### /YIIS6/1)**~----------------------------------------------------------------~-----** Form Approved.. O.M.B.**~o.** 2!l7O-OO12 AwovaJ, Expu:s 10-31-96. U. S. ENVIRONM:ENTAL PROTECTION AGENCY 

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# EAtA PREMANUFACTURE 

# o 1-\ NOTICE DateoEreceipt

\Nhl'n ('(11l1pll-tl'd "end tht'­ torm h. FORNEW CHEMICAL SUBSTANCES**OOCUMENTCONTROLO~CER** OFFICE OF POLLUTION PREVENTION

.AND TOXIC SUBSTANCES, 1401 U.S. E.P.A. 401 M STREET, SW WASHINGTON D.C. 20460 Enter the total number ofpages Docu:mentcontrol number EPA caMnum.bc in thePremanufa.cture Notice

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... CIIIlfIdatlaL ao-uc. Cmam IllUcltfDa ,noq....., .... , Oil pap3) EPA FORM**7ll~2S.** (Rev. 5-95) Replace!!I previous editions ofEPA Form**771~2S.** 

# IPublic reporting burden for this coilection ofinformation is estimated to average 110 hours per response, including time for reviewing instructions, searching existing data sources, gathering and maintaining the dataneeded.. and completing and reviewing the collection ofinformation. Send comments regarding the burden estimateor any other aspect of this collection of information, including suggestions for nldudng this burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St, S.W." Wlllhington, D.C 20460; and to the Office of Management and Budget, Paperwork Reduction Act (20'l().0012), Washinsrton, D.C 20503. 

### CERTIFICATION I certify that to thebest ofmyknOWledge and belief: 1. Thecompanynamed in Par! I, section A, subsection 1aof this notice form intends to manufacture orimportfor a commercial purpose, other than in smallquantities solely for research and development, the substance identified in Part I, Section B. 2. All information provided in this notice is complete and truthful as ofthe date of submission. 3. I am submitting with this notice all test data inmypossession orcontroland a description of all otherdataknown to or reasonably ascertainable byme as required by §720.50 ofthe Premanufacture Notification Rule. Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, orSNUN, check the following uaerfee certification statement that applies: 

# o The Companynamed. in Part I, Section A hasremitted the fee ofS2500 spedfied. in40 a:R. 700.45 (b), or • 

# o The Companynamed in Part I, Section A hasremitted the fee of$1000 for an IntermediatePMN (defined • 40 a:R.700.43) in accordance with 40 CFR 700.45(b), or 

# o The Company namedin Part I, SectionA is a smallbusiness concernunder40 a:R.700.43 and hasremitted a fee of$100 in accordance with40 CFR 700.45 (b). Ifyou are submittinga lowvolume exemption (L VE) applicationin accordance with40 CFR 723.50 (c) (1) ora Low release and low expoaure exemption (LoREX) application in accordance with 40 CFR 723.50 (c) (2), checkthefollowing certification statements: 

# o The manufacturer submittingthis notice intendsto manufactureorimportthe new chemical substancefor commercial purposes,otherthan in smallquantities solely for research and development,. underthe terms of40 CFR723.50. • .­ 

# o The manufactureris familiar withthetermsofthis section and willcomplywith those terms; and 

# o Thenew chemical substancefor which thenotice is submitted meets all applicableexemption conditions. 

# o Ifthis application is for anL VB in accordancewith40 CFR 723.50 (c)(1), the manufacturerintendsto commenC2 manufactureoftheexempted substa.nce forcommercialpurposes within 1year ofthedate oftheexpirationofthe 30 day reviewperiod.

The accuracyof the statemenlsyou make illthis noticeshould re£Iectyourbestpredidim ofthe anticif8tal

### fads regarding the chemical

s.ubstance d8scribed herein. Anyknowing and willful misinterpretationis subject to criminal peaa1typumuant 1018USC 1001.**r-----~** Coafide;ntial Signature and title of Authorized. Off:idal (0rigiDal Signature R.e'luized) Date

Date SignatureofapDt • (ifapplicable)

FORM EPA 7710-2S (Rev. 5-95) Page 2Ifyou havesubmitteda noticeof Bona fide Irttentto manufactureor -----.:)Ir... 

# D impartfCII' thechemic:alsubetanoe aweredbythisnotice.. enter the notice number assignedbyEPA. ,.. 2.

## D'~ 3 • 

# DBoth**D~** Mailing and street) Oty, State, ZIP Code

Nameofauthorized o.fficial

Mailing addrees (number and street) Qty, State. ZIP Code**Telep~** Ifyouaresubmitting this notice 81 part ofa joint submission. mark 00 box. -------------- ..... NameofauthCllized. offidal POlliliao

## (if~li~~**-C;;~----------------------~-----------------------------** Mailing adc:lrea (number and street) ety,State, ZIP Code

Name Company Mailingaddress (number and street) QtyiState, ZIP Code
Ifyou'have hada prenotice.cmnmunic:atl.an (PC) alIKI!rl1i.ng thisnalia! and EPA assigneda PCNumber 110 the notice. enter thenumber. Ifyoupreviously submitted 8D exe:mpt:Km appIk:atIoa fCII' the c:hemicIl

Type ofNotice

- Mark00 

# D Only

. Muufw::ture 1.


# . Opt:km FORM EPA 771G-2S (Rev,5-95) Telephone Position

Teleph
..e I I Mark00 ifnone 

### Part I - GENERAL INFORMAnON**~_S_e_c_ti_o_n_A** 

### __ _____ __

### __________ ______________________________________________**~~~**



### -_S_UB I_I_ER 

### nD CA ll_0N

1a. Person Submitting Notice • (in

### u.s.)

b. Agent (if applicable)

c. Jomt Submitter

2. Technical Contact (in

### U.s.>

3.
4. substancecovered by tJrls.notice. entertheexempI10Il number_peel Mark00 by EPA. Ifyou previously submitlalaPMNfCII' dUll IUbetance enterthe**-~.,...** 

# D
.... ifnane
. PMNnumberusiped by EPA 0.& withdrawn 01' b:Icomplete). ---:JI
......

### Part I - GENERAL INFORMATION

- Continued Section B _ CHEMICAL IDENTITY INFORMATION: YOIllllut provide a C1.I1TerItly com!Ct 0Iemic:al Abatracm (CA) name of the subitanCll! baaed on the nmth Collective Indc: (9Cl) ofCA nomencl.1ture rul. md amventiDna. Mark 00 the "Confidential" any item youclaim as confidential. Complete eitheritem 1 (Cl_ 1 or2 subltances) or 2 (Polymers) al appropriate. Complete another itema. if anotherperson will submit chemicalidentityinformation for you (for either item 1 or2), mark00 the box atthe.t 

# 0 the name, and add.re!lls of that in a continuation sheet. .. 1. Cla

.. 1 or2 chemicalwbstance. (for definitions of class land class 2 substances, seethe In.truction.Manua1) a. Oass of substance

- Mark 00 1 

# D Cla9s 1 or 2 

# 0 Oass2 b. Otemical name (Currently correct OtemicalAbstracts (CA) Name that isconsistent withT5CA Inventory.listings for similar substances. ForCasa 1 substances a CA Index Namemust be provided. For Oass2 subetances either a CA Index Nameor CA PreferredName be providecL whichever is appropriatebasedonCA 9CI nomenclaturerules andconventioNL).

c. Please identify which method you used to develop orobtain the spec::ified chemical indentityinformationreportedin thiB notice:

(check one). 

# D Method 1 (CAS InventoryExpert Service • a copyof theIdentification DMebxi 2 (OtherSource) report obtainedftom theCAS Inv",toryExpI:!rt ServiceIIUlIIt be submitted asan attachmentto this notice) d. Molecular formula andCAS Registry NumberOf a numberalready exists forthe substance)

CAS.

e. Fora cla
.. 1 substance, provide a complete andcorrect cbemic::al st:rud\lIediagram.. Far a daN 2 sublltance

- (1) Ust the immediate

precursor substances with theirrespective CAS Registry Numbers. (2) Describe the natureof the re.::tkm orprocea (3) lDdicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correct U!P!eae.ntative orpartialchemical structure diagram,as complete at CIIl1 be known, ifone CIIl1 be reuonablyascertained. 

# x

FORM EPA**1'11~2S** (Riw.5-95) P_.4

### I Part I - GENERAL INFORMA nON

- Continued 

# • Section B - CHE1VflCAL IDENTITY INFORMATION

- Continued 2. Polymers (For a definition ofpolymer, see the In.lnlcnon. ManuaL) Conticlential a. Indicate the number-average weight of the lowestmolecular weight composition of the polymer youintend to·manufacture. Indicate maximum weightpercentoflowmolecular weight species (not indudingresidual monomers,reactants, or solvents) below 500 and below 1,000 absolutemolecular weightof that compositiOl\. 

## • Describe the methocls ofmeasurementorthe basil for yoarestimatel: GPC 

## 0 OthI!'1'D : (Specify) n lowest number averagemol.ec:ul.ar weight mmaximumweight'" below 500 molecular weight iii) maximumweight '" below 1000 moloecul.ar weight 

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

b. You mustmake separate confidentiality claims for monomer orotherreactant identity, composition information, andresidual informatiOl\. Mark 00 the "Confidentialft box next to any item youclaim &!I confidential (1)

- Provide the specific chemicalname and CAS Registry Number (ifa number exists) ofeach. monomer orotherreactantused. in the 

# • manufactureof the polymer. (2)

- Mark 00 this column ifentryin column (1) is mnfidentiaL (3)

- Indicate the typical weight percent of each monomer orother reactantin thepolymer. (4)

- Mark DO the identity mlumn ifyou want a monomer orotherreadant uNd at two weightpercent orleuto be lilted aspartof the polymerdocription on theTSCA Chemical Substance Inventory. (5)

- Mark (X) this column if entriesin columns (3) and (4) areconfidential (6)

- Indicate the maximum weight percent of each mot.\omer or otherreactant thal:maybepresent ... residual in thepolymer ill manufactured. for commercial purposes. (7)

- Mark 00 thiscolumn ifentryin column (6) is mnfidential Monomer or other reactant and CAS Registry Number Conti- Typical Identity Ccnfi.. Maximum Conti­ 'deatW ampoaition Mark (X) dadal residual deatW (1) (2) (3) (4) (5) (6) 

## . (1) 9i. 9i. .9i. 9i.**~ ~** **.~** 9i.**~** 

# " '1& 'I:.**~** 

# " 

# o Mark (X) this box ifyou attach a amtinuatian sheet. c. Please identify which method. you used. todewlap orobtain the specifI.ed chemicalidentityinformatiim reported. in thisnotice. (c:hec:k one). 

## . D Method. 1 (CAS Inventory Service - a copy01 theidentification report 

## D Method. 2 (other source) obtained.from Inventory Expert Service mvatbe submittedas . '. an attachment to this notice) d. TIle currently correct O\emicalAbstracts (CA) namefex the polymerthat Is consistentwithTSCA Inventory Ustinp for similarpolymers.

e. Provide a mrrectrepresentativeor partial cbemic:al1trUdUrediagram,
.. complete ascan beknown. if oneem be re.onably ucertained...



# -



# o Mark (X) this box ifyou attach a**~tinUatton** sheet. PageS 

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

### Part I - GENERAL INFORMATION

- Continued**~** Section B - CHEMICAL IDENTITY INFORMATION

- Continued 30 Impuritis

(a) - Identify -=b. Imparity tlUIt lDay be _ubly utidpated ID be praeat Inthe chaaic:alnHUDca

__1IDdUed for aJ_en:W p1IrpOMa. Provide the CAS Rep.try NlUIlber If
..nibhle. Utheft
.. lIIlidentified Imparltl.., eater "uideatlfied. • (b) -EatbutetM awdmlUll weipt Yo of eKIa Imparity. U th
.... 1IJlidatified baputtiel, stba
.. theirIDtaI wetpt Yo. Maximum 

## . Impurity and CAS Registry**Num~** Coatipercent dential (a) (b) 'l{, 

# X 'l{, 'l{, 

# ,. ,. 'l{, 'l{, 

# o Mark (X) this box if you attach a continuation sheet. 4. Synonyau- Enter anyc:hemicalsynonyms for thenew chemical substanceidentified.in subsectlCll 1 ar 2.



## . 

# :1 X 

# o Mark (X) this box ifyou attach a continuation sheet. 5. Trade identifiatiGl

- List tradenamesfor the newc:hemicallUbetanc:e identified. insuteed:iCll 1 or2.**~-O ~"** **~.** c 

### I.
. IB'-\OGO 

### (p\"'\\'J**'Sv~lo..\'\c.e It'~;>**  

## n-butylc.duJ'\c\ :).
. . .**'~)L~** t / 

### I .etC 0 t C..L 

### tc:. :2.!"")/o) 

# o ( . -\tv\

## \3? C f tJ\

## tJ C:;LbS**t...c\.V~c...e** 

### IOD 

### %) -' Mark (X) this box ifyou attach a continuation sheet.
. 6. Generic chemicalname - U you claim chemical identity as amfidential. you muatprovide a generic chemical namefor your substance that reveals the spedfk: chemical identity of thenewchemicalsubetance to the maximum extent peBble. Refer to the TSCA Chemical Substance Inwntcry, 1985 EditiCll. Appendix B far guidanceon developing generic names. . 

## . 'S'q

# t--t. 

# --retr (AO \ .\:::

# fe G \'VI OV\\ 

# u. VV\ 

# o Mark (X) .this box ifyou attach a continuatlCll sheet. 7. Byproduct.

- :::;_._ anybyprodudsresultingfrom themanufacture,processing, UM. QI'Ctispc.l 01. the newchemical subetance. Provide theCAS

.... Number if available.
. Byproduct CAS RegiIItry Number Conti­ (2) (1) dential


### -. 

# x:

- 

# 0 Mark (X) this box ifyouattach a cxmtlnuatiCll. Ibeet.

.. FORM EPA**771~25** (ReV. 5-95) Pap'

### Part I - GENERAL INFORMA nON

- Continued Section C - PRODUCTION, IMPORT, AND USE INFORMATION: Mark 00 the**~Confidential"** boxnext to anyitem you daim asconfidential. L PJOduction volume

­ Estimate theuwdmum production volume duringthe first 12 months ofproduction. Also estimate the maximum productionvolume for anyconsecutive 12-monthperiod duringthe first three years ofproduction. Estimates should beon 100% newchemical substancebasis. For a Low Volume Exemption application, ifyou choose to have your notice reviewed at a lower productionvolume than 10,000 kg/yr, specify the volume and mark (x) in thebindingbox. Ifgranted, you are bound to this volume. Maximum first 12-monthproduction (kg/yr) Maximum 12-month production (kg/yr) Confi.. Bl:acHJII dential Optioa (100% new chemical substance basis) (100% newchemical substancebasis) MuXW 

# X )<

2. Use informanOD

- You mustmake separateconfidentialityc1aims for the deeaiptionof the category of UIIe, the percent ofproduc:tion volume devoted to eachcategory, the formulation of the newsubstance. andother useinformation. Mark 00 the "Confidential" Box next to anyitem you claim as confidenlial. a. (1)

- Desaibe each Intendedcategory of use of the newc:he:mk::al substancebyfunction and appUc:aticn. (2)

- Mark 00 this column ifentryin column (1) is confidentialbusinee**~tion** (CBD. (3)

­ Indicate your willingness to have the information provided in column 0)binding. (4)

­ Estimate thepercent of totalprocI.w:timl far the first thnIe yeandevotedto each category ofu-. (5)

­ Mark 00 this column ifentry'in column (4) is confidentialbusineeinformation (CBD. (6)

­ Estimate the percentof the newsubetanceasformulated. in mixtUres, suspenskma, emulslons, solutions, orgelIJ asmanufacturedfor commercialpurpoaes at sites underyourcontrol"""';ated with each category of UIe.. 

## m­ Mark 00 this column if entryin column (6) iscaniidentlalbusineeinformatklll (CBD. (8)

­ Indicate" of productvolumeexpec:ted. farthe 1J8ted. "UN" -=ton. Markmenthan onebox ifapptopriate. Mark 

### . 00 toindicate your willingnee tohavethe usetypeprovidedin (8) binding. (9)

­ Mark 00 this column ifentry(ies) incolumn (8) is (are) canfidlll1tial busineIIinformatiaa (CBD. Categoryof use (1) CBl 'IIiIldiai Produc- CBI**~in** CBI " ofIlUbetance expec:ted. per UIIe CHI (by function and applkationi.e.
.a dispersive dye for Opdaa tiaa
.. form­ (8) MoakW ulatloD finishingpolyester libera) 

# I:ted 

### eon-" Indua- Com- BiDding (2.) (3) (4) (5) (6) 

### m sumer trial merdal 0pti0D. (9) ')( ')<. 

# " )( 

# " X " 

### X )Z X 

# " " 

# " % 

# " %



### . 

# " " '" 

# " 

# " " ·Ifyou have identified a "consumer"

### use. pleueprovideona continuationsheeta detailed description ofthe use(s) ofthische.mical substance in consumerproducts. Inaddition includeestimatesofthe amcentration ofthenewchemical substance asexpected in consumerproducts and describe thechemical reactionsbywhichthis substance loses itsidentity intheamsumerproduct. 

# o Mark 00 this box ifyou attach a continuationsheet. b. Generic IfyoucbIim any categoryof UIIedelaiptkmin IUhtIectian 2a
.. amfidt!ntIaL entIlra pMIicdeIaiptkm ofthat use category. Read the inltr'actJaa.Muraalfoe examplesof generic: WIll delaiptiODL • description 

# C U r-'lv\({**O.C1~V\t**

# ~~ CoCt ''\:..\ 

## \;qS.



# - o Mark00 thisbox ifyou attach a c:ontlnuatkID sheet. 3. Hazard IDfonDatlaa- Include inthe noticea copy ofre.aDIlblefaaimiIe ofany hazard. wammg 1ltaalneDt.1abeI,. mata:lalaietydataIIbeet. 

# =: orotherinformatiaa which¥Iill beprovided toany pnDD who isnaooably ltblytobeexpoI8d totb.iII reprdIDgprotective JoUrk()l) I

### Dequipmentor practicesfar thesafehandling.transport. WIll,. ordJspoeal ofthenew su........ 1JItin part

### m hazard infonuation,you Include.**~** Marie 00 this box tfvou attach hazard ' - FORM EPA 7710-25 (Rev. 5-95) Pap7sites Mark (X) this box if b. TypeMark00 attach a continuation sheet: 

# o Manufacturing Hoursfbatc:b 

# o Use

Mark eX) to indicateyoarwUliop", tID haveyom: procna deKriptlaa

# biDdiD. PartJI

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE U>lUldJentllal box next to any item you Complete section A for each type ofmanufacture,proce..mg,ortiM operation iDvolviDg the new chemi&:almbttance at iDcta.ltri.allitei yoa controL Importen do nothave to complete tha HCtion for operation.oattide the U.S.;however, yoa may Rill have reporting requirementt ifthere are furtheriDdtl.trialprocuaiDgortiM operation.after imp«t.Yoa mu.t describe theM operation
.. See iDatruc:tion. manuaL 1. Operation description a. - Enter the identity of the site atwhich the willocx:ur. Name

City, County, State, ZIP Code
If the sameoperation willocx:ur atmore than one site, enter the numberofsites. Identify the additional sites on a continuation sheet, and Ifany ofthesiteshavesignificantly different**~** productionrates oroperations,include all theinformation requested in this lIedion for thOle

(1) Diagram themajorunitoperation stll!pl andchemical (Onvenionl. Indude intarim stonge andtranIpoIt <X1IltIIin4n (spedfy-e.g. S gallon paila,S5 gallon drum, rail car, tank truck. etc.). (2) Provide the identity, the approximate weight (by kg! day orkg/balch 01'1 a 1(){)CJI. newchemical subatancebasis),andentrypoUlt ofall starting materialt andfeedstoclal (including reactants, salvents. andcatalysts. etc.), andof

### an producta,rec:ycle aae.ms, and wuteL Include cleaning chemicals (note frequency ifnotused daily orperbatch.). (3) Identify by number the poUlts ofreIeue,includingsmall orintenrdttantrei
... to the environmentofthenewchemical substance.



# o Mark 00 this box ifyoulltt1lch • continuaIiaD IIbeet. FORM EPA 7110-2S (Rev. 5-95) Page 8, 

### Part II

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued**~** Section A ­ INDUSTRIAL SITES CO:r-.tlROLLED" BY THE SUBMIIIhK

­ Continued 2. Occupational Expomre

­ You must make separate confidentiality claims for the c1esc:riptiOl'l of worker activity, physical form ofthe new chemical substance, number of workertl expoeed. and duration of activity. Mark ()() the "Confidential" box next to anyitem youclaim asconfidential. (1)

­ Describe the activities (i.e. bagdumping, tote filling, unloadingdrums, sampling, deaning, etc.) in which workers may be expoaecl to the substance. (2)

­ Mark 00 this column if entry in column (1) is confidential businessinformatiOl'l (CDD. (3)

­ Describe an)l protectiveequipment and engineeringcontrols used to protect workers. (4) and (6)

- Indicate you willingness tohave theinformation providedin column (3) or (5) binding. " (5)

- Indicate thephysical formes) and % new chemical substance (ifin mixture) of the new chemical substance at the time of exposure (ie., solid: crystal, granule, powder, or dust) and % new chemicalsubstance (ifin mixture). (7)

­ Mark (X) this column ifentry in" column (5) is confidential businessinformation (CDD. (8)

­ Estimate the maximum number of workertl involvedin each activity for an sites combined. (9)

- Mark (X) this column ifentry in column (8) is confidential businessinformation (CDD. (10) and (11) ­ Estimate the maximum duration of the activity for any worker in hOUD per day and days per year. (12)

­ Mark (X) this column ifentries in columns (10) and (11) are confidential businessinformation (CDh. Worker activity CDI ProtectiveEquipment! BilldillS Physical farm(s BI.diIlS CBI 'of CBI MaximWl duration CBI (i.e
.. bag dumping,filling drums) Engineeri.n& Controia Optioa and';' new Optioa Workers Hn/day MlIrkW substance MlIrk(x) Expoeed Days/yr (1) (2) (3) (4) (5) (6) (7) (8) (9) nO} (I1) (12)



## .

. 

# 0 Mark 00 this box ifyouattach a continuation

### meat. 3. Environmental.ele
... and DiIpoal- Youmultmake.epuateamf1dentialBy daimI far the rei_ nUlllba- and. the amountofthe newchemical substancereleased. and otherrelease aad dispoeallnfc:nnatton. Mark 00 the "Confidential" boxnext toachitemyoudaim • confidential. (1)

- Enter thentmlberofeach releasepoint identlfied.in theproceI8delcriptloD. part II" II!IdIoaD A.1U.beectian Id(3). (2)

- Estimate the amount of the newsubstance rel.....:l (a) di.n!dly to theenvironmentor (b) into amtnlltedmology (inkgldayorkg/batch). (3)

- Mark 00 this column if entriesin col1.lDU1S (1) and (2) are confidential busineMlnfc:nnatlon (CBI). (4)

- Identify the media (stack air, fugitive air (option.al-Iee InstrudIon Manual), I!IUriac:e water, an-site 01' off-sitelaud ormcineratian, POlW, or other (specify» to whichthe new substance willbe released&om thatrel_point. (5)

- a. Desa::ibe control technology, if any, ,andcontnll elfkiency thatwill beusedtolimit therelease ofthe newsubstance to theenvironment. Forreleases disposedofon land,. cbaracter.lze the dispoealmethod and statewhether it is approvedfor dispcaal ofRCRA hazardous waste. On a continuation sheet , for each sitedescribe any additlOl'lal dispoeal methods that will beUIIed and whether the wasteis subject to secondaryor tertiary on-sitetreatment. 

### J'I. Estimate the amount reIeaaed to theenvironment after contnll technology (in kglday). (6)

- Mark 00 thiscolumn if entriesin columns (4) and (5) are confidentialbusineuinformation (CBI). (7)

- Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant0i.IIc:harge Eliminatkm System)numbersfor direct dischargers or NPDES**num~** oftbePOIW (Publidy Owned TreatmentWorks). Mark00 Ifthe POIWnameorNPDES, isconfidential businessinformatkm (CBn. Release Amount of-new subetance 

### em**r.;.~:** CoIltnll ta::bDoJogy and efficiency (youmaywiIb to optklrIaIly attach effidenc:y data) CDI Number relea!ll.!d (1) (2a) (2b) ... __.ur I=:' (3) (4) (51) I (Sb) (6) I I I "I I I 

# , I



### - I I 

# . I 1 1 

### m Mark 00 the 

## .0 POIWprovide name(s)below: CBI 

# 0:= o**~-Specify** provide NPDES. CB( destination(s) of 

# - releases to water. 

# D Mark00 this box ifyouattach a conttnuatlOl'llbeet.. FORM EPA 7710-2.5 <Rev. s...9S) Page 9­1 

### Part II

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued**~** Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section Bfor typical processing or use operationSinvolving the new chemical substance at sites you do not oontrol. Optionalfor typical manufacturing orproc:eui.ng operationsinvolvingthe new chemiCal substance at sites outside the U.s. thatyou do not control
... Inst:ructiOl'll Manual Complete a teparate section B for each type ofprocessing, oruse operation involvingthenew chemical substance. Uthe sameoperation is performed at morethan one site describe the typical operation amunon to these sites. Identify ad.dltional sUes on • continuation sheet. Optionalfor sites outsidethe United States.- See InstructionsManual 1. Operation OelCription

- To claiminformation in this section asc:onfid.ential. drde or bracketthespecific information thatyou daim • c:onfidentiaL (1)

- Diagram themajor unit operation steps and chemical conversions,.includinginterim storage and transport containers (spec:I..fy-S gaI10n pails, S5 gallon drums, rail cars, tank trucks, etc). On thediagram, identify byletterandbriefly describe each worker activity. (2)

­ Provide theidentity, the approximate weight (by kg/day or kg/batch, 1()(W. newchemical5Ubetanc:e basis), and entrypoint ofall feedstocb (indudingreadants, solvents and catalysts, etc) and ofall products,recycle streams, and wastes. lndude deaiU.ng c:hemicals (notefrequency ifnot used daily orper batch). (3)

­ Identify by numberthe points ofrelease, includingsmall. or intermittentreleases, to theenvironment ofthe newcbemic:al substance. Enter '1# of sites it ofSit.­ 

### CD Paint manufacturing Resines (1000Kg/B) ! 

### J 

# I Solvents ( 700Kg/B)**~** 

### I A. mixing Add; ti ves( 50Kg/B)**~** 

### Catalyst solution 

### I mixing 

# I lB. ( 

### 20Kg/B)**~** 

### (PMN subs tance 25% ) 

## I C. 

# I solvent 75% filtration**~** 

# I D. 

### bottling 1(cleaning) 

# . 0 Mark 00 this box if·youattach a continuatkm tbaat. 2. WCKkll' Expotan/EIl'firaluDlatalaeteue (1) ­ From thediapm above,prcwkle the kttarfer each worker a::tlvtty. Oxnplelll2-8fer each worker a::tlYity deIc:ribed. (2)

- Estimatethe numberofworkenexpoeeclfer all
.... a:mbinad. (4)

- Estimate the typical duratl.on ofexpoaure per workerin (a)bounperday IIIICl (b) daY" pery... (6)

- Desaibephysicalform ofexposure IIIICl " newcbemicalsubetance (Ifinmixture),and. anyp:otec:tlYe equipmentand. engineeringcontrols, if any usedtoprotect worlcers.. 

