/YIIS6/1) 


~----------------------------------------------------------------~----- Form Approved.. O.M.B. ~o. 2!l7O-OO12 AwovaJ, Expu:s 10-31-96. 

U. S. ENVIRONM:ENTAL PROTECTION AGENCY 

ft EAtA PREMANUFACTURE o 1-\ NOTICE 

DateoEreceipt 

\Nhl'n 

('(11l1 pll-tl'd 

"end tht'­

torm h. 

FOR NEW 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 of pages Docu:ment control number EPA caM num.bc 

in the Premanufa.cture Notice 

• YOIl mut PlDrida alllafo-..doe. noqll........ tIUa fo..to tIM __t tUt It t. kaowa to or__1y ~leby,.,.. Make __le tStiJuta If you do 

not lI....e iIIdaaI data. 

•'BefoN you _plete tbia foaa,. ,.,.IIlOUld .... tile "laIImIdiou Mlaul for .........atac:t1lft NotlftaIioe.- (tile lubv.diou M.umai. -.u.ble flom !heToxiC 

Sub..ca Coatml Ad (TSeA) bllonutioa Scrice'by caIlbIJ 202-SM-U04. 01' faxial 2.0:1-556-560:1. . 

·IfII _ fee ..... been nmdtted for tbia aotice ,to CFJl 700.45), l:adlcate ill tile "-aboft tile 'fS...ueI' feeld_ti.8C11ti_ .umbeI' YOIl laave l-emIed. 

Remember. your IIMrfee 10 alllJlbel' lJlut aI.Io II,.,... _ your CICII'IISfIOlldial fee .-J1Uaa, wIIidl ..tattoe EPA. HQ ACCINJIIiDI Opentiou Br.m.cIa (PM-266),

P.O. 360399M,. PIltlll:l'Ill'ail.PA lSlSl-6399, Alta. TSCA. UNrFee. 

Part 1- CENERAL INFORMATION 

Yo. IIUIlt prorida tIM ClUNIltly CDDIId CIa.ucal AlHttDdII (eA) Name of the 

Ilew claemkal.llbllt.mce. ev_ If YOIl daiIII tile (d_tlty. CIIlltlfidntiaL YOIl 1IUI)' 

lI.tIaodze aatll_ to nbm.lt chemkalldatlly Iafo-..doa for,.-. bat 

J'01lf nbmJal_ wID IIOl beCD.,....ad the -wwwID1IOl1Npt ..tII EPA 

recei_ tIlt.lafo....... A Ietiu..nppoct ofyo_~u-w 

Nf_yourTS ...fee Weatlfic:lIIioII ...... YOIl ...IRIIbaIt_1IdJIuI 

ad two CDpl. of tIUa Ilotloe I:ad1ldlq all...cWa. IfJa. c:I.UaM ..,. 

iafoaaatioa • collfidedal,. II IIlqIe...wad CtIIp7' ...aIIo IMIAIbaitIIrId. 

Part D - HUMAN EXPOSURE AND ENVIRONMENTAL I.EtEASE 

If tlaeN are""'" Ill_at........-mil- 01' _ 0JNIIIdl0u to 1Md.aibed I:a 

Part II. Mdtou A ud Bof tilt. _tioe:,. reprodllCll! tile IIed:IaIIII ••eeded. 

Part m-llST OF ATTACHMENTS 

Altadl additioaal ....... If tIleN • not ..oup .pace to __II qustiOD f1llI,.. 


Label eadl CDIltia'Utioalheet wttIa tlae comspoadl:al Mdt_ JaeIIdbls. la Part 

lII.list tIlae iItbdlmeabl,. ay tat data 01' atIl_ data ad lID,. optIODai 

informUioIl l:adllded I:a the aotic& 

OPTIONAL INFORMATION 

So-called "biDdI:aI" t.ox.. arel:adllded tIlmqIIIoat ddI ....-Ja.to IadkitttIoI 

your willlap_ to be bouad to 0IItai:a ............,.0Il....-I:a tIda aotiat,. HCIa 

.. lUIe. prodlldioa voilUlle, pI'OMdiYe ..iii,...... nu. optIo... bI*-ded to 

reQlICe delays tIl.u routinely _pa,. tile ~oI_torden 01' 

Silal.flcmt New u..R1IieI. Exczpt ht tile _ of _ptioa applJl:Mia_ 1Iuch. ... 

T'MEA, LVE, LOREX) wla_1CIItWa iDfcmIuItiOD proricfed I:a IIIIch aaWk:IIioa t. 

bl:adl:al_ tile .ablJliltarwUa tile AIacJ' ..,..... tile _ptioa appliaId-. 

cil.edc.illg II bhtdilll box I:a tIliIlIOtIce.....by UIelf pmldDlt IM __ 

DolllllW'd~fmIIl tile ia.6D1IIUItka (ampt cIaemr:alldatlty) npoftIId I:a theN~ -

CONFIDENllALlTY CLAIMS 

Y0II1IUI)' cI.t.bI_,. iafoauIIoa I:a tIda ___-"4-tW To --'II cW. 

OIl the form" auk 00 tile Cl)llfldewtlal box.atlID tile 111.........tUt,.. cWa 

• CIIIlfIdatlaL To -aII daia I:a _ c:iJcIe 01' billdral tile 

iafCiDllatloa Ja. cIaba _ u,... d.tm Ipfpmalln '" tM PAtio M 

qmfld.U.1 J9P mg. aIIp pard••_II. Y''''. gf ""Mllca.(lw;l,4m. 

attw;bmcpllU.. For: addltlo&al11lltnc:tiou _ daaial ............ 


CIIIlfId..u..l. rad tile laItndiaaII MIla..... 

. 

TEST'DATA AND O1HEK'DATA 

YH _ noq..u.d lID""" aD ... data Ia youpOINMIoa 01' CDDtmi • 

ad to pIO'f'We a ca.atptioa 01 aD otIMr data bowallD OI'..-ablf 

aoemI:a,hIe Dyyoa.1f a...data _ ....... to tile IMaltIa ud 

_riN~""'oIlM_~.--mll-dltbiktl_ 

Ia m--..-.. otdltpaMlof tile _ c:Iiweak.Il .......ee SbDdu.d 


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....... datIiDu.., emapIW.IMAlnaiItAId 4.tmttJep 

_data 

'" EDIBaJa) 

I:a tile 

.otapmmart. 

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pt4.. 


• .,. be ...."'" If tl!cy dO ..._ Ip the oga'lttpIpm YH IIlo1lW 

dHdy ldatlfywMtlaerIIIIt data t. OIl the ......OI'oe. ..aaaloa- AIM, 

tile cheaial_,-oo. 01 tile*-led aatedalIIlo1l.ld be c:Iua:'III:tesi 

Followiq __pia01 ...data ad otIiIerdat&. 'DatallbDuld be 

I1IIbIlI.itI8d __I..to tile JIIIII1II-eatt 018720.50 01 tile P_atac:t1lft 

NoIifiClitioa ......,to CFJl Put '12D). 

• Ea~eff..data 	 StrIIdaNiadivity n.latlauhl~ 

.PII~~-DY. T_data IIOlI:a tIM,-loe. 

01' c::DIdftII 01 tile IIIIItaiItcr 

-A pia"'" ad cIleaial piIIDJMIlia -all_t.1oc:atirId OIl tile 1at pap 


ofW.... 


T"rPE O.Nona 

tzl 

o 


o 


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o 

o 

o 

ISnDSACONSOlJDATE'DPMNT 0 
Y.

o , of ch«mkat. Mark 00 If~,.Iafo-..doa I:a tIUa llotica. c:Iahaed ... CIIIlfIdatlaL ao-uc. CmamIllUcltfDa ,noq....., ...., Oil pap 3) 

EPA FORM 7ll~2S. (Rev. 5-95) 	 Replace!!I previous editions of EPA Form 771~2S. I 

Public reporting burden for this coilection of information is estimated to average 110 hours per response, including time for reviewing instructions, 

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

comments regarding the burden estimate or any other aspect of this collection of information, including suggestions for 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 the best of my knOWledge and belief: 

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


commercial purpose, other than in small quantities 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 of the date of submission. 

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

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

Additional Certification Statements: 

If you are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following uaer fee certification 

statement that applies: 

o 	The Company named. in Part I, Section A has remitted the fee of S2500 spedfied. in 40 a:R. 700.45 (b), or • 

o The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined • 40 a:R. 700.43) in 

accordance with 40 CFR 700.45(b), or 

o The Company named in Part I, Section A is a small business concern under 40 a:R. 700.43 and has remitted 


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


Ifyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50 (c) (1) or a Low 

release and low expoaure exemption (LoREX) application in accordance with 40 CFR 723.50 (c) (2), check the following 

certification statements: 

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


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


of 40 CFR 723.50. • .­

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

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

o If this application is for an LVB in accordance with 40 CFR 723.50 (c)(1), the manufacturer intends to commenC2 


manufacture of the exempted substa.nce for commercial purposes within 1 year of the date of the expiration of the 


30 day review period. 


