### u.s. ENVIRONMENTALPROTECTIONAGENCY

...."01:;"1\;1ANTmArTURE 

# f II/III 

## JIIII JIIIIIIII

# III~ 111111111IIli11111111111111111111111111111 10 000 008 4 I S .q:.::':

YOl) mustprovide all information reljHeSled in this form to the extenl that itis known to orreasonably ascertainable by you. Makereasonable estimates ifyou dD not have actual data. Before yo\.! complete this fom1, you shouldread the "(nstructionsManual for Premanufacture Notification" (the Instructions Manual is availabiefrom tbe Toxic SubstancesCuntrol Act (TSCA) Inlormation Service by calling202.554·1404, or taxing 202.554-5603). [fa user fee hasbeenremitted for this notice (40 CPR 700.45), indicate in the boxes above theTS-user fee identiticution number you have generated, Remember, your user foe ID n,,,,,bermust also .Pl'earon ,,,rrespor,ding fee remittal'ce, which is sentto EPA, HQ Accounting Operations Branch (PM·216), P.O. 360399M, I'ittsburgh, PA 15251·6399, Artn. TSCA User
Part I GENERAL li';fORMATION You IHust pro\<;de the currently correct**Chem~cal** AbstrJcts (CA)Nameofthe new chemical substance, e';l';n claim**~he** idemlt)' as confiuentiaL Yuu may aUlhorize anoLhr::rperson to**S~llm(t** identity ioformatio:l for you, but suumissionwill r:ot ht: complete tlnd the review wil! notbeginuntil EPA receives information. A letter in support ofyour submission should reference yourTS user fre identification ilLHnbtf You must**~ubmit** an origInal and lwo ofthis notice including all testdatu. Ifyou daim':':d any ;nformalinl1 as conlidentiaL copy I!1IiS( also he 5ubrHincd.
Part II HUM,\N EXPOSURE AND ENVIRm'McNT/\LRiOLEASE IfthereareSevera: nlanufilCture, 1)l'Qt:es.splg, oruse oper,:ulGIlS bedescr;bed in Part I L ,!,e..,::ions A and 6 orthis noti!..:e••eproduce the secrior." ilS needed,

Part**Ilt~-~LlSTOf** ATrACHMENTS**Atta~h** udwtional shc..:ts ifrlu::rt: is not space to al13,ver {J quesoon**Lah~1** <:!ach continuatiull sheet with the section heading, (n P<.i11 la, these attachments, any tC] dataorotht:rdatn nod any OptiOl1'11 informationlnclut.led in rile notl;,;e.

OPTIONAL tNFORMI':rION Yuu ma)' inCludeany inrorm:Hion that you want EPA to considerin evaluating [he n-:w substance, .on page II ofthis lorrn, spacehas beenprovided for you to descnbed pollution prevention and recycling inlhrmatiol1 yOIl may h::1ve re"gardJng thl! new**~;uhstnnce. Sp~a!!ed** **··blnGil1~(** boxesareincluded throughout this form for you LO indicate your \;,tiHingness to be buund tocertain st:Hemenls yOu Olakein thiS section, :illCh as use. production**voltllll~.** prolectilie equipnl!;:nt
.**Tl1i~** optIOn IS intended to ro..lllce delays that routinely accompany thedevelopmentorconsent urders orSigl1iricanr New Use**Rlll~,** ExC.:pl in the**c~se** ofexemption :}Ptilic3tiOils (suc,h.Js TMEA. LVE. LOREX) where ;,;erHUI'l IhiiJrmation providedIII suchnotification is b:ndingonthesuomi**tt~r** when theAgency appr.:,ve.s the exemption checking a bindingbox III this nOtil:e by i:se1fpmhibit Lhe submitter hlter je'¥iating from the itltorm.:ttion (t;x..:epl Idel1rity) reported**~n** the form.
CONFIDENTiALITY CLAIMS Yuu mi\Y claim any IIllbnnatiOfl in tillS notIce as(;\.Hitidentiai. To ussert**~** cbun CIl !b:: form, mark the;contiJenri,,! uo:.;: nt:xt to theintDrmal:ion**th~t** youcIai III as lIssert 0. claim in an attic!uno::nL circ!t: orhradccrthe inform,llilln you

Mark (x) ;fany infonnationin [his notice i:i ClilillHXI as coniidrotiaL TEST DATA ANDOTHER DATA You arerequired to submit all test data in your or control and to descnplionofall otherdata known to or ascertainablebyyou, data arerelated to the health and environmental effectson the m3nufacmre, processing, dlSlribution in eommen:e, use, or disposaJ ofthe new chemical
. substance. S2Ildard literature citationsmaybesubmitted fordata ih the open
idenllfy whethertt;st data on the substanceor onan compos:tion ofthe testedmmerl:::l1 should becharacterized, are.examples of**~est** data and otherdata. Data shoulrJ be si.lbmitted according to the requirements 

### 0"5720.50 ofthe PremanulaCltUeNotiticationRule (40 CFRPan720).

Test Data (Check Below a,lY included in this notice)**En'llronmenl~'l** nile data 0 Yes Otherdata 0 Ye:; Heal!:1 effectS data**~** Yes Risk iHs,<::i';ilillents Ell""':**(~~l\mentJ.l** etTecls dOH 0 Yes**StruC'tlln:la'~ti** ",iry reilltionships ehysic<,J/Cl,emical PropertIes· 0 Yes**Te~t** du:a nol in the pCJsse)sion orcontrol ofthesubmitter

.. Aphysical ;j:ld ch::':01lcal**prop~nH'_"** worksheet lS located 01 the IDS( pageofthis form. TYPEOF NOTICE (Ched. Onl} One) PMN {Premanut'octure Notice)
INTERMEDiATE PMN (sublOltted in sequenC'e '.'lith f!'la: lln,(jtlc( PMN) SNUN (Significant New Use Notice) 

### D TMEA (Test MarXeting E'(emption ApplicJtion} 

### D LVE (Low Volume Exemptioll) ..to CFR723,)O!
...:)( 1) LOREX {Low Release/Low Expllsure**r::~,empUotlj** (fi! 40 CFR 72150{c)(':) LVE Modification LOREX Motiittcatioll 

### D D
IS TtllS A CONSOLIDATED PMN'! Yes 

# o
# ofchemlcals (Prenotice**Comn~l.Inica ton**  11 requira:!-, e.r.ter # on page 3)
EPA FORM 7710·25 (Rev. 5·95) Replaces previous editionsofEPAForm 7710·25.

### ~70Public reportingburden for this collection ofinfonnation is estimated to average 110hoursperresponse, includingtime for reviewing instructions, searching existingdata sources, gathering andmaintainingthe dataneeded, andcompletingandreviewing thecollectionofinfonnation. Send comments regardingtheburden estimate oranyotheraspect ofthis collectionofinfonnation, includingsuggestionsforreducingthis burden,to Chief, lnfonnation Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W.,Washington, D.C. 20460; andto the Office ofManagementandBudget,PaperworkReduction Act (2070-0012),Washington, D.C. 20503. CERTIFICATION 1certifythatto the bestofmyknowledgeandbelief: 1. The companynamedin PartI, section A, subsection I aofthis notice form intends to manufacture or import for a commercialpurpose, otherthan in small quantities solely for research and development, the substance identified in Part 1, Section B. 2. All informationprovidedin this notice is complete and truthful asofthe date ofsubmission. 3. Iam submitting with this notice all testdata in mypossessionorcontrol and adescription ofall otherdataknown to or reasonablyascertainable byme as requiredby§720.50 ofthe PremanufactureNotificationRule.
AdditionalCertificationStatements: Ifyou are submitting aPMN,Intermediate PMN, ConsolidatedPMN, orSNUN, check the following userfee certification statementthatapplies: [2'J TheCompanynamed in Part 1, Section A has remitted the fee of$2500 specified in 40 CFR700.45(b), or 

# o TheCompanynamed in Part 1, Section A has remitted the fee of$1000 for an IntermediatePMN (defmed @ 40CFR 700.43) in accordance with40 CFR700.45(b), or TheCompanynamed in Part 1Section A is a smallbusiness concern under40CFR700.43 andhas remitted a fee of$100in accordance with40CFR700.45(b). Ifyou are submitting a low volumeexemption (LVE) application in accordance with40CFR723.50(c)(l) oraLow release and low exposureexemption (LoRex) application in accordance with40 CFR723.50(c)(2), check the following certification statements: Themanufacturersubmitting this notice intends to manufactureorimport the new chemical substance for commercial purposes, other than in small quantities soleIy for research anddevelopment, under the terms of40CFR723.50. 

# o Themanufacturer is familiar with the terms ofthis section and will comply with those terms; and 

# o Thenew chemical substanceforwhichthe notice is submittedmeets all applicable exemptionconditions.

• 

## I

I Ifthis application is for an LVE in accordancewith 40 CFR723 50(c)(l) the manufacturer intends to commence , manufacture ofthe exempted substance forcommercialpurposes within 1year ofthe date ofthe expiration ofthe 30 day review period.
Theaecuracy oftbe statemetlts you make in this noticeshould n:tlecr yourbest predictionQrthe <tnticipated facts regardingthe chemical substance describedherein, Any kltowiug und willful misinterpretatiun is subject to crimlnal pt'naltypursuantto J8 USC 100L Contidentiul Signature ::lIld title ofAuthorized 0 nicial (Original Si&,'11aturl;! Required) Date X Signatureof:Jgent**~** (if appticuble) Date

PartI

-- GENERALINFORlVIATION

EPA FORM 7710-25 (Rev. 5-95) Page 2PartI-- GENERAL INFORMATION SectionA ­ SUBMITTERIDENTIFICATION
la. Person Submitting Notice (in U.s.)

b. Agent (if applicable) Oty,State,ZIPCode
Nameofauthorizedofficial Company Mailingaddress(numberandstreet) Oty,State,ZIPCode Telephone

c. Ifyouaresubmittingthisnotice as partofajointsubmission, mark (X) thisbox.

-----------------...... Joint Submitter (ifapplicable)

2. Technical

Contact (inU.S.) Nameofauthorizedofficial Company Mailingaddress (number and street) City, State,ZIP Code

Name Company Mailingaddress (numberandstreet) Oty,State, ZIPCode
3. ifyouhavehada prenoticecommunication(PC) concerningthis notice andEPA assigneda PC Numberto thenotice, enterthe number. Ifyoupreviouslysubmittedanexemption application for thechemical substancecoveredby thisnotice,entertheexemption numberassigned by EPA. Ifyou previouslysubmittedaPMNfor this substanceenterthe PMN numberassignedbyEPA (i.e. withdrawnorincomplete).**--,~** IfyouhavesubmittedanoticeofBonafideintentto manufactureor importfor thechemicalsubstancecoveredby thisnotice, enter the noticenumberassignedbyEPA. ,..**T~** ofNotice

- Mark(X)

FORM EPA 1710-25 tRev.5-95) 1. 

# D D Manufacture Only BindingOption Mark (x) Pag,,3 Position

2. Mark(X) ;... 

## ID ifnone
Mark(X) )­ 

## ID ifnone Mark (X) )­ 

## fLJ if none 

# lU Import On.ly 3. 

# D Both 

# D BindingOption Mark(x)

### x based on

I name correct AbstraeIS**;;;;~w;;i;;th;CTTo:srC"A\!.1**n:;;v;;e:;n",to;;;r:;;y:JITi s;;tii;n;;;g;;;sTn;:-;;]i;;;]r,;;:;;;;J;:;i;;;;;;;;::;--T-----j

c. Please· ndfy which method you used to develop or obtain the specified chemical identity**infarmati';·~** reported in this notice: (check one).**~** 

### 0 Method 2 (Other Source)
d.

e. p.'ecw·sol' substances their range ofcomposition and the diagram, as comolete as can be COrTeet structure diaglum. For a class 2substance

- (I) List the immediate CAS RegistryNumbers. i2) Describethe nature ofthereaction orprocess. {JJ Indicate the composition (whereuppropriate). (4) PrOVIde a correctrepresentative orpartial chemical stl1lcture

ifone canbereasonalAvascertained. Section B

-- CHEMICAL

ForClass I substances a CA Index Name must beprovided. ForClass 2 substanceseither a CAIndexName orCAPreferred Name must beprovided,which ever is appropriate based on CA 9CI nomenclarure rules and conventIOns).

EPA FORM 7710-25 (Rev. 5-95) Page4

# Confidential
Page4aPart 1- GENERAL**li~FORMATlON** -- Continued Section B

-- CHEMICAL IDENTITY JNFORMATION

- Continued 2. Polymers(For adefinition ofpolymer, seethe InstructionsManuaL)
a. Indicatethe number-average weight ofthe lowestmolecularweightcomposition arthepolymer you intendto manufacture, Indicate maximumweightpercent oflow molecular weight species(not including residual monomers, reactants, or solvents) below500 and below 1,000 absolute molecular weightorthat composition, DescIibe the methods ofmeasurementorthebasis for yourestimates: GPC Other 

## D: (Specify) i) lowest number average molecularweight: Confidential

ii) maximum weight % below 500 molecularweight: iii) maximum weight % below 1000 molecultifweight: 

### D Mark (Xl thisbox ifyou attach acontinuation sheet. b, You must make separateconfidentialityclaims for monomer or otherreactantidentity, composition information, and residual information. Mark (X) the "Contidential"boxnext to any item you claim as confidential (I) Providethe specilicchemical name and CAS RegistryNumber (ifa number exists) ofeach monomeror otherreactant used in the manufaclUre of thepolymer, (2) Mark (X) this column ifentry in column (I)is confidential. (3) Indicatethe typical weight percent ofeach monomerorotherreactant in the polymer. (4) Mark (X) the identity column ifyou want a monomerorotherreactant used attwo weightpercentor less tobe listed as partofthe polymer description on the TSCA Chemical Substance Inventory, (5) Mark(Xl this columnifentriesin columns (3) and(4) areconfIdential. (6) Indicatethe maximum weight percent ofeach mOnOmerorotherreactantthat may bepresent as aresidual in the polymer as manufactured for commercial purposes.

% % Mark this column if is confidential.

d,

e, a correct or ify which method you used to develop or obtc.in the specifiedc11emic.1 idcntilY informationreported Method I (CASInventory Expe., Service - a copyofthe identification repo., 

### D Method 2 (othersource) obtained from CAS InventoryExpert Service must be submitted as the polymerthat is consistent with TSCA (check one).

Iyascertained,

Mark (X) this box ifyou attach acontinutation sheet.

EPA FORM 7710-25 (Rev, 5-95) PageSImpurities (a) Identify eachimpurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose. Provide the CAS Registry Number ifavailable. Ifthere are unidentified impurities, enter "unidentified." Estimate the maximum % ofeach
. Ifthere are unidentified estimate their total Impurity and CAS Registry Number

Mark (X) this boxifyou allach acontinuation sheet. 0/0
0/0
%

Confidential

Tradeidentification· List trade names for the new chemical substanceidentified in subsection 1or 2.

Mark (X) this boxifyou allach acontinuatic>fl sheet.

·5',9',15,19auach acontinuation sheet. Genericchemical name· Ifyou claimchemicalidentify as yQU must a name your substance that reveals thespecificchemical identity ofthe newchemicalsubstanceto the maximumextentpossible. Refer to the TSCAChemical Substance Inventory, 1985 Edition, Appendix B for guidanceon developinggenericnames. CarbazoleViolet sulfonamide derivs.

new Chemical substance. CAS Number Confi· dential

PartI -- GENERAL INFORMATION _. Continued EPA FORM7710-25 (Rev. 5-95) Page

## 6PartI - GENERALINFORMATION

-- Continued Section C

-- PRODUCTION, IMPORT,AND USE INFORMATION: Mark (X) the "Confidential"boxnexttoany item you claim as confidentiaL 1. Production volume

-- Estimate the maximumproduction volumeduringthe flfst 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-monthperiod during the flfSt three years ofproduction. Estimates shouldbeon 100% new chemical substancebasis. For a Low Volume Exemption application, ifyou choose to have yournoticereviewed at a lowerproduction volumethan 10,000 kg/yr, specify the volume andmark (x) in thebindingbox. Ifgranted, you areboundto this volume Maximum first 12-monthproduction (kg/yr) I Maximum 12-monthproduction (kg/yr) (100% new chemical substancebasis) (100% new chemicalsubstancebasis) Confidential Binding Option Mark(x) X 2. Use Information- You mustmake separateconfidentialityclaims for thedescription ofthe categoryofuse, thepercent ofproduction volume devoted to each category, the fonnulation of thenew substance, and otheruse infonnation. Mark (X)the "Confidential" Box next to anyitem you claim as confidential. a. (I)

-­ Describe each intendedcategoryofuse ofthe new chemical substanceby function and application
.. (2)

-­ Mark (X) this colulnn ifentrycolumn (I)is contldential business information (CBI). (3)

-­ Indicateyour willingness to have the infonnationprovided in column (1)binding. (4)

-­ Estimate the percent oftotal production for the first three years devoted to each category ofuse. (5) -­ Mark (X)this column ifentryin column (4) is confidentialbusiness information (CBI). (6) -­ Estimate the percentofthe new substance as fonnulated in mixtures, suspensions,emulsions, solutions, orgels as manufacturedfor commercialpurposes at sites under your control associated with each category ofuse. (7)

-- Mark (X)this column ifentryin column (6) is confidentialbusiness infonnation (CBO. (8)

-- lndicate%ofproductvolumeexpected for the Iisted"use"sectors. Mark more than onebox ifappropriate. Mark (X)to indicate your WI1IlllgnesS to h h avet euse typepraVI e m m d d (8) b dmg. (9) -­ Mark (X)this column ifentry(ies) in column (8) is (are) confidential business ll1fonnation (CBI). Categoryoruse(I) CBII Bmoiug Produc- CBr %jo CBI %ofsubstanceexpected peruse CBI Ophon tion 0/0 Form­ (8) (by function and i.e. adlspersive dye Mark (.:1 illation Site- Con-· Indus- Com- Binding for finishing pclyester fibers) (2) 01 (4) (5) (6) (7) limited sumer trial mercial Option (9) X %. X % X X X 

### I % % % % 

## L 

# .' %

I % "/' - I I % % % 

# . Ifyou have identined a"consumer" use, pleaseprovide on acontinuation sheet a detaileddescription ofthe use(s) ofthis chemical substance inconsumerproducts,

In additionincludeestimates ortile concentration oftbe new chemical substance as expected in consumerproducts and describe the cherr,ical reactions by whichthis substancelosesits identityinthe consumerproduce DMark(Xlthis box ifyou attach acontinuation sheet. b. Generic Ifyou claim any category ofuse desctir.tion in subsection 2a ascontident;al. enter agenetic description ofthat category. Read the use Instructions Manual for examplesorgeneric use desoriptions, description Dispersion additive forprinting ink

D Mark (Xl this boxifyou attach acontinuation sheet. 3. Hazard Information Include inthe notice acopy ofreasonable facsimile ofany Ilazard warn;, statement. label. matelial safety data sheet. orother 8lOumg**lnrurm~lllvll** which -.vlii b-: pr;JV1J-.:J P":L:iUU v,hv l:i ica.sulIJbl, tikdy tv b..: tv LlIl:::.**plljl.\:",:tlv~** cttU1pmcllt
..11-**pr~h.:t;ct.'~**

for the safe handing, transport, use, ordisposal oflhe new substance. List inpan hazard ,"fonnalion you 

### LZJ Mark (Xlthis box .fyou attach hazardinformation.

- EPAFORM 7710-2) (Rev. 5-95) Page1PartII-- HUMAN EXPOSUREAND ENVIRONMENTAL RELEASE Section A -- INDUSTRIALSITES CONTROLLEDBY THESUBMITTER Mark claim for each type ofmanufacture, processing, or use operation involving thenew chemical substanceat industrialsites you do not have to complete this section for operationsoutsidethe U.S.; however, you maystill havereportingrequirements if after' describe these Seeinstructions manual Confiwill occur. dential 

### x
Site address and**~lreer)** City. Countv S'"'e ZIP code [ thesameoperation wi!! occurat more than onesite, enter the numberofsites. Identify the additional sites on a continuation sheet, and ifany ofthesites haye signiticantly different productionrates oroperations, include all theinformation requested in thl' section for those sites as attachments. 

### x Use Batches/year 

### x

(I) Diagram the majorunit operationstepsand chemical conversions. Includeinterim storage and transport containers(specify- e.g. 5 galionpails, 55 galion urLun, rail car, tank truck. etc.). (2) Provide the identity, the appro)(imate weight (by kg/dayorkglbatch on a 100% new chemical substancebasis), and entry point ofall starting materials and feedstocks (including reactants. solvents,catalysts. etc,), and ofall products, recyclestreams, and wastes. Include cleaning chemicals(note frequency ifnot used daily orperbatch,). (3) Identify by number thepoints ofrelease. including small or intermiUent releases, to theenvironment ofthe new chemical substance. I. Batch Maximum {IUO% newcf<lfliclil surn.r.u.:c) 2. Continuous d. Processdescription Mark (X) to indicateyour willingness to hove your processdescription bindmg.**~** Mark (Xl this boxifyou attach acontinuation sheet. EPA FORM 7710-25 (Rev.5-Q5) PageS

# Confidential
Page 8aPart 11- HUMAN EXPOSURE AND ENVIRONMENTALRELEASE

-- Continued Section A

-- INDUSTRIAL SITESCONTROLLEDBY THESUBMITTER

- Continued 2, Occupational Exposure

-- You must make separatecontidentiality claims for the description ofworker activity, physical form ofthe new chemical substance, number ofworks exposed, and duration ofactivity. Mark (X) the "Confidential" box next to any item you claim as confidential. (I)

-- Describe the activities (i.e. bag dwnping, tote filling, unloading drums, sampling, cleaning, etc.) in which workers may be exposed to the substance. (2)

-- Mark (X) this column ifenn-y in column (I) is contidential business information (CBI). (3)

-- Describe any protective equipment and engineering conn-ols used to protectworkers. (4) and (6)

-- Indicateyour willingness to have the informationprovided in column (3) or (5) binding. (5)

-- Indicate thephysical formes) ofthe new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance(ifpart ofa mixture) at the time ofexposure. (7)

--Mark (X) this column ifentry in column (5) is confidential business information (CBI). (8)

-- Estimatethe maximum number ofworkers involved in each activity for all sites combined. (9)

--Mark (X) this column ifenn-y in column (8) is confidential business information (CBI). (!O) and (II)

-- Estimate the maximum duration ofthe activity for anyworkerin hours per day and days per year. (12)

-- Mark(X) this colwnn ifentries in columns (10) and (II) are confidential business information(CBl). Worker activity CBI ProtectiveEquipment/**Bindin~** Physical roons(s) Bindrng CBI # of CBI Ma};'imu duration CBI m (i.e., bag dumping, tilling drums) Engineering Controls Opoun and % new Opllun Worker Hrslday Days'yr MiUk(lt) substance Mnrk(x) S (I) (2) (3) (4) (5) (6) (7) Expose (9) (10) (II) (12) d (8)

-­ X X X X
X ,­ X X X

- --: 

# r---x X X X

_.. -­ X X X X 

## D Mark (X) this box ifyou attach a continuation sheet. 3, Environmental Release and Disposal

-- You must make separateconfidentiality claims for the release numberand the amount ofthe new chemical substance released and other release and disposal information. Mark (X) the -'Contidential"box next to each item you claim as confidential. (I)

-- Enterthe nUlOber ofeach release pointidentified in the process description, part II, section A, subsection Id(J). (2)

-- Estimate the amount ofthe new substancereleased (a) directly to the environmem or (b) into control technology (in kg/dayorkg/batch). (3)

--Mark (X) this columnifenn-ies in colWllns (I) and (2) areconfidential business information (CBI). (4) --Identitythe media (stack air, fugitive air (optional-see Instl1lction Manual), surface water, on-site or off-site land or incineration, POTW, or other(specity)) to which the new substancewill be released fi'om that release point. (5) --a. Descl;be conn-ol technology, ifany, and control efficiency that will be used to limit the release ofthe new substance to theenvironment. For releases disposed ofon land, characterizethe disposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuationsheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary Or tertiary on-sitetreatment. b. Estimate the amount released to the environment aftercontrol technology (In kg/day). (6)

-- Mark (X) thiscolumn ifentries in columns (4) and (5) are contidential business information (CBI). (7) --Identitythe destination(s) ofreleases to water. Please supply NPDES (National PollutantDischarge Elimination System) numbers for direct discharges or NPDES numbers ofthe POTW (Publicly OwnedTreatment Works). Mark(X) ifthe POTW name orNPDES # is confidential bl.siness information(CBI). Relea Amount ofnew substance CBI Mediaof Control technology and efficiency (you may wish to optionally attach efficiencydata) CBI

se released rdease Numb

er (I) (2a) (2b) cg**~~CkilH B~nding**  (3) (4) (5a) Mark (X) (5b) (6)

- I-­ X X
r-­ - - - I-­ X X
I-­ X X

- X X (7) Mork (X) the 

## D POTW provide name(s) below: 

# n D Navigable 

## D Other

- Specity provideNPDES # CBY destination(s) of waterway**rcle:ls~s** tn**\V~ttT** I 

## D Mark (X) this boxifyou attach a continuation sheet.

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

### 9Part11- HUMANEXPOSUREAND ENVIRONMENTALRELEASE

-- Continued SectionB

-- INDUSTRIALSITES CONTROLLEDBY OTHERS CompletesectionB for typical processing oruse operationsInvolving the new chemical substance at sites youdo'not control. Imp0l1ersdo not have to complete this section for operations outside the U,S,; however,you must reportanyprocessing or use activines afterimport. Seethe InstructionsManual. Complete a sepamte sectiollBJoreach type oj processing, orlise operatioll involvingthenewchemicalsubstance. Ifthe sameoperationisperformed at more thanone sitedescribe the typical operation common to thesesites, Identifyadditional siteson a continuationsheet. 1. Operation Description To claim information in this section as confidential, circle orbracket the specific information thatyou claim as confidential. (1)

-- Diagram the majorunit operation steps and chemical conversions, including interim storage andtransportcontainers (specify

- e.g. 5 gallonpails, 55 gallon drums, rail cars, tank u'ucks, etc). On the diagram, identifybyletter and brieflydescribeeachworker activity. (2)

-- Providethe identity, the approximateweight (bykg/day orkg/batch, on an 100% new chemical sub,tancebasis), andentrypointofall feedstocks (induding reactants, solvents and catalysts, etc) and all products, recyclestreams, and wastes, Inelude cleaning chemicals (note frequency ifnot used daily or perbatch). (3)

-- Identifybynumber thepointsofrelease, induding small orintermittent releases, to the environment ofthe new chemical substance. (4) Please enter the # ofsites (remembertoidentifythe locarionsof these sites on acontinuationsheet): # ofsites See attached sheet (page 

## }Dq)

Mark (X) this boxifyou attach a continuation sheet. 2, WorkerExposurefEnvironment.1 Release (I) --From the diagram above,provide the letter for each worker activity, Complete2-8 for each worker activitydescribed. (2)

--Estimatethe number ofworkers exposed for all sites combined. (4)

-- Estimatethe duration ofe"posureperworkerin (a) hoursperdayand (b) days peryear. (6) --Descnbe form ofexposure and% new chemical substance(ifin mixture), and anyprotective "'luipmenr andengineering controls. ifany. used to protect (7) -- Estimatethe percentofthe new substanceas formulated whenpackagedOr used as a final product. (9)

--From theprocess diagram above,enterthenumberofeach release pomt. Complete9-13 for each release pointidentifled. (10)

-­ Estimate the amount ofthe new substance released (a) directly to the environment or (b) intocontrol technologytothe environment(in kg/dayorkg/bmch). (12)

-- Describe media ofrelease i.e. stack air, fugitive air InstructionsManual), surfacewater, on-siteOr off-siteland orincineratIOn, POTW, orother (specify) and control technOlogy, ifany, thatwill be iO limit the release ofthe new substanceto theenvironment. (14)

-­ Identifybyproductswhich mayresult from the operation. (3), (5), (8), (II), (13) and (15)

-- Mark (X) this column ifanyofthe proceeding entries are confidentialbusiness information (CBI). Letter #of CBI ! Duratioll CBI Protect;veEquip. I %in! CBI Release Amoumof CBI MOOia0rRelease CBI

of Workers of EngineeringControls!**For~n-** i !Number New & Comrol Act- Exposed Exposure Physical Form and % ulatlorl: Substance Technology I

### i~~Y new substance ! Released (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (lOa) (IOb)_ (II) (12) (13) I XI X , X 

## 71 T I X X I X -f­ - f­ 

# -h­ , " 

# r-x

I
I X X X X ,.'
I , - 

### - r-­ -­ X X X X X I I 

### - -,
(14)

-- Byproducts: (15)**~'Mark** (X) this box ifyou attach acontinuation sheet, EPA FORM 7710-25 (Rev 5-95) Page 10

# Confidential
Page lOa

### OPTIONALPOLLUTIONPREVENTIONINFORMATION To claim infonnation in this section as confidential circleorbracketthe specific information that you claim as confidential. In this section you mayprovideinformation notreported elsewhere in this form regardingyour efforts to reduce orminimizepotentialrisks associated with activities surroundingmanufacturing,processing, use and disposal ofthe PMN substance. Please include newinformationpertinent to pollutionprevention, including source reduction, recyclingactivities and saferprocessesorproducts available due to the newchemical substance. Source reduction includes the reduction in the amountortoxicityofchemical wastesbytechnological modification,process and procedure modification, productreformulation, raw materials substitution, and/or inventorycontrol. Recyclingrefers to the reclamation ofuseful chemicalcomponents from wastes thatwould otherwise be treated orreleased asairemissionsorwaterdischarges, orland disposal. Descriptionsofpollution prevention, sourcereduction and recycling should emphasizepotentialriskreduction subsequentto compliance with existingregulatoryrequirements and canbe eitherquantitative orqualitative. The EPA is interested in the information to assess overall netreductions in toxicityorenvironmental releases and exposures,not the shifting ofrisks to otherenvironmental mediaornon-environmental areas (e.g., occupationalorconsumerexposure). In addition, information on the relative costorperfonnancecharacteristicsofthe PMN substance to potential alternativesmaybeprovided. All informationprovidedinthis section will be takeninto considerationduring the reviewoUhis substance. See InstructionsManual and PollutionPreventionGuidancemanualfor guidance and examples. Describethe expectednetbenefits, such

## as (I) an overallreduction in risk to human healthorthe environment; (2) areduction in the volumemanufactured; (3) areduction in the generation ofwaste materials through recycling, sourcereduction orothermeans;(4) a reduction in potential toxicityorhuman exposure and/orenvironmental release; (5) an increase in productperformance, a decrease in the costofproductionand/or improved operationefficiency ofthe new chemical substance in comparison to existing chemical substances used in similarapplication; or(6) the extentto which the new chemical substancemaybe a substitute for an existing substance thatposes a greateroverall risk to human health ortheenvironment. ./­ 

### -_
....

## _--_ ..

# ­
\



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

EPA FORM 7710-25 (Rev 5-95) Page 

## IIi

i - 

## I 

# r
1 Part III -- LIST OF AITACHMENTS 

### I I Attach continuation sheetsfor sectionsofthe form and test dataandotherdata(includingphysical/chemicalpropertiesand structure/activityinfoIII1ation), and optionalinformation after this page. Gearlyidentify theattachmentandthesectionofthe form to which itrelates, ifappropriate. Numberconsecutively the pagesofthe attachments. in thecolumnbelow,entertheinclusivepage numbersofeach attachment. Mark (Xl the "Confidential"boxnext toanyattachment name youclaimascon.fidentlal. Read the InstructionsManualfor guidanceonhowto claim anyinfoIII1ationin an attachment as confidential. Youmustincludewith the sanitized copy ofthenotice form a sanitizedversionofanyattachmentin which youclaim information as confidential. I Attachment name i Material Safety;Data Sheet (MSDS)**~~** I.-

..