## m­ Estimatethepercentof thenew substance
..formulated whenpec:kaged orUMd asa Bnalproduct. (9)

-- From the process diagram above, enter the number ofeach reI.eue point. Ccmpleteg..13 fer each reI.eaIe point identified. (10)

-- Estimate the amountofthe newsubstance releued. (a) directly to theenvirclmneDt ell' (b) intocontrol tedmologyto the env:tnmment (inkg/day orkg/batch).
. (12)

-- Desc:ribe media ofrelease(stack air, fusi.tive IIlr(optiona1-see InstructionsMaiuaO, surfa:ewater,0IHi.te ell' oif-siteland CI'incineration, POIW, or other (spec:ify» and amtrol tec:hnology, ifany, that willbeused to Wt thereI.eaIe ofthenew subIbmce to theenviroDment. (14)

-- Identifybyproc:lUdS which may resultfrom theoperation.
. (3), (5), (8), (11), (13) and (15) • Mark (X) this column If any ofthe proceedingenl:det
.... c:onf1deDtialbusIneIa infc:a'matkm (CBI). Letter lof CBI DuratIca CBI Protdve Equip. I I'AI in CBI Releae AmOUlltof 

### em Ccotrol CBI

of Workers of**~BControial** FcJrm.. Numl:Ia' New

. Technology Act- Exposed .Expoauze Physical Perm and " ulatl.ml SubIItanc:a ivity new IIUbetance ReIeuecl I (10.) : (lOb) 0) (2) 

### B) (48) I (4b) (5) (6) (7) (8) (9) (11 (12) (13) 

# : )< I 

## y.. 

## 'X I I I

### "fI 

## Y I I 

### 'X I. I 

### - .. I 

# " I 

## 'X 

## Y. I I I I I I I , 'f... I 

## 1. .X I

### ~ 

# fX I 

# . I (14)

-- Byproduds: (15) 

# o Mark (X) this box ifyouattach a oontinuationsheet. FORM EPA 7110-25 (Rev. 5-95) Pap 10

## OPll0NALPOLLunON PREVENTION INFORMATION
To claim information In this section asconfidential circle orbracket the specji6c information that you claim as confidential.
I 

### In 

### this sectionyou may provide information notreported**~lsewhere** 

### in this form regarding your efforts to reduce or minimize

### potential risks associated**wi~** 

### activities sUlTounding manufacturing, processing, use and disposal ofthe P:MN spbstance. Please include new infonnation**pertinent~** 

### pollutionprevention, including lurcereduction, recycling activities and safer.processes orproducts available due to 

### the new ,emical substance. Source reduction inc1udesthe reduction in 

### the amountor toxicity ofchemiapl 

### wastesby technological modification, process and proceduremodification,**produ~ refo~ula:tion,**  

### rawmaterials substitution, and/orinventorycontrol' Recyclingrefers 

## tq the reclamation ofuseful chemical components from wastes thatwould otherwise

### be treated Qr released as air

### emissions orwater discharges, orland disposal. Descriptions ofpollution**pre~ention,** 

### source reduction and recycling should emphasizepotentialrisk reduction subsequent to**comp~ance** 

### with existing regulatoryrequirements and canbeeither quantitative or**~ualitative.** 

### The EPA is interested in 

### this information to 

### assess overall netreductions in toxictylorenvironmentalreleases and exposures, notthe shiftingofrisks to 

### other environmentalPtedia ornon-environmental areas (e.g., occupationalor consumerexposure). 

### Inaddition,infPzmation on therelative costor perfonnance characteristics oftheP:M.N substance to 

### poteIitial alternatives may

### be prOvided. All informationprovided in

### this 

### section will 

### be taken**in~o** 

### consideration during the review of this substance. See the revised InsttuctionsMaJlual**th~t** 

### includes a Pollution Prevention manual for guidance and examples; Describetheexpected netbenefitS, sw:h as (1)-an overall reduction in risktohumanhealth orthe environment; (2) a reduction in the volume**manula~;** (3) 

### a. nductlonin thegenerationofwastematerialaltmoush recycling, sourcereduction orother**m~Si** (4) a reduction inpotential toxicity orhuman

### exposure and/orenviro*,ental release; (5) aninaease. inproduct performance, a dec:rease inthec::ostofproduction_lor improved

### operation**~dency** ofthe newc::hemical substance in comparisonto existingchemical.ubatancesused inSimilar appUeationai or (6)!the extent to whichthe newchemical substance 

## maybe. substitute loranexisting sub8tancethatpo.. a greateroverallrisk

### to human health ortheenvironment.

,

, 

# o Marl< (X) this box If you attach • continuation sheet. ;

lORNEPA?'Tl04.S CRaY.NS) Page 11 ;

### Part IlI- LIST OF ATTACHMENTS I : ·1 Attach contlnuati"" sh
.... for: MCtiona atthe/arm arid tatdata arid oth
.. clata**(lnd~8** phr-"c:al/chem1c.al propertiesU'ld structurelactlvttyiinfonnatlon)•.arld optionalinIorJ:natlon aft.- tblIpage. Qarly tify theattachment arid the MCtion of thelortn to which Itre1at-.if1appropriate. Numberconsecutively the paS- ofthe attadunen In thecolwnn below, em_ the inclusivepage num" ofeachattachment. MarkGO the**"~tial** .. boxnext toany att.ac:Junent.name you claim
.. con.ftden Rad theInstructions Manualfor r=ciance onhowto daim anyinformation in arI attachment allCDnfidmtIaL You III 1Itinclude with the sanitlzed copyofthenotice orm a sanitized ofUly.ttachment in which yoU claim Wormalion
.. ... tial.
. I**A~ent** I Attacluru.,tname i 

### Conti· I , page number(s) dmtial 

### Material Safety Data Sheet (MSDS) 

# . i I 

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## Pr;~vy £y"ln";t 41 

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## R&J.bits

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### I.rT,tjio'Yl/Cornsirm S"j,I.IJ), 

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# S"'b~CA ;..... R.lII.bb.:ts 4h-bb

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### De!lAtt~ TDX,i<.ity 

## stcJ.J.yoi PM 

## tJ 

## $:,...J,itA."'d,..

-- RtNts 

## Ac...wU? Toxicity of PMfIi

### S"'''~G2.1:O**S'~Aj,.""," c~r;c.o**  ... rll.c:t""", 

### Pf"j,."tA 

## 8'? -110

, 

## Ac..wte Tox; city 

## .f Pf"'1N 

## s,J,stA
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## D~phniA..**,.,....~** ... til

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### li~...., 

## 148**A~e**

### FJow-Thro~f" 

## To-i:i1y 

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## To**~Nthow** 

### TrocJ;'" 

## B"'-c:t"erl J.. 

## R-e.. "-eA.S'e**H~qj:,'lPtt** 

## T oj- 

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# for**,..,~~ JiV)(po.s~** **".,.,..JJ-INI"i~"'~J.. r~o..se.** 



## .

, , 1 : ! , 

### I

, . ! i j I i

, 

# :**~**
i ! i ! I I 1 I I i , 

# 0 Mark00 tlda box ifyou attach a CDnlinuatlon sheet..

### Entei tbeatt.durlentnamektnumber. i FORMEl'Amo.zs .5- Pa 11 

### I i, 

### PHYSICAL AND OfEMICAL PROPERTIES WORKSHEET To assist .EIrA', review ofphysical and chemical propertiesdatil, pi
.... compIet. the following worksheet for data you provide and

### 1nd.uc:Mt it in the notice. Identify thepropertymeuured. the page of the::rt,ce on which the property appears, thevalue of the**pro~'** the unil:aiin which the property II meaured (u**~).** whether 01'not theproperty isclaimed IS conllden 

# Th. phy.lcal stateoftheneat IJUbItance .hould beprovkIed.

# Th; meuuredproperttea shouldbefor theneat (l()()f;, pure) chem1calsubstlll'lCll!. ProperUa that aremeasured lor mixturesorform tloJ'll shouldbe.so noted. ('JI. PMN substancein
_ ).You are not to submit thlII worksheet; h_• .EPA 'Ironglyncarrun..... that youdo so, .. it wI.1l simplify review and enaure that c:onfidential information IIproperlyprotectld. You should submit this Worksheet U I supplement to your subm.lssion oftelt data. This**~rksbeet** isnot I substitutefarIUbmillionoftat data.' , 

### , Property Value M
.....ttCI**Conft~** Mark 00 If 

### , Pas· t ! 01' dential provided number ' btIMe

- (a) l (0) (e) 

### Mark (X) 

### , (M'orE) (d) 

### , ; 

## X

Physical stateofneat substance , 

# le,) (1)_ (Jt) 

### M

, ' :**Vapo~essure** - fi emperature "C : Torr ,

; : Density/relative-density g/c:m3

;, Solubility 2-0 " @ Temperature "C )<. 

## 1'1**t:i~** I 

## u-o-o Solvent gIL :

- Soiubllityin water fiTemJ)er3.ture 

### 2-0 "C 

### X 

## 1 000

# a- 

# IL "C 

### IV1 

# x... ! 

## '15'" M 

# . ·c t Meltingtemperature

.. BoU!ng/sublinultfontemperature 

# .. torrpr.-ure Spectra 

# LR X It" 

## 1'-1 

## Xi Diaodation coJ'lltet
;
: Partide sizedistribution Oc:tanol/water partitionc:oeffident Henry's Lawconstant VoUtallzat:l.on from water VOUtallzatlOD &om soU

, 

### . pH CD concentration


### , : 

### , , flammability ! .Explodabillty : Adsorpt:I.on/coe.f.fldent
, Other

- Specify j**H~NM~** 

### X 

## II, 

## l"1 )(

, 

### ,**I~**i

; 

## Part II

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

­ Continued**~** Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS ; Complete section B for typic:a.l proce!IIIing or UlIII! operatiOnsinvolving the MW substance atsites you do not controLlmporters do nothave to complete thisMdlQn for oprrationsoubllide the tis.;however, YOI.lIl'lUltreport ;my**~g** or\1Mactiviti_ after import. S- the Instructions Manual. eom."lc,. II fUlTIllratc sa:ticm B(or au:Ja typIof1fI"'CIS$i1fg, orILW opmtitm iInIDl1lljor, 1M /VIPcMrriallncbstaJl«. Itthe sameoperation illperfon:ned. at.Il'IOnt than"onesitedescribethe typical operatiem coau:non to these sites. ldendfy-fditional sitesem a amtinu.tion sheet. 1. Operation Oacription

- To claim tnionnation in thiIMCtIon u c:oniidentiaJ, drde Ol'/.mK:ket thespedfic infonnation thatyou claim as confidential. (1) - Diagram the Il'IIIjor**~on** .. andchemicalC:XlnvlI!I'Iiona" including**in~** stonge and tnlNportcontainers (specify- e.g. 5 gallonpails, SSgaIlon cI.n.ima, nLilc::an. 1Itc). On the dIIIg:ram. Identify byletterand bri 

### yd-=ribe ..-h wade.-activity. (2) ­ Providetheidentity, the approximateweight (by kg! dayorkglbatch.on an 100'J110 new chemicalsubstan baIs),
..d entrypointofaD. feedstocks (lndudingreactants, solvents aDdcatalystl, etc:) aDd. 01 aU products, recyc1e streaa\s,and _lei. Include**~g** cheml.cala (notefrequency 1fnot UlIII!d dally orper batch). (3)- Id.eDtttybynumber thepolntl oll'l!!ieae, lndudlngmW1 orlntmNttent

### teIases. to theenvironment of thenewc:heU'I1ca1. substlUlc:e. (4) Pleaseenter the if 01 sites ( remember to idenllfy thelocations ofthesesites em a continuation sbeet): jot $ltes**~** 

### Coating car body
Paint described on page 10 - E. 

### Painting preparation**~2** 

### Cleaning (1900Kg/B) (Viscosity adjustment) the equipment So 1 vents ( 

### 100Kg/B) 

## I F. Coating (Machine Spray)**~3** 

### Cleaning 

## I the equipment



# - G. Cur;ng(140°C x 25m;n}


## - 

# I Coated car body 

# I

- " 

### - I..etter ,ot CDI DUlratlon**~I** 1ii:fuip.1 I" III em a...... AuIountot CDI Mec:Ua of RaI.eue CBI

of Worbn of B.npaeeringControlsl Fcxm­**~** New &r:Omtrol Act· Sxpoeed ExpoIure

. Physlcalterm mel '" alation ! sUb.t..ce TedmaJ.osy lvity new 1Ubstu\Ce ReIeued. I i (lOa) : (lOb) (l) (2) (3) , (4.) I C4b) (5) (6) (1) (8) 

### ; (9) (1l (12) (13) I L**~** ,;- /1'**~ ~**  . I 

## E I . . 

### ... ; I
.. I 

## 'f.. ' ... 

## X - 

# _.­ :,r .. <:l L X 

### 2**~,** 

### X­ X I , " .. :. 

### . ,. :**~** 1 . 

## .­

,,_ .......,I. I**~** I .v -.;. I L
____ 

### -...... 

# -­**~** " 

## X 

# F )( ! < I 1. 

### X 

## .. )( 

## 3 I ' 

### >< 

### r - •
.... 1'­ 

### .

# ­ ., I 

## - , 

## "'­ I ! I I 

### , 1 I ; I I .1 I ; t , I 1 , (l4)

- Byproducts: (15) 

# o MIIIk 00 this box ifyouattach • CDl\tinuadon
..... FORM EPA 771Q..2S (Kn.US) 

### &s '9 : 

## 14

# JS\Cd\~**~(\0~** 

# -**~**

### MATERIAL SAFETY DATA SHEET (FOR POWDER COATINGS AND RELATED MATERIALS) MANUFACTURER NAME: ADDRESS: 

### PHONE: ' IMPORTER NAME: 1 ADDRESS:.· u 

### PHONE: EMERGENCY PHONE (800)-424-9300 (CHEMTREC) 

### DATE OF PREPARATION: May 26. 1997 (5141-U) 

### SECTION I PRODUCT IDENTIFICATION CHEMICAL NAME 

## NIA TRADE NAME TMBP CHEMICAL FAMILY CATALYST(PMN SUBSTANCE) SECTION II INGREDIENTS OCCUPATIONAL INGREDIENTS CAS No. wt% 

### EXPOSURE LIMITS VAPOR TLV(TWA) PEL(TWA) PRESSURE
CATALYST(PMN SUBSTANCE) 

### proprietary 100.00 not est. not est. H/A



### NOTE: (RD)= Respirable dust. (c)- ceiling l1lit. (Skin)= skin penetrative. Ippcf= Millions of particles per cubic 

### foot.**fl~,8** 

### =Carcinogenic according to criteria established 

### ·bl (f=HTP 

### +=lA!C @=OSHA)

1 

## 17

### (5141-U) SECTION III PHYSICAL DATA BOILING RANGE: [N/A] % VOLATILE VOLUME: [<1%] S.G.: [Unknown] VAPOR PRESSURE: [N/A] MELTING POINT: [Unknown] VAPOR DENSITY: [N/A] EVAPORATION RATE: [N/A] SOLUBILITY IN WATER: [Soluble] APPEARANCE AND ODOR: [White powder] SECTION IV FIRE EXPLOSION HAZARD DATA FLAMMABILITY CLASSIFICATION: FLASH POINT: [N/A] LEL: [N/A] OSHA: [N/A] DOT : [N/A] EXTINGUISH MEDIA: [X] FOAM [ ] ALCOHOL [X] CO2 [Xl DRY CHEMICAL [Xl WATER FOG FOAM [ ] OTHERS UNUSUAL FIRE AND EXPLOSION HAZARDS: Dust can form explosive mixture in air (in the range of 10 gram/m3). SPECIAL FIREFIGHTING PROCEDURES: Wear self-contained breathing apparatus. SECTION V HEALTH HAZARD DATA SIGNS AND· SYMPTOMS OF EXPOSURE: Eye watering, reddening and coughing. EFFECTS OF OVEREXPOSURE: INGESTION Harmful if 

### swallowed. INHALATION : harmful if inhaled. SKIN EXPOSURE: May cause slight skin irritation. EYE EXPOSURE : Causes eye irritation. MEDICAL CONDITIONS PRONE TO AGGRAVATION BY EXPOSURE: No applicable information was found. PRIMARY ROUTE(S) OF ENTRY: [Xl DERMAL [Xl INHALATION [X] INGESTION EMERGENCY AND FIRST AID PROCEDURES: INGESTION Give 1 or 2 glasses of water and induce vomiting. INHALATION Remove to fresh air. SKIN EXPOSURE: Remove promptly by wiping followed by washing with soap and water. EYE EXPOSURE: Flush eyes thoroughly with plenty of water at least 15 minutes. SECTION VI REACTIVITY DATA STABILITY: [X] STABLE [] UNSTABLE HAZARDOUS POLYMERIZATION: [X] WILL NOT OCCUR [] MAY OCCUR HAZARDOUS DECOMPOSITION PRODUCTS; May produce hazardous products including carbon monoxide and fumes when exposed to extreme heat. CONDITIONS TO AVOID Do not expose to heat and humidity.

2 

### 19"

### (5141-U) 

### INCOMPATIBILITY'(MATERIALS TO AVOID) Avoid contact with, strong alkalies. strong mineral acids and strong oxidizing agents. SECTION VII SPILL OR LEAK PROCEDURES STEP TO BE TAKEN IN CASE MATERIAL IS RELEASED OR SPILLED: Return the material to the container if the material is not contaminated. WASTE DISPOSAL METHOD: Waste material must be disposed of in accordance with federal, state and local environmental control regulations. SECTION VIII SAFE HANDLING AND USE INFORMATION RESPIRATORY PROTECTION: Overexposure to the dust may be prevented by ensuring ventilation controls, exhaust or fresh air entry. NIOSH/MSHA(TC-2/C-) high efficiency dust and mist respirator may also reduce exposure. VENTILATION REQUIREMENT: Keep the concentrations of ingredients listed in Section II below the TLV. SKIN PROTECTION: ' Wear normal clothing covering arms and legs. EYE PROTECTION: Wear goggles or full face shields to handle large amount. HYGIENIC PRACTICES: Minimize skin contact. Wash hands with soap and water before eating drinking, smoking or using toilet facilities. Launder contaminated clothing before reuse. SECTION IX SPECIAL PRECAUTIONS PRECAUTIONS TO BE TAKEN IN HANDLING AND STORING: Store in a cool, dry and well-ventilated area. OTHER PRECAUTIONS: For industrial use only.
ABBREVIATIONS: not est. = 

### not established N/A = 

### not applicable U/I 

## = unknown information ('FORM NP92.1-30U)RepoitITMBP 

# Rlcerca Inc. 6925-96-(t172-TX-001


## STATEMENT OF NO DATA CONFIDENTIALITY CLAIM No claim

### ofconfidentiality is made 

### for any information contained in this 

### study on the basis 

### ofits 

### falling within 

### the 

### scope ofFIFRA § 

### 10 (d) 

### (1) (A), (B), 

### or (C)
..

'I
....._, __ .. __ ...**~** ___ .. .....w. 

### Company: Company Agent: 

### Title: President

Signature Date



### These data are the property 

### of_ . - - .::.. dDd, 

### as 

### such, are considered confidential for all

### purposes other

### than compliance 

### with

# JOJFRA. Section 10. Submission ofthese data 

### in

### compliance with F1FRA does not constitute a waiverofany right to confidentiality 

### that may 

### exist 

### under any other statute·or in any othercountry.

..



# -RepottITMBP 

# Rlcerca Inc. 8925-96-0172-TX-oD1


## STATEMENT 

# OF NO DATA CONFIDENTIALITY CLAIM No claim ofconfidentiality is made 

### for any information contained in this 

### study on the basis 

### ofits falling within the 

### scope ofFIFRA § 10 

### (d) (1) 

### (A). (B). 

### or (C). 

### Company:· 

### Nippon Paint Co. Ltd. 

### Company Agent: Japan T echnicallnformation Center 

### ntle: President

Signature Date



### These data 

### are the property ofNippon Paint 

### Co. Ltd. 

### and, as such, 

### are considered cOnfidential for all 

### purposes otherthan compliance with 

### FlFRA Section 10. 

### Submission ofthese data in

### compliance with FIFRA

### dOes not constitute a waiver ofany right 

### to confidentiality 

### that may existunder

### any other statute or in 

### any othercountry.• ReportlTMBP 

# RIcerca, Inc. 6925-96-0172-TX"()o1 

## COMPLIANCE STATEMENT The study 

### reported herein. ''Primary

### Eye Initation Study in Albino Rabbits with 

### TMBP:' Ricerca DocumentNumber 692S-96-0172-TX..Q01, 

### was conducted and 

### reported in compliance with the 

### United States Environmental

.ProtectionAgency. Title 40 Code of

### Federal Regulations Parts 160 (FIFRA) 

### and792 (l'SCA). Federal Register, 

### August 17, 1989; Japan Ministry 

### of Agriculture, Forestry andFuheries. 

### 59 NohSan, Notification No. 3850, Agricultural ProductionBureau, August 10, 1984; and "'The OECO Principles ofGood

### Laboratory Practice,"

### EnvironmentMonographNo. 45, Organisation for Economic Co-operation and Development, Paris 1992. All responsibility for specific information regarding the physical. chemical

. and stability properties ofthe test material 

### is 

### assumed by
..

Steven 

### K.. Shults, 

### B.A., Research Toxicologist StudyDirector ToxicologyandAnimal 

### Uetsbolillm L 

### Rlcen:a, Inc.

, . 

### Sponsor Date



## .... 

# . 

# . - - 

# --

# . Submitter Date

# Rlcerca,Inc..
APPROVALS ReporlfTMBP 6925-96-0172-TX..Q01



### Study Title: 

### Primary Eyelrrftation Study 

### in Albino RabbHs with TMBP Document Number: 

### 6925-96-0172-TX-001 

### Performing Laboratory: Toxicology and Animal Metabolism Ricerca, Inc. 7528 Auburn Road P. O. Box 1000 Painesville OH 440n-1000

SlewnK. Shults, 

### B.A., Research 

### Toxicologist StudyDirector
. Toxlt:ologyandAnimalMetabolism

Ann W. Brock, M.s., ReportWriter 

# R:andAnImIII",,­

.**'wJJn~**

.David M. Serrone, Ph.D., D..A.B.T., Manager

ToxicologyandAnimalMetabolism



### 2.3

# RlcercaInc.
QUALITY ASSURANCE STATEMENT ReportlTMBP .6925-96-0172-TX-001



### Based on a review of"Primary

### Eye Irritation Study in 

### Albino Rabbits with TMBP." Ricerca Document Number 6925-96-0172-TX-OOI. it is 

### concluded that 

### this 

### report accurately describes 

### the methods and standard 

### operating procedures. 

### and that the 

### reported results 

### accurately reflect the 

### raw data ofthe study. Listed below are the dates inspections were conducted by Quality 

### Assurance and the dates findings were reported to 

### the Study Director and to Ricerca Management. Dates 

### of 

### Phase of Dates Reported

### to Dates Reported to 

### inspection Study Study Director 

### Rlcerca Management

--. 

### August 8. 1996 

### In-tift; -:August 8. 1996 September 5, 1996 September 16. DatalDraft September 17, September 17, 1996 Report 1996 1996

Bonita

### J. Wei•• 

# dLa;Lfff ate' QualityABSura 

### ce Auditor

PIIf1tI5 

## 24ReportlTMBP 

# Ricerca Inc. 6925-96-0172-TX-oG1 

## ABSTRACT Study Title: Primary Eye Irritation Study in Albino Rabbits 

### withTMBP

This study was conducted to determine the 

### eye irritation produced by 

### a single application ofTMBP in albino rabbits. A dose of0.09 g ofthe material was administered to the 

### conjunctival sac ofone eye ofeach ofthree 

### New Zealand White rabbits. After each administration. the upper and lower eyelids were held closed for approximately one second to**minimj~** 

### loss ofmaterial. The eyes ofthe three 

### rabbits were not flushed with water. Two ofthe 

### three rabbits died within one to five minutes following dose administration. Clinical signs observed in these two rabbits priorto 

### death included salivation; tremors andprostration. Clinical 

### signs noted in the surviving rabbit included salivation. soft feces and 

### anogenital staining for approximately 30 minutes following dosing. Soft feces were observed at 

### the I-boor iIiterval. No abnormal clinical signs were noted in

### the rabbit after the 

### I-hourinterval. No abnormal tissues were 

### noted internally at necropsy in 

### the two

.rabbits which died during the 

### study. Ocularexaminations for irritation and lesions were performed on the one SUl'Viving rabbit at 

### approximately 1,24.48 and 72 hours after treatment and on Days 4, 7 and 10 ofthe study. Ocular

### examination following staining with fluorescein was perfonned at approximately 24.48 and 72 hours after treatment. The single ocular

### administration ofthe test material, 

### TMBP, in the 

### one surviving New ZealandWhite rabbitresulted in

### corneal effects. (opacity) and iridal 

### effects. Conjunctival redness, chemosis and discharge were observedby the I-hour interval in

### the rabbit. Minima) comeal opacity was noted in the one rabbit at the 72-hourinterval. Minimal conjunctival redness and discharge were observed inthe rabbit on Day 7 • No ocular effects 

### were noted 

### in the rabbit on Day 10 ofthe study. The maximum

### individual tota) score was 39 (out ofa maximum mean tota) score of 110), observed in the animal at 

### 48 hours post-treatment

Page

## 6ReportlTMBP 

# RicercaInc. 6925-96-0172-TX-oG1


## TABLE OF CONTENTS
'ITIlEPAGE
....................................................................................................................... 1

QUALITY ASSURANCE STA1'EMENT
.......................................................................... 

### S

Statistical 

### Analysis ...•....•.....••.••......................•.••...........••.............•.......•.....•.......... IS Smdy 

### Termination.....•••..•••....•...•.••...•.....•...•.....•...............•••.•..•.......••..•.........

__..... IS 

### STATEMENT OF NO DATA CONFIDENTIALITY CLAIM
................................ 2 COMPLIANCE ST A

### 1'EMENT
.......................................................................................... 3 

### APPROVALS•......•.•..•................................................................••.......•.........•....•.....•........•• 4
ABSmACf
........................................................................................................................ 6 

### TABLE OF CON1'.ENTS
....................................................................................................7 UST OFTABLES ..•........................................................................................ a •••••••••••••••••• 

### 9 liST OF

### APPENDICES
......................:............................................................................ 10 IN1'RODUcnON
............................................................................................................. 11 Title
................................................................................................................................ 11 Objective
............................................................................................................... 

### 11 Penorming I..abor:a.tory
.......................................................................................... 11 Study Director
....................................................................................................... 

### 11 Study Dates
............................................................................................................. 

### 11 Supportive Data. Protocol and Report
................................................................... 

### 11 

### STIJD:Y DESIGN....................•.................................................................•..........._ .•........ 12 'J'ES,T MA1'ERIALS
...............................................................;.....•..••...••..•......... ..••............• 12 '1'MBP
....................................................................................................................... _

••.. 

### 12 Ch.a:r"acteri.zation
......................................................................................... 12 Disposition
......................................................................................_.. _..••. 12 

### MA.1'ERIALS .AN'D**~O** 

### DS ••.....•••••••.•.....••.........................•....•..•....••..••.••......•_...... 12 

### TestMaterial. Prepamtion....•..••.........•...............•.•.......••.••..•.•.•....................•...•...•• 12 Test Material AdministtatiOll
...........•.••........••••....•••.••••..••......•..•••..................••..... 13 Animals .....•............................•..........................•...........................•.....................•• 13 Husban.dry'
......................................................................................................_._...... 13 Randomization and Assignment to Study and Preparation
...... ................_ ......... 14 AIiimal Obse:rY'ations
...•••••••••.•••.•.••••......•.•.................•.••..•••................••••.•.••.............. 14 Viability'
................................................................................................._...... 14 0cW8r' Iaitation
......................................................................., ..•.•..•..._......... 14•••• ReportlTMBP 

# Ricerca Inc. 8925-96-0172-1X-001


### RES'UL TS AND DISCUSSION
................................................................................., ...... 15 Anima1 

### Obse.f\1ations
...................................................**~**

### •.....•...••.••••..•••••••...•• .....,..•• IS 

### Viability.•.................•...•.••................................ ................•......•............... .... 15 Ocular Observations••.•..••••............••...•..........•....•......•....••.••...•••..•••.- ..•.. t 

### 15 Necropsy
............................................................................................, •... , .... 16 

### -CONCI...USION
............................................................................................................... , .... 17



# 27ReporVTMBP 

# Rlcerca, Inc. 6925-96-0172- TX;.oQ1 

## LIST OF TABLES Page Table 1: Individual Scores and Individual Weighted and Total Weighted Scores•.•.•..•••• IS Table 2: .Additional OcularFindings
.............................. _................................................ 19 Table 3: Results ofthe Examination ofTreated Eyes with Fluorescein Solution
............ 20 Table4: Summary ofIncidence ofPositive OcularEffects
............................................. 21Report/rMBP 

# Ricerca Inc. 6925--96-0172- TX-oG1 

## LIST OF APPENDICES "age Appendix A: Scale for the 

### Weighted Scores for Grading the 

### Severity ofOcular

### Lesions
.......................................................................... 

### 22 Appendix B: List of

### Contributors
........................................."
........,..................................... 25

:..:



### Pagll0

# RlcercaInc. ReportITMBP 6925-96-0172- TX-G01 

# . INTRODUCTION
TITLE 

### Primary Eye Irritation Study in

### Albino Rabbits with 

### TMBP OBJECTIVE 

### The objective ofthe study was to determine the primary 

### eye irritation produced

### by a single 

### application ofthe test

### material in albino rabbits. 

### This study 

### was petformed 

### according to the United States Environmental Protection Agency. Pesticide Assessment Guidelines. Subdivision F, Number 

### 81-4, and the protocol (and 

### one amendment) for a primary 

### eye irritation study, 

### RicercaDocument Number 692S
...

### 96-0172-TX-OOO
.

. PERFORMING LABORATORY 

### Toxicology and Animal 

### Metabolism 

### Ricerca, Inc. 7528 Auburn Road 

### P. O.Box 

### 1000 Painesville OH44077-1000 STUDY DIRECTOR 

### Steven K. Shults, B.A., ResearchToxicologist STUDY DATES 

### Study Initiation Date: August 1, 1996 Experimental Stan Date: August 6, 1996 Experimental Termination Date: August 16,1996 SUPPORTIVE DATA, PROTOCOL AND REPORT The 

### raY'. data, 

### original protocol and original laboratory

### report will

### be maintained inthe 

### Ricerca Archives. 

### Page 

### 11 30ReportITMBP 

# Ricerca Inc. • 6925-96-0172-TX..Q01 

## STUDY DESIGN

Test Material 

### Dose 

### Level (g) 

### Numberof

### AninaIs

## atInitiation Males Females TMBP 

### 0.09 3­ 

# - "Originol studydesign. requiredsi% 

### rabbits. Due to two deaths outof the first 

### three rabbits dosedwith TMBP, the design. was chonge to three rabbits. 

## TEST MATERIALS TMBP Common name: TMBP 

### Lot

### number: 

### 051796 Chemical name: SOUJ.'CC 

### ofmaterial:

... 

