 control 200 0(0.0) 1(0.5) 0(0.0) 1(0.5) 0(0.0) (l%-CMC-Na) 100 1 0 0 1 . 

## + 0 100 0 / 1 , 0 0 200 1(0.5) 1(0.5) 0(0.0) 1(0.5) 0(0.0) 100 0 0 1 0

- 1250 100 0 

### - 1 0 1 

## t 200 0(0.0) 1(0.5) 1(0.5) 1(0.5) 0(0.0) 100 0 1 1 0



### - 2500 100 0 

# - 0 0 0 

## t 200 0(0.0) 1(0.5) 1(0.5) 0(0.0) 0(0.0) 100 0 0 0 0



### - 5000 100 0 

# - 0 0 1 • Test 

## t 200 0(0:0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) substance 100 0 

### 0 0 

### 0 

## + 1250 100 0 

# - 0 0 0 

## t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 100 0 1 0 1 

## + 2500 100 0 

### - 0 0 0 0 I



## t 200 0(0.0) 1(0.5) 0(0.0) 1(0.5) 0(0.0) 100 0 0 0 0 

## + 5000 100 0 

# - 0 0 0 

## t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 100 1 0 0 0 Positive 

### - 20 100 0 

### - 1 0 0 control 200 1(0.5) 1(0.5) 0(0.0) 0(0.0) 0(0.0) 100 0 1 5 29 (BP) 

## + 20 100 0 

# - 1 2 30 200 0(0.0)

. 2(1.0) 7(3.5) 59(28.5) 4(2.0)_ * ctb: chromatid break. cte : chromatid exchange. csb : chromosome break. cse : chromosome exchange.

others: fragmentation(except pulverization) ** 

### X2-test : significantly different from negative control; +++(p(O.OOl) Cells were treated with S9 (5% of final conc.) or without S9 for 6 hours and recovered for 18 hours. 1%-CMC-Na : 1" sodium carboxymethylcellulose solution. BP : benzo(a)pyrene 

### t : undissolved test substance was observed in medium at cell tratment time. cse 0 0 0 0

0(0.0) 0 0 0 0

0(0.0) 0: 0 0 0

0(0.0) 0 0 0 0

0(0.0) 0 0 0 0(0.0) 0 0 0 0

0(0.0) 0 0 0 0

0(0.0) 0 0 0 0

0(0.0) 0 0 0 0

0(0.0) 2 1 2 3

4(2.0) Others* Total -g +g 0 0 0 0 1 2 0(0.0) 1(0.5) 2(1.0) 0 1 1 0 0 1 0(0.0) 1(0.5)
. 2(1.0) 0 1 1 0 1 2 0(0.0) 2(LO} 3(1.5) 0 1 2 0 0 0 0(0.0) 1(0.5) 2(1.0) 0 0 0 0 1 1 0(0.0) 1(0.5) 1(0,5) 0 0 0 0 0 0 0(0.0) 0(0.0) 0(0.0) 0 1 2 0 0 0 0(0.0) 1(0.5) 2(1.0) 0 0 0 0 0 0 0(0.0) 0(0.0) 0(0.0) 0 0 0 0 0 1 0(0.0) 0(0.0) 1(0.5) 0 

### 36 37 0 38 38 0(0.0) 74(37.0) 75(3].5) Judgement** /
/



# -

-

-



# -



# -



# -

....
+++

.- ,j:::..**~** 

### Li ,j:::.. v.:> en

### 4L435



### • 24hours treatment - 0 - 48hours treatment 

### 100

......... '-' 

### "0


# .. 

# *

.. >< 

# .. (1) 

# .. "'0 

# .. J:: 

# .. -.-4 

# .. ...c:: 

### .. 0
.. -+"' 

# .. 

# ..**~** 0 

### 50 

# .. 

# .. .. Io-! 

# .. .. 

# .. 

# .. bO 

# .. 

# .. .. r-1 r-1 (1) U


# o o 1000 2000 3000 4000 5000 Concentration (J.L 

### g/ml) Fig. 1 Cell growth inhbition treated with YDC-1312 (Without metabolic activation)

_With S9 mix 

### 100

50



# o o 1000 2000 3000 4000 Concentration (J.L 

### g/mD Fig.2 Cell growth inhibition treated with YDC-1312 (With metabolic activation) 

### 5000

1 9 1:""7 \.: '1

### 4L435 10.0

8.0



# - 6.0



# - '*

Q) u Q) =: 

### 4.0 "0 U =: 

### 2.0
0.0

10.0

8.0 

# -.

- '* 6.0 Q) u =: Q) "0 U =: 

### 4.0
2.0
0.0 

## r 

# • 24hours treatment

- 0- 

### 48hours treatment



### .. 0­ .­ 

# ......**~** 

# - 

# .. 

# -. .. .. 

# - .. 

# .. .. J' 

# .. 

# .. 

# .. ..;;. 

# .. .. 

# .. 

# - o 1250 2500 5000 Concentration (tlg/ml) Fig.3 Incedence of aberant cell treated with YDS-1312 (without metabolic activation)



# - 0- 

### Without S9mix 

# • With S9mix



# .--0-_.


# . ... 

# - - 

# - ­ .. 

# - 

# - - .. -**-~** 

# .....

# -­ o 1250 2500 5000 Concentration (Jl 

### g/mD Fig.4 Incidence of aberrant cell treated with YDC-1312 (with metabolic activation)



## 2 0

## 00'00,56

# --

# 000058

### Acute Oral Toxicity TestofYDC-1312 in Rats (English version)
Study No. 4L437 StudyTitle Acute OralToxicity TestofYDC-] 3] 2 in Rats Sponsor Tohto Kasei CO" LTD
This test was conducted in Kashima Laboratory. Mitsubishi Chemical Safety Inst.itute Ltd
.. 14 Sunayama. Hasaki-machi. Kashima-gun. Ibaragi. Japan.

This reportis the English version ofthe original. which was written in Japanese. The undersigned hereby dpciare that. thisversion faithfully reflects theoriginal to the best.of our knowledge.

Translated by
Takayoshi Hosono Manager for Technical Division Tokyo Head Office, Tohto Kasei CO., LTD

Approved by

I**~** , 

### . I I 

## =- .. r 7
__ --.)..:..' / 

### Dale: I 

### Koyo Murakami n(meral Manager for**1\~ehnieal** 

### Division Tokyo Head Office. 'I'ohlo KaHPi 

### CO
.. LTD



## 000.059

### COMPLIANCE WITH THE GLP STANDARDS
Study No. 4L437 StudyTitle Acut.e OralToxicity Test ofYDC-1312 in Rat8 Sponsor Toht.o Kasei CO., LTD

The study described in this report was conduct.ed in compliance with the Good Laborat.ory Pract.ice regulations recognized by the OECD GLP (1981).



### Facility Management. Sakae Koike Sealed date: March 14, 1995 Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

### oooo~;o

### QUALITY ASSURANCE STATEMENT Study No. 4L487 StudyTitle Acute Oral Toxicity Test ofYDC-l:312 in Rat.s Sponsor Tohto Kasei CO
.. LTD We hereby cert.ify that. the above Rl.udy has been performed in accordance with the Gooel Laboratory Pract.ice (GLP) regulat.ions recognized by the OECD GLP (1981).
Dates of inspect.ions and reportings to the Study Director and the Facility Managemenl are as rollows :
In-progress study Final report Date of Inspection November 15. 1994 January 11. 

### 1995 March 14, 1995 Date Reporting November 16. 1994 January 1:3, 

### 1995 March 14. 1995

Qualit.yAssurance Personnel Kunihiko Ofuchi Mit.subishi Chemical Safet.y Institute Ltd. (M.s.l.) Sealed date: March 14, 1995

### STUDY PERSONNEL St.udy Personnel S1udy Director: Masato Sudo Sealed Mayumi Kitami Sealed



# OOOOG2

### CONTENTS

SYNOPSIS TEST SUBSTANCE AND ITS EVALUATION METHOD 1 

### TEST SUBSTANCE 2 EXPERIMENTAL ANIMALS 

# a ANIMAL MANAGEMENT 4 PREPARATION OF DOSAGE 5 DOSE LEVEL G 

### METHOD OFADMINISTRATION 7 CLiNICAL OBSRVATIONS OF RATS 8 MEASURING BODY WEIGHTS OF RATS 9 NECROPSY

RESULTS AND CONCLUSION 1 NUMBER OF DEAD RATS 2 CLINICAL OBSERVATION :3 

### BODY WEIGHT 4 NECROPSY 5 CONCLUSION TABLE 1 

### INDIVIDUAL BODY WEIGHTS Page 

### G
7 

### 7 7 

### 8 8 8 8 9 9 9

10 10 10 

### 10 10 10
11



## OOOOf;3

### SYNOPSIS (1) 

### YOC-IaI2 was dosed orally once to SO strain rals(SPF) ofmales and females with a dose of2000mglkg and observations were made on the mort.ality and toxicological effect.s. (2) No animalR died during 14 days test period. (a) No abnormalities were**obSt~rved** 

### in any animals up to the end ofobservation. (4) Body weightsofall animals increased normally. (5) At necropsy. no nhnormalil.i0s were found in any animals. (6) YOC-la12 gave no toxicological effecls to 

### the rats by oral administ.ral.ion, and the lowestlethal dose ofYDC-l:312 was greater than 2000mg/kg.

( i 

# OOOOG4Test subst.ance and its evaluation method 1. Test subst.ance

## ---------------------~--------~---

2, Experimental animals Five males and females SD strain(Crj:CD) rats(SPF) were purchased from Charles River ,Japan Inc, on November 9, 1994. Aft.er quarantine and acclimatization for 6 days. it wasconfirmed that the rats were in goot! health condition. ancl the age ofanimals at administrat.ion was 5 week old. The rats were marked in their tails with oil base marker. so that. they were able to 

### IlP identified individually. TIlt' cages which kepI the ahove animals were identified with a lahellisting stu(ly number. name or thl' test substance. animal numbers. sex, an(1 dose level. Their body weights at t.he first administration ranged from I:3G to 149 g in maiN;; and**I()~** to 122 g in r('males,



### 7 

## 0000';5

### :3 Animal management :3.1 Housingcondit.ions Throughout. the study period. including t.he quarantine and acclimatization periods. t.he animal room was maint.ained at a temperature between 20·C and 25·C, and a relat.ive humidity betwe.en 40% and 70% with 12 fresh air ('hangesper hour and was lit for 12 hours per day (7:00-19:00). The animals were housed in polycarbonate cages(265WX 4260 X 200Hmm, Tokiwa Kagaku Kikai Co
..Lt.d.) wit.h hard wood chip bedding for experiment.al animals (Beta.Chip. Charles River Japan Inc.). Five rats were housed in each cage. The cages were placed on a steel rack(4-tiered. Tokiwa Kagaku Kikai 

### Co
..Ltd.). Stainless steel feeders for pelletdiet(rokiwa Kagaku Kikai 

### Co
..Ltd.) and polycarbonate watering bott.los(700mL. Tokiwa Kagaku Kikai Co
..Lt.d.) were m;ed. The cages (including bedding). feeders. and watering bottles were autoclaved and replaced once a week. :3.2 Diet Pellte diet for experimental animals(MF. OrientalYeast Co
..Ltd.) was given 

### adlibitum and was renewed once a week. Chemical analysis oft.he diet for contaminants such as pesticide residue certified that their concentrations were lower than the limitvalues determined in our Standard Operat.ion Procedure. 3.3 Drinking water Ta» water was given 

### adlibitum and was renewed once a week. The water was filtrated through a 5-j.1 m filter and irradiated by UV. The wat.er was analyzed periodically in accordance with the Waterworks Law ofJapan, and wascert.ified to meet the required standard. 4 Preparat.ion ofdosage YDC-l:312 which has been grounded with a mortar was dispersed in 0.5% aqueous CMC·Na solution to be an emulsion with a super high Hpeed homogenizer (Polyt.ron: Central Science TradingCo
.. Ltd.). The emulsion waH Hlljust.ed to he a**s(~lected** concent.ration and t.hen dosed. 5 OOl-:'e level Preliminary I.est.s wern carried out for two male rats with a dose of2000mg/kg rat. The results indicat.ed t.hat there wnre no ahnormal obst'rvations and**Hubl-;(~qlH'nt.ly** further**h~l-:'t** with a dose of 2000mg/kg rat which is the upper limit df'f'inpd in the guideline was conducted for fivn mall' nIls and five f<'maln oneK The clost' lewl waH t'quivalpnt to 1.0 

### milhody wl'ighl. WOg. () M<,thod 

# or administration All ralH wert'**faHIt~d h('for<~**  dOHe for ovprniJ.{ht. Oral dosage was given 10 their Hlom:whjo.; by usinJ,!; a stomneh tuhe. Tlw diet was wil.hlwld for thr('(' hoursaft,('r administ.ration.


## 000 0.;r' <-.**~)".)·o**

### 7 Clinical observationsofrats Clinical observat.ions ofeach rat. to be dosed were made 15. :30 min. and 1. :3, 

### and 6 hours later in the day when t.he dosage was given to the animal and subsequently once a day during 14 days. 8 Measuringbody weights ofrats Body weights ofall rat.s were measured by use ofelect.ronic balances (ES·5000. EB· 6200S Shimazu Corp.) in the int.ervalsjustbefore the dosage and on 

### 8. 7. and 14 days after administrat.ion. The day ofadminist.ration was designated as "0 day". 9 Necropsy At 14 dny oftest.. all rat.s were anest.hetized with a thiopent.al sodium anesthesia(Ravonal.Tanabe Seiyaku Co

.. 

### Ltd.) and t.hey were euthanized by severing t.he abdominal aorta and then subjected to necropsy.

!)

### Results and Conclusion (1) 

### No animals died during 14 days test period. (2) No abnormalities were observed in any animals up to the end orobservation. (3) Body weight.s ofall animals incrcaS0d normally. (4) At. necropsy. no abnormalities were found 

### in any animals. (5) YDC-l:312 gave no t.oxicological effect.s to the rats by oral administration. and the

.lowest lethal dose ofYOC·]:312 was greater than 2000mg/kg.

10 

## OOOGf;S

## (Study No. : 4L437)
Table 1 Individual body weights unit g Sex : Days after administration Dose level Animal Gain emg/kg) No. 0 3 7 14 (Od--14d) 

## Male : 1 135 184 223 300 165 2 138 176 215 278 140 2000 3 138 180 217 279 141 4 149 197 243 316 167 5 135 175 212 284 149 mean 139 182 222 291 152 S. D. 

## 5.8 8.9 12.4 16.3 12.9

N 

### 5 5 5 5 5 

## Fellale 1 109 139 158 192 83 2 113 152 181 213 100 2000 3 111 144 160 200 89 4 122 157 183 215 93 5 112 134 156 190 78 mean 113 145 168 202 89 S. D. 

## 5.0 9.4 13.2 11.6 8.6

N 

## 5 5 5 5 5

II 

# OOOOG9BOI0234 

## Translation Statement

Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.
Sponsor: Tohto Kasei Co., Ltd. Title: Twenty-Eight-Day Repeated Dose OralToxicity Study ofYDC-1312 in Rats Study number: 4lA36

This study was conducted in Kashima Laboratory ofMitsubishi Chemical Safety Institute Ltd. The original final report was written in Japanese. I hereby declare that this report faithfully reflects the original final report as accurately as possible.

Translator: 

### Date:

# t8r:J 1, '}.-f'¢

# l Kaname Takahashi Senior Research Scientist Toxicology Division ofKashima Laboratory Mitsubishi Chemical Safety Institute Ltd.4L436

Submitted to: Tohto Kasei Co., Ltd.



# FINAL REPORT
Twenty-Eight-Day Repeated Dose Oral Toxicity Study of YDC-1312 in Rats

(Study No. 4L436)

March 14, 1995

Mitsubishi Chemical Safety Institute Ltd.



## 0000 .14L436 

# Statement

Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.
Sponsor: TohlO Kasei Co., Ltd. Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study ofYDC-1312in Rats Study number: 4IA36

This study was conducted in compliance with the Good Laboratory Practice Standards applied to industrial chemicals ofJapan.

Management: Sakae Koike sealed Date: March 14, 1995



### 2 

## 0000'724L436 

## Quality Assurance Statement

Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.
Sponsor: Tohto Kasei Co., Ltd. Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study of YDC-1312 in Rats Study number: 4LA36
By the inspections and audits listed below, I hereby certify that this study was carried out in accordance with its protocol and the Standard Operating Procedures, that this report faithfully describes the methods and procedures employed, and that the reported results accurately reflect the
raw data ofthe study. Inspection or audit Date ofinspection oraudit Date ofreporting to the study director and management Study procedure

Study report October 13, 1994 October 21, 1994 November 8, 1994 November 11, 1994 February 16, 1995 March 14, 1995 October 13, 1994 October 21, 1994 November 8, 1994 November 11, 1994 February 22, 1995 March 14, 1995 Quality Assurance Manager: Kunihiko Ofuchi sealed Date: March 14, 1995

3 

## 0000'734L436 

## Study Outline

L Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study ofYDC-1312 in Rats (Study No. 4L436)

2. Purpose: The purpose ofthis study is to determine the toxicity ofthe test substance by observation ofthe changes in function and morphology in rats that received repeated oral administration ofYDC-1312 for 28 days.

3. Guideline: Japanese Guidelines on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.l039, 61 Kikyoku No. 1014, 1986)



### 4. GLP: Japanese GLP on Industrial Chemicals (Kanpogyo No.39. Yakuhatsu No. 229. 59 Kikyoku No.85, 1984)

5. Sponsor: (Name) Tohto Kasei Co., Ltd. (Address) 17-14, Higashikasai 3-chome, Edogawa-ku, Tokyo, Japan (Sponsor representative) Yoshiaki Nakamura

6. Organization under contract: (Name) Mitsubishi Chemical Safety Institute Ltd. (The company changed its name on October 1, 1994) (Address ofhead office) 1-30, Shiba 2-chome, Minato-ku, Tokyo, Japan (Testing facility) Kashima Laboratory (Address oftesting facility) 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, Japan

4 

## 0000'74:4L436

7. Study dates: (Initiation ofthe study) September 8, 1994 (Receipt oftest animals) October 5, 1994 (Start ofadministration) October 14,1994 (Necropsy at the end ofthe dosing period) November 11, 1994 (Necropsy at the end ofthe recovery period) November 25, 1994 (Issue offinal report) March 14, 1995



### 8. Study personnel: Study director Masato Sudo sealed March 14, 1995 Study contributor Mayumi Kitami sealed March 14, 1995 Pathologists Minoru Tsuchitani sealed March 14, 1995 Yoshimasa Okazaki sealed March 14, 1995 Clinical chemistry Naoto Toyota sealed March 14, 1995 Katsuyo Takano sealed March 14, 1995

9. Unforeseeable circumstances that may have adverse effects on the reliability ofthe study: None.

10. Retention: After the submission ofthe final report, all records and documents concerning to this study will be retained for 10 years in the archives ofthe Kashima Laboratory. Further retention will be discussed with the sponsor.



### 5 

## 0000754L436 

# Contents
Summary
.............................................................................................................................................8 Materials and Methods
........................................................................................................................

### 9 1. Test substance
.....................................................................................................................9 

### 2. Experimental animals
..........................................................................................................