I 

### I
Test Reports ACllte oral toxicityin the rat rabbit itation Magnusson & kligman maximisation study in the guinea pig Reverse mutation assay"ANIES lest" using salmonella typhimurium and escllerichiacoli Assessmentofready biodegradation;C02 evolution test



## D Mark (X) thisbox ifyouattadl acontinuationsheet. Enter the attachmentnameand nUtll1ber. FORM EPA 7710·25 (Rev. S.qSl Page i'2 Attachment Co tipagenumber(s) de!,"-.1 14-17 18-19 20 

### ,
I 21 

# 1 22 X 23-24

25 X 26 X 27 X 28 

# ! xi 29 29a xl 1 I 30-45 46-60 61-79 80-110 I ; I I 111-147 "1
148-166 

## I I : 

## i I I 

## I I

iPHYSICALAND CHEMICAL PROPERTIES WORKSHEET To**as.~ist** EPA's review ofphysical and chemical propertiesdata, please completethe following worksheet for data you provide and include it in the notice. Identifythe property measured, thepage ofthe noticeonwhich the property appears, the value ofthe property, theunits in Which thepropertyis measured (as necessary), and whether or not thepropertyis claimed as confidential. Thephysical state ofthe neat subs.anceshouldbeprovided. These measuredpropeItie:; shouldbe for the neat (100% pure) chemical substance. Propelties that are measured for mixtures or fonnulations should be so noted (% PMN substance in You are not required to submitthis worksheet; however, EPA strongly recommends that you do SQ, as it will simplify review and ensure that confidential informationisproperlyprotected. You should submit thisworksheet as a supplement to yoursubmission oftest data. Thisworksheet is not a substitute for submission oftest data.

I Prope,1y Mark (Xl if (a) provided Physical state ofneat substance Vaporpressure iaJ Temperature °C Densitylrelativedensity Solubility @ Temperature "C

Solvent Solubility in water @ Temperature 'C Melting temperature Boiling I sublimationtemperature@ pressure Spectra Dissociation constant Particle size distribution Octanollwater**~artition** coefficient Henry's Law constant Volatilization from water Volatilization from soil pH@ concentration Flammability FlashPoint Explodability Adsorption I coefficient Other

- Specify Page Value number

(b) (el

_ (s) (I) (g)
Torr

giL

-

- Measured 0 Confi-,I Estimate dcntiol Mar.(X) (MorE) (0)

X

X

X



### - X

- X

X

X

! I

ATTACHMENT PAGE**'-~**

## Material SafetyDataSheet

### Page 18 of 3 Date of Issue Nov, 6, 2009 

### MATERIAL SAFETY DATA SHEET 1. PRODUCT AND COMPANY IDENTIFICATION PRODUCT NAME :1 

### .....
______....1 

### SUPPLIER'S COMPANY ADDRESS TELEPHONE NO. .1 

### FAX NO. EMERGENCY PHONE NO. 

# [ 2. COMPOSITION/INFORMATION ON INGREDIENTS INGREDIENTS CAS NO**~---,-,---:---:---~~------,** 

### 3. HAZARDS IDENTIFICATION Classification: Not classified as hazardous preparation. Routes of entry: Inhalation. Ingestion and Skin. Harm to health: Contact with eyes may result in irritation. Contact with skin may resul t 

### in irritation. Ingestion may result in gastric disturbance. Harm to ecological: Biodegradation: extrapolate from similar compounds. cannot be considered to be readily biodegradable, 4. FIRST-AID MEASURES EYE CONTACT Immediately flush eyes with copious amounts of water. If irritation develops, seek medical attention. SKIN CONTACT Wash affected areas with soap and**wate~** 

### If irritation develops, seek medical attention. Remove and launder contaminated clothing before reuse. INHALATION Move to fresh**ai~** 

### Aid in breathing if necessary and seek medical attention. INGESTION If 

### swallowed, dilute with water and immediately induce vomiting. Never give fluids or induce vomiting if the victim is unconscious or having convulsion. Seek medical attention. FURTHER MEDICAL TREATMENT : Unknown 5. FIRE-FIGHTING MEASURES FLASH POINT : Not applicable FLAMMABLE LIMITS: Not applicable SUITABLE EXTINGUISHING MEDIA Water spray, Carbon dioxide, Foam, Dry powder extinguistier. 

### HAZARDS DECOMPOSITION PRODUCTS CO. CO". eIc. 

### FIRE FIGHTING PROTECTIVE EQUIPMENT Aself contained breathing apparatus and suitable protective clothing must be worn in fire conditions. 6. ACCIDENTAL RELEASE MEASURES Collect spilled material in the most convenient and safe manner and deposit in sealed 15

# [ Page 19 0f 3 Date of Issue Nov, 6, 2009 container for reclaiming or proper disposal, Avoid inhal ing dust. 7, HANDLING AND STORAGE HANDLING Avoid contact wi th skin and eyes. Avoid inhal ing dust. STORAGE : Keep container tightly closed and keep away from moisture. 8. EXPOSURE CONTROLS/PERSONAL PROTECTION ENGINEERING CONTROLS Adequate ventilation should be provided. RESPIRATORY PROTECTION NIOSH approved respirator. HAND PROTECTION Use latex or neoprene gloves. EYE PROTECTION Use safety glasses. SKIN PROTECTION : As required to avoid skin**ront~rt** ACGIH TLV (TWA) : ..

### 1 

# J OSHA PEL (TWA) '-

________. _____________ ----J 

### 9. PHYSICAL AND CHEMICAL PROPERTIES,--

__________--. FOR\1 COLOR ODOR pH

.

### , RELATIVE DENSITY SOLUBILITY IN WATER DECOMPOSITION TEMPERATURE 10. STABILITY AND REACTIVITY HAZARDOUS REACTIONS No data HAZARDOUS DECOMPOSITION PRODUCTS No data II. TOXICOLOGICAL INFORMATION EYE CONTACT Extrapolate from similar compounds, mild to moderate irritant (Class on a 1 to 8 scalel to the rabbi t eye. No symbol and risk phrase. SKIN CONTACT Extrapolate from similar compounds, mild irritant to rabbit skin. No symbol and risk phrase. Non-sensitiser to guinea pjg skin. No risk phrase. INHALATION ; Dust may be irritant to the upper respiratory tract. INGESTION ; UnlikelY to be hazardous if swallowed. Carbazole violet sui ronamide derivs. Extrapolate from similar compounds, Oral LDso (rat) greater than 2000mg/Kg MUTAGENICITY "AMES TEST" ; Extrapolate from similar compounds, non-mutagenic Carcinogenicity; No data 12.ECOLOGICAL INFORMATION ENVIRONMENTAL FATE AND DISTRIBUTION ; The product is essentially insoluble in water. TOXICITY ; linknown B

IODEGRADATION ; Extrapolate from simi lar compounds, 0% degradation after 28 days, Cannot be considered as readily hi adahle. 13.DISPOSAL CONSIDERATIONS )kJ Dispose of in accordance wi th local. state or nat lonal legislation, 14. TRANSPORT INFORMATION Not classified as nazardous for transport.

# [ J Page 20 of 3 Date of Issue Nov, 6, 2009 15. REGULATORY INFORMATION This product does not contain following toxic chemicals subject to the reporting requirements of Section 313 of the Emergency Planning and Community Right-to-know Act of 

### 1986 and 

### 40 CFR 

### 372. Follow all regulations in your country. 

### 16. OTHER INFORMATION

# ~~~~~i~TrRER'S COMPANY
.. :.:.; TELEPHON No. FAX NO.**~------------------------------------~** This MSDS is created based on the data which can be procured at present informal ion and data. It may be revised by revision and the new knowledge of a statute. When you handle this product. please refer to written contents and take the safety measures based on substance in a user's responsibility. This MSDS is not the guarantee and standard of safety and Quality.



## 17

# Confidential 

# Pages 18--29**r----~** 

## TestReports

# -L -

# -~

# l J 

# n n 

# n n '. I 

# n ACUTE ORAL TOXICITY (LIMIT 

### TEST) IN THE RAT 

# n PROJECT NUMBER: 163/129 , J 

# n n l ! AUTHOR: D.J. Allen



### ISSUED BY; Safepharm Laboratories Limited 

### P.O. Box No. 45 DERBY DEI 2BT 

### U.K. Telephone: 

### DERBY (0332) 792896 Facsimile: 

### (0332) 799018 PAGE 1 OF 16 PAGES Telex: 377079 SAFPHM G 

# n n o 

# n

# u -2­ 

### PROJECT NUMBER: 163/129 

# n
'I., ;


### QUALITY 

### ASSURANCE REPORT

_ J 

# r 

# n


# n The routine inspection of short term studies at Safepharm laboratories is carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently completed series of monthly inspections relevant to the study type(s) in this report are given below. Date(s) of Inspection and Reporting: 

# n 03, 

### 16, 24, 25 Hay 

### 1994 This report has been audited by Safepharm laboratories Quality Assurance Unit. It is considered to be an accurate account of the data generated and of the procedures followed. Date of Report Audit: 

# o 15 June 1994

J.R. Pateman C. Biol., M.l. Biol. 

### FOR SAFEPHARM QUALITY ASSURANCE UNIT 

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

# ~.
n 

# U 26. JUl. 1994 

# n
\.
.....i
Page 31

# lJ -3­ 

### PROJECT NUMBER: 163/129

GLP COMPLIANCE STATEMENT

I. the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the· quality or integrity of the study. I 

### consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated. The work described was performed in 

### compliance with the UK 

### Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GLP standards published as OECD Environment Monograph No. 45 (OCDE/GD(92)32); and are in conformity with, and implement, the requirements of Directives 87/18/EEC and n I . t !



### 88/320/EEC. 

# n These international standards are Environmental Protection Agency and Food 

# n I I l 	 . 

### the requirements of 40 

### eFR Part 160, 40 amended)
. 

# r !". .' 

# n l ) 

### acceptable to the United States and Drug Administration, and fulfil 

### eFR Part 792 and 21 CFR Part 58 (as

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

## ~\~ ......,. DATE:
........

## ~:7:~~.':t 

# ........... D.J. Allen B.Sc. (Hans) Study Director for Safepharm Laboratories 

# n , I

32 

## " I 

### U-4­ 

### PROJECT NUMBER: 

## G CON TEN T S l .'

# ~ 

# [ SUMMARY 1. 

### INTRODUCTION 

# n 2. 

### TEST MATERIAL 2.1 Description, Identification and Storage Conditions

, 

# n

. 

### 2.2 Method of Preparation 3 • 

### TEST SYSTEM 

### 3.1 Specification 

### 3.2 Husbandry 

### 4. 

### PROCEDURE
r- 

### 4.1 Range-Finding Study 

### 4.2 Main Study 

# l**~** 5. 

### EVALUATION OF DATA 

# n 6. 

### ARCHIVES 7. 

### RESULTS 7.1 Range-Finding Study 7.2 Main Study 8. CONCLUSION 

# n TABLES

.TABLE 1 Individual Clinical Observations and Mortality Data in the Range-Finding Study 

# n TABLE 2 Individual Clinical Observations and Mortality Data in the Main study 

# n TABLE 3 Individual Bodyweights and Weekly Bodyweight Gain in the Main Studyri ! 

### TABLE 4 Individual Necropsy Findings in the Main Study APPENDIX 

# n APPENDIX I GLP Statement of Compliance 

# n

I 

### 163/129

PAGE

5 6 6 6 7 7 7 7 

### 8 8 8 9 10 11 11 11 11 

### 12 13 14 15
16

Page 33-5­ 

### PROJECT NUMBER: 163/129 

### SUM H A R Y
STUDY SPONSOR 

### PROJECT NUMBER 163/129 

# n TEST MATERIAL 

# n 1. 

### A study was performed to assess the acute oral toxicity of the test material in the Sprague-Dawley strain rat. The method used followed 

# n [**~**

### ! that described in the OECD Guidelines for Testing of Chemicals No. 401 "Acute Oral Toxicity" (adopted 24 

### February 1987) and Method B1 of Commission Directive 92/69/EEC (which constitutes Annex Vof Council Directive 67/548/EEC). 

# r I , 'L.) 

### The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 91/325/EEC). 2. Following a range-finding study, a group of ten fasted animals (five males and five females) was given a single oral dose of test material, 

# n as a suspension in arachis oil B.P. at a dose level of 2000 mg/kg body­
, 

### . weight. The animals were observed for fourteen days after the day of d9sin9 and were then killed for gross pathological examination. 3. There were no deaths. No signs of systemic toxicity were noted during the study. 

# n 4. All animals showed expected gain in bodyweight during the study. 

# n 5. No abnormalities were noted at necropsy. 6. The acute oral median lethal dose (LOSO) of the test material in the Sprague-Dawley strain rat was found to be greater than 2000 mg/kg bodyweight. No symbol and risk phrase are required according to EC 

# n labelling regulations. , J

34 

# r l !-6­ 

# c=**~** ACUTE ORAL TOXICITY PROJECT NUMBER: 

### 163/129
(LIMIT TEST) 

### IN THE RAT 1. 

### INTRODUCTION The study was performed to assess the acute oral toxicity of the test 

# n material in the Sprague-Dawley strain rat (Safepharm Standard Method Number EC 1). The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals No. 401 "Acute Oral Toxicity" (adopted 24 February 1987) and Method 81 of Commission Directive ..--, ! 

### I 92/69/EEC (which constitutes Annex Vof Council Directive 67/548/EEC). L 

### The results may be used as a basis for**clas~ification** 

### and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 91/325/EEC). The test system was chosen Decause the rat has been shown to be a suitable model for this type of study and is recommended in the test method. The results of the study are believed to be of value in predicting the likely toxicity of the test material to man. 

# n The study was conducted in accordance with the internali

### ona11y 

### accepted general principles of Good Laboratory Practice and Safepharm Standard Operating Procedures. The study was performed between 26 April 1994 and 18 May 1994. 

# n 2. TEST MATERIAL

,. , 2.1 Description. Identification and Storage Conditions ,-, l , !

i 

### The test material was supplied by 

### the study sponsor as follows: Sponsor's identification 

# r Chemical name I c-; \ I \ 

# I

# ! I i 

# --,---­ Batch number 

# \ Date received Description 

# \ Container Storage conditions 

# -----,

# --~ Page 35

### ~l

# '
. i

# Li -7­ 

### PROJECT NUMBER: 163/129 2. TEST MATERIAL (contd) 2.1 

### Description, Identification and Storage Conditions (eontd) 

# r: Data relating 

### to the identity, purity and stability of 

### the test

c J 

### material are the responsibility of the sponsor. 2.2 Method of 

### Preparation For the purpose of the study the test material was freshly r 

### prepared, as required, 

### as a suspension at the appropriate 

### I : l _J 

### concentration in arachis oil B.P. Homogeneity was assured by the use of a Silverson Homogeniser. 

# n Determination by analysis of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a
("""'\ 

### requirement of the Test Guideline. 

# LJ 3. 

### TEST SYSTEM r

, ) I

.' 

### 3.1 Specification 

### Male and female Sprague-Dawley strain rats were supplied by Harlan 

# n U.K. Ltd., Blackthorn, Bicester, Oxon, 

### U.K. At the start of the main study the males weighed 145

- 164g, and the females 125

­ 139g, and were approximately five to eight weeks old. After a 

# n minimum acclimatisation period of at least five days the animals were selected at random and given a 

### unique number within the study by indelible ink marking on the tail and a number written on a 

### cage card. r 

# U 3.2 

### Husbandry The animals were housed in groups of five by sex in solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approxi­ 

# o mately two hours after dosing, free access to mains drinking water and food (Rat and Mouse Expanded Diet No.1, Special Diets Services 

# n Limited, Witham, Essex, U.K.) was allowed throughout the study. The animal room was maintained at a temperature of 19 23·C and 

# n relative humidity of 44

- 58%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled 

# n by a time switch to give 12 hours continuous light and 12 hours darkness. 

# n Page 36**l~**

# . -8­ 

### PROJECT NUMBER: 163/129 

# n 4. 

### PROCEDURE lJ 

### 4.1 Range-finding Study 

### Arange-finding study was performed to establish a 

### dosing regime as follows: 

### DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF RATS

mg/kg mg/ml ml/kg 

### MALE FEMALE 

### r 

## lj 2000 200 10 1


### The animals were observed for deaths or overt signs of toxicity 

### t, 1, 2 

### and 4 

### hours after dosing and subsequently once daily for 5 

# r days. Individual bodyweights were recorded on the day of dosing to allow 

# n

, ) 

### calculation of individual treatment volumes. No necropsies were performed. 

# n 4.2 

### Main Study: Based on the results of the range-finding study a further group of animals was treated as follows: 

# n

. , 

### DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF 

### RATS 

# r; l "

: 

### mg/kg mg/ml ml/kg 

### MALE FEMALE :I ! i 2000 200 10 5 5 

### , I

'


### All animals were dosed once only by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing. 

# I:

, : 

### The animals were observed for deaths or overt signs of toxicity j, 

# n 1, 2 

### and 4 

### hQurs after dosing and subsequently once daily for 14 

### days. --. \ 

### . l .' Page 37i I 

### U -9­ 

### PROJECT NUMBER: 163/129 

### 4. PROCEDURE (contd) 

# n 4.2 Main Study (contd) Individual bodyweights were recorded prior to dosing on Day 0 and 

# n on 

### Days 7 and 

### 14. 

# o At the end of the study the animals were killed by cervical dislocation and subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. 

### No tissues were retained. 

# n

n 

### 5. EVALUATION OF DATA 

### [1 Data evaluations included the relationship, if any, between the animals' exposure to the test material and the incidence and severity 

### of all

,

# n

, 

### abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological 

# n effects. USing the mortality data obtained, an estimate of the acute oral median lethal dose (LDSO) of the test material was made. 

# n The results were interpreted according to the Commission Directive 91/325/EEC which adapts Council Directive 67/548/EEC on the regulations relating to the classification, packaging and labelling of dangerous , " r, 

### substances as follows: 

### 1

## ~ i) 

### Interpretation according to Annex VI Section 3.2 

# n The test material will be classified and assigned the appropriate symbol and risk phrase as follows: 

# Ii l } 

### ACUTE ORAL CATEGORY LD50 SYMBOL 

### RISK PHRASE mg/kg 

# n**~** 1 

### VERY TOXIC**~25** 

### T+ R28 ·VERY TOXIC IF SWALLOWED" 

# r TOXIC >25 to 200 T R25 "TOXIC IF SWALLOWED" i. 

### I HARMFUL >200 to 2000 Xn R22 "HARMFUL IF SWALLOWED" 

# n Test materials with acute oral LDSO values greater than 2000 mg/kg require no symbol and risk phrase. 38

# l ! -10­ 

# n PROJECT NUMBER: 163/129
, J 

# n 6. ARCHIVES 

### Unless instructed otherwise by 

### the sponsor, all original data and a copy n 

### I , ji of the final report 

### will be retained in the archives of Safepharm 

# . ' 

### laboratories for a period of ten years. After this period, the Ii , I 

### sponsor's instructions will LJ n

## ~J 

# n

'
.. j


# n , J 

# n

: .I 

# n n

,......, i . I I c J 

# n 

# n
l j 

# II t ) 

# n l ; 

# II n 

### be sought.

Page 39 

### n 

### I ' \.. "I 

## L:I -11­ 

### PROJECT NUMBER: 163/129 7. RESULTS 7.1 Range-finding**Stud~** 

### Individual clinical observations and mortality data are given in Table 1. 

### There were no deaths. Signs of systemic toxicity noted in the male were hunched posture, lethargy and decreased respiratory rate. n

### l~ Based on this information, 

### a dose level of 2000 

### mg/kg bodyweight 

### r was selected for the main study. 

## [ j 7.2 Main Study Individual clinical observations and mortality data are given in Table 2. 

### ,...., 

## I \ a) 

### Mortality Data There were no deaths. b) 

### Clinical Observations

, ) 

# n No signs of systemic toxicity were noted during the study. 

# n c) 

### Bodyweight Individual bodyweights, together with weekly bodyweight gains are given in Table 3. 

### All animals showed expected gain in bodyweight during the study.
r--. 

### I I**~** j d) 

### Necropsy Individual necropsy findings are given in 

### Table 4. 

### No abnormalities were noted at necropsy. 

# n l; 8. 

### CONCLUSION 

# n ( J

I 

### The acute oral median lethal dose (LDSO) of the test material 

# ­

_____ .J in the Sprague-Dawley strain.rat was found to be greater than 2000 

### mg/kg bodyweight. No symbol and risk phrase are required according to EC labelling regulations. 

# n

f' I I ( ! 40r--­**~--=~~]** r] ',---) ,_. 

### =-J

____J**~J** 

### :....._J 

# ==l

o = no 

### signs of systemic toxicity L = lethargy H

### = hunched posture Rd = decreased respiratory -c ::0 

### E rate m**~** :z c::: 3: C::J m ::0
I-' 0\**~** ..... N \D**~ ~ ~** **~~ ~** **~ ~ ~~** **~**



### ': ACUTE ORAL TOXICITY (LIMIT TEST) IN HIE RAT**~** 

### TAB L E 2 

### INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA IN THE MAIN STUDY Effects Noted After Dosing Effects Noted During Period After Dosing Dose Animal (Hours) (Days) 

### level Number mg/kg 

### &Sex 1 2 4 1 2 3 

### 4 5 6 7 8 9 10 11 12 13 

### 14 3-0 

### Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-1 

### Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

'0 PJ cO ro 

## "'" N 2000 3-2 

### Male 3-3 

### Male 3-4 

### Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

## a 0 0 0 0 0 0 0 0 I 

# - w I
4-0 

### Fema1e 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4-1 

### Fema1e

4-2 

### e 4-3 

### Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -0 :::r::I 0 c.... 1"1'1 n -t :;z: c: 3: CCI 1"1'1 :::r::I 

# .. 4-4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

### Female

...... en w


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

### no signs of systemic**toxi~;ty** ...... N ID

### '::.-:-J**~_.) ~=]**  ,___

### J : .J**~._.J** :""_J 

### ,_:J



### \: ACUTE ORAL TOXICITY (LIMIT TEST) IN THE RAT TAB L E 3 INDIVIDUAL BODYWEIGHTS AND WEEKLY BODYWEIGHT GAIN IN THE MAIN STUDY
Dose Level Animal Number Bodyweight (g) at Day Bodyweight Gain (9) During Week mg/kg 

### &Sex 

### o 7 14 1 2



### 3-0 Male 164 218 

### 252 54 34 '0 I PJ 3-1 

### Male 145 197 

### 235 52 38 t.Q
....... CD**~** 3-2 

### Male 162 224 285 62 61 I 

### "'" w 

### 3-3 Male 151 202 250 

### 51 48 3-4 

### Male 145 206 252 61 46 

### 2000 4-0 Female 139 173 

### 190 34 17 ""0 

# e: c... 

### 4-1 Female 128 162 180 34 IS IT! n -I 

### 4-2 Female 125 161 

### 178 36 17 :z c::: 3: co 

### 4-·3 Female 134 168 186 

### 34 18 1"11 :;0 

### 4-4 Female 128 

### 147 159 19 12

....... Q"I W

......... ...... N \0'm_J : 

### _J =::::J 

### :=:::J**~** 

### J

__J



### ): ACUTE ORAL TOXICITY (LIMIT TEST) IN THE RAT 

# ------' TAB L E 4 INDIVIDUAL NECROPSY FINDINGS 

### IN THE 

### MAIN STUDY
Dose Level Animal Number Ii

### me of 

### Death Macroscopic Observations mg/kg 

### &Sex

3-0 Male Killed day 14 No abnormalities detected 3-1 Male Ki11ed day 14 No abnormalities detected I

....... I 

### 3-2 Male Ki11ed day 14 No abnormalities detected U'I 

## "" "" 3-3 Male Ki 11ed day 14 No abnormalities detected 3-4 Male Killed day 14 No abnormalities detected 2000 

### 4-0 Female Ki 11ad day 14 No abnormalities detected '"0 

## e; c... 

### 4-1 Female Killed day 14 No abnormalities detected rr1 ("") -I 

### 4-2 Female Killad day 14 No abnormalities detected % I=: 

### 4-3 Female Killed day 14 No abnormalities detected 3: o:l rr1 ::c 

# .. 4-4 Female Ki11ed day 14 No abnormalities detected



## .... O'l W

......... 

### ..... N I.C

# lj -16­ 

# n
n : i '.**~** 

# n 

# n n n 

# n n PROJECT NUMBER: 163/129 

### A PPEN D I X I

TIlEDEPARTl\1ENfOF

### HEAI..J'HOFTHE GOVERNMENT OF

## nmUNITED KINGDOM
GOOD LABORATORY PRAcnCE
SfAlEMENTOFCOMPIlANC£ IN ACCORDANCEWITH DlRECIlVE 88/320 EEC iABORATORY SafePharm Laboratories Limited POBox No 1.5 Derb.v DEl 2BT

DAlE OF [NSP£GnON
31 January 1994 A general inspoctiol1 for compliance with the Principles ofGood Labonlfory Practice was carriedoutatthe abo-ye labonitory as partofthe UKGLP Complil.nce Progmnme. At the time ofIhe inspection no deviations were found of$Ufficient magnitude to affect tile validityofstudies perfonned at these facilities.


