u.s. ENVIRONMENTAL PROTECTION AGENCY 

...."01:;"1\;1 ANTm ArTURE 

f II/IIIJIIIIJIIIIIIII III~ 1111 11111 IIli 11111 111111111111111111111111 10 000 008 4 I S 

.q:.::': 

YOl) must provide all information reljHeSled in this form to the extenl that it is known to or reasonably ascertainable by you. Make reasonable estimates if you dD not 

have actual data. 

Before yo\.! complete this fom1, you should read the "(nstructions Manual for Pre manufacture Notification" (the Instructions Manual is availabie from tbe Toxic 

Substances Cuntrol Act (TSCA) Inlormation Service by calling 202.554·1404, or taxing 202.554-5603). 

[f a user fee has been remitted for this notice (40 CPR 700.45), indicate in the boxes above the TS-user fee identiticution number you have generated, Remember, 

your user foe ID n,,,,,ber must also .Pl'ear on ,,,rrespor,ding fee remittal'ce, which is sent to 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) Name ofthe 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 suumission will 

r:ot ht: complete tlnd the review wil! not begin until EPA receives information. A 

letter in support of your submission should reference yourTS user fre identification 

ilLHnbtf You must ~ubmit an origInal and lwo of this notice including all test datu. 

If you daim':':d any ;nformalinl1 as conlidentiaL copy I!1IiS( also he 

5ubrHincd. 

Part II HUM ,\N EXPOSURE AND ENV IRm'McNT/\L RiOLEASE 

Ifthere are Severa: nlanufilCture, 1)l'Qt:es.splg, or use oper,:ulGIlS be descr;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] data or otht:r datn nod any OptiOl1'11 information lncl ut.led in rile 

notl;,;e. 

OPTIONAL tNFORMI':rION 

Yuu ma)' inClude any inrorm:Hion that you want EPA to consider in evaluating [he n-:w 

substance, .on page II of this lorrn, space has been provided for you to descnbed 

pollution prevention and recycling inlhrmatiol1 yOIl may h::1ve re"gardJng thl! new 

~;uhstnnce. 

Sp~a!!ed ··blnGil1~( boxes are included throughout this form for you LO indicate your 

\;,tiHingness to be buund to certain st:Hemenls yOu Olake in thiS section, :illCh as use. 

production voltllll~. prolecti lie equipnl!;:nt . Tl1i~ optIOn IS intended to ro..lllce delays 

that routinely accompany the development or consent urders or Sigl1i ricanr New Use 

Rlll~, ExC.:pl in the c~se of exemption :}Ptilic3tiOils (suc,h.Js TMEA. LV E. LOREX) 

where ;,;erHUI'l IhiiJrmation provided III such notification is b:nding on the suomi tt~r when 

the Agency appr.:,ve.s the exemption checking a binding box III this nOtil:e 

by i:se1f pmhibit 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 the in tDrmal:ion th~t you cI ai III as 

lIssert 0. claim in an attic!uno::nL circ!t: orhradccr the inform,llilln you 

Mark (x) ;f any infonnation in [his notice i:i ClilillHXI as coniidrotiaL 

TEST DATA AND OTHER DATA 

You are required to submit all test data in your or control and to 

descnplion of all other data known to or ascertainable by you, 

data are related to the health and environmental effects on the m3nufacmre, 

processing, dlSlribution in eommen:e, use, or disposaJ of the new chemical . 

substance. S2Ildard literature citations may be submitted for data ih the open 

idenll fy whether tt;st data on the substance or on an 

compos: tion of the tested mmerl:::l1 should be characterized, are. examples 

of ~est data and other data. Data shoulrJ be si.lbmitted according to the requirements 0" 5720.50 of the PremanulaCltUe Notitication Rule (40 CFR Pan 720). 

Test Data (Check Below a,lY included in this notice) 

En'llronmenl~'l nile data 0 Yes 	 Other data 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 

or control of the submitter 

.. A physical ;j:ld ch::':01lcal prop~nH'_" worksheet lS located 01 the IDS( page of this form. 

TYPEOF NOTICE (Ched. Onl} One) 

PMN {Premanu t'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 LV E (Low Volume Exemptioll) ..to CFR 723,)O! . ..:)( 1) 

LOREX {Low Release/Low Expllsure r::~,empUotlj (fi! 40 CFR 72150{c)(':) 

D 	 LVE Modification D LOREX M otii ttcatioll

IS TtllS A CONSOLIDATED PMN'! o Yes

# of chemlcals 


(Prenotice Comn~l.Inicaton 11 requira:!-, e.r.ter # on page 3) 


EPA FORM 7710·25 (Rev. 5·95) 	 Replaces previous editions of EPA Form 7710·25. 

~70 
Public reporting burden for this collection ofinfonnation is estimated to average 110 hours per response, including time for reviewing instructions, 

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

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

Chief, lnfonnation Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W., Washington, D.C. 20460; and to the Office 

of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

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

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

commercial purpose, other than in small quantities solely for research and development, the substance identified in Part 1, 

Section B. 

2. All information provided in this notice is comp lete and tru thful as of the date of submission. 

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

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

Additional Certification Statements: 

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

state men t that app lies: 

[2'J The Company named in Part 1, Section A has remitted the fee of$2500 specified in 40 CFR 700.45(b), or 

o The Company named in Part 1, Section A has remitted the fee of $1000 for an Intermediate PMN (defmed @ 40 CFR 

700.43) in accordance with40 CFR 700.45(b), or 

The Company named in Part 1 Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of$1 00 in 

accordance with 40 CFR 700.45(b). 

If you are submitting a low volume exemption (LV E) application in accordance with 40 CFR 723.50(c)(l) or a Low release 

and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50( c)(2), check the following certification 

statements: 

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

purposes, other than in small quantities soleI y for research and development, under the terms of40 CFR 723.50. 

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

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

• 

I 

I 

If this application is for an LVE in accordance with 40 CFR 723 50(c)(l) , the manufacturer intends to commence

manufacture of the exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day 

review period. 

The aecuracy of tbe statemetl ts you make in this notice should n:tlecr your best prediction Qr the <tnticipated facts regarding the chemical substance 

described herein, Any kltowiug und willful misinterpretatiun is subject to crimlnal pt'nalty pursuant to J8 USC 100L Contidentiul 

Signature ::lIld title of Authorized 0 nicial (Original Si&,'11aturl;! Required) Date 

X 

Signature of :Jgent ~ (i f appticuble) Date 

Part I -- GENERAL INFORlVIATION 

EPA FORM 7710-25 (Rev. 5-95) Page 2 
Part I -- GENERAL INFORMATION 

Section A ­ SUBMITTER IDENTIFICATION 

la. Person 

Submitting 

Notice 

(in U.s.) 

b. Agent (if 

applicable) 

Oty, State, ZIP Code 

Nam e of authorized official 

Company 

Mailing address (number and street) 

Oty, State, ZIP Code 

Telephone 

c. If you are submitting this notice as part of a joint submission, mark (X) this box. -----------------...... 