### 9 3. AniInaI management ...........................................................................................................9 4. Administration
.................................................................................................................. 10 

### 5. Preparation ofthe dosing solution
..................................................................................... 11 6. Recovery period
................................................................................................................ 11 7. Constitution oftreatment groups....................................................................................... 12 

### 8. Clinical signs
..................................................................................................................... 12 

### 9. Body weight
...................................................................................................................... 12 10. Food consumption
............................................................................................................. 12 11. HeInatological examination
.............................................................................................. 12 12. Blood chemistry examination
........................................................................................... 13 13. Urinalysis
.......................................................................................................................... 14 14. Pathological examination
.................................................................................................. 14 2.15 Statistical analysis
............................................................................................................. 15 Results
............................................................................................................................................... 16 1. Dead animals
..................................................................................................................... 16 2. Clinical signs
..................................................................................................................... 16 3. Body weight
...................................................................................................................... 16 4. Food consumption
............................................................................................................. 16 5. Hell1atological examination
.............................................................................................. 16 6. Blood chemistry exatnination
........................................................................................... 16 7. Urinalysis
.......................................................................................................................... 17 8. Organ weight
..................................................................................................................... 17 9. Necropsy
........................................................................................................................... 17 10. Histopalhologicall·xaminalioJl
....................... .................................................... 18 6 

## 0000'764L436

Discussion
......................................................................................................................................... 19 References
.........................................................................................................................................20
Figure and Tables Figure Table Table 2 Table 3 Table 4,5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 

## " Appendices Appendix Appendix 2 Appendix 3 Appendix 4,5 Appendix 

### 6 Appendix 7 Appendix 8 Appendix 9 Appendix 10 Appendix II
PholOgraphs Body weight changes in rats treated orally with YDC-1312 for 28 days
....... I Incidence ofclinical signs
.............................................................................. 3 Body weight

- group mean values
.................................................................. 7 Food consumption

- group mean values
......................................................... 9 Hematology

- group mean values
................................................................. 11 Clinical chemistry

- group mean values
....................................................... 19 Urinalysis

- group mean values
.................................................................... 27 Organ weight (absolute)

- group mean values
.............................................. 29 Organ weight (relative: percentage ofbody weight)

- group mean values
... 33 Incidence ofnecropsy findings
.................................................................... 37 Incidence ofhistological findings
................................................................ 38

Incidence ofclinical signs
.............................................................................. 1 Body weight

- individual values
..................................................................... 9 Food consumption

- individual values
......................................................... 17 Hematology

- individual values
................................................................... 25 Clinical chemistry

- individual values
.......................................................... 49 Urinalysis

- individual values
....................................................................... 73 Organ weight (absolute) individual values
................................................ 81 Organ weight (relative: percentage ofbody weight)

- individual values
..... 93 Individual necropsy findings
...................................................................... 105 Individual histological findings
.................................................................. 108



### 7 

## onon··"'i'·..., U I uU I4L436 

# Summary
(1) YDC-1312 was administered orally to male and female SD strain rats for 28 days. A recovery period of 14 days was also arranged for the control and 1000 mglkg groups. (2) Thetest substance was suspended in a 0.5% sodium carboxymethylcellulose solution, and administered to the rats with a stomach tube. The dose levels were 20, 140, and 1000 mglkg, and the control group was administered the vehicle only. The dosing volume was set at 1.0 mlJloog body weight in each group. (3) No death was observed throughout the study period. (4) No abnormalities were observed in clinical signs, body weight, food consumption, hematological examination, or urinalysis. (5) Inthe blood chemistry examination, significantly increased albumin was observed in the males ofthe 1000 mglkg group. (6) In the pathological examination at the end ofthe dosing period, significantly increased weight (relative weight) ofthe ovaries was observed in the females ofthe 1000 mglkg group. An increase in hyaline droplets in the proximal tubular epithelium in the kidneys was observed in the males ofthe 1000 mglkg group. After the end ofthe recovery period, significantly increased weight (absolute weight) ofthe kidneys was observed in males ofthe 1000 mglkg group. Basophilic change ofthe tubular epithelium accompanied with hyaline casts in the kidneys was also observed; however, incidence ofthe increase in hyaline droplets in the proximal tubular epithelium decreased. (7) Fromthe above results, it was estimated that the no observed effect level of YDC-1312 under these study conditions was 140 mglkg in both sexes.

8 

# 0000784L436 

# Materials and Methods

I. Test substance

# ~--~---------------~ 2. Experimental animals Thirty-six male and female SD strain (Crj:CD) rats (SPF) were purchased from Charles River Japan Inc. on October 5, 1994. After quarantine and acclimatization for 9 days, it was confirmed that the rats were in good general condition, and the administration was started at 5 weeks old. The rats were grouped bythe stratified-by-weight randomization method one-day before the first administration. The animals were identified by the earpunch method and each cage was identified with a label listing study number, name ofthe test substance, animal numbers, sex, and dose level. Theirbody weights at the first administration ranged from 171 to 198 g in males and 133 to 167 g in females. It was confirmed that the individual body weight was within mean body weight ± 20%

3. Alimal management 3.1 Housing conditions Throughout the study period, including the quarantine and acclimatization periods, the animal
room was maintained at a temperature between 20· and 

### 2ye. and a relative humidity between 9 

## 0000'794L436
40% and 70% with 12 fresh air changes per hour and was lit for 12 hours per day (7:00-19:00). The animals were housed in polycarbonate cages (265W x 4260 x 200H mm, TR-PC-2oo: Tokiwa Kagaku Kikai Co., Ltd.) with hard wood chip bedding for experimental animals (BetaChip, Charles River Japan Inc.). Two rats ofthe same sex were housed in each cage. The cages were placed on a steel rack (4-tiered, Tokiwa Kagab Kikai Co., Ltd.). Stainless steel feeders for pellet diet (Tokiwa Kagaku Kikai Co., Ltd.) and polycarbonate watering bottles (700 

### mL, T-564: Tokiwa Kagaku Kikai Co., Ltd.) were used. Thecages (including bedding), feeders, and watering bottles were autoclaved and replaced
once a week. 3.2 Diet Pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.) was given adlibitum and
was renewed once a week. Chemical analysis ofthe diet for contaminants such as pesticide residue certified that their concentrations were lower than the limit values determined in our Standard Operation Procedure. 3.3 Drinking water Tap water was given adlibitum and was renewed once a week. The water was filtered through
a 5-J.lm filter and irradiated by UV. The water was analyzed periodically in accordance with theWaterworks Law ofJapan, and was certified to meet the required standard.

4. Administration 4.1 Dose levels and basis for dose setting No abnormalities were found as the results ofa 7-day repeated dose preliminary study conducted at dose levels of500 and 1000 mg/kg. From the results ofthis preliminary study. thehigh dose level was determined as 1000 mg/kg, which was the highest dose described in the guideline, and the middle and low dose levels were set as 140 and 20 mg/kg. respectively. The control group was also arranged. The control group was received the vehicle only.

10 

# 0000004L436

4.2 Dosing period The administration was performed for 28 days, once a day in the morning. The start ofthe administration was designated as 

### "0 day after commencement ofthe treatment" in this report. 4.3 Method of administration The dosing solution was administered into the stomach by a syringe with a stomach tube. 4.4 Dosing volume Dosing volume was set at 1.0 mUlOO g body weight. IndiVidual dosing volume was calculated by the most recent weighed individual body weight.

5. Preparationofthe dosing solution An appropriate amount ofthe test substance was weighed and ground in a mortar. The dosing solutions wereprepared by suspending the test substance in a 

### 0.5% carboxymethylceUulose sodiumaqueous solution. Thepreparation was performed once a week and the dosing solutions were kept inbrownbottles and the bottles were stored in a refrigerator protected from light untiljust before dosing. The stability ofthe test substance in the dosing solution stored in a refrigerator for 

### 8 days was confirmed by analysis ofthe dosing solutions. (Unit: mg/mL)

# r~ oday 

# , 8 day • Concentration 2 1.86 1.78
100 95.4 

# I 101 i

6. Recovery period A recovery period of 14 days was arranged in the control and 1000 mg/kg groups after the dosing period.

II4L436

7. Constirution oftreatment groups Constitution oftreatment groups is described below. Group name Dose level (mglkg) Male Control group 0 6 6 Low dose group 20 6 6 6 Middledose group 140

High dose group 1000 6 6 6 6 Total number 24 24 12 12 a Animals were necropsied on the day after the last administration. b Animals were necropsied after the recovery period of 14 days.
8. Clinical signs Observations ofall rats on their fate, appearance, and behavior were made once a day. More detailed observations including palpation were also made oncea week.

9. Body weight All rats were weighed onthe day ofthe ftrst administration and once a weekthereafter with an electronic balance (EB-5000, Shimadzu Corp.).

10. Foodconsumption All feeders including food was weighed onthe day ofthe first administration and once a week thereafter with an electronic balance (EB-5000, Shimadzu Corp.). Daily consumption per rat
was then calculated.

11. Hematological examination Hematological examinations were performed at each scheduled sacrifice. Blood samples were obtained from the inferior vena cava ofthe rat without fasting under intraperitoneal thiopental sodium anesthesia (Ravonal®, Tanabe Seiyak.'U Co., Ltd.). The items listed below were measured using the blood collected. As the anti-coagulant, 3.13 %sodium citrate aqueous solution was used for the measurement ofthe prothrombin time and activated partial thromboplastin time, and EDTA-2K was used for the other measurements. Items 1 7 and 10 -12 of one male in the 140 mglkg group (MeOOS) could not be measured because ofclotting.
12

### OOOO~i24L436

Items 8 and 9 ofone female in the 140 mglkg group (FCOO 1) also could not be measured because ofclotting.

Items Measurement/calculation 1 Erythrocyte count Sheath flow DC impedance detection method 2 Leukocyte count RFIDC impedance detection method 3 Platelet count Sheath flow DC impedance detection method 4 Hemoglobin concentration SLS hemoglobin method 5 Hematocrit Cumulative pulse height detection method 6 Differential leukocyte count Microscopic examination after Wright's staining 7 Reticulocyte count Flow cytometry by argon laser method 

### 8 Prothrombin time Modified Quickmethod 

### 9 Activated partial thromboplastin time Activated cephaloplastin method 10 Mean corpuscular volume Calculated from 1 and 5 11 Mean corpuscular hemoglobin Calculated from 

### 1 and 4 12 Mean corpuscular hemoglobin Calculated from 4 and 5 concentration Measuring instruments I - 5: Automated hematology analyzer (NE4500, Toa Medical Electronics Co., Ltd.) 6: Auto cell analyzer (MICROX HEG-70A, OmronTateishi Electronic) 7: Automatedreticulocyte analyzer (R-2000, Toa Medical Electronics Co., Ltd.) 8, 9: Coagulometer (KC lOA, Amerung, GmbH)

12. Bloodchemistry examination The blood samples simultaneously collected with those for the hematological examination
were used. The serum samples were obtained after centrifugation at 3000 rpm for 10 min (maximum centrifugation gravity, 1570

- 2050 g) ofblood which had been left to stand at
room temperature for about 30 min and the measurements ofeach item listed below were performed.

Items 1 Total protein 2 Albumin 3 NG ratio 4 Glucose 5 Triglycerides 6 Total cholesterol 7 Urea nitrogen 8 Creatinine 9 Calcium 10 Inorganic phosphorus 1 I GOT (AST) 12 GPT (ALT) 13 y-GTP 14 ALP 15 Sodium (Na) 16 Potassium (K) Measurement/calculation Biuret method BCG method Calculated from 

### I and 

### 2 Enzymatic-UV method (GK-G6PDH method) Enzymatic method (LPL-GK-G3PO-POD method) Enzymatic method (CES-CO-POD method) Enzymatic-UV method (Urease-GLDH method) Jaffe method O-CPC method UV method UV-Rate method (modified SSCC method) UV-Rate method (modified SSCC method) y-glutamyl-p-nitroanilide substrate-method (modified SSCC method) p-nitrophenyJphosphale substrate method (modified GSCC method) lon-selective electrode method lOll-selective electrode method 13 

## 000053

### 4lA36
17 Chlorine (CI) Ion-selective electrode method Measuring instrument: Automatic analyzer (Hitachi 736- 10, Hitachi Ltd.)

13. Urinalysis Twenty-five days after conunencement oftreatment, fresh urine samples collected from all rats
were examined for items listed below by a paper test method (Multistix 4!1, Miles-Sankyo Co., Ltd.) using a analyzer (Clinitek 10, Miles-Sankyo Co., Ltd.). I pH; 2 Occult blood; 3 Protein; 4 Glucose; 5 Ketone bodies; 6 Bilirubin; and 7 Urobilinogen.

14. Pathological examination 14.1 Necropsy All surviving animals at each scheduled sacrifice were euthanized after blood collection by severing the abdominal aorta under thiopental sodium anesthesia (Ravonale, Tanabe Seiyaku Co., Ltd.) and then subjected to necropsy. 14.2Organ weight The following organs were weighed from all animals at each scheduled sacrifice with an electronic balance (ED-H60, Shimadzu Corp.), and the ratio oforgan weight to the final body weight (relative organ weight) was calculated: the brain, liver, kidneys, adrenals and testes or ovaries. 14.3Histopathological examination The following organs ofall animals at each scheduled sacrifice were removed and preserved in
a 10% neutral phosphate-buffered formalin (except for the eyes and Harderian gland which
were fixed in Davidson's fluid): the brain, pituitary, eyes (including Harderian glands), lungs, stomach, thyroids (including parathyroids), hean, liver, spleen, kidneys, adrenals, urinary bladder, testes or ovaries, and bone marrow (femur). Histopathological examination was performed on the heart, liver, spleen, kidneys, and adrenals of both sexes ofthe control and 1000 mglkg groups at the end ofthe dosing period. Additionally, the ovaries ofthe control and 1000 mglkg groups at the end ofthe dosing period
14

### 4U36

was examined histopathologically since a change in organ weight was observed. Moreover, since gross lesions were observed at the necropsy, the following organs were examined: the pituitary ofone male ofthe 20 mglkg group, the lung ofone female ofthe 20 mglkg group, the liver ofone male ofthe 140 mglkg group, and the lung 01 one male ofthe 1000 mglkg group. Since this histopathological examination revealed the changes attributable to the administration ofthe test substance in the kidneys ofmales, further examination in the kidneys ofthe males ofthe middle and low dose groups at the end ofthe dosing period and ofall males at the end ofthe recovery period were carried out. Additionally, the liver ofboth sex ofthe middle and low dose groups at the end ofthe dosing period and ofall animals at the end ofthe recovery period were also examined since hypertrophy ofhepatocyte was suspected in the both
sexes ofthe 1000 mglkg group at the end ofthe dosing period.

15. Statistical analysis Numerical data obtained in the study were analyzed by multiple comparison tests. They were first analyzed by Bartlett's test. When the group variance was determined to be homogeneous, 

## "" groups were compared by a one-way analysis ofvariance. When intergroup differences were significant, Dunnett's multiplerange test (for groups with equal number) or Scheffe's test (for groups with unequal number) were used. When Bartlett's test indicated heterogeneous group variance, comparisons were done by the Kruskal-Wallis test and when intergroup differences
were significant, Dunnett's rank sum test (for groups with equal number) or Scheffe's test (for groups with unequal number) was used. The categorical data obtained by urinalysis was analyzed by Armitage's Chi-square test.

154L436 

# Results

I. Dead animals No death was observed in either the dosing orrecovery periods.

2. Clinical signs (fABLE 1) No changes considered caused by the administration ofthe test substance were observed.

3. Body weight (FIG. 1, TABLE 2) No changes considered caused by the administration ofthe test substance were observed.

4. Food consumption (fABLE 3) No changes considered caused by the administration ofthe test substance were observed.

5. Hematological examination (fABLE4,5) Although significantly extended prothrombin time was observed in the females ofthe 1000 mglkg group atthe end ofthe recovery period, it was considered that this change was not caused bythe administration ofthe test substance since this was not observed at the end ofthe dosing period and the prothrombin time ofsome control animals were almost the same as those ofthe 1000 mg/kg group. There were no significant changes in other items.

6. Blood chemistry examination (TABLE 6) At the end ofthe dosing period, significantly increased albumin in the males ofthe 1000 mg/kg group and decreased AlG ratio in the males ofthe 20 mg/kg group were observed. In these changes, decreased AlG ratio in the males ofthe 20 mg/kg group was not considered related to the test substance because this change was not observed in the 140 and 1000 mg/kg groups.

16 

# 0000564L436

At theend ofthe recovery period, significantly increased total protein and inorganic phosphorus were observed in the males ofthe 1000 mglkg group and significantly increased albumin was observed inthe females ofthe 1000 mglkg group; however, they were not considered caused by the administration ofthe test substance since they were not observed at the end ofthe dosing period.

7. Urinalysis (fABLE7) Inthe examination 25 days aftercommencement oftreatment, significantly increased urine protein was observed in the males ofthe 20mglkg and increased urobilinogen was observed in the females ofthe 140 mglkg group; however, they were not considered related to the administration ofthe test substance since these changes were not observed in the 1000 mglkg group.

8. Organ weight (fABLE 8, 9) At the end ofthe dosing period, significantly increased weight ratio ofthe adrenals was observed inthe males ofthe 20 mglkg group and increased weight ratio ofthe ovaries was observed in the females ofthe 1000 mglkg group. In these changes, increased relative adrenal weight ofthe males was not considered related to the administration ofthe test substance since this change was not observed in the 1000 mglkg group. At the end ofthe recovery period, significantly decreased weight ratio ofthe brain and increased weight ratio ofthe kidneys were observed in the males ofthe 1000 mg/kg group. In these changes, decreased relative brain weight was not considered related to the administration ofthe test substance since this change was observed at the end ofthe recovery period only.

9. Necropsy (TABLE 10) No changes attributable to the administration ofthe test substance were observed at the end of either the dosing or recovery periods.

17 

## 00008';4L436

Incidental changes such as impression, yellow patch, and hepatodiaphragmatic nodule ofthe liver, white patch ofthe pituitary, and edema and red patch ofthe lungs were observed sporadically.

10. Histopathological examination (fABLE 11) Changes consideredrelated to the administration ofthe tes; substance was observed in the kidneys ofthe males. At the end ofthe dosing period, increase in hyaline droplets in the proximal tubular epithelium
was observed in five males ofthe 1000 mglkg group. At the end ofthe recovery period, increase inhyaline droplets in the proximal tubular epithelium was observed in one male ofthe 1000 mglkg group. Basophilic change ofthe tubular epitheliumwas enhanced in two males ofthe 1000 mglkg group. Hyaline cast was also observed in their tubular epithelium. These changes were not observed in the kidneys of the females. Although significantly increased weight ratio ofthe ovaries was observed in the females ofthe 1000 mglkg group, no histopathological change was observed in the ovaries. The liver ofboth sexes ofthe middle and low dose groups at the end ofthe dosing period and ofall animals at the end ofthe recovery period were additionally examined since hypertrophy ofhepatocyte was suspected in the both sexes ofthe 1000 mglkg group at the end ofthe dosing period. After all examinations, it was concluded that hypettrophy ofthe hepatocyte in the liver did not occurr in any animals. In addition to the above changes, spontaneous changes frequently seen in rats were observed; however, there was no enhancement ofthese changes in the treatment groups.

18 

## 0000884L436 

## Discussion

YDC-1312 was administered orally to male and female SD rats for 28 days to investigate the changes on biological function and morphology. Changes considered related to the administration ofthe test substance were as follows: significantly increased serum albumin and increase in hyaline droplets in the proximal tubular epithelium in the kidneys in the males ofthe 1000 mglkg gwup and significantly increased weight ratio ofthe ovaries in the females ofthe I()()() mglkg groups. At the end ofthe recovery period, changes ofserum albumin and the ovaries were not observed. There were no histopathological changes in the ovaries. Increase in hyaline droplets inthe proximal tubular epithelium in the kidneys was considered to be a similar change as alpha 2u globulin nephropathy [1, 2] because it was observed in males only and ofits morphologic characteristics. This nephropathy was reported to be induced by a diverse group ofhydrocarbons which was similar to the test substance and that only male rats developed this lesion [3]. Thereversibility ofthis nephropathy was observed since this finding was reduced after the recovery period and basophilic change ofthe tubular epithelium, which was considered to be regeneration, was observed at the end ofthe recovery period. Significantly increased ratio ofkidney weight of the males in the 1000 mg/kg group also considered to reflect the regeneration from the nephropathy. On the basis ofthe above, the non observable effect level of YDC-1312 was estimated to be 140 mglkg in both sexes.