The accuracy of the statemenls you make ill this notice should re£Iect your best predidim of the anticif8tal fads regarding the chemical 

s.ubstance d8scribed herein. Any knowingand willful misinterpretation is subject to criminal peaa1ty pumuant 1018 USC 1001. r-----~ Coafide;ntial 

Signature and title of Authorized. Off:idal (0rigiDal Signature R.e'luized) Date 

Signature of apDt • (if applicable) Date

FORM EPA 7710-2S (Rev. 5-95) 	 Page 2 
If you have submitted a notice of Bona fide Irttent to manufacture or 

impart fCII' the chemic:alsubetanoe awered by this notice.. enter the 
 -----.:)Ir... D

notice number assigned by EPA. ,.. 


2. D'~ 3 • DBoth D~ 

Mailing and street) 

Oty, State, ZIP Code 

Name of authorized o.fficial 

Mailing addrees (number and street) 

Qty, State. ZIP Code 

Telep~ 

If you are submitting this notice 81 part of a joint submission. mark 00 box. --------------..... 

Name of authCllized. offidal POlliliao 

(if~li~~ -C;;~----------------------~-----------------------------

Mailing adc:lrea (number and street) 

ety, State, ZIP Code 

Name 

Company 

Mailing address (number and street) 

QtyiState, ZIP Code 

If you'have had a prenotice .cmnmunic:atl.an (PC) alIKI!rl1i.ng this nalia! 

and EPA assigned a PC Number 110 the notice. enter the number. 

If you previously submitted 8D exe:mpt:Km appIk:atIoa fCII' the c:hemicIl 

Type of Notice - Mark 00 D Only . Muufw::ture 1. . Opt:km 

FORM EPA 771G-2S (Rev,5-95) 

Telephone 

Position 

Teleph..e I 

I 

Mark 00 

if none 

Part I - GENERAL INFORMA nON 

~_S_e_c_ti_o_n_A__ __ -_S_UB ___ I_I_ER __nD_______CA___ ll_0__N ____________________________________________~~~

1a. Person 


Submitting 


Notice 
 • 	 (in u.s.) 

b. 	 Agent (if 


applicable) 


c. 

Jomt 

Submitter 

2. 	 Technical 


Contact 


(in U.s.> 


3. 

4. 


substance covered by tJrls.notice. enter the exempI10Il number _peel 
 Mark 00 by EPA. If you previously submitlala PMN fCII' dUll IUbetance enter the ifnane . -~.,... .... D

PMN number usiped 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) of CA nomencl.1ture rul. md amventiDna. 

Mark 00 the "Confidential" any item you claim as confidential. 

Complete either item 1 (Cl_ 1 or 2 subltances) or 2 (Polymers) al appropriate. Complete an other itema. 

if another person will submit chemical identity information for you (for either item 1 or 2), mark 00 the box at the.t 0 

the name, and add.re!lls of that in a continuation sheet. .. 

1. Cla..1 or 2 chemicalwbstance. (for definitions of class land class 2 substances, see the In.truction. Manua1) 

a. 	Oass of substance - Mark 00 1 D Cla9s 1 or 2 0 Oass 2 

b. 	Otemical name (Currently correct Otemical Abstracts (CA) Name that is consistent with T5CA Inventory .listings for similar substances. 

For Casa 1 substances a CA Index Name must be provided. For Oass 2 subetances either a CA Index Name or CA Preferred Name 

be providecL whichever is appropriate based on CA 9CI nomenclature rules and conventioNL). 

c. Please identify which method you used to develop or obtain the spec::ified chemical indentityinformation reported in thiB notice: 

(check one). D Method 1 (CAS Inventory Expert Service • a copy of the Identification DMebxi 2 (Other Source) 

report obtainedftom the CAS Inv",tory ExpI:!rt Service IIUlIIt be 

submitted as an attachment to this notice) 

d. Molecular formula and CAS Registry Number Of a number already exists for the substance) 

CAS. 

e. 	For a cla .. 1 substance, provide a complete and correct cbemic::al st:rud\lIe diagram.. Far a daN 2 sublltance - (1) Ust the immediate 

precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of the re.::tkm or procea (3) lDdicate the 

range of composition and the typical composition (where appropriate). (4) Provide a correct U!P!eae.ntative or partial chemical 

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 of polymer, see the In.lnlcnon. ManuaL) Conticlential 

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

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

500 and below 1,000 absolute molecular weight of that compositiOl\. • 

Describe the methocls of measurement or the basil for yoar estimatel: GPC 0 OthI!'1'D :(Specify) 

n lowest number average mol.ec:ul.ar weight 

mmaximum weight'" below 500 molecular weight 

iii) maximum weight '" below 1000 moloecul.ar weight o Mark (X) this box if you attach a continuation sheet. 

b. You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual informatiOl\. 

Mark 00 the "Confidentialft box next to any item you claim &!I confidential 

(1) - Provide the specific chemical name and CAS Registry Number (if a number exists) of each. monomer or other reactant used. in the 

• manufacture of the polymer. 

(2) - Mark 00 this column if entry in column (1) is mnfidentiaL 

(3) - Indicate the typical weight percent of each monomer or other reactant in the polymer. 

(4) - Mark DO the identity mlumn if you want a monomer or otherreadant uNd at two weight percent or leu to be lilted as part of the 

polymer docription on the TSCA Chemical Substance Inventory. 

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

(6) - Indicate the maximum weight percent of each mot.\omer or other reactant thal:may be present ...residual in the polymer ill 

manufactured. for commercial purposes. 

(7) - Mark 00 this column if entry in 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 if you attach a amtinuatian sheet. 

c. Please identify which method. you used. to dewlap or obtain the specifI.ed chemical identity informatiim reported. in this notice. (c:hec:k one). . D 	 Method. 1 (CAS Inventory Service - a copy 01 the identification report D Method. 2 (other source) 

obtained. from Inventory Expert Service mvat be submitted as . '. 

an attachment to this notice) 

d. TIle currently correct O\emical Abstracts (CA) name fex the polymer that Is consistent with TSCA Inventory Ustinp for similar polymers. 

e. Provide a mrrect representative or partial cbemic:al1trUdUre diagram, .. complete as can be known. if one em be re.onably ucertained... 

-

o Mark(X) this box if you 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 _ublyutidpated ID be praeat In the chaaic:alnHUDca __1IDdUed for aJ_en:W p1IrpOMa. 


Provide the CAS Rep.try NlUIlber If ..nibhle. U theft ..lIIlidentified Imparltl.., eater "uideatlfied. • 
 (b) -Eatbute tM awdmlUll weipt Yo of eKIa Imparity. U th....1IJlidatified baputtiel, stba..their IDtaI wetpt Yo. 

. 	 Impurity and CAS Registry Num~ Maximum Coatipercent (a) dential (b) 

'l{, X 

'l{, 

'l{, 

,. 


,. 


'l{, 

'l{, 

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

4. Synonyau -	 Enter any c:hemicalsynonyms for the new chemical substance identified. in subsectlCll 1 ar 2. . :1 

X 

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

5. Trade identifiatiGl - List trade names for the new c:hemicallUbetanc:e identified. in suteed:iCll 1 or 2. 

~. 

~-O ~" c I. . IB'-\OGO (p\"'\\'J 'Sv~lo..\'\c.e It'~;> n-butylc.duJ'\c\ 

:). . . 	 . '~)L~ t / I . etC0 t C..L tc:. :2.!"")/o) o ( . 	-\tv\ \3? Cf tJ\tJ C:;LbSt...c\.V~c...e IOD %) 

-' 

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

6. 	Generic chemical name - U you claim chemical identity as amfidential. you muat provide a generic chemical name for your substance 


that reveals the spedfk: chemical identity of the new chemicalsubetance to the maximum extent peBble. 


Refer to the TSCA Chemical Substance Inwntcry, 1985 EditiCll. Appendix Bfar guidance on developing 


generic names. . 
 . 

'S'q t--t. --retr(AO \ .\::: fe G \'VI OV\\ u. VV\ 

o Mark (X) .this box if you attach a continuatlCll sheet. 

7. 	Byproduct. - :::;_._ any byproduds resulting from the manufacture, processing, UM. QI' Ctispc.l 01. the new chemical subetance. Provide the CAS 

....Number if available. . 

Byproduct CAS RegiIItry Number Conti­

(1) (2) dential 

-. x: 

-

0 Mark (X) this box if you attach 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" box next to any item you daim as confidential. 