Joint 

Submitter 

(if applicable) 

2. Technical 

Contact 

(in U.S.) 

Name of authorized official 

Company 

Mailing address (number and street) 

City, State, ZIP Code 

Name 

Company 

Mailing address (number and street) 

Oty, State, ZIP Code 

3. if you have had a prenotice communication (PC) concerning this notice 

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

If you previously submitted an exemption application for the chemical 

substance covered by this notice, enter the exemption number assigned 

by EPA. If you previously submitted a PMN for this substance enter the 

PMN number assigned by EPA (i.e. withdrawn or incomplete). 

--,~ 

If you have submitted a notice of Bona fide intent to manufacture or 

import for the chemical substance covered by this notice, enter the 

notice number assigned by EPA. ,.. 

T~ of Notice - Mark (X) 

FORM EPA 1710-25 tRev.5-95) 

1. D 

D 

Manufacture 

Only 

Binding Option 

Mark (x) 

Pag,,3 

Position 

2. 

Mark (X) ;... if none ID

Mark (X) )­ if none ID

Mark (X) )­ if none fLJ

lU Import 

On.ly 3. D Both 

D BindingOption 

Mark (x) 
x 

based on 

I name corre ct Abst rae IS ;;;;~w;;i;;th;CTTo:srC"A\!.1n:;;v;;e:;n",to;;;r:;;y:JITis;;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 of composi tion and the 

diagram, as comolete as can be 

COrTeet structure diaglum. For a class 2 substance - (I) List the immediate 

C AS Registry Nu mbers. i2) Describe the nature of the reaction or process. {JJ Indicate the 

composition (where uppropriate). (4) PrOVIde a correct representative or partial chemical stl1lcture 

if one can be reasonalAv ascertained. 

Section B -- CHEMICAL 

For Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA Preferred Name must 

be provided, which ever is appropriate based on CA 9CI nomenclarure rules and conventIOns). 

EPA FORM 7710-25 (Rev. 5-95) Page 4 

Confidential 


Page 4a 

Part 1- GENERAL li~FORMATlON -- Continued 

Section B -- CHEMICAL IDENTITY JNFORMATION - Continued 

2. Polymers (For a definition of polymer, see the Instructions ManuaL) 

a. Indicate the number-average weight ofthe lowest molecular weight composition arthe polymer you intend to manufacture, 

Indicate maximum weight percent of low molecular weight species (not including residual monomers, reactants, or solvents) below 500 and 

below 1,000 absolute molecular weight orthat composition, 

DescIibe the methods ofmeasurement or the basis for your esti mates: GPC Other D: (Specify) 

i) lowest number average molecular weight: 

Confidential 

ii) maximum weight % below 500 molecular weight: 

iii) maximum weight % below 1000 molecultif weight: 

D Mark (Xl this box if you attach a continuation sheet. 

b, 	 You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the 

"Contidential" box next to any item you claim as confidential 

(I) 	 Provide the specilic chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufaclUre of 

the polymer, 

(2) 	 Mark (X) this column if entry in column (I) is confidential. 

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

(4) 	 Mark (X) the identity column if you want a monomer or other reactant used at two weight percent or less to be listed as part ofthe polymer 

description on the TSCA Chemical Substance Inventory, 

(5) 	 Mark (Xl this column ifentries in columns (3) and (4) are confIdential. 

(6) 	 Indicate the maximum weight percent of each mOnOmer or other reactant that may be present as a residual 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 specified c11emic.1 idcntilY information reported 

Method I (CAS Inventory Expe., Service - a copy of the identification repo., D Method 2 (other source) 

obtained from CAS Inventory Expert Service must be submitted as 

the polymer that is consistent with TSCA 

(check one). 

Iyascertained, 

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

EPA FORM 7710-25 (Rev, 5-95) PageS 
Impurities 

(a) Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose. Provide the 

CAS Registry Number if available. If there are unidentified impurities, enter "unidentified." 

Estimate the maximum % of each . If there are unidentified estimate their total 

Impurity and CAS Registry Number 

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

0/0 

0/0 

% 

Confidential 

Trade identification· List trade names for the new chemical substance identified in subsection 1 or 2. 

Mark (X) this box if you allach a continuatic>fl sheet. 

·5' ,9' ,15,19auach a continuation sheet. 

Generic chemical name· 	 If you claim chemical identify as yQU must a name your substance that reveals 

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

TSCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing generic names. 

Carbazole Violet sulfonamide derivs. 

new Chemical substance. 

CAS Number Confi· 

dential 

Part I -- GENERAL INFORMATION _. Continued 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 6 
Part I - GENERAL INFORMATION -- Continued 

Section C -- PRODUCTION, IMPORT, AND USE INFORMATION: 

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

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

production volume for any consecutive 12-month period during the flfSt three years of production. Estimates should be on 100% new chemical 

substance basis. For a Low Volume Exemption application, if you choose to have your notice reviewed at a lower production volume than 10,000 

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

Maximum first 12-month production (kg/yr) 

I 

Maximum 12-month production (kg/yr) 

(100% new chemical substance basis) (100% new chemical substance basis) 

Confidential 

Binding 

Option 

Mark (x) 

X 

2. Use Information- You must make separate confidentiality claims for the description of the category of use, the percent of production volume 

devoted to each category, the fonnulation of the new substance, and other use infonnation. Mark (X) the "Confidential" Box next to any item 

you claim as confidential. 

a. (I) -­ Describe each intended category of use of the new chemical substance by function and application .. 

(2) -­ Mark (X) this colulnn if entry column (I) is contldential business information (CBI). 

(3) -­ Indicate your willingness to have the infonnation provided in column (1) binding. 

(4) -­ Estimate the percent of total production for the first three years devoted to each category of use. 

(5) -­ Mark (X) this column if entry in column (4) is confidential business information (CBI). 

(6) -­ Estimate the percent of the new substance as fonnulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for 

commercial purposes at sites under your control associated with each category of use. 

(7) -- Mark (X) this column ifentry in column (6) is confidential business infonnation (CBO. 

(8) -- lndicate % of product volume expected for the Iisted "use" sectors. Mark more than one box if appropriate. Mark (X) to indicate 

your WI 1IlllgnesS to h ave t e h use type praVI d d e m (8) b mdmg. 

(9) -­ Mark (X) this column if entry(ies) in column (8) is (are) confidential business ll1fonnation (CBI). 