19 

## OOOOE'j94L436 

## References [I] Alden, C.L. and Frith, C.H.: Urinary system, Inducible alpha 2u globulin nephropathy syndrome. 

### In: Handbook oftoxicologic pathology. W.M. Haschek and C.G. Rousseaux, eds., pp.293-294, Academic Press, San Diego, 1991 [2] Swenberg, J.A., et al.: The comparative pathobiology of a 2u-globulin nephropathy. Toxicol. Appl. Pharmacol., 97: 35-46, 1989. [3] Alden, C.L.: A review ofunique male rat hydrocarbon nephropathy. Toxicol. Pathol., 14, 109-111, 1986.

20 

## 0000.:;0

### 4L436



## Figure and tables

OOOO:J.l

### BODY WEIGHT (G)
500 450 400 350 300 250 200 

## o MG/KG GROUP

...... : 20 MG/KG GROUP •••• : 140 MG/KG GROUP 150 

### ••_•• : 1000 MG/KG GROUP
100 50 

### ADMINISTRATION 

## -------------------------+ +-------- 

### RECOVERY 

## ------------+



## o**~---------------------------------------------------------------------------** 

## o 7 

### 14 21 28 35 

### 42 DAYS AFTER COMMENCEMENT OF TREATMENT 

# o o o FIG. 1 - M BODY WEIGHT CHANGES 

## C) !**~\** 

# .""" N 

### IN RATS TREATED ORALLY WITH YDC-1312 FOR 28 DAYS. [MALE]

- 1­

# - - 

# - - 500 

### BODY WEIGHT (G)

450
) 400 350 300**~.,...** .. .",.-.

### ......... 250 

# .".- oMG/KG GROUP



# ...... : 

### 20 MG/KG GROUP •••• : 140 MG/KG GROUP 100 

### -_•• : 1000 MG/KG GROUP 50 

### ADMINISTRATION RECOVERY

0 0 

### 7 

### 14 21 28 35 

# o DAYS AFTER COMMENCEMENT OF TREATMENT 

# o o FIG. 1 - F BODY WEIGHT CHANGES IN RATS TREATED ORALLY WITH YDC-1312 FOR 28 DAYS. [FEMALE] c.:,) (J:)



### - 2­ W 200 150



### 42TABLE 1 - M - 1 STUDY NO. 4L436CEO INCIDENCE 

### OF CLINICAL SIGNS

TEST ARTICLES : YDC-1312 SEX : MALE
Dose Clinical Days After Commencement of Treatment Level Signs (mg/kg) N 

## o 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 20 6 No Abnormality 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6
140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6
1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 N Number of Animals.



# o o o C) (;)


# .. *'" 3 - TABLE 1 - M - 1

### TABLE 1 - M - 2 STUDY NO. 4L436CEO 

### INCIDENCE 

### OF CLINICAL SIGNS

TEST ARTICLES : YDC-1312 SEX : MALE
Dose Clinical Days After Commencement I Cessation of Treatment Level Signs (mg/kg) N 22 23 24 25 26 27 28/0 29/1 30/2 31/3 32/4 33/5 34/6 35/7 36/8 37/9 38/10 39/11 40/12 41/13 42/14 o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6**6/~** 6/6 6/6 6/6 6/6
20 5 No Abnormality 6/5 6/6 6/6 6/6 6/6 6/6 6/6
140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6
1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 N Number of Animals.



# o o o C) r.:; (,ii 4 - 

### TABLE 1 - M - 2TABLE 1 - F - 1 STUDY NO. 4L436CEO INCIDENCE 

### OF CLINICAL SIGNS

TEST ARTICLES : YDC-1312 SEX : FEMALE

Dose Clinical 

### Days After Commencement of Treatment level Signs (mg/kg) N 

### o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

### o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12
20 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6
140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 N Number of Animals.



## o o o C """ 

## "" \1:; 

### 0) 5 - TABLE 1 - F - 1Dose level (mg/kg) N Clinical Signs


### o 12 No Abnormality 20 6 No Abnormality
140 6 No Abnormality
1000 

### 12 No Abnormality N Number of Animals.



## o 

# o o C)



### .....**~**

....j 

### TABLE 1 - F - 2 INCIDENCE 

### OF CLINICAL SIGNS

TEST ARTICLES : YDC-1312 SEX : FEMALE STUDY NO. 4L436CEO

Days After Commencement I Cessation of Treatment 22 23 24 25 26 27 28/0 29/1 30/2 31/3 32/4 33/5 34/6 35/7 36/8 12/12 12/12 12/12 12/12 12/12 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 37/9 6/6 38/10 39/11 40/12 41/13 42/14 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 12/12 12/12 12/12 12/12 12/12 

### 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6

6 - 

### TABLE 1 - F - 2

## ------------------------------------------------------------------------------------------------------------

# ---------------------------- TABLE 2 - M- 1 BODY WEIGHT

- GROUP MEAN VALUES

TEST ARTICLES: YDC-1312 SEX: MALE STUDY NO. 4L436CEO

UNIT G DOSE LEVEL

MG/KG 

### o 7 

### 14 DAYS AFTER COMMENCEMENT 21 281 

### 0 351 7 I 

### CESSATION OF 42114 

### TREATMENT

0 MEAN 184. 249. 311. 366. 405. 445. 479. S.D. 8.4 16.4 22.8 26.6 30.4 14.5 16.4

N 

### 12 12 12 12 12 6 6 20 MEAN 185. 247. 311. 369. 411. S.D. 8.6 16.8 22.2 25.7 27.9

N 

### 6 6 6 6 6 140 MEAN 184. 251. 317. 376. 417. S.D. 9.1 16.6 20.3 27.1 32.7

N 6 6 6 6 6 1000 MEAN 185. 249. 314. 371. 412. 445. 474. S.D. 7.2 11.5 15.4 21.2 25.8 21.0 24.0

N 

### 12 12 12 12 12 6 6



## o o o C) (.t:; Q)

7 - 

### TABLE 2 -M- 1

# ---------------------------------------------------------------------------------------------------------------------------------------- TABLE 2 - F - 1 STUDY NO. 4L436CEO 

### BODY WEIGHT

- GROUP MEAN VALUES

TEST ARTICLES: SEX: YDC-1312 FEMALE UNIT G DOSE LEVEL

MG/KG 

## a 7 

### 14 

### DAYS AFTER COMMENCEMENT I 

### 21 261 0 351 7 

### CESSATION OF TREATMENT 42114



### 0 MEAN 151. 184. 213. 240. 263. 276. 291. S.D. 7.5 10.2 14.2 21.4 20.6 13.7 17.5

N 12 12 12 12 12 6 6 20 MEAN 148. 180. 203. 235. 254. S.D. 9.2 9.4 16.2 16.4 21.0

N 6 6 6 6 6 140 MEAN 147. 174. 207. 234. 260. S.D. 6.1 

### 10.1 13.9 16.7 20.3

N 6 6 6 6 6 1000 MEAN 152. 179. 207. 236. 255. 279. 291. S.D. 6.7 7.7 9.3 16.0 12.6 16.3 20.5

N 12 12 12 12 12 6 6



## o o o C) (f) (.&)

8 ­ 

### TABLE 2 -F- 1

# ---------------------------------------------------------------------------------------------------------------------------------------- TABLE 3 - M - 1 STUDY NO. 4L436CEO FOOD CONSUMPTION

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE UNIT G/ANIMAL/DAY DAYS AFTER COMMENCEMENT I 

### CESSATION OF TREATMENT DOSE LEVEL

MG/KG 7 

### 14 21 25 351 7 38/10


### 0 MEAN 22.6 26.1 27.9 28.5 28.1 28.3 S.D. 1.07 1.39 1.27 1.61 1.15 0.72



### N 6 6 6 6 3 3 

### 20 MEAN 22.2 25.7 28.2 28.4 S.D. 0.52 0.29 0.67 0.56



### N 3 3 3 3 140 

### MEAN 22.9 26.5 28.5 28.7 S.D. 1.54 1.89 2.39 1.86

N 3 3 3 3 

### 1000 MEAN 22.6 26.2 28.2 28.5 27.4 26.1 S.D. 1.03 1.20 

### 1.94 2.21 1.50 0.98

N 

### 6 6 6 6 3 3



## c o 

# o F**~)** 

### C
9 - 

### TABLE 3 -M- 1

# ---------------------------------------------------------------------------------------------------------------------------------------- TABLE 3 - F ­ FOOD CONSUMPTION

- GROUP MEAN TEST ARTICLES: YDC-1312 SEX: FEMALE 1 VALUES STUDY NO. 4L436CEO

UNIT G/ANIMAL/DAY DOSE LEVEL

MG/KG 7 

### 14 21 DAYS 25 AFTER COMMENCEMENT 351 7 38/10 I 

### CESSATION OF TREATMENT

) 

### 0 MEAN 17.0 18.7 20.0 20.7 20.1 20.2 S.D. 0.91 1.57 2.12 1.79 0.45 1.54

N 6 6 6 6 

### 3 3 20 MEAN 16.8 18.1 20.0 20.0 S.D. 0.31 0.74 0.32 0.23

N 3 

### 3 3 3 140 MEAN 16.2 

### 18.5 19.8 20.7 S.D. 0.95 1.21 1.14 0.70

.N 3 

### 3 3 3 1000 MEAN 16.8 18.3 19.5 20.0 21.8 20.3 S.D. 0.77 1.13 1.02 1.22 1.50 3.00

N 

### 6 6 

### 6 6 3 3



# o c::.> 

# o**!-~** 

# o j-i;, 

### 10

- TARI 1= "Ii -1=- 1 I

# ----------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------- TABLE 4

- FS - M- 1 STUDY NO. 4L436CEO HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES : YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE ( 4 \.lEEK) RBC H8 

### RETICULO- PLATELET DOSE LEVEL COUNT HT CONC. CYTE MCV MCH MCHC COUNT PT APTT

MG/KG COUNT (x**10},~U** (X) 

### (G/DU ( X. ) { (1M' ) 

### (PG) (X) (x 

### 10Yp.U (SEC) (SEC)
0 MEAN 742. 44.4 15.1 26. 59.8 20.3 33.9 102.3 12.4 17.5 S.D. 20.2 1.17 0.35 3.5 1.61 0.50 0.22 9.24 0.41 1.02

N 6 6 6 6 6 6 6 6 6 6 20 MEAN 746. 44.6 15.1 30. 59.8 20.2 33.8 109.3 12.2 16.5 S.D. 19.8 1.06 0.42 2.3 2.35 0.90 0.24 7.86 0.19 0.74

N 6 

### 6 6 6 6 6 6 6 6 6 140 MEAN 742. 44.3 14.8 29. 59.7 20.0 33.5 101.3 12.0 17.2 S.D. 25.4 0.46 0.23 2.7 1.96 0.44 0.47 6.73 0.26 0.49

N 5 5 5 5 5 5 5 5 6 6 1000 MEAN 730. 44.4 15.2 29. 60.S 20.8 34.2 101.4 12.0 16.7 S.D. 35.4 1.58 0.50 5.0 1.63 0.45 0.29 5.56 0.21 1.39

N 6 6 6 6 

### 6 6 6 6 6 

### 6



# o <:) 

# o**~** (;) 

### N

11- 

### TABLE 4 -FS-M- 1

# ----------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------- TABLE 4

- FS - F - 1 STUDY NO. 4L436CEO HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES : YDC-B12 SEX : FEMALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) RBC 

### HB RETICULO- PLATELET DOSE LEVEL COUNT HT CONC. CYTE MCV MCH MCHC COUNT PT APTT

MG/KG COUNT (x 

### 10 rILL) (X) 

### (G/DU**(~** ) ( 11M' ) 

### (PG) (X) (x 

### 10**r~L)** 

### (SEC) (SEC)
0 MEAN 705. 41.3 14.3 23. 58.7 20.3 34.6 94.4 13.0 15.5 S.D. 23.5 1.60 0.43 3.8 1.90 0.63 0.45 6.58 0.46 1.47

N 6 6 

### 6 6 6 6 

### 6 6 6 6 

### 20 MEAN 707. 41.9 14.4 24. 59.2 20.4 34.4 100.2 13.3 15.4 S. O. 30.6 1.55 0.57 3.1 1.46 0.62 0.30 8.68 0.56 1.02

N 6 6 6 6 6 6 6 6 6 6 140 MEAN 710. 41.8 14.4 21. 58.9 20.3 34.4 88.3 13.6 14.8 S.D. 28.1 2.06 0.73 2.6 1.69 0.70 0.25 20.43 0.54 1.48

N 

### 6 6 6 6 6 6 6 6 5 5 1000 MEAN 714. 43.0 14.8 23. 60.3 20.8 34.5 95.5 13.4 15.1 S.D. lS.7 1.41 0.48 2.6 1.90 0.64 0.38 9.56 0.42 0.67

N 

### 6 6 6 6 6 6 6 6 6 6



# o o o 1-;;, 

# o (;..) 

### 12 - 

### TABLE 4 -FS-F- 1

# ------------------------------------------------------

# ---------------------------------------------------------------------------------- TABLE 4 - RS - M - 1 STUDY NO. 4L436CEO HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) RBe HB RETICULO- PLATELET DOSE LEVEL COUNT HT eONC. CYTE MCV MCH MCHC COUNT PT APTT

MG/KG COUNT (x 

### 10i',uU (X) 

### CG/DU**(~)** ( 144' ) 

### (PG) (X) (X 

### 10 

### rllu (SEC) (SEC)
0 MEAN 756. 43.7 14.9 26. 57.8 19.7 34.1 98.7 13.1 16.7 S.D. 32.0 1.61 0.58 3.2 0.72 0.23 0.32 9.74 0.19 2.02

N 6 6 6 6 6 6 6 6 6 6 1000 MEAN 768. 43.8 14.9 25. 57.1 19.5 34.1 99.8 13.1 16.9 S.D. 19.3 1.09 0.42 2.2 2.04 0.73 0.22 6.05 0.16 1.30

N 

### 6 6 6 6 6 6 6 6 6 6



## o 

# o o !-A 

## o

... M-< 13- 

### TABLE 4 

### -RS-M- 1

### TABLE 4 - RS - F - 1 STUDY NO. 4L436CEO HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) RBC HB RETlCULO- PLATELET DOSE LEVEL COUNT HT CONC. CYrE MCV MCH MCHC COUNT PT APTT

MG/KG COUNT (x 10,;s

### L> 

## ex) (G/OL> ( %. ) (PM' ) 

### (PG) (X) (X 

### 10,;sL> (SEC) (SEC)
0 

### MEAN 717. 41.1 14.2 27. 57.3 19.9 34.6 88.1 13.6 15.5 S.D. 21.9 1.23 0.43 4.9 1.39 0.45 0.24 4.78 0.31 1.57

N 6 6 6 6 6 6 6 6 6 6 

## * 1000 

### MEAN 742. 42.1 14.6 24. 56.8 19.7 34.7 90.9 13.9 15.8 S.D. 23.9 0.67 0.32 3.1 1.78 0.55 0.39 6.32 0.17 0.87

N 6 6 

### 6 6 

### 6 6 6 6 6 

### 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

# * , P<0.05.



## o o 

# o**~** 

## c'**'-;~** 

### 14

- TABLE 4 

### -RS-F- 1

### TABLE 5 - FS - M- 1 HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON 

### SCHEDULE ( 4 WEEK) STUDY NO. 4L436CEO

DOSE LEVEL

MG/KG 

### WBC COUNT (x 

### 10 yp.L) 

### LYMPHOCYTES DIFFERENTIAL COUNT OF NEUTROPHILS SEGMENTED BAND LEUKOCYTESEOSINOPHILS 

## ex OFBASOPHILS TOTAL COUNTEDMONOCYTES CELLS)

0 MEAN S.D.

N 

### 112. 18.3

6 

### 86. 5.6 6 10. 6.0 6 $ 

### O. 0.4 6 1­ 

### 1.3 6 

### O. $ 

### 0.0 6 

### 3. 

### 2.2 6 

### 20 MEAN S.D.

N 128. 24.9

6 92. 

### 3.7 6 6. 

### 2.9 6 O. 0.0 

### 6 1. 0.8 6 

### O. 0.0 6 

### 2. 1.4 6 140 MEAN S.D.

N 

### 119. 20.7

5 94. 2.4 5 4. 2.8 5 

### O. 0.4 5 

### 1. 0.8 5 

### O. 0.0 5 1. 1.0 5 1000 MEAN S.D.



### N 131. 26.4

6 91. 4.4 6 6. 

### 2.7 6 O. 0.4 6 1­ 

### 0.8 6 

### O. 0.0 6 

### Z. 2.1 

### 6 $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



# o o 

# o 1-4. 

# o 

# en 15

- TABLE 5 -FS-M- 1

### TABLE 5 - FS - F ­ 1 HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) STUDY NO. 4l436CEO

DOSE LEVEL

MG/KG \.JBC COUNT (x 

### 10 y,.U 

### LYMPHOCYTES DIFFERENTIAL COUNT NEUTROPHllS SEGMENTED BAND OF LEUKOCYTESEOSINOPHILS 

### (X OFSASOPHILS TOTAL COUNTEDMONOCYTES CELLS)

0 MEAN S.D.

N 

### 105. 25.7

6 91. 3.8 6 7. 

### 2.3 6 O. 0.4 6 

### 1. 1.5 6 

### O.

## s 0.0 6 

### 2. 2.0 6 

### 20 MEAN S.D.

N 89. 13.5



### 6 92. 4.7 6 6. 

### 4.7 6 

### O. 

### 0.4 6 

### O. 0.5 6 

### O. 0.0 6 

### 2. 1.4 6 

### 140 MEAN S.D. 'N 103. 17.8

6 92. 

### 4.4 6 4. 3.9 6 

### O. 0.4 6 1. 

### 0.8 6 

### O. 0.0 6 

### 4. 2.5 6 

### 1000 MEAN S.D.

N 110. 15.9

6 95. 3.1 6 

### 3. 1.4 6 

### O. 0.4 6 

### 1. 0.8 6 

### O. 0.0 6 

### 2. 1.6 6 $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



# o (:J 

## o !w..,;. (..)**~,j**
16

- 

### TABLE 5 

### -FS-F- 1

### TABLE 5 - RS - M - 1 STUDY NO. 4L436CEO 

### HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) 

## wac DIFFERENTIAL COUNT OF LEUKOCYTES**(~OF** 

### TOTAL COUNTED CELLS) DOSE LEVEL 

### COUNT

MG/KG LYMPHO­ NEUTROPHILS EOSINO­ BASO­ MONO­ (x10Y/.Il) 

### CYTES SEGMENTED BAND PHILS PHILS CYTES $ 

### 0 MEAN 81. 88. 8. 1. 1. 

### O. 2. S.D. 23.6 6.0 4.5 0.5 1.0 0.0 1.9

N 6 6 6 6 6 6 6 1000 MEAN 94. 89. 9. 