L PJOduction volume ­ Estimate the uwdmum production volume during the first 12 months of production. Also estimate the 

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

100% new chemical substance basis. For a Low Volume Exemption application, if you choose to have your notice reviewed at a lower 

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

Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confi.. Bl:acHJII 

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

X )< 

2. Use informanOD - You must make separate confidentiality c1aims for the deeaiption of the category ofUIIe, the percent of produc:tion volume 

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

claim as confidenlial. 

a. (1) - Desaibe each Intended category of use of the new c:he:mk::al substance by function and appUc:aticn. 

(2) - Mark 00 this column if entry in column (1) is confidential businee ~tion (CBD. 

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

(4) ­ Estimate the percent of total procI.w:timl far the first thnIe yean devoted to each category of u-. 

(5) ­ Mark 00 this column if entry 'in column (4) is confidential busineeinformation (CBD. 

(6) ­ Estimate the percent of the new subetance as formulated. in mixtUres, suspenskma, emulslons, solutions, or gelIJ as manufactured for commercial purpoaes at sites under your control"""';ated with each category of UIe.. 

m­ Mark 00 this column if entry in column (6) is caniidentlal businee informatklll (CBD. 

(8) ­ Indicate" of product volume expec:ted. far the 1J8ted. "UN" -=ton. Markmen than one box if apptopriate. Mark . 00 to indicate your willingnee to have the use type provided in (8) binding. 

(9) ­ Mark 00 this column if entry(ies) in column (8) is (are) canfidlll1tial busineII informatiaa (CBD. 

Categoryof use (1) CBl 'IIiIldiai Produc- CBI ~in CBI " ofIlUbetance expec:ted. per UIIe CHI 

(by function and applkation i.e. .a dispersive dye for Opdaa tiaa .. form­ (8) MoakW ulatloD finishing polyester libera) I:ted 

eon-" Indua- Com- BiDding (2.) (3) (4) (5) (6) m sumer trial merdal 0pti0D. (9) 

')( ')<. " )( " X " X )Z X 

" " 

" % 

" % . 

" " 

'" " 

" " 

·If you have identified a "consumer" use. pleue provide on a continuation sheet a detailed description of the use(s) of this che.mical 

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

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

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

b. Generic If you cbIim any category of UIIe delaiptkm in IUhtIectian 2a ..amfidt!ntIaL entIlr a pMIic deIaiptkm ofthat 

use category. Read the inltr'actJaa. Muraal foe examples of generic: WIll delaiptiODL • 

description 

C U r-'lv\({ O.C1~V\t ~~ CoCt ''\:..\ \;qS. 

- o Mark 00 this box if you attach a c:ontlnuatkID sheet. 

3. 

or 

Hazard 

other 

IDfonDatlaa 

informatiaa 

-

which 

Include

¥Iill 

in 

be 

the 

provided 

notice a 

to 

copy 

any 

of

pnDD 

re.aDIlble 

who is 

faaimiIe 

naooably

of any

ltbly 

hazard. 

to be 

wammg

expoI8d 

1ltaalneDt.1abeI,. 

to tb.iII 

mata:lal 

reprdIDg 

aiety 

protective 

data IIbeet. =:

JoUrk()l) 

I Dequipment or practices far the safe handling. transport. WIll,. or dJspoeal of the new su........ 1JItin part mhazard infonuation,you Include. 

Marie 00 this box tf vou attach hazard ' - ~ 

FORM EPA 7710-25 (Rev. 5-95) Pap7 
sites 

Mark (X) this box if 

b. TypeMark 00 

attach a continuation sheet: 

o Manufacturing 

Hoursfbatc:b 

o Use 

Mark eX) to indicate yoar wUliop", tID have yom: procna deKriptlaa biDdiD. 

PartJI - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

U>lUldJentllal box next to any item you 

Complete section A for each type of manufacture, proce..mg, or tiM operation iDvolviDg the new chemi&:almbttance at iDcta.ltri.allitei yoa controL 

Importen do not have to complete tha HCtion for operation. oattide the U.S.; however, yoa may Rill have reporting requirementt if there are 

further iDdtl.trialprocuaiDg or tiM operation. after imp«t. Yoa mu.t describe theM operation .. See iDatruc:tion. manuaL 

1. Operation description 

a. - Enter the identity of the site at which the will ocx:ur. 

Name 

City, County, State, ZIP Code 

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

additional sites on a continuation sheet, and If any of the sites have significantly different ~ 

production rates or operations, include all the information requested in this lIedion for thOle 

(1) 	Diagram the majorunit operation stll!pl and chemical (Onvenionl. Indude intarim stonge and tranIpoIt <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 or kg/balch 01'1 a 1(){)CJI. new chemical subatance basis), and entry poUlt of all starting 

materialt and feedstoclal (including reactants, salvents. and catalysts. etc.), and of an producta, rec:ycle aae.ms, and wuteL Include cleaning 

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

(3) Identify by number the poUlts of reIeue, including small or intenrdttant rei...to the environment of the new chemical substance. 

o Mark 00 this box if youlltt1lch • 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 SUBMIII hK ­ Continued 

2. Occupational Expomre ­ You must make separate confidentiality claims for the c1esc:riptiOl'l of worker activity, physical form of the new chemical 

substance, number of workertl expoeed. and duration of activity. Mark ()() the "Confidential" box next to any item you claim as confidential. 

(1) ­ Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling, deaning, etc.) in which workers may be expoaecl to the 

substance. 

(2) ­ Mark 00 this column if entry in column (1) is confidential business informatiOl'l (CDD. 

(3) ­ Describe an)l protective equipment and engineering controls used to protect workers. 

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

(5) - Indicate the physical formes) and % new chemical substance (if in mixture) of the new chemical substance at the time of exposure (ie., solid: 

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

(7) ­ Mark (X) this column if entry in" column (5) is confidential business information (CDD. 

(8) ­ Estimate the maximum number of workertl involved in each activity for an sites combined. 

(9) - Mark (X) this column if entry in column (8) is confidential business information (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 if entries in columns (10) and (11) are confidential business information (CDh. 

Worker activity CDI Protective Equipment! 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 

MlIrkW substance MlIrk(x) Hn/day Expoeed Days/yr 

(1) (2) (3) (4) (5) (6) (7) (8) (9) nO} (I1) (12) 

. . 

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

3. Environmental.ele... and DiIpoal- You mult make.epuate amf1dentialBy daimI far the rei_nUlllba- and. the amount of the new chemical 

substance released. and other release aad dispoeallnfc:nnatton. Mark 00 the "Confidential" box next to ach item you daim • confidential. 

(1) - Enter the ntmlber of each release point identlfied. in the proceI8 delcriptloD. part II" II!IdIoaD A.1U.beectian Id(3). 

(2) - Estimate the amount of the new substance rel.....:l (a) di.n!dly to the environment or (b)into amtnll tedmology (in kglday or kg/batch). 

(3) - Mark 00 this column if entries in col1.lDU1S (1) and (2) are confidential busineM lnfc:nnatlon (CBI). 

(4) - Identify the media (stack air, fugitive air (option.al-Iee InstrudIon Manual), I!IUriac:e water, an-site 01' off-site laud or mcineratian, POlW, or 

other (specify» to which the new substance will be released &om that rel_point. 

(5) - a. Desa::ibe control technology, if any, ,and contnll elfkiency that will be used tolimit the release of the new substance to the environment. 

For releases disposed of on land,. cbaracter.lze the dispoeal method and state whether it is approved for dispcaal of RCRA hazardous waste. 

On a continuation sheet , for each site describe any additlOl'lal dispoeal methods that will be UIIed and whether the waste is subject to 

secondary or tertiary on-site treatment. J'I. Estimate the amount reIeaaed to the environment after contnll technology (in kglday). 

(6) - Mark 00 this column if entries in columns (4) and (5) are confidential busineu information (CBI). 

(7) - Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant 0i.IIc:harge Eliminatkm System) numbers for direct 

dischargers or NPDES num~ of tbePOIW (Publidy Owned Treatment Works). Mark 00 If the POIW name or NPDES, is confidential 

business informatkm (CBn. 

Release Amount of-new subetance em r.;.~: CoIltnll ta::bDoJogy and efficiency (you may wiIb 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 POIW provide name(s) below: CBI 0:= o ~-Specify provide NPDES. CB( 

destination(s) of - releases to water. 

D Mark 00 this box ifyou attach a conttnuatlOl'llbeet.. 