Category oruse (I) CBII Bmoiug Produc- CBr %jo CBI % of substance expected per use CBI Ophon tion 0/0 Form­ (8) 

(by function and i.e. a dlspersi ve 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

% %

.If you have identi ned a "consumer" use, please provide on a continuation sheet a detailed description of the use( s) of this chemical substance in consumer products, 

In addition include estimates or tile concentration oftbe new chemical substance as expected in consumer products and describe the cherr,ical reactions by which this 

substance loses its identity in the consumer produce 

DMark (Xl this box if you attach a continuation sheet. 

b. Generic If you claim any category of use desctir.tion in subsection 2a as contident; al. enter a genetic description of that category. Read the 

use Instructions Manual for examples or generic use desoriptions, 

description Dispersion additive for printing ink 

D Mark (Xl this box if you attach a continuation sheet. 

3. Hazard Information Include in the notice a copy of reasonable facsimile of any Ilazard warn;, statement. label. matelial safety data sheet. or other 8lOumg 

lnrurm~lllvll which -.vlii b-: pr;JV1J-.:J P":L:iUU v,h v l:i ica.sulIJbl, tikdy tv b..: tv LlIl:::. plljl.\:",:tlv~ cttU1pmcllt .. 11- pr~h.:t;ct.'~ 

for the safe handing, transport, use, or disposal oflhe new substance. List in pan hazard ,"fonnalion you 

LZJ Mark (Xl this box .fyou attach hazard information. 

- EPA FORM 7710-2) (Rev. 5-95) Page1 
Part II-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark 

claim 

for each type of manufacture, processing, or use operation involving the new chemical substance at industrial sites you 


do not have to complete this section for operations outside the U.S.; however, you may still have reporting requirements if 


after' describe these See instructions manual 

Confiwill occur. dential 

x 

Site address and ~lreer) 

City. Count v S'"'e ZIP code 

[ the same operation wi!! occur at more than one site, enter the number ofsites. Identify the 

additional sites on a continuation sheet, and if any ofthe sites haye signiticantly different 

production rates or operations, include all the in formation requested in th l' section for those 

sites as attachments. 

x 

Use 

Batches/year x 

(I) 	 Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5 galion pails, 55 galion 

urLun, rail car, tank truck. etc.). 

(2) 	 Provide the identity, the appro)(imate weight (by kg/day or kglbatch on a 100% new chemical substance basis), and entry point of all starting materials and 

feedstocks (i ncludi ng reactants. solvents, catalysts. etc,), and of all products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not 

used daily or per batch,). 

(3) 	 Identi fy by number the poi nts of release. including small or i ntermiUent releases, to the environment of the new chemical substance. 

I. Batch 

Maximum {IUO% newcf<lfliclil 

surn.r.u.:c) 

2. Continuous 

d. Process description Mark (X) to indicate your willingness to hove your process description bindmg. 

~ Mark (Xl this box i f you attach a continuation sheet. 

EPA FORM 7710-25 (Rev.5-Q5) PageS 
Confidential 


Page 8a 

Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER - Continued 

2, Occupational Exposure -- You must make separate contidentiality clai ms for the description of worker acti vity, physical form of the new chemical substance, 

number ofworks exposed, and duration of acti vity. Mark (X) the "Confidential" box next to any item you claim as confi dential. 

(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 if enn-y in column (I) is contidential business information (CBI). 

(3) -- Describe any protective equipment and engineering conn-ols used to protect workers. 

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

(5) -- Indicate the physical formes) of the new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance (if part of a mi xture) 

at the time of exposure. 

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

(8) -- Estimate the maximum number of workers involved in each activity for all sites combined. 

(9) -- Mark (X) this column if enn-y in column (8) is confidential business information (CBI). 

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

(12) -- Mark (X) this colwnn if entries in columns (10) and (II) are confidential business information (CBl). 

Worker activity CBI Protective Equipment/ 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 

- --: X X X r---x _.. -­ X X X X 

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

3, Environmental Release and Disposal -- You must make separate confidentiality claims for the release number and the amount of the new chemical substance 

released and other release and disposal information. Mark (X) the -'Contidential" box next to each item you claim as confidential. 

(I) -- Enter the nUlOber of each release point identified in the process description, part II, section A, subsection Id(J). 

(2) -- Estimate the amount of the new substance released (a) directly to the environmem or (b) into control technology (in kg/day or kg/batch). 

(3) -- Mark (X) this column ifenn-ies in colWllns (I) and (2) are confidential business information (CBI). 

(4) --Identity the 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 substance will 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 of the new substance to the environment. For releases 

disposed of on land, characterize the disposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for 

each site describe any additional disposal methods that will be used and whether the waste is subject to secondary Or tertiary on-site treatment. b. Estimate the 

amount released to the environment after control technology (In kg/day). 

(6) -- Mark (X) this column if entries in columns (4) and (5) are contidential business information (CBI). 

(7) --Identity the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or 

NPDES numbers ofthe POTW (Publicly Owned Treatment Works). Mark (X) ifthe POTW name orNPDES # is confidential bl.siness information (CBI). 

Relea Amount of new substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CBI 

se released rdease 

Numb 

er 

(I) (2a) (2b) c g ~~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 provide NPDES # CBY 

destination(s) of waterway 

rcle:ls~s tn \V~ttT 

I 

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

EPA FORM 7710-25 (Rev. 5-95) Page 9 
Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operations Involving the new chemical substance at sites you do' not control. Imp0l1ers do not have to complete this 

section for operations outside the U,S,; however, you must report any processing or use activines after import. See the Instructions Manual. Complete a sepamte 

sectioll B Jor each type ojprocessing, or lise operatioll involving the new chemical substance. Ifthe same operation is performed at more than one site describe the 

typical operation common to these sites, Identify additional sites on a continuation sheet. 

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

(1) -- Diagram the major unit operation steps and chemical conversions, including interim storage and transport containers (specify - e.g. 5 gallon pails, 55 gallon 

drums, rail cars, tank u'ucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2) -- Provide the identity, the approximate weight 

(by kg/day or kg/batch, on an 100% new chemical sub,tance basis), and entry point of all feedstocks (induding reactants, sol vents and catalysts, etc) and all 

products, recycle streams, and wastes, I nelude cleaning chemicals (note frequency if not used daily or per batch). (3) -- Identify by number the points of release, 

induding small or intermittent releases, to the environment of the new chemical substance. (4) Please enter the # of sites (remember to identi fy the locarions of 

these sites on a continuation sheet): 

# of sites 

See attached sheet (page }Dq) 

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

2, Worker ExposurefEnvironment.1 Release 

(I) --From the diagram above, provide the letter for each worker activity, Complete 2-8 for each worker activity described. 

(2) -- Estimate the number ofworkers exposed for all sites combined. 