# n D. F. Moore Director UKGLP MonitoringUnic 

# .l, 

# 1 Page 45 

# r

# lj 

# n n n 

# n n 

# c, n ACUTE DERMAL IRRITATION TEST IN THE RABBIT 

# n PROJECT NUMBER: 163/130 

# n n 

# n n AUTHOR: D.J. Allen 

# n n n STUDY SPONSOR: ISSUED BY: / Safepharm Laboratories Limited 

# n P.O. Box No. 45 DERBY DEI 2BT 

# n U.K. Telephone: DERBY (0332) 792896 

# n Facsimile: (0332) 799018 PAGE 1 OF 15 PAGES Telex: 377079 SAFPHM G 

# n Page 46

# C , 

## I .'

J 

## ; J 

# n 

# n r , ! 

# n
n
n 

# n n '1 l.J 

# n D


# n -2­**PRO~IECT** NUMBER: 163/130



### QUALITY ASSURANCE REPORT

The routine inspection of short term studies at Safepharm Laboratories is carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently completed series of monthly inspections relevant to the study type(s) in this renort are given below. Oate(s) of Inspection and Reporting: 04, 24, 25, 26 May 1994 This report has been audited by Safepharm Laboratories Quality Assurance Unit. It is considered to be an accurate account of the data generated and of the procedures followed. Date of Report Audit: 16 June 1994

J.R. Pateman C. Bio1., M.l. Biol. 

### FOR SAFEPHARM QUALITY ASSURANCE UNIT

2.6. JUL 1994
Page 47

## I , )

### ! 

# n
( .j 

# n
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# l; 

# L 

# n l. i 

# n 

# n n n 

# n 

# o

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# U -3­ PROJECT 

### NUMBER: 163/130

GLP COMPLIANCE STATEMENT

I, the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study, I consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated. The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GLP standatds published as OECD Environment Monograph No. 45 (OCDE/GD(92)32); and are in conformity with, and implement, the requirements of Directives S7/IS/EEe and 88/320/E£C. These international standards are acceptable to the United States Environmental Protection Agency and Food and Drug Administration, and fulfil the requirements of 40 CFR Part 160, 40 CFR Part 792 and 21 CFR Part 58 (as amended)
. 

## 9.5"-1-

- ct.<..I­ DATE:

.........**~** ....**~** ....... ".J
.......**~** •••• D.J. Allen 

### B.Sc. (Hons) Study Director for Safepharm Laboratories

Page 48-4­ 

### PROJECT NUMBER: 163/130

CON TEN T S



### PAGE SUMMARY 5 1. 

### INTRODUCTION 6 

### 2. TEST MATERIAL 6 2.1 Description, Identification and Storage Conditions 6 2.2 Method of Preparation 7 

### 3. TEST SYSTEM 7 3.1 Specification 7" 3.2 Husbandry 7 4. 

### PROCEDURE 8
Ii 

### 5. INTERPRETATION OF RESULTS 9 of the Irritancy Potential using the Draize scheme 

### I I \ j 5.1 Calculation of Primary Irritation Index and Grading 9 

# o 5.2 Interpretation According to Annex 

### VI of Council Directive 10 

# n 67/548/EEC, Relating to the Classification, Packaging and Labelling of Dangerous Substances

• J 

### 6. ARCHIVES 11 ,"l 

### I \. I

) 

### 7. RESULTS 12
n 

### 8. CONCLUSION 12 

### l

## j TABLES TABLE 1 Individual Skin Reactions 13 

### TABLE 2 Individual Daily and Individual Mean Scores 1'1 for Dermal Irritation Following 4-hour Exposure 14 

# l j Required for EC Labelling Regulations 

### APPENDIX APPENDIX I GLP Statement of Compliance 15
r 

# l ; Page 49 

# , r­

## I ' 

## IL -5­ II I ' PROJECT NUMBER: 163/130
r r \ J 

## l 

## n



# i SUM MAR Y

STUDY SPONSOR 

# In PROJECT NUMBER 163/130

TEST MATERIAL 

# n U 

# n

I l ; 1. A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method used I; followed the recommendations of the DECO Guidelines for Testing of 

## 11 

# n Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 17 July 1992) and Method 84 in Commission Directive 92/69/EEC (which constitutes Annex Vof Council Directive 67/548/EEC). 

# n I. i The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 91/325/EEC). 

# n 2. A single 4-hour, semi-occluded application of the test material to the 

# n intact skin of three rabbits produced very slight to well-defined erythema and very slight oedema. Desquamation was confined to one \. J treated skin site seven days after treatment. 3. The test material produced a primary irritation index of 1.3 and was classified as a mild irritant to rabbit skin according to the Draize 

# n classification scheme. No corrosive effects were noted. 'l The test material did not meet the criteria for classification as " I •

) 

# c irritant or corrosive according to EC labelling**regulation~.** No symbol and risk phrase are required. Page 50 

# n-6­ 

### PROJECT NUMBER: 163/130 

# n --------;**A~EitMAL** IRRITATION TEST IN THE RABBIT 

# r

, I 1. INTRODUCTION U The study was performed to assess the irritancy potential of the test material following a single, 4-hour, semi-occluded application to the intact rabbit skin (Safepharm Standard Method Number OECD 4). The method used followed the recommendations of the DECO Guidelines for 

# n Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 17 July 1992) and Method B4 in Commission Directive 92/59/EEC 

# o (which constitutes Annex Vof Council Directive 67/548/EEC). 

## n The results may be used as a basis for classification and labelling 

### l J under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 91/325/EEC). 

# n l J The test system was chosen because the rabbit has been shown to be a suitable model for this type of study and is recommended in the test 

# n method. The results of the study are believed to be of value in pre dieting the likely skin irritancy potential of the test material to man. The study was conducted in accordance with the internationally accepted general principles of Good Laboratory Practice and Safepharm Standard Operating Procedures. 

# n The study was performed between 26 April 1994 and 3 May 1994. 

# n 2. JEST MATERIAL 2.1 Oescription. Identification and Storage Conditions 

# n The test material was supplied by the study sponsor as follows: Sponsor's identification Chemical name 

# n Lot number 

### \ Date received 

## I Description 

# n \ I \ Container Storage conditions 

# n Page' 51

# c -7­ 

# n PROJECT NUMBER: 163/130 

### 1;

2. 

### TEST MATERIAL {contd} 

# n 2.1 Description, Identification and Storage Conditions (contd) Data relating to the identity, purity and stability of the test material are the responsibility of the sponsor. 2.2 Method of Preparation For the purpose of this study the test material was moistened with distilled water immediately before application. 

# n 3. 

### TEST SYSTEM 

# n 3.1 Specification Three New Zealand White rabbits were supplied by David Percival Ltd., Moston, Sandbach, Cheshire, U.K. At the start of the study the animals weighed 2.31

- 2.44 kg and were twelve to sixteen weeks 

# n old. After a minimum acclimatisation period of five days each 

### lJ animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear 

# n and on the cage label. 

# n 3.2 Husbandry The animals were individually housed in suspended metal cages. 

# Ii , 1 Free access to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets Services Ltd.) Witham, Essex, U.K.} was allowed 

# n throughout the study.



# n 

# n The animal room was maintained at a temperature of 18

- 23'C and relative humidity of 51 67%. The rate of air exchange was apprOXimately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. l .: Page 52

# II -8­ PROJECT NUMBER: 163/130

4. PROCEDURE On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study. On the day of the test a suitable test site was selected on the back of each rabbit. Aquantity of 0.59 of the test material, moistened with 0.5 ml of distilled water, was introduced under a 2.5 cm x 2.5 em cotton gauze patch and placed in position on the shorn skin. The patch was 

# n secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 em x 4.0 em). To prevent the animals interfering 

# n with the patches, the trunk of each rabbit was wrapped in an elasticated l J corset {TUBIGRIP} and the animals were returned to their cages for the duration of the exposure period. 

# n Four hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water followed by diethyl ether. 

# n Approximately one hour following the removal of the patches, and 24, 48 

# n and 72 hours 1ater, the test sites were examined for evidence of primary 

### , , irritation and scored according to the following scale from Draize J.H., 

# [j (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of 

### " Sciences, Washington D.C. p.31. 

### I

. \ ,

[

n 

### EVALUATION OF SKIN REACTIONS 

## tj Erythema and Eschar Formation Value 

# n No erythema
..................,...................,................ 0 Very slight erythema (barely perceptible)
......................... 1 well-defined ei"ythema
............................................. 2 Moderate to severe erythema
....................................... 3 Severe erythema (beet redness) to slight eschar formation

{injuries ; n depth}
............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Page

## tj -9­ 

### PROJECT NUMBER: 163/130 

# c 4.**PROCEDUR~** 

### (contd) Oedema Formation 

# n No oedema .........•......................."........................ 0 [ j Very slight oedema (barely perceptible)
........................... 1 Sl ight oedema (edges of area well-defined by definite raising)
.... 2 Moderate oedema (raised approximately 1 millimetre) ..........•.... 3 Severe oedema (raised more than 1 millimetre and extending" 

# r li beyond the area of exposure)
..................................... 

### 4 Any other skin reactions, if present, were also recorded. An additional observation was made on day 7 to assess the reversibility of skin reactions. 

### 5. INTERPRETATION OF RESULTS 

# n 5.1 Calculation of Primary Irritation Index and Grading of the



# n Irritancy Potential Using the Draize Scheme 

# n The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by 6 to give the primary irritation index of the test material. The test material was classified according to the following scheme devised by Draize J.H. (1959) "Dermal Toxicity" 

# n In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United t l States, Austin, Texas, p.47: Primary Irritation Index Classification of Irritancy 0 Non-;rritant 

# n
, ; > 0 2 Mild irritant > 2 5 Moderate irritant 

# n > 5 

# - 8 Severe irritant 

## n

i If irreversible alteration of the dermal I tissue is noted in any l . ) 

# n rabbit, as judged by the Study Director, which may include ulceration and clear necrosis or signs of scar tissue, the test material is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be**applicable~** 

# n Page 54I 1 u -10­

......

### , PROJECT 

### NUMBER: 163/130 

### t J 

# n 5. INTERPRETATION OF RESULTS (contd) 

## " 5.2 Interpretation According to Annex VI of Council Directive I I CJ 67/548/£EC, Relating to the Classification, Packaging and Labelling of Dangerous Substances The results were interpreted according to Commission Directive 91/325/EEC which adapts Council Directive 67/548/EEC on the regulations relating to the classification, packaging and labelling II of dangerous substances as follows: 

# U



# n i) Interpretation according to Annex VI Section 3.2.5 

# n The test material is considered to be corrosive if it produces full thickness destruction of skin tissue on at least one animal. The symbol 

### 'e' and indication of danger "corrosive" will be required. Risk phrases shall be assigned in accordance with the following criteria. R35 "CAUSES SEVERE BURNS" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to three 

# n minutes exposure, or if this result can be predicted. 

# n R34 "CAUSES BURNS" 

# c If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to four hours exposure, or if this result,can be predicted. 

## n 

## II ii} Interpretation according to Annex 

### VI Section 3.2.6 

# n Non-corrosive test materials shall be classified as irritant l J and assigned the symbol "Xi" and the indication of danger "irritant" if inflammation of the skin is noted, which persists 

# n for at least 24 hours after an exposure period of up to four hours, and if either erythema and eschar formation, or oedema iOr'mation, e4uiva1ent to a mean value of two or more, calculated for each animal separately, is observed in two or more animals. e 55 

# n

# l J 

# : -11­ 

# n PROJECT 

### NUMBER: 163/130

! 

# n 5. INTERPRETATION OF RESULTS (contd) 5.2 Interpretation According to Annex VI of Council Directive 67/548/EEC. Relating to the Classification, Packaging and**~** labelling of Dangerous Substances (contd) ii) Jnterpretation accordlng to Annex VI Section 3.2.6 (eontd) 

# n The scores at the 24, 48 and 72-hour readings are used in calcu1ating the respective mean values. The following risk phrase sha1l be assigned in accordance with the criteria given: R 38 ItIRRITATING TO SKIN" If, when applied to healthy intact animal skin for up to four hours, significant inflammation is caused which persists for 24 hours or more after the end of the exposure period. Inflammation is significant if the mean score for either erythema and eschar formation or oedema formation, observed in two or more animals, is equivalent to the value of two or more. 

# n 6. 

### ARCHIVES Unless instructed otherwise by the sponsor, all original data and a copy of the final report will be retained in the archives of Safepharm Laboratories for a period of ten years. After this period, the 

# n sponsor's instructions will be sought. 

## I n ! , ) 

# n

Page 56 

# ;n : I-12­ 

# n 

# n

f' 

# l J



# n 

# n n

, ) 

# n

f1

n 

## l \ 

# n l j 

# n \ , 

## n ( j 

# n l ; 

# n PROJECT NUMBER: 

### 163/130
7. RESULTS The individual scores for erythema/eschar and oedema, are given in Table 1. Mean values required for EC labelling regulations are given in Table 2. Faint purple-coloured staining caused by the test material was noted at all treated skin sites following patch removal. The staining did not affect evaluation of skin responses. Very slight erythema was noted at one treated skin site one hour after patch removal. Very slight to well-defined erythema was noted at all treated skin sites at the 24 and 48-hour observations. Very slight erythema was noted at two treated skin sites at the 72-hour observation. '­ Very slight oedema was noted at one treated skin site at the 24, 48 and 72-hour observations. Desquamation was confined to nne treated skin site seven days after treatment. 

### 8. CONCLUSION The test material, ) produced a primary irritation index _-,-

_____...... J of 1.3 and was classified as a MILD IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. The test material did not meet the criteria for classification as irritant or corrosive according to'EC labelling regulations. No symbol and risk phrase are required.

Page 57----"-----­**~ ~.J**  

### =-.-=:J**~J =~**  

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__

### J =:J ==:J.J**~._.J ~** J 

### :...._J**~_J** __

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## ="l**~_._)** 

### '::=J

# ~: ACUTE DERMAL IRRITATION TEST IN THE RABBIT TABLE 1 INDIVIDUAL SKIN REACTIONS Individual Scores

- Rabbit Number and Sex (Bodyweight Kg) Skin Reaction Observation Time Total 68 FEMALE 72 MALE 73 MALE

(2.31) (2.44) (2.42) 1 Hour OSTA OSTA ISTA I 24 Hours ISTA 2 ISTA 4 Erythema/Eschar Formation 48 Hours I 2 1 4 'U 72 Hours 

### o 1 1 2 OJ I lQ 7 Days 

### o 00 

## o o
...... ro W I (Jl 0) 1 Hour 

## o o 

## o o 24 Hours 

## o 1 

### o I Oedema Formation 48 Hours 

### o 1 

### o 1 72 Hours 

### o 1 

### o I

7 Oays 

### o a 

### o o -0 

### E5 Sum of 24 and 72-Hour Readings (S) 8 

### ,..., n -i :z: Primary Irritation Index (S/6) 8/6 1.3 c: % o::J 

### ,..., ::c Cl assification 

### MILD IRRITANT

...... 01 W

.........
...... ( ) = total values not used for calculation of primary irritation**inde~** 

## o desquamation w o STA = faint purple-coloured staining-------- 

### :::-=J 

### :__J
___J
....J


### ): ACUTE DERMAL IRRITATION TEST IN THE RABBIT TAB L E 2 INDIVIDUAL DAILY AND INDIVIDUAL MEAN SCORES FOR DERMAL IRRITATION FOLLOWING 4-HOUR EXPOSURE FOR EC LABELLING REGULATIONS

Skin Reaction Reading (Hours)

24 

### Erythema/Eschar Formation 48 '1J 72 PJ \Q ro lJ1 

### Total ill 

### Mean Score 24 

### Oedema Formation 48 72 

### Total Mean ScoreI 

# lj

, " 

# n n 

# n n n n l 1 

# II 

# n



# n n 

# n n 

# r n L.! 

# n Ii -15­ 

### PROJECT NUMBER: 163/130 A PPEN D I X I

THEDEPARTMENTOFHEAIXHOFTHE GOVERNMENT OF1HEUNITED KINGDOM
GOOD LABORATORY PRACIlCE STATEMENTOFCOMPLIANCE INACCORDANCEWITHDIRECI1VE 88/320 EEe LABORATORY SafePharm Laboratories Limited POBox No 45 Derby DEl 2BT

DATE OF INSPECllON 31 January 1994 A general illSpCCtiol1 Cor compliance with the Principles ofGood Laboatory Pactice wascarriedoutatcbe above laboritol)' U**~f(** oftheUKGLP Compliance Programme. At. the time ofme iJupccdon no deviarions were found of**u{flCi.en (**  magnitude (0 affect thevaliditY ofstudies perfonnedatthesefacilities.

D. F. Moore Page 60 Director UKGLP Monitoring Unit

# l : 

# n 

# n n 

# n n n ACUTE EYE IRRITATION TEST IN THE RABBIT 

# n PROJECT NUMBER 163/131 

# fl , i 

# n

AUTHOR: D.J. Allen 

# l! n 

# n n STUDY SPONSOR: ISSUED BY: Safepharm Laboratories Limited 

### P.O. Box No. 45 

### DERBY DEI 2BT 

### U.K. Telephone: DERBY (0332) 792896 

# n l. Facsimile: (0332) 799018 

### PAGE 1 OF 19 PAGES Telex: 377019 SAFPHM G 

# n , ) 

# n n

Page 61 

# n,

## l ; I -2­ 

### PROJECT NUMBER: 163/131



### n QUALITY ASSURANCE REPORT 

## l ; 

# n , J



# n The routine inspection of short term studies at Safepharm Laboratories is carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently'completed series of monthly inspections relevant to the study type(s) in this report are given below. Date{s} ofInspection and Reporting: 

# n 03, 24, 26 May 1994 

### 1) t ! This report has been audited by Safepharm Laboratories Quality Assurance Unit. 

# n It is considered to be an accurate account of the data generated and of the procedures followed. 

# n Date of Report Audit: 16 June 1994 

# n

J.R. Pateman C. 8iol., M.I. Biol
.

# ~o~ l I 

# ...............tp\\(
... ...

, FOR SAFEPHARM QUALITY ASSURANCE UNIT

26. JUl. 1994 

# n Page 62 I (--

!

# II

n I i c J 

# n 

# n I, the 

## n I I protocol \. .. quality valid. -3­ 

### PROJECT NUMBER: 163/131



### GLP COMPLIANCE STATEMENT

undersigned, hereby declare that the objectives laid down in the

were achieved and as nothing occurred to adversely affect the or integrity of the study, I consider the data generated to be

This report fully and accurately reflects the procedures used and data generated. 

# n The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GLP standards published as OEeD Environment Monograph No. 45 (OCDE/GD(92)32); and are in ! 

### I·: n conformity with, and implement, the requirements of Directives 

### 87/18/EEe and

, 88/320/EEC. '1 

# L 

# !n These international standards are acceptable to the United States Environmental Protection Agency and Food and Drug Administration, and fulfil the requirements of 40 CFR Part 160, 40 CFR Part 792 and 21 CFR Part 58 (as amended)
. 

### 'CI:;-- - 'f­

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

# ~\~ .......... DATE: '" ...... '" 

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

### ........... r D.J. Allen B.Sc. (Hons) I. J Study Director for Safepharm Laboratories 

# n
c :



# n l \ Page 63

# II

f' I 

### I I ' , J 

# n I,

, , 

# n

\- -'

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' ,'--' 

### I I l .I


# n 

# r

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# r l 1 -4­ 

### PROJECT NUMBER:


### CON TEN T S

SUMMARY 1. 

### INTRODUCTION 2. TEST MATERIAL 2.1 Description, Identification and Storage Conditions 2.2 Method of Preparation 3. 

### TEST SYSTEM 3,] Specification 3.2 Husbandry 4. 

### PROCEDURE 5. INTERPRETATION OF RESULTS 6. 

### ARCHIVES 7. 

### RESULTS 8. 

### CONCLUSION KEY TO TABLE 1 

### TABLES TABLE 1 TABLE 2 TABLE 3 

### APPENDICES APPENDIX [ APPENDIX II APPENOIX IIIIndividual Scores and Individual Total Scores for Ocular Irritation Individual Total Scores and Group Mean Scores

for Ocular Irritation Individual and Mean Scores for Cornea, Iris and

Conjunctivae Required for EC Labelling Regulations

Draize Scale for Scoring Ocular Irritation Modified Kay and Calandra Interpretation of

Eye Irritation Test GLP Statement of Compliance
Page 64 

### 163/131

PAGE
5 6 6 6 7 7 7 7 7 8 ]0 11 11 12
13 14 15
16 17 18 19

# lj -5­ 

### PROJECT NUMBER: 163/131 

## I r>

i \ / S U H H A R Y .-- 

## I i , } 

### STUDY SPONSOR

PROJECT NUMBER 163/131 

# n TEST MATERIAL 

# n 1. A study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method used 

# in followed that described in the O£CO Guidelines for Testing of Chemicals

L ; No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 February 1987) and Method 85 in Commission Directive 92/69/EEC (which constitutes Annex V <- 

## I i '.- of Council Directive 67/548/EEC). r-; I .**~** ) The results may be used as a'basis for classification and labelling l"! under Annex VI of Council Directive 67/548/EEC (as adapted to technical
'. I I**~**

I
, progress by Commission Directive 91/325/EECj. [Ii. 2. A single application of the test material to the non-irrigated eye of three rabbits produced iridial inflammation and/or minimal to severe conjunctival irritation. Dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment. No r: ocular effects were noted in two treated eyes 48 hours after treatment. 

### I ! Staining of the cornea and iris persisted in one treated eye seven days after treatment. 3. The test material produced a maximum group mean score in the range of r-\ , I 13.0

- 15.0 and was classified as a mild to moderate irritant (Class 4 . 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The test material did not meet the criteria for classification as irritant according to EC labelling regulations. No symbol and risk phrase are required. Page 65

## t': -6­ 

# n PROJECT NUMBER: 163/131**~----.------** rI

# ~Tr-rYE IRRITATION I : ( J 

### TEST IN 

### THE RABBIT 1. 

### INTROOUCTION The study was performed to assess the irritancy potential of the test material following a single application to the rabbit eye (Safepharm Standard Method Number EC 5). The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals No, 405 "Acute Eye Irritation/Corrosion" (adopted 24 February 1987) and Method B5 of Commission Directive 92/69/EEC (which constitutes Annex 

### Vof Council 

# n Directive 67/548/EEC). 

### l J The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical

r-. progress by Commission Directive 91/325/EEC). I , I , I, , The test system was chosen because the rabbit has been shown to be a suitable model for this type of study and is recommended in the test 

# I'll . I method. The results of the study are believed to be of value in predicting the likely eye irritancy potential of the test material to 

# 1 

# L man. The study was conducted in accordance with the internationally accepted general principles of Good Laboratory Practice and Safepharm Standard Operating Procedures. The study was performed between 3 May 1994 and 10 May 1994. r I I l ; 2. TEST MATERIAL n 2.1 Description, Identification and Storage CO!lQitions 

# I l The test material was supplied by the study sponsor as follows: Sponsor's identification 

# (----­ Chemical name

Lot number 

# \ Date received Description Container 

# l
Storage conditions i i 

### 66'-

__---------­

# , : 

### t ! -7­ 

## i 

# n

I PROJECT**NlIMBER~** 163/131 2. TEST MATERIAL (contd) 

# 1I L 2.1 Description. Identification and Storage Conditions (contd) 

# n Data relating to the identity, purity and stability of the test l
..' material are the responsibility of the sponsor. 2.2 Method of Preparation For the purpose of this study the test material was used as supplied. 3. TEST SYSTEM 3.1 Specification 

# n Three New Zealand White rabbits were supplied by David Percival Ltd., Maston, Sandbach, Cheshire, 

### U.K. At the start of the study the animals weighed 2.50

- 3.18 kg and were approximately twelve to sixteen weeks old
..After a minimum acclimatisation period of five 

# [ days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface 

## I i. 

# "
C .\ of the ear and on a cage label. 

# r: 3.2 Husbandry 

### l .'



### ' The animals were individually housed in suspended metal cages. Free access to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets Services Ltd., Witham, Essex, 

### U.K.) was allowed throughout the study. The animal room was maintained at a temperature of 18

- 23"C and relative humidity of 47

- 67%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. 4. PROCEDURE

l. Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocu1ar irritation or defect with the aid of a 1ight source from a standard ophthalmoscope. Animals showing evidence of ocular 1esions were rejected and replaced. 67J i '-

­ -8­ 

# " PROJECT 

### NUMBER: 163/131 

# 1J 4. 

### PROCEDURE (contd) One rabbit was initially treated. A volume of 0.1 ml of the test material, which was found to weigh approximately 74 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after n . ! application, to prevent loss of the test material; and then released. '- J 

# n The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made. Confirmation of corneal opacity was made by examination of the eye 'under ultra-violet illumination using Sodium Fluorescein B.P. (FLUORETS: Smith 

### &Nephew Pharmaceuticals Ltd.), if required. 

# n l ; After consideration of the ocular responses produced in the first ,.--., 

## ! I treated animal, two additional animals were treated
..In order to l. ; minimise pain on instillation of the test material, one drop of local anaesthetic ("Ophthaine", 0.5% proxymetacaine hydrochloride, CR. Squibb 

### & Sons, Limited Hounslow, Middlesex, U.K.) was instilled into both eyes of these animals 1 - 2 minutes before treatment. 

# n II 

# n Assessment of ocular damage/irritation was made approximately 1 hour and**24~** 48 and 72 hours following treatment. according to the numerical evaluation given in Appendix I, (from Draile J.H. (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington D.C. p.48-49). 

## I' Any other ocular effects were also noted. Examination of the eye was LJ facilitated by use of the light source from a standard ophthalmoscope.

## ~ An additional observation was made in one treated eye on day seven to assess the reversibility of the ocular effects. 

### 5. INTERPRETATION OF RESULTS l." 

# fl The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were 

# n .Page. 68 ,-9­ 

# n PROJECT NUMBER: 163/131

- , 5. INTERPRETATION OF RESULTS (eontd) 

# n those relating to the iris designated by the letter 0 and those relating 

# r to the cornea by the letters E (degree of opacity) and F (area of opacity). For each tissue the score was calculated as follows:

'- -' ,-., Score for conjunctivae (A + B + C) x 2 

# u Score for iris 

## ox 5 Score for cornea (E 

### x F) 

### x 5 I 

# I: I l
.. Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C., J. Soc. Cosmet.**Ch~m.,** 1962 13 

# in:

, ' i " 281-289 (see Appendix II) was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores 'for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled I' 

### I I classification of the eye irritancy potential of the test material. l -' 

# n The results were also interpreted according to Commission Directive 91/325/EEC which adapts Council Directive 67/548/EEC on the regulations relating to the classification, packaging and labelling of dangerous 

# n substances, as follows: Interpretation according to Annex VI Section 3.2.6, Subsection 2 The test material will be classified as irritant and will require the appropriate "Xi" symbol if ocular lesions occur within 72 hours after exposure, persist for at least 24 hours and correspond to one or more of 

# n the following mean values in two or more animals: corneal opacity 2 or more iridial 1esion or more r-, redness of conjunctivae 2.5 or more 

### I l ; chemosis of conjunctivae 2 or more The 24, 48 and 72-hour readings for each anima1 wi 11 be used to ca1culate the mean values. Page 69 

# n

### -10­ PROJECT NUMBER: 163/131
5. 

### INTERPRETATION OF RESULTS (contd) Interpretation according to Annex VI Section 3.2.6, Subsection 2 (contd) In addition the following risk (R) phrases will be assigned to the test material, if appropriate, according to the criteria indicated as follows: R 36 ItIRRITATING 

### TO EYES" If, when applied to the eye of three rabbits, significant ocular lesions are caused which are present 24 hours or more after the 'instillation of the test material in two or more animals. Ocular lesions are significant if the mean of the 24, 48 and 72-hour readings comply with any of the following criteria: 

# n , )

- corneal opacity equal to or greater than 2 but less than 3 

### ,"""" j I c _

- iridial lesion equal to or greater than 1 but less than 2 n 

# IU conjunctival redriess equal to or greater than 2.5

- conjunctival chemosis equal to or greater than 2 R41 

### "RISK OF 

### SERIOUS DAMAGE TO 

### EYES" 

# 'I I , If, when applied to the eye of three rabbits, severe ocular lesions are caused in two or more animals which are present 24 hours or more after instillation of the test material. Ocular lesions are severe if the mean of the 24, 48 and 72-hour readings comply with either of the following,criteria: 

# n

- corneal opacity equal to or greater than 3 iridial lesion equal to 2 

# n

, ; 6. 

### ARCHIVES Unless instructed otherwise by the sponsor, all original data and a copy of the final report will be retained in the archives of Safepharm J I Laboratories for a period of ten years. After this period, the sponsor's instructions will be sought. 