FORM EPA 7710-2.5 <Rev. s...9S) Page 9­
1 Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued 

~ Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operationS involving the new chemical substance at sites you do not oontrol. Optional for typical 

manufacturing or proc:eui.ng operations involving the new chemiCal substance at sites outside the U.s. that you do not control...Inst:ructiOl'll 

Manual Complete a teparate section B for each type of processing, or use operation involving the new chemical substance. U the same operation is 

performed at more than one site describe the typical operation amunon to these sites. Identify ad.dltional sUes on • continuation sheet. Optional for 

sites outside the United States.- See Instructions Manual 

1. Operation OelCription - To claim information in this section as c:onfid.ential. drde or bracket the specific information that you daim • c:onfidentiaL 

(1) - Diagram the major unit operation steps and chemical conversions,. including interim storage and transport containers (spec:I..fy-S gaI10n pails, S5 

gallon drums, rail cars, tank trucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2) ­ Provide the identity, 

the approximate weight (by kg/day or kg/batch, 1()(W. new chemical 5Ubetanc:e basis), and entry point of all feedstocb (induding readants, 

solvents and catalysts, etc) and of all products, recycle streams, and wastes. lndude deaiU.ng c:hemicals (note frequency ifnot used daily or per 

batch). (3) ­ Identify by number the points of release, including small. or intermittent releases, to the environment of the new cbemic:al substance. 

Enter '1# of sites 

it of Sit.­

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 su bs tance 25% ) I C. I solvent 75% filtration 

~ 

ID. bottling 1(cleaning) 

. 0 Mark 00 this box if·you attach a continuatkm tbaat. 

2. WCKkll' Expotan/EIl'firaluDlatal aeteue 

(1) ­ From the diapm above, prcwkle the kttar fer each worker a::tlvtty. Oxnplelll2-8 fer each worker a::tlYity deIc:ribed. 

(2) - Estimate the number of worken expoeeclfer all ....a:mbinad. 

(4) - Estimate the typical duratl.on of expoaure per worker in (a) boun per day IIIICl (b) daY" per y ... 

(6) - Desaibe physical form of exposure IIIICl " new cbemical subetance (If in mixture), and. any p:otec:tlYe equipment and. engineering controls, if any 

used to protect worlcers.. 

m­ Estimate the percent of the newsubstance .. formulated when pec:kaged orUMd as a Bnalproduct. 

(9) -- From the process diagram above, enter the number of each reI.eue point. Ccmplete g..13 fer each reI.eaIe pointidentified. 

(10) -- Estimate the amount of the new substance releued. (a) directly to the envirclmneDt ell' (b) into control tedmology to the env:tnmment 

(in kg/day or kg/batch). . 

(12) -- Desc:ribe media of release (stack air, fusi.tive IIlr (optiona1-see InstructionsMaiuaO, surfa:e water, 0IHi.te ell' oif-site land CI' incineration, POIW, 

or other (spec:ify» and amtrol tec:hnology, if any, that will be used to Wtthe reI.eaIe of the new subIbmce to the enviroDment. 

(14) -- Identify byproc:lUdS which may result from the operation. . (3), (5), (8), (11), (13) and (15) • Mark (X) this column If any of the proceeding enl:det.... c:onf1deDtial busIneIa 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 Permand " ulatl.ml SubIItanc:a 

ivity new IIUbetance ReIeuecl 

I 

0) (2) B) (48) I (4b) (5) (6) (10.) : (lOb) (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 if you attach a oontinuation sheet. 

FORM EPA 7110-25 (Rev. 5-95) Pap 10 
OPll0NALPOLLunON PREVENTION INFORMATION 

To claim information In this section as confidential circle or bracket the specji6c information that you claim as confidential. 

I 

In this section you may provide information not reported ~lsewhere in this form regarding your 

efforts to reduce or minimize potential risks associated wi~ activities sUlTounding 

manufacturing, processing, use and disposal of the P:MN spbstance. Please include new 

infonnation pertinent~ pollution prevention, including lurce reduction, recycling activities 

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

inc1udesthe reduction in the amount or toxicity of chemiapl wastes by technological 

modification, process and procedure modification, produ~ refo~ula:tion, raw materials 

substitution, and/or inventory control' Recycling refers tq the reclamation of useful chemical 

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

discharges, or land disposal. Descriptions of pollution pre~ention, source reduction and 

recycling should emphasize potential risk reduction subsequent to comp~ance with existing 

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

this information to assess overall net reductions in toxictylor environmental releases and 

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

(e.g., occupational or consumer exposure). In addition, infPzmation on the relative cost or 

perfonnance characteristics of the P:M.N substance to poteIitial alternatives may be prOvided. 

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

of this substance. See the revised Insttuctions MaJlual th~t includes a Pollution Prevention 

manual for guidance and examples; 

Describe the expected net benefitS, sw:h as (1)-an overall reduction in risk to human health or the environment; (2) a reduction in 

the volume manula~; (3) a. nductlon in the generation of waste materialaltmoush recycling, source reduction or other 

m~Si (4) a reduction in potential toxicity or human exposure and/or enviro*,ental release; (5) an inaease.in product 

performance, a dec:rease in the c::ostof production _lorimproved operation ~dency ofthe new c::hemical substance in 

comparison to existing chemical.ubatances used in Similar appUeationai or (6)!the extent to which the new chemical substance 

maybe. substitute lor an existing sub8tance that po..a greater overallrisk to human health or the environment. 

, 

, 

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

lORN EPA?'Tl04.S CRaY.NS) Page 11 ; 
Part IlI- LIST OF ATTACHMENTS 

I 


Attach contlnuati"" : sh.... for: MCtiona at the/arm arid ·1tat data arid oth ..clata (lnd~8 phr-"c:al/chem1c.al properties U'ld 

structurelactlvttyiinfonnatlon)•.arld optional inIorJ:natlon aft.- tblI page. Qarly tify the attachment arid the MCtion of thelortn to 

which It re1at-.if1appropriate. Number consecutively the paS- of the attadunen In the colwnn below, em_ the inclusive page 

num" of each attachment. 

Mark GO the "~tial.. box next to any att.ac:Junent.name you claim .. con.ftden Rad the Instructions Manual for 


r=ciance on how to daim any information in arI attachment all CDnfidmtIaL You III 1It include with the sanitlzed copy of the notice 


orm a sanitized of Uly.ttachment in which yoU claim Wormalion .. ... tial. . 


I 


I 
 Attacluru.,t name i 
 A~ent Conti· I


, page number(s) dmtial 

Material Safety Data Sheet (MSDS) . 

i 


I 
 17-- 19 


Phy sic ~ c.-"t. "he-.i c ~ pro F;-.o ~,. rk S" f.,......t"" i 
 (3 

i
 IR ch~ of pM III I.4.b:s'TA>Mc.L i 

; 
 1!;-, X 
 I • 

'H--I\IMR s p-ecfn.t....... ot CDfl~ So\"t,,,,,,,, of PNN S'",~.s.tIit.,.C.(. 16 
 X 


Pr;~vy £y"ln";t41tiD''' st",J.y ;..., Alb;YlO R&J.bits wi~ P~ IV ... '" bA"~,e. .2..0 - 4-5" 

Ac.v.te E..)' e· I.rT,tjio'Yl/Cornsirm S"j,I.IJ), oj PMtJ S"'b~CA ;..... R.lII.bb.:ts 4h-bb 

A~e De!lAtt~ TDX,i<.ity stcJ.J.yoi PM tJ $:,...J,itA."'d,.. -- RtNts ,,, - ~b

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...tJl ~ D~phniA.. ,.,....~ ... til -- 128' i 


A~e FJow-Thro~f" To-i:i1y I)f pt--fN s"'Lrd.....u.. To ~Nthow TrocJ;'" li~...., 148


B"'- c:t"e rl J.. R-e.. "-eA.S'e H~qj:,'lPtt T oj- PH IJ s~/u·1A.;.. t..A.. 14Q-17o 

Co~ti~tA.~O"M S'h-vit" for ,..,~~ JiV)(po.s~ ".,.,..JJ-INI"i~"'~J.. r~o..se. 14­ X


. 