(4) -- Estimate the duration of e"posure per worker in (a) hours per day and (b) days per year. 

(6) --Descnbe form of exposure and % new chemical substance (i fin mixture), and any protective "'luipmenr and engineering controls. if any. used to 

protect 

(7) -- Estimate the percent of the new substance as formulated when packaged Or used as a final product. 

(9) -- From the process diagram above, enter the number of each release pomt. Complete 9-13 for each release point identi fled. 

(10) -­ Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kg/bmch). 

(12) -- Describe media ofrelease i.e. stack air, fugitive air Instructions Manual), surface water, on-site Or off-site land or incineratIOn, POTW, or other 

(specify) and control technOlogy, ifany, that will be iO limit the release of the new substance to the environment. 

(14) -­ Identify byproducts which may result from the operation. 

(3), (5), (8), (II), (13) and (15) -- Mark (X) this column ifany of the proceeding entries are confidential business information (CBI). 

Letter # of CBI ! Duratioll CBI Protect; ve Equip. I %in! CBI Release Amoumof CBI MOOi a 0 rRelease CBI 

of Workers of Engineering Controls! 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 if you attach a continuation sheet, 

EPA FORM 7710-25 (Rev 5-95) Page 10 
Confidential 


Page lOa 

OPTIONAL POLLUTION PREVENTION INFORMATION 

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

In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce 

or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal 

of the PMN substance. Please include new information pertinent to pollution prevention, including source 

reduction, recycling activities and safer processes or products available due to the new chemical substance. 

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

modification, process and procedure modification, product reformulation, raw materials substitution, and/or 

inventory control. Recycling refers to the reclamation of useful chemical components from wastes that would 

otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions of pollution 

prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance 

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

information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting 

of risks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In 

addition, information on the relative cost or perfonnance characteristics of the PMN substance to potential 

alternatives may be provided. 

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

substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and 

examples. 

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

volume manufactured; (3) a reduction in the generation of waste materials through recycling, source reduction or other means; (4) a 

reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in 

the cost ofproduction and/or improved operation efficiency of the new chemical substance in comparison to existing chemical 

substances used in similar application; or (6) the extent to which the new chemical substance may be a substitute for an existing 

substance that poses a greater overall risk to human health or the environment. 

./­ -_.... _--_..­

\ 

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

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

i 

- I r 

1 Part III -- LIST OF A ITACHMENTS I I 


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

structure/activity infoIII1ation), and optional information after this page. Gearly identify the attachment and the section of the form to 

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

numbers of each attachment. 

Mark (Xl the "Confidential" box next to any attachment name you claim as con.fidentlal. Read the Instructions Manual for 

guidance on how to claim any infoIII1ation in an attachment as confidential. You must include with the sanitized copy of the notice 

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

I 

Attachment name i 

Material Safety; Data Sheet (MSDS) 

I.- .. ~~

I 

I 

T est Reports 

ACllte oral toxicity in the rat 

rabbit 

itation 

Magnusson & kligman maximisation study in the guinea pig 

Reverse mutation assay "ANIES lest" using salmonella typhimurium and escllerichia coli 

Assessment of ready biodegradation; C02 evolution test 

D Mark (X) this box if you attadl a continuation sheet. Enter the attachment name and 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 

i 
PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

To as.~ist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and 

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

the units in Which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical 

state of the neat subs.ance should be provided. These measured propeItie:; should be 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 

submit this worksheet; however, EPA strongly recommends that you do SQ, as it will simplify review and ensure that confidential 

information is properly protected. You should submit this worksheet as a supplement to your submission of test data. This worksheet 

is not a substitute for submission of test data. 

I 

Prope,1y Mark 

(Xl if 

(a) provided 

Physical state of neat substance 

Vapor pressure 

iaJ Temperature °C 

Densitylrelative density 

Solubility 

@ Temperature "C 

Solvent 

Solubility in water @ Temperature 'C 

Melting temperature 

Boiling I sublimation temperature@ pressure 

Spectra 

Dissociation constant 

Particle size distribution 

Octanoll water ~artition coefficient 

Henry's Law constant 

Volatilization from water 

Volatilization from soil 

pH@ concentration 

Flammability Flash Point 

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 Safety Data Sheet 

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 med ical at ten t ion. 

SKIN CONTACT Wash affected areas with soap and wate~ If irritation develops, seek medical 

attent ion. 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 attent ion. 

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 ext inguistier. 

HAZARDS DECOMPOSITION PRODUCTS CO. CO". e I c. 

FIRE FIGHTING PROTECTIVE EQUIPMENT A self 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 0 f 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 respi rator. 

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 essent ial ly insoluble in water. 

TOXICITY ; lin known 

BIODEGRADA TI ON ; 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 INFORMA TI ON 

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----~ 

Test Reports -L - -~ 
l J 

n 


n 


n 

'. nI 

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 'I., 

n; 

QUALITY ASSURANCE REPORT 

r _ J

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 Study ri ! 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 V of 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 , J labelling regulations.

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 V of 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 internal i ona11 y 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 

A range-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 

, I ' 2000 200 10 5 5

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 37 
i 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+ R 28 ·VERY TOXIC IF SWALLOWED" 

r TOXIC >25 to 200 T R 25 "TOXIC IF SWALLOWED" 

i. I HARMFUL >200 to 2000 Xn R 22 "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 

, j .i ' 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 

: n.I

n 

,......, 

i . 

I I c J 

n 


n
 l j 

IIt ) 

nl ; 

IIn 

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 

( ! 

40 

~--=~~] r] r--­ ',---) ,_. =-J ____J ~J :... .. _J ==l 

o = no signs of systemic toxicity L = lethargy 

H = hunched posture Rd = decreased respiratory 

-c 

::0 

rate E

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 

Mal e 

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 

t.Q 3-1 Male 145 197 235 52 38 ....... 
 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 

Iime of Death Macroscopic Observations 

mg/kg &Sex 

3-0 Male Killed day 14 No abnormalities detected 

3-1 Male Ki 11 ed day 14 No abnormalities detected I 

....... 


I 3-2 Male Ki 11 ed day 14 No abnormalities detected U'I 

"" "" 

3-3 Male Ki 11 ed day 14 No abnormalities detected 

3-4 Male Kill ed day 14 No abnormalities detected 2000 

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

e; 

c... 4-1 Female Kill ed day 14 No abnormalities detected rr1 

("") 

-I 

4-2 Female Kill ad day 14 No abnormalities detected % 

I=: 

4-3 Female Killed day 14 No abnormalities detected 3: 

o:l 

rr1 

::c.. 4-4 Female Ki 11 ed day 14 No abnormalities detected .... 

O'l 

W 

......... 


..... 