### Date material received: July 

### 19, 1996 Storage

### conditions: Ambient (room temperature) 

### Characterization All responsibility for specific information regardingthe 

### physical, chemical, purity and 

### stability properties of

### the test

### material is 

### assumed by the 

### Sponsor. 

### Disposilion After

### completion ofthe 

### study, the

### remainder ofthe 

### material, 

### exceptfor a retention sample, will

### be n:ttImcci to the 

### sotm:e. 

## MATERIALS AND METHODS
TESTAlATERIAL PREPARATlON Forthe 

### purpose of

### this study. the 

### test material

### was CODSiden:d.

### to be 

### 100% TMBP and was used 

### inthe

### form. as 

### received. The 

### solidmaterial was 

### ground to a 

### fiDe 

### powderprior

### to 

### admiaistration. '


### "".12 

# 3\ReportlTMBP 

# Ricerca Inc. • 6925-96-0172-TX-001


### TEST MATERIAL ADMINISTRATION 

### Each rabbit was fIrmly but gently restrained .until quiescent. A dose of0.09 g ofthe 

### ground test material 

### was administered into the 

### lower conjunctival sac of the right eyeofeach rabbit. The dose of0.09 g 

### was determined 

### to be 

### equivalent inweight to an estimated volume of0.1 

### mL. Immediately after 

### each administration, the upper and lowereyelids were held together for approximately one second to minimize loss ofthe 

### test material. The

### lefteye was untreated and was utilized 

### for control purposes. The treated eyes ofthe three 

### rabbits remained unwashed. All animals were returned to their individual cages to 

### await the observation intervals. Tbe 

### original study design 

### required six rabbits. Due to 

### two deaths 

### out ofthe

### first three 

### rabbits dosed with TMBP, the 

### design was change to three

### rabbits. ANIMALS 

### The three 

### male New Zealand White (young adult) rabbits used 

### for this srudy were selected from a group 

### ofrabbits received on July 29, 1996 from·Mohican Valley RabbitJ:y. Loudonville, Ohio. 

### Upon receipt, 

### these animals werehoused individually 

### in suspended stainless steel cages.

. HUSBANDRY 

### The animals were housed for a minimum S-day acclimation period prior to the study initiation. During the 

### acclimation period. the animals were observed twice daily for mortality and moribundity. The air

### flow in the 

### animal room was equal 

### to ten 

### ormore fresh air

### changes per hour. During the acclimation and study periods, the temperature 

### and relative humidity generally were within the range of61 to

### 70 

### OF and40to 60%, 

### respectively. Relative humidities 

### inthe range of

### 61 to 63% were recorded on three 

### days during the 

### accUmation period and

### relative humidities in the range of61 to 72% were recorded on thJ:ee 

### days during the 

### study 

### period. These 

### deviations from protocol specifications were not considered to have any 

### adverse effect on the results ofthe 

### study. A 12-hourlight/darkcycle 

### was maintained during the acclimation and study periods.
. The animals received PMI Feeds, 

### 1nc••TM Lab 

### Diet,TN 

### the Richmond**Stan~** TN

### Laboratory RabbitDiet 

### IfF, #5326. The feed was available ad 

### libitum during both 

### the acclimation and study periods. Tap water (supplied by the City ofPainesville) was available adlibitum 

### through an automatic watering system during both the 

### acclimation and study periods. I'Ige 

### 13 

# 31. :." ;**~**ReportlTMBP 

# Ricerca, Inc. • 6925-96-0172-TX-oD1
RANDOMIZATIONAND ASSIGNMENT

### TO STUDYAND PREPARATION i· ,. 

### Prior to

### the 

### initiation ofthe study, a physical examination ofthe 

### animals was j. 

### conducted to

### determine the suitability ofthe animals 

### for placement on study. All animaJs were consideredto be

### healthy and acceptable for use 

### on the 

### study,

.pending the 

### determination ofbody 

### weight and an ocular

### examination. On the 

### day before test material administration, six rabbits were· selected for 

### use. Each ofthe animals was 

### identified permanently with an 

### ear tag, imprinted with aunique number. and 

### by cage card. The 

### body weight ofeach 

### animal was recorded and a macroscopic examination ofeach ofthe eyes was conducted and recorded. This 

### examination included the use offluorescein sOlution and ultraviolet light. 

### Body weights ofthe six

### rabbits ranged from 

### 2459 to 

### 2691. Body weights ofthe three 

### rabbits actually utilized on the study ranged 

### from 2605 to 

### 2691 grams. The ocular

### examinations ofthe rabbits demonstrated the absence ofeye initation, ocular 

### defects and pre-existing corneal injury.
. ... 

### To control bias, animal selection for this 

### study was based on acomputergenerated process ofrandomization ofanimals utilizing random numbers.
ANIMAL OSSERVA "f.IONS 

### Viability All animals were observed 

### twice daily for mortality and moribundity during tbestudy. 

### Ocular Irritation Observations for initation and ocular lesions were performed at approximately 1.24.48 and 72 hours after

### treatment and on Days 4. 7 and 10of

### the study. An 

### examination. utilizing an ultraviolet light source. ofall treated 

### eyes following staining with fluorescein strips 

### and eyewash solution was made at 

### approximately 24. 48 and 72 hours after treatment order to detect corneal epithelial damage. 

### if

### present. Grading and scoring ofinitation was performed in 

### accordance with the Draize 

### system (Appendix A). All observations were appropriately recorded. .Any oculareffects notincluded in

### the Draize 

### system were reported, 

### if

### present.



### ,.,.14 

## 33

### I 

## i**Repo~BP** 

# RIcercaInc. 6925-96-0172-YX..Q01 

## I STATISTICAL ANAL VSIS 

## i No statistical analysis were performed on the data

### generated in this**stud~** STUDY TERMINATION 

### The one surviving rabbit was 

### killed at 

### termination of

### the study by an . intravenous injection ofan euthanasic agent, Sleepaway.- A 

### necropsy Jasnot conducted on this rabbit. The two rabbits which died during the study

# wb sUbjected to a gross necropsy. 

### AIl necropsy observations were recorded.l· No tissues were retained.
. , 

## RESULTS AND DISCUSSION
ANIMAL OBSERVATIONS 

### Viability 

### Two ofthe three 

### rabbits died within one to

### five minutes following dose administration. Clinical 

### signs observed in these 

### two rabbits priorto 

### death included salivation;tremors and

### prostration. Clinical signs noted in the 

### surviving rabbit included salivation. soft feces and anogenital staining for
. approximately 30 

### minutes following dosing. Soft 

### feces were observed the 1­ 

### hour interval. No abnormal clinical 

### signs 

### were noted in the rabbit after the one-hourinterval. Ocular Observations The individual corneal. 

### iridal and conjunctival scores 

### can be 

### found in Table 1. 

### An itemization ofadditional ocular

### findings can be 

### found in Table 

### 2. The results ofthe 

### eye examinations using fluorescein solution can be 

### found in Table 3. A summary ofincidence ofpositive ocular

### effects can be 

### found in Table 4. The test 

### material elicited ocular effects in the one surviving rabbit following test material administration. Corneal opacity was observed

### in the 

### iabbit from the l·hour

### interval through the 

### 72-hour intervaL No corneal opacity

### was noted in the rabbit after the

### 72-hourinterval ofthe 

### study. The results ofthe eye examinations using fluorescein solution indicated corneal epithelial effects involving up to approximately SOCJ, ofthe corneal Page 16 

### 34

# Ricerca Inc. ReporflTMBP • 6925-9&-0172-lX-oG1


### surface in the rabbit at 

### the 24-hour interval. Corneal epithelial effects up 

### to approximately 25% ofthe 

### corneawere noted in the rabbit at

### 48 hours. No corneal epithelial effects were observed in the rabbit after the 

### 48-hourinterval ofthe study. An 

### epithelial lesion was observedin the 

### rabbit atthe 1 and 24-hour intervals ofthe study. Iridal 

### effccts (congestion and swelling) were observed in the rabbit from. 

### the 24-hour interval through the 72-hour interval. No iridal 

### effects were observed in the rabbit after the 72-hour interval ofthe study. Conjunctival redness, chemosis and discharge were observed in the rabbitby the I-hour interval. Draize 

### grades of2 to 

### 3 were noted from the I-hour interval through 48 hours and Draize grades 

### of 1to 

### 3 were observed 81

### the 72­ hour interval. Minimal**conjunctiva~l ~dness**  

### and discharge persisted in the 

### rabbit through Day 10. The minima] (Grade 1) redness and discharge were 

### not considered to be 

### a positive finding 

### in accordance with the United States Environmental Protection Agency guidelines for this 

### study as noted inTable 6. Conjunctival blistering was noted in the rabbit at

### 24 and 48 hours. Conjunctival blanching was observed in the rabbit at 24 hours throughDay 4 •

. No positive conjunctival fmdings were noted in any ofthe 

### rabbits on Day 

### 10

ofthe study.
. Yellow crusting around the 

### edges ofthe 

### eyelid was noted 

### inthe rabbit at

### 48 hours. The maximum 

### individual total score was 

### 39, observed 

### in the animal 8148 

### hours post-treatment. 

### Necropsy Findings noted atexternal examinations during necropsy 

### in the two rabbits which died during the 

### study included wet tan/clear material around nose, mouth and right 

### eye. The 

### treated eye ofone rabbit was cloudy. No 

### abnormal tissues were noted intemally at 

### necropsy 

### in the 

### two rabbits which died during the study.



### ".16

# ------------------------------ Ricerca, Inc. Repol1JTMB P &925-96..()172-TX

..**<X"'~** 

## CONCLUSION Two of

### the three rabbits 

### died within approximately 5minutes foHowing**dC)E~** 

### administration. The single ocular administration of

### the test material, 

### TMBP. in 

### the one smviving New Zealand White rabbit resulted in 

### corneal effects (opacity) anl1 iridal 

### effects. Conjunctival redness, chemosis and

### discharge were obsened,**b~)** 

### the 1-h4lurinterval in the rabbit. 

### Minimal corneal opacitY 

### was noted in the 

### one rabbit at the 

### 72-hour interval. Minimal 

### conjunctival redness and 

### discharge were observed in

### the rabbit on 

### Day 7. No ocular effects were :JOi.';!C 

### in the rabbit on 

### Day 10 ofthe 

### study. The 

### maximum individual total score was 39 {out ofamaximum mean tNaL 

### score (If 

### 110), observed in

### the animal at48 

### hours post-treatment.

10l8l96lces

Page 1

### i

# RlcercaInc. ReportITMBP • 6925-96-0172-TX-G01



### Table I: Individlllll Scores andIndividual WeighUdIIlUl Total WeightedScores, AninuzlNumber: 205397, (M,.J,



### Observation Observation

### HwvaIs HoIn Days 1 

### 24 48 72 

### 4 7 

### 10 14 Cornea (Opacity) Density Area Weighted Score 

### 1 

### 4 20 1 2 

### 10 2 2 

### 20 1 1 

### 5 0 0 0 

# -

-

- -

-

- -



# -

- IrisWeigbted Score 

### 0 0 1 

### 5 I 

### S I 

### 5 0 0 

# -

- 

# -

- 

# -

- Conjunctivae Redness Chemosis Discharge Weigbted Score 

### 3 2 

### ·3 16 3 3 3 18 3 2 

### 2 14 3 1 

### 1 10 2 1 

### 1 

### 8 1 0 1 4 

### 0



## - 0 0 

# -

-

-

- Total WeightedScore 

### 36 33 39 20 

### 8 4 

## - 

# - Calculations 

## of Weightedand Total WeightedScores were determinedas follows:**~** 

### Cornea WeightedScore 

# = Density ScorexArea Scorex 5 

## Iris Weighted Score 

# = Iris Score x 5

.Conjunctivae Weighted Score 

### = (Redness Score 

### + Chemosis Score 

### +Discharge Score)x 2

Total Weighted Score = Sum of WeightedScores



### "..18

# R1cerca, Inc. ReportITMB.P • 6925-96-0172--TX-G01

Ttlble 

### 2: Additiolllll OcularFindings



### Animal No. (Sex) Observation Intervals Ibn 

### Days 

### 1 24 48 72 

### 4 7 

### 10 14
205397 (M) 

### EL 

## EL.BL BS BL.BS YC 

## BL BL


# - - - M=Male 

### EL 

# = Epitheliallesion BL 

# = Conjunctival blanching BS 

# = Conjunctival blistering YC 

# = Yellow crusting arourul edges 

### of eyelid

Page 19

# -- I "**,~.** ." •**i~" ~** **~~'" ~j'H**  I II,:' , \;,' " ",-:J,lr ' ." t 1 _, '.', ';, ,. '"**~** ,1<, r' • •**,.~** • , •



# -----_.- ReportlTMBP 

# Ricercalnc • 6925-96-0172-TX-Q01 

## ._-,-""----------------------------­

Table**3~** 

### Results of the

### Examination of Treated

### Eyes with Fluorescein Solution**r--"~·'~"·"** 

### ObservatIon

### Intervals ( ( Animai Number 

### (Sel) 

### 24 Hours 48 

### 72

j \M) 2 1 0**~i** No "taimn**~** Srainm.r'**i~p** to appro,Timately 25% 

### tfftlu! comealsuiface 7 Staining ofup to approximately5()%**~jihe** 

### cornealsuiface**~tainintr (~'UP**  to 

### appro.Timo.tely 75% 

## of the 

### comealsUiface :+; )/ainil'H' !ilJ to 

### approtimately 

### 100% 

## of the 

### cornealsuiface

---,..

# _-'------------------------­

# - .......

# -­**Rlcer(~2L** 

## Inc. ReportlTMBP '6925-96-0172-TX-001 

## _._.__.D.

___­

_______________________

Tau 

### 4: Sum'llUll1 o/Incidence 01

### Positive Oculllr 

### Effects 

### Hours Days Obsefwticlft 

### 1 24 

### 48 72 

### 4 7 

### 10 14 C,lmel 

# _._-_
.. "','....

# _­ Opacity 

### 111 

## III 111 111 011 

# - - - 011 

## III 111 111 011 

# - - 

# -

.>**.~** I 

### Redness 

### 111 111 

### 111 111 111 011 011


# - ChCIt:lO:i**~s** 

### 111 111 

### 111 011 

## I 011 011 

# - - ie 

### 111 111 111 Olt­ 

### 011 011 011 

# - 

# .................

# ­ tl

### Un itt'd**~;'Grt** r'!S 

### Environmental ProtectionAgency (USEPA), PesticideAssessment**Guidelir~'~s.** 

### Subdivision F. 

### HazardEvaluation: Human andDOIMsticAnimals, Series 81-4. 

### For tbi:>**.~ Ul'NM.ry** **.** 

### in 

### accordance with the guidelines. thefollowing grades for each tissue a re**c(.llw'~!J'ed** 

### positive. The

### positive'scores for conjunctivaldischarge were established based 011 

### pe**~sonal** 

### communication wilh USEPA. Pesticide Programs. Registration DivislOIi. Opacity (Density): Grades 1. 2. 3 and

### 4 

### Iris: 

### Grades 

### 1 and

### 2 

### C

### c,.,junctivae (Redness): Grades 2 and

### 3 (Chemosis): Grades 

### 2. 3. and

### 4 (Discharge): Grades 2 and

### 3



# ----.. '

# .._.'

... 

# _------------------------­ Paga21 

## 40

# Ricerca, Inc.
APPENDIX A Reportn'MBP • 6925-96-0172- TX-G01



## Scale for the Weighted Scores for Grading the Severity of Ocular Lesions

41

# - Area of 

### Cornea Involved: quarter (or less) but not zero 

### 1 

# "­ 'Ireater than one quarter. less than one-half 

### 2



# - ':ater than one-half, less than three quarters 

### 3



# - :at.er than three quarters 

### up to whole 8Ie8. 

### 4



# - Score equals AX

### BX

### 5 

# I Total Possible Maximum

# = 80**."-.-"~-"----------------------------......,** 

### iris 

# ,--."
...**~.----------------------------I** ValUE..lS:. " Nom18l 

## o Folds above normal, congestion. swelling. circum-comea1 injection (anyone J{' ,ll! ofthese). iris still reacting 

### to light (sluggish reaction is positive) 

### 1**,,,,.,,,~.-.~** ',"""-",, 

# Ricer::a Inc. ReportlTMBP 6925-96-0172- TX-001


# ----------------------------------------------------- SCALE FOR THE WEIGHTED SCORES 

# Fon GRADING THE SEVERITY OF OCULAR LESIONS



# - Cornea 

# "- (Degree of Densityarea

### which Is most

### dense

### Is

### takenfor reading): 

# '- Opac:ity, 

### 0 ttered or diffuse area •details ofiris clearly visible 1 ily discernible translucent areas, details ofiris slightly obscured 

### 2



# - llescent areas, no details of

### iris visible, size ofpupil barely discernible 

### 3



# - lque, 

### iris invisible 

### 4

.-- ...

... reaction tolight, hemorrhage, gross destruction (anyone orall ofthese) 

### 2 ""-",,.,-«---...;;..---...;;;.........::.._-,----...;;;..-----...;.....---1 

### ScoreAX

### 5 

# I Total Possible Maximum

# = 10 

# "_._,
.., ..,,.,-------------_----1_---------------_
......**-.~'""'** ..".,...**".~-----------------------** 

## 41

## Ricerca. Inc: ReportlTMBP • 6925-96-0172-TX..(JOt 

# -----_._-----,,,,,,_._------------------­

--. Conlunctivae

..........**~1'.** Redness (refers 

### to palpebraland bulbarconjunctivae only): 

# --_.­ " \ies:;e!s NO! "Utai. 

# _._._-"",,_.­**,~** Vessds defiDitely 

### iIlJected above nomtal



# ._-_.­ e, deeper crimson red. individual vessels not easily discernible



## ---,."**~,** 

### Diflilse beefyrl.'d Chemosis: 

# _._. .'_M

__...___ 

### 1; aboyc 

### normal (includes nictitating membrane) 

# ,_....

# __ . sw

### rUing with partial evcrsionofthe lids 

# ._-,,,"
• Sw 

### wi

### th Hds about 

### halfclosed 

# --"""**~,** 

### Swelling wi

### lb lids about halfclosed to completely closed 

## .-_--,,_. Discharge: 

## ----'._

.. 

### Fe

.

# __..

# _"

..

# _­ ,A,rry amoun ob'served inT

### different from normal (docs 

### not include small amounts inner <:;tntbus ofnormal animals) 

# ---,.­

.. [Ii scharge v..

### ritn meistening ofthe lids 

### and hairs

### just adjacent to 

### the lids 

# .--­ • Di scharge v..,riUl 

### m()istening ofthe lids 

### and hairs 

### andconsiderable area around eye



# ---- Sec

### re(A+B+C)x2 

# I Total 

### Possible ,MaxImum

# = 3) 

### Score

# = Sum of 

### all scores 

### obtainedforthe

### cornea, iris, andconjunctivae



# -­**L_~nimum!~** 0 1 2 

### 3
0 1 

### 2 3 

### 4
0 

### 1 

### 2 

### 3

I lL Draiz.e.**'Dl~nna!** 

### Toxicity," Appraisal of the

### Sa/ety ojChemicaLs in 

### Foods. Drugs and Cosmetics, 

### TIfe A"$OClam.m 

### of Food and

### Drug 0jJici0/.s 

### of the 

### United States. 46-59, .I



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

### ...

# ----------------------­ 43 Page

### 24

# _11...... II Rkerca,Jnc ReportlTMBP 692S-96-0172-TX.Q01



# ----------------------------------­ APPENDIX B List of Contributors**'~·'''-,.,'-·**

### ...___...., ....**~,"._.Il_.l** 

### _________________ 

## 44 

# '.25

# _.B. .. ReportlTMBP tl 925--96-0t72
..TX-001


## LIST 

## OF CONTRIBUTORS**:te"e~l** K. bJJ15.3.A. law,,; [
...**F'(~~:n::aJ,** A.H.T, LAT**<.athJl~en** ;',1issy B.S., ALAT



## .**~..I:**

## ...\.T .lenise J. K::T:r. B.S., LAT ;Itacy E.S, LAT'

QllaHty .1\SSUr-.J.)]C(' 

### Seryins: Ri'p(;rt**Pl''t~paration,**

Archill:tl 

### Report !Jistnbmion 'Vendv PaLtgv, B.S,**~:herr:** Parker 

### nen:,.a .n,)Un:cool, 

### B.S., LAT

.;.nnmarie B.S., LAT 

### Ltavid ( . 'I! ,d I
5tevl!::' K. Shul!:;" B.A. !\nn "/**B[CIC'~** ..\1.:{ 

### \taril,)n "[ :1(l:H1er Cner}i S,'lrnn,.k ·\nn W Brock,**:~'\"LS.** Rosebud Lefmet: 

### "'.lice K. LCIl1Hl, 

### CPS 

### Stepham PreST.'JI

"'""........**','~'**..

# -

......'

# .. ,.."'...

# ""----

..."'
......

# -"-

......

# -""

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# -,
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# -
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## .. ,

### ............

# _ ...

# _---...--
...

## ,--,-,.,....

# ,'.'--

.... _._.

# ----­ Pagl26

## Statement ofEnglish Translation
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd. Sponsor Title Acute Eye Irritation/Corrosion Study ofTMBP in Rabbits Study No. 6L738



### The study was conducted in. Kashima Laboratory of Mitsubishi Chemical Safety Institute Ltd. The original**s~dy** 

### report was written 

### in Japanese. I hereby declare that this report faithfully reflects the original study report as accurately as possible to my knowledge.



### Translator:

# (),,~ Date : March 6. 1997

# JU~ Oigawa, B.&c. Research Scientist, Toxicology Group 2 ofKashima Laboratory

### Submitted to :



# [REPORT]

Acute Eye Irritation/Corrosion Study of 

### TMBP in Rabbits (Study No. 6L738)

January 16, 1997 Mitsubishi Chemical Safety Institute Ltd.

### Statement
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.

Sponsor Title Acute Eye Irritation/Corrosion Study ofTMBP in 

### Rabbits Study No. 6L738·

To the best ofmy knowledge and belief, the study in this report was conducted in compliance with the following Good Laboratory Practice Standards.
OECD Principles ofGood Laboratory Practice (1981)

Management: Osamu Fujii sealed· Date : January 16.· 1997 Head ofKashima Laboratory

# ---------------------------------------------------------- Quality Assurance Statement
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.

Sponsor Title Acute Eye Irritation/Corrosion Study ofTMBP in Rabbits Study No. 6L738



### 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 ofthe study.

Inspection or audit Date ofinspection or audit Date of reporting to the study director and to the management Study protocol November 1, 1996 

### November 1, 1996 

# -' Study procedure November 

### 11, 1996 

### November 11, 

### 1996 

### November 12, 1996 

### November 12, 1996 

### Study report January 7, 1997 

### January 7, 1997

## ---------------~-~~~~----L~-1~~~-----~~~~- 

# __**J2~_1~~~** 

# ___.

Quality Assurance Manager: KunihikQ Ofuchi sealed Date: January 16. 1997 

### Quality Assurance Personnel : Akiko 

### Esaki Sealed Date: January 16. 1997



## 4

### Outline ofthe Study
1. Title Acute Eye Irritation/Corrosion Study ofTMBP in Rabbits (Study No. 6L738)


### 2. 

### Purpose To assess acute eye irritation or corrosion effects ofTMBP in rabbits
3. 

### Guidelines: OECD guideline (No. 405, 1987)


### 4.GLP OECD GLP (1981)


### 5. 

### Sponsor 

### '.


### .... Responsible Person : Kei AoId
6. 

### Organization under contract: Mitsubishi Chemical Safety Institute Ltd. 1-30, 

### Shiba 2-chome. Minato-ku, Tokyo,Japan
7. 

### Testing facility: Mitsubishi Chemical Safety Institute Ltd. Kashima Laboratory 14 

### Sunayama, Hasaki-machi, Kashima-gun; Ibaraki, 

### Japan
8. 

### Study contributors: Study director:**&.:Ju.u.nwk"""o...1lo0uio:ga&.lw~a:L...-**

### __...w;::se",,alLllle'-ld.d Date : Ianuary 16. 1997


### Other contributor:
.... ........ Date : ,January 16. 1997 

### M_aki**Ao...M.IJim~i**

### ____-.:selQlawlell:,)t.d
Masashi Murakami sealed Date: January 16. 1997



# so 49. Study date: Initiation ofthe study November I, 1996 Receipt ofanimals November 6, 1996 Administration (1%,2% group) November 

### 11, 1996 (25% group) November 

### 18, 1996 Final observation (1%,2% group) November 

### 14, 1996 (25% group) November 21, 1996 Issuance ofthe final report January 16, 1997
10. Unforeseeable circumstances that may have 

### ill effects on the reliability ofthe study: None
11. Retention :. The protocol, 

### all data, documents, a sample ofthe test substance, and the final report will be retained in the archive of Kashima Laboratory. The sponsor and the testing facility 

### will discuss regarding further retention ofthe data five years after submission of the final report.



# 1)1 5

### Contents Summary ..................................................... '0 •••• 7 Materials and Methods 1. Test substance ................................................ 8 

### 2. Experimental animals ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 . 

### Animal husbandlY' .........**~** 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8 4. Preparation ofthe test substance •.•••••••••••••••••••••••.•••••••• 9 5. Group constitution and number ofanimals' • • • • . • • • • • • • • • • • • • • • • • • • • 9 6**~** 

### Administration' . · .. · ........... · . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 7. Observation, criteria for scoring and classification ofeye irritation •••.•. 10 8. Body 

### weight measurement· ..................................... 11 Results and. Conclusion 

### 15 

### Ocular reactions · . · · ........................ · . . . . . . . . . . . . . . . .. 12 2. Classification ofeye irritation' ••••••••••• • ••••••••.•••• • • • • . • • • . • 12 3. 

### Clinical signs' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 4. Body 

### weights ...•......................**~** 

### .. · ...... · ... · ..... .. 12 

### 5. Conclusion' ... · ........................................ · . . .. 12 References ........................................................ 13
Annex. 1 Grades for ocular lesions •••• . . . . . . • • . • • • . • . . . . . . • • • • . . • • . .• 14 Annex. 2' Scale ofweighted score for grading the severity ofocular lesions ••. 15 Annex. 3 Classification ofcompounds based on eye irritation properties' • • • •• 16 Table 1 Individual scores for ocular reactions •••••••••••••••••.•••••.• 17 Table 2 Weighted scores for ocular reactions according to Draize method •• 18 Table 3 

### Clinical signs' ... · .... · . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19 Table 4 

### Individual body weights .................................... 20

6

### Summary

.The acute eye irritation!corrosion effects ofTMBP were assessed intotal ofnine male Japanese White rabbits (Kbl: 

### JW, SPF) in accordance with the OEeD 

### guideline (No. 405, 1987). 

### The test substance (TMBP) supplied by the sponsor was dissolved in physiological saline to 1%, 2% 

### and 25% 

### (w/v) for use. Application ofthe test substance caused no eye irritation effects and abnormal clinical signs in any animal ofthe 1% 

### and 2% 

### groups. In**~he** 25% 

### group, there were slight conjunctival redness and discharge, and no iris reaction to light. These symptoms disappeared by 24 hours after application. Further, there were signs oflicking chops, salivation, miosis, increase ofrespiration rate and tremor in clinical observation. The maximum ofweighted mean total score after Draize was 16.0 in the 25% 

### group. In accordance with this score, the eye irritation of the test substance was classified as



## "Mildly irritating" in 

## conformity with Kay and Calandra's method. 

## These results have led to the conclusion that 25% TMBP has reversible and mild' irritation potential, and 1 % 

### and 2% 

### TMBP have no irritation potential to the eyes of rabbits under the conditions ofthis study.



## 53 7

## Materials and Methods
1. Test substance TMBP 

### (lot no. 10-lSIPA, 

### purity 96.6% 

### or over) was supplied by The test substance is colorless flaky 

### crystals and it's chemical name, structural formula and molecular weight are shown. Data on the stability ofthe lot ofthe test substance is guaranteed by sponsor and 

### was provided. Chemical name -"**'--~,--"** 

### Structural formula :

Molecular weight 359 Impurity water 3.4% and less
2. Experimental animals Nine male Japanese White rabbits (Kb1:JW, SPF) were purchased from'K.itayama LABES Co., Ltd. on November 6, 1996. The animals were quarantined and acclimatized to confirm good health condition for five days in the 1% and 2% groups, and twelve days in the 25% group. About four hours before application, their eyes were examined and animals without abnormalities in their eyes were used for this study. They weighted from 2.1 to 2.6 

### kg and were 10 

### to 11 weeks old on the day of application. The animals were identified by a number marked with an oil-based ink on one earlobe. Cages were also identified by a label listing the study number, name ofthe test substance, group name, animal number. sex, the day ofapplication, name ofstrain andspecies.