# n 70

### I I 1..; -11­ 

# n PROJECT NUMBER: 163/131 l.J 

### 7. RESULTS Individual and group mean scores for ocular irritation are given in 

# n 

# i[ l; Tables 1 and 2. The indiyidua1 mean scores as required for the EC labelling regulations are presented in Table 3. Residual test material was noted around the treated eye of all animals 

## I throughout the study. 

# o 

# o Faint purple-coloured staining was noted in two treated eyes during the study.


# n Dulling of the normal lustre of the corneal surface was noted in one treated eye one hour after treatment. Heavy purple-coloured staining over approximately**~** of the eye prevented accurate evaluation of the corneal effects in one animal at the 24, 48 and 72-hour observations. Sodium fluorescein B.P. was placed in this treated eye at the 24, 48 and 72 hour observations to aid confirmation of corneal opacity. Iridial inflammation was noted in one treated eye one hour after treatment. Iridial inflammation developed in another treated eye at the 24 and 48-hour observations. No other iridial effects were noted.


# n Minimal to moderate conjunctival irritation was noted in all**tre~ted** eyes one hour after treatment. Minimal conjunctival irritation was noted in one treated eye at the 24-hour observation. The reaction increased to severe conjunctival irritation in one treated eye at the 24-hour observation with moderate conjunctival irritation at the 48-hour observation and c ) minimal conjunctival irritation at the 72-hour observation. 

# n No, ocular effects were noted in two treated eyes '48 hours after \. : treatment. Staining of the cornea and iris persisted in one treated, eye 

# n seven days after treatment. to ) 8. CONCLUSION The test mateda1,'-. 

# J produced a maximum group mean score in the range of 13.0 15.0 and was classified as a MILD TO MODERATE IRRITANT (CLASS 4 - 5 ON A 1 TO 8 SCALE) to the rabbit eye according to a modified Kay and Calandra classification system. The test material produced positive criteria in 1/3 rabbits but did not meet the criteria for classification as irritant according to £C labelling regulations. No symbol and risk phrase are therefore required. Page 71

### , ' I '. -12­ PROJECT 

### NUMBER: 163/131 

# n KEY TOT A BlEI 

# n n , I l ) d dulling of the normal lustre of the corneal surface 

# [ dydays 

# o F due to heavy purple-coloured staining over the cornea sodium

, , , ' fluorescein 

### B.P. was used to aid confirmation of corneal opacity 

### "' r l hr hour(s) IPR initial pain reaction Re residual test material around treated eye r I I 

### LJ 

## s faint purple-coloured staining 

# n ?s heavy purple-coloured staining over 

### %of the eye preventing accurate evaluation of corneal effect 

# n + one drop of ophthaine instilled into each eye 1 - 2 minutes before 11 dosing 

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

# n n \. J 

# n I. .J 

# n , I ( ... 

# n 

# ,. Page 72 I I

# ---- =J 

## ='J :=-:J ,

ACUTE EYE IRRITATION TEST IN THE RABBIT TAB L E 1 

### rNDIVIDUAL SCORES AND INDIVIDUAL TOTAL SCORES FOR OCULAR IRRITATION (**~1:**



# -----­

IPR = 

### 3 IPR = 0 

### IPR 0 

### Rabbit Number and Sex 28 

### FEMALE 80 

### MALE 84 

### MALE + 

### +**(Body\~eight** 

### Kg) (2.53) (2.50) (3.18) I 

### 24 48 72 7 1 24 48 72 24 48 72 

### Time ll.fter Treatment hr 

### hr hr hr dy hr hr hr hr hr 

### hr hr hr CORNEA E [)egree of Opacity oS ?sF ?sF ?sF oS dS 0 0 0 0 

### 0 0 0 

### F = 

### Area of Opacity 0 1 0 

### 4 0 0 0 0 0 0 0 rei I**~** 

### Score (E x F) x 5 0 5-20 

### 5-20 5-20 0 0 0 0 0 

### 0 0 0 0 

### ..... w (() I 

### -...) IRIS w­ 0 

### OS IS IS OS oS 1S 0 0 0 0 

### 0 0 0 Score (0 x 5) 0 5 5 0 

### 0 5 0 0 0 0 

### 0 0 0 

### CONJUNCTIVAE "1;l**~** 

### 2S 25 c... A 

### Redness 2 2 0 0 0 0 0 0 rr'l ('"') B 

### Chemosis 2 

### 3 2 0 2 0 0 0 0 0 -t z 

### 3Re 3Re ORe ORe ORe 

### 3Re ORe 

### ORe .0Re IRe ORe oRe 

### ORe c: co C 

### Discharge 3: tTl 

### Score (A + 

### B+ 

### C) x 2 14 16 8 4 

### 0 14 4 0 0 6 

### 0 0 0 

# .. ;:IC



## .... Total Score 14 26-41 18-33 9-24 0 19 4 0 0 6 0 0 0 en**~** ...... (.oJ 

## ....,.--­**~.--:J** 

### : _ J

# ~: ACUTE EYE IRRITATION TEST IN THE RABBIT
TAB l E 2 INDIVIDUAL TOTAL SCORES AND GROUP MEAN SCORES FOR OCULAR IRRITATION Individual Total Scores At: 

### Rabbit Number and Sex Hour 24 Hours 48 

### Hours 72 

### Hours 7 

### Days
28 FEMALE 14 26-41 18-33 9-24 0 '"0 (!J I cO
.....**~** (!l 80 

### MALE 19 4 0 0 I -.J 

## "'" 84 

### MALE 6 0 0 0

"'CJ :;I::J c:... 

### Group Total 39 30-45 18-33 9-24 0 0 /"1"1 n -I :z c:: 13.0 10.0-15.0 6.0-11.0 3.0-8.0 3: 0.0 o;l 

### Group Mean Score rn



### . Hour 24 

### Hours 48 

### Hours 72 Hours 7 Days :;I::J 

# ..

..... C'\ 

### no data, observation not perfQrmed w

....... 

### ..... w


## ....-,---. ::"--:J 

## =-=:J r ____

### J (

__.J ,__ -:::1 :

___

### J [ .J L
____

# ~: ACUTE EYE IRRITATION TEST 

### IN THE RABBIT TAB L E 3 

### INDIVIDUAL 

### & MEAN SCORES FOR CORNEA, IRIS 

### &CONJUNCTIVAE REQUIRED FOR EC LABELLING REGULATIONS Rabbit Number Time After Corneal Iridial Conjunctival Conjunctival 

### &Sex Treatment Opacity Inflammation Redness Chemosis (Bodyweight Kg) 28 FEMALE 24 Hours (1-4)?s 1 2 3 

### 48 Hours (1-4)15 1 

### 2 2 (2.53) 72 Hours (1-4)?s 0 1 1 

### Total 3-12 2 5 6 

### Mean 1.0-4.0 0.7 1.7 2.0+ 24 Hours 0 

### 0 1 1 'U 

### 80 MALE III I to 

### 48 Hours 0 0 

### 0 0 (]) 

### (2.50) 

# - I 

### 72 Hours 0 

### 0 0 0 U'1 --l lJ1 

### Total 0 

### 0 1 Mean 0.0 0.0 0.3 

### 0.3 84 

### MALE

(3.18) Total 24 Hours 48 Hours 72 Hours 0 0 0 0 0 

### 0 0 0 0 0 

### 0 0 0 0 0 0 

## " :::c 

### E! rr1 n -I 

### Mean 0.0 0.0 0,0 0.0 z c: 3: Cl:l m :::c 

# .. + 1s**~** 

### positive criterion 

### =heavy purple-staining prevents accurate evaluation of corneal opacity 

# - 0'1 w

....... 

## .... w 

## ....i I : ! : L ....


# n tJ 

### n 

### 1j Ii 

# l ] n I . l J

r-' ! I I



### . )

!**I~** C }

I 

# n l ; 

# n n Ii

- ) 

# n , I

: t ) 

# n n I 

## Ii c. ,I 

## n • I 

### l .i 

# n -16­ PROJECT NUMBER: 163/131



### A P PEN DIe E S

Page 76-17­ 

# n PROJECT NUMBER: 163/131 , ) 

### A P P E N D I X I 

### f' i I I; 

### DRAIZE SCALE FOR SCORING OCULAR IRRITATION 1. CONJUNCTIVAE 

# n (A) Redness (refers to p<ltpebrlll Vess.els normal and bulber conjlrlCtlvlle e)(cluding cornea and Ids) o Vessels definitely inJ.cted**abo~** no"..l 1 Hore diffuse, deeper crt..on red, indiviau.1 vessels not easily discernible 2 Diffuse beefy red 3 

## n I I l , 

### (B) Chemosis NQ swelling o Any swelling above nol"llllll (includes nictitating II!e!l'brane) 1 

# n**~.** J obvious swelling with portia! eversion of lids swelling with lids about half closed Swelling with lids helf closed to completely closed :2 3 4 n ; (C) Discharge I.. .J No discharge o 

# n Any amount different frOM r1Of"lIIIIl (don not include small I!IftO<XIts observed in imer canthus of roormel an;1IIII19) Discharge with moistening of the lids and hairs just**adjac~t** to lids 2 Discharge with moistening of the lids and hairs a considerable area around the eye 3 

### THE TOTAL SCORE (A 

### + B 

### + C) X 2 

### MAXIMUM TOTAL 20 

# " : ) 2. 

### IRIS (O) Valges 

# n I ; c J Normal Folds above OOrTIIIIl, cCJngelltion, ,"wIling, circuoncorneal injection o

(any or all of thHe or cOlllbil'Wltion of any thereof) iris still reacting to light (sluggish reacHon is positive) Mo reaction.to light,**hlleMOrrh~e,** gross destruction (any or all of these) 2 

### THE TOTAL SCORE D X 5 

### .MAXIMUM TOTAL 10 3. 

### CORNEA 

# n (E) Degree of Opacity (lnOst deN. area used) 110 opacity o Scattered or diffuse areas, details of iris clearly visible 1 n 

# Ij Easily discernible translucent areas, details of iris slightly obsclJre<l Opalescent areas, no details of iris visible, size of pupil barely discernible Opaque, iris invisible 2 3 4 

# r L) (F)**~rea** of Cornea involved One quarter (or less) but root zero 1 Grellte. than one quarter but less than half 2 Greater than half but lass than three quarters 3 Greater than three quarters, up to unole .re. 

# " 

# n THE TOTAL SCORE (E X F) X 5 page 77 

### MAXIMUM TOTAL 80-18­ 

### PROJECT NUMBER: 163/131 

# n A PPEN 0 I X I 

### I 

# n 

# n n \ J

'. ) 

# n I I , I \ .' 

# II 

# n n fJ n l j 

# n 

# n n n MODIFIED KAY AND CALANDRA INTERPRETATION OF EYE IRRITATION TEST

MAXIIU4 lIEAll' DESCRIPT10M RATING SCORE PERS1STENCE Of SCORE
. (AIIO CLASS) 

### ! 0.0to 0.5 Group mean total score at 24 hours :: 0 Non-lrritant (1) Group mean total score at 24 hours > 0 Practically non-irritant (2). 0.5to 2.5 Group mean total score lit 24 hO<Jrs
.. 0 Practically non-irritant (2) Group mean total score at 24 hours > 0 Minimal irritant 

### en 2.5to 15 Group mean total score at 48 hours:; 0 Minimal irritant (3) Group mean total score at 48 hours> 0 Mild irritant Ct.} 15 to 25 Group mean total score at 

### n hours" 0 H ild irritant (4) Group mean total score at 

### n hours> 0 Moderate irritant (5) More than half of the individual**~total** SCores at 7 days 10 or less Moderate irritant (5) 25 to 50 Group mean total SCore at 7 More than half of the individual days 20 or less total scores at 7 days > 10 but no individual total score at '7 days> 30 Moderate irritant (5) Hore than.half of the individual total scores at 7 days > 10 and any Individual score at 7 days > 30 Severe irritant (6) Group mean total score at 7 days ,. 2D Sellere irritant C6} More than half of the individual total scores at 7 days 30 or less Severe irritant (6) 50 to 80 Group mean total score at 7 More than half of the individual days 40 or less total scores at 7 days ,. 30 but no individual total scores at 7, days > 60 Severe irritant (6) More than half of the irdividuaL total scores at '7 days > 30 and individual total score at 7 days > 60 Very severe lrritant (7) Group mean total score at 7 days ,. 40 Very severe irritant (7) Hore than half of the individual less**~total** scores at 7 days 60 or Very severe irritant (7) 80 to 100 Group mean days 80 or total score at 7 Hore than half of the individual less total scores at 7 days ,. 60 but no irdividual total score at 7 days> 100 Very severe irritant (7) ,More than half of the individual total scores at 7 days ,. 60 and any individual total score at '7 days ,. 100 Extremely severe irritant (8) Grcup mean total score at 7 days ,. 80 Extremely severe irritant (8) 100 to 110 Group mean total score at 7 days 80 or less Very severe irritant (7) Group mean total score at 7 days > SO Extremely severe irritant (8) I?age 78 

# n

# n n 

# n fl I ! 

### , : 

# n l.!



# n

n I" ) 

# II 

# n

("""1 l.J 

### -19­ PROJECT NUMBER; 

### A PPEN D I X I I I



### TIlEDEPARTMENTOF

### HEAI.:I1I OF1HEGOVERNMENT OFTImUNTIED KINGDOM
GOOD LABORATORY PRACIlCE
STAn:MENTOF COMPIlANCE INACCORDANCEWITHDlRECllVE88/320 EEC UBORATORY SafePharm Laboratories Limited POBox No 4.5 Derb,v DEl 2BT

DATE OFINSPECIlON 31 January 1994 A general inspection for compliance with the Principles of Good LaborllflUY Practice wu carriedout atthe above laboratoryaspanofthe UKGLP Compliance Progrllmme. At. the time ofthe inspection no deviations were found ofsufficient magnitude to .affect thevalidiryofstudies perfonned atthese facilities.

Page 79 UKGLP MonitoringUnit D. F.Moore Din:aor 

### 163/131

# l J 

# 1 l , 

# in n n 

# n**c~** l MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

# n PROJECT NUMBER: 163/132 

# n 

# n n 1 I , , J 

### AUTHOR: O.J. Allen 

# n n t. I 

### STUDY SPONSOR: ISSUED BY: 

# n ____ --

____ __**----;r-~** _ J 

### Safepharm Laboratories Limited 

### P.O. Box No. 45 DERBY DEI 2BT 

### U.K. 

# n , I Telephone: DERBY (0332) 792896 

# n Facsimi1e: (0332) 799018 

### PAGE 1 OF 31 PAGES Te1ex: 377079 SAFPHM G 

# n Page 80 

# ,n

# li -2­ 

# n PROJECT NUMBER: 163/132 , J 

# n**~** QUALITY ASSURANCE REPORT 

### l .

n 

### I I L .' 

### The routine inspection of short term studies at Safepharm laboratories is Ll 

### carried out as 

### a continuous process designed to encompass all major phases of



## -**~I** 

### each study type once per-month. Dates of the most recently completed series of monthly inspections relevant to the study type(s) in this report are given below. 

# n Date(s} of Inspection and Reporting: 03, 04, 16, 24 

### May 1994 

# n This report has been audited by Safepharm Laboratories Quality Assurance Unit.

# n

# n It is considered to be 

### an accurate account of the data generated and of the procedures followed. 

# n Date of Report Audit:


## r: 17 

### June 1994

### ~ 1 

# n

I I c ; J.R. 

### Paternan C. Biol. I M. I. 

### Bio1. 

# n FOR 

### SAFEPHARM QUALITY ASSURANCE UNIT L • 

# n JUL_ 1994 

### DATE: 

# n

c J 

# n Page 81

### n t

### j 

# n 'i 

### I i LJ r: 

# l
1\ 

## I : '. J r 

## I

, ) I**~**

• 

### l j 

# n

­ )



# n 

# n

.... I ! ", J -3­ 

### PROJECT NUMBER: 163/132



### GLP COMPLIANCE STATEMENT

I. the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study. I 

### consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated. The work described was performed in compliance with the UK 

### Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GlP standards published as OECO Environment Monograph No. 45 

### (OCDE/GO(92)32); and are "in conformity with, and implement, the requirements of Directives 87/18/EEe and 88/320/EEC. These international standards are acceptable to the United States Environmental Protection Agency and Food and Drug Administration, and fulfil the requirements of 40 CFR Part 160, 40 eFR Part 792 and 21 

## erR Part 58 (as amended)
.**Q.s--~,q't**

.......... ,. ........ t .... , ....**~** ................. .. 

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

## ~~~~. '" 

## ...... DATE: D.J. Allen B.Sc. {Hans) Study Director for Safepharm Laboratories

82 

# c

# u -4­ 

# n PROJECT NUMBER: 

# In l ,

C 0 H T E H T S 

# n 

# o

... ) 1**~J** 

### SUrt4ARY 1. INTRODUCTION r; 

# lj 2. TEST MATERIAL 

# n 2.1 Description, Identification and Storage Conditions l J 2,2 

### Method of Preparation 

# r I : , , 3. TEST SYSTEM 3.1 

### Specification 

## r i 

### I ( ; 3.2 

### Husbandry 

### 4. PROCEDURE 

# l

# i 4.1 

### Selection of Concentrations for Main Study (Sighting Tests) 4.2 Main 

### Study 5. 

### EVALUATION Of DATA 

# n 6. 

### ARCHIVES 7. RESULTS 

### I r i 

### 7.1 Intradermal and Topical Sighting Tests "",.,, 7.2 

### Main Study 7.3 Bodyweight 

### 8. CONCLUSION


### n 

# lj
Page 83 

### 163/132



### PAGE

6 7 8 8 8 8 8 9 9 9 10 13 

### 14 

### 15 15 15 16 16


# oI : I '

...**~)** -5­ 

# n PROJECT NUMBER: 163/132 

# n C 0 H

### TEN T S {continued}
PAGE 

# n TABLES TABLE 1 Individual Skin Reactions in 

### Test Animals at Challenge 17 TABLE 2 Individual Skin Reactions in Control Animals at Challenge 18 

# n APPENDICES APPENDIX I 

### APPENDIX II APPENDIX III APPENDIX IV APPENDIX V APPENDIX VI 

# [J
APPENDIX VII

c. , 

# n APPENDIX VIII APPENDIX IX APPENDIX X APPENDIX XI APPENDIX XII 

## 1: L )



## r I I I ) 19 

### Intradermal Sighting Test

- Summary of Results 20 

### Topical Sighting Test For Induction Application

(48-hour Exposure)

- Individual Skin Reactions - 21 

### Topical Sighting Test For Challenge Application

(24-hour Exposure)

- Individual Skin Reactions - 22 

### Intradermal Induction

in Test Animals Individual Skin Reactions 23 

### Intradermal

in Control Induction Animals - Individual Skin Reactions 24 Topical Induction

in Test Animals - Individual Skin Reactions 25 

### Topical Induction

- Individual

in Control Animals Skin Reactions 26 

### Individual Bodyweights and Bodyweight Gains

of Test Animals 27 

### Individual Bodyweights and Bodyweight Gains

of Control Animals 28 

### Evaluation of Skin Reactions 29 

### Summary of Positive Control Data 30 

### GLP Statement of Compliance 31

Page 84

## l J

r"'\ -6­ I : 

### PROJECT NUMBER: 163/132 I ' , ' 

# n SUMMARY c ; 

# n STUDY SPONSOR 

# n PROJECT NUMBER 163/132


### TEST MATERIAL

1. A 

### study was performed to assess the skin contact sensitisation potential r-: 

### , I l j 

### of the test material in the albino guinea pig. The method used followed that ,described in the OECD 

### Guidelines for Testing of Chemicals No. 406 

### "Skin Sensitisation" (adopted 17 

### July 1992) 

### and Method 86 

### of Commission Directive 92/69/EEC 

### (which constitutes Annex 

### Vof Council' Directive 67/548/EEC)
. The results may be uied as a basis for classification and labelling under Annex 

### VI of Council Directive 67/548/EEC (as adapted to technical f! 

### progress by Commission Directive 91/325/EEC). I I '

### .. 2. 

### Ten test and five control animals were used for the main study. Based on the results of sighting tests, the concentrations of test mater;a1 

### for the induct;on and challenge phases were selected as follows: ,.-, 

# U Intradermal Induction 10% w/v in arachis oil 

### B.P. 

# r' Topical Induction 50% w/w 1n arachis oil 

### B.P. 

### , I l . 

### Topica1 Challenge 25% and 10% wjw in arachis oil B.P. 3. The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material 

# n dld not meet the criteria for classification as a sensitiser according to EC 

### labelling regulations. No risk phrase is required. 

# n

Page 85 

## I r 

## i "I I L ) 

# n -7­ 

### PROJECT NUMBER: 163/132 

# n MAGNUSSON 

### &KLIGMAN MAXIMISATION 

# n STUDY 

### IN THE 

### GUINEA PIG**~** I i 

### U 

# n 1. INTRODUCTION 

# n This study was performed to assess the skin**cont~ct** 

### sensitisation potential of the test material (Safepharm Standard Method Number EC 6). The method used followed the recommendations of the OECD Guidelines for the Testing of Chemicals No. 406 "Skin**Sens~tisationn** 

### (adopted 17 Jtily '1992) and Method 86 of Commission Directive 92/6g/EEC (which constitutes 

# n Annex Vof Council Directive**67/548/EEC)~** l : 

# n The results may be used as a basis for classifi:cation and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 91/325/EEC). The test system was chosen because the guinea pig has been shown-to be a suitable species for this type of study and is recommended in the test method. The strain used in these laboratories has been shown to produce a satisfactory sensitisation response using known positive sensitisers (see Appendix XI). The results of the study are believed to be of value in predicting the likel,Y contact sensitisation potential of the test material to man. 

# r l ! 

### The study was conducted in accordance with the internationally accepted general principles of Good Laboratory Practice and Safepharm Standard Operating Procedures. The study was performed between 21 April 1994 and 27 May 1994. 

# n l; 

# r Lj Page 86I i 

### tJ -8­ 

### PROJECT NUMBER: 163/132 

# n 2. 

### TEST MATERIAL 2.1 Description, Identification and Storage Conditions 

# [) The test material was supplied by the study sponsor as follows: 

# r
t. J 

### Sponsor's identification Chemical name 

## r. I ;

. ) 

# r Lot number**l~** 

# n Date received Description 

# r Container Storage conditions**'~** ; 

### Data relating to the identity, purity and stability of the test 

# n material are the responsibility of the sponsor. l ,I 2.2 Method of Preparation 

### For the purpose of this study the test material was freshly prepared as follows: l : 

### Intradermal Induction 10% w/v in arachis oil B.P. 10% w/v in a mixture of Freund's Complete 

# n Adjuvant plus distilled water (1:1)

,....., 

### Topical Induction 50% w/w in arachis oil B.P. I j 1 ) 

### Topical Challenge 25% and 10% IH/w 

### in arachis oil B.P. Determination by analysis of the concentration, homogeneity and 

# n stability of the test material formulations was not 'appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline. 

# ji 3. TEST SYSTEM 3.1 Specification 

### Twenty-three female, albino Dunkin-Hartley guinea pigs were supplied by David Hall Limited, Burton-an-Trent, Staffordshire, ('! , I 

### U.K. At the start of the main study the animals weighed 347

- 4109, l J 

### and were approximately eight to twelve weeks old. After a minimum acclimatisation period of five days, each animal was selected at 

# n random and given a number unique within the study which was written on a small area ·of clipped rump using a black indelible marker-pen. 

# n
L**~** Page 87

# n -9­ 

### PROJECT NUMBER: 163/132 

# n

I 

### 3. TESTSYSTEM (contd) l 

### 3.2 Husbandry 

# r: I ! l ) 

### The animals were housed in groups of up to two in solid-floor

,...., 

### polypropylene cages fUrnished with woodflakes. Free access to ! 

## II [ ! 

### mains tap water and food (Guinea Pig FOI Diet, Special Diets Services Limited, Witham, Essex, 

### U.K.) was allowed throughout the study. 

# o The animal room was maintained at a temperature of 20 23°C and relative humidity of38

- 62%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled 

# n l I )

### I



### .by a time switch to give 12 hours light and 12 hours darkness. 4. 

### PROCEDURE n 

### The method used for assessing the sensitising properties of the test I I 

### l J 

### material was based on the Guinea Pig**Maximisati~n** 

### test of Magnusson B. & 

### Kligman A.M., J. 

### Invest. Oermatol. (1969) 52: 268 276. 

## n 

# lJ 4.1 Selection of Concentrations for Main Study 

### (Sighting Tests) 

# n The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of 

# [1 guinea pigs were treated with various. concentrations of te$t material. The procedures were as follows: 

# n a} Selection of Concentration for Intradermal Induction

## ~ Four animals were intradermally injected with.preparations of 

## l . test material (1%, 5%, 10% or 25% w/v in arachis oil B.P.). 

# n The degree of erythema at the injection sites was assessed 

### l ; Ii 

### approximately 24, 48 and 72 hours and 7 days after injection
I , 

### according to the Draize scale shown in Appendix X. The degree i. :. 

### of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that did not cause 

# n L . 

### local necrosis, ulceration, severe irritation or systemic

.toxicity, was selected for the intradermal induction stage of 

# n the main study.

Page 88 

## r t.:

# u -10­ 

# n PROJECT NUMBER: 163/132 

# n 4. PROCEDURE {contd} 

### 4.1 Selection of Concentrations for Main Study (Sighting Tests) (contd) b) 

### Selection of Concentration for Topical Induction 

## r lJ 

### Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fifteen days earlier) were treated with four preparations of the test material (50%, 2S%, 10% and 5% w/w in 

### arachis' n 

### I ' oil B.P.). '. ! r , I 

### The highest concentration producing only mild to moderate LJ 

### dermal irritation after a 48-hour occlusive exposure, was 

# n

. ) 

### selected for the topical induction**sta~e** 

### of the main study. 

# n l; 

### c) Selection of Concentration for Topical Challenge Four preparations of the test materia1 (50%, 25%, 10% and'S% 

# n \ : 

### w/w in arachis oil 

### B.P.) of two guinea pigs 

### for a 

### were appl ied occlusively to the flanks period of 24 hours. These guinea pigs did not form part of toe main study. but had been treated 

# n identically to the control animals of the main study, 14. 

### The highest non-irritant concentration of up 

### to Day the test 

# n material and one lower concentration were topical cha11enge stage of the main study: selected for.the
4.2 

### Main Study A group of fifteen guinea pigs was used for the main study,ten l ; 

### test and five control. The bodyweight of each animal was recorded at the start and end of the study. (l 

### 1

### j Two main procedures were involved in the maximlsation test; (a) an induction of a response and (b) a challenge of that response. 

## r a) Induction 

### 11 Induction of the Test Animals: Shortly before treatment on Day 

## o the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. 

# n A row of three injections (0.1 ml each) of the mid-line. The injections were: was made on each side

Page 89 

# r 1 .'

# tJ -11­ 

### PROJECT NUMBER: 163/132 

# l n

# ! 4. PROCEDURE (contd) 

# n 4.2 

# n 

# n n l :. 'I 

## I ; l J 

# n I

## !

n 

## I ;

(I , I I I t • n , , ; i 

## ,

# . n , I ( J n i I i . '-. ) 

# n n ( J 

## r 

## !J Main Study (contd) a} Induction (contd) Induction of the Test Animals (contd) i) 

### Freund's Complete Adjuvant plus distilled water in the ratio 1:1. ii) 

### a 10% w/v dilution of test material in arachis oil B.P. iii) 

### a 10% w/v di1ution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water; Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (i.e. injection site iiJ 

### was evaluated according to the scale shown in Appendix X. One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation (50% w/w in arachis oil B.P.). The test material formulation (0.2

- 0.3 ml) was applied on filter paper (WHATMAN No.4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered wit,h an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours. The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches using the scale shown in Appendix X. Any other reactions were also recorded. Induction of the Control Animals: Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were: Page 90

# I J -12­ 

# II PROJECT 

### NUMBER: 163/132 \ ) 

### fl I 

### 1 j 

### 4. PROCEDURE (contd) 4.2 Hain Study (contd) 

# n a) Induction (contd) Induction of the Control Animals: (contd) 

# I' J 

# n i) 

### Freund's Complete Adjuvant plust distilled water 1n the ratio 1:1. ii) 

### arachis oil 

### B.P. 

# n iii} 50% 

### w/v formulation of arachis oil B.P. in a mixture of l -' 

### Freund's Complete Adjuvant plus distilled water in the ratio 1:1. 

# n The topical applications followed the same procedure as for 1 ; 

### the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals. 

# n b) 

### Challenge Shortly before treatment on Day 21, 

### an area, approximately 50 

### mm x 70 

### mm on 

### both flanks of each animal, was clipped free of hair with veterinary clippers.



# n A quantity of 0.1

- 0.2 

### ml of the test material formulation 

### (25% w/w in arachis oil 

### B.P.) was applied to the shorn right flank of each animal on a square of filter paper (WHATMAN No.4: approximate size 20 

### mm x 20 

### mm) which was held in place by a 

### strip of surgical adhesive tape (BLENDERM: approximate size 40 

### mm 

### x 50 

### mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% w/w in arachis oil B.P. was also similarly applied to a separate skin site on the right shorn flank. The


### n vehicle alone was similarly applied to the left shorn flank. , I 

### The patches were occluded with an overlapping length of 

### t j 

### aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in 

# n a double layer around the torso of each animal.

Page 91

## I ! . I

....j -13­ 

# n PROJECT NUMBER: 163/132 

### tj n 

### 4. PROCEDURE (contdJ I ' l 

### 1 4.2 Main 

### Study (contd) r 

### b) Challenge 

### (contd) 

### L) ! 

### I After 24 

### hours, the dressing was carefully cut using blunt

### n tipped scissors, removed and discarded. The challenge sites 

## l J 

### were swabbed with cotton wool soaked in diethyl ether to remove 

# n

! 