N 

I.C 
l j 


-16­

n 


n 


: i '. ~ 

n 


n 

n 

n 

n 

n 


PROJECT NUMBER: 163/129 

A P PEN D I X I 

TIlE DEPARTl\1ENf OF HEAI..J'H OF THE GOVERNMENT 


OF nm UNITED KINGDOM 


GOOD LABORATORY PRAcnCE 

SfAlEMENT OF COMPIlANC£ 

IN ACCORDANCE WITH 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 of Good Labonlfory Practice 

was carried out at the abo-ye labonitory as part of the UK GLP Complil.nce Progmnme. 

At the time of Ihe inspection no deviations were found of $Ufficient magnitude to affect 

tile validity ofstudies perfonned at these facilities. 

n D. F. Moore 

Director 

UK GLP Monitoring Unic 

.l,1 

Page 45 

r 
l j 

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 
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-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 

l 

n
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-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 

nI 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 V of 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 V of 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. A quantity 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 l J with the patches, the trunk of each rabbit was wrapped in an elasticated

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 

t j 

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 


{i njuri es ; n depth} ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 


Page 
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-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 

nI i If irreversible alteration of the dermal 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 54 
I 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. 

R 35 "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 R 34 "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 It IRRITATING 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 

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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 =~ .. J :='-=:1 '__J =:J ==:J.J ~._.J ~ J :...._J ~_J __J ="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 Score 
I l j 

, " 

n 


n 

n 

n 

n 

n

l 1 

II 


n 

n 


n 


n 


n 


r 


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L .! 

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Ii 

-15­

PROJECT NUMBER: 163/130 

A P PEN D I X I 

THE DEPARTMENT OF HEAIXH OF THE GOVERNMENT 

OF 1HE UNITED KINGDOM 

GOOD LABORATORY PRACIlCE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECI1VE 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 of Good Laboatory Pactice 

was carried out at cbe above laboritol)' U ~f( of the UK GLP Compliance Programme. 

At. the time of me iJupccdon no deviarions were found of u{flCi.en( magnitude (0 affect 

the validitY of studies perfonned at these facilities. 

D. F. Moore 

Page 60 Director 

UK GLP 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 

nl. Facsimile: (0332) 799018 

PAGE 1 OF 19 PAGES Telex: 377019 SAFPHM G

n 

, ) 

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n 

Page 61 

n 

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-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} of 	Inspection 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 

, FOR SAFEPHARM QUALITY ASSURANCE UNIT ...............tp\\(... ...

26. JUl. 1994 

n 

Page 62 

I 

(--

! 
II 

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n 

n 

I, the 

nI 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 
 nc : 

nl \ 

Page 63 
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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 III 

Individual 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 
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-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 requ ired. Page 65 
t ': 


-6­

n 	 PROJECT NUMBER: 163/131 ~----.------

rI 

I : ~Tr-rYE IRRITATION

( 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

1L 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 

Storage conditions l 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. 

67 
J 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. nl ; 

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 iri s o x 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 1 es ion 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 

foll ows: 

R 36 	 It IRRITATING 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 

R 41 	 "RISK OF SERIOUS DAMAGE TO EYES" 'II , 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 i ri s persisted in one treated, eye 

n seven days after treatment. to ) 

8. 	 CONCLUSION 

The test mated a1, '-. J produced a maxi mum 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 B lEI 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 

nI. .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 

w­

-...) IRIS 

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 

~ 2 A Redness S 2 2 0 	 25 	 0 0 0 0 0 c...

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 

Group Total 	 39 30-45 18-33 9-24 0 c:...

0 

/"1"1

n 

-I 

:z

c:: 13.0 10.0-15.0 6.0-11.0 3.0-8.0 	 0.0 	 3:

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 I nfl ammat ion 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+ 

'U 80 MALE 24 Hours 0 0 1 	 1

III 	 I 

to 	 48 Hours 0 0 0 0 (]) (2.50) 	 72 Hours 0 0 0 0 	 -I

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 

t J 

n 

1j 

Ii l ] 

nI . 

l J 

r-' 

! I I

. ) 

! 

I~ C } I 

n
 l ; 

n 


n 


Ii

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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 swell ing 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 

I ; n

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 n PROJECT NUMBER: 163/131

A P PEN 0 I X I I 

n 


n 


n
 \ J 

'. ) 

nI 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) ! 

0.0 	to 0.5 Group mean total score at 24 hours :: 0 Non-l rri tant (1) 

Group mean total score at 24 hours > 0 Practically non-irritant (2). 

0.5 	to 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 

2.5 	to 15 Group mean total score at 48 hours:; 0 Minimal irritant (3) 

Group mean tota l score at 48 hours> 0 Mild irritant Ct.} 

15 to 25 	 Group mean total score at n hours" 0 Hild irritant (4) 

Group mean total score at n hours> 0 Moderate irritant (5) 

More than hal f of the individual 

~total SCores at 7 days 10 or less 	 Moderate i rri tant (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 hal f of the irdividuaL 

total scores at '7 days > 30 and 

individual total score at 7 

days > 60 Very severe lrri tant (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 hal f of the individual 

total scores at 7 days ,. 60 and 

any individual total score at '7 

days ,. 100 Extremel y 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 P PEN D I X I I I 

TIlE DEPARTMENT OF HEAI.:I1I OF 1HE GOVERNMENT 


OF TIm UNTIED KINGDOM 


GOOD LABORATORY PRACIlCE 

STAn:MENT OF COMPIlANCE 

IN ACCORDANCE WITH DlRECllVE 88/320 EEC 

UBORATORY 

SafePharm Laboratories Limited 

POBox No 4.5 


Derb,v 


DEl 2BT 


DATE OF INSPECIlON 

31 January 1994 

A general inspection for compliance with the Principles of Good LaborllflUY Practice 

wu carried out at the above laboratory as pan of the UK GLP Compliance Progrllmme. 

At. the time of the inspection no deviations were found of sufficient magnitude to .affect 

the validiry of studies perfonned at these facilities. 

Page 79 

UK GLP Monitoring Unit 

D. F.Moore 

Din:aor 

163/131 

l J 

l , 1

in 

n 


n 


l 


n 


MAGNUSSON c~ &KLIGMAN MAXIMISATION 


STUDY IN THE GUINEA PIG 

n 
 PROJECT NUMBER: 163/132 


n 

n 


n 


1I , 

, 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 
l i 


-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. nn 

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 


nI 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 


'iI 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;l j 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 li 

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
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PAGE 


6 

7 

8 

8 

8 

8 

8 

9 

9 

9 

10 

13 

14 

15 

15 

15 

16 

16 

o 

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-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 

n 	

c. , 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 
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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 

foll ows: 

,.-, 

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 

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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 V of 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. 

nl; 

r

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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 ~ 

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PROJECT NUMBER: 163/132 

n I 3. TEST 	SYSTEM (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 of 	38 - 62%. The rate of air exchange was 

n approximately 15 changes per hour and the lighting was controlled

l

I )I . by a time switch to give 12 hours light and 12 hours darkness. 