3. Animal husbandry Throughout the study including quarantine-acclimatization period, the rabbit room was maintained at a temperature of 22±2°C. and at a relative humidity of

### 55±15%. Air exchange was approximately 12 times per hour and lighting was from 7:00 a.m. to 7:00 p.m. The animals were individually housed in an aluminum cages (32W x 48D x 33H em, Tokiwa Kagaku Kikai Co., Ltd.) furnished with a mental mesh flooring. The cages were mounted on an automatic cascade type maintenance device (Tokiwa Kagaku Kikai Co., Ltd.). Excrement was washed out automatically with water. Diet was supplied with a stainless feeder (Tokiwa Kagaku Kikai Co., Ltd.) and water was from an automatic watering system (Tokiwa Kagaku Kikai Co., Ltd). Cages and feeders were disinfected with steam and were changed weekly. A pellet diet for experimental animals (LRC4, Oriental Yeast Co., Ltd.) and orqinary tap water filtered with 5 11m filter and irradiated with UV, were given to the animals 

### ad libitum. A fresh diet was added daily and renewed once a week. An 

### analytical report on hazardous contaminants in the diet showed that the diet met our laboratory's·specifications. The water was analyzed periodically in accordance with the Waterworks Law ofJapan, and met the required specifications. 

## 4. Preparation ofthe test substance According to the sponsor's instructions the test substance (crystal) was dissolved in physiological saline (Otsuka Pharo Fac. Inc., lot No. K6H96) in the concentration of 1%, 2% and 25% (w/v) shortly before use. 5, Group constitution and number ofanimals

. Group Test article Number ofanimals Animal number 1% l%TMBP 3 I -.. 3 

## 2% 2%TMBP 3 4 '" 6 25% .. 

### 25%TMBP 3 7

-­ 9

96. Administration 6.1 Dose level and frequency ofadministration 0.1 

### mI, once 6.2 Treatment The test article was instilled into the lower conjunctival sac ofthe right rabbit eye. The eyelids were then gently held together for one second to prevent loss ofthe test article. The treated eye remained unwashed for 24 hours following application. The left eye ofeach rabbit was not treated and served as a control. I 

### An Elizabethan collar was fitted around the neck to protect the applied eye from sh<;>rtly before application until 24 hours after application.
7. Observation, criteria for scoring and classification ofeye irritation 7.1 Observation Eyes were examined at I, 24, 48 and 72 hours after application. Clinical signs were also observed everyday. Abnormalities 

### in the cornea, iris and conjunctivae were examined with a portable slit lamp (SL M 5: Kowa Co., Ltd.). At 24 hours after application, 1% sodium fluorescein (Wako Pure· Chemical Ind. Ltd.) aqueous solution was instilled in the treated eye and rinsed out with purified water, and cornea was fully**exa~ned** for any lesion under UV lighting 

### (UVM M 57, Funakoshi Co., Ltd.). 7.2 Criteria for scoring Opacity of the cornea, congestion and swelling of the iris, and redness and swelling ofthe conjunctivae were scored according to the grades ofOECD guideline (No. 405) shown in Annex 1. Additionally, area ofcorneal opacity and discharge in the conjunctivae were scored according to Draize methodI) shown in Annex 2. 7.3 Classification ofeye irritation The weighted score were calculated for each animal according to the formula shown in Annex 2. Mean total score was calculated for each examination time and the maximum of mean score until the 72 hours was obtained. In accordance with the maximum, eye irritation property was classified in conformity with Kay and Calandra's methoi) shown in Annex 3.

10

### 8. Body weight measUrement Body weights of all animals were measured with an .electric balance (EB-16KS, Shimadzu Corp.) shortly before application and at the final 

### observation.



## S"7 11

## Results and Conclusion
1. Ocular reaction (Table 1) In the 1 % 

### and 2% groups, no eye irritation reactions were observed in any animal until the final observation. In the 25% group, no reaction to light of iris (score 2), conjunctival redness ofscore 

### 1 and discharge ofscore 2 were observed in all animals at 1 hour after application. These symptoms disappeared by 24 

### hours after application.
2.**Cla~sification** 

### ofeye irritation (Table 2) The maximum ofmean total score in the 25% group was 16.0 at 1 hour after application. The eye irritation potential ofthe test article was classified as "Mildly irritating"
. 3. Clinical signs (Table 3) In 

### the 1 % and 2% groups, all animals showed no abnormal clinical signs during the observation period. In the 25% group, licking chops were observed in all animals immediately after application, and slight salivation was observed in one animal. In addition;increased respiration rate, miosis and tremor were observed in**~1l** 

### animals by 30 minutes after application. These symptoms disappeared by 24 hours after

.application and thereafter all 

### animals showed no abnormal clinical signs until the final observation.
4. Body weights (Table 4) Administration ofthe test substance caused no abnormal change in body weights ofall animals.
5. Conclusion These results have led to the conclusion that 25% TMBP has reversible and mild irritation potential, and 1% and 2% TMBP have no irritation potential to the eyes of rabbits under the conditions ofthis study.

12

## References 1) Draize, J. 

### H., Woodard, G., Calvery, H. O. (1944) : Methods for the study of irritation and toxicity of substances applied topicaly to the skin and mucous membranes, J. 

### PharmacoL Exp. Therap., 

# .u. 377-390. 2) Kay, J. 

### H. & 

### Calandra, J. 

### C. (1962) : Interpretation of eye irritation tests, J. 

### Soc. Cosmo Chem.,.u. 

### 281-289.

13Annex I Grades for ocular lesions CORNEA· Opacity: degree of density (area most dense taken for reading) No ulceration or opacity

-- ----- -- ---- -- -- -- --**-~** ------ ---- ------ ---- -.---- ------- 0 Scattered or diffuse areas of opacity (other than slight dulling ofnormal lustre). details of iris clearly visible

------------------------------------------------------ 1 Easily discernible translucent area, details of iris slightly obscured

----------------- 2 Nacrous area, no details ofiris visible. size ofpupil barely discernible
. -.---------- 3 Opaque cornea, iris not discernible through the opacity .--------------------------- 4

Normal

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

### 0 Markedly deepened rugae, congestion, swelling. moderate circumcorneal 

### hyperaemia, or injection, any 

### ofthese or 

### combination ofany 

### thereof, iris still reacting to light (sluggish reaction is positive) . ---------------------------­ Noreaction to light, haemorrhage. gross destruction (any or all of these)

---------- 2 CONJUNCTIVAE Redness (refers to palpebral and bulbar conjunctivae, cornea and iris) Blood vessels normal .---- ------- ---- ---- ------ --- ---- ---- ------ ------- --.- ----- - 0 Some blood vessels definitely hyperaemic (injected)

------------------------------ I Diffuse, crimson colour, individual vessels not easily discernible

------------------ 2 Diffuse beefy red

--- -- --- -- ---_. - ..-••._---- ---- ---- -- ----- ------- ---- ----- ---- 3 Chemosis: lids and/or nictating membranes No swelling . ------------------------------------------• ----------------------- 0 Any swelling above normal (includes nictating membranes) ----------------------- 1 Obvious swelling with partial eversion of lids .----------------------------------- 2 Swelling with lids about half closed .---------------------------------.---------- 3 Swelling with lids more than halfclosed

-------- ------ ------ ----- --- ------------- 4 ·The area ofcorneal opacity should be noted

I 4 

# GoAnnex 2 Scale ofweighted score for grading the severity ofocular lesions
I. Cornea A. Opacity

- degree ofdensity (area most denSe taken for reading) No opacity

-- -- ------ --------- --- ---- ----- ------------ --- ---- ----- --- ------ 0 Scattered or diffuse area, details of iris clearly visible

------------------**~**---- 1 Easily discernible translucent areas, details 

### of iris slightly obscured . ----------- 2**Opalesce~t. ~as~**  

### ':10 details of iris visible, size ofpupil barely discernible . ---- 3 Opaque, uns invisible

------------------------------------------------------ 4 B. Area ofcornea involved One quarter (or less) but not zero .------------------------------------------ 1 Greater than one quarter, but less than half

-- ---- ----- ---- -- ------- --- --- ---- 2 Greater than half less than three quarters .--- ----- ------ ------- ---- ------ ----- 3 Greater than three quarters up to whole area ------------.-------------------- 4 Score equals A x B x 5 Total maximum 

## = 80 

## n.Iris A. Values Normal .------------------------------------------------------------------- 0 Folds above normal, congestion, swelling, circumcorneal injection (anyone or all ofthese or combination ofany thereof), iris still reacting to light (sluggish reaction is positive)

-••--.--------------.- .•••---.--.----.--.------ I No reaction to light, hemorrhage; gross destruction (anyone or all ofthese) 2 Score equals A x 5 Total maximum = 10 

## m. Conjunctivae A. Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris) Vessels normal
.-------- ---.-- -- ----------.-- --- --.----- -- -- ---- ----- ---- -- 0 Vessels definitely injected above normal

------------------------------------- 1 More diffuse, deeper crimson red, individual vessels not easily discernible ----- 2 Diffuse beefy red

-- ---- -- ---- ------------- ---- --- -- ------ -- ---- ---..----- -- 3 B. Chemosis No swelling

----- -- -- ------------- ---- ----- --- ------ -- ----- ---- --- ----- ---- 0 Any swelling above normal (includes nictitating membrane) .------------------- 1 Obvious swelling with partial eversion of the lids .- -- --- ----**---~** ---- ---- ----- 2 Swelling with lids about half closed

---------------------------------------- 3 Swelling with lids about half closed to completely closed --------------------- 4 C. Discharge No discharge

-------------------------------------------------------------- 0 Any amount different from normal (does not include small amount observed in inner canthus ofnormal animals)

--------------------------------- I Discharge with moistening of the lids and hairs just adjacent to the lids -------- 2 Discharge with moistening ofthe lids and considerable area around the eye 3 Score CA+B+C) X2 Total maximum = 20 The maximum total score is the sum of all scores obtained for the cornea, iris and conjunctivae.



## &1 1 

### SAnnex 3 Classification ofcompounds based on eye irritation properties (Steps 1 and 2) Step 1 Find the maximum, mean total score for 

### a11 three tissues occurring within the first 96 

### hr. following instillation for which the incidence of this score plus or minus 

### 5points is at least40 per cent Step 2
. Choose an initial or tentative rating on the basis ofthe score found in step 1 as follows: Seore from step 1 Tentative eye irritation rating

0.0 to 0.5 points Nonirritating (N) 0.5 to . 2.5 points Practically nonirritating (PN) 2.5 to 15 points Minimally irritating (M 1 ) 15 to 25 points
.Mildly irritating (M 2) 25 to 50 points Moderately irritating (M 3) 50 to 80 points Severely irritating (S) 80 to 100 points Extremely irritating (E) 100 to 110 points Maximally irritating (Mx) For borderline scores choose the higher rating
(Step 3) Tentative rating Requirement for maintenance N MTSu 

## =0; forMTSu > 0, raise one level PN As for N 

### Ml MTS
... 

## =0; for 

### MrS. 8 

### > 

### 0, raise one level 

### M2 

### MrS 88 ,.. 

### 0; for 

### MTS 8 8 

### > 

### 0, raise one level Ma

. (1) MTS f**~** 20; for MTS f > 20, raise one level (2) ITSf**~** 10 (60%); ifnot true then no rabbit may show ITS f > 30; otherwise raise one level S (1) As for Ms except use MTS f ;:;; 40 (2) As for Ma except use ITS f**~** 30 (60%) and 60 for high E (1) As for Ma except use MTS f**~** 80 (2) As for M3 except use ITS I ;:;; 60 (60%) and 100 for high Mit (1) MTS f > 80; for MTS f;:iii; 80, lower one level (2) ITS f > 60 (60010); otherwise lower one level Symbols: MTS ,.. Mean total score. ITS == Individual rabbit total score. Subscripts denote scoring interval: 24, 48 or 96 hr. f;' final score (1 days) "Kay, 

### I. 

### H. & Calandra, 

### I. C.; Interpretation of eye irritation tests, 

### I. Soc. Cosmo Chem., 

### il, 281-289 (1962)"

1 6Study No. 6L738
Table 1-1 Individual scores for ocularreactions Group:**1~** 0 Animal

No. Observation 

### Time Score for ocular reaction Other 

### findings Cornea Opacity Area Iris Conjunctivae 

### Redness 

### Discharge Chemosis 1 0 0 0 0 0 0 

# - 1 

### 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# - 1 0 0 0 0 0 0 

# - 2 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# -

1 0 0 0 0 0 0 

# - 3 

### 24 Hours 

### O· O· 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# - *: Examined with fluorescein -: No findings

Table 1-2 Individual scores for ocular reactions Oroup: 2% 0 Animal

No. Observation Time Score for ocular reaction Other findings Cornea Opacity Area Iris Conjunctivae Redness Discharge Chemosis 1 0 0 0 0 0 0 

# - 4 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# -

1 0 0 0 0 0 0 

# - 5 

### 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# -

1 0 0 0 0 0 0 

# - 6 

### 24 Hours 0*, 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# - *: -: Examined with fluorescein No findings 17 

### /;,3Study No. 6L738
Table 1-3 Individual scores for ocular reactions Grou2 : 25o/c 0 Animal

No. Observation Time Score for ocular reaction Other 

### findings Cornea Opacity Area Iris Conjunctivae 

## Redness Discharge Chemosis 1 0 0 

### 2 1 0 

### 2 M 7 

### 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# -

1 0 0 

### 2 1 0 2 M 

### 8 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# -

1 0 0 

### 2 1 0 2 M 9 

### 24 Hours 0* 0* 0 0 0 0 

# - 48 0 0 0 0 0 0 

# - 72 0 0 0 0 0 0 

# - *: Examined with fluorescein M: Miosis



### .: No 

### findings

Table 2 Weighted scores for ocular reactions according to Draize method 

### Oroup: 

### 25o/c 0 

### Animal 

### Score·



# No. 1 

## Hours 24 48 72 7 16 0 0 0 8 16 0 0 0 9 16 0 0 0 

### Mean 16.0 0 0 0 • The maximum score : 110


# 64 18Study No. 6L738
Table 3 Clinical signs

Group 

### Animal Findings Time (Dose) No. 0 5M 10M 30M IH 24H 48H 72H 1 No abnonnality 1% 2 No abnonnality 3 No abnonnality 4 No abnonnality 2% 5 No abnonnality 6 No abnonnality 7 Licking chops 

### + + + Miosis 

### + + + Increased

respiration rate 

### + + + Tremor 

### +' 25% 8 Licking chops 

### + + + Miosis 

### + + + + Increased



## respiration rate + 

## + + Tremor 

### + + 9 Licking chops 

### + + + Salivation 1 1 Miosis 

### + + + Increased.

respiration rate 

### + + Tremor 

### + Time 0: Immediately after application, M: minutes. H: hours 

### +: Positive

1: Slight



## 65" 19

### Study No. 

### 6L738
Table 4 Individual body weights (k) cg; Group Animal No. Before application 72 hrs after application


### 1% 1 

### 2 3 2.1 2.2 2.2 2.2 2.3 2.3
2% 4 

### 5 6 

### 22 

### 2.1
. 2.1 2.3 2.1

. 2.2

25% 7 8 

### 9 

### 2.4 2.2 2.6 2.5 2.2 2.7



# bb 20

# Statement ofEnglish Translation
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd. Sponsor :**~ .~**  

### --.:**''':~** 

### .. Ltd. 

### Title: Acute,Dermal Toxicity Study ofTMBP 

### in Rats· Study No.: 6L688

The study was conduCted**~** 

### Kashima·Laboratory ofMitsubishi Chemical Safety Institute Ltd. The original study report was written 

### in Japanese. I hereby declare that this report faithfully reflects the original study report as accurately as pos·sible to my knowledge.



### Translator:

# ~(24I.4e:. Date: (f 

# OnN,,-J{ 

# 1m Koutarou Yamashita, B.Sc. Researcher, Toxicology Group 3 ofKashima Laboratory

### Submitted to :



# [REPORT]

Acute Dermal Toxicity Study ofTMBP 

### in Rats (Study No. 6L688)



# '-­

January 

### 29, 1997 Mitsubishi Chemical Safety Institute Ltd.

1

# ---------------------------------------------------------- Quality Assurance Statement
Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd.

Sponsor: Title: Acute Dermal Toxicity Study ofTMBP 

### in Rats Study No.: 6L688

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 ofthe study.



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

# ---."

Inspection or audit Date ofinspection oraudit Date of reporting to the study director and to the management



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

# --_.

Study protocol October 18, 1996 October 18, 1996 Study procedure October ,30, 1996 October '30, 1996 Study report November 

### 25, 1996 November 25, 1996 January 29, 1997 January 29, 1997 Quality Assurance Manager: Kunihiko Ofuchi sealed Date: 

## January 29. 1997 Quality Assurance Personnel: 

### Mjkiko Suzuki sealed Date: JanuatY 29. 1997

3

## Outline ofthe Study
1. 

### Title: Acute Dermal Toxicity Study ofTMBP in Rats (Study No. 6L688)
2. Purpose: To assess acute dermal toxicity ofTMBP in rats
3. Guidelines: OEeD guideline (No. 402, 1987)


### 4. GLP: OECD GLP (1981)
S. 

### Sponsor: Responsible Person: Kei Aoki
6. Organizati?n under contract : Mitsubishi Chemical Safety Institute Ltd. 1-30, Shiba 2.chome, Minato-ku, Tokyo, Japan
7. Testing facility : Mitsubishi Chemical Safety Institute Ltd. Kashima Laboratory 14 Sunayama, Hasaki-machi, 

### Kashima-gun, Ibaraki. Japan


### 8. Study contributors : Study director : Koutarou Yamashita sealed Date : January 29. 1997
Other contributor : 6,lIK
.. aY,uQ"",FlI..lu ouo iil.l.i

____**--il:selldialwe~d** 

### Date : lanuaQ' 29. 1997



## .. 

# '7/ 4

### 9. Study date: Initiation ofthe study October 18, 1996 Receipt ofanimals October 23, 1996 Administration October 30, 1996 Necropsy November 13, 1996 Issuance ofthe final report January 29, 1997
10. Unforeseeable circumstances thatmay have 

### ill effects on the reliability ofthe study: None
11. Retention : The protocol, 

### all data, documents, a sample ofthe test substance, and the final report will be retained in the archive of Kashima Laboratory. The sponsor and the testing facility will discuss regarding further retention ofthe data five years after submission of the 

### final report.



### 5

## Contents

,,",. Summary
..................................................................................................... 7**M~terials** 

### and Methods"
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8 Test substance
........................................................................................ 8 1. 

### Experimental animals
.... .. .. .. .. .. .. .. .. .. .. .. .. .. •.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8 2. .Animal husban.dry
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8 

### 3. Administration
................................................................................ .. 9 

### 4. Treatment group"
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 10 

### 5. Observation and measurement • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• 10 

### 6. Clinical observation
......................................................................... 10



### 6.1 Body weight
........................................................................................ 10 

### 6.2 Necropsy
.............................................................................................. 10 

### 6.3
Results and Conclusion 

### 1. Dead animals •••••••••••••••.•••••••••••••••••.•••••••••• 11 2. Clinical 

### sign.s •••••••••••••••••••••••••••••••••••••••••••• 11 3. Body weight ••••••.•••••••••••••••••••.••••••••.•.•••••• 11 4. Necropsy findings ..••.••..••.....•••..•.••••.•••.••.•• 11 9 •• 

### 5.. Conclusion ••••••••••••.•••..•••••••••••••••••••••••••••• 11 Tables Appendices



## 73 6

### Summary
Acute dermal toxicity ofTMBP was assessed in five male and female SD rats. The test substance was administered to rats at only one dose level of2000 mg/kg body weight. The animals were observed for 14 days after administration. No animals died. Reddish tear was observed on the day of dosing and irregular respiration on Day 2. Body weight offemale decreased on Day 

### 4. No abnormalities were found in any animal in necropsy. Lethal dose lowest is greater. than 2000 mg/kgbody weight in both male and female rats, when TMBP is administered dermaly at 2000 mg/kg body weight.



### 7 

# '14

## Materials and Methods
1. Test substance TMBP (lot no. 10-1SIPA, 

### purity 96.6% 

### or over) was supplied by Nippon Paint Co., Ltd.. The test substance is colorless flaky 

### crystals and it's chemical name, structural formula and molecular weight are shown. Data onthe stability ofthe lot ofthe test substance is guaranteed by sponsor and was provided. Chemical name:. _. -:-...-- 

# .. , 

### Structural formula:

Molecularweight·: 359 

### Impurity: water 3.4% and less


### 2. Experimental animals SD strain albino rats (CIj : CD, 

### SPF) were purchased from Charles River Japan, Inc. on October 23, 1996. 

### After quarantine and acclimation for 7 

### days,**he~thy** 

### rats ofboth.sexes were used in this study. Males were 

### 7 weeks old and females were 

### 10 weeks old and weighed 270-274 g in males and 203-226 g in females on the day of administration. 

### Animals were identified by marking on the tail with 

### an oil-based ink:. Cages

. 

### were also identified with a label listing the study number, name ofthetest substance, animal number, the sex, the dose level, the day ofadministration, the name ofspecies and strain.


### 3. 

### Animal husbandry Throughout the study including quarantine and acclimation, the. animal room was maintained at a.temperature of22±2°C, and at a relative humidity of 55±15%. 

### Ventilationwas about 12 changes perhour (all fresh filtered air) and lighting was 7:00

### a.m. to 7:00 p.m. (12 hours per day). Animals were housed in polycarbonate cages (265W x 426D x 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with hard wood chip bedding for experimental animals (Beta-Chip, Charles River Japan, Inc.) on a steel rack (Tokiwa Kagaku Kikai Co., Ltd.). Three rats ofthe same sexes (before fur removing) or 1 rat (after fur removing) were housed in one cage. A pellet diet and water were supplied with stainless steel feeders (Tokiwa Kagaku Kikai Co., Ltd.) and polycarbonate bottles (700 mL, 

### Tokiwa Kagaku Kikai Co., Ltd) respectively. The cages (including bedding), feeders and watering bottles were autoclaved and were renewed once a week. A pellet diet for experimental animals (MF : 

### Oriental Yeast Co., Ltd.) and ordinary tap waterwere given 

### adlibitum 

### and were renewed once a week. The water was filtered with a 5 J.lm filter and irradiated with UV. Analytical reports on hazardous contaminants showed that the diet and bedding met our laboratory's specifications. The water was analyzed periodically in 

### accordance with the Waterworks Law ofJapan, and met the required specifications.
4. Administration The test substance was given dennally in a single administration. Dosing was performed in accordance with the OECD guideline (No. 402, 1987). Approximately 24 hours before test, the fur was removed from the dorsal area ofthe trunk (4 em x 5 cm : approximately 10% ofthe body surface area) by hair chippers (THRIVE 900 ; Daito Electric Machine Industry Co., Ltd.). After pulverizing in a mortar, the test substance was applied uniformly on a wet gauze dressing moistened with 0.3 mL 

### of water. The gauze dressing was· applied on the skin where the fur had been removed. The test site was further covered with a sticking plaster (ELATEX, ALCARE Co., Ltd.) having a vinyl film backing and with an additional sticking plaster (SILKYTEX. ALCARE Co., Ltd.) to hold the gauze dressing and test substance. After 24 hours exposure, the test site was washed with water. In a preliminaq study employing dose levels of 2000 and 1400 mg/kg body weight and three male and female rats in each dose level, none of animals died at 9 

# 76either dose level. The dose level was therefore set at one dose of2000 mg/kg in a single administration. Dose volume was calculated based on the body weight

. measuredjust before administration.
5. Treatment group The study was carried out using one group of5 male and 5 female rats. Animal number was 10101-10105 

### in males and 50101-50105 in females
..
6. Observation and measurement The following items were examined. The day ofadministration was designated as Day 1. 6.1 Clinical observation All animals were observed 15 and 30 minutes, 1, 3 and 6 hours after administration, and thereafter once a day for 14 days. 6.2 Body weight Body weights ofall rats were measured shortly before administration, and Day 4, 8 and 15 with an electronic balance (BB-5000 : Shimadzu Corp.). 6.3 Necropsy All rats were sacrificed after the final observation (Day 15) by severing abdominal aorta under anesthesia with thiopental sodium (RA VONAL : Tanabe Seiyaku Co., Ltd.) and were necropsied.



# -

10 

# 77

### Results and 

### Conclusion 1. Dead animals No animals died.
2. Clinical signs ( Table 1 ) Reddish tear was observed at 15 or 30 minutes on the day ofdosing in one male and one female. Irregular respiration was observed on Day 2. 

### in 3 males and one females. Thereafter no abnormalities were found in all rats.
3. Body weight ( Table 2 and 3 ) Body weight decreased Days 4 in females, but increase after Day 8. Males showed normal weight gain throughout the observation period.


## 4. Necropsy findings 

## No abnormalities were found in 

## all animals.
5. Conclusion Lethal dose lowest is greater than 2000 mg/kg body weight in both male and female rats, when TMBP is administered dermally at 2000 mg/kg body weight.



# .­

11**7~**

# Tables

Table 1 Clinical Sign. Sununary
.................................................................

## ····························1 Table 2 Body Weight· Summary

........................................................................................·....2 Table 3 Accumulative Body Weight Gain - Sumnuu:y
..··..···· ....·..·................·····.... ····· ..··· ......3



# -

79

## i

Table I Clinical SliD

- Su•• ary Study Ko. &LUB Test Substance; TIIBP Su Dose FludiuU Day I ! 

### :s .( 5 6 'l 8 9 10 II 12 13 f.t 15 (ai/k,) rlu ISa 

### :soa Ib 3b ib Kale !OOO Ku.ber 01 Aniaals 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 5 5 5 No Aboorul i ty leU lsI! tear 5 0 .( 1 5 0 5 0 5 0 2 0 5 0 6 0 5 0 5 0 5 0 5 0 5 0 . 5 0 5 0 5 0 5 0 5 0 5 0 Irreiular respiration 0 0 0 0' 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 Peuh 2000 Kaaber 01 Aniaals 5 5 5 5 5 6 5 5 6 6 5 5 5 5 5 5 6 5 5 Ko Abooruilly leddlsh tear .( I 5 0 5 0 5 0 5 0 .( 0 5 0 5 0 6 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 6 0 5 0 5 D Irregalar respiration D D D D D I 0 D 0 0 D 0 D D D 0 0 0 0

oQ <::)Table Z· Body Ie Igb t - Sonarv Study Ko. ILU8 Test Substuce TIIBP Un it : g Su Dose Dav 4 8 15 <a,/kg) III I e %OlD lean %71. I %89.8 326. & 389.8 S. D. t.1 1.8 3. I 8. 1 n 5 S 5 5 Fe.,1 e %000 lean %19.4 218. 0 224. Z %43.6 S. D. 9.3 6. 5 I. & 9.9 n S 5 5 5

oQ

2

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# - .. ... 

# .. 0111 !II::



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## • ""'

## Appendices

Clinical Sign
...........................................................................···..........·····..··..··..···1 

### Appendix 1 Body Weight
..............................................................................·.. ·········.... ·..........2 

### Appendix 2 Appendix 3 Accumulative Body Weight Gain
........................................................................3

### ( 

## r

Appendix I eli a i cal S Iin Sindy 110. ILUS Test**Sn~stance** : TIBP Sex Dose (1II/h) Kale 2000

Fuale %000

Positive· Ani
.. 1 Xulliler 10101 10102 10 I03 I

### a**1O~** 10105 50101 5010% 50103 5010. 50105 Pi ad IaiS Ko Ahormallty 110 Abnormality reddish lear Irre,ular respiration [rre,ular respiration [rre,ular respiration No Abnormality leclcllsb tur [cre,nlar respiration 110 Abnor
.. llty 110 Abnor
.. llty Day Tiae I Ih 3011

t

t Ih 3h 6b 2

t t t

t 3**~** 5 & 7 8 9 10 II 12 13 14 15

## ~( 

### (

Appendix % Body leight S Iud, Mo. ILU8 Test**SQhsta~ce** : flBP Unit : g Sex Dose Aniul haber Day 4­ 8 16 (a,/kld laic ZOOO (Q(QI 270 289 UZ 383 10102 272 Z90 318 389 10lOS 270 287 3%8 399 10104. 272 

### no 3U 381 10105 %14. Z9Z SSI 397
Fuah ZOOO SOlO I 221 Z.19 IU U9 501 02 ZU ZlO 232 lSi 50103 ZU 217 IU %39 50104­ ZOS 288 214. 