### 1. 1. 

### O. 1. S.D. 18.3 4.2 4.9 0.5 0.8 0.0 0.8

N 6 6 6 6 6 6 6 $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



## o 

# o o 1-& 

# o ()J 

### 17 - 

### TABLE 5 -RS-M- 1

### TABLE 5 - RS - F - 1 STUDY NO. 4L436CEO HEMATOLOGY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) WBC DIFFERENTIAL COUNT OF LEUKOCYTES (X 

### OF TOTAL COUNTED CELLS) DOSE LEVEL COUNT

MG/KG 

### LYMPHO­ NEUTROPHILS EOSINO­ BASO­ MONO­ (x 

### 10 }II'U 

### CYTES SEGMENTED BAND PHILS PHILS CYTES
J S 0 

### MEAN 77. 

### 90. 7. 1. 1. 

### O. 1. S.D. 16.9 2.8 3.6 0.8 1.0 0.0 0.8

N 

### 6 6 6 6 

### 6 6 6 

### 1000 MEAN 86. 90. 8. 

### O. 1. O. 2. S.D. 35.1 2.1 2.7 0.4 0.9 0.0 1.3

N 6 6 

### 6 6 6 6 6 $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



## o 

# o o**I-~** 

# o W


### 18

­ TABLE 5 -RS-F- 1

### TABLE 6 - FS - M- 1 CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) STUDY NO. 4L436CEO

DOSE LEVEL



### MG/KG GOT (lUlU 

### GPT (lUlU 

### r-GTP (lUlU 

### ALP <lUlU 

### UREA NITROGEN {MG/DU CREATININ (MG/DL) GLUCOSE (MG/DL> TOTAL CHOL. (MG/DL> TRIGLYCERIDE

(MG/DU TOTAL oROTEIN

{G/DU ALBUMIN (G/DU 

### AlG RATIO



## o MEAN S.D.

N 

### 100. 21.2

6 

### 35. 6.2 6 

## s 

## o. 0.0 6 

### 570. 60.8

6 

### 17.8 2.34

6 

### 0.4 0.04

6 

### 154. 12.8

6 

### 63. 6.4 6 

### 133. 61. 7

6 

### 6.60 0.225 6 

### 3.68 0.109 6 

### 1.27 0.048 6 

### 20 MEAN S.D.

N 

### 90. 11.9

6 

### 31. 1.8 6 

### O. 0.0 6 

### 545. 64.8

6 

### 15.5 1.61

6 

### 0.4 0.05

6 

### 156. 10.6

6 

### 70. 7.1 6 172. 

### 35.1

6 

### 6.70 0.212 6 

### 3.62 0.111 6 

### 1.18

# ** 0.027 6 

### 140 MEAN S.D.

N 

### 87. 24.0

6 

### 32. 3.5 6 

### O. 0.0 6 623. 

### 122.2

6 17.8 

### 1.53

6 

### 0.4 0.04

6 

### 148. 6.9 6 63. 

### 5.2 6 114. 

### 57.0

6 6.73 

### 0.209 6 

### 3.77 

### 0.127 6 

### 1.27 0.043 6 

### 1000 MEAN S.D.

N 

## n. 17.0

6 

### 34. 5.2 6 

### O. 0.0 6 642. 

### 100.3

6 16.9 3.11

6 

### 0.4 0.05

6 

### 150. 12.4

6 63. 

### 14.3

6 

### 153. 35.3

6 

### 6.84 0.236 6 

### 3.85

# * 0.074 6 1.29 

### 0.056 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<0.05 ; 

## ** , P<O.Ol ; S , STATISTICAL ANALYSIS IMPOSSIBLE.



# o o o !-J:. It-, , 

# o
19

- 

### TABLE 6 -FS-M- 1

## ------------------------------------------------------------------------------------------------------------

# ----------------------------- TABLE 6 - FS - M- 2 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) INORGANIC DOSE LEVEL CALCIUM PHOS. NA K 

### CL

MG/KG 

### (MG/DL) (MG/DL) (MEQ/L) (MEQ/L) (MEQ/L)
0 MEAN 9.8 9.4 144. 4.5 100. S.D. 0.21 0.43 1.5 0.17 1.4

N 6 6 6 6 6 20 MEAN 9.9 9.2 143. 4.7 100. S.D. 0.20 0.41 0.8 0.08 1.2

N 6 6 6 6 6 140 '1EAN 10.1 9.3 144. 4.6 100. S.D. 0.31 0.31 1.0 0.15 1.4 'N 

### 6 6 6 6 6 1000 MEAN 10.0 9.0 143. 4.6 100. S.D. 0.27 0.41 1.2 0.24 2.6

N 6 6 6 6 6



# o o 

# o l-'­ I---li.**~** 

### 20 - TABLE 6 -FS-M- 

### 2

# ----------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------- TABLE 6 - FS - F - 1 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES : YDC-1312 SEX : FEMALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) UREA TOTAL TRI- TOTAL A/G 

### DOSE LEVEL GOT GPT T- 

### GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO

MG/KG 

### CIU/U (LUlU (LUlL> (lUlL> 

### (MG/DL> (MG/DL> (MG/DL> (MG/DL> (MG/DU (G/DU 

### (G/DU S S 

### 0 MEAN 78. 27. O. 352. 18.8 0.5 150. 65. 83. 6.68 3.84 1.35 S.D. 7.3 3.7 0.0 86.6 3.17 0.04 10.8 2.9 45.5 0.243 0.136 0.078

N 6 6 6 6 6 6 6 6 6 6 6 6 20 MEAN 86. 27. 

### O. 364. 18.7 0.5 150. 69. 38. 6.62 3.79 1.34 S.D. 13.6 7.8 0.4 100.4 2.99 0.06 9.7 7.5 11.2 0.121 0.096 0.049

N 6 

### 6 6 6 6 6 6 6 6 6 6 6 

### 140 MEAN 86. 27. 

### O. 353. 17.1 0.5 150. 63. 105. 6.61 3.84 1.38 S.D. 15.7 2.7 0.0 101.2 2.00 0.08 5.4 11.1 50.0 0.450 0.254 0.078

N 6 

### 6 6 6 6 6 6 6 6 6 6 

### 6 1000 MEAN 79. 29. O. 353. 18.0 0.5 141- 63. 38. 6.63 3.83 1.37 S.D. 10.5 4.5 0.0 65.3 2.65 0.00 8.0 7.8 10.6 0.079 0.097 0.067

N 6 6 6 6 6 6 6 6 

### 6 6 

### 6 6 S , STATISTICAL ANALYSIS IMPOSSIBLE.



## o o 

# o 10-"::'**t~!.** 

### N
21 - TABLE 6 -FS-F- 1

### DOSE LEVEL MG/KG
0 MEAN S.D.

N 20 MEAN S.D.

N 140 MEAN S.D. . 	N 

### 1000 MEAN S.D.

N



# o o <:) 

### l-Ji. r.', Vd 

### CALCIUM CMG/DU 9.8 0.18

6 9.6 0.14

6 9.9 0.37

6 9.7 0.43

6 INORGANIC PHOS. (MG/DU
8.3 0.57

6 8.2 0.55

6 

### 8.6 0.87

6 8.1 0.99

6 

### NA (MEG/U
143. 1.5 6 

### 143. 1.1 6 

### 142.

0.8 6 144.

0.8 6 

### TABLE 6 - FS - F - . 2 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK)

K 

### CL (MEG/U (MEG/L>
4.2 102. 0.20 2.1

6 6 

### 4.0 103. 0.29 0.8

6 6 

### 4.2 101. 0.17 1.7

6 6 

### 4.1 104. 0.21 1.4

6 6



### 22 - TABLE 6 FS-F- 2

# ------------------------------------------------------

# ------------------------------------------------------

# ---------------------------- TABLE 6 - RS - M - 1 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 

### SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) UREA TOTAL TRI- TOTAL AlG 

### DOSE LEVEL GOT GPT T- 

### GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO

MGIKG <lUlU (lUlU (lUlU (lUlU (MG/DU (MG/DU (MG/DU (MG/DU CMG/DL> (G/DL> 

### CG/DL> $ 

### 0 MEAN 91. 

### 27. O. 

### 472. 20.3 0.5 162. 58. 178. 6.79 3.60 1.13 

### S.D. 17.6 7.0 0.0 163.1 0.83 0.05 20.7 9.3 40.7 0.187 0.071 0.045

N 6 6 6 6 6 6 6 6 6 6 6 6 

## * 1000 MEAN 92. 29. O. 472. 19.1 0.5 148. 68. 178. 7.05 3.69 1.10 S.D. 22.1 9.5 0.0 80.6 1.62 0.05 11.4 15.6 112.3 0.181 0.072 0.057

N 6 6 6 6 6 6 6 6 6 6 6 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<0.05 ; $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



# o o o bJr. r··.-t. 

### ;;.:, 23

- TABLE 6 -RS-M- 1 I

### TABLE 6 - RS - M - Z 

### STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) INORGANIC DOSE LEVEL CALCIUM PH

### OS • NA K 

### CL

MG/KG (MG/DU (MG/DU (MEG/L> CMEG/L) (MEG/L) 0 MEAN 9.3 6.9 143. 4.5 102. S.

### D. 0.18 0.64 0.8 0.21 0.8

N 6 6 6 6 6 

# * 1000 MEAN 9.4 7.7 

### 143. 4.6 101. S.D. 0.26 0.56 0.9 0.18 1.4

N 

### 6 6 6 6 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

# * , P<0.05.



## o o o 1-.;" 

### t·..t

24

- TABLE 6 -RS-M- 2 C.ii

# ------------------------------------------------------

# ---------------------------------------------------------------------------------- TABLE 6 - RS - F - 1 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES rEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) UREA TOTAL TRI- TOTAL A/G 

### DOSE LEVEL GOT GPT T- 

### GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO

MG/KG (LUlL) 

### (lUlU (lUlU (lUlU (MG/DU (MG/DU (MG/DU (MG/DU (MG/DU (G/DU (G/DL> $ 

### 0 MEAN 84. 21. 

### O. 350. 23.5 0.5 150. 72. 56. 6.67 3.73 1.27 S.D. 11.7 3.0 0.0 78.9 5.79 0.05 9.5 5.8 15.4 0.208 0.111 0.045

N 6 6 6 6 6 6 6 6 6 6 6 6 

## * 1000 MEAN 87. 26. 

### O. 315. 20.6 0.6 144. 75. 81. 6.99 3.92 1.28 S.D. 12.4 5.8 

### 0.4 83.2 3.05 0.04 7.0 8.4 25.6 0.349 0.156 0.049

N 6 6 6 6 6 6 6 6 6 6 6 

### 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<0.05 ; $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



## o 

# o o**~ t~~**  (J'; 

### 25 - TABLE 6 -RS-F- 1

### TABLE 6 - RS - F - 2 STUDY NO. 4L436CEO CLINICAL CHEMISTRY

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) INORGANIC DOSE 

### LEVEL CALCIUM PHOS. NA K 

### CL

MG/KG (MG/DU (MG/DU (MEG/U (MEGIll (MEGIll

j 

### 0 MEAN 9.1 6.8 

### 143. 4.0 103. S.D. 

### 0.29 0.76 1.3 0.28 1.4

N 6 6 6 6 6 

### 1000 MEAN 9.3 6.8 142. 4.0 101. S.D. 

### 0.29 0.75 1.6 0.11 1.9

N 6 

### 6 6 6 

### 6



## o o o l-.\**;'~.u6**



### "' '-1

26 - 

### TABLE 6 -RS-F- 2----------------- 

### DOSE lEVEL

MG/KG
0
20
140
1000



## o o 

# o**~ ~_<a**  I
........ ..", 

### TABLE 7 - Cl - M - 1 URINALYSIS

- GROUP 

### MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE 3 WEEKS AFTER COMMENCEMENT OF TREATMENT NUMBER PH 

### PROTEIN GLUCOSE KETONES BILIRUBIN EXAMINED 

### (MG/DU CG/DU CMG/DU 7.5 8.0 8.5 9.0



### - TR 30 100**~300** 

### - 0.1 0.25 0.5**~1** 

### - 5 15 40**~80** 

### - 1+ 2+ 3+ 12 1 2 7 

### 2 0 2 6 

### 3 1 11 

### 1 0 0 0 

### 1 5 6 

### 0 0 11 

### 1 0 0 

# * 6 0 1 5 0 0 

### 0 0 6 0 6 0 0 0 0 0 

### 1 4 

### 1 0 6 0 0 0 

### 6 0 

### 2 4 0 0 

### 0 3 3 0 6 0 0 0 

### 0 0 

### 1 5 0 0 

### 6 0 0 0
12 0 

### 3 5 

### 4 0 0 

### 4 8 0 

### 12 0 

### 0 0 0 0 

### 6 6 0 0 

### 12 0 0 0



### , NEGATIVE ; TR, TRACE ; 1+ , SLIGHT ; 2+, MODERATE ; 3+, SEVERE. 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<O.OS ; S , STATISTICAL ANALYSIS OCCULT BLOOD

- TR 1+ 2+ 3+ 12 0 0 

### 0 0 S 

### 6 0 0 0 0 $ 

### 6 0 0 0 0 S 

### 12 0 0 0 0


### IMPOSSIBLE. STUDY NO. 4L436CEO

UROBILINOGEN CEU/DU 0.1 1 2 4 8

8 

### 4 0 0 0 

### 2 4 0 0 0
2 

### 4 0 

### 0 0 

### 6 6 0 0 0

27

- 

### TABLE 7 -C1-M- 1

# ---------------------------------------------------------------------------------------------------------------------------------------------------

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

--------------------------------------------------------------------------------------------------------------------------------------------------- TABLE 7


### - Cl - F - 1 STUDY NO. 4L436CEO URINALYSIS

- GROUP MEAN VALUES TEST ART!CLES : YDC-1312 SEX : FEMALE 3 WEEKS AFTER COMMENCEMENT OF TREATMENT NUMBER PH 

### PROTEIN GLUCOSE KETONES BILIRUBIN OCCULT BLOOD UROBILINOGEN DOSE EXAMINED (MG/DL> (G/DL> (MG/DL> (EU/DU LEVEL


# ------------------------- ---------------- ----------------- -------------- ---------- ------------- 

# -------------­ MG/KG 

### 6.5 7.0 7.5 8.0 8.5 9.0
- TR 30 100 It 300

- 0.1 0.25 0.5 Itl 

# - 5 15 40 1t80

- 1+ 2+ 3+

- TR 1+ 2+ 3+ 0.1 1 2 4 8
0 

### 12 0 0 

### 0 1 6 

### 5 1 7 4 

### 0 0 12 0 

### 0 0 0 7 

### 5 0 0 0 12 0 0 0 12 0 0 0 0 8 4 

### 0 0 0 $ $ $ 20 6 0 

### 0 0 Z 

### 2 2 

### 0 5 0 

### 1 0 6 0 

### 0 0 0 1 5 0 0 0 6 

### 0 0 0 6 

### 0 0 0 0 

### 2 4 

### 0 0 0 $ $ $ 

## ** 1l.0 

### 6 0 0 '1 0 4 1 0 

### 1 5 

### 0 0 6 0 

### 0 0 0 

### 1 5 

### 0 0 0 6 

### 0 0 

### 0 6 

### 0 0 0 0 0 6 0 0 

### 0 $ $ I 

### 2 1000 12 .1 

### 0 0 2 

### 3 6 2 

### 1 0 12 0 0 0 0 8 4 

### 0 0 0 

### 12 0 0 0 11 0 

### 1 0 0 8 4 0 0 0



### , NEGATIVE ; TR, TRACE ; 1+ , SLIGHT ; 2+, MODERATE ; 3+, SEVERE. 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

# ** , P<O.Ol ; $ , 

### STATISTICAL ANALYSIS IMPOSSIBLE.



## o o o lor';' 

### p, 

### <.D 28

­ 

### TABLE 7 -Cl-F- 1

### DOSE LEVEL MG/KG

0 MEAN S.D.

N 20 MEAN S.D.

N 140 MEAN S.D.

N 1000 MEAN S.D.

N



# o 

# o o }--1.t. t· ; 

# o TABLE 8 - FS - M- 1 ORGAN WEIGHT (ABSOLUTE)

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) STUDY NO. 4L436CEO



### FINAL BODY WEIGHT

(G) 

### BRAIN (G) 

### LIVER (G) 

### KIDNEYS (G) ADRENALS (MG) TESTES (G)
402.

42.7

6 2.05 0.101 6 15.78 1.823

6 2.87 0.320 6 59.3 10.42

6 2.98 0.253 6 410.

27.8

6 2.04 0.067 6 17.00 1.185

6 2.83 0.372 6 50.2 2.90

6 3.10 0.253 6 417.

32.7

6 2.09 0.057 6 16.68 1.961

6 3.08 0.279 6 55.9 5.89

6 3.15 0.312 6 414.

33.7

6 2.08 0.076 6 16.82 2.173 6 2.85 0.239 6 53.2 5.48

6 3.11 0.170 6

29 - 

### TABLE 8 

### -FS-M- 1

### DOSE LEVEL MG/KG

0 MEAN 

### S.D.

N 20 MEAN 

### S.D.

N 140 MEAN 

### ,S.D.

N 1000 MEAN 

### S.D.

N



# o o o p..;.. 

### iJ**~.** 

### TABLE 8

- FS - F ­ 1 STUDY NO. 4L436CEO 

### ORGAN WEIGHT (ABSOLUTE) TEST ARTICLES: - GROUP MEAN YDC-1312 VALUES SEX: FEMALE ANIMALS KILLED ON SCHEDULE ( 4 

### WEEK) FINAL BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES WEIGHT

(G) 

### CG) (G) (G) (MG) (MG)
266. 1.94 10.14 1.87 76.2 107.2

27.8 0.044 1.343 0.160 9.02 10.96

6 6 6 6 6 6 255. 1.91 9.49 1.89 75.9 118.5

21.4 0.129 1.184 0.151 10.20 18.14

6 6 6 6 6 6 260. 1.92 9.67 1.80 67.5 106.3

19.9 0.065 0.916 0.088 6.00 7.31

6 6 6 6 6 6 25I. 1.97 9.10 1.84 73.4 120.2

10.5 0.058 0.300 0.113 6.44 9.82

6 6 6 6 6 6

'1:1'\
_ TARI**~** R -1="-1=- 1

### TABLE 8 - RS - M - 1 STUDY NO. 4L436CEO ORGAN WEIGHT (ABSOLUTE)

- GROUP MEAN VALUES TEST ARTICLES: YOC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) FINAL 

### DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES

MG/KG WEIGHT (G) (G) (G) 

### (G) (MG) (G)
0 MEAN 479. 2.16 19.38 2.97 56.3 3.32 S.D. 16.4 0.071 1.781 0.173 2.51 0.172

N 6 6 6 6 6 6 1000 MEAN 474. 2.05 19.52 3.20 57.S 3.21 S. D. 23.8 0.116 1.338 0.246 6.32 0.245

N 6 6 6 6 6 6



# o o 

# o l-" \',

## " t ',,' (\)


### 31

- TABLE 8 -RS-M- 1

## ....,**:"~** 

### TABLE 8 - RS - F - 1 STUDY NO. 4L436CEO ORGAN WEIGHT (ABSOLUTE)

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) FINAL DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES

MG/KG WEIGHT (G) (G) (G) (G) 

### (MG) (MG)
0 MEAN 291. 1.97 10.45 1.82 77.2 106.0 S.D. 17.3 0.048 1.244 0.071 11.05 16.37

N 6 6 

### 6 6 6 6 

### 1000 MEAN 291. 1.95 10.79 1.85 79.1 103.8 S.D. 20.6 0.029 1.232 0.165 11.88 12.06

N 6 6 6 6 6 6



## o o o 1-1c. {";J 

### c:.J 32

- TABLE 8 -RS-F- 1

### TABLE 9 - FS - M- 1 STUDY NO. 4L436CEO ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT)

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE ( 4 WEEK) FINAL DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES

MG/KG WEIGHT (G) (x 

### 10 ")
0 MEAN 402. 0.51 3.92 0.72 14.7 0.74 S.D. 42.7 0.034 0.104 0.035 1.56 0.019

N 6 6 6 6 6 6 

# * 20 MEAN 410. 0.50 4.15 0.69 12.3 0.76 S.D. 27.8 0.038 0.108 0.066 1.05 0.060

N 6 6 6 6 6 6 140 MEAN 417. 0.50 4.00 0.74 13.5 0.76 S.D. 32.7 0.030 0.208 0.063 1.78 0.089

N 6 6 6 6 6 6 1000 MEAN 414. 0.50 4.05 0.69 12.9 0.75 S.D. 33.7 0.045 0.238 0.062 1.00 0.079

N 6 6 6 6 6 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<0.05.