4. PROCEDURE 

n 

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

l J material was based on the Guinea Pig Maximisati~n test of Magnusson B. & 

n Kligman A.M., J. Invest. Oermatol. (1969) 52: 268 276.

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 

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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 

L J dermal irritation after a 48-hour occlusive exposure, was 

n. ) 

selected for the topical induction sta~e of the main study. 

nl; 

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 

1j 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 

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PROJECT NUMBER: 163/132 l 

n 

! 

4. PROCEDURE (contd) 

n 4.2 

n 

n 

l n:. 

'II ; 

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 
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-12­ II PROJECT NUMBER: 163/132 

\ ) 

fl I 1j 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 
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n PROJECT NUMBER: 163/132 t j 

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 bluntn 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 ) 

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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 

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PROJECT NUMBER: 163/132 

r 

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, , 

~ 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. 

Topi cal 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 

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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 

n control group animals at the 24 and 48-hour observations. The . ) 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 ; 

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~ 

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 

II l. ,J 

Page 98 

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==: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. 

Animal Time Concentration of of Evidence of Local Evidence of 

Test Materi a 1 ldentl fi cat ion Observation 	 Necros is· Systemic Toxicity (% w/v) 

24 hours None None 

A 48 hours None None

72 hours None None 

7 days None None 

24 hours None 

'1J 

III B 	 48 hours 5 None

to 

(1) 	 72 hours None 

7 days None I.D 

I.D 

24 hours None 

C 48 hours 10 None

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 

D 	 48 hours 	 25 None None 

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 

i 
l __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 

1.Q N 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 

F 25 ?s 0 I 0 STA 0 0 STA

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 SIT! 

~ 

< 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 
------., .=::-J r­ :_-_J ~ ~.-::-:J =) -~~ , J -,--I ,___ J :-:=J ~-~ C) r __J 	 =~ :=J 	 =J ~--=---=-) ~-=-J ~-=-~ :-=-~ l__. 

..------- ----­ ~_ ' : 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 :-=:J c-]

\ :: 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. 

---- ..; 

' 

Animal Number Skin Reactions at Observation Time:

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-' 

o 3 ?5 ?s ?s ?5 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 
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,,--<~ 

___ I : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

A P PEN 0 I X V INTRADERMAL INDUCTION - INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS 

VEHICLE: ARACHIS OIL B.P. 

Animal Number Skin Reactions at Observation Time:

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 
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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 

N 
r---, =.J =-=-J -:. ..... ____ .J J =-.---:--:J - 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 P PEN 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 
------------

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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 

N 
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-29­

PROJECT NUMBER: 163/132 IIn 

A P PEN D I X x 

n

l; EVALUATION OF SKIN REACTIONS* 

n 

n Erythema and Eschar Formation 

l ) 

No erythema .........................•.......................•........... 0 


Very sl ight erythema (barely perceptibl e) ....................'............ 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 

u 
C:-::-J =---=:-J ~ ~ ,..=:1 ~=-=-:J 

( MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG 

A P PEN 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,c innamaldehyde Tech 85% 

2-mercaptobenzo- . thiazole 

(X- hex'ac innamaldehyde 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 

arachi s oil 

B.P. 

Topical 

100% 

50% in 

arachis 

oil B.P. 

100% 

50% in 

95% 

aqueous

ethanol 

U.K. 

Chall enge 

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 80% {SIlO} I

70% (l/10) 

8 

-c 

m 

n 

-I 

:z 

80% (S/lO) i 

c:l 

m 

..

;:tl 

...... 

en 

, 

w

..... 

W 

N 
I I 

· ! l -' 

-31 


n 


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PROJECT NUMBER: 163/132

A P PEN D I X X I I 

• 


lliE DEPARTMENT OF HEAL1H OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORAroRY PRACIlCE 

SfATEMENT OF COMPlIANCE 

£N ACCORDANCE 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 of Good LaboraroIJ Pradice 

was c:a.rried out at abe above laboratory as pa,rt of the UK GLP Compliance Programme 

At the time of abe inspc:ction nO deviations wece found or sufficient magnitude to affect 

the validity ofsrudies performed at lhese facilities. 

D. F. Moore 

Director 

UK GLP Monitoring Unit 

Page 110 

.. 
Ij 

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(--~ 

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 

Facsimil e: (0332) 799018 

Telex: 377079 SAFPHM G 

111 

l) 

n -2­ \ , 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. 

FOR SAFEPHARM 

Paternan 

QUALITY 

C. Biol., 

ASSLIRANCE 

M.I. B101. 

UNIT .. ~......... "'fj£... .................. ~ . 


[J 

10. JUN. 1994 

DATE: I 0 .. .. .. .. ... .. .. ... " .......................... -.................. . 


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 

qual ity or integrity of the study, I consider the data generated to be 

valid. Th is repOl't fully· and accuratel y refl ects 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 i nternat i ona1 standards a ra acceptab1e to. the-·J apanese Min i stry 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 

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l~ 

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. .....'.c;... ....... .. !!:T.'-!": ..... . 


Page 113 
u 


n -4­

,

I )i PROJECT NUMBER: 163/133 

CONTENTS 

PAGE 

SUl91ARY 5 


INTRODUCTION 6 


TEST MATER1AL r 


METHODS 7
 11U 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) Prel iminary 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 C J PROJECT NUMBER: 163/133

r. , I 

U 

REVERSE MUTATION ASSAY "AMES TEST" o 

USING SALMONELLA TYPHIMURIUH 

AND ESCHERICHIA COLI. 

rJLJ 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.' 

Iil; 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 Viol et was therefore tested up to the maximum recommended dose 

level of 5000 Jig/plate. A precipitate 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) guidel in,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 
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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/pl ate 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'centrati ons: 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 State bourne 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 MGlucose-5-phosphat~ 2.5 ml ,--. 	 0.1 M NADP 2.0 1111 1 i 

u 	 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml 

Sterile distilled water 14.5 ml 

Page 118 r 

Lj 
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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 

Escheri ch i a coli stra i n 5 ml of 1. a 111M tryptophan was added to each l J 

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 E and 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 prel iminary 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 

n histidine (or tryptophan in the case of WP2uvrA-) supplemented top 

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 119 
n 	 -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 containn 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 
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PROJECT NUMBER: 163/133 


METHODS (contd) 

nd 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 confi rm the correct response. To b,e cons i dered . negat i ve the 

n number of induced revertants compared to spontaneous revertants should

l J 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 

11U 

Page 121 
I. ,~ 

-12­ n PROJECT NUMBER: 163/133 

r-, RESULTS 

I I 

u a) preliminary Toxicity Study 

The dose range of L )used in the prel imi nary toxici ty 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 

i

1 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 nl) 

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 

., 

IjI , . 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 

n for positive controls used in this assay are shown in Tables 1 to 4.