### residual material. The vehicle sites were**si~ilarly** 

### swabbed.
, \. ) 

### The position of the treatment sites was identified by 

### using a black indelible marker-pen. 

# n L J 

### Prior to the 24-hour observation the flanks were clipped· using 

# n T ' 

### veterinary clippers to remove regrown hair. l. j 

# n l i 

### c) Evaluation of Skin Reactions 

# n Approximately 24 

### and 48 hours after challenge dressing removal , the degree of erythema and oedema was quantified using the scale shown in Appendix 

### X. 

# fl Any other reactions were also recorded. 

# n 5. EVALUATION Of DATA 

# n The percentage of test anima1s that showed a more severe reaction at the , ! 

# n test material challenge site than the most severe reaction seen in the control animals, was compared with 

### the following scale:

\. j 

# n %

### of animals Classification of 

### sensitised sensitisation potential , ) 

### n 

## a**non-sensitisel~** 

## l ! > 0 

## - 8 

### weak sensitiser n > 8 - 28 

### mild sensitiser [ I J > 28 

## - 64 moderate sensitiser > 64


### - 80 

### strong sensitiser > 80

- 100 

### extreme sensitiser 

### n I ' 

### The data obtained may be used to classify the test material under the ( I 

### Council Directive 67/548/EEC. as amended by Commission according to 

## r; Directive 91/325/EEC, on the classification, packaging and labelling of**~** : L ) Page 92

### I I J • , ) -14­ 

### PROJECT NUMBER: 163/132 

# Jl 5. EVALUATION OF DATA (contd)

# I~ dangerous substances. These classification criteria are also in the UK Approved Code of Practice "Classification and Labelling of Substances Dangerous for Supply". n I l .:

. A 

### response in 

### at least 30% of 

### the animals is considered positive and 

### the risk phrase R 43**"r~AY** 

### CAUSE SENSITISATION BY 

### SKIN CONTACT" is 

### required. 

# n

;

. J 6. 

### ARCHIVES Unless instructed otherwise by the sponsor, all original data and a copy of the final report will be retained in the archives of Safepharm Laboratories for a period of ten years. After this period, the sponsor's instructions will be sought.



# n n ! j 

# n

n l ! 

# I: I J Page 93 

# l !I I I,

# n -15­ 

### PROJECT NUMBER: 163/132 

### r j

, ,**~** 7. 

### RESLILTS 7.1 Intradermal and Topical Sighting Tests 

# n A summary of the results of the intradermal sighting test and the individual skin reactions observed in the topical sighting tests 

# n are given in Appendices I, II and III. 

# n

.J Based on these results, the following concentrations were selected' for the main study: , 

# n n Intradermal Induction 10% w/v in arachis oil B.P. Topical Induct;on 50% w/w in arachis oil B.P. { , Topical Challenge 25% and 10% w/w in arachis oil B.P., 

### 7.2 Hain Study a} Skin Reactions Observed After Intradermal Induction 

# n Individual reactions observed at the intradermal induction sites of test and control group animals are given in Appendices IV and V, 

# n Staining was noted at the intradermal induction sites of all test group animals at the 24 and 48-hour observations. The 

# n sta'ining prevented accurate evaluation of erythema in all test group animals at the 24 and 48-hour observations. 

# n n Very slight erythema was noted at the intradermal induction sites of two control group animals at the 24 and 48-hour L j observations. 

# n b) Skin Reactions Observed After Topical Induction Individual reactions observed at the topical induction sites of 

# n test and control group animals are given in Appendices VI and VI I. 

# n 

# n Staining \"as**not~d** at the induction sites of all test group animals at the and 24-hour observations. The staining prevented accurate evaluation of erythema in all test group animals at the 1 and 24-hour observations. 

# n Page 94i I 

## l J 

# n -16­ 

### PROJECT NUMBER: 163/132 

# n 7. 

### RESULTS (contd) 

# n 7.2 Main Study (contd) b} Skin Reactions Observed After Topical Induction (contd) 

# r l :

) No 

### skin reactions were noted at the treatment sites of control group animals at the 1 

### and 24-hour observations. 

# n
, j 

### c) .skin Reactions Observed After Topical· Challenge 

# n Individual reactions at the challenge sites of test and control group animals are given in Tables I 

### and 2. 

# n I. i 

### Staining was noted at the challenge sites of all test and control group animals at the 24 and 48-hour observations. The 

# n

. ) 

### staining did not affect evaluation of**ski~** 

### responses. 

# n No skin reactions were noted at the challenge sites and vehicle control sites of the test or control group animals at the 24 

# n and 48-hour observations. 

# n 7.3 

### Bodyweight , J 

### Individual bodyweights and bodyweight gains of test and control 

# n group animals are given in Appendices VIII and 

### IX., Bodyweight gains of guinea pigs in the test group, between Day 0 

# '[I and Day 24, were comparable to those observed 'in the control group animals over the same period. 

# n 

# n 8. CONCLUSION 

### The test material,

# 1=--'~prOduced a 0% (OlIO) 

### sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin. 

# n, The test material was a150 classified as a non-5ensitiser according to 

# n EC labelling regulations. No risk phrase is therefore required. 

## r. I ;

Page 95 

### :1. l.j**----------~--** ---------

-- ---- ---**--~- ~** 



### MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG TAB L E 1 

### INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CHALLENGE CHALLENGE CONCENTRATIONS:**25~** AND**1~** 

### w/w VEHIClE: ARACHIS OIL B.P. Skin Reactions (Hours After Removal of Dressing) 24 hours 48 hours Animal 25% 10% Vehicle 25% 10% 

### Vehicle Number Er Oe Other Er De Other Er Oe Other Er Oe Other Er Oe Other Er Oe Other 0 0 

### STA 0 0 

### STA 0 0 0 

### 0 STA 0 

### 0 STA 0 

### 0 2 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 I -..c 3 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 

# - I <..0 0'1 4 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 5 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 6 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 7 

### 0 0 STA 0 0 

### STA 0 0 0 0 

### STA 0 

### 0 STA 0 0 "C:I 8 0 

### 0 

### STA 0 0 

### STA 0 0 

### 0 0 

### STA 0 0 

### STA 0 0 9 0 

### 0 STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 

# E**~** 10 0 

### 0 STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 -I :II!: 

### i o:l 1"1"1 ;a::::I 

# .. Er 

### =erythema STA 

### = staining at 

### De 

### =oedema - = 

### no other dermal reactions noted 

# ­**~** W 

# - Nt---_..._- ­**~1** 

## :-:::::J ,- =J**:~:.-_) :=~]** **~-~** 

# \..

____ J: MAGNUSSON 3: 

### KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG TABLE 2 INDIVIDUAL SKIN REACTIONS IN 

### CONTROL ANIMALS AT CHALLENGE CHALLENGE CONCENTRATIONS:**2S~** 

### AND 10% w/w 

### VEHICLE: ARACHIS OIL B.P. Skin Reactions (Hours After Removal of Dressing) 24 hours 48 hours Animal 25% 10% Vehicle 25% 

### 10% 

### Vehicle Number '"d 

### Er Oe Other Er Oe Other Er Oe Other Er Oe Other Er Oe Other Er Oe Other PJ 

### , to OQ (l) 

### """ I ill --.l 11 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 

### 12 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 13 

### 0 0 

### STA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA 0 0 14 0 0 

### SlA 0 0 

### STA 0 0 0 0 

### STA 0 0 

### STA O· 0**~** 

# E ,.., 0 0 n 15 0 0 

### STA 0 0 

### STA 0 

### o. STA 0 0 

### STA 0 0 -I :z 

# i**~** Er 

## =erythema STA 

### = staining 

# ­ Oe 

### =oedema no other dermal reactions noted w

....... W 

## """ N

### -19­ PROJECT NUMBER: 163/132


# n

n I l..

### i 

# r1 u A P PEN 0 ICE S· 

# n \ , 

# n

n I ! c • 

# n n
r, 

### [I " I r-. I
l j 

# I I l. ,J Page 98 

# iU "

### ==:J 

### =1**~** r-­ 

# =1**~~):** MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG APPENDIX I 

### INTRADERMAL SIGHTING TEST

- SUMMARY OF RESULTS VEHICLE: ARACHIS OIL B.P. Concentration of Animal Time of Evidence of Local Evidence of Test Materia 1 ldentlfication Observation Necrosis· Systemic Toxicity 

### (% 

### w/v) 24 hours None None 48 hours None None A 

### 72 hours None None 7 days None None 24 hours None '1J 

### 48 hours None III B 5 to (1) 

### 72 hours None 7 days None I.D I.D 

### 24 hours None 48 hours 10 

### None C 

### 72 hours None 7 days None None None None I Nt 

### None Q I 

### None None None None '"1:7

## ~ 24 hours None None 1"1"1 

### 48 hours None None D 25 

### 72 hours None None :z:



### 7 days Eschar None**~** m**~** 

### The concentration of the test material selected for the intradermal induction stage of the main study was 10% w/v I-' 

### in arachis oil B.P. en**~** 

### ..... W N 

### il

__l '. 

### =:J :. J**~) ~J** **~** ) 

# : ) 

### :_J 

# (**---->-:~__;:** MAGNUSSON & 

### KLIGMAN MAXIMISATION STUDY IN 

### THE GUINEA PIG 

# - A P PEN D I X I I TOPICAL SIGHTING TEST FOR INDUCTION APPLICATION (48-HOUR EXPOSURE) INDIVIDUAL SKIN REACTIONS VEHICLE: ARACHIS OIL B.P. Skin Reactions Concentration of Animal (Hours After Removal of Patches) Test Material Identification 1 

### 24 48 (% 

### w/w) Er Oe Other Er 

### Oe Other Er 

### De Other

I )lJ '0 50< 

### ?s 1 1 0 

### STA 0 0 

### STA N 1.Q 25 

### ?s 0 1 0 

### STA 0 0 

### STA 

### ..... (D 

### E 10 

### ?s 0 0 0 

### STA 0 0 

### STA I 0 1-' 5 ?s 0 0 0 

### STA 0 0 

### STA 0 50< 

### ?s 0 I 0 

### STA 0 0 

### STA 25 

### ?s 0 I 0 

### STA 0 0 

### STA F 10 

### ?s 0 0 0 

### STA 0 0 

### STA 5 

### ?s 0 0 0 

### STA 0 0 

### STA -0 

# 8 IT! n 

### Er erythema STA staining -I 

### Oe oedema ?s staining prevents accurate evaluation of erythema no other dermal reactions noted 

# S IT!**~** 

### < Maximum attainable concentration SUitable for topical application 

# ..

..... Ol 

### The concentration of the test material'selected for the main study topical induction was 50% 

### in arachis oil (.oJ

......... B.P. ' 

### ..... (.oJ N------., r­ 

### .=::-J 

### :_-_J**~ ~.-::-:J**  =)**-~~** 

### , J -,--I 

### ,___J 

## :-:=J**~-~** C) r

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### J l

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## :=J**~--=---=-) ~-=-J** **~-=-~ :-=-~**  

# =J

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# ----­**~_** 

### ' : MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG**.---------<~** 

### A P PEN 0 I X I I I TOPICAL SIGHTING TEST FOR-CHALLENGE APPLICATION (24-HOUR EXPOSURE)

- INDIVIDUAL SKIN REACTIONS VEHICLE: ARACHIS OIL B.P. Skin Reactions Concentration of Animal (Hours After Removal of Patches) Test Material Identification (% 

### w/w) 1 24 48 

### Er Oe Other Er Oe Other Er De Other '"d PJ to (]) 50< 2 1 

### STA 1 0 

### STA 0 0 

### STA N I G 25 1 0 

### STA 0 0 

### STA 0 0 

### STA N I o f-' 10 2 1 

### STA 0 D 

### STA 0 0 

### STA f-' 5 - 2 1 

### STA 0 0 

### STA 0 0 

### STA 50< 1 0 

### STA 1 0 

### STA 0 0 

### STA H 25 1 0 

### STA 0 0 

### STA 0 0 

### STA 10 1 0 

### STA 0 0 

### STA 0-· 0 

### STA "0

5 1 0 

### STA 0 0 

### STA 0 0 

### STA

## ~**~** -i 

### Er erythema no other dermal reactions noted :z 

### De oedema STA very slight blue/purple-coloured staining 

### i c::I ITI < 

### Maxirnum attainable concentration suitable for topical application 

# ..

.... The concentrations of the test material selected for the main study topical challenge were 25% 

### and 10% 

### w/w in C7I 

### arachis oil B.P. 

# - W N

# ----------- ,---'l**~=)** 

## :_=J :_-__J :-=:J 

# c-]
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\ :: MAGNUSSON &. KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

...**-,--~** 

### A P P L N 0 I 

### X I V INTRADERHAL INDUCTION

- 

### INDIVIDUAL SKIN REACTIONS 

### IN TEST 

### ANIMALS INTRADERMAL INDUCTION CONCENTRATION: 10% 

### w/v 

### VEHICLE: ARACHIS 

### OIL B.P. 

# ---- ..; '



### Skin Reactions at Observation Time: Animal Number 24 hours 48 

### Left Side Right Side Left Side Right Side
1 

### ?5 ?s ?5 15 I 2 

### ?5 ?5 ?$ 

### ?s N W I f-' 3 

### ?5 ?s ?s ?5 o N 4 

### ?s 15 1s 15 5 ?5 

### 15 ?s 15 6 

### '?5 15 ?s , " '?$ 7 

### 15 ?s ?5 "'0 8 ?s 

### ?s ?5 ?s 

# 8 9 

### ?5 ?s ?s ?5 

# 6 10 

### ?5 ?s 1s 15 z 

### i 1:1:1 m ;a 

# ..

..... ?s staining prevents accurate evaluation of erythema

## ~ ...... W N,---­**=:~] ~--:---:J**  

### ,_ 

### J**C-=-~J** 

## ::=:J C=:J l

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### J**,,--<~**

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### : MAGNUSSON &KLIGMAN 

### MAXIMISATION STUDY IN 

### THE GUINEA 

### PIG A PPEN 0 I X V INTRADERMAL INDUCTION

- INDIVIDUAL SKIN REACTIONS IN 

### CONTROL ANIMALS VEHICLE: ARACHIS OIL B.P. Skin Reactions at Observation Time: Animal Number 24 

### hours 48 hours hj 

### Left Side Right Side Left Side Right Side Pl I N \.Q (]) .po. I I-' o w 11 0 0 0 0 

### 12 0 0 0 0 13 0 1 0 1 "'C :;0 14 0 0 0 0 a e:.­ rr"! n 

### 15 0 1 0 -t :z c:: :3: OJ rr"! :;0

....... m w "­

....... W N,-­ 

### ;-:.-:-J 

### =-:J**~.-:J** 

## :---:-J =::J :._ ) 

### =.=:1 :=--..::J**~-~** 

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### : MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

### A P PEN D I X V I TOPICAL INDUCTION - INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS INOUCTION CONCENTRATION: 50% 

### w/w 

### VEHICLE: ARAOIIS OIL B.P.
Animal Number Er Skin Reactions 1 hour

De Other (Hours After Removal 

### of Dressing) 24 

### hours Er De 

### Other 1 ?s 0 

### ?s 0 2 

### ?s 0 

### ?s 0 N I U'1 

### , 3 

### ?s 0 

### ?s 0 I-' 0**~** 4 

### ?s 0 

### ?s 0 5 

### ?s 0 

### ?s 0 6 

### ?s 0 

### ?s 0 7 

### ?s 0 

### ?s 0 -u 8 

### ?s 0 Ts 0 

# E m 9· 

### ?s 0 

### ?s 0**~** 10 

### ?s 0 

### ?s 0 :z 

### i 

# = I"T1 :::tI 

### Er erythema no other reactions noted

...... en Oe 

### oedema· ?s staining prevents accurate evaluation of erythema**~** ...... W Nr---,

.....____.J 

### =.J 

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### - L._ '-- 

### :.J**~_** 

### J C=J



### ): MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN 

### THE GUINEA PIG
, 

### A P PEN D I X V I I TOPICAL INDUCTION

- INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS VEHICLE: ARACHIS OIL B.P. Skin Reactions (Hours After Removal of Dressing) , Ani Number 

### 1 hour 

### 24 hours Er Oe Other 

### Er Oe Other 'U (lJ I to N ro 0"1 I f--' 11 

## o o 0 0 o (J1 12 

## o o 0 0 13 

## o o 0 0 14 

## o o 0 0 '"0

## ~ 15 

## o o 0 0 !T1 

### Sl :z 

### i co ITl :;:g 

### Er er'ythema no other dermal 

### reactions noted 

# .. Oe oedema 

### ..... 0"1**~** 

## .... W N-----------,**:--~** 

## =--:-]**~=:1** = ..**_~J ~.,_J**  :"
__

### J r 

# =J**~---<** 

### : MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

# - A PPEN 0 I X V I I I INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF TEST ANIMALS fiodyweight (9) Bodyweight (g) Animal Number Day 0 _Day 24 Increase
1 347 547 200 I N 2 394 626 232 

### ..... I f-' 0 3 352 553 201 0'\ 4 353 592 239 5 402 622 220 6 382 572 190 7 363 563 200 -0 

### E5 8 392 '577 185 c:... J"T'I ("'l 9 379 593 214 -r .z 10 407 626 219 c::: 3: t:l:1 J"T'I ::0 

# ..

..... 01 W 

### -.....
..... W N

# ------------ :::=-::J 

### :::-=J 

## :=-:J**'_-_-=-.~** 

### :::=J ==:J



### MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN 

### THE GUINEA PIG
. A P 

### PEN D I X I X INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF CONTROL ANIMALS Bodyweight (g) Bodyweight (g) Animal Number Day 0 Day 24 Increase

J N co 

### , f-' 0 11 410 607 197 -..j 12 378 662 284 13 380 ,600 

### 220 14 409 623 214 '"'0**~** c.... 

### 15 376 536 160 ,.." n 

# .... :z c ::J: c::J m 

# ..

..... O'l W

.......... ...... W NI 

# I:

, ,. -29­ 

### PROJECT NUMBER: 163/132 

# II n 

### A PPEN D I X 

# x 

# n l; EVALUATION OF SKIN REACTIONS* 

# n n Erythema and Eschar Formation l ) No erythema .........................•.......................•........... 0 Very slight erythema (barely perceptible)
....................'
............ 1 Wel1 1 defined erythema
........**~** ....•....•.............,.. ,.••. ,•....'
...... 2 

# n l J Moderate to severe erythema ......•.•...............'..................... 3 Severe erythema (beet redness) to slight eschar formation

(injuries in depth)
........ , .......•................................... 4 

# n l J Oedema Formation No oe<lema
............ , ............................" ......•,............ 0 Very slight oedema (barely perceptible)
...........'
..................... 1 Slight oedema (edges 

### of area well-defined by definite

raising) .....•...............................•...,. :,',
............., .... 2 t·loderate oedema (raised approximately 1 millimetre)
..**~** ..............;... 3 I '--1 Severe oedema (raised more than 1 millimetre and extending 

### l )

1 beyond the area of exposure)
.. , ....•.•................................. 4



### * Oralze J.H., (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, Nationa1 Academy of Sciences, Washington D.C. p.31 .n 

# l: 

# n l J 

# n Page 108 

# n uC:-::-J 

### =---=:-J**~ ~**  

### , ..=:1**~=-=-:J** 

### ( MAGNUSSON 

### &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

### A PPEN D I X X I SUMMARY OF POSITIVE CONTROL DATA FOR THE MAGNUSSON AND KLIGMAN MAXIMISATION STUDY (JANUARY 1993 TO MARCH 1994) Project Number
039/44

I-' o 

### 039/45 '-D



### 039/62

039/70 Date Start

., . 

### 21/01/93

22/01/93

19/10/93

01/03/94 Date End
14/02/93

15/02/93

12/11/93

25/03/94 Number of Animals* Test

10 Control 10

10 10

10 5



### 10 5 Positive Control Material

(X-

### hexy,cinnamaldehyde Tech 85%
2-mercaptobenzo-
. thiazole

(X-

### hex'acinnamaldehyde Tech. 85%

..


### 2-mer'captobenzothiazole, - 

### Concentration Induction



### * All animals supplied by David Hall ted.**Burton~on-Trent)** 

### Staffordshire, Intradermal 25% in arachis oil 

### B.P.
5% in arachis oil 

### B.P.
25% in arachis oil' 

### B.P.
10% in arachis oil 

### B.P. Topical 100%

50% in arachis oil B.P.
100%

50% in 95% 

### aqueous ethanol U.K. Challenge 100% and 75% in arachis oil 

### B.P. 50% and ' 25% in

arachis oil B.P. 100% and 75% in arachis oil 

### B.P. 

### 25% in 95% aqueous ethanol**~J**



### Incidence of Sensitisation

30% {3/10}

I W o I 

### 80% 

### {SIlO}

70% (l/10) 

# 8 -c
m n -I :z 

### 80% 

### (S/lO) 

### i c:l m


# .. ;:tl

...... en w 

# ,

..... W NI I · ! l -' 

### -31 

# n 

# n n

, ) 

# n n li

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.

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# r I , 

# n l / 

# n 

# n n n l ; 

# r: 

# n PROJECT NUMBER: 163/132 

### A PPEN D I X X I I

•

lliE

### DEPARTMENTOFHEAL1H OFTHE GOVERNMENT OFTHE UNITED KINGDOM
GOOD LABORAroRYPRACIlCE SfATEMENTOFCOMPlIANCE £NACCORDANCE WITH DlRECllVE 88/326 EEe LABORAmRY SafePharm Laboratories Limited POBox No 45 Derb.v 

### DEl 2ET

DAtF, OF INSPECIlON
31 January 1994 A genem inspection for compliance with the Principles ofGood LaboraroIJ Pradice was c:a.rried outatabe above laboratoryas pa,rtofthe UKGLPComplianceProgramme At the time ofabe inspc:ction nO deviations wece found

## orsufficient magnitude to affect the validityofsrudies performed atlhese facilities.

D.F. Moore Director UKGLP MonitoringUnit Page 110



## ..

# I j 

# n 

# n 

# n
n l J 

# I r"

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## j

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# n I J r' J 

### I t J 

# n

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## i rj 

## I I 1 

### n i I l!

# (--~ REVERSE MUTATION ASSAY -AMES TESTUSING SALMONELLA TYPHIHURIUH 

### AND ESCHERICHIA COLI 

### PROJECT NUMBER 163/133

Date Started: 22 

### March 1994 

### Experimental Procedures:

STUDY SPONSOR:**-~**

# L)-~I

PAGE I OF 

### 37 PAGES 

### Date Completed: 12 May 1994

ISSUED BY: Safepharm Laboratories Limited P.O. Box No. 45 DERBY DEI 2BT 

### U.K. Telephone: DERBY (0332) 792896 Facsimile: (0332) 799018 

### Telex: 377079 SAFPHM G

111

### l) -2­ 

# n \ , 

### PROJECT,NUMBER: 163/133

QUALITY ASSURANCE REPORT

r""', 

# lj
The routine inspection of short term studies at Safepharrn Laboratpries**,i~** 

### carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently completed series 

# n of monthly inspections relevant to the study type{s) in this report are given 'below. 

### Date(s} of 

### Inspection and Reporting: 07, II, 13, 20, 26 

### April 1994
Th'is**repOi~t** 

### has been audited by Safepharrn Laboratories Quality Assurance Unit. 

# r It is cons'idered to be on accurate account of the data generated and of the procedures followed. 

# rJ Date of Report Audit: 26 ,May 1994



### J.R. Paternan C. Biol., 

### M.I. B101. 

# "'fj£**~** FOR SAFEPHARM QUALITY ASSLIRANCE UNIT 

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

# ... 

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

# [J
10. JUN. 1994 

### DATE:
.. .. .. .. 

## . .. .. .. 

### . .. "

### .......................... -

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

# I0 

# n Page 112

# l -3­ 

### PROJECT NUMBER: 163/133 

# o GLP COMPLIANCE STATEMENT

r 

# lJ

r I, 

### the undersigned, hereby declare that the objectives laid down in the 

# lj protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study, I 

### consider the data generated to be valid. This repOl't fully·and accurately reflects the procedures used and data generated. 

# n lJ 

### The work described was performed in compliance with the UK Principles of Good r 

### Laboratory Practice (The United Kingdom Compliance Programme, Department of**~** ". 

# l ) Health 1989). These Principles are in**~ccordah~~** 

### with GLP standards published as OECD 

### Environment Monograph No. 45 

### (OCDE/GD(9Z)32); and are in conformity with, 

### and implement, the requirements of Directives 87/18/EEC and 88/320/EEC. 

### These internationa1 

### standards ara acceptab1e to. 

### the-·Japanese Ministry of 

### Agriculture, Foresti"y and 

### Fisheries (59 NohSan, Notification No. 3850, 

### Agricultura-I Production Bureau)

- confirmed 

### py an Arr,angernent between the Ministry and the 

### UK Department of 

### Health; and the Japanese Ministry of 

### Health and Welfare (Notification N.c. 313, 

### Pharmaceutical Affairs Bureau "as· 

### amended; and Kanpogyo No. 39**Environmental'Agency~** 

### Yakuhatsu No. 229). 

# I' \ _ 1I



# .., .......**9.Sl~!.~**

# ....: ......... DATE: 

# n P.W. Thompson H.N.C. 

## L, Study Director for Safepharm Laboratories 

# n , I

I 

# r:I l .•**l~** 

# U 

# r l , ......'.c;
... ....... ..

### !!:T.'-!":
......

Page 113

# u -4­ 

# n

, I )i 

### PROJECT NUMBER: 163/133 

### CONTENTS
PAGE 

### SUl91ARY 5 

### INTRODUCTION 6 

### TEST MATER1AL 

## r METHODS 7 

## 11 U 1 ' 

### 1. Tester Strains 7 n 

### 2. Preparation 

# of Test 

### and Control Materials 7 

# U 3. 

### Microsomal Enzyme Fraction 8 

## I 4
... S9 

### Mix**~nd** 

### Agar : 8
.
t..**~**

.1 

## i 5. 

### Test Procedure 9 

### a) Preliminary Toxicity Study 9 . 

# [1 b)
.. Mutation Study 9 

### 6. Interpretation of Results 11
I . 

### RESULTS 

# r
t. ;. 

### a} Preliminary Toxicity Study 12 b) Mutation Study 12 

### CONCLUSION

.13 

### ARCHIVES 13 

### REFERENCES 13 

# n KEY TO TABLE OF TEST RESULTS 14 r 

# II TABLES 1 

### Experiment 1 - Without Metabolic Activation 15 2 

### Experiment 1 - With Metabolic Activation 16 3 

### Experiment 2 - Without Metabolic Activation 17 

### n 

### I 4 

### Experiment 2 With Metabolic Activation· 18

i ',>-.1 APPENDICES 19 

### Report of Results in Mutagenicity Test Using 20



### Nicro-organisms (Industrial Safety and Health Law) II 

### Dose Response Curve 33 

### III GLP Certificate of Compliance 37
114**~** 

### -5­ I I 

### PROJECT NUMBER: 163/133 C J r. , I 

## U REVERSE MUTATION ASSAY "AMES TEST" 

# o USING SALMONELLA TYPHIMURIUH AND ESCHERICHIA COLI. rJ 

# LJ SUMMARY 

# n 1. 

### Salmonella typhimurium strains TAl535, . 

### 'TA1537, . 

### TA9S and TAlOO and I : I"
.....

: 

### Escherichia coli strain WP2uvrA- were treated with**No~** 

### 650 Violet by the (\ 

### Ames' plate incorporation method at five dose levels, in tripHcateboth I i t**~** 

### with and without the addition of a rat liver ,homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to 

# c the guide1ines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW,MOL 

### and MAFF. It 

# n also meets the requirements of the OECD, HC and USA, EPA (TSCA) guide­ 

### , .' 

# n lines. The dose range was determined in a preliminarytox.icHy assay and was 8 to 5000 Jig/plate in the first,experiment. The experiment was t ) 

### repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In 

### this case the dose range of No. 650 Violet was**312~5** 

### to 5000 Jig/plate.' 

## Ii l; 2. The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. 3. All positive control chemicals produced marked increases in the numbers 

### r of revertant colonies, both with and without the metabolising system. I 

### I 1..1 

### 4. No. 650 Violet caused no visible reduction in the growth of the bacteriil
. lawn at any dose level either with or without metabolic activ'ation. No.. 650 Violet was therefore tested up to the maximum recommended dose level of 5000 Jig/plate. Aprecipitate was**observ~d** 

### at and above 1000 pg/plate in both experiments, this did not interfere with the scoring of

## r~ revertant colonies. l . 

### 5. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of No. 650 Violet, either with or without metabolic activation. No. 650 Violet was found to be non-mutagenic under the conditions of this test.
,--., ; . 115 I ' 

### ,-,-6­ 

### PROJECT NUMBER: 163/133 ( 

### ---.-­ REVERSE HUTATION ASSAY "AMES TEST" 

# o USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI r. I ! , , LJ 

### INTRODUCTION 

# n , , 

### This study was conducted according to Safepharm Standard Method Number JMOL '-

### . 

# c OIG and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study .was based, on the in vitro technjque described by Ames and his co-workers (1, 2, 3) and Garneret al (4)
,-. 