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, 

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, 0 Mark 00 tlda box if you attach a CDnlinuatlon sheet.. Entei tbeatt.durlent name ktnumber. 

i 


FORMEl'Amo.zs i 
 .5- Pa 11 I

, PHYSICAL AND OfEMICAL PROPERTIES WORKSHEET 

To assist .EIrA', review of physical and chemical properties datil, pi....compIet. the following worksheet for data you provide 

and 1nd.uc:Mt it in the notice. Identify the property meuured. the page of the::rt,ce on which the property appears, the value 

of the pro~' the unil:aiin which the property IImeaured (u ~). whether 01' not the property is claimed IS 

conllden Th. phy.lcal state of the neat IJUbItance .hould be provkIed. Th; meuured properttea should be for the neat (l()()f;, 

pure) chem1calsubstlll'lCll!. ProperUa that are measured lor mixtures or form tloJ'll should be.so noted. ('JI. PMN substance in _ ).You are not to submit thlII worksheet;h_•.EPA 'Irongly ncarrun..... that you do so, ..it wI.1l simplify review and enaure 

that c:onfidential information II properly protectld. You should submit this Worksheet U I supplement to your subm.lssion of telt 

data. This ~rksbeet is not I substitute far IUbmillion of tat 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) , 

; 

, Physical state of neatsubstance X le,) (1)_ (Jt) M , ' 

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

; 

: Density/relative-density g/c:m3 

;

, Solubility 2-0 " 

@ Temperature "C )<. 

t:i~ I u-o-o 1'1

Solvent gIL : 

- Soiubllityin water fiTemJ)er3.ture 2-0 "C X 1000a- IL "C IV1 

x... ! 

. 

t Melting temperature '15'" ·c M

.. 

BoU!ng/sublinultfontemperature .. torrpr.-ure 

Spectra LR X It" 1'-1 Xi 

Diaodation coJ'lltet ; 

: Partide size distribution 

Oc:tanol/water partition c:oeffident 

Henry's Law constant 

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! operatiOns involving the MW substance at sites you do not controLlmporters do not have to 

complete this MdlQn for oprrations oubllide the tis.; however, YOI.lIl'lUlt report ;my ~gor \1M activiti_ after import. S-the Instructions 

Manual. eom."lc,. II fUlTIllratc sa:ticm B(or au:Ja typI of1fI"'CIS$i1fg, orILW opmtitm iInIDl1lljor, 1M /VIP cMrriallncbstaJl«. It the same operation ill perfon:ned. 

at.Il'IOnt than" one site describe the typical operatiem coau:non to these sites. ldendfy -fditional sites em a amtinu.tion sheet. 

1. Operation Oacription - To claim tnionnation in thiI MCtIon u c:oniidentiaJ, drde Ol' /.mK:ket the spedfic infonnation that you claim as confidential. 

(1) - Diagram the Il'IIIjor ~on..and chemical C:XlnvlI!I'Iiona" including in~ stonge and tnlNport containers (specify- e.g. 5 gallon pails, 

SSgaIlon cI.n.ima, nLil c::an. 1Itc). On the dIIIg:ram. Identify by letter and bri yd-=ribe ..-h wade.-activity. (2) ­ Provide the identity, 

the approximate weight (by kg!dayorkglbatch. on an 100'J110 new chemical substan baIs), ..d entry point of aD. feedstocks (lnduding reactants, 

solvents aDd catalystl, etc:) aDd. 01 aU products, recyc1e streaa\s, and _lei. Include~gcheml.cala (note frequency 1f not UlIII!d dally or per 

batch). (3)-Id.eDttty by number thepolntl oll'l!!ieae, lndudlng mW1 or lntmNttent teIases. to the environment of the new c:heU'I1ca1. substlUlc:e. 

(4) Please enter the if 01 sites ( remember to idenllfy the locations of these sites 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 

So1 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 ofRaI.eue CBI 

of Worbn of B.npaeeringControlsl Fcxm­ ~ New &r:Omtrol 

Act· Sxpoeed ExpoIure . Physlcal term mel '" alation ! sUb.t..ce TedmaJ.osy

lvity new1Ubstu\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 I , ~, X­ X " .. :. .,. : ~ 1 . .­ ,,_ .. .....,I. I ~ 

I .v -.;. I L ____ -...... -­ ~ " F )( ! < I 1. X X .. )( 3 I ' r - • .... 1'­ . ­ >< ., 

I - ,

"'­

I ! I 

I , 1 

I ; I 

I .1 

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(l4) - Byproducts: (15) 

o MIIIk 00 this box ifyou attach • 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:.· 

PHONE: u

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] CO 2 [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 of its falling within the scope ofFIFRA § 10 (d) (1) (A), (B), or 

(C).. 

Company: 'I ....._,__.. __ ... ~ ___ .. .....w.

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 of these data in compliance with F1FRA does not 

constitute a waiver ofany right to confidentiality that may exist under any 

other statute ·or in any other country. 

.. 

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

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM 

No claim of confidentiality is made for any information contained in this study 

on the basis of its 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 of Nippon Paint Co. Ltd. and, as such, are 

considered cOnfidential for all purposes other than compliance with FlFRA 

Section 10. Submission of these data in compliance with FIFRA dOes not 

constitute a waiver of any right to confidentiality that may exist under any 

other statute or in any other country. 
• ReportlTMBP RIcerca, Inc. 6925-96-0172-TX"()o1 

COMPLIANCE STATEMENT 

The study reported herein. ''Primary Eye Initation Study in Albino Rabbits 

with TMBP:' Ricerca Document Number 692S-96-0172-TX..Q01, was 

conducted and reported in compliance with the United States Environmental 

. Protection Agency. Title 40 Code ofFederal Regulations Parts 160 (FIFRA) 

and 792 (l'SCA). Federal Register, August 17, 1989; Japan Ministry of 

Agriculture, Forestry and Fuheries. 59 NohSan, Notification No. 3850, 

Agricultural Production Bureau, August 10, 1984; and "'The OECO Principles 

ofGood Laboratory Practice," Environment Monograph No. 45, Organisation 

for Economic Co-operation and Development, Paris 1992. 

All responsibility for specific information regarding the physical. chemical 

. and stability properties of the test material is assumed by .. 

Steven K.. Shults, B.A., Research Toxicologist 

Study Director 

Toxicology andAnimal 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 

Study Director . 

Toxlt:ology and Animal Metabolism 

Ann W. Brock, M.s., Report Writer R:andAnImIII",,­

. 'wJJn~ . David M. Serrone, Ph.D., D..A.B.T., Manager 

Toxicology and Animal Metabolism 

2.3 

Rlcerca Inc. 

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 of the 

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' 

Quality ABSura ce Auditor 

PIIf1tI5 24 
ReportlTMBP 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 of 0.09 g of the material was administered to the 

conjunctival sac ofone eye of each of three 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 of the 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 prior to 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 at the I-boor iIiterval. No abnormal clinical signs were noted in the rabbit after 

the I-hour interval. No abnormal tissues were noted internally at necropsy in the two 

. rabbits which died during the study. 

Ocular examinations 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 of the test material, TMBP, in the one surviving New 

Zealand White rabbit resulted in corneal effects. (opacity) and iridal effects. Conjunctival 

redness, chemosis and discharge were observed by the I-hour interval in the rabbit. 

Minima) comeal 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 noted in the rabbit on Day 10 of the study. 

The maximum individual tota) score was 39 (out of a maximum mean tota) score of 110), 

observed in the animal at 48 hours post-treatment 

Page 6 
ReportlTMBP Ricerca Inc. 6925-96-0172-TX-oG1 

TABLE OF CONTENTS 

'ITIlE PAGE ....................................................................................................................... 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 OF TABLES ..•........................................................................................ 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 ~ODS ••.....•••••••.•.....••.........................•....•..•....••..••.••......•_...... 12 


Test Material. 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'ULTS AND DISCUSSION................................................................................. , ...... 15 


Anima1 Obse.f\1ations...................................................~ •.....•...••.••••..•••••••...•• ..... , ..•• IS 


Viability.•.................•...•.••............ .......... ................ ..... .....•......•............... .... 15 


Ocular Observations ••.•..••••............••...•..........•....•......•....••.••...•••..•••.-..•..t 15 


Necropsy............................................................................................ , •... ,.... 16 


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


27 

ReporVTMBP 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 Ocular Findings .............................. _ ................................................ 19 

Table 3: Results ofthe Examination ofTreated Eyes with Fluorescein Solution ............ 20 

Table 4: Summary ofIncidence of Positive Ocular Effects ............................................. 21 
Report/rMBP Ricerca Inc. 6925--96-0172-TX-oG1 

LIST OF APPENDICES 

"age 

Appendix A: Scale for the Weighted Scores for Grading the 

Severity of Ocular Lesions .......................................................................... 22 

Appendix B: List of Contributors ......................................... " ........ , ..................................... 25 

:..: 

Pagll0 
Rlcerca Inc. ReportITMBP 

6925-96-0172-TX-G01 . 

INTRODUCTION 

TITLE 

Primary Eye Irritation Study in Albino Rabbits with TMBP 

OBJECTIVE 

The objective of the study was to determine the primary eye irritation 

produced by a single application of the 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, 

Ricerca Document 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., Research Toxicologist 

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 in the Ricerca Archives. 