I. 	 J 

n Information regarding the equipment and methods used in these experi­ , ' 

U 	 ments and the details of the personnel involved, as required by the 

Japanese Ministry of Labour, are given as Appendix 1. 

t 

rI , ) 

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 nl.._ 	 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 Ll 	 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. Reportr~ Part III (1989) Cambridge University Press. 

o 

nu Page 123 
i I 

LJ 

-14­

PROJECT NUMBER: 163/133 

nd 

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'enthes is. 

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 , Ji 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 

nl J 

Page 124 
-15­

on 	 PROJECT NUMBER: 163/133 c ) 

TAB LEI 

Ii I : 

U 

n 	 TABLE OF TEST RESULTS: EXPERIMENT 1 

L.. 

n 

L J 

I : 	

NAME OF TEST SUBSTANCE:(~ =========~==~====~=============~~~::~~~~:=~~~=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 TAl 00 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 

nL ) 

!II 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") 

ni \ 

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 

TAB 	L 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 

nl 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· PA I R SUBST IT.UTI ON 

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 FRAHESH IFT 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' 

l nJ ... .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 128 
I l J 


-19­ n PROJECT NUMBER: 163/133 

n 

\l, 

, ) 

II 

'1 

r-' 

! I 

I ; 

n 

;l A P P EN 0 ICES

L ) --=~=====~=~

n 

I, .I 

n 

( n. REVERSE HUTATION ASSAY HANES TEST" REPORT 

n

l.1 

rl 


n 


n
 , J 

n 


n 


n 


129 
II 

-20­

n 


n 


nI ; 

n 


n 


n 


l: 

[1 


n 


l

n, 

n 

l : 

n 


< 

n
I l , 

n 

\ j 

f: 

l J 

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 compl eted 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 

suspens i 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 strai n 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 

.'-, . ..: 

r '(3) Composition of S9 Mix

L 

Constituents Amount in Iml 

S9 Mix 

S9 0,1 ml

MgC12 8.0 tlmol

KC? 33,0 tlmo1 

n-. I Glucose-6-phosphate . 5.0 .umol 

u 

-Gl ucose-6-PhOSPh9 

dehydrogelHlse 

Const ituents 

. 

NADPH 

NADH 

Na-phosphate...buffer 

Others (NADP) _. 

.. 

Amount in 1 ml 

59 Mix 

tlmol 

tlmol 

100.0.umo l. 

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 S1Gt·1A lO6F-3691 Technical 97 DMSO 

control - I 

4NQO SIGMA 36F-0015 TechrHcal '99 DMSO 

9AA SIGt~A 96F 5641 Tecbnical 98 DMSO 

2AA SIG~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~ 

1l, 

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) " , '. .... 

Name Supplier ,lot 	No., Grade, Purity(%} 

, 

Dimethyl Solvent used SIGMA' 83H0557 99.5 sulphoxide « •••••

.' Stabil ity 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 puri ty 

6. 	 cormITIONS HC. OF PRE-CULTURE 

(1) 	Conditions 

~.r=e== '1('-- f"""t7~~~; 

Name 

Nutrient broth ., Oxoid Oxoid Ltd. .350 51940 11/97 - -

Period of pre-culture 10 hrs ; 

e' 	 ~o ~'"!-~-- ,

.'~=-=:'l:::;=:"-=:t~ . 

Manufactl!rer Lot 	 No. .. ' --..:.--:. . 

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 ' 

l

I ' 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

(x 10g/mT) study 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.' 

nI 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 .Pl ate Method -

Composition 

" 

Bacteri al suspension Hil Od ml 

Test substance solut'ion ml 0.1 ml -

Na-phosphate buffer ml 0.5 ml ,. - S9 Mix (i n case of metabo­ ml 0.5" ml lic activat'ion method) - Top agar solution ml 2.0 ml -----~"... . Others ( ) .. 

Pre-incubation Temperature ·C.. ,

-

Time rni n . , . 

Incub,at fan Temperature ~C 37"C ..... . ..' 

-,--------. 

Time hours 48 hours ........ 
 .. ~ '. .. =. = = ­

10. COUNT MElHOD or COLONIES (Enci rei eland 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 

, 

,

, ~...} 

flI 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:- A statistical analysis of the data l~ was not 'requ; red to determi ne the result of the test. : A predp Hate was 

observed at and above 1000 pg/p 1 ate, ' thi s di d 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 level s in the rat 1 iver 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 

- - III 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 Qual ity Name C. Bi 01 . I M.L B10,1 . Assurance ., 

Study 

Title Director - Name P.W. Thompson H.N.C. 

Study Director Genetic 

Toxicology () n' . 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 

, - Titl e 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) 

IIu 

KEY TO TABLE OF TEST RESULTS. 

LJ n

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. 

fl."_ 4. Th~_f~llowing postfixes. are used when required:­

n. c ~ contanli nated I..,j 

P precipitate 

x s plate unscarable 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 o PROJECT NUMBER: 163/133 A P PEN 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 

nI I " ) 

Ji i i. 

" i I 

U 

[ 


138 

u 

-29­ o 	 PROJECT NUMBER: 163/133 

o 

A P PEN 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 

CON CeNTRAl iON 

(ug/plate) 

Tlll00 

BASE-.PAI R SU8STlTUTlON TYPE 

TA.1535 IIP2uvO.·· , 

FRAMES"! fT· TYPE 

TA98 T... ,537 

nl 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) 

nu 

+ 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

nI 

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 .
 nL J 


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 o PROJECT NUMBER: 163/133 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

l J + .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-ITRATl ON 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 + COLOIH ES/PLA TE 420 ( 411.7 ) 113 ( 119.7 ) 153 ( 155.7 ) 150 ( 154.7 ) 117 ( 111.0 ) 

417 124 166 166 

=~~======================~~~===~=========~=================s====::====================~====:======================:.:==~=~ 

o 

Page 141 

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118 
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-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-findi n9 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 

WP2uvrATA1535 

N/A 

N/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 =:.-.~ =-:J C-=-: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 

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W 


L.1 

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:-~ 

.__J ~=-':::J =J '~::=J c.~

~" ~~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- -t 59 EXP[RIM ENI 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 L-J :~ __::J

~--'.-

~ ___ ~DOSE-RESPON I 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 

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, 

w 

CTI 

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F ==: 1"1'1 • ~~: : 

E 

20 n 

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~ co 

1'1"1 312.5 625 1250 2500 5000 :::c 

UG/PLAT£ ...... 