### no 50105 ZU 2%6 %27 IH

00 

### "l

2Appendix 3 AccuBulalive Body leight Gain Stndy No. 6LU8 Tesl SubstaDce : TMBP Unit: g SU Dose ADinl NUllber Day 4 8 15 <_g/kg) Ifa Ie 2000 10101 19 52 113 1010% 18 56 117 10103 17 58 1%9 10104 18 52 109 10105 18 57 123 Peule 2000 5010 I -% 3 %8 5010% -3 9 33 50104 5 11 %7 50103 -7 0 I 5 50105 0 I 18

OQ 0­

3

### Laboratories Columbia, Missouri

ABC Laboratoriel California 32380 Avenue 10 Madera. CA 93638 USA Tel: 209/675-0889 Fax: 209/875-0884 

### SPONSOR

T' •



### STUDY TITLE Acute Toxicity of TMBP to 

### Selenastrwn capricomutwn Printz DATA REOUIREvfENTS U.S. EPA-TSCA, 40 CPR, Part 797 Guideline 797. 1050 AUTIIORS Stephen L. Hicks Scientist/Study Director Thomas A. Ziegler Technical Scientist STUDY COMPLETION DATE December 4, 1996 PERFORMING LABORATORY ABC Laboratories, Inc. Environmental Toxicology 7200 E. ABC Lane Columbia, Missouri 65202 ABC LABORATORIES' PROJECT ID Final Report #43360 Page 1 of 131

ABC Laboratorlel Millourl ABC Laboratorlel Europe 7200 E. ABC Lane 38 Castleroe Road Columbia, MO 65202 USA Coleraine. N. Ireland BT51 3RL Tel: 573/474-8579 Fax: 573/443-9033 Tel: 44 (01265) 320839 Fax: 44 (01265) 320853

### STUDY COMPliANCE STATEMENT Study Compliance Statement for ABC Laboratories' Report #43360, entitled"Acute Toxicity of 

### TMBP to Selenastrwn 

### capricomutum Printz," for**r~:** 

## ..**_~** 

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

## - .. ... ,"(:;.:.:::

...**-~** 1"'".,.., 1­

.**---~,** 

### ABC Laboratories' study director for the above test herein confmns that the study was conducted in compliance with the U.S. EPA Toxic Substances Control Act: Good Laboratory Practice Standards (40 CFR, Part 792). Characterization, stability, and solubility were the sponsor's responsibility. All 

### data in support of this report, original and certified exact copies, were provided to :.:...**~** 

### -.**~ ~**  

### :., with the fmal report. A copy of the raw data and report, along with facility records, were retained at 

### ABC Laboratories, Inc.

Stephen
. Hicks Date ABC Laboratories' Study Director

ABC LABS #43360-2

### OUALITY ASSURANCE STATEMENT ABC Laboratories' Quality Assurance Unit reviewed Study #43360, "Acute Toxicity of 1MBP to 

### Selenastrum capricomutum Printz," for .I.'
_ _ :-**-:.~!** 

### :-'..... - ·c '. 

### The following inspeCtionslaudits were conducted on this study. Date Reported to Date Reported to Date Qf InM2ection PhaSe Inmected**Stud~** 

### DirectQr Mmagement 09/09/96 

### Preparation of Test 09109196 09/09/96 

### Solutions 09/26/96 

### Draft Report and 09126196 09127/96 

### Raw Data 11119196 

### Final Report 11119/96 12/04/96



### The undersigned conducted the 

### draft and 

### fmal report audits. These audits indicate that the report is an accurate reflection of the study as it was conducted by ABC Laboratories, Inc.

Kimberly S. Frazier Date Quality Assurance Officer I

ABC LABS #43360-3SIGNATURE PAGE
Submitted by: ABC Laboratories, Inc. 7200 E. ABC Lane Columbia, Missouri 65202 (573) 474-8579 Prepared by:

Date

Date

Approved by:



### William A. McAllister Date Manager, Environmental Toxicology



### Kifi1l);r1y Frazier Date Quality Assurance Officer I :dlj

ABC LABS #43360-4TABLE OF CONTENTS Page TITI..E PAGE
.............................................. 1 STUDY COMPUANCE STATEMENT.
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 QUAUTY ASSURANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3 SIGNATURE PAGE
......................................... 4 TABLE OF CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 UST OF ILLUSTRATIONS
.................................... 7 SUMMARY 8 ABSTRACT 

### 9 INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 MATERIALS AND METIIODS
.................................. 10 1. Test Substance
..................................... 10 A. Receipt of Test Substance
........................ , 10 B. Preparation of Standards and Test Solutions
.............. 10 II. Algal Nutrient Medium
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 

### m. Test Species
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 IV. Test Vessel and Test System
. . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 V. Biological Test Method
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 A. Algal Cell Counting
....................... '.'
. . .. 12 B. Preliminary Testing
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 C. Definitive Testing
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 VI. Statistical Analysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 A. Analysis of Variance

- Dunnett's Comparison to Control.
. . . .. 13 B. EC Calculations and 95 

### % Confidence Limits
............. 14 RESULTS AND DISCUSSION.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 15 CONCLUSION
............................................ 16 REFERENCES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 24 APPENDICES

- RAW DATA.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25 APPENDIX I - PROJECT PERSONNEL
. . . . . . . . . . . . . . . . . . . . . . . . . . .. 26 APPENDIX II

- BIOLOGICAL RAW DATA
......................... 30
ABC LABS #43360-5TABLE OF CONTENTS (continued) Page No. A. Preliminary Test Data
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31 B. Definitive Test Data
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 42 C. Records and Receipts
................................ 58 APPENDIX III

- STATISTICAL ANALYSIS DATA
..................... 72 A. ANOVA and Dunnett's Multiple Mean Test Data
............... 73 B. EC Calculations
................................... 99 APPENDIX IV

- CORRESPONDENCE AND PROTOCOL
................ 112

ABC LABS #43360-6

### UST OF ILLUSTRATIONS Table No, Page No,
I Chemical Characteristics ofAlgal Nutrient.Medium Used by ABC Laboratories' Environmental Toxicology Division " , , ,
......... , 17 II Environmental Chamber Data for the 96-Hour Acute Toxicity Study of TMBP to 

### Selenastrum capricomutum Printz , , , . . . . . . . . ,. 18 ill 

### Mean Cell Counts for 

### Selenastrum capricomutum Printz During the Exposure to TMBP ....,."....,.. , . . , . . . , . . . , , . .. 19 IV EC

### so Values, 95 % 

### Confidence Limits, and No-Observed Effect Concentrations of TMBP Exposed to 

### Selenastrum capricomutum Printz and Goodness of Fit Values
... "
...... ,............ 20 V Water Quality Measurements During the Exposure of

### Selenastrum capricomutum Printz to TMBP
.......................... 21 Figure No. Page No. 1 Cell Counts of 

### Selenastrum capricomutum Printz for Each Nominal Concentration (mg/L) During the 96-Hour Acute Toxicity Study with TMBP
.................................. 22 2 Scatter Plot of Observed and Predicted Percent Inhibition Based for TMBP from Control for 96-Hour Cell Counts vs. LoglO ' 

### Concentration (mg/L)

.... . . . . . . . . . . . . . . . . . . . . . . . . . . .. 

### 23

ABC LABS #43360-7

### Su.MMARY Subject: "Acute Toxicity of TMBP to 

### Selenastrum capricomutum Printz, It 

### ABC Laboratories' Report #43360 Sponsor: 

### :...• .:1. Testing Facility: ABC Laboratories, Inc. 7200 E. ABC Lane Columbia, Missouri 65202 (573)-474-8579 Location of Final Report and Original Raw 

### Data:

.... .., I
....



### Test Material: TMBP Nominal Test Concentrations: Control, 50, 100, 200, 400, and 800 mg/L Dilution Media: Algal media (PH 7.5) Experimental Test Dates: Initiation

- September 9, 1996 Tennination

- September 13, 1996 Length of Study: 96 Hours Results Based on Nominal Test Concentrations: 96-Hour ECto 

### = 

### 170 mg/L 

### (95 % confidence limits = 

### 130 and 200 mg/L) 96-Hour ECso 

# = 310 mg/L (95% confidence limits 

# = 290 and 330 mg/L) 

### 96-Hour EC90 

# = 570 mg/L (95 % confidence limits 

# = 500 and 630 mg/L) 

### 96-Hour NOEC 

### = 100 mg/L Test Species: 

### Selenastrum capricomutum Printz Source of Organisms: Department ofBotany, Culture Collection of Algae, University of Texas at Austin Age of Culture at Study Initiation: 3 days old

ABC LABS #43360-8ABSTRACT The primary objective of this test was to evaluate the acute toxicity of TMBP to 

### Selenastrum capricomutum Printz under static conditions following the procedures outlined in ABC Protocol No. TSCA 797.1050 (ABC Study #43360). The test was designed to yield EC

### so values following 24, 48, 72, and 96 hours ofexposure and a 96-hour no-observed effect concentration. The defInitive study was conducted from September 9, 1996, to September 13, 1996. The 96-hour acute toxicity test was conducted by exposing 

### Selenastrum capricomutum Printz to the following nominal exposure concentrations: 50, 100,200,400, and 800 mg/L TMBP along with a control. The test was conducted in triplicate, 250-mL Erlenmeyer flasks (A,B, and C) which contained 100 mL of the appropriate test solutions. Each replicate received 1.0 mL of algal inoculum containing approximately 1.0 X 106 cells/mL, resulting in approximately LO X 10 4 cells/mL for each flask. Water qUality parameters oftemperature and pH were measured at 0 and 96 hours ofthe study. The temperature of the test solutions ranged from 22 to 24°C and the pH values of the test solutions ranged from 7.4 to 7.8. The 96-hour no-observed-effect concentration was determined to be 100 mg/L. The 96-hour EC lO , EC

### so, and EC90 values were 170 mg/L (95% confIdence limits = 130 and 200 mg/L), 310 mg/L (95 % confIdence limits 

## = 290 and 330 mg/L), and 570 mg/L (95 % confIdence limits = 500 and 630 mg/L), respectively.

ABC LABS #43360-9

### INTRODUCTION The defInitive study was perfonned following the procedures outlined in ABC Protocol No. TSCA 797.1050 (ABC Study #43360) as approved by a representative ofl\T' A**~** ,-**~ ~**  _ •**~** 

### on May 31, 1996, and ABC Laboratories' study director on June 25, 1996. The test was designed to yield ECso values following 24, 48, 72, and 96 hours of exposure along with a 96-hour no-observed-effect concentration (NOEC). The defInitive study was conducted from September 9, 1996 to September 13, 1996. MATERIALS AND ME1HODS 

### I. Test Substance A. Receipt of Test Substance The TMBP test material (lot #051796) was received from :-, • on June 26, 1996. The sample was observed to be a white powder and was stored at room temperature. This sample was assigned ABC reference #TS-9220. Sample purity was reported to be· 98.9% by the study sponsor. All test concentrations for the preliminary and defmitive studies were prepared based on total product. The TMBP test material was used to prepare the preliminary and defInitive test solutions. B. Preparation of Standards and Test Solutions For defmitive testing an 800-mg/L primary standard (level 5) was prepared by quantitatively transferring 0.8002 g ofTMBP to a sterile 1-L glass volumetric flask containing algal nutrient media. The flask was then brought to volume with the algal nutrient medium to' produce the 800 mg/L test solution. The 8oo-mg/L primary standard was used to prepare the 50, 100, 200, and 400 mg/L test solutions by transferring volumes of 31.3, 62.5, 125 and 250 mL, respectively, to sterile 500-mL glass volumetric flasks. The flasks were then brought to volume with the algal nutrient medium. All solutions were observed to be clear with no visible precipitate following preparation. II. AJgal Nutrient Medium The test medium was composed of 1.0 mIJL of each of the following nutrient solutions in autoclaved ABC reagent water. ABC reagent water is defIned as reverse osmosis/deionized water passed through carbon, ion-exchange, and organic adsorption cartridges and fIltered through a 0.2-J.'m hollow fIber fmal fIlter to produce 16-18 megohmoem water. The following nutrient amounts were target weights. The actual weights for each ofthe follo'Ying nutrients are included in Appendix II. ABC LABS #43360-10Macronutrient Stock Solutions NaN03 25.500 g NaHC03 15.000 g MgS04·7H2 0 14.700 g 

### (each in 1000 

### mL ABC reagent water) MgCI2·6H2 0 12.164 g CaC12,2H2 0 4.410 g K2HP0 4 1.044 

### g Micronutrients Stock Solution MnCI2·4H2 0 415.4 mg H3B03 185,5 mg FeCI3,6H2 0 159.8 mg Na2Mo04,2H20 7.3 mg 

### (in 1000 

### mL ABC reagent water) ZnC12 3.3 mg CoCI2·6H2 0 1.4 mg CuCl2,2H2 0 12,0 p.g Na2EDTA·2H20 300.0 mg The pH ofthe media was 7.5 

### ± 0.1. Following pH detennination, the medium was resterilized by passing the media through 0.45-p.m Millipore filters. A total of4 L had been prepared prior to initiation ofthe deftnitive test. Table I contains results of the most recent screening for**sel~ted** water chemicalJphysical parameters and contaminants in the medium. 

## m. Test Species The parent stock of

### Selenastrum capricomutum Printz used in the deftnitive test was obtained from the Department of Botany, Culture Collection of Algae, University of Texas at Austin, on May 21, 1996. The parent stock of algae (UTEX-1648) was received growing on an agar slant, contained in a 50-mL culture tube, and was assigned ABC lot #96-1648-1. The algae culture was identified as 

### Selenastrum capricomutum Printz on the culture tube label. Periodically, new 

### Selenastrum capricomutum Printz cultures were initiated using the parent stock or cloned from an existing culture in 100 mL of sterile culture medium derived from the parent stock. Cultures of 

### Selenastrum capricomutum Printz at ABC Laboratories were maintained under the same conditions as those for the toxicity test. The algal culture (96-D 16) used for this toxicity test was 3 days old at test initiation. IV. Test Vessel and Test System The algal toxicity study was conducted in 250-mL Erlenmeyer flasks that were stopped with foam plugs and sterilized in an autoclave prior to the test. All test flasks were labeled with a felt marker as to ABC study number, concentration, replicate, and grid position. Following the transfer ofthe test solutions and algal inoculation, the test vessels were positioned in a random fashion and incubated for 96**hour~** at 24 

### ± 2°C under continuous cool-white fluorescent lighting and ABC LABS #43360-11 

## 9'7constant rotary agitation. Light intensity was maintained at 400 

## ± 10% footcandles (approximately 4300 lux) and the agitation rate was approximately 100 rpm. Temperature, light intensity, and oscillation rate were monitored throughout the study as shown in Table

# n. A continuous temperature recording of the test chamber was maintained during the study and is reported in Appendix 

## n. V. Biological Test Method A. Algal Cell Counting The algal cell counts were accomplished utilizing a hemacytometer and an Olympus Model BH-2 microscope. The hemacytometer has two chambers each with nine squares, 1 mm on a side. The average number ofcells per I mm2 was designated as Q. The center square was subdivided into twenty-five 0.20-mm squares. The average number ofcells per0.04 mm2 was designated as R. The cell density (d) for a given suspension of algal cells could be calculated from either of the following equations: A. d(cells/mL) = 104 X Q (average number of cells/I mm2) B. d(cells/mL) = 106 X 

### JAR (average number of cells/0.04 mm2) In general, equation B was only used with very dense cell populations such as those encountered with a 4- to 7-day-old algal culture typically used in the preparation of an algal test inoculum. Algal cell counts conducted during the definitive study used equation A. When the average number of cells per I-mm square was less than 11 algal cells, all nine I-mm squares were counted and divided by 9 to obtain average number of cells per l-mm square (Q) or the replicate mean value. For all suspensions with at least 11 cells per l-mm square, the four comer l-mm squares could be counted and averaged to obtain Q (replicate mean value). The cells/milliliters for each replicate were calculated and recorded. B. Preliminary Testing Prior to the initiation of the definitive study, range-finding studies with TMBP were conducted to detennine the concentration range for the definitive study. The first range-fmding study was conducted June 27, 1996, to July 1,1996, using nominal concentrations of0.10, 1.0. 10, 100, and 1000 mg/L. Following 96 hours of exposure, the 0.10, 1.0, 10, 100, and 1000 mg/L exposure solutions exhibited 0, 0, 1.4, 29, and 99% inhibition of cell growth, respectively, as compared to the control. A second range-fmding study was conducted July 5, 1996, to July?, 1996, using nominal concentrations of 0.50 and 500 mg/L.
ABC LABS #43360-12Following 96 hours of exposure, the 0.50 and 500 mg/L exposure solutions exhibited 0 and 91 

### % inhibition, respectively, as compared to the control. The results obtained from these preliminary studies were used to select the concentration range for the defmitive study. C. Defmitive Testing Based on preliminary test results, five nominal concentrations of TMBP, including a control, were selected for the defmitive algal assay. Nominal test concentrations for the defmitive study conducted September 9, 1996, to September 13, 1996, were 50, 100, 200, 400, and 800 mg/L. Each treatment level and control were prepared in triplicate using 100 mL of the appropriate concentration for each test vessel. Each test flask received 1.0 mL of algal inoculum containing approximately 1.0 

### x 106 cells/mL, resulting in approximately 1.0 

### x 104 cells/mL for each flask. Initial cell counts ofthe control flasks resulted in an actual mean cell count of 8.5 

### x 

## lW cells/mL. At 0 and 96 hours, measurements oftemperature and pH were taken in the control and all treated concentrations. O-hourtemperah;1re and pH measurements were performed on each parent solution and 96-hour measurements were performed on replicate A of each exposure solution. VI. Statistical Analysis The SAS program (1) prints out the raw data, plots, charts; computes the mean number of cells by treatment and hour; and tests for significant differences from the control and each test level mean at each time point. A. Analysis of Variance

- Dunnett's Comparison to Control A one-way analysis of variance (ANOVA) (using PROC GLM in SAS) was conducted for each timepoint with a Dunnett's comparison to the control. A one-tailed Dunnett's test was conducted at the 0.05 level of significance with the alternate hypothesis being that the mean number of cells was reduced in comparison to the control population. Prior to the Dunnett's test, a Shapiro-Wilk's test (2) and a Levene's test (3) were conducted to test for normality and homogeneity ofvariance, respectively, over treatments at each hour. Ifthe

### p values from the Shapiro-Wilk's and Levene's test were greater than 0.01, indicating normality and insignificant heterogeneity, then the analysis was performed on the nontransformed raw data. If the p values were less than 0.01 for some hour(s), then the raw data for each replicate were transformed using the square root of the cell count, the recommended transformation for count data (4). If both the raw data and transformed data showed nonnormality
ABC LABS #43360-13 

# ?9or inequality of variance, then a nonparametric analysis of variance was perfonned using the ranks of the values (2). B. EC Calculations and 95 % Confidence Limits The SAS program prints out the raw data; calculates percent inhibition; and estimates the EC values for conditions where the data pennit along with 95 % confidence limits. A logistic (sigmoid-shaped) model is fit to the data with percent inhibition as the dependent variable and concentration as the independent variable. The percent inhibition is calculated based on the cell density. The model used to describe the response to increasing concentrations is the four-parameter logistic model with twoparameters fixed, the minimum percent inhibition (A) at 0 %, and the maximum percent inhibition (D) at 100%. The model is only fit in instances where the mean percent inhibitions at the highest test concentration are greater than 45 % . The model is: Percent inhibition 

## = D 

## + «A-D) / (1 

## + (CONC**(B)) * (ECso**(-B)))), where: CONC 

# = test concentration 

### B 

# = slope 

## = concentration corresponding to a response halfway ECso between the minimum and maximum, 50% inhibition in this case. The SAS nonlinear modeling procedure (pROC NUN) is used to estimate Band ECso. Weighted regression, proportional to the predicted percent inhibition, is used since the variability among replicates is greater at lower concentrations having smaller percent inhibition values. The fonnula can be solved for:

= * «(A-lO)/(lO-D))**(1/B)) EC IO ECso = ECso * «(A-90)/(90-D»**(lIB») EC90 The distribution of x hat method (5) is used to estimate the 95 % confidence limits. An approximate F-test for significance of regression is also perfonned. It is used to test the null hypothesis that there is no significant relationship between percent inhibition and concentration.

ABC LABS #43360-14 

### /00Two measures ofgoodness of fit are calculated, R-squared (R_SQUARE) and the root mean square error (RMSE). R_SQUARE is calculated in order to describe the percent ofvariation about the mean explained by the logistic model. The RMSE is presented since it describes the average distance (in units of percent inhibition) between the observed data values and the estimated model fit to these values. It is one measure of the closeness ofthe data points to the logistic model curve. Itis desirable for the value of R_SQUARE to be large (100% being the maximum) and for the value of the RMSE to be small (0 being the minimum). RESULTS AND DISCUSSION A 96-hour static acute algal study with TMBP was completed on September 13, 1996. Based on the results of range-fmding studies, nominal test concentrations selected for the defmitive study were 50, 100, 200, 400, and 800 mg/L along with a control. The growth data (cell counts) from the defmitive test are presented in Table ill and Figure 1. Logarithmic phase growth was confmned at 96 hours with a mean count of9.5 

### x 105 cells/mL in the control, which was a lI2-fold increase from the initial 8.5 

## x 103 cells/mL for the control. The growth data were subjected to an ANOVA and multiple means test (Dunnett's test). The multiple means test showed a significant inhibition effect (p**~** 0.05) on growth in the 200, 400, and 800 mg/L test concentrations, as compared to the control after 96 hours. The EC values, NOEC values, and 95 % confidence limits for each observation period are presented in Table IV. The 96-hour EC lO , ECso, and EC90 values were estimated to be 170, 310, and 570 mg/L, respectively. The 96-hour NOEC was detennined to be 100 mg/L. Figure 2 shows a scatter plot of the 96-hour observed and predicted percent inhibitions based on cell density. Temperature and pH were measured in the parent solutions of the control and each test concentration at 0 hour, and were measured in replicate A of the control and each exposure concentration at 96 hours (Table V). The temperature ranged from 22 to 24°C and the pH values ranged from 7.4 to 7.8. The defmitive algal assay was perfonned following ABC Protocol No. TSCA 797.1050, which conforms with the U.S. EPA-TSCA Guidelines. However, the nutrient solution used for culturing and testing of

### SeleTUlStrum capricomutum Printz contains the chelate EDTA that is not recommended by the guidelines. Studies perfonned by ABC Laboratories and others (6) have shown EDT A to be essential to reach logarithmic phase growth and is necessary in the culturing of

### SeieTUlStrurn capricomutum Printz. The studies carried out by ABC Laboratories consisting ofa control group containing EDTA vs. non-EDTA solution have conclusively indicated that the presence of. EDT A was necessary for growth. Since organic chelators are a natural environmental constituent, the presence of EDTA in the nutrient media is both reasonable and necessary (6). For the preliminary and defmitive studies, the nutrient solution contained 300 p.g/L of EDTA, which is necessary for algae growth.

ABC LABS #43360-15 

## /01

### The study was conducted following the Good Laboratory Practice regu1ations (7) 

### and the draft and final reports were reviewed by ABC Laboratories' Quality Assurance Unit. All data in support of this report, original and certified exact copies, were provided to**~-'** 

### .. A



### -

___;. -**~** 

### , ; .... , with the fmal report. A copy of the raw data 

### and fmal report, along with facility records, were retained at ABC Laboratories, Inc. CONCLUSION The nominal test concentrations ofTMBP tested for the definitive srudy were 50, 100, 200, 400, and 800 mglL. The 96-hour NOEC value was detennined to be 

### 100 mglL. The 96-hour EC lO , 

### EC

### so, and EC90 

### values were estimated to be 

### 170, 310, and 570 mg/L, respectively.

PUBC**~S** 

### #43360-16TABLE I Chemical Characteristics of Algal Nutrient Medium Used by ABC Laboratories' Environmental Toxicology Division July 

### 1996 Screens (mglL) Total Organic Carbon: 

### < 1.0 Suspended Solids: 

### 0.50 January 

### 1996 Screens Elements (mg/L) Chlorinated Hydrocarbons (ug/L) Aluminum 

### 0.021 DDE 

### <0.002 Arsenic 

### <0.0023 DDD 

### <0.004 Boron 

### 0.041 DDT 

### <0.009 Cadmium 

### <0.00012 Dieldrin 

### <0.003 Chromium 

### <0.00089 Endrin 

### <0.008 Cobalt 

### <0.0047 a-BHC 

### <0.002 Copper 

### <0.0042 ,B-BHC 

### <0.004 Fluoride 

### 0.020 "Y-BHC-Lindane 

### <0.001 Iron 

### 0.025 Ll-BHC 

### <0.002 Lead 

### <0.0017 Heptachlor 

### <0.003 Mercury 

### <0.00003 Heptachlor Epoxide 

### <0.004 Nickel 

### <0.011 Aldrin 

### <0.005 Silver 

### <0.00019 Chlordane 

### <0.07 Selenium 

### <0.0015 Methoxychlor 

### <0.05 Zinc 

### 0.0025 Toxaphene 

### <0.7 Endosulfan Sulfate 

### <0.008 Endosulfan I 

### <0.008 Endosulfan IT 

### <0'.008 HCB 

### <0.003 Mirex 

### <0.009 Telodrin 

### <0.001 PCBs 

### <0.1 Organaphoaphate 

### U£gIL) Miscellaneous (mg/L) Diazinon 

### < 0.001 Chemical Oxygen Demand 

### 11.0 Methyl Parathion 

### <0.005 Un-Ionized Ammonia 

### <0.04 Ethyl Parathion 

### <0.007 Nitrite N 

### <0.02 Ronnel 

### <0.003 Nitrate N 4.4 Malathion 

### <0.01 Total Phosphorus as P 

### 0.15
. Etmon 

### <0.003 Ammonia Nitrogen 

### <0.04 Note: Results of tbe chemical analyses supporting these data are on fIle at ABC Laboratories.



### I 17

## J ABC LABS 

### #43360-17TABLEn Environmental Chamber Data for the 96-Hour Acute Toxicity Study of TMBP to 

### Selenastnun capricomurum Printz Light**Readin~** Oscillationc StudX HQur lux 

## JK. Teml2.b °C Rate (mm) O-hour 4347 404 24 100 24-hour 4322 402 24 100 48-hour 4245 394 24 100 72-hour 4160 386 23 100 96-hour 4125 383 25 100

a U-COR Model U-189 Quantum/Radiometer/Photometer (ABC Material Control #163-341) and U-COR Model Photometric Sensor (Serial PH-4447) ftc

- footcandles 

## = lux x 0.0929 b Mercury thermometer

C Oscillation rate taken directly from LED indicator on orbital shaker (ABC Material Control # 163-1111) in environmental chamber (ABC Material Control #163-1122)

ABC**~S** #43360-18 I 0

### if­TABLBm Mean Cell Counts for 

### Selenaslrum capricomulum Printz During the Exposure to TMBP

Nominal Test Cone.

(mg/L) O-Hr 24-Hr Mean Cell Counts (104 cells/mL) 48-Hr 72-Hr 96-Hr Control 0.85 1.6 3.3 22 95 50 1.3 3.9 21 98 100 1.2 2.7 21 94 200 1.1 * 2.2* 21 84* 400 1.0* 2.0* 11* 25 * 800 1.0* 1.6* 3.6* 7.3* * Denotes a significant (p**~** 0.05) inhibition effect from the control as determined using cell

counts and Dunnett's test



### I 0 

### $- ABC LABS #43360-19TABLE IV Values, 95 % Confidence Limits, and No-Observed Effect Concentrations of Tl\t1BP ECso Exposed to Selenastrnm capricomulum Printz and Goodness of Fit Values
Study Hour 24 48
72 ECso (95 % Confidence Limits) >800 mg/L (a) 700 mg/L (410 and 1000 mg/L) 410 mg/L (390 and 440 mg/L) 310 mg/L (290 and 330 mg/L) NOEC (mg/L) 100 100
200 100

a Could not be determined b 

## = 170 mg/L (95 % confidence limits = 130 and 200 mg/L) EC IO = 570 mg/L (95 % confidence limits = 500 and 630 mg/L) EC90 Values Indicating the Goodness of Fit of the Model for Percent Inhibition Based on Number of Cells Hour R-Square(%)A RMSEb DF

24 23.2 11.51 13 48 61.5 17.00 13 72 95.8 6.94 13 96 98.5 5.27 13 A

Desirable maximum = 100% b Desirable minimum = 0

I () 

## b ABC LABS #43360-20TABLE V Water 

### QUality Measurements During the Exposure of 

### Selenaslrum capricomutwn Printz to TMBP 

# oHour (parent Solution) 96 Hour (Replicate A) Nominal Test Cone.

(mg/L) Control 50 100 200 400 800 Temp. a °C 24 24 23 23 23 23 pHb 7.5 7.5 7.5 7.5 7.4 7.4 Temp. a °C pHb 22 7.8 23 7.8 23 7.8 23 7.7 23 7.7 23 7.6



# -- <::) a Mercury thermometer "'-J' b Beckman 

### .p 34 pH meter (ABC material control #163-640) and Beckman 39841 electrode

# ~

# ~**~** w w



# .... 

# ZFIGURE 1 Cell Counts of 

### Selenastrum capricomutum Printz for Each Nominal Concentration (mg/L) During the 96-Hour Acute Toxicity Study with TMBP

# 100~~~~~~~~~~~~~~~~~ /"

### ~**~** 0

# /~ 0 

### 1 0 0 0**~~77~~-------------= ~**  X 

### ::;:::;r/?/**~** ---l

# ~~ 

# -----­ E 

## "­ (II Q

# ~~ 

# - 00() cv ()

## ~ n 0.1**~--~--------~--------~--------~--------~--~** 

# E o 24 48 

### 72 96 en Hours**~** I....l I....l

--- Control --+- 

### 50 mg/L --+- 

### 100 mg/L -B- 

### 200 mg/L -x- 

### 400 mg/L -A- 

### 800 mg/L

# ~FIGURE 2 Scatter Plot of Observed and Predicted Percent Inhibition Based for TMBP from Control for 96-Hour Cell Counts vs. Log 1o Concentration (mg/L)

## ~**O+-------------------~--------------------4** 

### n 

### 1 

### 3

# ~ Log Concentration (mg/L) en 10 

## l I..W I..W

## ~ N I..W

## L~Actua' -+- 

### Predicted

### REFERENCES (1) The SAS System for Windows, Release 6.10. Inc., Cary, North Carolina 27513, USA. Copyright 1991, 1994 by SAS Institute (2) Conover, W.J. 