### lnwhich mutagenic activity is assessed byexpostng histidine auxotrophs of ) ! \ ' 

### Salmonella typhimurium to various concentrations of**the~est** 

### material. This method conforms to the guidelines for bacterial mutagenicity, testing 

# o published by the major Japanese Regulatory Auth.or,i,t,ies incl;udingMITI, MHW, MOL and MAFF. This method also conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 47l r 

### .Method B14 i.n EEe Commission Directive 92/69/EEC and the USA, EPA (TSCA) guidelin,es., 

# o These mutant strains of Salmonella are incapable of synthesising histidine and are, therefore, dependent for growth on an external source of this 

## " 

# U particular amino acid. When exposed to a mutagentc agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected 

# o by their ability to grow' on a histidine deficient mf)dium. Using various strains of this organism, revertants produced after exposure to a**che~ical** 

# fl mutagen may arise as a result of base-pair substitution in the genetic , J 

### material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by 

### chemical and physical agents several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB) which renders the organism incapable of DNA excision repai( and a deep rough mutation (rfa) which increases the permeability of the cell wall. In addition, a mutant strain of Escherichia coli (WP2uvrA-), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the Salmonella strains. Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver microsomal fraction (S9) obtained from rats previously treated with a 

# ·c compound known to induce an elevated level of these enzymes. 116

# n L.J
r 

# U 

# o o

r-.. 

### I I 

### U 

# n _J**I~** 

# U (, , I 

## L·, 

# n

... ) 

### n 

## U
n 

# U r1 

### I : { } 

# n

n I 

## l
{ > 

# 'l 

# L; 

# n**~)**

r. 

# I ; -7­ 

### PROJECT NUMBER: 163/133 TEST MATERIAL --"'\ 

### J Sponsor's identification Chemical name
Label (if different from above) Lot number Purity Date Received Description Container Storage conditions :{

# - Data relating to the identity, purity and the re'sponsibil1ty of the sponsor. METHODS 1. Jester Strains 

### stability of the test materia1 are

Salmonella typhimurium TA1535, TA1537, TA9S and iAIOO Escherichta coli WP2uv\"A

­ These strains were obtained. from. the British Industrial Biologica1 Research Association on 14th August 1987 

### and were**~tored** 

### at 196"C in a Statebourne liquid nitrogen freezer, Il)!>del SXR 34. Prior to being used, characterisation checks w('re carried out to detet'mine the amino-acid requirement, presence of 

### rfa, 

### R factors, uvr mutation and the spontaneous reversion rate. In this assay, overnight sub-cultures. of 

### the appropriate coded stock cultures were prepared in nutrient broth (OxQid limited) and incubated at 37'C for 10 hours. 2. Preparation 

### of Test 

### and Contro"' Materials 

### No. 650 Violet was accurately weighed (with an allowance made for a purity of 96% in both experiments) and dissolved in dimethyl sulphoxide and appropriate dilutions made. Analysis of the test material preparations for homogeneity, stability and concentration 'is not a requirement of 

### the Test Guideline and is therefore not appropriate. Page 117-8­ 

### PROJECT NUMBER: 163/133 

## [J METHODS (contd) 2. 

### Preparation of Test and Control Materials (contd) f' \1 ,-.J 

### Negative and positive controls were used in parallel with the test material. A solvent treatment group was used as the negative control and the positive control materials were as follows: 

# n N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) 2 

### ,ug/plate for WP2uvrA-, u 

### 3 ,ug/plate for TAIOO and 5 ,ug/plate for TA1535 9-Aminoacridir.e (9AA) 80 ,ug/plate for TAI537 

# I'I I L.' 

### 4-Nitroquinoline-l-oxide 

### (4NQO) 0.2 ,ug/plate**f~r** 

### TA98 In addition the materials 2-Aminoanthracene (2M) 

### and benzo(a)pyrene (BP) were used in the S9 series of plates at the following ,..-, 

### con'centrations: 

## II,
'- : 

### 2AA at 2 ,ug/plate for TA1535 and 10 ,ug/plate for WP2uvrA­ 

# [j BP at 5 ,ug/plate for TAlOO, TA1537 and'TA98 

# C 3. 

### Microsomal Enzyme 

### Fraction Lot No. Aro. S9/24/03/94, prepared on 24/03/94, and 03/Q2/94 prepared on 03/02/94, were obtained from the British Industrial**~iological Re~earch**  " . ".. '...
. 

### Association on 12/04/94. They were prepared from the livers of male 

# n I
..' 

### Sprague-Dawley rats weighing

- 200g. These had received a single i.p.

: " . 

### injection of Aroclor 1254 at 500 mg/kg, 5 days**b~fore** 

### 59 preparation. 

## I' t J 

### The S9 was stored at -I95"C in a Statebourne liquid nitrogen freezer, model SXR 34. 

### r, 

### \ ! 4. •59 Mix and Agar**l~** ..: 

### The S9 Mix was prepared at 4·C as follows: 

# n S9 5.0 ml 

## (, I ' 

### 1.55 MKC1/O.4 MMgC12 1.0 ml l.j 

### 0.1 M**Glucose-5-phosphat~** 

### 2.5 ml
,--. 

### 0.1 MNADP 2.0 1111 1 i 

## u 0.2 MSodium phosphate buffer (pH 7.4) 25.0 ml Sterile distilled water 14.5 ml Page 118 

# r Lj

# u 

# o -9­ 

### PROJECT NUMBER: 163/133 METHODS (centd) 

# o 4. 59 

### Mix and 

### Agar (cantd) Top agar was prepared using 0.6% 

### Difco Bacta agar and 0.5% 

### sodium 

# n chloride. For the Salmonella strains 5 

### ml of 1.0 111M 

### histidine/l.O mM



### . biotin solution was added to each 100 

### ml of top agar and for the 

## r Escherichi a coli strai

### n 5 

### ml of 1.

## a 111M 

### tryptophan was added to each 

## lJ 100 ml 

### of top agar. Base agar plates were prepared using 1.2% 

### Oxoid 

# n

.Agar technical No.3 with Vogel-Bonner**Me~i~m** 

### Eand 20 

### mg/ml**D-gl~cose.** LJ 

# o 5.
.

### Test Procedure a) 

### Preliminary Toxicity Study 

# Q In order to select appropriate dose; levels for use in the main

. study, a preliminary test I'las carried out to determine the toxicity

## ~I of the test material to the**t~ster orga~isms:**  

### 0.1 ml of bacterial '- } 

### suspension (TAIOO or WP2uvrA-) was mixed with 2 ml of molten, trace histidine or tryptophan supplemented media (histidine/biotin'or tryptophan and top agar) followed by D.lml of'test solution and 0.5 

### ml phosphate buffer and overlayed onto sterile plates of VogelBonnm- minimal agar (3D 

### ml/plate). Five. doses o.f .thetest·material and a solvent control (dimethyl sulphqxide) were. tested in 

# f1 duplicate. After 48 hO'urs of incubation at 37"Cthe plates were scored for revertant colonies and examined for a thinning of the background lawn. 

### b) Mutation Study EXPERIMENT 1. 

### (RANGE-FINDING STUDY) 

# [1 Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation 

# n, method in accordance with the standard methods for mutagenicity tests using bacteria. 

# n

.... ./ 

### Test Material and Negative Controls 0.1 ml aliquots of one of the bacterial suspensions**we~e** 

### dispensed into sets of sterile test tubes containing 2.0 ml of molten trace histidine (or tryptophan in the case of WP2uvrA-) supplemented top 

# n u agar at 45"C. These sets comprised two test tubes for each bacterial tester strain. 0.1 ml of the appropriately diluted test 

# o material or negative control solution was also added to each of the Page 119n -10­ 

## I : PROJECT NUMBER: 163/133 '- .J 

### METHODS (contd) 5. Test Procedure (contd) b) Mutation Study (contd) Test Material and Negative Controls (contd) 

### two tubes followed by either 0.5 

### ml of 

### the S9liver microsome mix or 0:5 

### ml of pH 7.4 

### buffer. The contents of each test-tube were mixed and equally distributed onto the**sur~ac~** 

### of**Vogel-Borin~r** 

### agar plates (one tube per plate). This procedure was repeated, in triplicate, 

# Ii

) 

### for each bacterial strain and for eaeh ,concentration of test

, . .. " . 

### material. Positive Controls i) 

### Without Activation 0.1 ml of 

### one of the positive control 'soluttons (ENNG, 9AA, or 

### n

, 

### L.I

i 4NQO) 

### was added to a 

### test tube containing 2.0 

### ml of molten, trace histidine'or tryptophan**supplement~dtop** 

### agar aod 0.1 

### ml of the appropriate bacterial suspension. FinallY, 0.5. 

### m1 of pH 7.4 buffer was added to the tube, the**content~** 

### mixed and poured onto an agar plate. This procedure wa$,**th~nrepeated** 

### in triplicate, for each of the positive controls. 

# fl l.' 

# in Wit!:! 

### Activatin!! 0.1 ml of 2AA or BP solution was added to a test tube contain

# n ing 2.0 ml of molten, trace histidine or**tr~ptophan** 

### supplemented top agar and 0.1 ml of one of the test bacterial 

# n suspensions. Finally, 0.5 

### ml of S9 

### mix was added to the test lJ 

### tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester J 

### strain. The plates were incubated at 37·C for 48 hours and the number of revertant colonies counted. I! 

# U EXPERIMENT 2 (MAIN STUDY) The second experiment was performed as described for Experiment 1 using fresh bacterial cu1tures, test material and contro1 solutions in triplicate. Page 120

### ) i .J 

# n l.J -11­ 

### PROJECT NUMBER: 163/133

METHODS (contd) 

# n d 6. Interpretation of Results For a substance to be considered positive in this test system, it should have induced a dose-related and statistically (5) significant increase in mutation rate (of at least twice the spontaneous reversion rate) 

### in' one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the 

## n two experiments give conflicting results then a third experiment may be 

## ,I.; used to confirm the correct response. To b,e consi

### dered
.negati

### ve the number of induced revertants compared to spontaneous revertants should n 

# lJ 

### be less than twofold at each dose level employed, the intervals of which

..-\ 

### should be between 2 and 5 fold and extend to the**limit~** 

### imposed by 1 l 

### I

, 

### toxicity or solubility or up to'the maximum recommended**d~~e** 

### of 5000 ttg/plate. 

### In this case the limiting factor was the maximum recommended dose.

r-. 

# U. 

# f1 

# n 

# n 11 

## U

Page 121I.

## ,~ -12­ 

# n PROJECT NUMBER: 163/133 

## r-, RESULTS 

### I I u 

### a) preliminary Toxicity Study The dose range ofL 

### )used in the preliminary toxicity study was 0, 

### 312.5. 625, 1250, 2500 

### and 5000**~g/plat~.** 

### No. 650 

### Violet was Ii 

### non-toxic in the strains of bacteria used (TAIOa and WP2uvrA-). 

### L. 

# ,! The mean numbers of revertant colonies for the toxicity assay were: 

# n d Dose**'(~g/pl**

### ate) Strain 0 312.5 

### 625 1250 2500 5000 

## i1 

### TAIOO 127.0 151.0P 132;5P 124.0P 123.5P lU.5P l J 

### WP2uvrA- 24.0 19.5P 29;5P 27-.0P 16.5P 19.5P


### P 

### =pi'ecipitate 

# n l) b)**~ytation** 

### Studv The results of the chetks for characteristics, viability and spontaneous reversion rate for each. t8ster strain were all found to be satisfactory. 

# n Individual plate counts together with the mean number of revertant

., 

## Ij I ,



### . 

### colonies obtained for each tester strain following incubation with the test material, with and without metabolic activation, are shown in n 

### Tables 1 and 2 for Experiment 1 and Tables 3 and 4 for Experiment 2. 

## 1.1 The results are also expressed graphically as Appendix II. The results for positive controls used in this assay are shown in Tables 1 to 4. 

# n I. J 

### Information regarding the equipment and methods used in these experi­ 

### n , ' 

### U ments and the details of the personnel involved, as required by the Japanese Ministry of Labour, are given as Appendix 1. 

# r t I , ) 

### No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 1000**~g/plate** 

### in both experiments, this did not interfere with the scoring of revertant colonies. 

# Ii " . J 

### No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any 

# n l.._ 

### dose level, either with or without metabolic activation. Page 122 I !

# Ll 

# n -13­

.....' 

### n 

### I I c " 

### RESULTS (contd) 

# Il b) Mutation study (contd) L_. 

### PROJECT NUMBER: 163/133


# o The positive control sUbstances all produted marked increases in the number of revertant colonies and the activity of the S9 

### fraction was found to 

### be satisfactory. 

# n r \ 

### CONCLUSION

"­ 

# n The test material,

# {--~ was found to be non-mutagenic under the I. j 

### conditions of this

### te'st.--:------"~~

Unless instructed otherwise by the spohsor, all**origi~al** 

### data and a c6py of the final report will be retained in the archives of Safepharm LabQratories n , , I 

### for a period of ten years. After this period, the sponsor's instructions C 

### will be sought. 

# r: l J 

# o REFERENCES 1. Ames, B.N., Ourston, W.E., Yamasaki, E., and Lee, F.D. Proc. Nat. Acad. Sc:1. USA (1970), LQ, 2285. 

### 2
.. Ames, B.N., McCann, J. 

### and Yamasaki, E"**~lutatiQn** 

### Research (1975), 

### 1L 

# L l 347.


## " I ; I J 

### 3. McCann, J., 

### Co;, E., Yamasaki, E., and Ames, B.N. Proc. Nat. Acad. Sci. USA (1975) 75, 

### 5135. 

# n 4. Garner., R.C., 11iller, Le., 

### and**r~;ller,** J.J\. 

### Cancer' Res. (1972), 33, 2058. 

### 5. Kirldand, D.J., (Ed). Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report# r~ Part III (1989) Cambridge University Press. 

# o n u Page 123i I 

### LJ -14­ PROJECT NUMBER: 163/133 

# n d n KEY TO TABLE OF TEST
.RESULTS 

## U 

# r. l j 

### NOTES: ==--.::;::::>I!Il. 

### n I I \., } 

### 1. When bacterial growth inhibition is.

### fovndjthe applicable value 

### is marked 

## n with an asterisk. 

# U 2. 

### The average number of colonies for each concentration is 

### recorded in pai'enthesis. 

### n 

## I J 3. 

### "Number of revertants"

- The observed values and ayerage value are shown 

### in order, beginning with the lowest**concentration~f** 

### the test substance. 

# n ,'.', . 

### l J 

### 4. The following postfixes are used when required:­ 

### C contaminated 

# n ( ) 

### P '" precipitate 

### n I , J

i 

### x =plate unscorable 

# n ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 

### 4NQO 4-Nitroquinoline-l-oxide

9AA 

### 9-Aminoacridine
I I 

# " 2AA = 

### 2-Aminoanthracene 

### t \ 

# n BP Benzo(a)pyrene 

## u 

# fl LJ 

# n l J Page 124-15­ 

# on PROJECT NUMBER: 163/133 c ) 

### TAB LEI Ii I : U 

# n TABLE OF TEST RESULTS: EXPERIMENT 1 L
.. 

### n LJ I :

# SUBSTANCE:(~ NAME OF TEST

### =========~==~====~=============~~~::~~~~:=~~~=c===J~====~=~========~.~_~:x. 

### __••**=.~=====~==~~.~~a.===~===;:====** 

# [1 HUMBER OF REVERTANTS (number of colonies/plate) VITH (oJ OR TEST SUSSTANCE BASE-PAIR SUBSTITUTION TYPE FRAMESHIFT TYPE VITHOUT C-) 'CONCENTRATION TAl00 TA1535 1iP2uvrA- 1A98 T..,1537 r-., 59 MIX Cug/plate) I ' 

# . L i ) 150 16 25, 19 11 

# n o 156 ( 145.0 ) 19 ( 17.3 ) 29 ( 22.3 ) 15 18.0 14 13.3 ) u 129 17 13 20 15
158 17 16 16 13 B.O 132 ( 137.7 ) 13 ( 15.3 15 15.7 ) 13 ( 16.7, ) 16 ( 13.0 ) 123 16 16 21 10 

### n 1 \ 142 11 16 12 10 

# n 40 175 ( 148.3 ) 15 ( 13.7 } 21 ( 19.:1 20 ( 14.7 ) 12 ( 12.3 ) I I , I '. I 128 15 21 12 15 

# n L )



### !I I I l 1 lOaD 165 137 { :57.0 > 169
1561' 139P ( 11,6.0 ) 1431' 20 C 16
13P 121' 13P (

( lB.O)

12.7 ) 15 20 24

22P 151' 19P ( 19.'7) ,13 21 ( 17.7) 19 12 11 ( 10.7) 9**.~~----'----.-------** 2SP 14P ( IB.7 } 131' ( 19.7 ) UP ( 10.7 ) 211' 71' 

# n ") 

# n i \ l ! 

# n I. J POSITIve 5000

NAHE 1421' 1351' 125P

ENNG ( 134.Q ) 151' 16P 17'"

EIING ( 16.0 ) 121' 13P( 16P

ENNG 13.7 ) C 13P ,< 101'
4NQO 11.5 ), lOP lOP 6P

9M ( B.7 )

CONTROL COIlCENTRATlOH 3.0 5.0 2.0 0.2 80.0 

### n LJ NOT (ug/pIMe) REQUIRING
___________ _____________ ______

59 MIX 

### n I \ (j NUMSER OF COLOIlIES/PLATE 687 637 (671.0) 689 232 247 t 258 245.7 ) 243 249 (265.7) 305 98 93 99 ( 96.7) 428 315 395 (379.3) 

# n L J 125

## I • L_' -16­ 

# n lj PROJECT NUMBER: 163/133 TABL E 2 n 

## t .i 

### TABLE OF TEST RESULTS: EXPERIMENT 1 {contd} 

# n n**~fr'"-""'-·'~"."_** 

### l; 

### NAME OF TEST SUBSTANCE:
.

# --:.~-**~ ;=;==;~~;;=-===~======~=====~~~===;===========;~=;=~==;==~==~=a=**  

### ••**~=~#=a~=:~:~~======~=~?~=======~===========** 

### n NUMBER OF REVERTANTS (number of colonies/plate) 

### lJ WITH C.) OR reST SUBSTANCE BAse·PAIR sueSTI.TUTION TYPE fRAMES!!! FT TYPE WITHOUT (-) CONCENTRATION TA100 T"1535 WP2.uvrA- TA96**T~1537** S9 MIX' (ull/plate)
152 20 29 25 15


# .. 0 to6 130.0 ) 17 ( 17.3 ) 26 ( 24.7 ) 28 ( 26.3 } 16 15.3 ) 132 15 19 26 15.
125 21 21 22 7 8.0 ".7 ( 134.7 ) 16 ( 18.7) 30 ( 26.3 ) 24 ( 25.3 ) 16 ( B.3 ) 132 19 28 30 17 ) 

## 11

, • J 1'.7 16 30 22 15
.. + ;40 134 ( 136.3 ) 17 ( 15.0 ) 19 23.3 ) 19 .( 25.3 ) 15 ( 15.7 ) 128 12 21 35 17 

### n I i 130 2B 15 31 12 l ) + 200 102 ( 122.7 ) 25 ( 24.7 ) 2B ( 21.3 ) 20 (**24~O** ) 13 12.0 ) ! 136 21 21 21 11 

## [l 

# n 1291" 151" 13P 16P 7P 1000 113P ( 116.7 ) llP ( 13.7 ) 201" ( 17.0 ) 19P ( 19.7 } BP ( 8.3 ) 108P 1SP HIP 24P lOP 

# n 1511" 8P 14P 23p 10P l ) + 5000 1521" ( 143.0 ) 20P ( 14.7 ) 22P ( 10.3 ) 231" ( 20.0 ) J1P ( 10.0 l 126P 161" 13P 14;> 9P 

# n**:=:==::.;:;:;:t1.:::==:::;:;;;::;:=:.:=;:.;::;:=-=:;;,;;::::::===::;;;::::::::::::=======::;::==== ============'~w==='~;:::======.===~=:::===="=~===========::=** **====:_===~==:.::::=** 

### , \ l , NAME Sp 2M ZAA BP 6P 

### n POSITIVE 

## l 1 CONTROL CONCENTRATION 5.0 2.0 10.0 5.0 s.o REQUlRING (ug/plate) Ii \ I 59 MIX ( ) NUMBER OF 446 182 169 91 107 

# n + COl<>NIES/PlATE 498 (468.0)**19~** (183.0) 168 (169.7) 109 (104.0) 93 ( 97.7) li 460 173 172 '12 93

126 

# n

# L. 

# n -17­ 

### PROJECT NUMBER: 163/133 TABLE 3
TABLE OF 

### TEST RESULTS: EXPERIMENT 2 

## , 

# n

)**~---.---------** 

# n l . NAME OF 

### TEST SUBSTANCE: ( )**.::,1le..t:::::;;;.;;=======:::;;:.===:::;.-:.:::::::======::;==~==:.=:::======::o:.~'::::::'==~a::CI::;.::a~** ..:wt.n===;.;::.:a:.:It=======::::::'==:;:::21=:::::!'::':"';:=====;;Oo:iIia.=&:a'=:':=====:;=:;:: NUMBER OF REVERTANTS (number of colonies/pLate) WITH (+) OR TEST SUBSTANCE BASE-PAIR SUBSTITUTION TlPE· FRAME5HIFT TYPE. WITHOOT (-) CONCENTRATION TA100 1A1535 IIP2uvrA- TA9S TI\.1537 59 MIX (ug/plate) 

# o
145 21 33 19 11 0 121 ( 129.7 ) 19 ( 21.3 ) 21 C 27.7 ) 25 ( 23.0 ) 11 ( 11.3 ) r 

### I : I , )

• 1n 24 29 25 12

,-.. I i ! I 130 16 29 20 15 i) 312.5 121 ( 122.7 ) 22 20.0 ) 32 ( 29.7 ) 21 ( 20.0 ) 12 ( 13.0 ) 117 22 28 19 12 

# n

, ) 136 22 29 25 B 

## n 625 137 ( 133.0 ) 13 ( 15.7 ) 29 ( 25.7 ) 19 ( 19.7) 17 ( 11.3 ) 

### [: 126 12 19 15 9 

# n 112? llP 25? 26? 15P 1250 99P ( 108.0 ) 22P ( 19.0 ) 25p·{ 27.3 )**.~p** ( 22.0 ) 12P ( 14.3 ) 113P 24r 32P .25P 16P 

# n l J 125P 20P 25P Wi> UP .-. 2500 126P ( 126.0 ) IS? ( 17,:' ) 26P ( 27.0 ) 17P ( 18.7) 14P ( U.3 ) 

# I**~** )

i 1llP 18P 30P aop 9P

147P 16P 19P lS? lOP 5000 143P ( 141.0 ) 2lP ( 19.7 ) 17P ( 23.0 ) 14P ( 16.7 ) 7P ( 8.7 ) 1331' 22P 33P ZlP 9P 

# n**======-:;;:=~,==;'::;=::==;==:::;:=:::::=:~.:========,:;==::::.::.=============~~==;_:;::::;:.-=:::?==::::.;:=.:;===_,::::!:::======::.::;:::r:::t:::I11====::'====:;:;;;:::;~-:':_;;;;;,;::;;;;;;::::;;;;:=;;:;=** 

### t )**EN~G** EIING ENIIG 41100 9AA.**---------~-----------------------------------------** POSITIVE CONTROL CONCENTRATION 3.0 S.O 2.0 0.2 80.0

NOT (Ltg/plate) 

## n , I R£QUIR!NG U 59 MIl( HUMBER OF 480 170 170 119 3Gd 

# I; COLONIES/PLATE 477 (466.3) 161 (170.3) 199 (197.3) 128 (125.7) 339 (331.7 1 223 130 348 l ! 442 180 

## n 1 ] 127------- I I 

### I
..

## J 

# n l J 

### TAB L E 4 

# n n 

# n**~ITH** (+) OR TEST SUBSTANCE WITHOUT C·)**CO~CENTRAT)ON** S9 MIX (ug/plate) -18­



### TABLE OF TEST RESULTS: PROJECT NUMBER: 163/133



### EXPERIMENT 2 (contd)
NAME OF TEST SUBSTANCE:
NUMBER BASE·PAI RSUBSTIT.UTION TA100 TA1535 

# n 159 22 I'
... 0 128 ( 139.7 ) 13 ( 16.7 ) I I l) 132 15
123 15 + 312.5 117 118.7 ) 21 17.7 ) 116 17 

# n 115 22 

# n ... .625 149 ( 130.7 ) 11 ( 16.7 ) 128 17 OF REVERTANTS (number of colonies/plate) TYPE FRAHESHIFT TYPE**~P2uvrA"** lA98 lA1537

26 25 24 C**~5.0** } 29 22 32 ( 27.7 ) 15 13 13 ( 13.7 )

28 17 38 ( 27.7 ) 33 16 39 ( 29.3 ) 15 15 14 14.7 )

22 30 14 26 ( 22.7 ) 2¢'i ( .26\-7.) 12 ( 14.0 ) 20 24 16
1261' 171' 26p 211' 91' + 1250 100f' 1'10.0 ) 19P Ill.7 ) 221' C 22.7 ) 171' C 22.3 ) 19P ( 13.3 ) 1041' 20P 201' 291" 121' 

### n I i \ .' 1191' 131' 2()p 251' 111'.



## .. 25:l.0 1241' ( 12u.O ) 111' ( 11.0 ) 201' 2C.3 ) 241' 26.7 ) 151' ( 13.3 } 1171' 91' 211' 311' 141' 

# n l J ... .5000 1201' 1221' ( 118.7 ) 101' 141' ( 12.7 ) 291' 141' .( 20.7 ) 241' 251' ( 26.3 ) 91' 131' ( 11
..7 ) 1141' 141' 191' 301'
. 13P 

# n**=======:'==--~===:';:;:========:'::::=::=-='::;===========::==':=~==:'=:J==C=O:;:'::;;===;;;;:;=::::.=~===::=====:::======;:;:===":==========~.=========~==.==:::** NAME Bp 2M 2AA Bp
... SP 

## [l POSITIVE CONTROL CONCEIITRATION 5.0 2.0 10.0 5.0 5.0 REQUIRING (ug/plate) 

# n 59 MIX l. NUi1BER Of 398 122 i4i.J 148 98 + COLONIES/PLATE 420 ( 411.7 ) 113 ( 119.7 ) 153 ( 155.7 ) 150 ( 154.7 ) 117 ( 111.0 ) 417 124 166 166 118**=========================~====================;~================:;2=============~========================~2~====~=======** 

# n Page 128I 

# l J -19­ 

# n PROJECT NUMBER: 163/133 

# n \l

### , 

# , ) 

# II '1

r-' ! 