## o o o**~** \.-- \**~** 33 - 

### TABLE 9 

### -FS-M- 1 IV

# ---------------------------------------------------------------------------------------------------------------------------------------- TABLE 9 - FS - F - 1 STUDY NO. 4l436CEO ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT)

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: FE"ALE ANI"ALS KILLED ON SCHEDULE ( 4 WEEK) FINAL DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES

MG/KG WEIGHT (G) (X 

### 10 ") (X 

### 10 ")
J 

### 0 MEAN 266. 0.74 3.82 0.70 28.8 40.5 S.D. 27.8 0.073 0.268 0.048 3.02 3.87

N 6 6 6 6 6 6 20 MEAN 255. 0.76 3.72 

### 0.74 30.1 46.7 S.D. 21.4 0.092 0.166 0.034 5.80 7.10

N 

### 6 6 6 6 6 6 140 MEAN 260. 0.74 3.71 0.69 26.1 40.9 S.D. 19.9 0.079 0.145 0.039 3.45 2.31 'N 6 6 6 6 6 6

## * 1000 MEAN 251. 0.79 3.63 0.74 29.4 48.0 S.D. 10.5 0.033 0.146 0.071 2.55 3.90

N 6 6 6 6 6 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

## * , P<0.05.



# o o o**~ ~.,)**  Cil 

### 34 - TABLE 9 -FS-F- 1

### TABLE 9 - RS - M- 1 STUDY NO. 4L436CEO ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT)

- GROUP MEAN VALUES TEST ARTICLES: YDC-1312 SEX: MALE ANIMALS KILLED ON SCHEDULE (RECOVERY) fINAL DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES

MG/KG 

### WEIGHT (G) (x 

### 10 ")
0 

### MEAN 479. 0.45 4.04 0.62 11.8 0.69 S.D. 16.4 0.008 0.294 0.029 0.56 0.033

N 

### 6 6 6 6 6 6 

## * * 1000 MEAN 474. 0.43 4.12 0.68 12.3 0.68 S.D. 23.8 

### 0.016 0.247 0.052 1.56 0.041

N 6 6 6 6 6 6 

## * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , 

# * , P<0.05.



# o o o**~** "',", 

# ..... r:J)
35

- TABLE 9 -RS-M- 1

### DOSE LEVEL 

### MG/KG
0 MEAN S.D.

N 

### 1000 MEAN S.O.

N



## o o o**~** 

### " 

# , t
..... 'j 

### ORGAN WEIGHT TABLE 9 - RS - F - 1 (RELATIVE : PERCENTAGE OF BODY WEIGHT)

- GROUP MEAN TEST ARTICLES: YDC-1312 SEX: FEMALE ANIMALS KILLED ON SCHEDULE (RECOVERY) VALUES STUDY NO.**~L436CEO**



### FINAL BODY WEIGHT

(G) 

### BRAIN LIVER KIDNEYS ADRENALS 

## ex 10 ") OVARIES 

## ex 10 ") 291.

17.3

6 

### 0.68 0.038 6 

### 3.59 0.287 6 0.62 0.042 6 26.7 4.67

6 36.S 

### 6.11

6 

### 291­

20.6

6 0.67 0.047 6 3.71 

### 0.309 6 0.63 

### 0.029 6 27.3 3.84

6 35.6 1.89

6

36

- TABLE 9 -RS-F- 1

### TABLE 10 STUDY NO. 4L436CEO INCIDENCE OF NECROPSY FINDINGS TEST ARTICLES : YDC-1312 Period 28 days Recovery Sex Male Female Male Female Organ

Findings Dose level(mg/kg) Number of animals 

# o 6 

### 20 140 1000 666 

## o 6 

### 20 6 

### 140 6 

### 1000 6 

## o 6 

### 1000 6 

# o 6 

### 1000 6 

### Liver

Hepatodiaphragmatic nodule Impression Yellow patch 0 1 1 0 0 

### 0 1 0 0 

### 0 0 0 

### 0 0 0 

### 0 0 0 

### 0 0 

### 0 0 0 

### 0 0 0 0 0 0 0 

### 0 0 0 

### 0 0 

### 0 Lung

Edema 0 

### 0 0 

### 0 0 1 0 0 0 0 0 0 Red patch 0 0 0 

### 1 0 0 0 0 

### 0 0 0 0 Pituitary

White patch 0 

### 1 0 0 0 0 0 0 0 0 0 

### 0



# o o 

## o**~** 

# ,**~'.**

### ....



# , 00

- 37 - 

### TABLE 10

## TABLE 11 - 1 STUDY NO. 4L436CEO INCIDENCE OF HISTOLOGICAL FINDINGS TEST ARTICLES : YDC-1312 Period 28 days Recovery Sex Male Female Male Female Organ Dose level(mg/kg) 

# o 20 140 1000 

# o 20 140 1000 

## o 1000 

## o 1000



### Findings Number of animals 6 

### 666 6 

### 666 6 6 

### 6 6

•••••••••••• _ ••••••••.••••••••••••**~** ••• •••••**~** •••••• _ ••• .......... •••• _ ....**~** ..... ..... "
.... *
......................... ......**~_** .....**~** ... _ .. •• •• __ w. __ ........_ ..........**~** .. ..•._._**~** ••**_.~ _~** **_.~** ••**_~~._"._.~ _.~** **~ _~** **~ _~._** * 

### Heart (Grade)

Inflammatory cell infiltration, focal (+) 

### 2 2 

## o 1 

## * * * 

## * * * 

## * * Liver

Bile duct hyperplasia (+) 

## o o 1 

## o o 

## o 

# o o 

## o 

# o 

# o o Fibrosis, focal (+) 1 

# o 1 

# o o o 

# o o 

## o o 

# o o Inflammatory cell infiltration, focal (+) 2 

### 1 

### 3 1 

### 2 2 

### 3 1 1 

### 2 2 

## o Microgranuloma (+) 

### 6 3 

### 2 

### 5 6 

### 2 3 

### 5 3 4 11 

### 5 

### Necrosis,·focal (+) 

# o o 1 1 

## o o o o 

# o o 

## o o Vacuolation, hepatocyte, focal (+) 

# o o o 

# o o 1 

## o 1 

## o 

# o o o Kidney

Basophilic change, tubular epithelium (+) 

### 5 4 

### 5 6 4 4 

### 5 3 

## * * * 

## * (++ ) 

## o 

# o o o 

# o o o 2 

## * * * 

## * Calcification, corticomedullary

junction (+) 

## o 

# o 1 

# o o o o o 

# * * * 

## * Cast, hyaline (+) 

## o o 

# o o 

# o * * o o 2 

## * * Cyst (+) 

# o o o 

# o o * * 1 

## o o * * Increase of hyaline droplet,

proximal tubular epithelium (+) 

## o 

# o o 4 

# o o o 1 

## * * * 

## * (++ ) 

# o o 

## o 1 

# o o o o 

## * * * * Inflammatory cell infiltration, focal (+) 2 2 1 

### 1 

# o 1 

# o 1 

## * * * 

## * Adrenal 

# o Vacuolation, cortical cell (+) 1 1 

# o o 

## * * 

## * * * * * * 

# o**(~** 

### Spleen NO 

## NO NO 

### NO 

## * 

# * * * 

## * * * ***~ ~\,j**  + 

### Slight, ++ : 

### Moderate. 

### <.D 

## * Not examined, NO : No abnormalities were detected. 38

- 

## TABLE 11 - 1

### TABLE 11 - 2 STUDY NO
.. 4L436CEO INCIDENCE OF HISTOROGICAL FINDINGS TEST ARTICLES : YDC-1312 Period 

### 28 days Recovery Sex Male Female Male Female

.. '" ...**~** ...**~** '" " 

### . ".......... " ................ ".......... ....................................................... ....................... . .................. 

### Organ Dose level(mg/kg) 0 20 

### 140 1000 

# a 20 140 1000 

## a 1000 0 1000

Findings Number of animals 6 6 6 6 6 6 6 6 6 6 6 6

.. . ............ "..
. ... ........ . .......... " .. " .. ........................ , .................. " ...................................... " .......................... " ........................................ " ........ , .. .. .... .. ............ " .. -......**~** 

## ... .. .. .... .. ....... " ........... " ......... '" ..... " .. "**~** 

### (Grade) Ovary NO 

## * * NO 

## * * Lung

Accumulation of foam cell 

## (+) 

## * * 

## * l/U 

## * O/U 

## * * * * * * Edema, focal 

## (+) 

## * * * 011* 

## * 1/U 

## * * * * * * Pituitary

Cyst. anterior lobe 

## (+) 

## * l/U 

## * * * * * * * * * 

## *
+ 

### Slight. 

## * : Not examined. # 

### Number of animals with lesion / number of animals examined, NO No abnormalities were detected.



# o o 

# "**~** t' • V! 

# o 39 - 

### . TABLE 11 - 2

### Ready BiodegradabilityTestofYDC-1812 (English version)
StUf1y No. 

### 4FOO8(:3)G StudyTitle Ready BiodegradabilityTestofYDC-l:312 Sponsor Tohto Kasei CO., LTD
This test was conducted in Kitakyusyu Laboratory, Mitsubishi-Kasei Institute of Toxicological and Environmental Sciences LTD, I-I 

### Kurosakishiroishi. Yahatanishi-ku, Kitakyusyu-shi, Fukuoka 806, Japan.

This reportis the English version ofthe original, which was written in Japanese. The undersigned hereby declare thatthisversion faithfully reflects the original to the best. of our knowledge.

Translated by /.2 

## , 2... C' ,') 

### I Takayoshi Hosono Manager for Technical Division Tokyo Head Office Tohto Kasei CO., LTD

Approved by

-"1 )\::1

### --~.J-'[_"1-..'

__ X-_-'

__ 

## !:J_""_-,-lJr.-LI7J"'--___ Date . 

### Koyo Murakami 

### General Manager 

### for Technical Division Tokyo 

### Head Office, 

### 'Ibhto Kasei 

### CO
.. LTD



## 0001:3.1

### COMPLIANCE WITH THE GLP STANDARDS
St.udy No. 4F008(:3)G St.udy Tit.le Ready Biodegradability Test.ofYDC·1812 Sponsor Toht.o Kaflei CO., LTD

The st.udy described in this report was conduct.ed in compliance with t.he Good**L~lborat.ory** 

### Practice (GLP) regulations recognized by the Basic Indust.ries Bureau oft.he Japanose Minist.ry ofIntklrnat.iona\ Trade and Indust.ry
.

Facility Management Tadashi Entlo Sealed dat.e :June 22, 1994 Mitsubishi-Kasei Institute ofToxico\ogical and Environmental SciencesLTD(MITES)



## 0001.,32QUALITY ASSURANCE STATEMENT Study No. 4F008(:3)G St.udy Tit.le Ready Biodegradabilit.y TestofYDC·l:312 Sponsor Tohto Kasei CO
.. LTD We hereby cert.ify t.hat. the above study has been performed in accordance with the Good Laboratory Practice (GLP) regulations recognized by t.he Basic Industries Bureau ofthe Japanese Minist.ry ofInternational Trade and Industry.

Dat.es of inspections and**reporting~** to t.he Study Director and t.he Facility Management. are as follows:



### Jn·progress study Final report Date of Inspection May 

### 13. 1994 June 10. 

### 1994 June 

### 22, 1994 Date of Report.ing May 

### 1:3. 1994 June 10, 

### 1994 June 

### 22. 1994 Quality Assurance Personnel Takeshi Miura Sealed date: June, 

### 22 1994STUDY PERSONNEL Study Personnel St.udy Supervisor: Deputy Facility Management Katsumi Inoue sealed St.udy Director: Shiro Furukawa sealed Yoshihiro Kusumoto sealed



## 0001.34

### CONTENTS Page THE TEST PROGRESS 6 SYNOPSffi 7 ] TITLE. PURPOSE 8

1.1 TITLE OF STUDY 8 1.2 AIM OF THE STUDY 8 

### 1.:3 TEST METHOD 8 1.4 COMPLIANCE WITH GLP 8 1.5 TEST PERIOD 8 ].6 STORAGE OF THE DOCUMENTS AND SAMPLE 8 2 TEST SUBSTANCE 8

2.1 CHEMICAL NAME, CHEMICAL STRUCTURAL. PHYSICAL 8 AND CHEMICAL PROPERTIES 2.2 SAMPLE TO BE SUPPLIED 9 2.:3 IDENTIFICATION OF THE SAMPLE 9 2.4 CONFIRMATION OF SAMPLE'S STABILITY 9 3 TEST METHODS 10

:3.1 TEST CONDITIONS FOR BIODEGRADABILITY MEASUREMENT 10 3.2 BOD MEASUREMENT 11 3.3 PRETREATMENT PROCEDURE FORTHE TEST SOLUTION 11 AFTER ITS BOD MEASUREMENT 3.4 pH MEASUREMENT 12 :3.5 DOC MEASUREMENT 12 :3.6 RESIDUALYDC·1312 MEASUREMENT 12 3.7 CALCULATION FOR BIODEGRADABILITY ]3 4 RESULTS AND DISCUSSION 14

4.1 ENVIRONMENTAL FACTORS WHICH HAVE AFFECTED 14 TEST RESULTS RELIABILITY 4.2 OBSERVATIONS OFTEST SOLUTIONS DURING 14 BOD MEASURING TESTS 4.:3 RESULTS OFpH MEASUREMENT 14 4.1 BIODEGRADABILITY PERFORMANCE BY ACTIVATED SLUDGE 14 4.5 BOD BASIS BIODEGRADABILITY ] 4 4.6 DOC BASIS BIODEGRADABILITY 15 4.7 RESIDUALYDC·]:312 CONCENTRATION SASIS 15 BIODEGRADABILITY Ij 

### CONCLUSION 11)



### TABLES AND FIGURES 

### 16·:32



# OOO:1asTHE TEST PROGRESS 

### The biodegradability t.est. of YDC-l:312 was at. first started as test. number of 4FOO8G on April 12th. 1994 and measurement.s of BOD values start.ed on April 18t.h. 1994.However. on day 7 of BOD measurement, April 25t.h. BOD basis decomposition of aniline as reference substance of t.he decomposition did not exceed 40 % and subsequently t.he decomposit.ion test was terminat.ed on the above same day. Furt.hermore, t.he second biodegradabilit.y t.est of YDC-1312 as t.est number of 4FOO8(2)G was start.ed on April 26th, 1994 and measurementsofBOD values started on April 27th, 1994. However. on day 7 of BOD measurement, May 4th, BOD basis decomposit.ion of aniline as reference substance of the decomposition was did not. exceed 40% and subsequently the decomposit.ion test was terminated on the above same day. The study with test number of 4FOO8(3)G for biodegradabilit.y test of YDC-1312 was carried out by use oft.he same test met.hod as the test number of4FOO8G and the same lot number oftest sample as the study. Results concerning the identification of test substance YDC·1312, the calibrat.ion and recovery test ofYDC-1312 described in this report were obtained from test number of4FOO8G and included in this study report.



### (;SYNOPSIS Title ofStudy Ready biodegradability test ofYDC·l:31 2(Test No. : 4F008(:3)G) Test method The test.s were conducted in accordance wit.h t.he test. met.hod concern:ng Chemical Substances Cont.rol Law (: the not.ice defined by Japanese Law 117, 197:3). (Content.s oftest.s) No.1 : Confirmative testofbiodegradabilit.y by using a system ofaniline. activated sludge and basal medium No.2 : Basic respirat.ion testby using a system of acHvated sludge and basal medium No.:3
........5 : Biodegradabilit.y tests ·1,2,and3 ofYDC·1312 by using a syst.em YDC13]2, activat.ed sludge and basal medium No.6 : Stabilit.y ofYDC-1312 in water by using a system ofYDC·]312 and water (Items to be measured) (a) Biochemical oxygen demand(BOD) (for 28days) (b) Dissolved organic carbon(DOC) (After 28day test) (c) Residual YDC·1312 content.by HPLC (After 28day test) [measured by high performance liquid chromatography] 28day-Testresults "'" Measured Results(A Measurement fter 2&lay test) Biodegrad 

### 1 abilit.y 

### 2 Test

### * 3 Stabilit.y in water Theoretical value BOD, mg 3.1 

### -0.9 -1.7 0.8 

### 73.2 DOC, mg/l 

### 0.0 3.2 2.4 1.] 73.9 YDC·1312, mg/l 

### 102.1 102.2 103.5 100.:3 100.0 *) Calibrated values to have compensate basic respiration basis value for each t.est Biodegradahility aft.er 28 day test. Degradabj)ity Biodegradability Test BOD basis value 

### 4 -1 

### .:2 DOC basis value No calculat.ion**du(~** to its difficult solubility in waIN YDC·]:312 basis value 

### -:2 -:2**(~oncl** usion

. II has come to a conclusion that. YDC-l:312 waF> not. wadily biod<'gradahle because

it.s 28 day BOD hasis <!egradahilily is 

### 4% or less and r(>si(\ual YDC·]3]2 basis one is -2% or Ipss.

7

### 1. TITLE, PURPOSE 1.1 Title ofstudy Biodegradability te8t

### ofYDC-1812 1.2 Aim ofthe study The study was performed t.o obtain data on biodegradability and stabilit.y in water required for the application ofYDC·1:312 as new chemical substance in accordance with t.he Chemical Substance Control Law. 1.:3 Test method The t.e8tS 

### were carried out in accordance with the test method concerning the Chemical Substance Cont.rol Law (Japanese Law] 17, 197:3). 1.4 Compliance wit.h GLP The tests were conducted in compliance with Good Laboratory Practice (GLP) defined by t.he above mentioned Law. 1.5 Testperiod May 13, to June 22, 1994 (Where BOD measurements were made from May 13, to June 10, 1994) 1.6 

### Storage ofthe documents and samples The documents and sample which will be 

### archived atthe Kitakyushu Laboratory ofMitsubishi chemical Safety Institute Ltd. For ten years are described in the following:

1) 

### Protocol. 2) 

### Testreport. :3) 

### Raw data. 4) Quality assurance reports. 5) Sam])le (about 2g) 6) Miscellaneous 2. Test substance

H 

## 0001.38

# ~\a~~**~C\~~**

,



## 000:1398. Test Met.hods The test.s were carried out. in accordance with t.he test. method as requ,jred hy the Chemical Subst.ance Cont.rol Law (Japanese Law 117. 197:3). The test. subst.ance was exposed to the act.ivated sludge in a closed-system oxygen consumpt.ion measuring apparatus. The biochemical oxygen demand (BOD) was measured over a 28 day period. After this period, the concen:.rations of the dissolved organic carbon (DOC) and the residual test subst.ance in t.he test. bottleswere measured. The biodegradability oft.he test subst.ance was evaluated from t.hese result.s. 8.1 Testconditions for biodegradabilit.y measurement. Biodegradability tests were conduct.ed in the following conditions. (SbmtiardAct.ivat.ed Sludge) l)Mixed liquor suspended solid (MLSS) : 5700mg/l 2)Source oCthe sludge Chemical Biotesting Center. Chemical Inspect.ion and Testing Institut.e. Ja})an :3) Date ofreceipt. May 10, 1994 (restcondit.ion) 1) Temperature 25±1 

### °C 2) Exposure period 28 days 8) Testvolume 300ml 4) Concentrations YDC-1312 (test substance) 100mgIl Aniline*1 100mgil Activated sludge 30mgIl *1 :Aniline: Reference substance for the biodegradabilit.y for YDC-1312. KANTO CHEMICAL Co., INC. Special grade. 