### Practical Nonparametric Statistics. 1980. 2nd 

### Edition. (3) Milliken, G.A. and D.E. Johnson. 

### Analysis of Messy Data. 1984, Vol. 1, p. 22. (4) Zar, J.H.; 

### Biostatistical Analysis. 1984, 2nd edition, p. 241. (5) Schwenke, J.R. and G.A. Milliken. 

### Biometrics 47 (June 1991), pp. 563-574. (6) Miller, W.E., J.C. Greene, and T. Shiroyama. 1978. 

### Selenastrum capricomutum Printz Algal Assay Bottle Test: Experimental Design, Application and Data Interpretation Protocol. EPA-600/9-78-018. Corvallis, Oregon. (7) 

### U. S. Environmental Protection Agency. Toxic Substances Control Act; Good Laboratory Practice Standards; Final Rule (40 CFR, Part 792). 

### Federal Register.

ABC LABS #43360-24


### 110

## Vorkingfor You Columbia, Missouri

ABC Labontories., Pm-AgDhisIon 32380 Avenue 10 Madera.. CA 93638 USA Tel: 209/675-0889 Fax: 209/675.{)884 

### SPONSOR
1'.1' _:::**~"'r** - -J
.

• - Y •.. :

___**~_ .~**  __ 

### whi
STUDY TITLE Acute Toxicity of TMBP to 

### Daphnia magna DATA REOUIREMENTS U.S. EPA-TSCA, 40 CFR, Part 797 Guideline 797.1300 AUTHOR Jane Bowman Scientist/Study Director STUDY SUBMITTED ON May 13, 1997 PERFORMING LABORATORY ABC Laboratories, Inc. Enviromnental Toxicology 7200 E. ABC Lane Columbia, Missouri 65202 ABC LABORATORIES' PROJECT ID Final Report #43359 Page 1 of 79

ABC Labotatories, Inc. ABC lAboratodes Europe.IJd.

7200 E. ABC Lane 38 CastIeroe Road Columbia, MO 65202 USA Coleraine, Nortbcm Ireland BT51 3RL TrJ: 314/474-&579 Far. 314/443-9033 Tel: 44 (01265) 320639 Far. 44 (01265) 320653 

## 1/1

### STUDY COMPUANCE STATEMENT Study·Compliance Statement for ABC Laboratories' 

### Final Report #43359, entitled "Acute Toxicity of TMBP to 

### Daphnia magna't for
_**~'~** 

### _ -- ::.---**~~.,** 

### _**~'::** 

### .• 

### "':1'=-;;'::"- .-.-..,_...., ABC's study director for the above test herein confmns that the study was conducted in compliance with the U.S. EPA Good Laboratory Practice Standards; Toxic Substances Control Act (40 CFR, Part 792). The solubility, stability, and characterization of the test chemical was the responsibility of the study sponsor. The study sponsor was also responsible for the retention of test chemical samples. This statement of compliance does not extend to supplementary analysis of the fish food contaminant screen conducted by Lancaster Laboratories, Lancaster, Pennsylvania. Results of these analyses are kept on file at ABC Laboratories Missouri. All raw data in support of this report, both originals and certified exact copies, were provided to**-~ ~ ~** **~ ~i:..:**  - -,., 

### with the fmal report. A copy of the report, along with facility records. were retained at ABC Laboratories.

## ~wman ABC Laboratories' Study Director

ABC LABS #43359-2 

## 1/).

### QUALITY ASSURANCE STATEMENT ABC Laboratories' Quality Assurance Unit reviewed Study #43359, "Acute Toxicity of TMBP to 

### Daphnia magna," for ._. - . ___ '.**l-..T~** ••_-- '" i\

..**~'-** • 

### The following inspections/audits were conducted on this study. Date Reported to Date Reported to Date of InsJ2ection Phase InsJ2ected Study Director Management December 11, 1996 Test Solution December 11, 1996 December 11, 1996 Preparation February 12, 1997 Draft Report and February 12, 1997 February 13, 1997 Raw Data April 18, 1997 Final Report April 18, 1997 April 21, 1997

The undersigned conducted the draft and final report audits. These audits indicated that the report submitted was an accurate reflection of the study as it was conducted by ABC Laboratories, Inc.

Draft Report:

# ';?fbtottu~ b'1 

# Nnu+ Ronda C. Stewart Quality Assurance Officer I



### Final Report:

# ~7.1t**~** cy . 

### Hunt Senio QualIty Assurance Specialist

ABC LABS #43359-3 

## 113SIGNATURE PAGE Submitted by: ABC Laboratories, Inc. 7200 E. ABC Lane Columbia, Missouri 65202 

### (573) 474-8579 Prepared by:

e Bowman Scientist/Study Director Approved by:

Marc C. Sword Date Scientist/Team Leader Environmental Toxicology

cy . Hunt 

### 7 Date Senio Quality Assurance Specialist

William A. McAllister Manager, Principal Scientist Environmental Toxicology :amk

ABC LABS #43359-4TABLE OF CONTENTS

TITLE PAGE
.............................................. 1 STUDY COMPLIANCE STATEMENT.
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 QUALITY ASSURANCE STATEMENT
........................... " 3 SIGNATURE PAGE
......................................... 4 TABLE OF CONTENTS.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 LIST OF TABLES
.......................................... 6 LIST OF FIGURES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6 INVERTEBRATE ACUTE TOXICITY COMPENDIUM
................... 7 ABSTRACT
.............................................. 8 METHODS AND MATERIALS
.................................. 9 I. Test Organisms
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 9 II. Test Substance
.................................... 9 A. Substance Receipt
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 9 B. Preparation of Biological Test Solutions.
. . . . . . . . . . . . . . .. 10 III. Test Method.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 A. Test Vessel and Test Apparatus.
. . . . . . . . . . . . . . . . . . . .. 10 B. Test Design.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1. Biological.
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 2. Chemical and Physical
.................. '.
. . .. 11 IV. Statistics
........................................ 11 RESULTS AND DISCUSSION.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 CONCLUSIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 REFERENCES.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18 APPENDICES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19 PROJECT PERSONNEL
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20 APPENDIX I - BIOLOGICAL RAW DATA.
. . . . . . . . . . . . . . . . . . . . . . . .. 23 A. Definitive Study
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 24 B. Records.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 38 C. Range-Finding Studies
........................... 46 APPENDIX II

- PROTOCOL AND CORRESPONDENCE
. . . . . . . . . . . . . . . .. 53 ABC LABS #43359-5

### LIST OF TABLES Table No. I Chemical Characteristics ofHard Blended Water Used for Aquatic Toxicity Studies at ABC Laboratories
.. , , , . , . . . . . . . . . . . . . . . . 13 II Water Quality Measurements During the Acute Toxicity Test of TMBP to 

### Daphnia magna , . . . . . . . . . . . . , , ' , . , , . . . . . . . . , . 14 

### III Immobility and Behavioral Observations During the Acute Toxicity Test of TMBP to 

### Daphnia magna ."
.... , .. , ... , ... "
..... 15 IV Acute Toxicity of TMBP to 

### Daphnia magna . , , "
..... , . . . . . . . . . 16 LIST OF FIGURES Figure No, 1 The 48-Hour Dose-Response Line for 

### Daphnia magna Exposed to TMBP , , . , ..... , , . , , , , .. , .... , ....... , .......... 17

ABC LABS #43359-6 

## I/b

### INVERTEBRATE ACUTE TOXICITY COMPENDIUM Subject: Acute Toxicity of TMBP to 

### Daphnia magna, ABC Laboratories' Report #43359 Sponsor: 

## r**~**

## _ .. _ Test Facility: ABC Laboratories, Inc. Environmental Toxicology 7200 E. ABC Lane Columbia, Missouri 65202 (573) 474-8579 Location of Original Raw Data and Final Report:

....

## -­ . -**~"'.** ,....l 

### Test Material: TMBP Nominal Test Concentrations: Control, 6.3, 12.5, 25, 50, 100 and 200 mg/L Dilution Water: 150 mglL as CaC03 

### (total hardness), 160 mglL as CaC03 

### (total alkalinity), 8.39 (pH), and 300 JLmhos/cm 

### (conductivity) Experimental Test Dates: Initiation

- December 11, 1996 Termination

- December 13, 1996 Length of Study: 48 hours



## .. Results: 48-hour ECsO = 

### 75 mglL (95 

### % C.L. 

# = 59 and 96 mglL) 48-hour NOEC = 12.5 mg/L 48-hour slope of dose-response line 

# = 6.0 Test Species: 

### Daphnia magna Source of Organisms: ABC Laboratories' in-house culture Age of Organisms at Study Initiation: Neonates (

### <24 hours old)

ABC LABS #43359-7 117

### ABSTRACT The acute toxicitY ofTMBP to 

### Daphnia magna was assessed using the methods outlined by ABC Protocol No. TSCA 797.1300 (for**r~.** 

## -'. r**.:...~** -). 

### Water quality parameters of temperature, dissolved oxygen, and pH were measured at 0 and 48 hours and were within acceptable limits. The dapbnids used for this study were fJISt-instar dapbnids (

## < 24 hours old) and were cultured at ABC Laboratories. Culture records indicated the adult dapbnids were in good condition. The study was conducted at the nominal concentrations of6.3, 12.5,25,50, 100 and 200 mg/L. There were 2 replicates ofeach test concentration and control. Ten dapbnids were added to each replicate beaker for a total of 20 dapbnids per test concentration and control. The 24- and 48-hour EC

### so values were >200 and 75 mg/L. The slope of the 48-hour doseresponse line was 6.0. The results indicated the 48-hour no-observed effect concentration (NOEC) was 12.5 mg/L, based on the lack of immobility or abnormal effects at this concentration. There were no abnormal effects in the control, 6.3- and 12.5-mg/L test concentrations. The abnormal effects of immobility, trailing extraneous material, quiescence and/or on bottom of the test chamber were noted in the 25-, 50-, 100- and 2oo-mg/L concentrations. All solutions in the test chambers were clear, with no visible surface 

### film or precipitate throughout the 48-hour test period.

ABC LABS #43359-8 /1 

### 'g

### METHODS AND MATERIALS The static Daphnia magna bioassay was conducted following the procedures outlined in ABC Protocol 

# N<:: !S<:,A**7~7_.1300** 

### (for 1. .--;---- ,,",.' - _ -, ,,) 

### as**approv~d** 

### ?y a**reI2~sentative** 

### of L _ _
__ 4 ""
..**~__** _, 

### on May 31, 1996, and by ABC Laboratones study1:iIrector on • 

### June 26, 1996. The definitive study was conducted from December 11, 1996, to December 13, 1996. 1. 

### Test Organisms Test specimens of Daphnia magna were obtained from an in-house daphnid culture that ABC Laboratories has maintained. The daphnids were identified to species using the taxonomic key provided by Pennak (1). All daphcids were cultured in a temperature-controlled area at 20 ±2°C. The lighting was maintained at approximately 54-56 footcandles (577-598 lux) on a 16-hour daylight photoperiod with 30-minute transition periods. Light reading measurements are kept on file at ABC Laboratories. The daphcid cultures were maintained in 1.5-L glass containers and were cultured 

### in hard blended water. Hard blended water is a combination of well water and reverse osmosis water blended to a hardness of 130 to 160 mg/L as CaC03 

### (Table I). 

### The hard blended water was biologically aged prior to use. That is, the water was held in a tank 

### containing aquatic organisms. The water was filtered with a sediment filter prior to use. The total organic carbon content of this water was less than 2.0 mg/L when measured. During the holding period, the daphcids were fed a suspension of at least one algae species cultured at ABC: 

### Selenastrum capricomutum and/or 

### AnkistrodesmusJalcatus at least once a day. Along with the algae, the daphnids were fed an artificial diet supplement. Results of the analysis of the fish food used in the artificial diet are kept on file at ABC Laboratories. First-instar daphnids (

## < 24 hours old) were selected for the test. To provide test daphnids that were 

### < 24 hours old, the adult daphcids were isolated by transferring to a fresh culture water/food suspension the previous day. All test daphnids were provided by ABC culture #96-F 22 

### on December 11, 1996. The adults from this culture had been cultured for 13 days and were considered acceptable with no signs ofstress, disease, orphysical damage. Since the culture and test parameters oftemperature, dilution water and lighting were the same, no acclimation period was necessary. The daphcids were not fed during the test. II. Test Substance A. Substance Receipt The TMBP test substance (lot #051796) was received from -: _ :-...."':
....**;~-;.-"'~** :"--:----=:Uil.;l"!:

- 

### on June 26, 1996, in good condition. The sample was observed to be a white powder and was stored at room ABC LABS #43359-9

### temperature. Sample purity was specified as 98.9%. The test concentrations were based on the total product, i.e. not corrected for purity. Sample stability was listed as stable in the MSDS and was the responsibility of the study sponsor. B. Preparation of Biological Test Solutions A 0.2-mg/mL primary standard, equivalent to the high concentration (level 6), was prepared for the definitive test by weighing out 0.200 g of TMBP. This sample was then diluted to 1000 mL with hard blended water. Aliquot volumes of 15.7, 31.3, 62.5, 125 and 250 mL of the primary standard were used to prepare stock solutions for levels 1-5. Levels 1-5 stock solutions were brought to a fmal volume of500 mL with hard blended water. Each replicate test beaker was filled with 200 mL of the appropriate stock solution. III. Test Method A. Test Vessel and Test Apparatus The static 

### Daphnia magna bioassay was conducted in 250-mL glass beakers containing a 2oo-mL test volume. The depth and surface diameter for the test solution in the test vessels were both 6.3 cm. No special conditioning of the test vessels was done. All test vessels were covered with loose-fitting Petri dish covers to minimize evaporation and prevent contamination during the study. These vessels were kept at 20 

### ± 2°C in a temperature-controlled waterbath. Cool, fluorescent lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods. The light intensity was 66-74 footcandles (705-797 lux) when measured at 0 and 48 hours. B. Test Design 1. Biological The first range-fmding study was conducted using five 

### Daphnia magna in exposure concentrations of 0.1, 1.0, 10 and 100 mg/L with a concurrent control chamber. No immobility or abnormal effects were observed in the control, 0.1- and lO-mg/L test chambers. Two daphnids were immobile in the 1.0-mg/Lchamber after 48 hours. The loo-mg/L chamber had 100% immobility after 48 hours. All solutions were clear with no visible precipitate or surface film. A second range-finding study was conducted with concentrations of 1.0, 3.2, 10, 32, 100 and 320 mg/L with a control chamber and five daphnids per chamber. There was no immobility or abnormal ABC LABS #43359-10 

### /lDeffects in the control, 1.0-, 3.2- and 

### 10-mg/L chambers. There were 1, 3 and 5 immobile daphnids at 48 hours in the 32-, 100­ and 

### 320-mg/L chambers, respectively. All solutions were clear and colorless with no visible precipitate or surface film. The definitive test concentrations were set at 

### 6.3, 12.5, 25, 50, 100 and 

### 200 

### mg/L after consultation with the study sponsor. Two replicates of the test concentrations and control were prepared. Ten Daphnia 11Ulgna (less than 

### 24 hours old) per beaker were used for the definitive bioassay for a total of 

### 20 daphnids per concentration. Daphnids were impartially added to test beakers within 

### 30 minutes of test solution preparation. All chambers were observed at 

### 0,24, and 

### 48 hours for immobility and other abnormal effects such as surfacing, trailing extraneous material, quiescence, and tending to the bottom of the test chambers. 2. Chemical and Physical Water chemistry parameters oftemperature, dissolved oxygen, and pH were performed in all replicates of the test concentrations and control at 0 hour before the addition oforganisms and at 48 hours. IV. Statistics Statistical analysis ofthe concentration vs. effect data (immobility) was performed using a computerized EC50 program developed by Stephan et al. (2). This program is designed to calculate the EC

### so statistic and its 

### 95 % confidence limits using the binomial, moving average and probit tests. Three different methods of analyzing the data were used since no one method of analysis is appropriate for all possible sets ofdata that may be obtained. The method ofcalculation selected for presentation in this report was that which gave the narrowest confidence limits for the EC50 (2, 3), although all three models are valid. However, if no immobility occurred or if a dose response could not be demonstrated over a reasonable range 

## « 37 to 

### > 63%), the EC

### so and/or its 95% confidence limits could not be calculated. The slope of the dose-response line was calculated by transforming percent immobility to probit values and plotting probit values against the log of the concentration. Linear regression was used for the calculation of the slope of the dose-response line. RESULTS AND DISCUSSION The nominal test concentrations of this toxicity test with TMBP to Daphnia 11Ulgna were 6.3, 

### 12.5, 25, 50, 100 and 

### 200 mg/L. Table 

### II presents the water quality parameters measured 

### ABC LABS 

### #43359-11 /2./

### during the definitive test. Temperature was 20.4 to 21.5°C when measured at 0 and 48 hours. The pH values ranged from 8.3 to 8.4. Dissolved oxygen concentrations ranged from 8.3 to 8.6 mg/L during the test. These values represented 95 to 101 % 

### saturation at 20 and 21°C, respectively. The dissolved oxygen was considered adequate for testing (4); therefore, the test chambers were not aerated during the test. All results were based on the nominal concentrations. Table ill presents the individual immobility and behavioral observations for dapbnids exposed to TMBP. There were no abnormal effects in the control, 6.3- and 12.5-mg/L test concentrations. The abnormal effects of immobility, trailing extraneous material, quiescence and/or on bottom of the test chamber were noted in the 25-, 50-, 100- and 200-mg/L concentrations. A 48-hour no-observed effect concentration for this study was determined to be 12.5 mg/L, based on the lack of immobility and abnormal effects at this concentration. All solutions in the test chambers were clear with no visible precipitate or surface film 

### throughout the study. The 24- and 48-hour EC

### so values were 

### > 200 and 75 mglL (Table IV). The 48-hour slope of the dose-response line was 6.0. The dose-response line is shown in Figure 1. The study was conducted following the Good Laboratory Practice regulations (5) and the draft and final reports were reviewed by ABC Laboratories' Quality Assurance Unit. All original raw data were provided to =-'
__....**~** :. .. - .._ 

### ,with the final report. A copy ofthe report was retained at ABC Laboratories, Inc. CONCLUSIONS The nominal test concentrations for this study were 6.3, 12.5,25,50, 100 and 200 mg/L. 

### The 48-hour ECsO 

### for 

### Daphnia magna exposed to 

### TMBP was shown to be 75 mg/L with 95 

### % confidence limits of59 and 96 mg/L. 

### A 48-hour no-observed effect level was determined to be 12.5 mg/L in this study. The slope ofthe 48-hour dose-response line was 6.0. All results were based on the nominal concentrations.

ABC LABS #43359-12 /22TABLE I Chemical Characteristics of Hard Blended Water Used for Aquatic Toxicity Studies at ABC Laboratories Monthly/Daily Screens Dilution Water Screena Monthly Screens 

### (mg/Ub Hardness 150 mg/L (as CaC03) Total Organic Carbon <1.0 Alkalinity 160 mg/L (as CaC03) Suspended Solids 0.0 pH 8.39 Conductivity 300 ",mhos/cm July 1996 Screen Elements (mg/L)

Organophosphate (p.g/L) Chlorinated Hydrocarbons (p.g/L) DDD <0.004 DDT <0.009 Dieldrin < 0.003 Endrin < 0.008 a-BHC < 0.002 t3-BHC 

### < 0.004 -y-BHC-Lindane < 0.001 .:1-BHC < 0.002 Heptachlor < 0.003 Heptachlor Epoxide 

### < 0.004 Aldrin < 0.005 Chlordane <0.07 Methoxychlor <0.05 Toxaphene <0.7 Endosulfan Sulfate <0.008 Endosulfan I <0.008 Endosulfan II < 0.008 HCB <0.003 Mirex <0.009 Telodrin < 0.001 PCBs <0.1 Miscellaneous (mg/L) Chemical Oxygen Demand 4.0 Un-Ionized Ammonia <0.1 Chlorine Residual <0.020 Nitrite N 0.02 Nitrate N 0.15 Total Phosphorus as P <0.04 Ammonia Nitrogen <0.04 Aluminum Arsenic Boron Cadmium Chromium Cobalt Copper Fluoride Iron Lead Mercury Nickel Selenium Silver Zinc 0.053 <0.0015

0.260 <0.00008

0.00087 <0.0055 <0.0038

0.5 0.0086 <0.0012

0.000051 <0.0054 <0.0014 <0.00018

0.0171

Diazinon Methyl Parathion Ethyl Parathion Malathion Ronnel Ethion Trithion Vapona <0.001 <0.005 <0.007 <0.01 <0.003 <0.003 <0.01 <0.04

a Represents the values measured on dilution water used for the test b Represents the values obtained from the screens of November 1996 Note: Results ofthe chemical analyses supporting these data are on file at ABC Laboratories. ABC LABS #43359-13 

## J23TABLE II Water Quality Measurements During the Acute Toxicity Test of TMBP to 

### Daphnia magna 

## oHour 48 HQurs Nominal Concentration Tempa DOb Temp. DO (mg/L)**~** mg/L U Hc**~** mg/L 

## --IllL Control A 20.8 8.3 8.4 20.4 8.4 8.3 Control B 20.8 8.3 8.4 20.4 8.3 8.4 6.3 A 21.3 8.4 8.4 20.4 8.3 8.4 6.3 B 21.3 8.4 8.4 20.5 8.3 8.4 12.5 A 21.1 8.4 8.4 20.4 8.3 8.4 12.5 B 21.1 8.4 8.4 20.5 8.3 8.4 25 A 21.2 8.6 8.4 20.4 8.3 8.4 25 B 21.3 8.6 8.4 20.5 8.3 8.4 50 A 21.3 8.5 8.4 20.5 8.3 8.4 50 B 21.3 8.5 8.4 20.5 8.3 8.4 100 A 21.5 8.5 8.4 20.4 8.4 8.4 100B 21.5 8.5 8.4 20.5 8.4 8.4 200 A 21.4 8.5 8.4 20.5 8.4 8.4 200 B 21.5 8.5 8.4 20.5 8.4 8.4
a Temperature measured with a mercury thermometer b Dissolved oxygen concentration

- YSI Model 54 A dissolved oxygen meter

C pH

- Beckman Model <lJ34 pH meter Note: Dissolved oxygen saturation corrected for altitude at the test temperatures of 20, 21 and 22°C are 8.7, 8.5 and 8.4 mg/L, respectively.

ABC LABS #43359-14 

## /24

### TABLE III Immobility and Behavioral Observations During the

Acute Toxicity Test of TMBP to Daphnia magna 

### Nominal Number 24 

### Hours 48 Hours Test Placed Concentration 

### in Test (mg/L) Vessel Imm. Observations Imm. Observations Control A 

### 10 0 10 N; CLR 

## o 10 N; CLR Control B 10 0 10 N; CLR 

## o 10 N; CLR 6.3 A 10 0 10 N; CLR 

## o 10 N; CLR 6.3 

### B 10 

## a 10 N; CLR 

## o 10 N; CLR 

### 12.5 A 10 

### 0 10 N; CLR 

## o 10 N; CLR 12.5 B 10 0 

### 10 N; CLR 

## o 10 N; eLR 25 A 10 0 10 N; CLR 

## o 3 OB; 7 N; CLR 25 B 10 0 2 Q; 8 N; CLR 

## o 5 OB; 3 N; 

### 2 

### OB/Q; CLR 50 A 10 0 2 Q; 8 N; CLR 3 

### 2 OB; 

### 2 N; 3 

### OB/Q; CLR 50 

### B 10 0 

### 2 Q; 

### 8 N; CLR 

## o 60B/Q; 

### 4 OB; CLR 100 A 10 3 2 Q; 5 N; CLR 8 2 

### OB/Q; CLR 100 

### B 10 3 3 Q; 4 N; CLR 

### 10

--­ <ZLR 200 

### A 10 

### 3 7 OB; CLR 8 

### 10B/TR; 1 OB; CLR 200 B 10 5 3 OB; 2 Q; CLR 9 1 

### OB/Q; CLR Key to Abbreviations: Imm. 

## = Immobility; N 

### = Normal; TR = Trailing Extraneous Material; OB = On Bottom of Test Chamber; Q 

### = Quiescent; CLR = Clear Solution

ABC LABS #43359-15 {2 

## '5TABLE IV Acute Toxicity of TMBP to 

### Daphnia magna ECSO in mg/L based on nominal concentrations Compound 24-Hour 48-Hour

TMBpa 

### >200b 75c (59 and 96)d N = 20 daphnids per concentration

a Bioassay conducted at 20 

### ± 2°C b Insufficient immobility to calculate an EC

### so
c EC

### so calculated using moving average method d 95 % confidence limits The 48-hour no-observed effect concentration for this study was determined to be 12.5 mg/L, based on the lack of immobility or abnormal effects at this concentration.

ABC LABS #43359-16

### FIGURE 1 The 48-Hour Dose-Response Line for

Daphnia magna Exposed to TMBP

.0 

### o 6.5

6
5.5

5

### ~ 4

35

3


### 2.5



### 2

### I

I 

### 1.3 V 

# /! / 

# / / / 

# /
/ 

# IV

1.5 1.7 / 

# / 

# /

I i 

### 19 

### Log of Concentration (mg/L)

AUBC LAUBS #43359-17 

## 127

### REFERENCES (1) Pennak, R.W. 1978. Freshwater lnvenebrates ofthe United States, 

### 2nd 

### ed. 365 p. (2) Stephan, C.E., K.A. Busch, R. Smith, J. Burke and R.W. Andrew. 1978. A Computer Program for Calculating an LC

### so. U.S. Environmental Protection Agency, Duluth, Minnesota, pre-publication manuscript, August 1978. (3) Stephan, C.E. 1977. Methods for Calculating an LC50, 

### p. 65-84. In F.L. Mayer and J.L. HameJink (Eds.). Aquatic Toxicology and Hazard Evaluation. 

### ASTM Special Technical Publication 634. ASTM. Philadelphia. (4) Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methodsfor Acute Toxicity Tests with Fish, Macroinvenebrates and Amphibians. 

### Environmental Protection Agency, Ecological Research Series EPA-660/3-75-009. (5) U.S. Environmental Protection Agency. Toxic Substances Control; Good Laboratory Practice Standards; Final Rule (40 

### CFR, Part 792). Federal Register.

ABC LABS 

### #43359-18

## Workingfor You Columbia, Missouri

ABC Laboratories, PIII-Ag IJiWdon 32380 Awnue 10 

### SPONSOR

STUDY TITLE Acute Flow-Through Toxicity of TMBP to Rainbow Trout (Oncorhynchus mykiss) 

### DATA REOllREMENT U.S. EPA

- TSCA, 40 CPR, Part 797 Guideline 797.1400 AUTHOR Jane Bowman Scientist/Study Director REPORT SUBMITTED ON May 15, 1997 PERFORMING LABORATORY ABC Laboratories. Inc. Environmental Toxicology 7200 E. ABC Lane Columbia, Missouri 65205 ABC LABORATORY PROJECT ID Final Report #43358 Page 1 of 115

ABC Labonlorifs, Inc.

7lOO E. ABC laDe

### STUDY COMPLIANCE STATEMENT Study Compliance Statement for ABC Laboratories' Final Report #43358 entitled "Acute FlowThrough Toxicity of TMBP to Rainbow Trout (Oncorhynchus mykiss)" 

### for 

### t-T:;; .-:- - '.:_

- ., ABC Laboratories' study director for the above test herein confinns that the study was conducted in compliance with the U.S. EPA Good Laboratory Practice Standards; Toxic Substances Control Act (40 

### CFR 792). 

### No analytical confinnation of test concentrations was done nor was the stability of the test material under test conditions investigated. This was the responsibility of the study sponsor. This statement of compliance does not extend to supplementary analyses of the fish food contaminant screen conducted by Lancaster Laboratories, Lancaster, Pennsylvania. Results of these analyses are kept on file at ABC Laboratories. All data in support of this report, original and certified exact copies, were provided to ...--. :-..__•.::_ . , ".... ., with the final report. A copy of the report text and the raw data, along with facility records, were retained at ABC Laboratories, Inc.