### I I ; 

# n A P P EN 0 ICES 

## ;l**--=~=====~=~** L ) 

### n I , .

# I 

# n n ( . REVERSE HUT ATION ASSAY HANES TEST" REPORT



## n 

# l.1 

# rl 

# n 

# n , J 

# n 

# n n 129

# II -20­ 

# n 

# n n I ; 

# n n n l: 

# [1 

# n

l 

# n

,



### n 

## l : 

# n

n

< I l , 

# n \ j 

# f: lJ 

# n o PROJECT NUMBER: 153/133 A P PEN D I X I

< 

### REPORT OF RESULTS IN MUTAGENICITY TEST USING MICRO-ORGANISMS l1NDUSTRIAL SAFETY AND HEALTH LAW}

1. GENERAL IT01S 

### (to be completed by 

### the sponsor)

Name of the nel.,. 

### chemical substance (IUPAC nomenclature) ------------t--"+



### Other name

# -~ Structural formula or**~** 

### rational formula (or**outli~e** 

### ofmanufacturin method, in case both are unknown)
. 

# ( I i

- 

### Purity of the new

chemical substance tested 

### Name of impurities 

### CAS No. 

## t Molecular weight 

# I I**~1elting** point i**~** 

### Boil i

### ng point Stability 

# \ Degree of solubility in so1vent 

# \. ( I



### ­ I \

- <-"/
.......,,­ 

### '---­ Page 130

### -21 PROJECT NUMBER: 163/133 A P PEN 0 I X I (continued) 

# n



# n n L ;
r> ! I I , , J 

### fl I l ,

### I ,-' : i 

### l J 

# n 

# o**r~"':** I i I I l ; 

# n D
n L"J 

### 2. 

### TESTER STRAINS (1) 

### Procurement
Test strain



## --­ Obtained from 

### Date obtained Strain check date for coded stock cultures used for this study TAIOO BIBRA* 14 August 1987 9 March 1994 TA1.535 BIBRA* 14!\ugust 1987 9 March 1994

, "',**WP2uvrA~** 

### BIBRA* 14 

### August" 1981, 9 March 1994 TA9S



## - BIBRA* 14 A"ugust '1987 , 9 March 1994 TA1537 BIBRA* 14 August "1987 9 Manh 1994 

### * BI8RA: British Industrial 8iological Research Association**~2)** 

### Storage (Encircle the applicable number and fill in**therelev~nt** 

### entries) Freezing in small aUql,lot Storage method 2. 

### Freezing in large aliquot ,3. Others(**----------------~--4---------------------------------~---** 

### Storage temperature Composition Bacterial suspensi

### on**-1~6·** C 8;0 0.7 1Il1 ' ) 

### 3 S9 MIX (1) 

### Source of 59 (Encircle the applicable number and "fill in 

### the relevant**~ntri~s)** 

### Made in-house or purchase 1.

### I~ade in-house 

# ,0 Purchase 

### '-S:J. (supplier BIBRA*,) Prepared on Lot No. (in case of purchase) 24/03/94 (range-finding study) 03/02/94 (main study) Aro. S9/24/03/94 (range-finding study} Aro. S9/03/02/94 (main study) Storage temperature -196·C

131

### ---- 

## I I l.J 

# n '- J 

# [J A P 

### PEN D I X I (continued) 3. S9 MIX (continued) (2) Preparation of S9 -22­ 

### PROJECT NUMBER: 163/133



# o
n I I 

### Animal used Inducing substance Species strain 

# Is, Rat Name AROCLOR 1254 prague-Dawley
-, 

### Sex Male Administration method SINGLE LP; 

### INJECTION Age (i

### nl'leeks) 7 weeks Administration period 5 days and amount 

### Weight

- 200g 

### (g/kg body weight) 0.5

.'-,...: 

### '(3) Composition of S9 Mix 

### r 

## L Constituents Amount in Iml 

### S9 Mix 0,1 

### ml S9 

### 8.0 tlmol 

### MgC12 KC? 33,0 tlmo

### 1 

# n

-. I 

### Glucose-6-phosphate
. 5.0 .umol 

# u -Glucose-6-PhOSPh9 dehydrogelHlse Constituents



### .NADPH

NADH Na-phosphate
...buffer Others (NADP)

_.

.. 

### Amount in 1 ml 59 Mix tlmol tlmol 100.0.umol.



### 4.0 tlmol

Ii 4. POSITIYE CONTROLS AND SOLVENT FOR POSITIVEJONTROLS 

### I 

### I**~** .I 

### (Encircle the applicable number and fill in the relevant entries) 

# n 

# u
., I I 

## U -""'1"" ==--=- -- 

### Substance name Supplier Lot No. Grade Purity(%) Solvent Pos iti

### 'Ie 

### ENNG I 

### S1

### Gt·1A lO6F-3691 Technical 97 DMSO control 

### - 

# I 4NQO 

### SIGMA 36F-0015 TechrHcal '99 DMSO 

### 9AA S**IGt~A** 

### 96F 5641 Tecbnical 98 DMSO 2AA S**IG~Il\** 

### 121H3475 Technical 97
..5 DMSO

---



### BP SIGMA cal 99.8 DMSO
Solvent DMSO 

### SIGMA 99.5**-----l----------i---.--J..-----t------+~---** 

### Preparation and storage of positve control 1. Freshly prepared Storage in small aliquot 3. Others{ 132 

### (Storage temperature -20·C) } 

# o

# u 

# n 

# o n**L~** 

# 1 l, n I : ,J , 

# n []
n lJ 

# n n n n rr l. J 

# o -23­ 

### PROJECT NUMBER: 163/133 A P 

### PEN 0 I X I (continued)


### 5. PREPARATION OF THE TEST MATERIAL IN 

### SOLUTION (Encircle the applicable response regarding purity conversion)

" , '. .... ,lotNo., 

### Name Supplier Grade, Purity(%}

, 

### Dimethyl Solvent used 

### SIGMA' 83H0557 99.5

« ••••• 

### sulphoxide 

## .' Stability of test 

# ..**sub~tance** 

### in solvent



## - .. " 

### Reason for selection Soluble at 50 mg/ml of solvent
, 

## ----- " 

# -

.. 

### Suspension and other methods when test substance ",' 

### difficult to dissolve
, 

### Storage time and tempera­**~** 

### hr 10 mins room temperature (range­ inding study), " . ture of solution from preparation until use 1 

### hr room temperature (main study)**---~-** 

### Con·vers.ion of purity

6. cormITIONS HC. OF 

### PRE-CULTURE (1) 

### Conditions**~.r=e==** '1('--**f"""t7~~~;** 

### Name Nutrient broth

., .350 51940 11/97 

### Oxoid Ltd. Oxoid

- 

### - Period of pre-culture 10 hrs ; 

# e'**~o** ,**~'"!-~-- .'~=-=:'l:::;=:"-=:t~**  . 

### Lot No. Manufactl!rer

.. ' --..:.--:.
.


### storage time and temperature of cultures from inoculation until shaking**~p'proXimatelY** 

### 79 hours at 4Q

### C (range­ inding study)
. Approximately 56i hours at 4·C (main study) .­ 

### Storage time and temperature of cultures from shaking to Up to**3~** 

### hours at room temperature harvest

..**~-.** 

### Shaking method (shaking type and fr'equency) Orbital shaker 130 revs/min, 

### 3rC Culture flask (form, size) 

### Plastic universal container, 30 ml Amount of culture medium 5 ml Amount of strain inoculated 20 .ul 

# r u Page 133 r I ' lI '

j

# u -24­ 

### A P P [ N D I X I (continued) 6. 

### CONDITIONS ETC. OF 

### PRE-CULTURE (continued) (2) Cell viability at the end of pre-culture 

# n ',. ) Cell 

### number Range-finding study (x 

### 10g 

### /mT) 

# o

... 

### Main study



## - Count method (Encir.cle the applicable

number}
., 

# rl ___**~'T ~~~=-=oo::;';.**  '''"/"

t J 

### 7. AGAR PLATE MEDIUM (1)"Tup 

### Agar 

# n I.**~** 

### Base-pair substitution TAIOO

4.1 3.2

1. 

## © 3. 

### TAl535 

### 1.2 1.5
. WP2uvrA3.5, 5,6 

### type

., . 

### TA98 1.9 

### 1.7
Conversion. from 0.0. 

### value Stepwise d1iution method Dthers(
=
.. 

### PROJECT NUMBER: 163/133

Frameshift type TAl537

1.5
, ."_.,



### 1.4

. .

., . )



### .

r,

# Agar~l~.:::--:-:-:-c-tu-r-e-~-l=r------:-:-;-::_-_B_a_ct_o__ ag_a_r

____

# ~~--~------------ 1!

__...I.I_L,.,?t No.

### ~~ 37430AJE 11/98 

# 0 (2) 

### Minimum Glucose Agar Plate 

### Medium 

# n ('Encircle the app1 icable number and n,ll 

### in the relevant entries)
, I }

' Made 

### in-house or purchase 

### '2 " Purchase (Suppl

### ier Prepared 

### on 22/04/94 (range-finding study) 29/04/94 (main study) 

### lot No.**i~** 

### case of purchase 

### ---,--.--:-------------­

Name ·of ,supplier and 

### lot

No. of 

### the used 

### Agar 

### +----------_._------------------------ Oxoid Ltd. '.'
. Oxoid Technical No.3, Lot 

### Nos, 8326 76158 10/98 

### (range-finding study) B040 76495 10/98 

### (main study) ,.-.. I • 

# ·u ·8. 

### STERILITY**T~ST** 

### (Encircle the applicable response in the right hand 

### column)**==-==-="=.=-=======-=---=-~-=~=----------=~==============---** 

### Bacterial growth other than expected Test substance solution 

### Yes S9 

### Mix Yes



## " i . 134 

### u

# -- 

# u -25­ 

# n 

# n 

# o

n 

# LJ

r] l.' 

## n I I 

### U 

# I:,

L : j; I 

### I l j r'" 

### I j l... j 

# n l 1 PROJECT NUMBER: 163/133


### A P PEN D I X I (continued)

9. TEST METHOD

, .

..

, . 

### Pre-incubation 

### .Plate Method



### -Composition

" 

### Bacterial suspension Hil 

### Od ml Test substance solut'ion 

### ml 0.1 

### ml



### -Na-phosphate buffer 0.5 

### ml ml

,. 

## - S9 

### Mix (in 

### case of metabo­ 0.5" ml 

### ml lic activat'ion method)



# - Top agar solution ml 2.0 

### ml**-----~"** ... 

## . Others ( )

.. 

### ·C 

### Temperature Pre-incubation

.. , - 

### Time rnin .

, .**~C** 

### 37"C Temperature I

### ncub,atfan

..... . ..' -,--------. 

### hours 48 

### hours

Time



## ........
.. '.**~** .. =. = = 

# ­
10. COUNT MElHOD 

## or COLONIES (Enci

### reieland 2 i

### Fboth meHiods were used)

Reason 

### why both methods were used Name, type and manufacture of colony counter Correction method SEESCAN COLONY COUNTER 1. No correction Miscount-correction 2. Area-correction Area and miscount-correction

, , ,**~...}**


### fl I i , J 135

# u -26­ PROJECT NUMBER: 163/133 A P PEN D I X I (continued) 

# o 11. TEST RESULTS 

### (1) The results should be reported on the attached form 

# o (2) Judgement of the results Judgement {encircle one} Positive

### CNegati~ 

# o Reason for judgement and referential matters: No significant increases in the frequency of revertant....J;plQojes wey'e recorded for any bacterial strain used with any dose ofl

- lin two separate experiments either 

# n with or**~ithout** 

### metaboliC**act'"I\I<1~IUII:-** 

### Astatistical analysis of the data**l~** 

### was not 'requ;red to determine the result of the test. : ApredpHate was observed at and above 1000 pg/p1ate, ' this did not j

### nterfere with the' 

### G scoring of revertant colonies. An allowance for the, purity'of No. 650' 

### LJ Violet was'made when the test material formulations were**prepared~~** 

### , NADPH was not added to the S9 co-factors, however, NADPH is generated,' in-situ by the addition of NADP and excess G-6-P and this is**con5id~red** 

### : to' be equivalent to direct addition of NADPH. Aroclor 1254 

### was used to

,...... 

### induce higher enzyme levels in the rat 1iver S9. Aroclor 1254 is considered to be equally effective as the use of the**eq~ivalent** 

### combination l 

### ) !

) 

### of phenobarbital,5,6-benzoflavone and methylcholanthrene. ' 12. OTHERS



# - - II I I I ,,'

.... G" 

### I : l.)

n 

# LJ 

# n L;

### ~ I \ \. ,.,:



# n Name Safepharm Laboratories Ltd. Testing nistitution 

# - - Address P.O. 

### Box 45) 

### Derby, Unitl'ld Kingdom --', 

### Head of P.C. 

### Jenkinson

Administrator Title Genetic Name B.Sc. , 

### Ph.D. Toxicology



## -­ "--.

### -..:......-----­ - 

### ----­ Head of

. J.R. Pateman t\rchives Director Title Quality Name C. 

### Bi01
. I M.L B10,1
. 

### Assurance

., 

### Study Title Director

- Name P.W. Thompson 

### H.N.C. Study Director Genetic 

# () n'
. Toxicology Years of experience 7 \ v->**\J~** ---yr.,... 

### Leading U' 

### Title Technician - , Name J. 

### Kemp Genetic Toxicology Personnel engaged Years of experience 3 (S(OU[) t 

### in the study 

# I Technician 

# ,

- Title 

# I Genetic 

### Name 

### D. Parton Toxicology Years of experience 2 

# flJIc.J. Test dates From 22/04/94 

### To 12/05/94 

### Study No. 163/133 136

# u -21­ 

# n PROJECT NUMBER: 163/133 l..J 

# o A P PEN D I X I (continued) 

# II u KEY TO TABLE OF TEST RESULTS. 

# n LJ 

### n 

# U NOTES: r-\ 

## LJ 1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk. n I I 

### C: 

# o 2. The average number of colonies foreachconcerltration is 

### recorded in parenthesis. 3. 

### llNumber of**revertants~** 

### - The observed 

### values and average value are shown 

### n • ! LJ 

### in order, beginning with the 1owest, concentration of the test substance. 

# f

l." 

### _ 4.**Th~_f~llowing** 

### postfixes. are used when required:­ 

# n. c**~** 

### contanlinated I..,j P 

### precipitate 

## x plate unscarable s rl {.,.: 

## I ! 

# n ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 

## " 4NQO 4-Nitroquinollne-l-oxide 

# t ) 9AA 

### 9-Aminoacridine 

# n ,-_,I 2AA 

### = 2-Aminoanthracene**r~** 

# U BP Benzo(a)pyrene 

# n u

137

# c J 

## U -28­ 

### PROJECT NUMBER: 163/133 

# o A PPEN 0 I X I 

### (continued) n 

### I I U 

### TAB l E 1 

# n TABLE.OF TEST RESULTS: EXPERIMENT 1

\. j 

# o NAME OF TEST SUBSTANCE: NUMBER OF REVERTANTS (number of coloniesfplate) YITH (+) OR TEST SUBSTANCE BASE-PAIR SUBSTITUTION TYPE. FRAJotESHIFT TYPE**~ITHOJT** (-) CONCENrRATION TA100 TA1535 1II'2uvrA­ TA9a TA1537 59 MIX (ugfplate)



# n u

"" 

### I i l 1


# n I 

### I " ) 

## Ji i i.



### " i I U 

# [ 138

# u -29­ 

# o PROJECT NUMBER: 163/133 

# o A PPEN 0 I X I 

### (continued) TAB l E 2 

### TABLE OF TEST RESULTS: EXPERIMENT 1 (contd)

r: \ ! I I ••-.1 YITH (.) OR loHTHWT (-) S9 MIX TEST**SU8ST~ijCE** CONCeNTRAliON (ug/plate) Tlll00 BASE-.PAI R SU8STlTUTlON TYPE TA.1535 IIP2uvO.·· , FRAMES"!fT·TYPE TA98 T
...,537 

# n l J + 0 152 106 ( 132 130.0 ) 20 17 15 ( 17.3 ) 29 26 19 ( 24.7 ) 25 28 26 ( 26.3.J 1S· 16 15 15.3)


# n u + H.U 125 147 132 ( 134.7 ) 21 16 19 18.7 ) 21 30 28 ( 26.3 ) 22 24 30 ( 25.3 ) 7 16
. ( . 13.3 ) 17 

# n l.J + 40 147 134 ( 128 136.3 ) 16 17 12 ( 15.0 ) 30 19 21 ( 23.3 ) 22 19 35 ( 25.3 ) 15 15 ( 1i'
. IS.7.J

130 28 15 3·1 12 

# I )

l + 200 102 ( 122.7 ) 25 ( 24.7 ) 28 21.3 ) 20 24.0 ) . 13 12.0 ) 136 21 21 21 11 I 

# n

I 

# n , ) 1291> 151' 131' 161' 7l' " 1000 IBr ( 116.7 ) 111' ( 13.7 ) 201' ( 17.0 ) 191' ( 19.7 ) 81' .( 8.3 ) L) 1081' ISP 181' 24p 101'
1511' 8P 141' 231' 101'



# .. 5000 1521' ( 143.0 ) 201' ( 14.7 ) 221' ( 16.3 ) 231' ( 20.0 ) 111' ( 10.0 ) 

# r: l )

' 1261' 161' 131' 141' 9P**======~=:::::=,===::::====!!::-=!::::===::::===:=::-::::!::=========::======= ====.::::===t:::::::=!:::=====::==-=====::==:====~==========.===:;:=:==** **============-:::** NAME Sp 2M 211A BP BP il I ' POSITIVE CONTROL CONCENTRATION 5.0 2.0 10.0 5.0 5.0 REQUIRING (ug/plate)

59 MIX 

# n NUfoISER OF 446 182 169 91 107 COLONIES/PLATE 498 (468.0) 194 (183.0) 168 (169.7) 109 (104.0) 93 ( 97.7) \...J 460 173 172 112 93
Page 139 

## n . I 

### U-30­ 

# n PROJECT NUMBER: 163/133 

### u A P 

### PEN 0 I X I 

### (continued) 

# 0 TAB l E 3 TABLE OF TEST RESULTS: EXPERIMENT 2 

# r ,J 

# 0 NAME OF TEST SUBSTANCE: r--; NUMBER OF REVERTANTS (number of colonies/plate) 

# LJ WITH (+) OR TEST SUBSTANCE SASE-PAIR SUBSTITUTIO+I TYPE fRAMESHlfT TYPE**~ITHOU1** C-) CONCENTRATION TA100 TA1535
. Wp2lNrA- 1A98 TA1537 

### ,-' 59 HXX (ug/pll'lte) I [ 

### l.J 145 21 33 19
. 11 

# C '). a 121 ( 129.7 ) 19 ( 21.3 1 _I ( 27.7 ) 25 (**23~0** .) 11 ( 11.3 ) 123 24 29 25 12**~...,** 

# lJ no 15 29 20 15 i 312.5 121 r. 122.7 ) n 20.0 1 32 ' 29.7 ) 21 20.0 ) 1? ( 13.0 ) 117 22 28 19 12
. 

# n LJ 136 22 29 25 ' a '--1 625 137 ( 133.0 ) 13 ( 15.7 ) 29 ( 25.7 j 19' 19.7 ) 17 ( 11.3 ) 

## 11 126 12 19 15 9
'121' 111' 251' 261' 151' ·1250 991' ( 108.0 ) 221' ( 19.a l**25~** ( "27.3 } 151' ( 22.0 ) 121' ( 14,3 ) 1131' 241' 321' 251' 161' 125p 201' 251' 19P' 111' 2500 126P ( 126,0 ) 15? 17,7 ) 261' ( 27.0 ) 17p**~** lB.7 ) 14P( 11.3 ) 127P 181' 301" 201' 91'
1471' 161' 191' 15P 101' 5000 143P ( 141.0 ) 211' ( 19.7 ) 171' ( 23.0 ) 14P ( 16.7 ) 7P ( 8.7 ) 1331' 22P 331' ZIP 91' 

# n lia':;;;;;=====:::=======::==
....**:;:r::======o=:::::::::::::========;'!'l::.e====;.:=====;:;;::==:::.;~':;=,=====':::==_~=-====:=!:::='=:S:~I':.'l=)!lI::lI!:;:t=~=::==:::::t:::t~=::;:;a:::,::;=:.::=a.!:::::ta=:::::;:;;.:;t::.::li;;;;==:==** l.I NAME EHHG EN!;G ENIIG 4HOO 9AA 

# o -----_. POSITIVE CONTROL CONCENTRATION 3.0 5.0 2.0 0.2 80.0

NOT (ugjplate) ilEClUIR1Nti

59 MIX NUMBER OF 480 170 170. 119 30B COLONIES/PUlTI:: 477 ( 466.3 ) 161 ( 170.3 ) 199 ( 197.3 ) \28 ( 125.7 ) 339 ( 331.7 ) 442 180 223 130 348**==:::t=======~** ••**=======:=~=========:~==~============~===~;::===:=====~======;==~~========~===========~~a=a~===============** 

# n L
.. Page 140 

# c

## ----- 

# u -31­ 

### PROJECT NUMBER: 163/133 

# o A P PEN 

### P

### I X I 

### (continued) 

# o o TABlE---.! TABLE OF TEST RESULTS: EXPERIMENT 2 (contd)
NAME OF TEST SUBSTANCE:,

__**._~....** _._...**,,"'i""~ =::;';;.::~=====_;:::=~:::==_==::z==:=========::====="='::::========.::=======:I:a==,liIl~==::I~==:::~=:::====.=;;t==':~;I.aZ71G===;:=====::===::==,:;:::::;;:;:::;::s**  

# n NUM8ER OF REVERTANTS (number of colonies/plate) 

### lJ WITH (+) OR TEST SUBSTAUCE BASE·PAJR suaSTJJUTIOII TYPE FRAHESHIFT :rYPE**~lTHOUT** (-) CONCENTRATiON TA100 TA1535 IIP2\,IVrA- TA98 T41537 59 MIX (ug/plate) 

# o
159 22 26 29 15 + 0 128 ( 139.7 ) 13 ( 16.7 ) 25 ( 25.0 ) 22 ( 27.7 ) 13 ( 13.7 ) 132 15 24 32 13
123 15 28 33 15,

., + 312.5 117 ( 118.7 ) 21 17.7 ) 17 ( ,(7.7 ) 16. ( 29.3 ) 15 ( 1l'+.7 ) 116 17 38 39 14 

# n _.J 115 22 22 30, 14 

# [l .. 625 149 128 ( 130.7 ) 11 17 ( 16.7 ) 26 20 ( 22.7 ) 26 24 ( 26.7 ) 12 16 ( 14.0 ) 

# r lJ + .1250 126P 1001' ( 110.0 ) 171' 19ft ( 18.7 ) 26P 221' ( 22.7 > 211' 1(1) ( 22.3 > 91' 191' ( 13.3
..) 

# n I

. )r 

### --......

# _­ 104P 201' 201' 291' 121'
1191> 1;)1' 201' 251' 1\1' 

## " I ! + 2500 1241' ( 120.0 ) 111' ( 11.0 ) 20P (­ 20.3 ) 241' ( 26.7 ) 151' ( 13.3 ) I} 1171' 9P 211' 311' 14P
1201' 101' 29p 241' 91' + 5000 1221' ( 118.7 ) 14P ( 12.7 ) 141' ( 20.7 ) 25P.(**2~.3** ) 131' ( .11.7 ) 1141' 141' 191' 30P 131' r;**:;;;;;;;;:;;:;=:.:;;.====;:;-::.::;o:=~:::==~~-==.::;:::;:::::::.:;~=.::;;:.::;:;;;:,;:;:.::===:.:=::;:;~==.::=======::=_:~===:=;=~l:t:::l:llll'liI:=,;;":;;;;:_;;;';;;;:;;:;;;:=;;'====:=:;=~_==;;;:::;==='::=;';===:::;;:;;=:=;=;~==;:;:.:-** . \ I l. > NAME BI' 2M 2M SP Bp POSITIVE CONTROL CONCEI-ITRATlON 5.0 2.0 10.0 5.0 5.0 

### 1'", RECUIRING (lI9/plate)

, , 59 MIX 

## J NUMI!Ei! Of 398 122 148 148 96 

# o + COLOIHES/PLATE 420 ( 411.7 ) 113 ( 119.7 ) 153 ( 155.7 ) 150 ( 154.7 ) 117 ( 111.0 ) 417 124 166 166**=~~======================~~~===~=========~=================s====::====================~====:======================:.:==~=~** 

# o Page 141 

### n I ' L.J 118

# c f( 

# o n**~J** 

# r . • I ; l J



### n 

# U 

# n , J 

# n l J 

# n u 

# l-1
. 

# [l 

# rr [ J 

# n n Lj 

# n**-32~** PROJECT 

### NUMBER: 163/133 

### A P PEN D I X I (continued)

TABLE OF TEST RESULTS (RELATIVE ACTIVITy}


### TEST SUBSTANCE:

._...**~** '.



### - S9 Mix + 

### 59 Mix

.... ' 

### Test strain Relative Dose levels Relative Dose levels used to used to activity calculate activity calculate Range-findin9 

### TAIOO N/A N/A Study TA1535 

### N/A N/A WP2uvrA­ N/A N/A TA98 N/A. .. 

### N/A

,. 

### TA1537 

### N/A

- 

### Main 

### Study TAlOO N/A 

### N/A TA1535 N/A

# ~WP2uvrAN/A 

### N/A TA9S NjA 

### N/A**TA153~---** 

### -.
..



### . N/A N/A

.. ,



### n N/A**~** not applicable 

### as no evidence 

### of mutagenic activity was observed 

# lJ
Page 142**~) =~J** 

140

120

UJ 100 !;;{ ...-l 0..

.......... OJ 

# '" (I) <a CD 

### 'Z 80**~** 

### ..,. 0::: w**~** w 0::: 60


### 40 -_._._.

## __.-- ----------­ r---,**~~._~J** 

### :=::J =--=--:-J 

### =-:::J**=:.-.~** 

### C-=-:J 

# =-:J

./ 

# --------­**~OSE-RESPONSECURVE**



# ------
..

# ------

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

.. 

# -------

# ------.------.

# -------. r'" TA100 -59 EXPERIMENT 1



# * TA1535 S9 EXPERIMENT 1 

### I ... WP2uvrA- S9 EXPERIMENT



## -<>- TA98 -S9 EXPERIMENT 1

-- TA1 -59 EXPERIMENT 1 

## )**:.~~:** '..;-;' ;."
UGjPLATE 

### ::---=:::J

;J> "'C "'C 1"'"1 :z:



### ..... ><

.....



### .....

I w W I

'"ti

## ~ r""'I**~** :z: 

# = i IT1 XI
>-' 0'1**~** I-' W L.1 c**:-~** 

### . __J**~=-':::J '~::=J** **c.~** 

# =J**~" ~~DOSE-RESPON**  

### CURVE 160T
140+

120+

w 100 

# :s f­ (L ""­ ro (/) OJ fcO Z 80 CD

## ~ W**~** > w**~** Q:: 60


### 40

# I~ 1"'t:I ,..,., :z 

## b Ix

# ~./**~ ~**  

# I:



# -

... TA100 +S9 EXPERIMENT 1 n 0 ::l c+ TA1535 +S9 EXPERIMENT 1 

### I, c.. 

# - -Glf- WP2LNrA- -t59 EXP[RIMENI 1 -0- lA98 +S9 [XPERIIJENT 1 I
-- TA1537 +S9 EXPERIMENT '_ ) !.oJ.tI



# e "'C

,..,.,

# 2D~ :;;: 

# =**~~** £ n -l :z c:: 3: 0+1--------------+-----­ co ,..,., 

## o 8.0 UG/PLATE


# - en**~** 

## .... w w XI

### :::::=J**~_._J** 

## :-....::] :___J 

### =:-J

# C~DOSE-RESPONSE CURVE 160T
140+

120+

w 100t

# ~ /TI I:z**..___** 

### 1° 

# - ><

## ~ 

# ­**~.** 

# -

- n Q '=' rT 0.. 

# -

___ TA100 - S9 EXPERfMENT 2 -k lA1535 -$9 EXPERIMENT 2 I w • WP2LNrA- -.S9EXPERIMENT

# 21 (J"I I -0- TA98 -S9 EXPERIMENT 2

-- TA1537 -59 EXPERIMENT 2

"'C**~** c.... rn**~** :z c: 3: I ! c:l 2500 5000 ;;c

# ~/~~E /TI**~** en w 

# :s D­ "­ (J1 '1j PJ**~** 80 cO rD

## ~ W r-' ,p. > w U1 IX 60

40



## o

-...... >-' W W

# -- .----, ;­**~l** _____J 

### :==:J**:~**

### __::J '

__ .. ..J**~:-=J** 

### L-J**~--'.-**

___ I

# ~**~DOSE-RESPON** CURVE :> 160

14

# "'~ 120

w 100 

# 3 a...

........... (fl**~**

### ~ 80
I-' 

### ,... > w w C!\ a:: 60
40

# ~ ,., I -0 1'1"1 :z o ><

.......