Page 11 30 
ReportITMBP Ricerca Inc. • 6925-96-0172-TX..Q01 

STUDY DESIGN 


Test Material 

Dose Level 

(g) 

NumberofAninaIs at Initiation 

Males Females 

TMBP 0.09 3­ - "Originol study design. required si% rabbits. Due to two deaths out ofthefirst 

three rabbits dosed with TMBP, the design. was chonge to three rabbits. 

TEST MATERIALS 

TMBP 

Common name: TMBP 


Lot number: 051796 


Chemical name: 


SOUJ.'CC of material: ... 

Date material received: July 19, 1996 

Storage conditions: Ambient (room temperature) 

Characterization 

All responsibility for specific information regarding the physical, chemical, 

purity and stability properties of the test material is assumed by the Sponsor. 

Disposilion 

After completion ofthe study, the remainder ofthe material, except for a 

retention sample, will be n:ttImcci to the sotm:e. 

MATERIALS AND METHODS 

TEST AlATERIAL PREPARA TlON 

For the purpose ofthis study. the test material was CODSiden:d. to be 100% 

TMBP and was used in the form. as received. The solid material was ground 

to a fiDe powder prior 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 

of the ground test material was administered into the lower conjunctival sac of 

the right eyeof each rabbit. The dose of 0.09 g was determined to be 

equivalent in weight to an estimated volume of0.1 mL. Immediately after 

each administration, the upper and lower eyelids were held together for 

approximately one second to minimize loss of the test material. The left eye 

was untreated and was utilized for control purposes. 

The treated eyes of the 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 of the 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 of rabbits received on July 29, 1996 from· Mohican 

Valley RabbitJ:y. Loudonville, Ohio. Upon receipt, these animals were housed 

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 or more fresh air changes per 

hour. During the acclimation and study periods, the temperature and relative 

humidity generally were within the range of 61 to 70 OF and 40 to 60%, 

respectively. Relative humidities in the range of61 to 63% were recorded on 

three days during the accUmation period and relative humidities in the range 

of 61 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 of the study. A 12-hour light/dark cycle was 

maintained during the acclimation and study periods. . 

The animals received PMI Feeds, 1nc••TM Lab Diet,TN the Richmond 

Stan~TN Laboratory Rabbit Diet IfF, #5326. The feed was available ad 

libitum during both the acclimation and study periods. Tap water (supplied by 

the City of Painesville) was available ad libitum through an automatic 

watering system during both the acclimation and study periods. 

I'Ige 13 31. 

:." ; ~ 
ReportlTMBP Ricerca, Inc. • 6925-96-0172-TX-oD1 

RANDOMIZATION AND ASSIGNMENT TO STUDY AND PREPARATION i·

,. 

Prior to the initiation of the study, a physical examination of the animals was j. 


conducted to determine the suitability of the animals for placement on study. 


All animaJs were considered to be healthy and acceptable for use on the study, 


. pending the determination of body weight and an ocular examination. 

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


use. Each of the animals was identified permanently with an ear tag, 


imprinted with a unique number. and by cage card. The body weight of each 


animal was recorded and a macroscopic examination of each of the eyes was 


conducted and recorded. This examination included the use of fluorescein 


sOlution and ultraviolet light. Body weights of the six rabbits ranged from 


2459 to 2691. Body weights of the three rabbits actually utilized on the study 


ranged from 2605 to 2691 grams. The ocular examinations of the rabbits 


demonstrated the absence of eye initation, ocular defects and pre-existing
 corneal injury. . ... 

To control bias, animal selection for this study was based on a computergenerated process of randomization of animals 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 10 of the study. 


An examination. utilizing an ultraviolet light source. of all 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. ifpresent. Grading and scoring of initation was performed 


in accordance with the Draize system (Appendix A). All observations were 


appropriately recorded. .Any ocular effects not included in the Draize system 


were reported, ifpresent. 


,.,.14 33 
I 

i Repo~BP RIcerca Inc. 6925-96-0172-YX..Q01 

I 

STATISTICAL ANALVSIS 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 of an euthanasic agent, Sleepaway.- A necropsy Jas not 

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 of the three rabbits died within one to five minutes following dose 

administration. Clinical signs observed in these two rabbits prior to 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-hour interval. 

Ocular Observations 

The individual corneal. iridal and conjunctival scores can be found in Table 1. 

An itemization of additional ocular findings can be found in Table 2. The 

results of the eye examinations using fluorescein solution can be found in 

Table 3. A summary of incidence 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-hour interval ofthe study. 

The results of the 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% of the cornea were noted in the rabbit at 48 hours. No 

corneal epithelial effects were observed in the rabbit after the 48-hour interval 

of the study. 

An epithelial lesion was observed in the rabbit at the 1 and 24-hour intervals 

of the 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 rabbit by 

the I-hour interval. Draize grades of 2 to 3 were noted from the I-hour 

interval through 48 hours and Draize grades of 1 to 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 in Table 

6. Conjunctival blistering was noted in the rabbit at 24 and 48 hours. 

Conjunctival blanching was observed in the rabbit at 24 hours through Day 4 • 

. No positive conjunctival fmdings were noted in any ofthe rabbits on Day 10 

of the study. . 

Yellow crusting around the edges of the eyelid was noted in the rabbit at 48 

hours. 

The maximum individual total score was 39, observed in the animal 8148 

hours post-treatment. 

Necropsy 

Findings noted at external 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 of one 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 5 minutes 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-h4lur interval 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 of the study. 

The maximum individual total score was 39 {out of a maximum mean tNaL 

score (If 110), observed in the animal at 48 hours post-treatment. 

10l8l96lces 

Page 1i 

Rlcerca Inc. ReportITMBP 

• 6925-96-0172-TX-G01 

Table I: Individlllll Scores and Individual WeighUd IIlUl Total Weighted Scores, 


Aninuzl Number: 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 

-

-

-

-

-

-

-

-

-

Iris 

Weigbted 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 Weighted Score 36 33 39 20 8 4 - - Calculations ofWeighted and Total Weighted Scores were determined as follows: 

~ Cornea Weighted Score =Density Score x Area Score x 5 

Iris Weighted Score =Iris Score x 5 

. Conjunctivae Weighted Score = (Redness Score + Chemosis Score + Discharge Score) x 2 

Total Weighted Score = Sum ofWeighted Scores 

"..18 

R1cerca, Inc. ReportITMB.P 

• 6925-96-0172--TX-G01 

Ttlble 2: Additiolllll Ocular Findings 

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 ofeyelid 

Page 19 
--

I 

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

-----_.- ReportlTMBP Ricercalnc • 6925-96-0172-TX-Q01 ._-,-""----------------------------­

Table 3~ Results ofthe Examination ofTreated 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 of up to approximately 5()% ~jihe corneal suiface 

~tainintr (~'UP to appro.Timo.tely 75% ofthe comeal sUiface 

:+; )/ainil'H' !ilJ to approtimately 100% ofthe corneal suiface 

---,.._-'------------------------­
- .......-­ Rlcer(~2L Inc. ReportlTMBP _._.__ '6925-96-0172-TX-001 .D.___­_______________________ 

Tau 4: Sum'llUll1 o/Incidence 01Positive OculllrEffects 

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 Protection Agency (USEPA), Pesticide Assessment 

Guidelir~'~s. Subdivision F. Hazard Evaluation: Human and DOIMstic Animals, Series 

81-4. 

For tbi:> .~Ul'NM.ry. in accordance with the guidelines. the following grades for each tissue 

a re c(.llw'~!J'ed positive. The positive' scores for conjunctival discharge 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 

Cc,.,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

"­

'Ire ater 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 AXBX5 I Total Possible Maximum =80 

."-.-"~-"----------------------------......, 

iris ,--."...~.----------------------------I 

ValUE..lS :. 

" Nom18l o 

Folds above normal, congestion. swelling. circum-comea1 injection (anyone 

J{' ,ll! of these). 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 Density area which Is most dense Is taken for 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 of pupil barely discernible 3 -

lque, iris invisible 4 .--... 

... reaction to light, hemorrhage, gross destruction (anyone or all ofthese) 2 

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


Score AX5 I Total Possible Maximum =10 "_._, ..,..,,.,-------------_----1_---------------_...... 


-.~'""'..".,...".~-----------------------

41 
Ricerca. Inc: ReportlTMBP 

• 6925-96-0172-TX..(JOt -----_._-----,,,,,,_._------------------­

--. 

Conlunctivae ..........~1'. 

Redness (refers to palpebral and bulbar conjunctivae only): --_.­

" \ies:;e!s NO!"Utai. _._._-"",,_.­ ,~ Vessds defiDitely iIlJected above nomtal ._-_.­

e, deeper crimson red. individual vessels not easily discernible ---,." ~, Di flilse beefyrl.'d 

Chemosis: 

_._. 