### Lot No. 503E1288) (Contents oftest.s and chemicals) Test No.1 : Confirmative test for biodegradability in the system ofaniline. act.ivated sludge and hasal medium. Procedure for No.1 (1) Take baf.:al medium*2 into a culturing bottIe. (2) Add weighed 29,u1(30 mg) aniline to the medium and mix t.hem. (8) Add standard activated sludge to t.he mixture. TtH;l No.2 : Basic I'ef.:piration test in t.he system ofact.ivated sludge and basal medium. Procedure for No.2 (1) 1'Hke basal medium*2 into a culturing bottle. (2) Ad(l st.andard aetivat.ed sludge to the mixt.ure. Test No.:)- No.5: Biodpgradabilily test. for 

### YDC-l:312 in 

### tlw system ofYDC-1 :312. activat.ed sludge and basal medium. Pl'oeedure for No.a 10 No.5 (J) 

### Take hasal mp(lium*2 into 

### a culturing 

### boull', (2) Ad(l w('iglwd:30 mg YDC-l:312to Ill!' hasal mC'dium and
10 

## 0001-::0mix t.hem. (:3) Add s1.amhml activated sludge fo the mixture. Test No.6 : Stability ofYDC·l:312 in water by using a system ofYDC-l:312 ami water Procedure for NO.6 :30mg ofYDC·l:312 was aclded to 300 ml ofdouble-disfillecl water (purified by Milli.Q. >10 Mohm·cm). *2Quantjty ofbasal medium is adjust.ed to be the one ofwhich is equal fo 300ml

volume ofact.ivated sludge to 

### be added. 3.2 BOD measurement. BOD was measured by use of the following equipment and also observations were made for t.he contentinside t.he culturing bottle during t.he measurement. 1) BOD measurement unit: closed type ofoxygen consuming measuring unit Ohkura Electric Co
.. Model OM·2001 (MITES 

### 10 No.B) 3.3 Pretreatment.procedure for the t.est solution after its BOD measurement Procedures for the pret.reatmentwere made in order to measure 

### pH. DOC, residual YDC·l:312 in the test. solution in accordance with the following scheme. 300ml t.est. solutjon Sample for DOC measurement Procedure for DOC measurement. (1) Take 10m) the test solution into lOml centrifuge tube by use of lOmlllipet. and centrifuge itfor lOminutes. (2) Take 5ml centrifuged solution into Saml»)e bottle and inject itinto DOC meter. Sample for 

### pH measurement Procedure for DOC measurement (1) Take 20m) test solut.ion to a suit.able size ofbeaker anel measure it wit.h pii me1.er. (2) Transfer t.he solution to t.he funnel forYDC·]:312 measurement. Sample for residual YDC-l:312 measurement. Procedure for residual YDC-1312 measurement. (1)Mix the above centrifuged test solution and activafed sludge and the residual test. solution before it.s centrifuge. (2)Transfer the mixture into 500ml liquid separation f'unnpJ and ext.ract it wit.h IOOml chloroform for 5 minut{'s shaking twice. (:3) Filtrate the chluruform lay<'r lind dewau>r it through a 11G:3type giasH filter muunted wit.h 40g ofsodium f>ulfate anhydrous. (4) Transfer the filtrate 10 a rotary evaporalor and concentrate to dryness at b'mIWrnluf(l lowol' than 4ctC using wal.er hath. (fi) Dissolve t1w re!-;idue with ac(>lonitril<' to 25 In\.


### 11 

## 0001. :1

### (5) Take acetonitrile solution t.o 2ml samilling bottle and inject it to HPLC t.o measure YDC·1812. :3.4 pH measurement pH value was measured t.o confirm pH change during BOD measurement: Apparat.us : pH met.er . Table type pH meter (fOA Denpa Co
.. Model HM·:30S) :3.5 DOC measurement The measuring unit. for DOC is described t.ogether with 

### it'> operat.ing condibons in 

### t.he following. (I)Apparatu8 Tot.al Organic Carbon analyzer. Shimazu Corp.(Japan) Model TOC-5000 (2)Measuring Furnace temperat.ure 680"(; (fC). Conditions Air flow rate 150mllmin. Measurement. sensitivity level 

# xa Injection quantity lOttl (:3) 

### Calibrat.ion curve The following st.andard solutions were injected int.o the TOC analyzer. The calibrat.ion curve was prepared by t.he data processor ofthe analyzer. St.andard solutions TC(t.otal carbon) 20 and 50 mg CII aqueous solutions ofpotassium biphthalate. I 

### C(inorganic carbon) 

# omg CII deionized water (purified by Milli-Q) and 10 mg

# cn aqueous solution ofsodium hydrogen carbonate and sodium carbonate. (4) 

### Measurement. Pretreated test solution in accordance with 

### § 

### a.:3 was used for DOC measurement. 8.6 ResidualYDC·1312 measurE'ment Residual YDC·131 2 after itsbiodegradability test was determined by a HPLC system which is de-scribed in t.he following: (1) 

### Apparat.us HPLC Pump Yokogawa Electric Co., Model HP79851A Ultra viol(>t det.ect.or Yokogawa Electric Co., Model HP79853C Automatic sampler System 

### I nst.rument Co.. Model 2:3 LC work !-:ilalion Yokogawa Electric Co., Model WSI050 (2) 

### M(>asuring ('onditions Column YMC 

### Co.. YMC·Pack ODS·A A·302, 4.6mmLd. 

### x 150mm Eluent Acelonitrile/double·distilled wa1.er 

### = 80/20 

### Flow**nth~** 

### Iml/min. D('h>clion wav('I(>ngth 280nm Allpnualioll (deh>el.or) 2 

### AU/FS**I~** 

## 0001/12Inject.ion quantity 

### 20.£11 (:3) Calibrat.ion curve The st.andard solutions of t.he test substance wit.h 

### O. 500. 1000. and 2000 mg/l in Acetonit.rile were prepared and analyzed. The peak area oft.he YDC·1812 was plotted against t.he concentration. The correlation coefficient. was calculated to be 1.0 by the method described in Japanese Indust.rial Standards Z9041-1968. Concentration of the YDC·1812 for each Test No. was calculated from ratio of peak area for t.he sample t.o t.hat. for the standard solution wit.h 1000.0 mg/l in Acet.onitrile. [Figure :3 (P ·21) and Figure 4 (P·22-2:3)] (4) Recovory t.est Each 

### testbottle (described in 

### § 3.1) were kept in a closed-syst.em oxygen consumptjon measuring apparat.us for 30 minut.es
. Each test. solut.ion was treated by the procedures deRCribed in 

### §

## a.:3. The concent.ratjon of**t.h(~** YDC·l:312 was measured by HPLC. The average recovery of the YDC·1312 was 96%. The concent.ratjon ofthe residual YDC·l:312 were corrected with the average recovery value. [Table :3 (P·18) and Figure 5 (p·24-25)] (I) Det.ectjon limit The detection limitoft.he test substance was calculated to 

### be 1 mgll based on t.he minimum detectable peak area of 10 mAU
. sec for the peak in the HPLC chromatogram. [Table 3 (P-18) and Figure 5 (P·24-25)] (6) Measurements Pretreated test solution in accordance with 

### § :3.3 was used for residual YDC·l:312 measurement.. :3.7 Calculation for biodegradability Three different kinds ofcalculation formulas are used for the biodegradability in the following: 1) BOD basis biodegradability (for 7.14,21, and 28 <lays t.est.s) Biodegradability(%) = (BODs

- BOOb) IThoD x 100 where BODs Oxygen commmp1.ion (mg) for t.he confirmat.iv(> 

### test or for YDC·l:312 biodegradahility. 

### BOOb Oxygen commmpt.ion (mg) for the basic re8pirat.ion of act.ivated sludge. ThaD TheorC'lical oxygen opmlmd (mg) for aniline or YDC·1312. Calculat.ion for 1.heoret.ical oxygl>n demand(ThoD) 

### Aniline : 

### nO.2 mg**O~I** 

### :30 mg·aniline Aniline iR at;sumed In 1)(' oxidized in acconlanc(> wilh 

### tilt-! following



## 0001/13chemical react.ion. C.,H7N 

## + 85/402**~** 

### 6COz 

## + 7/211. 2

### 0 

## + N02 YDC-1812 : 7:3.2 mg O2/80 mg-YDC-1312 YDC-l :312 is assumed to be oxidized in accordance with the following C 2oH 3 /J04 

## + 511202**~** 

### 20CO;z 

## + 15H20 2) DOC basis biodegradation for 28 days test. Biodegradabilit.y(%) = [1

- (DOCs

- DOCb) 

### 1DOCc]

### x 100 DOCs DOC (mg/l) for YDC-1:312 DOCl> DOC (mgll) for 1J1e basic respiration DOCc DOC (mg/l) for t.he stability t.est: in water 

### :3) YDC-1812 basis biodegradability for 28 days t.est Biodegradability(%)

# =(1 - Cs

### 1Cc )

## x 100 Cs YDC-1312 concentrat.ion (mgll) aft.er itsbiodegradability test Cc : YDC·1312 concentrat.ion (mgll) after its stabilit.y test in water 4 Results and Discussion 4.1 Environmental factors which have affected test results reliability Any environment.al phenomena which have affected test. results qualit.y were not observed. 4.2 Observations oftest solut.ions during BOD measuring tests 1) Color oftest solution was pale yeJJow for No.1 confirmat.ive test. in t.he system ofaniline, activated sludge and basal medium. 2) The test solution wascolorless and transparent for No.2 test for basic respiration by activated sludge, No.3, 4, 5 t.ests for YDC-1812 biodegradability and No.6 test for the stabilit.y t.('.st ofYDC-1312 in the water. :3) Growth ofthe sludge was observed in confirmatjve testbott.le compared 10 t.he basic respiration system(No.2). No growth oft.he sludge was observed in YDC- 1312 degradability systems, No.3,4,5. 4.3 Result.s ofpH measurement The 

### pH values were 6.4, G.8. and 7.1 for No.3. No.4. and No.5 tests. The pH values was 7.2 after the st.ability test in the water. ITable 1 (P-16) 

## 1 4.4 Biodegradability )lPrformance by activated l;lu<lge BOD basif.; biodegradability was 60% based on 7 days confirmative 1£':;;1. u!-;ing anilinp. The result indicated I.lulI l.Iw f.;ludge hl-ld good peri'orml-lnc(' f()r aniline de!{radability. ITHbl<> 1 (P·16) l-lnd Figun' G(P·2G--2D)] 4.5 BOD baHi!> biodegradability (1) Tlworptical vahH' (lfBOJ) tl8 <h!y!') for YDC·1312 is 

### n.2 mg. (2) Act.ual 

## non**valu<~** ohtainpd frum tlw h'st.s fur YDC·l:312 were**:~.I,**
11 

## 0001.'145 ·0,9, and -1.7 mg after calibration using sludge basic respiration value, (:3) BOD value for the stability test using YDC-1312 was 0,8 mg, (4) BOD basis biodegradabiliti(ls were calculated to be 4
.. 

### 1. and -2 

### % by

use ofthe above valu(l, [Table 1 (P ·16) and Figure 6 (P·26-29)] 4,6 DOC basis biodegradability (1) DOC ofsample to be used was 78,9 mg/I. (2) On the other hand, DOC for biodegradable tests for YDC·l:312 was 0,0, :3,2, and 2.4 mg/l after t.he same calibration as the above one, (3) DOC for the stability t.est ofYDC-1312 in water wasmeasured to be 1.1 mglJ, (28 days test) (4) However, DOC basis biodegradability was not calculaled because YDC-1312 was almost insoluble in water, [Table I (P·16) and Table 2 (P-I7)] 4,7 Residual YDC·1312 concentration basis biodegradability (1) YDC·1312 concentratjon to be used for the testwas 100 mg/1. (2) Remaining YDC·l:3l2 concentration after the tests were 102.1, 102,2, and 103.5 mgll, (3) Remaining YDC-] 312 after the stability testwas measured to be 100,:3 mgll. (4) YDC-13l2 basis degradability was calculated to be -2, ·2, and -3 

## %. " [Table 1 (P·16), Table 4 (p·18) and Figure 7 (P·30-32)} Conclusion It has come t.o a conclusion thatYDC·1312 was notreadily biodegradable because its 28 day BOD basis degradabilit.y is 4% or less and residual YDC·1312 basisone is -2% or less.

I;' 

## 0001/15

## ----



# ---- Table 1 

## Test results

Test No. Test systems Test samples ThOD BOD basis dee-adability added. mg 7 days 14 days mg BOD Degrad- BOD Degradmg ability " mg ability " No.1 Confirmative test aniline 90.2 55.6 60 67.9 68 ofbiodegradabilitv 30.0 No.2 Basic respiration 

# ---­ -­ 1.9 

# -­ 6.5 

## -­ test ofactivated sludf:es No.3 Biodegradability YDC-1312 73.2 1.9 0 

### 6.9 1 tests-l of YDC-1312 30.0 No.4 Biodegradability YDC-1312 73.2 0.5 -2 5.0 -2 tests-2 ofYDC-1312 30.0 No.5 Biodegradability YDC-1312 73.2 1.9 

### a 6.0 -1 test8-3 ofYDC-1312 30.0 No.6 Stability of YDC-1312 73.2 0.0 

# -­ 0.2 

# -­

_ 21>Q=-1312
_____ 30.0



## - 21 days 28 days BOD

ma­ . 70.0

7.3 8.4 5.4 6.2 0,3

-- Degrad- BOD Degradability " mg abilitv " 69 73.7 71



# -­ 10.0 

# -­ 2 13.1 4 -3 9.1 -1 -2 8.3 -2



# -­ 0.8 

# -­

- pH I Start Rnish



# -­ 6.9 7.0 7.1



# -­ 6.4



## -­ 6.8



## -­ 7.1

6.2 7.2 Test sample: aniline for No.1, YDC-1312 for No.3-No.6 Where % degradability was calculated to be negative. this value is shown in brackets.
Test No.
No.2 No.3 No.4 No.5 No.6 Test systems
Basic respiration test ofactivated sludges Biodegradability tests-1 ofYDC-1312 Biodegradability tests-2 ofYDC-1312 Biodegradability tests-3 ofYDC-1312 Stability of YDC-1312 Origanic carbon added mg/l
73.2 73.2 73.2 73.2 DOC basis degradability TC DOC' mg/l IC mg/l mg/I 1.1 0.0 1.1 1.1 0.0 1.1 0.0 4.3 4.3 0.0 3.5 3.5 0.0 1.1 1.1 1 Degradability 

# " 1

. No calculation

, No calculation j I No calculation

. Residual YDC-1312 basis degradabilitv 

### I concentration degradability I mf:/1 % <1 -2 102.1 -2 102.2 -3 103.5 100.3

-_._....... _--­ '-­ DOC basis biodegradability was not calculated due to the insolubility ofYDC-1312 in water. Where % degradability was calculated to be negative, 

# o this value is showt. in brackets. 

## o 

# o**~ ~** **a·~**

# ----------------

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

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

# ---------------- Table 2 DOC measurements (28 days) standard solution No.2 test system No.4 test system No.6 test system 

## TC 

# 50mg/l 

# TC TC TC STD-HI 50.0 St1PL01 

### sr1PL03 

### SMPU35

TC 01 010016 

### TC 01 

### 000000 1.1 

### TC 01 000741 

### 4.7 TC 01000000 1.1 TC e2 010249 

### TC 02 

### e00ea0 1.1 

### TC 02 000&14 4.1 TC 02 000000 1.1 TC 03 009861 

### TC 03 000000 1.1 

### TC 03 e00611 4.1 TC 03 000008 1.1

i1

### Ehi-I 

### 0-H3

# e J 

### 2 MEAN 080088 1.1 

### MEAN 000&55 

### 4.3 tlEAN 

### e0008e 'J • -I 

## T C ZOn/) 

### I C 

## I C I C ;:TCI-LO 

### 20.e :::r-1F'L0-1 

### SHPL03 Sr-tPL05

TC 01 0039'14 IC 01 0aB00a 0.8 IC 01 000000 0.0 IC 01 90000e 0.0 

## ;c**~:2** 012:!03 

### IC 02 000000 0.8 IC 02 000000 0.0 IC 02 000000 0.8 

## rc 03 (H33945 

### IC 03 000000 0.0 IC 03 0000000.0 IC 03 000000 8.8 

### TC+i.34 003770 _t:j", No use due- to ....J '.'"
.abnorma1 value. 

### MEAN ee0000 0.0 

### MEAN 00000e 0.0 MEAN 000880 a.e i'1EM1 00387E. 

### I C lOmg/l 

## No.3 test system NO.5 

## test system Final results (mg/l) ST(I-HI -1 €I •'3 

## TC 

## TC SMPL01 iC '1. ")' 

### No.2

## Tes~ systel!l
. 

# o baslc resplratlon SHPL02 

### SNPL04 IC 

### by sludge IC 81 OB44JB TOC 1 • 1 TC 0 -1 

### 000000 1.1 

### TC e1 000467 3.4 IC 02 084188 

### SMPLC12 TC 

### L 1'; No.3 Test system TC 02 000000 1. '1 

### TC 02 000530 3.7 

# e YDC-1312 degradability IC 

### BJ 00415B IC TC 03 0eaaee 1 • -1 

### TC 03 000506 3.6 TOC -1. 1**~tEAN** 

### 0134256 SMPL03 TC 

# 4.3; No.4 Test system

MEAN 000000 1. 1 MEAN 

### 000501 3.5 

## o YDC-1312 degradability IC 

### I C Omg/ I 

### I C I C 

# 4.3 TOC 

### SMPL04 

### SMPL..04 TC 

# 3.S\- No.5 Test system SMPL02 

### STO-LO e.0 

## Ie**3.~~** 

### YDC-1312 degradability IC 01 00ee0e a.e 

### TOC IC 01 000000 0.0 IC 

### 01 00Beee 

### IS 92**~B99"e** 

### BieT sr1PL05 TC 

### No.6 Test system IC 02 000000 0.0

. o'tJSe ue to 

# L-t, o Ie 82 (H3eeee 

### Stability of YDC-1312 -02 abnormal value • 

## IC 8 

### Ie 03 

### 000000 0.0 

# o Ie 03 (I(1eB00 in 

### water Ie 92 000000 0.0 TOC 

### "'-1 

# o ---------------­ IC 03.000000 0.0**.~** 

### MEAN 088880 0.8 ttErHl 013130£10**~** 

### ..,1 MEAN 800000 0.0

## Table 3 Calculation-ofrecovery and detection limit Test systems Peak area mAU·sec Concentration in solution m

### til Recovered mass

mg (D) 

### Added mass

mg (E) 

### Recoverry 

### % (F) 

### Detection limit mgll (G) 

### (A) for HPLC analysis 

### (8) in culturing bottle

(0) 

### Recovery test 1 10063.03 1172.5 97.7 29.3 30.0 98 --I 

### Recovery test 2 9816.39 1143.8 95.3 28.6 30.0 95 

# - Blank <10.00 <1.2 <0.1 

# -­ 0.0 

# -­ 1 ·Average recovery; 100% Standard solution Concentration (H) : l000mgll Peak area (I) : 

### 8582.61mAU•sec Volume of solution for HPLC analysis (J) : 

### 0.0251 Volume of test solution for HPLC analysis (K) : 0.300 I Equation :B=HxA+I C=HXA+tXJ+K O=B x J F=

### 0+ EX 100 G=C raise decimal fractions unit. 

## Table 4 Residual YDC-1312 ::x: 

### Test No. Test systems Peak ar.ea Concentration in solution mAU·sec mu'l for HPLC in culturing

analysis bottle (A) 

### 18) (C) No.2 Basic respiration test of activated sludges <10 <1 

# <1 No.3 Biodegradability tests-l ofYDC-1312 11486.75 1156.9 102.1 No.4 Biodegradability tests-2 ofYDO-1312 11500.23 1158.3 102.2 No.5 Biodegradability tests-3 ofYDC-1312 11643.54 1172.7 103.5 No.6 Stability ofYDC-1312 11280.56 1136.1 I­ 

### 100.3 Standard solution Concentration (D) : 

### 1000mg/l Peak area (E) : 

### Volume of solution for HPLO analysis (F) : 

### Volume of test solution for HPLC analysis (G) : 

### Average recovery (H) : 

# c Equation : B=A x D+E C=AxO+ExF+G+H

## x 100 

# o <::>**~** 

### ..;.::. CX; 

### 9928.84mAU·sec 0.0251 0.295 I 

### 96%

## 0001/19

## "2.1

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## +'- I' 

### f- .:...i. 

### -I---,---,--;""""'--+...ij.....!'1

# :,=~j:'C:;:: 1 No, 1 test system 

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# ~/~**~8/~ ~:::I::::::**  

# ,+:: 'i

# :::~ t:-

# !='.--'

# -t:ttt::±i'.=ff! :::1:$:

## -j-:';:-': : : 7 , : , -7'30 2 No.2testsystem 30'0 --t :-- . ",
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## ,=4:l--l+-
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### --+-r"l-
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### l-+/i' "-+.-' !_
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. 