### r1a~man ABC Laboratories' Study Director

ABC LABS 

### NO. 43358-2

### QUALITY ASSURANCE STATEMENT ABC Laboratories' Quality Assurance Unit reviewed Study #43358, "Acute Flow-Through Toxicity of TMBP to Rainbow Trout (Oncorhynchus mykiss) , " 

### for**~** 

### ....--___ - " 

### J.,**~~** :./**#.~,,",,' ~**  •• -.,**O~ ~--..** **.~** 

### The following inspections/audits were conducted on this study. Date Reported to Date Reported to Date of Inspection Phase Inspected Study Director Management January 

### 10, 1997 Fish Addition January 

### 10, 1997 January 

### 24, 1997 February 

### 06, 1997 Draft Report and February 

### 06, 1997 February 

### 11, 1997 Raw Data April 

### 18, 1997 Final Report April 

### 18, 1997 April 

### 21, 1997 The undersigned conducted the draft and final report audits. The audits indicated the report is an accurate reflection of the study as it was conducted by ABC Laboratories, Inc.
5-/A{q7 

### Draft Report:

# ~.~ R nda 

### C. Stewart Date Quality Assurance Officer 

### I

Final Report:

# ~~**~L** t:r-1"'1-9

## Z aney . Hunt Date Senio Quality Assurance Specialist

ABC LABS NO. 

### 43358-3SIGNATURE PAGE Submitted by: ABC Laboratories, Inc. 7200 E. ABC Lane P.O. Box 1097 Columbia, Missouri 65205 (573) 474-8579 Prepared by:

e Bowman

## )5~9? Date Scientist, Study Director Approved by:

Marc C. Sword Date Team Leader, Aquatic Toxicology

William A. McAllister Date Manager, Aquatic Toxicology

)A./. 

# fflIty 97 Date Senior Quality Assurance Specialist

:amk



### I

# 3J,.. ABC LABS NO. 43358-4TABLE OF CONTENTS Page No. TITLE PAGE 1 STUDY COMPLIANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 QUALITY ASSURANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3 SIGNATURE PAGE
......................................... 4 TABLE OF CONTENTS.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 LIST OF TABLES
.......................................... 6 LIST OF FIGURES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6 FISH ACUTE TOXICITY COMPENDIUM
. . . . . . . . . . . . . . . . . . . . . . . . . .. 7 SUMMARY
........................................ " .
... 8 INTRODUCTION
.......................................... , 9 MATERIALS AND METHODS
.................................. 9 I. Test Fish.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 9 II. Test System
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 III. Test Material
..................................... 10 IV. Test Procedure

- Biological
. . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 V. Test Procedure

- Chemical and Physical
.................... 11 VI. Biological Data Analysis
.............................. 11 RESULTS.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 CONCLUSIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 REFERENCES
............................................. 20 APPENDICES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21 PROJECT PERSONNEL
..................................... " 22 APPENDIX I

- BIOLOGICAL RAW DATA
........................ " 25 A. Definitive Study
..................................... 26 B. Records
.......................................... 50 C. Range-Finding Studies
................................. 85 APPENDIX II

- PROTOCOL AND CORRESPONDENCE
...... . . . . . . . . . .. 93 

### 13"3 ABC LABS NO. 43358-5LIST OF TABLES

I Chemical Characteristics ofHard Blended Water Used for Aquatic Toxicity Studies at ABC Laboratories
. . . . . . . . . . . . . . . . . . . . . . . 14 

### II Water Quality Measurements During the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout (Oncorhynchus mykiss)

.......................................... 

### 15 III The LC

### so Values for the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout (Oncorhynchus mykiss)

. . . . . . . . . . . . . . . . 17 IV Mortality and Behavioral Observations During the Acute FlowThrough Toxicity Test ofTMBP to Rainbow Trout (Oncorhynchus mykiss)

.......................................... 

### 18
LIST OF FIGURES

1 The 96-Hour Dose-Response Line for Rainbow Trout (Oncorhynchus mykiss) Exposed to TMBP
. . . . . . . . . . . . . . . . . . . . 19



### ABC LABS NO. 43358-6 

## 134

### FISH ACUTE TOXICITY COMPENDIUM

... .....J

- ...:.. 

### Subject: "Acute Flow-Through Toxicity ofTMBP to Rainbow Trout (Oncorhynchus mykiss) , II 

### ABC Laboratories' Report #43358



### ..... Sponsor: ' Testing Facility: ABC Laboratories, Inc. Environmental Toxicology 7200 E. ABC Lane Columbia, Missouri 65205 (573-474-8579) Location of Original Raw Data and Final Report:

--'

..**~-** - 'r-.... .( 

### Test Material: TMBP Nominal Test Concentrations: Control, 63, 125, 250, 500 and 1000 mg/L Dilution Water: Hardness

- 140 to 154 mg/L as CaC03 

### Alkalinity

- 152 to 164 mg/L as CaC03• 

### pH

- 8.16 to 8.25 Conductivity

- 300 to 340 p,mhos/cm 

### Experimental Test Dates: Start

- January 10, 1997 Termination

- January 14, 1997 Length of Study: 96 Hours Results: 96-Hour LCsO = 

### 810 mg/L (95% C.1. = 

### 500 to 1000 mg/L) 96-Hour NOEC 

# = 250 mg/L 96-Hour Dose-Response Slope 

# = 11 Test Species: Rainbow Trout (Oncorhynchus myldss) 

### Source of Organisms: Mt. Lassen Trout Farm Red Bluff, California Mean (+ 

### SD) Wet Weight: 2.46 (± 

### 0.51) g Mean (± 

### SD) Standard Length: 56 (± 

### 3) mm Mean (± 

### SD) Total Length: 64 (± 

### 4) nun ABC LABS NO. 43358-7

### SUMMARY A 96-hour flow-through toxicity study of TMBP to rainbow trout (Oncorhynchus mykiss) 

### was conducted for"
... r- c-..: -, ., 

### from January 10, 1997, to January 14, 1997. Ten fish were exposed to each test concentration replicate and to a hard blended water control replicate for a total of 20 fish per concentration or control. The nominal dosing regime of TMBP was 63, 125, 250, 500 and 1000 mglL. All control and test chambers were clear and free of precipitates or surface films during the study. Water chemistry measurements of dissolved oxygen, pH and temperature were consistent throughout the experiment and were within the protocol specifications. Ten rainbow trout (Oncorhynchus myldss), 

### with a mean (± 

### SD) wet weight of2.46 (± 

### 0.51) g, a mean (± 

### SD) total length of 

### 64 (± 

### 4) 

### mm and a mean (± 

### SD) standard length of 56 (± 3) 

### mm, were placed in each test chamber for a total of twenty fish exposed to each concentration and control for 96 hours at 12 

## ± 1°C. The fish used in this study were supplied by Mt. 

### Lassen Trout Farm, Red Bluff, California. Culture and acclimation records indicated the fish were in good condition for testing. Total organic carbon (TOC) values measured in triplicate on a sample taken from control 

### A at 

# o hour before fish were added to the test chambers were 

### < 1.0 mg/L. TOC values measured in triplicate on a sample taken from control B 

### at 96 hours were 

## < 1.0 mg/L. Analyses supporting these data are on file at ABC Laboratories. Suspended solids measured in duplicate on a sample taken from the control 

### A at 0 hour before fish addition were 0.2 and 0.0 mg/L (x = 

### 0.1 mg/L). Suspended solids measured in duplicate on a sample taken from control B 

### at 96 hours were 0.0 and 0.0 mglL (x 

### =**O~O** 

### mglL). Total hardness (mg/L as CaC03) 

### measured on the control 

### A test chamber at 0 hour was 146 mg/L. Total hardness measured on the control B 

### test chamber at 96 hours was 144 mg/L. Total alkalinity (mg/L as CaC03) 

### measured on the control 

### A test chamber at 0 hour was .148 mg/L. Total alkalinity measured on the control B 

### test chamber at 96 hours was 150 mg/L. Dissolved oxygen ranged from 8.6 mg/L (85% of saturation at 13°C) to 10.2 mg/L 

### (101 % of saturation at 13°C), pH ranged from 8.08 to 8.34 and temperature ranged from 12.0 to 12.9°C. The test solutions were not aerated during the test. From the data collected during this study, a 96-hour LC

### so was determined to be 810 mglL based on the nominal concentrations of TMBP. The slope of the 96-hour dose-response line was 11. The abnormal effects ofmortality and/or fish on the bottom of the test chamber were observed in the 500 and 1000 mg/L test concentrations during the study. As determined by this study, a 96-hour NOEC ofTMBP toxicity to rainbow trout was 250 mg/L based on a lack of sublethal responses at this concentration.

ABC 

### LABS NO. 43358-8

### INTRODUCTION**J'~'.** • 

### contracted the Environmental Toxicology division of ABC I 

### Laboratories, Inc., to conduct a five concentration and control flow-through toxicity test to determine the toxicity ofTMBP to rainbow trout (Oncorhynchus mykiss). 

### The primary objective of this study was to estimate the acute toxicity of TMBP to rainbow trout by determining the median lethal concentration (LCsO)' 

### if possible, and examining sublethal/behavioral responses during the course of the test. This was accomplished by exposing 20 rainbow trout to each concentration and dilution water control. Each chamber was monitored for mortality and sublethal/behavioral responses every 24 hours over a 96-hour exposure duration. The term "LC

### so'" as used 

### in this report, is that concentration ofTMBP causing 50% mortality after a specified exposure period. The study was conducted from January 10, 1997, to January 14, 1997, following the procedures outlined in ABC Protocol No. TSCA 797.1400 FT for i' • - __ . 

### The protocol was approved by a representative of**~** --r -.­ C 

### on May 31, 1996, and by the study director on June 19, 1996. This report presents ,T -"' 

### the results of the toxicity study and describes the technical approach used to meet the project objectives. MATERIALS AND METHODS The biological methods, as well as the recommended statistical analyses, used in this study were those described by the U.S. Environmental Protection Agency (1). I. 

### Test Fish The 120 rainbow trout (lot #3996) were obtained from Mt. Lassen Trout Farm in Red Bluff, California. The fish were received as green eggs and sperm and were fertilized on July 3, 1996. The culture room was maintained on a 16-hour daylight photoperiod with 30-minute transition periods. The fish were fed newly hatched brine shrimp (Artemia 

### sp.) and/or a commercially available fish food one to three times per day. The fish food was received from Zeigler Bros., Inc., in 

### Gardners, Pennsylvania. The brine shrimp cysts were received from Mackay Marine Brine Shrimp Co., Inc., (providence, Utah). All food was screened for contaminants and the results of those analyses are on fIle at ABC Laboratories. Fish culture techniques were basically those described by Brauhn and Schoettger (2). Daily mortality and feeding records were maintained during the culture period. A sublot ofrainbow trout was acclimated to the test temperature and 

### test dilution water for approximately 48 hours before the defInitive test. The fish were not fed during the acclimation and test periods. The fish were distributed to the test chambers in an impartial manner at test initiation. The rainbow trout (Oncorhynchus mykiss) 

### used as the control group during this study had a mean (± 

### SD) wet weight of 2.46 (± 

### 0.51) g, a mean (± 

### SD) total length of 64 (± 

### 4) mm and a mean (± 

### SD) standard length of 56 (± 

### 3) mm when measured at the end of the test. The biomass loading rate was 0.24 g/Uday. ABC LABS NO. 43358-9 

## 137

### The biomass loading rate at any point in time was 1.64 

### gIL. The control group was believed to be representative of all fish used in the definitive test. II. Test System A proportional diluter system described by Mount and Bmngs (3) and a Hamilton Model 420 syringe dispenser were used for the intermittent introductionofTMBP test solutions and dilution water into each test chamber. Five concentrations of the test material and a dilution (hard blended) water control comprised the test design. Each concentration was replicated twice. Ten fish were added to each test chamber and control chamber at test initiation for a total of 20 fish per concentration. Chemical characteristics, typical of hard blended water, are provided in Table I. A diluter stock solution (99 mg/mL) 

### was prepared by dissolving 198 g ofTMBP in 2 L ofhard blended water. The injector volume was adjusted to deliver 20 

### mL of stock to 1.98 L ofdilution water. The resulting nominal concentration regime of TMBP was 63, 125, 250, 500 and 1000 mg/L. 

### The diluter delivered 0.5 L of test solution or control water to the test vessels at an average of 8.6 times per hour over the course of the study. TIlls flow rate resulted in 103 L of water flowing through the 15-L aquaria per day. TIlls represented 6.9 tank volume replacements flowing through each chamber every day. The test aquaria were immersed in a circulating waterbath that was thermostatically held at 12 

## ± 1°C. The test containers were glass aquaria that measured approximately 29.0 cm length 

## x 26.8 cm width x 31.1 cm height. The aquaria were filled to a depth of approximately 19.3 cm, which was equivalent to approximately 15 L. III. Test Material The TMBP samples were received on June 26 and December 6, 1996, from**t~:.**

# _ - . ..... -:. in good condition. The sample received on June 26, 1996, (lot #051796) was assigned ABC reference HTS-9220 and was received in four bottles which were labeled A through D. The sample received on December 6, 1996, (lot #111596) was assigned ABC reference #TS-9611 and was received in five containers which were given the letters A through E. The samples were observed to be a white solid or powder and were stored at room temperature. Sample purity was 98.9% for TS-9220 and was not specified for TS-9611. 

### All test concentrations were not corrected for sample purity, i.e., based on the total product. The fIrst sample (TS-9220) was used for the rangefinding studies and the second sample (TS-9611) was used for the defmitive study.



### ABC LABS NO. 43358-10IV. Test Procedure

- Biological Static 96-hour range-finding tests were conducted to deterinine the concentration range of TMBP to use in the definitive test. Five fish were added to each test concentration in the range-fmding tests. The first preliminary test was conducted from June 28, 1996, to July 2, 1996, at the concentrations of 0.1, 1.0, 10 and 100 mg/L. No mortality was noted in any test concentration. A second range-fmding test at the concentrations of 320 and 1000 mg/L was conducted from July 17, 1996, to July 21, 1996. No mortality was observed in the 320-mg/L concentration. The lOOO-mg/L concentration caused 2 of the 5 fish to die at 48 hours. No additional mortality occurred after 96 hours. The definitive test was conducted from January 10, 1997, to January 14, 1997, at nominal test concentrations of 63, 125, 250, 500 and 1000 mg/L with a concurrent control chamber. Each concentration was conducted in duplicate. The test was initiated after the test solutions had been flowing through the aquaria approximately 25 hours. One to two rainbow trout were added sequentially to each chamber in an impartial manner until each chamber contained its complement of 10 fish (20 fish per concentration). Observations for mortality and sublethal responses were made once every 24 hours during the 96-hour test. V. Test Procedure

- Chemical and Physical Temperature (OC), dissolved oxygen (mg/L) and pH were measured in all test concentrations at 0, 24, 48, 72 and 96 hours of the test. In addition to these measurements, total organic carbon (mg/L), suspended solids (mg/L), total hardness (mg/L as CaC03) and total alkalinity (mg/L as CaC03) were measured on the control chamber at 

## a and 96 hours of the study. A 16-hour daylight photoperiod with 30-minute transition periods was maintained in the test area. The light intensity over the diluter was an average of 87 footcandles (882-978 lux). Light measurement data are kept on file at ABC Laboratories. VI. Biological Data Analysis Statistical analysis of the concentration vs. effect data (generally mortality) was obtained by employing a computerized LCSO program developed by Stephan et al. (4). This program calculated the LCSO and its 95 

### %confidence limits by using the binomial, moving average and probit tests. Three different methods of analyzing the data were used since no one method of analysis is appropriate for all possible sets ofdata that may be obtained. However, ifno mortality occurred or if a dose response could not be demonstrated over a reasonable range 

## « 37 to 

### > 63 

### %), an LCSO and/or its 95 

### % confidence limits could not be calculated. 

### If the program calculated an LC

### so by more than one method, the method of calculation selected for presentation in this report was the method that gave the narrowest confidence interval for the LC

### so, although all three methods are valid. All calculations were performed using the nominal concentrations of TMBP.

ABC LABS NO. 43358-11

### The slope of the dose-response line was calculated by transforming percent mortality to probit values and plotting probit values against the log of the concentration. Linear regression was used for the calculation of the 'slope of the dose-response line. RESULTS Water chemistry parameters measured at 0, 24, 48, 72 and 96 hours were within the specified limits for conducting aquatic toxicity tests (1). The temperature ranged from 12.0 to 12.9°C and pH ranged from 8.08 to 8.34 (Table 

# m. Dissolved oxygen ranged from 8.6 to 10.2 mg/L. These values represented 85 and 101 

### % saturation at 13°C, respectively. The test solutions were not aerated during the study. Total organic carbon (TOC) values measured in triplicate on a sample taken from the control A solution collected at 0 hour before fish were added to the test chambers were 

## < 1.0 mg/L. TOC values measured in triplicate on a sample taken from the control B solution collected at 96 hours were 

## < 1.0 

### mg/L. Analyses supporting these data 

### are on fIle at ABC Laboratories. Suspended solids measured in duplicate on a sample taken from the control A test chamber at

. 0 hour before fish addition were 0.2 and 0.0mglL (x 

### = 0.1 mg/L). Suspended solids measured

in duplicate on a sample taken from the control B test chamber at 96 hours were both 0.0 mg/L (x 

### = 

### 0.0 mg/L). Total hardness (mg/L as CaC03) values measured on the control A test solution at 0 hour was 

### 146 mg/L. Total hardness measured on the control B test solution at 96 hours was 144 mg/L. Total alkalinity (mg/L as CaC03) 

### measured on the control A test solution at 0 hour was 148 mg/L. Total alkalinity measured on the control B test solution at 96 hours was 150 mg/L. From the data 

### collected during this flow-through study, the 24-, 48-, 72- and 96-hour LC50 

### values were 

## > 1000, 

## > 500, 880 and 810 mg/L, respectively, based on the nominal concentrations of TMBP (Table IIl). 

### The slope of the 96-hour dose-response line was 11 (Figure 1) as calculated by least-squares regression analysis. The 96-hour no-observed effect concentration ofTMBP toxicity to rainbow trout (Oncorhynchus mykiss) 

### was determined to be 250 mg/L, based on the lack of mortality or observed sublethal/behavioral effects at this concentration. The abnormal behavioral effects ofmortality andlor fish on the bottom ofthe test chamber were observed in the 500 and 1000 mglL test concentrations during the study. Mortality and behavioral effects noted during the study are presented in Table IV. The study was conducted following Good Laboratory Practice regulations (5) and the draft and final reports were reviewed by ABC Laboratories' Quality Assurance Unit. All original raw data were provided to**!~~"...** 

### with the [mal 

### report. A copy ofthe report was '.4 -::**~_.** • • -.:., 

### retained at ABC Laboratories, Inc.

ABC LABS NO. 43358-12 

## \4-0CONCLUSIONS The nominal test concentrations for this study were 63, 125, 250, 500 and 1000 mg/L. The 96-hour for rainbow trout (Oncorhynchus mykiss) exposed to TMBP was shown to LC50 be 810 mg/L. A 96-hour no-observed effect level was determined to be 250 mg/L in this study. 

### All results were based on the nominal concentrations.

ABC LABS NO. 43358-13 

# It 1TABLE I Chemical Characteristics of Hard Blended Water Used for Aquatic Toxicity Studies at ABC Laboratories

Dilution Water Screena Monthly/Daily Screens Monthly Screens (mg/Ub Hardness Alkalinity pH 140-154 mg/L (as CaC03) 152-164 mg/L (as CaC03) 8.16-8.25 Total Organic Carbon Suspended Solids 

### < 1.0 0.1 Conductivity 300-340 ILmhos/cm July 1996 Screen Elements (mg/U Aluminum 0.053 Arsenic <0.0015 Boron 0.260 Cadmium <0.00008 Chromium 0.00087 Cobalt <0.0055 Copper <0.0038 Fluoride 0.5 Iron 0.0086 Lead <0.0012 Mercury 0.000051 Nickel <0.0054 Selenium <0.0014 Silver <0.00018 Zinc 0.0171 Chlorinated Hydrocarbons (ug/U

Organophosphate (lLg/L) DDD DDT Dieldrin Endrin a-BHC tJ-BHC ,),-BHC-Lindane d-BHC Heptachlor Heptachlor Epoxide Aldrin Chlordane Methoxychlor Toxaphene Endosulfan Sulfate Endosulfan I Endosulfan II HCB Mirex Telodrin PCBs Miscellaneous (mg/L) Chemical Oxygen Demand Un-Ionized Ammonia Chlorine Residual Nitrite N Nitrate N Total Phosphorus as P Ammonia Nitrogen <0.004 <0.009 <0.003 <0.008 <0.002 <0.004 <0.001 <0.002 <0.003 <0.004 <0.005 <0.07 <0.05 <0.7 <0.008 <0.008 <0.008 <0.003 <0.009 <0.001 <0.1

4.0 <0.1 <0.020

0.02 0.15 <0.04 <0.04 Diazinon Methyl Parathion Ethyl Parathion Malathion Ronnel Ethion Trithion Vapona <0.001 <0.005 <0.007 <0.01 <0.003 <0.003 <0.01 <0.04

a Represents the values measured on dilution water used for the test b Represents the values obtained from the screens ofJanuary 1997 Note: Results of the chemical analyses supporting these data are on file at ABC Laboratories.
ABC LABS NO. 43358-14 

## 14-2.TABLE 

### II Water Quality Measurements During the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout (Oncorhynchus mykiss) Water Quality Nominal

Cone. mg/L Temp.a**~** O-Hour DOb mgLL 

## .Jili.:.. Temp.**~** 24-Hour DO mg/L 

### Iili Temp.**~** 48-Hour DO mg/L 

### .lili.. Control Ad Control B 12.8 12.8 9.6 9.6 8.16 8.21 12.5 12.5 8.9 9.1 8.21 8.25 12.3 12.4 9.3 9.2 8.18 8.17 63 A 63 B 12.8 12.8 9.7 9.7 8.21 8.22 12.5 12.5 9.2 9.3 8.28 8.30 12.3 12.3 9.1 9.3 8.19 8.19 125 A 125 B 12.9 12.8 9.6 9.6 8.22 8.22 12.5 12.5 9.0 9.0 8.26 8.26 12.3 12.3 9.1 9.1 8.19 8.17 250 A 250 B 12.9 12.8 9.5 9.6 8.20 8.20 12.5 12.5 9.1 9.1 8.28 8.28 12.3 12.3 9.1 8.9 8.18 8.18 500 A 500 B 12.9 12.9 9.5 9.5 8.20 8.21 12.5 12.5 8.8 8.9 8.25 8.23 12.3 12.3 8.8 8.8 8.16 8.17 1000 A 1000 B 12.8 12.9 9.4 9.6 8.22 8.20 12.5 12.5 8.8 9.2 8.24 8.30 12.3 12.3 8.7 8.9 8.12 8.18
a Temperature measured using a mercury thermometer b Dissolved oxygen concentrations

- dissolved oxygen meter (YSI Model 54 A)

C pH- pH meter (Beckman Model <J?34) d Hardness and alkalinity in the control A solution at 0 hour was 146 and 148 mg/L as CaC03,

respectively. Hardness and alkalinity measured using titrimetric method adapted from Standard Methods. Suspended solids in a sample taken from the control A chamber at 0 hour averaged 0.1 mg/L. Suspended solids were measured by gravimetric determination. TOC measured in triplicate from a sample collected from the control A solution at 0 hour before fish addition was 

### < 1.0 mg/L. TOC determined by Engineering Surveys and Services in Columbia, Missouri. Note: Altitude-adjusted dissolved oxygen saturation values at the test temperatures of 12 and l3°C are 10.3 and 10.1 mg/L, respectively.

ABC LABS NO. 43358-15 

## 14-3TABLE 

### II (cont'd) Water Quality Measurements During the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout 

### (Oncorhynchus mykiss) Water Quality 72-Hour 96-Hour Nominal Temp. a Cone. mg/L

Control A 12.7 Control Bd 12.7 63 A 12.7 63 B 12.7 125 A 12.7 125 B 12.7 250 A 12.7 250 

### B 12.7 500 A 12.7 500 B 12.7 1000 A 12.7 1000 B 12.7 DOb mg/L 9.4 9.1 9.5 9.4 9.6 9.5 9.4 9.4 8.6 9.2 10.1 9.7 pHc 8.10 8.10 8.15 8.17 8.17 8.17 8.17 8.16 8.08 8.13 8.24 8.20 Temp.**~** 12.4 12.3 12.2 12.0 12.1 12.1 12.6 12.6 12.5 12.6 12.6 12.5 DO mg/L 9.6 9.2 9.6 9.6 9.8 9.6 9.6 9.5 8.9 9.3 10.2 10.1 

### ..PH 8.23 8.18 8.24 8.24 8.26 8.25 8.26 8.22 8.18 8.21 8.34 8.31
a Temperature measured using a mercury thermometer b Dissolved oxygen concentrations

- dissolved oxygen meter (YSI Model 54 A)

C pH

- pH meter (Beckman Model**~34)** d Hardness and alkalinity in the control B solution at 96 hours was 

### 144 and 

### 150 

### mg/L as

CaC03, respectively. Hardness and alkalinity measured using a titrimetric method adapted from Standard Methods. Suspended solids in a sample taken from the control B chamber at 96 hours averaged 

### 0.0 mg/L. Suspended solids were measured using a gravimetric determination. TOC measured in triplicate from a sample taken from the control B chamber at 96 hours was 

### < 1.0 

### mg/L. TOC determined by Engineering Surveys and Services in Columbia, Missouri. Note: Altitude-adjusted dissolved oxygen saturation values at the test temperatures of 

### 12 and 13°C are 10.3 and 10.1 

### mg/L, respectively.

ABC LABS NO. 43358-16TABLE 

### III The LCSO Vaiues for the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout (Oncorhynchus mykiss)

LC50 

### in mg/L Compound 24-Hour 48-Hour 72-Hour 96-Hour 

### > 1000b >500C (500 and l000l

N = 20 fish per concentration.
a Bioassay as conducted at 12 (± I)OC, mean (± SD) fish wet weight, total length and standard length, 2.46 (± 0.51) g, 64 (± 4) and 56 (± 3) mm. b Since no mortality occurred an LC50 could not be determined.

C Insufficient mortality to determine an LC50. LC50 estimated as 1000 mg/L. d Insufficient mortality to calculate a reliable LC50 and 95 % confidence limits.
e LC50 determined using binomial method. f 95 

### % confidence limits The 96-hour NOEC could be estimated at 250 mg/L, based on the lack of mortality and abnormal effects at this concentration.

ABC LABS NO. 43358-17**14~**TABLE IV Mortality and Behavioral Observations During the Acute Flow-Through Toxicity Test of TMBP to Rainbow Trout (Oncorhynchus mykiss)

Nominal Concentration ml!/L Number Placed in Test Mort. 24 Hours Observations Cum. Mort. 48 Hours Observations Cum. Mort. 72 Hours Observations Cum. Mort. 96 Hours Observations Control A Control B 10 10 0 0 ION 10 N 0 0 ION ION 0 0 10 N 10 N 0 0 ION ION 63 A 63 B 10 10 0 0 ION ION 0 0 10 N ION 0 0 ION 10 N 0 0 ION ION 125 A 125 B 10 10 0 0 ION ION 0 0 ION ION 0 0 ION ION 0 0 ION ION


### .p 0­ 250 A 250 B 500 A 500 B 10 10 10 10 0 0 0 0 ION ION ION ION 0 0 0 0 ION ION ION ION 0 0 0 0 ION ION 6 OB; 4 N 2 OB; 8 N 0 0 0 0 10 N 10 N 6 OB; 4 N 3 OB; 7 N

## ~ n

## ~ t:d CI:l Z 0 1000 A 10 1000 B 10 Key to Observations: N = 0 ION 5 2 OB; 3 N 0 ION 5 5N Nonnal; OB = On Bottom of Test Vessel 8 5 2N 5N 8 8 2N 2N

.j:>.. tu tu VI 00 I 

## .... 00

### FIGURE 1 The 96-Hour Dose-Response Line for Rainbow Trout (Oncorhynchus mykiss) 

### Exposed to TMBP

5.8 5.6 5.4 5.2

5 4B 4.6**~ ~**  4.4 jJ 0 4.2 l (l lB 16 14 12

2.B 2ii I
i I 

# V 

# V /

i I I 

# / / / / / / / / / / / i I 

### V i 2.6B 2./2 2.)5 2,B 2,84 2.BB log of Concentration (ngll) 2.92 2.96 3

ABC LABS NO. 43358-19 

### 147

### REFERENCES (1) 

### Committee on Methods for Toxicity Tests with Aquatic Organisms. (C.E. Stephan, Chairman). 1975. Methods of Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians. Environmental Protection Agency, Ecological Research Series EPA-660/3-75-009, 

### April 1975, 61 p. (2) Brauhn, J.L. and R.A. Schoettger. 1975. Acquisition and Culture of Research Fish: Rainbow Trout, Fathead Minnows, Channel Catfish and Bluegills. Environmental Protection Agency, Ecological Research Series EPA-660/3-75-011, 

### May 1975. 45 p. (3) Mount, D.1. and W.A. Bmngs. 1967. A Simplified Dosing Apparatus for Fish Toxicological Studies. Water Res. 1:21-29. 