.......

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

-. TA100 +59 EXPERIMENT 2 

# * TA1535 +S9 EXPERIMENT 2 -ill)- WP2LNrA- +S9 EXPERIMENT 2 

### 1MB +S9 EXPERIMENT 2 ­
--. lA1537 +59 EXPERIMENT 2 

# . ­ n o :::I Q. 

### ""'" '"-'



### , w CTI I



# ., 

# ==:

1"1'1 

# F •

# ~~: : 

# E 20 n -I :z:**~** co 1'1"1 312.5 625 1250 2500 5000 :::c UG/PLAT£

...... CTI**~** 

### ..... w w

# l J -37­ 

# n PROJECT NUMBER: 163/133 

### A PPEN D I X I I I 

# n 

# n 

# n n I! l 

### I

i 

# n

- ) 

### " i l )



## i THE DEPARTMENTOF

### HEAI.J.'HOFTIIEGOVERNMENT OF

### THE UNITED KINGDOM GOOD lABORATORY PRACTICE SfATEMENTOFCOMPUANCE INACCORDANCE WITH DIRECI'IVE 88/320 ERe
LABORATORY SafePharl11 Labora tori es Li111ited p 

### a Box No 65 Derby DEl 2BT

DATE OF INSPECTION
31 ]anuar.v 1994 A general inspo::tion for compliance with 

## me Principles of Good Laborarory Practice was carried outatthe above labonitory as partofthe UKGLP ComplianceProgramme. Al !:he time ofthe inspection no deviations 'M:re found ofsufficient magnitude to affect thevalidityofstudies perfonned at these**faciliti~**

D.F.Moore 

# n Director UKGLP MonitoringUnir Page 147 

# n! l) 

# n

n 

# n
ASSESSMENT OF THE READY BIODEGRAOAB'ILlTY,

### ~- -\USING THE CO EVOLUTION TEST 2

### '--~ 

# II , I 

### {MODIFIED STURM TEST} PROJECT HUMBER : 163{134 

# n l ) 

### [l 

# n
, J 

# n

I , l j ,I\UTHORS':' 1 • G .-Se\<le11 

### C. Mead
ISSUED BY: Safepharm,LaboratoriesLimjted P.O. Box .No. 45 DERBY DEl 2ST UNITED KINGDOM 

### Telephone DERBY (0332) 792896 Facsimile DERBY (0332) 799013 Telex 377079 SAFPHM G Page 1 of 19 Pages 

# n Page 148 

# n

### I 

# l i I : 

# n

'-, 

### [ j

,....., 

# l J 

# n L!**,...~** I ' 1 I I. , I 

### n, l ; 

# n fi I I I , 

# n Ii

• .i 

# r , J



# n 

# n 

# o -2­ 

### PROJECT NUMBER 163/134 QUALITY ASSURANCE REPORT The routine inspection of short term studies at Safepharm Laboratories is carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently completed series of monthly inspections relevant to the study type(s)

Date(s)" Of 

### Inspection and Reporting: 11. 12, 29 Apri 1 

### 1994 in this report are given below:

This 

### report has been audited by Safepharm Laboratories Quality Assurance Unit. It 

### is considered to be an accurate account of the data generated and of the procedures followed.

Date of 

### Report Audit: 31 May 

### 1994



### J.R.PATEMAN C.Bio1., M.l. Biol. 

## EOB SAFEPHARM QUALITY ASSURANCEUNII 

# ...: ....: ...**&2~~** 

# ..
27.**JUI~.!994**

149

# I : 

# n -3­ 

### PROJECT HUMBER 163/134 

# n 

# n 

# n 'l 

## I : l ; 

### TIlEDEPARTMENTOFHEALTII OF

### mEGOVERNMENT OFTIIE UNITED KINGDOM
GOOD lABORATORY rRAGTICE SfATEMENTOF COMPllANCE IN ACCORDANCEWITH D1RECI1VE88/320 EEC 

# n LABORATORY SafePharm Laboratories Limited
. POBox No 1..5 Derby DEl 2BT 

# Il

DATE OF INSPECl10N 

# n 31 January 1994 

# l! A general inspection for compliance with the Principles of G00d Laboratory P:acrice was carried out at the abovelaboratory as part ofthe UK GLP Compliance Programme. At the time ofthe inspection no deviations were found of sufficient magnirude to affect thevalidity ofsrudies performed at these facilities. 

# n (J/
..

, J 

# I: 

# 'IJ7/f-<­ n 

# 10(,11-·**FD~::** 0 UK GLP Monitoring Uni! Page 150

### I i \ ,I 

# n -4­ 

### PROJECT NUMBER 163/134 GLP COMPLIANCE STATEMENT I, the undersigned, hereby declare that the objectives laid down in the protocol 

# n were achieved and as nothing occurred to adversely affect the quality or**integ~ty** of the study, I consider the da.ta generated to be valid. This report fullyahd accurately reflects the procedures used and data generated. 

### r: The work described was performed in compliance with the UK Principles of Good I ,I l} Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GLP standards published as OECD 

# r; Environment**~lonograph** No.45 (OCDE/GD(92)32); and arei'nconTor.m.(ty wHh, and c I implement, the requirements of Directives 87/18/EEC and 88/320/EEC. 

# n l , These internationa

### 1 standards are acceptable to the United State's Environmenta

### 1 Protection Agency and Food and Drug Administration, and fulfil the requirements of 

## Il 40 CFR Part 160, 40 CFR Part 192 and 21 CFR Part 58 (as**amend~d);.and** to the l ; Japanese Mini stry of Agriculture; Forestry and Fi sheries**(5~** NohSan', Notifi,calion No.3850, Agricultural Production Bureau) confirmed by an Arrangement between the Ministry and the UK Department of Health; the Japanese Ministry of Health and It/eHare (Notification No.313, Pharmaceutical**Affair~:** Bureau

- as amended; and f"""' Kanpogyo No.39 Envil"Onmental Agency, Yakuhatsu No.229); and the J.apanese Mini.stx:y 

## I : L I of International Trade and Industry (Chemical Substances Control Law, Kanpogyo No.39 Environmental A.geney, Kikyoku No.85). 

### ,;..'+'<:; c1(? Date: .......•.................. 

### r, i I 

# ;

# :G:e~::·**~:~~:. (~~;d;-** **~;~~~;;;)** Deputy Head of Aquatic Tox;co1ogy 

## ri for Safepharm Laboratories Limited l : rl 

# l! The following person was involved in the study in the capacity as indicated:

I ' i '. r



# .....!Ii!.?
. ......._ ................. Date:
.....**ZJ-.-~: 'J~**  

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

# n c. Mead Study Supervisor 

# r

r 

# LJ Page 151

# -

# -------- I 

## I \ / 

# n \ ; 

# n

,.-., r • 

### lj 

# n Ii ,-,.' 

### r; I I l ; 

# n l :
r; I ' L:' 

# [1 'II \. ) r; 

## 11**f~** J , 

# n n 

# n n**~** 

## I , I

i , f


## n I ' I I CONTENTS 

### TITLE PAGE QUALITY ASSURANCE REPORT GOOD LABORATORY PRACTICE STATEMENT OF 

### COMPLIANCE GOOD LABORATORY PRACTICE STATEMENT SUMMARY INTRODUCTION METHODS ANDMAT£RIALS DATP,**EVALlJATIO~**

'

### .. RESULTS TABLES L 

### Inorganic carbon values on each samp1ing 9ccasion in a Modified Sturm test with sodium**benioat~** 

### and"**~** '*') 

# '--­ 2. 

### Percentage Dl0degradation values from a Modified ' 

### Stufm , 

### test with sodium benzoate andl

___

### ~~~ 3. 

### Oxygen consumption rates and percentage inhibition for 3 hours contact time for 3,5-dichlorophenol andre from a 

### preliminary investigational**~tudy.** 

### FIGURE 1. Biodegradation curves for sodium benzoate and


### APPENDICES 1. 

### Environmental measurements 2. 

### Culture medium Page 152 -5­ 

### PROJECT NUMBER : 163/134 PAGE 1 2 3 4 6 7 7-10 11-12 '
13

14 15

17

18 19

# -- I I , ! \ .' 

# n
n STUDY SPONSOR 

### !j 

# n PROJECT NUMBER TEST MATERIAL TEST TYPE**~"** \ 

### Ii 

## l \ 

# [1

" 

### " INOCULUM

I 

# II DUPATION 

## II L.: CRITERIP. n ,I ! \ 

## r RESULTS. 1

## i**--~-:-:--.-"**

r-"\ 1 : \ ) -6­ 

### PROJECT NUMBER 163/134 SUMMARY

163/134

Assessment of ready biodegradabi1ity according to DECD

### Guid~lin~ 

## No 3018 

### ·C02 Evolution Test (Modified Sturm -rest)" referenced 

### as Mr:thod 

### C.4-C of 

### Commission 

### Directive 92/69/EEC (which**:co~stitutes** Annex 

### Vof Council Directive 67/548/EEC). A mixed population of 

### micro-o·y"ganisms. 28 days. Production of carbon dioxide.

attained 0% degradation at a eoncen'f('atidn of 18.6 mg 

### e/1 and so, ;, . therefore, cannot be 

### consider'ed as readily biodegradable under the stt'iet terms and conditions of DECO GUideline No. 301B. 

# r

f-: , I 1'.

Page 153-7­ 

### PROJECT NUMBER 163/134 

# n ASSESSMENT OF THE READY BIODEGRADABILITY



## ..-.'-">. OF 

### !USING THE CO2 _EVOLUTION TEST (MODIFIED STURM TEST) 

# n**-~.** ,-,,-,\ .. '**---~----** 

### .INTRODUCTION r, 

### This report describes the methods used and 'results obtained during an assessment I i 

### of the ready

. biodegradability 

## 0(----**~,** '- } r 

### ,

# '-----.7"'
....... I i <.J 

### The method**follo~ed** 

### the recommendations of 

### the 

### OECD Guidelines for Testing of Chemicals (1992) Uo. 3018 

### "Ready BiodegradabflH:Yi CO2 

### Evolution Test (Modified Sturm Test)" referenced as Method C.4-C 

### of Commission Directive 92/69/£EC (which constitutes.Annex V

### of Council Directive 67/548/EEC). r, I I , 1 ; I 

### The study 

### was commissioned 11 

### April -E 

### May 1994. 

## n I I U by 

### Toya 

### Ink Mfg. Co., Ltd. and was undertaken between
The study was performed according to Safepharm Standard Test Method ID.BIOOEG. r-, 

### L I I**MEt~ODS** 

### AND MATERIALS Test Material 

## . I 

# n

\•..j 

### Identification 

# .' I ! Description \ J r; I l I I l J 

### I Purity 1 

### Date Re.ceived. 

### Container 

# \ Storage Conditions 

# n i 

### Method of Preparation 

# n Analysis of the concentration, homogeneity and stability of the test material 

# n preparations was not appropriate to the test gUideline. Data relating to the identity, purity and stability of the supplied test material are the 

# [] responsibility of the Sponsor. 

# [l

Page 154 

### l' J 

### I

# I! 

# "--'**~8** 

# n

.....**~'** 

# Ii 

# c

r ! 

### ! LJ

1 

### +

# nl l. , 

# n
\**r~"** i : c J

r-· i : 

## 1**~**


# n ( 

### .. 

# n

.... ;

r-; 

## I ) "
., I I Standard Material 

### Name Inoculum Type Source

Date of collection Pre-Treatment Usage.rate .

Dllration Vessels Culture medium
Test concentration Standard material Test seril::!s PROJECT 

### NUMBER 163/134 METHODS 

### AND MATERIALS**(c~nt)** 

### Sodium benzoate (C6 H

5.COONa).
Amixed population of .activated sludge microorganisms, The aeration stage of the Severn Trent Water Plc sewage treatment plant at Bel

# per, Derbyshire ' t 

### treating predominantly domestic 

### sewage: 11 

### April 1994. .; , 

### The activated sludge was homogenised for approximately 2minutas prior to being 

### cemtr.ifuged and the supernatant used for testiRg.**1~'** 

### inoculum.

28 

### days. 5 litre**gl~ss** 

### culture vessels containing 3 litres. As defined in Appendix 2. 1% i

### nocu1 umper

## 'fest vessel. 18.6 mg carbon/1 in duplicate. 10 mg carbonll sodium oenzoate, in duplicate. 1. Culture medium with inoculum in duplicate. 2. Culture medium with inoculum and sodium benzoate i!l 

### duplicate 1. 

### Culture medium with inoculum and No. 650 Violet, in duplicate. 4. Culture medium with**i~oculum** 

### and No. 650 Violet, poisoned by 

### the addition. of 10 ml, of a. 10 gIl mercur'ic c.hloride solution to act as an abiotic control (one vessel only)
.

Page 155 I**~9** c 

## I •

J PROJECT NUMBER 

### 153/134 

## n I I • J 

### METHODS 

### AND MATERIALS (cant) 

### Exposure (cont) 

### Temperature 2I c

## e. Light regime The study was carried out in darkness. Procedure Approximately 24 

### hours prior to the start of the study the vessels were filled with 2400 ml of culture medium and 30ml of inocUlum and aerated overnight. On day 0 the test and standard materials were added and 

### the vo1

### umein all vessels made up to 3 1

### itres by tne addition of culture medium. The CO2 absorption bottles were connected to the outlet of the vessels on dayO. Aeration The culture vessels were sealed and CO2

### -free air bubb:led f-: 

### through the solution at a rate of between approximately \ I I ,

I
, 

### 40-50 ml/min and stirred continuously by magnetic stirrers. CO 

### -free air was produced by sparging compressed air· 2 

### through the following series: 

# n - 3 

### x 500ml Dreschel bottles filled with 350 

### ml 10 N

### NaOH. 1 x 

### 500 ml Dreschel bottle filled with 350 

### ml 0.025 NBa(OH)2 1 xSOO ml empty 'Oreschel bottle to prevent liquid carry-over. 

### (i CO2 absorption 2 x 500 ml Dreschel.bottles filled with 350 ml

, ' , . l ) 

### 0.05 MNaOH. All CO

### 2 absorbing solutions were prepared**usin~ p~rified**  

### de-gassed water. Sampling Samples (2 

### ml) were taken from the first CO2 

### absorber vessel or. days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 

### and 29. 

### The second absorber vessel was r, 

### sampled on days 0 and 29. J 	 : , 	 ! 

# n On day 28 1 

### ml of concentrated Hel added to drive off inorganic carbonate. The vessels were aerated overnight

, .' 

### and the final samples were taken from both absorber

, 

### vessels on day 29.
, ..\ 

### ,tl.pparatu5 Ionic5 555 TOC Analyser. Samples taken from the CO2 absorbers were injected into the 

## Ie (Inorganic Carbon) 

# n Channel of the Total Carbon Analyser for direct analysiS '- , 

### of evolved CO2, 

### Each analysis was carried out in triplicate.Page 156

# u
-10­ PROJECT NUMBER 163/134 

### ,", 

# U METHODS AND MATERIALS (cont) 

## [J Exposure (cont) Toxicity

determ'jnations Preliminary investigational work 

### was carried out to assess any toxic effect of 

### the test material on sewage 

### '"' I : 

### sludge micro-organisms. The method followed that CJ 

### described in 

### OECO Guideline 

### No. 209 "Activated Sludge Resp'iration Inhibition test", The 

### test material was aerated for 

### a period 

### of 3 

### hours at 21"C 

### in the presence of domestic sewage sludge with the additionof··a synthetic 

### sewage as a respiratory substrate.**Th~** 

### concentrations employed were 4.65, 9.30 and 18.60 

### mg carbon/litre. After 3 

### hours aeration the rateof l"espiration was measured for a 10 

### minute period a;ld: compared to data for the control and a reference**materi~l,** 

### 3,5-dichlorophenol. Archives, 

### Unless instructed otherwise by the sponsot, all original data and a copy 

### of the 

# ,.. I : 

### final report will be retained 

### in the ilrchives 

### of Safepharm 

### Laboratories for.a 

## l ! pei'j

### od of 

### ten years. After thi

### s period, the sponsor's instructj

### ons wi'll be 

### sought.



### , 

# r

'


# n [', 

# n l :

.' G j i**"--~'** 157

# u -11­ 

# o PROJECT NUMBER 163/134



### I i \.,) b) DATA EVALUATION 

# n , , u a} 

### Calculation of the Amount of Carbon Present The theoretical amount of carbon present as test material for No. 650 

### Violet is calculated as follows:-' 

### r: No. 650 Violet consists of-2**compon~nts**

, , 

## t,: a) C4SHS4C1ZNS06S2' 

### percentage composition oftest material == 

### '40% r-: 

# r: The percentage carbon present is calculated by: L ; 

### N,o of C atoms x mol.wt. of C 

# n x 

### 100% : ;, :

I 

### mol.wt. of test material The percentage carbon for component a) 

## n

, 

# c)

I ,48 

### x 12.011



### ----- x 100 

### '"' 59.19% 974.04 

### for a purity of 40'" 

### the %

### carbon '" 23.68% The percentage carbon for component b)**~** ,55 x 12.011

----- x 100 

### '" 56.64%

, 1166.32

::; < 

### for a purity of60% the %

### carbon 33.98% l l 

### The averal purity for the test material is 96%. Therefore the percentage r-, 

### carbon for the test material 

### = (23.6B + 

### 33.9B} x 0.96 '" 55.35%. Therefore 

### I : l .I 

### for a test concentration of lS.6 

### mg 

### Cil (ie. a total of 100.8 mg) the total organic carbon present will be 55.8 mg C. 

# n b) 

### Percentage Degradation The percentage degradation or percentage of Theoret;ca'] Amount of Carbon Dioxide (ThC02) produced 

### fs calculated by substituting the inorganic carbon values given in Table 1 in the equation below. The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation. 

# n r,· %

### ThC02 (: %

### degradation) 

### ' ;

j 

# " 

# U mg 

### Ie from testflask

- mg 

### Ie in control x 100% 

### mg TOC as testmaterial
Page 158 

### L-12­
,.--., 

### PROJECT NUMBER 163/134 

# lJ DATA EVALUATION (cont) 

# o c) 

### Abiotic degradation The percentage abiotic degradation is calculated 

### from the, equation below. The inorganic 

### carbon measurements (see Table 1) for the 

### poisoned test 

### vessel r, i I 

### are corrected for the control values prior to'the calculation of abiotic l ' 

### degradat'jon.



### " %

### abiotic degradation CO2 formation in 

### the sterile 

### flask (mg) x 100 

# \:

- , 

### ThC02 (mg) ,-' i i l :

rl , , 

## tj**~-,** i , l ) 

# n I i

. )


# n l i.



### r; I ' , I l " 

### r, 

### I .**~** ) 

# 1: n l 1
rJ ! I l 159

# -- -13­ PROJECT NUMBER 163/134 RESLILTS Inorganic carbon values for the test material J 

### ;tandard

..... 

### material, sodium benzoate, control and abiotic control vessels at each sampling occasion are given in Table 1. Percentage biodegradation of the test and standard materials is given in Table 2 and the biodegradation curves for the 

# n test and'standard materials are presented in Figure 1., 

# n**---~** I ' 

### \

__attai

### ned 0% degradati

### on after 28 days and so, therefore, cannot l J

### ~e 

### consldered as readily biodegradable under**the~trict** 

### terms and £onditions of , r-' , 

### the 

### OEeD Guidelines. 1 . l J 

### The results of the inorganic carbon analysis of both absorber vessels on day 29 j'j' ' I ' 

### confirmed that no significant amounts of CO2 were present in solution in the culture vessels as inorganic carbonate and that there was no significant carry­ ) r-;

: 

### over of CO2 into the second absorber vessel. 1 :



### , .' 

### Additional investigational work was carried out using the Activated Sludge 

# r: I ! , } 

### Respiration Inhibition test method (OECO Guideline No. 209), see Table 3, which shm
...ed that**'---"-'--~,did** 

### not significantly 'inhibit the respiration of**~,** \ I 

### sewage sludgemicrci":Qrganlsms atthe test concentration employed in the test. 1 ,!

. 

## --............. 

### In the light of this the inhibition control vessel ( plus sodium 

### n I ' 

### benzoate) was Qm'itted from the test. J I

, ' 

### Sodium benzoate attained 84% degradation after 28 da.ys ther'eby confirming the suitabil ity 

### of the inoculum and test conditions.



# n l j



## j' l J

160 r I i iro OJ cQ (D I-' m I-' '-
­

DAY

0 t 2 3 6 8 10 12 14 16 18 20 22 24 27 28 29


### Rl

­ 

### " ..----"1**~-J** I**~=:J** ......-.

### =~

### =-~ ,­ TABLE 1 

### Inorganic Carbon Values on Each Sampling Occasion in a Modified Sturm test

Control (mg Ie) R, 

## I 1<2Abs 2 AbS 1 Aos 1 "'bs 2 0.35 0.70 

# I 0.35 0.82 i
.. 0.70 0.70 

### - I 1.04

.. 1.38 ·

.. 1.72 1.83

- 3;08 3.42 3.1,0 3.51 

## - I 4.39 4.39 · 

# I 5.71 5.60 ·

.. 5.68 5.68 ! 6.97 

# I · · 7.41 I ·

.. 8.14 7.70 9. 1,0 9.18 

### · 

## - I

, 9.56 9.67

, 9.50**9.~0** ·

, 10.20 10.20 10.56 10.45 

### - 

## - 1.39 10.18 0.70 10.18


### R2 

### =Replicates

value not determined. 

# ------....... with Sod; urn Benzoate 

### ana;**~**



### - Sodium a"nz:l>.fe'**(!l\~** IG) 110. 650 Violet {mg lei No. 650 violet (ma !C)

Il, R,**R~** +--. Abs 1 Abs 2 0.70 

# I 0.35 1.04 · 1.38 .. 2.06 -2.74 · 2.72 · 3.27 3.70 

### · 3.34 · I·6S 

### ·

..29 , 5,25 6.30 - 6.16 

### - 

# ;:::," : 7.?;1 D.70 Abiotic control R, R, Abs 1 Abs 2 Abs 1

.. 0.70 0.35 C.70 I 1.04 

### - 1.04 1.36 1.38 

### - 1.72 1.72 

### - 2;39 

# I 1.94 2.04 2.49 2.70 3.36 3.01 3.43 1;.07 5.25 6.52: 6.16 6.98 7.57 7.00 · 

# I 2.03

.. 2.69 

### · 

# I 2.00

.. 2.32 

### · I

..?31 

## ·**~.62**



### - 3.15

.. 2.27 "

­ 2,58 

# ! 2.68 0.'35
_ .2.97 

### --- Abs 2 0.35



## -

- · 

# . -



## -

..

..

· · .....**~** I



## -

-

· 0.35 "'CI :;10 o t;., rrt**~** :z c: :lI: to !;J



### ..... en W

........ ..... W .J:> Abs 1

0.70 3.48 11.99 15.94 23.71 26.29 flbg 2 Abs 1 Abs 2 0.,'0

0.35 D.35

· 5.22 I ·
· 15.45 

## ­ 

# I 18.81 ·

.. 25.54 I 

# I 28.11

, 27.94 • 29.29 

### -

.. I 29.57 

### -**31.~7**

.. .. I 31,51 30.17

.. 32.98 32.31 

# I ·

· .. " 31;.10 34.65 · 35.53 36.19 36.73 36.19 

### - 36.07 36.07 

### - ·

- ,37.46

### ~."

# I . 36.16 ' 35.52 36.04 1.74 36.78
___ 1.74

- 

### Abs 

## =COZabsorber vessel.

# I.i -15­ 

# n PROJECT NUMBER 163/134 

# n n n Ii l ! 

# n I J . J 

# rl r, I , 

## I i 

# r: ( )



### n 

## l .! r: 

### I : , J

# ~! TABLE 2 Percentage Biodegradation Values from a "!.qdHied Sturm test with Sodium Benzoate**an~**



### Day

1 2 3 6 3 10 

### lZ ,14 16 18 20

. 22

24 27 

## I 0 28 84 

### o· 29* 90 I %

### Degradati

### on' %

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

### Sodium Benzoate 

# L._
...-....-._. ,j
12 1 0 42 0 52 0 71 0 79 ; ·0 81 83 ·0 0', 84 ,0 85 0 88 0 89 0:
. " 89
" 0 89 0 89 %

### Abiot.ic degradation

-

### -"**-~---:---:---** J . -_
..- .' 

### Abiotic Control I 0 .0 0 0

... 0 0 

# a 0 0 ...



### .. 0

0

. , '0

0 0 0

- 

### Day 29 values corrected to include any carry-over of COz detected in 

# n * I! 

### absorber 2 on Day 29.



### " I i 

### i .i 

# r: I**~**

j

Page 162

i 

# n ) I

# ----

---- 

# n , J 

### n**~** i l,1 

# r l ) 

## n !, :


# r: I :

, , 

# n t i 

### n ( i , J



# n r' j ( ) 

## r; I ) 

# r:
n j , 

### t J -16­ 

### PROJECT NUMBER 163/134 TABLE 3 Oxygen Consumption Rates and Percentage Inhibition 

### for 3 hours Contact Time for 3,5-dichlorophenol and( 

### from the Preliminary Investigational Work for a Modified sturm test


### Nomina1 

### Concentration Control Rl

4.65 mg 

## Gil 9.30 mg e/l 18.60 rng 

## eil 3,5-dichlorophenol 3.2 mg/l 

### 32 mg/l 

# r I I
I**A~** with( 02 

### Consumption rates (mg O/l/min) 0.42 0.38

0.39 0.37 0.34

0.32 0.05 I %

### inhibition

3 8 15

20 88**~---------------------~-- ------~--------~**  

### Replicates 1 and 2

Page 163 i

# l,l 

# n n 

# n n 100 

# n 

# rj 90 

# n

, 80

, r-, I I 

### l,1 70 

# lJ 60 

# lJ c a :;::; o Ii "0 a 

## I i 50 L . I 01 Q) Cl 

# n -17­ 

### PROJECT NUMBER : 163/134 FIGURE 1 

## Biodegradation Curves for Sodium Benzoate and

# c=--=~"~~,.**~",:-.~-_-**

# ___,_---)
r---r----,---,---.----..,.---.-----.--,---.----,'--,---y--' ,'--,---, 0- 0 Sodium benzoate 8- 8 No.(

----..**~** \7- -\7 No. '"\ JAbiotic Control**----".40_~** ... 

## o

- 

## o 

# J /

- 

# //

# ~ 

# I

40

# r I
..

# I [ l

-' 

# n 30 

# n 20 

# r l.i 

## o 10 

### n 

# l j**O~·~~~-** 

## __ __**-~-,** 

## __**~~~~** 

## __**-~-~~** 

## __**-~~~** 

# n o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (Days) 

# n Page 164 

# n I I-18­ 

# n PROJECT NUMBER 163/134 

# l j 

# n**~** 

# n n l 

### J 

# n n

II c , 

### n 

# II ,i ! 

## I '

.. fl 

## I J 

# li 'I 

# n
\ ) 

## ! i 7 

### I 8 9 10 11 

### 12 13 

## I 14 15 16 17 i8 19 I 

### 20 21 22 23 24 25 26 27 28 29 I 

### APPENDIX 1 Environmental Measurements Day 

### Room Temperature 

## ·c -1 21 0 21 1 21 2 21 3 21 4 21 5

.. 21

I 6 

# I 21 

### 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 

### 21 21 21 21 21 21 21 21
Page 165

# l ,i -19­ 

### PROJECT NUMBER 163/134 APPENDIX 2
Culture medium:
Solution a KH2P 04 8.50 g/l K

2HP04 21.75 g/l Na2HP04·2H20 33.40 gil NH4Cl 

### 0.50 g/l 

## n I i 

# n pH 7.4 , I 

### Solution b 

### CaC12 27.5 gil Solution c 

### MgS04·7H2 0 22.5 gIl 

# r. I j 

### Solution d 

### FeC13·6H2 0 

### 0.25 gIl 

# n To 1 litre (final volume) of purified water* is added the following volumes of solutions a-d. 

# n n

. \ 

### -10 ml of**S~lution** 

### a -1 of Solution b

1 ml of Solution c I, 

# n ml 

# [1 -1 ml of Solution d 

# n * Ion exchange and reverse osmosis treated tap water. 

# I' 

# n

r---I I ! , . 166