.'_M__...___ 

1; aboyc normal (includes nictitating membrane) ,_....__ . 

swrUing with partial evcrsionofthe lids ._-,,," 

• Sw with Hds about half closed --""" ~, Swelling wilb lids about half closed to completely closed 

.-_--,,_. 

Discharge: ----'._.. 

Fe 

.__.._".. _­

,A,rry amoun 

ob'served in 

T different from normal (docs not include small amounts 

inner <:;tntbus of normal animals) 

---,.­ .. [Ii scharge v..ritn me istening of the lids and hairs just adjacent to the lids

.--­

• Di scharge v..,riUl m()istening ofthe lids and hairs and considerable area around eye ---- Secre(A+B+C)x2 I Total Possible ,MaxImum =3) 

Score =Sum of all scores obtained for the cornea, iris, and conjunctivae

-­ L_~nimum!~ 

0 

1 

2 

3 

0 

1 

2 

3 

4 

0 

1 

2 

3 

I lL Draiz.e. 'Dl~nna! Toxicity," Appraisal ofthe Sa/ety ojChemicaLs in Foods. Drugs 

and Cosmetics, TIfe A"$OClam.m ofFood and Drug 0jJici0/.s ofthe 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 

tl925--96-0 t 72..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! ,dI 

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 

"'""........','~'..-......'..,.. "'... ""----..."'...... -"-......-"".....-,....'"..-..".......--.. ,............_..._---...--...,--,-,.,....,'.'--...._._.----­ Pagl26 
Statement of English 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 of my knowledge and belief, the study in this report was conducted in 

compliance with the following Good Laboratory Practice Standards. 

OECD Principles of Good 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 of the 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: .... M_ak........ i Ao...M.IJim~i____-.:selQlawlell:,)t.d Date : ,January 16. 1997 


Masashi Murakami sealed Date: January 16. 1997 

so 

4 
9. Study date: Initiation ofthe study November I, 1996 

Receipt of animals 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 of the study: 

None 

11. Retention :. The protocol, all data, documents, a sample of the 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 of the 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 of animals' • • • • . • • • • • • • • • • • • • • • • • • • • 9 


6 ~ Administration' . · .. · ........... · . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 


7. Observation, criteria for scoring and classification of eye irritation •••.•. 10 


8. Body weight measurement· ..................................... 11 


Results and. Conclusion 

15 Ocular reactions · . · · ........................ · . . . . . . . . . . . . . . . .. 12 


2. Classification of eye 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 of compounds 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 of TMBP were assessed in total of nine 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 of weighted 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 of this 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 of the 

test substance, group name, animal number. sex, the day of application, name ofstrain 

and species. 

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 of Japan, 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 of animals 

. Group Test article Number of animals Animal number 

1% l%TMBP 3 I -.. 3 

2% 2%TMBP 3 4 '" 6 

25% 

.. 

25%TMBP 3 7 -­ 9 

9 

6. Administration 

6.1 Dose level and frequency of administration 

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 of the test 

article. The treated eye remained unwashed for 24 hours following application. 

The left eye of each 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 of eye 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 (UVMM 57, Funakoshi Co., Ltd.). 

7.2 Criteria for scoring 

Opacity of the cornea, congestion and swelling of the iris, and redness and 

swelling of the conjunctivae were scored according to the grades of OECD guideline 

(No. 405) shown in Annex 1. Additionally, area of corneal opacity and discharge in 

the conjunctivae were scored according to Draize method I) shown in Annex 2. 

7.3 Classification of eye 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 1and discharge ofscore 2 were observed in all 

animals at 1 hour after application. These symptoms disappeared by 24 hours after 

application. 

2. Cla~sification of eye irritation (Table 2) 

The maximum of mean total score in the 25% group was 16.0 at 1 hour after 

application. The eye irritation potential of the 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 

of all 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 of this 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. 

13 

Annex 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 of normal lustre). 


details of iris clearly visible ------------------------------------------------------ 1 


Easily discernible translucent area, details of iris slightly obscured ----------------- 2 


Nacrous area, no details of iris visible. size of pupil 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 of these or combination of any thereof, 

iris still reacting to light (sluggish reaction is positive) . ---------------------------­

No reaction 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 half closed ---------------------------- ------------- 4 

·The area of corneal opacity should be noted 

I 4 Go 

Annex 2 Scale of weighted score for grading the severity of ocular lesions 

I. Cornea 

A. 	Opacity - degree of density (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 of pupil barely discernible . ---- 3 

Opaque, uns invisible ------------------------------------------------------ 4 

B. 	 Area of cornea 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 of these or combination of any thereof), iris still reacting to light 

(sluggish reaction is positive) -•• --.--------------.-.••• ---.--.----.--.------ I 

No reaction to light, hemorrhage; gross destruction (anyone or all of these) 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 of normal animals) --------------------------------- I 

Discharge with moistening of the lids and hairs just adjacent to the lids -------- 2 

Discharge with moistening of the 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 S 
Annex 3 Classification of compounds 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 5 points is 

at least 40 per cent 

Step 2 . 

Choose an initial or tentative rating on the basis of the 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 (M2) 

25 to 50 points Moderately irritating (M3) 

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%); if not 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 6 
Study 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 

/;,3 
Study No. 6L738 

Table 1-3 Individual scores for ocular reactions 

Grou2 : 25o/c0 

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/c0 

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 18 
Study 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 of English 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 of Mitsubishi 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:(fOnN,,-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 or audit 	 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 of the 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..luouoiil.l.i____--il:selldialwe~d Date : lanuaQ' 29. 1997 

.. 

'7/

4 
9. Study date: Initiation ofthe study October 18, 1996 

Receipt of animals 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 of the 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. 

6. 
 Observation and measurement • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• 10


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 ..••.••..••.....•••..•.••••.•••.••.•• 9 •• 11


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 of 2000 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 of female decreased on Day 4. No abnormalities 

were found in any animal in necropsy. 

Lethal dose lowest is greater. than 2000 mg/kg body 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 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 

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 

of both.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 ofthe test substance, 

animal number, the sex, the dose level, the day of administration, the name ofspecies 

and strain. 

3. Animal husbandry 

Throughout the study including quarantine and acclimation, the. animal room 

was maintained at a. temperature of 22±2°C, and at a relative humidity of 55±15%. 

Ventilation was about 12 changes per hour (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 of the 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 water were 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% of the 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 76 
either dose level. The dose level was therefore set at one dose of 2000 mg/kg in a 

single administration. Dose volume was calculated based on the body weight 

. measured just before administration. 

5. Treatment group 

The study was carried out using one group of 5 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 of administration 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 of all 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 of dosing 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 

--

-


--
.....


.. 


0111 

!II:: 

... 

.... ­ "".­

-.. 
 .... I:> 

...

...

•

•

fill 

..,"" 

.. 


c.:o •

.. 

.... 

.. 

III 

.... "'" 

0 

I:C 

u

,.. .... -:.

...... 

•'" CO

..

.. ..

.. 
 -< •

OS 

... 

.... .... ..,"" .. 


... ... • .. ........ 


... 


00 

.. 


•

"'" 

-=­

be .. .... ... ....... 


'" too Q ~ 

..


.. 


." 

.. 0> ..... oOc-.: .. ~ ... .... ... 

o GO 00 C/II

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

o Ii­ .... oDo ..:. ... ... I ... 

OS .. • ,.,j ",,.,j 

.

u .,,; .. .

.. .,,; .. 

0 0 '0 ... ... ... .... ~ 

.. -

.. ..• • ~2.

:.• ""' 
Appendices 

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


Appendix Body Weight .............................................................................. · ..·········....· .......... 2
 2 


Appendix 3 Accumulative Body Weight Gain ........................................................................3 

( r 

Appendix I eli a i cal S I in Sindy 110. ILUS 

Test Sn~stance : TIBP 

Sex Dose 

(1II/h) 

Kale 2000 

Fuale %000 

Positive· 

Ani ..1 

Xulliler 

10101 

10102 

10 I 03 

Ia1O~ 

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 SIud, Mo. IL U8 

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 

2 

Appendix 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 Castle roe 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 inspeCtionsl audits 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 #43360-3 

SIGNATURE 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 #43360-4 

TABLE 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 #43360-5 

TABLE 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 of Algal 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 ECso 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 of Botany, Culture Collection of Algae, 

University of Texas at Austin 

Age of Culture at Study Initiation: 3 days old 

ABC LABS #43360-8 
ABSTRACT 

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 ECso values 

following 24, 48, 72, and 96 hours of exposure 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 104 cells/mL for each flask. 

Water qUality parameters of temperature and pH were measured at 0 and 96 hours of the 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 

EClO, ECso, 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 of l\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 megohm oem water. 

The following nutrient amounts were target weights. The actual weights for each 

of the follo'Ying nutrients are included in Appendix II. 