### (4) Stephan, C.E., K.A. Busch, R. Smith, J. Burke and R.W. Andrew. 1978. A Computer Program for Calculating an LC

### so. U.S. Environmental Protection Agency, Duluth, Minnesota, pre-publication manuscript, August 1978. (5) U.S. Environmental Protection Agency. 1989. Toxic Substances Control Act: Good Laboratory Practice Standards; Final Rule (40 CFR, Part 792). Federal Register, 

### VoL 54, No. 158:34043-34050.

ABC LABS NO. 43358-20Receipt No. T94-2190 Report No. T-4162 STIJDY CODE: 

### KO 1-1583

FINAL REPORT

BACTERIAL REVERSE MUTATION TEST OF TMBP

April, 1995

Rita Research Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute Japan
.KOl-1583



### I, the undersigned, hereby declare that this provides a correct English translation ofthe Final Report. (Study Code No. KOl-1583 issued on April 28, 1995)

(date)

## ['q:J~k ) 

# 7; / 1it"

# (Signatur~**~I"'<..** Yoshitsugu Kajiwara, B.S. Rita Research Laboratories' Chemical Biotesting Center Chemicals Inspection & Testing Institute, JapanKOI-1583

GLP STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan
Sponsor: NIPPON PAINT CO
.. LTD. Title: BACTERIAL REVERSE MUTATION TEST OF TMBP Study Code No.: KOI-1583 I, the undersigned, hereby declare that this test was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's ML, No.16, September 1, 1988) and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Tests related to the New Chemical Substances and of the Toxicity Investigations related to Designated Chemical Substances" (Japan's EA, MHW and MITI, No.39 ofKanpogyo, No.299 of Yakuhatsu and No.85 of 59 Kikyoku, March 31, 1984; No.233 ofKankiken, NO.38 ofEisei and NO.823 of63 Kikyoku, November 18, 1988). Management: Signed in original April 28, 1995 Shigetaka Yamane, Ph.D.



### I ;- rK01-1583

QUALITY ASSURANCE STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan Sponsor: Title: BACTERIAL REVERSE MUTATION TEST OF IMBP Study Code No.: KOl-I583
This test was audited by the Quality Assurance Section and the study procedures were inspected on the following dates.
Dates ofInspections and Audits Dates ofReports to 

# I Study Director Dates ofReports to Management April 10, 1995 April 10, 1995 April 10, 1995 April 18, 1995 April 18, 1995 April 18, 1995 April 27, 1995 April 28, 1995 April 28, 1995 April28--L 1995 April 28, 1995 April 28, 1995 I, the undersigned, hereby declare that this report provides an accurate description ofthe methods and procedures used in this study and that the reported results accurately reflect the raw data obtained. Section Chief, Quality Assurance: Signed in original April 28, 1995 Keiji Shiraishi, B.S.

### KOI-I583
Study code: KOI-I583 Test substance code: HR29I9 Sponsor code: N-460

TITLE Bacterial reverse mutation test ofTMBP SPONSOR

............ _ -..-/.!.. I**~L** " ....... "' ...... _ "'_



### TESTING FACILITY Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 822, 3-chome, Ishii-machi, Rita, Oita 877, Japan PURPOSE OF STUDY The purpose of this study was to determine the mutagenic potential of the test substance using Salmonella typhimurium 

### and Escherichia coli. 

### TESTlNG METHOD The test was conducted 10 

### accordance with the "Standards for Toxicity Investigations" (Japan's ML, No. 77, September 1, 1988) and the "Notification on Partial Revision ofTesting Methods Relating to New Chemical substances" (Japan's EA, MHW and MITI, No.700 ofKanpogyo, No.I039 ofYakuhatsu and No.IOI4 of 61 Kikyoku, December 5, 1986). GLP COMPLIANCE The study was in conformity with the "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's ML, No.76, September 1,1988) and the "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items of Tests related to New Chemical Substances and of Toxicity Investigations related to Designated Chemical Substances" (Japan's EA, MHW 

### and MITI, No.39 of Kanpogyo, No.229 of Yakuhatsu and No.85 of 59 Kikyoku, March 31, 1984; No.233 ofKankiken, No.38 ofEisei and No.823 of63 Kikyoku, November 18,1988). 

### I**~3**KOI-1583

PERIOD OF STUDY Commencement oftest: April 10, 1995 Dose range finding test: April 11, 1995 End ofmain test: April 20, 1995 Presentation offinal report: April 28, 1995 LOCATION AND PERIOD FOR RETENTION OF RAW DATA Data and test substance are retained in the archives and the test substance storage room ofHita Research Laboratories for 10 years following the date ofthe notification specified under Item 1 ofArticle 57-2 ofIndustrial Safety & Health Law. After termination of the retention period, any measures taken are done so with the approval ofthe sponsor.
PERSON CONCERNED WITH STUDY Study Director: Signed in original Tsunehiko Inai, B.S. Hita Research Laboratories Mutagenicity Section Staff: Yuko Koda, Seiko Oda Retention Responsible Personnel: Katsushi Nishimura AN UNEXPECTED SITUATION AND DEVIATION FROM PROTOCOL There were no unexpected situation which might have affected the test results and deviation from protocoLKOl-1583

CONTENTS

SUMMARY MATERIALS AND METHODS

1. TEST SUBSTANCE AND POSITIVE CONTROLS
............. .••.• 2

....................................... 4 2. BACTERIAL STRAINS 3. MEDIDMANDS9MIX 5 4. P"RE,-CULTURES •'"
..... '""'.'""'•••'" '""'•............•••.•••• 6 5. P"RE,PARATION OF TEST SUBSTANCE AND POSITIVE 

### CONTROLS ."'.................................... . 6 6. METHODS 7 7. MICROSCOPIC OBSERVATION AND COLONY COUNTING ....•••••..•..•.•• 8 8. INTERP"RE,TATION OF RESULTS
....................................... 8 "RE,SUL

### TS ..... "'. .................. ... ........... 8· CONCLUSION ................ "' .. "' ................. "'. 9 REFERENCES '""'•. '"'"'"'" '"'" ... "'.••••'"'"'""'.'" '" '"'" '" '"'""' .•"'•. '" '" 9 APPENDIX TABLES AND FIGURES
....................................... 1
..... 6KOI-1583

SUMMARY

The reverse mutation test of TMBP was performed in Salmonella typhimurium strains TAlOO, TA1535, TA9S, TA1537 and Escherichia coli strain WP2 uvrA using the preincubation method with and without metabolic activation system. The results showed the numbers oftheir revertant colonies for all strains in groups which were treated with the test substance were less than twice that of each negative control, with or without S9 Mix. The numbers of the revertant colonies in the negative control and the positive controls were within a background data in our laboratories.
In conclusion, TMBP wasjudged to be non.mutagenic under the present test conditions.

- 1 ­

### KOI-IS83

MATERIALS AND METHODS
1. TEST SUBSTANCE 

### AND POSITIVE CONTROLS 

### 1.1 Test substance (indicated by the sponsor) 

### 1) Name

--**~** '!
..**;....;':':~.-,,:** ..**~** - 4;
..... _ ..... J
.._ ..___ " It • •••• Other name: TMBP CAS No.: 2) Lot No. 100-IS 3) Supplier 4) Structural fonnula or rational fonnula (or outline of manufacturing method, in case both are unknown)

. 1

T (Molecular fonnula: r 5) Purity 

### >99% 6) Impurities Sodium di-n-butylphosphate 

## < 1

### % 7) Physicochemical properties Appearance at ordinary temperature: Molecular weight: Stability: Melting point: Boiling point: Vapor pressure: Partition coefficient: Solubility: Degree ofsolubility: White crystal 359.45 Stable in water, heat and light 95°C

soluble in water and oil soluble freely in water 20% or more in DMSO 20% or more in acetone

- 2 ­KOI-1583
8) Storage conditions A cold and dark place 9) Care on handling Generally toxic because it is a quaternary ammonium salt. Gloves, a mask, a head cap and a lab coat were worn when handling. Slightly hygroscopic 1.2 Positive control substances 1) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) Manufacturer: Wako Pure Chemical Industries, Ltd. Lot No.: PTQ1296 Properties: reddish-orange crystalline powder Purity: 98.8% Grade: Special grade 2) Sodium azide (NaN3) Manufacturer: Wako Pure Chemical Industries, Ltd. Lot No.: KCR6116 Properties: white crystalline powder Purity: 93.4% Grade: practical grade 3) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine • 2HCl (ICR-191) Manufacturer: Poiysciences, Inc. Lot No.: 432098 Properties: yellow crystalline powder Purity: > 95% Grade: 4) 2-Aminoanthracene (2AA) Manufacturer: Wako Pure Chemical Industries, Ltd. Lot No.: TWH23SS Properties: yellowish-green-brown powder Purity: 98.0% Grade: 5) Storage conditions A cold and dark place 6) Care on handling Gloves, a mask, a head cap and a lab coat were worn when handling.


### - 3 ­KOI-1583

2. BACTERIAL STRAINS 2.1 Strains selected Salmonella typhimurium TAIOO, TA98, TA1535 and TA1537 obtained from Dr. B.N. Ames, University ofCalifornia, u.S.A. , on June 20, 1990. Escherichia coli WP2 uvrA obtained from Dr. T.Matsushima, Institute ofMedical Science, University ofTokyo ,on June 13, 1987. 

### S. typhimurium strains TAl00, TA1535 and E. coli strain WP2 uvrA were used for the detection of base-pair substitution mutation, while 

### S. typhimurium strains TA98 and TA1537 for the detection offrameshift mutation. 2.2 Storage The test strains were stored as frozen stock cultures (0.045 

### ml ofDMSO*/O.S 

### ml ofbroth culture) at -80'C (MDF-29l, SANYO Ultra-deep freezer). '" purity> 99.0%, Dojindo Laboratories 2.3 Characterization ofthe strains 1 ) Characteristics ofthe strains Mutation on Mutation on Membrane R-factor Strains synthesis of excIsIon mutation (pKM10l) amino acid repaIr (LPS) Salmonella typhimurium TA1535 hisG46 b.uvrB rja TA1537 hisC3076 b.uvrB rja TA98 hisD3052 b.uvrB rja 

### + TAlOO hisG46 b.uvrB rja 

### + Escherichia coli WP2 

### uvrA trp b.uvrA + The amino acid requirement for growth was demonstrated by using histidine for 

### S. typhymurium strains and tryptophan for 

### E. coli strain. The presence ofR-factor, membrane mutation and mutation on the ability to repair DNA lesions were confirmed by ampicillin resistance, sensitivity to crystal violet and UV sensitivity, respectively.



### - 4 ­KOI-I583

2) Date ofcharacterization

Salmonella typhimurium TA1535 April 12, 1994 TA1537 April 12, 1994 TA98 April 7, 1994 TAI00 April 8, 1994 Escherichia coli WP2 uvrA March 29, 1994
3. MEDIUM AND S9 MIX 3.1 Medium 1) Minimal glucose agar plate (prepared in our Laboratories) The medium was prepared as follows, and poured 30 mt into a petri dish. Components Amount included in one litre 20 x Vogel-Bonner E 50 m1 40 w/V'1o Glucose 50 m1 Agar 15 g (1) Agar: Bacto-Agar (Difco Laboratories) Lot No. 48933AJB (2) Manufacturing date: dose range finding test on March 16,1995 main test on March 22, 1995 2) Soft agar The solution containing 0.5 mM histidine and 0.5 mM biotin for Salmonella strains or 0.5 mM triptophan for 

### E. coli strain was added to soft agar solution containing 0.6 w/V'1o agar (Bacto-Agar, Lot No. 29943AJA, Difco Laboratories) and 0.5 w/V'1o NaCl in a ratio of 1 : 10. 3.2 S9 Mix S9 Mix, which contained the following components, was prepared using rat liver S9. 1) Rat liver S9 (prepared in our laboratories) Animals: Sprague-Dawley rats, 7-week-old, male (161-216 g) Administration: intraperitoneal injection Induction method: 30 mg/kg x 1 time, 60 mg/kg x 3 times with phenobarbital 80 mg/kg x 1 time with 5,6-benzofuravon Lot No.: 9403 (manufactured on December 2, 1994) Storage temperature: -SO·e 

### 1(,0 - 5 -KOI-1583
Storage equipment: Ultra-deep freezer (MDF-291, SANYO) 2) Cofactor for S9 

### Mix (Oriental Yeast Industries, Ltd.) Lot No.: 999403 Storage temperature: _20

# oe Storage equipment: Bio-freezer (GS-2603, Nippon freezer Ltd.) 3) Composition ofS9 

### Mix 1 ml of S9 

### Mix contains 811M MgCh, 33 11 M KCI, 5 11 M G-6-P, 4 11 M NADPH,4 11M NADH, 100 11M of0.2 M Na-phosphate buffer (pH 7.4) and 0.1 

### rnI S9.
4. PRE-CULTURES From the stock cultures, 20 111 of bacterial suspension was inoculated to L-tube containing 10 

### rnI nutrient broth No.2 (Lot No. 154 52789, OXOID Ltd.), the bacterial culture was incubated at 37 ± 0.5°C for 8 h with shaking at 50 times/min by the Monod shaker (MONOSIN- 

### IT A, TAITEC CORPORATION). The viable cell counts calculated from the values determined by spectrophotometry at 660 nm (Novaspec, LKB Japan) at the end ofincubation are shown below. Base-pair substitution type Frameshift Type TAlOO TA1535 WP2 

### uvrA TA98 TA1537 No. of viable cells I{x 109 /ml) Dose range finding test 1.8 2.4 2.5 2.0 2.0 Main test 1.8 2.4 2.0 2.0 1.9 5. PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROLS 5.1 Test substance 1) Preparation The test substance was dissolved in distilled water (distilled water for injection, Lot No. K4I81, Otsuka Pharmaceutical Factory) to make 5 w/v% concentration and diluted with the same solvent to give lower concentrations. 2) Preparation time Freshly preparedjust before use. 3) Stability oftest solution No denaturation oftest solution was observed until 2 hours after the preparation.


## I "

## I - 6 ­KOl-IS83

S.2 Positive controls 1) Preparation NaN3 was dissolved in distilled water (Lot No. K4181). AF-2, ICR-191 and 2AA were dissolved in DMSO (Lot No. YYl37). 2) Preparation time and storage condition Prepared on every 3 months and stored at -80°C (MDF-291, SANYO Ultra-deep freezer).
6. :METHODS The test was earned out for S. 

### typhimurium strains TAlS3S, TAlS37, TA98, TAI00 and 

### E. coli strain WP2 

### uvr A using the pre-incubation method both with and without metabolic activation system, represented S9 Mix(+) and S9 Mix(-), respectively. The plating was done in triplicate for the negative control and in duplicate for the test substance and the positive controls. 6. 1 Procedures After 0.1 m1 of the test substance solution, O.S m1 of 0.1 M sodium phosphate buffer (pH 7.4) or S9 

### Mix, and 0.1 ml ofthe bacterial culture was added to a tube, the mixture was incubated for 20 min at 37 ± O.S°c. Two m1 ofthe soft agar was then added to each tube and poured onto a minimal glucose agar plate. After incubation for 48 h at 37 ± O.SoC, the number of revertant colonies were counted. As the sterility test, each 1 m1 ofeach bacterial strain, the test substance solution, the S9 Mix or 0.1 M sodium phosphate buffer (pH 7.4) are smeared on a minimal glucose agar plate and incubated at 37 ± O.SoC for 48 h, and then checked the bacterial contamination. Distilled water was used as the negative control, and the following positive controls were used for each bacterial strains. 11 g1Elate TAIOO TAl535 WP2 

### uvrA TA98 TAlS37 S9 Mix (-) AF-2 NaN3 AF-2 AF-2 ICR-191 0.01 O.S 0.01 0.1 1 S9 Mix (+) 2AA 2AA 2AA 2AA 2AA 1 2 10 0.5 2**\b~** - 7 ­KOI-ISS3

6.2 Dose selection I) Dose range finding test The test was carried out at seven doses of 50, 100, 200, 500, 1,000, 2,000 and 5,000 tL g/plate. 2) Main test Based on the results of the dose range finding test, main test was performed at the highest dose of 5,000 tL g/plate and 4 lower doses diluted with a geometric progression of2. 7. IvITCROSCOPIC OBSERVATION 7. 1 Microscopic observation The state ofrevertant colonies (size and number ofcolonies), deposition ofthe test substance and the growth inhibition were examined with a stereo microscope. 7.2 Colony counting The numbersofcolonies were counted with a manual counter or a colony analyzer (CA-7, Toyo-sokki Co., Ltd.). Correction for counting errors was made for measurements with the colony analyzer. Each plate was measured three times, and the average ofthese three measurements was adopted as the number or revertant colonies on the plate. The average for each dose was calculated from the values of the plates used. Decimals ofthe average figures were rounded off. 8. INTERPRETATION OF RESULTS The test substance was judged to be positive, when the number of revertant colonies was twice or more ofthe negative control, and the dose-relationship and the reproducibility were obtained. Any statistical procedure was not adopted.
RESULTS The numbers oftheir revertant colonies for all strains 

### in groups which were treated with the test substance were less than twice that ofeach negative control, with or without S9 Mix. The positive controls showed the distinct increase ofrevertant colonies, and the positive and negative controls were within the range ofthe background data in our laboratories.


### rb3 - 8 	­KOI-1583

There were no fluctuations which affected the test results because the sterility test confirmed the absence ofmicro-organisms.
CONCLUSION
In conclusion, TMBP was judged to have no reverse mutagenic potential under the present test conditions.
REFERENCES 1. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using Trp+ reversion in Escherichia coli, Mutation Res,. 38: 3-32.
2. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res,. 113: 173-215.
3. Ministry ofHealth and Welfare (1992) Guideline for toxicity Testing ofChemicals, Revised Edition, published by the Chemical Daily Co., Ltd.
4. Ministry of Labor (1991) Guidebook on Mutagenicity Tests using Microorganisms, New Edition (in Japanese) published by Japan Industrial Safety and Health Association.

- 9 ­KOl-15B3 

### Dose 

## range finding test Test substance: T 

### M B P


### With(+) 

### or Test substance 

### Number of revertants (olDDber of colonies/plate) without(-) concentration Base-pair substitution type Frameshift type

89 Mix 

### ()J.g/plate) 

### TA 100 TA 1535 107 8 negative 113 ( 118) 1O( 9) control 135 8 50 112 ( 116)

. 119 10 ( 13 12) 100 117 ( 113) 108 10 ( 12 11) 

### 89 Mix 200 133 ( 127) 14 ( 12) 120 9· (-) 111 ( 115) I 9 13) 500 118 16 ( 1000 113 ( 113) 112 9 ( 8 9) 2000 122 ( 130) 138 10 ( 8 9) 5000 108 ( 102) 95 10 ( 9 10)
I 119 10 negative 128 ( 131) 13 ( 11) control 145 11 50 118 ( 121) 123 12 ( 9 11) 100 125 ( 116) 106 15 ( 19 17) 

### 89 Mix 133 ( 126) 6 9) 200 119 11( (+) 

# ,**ii~** 

# (118) I 11( 500 11) 11 1000 112 ( 123) i

. 134 14 ( 10 12) 2000 115 ( 121) 127 1l( 9 10) 127 ('129) 9 10) 5000 131 I 11( 

### Positive i 

### Name AF-2 NaNa 

### control not**Conc~~trat1on** 0.01 0.5



### requiring (1lR/Dlate) !

89 Mix 

### Number of 449 ( 433) 246 ( 251) 

### colonies/nlate 417 255 

### Positive Name 2M 2M 

### control -uoncentratlon 1 2 

### (llR/nlate) requIrIng Number of 555 ( 559) 131 ( 127)



### 89 Mix 

### colonies/Dlate 583 123 

### WP2uvrA TA 98 TA 1537 40 19 11 39 ( 41) 20 ( 20) 7 ( 10) 45 21 11 55 ( 56 56) 23 ( 27 25) 10 ( 13 12) • 40 ( 49 45) 23 ( 20 22)
. 8 ( 8 8) 56 ( 47 52) 27 ( 21 24) 15 ( 8 12) 45 ( 51 48) 23 ( 18 21) • 14 ( 8 11) 50 ( 43 47) 25 ( 22 24) 9 ( 8 9) i 51 (**~?** 52) 20 ( 24 22) I 10 ( 9 10) 57 ( 57 57) 18 ( 25 22) 11( 12 12) 54 30 16 50 ( 52) 24 ( 26) 23 ( 19) 51 24 18 48 ( 54 51) 35 ( 28 32) 22 ( 19 21) • 47 ( 51 49) 25 ( 36 31) 13 ( 22 18) 60 ( 44 52) 34 ( 28 31) 16 ( 18 17) 54 ( 22 ( 17( ! 54) 26) 19) 54 30 21 

### I 58 (

67 63) 29 ( 33 31) 19 ( 19 19) 57 ( 57 57) 33 ( 35 34) 17 ( 19 18) 50 ( 42 46) 32 ( 28 30) 19 ( 12 16) AF-2 AF-2 rCR-191 0.01 0.1 1 530 ( 553) 576 543 ( 541) 538 2232 (2229)
. 2226 2M 2M 2M 10 Q.5 i 2 444 230 100 ( 105) 429 ( 437) I 195 ( 213) 109 Notes Parenthesis shows the mean of each plate
.



### .AF-2: 2-(2-Furyl)-3-( 5-nitro-2-furyI)aerylamide ·NaN3 : 

### Sodium azide 

### 'IeR-19I: 2-Methoxy-6-chloro-9-(3-(2-ehloroethyl)-aminopropylamino) acridine.2HCl ·2AA: 2-Aminoanthraeene 

## -1­KOl-15B3 

## Main test
Test substance: T 

### M B P I 

### Witb(+) or Test substance Number of revertants (nlllDber of colonies/plate) without(-) concentration Base-pair substitution type Frameshift type

89 Mix**(~Jplate)** 

## TA 100 TA 1535 WP2uvrA 

## TA 98 TA 1537 negative 120 11 42 28 7 122 ( 118) 13 ( 12) 48 ( 44) 21 ( 26) 9 ( 9) control 113 11 42 29 10 313 128 ( 121) 113 15 ( 8 12) 39 ( 45 42) 29 ( 27 28) 6 ( 8 7) 

### 89 Mix 625 112 ( 112) 12 ( 11) 51 ( 49) 28 ( 31) 7 ( 7) 111 10 47 33 6 (-) 1250 122 ( 112) 10 ( 14) 45 ( 49) 30 ( 26) 7 ( 8) 101 17 52 21 8 2500 112 ( 113) 113 10 ( 11 11) 50 ( 49 50) 24 ( 19 22) 7 ( 9 8) 5000 128 ( 124) 119 11( 10 11) 68 ( 44 56) 20 ( 34 27) 13 ( 7 10) negative 125 15 47 33 19 127 ( 125) 13 ( 14) 57 ( 52) 32 ( 32) 22 ( 20) control 123 14 53 32 20 313 123 ( 122) 121 17 ( 13 15) 69 ( 49 59) 30 ( 43 37) 13 ( 21 17) 

### S9 Mix 625 127 ( 116) 10 ( 11) 54 ( 54) 31 ( 35) 26 ( 24) 104 11 53 38 22 

### (+) 1250 132 ( 125) 16 ( 14) 49 ( 59)

# ~~ ( 32) 21 ( 19) 118 11 68 16 2500 125 12 ( 14) 21)

. 124 ( 125) 15 59 21 5000 136 ( 135) 134 20 ( 17 19) 

# 61(lli20(

57 ( 18 ( 71 15 17) 

### I 

### Positive 

### Name AF-2 NaNs 

### control not Goncentratlon 0.01 0.5



### requiring**(~/plate)**



### 89 Mix 

### Number of 474 ( 456) 269 ( 264) 

### colonies/plate 438 258 

### Positive Name 2AA 2AA 

### control Concentration 1 2 

### requiring (1J,g/piate)

89 Mil Number of 536 ( 542) 137 ( 143) 

### colonies/plate 547 149 AF-2 AF-2 ICR-l91 0.01 0.1 1 533 ( 540) 547 488 ( 508) 527 2105 (2103) 2100 2AA 2AA 2AA 10 0.5 2 418 ( 419) 419 168 ( 146) 124 106 ( 111) 115 I Notes Parenthesis shows the mean of each plate. 

### -AF-Z: Z-(Z-Furyl)-3-(5-nitro-Z-furyl)acrylamide -NaN3 : 

### Sodium azide ·ICR-191: 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)-aminopropylamino) acridine·2HCl 'ZAA: 2-Aminoanthracene



## -2­

### 50 

### Dose range 

### finding test 

### KOl-1583

<l) ....,



### ..... '" Co t... <l) Co til <l)
...... c: 0 

### ..... 0 u 

### ...., c: cd 

### ...., '­ <l) > <ll c:: 

### O:TA 

### 100 150 O:TA 

### 1535**~:WP2uvrA**



### 100

<l) ....,



### ..... 

## '" Co '­ <l) Co til <ll
...... c: 0 

### ..... 0 u 

### ...., c: ...., 

## '" '­ <l) > <l) c::**Ol~----~-----.------~-------.------.------.------~** 

### 50 100 200 500 1000 2000 5000 (J,£g/p 1ate) 

### Fig. 1 Dose-response curve without S9 Mix

O:TA 100 150 O:TA 

### 1535**~:WP2uvrA**


### 100

50**Ol~----~-----.------~-------.------~----~------~** 

### Fig. 50 

### 2 100 200 

### Dose-response curve SOD 

### with S9 1000 

### Mix 2000 5000 (J,£g/plate) 

## 167 -3­

### Dose range finding test 

### KOl-1583
O:TA 98 

### 30 

### D:TA 1531

<1.1 

### ....,

....... 

## '" c. 

### 20 '­ <1.1 C. til <1.1 

### ..... c:: 0

....... 0 u 

### ...., 

# '" c:: 

### 10 ...., '­ <1.1 :> QJ oc

<1.1 

### ....,

....... 

# '" c. '­ QJ C. til <1.1 

### ..... c: 0

....... 0 u 

### ...., c:: 

### ...., 

## '" '­ QJ :> <1.1 oc**Ol~--- -~----~------** **~------~ ------~-----'** **-------'** 

### 50 100 200 500 1000 2000 5000 

### Fig. 3 Dose-response curve without 89 Mix (ttg!p 1

### ate)

40



### 30

20



### 10**O~----,-----,------.--------.------.----~--------.** O:TA 98 

### D:TA 1531

50 100 200 500 1000 2000 5000 

### (Ltg/plate) Fig. 4 

## Dose-response curve with 89 Mix


## -4­

### KOl-1583 

### Main test O:TA 100 150 O:TA 1535 .o.:WP2uP'rA

Q.)


### ..... 

## '" 

# -c

### lOD c... Q.) 0­ m Q.) 

### ..... .:: 0 

# - 0 (.l



### ..... .::



### ..... 50 

## '" c... Q.) >**~ Ol~----~--------~r----------.----------'---------~**  313 

### 625 1250 2500 5000 

### (Jig/plate) Fig. 5 Dose-response curve without 89 Mix

150 O:TA 100 O:TA 

### 1535 .6.:WP2

### uvrA 3

....... 

## '" 0­ c... 

### 100 Q.) c.. m Q.) 

### ..... .:: 

## ..s o (.l



### ..... .::

..... 

## '" c... Q) >**~ O·~----.----------.----------~----------~--------~**  

### 313 625 1250 2500 5000 

### (}1g/p 1 at 

### e) Fig. 6 Dose-response curve with 89 Mix


## -5­

### 40 

### Main test 

### KOl-1583
O:TA 98 D:TA 1537

ClJ 

### 30 ...., aj

....... c.



### .... ClJ C. ttl ClJ 

### ..... == 

### 20 0

....... 0 <.J 

### ...., c:: 0:1 

### .... .... ClJ > ClJ cc: 

### 10**o~----~--------~----------~----------~---------.** 313 625 1250 2500 5000 

### (p.g/plate) 

### Fig. 7 Dose-response curve without 89 Mix

O:TA 98 40 D:TA 1537

ClJ 

### 30 ...., aj

....... c.

.... ClJ C. ttl ClJ 

### ..... == 

### 20 0

....... 0 <.J 

### ...., == 0:1 

## ....
.... ClJ ClJ > 10 c::**O~----,----------.-----------.----------.---------~** 313 

### 625 

### 1250 2500 

### 5000 (p.g/plate) 

### Fig. 8 Dose-response curve with 89 Mix


### -6­