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## 000:159 L

## 0001.60**0001~J:l**

## Bioconcentration Study of YDC·1312 with Carp



## (Englisb version)

Sponsor Tohto kasei Co.,Ltd. Study Title Bioconcentration study of YDC-1312 with Carp Study No. AOI0070
This report is the English version of the original, which was written in Japanese. The undersigned hereby declare that this version faithfully reflects the original report to the best of our knowledge.
Translated by 

# /)**~** 

# - ".1 ( p .1.

# O~ 

# !**.~.~~** ......

# ~~~ .......

### ---.---. Date :__**~.f!.~~** 

### ..___.______L.
____..___. _ ..... Motoshi MASUDA, M.Sc.

Ecochemistry Division of Yokohama Laboratory, Mitsubishi Chemical safety Institute Ltd. Approved by



# ..**-.~** 

# ..tSJ;
....- .....------.--.------...--------.....-Date:

# .--AP-t~-g 

# ...._LJ
..:

...1::
..

# ~-~-!--

# ..- Yasuo SATO

Research Scientist. Ecochemistry Division of Yokohama Laboratory, Mitsubishi Chemical Safety Institute Ltd.



## 0001.53

## Report No. 4B376G Bioconcentration Study of YDC-1312 with Carp

Submitted to:
Tohto Kasei Co.,Ltd.

Prepared by:
Mitsubishi Chemical Safety Institute Ltd. February 21, 1995



## 000:1G4

## Compliance with the GLP Standards

Yokohama Laboratory. Hitsubishi Chemical Safety Institute Ltd. 

### Sponsor: Tohto Kasei Co.,Ltd. Study Title: Bioconcentration Study of YDC-1312 with Carp Study No.: 4B376G
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:

Jun TORIYA, B.Sc. Sealed date: February 21, 1995 Facility Management Head of Yokohama Laboratory



### 2 

# 0001G5

## Quality Assurance Statement

Yokohama Laboratory. Mitsubishi Chemical Safety Institute Ltd. Sponsor: Tohto Kasei Co.,Ltd. Study Title: Bioconcentration Study of YDC-1312 with Carp Study No.: 4B376G

We hereby certify that the above study has been performed in accordance with the Good Laboratory Practice (GLP) regulations recognized by the Basic Industries Bureau of the Japanese Ministry of International Trade and Industry (MITI)
. Dates of inspections and findings reported to the Study Director and the Facility Management are as follows:

Date of Date of Inspection Reporting In-progress study: November 28, 1994 December 1, 1994 December 26, 1994 December 28, 1994 January 9, 1995 January 9, 1995 Final report: February 21, 1995 February 21, 1995
Quality Assurance Personnel:
Junko KATOH, Ph.D. Yoshihiro MIURA, M.Sc. Sealed date: Sealed date: February 21, February 21, 1995 1995



### 3

### Introduction Study Title: Bioconcentration Study of YDC-1312 with Carp
Purpose: This study was conducted to evaluate the bioconcentration potential of the test substance, YDC-1312, for notification under the Chemical Substances Control Law of Japan.

Test Guideline: The study was conducted according to the testing method for New Chemical Substances, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law of Japan.
GLP Compliance: The study was conducted according to the Good Laboratory Practice (GLP) standards applied to industrial chemicals under the Chemical Substances Control Law of Japan. Sponsor: Tohto Kasei Co.,Ltd.
Testing Facility: Yokohama Laboratory, Mitsubishi Chemical Safety Institute Ltd., 1000, Kamoshida-cho, Aoba-ku, Yokohama, Japan
Study Period: November 22, 1994 to February 21, 1995



### 4The person in charge of the study Tadayoshi SHIGEOKA, Ph.D. Sealed date: February 21, 1995 Study Management, Chief Research Scientist of Environmental Science Division Ayao NISHIKAWA, Sealed date: February 21, 1995 Study Director, Research Scientist of Environmental Science Division Eiichiro MURAKAMI Sealed date: February 21, 1995 Environmental Science Division (Experimental work and Reporting) Storage and Retention of 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 the Yokohama Laboratory 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. The 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 in freezer) 6) Other documents required under the GLP standards**0001~8**

### Content
page SUMf.IARY 8 1. Test Substance
.................................................................................................9

1.1 Identification 9 1.2 Source 9 1.3 Confirmation of Identity 10 1.4 Confirmation of Stability 10
2. Outlines of Bioconcentration Test
................................................. 11
3. Acute Toxicity Tes 1

3.1 Test Fish 11 3.2 Test Conditions 12 3.3 preparation of Test Water 12 3.4 Results 13 

### " 4. Analytical Methods
......................................................................................14

4.1 Apparatus and Reagents 14 4.2 HPLC System and Conditions 15 4.3 Calibration Curve 15 4.4 Analytical Method of Test Substance in Test Water 16 4.5 Analytical Method of Test Substance in Test Fish 17 4.6 Blank and Recovery Test 18
5. Bioconcentration Test Methods
............................................................19

5.1 Test Fish 19 5.2 Test Conditions 20 5.3 Test System 20 5.4 Preparation of Test Water 21 5.5 Stability under Test Conditions 22 5.6 Introduction of Fish. Observations and Environmental Control 22 5.7 Measurement of Test Substance 23 5.8 Bioconcentration Factor 23


### 6**0001~~9**

### 6. Results and Discussion of Bioconcentration Test
.....................24 6.1 Circumstances That May Have Affected the Reliability of the Test Results 24 6.2 Monitoring of Test Conditions 24 6.3 Concentration of Test Substance in Test Water 24 6.4 Concentration of Test Substance in Test Fish and BCF 24

Tables 1-9 25-31 Figures 1-14 28,29,32-42
Appendixes

1. Infrared absorption spectrum of the test substance (Data provided by the sponsor) 2 pages 2. HPLC chromatograms (analysis of test water) 18 pages 3. HPLC chromatograms (analysis of test fish) 9 pages



### 7 

## 00011'0

### SUMMARY Sponsor: Tohto Kasei Co.,Ltd. Study tittle: Bioconcentration Study of YDC-1312 with Carp Stuiy number: 4B376G Stuly period:**Nov~mber** 22, 1994 to February 21. 1995 Methods: The study was conducted according to the testing methods. for New Chemical Substances (1974), which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law of Japan. 

### * Supply of test water: Continuous flow-through diluter system (800 L/day) 

### * Exposure level: 0.1 Ilg/mL (High exposure level) (nominal concentration) O.Olllg/mL (Low exposure level) 

### * Analytical method: High performance liquid chromatography (HPLC) 

### * 48hr-LC50 to Medaka: 91 Ilg/mL 

### * Fat content: 4.6\(n-4. 3.8

- 5.5\)
Results Bioconcentration factors (BCFs) obtained in this study were as follows: Exposure period (weeks) 2 4 6 8 High Mean conc. (llg/mL) in water 0.097 0.100 0.099 0.099 exposure level BCF 

### « 6) No.1 No.2 4 2 2 2 2 1 3 6 Low Mean conc. ( 11 g/mL) in water 0.0094 0.0097 0.0097 0.0097 exposure level BCF 

### « 11) No.1 No.2 <10 <11 < 9 <10 < 9 <10 <11 <11
Discussion On the basis of the BCFs obtained. it can be concluded that the test substance is not bioaccumulative in carp under the prescribed test conditions.



### 8 

## OOOA!**I~,"~** .A. 1 . .IL..

# 9 000:1."722. OUTLINES OF BIOCONCENTRATION TEST
The study was conducted in accordance with the testing methods for New Chemical Substances, which prescribes the procedure for testing new chemical substances, as required by the Chemical Substances Control Law of Japan. The bioconcentration test system shown in Fig.4 (p.34) was set up in triplicate. In addition to the control, two groups of carp were exposed to the test substance at different concentration levels.During the exposure period, the concentrations of the test substance in water and fish were measured periodically. The bioconcentration factors (BCF) calculated from the results were used to evaluate the potential for the test substance to accumulate in the tissues of carp. Before starting the bioconcentration test, an acute toxicity test was conducted for 48hours using Medaka and a 50\ lethal concentration (LC50) was determined. The bioconcentration test was carried out at two exposure levels not exceeding a hundredth and a thousandth of the 48hour LC50.

3. ACUTE TOXICITY TEST
The acute toxicity test of the test substance to f ish was conducted according to the Japanese Industrial Standard Method "JIS K0102-1986, Industrial Waste Water Testing Method, 71, Acute toxicity study using fish".
3.1 TEST FISH l)Test fish

Species Medaka (Oryzias latipes) Source Sankyo Suisan Co. (1-1 Ichigayatamachi, Shinjuku ku, Tokyo) Date of purchase: November 7, 1994 Lot 

### Nu 94·H-1107 Body length approx. 2 cm


### IIBody weight approx. 0.2 g 2)Acclimation

Aquarium No. : 0-1 Water temperature : 25

### ± 2

### t Period over 1 week Feeding Feed (Tetramin, Tetra Werke, Germany) of approx. 2
... of the body weight was given to fish everyday except holidays and for 48 hours before testing. Mortality of fish during a week before testing 

### < 0
... 3.2 TEST CONDITIONS Test substance conc. : 0 (control), 10. 18, 32, 58, 100#g/mL The number of fish: 10 per aquarium Volume of test water 2 L Water temperature : 25 

### ± 

## 2t Renewal of test water: every 24 hours Aeration continuous Dissolved oxygen 

### > 4.0 mg/L
3.3 PREPARATION OF TEST WATER l)Exposure Level The test substance, 1.00g, was dissolved in 100mL of dimethyl sulfoxide. This solution was added dropwise to 1500mL of 20 w/v\ HCO- 30 in Millipore water to disperse the test substance and diluted to 2000mL with Millipore water. Each of 40, 72, 128, 232 and 400mL from the solution (concentration of test substance: 500#g/mL) was diluted to 2000mL with dilution water (to produce this water, Yokohama municipal tap water was treated with active charcoal and dechrolinated), resulting in test concentration with each of 10, 18, 32, 58 100 # g/mL test substance. 2)Control Level Twenty milliliters of dimethyl sulfoxide was dissolved with 300mL of 20\ HCO·30 in Millipore water and diluted to 2L with dilution water. But. concentrations of HCO-30 and dimethyl
12 

## 0001'75sulfoxide are the same of concentration in the highest exposure level.

3.4 RESULTS The 48hr-LCso value of the test substance was 91 Jl g/mL as follows : r Test substance conc. (Jlg/mL) Number of dead fish Mortality ! (III) 0 (control) 0 I 0 i 10 0 0 18 0 0 32 0 0 58 0 0 100 6 60 From this result, the nominal concentrations to be used in the bioconcentration test were set at 0.1 and O.OlJlg/mL in high and low exposure levels, lower than a hundredth and a thousandth of the 48hour LC50, respectively. And measured water temperature and dissolved oxygen. they are appropriate during the exposure period. test water 24.9

- 25.

## ot dissolved oxygen 6.0

- 7.8mg/L

13 

## 000j.'764. ANALYTICAL METHODS
4.1 APPARATUS AND REAGENTS 1) Apparatus

Even electrobalance Mettler Model AE163 (No.1 ) Automatic even Balance Mettler Model PL1200 (No.1) Homogenizer Nihon Seiki Co. Model AM·S (No.1) Aspirator Yamato Kagaku Co.Model BP·51 (No.2) Evaporator Buchi Model RE-111 (No
_1) Shaker Taiyokagaku Co. Model SR-ll (No.1) 2) Reagents

Acetonitrile Kokusan Chemical Co., Guaranteed Reagent n-Hexane Kishida Chemical Co., Guaranteed Reagent Ethyl acetate : Kokusan Chemical Co., Guaranteed Reagent IN-Soduim Hydroxide Solution Kishida Chemical Co. Sodium Chloride Kishida Chemical Co., Guaranteed Reagent Anhydrous**Sodiu~** Sulfate : Kokusan Chemical Co
.. Guaranteed Reagent



### 144.2 HPLC SYSTEM AND CONDITIONS 1)Apparatus Shimadzu Co. , Model LC-10AWS (No.3) (with column switching apparatus) Pump Shimadzu Co. , Model LC-10AD (2 sets) Detector Shimadzu Co. , Model SPD-10AV Data Processor Shimadzu Co. , Model CLASS-LC10 six course valve: GL-Science Inc., Model SPV-N-6 (No.4) 2)Condition

Column (for analysis): GL-Science Inc., Inertsil C8,**5~m** 4.6 mm i.d.X150 mm Column (for concentration) : GL-Science Inc., Inertsil ODS-2, 4.6 mm i.d.X 1 cm Mobile phase (for analysis) : acetonitrile / water • 62 : 38 Mobile phase (for concentration) : Millipore water Flow rate (for analysis) : 1 mL/min. Flow rate (for concentration) : 2 mL/min. Wavelength : 285 run Attenuation : 2 Injection volume 40**~L** (except test water and test fish analysis )
4.3 CALIBRATION CURVE The solution of the test substance with 100**~** g/mL in acetonitrile was diluted with acetonitrile to prepare standard solution of three concentrations 0.5, 1.0, 2.0**~** g/mL. These standard solutions were analyzed by HPLC. The total peak area (**~** V' sec) for the test substance in each solution was plotted against its concentration. The calibration curve yielded a straight line passing through the origin and its correlation coefficient was calculated to be 1.000 by the least square method. [Fig.5 - 6 (p.35
. 36)] At each measurement of the test substance, peak area for the standard solution (conc. 1.0**~** g/mL) was measured and a concentration of the test substance was determined from a ratio of peak area of sample to the standard solution.



### 154.4 ANALYTICAL METHOD OF THE TEST SUBSTANCE IN TEST WATER The test water was analyzed by HPLC with column switching apparatus. Test water sample was pumped to concentration column and the test substance concentrated in concentration column was pumped to analytical column by switching of valve and analyzed. It is checked that the HPLC system is running under the conditions given in Section 4 2 (the switching valve must being the "analysis" position 

# I 4000 

### IlL (400 

### IlL)' of test water is taken with a mjcrosyringe 

# I Immediately after the valve is rotated to the "concentration" posjtjon, the sample is injected 

# I The HPliC system is run for about 3 minutes to trap the test substance in concentratjon column 

# I The valve is returned to the "analysis" posjtion to start

HPliC analysi s 

### * : For the testing at high exposure level.



### 16 

## 0001.'794.5 ANALYTICAL METHOD OF THE TEST SUBSTANCE IN TEST FISH Prior to HPLC analysis, test fish was treated according to the following procedure : One fish of ahout 2Sg. weighted Cut jnto pieces with scissors

- Repeated once again I-acetonitrile SOmL Homogenized for 5 mjn at about 10000rpm Filtered with suction though a sheet of S.60 filter paper on a Buchner funnel 

# I Acetonitrile layer Residue -Millipore water 500mL Discarded -Sodium Chloride 25g -IN·Soduim Hydroxide Solution 5mL Shaken for 10 mjn for extraction with n·Hexane (JOOmI.) (SR·IT Shaker, Setting the dial at 6) 

# I I n·Hexane layer Water. acetonitrjle layer 

# I Dehydrated and filtered Discarded (by passing through a glass-filter plugged with 50g of anhydrous sodium sulfate) 

## I Djluted to 200m1. with n.Hexane Collect 4mL (Sampling factor' 50) passed through a SEP-PAK FlorisiJ cartridge (then 6mL of n-Hexane was passed and discarded) BmI. of n-Hexane/ethyl acetate
- 95/5 mjxture was passed 

## I Eluate 

# I Evaporated to dryness(at 40t: by rotary evaporator)**I~Acetonitrile/water=62/38** mixture, 5mL dissolved Collected**200~L** and analyzed by HPLC



### 17 

## 000:1.04.6 BLANK AND RECOVERY TEST The minimum detectable peak area was set at 1500 11 V • sec. which could be recognized visually. This value corresponded to about 0.06ll1g/mL on the calibration curve. l)Test water • Blank test

Twice in the exposure period. control water was analyzed by the method of low exposure level shown in §4.4. The absence of interfering peaks in the HPLC analysis was confirmed and detection limit concentration of the test substance in control water was O.OOll1g/mL. [Table 1 (p.2S), Fig.? (p.3?)]
• Recovery test

The standard solution (the test substance conc. : 1.0 I1g/mL in acetonitrile) of 0.5 mL was dispersed in control water and diluted to 100 mL. The resulting concentration was O. 0111 g/mL, the same value as the nominal concentration in test water of low exposure level. This solution was analyzed by the method of low exposure level shown in §4.4 to evaluate recovery. Recovery was measured twice and averaged 99.6 \. Values analyzed for test water were corrected by this factor. [Table 2 (p.25)' Fig.8 (p.38)] 2)Test fish • Blank test

Two control fish were analyzed by the method shown in §4.5 at the beginning and the termination of the exposure period. respectively. The absence of interfering peaks in the HPLC analysis was confirmed and detection limit concentration of the test substance in control fish was below 0.14I1g/g. [Table 3 (p.26), Fig.9-l0 (p.39-40))
• Recovery test

One milliliter of standard solution (conc. 100 11 g/mL in acetonitrile) was added to one control fish. This fish was analyzed by the method shown in §4.5 to evaluate recovery. When the fish weight is 25 g. the test substance concentration in the fish body will be 2 11 gig, corresponding to 20 times the nominal value in test water of high exposure level. Recovery was measured in two individual fish and averaged 94.8 %. Values analyzed for test fish were corrected by this factor. [Table 4 (p.26), Fig.ll (pAl))

18

## 5. BIOCONCENTRATION TEST METHODS
5.1 TEST FISH l)Test fish Species Carp (Cyprinus carpio) Source Sankyo Suisan Co. (1·1 Ichigayatamachi, Shinjuku-ku, Tokyo) Date of receipt: August 17, 1994 Lot No. 94-K-0817 Body length approx. 10 cm (measured with the eye) Body weight 20-40 g (measured with the eye) 2)Acclimation

Aquarium No. : B-3 Water temperature : 25±2 

### t Period over one week Feeding Feed (Minipet, Kyorin Co., Ltd., Japan) of approx. 2 , of the body weight was given everyday except holidays. Mortality of fish during the week before use 0\

195.2 TEST CONDITIONS l)Nominal concentrations of the test substance in water High exposure level 0.1 tlg/mL Low exposure level O.Oltlg/mL Control 

## o tlg/mL 2)Flow rate of dilution water : aoo L/day (Turnover rate :10 times/day) 3)Flow rate of feed solution : 160 mL/day 4)Population density (at the beginning of the exposure period) High exposure level 15 fish 

### / aO-L test water Low exposure level 15 fish 

### / aO-L test water Control 12 fish 

### / aO-L test water 5)Water temperature : 25±2 

### 't: 6)Dissolved oxygen 

### > 4 mg/L 7)Exposure period a weeks
5.3 TEST SYSTEM The outline is shown in Fig.4 (p.34).



### 205.4 PREPARATION OF TEST WATER l)Preparation of feed solutions The feed solution of each exposure level was prepared according to the following procedures : (High exposure level)

Test substance 1.00g I-Dimethyl sulfoxide 100mL Disso]ved mixed with 20 

### w/v' HCO-30 jn Millipore water (1500 mI.) to disperse the test substance I-Millipore water Diluted to 2000mL Concentration of test substance 0.50mg/mL Concentration of HCO-30 150mg/mL (Low exposure level)

Feed solution of high exposure 1 eve] (200mL) I-Millipore water Diluted to 2000mL Concentration of test substance 0.050mg/mL Concentration of HCO-30 15mg/mL (control)

MiJ 1jpore water of 20 w/V' HCO- 30 (J 500 mI.) I-Dimethyl sulfoxide (lOOmL) Djssolved mjx and djsperse I-Millipore water Diluted to 2000mI. Concentration of HCO-30 150mg/mL Each of these solution was renewed at about lO-day intervals. 2) Preparation of test water The feed solutions prepared in §S.4 1) were supplied to their respective mixing tubes by pumps to be mixed with dilution water so that concentrations of the test substance could reach their respective nominal values. The test water thus prepared were continuously supplied to their respective test aquarium. [Fig.4 (p.34)]


### 21 

## 000:1. ,15.5 STABILITY UNDBR THB TBST CONDITIONS 1)Stability in feed solution The feed solution of low exposure level concentration of the test substance 50 /.1 g/mL in aqueous solution (see § 5.4 1» was used as a test solution. A portion of the**tes~** solution left at room temperature was diluted 50 fold on days 0, 1, and 14. Each sample was analyzed by HPLC. and variation of concentration with time was evaluated. On day 14. 93.8 \ of the initial concentration was detected (48.2/.1g/mL on day 0 - 45.2 /.1 9 / mL on day 14)
. On the basis of this result, it is concluded that the test substance in the feed solution is sufficiently stable to allow approx. 14 days use. [Table 5 (p.21). Fig.12 (p.42») 2)Particle size The 1 /.1 g/mL water dispersion prepared in § 5.5 1). was filtered with a membrane filter of pore size 0.4S/.1m
. The test substance in the filtrate was analyzed by the HPLC. The particle size of the dispersion was determined from the concentration of the test substance in the filtrate. On the basis of the result that particles below 0.45/.1m were 91.5 \. it ""\ was concluded that the dispersion was satisfactory for the test solution.
5.6 INTRODUCTION OF FISH. OBSBRVATIONS AND BNVIRONMBNTAL CONTROL The test system was operated for over 4 days prior to the introduction of fish. It was confirmed that the test substance in water was kept at nominal concentrations during this period. Then fish were introduced into the test system. Feeding, observation of fish and control and measurement of water quality parameters were carried out as follows : 1) Feeding Feed (Minipet, Kyorin Co.Ltd.) of approx. 2\ of the body weight were given to fish everyday except holidays. 2) Observation of fish Conditions of fish were observed. 3) Dissolved oxygen concentration and temperature : Dissolved oxygen concentration and temperature were measured and recorded twice a week. 4) Measurement of fat content : Fat content of 4 test fish in the control aquarium were measured during the exposure period. 