ABC LABS #43360-10 
Macronutrient Stock Solutions 

NaN03 25.500 g 

NaHC03 15.000 g 

MgS04·7H20 14.700 g (each in 1000 mL ABC reagent water) MgCI2·6H20 12.164 g 


CaC12,2H20 4.410 g 


K2HP04 1.044 g 


Micronutrients Stock Solution 


MnCI2·4H20 415.4 mg 


H3B03 185,5 mg 


FeCI3,6H20 159.8 mg 


Na2Mo04,2H20 7.3 mg 
 (in 1000 mL ABC reagent water) ZnC12 3.3 mg 


CoCI2·6H20 1.4 mg 


CuCl2,2H20 12,0 p.g 


Na2EDTA·2H20 300.0 mg 


The pH of the 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 

of 4 L had been prepared prior to initiation of the 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-D16) 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 of the 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 

9'7 
constant 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 of cells per 

I mm2 was designated as Q. The center square was subdivided into 

twenty-five 0.20-mm squares. The average number of cells per 0.04 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 of 0.10, 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 #43360-12 
Following 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 of the control flasks 

resulted in an actual mean cell count of 8.5 x lW cells/mL. At 0 and 96 

hours, measurements of temperature and pH were taken in the control and 

all treated concentrations. O-hour temperah;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 (ANOV A) (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 of variance, respectively, 

over treatments at each hour. If the 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 ?9 
or 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 two parameters 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 

EC = concentration corresponding to a response halfway so 

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: 

EC = * «(A-lO)/(lO-D))**(1/B)) IO ECso 


EC = ECso * «(A-90)/(90-D»**(lIB») 
 90 

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 

/00 
Two measures of goodness 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 of variation 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 of the data points to the logistic model curve. It is 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 of 9.5 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 EClO, 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 EDT A 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 

of a 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 of TMBP 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, 

ECso, and EC90 values were estimated to be 170, 310, and 570 mg/L, respectively. 

PUBC ~S #43360-16 
TABLE 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 #43360-17 
TABLEn 

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 if­
TABLBm 

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 #43360-19 
TABLE IV 

EC Values, 95 % Confidence Limits, and No-Observed Effect Concentrations of Tl\t1BP so 

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 EC = 170 mg/L (95 % confidence limits = 130 and 200 mg/L) 
 IO 


EC = 570 mg/L (95 % confidence limits = 500 and 630 mg/L)
 90 

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 #43360-20 

TABLE 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 

....

Z 
FIGURE 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 


0 

1 0 /~ 0 
 ~~77~~-------------= ~ 

X ::;:::;r/?/ ~ 

---l 

E ~~-----­ "­ -

00() 

Q 

() 

(II

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. Log1o Concentration (mg/L) 


n 

~ O+-------------------~--------------------4

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 ofMessy 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-Ag DhisIon 


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 LAB ORA TORIES' 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 

113 
SIGNATURE 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 #43359-4 

TABLE 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 of Hard 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 of TMBP 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 of 6.3, 12.5,25,50, 100 and 200 mg/L. 

There were 2 replicates of each 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 ECso 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 

Ankistrodesmus Jalcatus 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-F22 on December 11, 1996. 

The adults from this culture had been cultured for 13 days and were considered 

acceptable with no signs of stress, disease, or physical damage. Since the culture 

and test parameters of temperature, 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 of 500 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/L chamber 

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 

/lD 
effects 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 of temperature, dissolved oxygen, and 

pH were performed in all replicates of the test concentrations and 

control at 0 hour before the addition of organisms and at 48 hours. 

IV. Statistics 

Statistical analysis of the 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 ECso 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 of data that may be obtained. The method of calculation 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 ECso 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 ECso 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 of the 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 of 59 and 96 mg/L. A 48-hour no-observed effect level was determined to be 

12.5 mg/L in this study. The slope of the 48-hour dose-response line was 6.0. All results were 

based on the nominal concentrations. 

ABC LABS #43359-12 

/22 
TABLE 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 of the chemical analyses supporting these data are on file at ABC Laboratories. 

ABC LABS #43359-13 

J23 
TABLE 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 UHc ~ 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 '5 

TABLE 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 ECso 

c ECso 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 of the 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 LCso. 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. Methods for 

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 

Working for 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. 

Draft Report: ~.~ 5-/A{q7

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. 43358-3 

SIGNATURE 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. 43358-4 

TABLE 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. 43358-5 

LIST OF TABLES 


I Chemical Characteristics of Hard 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 LCso 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 of TMBP 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 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, 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 

ohour 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 LCso 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 of mortality 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 of TMBP 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'~'. • I contracted the Environmental Toxicology division of ABC

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 "LCso'" as used in this report, is that concentration of TMBP 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 ,T -"' on May 31, 1996, and by the study director on June 19, 1996. This report presents 

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 sub lot of rainbow 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 introduction of TMBP 

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 of TMBP 

in 2 L of hard blended water. The injector volume was adjusted to deliver 20 mL 

of stock to 1.98 L of dilution 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. 43358-10 

IV. 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 of data that may be obtained. However, if no 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 LCso 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 LCso, 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 0.0 mglL (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 of TMBP 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 of mortality andlor fish on the bottom of the 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 !~~"... '.4 -::~_. • • -.:., with the [mal report. A copy of the report was 

retained at ABC Laboratories, Inc. 

ABC LABS NO. 43358-12 \4-0 
CONCLUSIONS 

The nominal test concentrations for this study were 63, 125, 250, 500 and 1000 mg/L. The 


96-hour LC for rainbow trout (Oncorhynchus mykiss) exposed to TMBP was shown to 
 50 

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 1 

TABLE 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 of January 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 14-3 
TABLE 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. 43358-16 

TABLE 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 LCso. 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. 43358-20 

Receipt 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 of the 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, Japan 
KOI-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 of Kanpogyo, No.299 of Yakuhatsu 

and No.85 of 59 Kikyoku, March 31, 1984; No.233 of Kankiken, NO.38 of Eisei and 

NO.823 of63 Kikyoku, November 18, 1988). 

Management: 	 Signed in original April 28, 1995 

Shigetaka Yamane, Ph.D. 

I ;- r 
K01-1583 


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 of Testing 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 of the 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 of main 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 ofHit a Research Laboratories for 10 years following the date of the notification 

specified under Item 1 of Article 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 protocoL 
KOl-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,SULTS 	 ..... "'. .................. ... ........... 8· 


CONCLUSION 	 ................ "' .. "' ................. "'. 9 


REFERENCES 	 '" "' •. '" '" '" '" '" '" ... "' .••••'" '" '" "'. '" '" '" '" '" '" '" "' .• "' •. '" '" 9 


APPENDIX TABLES AND FIGURES ....................................... 1..... 6 

KOI-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 of their 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 was judged 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 of solubility: 


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 of California, u.S.A. , on June 20, 1990. 

Escherichia coli WP2 uvrA obtained from Dr. T.Matsushima, Institute of Medical 

Science, University of Tokyo ,on June 13, 1987. 

S. typhimurium strains TAl 00, 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 

of broth culture) at -80'C (MDF-29l, SANYO Ultra-deep freezer). 

'" purity> 99.0%, Dojindo Laboratories 

2.3 	 Characterization of the 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 of R-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 of characterization 

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: _20oe 

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 of 0.2 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 prepared just before use. 

3) Stability oftest solution 

No denaturation of test 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 of each 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 of revertant colonies (size and number of colonies), deposition of the test 

substance and the growth inhibition were examined with a stereo microscope. 

7.2 	 Colony counting 

The numbersof colonies 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 of these 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 of the 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 of the negative control, and the dose-relationship and 

the reproducibility were obtained. Any statistical procedure was not adopted. 

RESULTS 

The numbers of their 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 positive controls showed the distinct increase of revertant colonies, and the positive 

and negative controls were within the range of the 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 of micro-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 of Health and Welfare (1992) Guideline for toxicity Testing of Chemicals, 

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 9 500 13) 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) 6 200 9) 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 5000 10) 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 89 Mix 555 ( 559) 131 ( 127) 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 

-1­
KOl-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(lli 20( 

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 1ate) 

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
Main KOl-1583 test 

O:TA 100 150 O:TA 1535 

.o.:WP2uP'rA 

Q.) 

..... 

-'"cc... lOD 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 a t 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

..... 

== 0 20 ....... 


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

..... 

0 == 20 ....... 


0 

<.J 

...., 


0:1==
 .... 

.... 

ClJ 

ClJ 

> 

c:: 10

O~----,----------.-----------.----------.---------~ 

313 625 1250 2500 5000 

(p.g/plate) 

Fig. 8 Dose-response curve with 89 Mix 

-6­