CTI 

~ ..... 

w 

w 
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-37­

n PROJECT NUMBER: 163/133

A P PEN D I X I I I 

n 


n 


n 


n 


I! 


l I 

n
i - ) 

" i 

l ) i 

THE DEPARTMENT OF HEAI.J.'H OF TIIE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD lABORATORY PRACTICE 

SfATEMENT OF COMPUANCE 

IN ACCORDANCE WITH DIRECI'IVE 88/320 ERe 

LABORATORY 

SafePharl11 Labora tori es Li111 ited 


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 out at the above labonitory as part of the UK GLP Compliance Programme. 

Al !:he time of the inspection no deviations 'M:re found of sufficient magnitude to affect 

the validity ofstudies perfonned at these faciliti~ 

D. F. Moore 

n Director 

UK GLP Monitoring Unir 

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 nl ) 

[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 
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-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 

'lI : 

l ; 

TIlE DEPARTMENT OF HEALTII OF mE GOVERNMENT 


OF TIIE UNITED KINGDOM 


GOOD lABORATORY rRAGTICE 

SfATEMENT OF COMPllANCE 

IN ACCORDANCE WITH 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 above laboratory as part of the UK GLP Compliance Programme. 


At the time of the inspection no deviations were found of sufficient magnirude to affect 

the validity of srudies performed at these facilities. 

n (J/ .. , J 

I: 

n 

10(,11-· 

'IJ7/f-<­

0 FD~:: 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 internat i ona1 standards are acceptabl e to the United State's Envi ronmenta1 

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 Mi ni 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 : 

rll! 	 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 
- --------

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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 

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CRITERIP. 

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r RESULTS. 

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-6­

PROJECT NUMBER 163/134 

SUMMARY 

163/134 

Assessment of ready bi odegradabi 1 i ty 

according to DE CD 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 V of 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 . '- } biodegradabil ity 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. 

nI 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 \ 

n 	 Storage Conditions 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 

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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 (C6H5.COONa). 

A mixed population of .activated sludge microorganisms, 

The aeration stage of the Severn Trent Water Plc 

sewage treatment plant at Bel per, Derbyshire ' t 

treating predominantly domest ic 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 cI • 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 

vo 1umei n all vessels made up to 3 1 itres by tne 

addition of culture medium. The CO 2 absorption bottles 

were connected to the outlet of the vessels on dayO. 

Aeration The culture vessels were sealed and CO 2-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 N Ba(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 M NaOH. 

All CO2 absorbing solutions were prepared usin~ p~rified 

de-gassed water. 

Sampling 	 Samples (2 ml) were taken from the first CO 2 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) 

Toxi city 

determ'j nat ions 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 rate of 

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 th i s peri od, the sponsor's instruct j ons wi'll be 

sought. 

,

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n[', 

n 

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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 of test 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 of 	60% 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 degradat i on 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 test 	flask - mg Ie in control 

x 100% 

mg TOC as test 	material 

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'j on. " 

% abiotic 


degradation CO2 formation in the sterile flask (mg) 
 x 100 \:

- , ThC02 (mg) 

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-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 ' \ 	 __atta i ned 0% degradat i 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 

j'j' ' The results of the inorganic carbon analysis of both absorber vessels on day 29 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 sludge micrci":Qrganlsms at the test concentration employed in the test. 1 ,! . 	 --............. 


In the light of this the inhibition control vessel ( 	 plus sodium 

nI ' 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 

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160 r 

I 

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ro 

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<2 


Aos 1 "'bs 2 AbS 1 Abs 2

0.82 0.35 0.70 I 0.35

i .. 0.70 - 0.70 I 

1.04 

.. 

1.38 · .. 1.83 1. 72

3.42 3;08 -

3.51 3.1,0 - 4.39 I 4.39 · 

I 5.60 5.71 · 

.. 5.68 5.68 ! 

6.97 I · 

· 

7.41 I · 

.. 8.14 7.70 


9. 1,0 
 - 9.18 · , I 9.67 9.56

, 9.~0 · 9.50

, 10.20 10.20

10.45 - 10.56 - 10.18 0.70 10.18 1.39

R2 = Replicates 

value not determined. 

------.......
 wi th 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 I0.35 

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 

Abs 2 Abs 1 flbg 2

0.35 0.,'0 D.35 

· 5.22 I · 

· 15.45 I ­ 18.81 · 

25.54 .. I

I 28.11 , 27.94 • 29.29 - .. 29.57 I 31.~7 - .. .. 30.17 I 31,51

.. 32.31 32.98

I 

· 

· 

.. 34.65 · 31;.10 "

35.53 36.19 

36.19 - 36.73

36.07 · 36.07 - ,37.46 -

36.16 ~." I . ' 35.52 

36.78 ___ 1.74 36.04 1.74 -

Abs =CO Z absorber vessel. 
I.i 

-15­

n PROJECT NUMBER 163/134 


n 


n 


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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 28 84 0

29* 90 o· I 

% Degradation % Degradat i on' --~.----~ 

Sodium Benzoate L._...-....-._. ,j 

12 1 

42 0

52 0

71 0

79 0; 

81 ·0

83 ·0 

84 0',

85 ,0

88 0

89 0

0: . 89 "

" 

89 0

89 0

% Abiot.ic 

degradation - -" -~---:---:---

J . -_ ..- .' 

Abiotic 

Control 

I 

0 

.0 

0 

0 ... 

0 

0 

a 

0 

0 ... 

.. 0 

0 . , 

'0 

0 

0 

0 -

n Day 29 values corrected to include any carry-over of COz detected in * 

I! absorber 2 on Day 29. 

"I i 

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Page 162 

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-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 

Nomi na 1 

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 L 50


. 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

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[

l 
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-' 

n 

30 


n 20 


rl.i o 

10 


n

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O~·~~~-__ __-~-,__~~~~__-~-~~__-~~~ n o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 


Time (Days) 


n 

Page 164
 n 

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n 	 PROJECT NUMBER 163/134 


l j 

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II 


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8 


9 


10 


11 


12 


13 


I 	 14 


15 


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17 


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19 
 I 


20 


21 


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23 


24 


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26 


27 


28 


I 
 29


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 

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PROJECT NUMBER 163/134 

APPENDIX 2 

Culture medium: 

Solution a 	 KH 2P04 8.50 g/l 


K2HP04 21. 75 g/l 


Na2HP04·2H20 33.40 gil 


NH4Cl 	 0.50 g/l n 

I i 

n pH 7.4 

, I 

Solution b CaC1 2 27.5 gil 


Solution c MgS04·7H2

0 22.5 gIl
 r. 

I j Solution d FeC1 3·6H2

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 

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166