### 22 

## 0001.555.7 MEASUREMENT OF THE TEST SUBSTANCE l)water Test water was analyzed twice a week, regularly, as described in § 4 .4
. 2) fish Three fish were collected and two of tilem were analyzed on weeks 2, 4, 6 and 8 of exposure period as described in §4.5.
5.8 BIOCONCENTRATION FACTOR l)Calculation of bioconcentration factors (BCFs) Bioconcentration Factors (BCF) were calculated from the following calculation equation. BCF

- (Cf

- Cfo) 

### I Cw where Cf : Concentration in treated fish body (Ug/g) Cfo: Concentration in control fish body (Ug/g) Cw Mean concentration in treated test water(Ug/mL) Cfo equals 0 in this study because blanks confirmed the absence of interfering peaks. 2)Detection limit As described in Section 4.6.2, the test substance in fish is detectable down to 0.14 U gig, indicating that bioconcentration factor can be obtained when of 0.1 and 0.01 U g/mL are concentrated by factors of about 1 and 14, respectively.



### 23

## 6. RESULTS AND DISCUSSION OF BIOCONCENTRATION TEST
6.1 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE RELIABILITY OF THE TEST RESULTS 'There was no significant matter that may have affected the reliability of the test**~esults** during the expusure period of 8 weeks.
6.2 MONITORING OF TEST CONDITIONS There was no dead fish or abnormal condition during the exposure period of 8 weeks. Condition of fish There was no abnormality in shape of the body or in swimming and feeding behavior. Body weight 22.36

- 37.34 g Body length 9.5

- 11.4 cm Water temperature 23.1

- 25.3 

### 'C Dissolved oxygen 5.3

- 7.9 mg/L Fat content 4.6 \ (n-4. 3.8
. 5.S \)
6.3 CONCENTRATIONS OF THE TEST SUBSTANCE IN WATER The mean concentrations of the test substance during the exposure period of 8 weeks were 0.099 11 g/mL for high exposure level and 0.0097 11 g/mL for low exposure level. which almost corresponded to the nominal values. The coefficients of variation were calculated to be 8.6 \ for high exposure level and 6.1 \ for low exposure level. [Table 6 ·7 (p.28 ·29), Fig.13
. 14 (p.28

- 29), Appendix-2]
6.4 CONCENTRATIONS OF THE TEST SUBSTANCE IN FISH AND BCF The concentrations of the test substance in fish were calculated to be below 0.57I1g/g at high and below 0.11I1g/g low exposure level. The bioconcentration factor (BCF) were determined to be below 6 for high exposure level and below 11 for low exposure level. [Table 8 - 9 (p.30 31), Appendix-3]

On the basis of the results, it was concluded that the test substance was not bioaccumulative in carp under the prescribed test conditions. 

### 24 

## 000:1

### Tab. 1 Blank test for water A B C sample Na. 

### peak area J.L

### V·sec conc. in water 1 samEle <1500 . std. 24395 /.1.glmL <0.001 2 <1500 24395. <0.001 Concentration of std. solution (Cstd) Injection volume of std. (Vst) Injection volume of sample (Vsa) Calculations: C=A+BxCstdxVst+Vsa 1.0/.1.g/mL 40 J.LL 4000 J.LL

Tab.2 Recovery test for water A B 

### C D R 

### sample test peak area measured recovery No. 

### substance amount added J.LV·sec

/.1.g samEle std. /.1.g % 1 

### 1.0 24313 24395 0.997 99.7 2 1.0 24266 24395 0.995 99.5 Average,n=2 99.6 Concentration of std. solution (Cstd) 1.0 J.Lg/mL Amount of test water (W) 100 ml Injection volume of std. (Vst) 40 /.1.L Injection volume of sample (Vsa) 4000 /.1.L Calculations: D=B+CxCstdxVst+VsaxW R=(B+CxCstdxVst+VsaxW)+Ax 100

25 

## 000:158

### Tab. 3 Blank test for fish
W 

### A B C D sample fish weight peak area tJ.

### V· sec cone. in final cone. in fish solution No. 

### g samEIe std. jj.g{aL jj.gLg



### 1 22.36 <1500 24380 <0.012 <0.14 2 23.03 <1500 24380 <0.012 <0.13 3 25.40 <1500 24230 <0.012 <0.12 4 28.75 <1500 24230 <0:012 <0.11 Concentration of std. solution (Cstd) 1.0 tJ.g/mL Injection volume of std. (Vst) 40 tJ.L Injection volume of sample (Vsa) 200 jj.L Final volume (FV) 5mL Sampling factor (SF) 50 Calculations: C=A+BxCstdxYst+Vsa D=(A+B

### xCstdxVst-:-Ysa) xFYxSF-:-W


### Tab.4 Recovery test for fish W 

### A B 

### C sample fish test peak area weight substance tJ.

### V·sec added No. 

### g ,u.g samEle

1 29.70 50.0 24019 2 26.81 50.0 22207 std. 24380 24380 D 

### conc. in final solution

,u.gLmL 0.1970 0.1822 Concentration of std. solution (Cstd) Injection volume of std. (Vst) Injection volume of sample (Vsa) Final volume (FV) Sampling factor (SF) Calculations: D=B-:-CxCstdxVst+Vsa Average,n=2 1.0 tJ.g/mL 40 jj.L 200 tJ.L 5

### ml 50 R=(B+C xCstdxVst-:-Vsa) xFVx SF-:-Ax 100 E 

### measured

8.IIount

,u.g 49.3 45.5 R 

### recovery % 

### 98.5 91.1 94.8

26 

## 000:189

### Tab.5 Stability of the test substance in feed solution A 

### B 

### C D period peak area conc. in feed ratio to day



### 0 J,J.V·sec**s3Jll~le** 

### std. 23210 24071 solution JJ.gLmL

48.2 initial conc. 

## % 100 7 23183 24395 47.5 98.6 14 22033 24369 45.2 93.8



## . Concentration of std. solution (Cstd) 1.0 J,J.g/mL Dilution ratio (DR) 50 CalcuIa ti

### ons : C=A-:-B xCstdxDR, D=Ci -:-

### Cox 100 i : Number of analysis

27 

## 0001. 0

### Tab.6 Analysis of the test substance in water
- High exposure level 

## i A B 

### C D 

### samplirur period number peak area conc. mean conc.

date of /.J.V· sec in water in water dal week anallSis samI!le std. Il:.gimL Il:.gl..mL 

### 1994/11/28 0 0 1 22863 24563 0.093 0.093 1994/12/01 3 2 23404 24395 0.096 0.095 1994/12/05 7 1 3 

### 26543 24623 0.108 0.099 1994/12/08 10 4 23041 24369 0.095 0.098 1994/12/12 14 2 5 22932 25017 0.092 0.097 1994/12/15 17 6 25097 24231 0.104 0.098 1994/12/19 21 3 7 26961 24420 0.111 0.100 1994/12/22 24 8 25216 24301 0.104 0.100 1994/12/26 28 4 9 22979 24517 0.094 0.100 1994/12/29 31 10 

### 23914 24756 0.097 0.099 1995/01/02 35 5 11 28254 24632 0.115 0.101 1995/01/05 38 12 23887 24725 0.097 0.101 1995/01/09 42 6 13 20228 24408 0.083 0.099 1995/01/12 45 14 26309 24567 0.108 0.100 1995/01/16 49 7 15 22622 24814 0.092 0.099 1995/01/19 52 16 26003 24307 0.107 0.100 1995/01123 56 8 17 22822 24650 0.093 0.099 Standard deviation 0.009 J..l(!./mL Coefficient of variation : 8.6**~** 

### Concentration of std. solution (Cstd.) 1.0 /.J.g/mL Injection volume of std. (Vst) 40 /.J.L Injection volume of sample (Vsa) 400 /.J.L Recovery (R)**99.6~** 

### Calculations: C=A+BXCstdxVst+Vsa+(R+lOO). D=kCi+i Fig.13 Concentration of the test substance in water
- High exposure level concentration (ttg/aL)

0.200 ----.-- analyzed value

- .. - - - Iminal value 

# I.
0.150

0.050 0.000 -I----_+_--+----+-------;O--,---+----+----+--__+_­ 

### o 7 14 21 28 35 42 49 56 

### exposure period (day)

28 

## 000.1:3.1

### Tab.7 Analysis of the test substance in water
- Low exposure level i 

### A B 

### C D 

### saDlpliM period number peak area conc. mean conc. date of j.J.

### V'sec in water in water dal week anallsis saDl:Rle std. j.J.gLmL Il:.gLmL 1994/11/28 0 0 1 22579 24563 0.0092 0.0092 1994/12/01 3 2 21393 24395 0.0088 0.0090 1994/12/05 7 1 3 20639 24623 0.0084 0.0088 1994/12/08 10 4 25146 24369 0.0104 0.0092 1994/12/12 14 2 5 25542 25017 0.0103 0.0094 1994/12/15 17 6 26081 24231 0.0108 0.0097 1994/12/19 21 3 7 24519 24420 0.0101 0.0097 1994/12/22 24 8 22265 24301 0.0092 0.0097 1994/12/26 28 4 9 24042 24517 0.0098 0.0097 1994/12/29 31 10 22988 24756 0.0093 0.0096 1995/01/02 35 5 11 

### 24304 24632 0.0099 0.0097 1995/01/05 38 12 24242 24725 0.0098 0.0097 1995/01/09 42 6 13 24092 24408 0.0099 0.0097 1995/01/12 45 14 24484 24567 0.0100 0.0097 1995/01/16 49 7 15 23488 24814 0.0095 0.0097 1995/01/19 52 16 23644 24307 0.0098 0.0097 1995/01/23 56 8 17 23923 24650 0.0097 0.0097



## . Standard deviation 

## . 0.0006 J.1f!./mL 

### Coefficient of variation : 6.1 

### % Concentration of std. solution (Cstd.) 1.

### 0 j.J.

### g/mL Injection volume of std. (Vst) 40 j.J.L Injection volume of sample (Vsa) 4000 j.J.L Recovery (R) 99.6 

### % Calculations: C==A-:-BXCstdxVst-:-Vsa-:-(R-:-100). D=2:Ci-:-i Fig.14 Concentration of the test substance in water
- Low exposure level concentration (tLg/aL)

0.0200 • analyzed value

- - - - - roIIinal value 0.0150

0.0050 0.0000 l-----+-----4----t----I------t--_--+----+-­ 

### o 7 14 21 28 35 42 49 56 

### exposure period (day)



### 29

### Tab.8 Analysis of the test substance in fish and bioconeentration factor (BCF)

- High exposure level
W 

### A 

### B C 

### D 

### E F 

### exposure fish peak. area cone. cone. BCF**me~**



### period weight in final in fish cone. j.1V· 

### sec solution body in water week No. g 

### sample std. ttg/mL /.J,g/g 

### /.J,g/mL



### 2 1 30.31 5333 24450 0.044 0.38 0.097 4 

### 2 2 28.52 2906 24450 0.024 0.22 0.097 2 

### 4 1 34.47 3295 24562 0.027 0.21 0.100 2 

### 4 2 33.53 3745 24562 0.030 0.24 0.100 2 

### 6 1 32.34 2473 24460 0.020 0.16 0.099 2
". 

### 6 2 30.45 1827 24460 0.015 0.13 0.099 1 

### 8 1 30.66 4826 24230 0.040 0.34 0.099 3 

### 8 2 32.40 8548 24230 0.071 0.57 0.099 6 

### Concentration of std. solution (Cstd.) 1.

### 0j.1g/mL Injection volume of std. (Vst) 40 ttL Injection volume of sample (Vsa) 200 j.1L Final volume (FV) 5IlL Sampling factor (SF) 50 Recovery (R) 94.8 ,; Calculations:C=A7BxCstdxVst7Vsa D=(A7B X.QstdXVst7Vsa) XFVXSF7(R7100)7W. F=D7E

30

### Tab. 9 Analysis of the test substance in fish and bioconcentration factor (BeF)

- Low exposure level W 

### A 

### B C 

### D E 

### F exposure fish peak area conc. ronco mean BCF

period weight in final in fish conc. j.J.

### V·sec solution JQdy 

### in water week No. g 

### sample std. /J.g/IIL Ltg!g /J.g!mL



### 2 1 35.52 <1500 24450 <0.012 <0.09 0.0094 <10 2 2 31.62 <1500 24450 <0.012 <0.10 0.0094 <11 4 1 

### 36.76 <1500 24562 <0.012 <0.09 0.0097 <9 4 2 33.37 <1500 24562 <0.012 <O.lD 0.0097 <10 6 1 34.64 <1500 24460 <0.012 <0.09 0.0097 <9 6 2 33.21 <1500 24460 <0.012 <0.10 0.0097 <10 8 1 30.58 <1500 24230 <0.012 <0.11 0.0097 <11 8 2 30.79 <1500 24230 <0.012 <0.11 0.0097 <11 Concentration of std. solution (Cstd.) 1.

### 0 j.J.

### g/

### mL 

### Injection volume of std. (Vst) 40 j.J.

### L Injection volume of sample (Vsa) 200 j.J.L 

### Final volume (FV)

. 

### 5 IlL 

### Sampling factor (SF) 50 Recovery (R) 94.8 % 

### Calculations:C=A-:-BxCstdxVst-:-Vsa D=(A-:-B xCstdxVst-:-Vsa) xFVxSF-:- (R-:-100)-:-W, F=D-:-E

31**~ c~r­** **ooo** J' _L . .l,.:}

### Fig.4 Bioconcentration test system
tap water

air 

### pump pump
feed solution o 

### activated charcoal (dechlorinat ion) dilution water

(25'"'C) 

### mixing tube

to waste treatment

system 85L-glass aquarium (80L water) 

### (Apparatus)

Pump (dilution water supply) : Exposure : FMI Co.,QD-2CSC (No.l,2) Control :FMI Co.,RP-D 2CKC (No.1) Pump (feed solution supply) : Exposure : Nihon Seimitu Kagaku Co.,NP-FX-l(02)**(N~I,2)** 

### Control : FMI Co.,QG 6-RHOOSTY (No.1-i)

34

### Fig. 6 Calibration curve of the test substance Curve fitting (Least square method) concentration peak area ,ug/ml ,uV-sec 

# o o 0.5 12357 1.0 24293 2.0 50136 R: 1.000

60000 

# ,. 50000 ,/ ,/ ,/ 

# - (.) ,/**~** 

### 40000 ,/ ,/ >­ ,/ 

### ::t ,/ 

### :; 30000 Q)**~** 

## A <:


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,/ 

## 7J 20000 Q)**~** ,/ ,/ 

### 10000 ,/ ,/ 

# o

______ ______ ______L­

____**~** 

# o 0.5 1 

### 1.5 2 

### Concentration (.l1g/mL)

36**OO~Q9** '- ..;\ • •..1

## ), "/

# --.------­

# 3;

### L{-I

## APPENDIX-l

Infrared absorption spectrum of the test substance (Data provided by the sponsor) (2 pages)

# B**,C~"**\Z. lA

# S <L. 

# -------.

L

## APPENDIX-2

HPLC chromatograms (analysis of test water) (18 pages)



# 000208

## 000209

## 000:2"1:1

## O()O",-:>

..**~** r> U '"
..**~** ."-'

# 0002t3

## /0

# 000219

### I(

### r') /'

# l7 Or.

## 0
........-., 1:' ••,' ",. f,**~** ,,"-¥ .. ...,

# '--------­

### APPENDIX-3

HPLC chromatograms (analysis of test fish) (9 pages)

# ~L\6y ..

### 5 

### 'L2')- 

## L ')

# ~**~\c~\\.~**

## I I**0002;~.::;**

# ~1/~1

", .: J I 

### April 27, 2001 

# TD**p~** 

# 0

# 1,- II

# c.f SANITIZED 

# 5DO (000 37Cfh 1


### Ms. Geraldine Hilton Office ofPollution Prevention

and Toxic Substances, 7407 U.S.B.P.A 401 M Street, S.W. Washington, D.C. 20460
Re: Selection ofRegulatory Option and Submission ofAdditional Information Our

### Ref: TS-600212 YourRef: P-0l-114 DearMs. Hilton: Pursuant to my recent telephone message, attached please fmd a completed Selection of Regulatory Option form for the above-identified case, as well as additional information together with four translations oftest reports which were recently discovered (acute oral toxicity test in rats, twenty-eight day repeated dose oral toxicity study in rats, ready biodegradability test, and bioconcentration study with carp). The Selection ofRegulatory Option form authorizes a 6O-day suspension, based on your indication. Since the submitter wishes to import the 

### new chemical substance as soon as possible, expedited consideration by EPA is respectfully requested, as is the 

### revocation ofany remaining suspension time once thePMNis found byBPA to be acceptable, 

### Ifyouhave any questions, pleasedo nothesitate to contact us. Very truly yours,

I C:l 

### Technical Contact



## Company Sanitized
? 

### 2 (' p<-- / )

### -7­
: .. ',)**~;"-; ~**  • ' t " " -­ 

### SELECTION OF REGULATORY OPTION
FOR PMN # 

### P-01-114
I have reviewed the Agency's letter dated

## ~~.2$, ?-DD 

### I , 

### which outlines the regulatory options for the**ab~ferenced** 

### PMN, and I select the following option: [

# J~ Option 1 upfront submission of additional information before commencement of manufacture (suspension as appropriate) Option 2 upfront ,subm.ission of specified data**b~fore'commencEilnerit;of** 

### manufacture ,CsuspEmsi'orfas 'appropitiate)

..... ', :**~~** .•**'\~. ~,'.'**  ,'":" '"\ "\**~-f"-' ~**  ..•**~., (,:".;;-~,:**  t 

## .<).::,'/'" " ' d f**~ ~:**  

### Option 3 withdrawal of PMN per 40 CFR 720.75(e) 

### (no suspension necessary)
To provide time for implementation of the selected option, I request, pursuant to 40 CFR 720.75(b), a suspension of the PMN review period for be 

### days or until the following date:

# '-7,~1 Dat