Fa= Approved. O.M..B.**~o.** 2070-0012 Approval Expu-eo 10-31-96.


### U. S. ENVIRONMENTAL PROTECTION AGENCY 

## A 

# EAtA PREl\iA.NUFACTURE 

# 0 1-\ NOTICE AGENCY USE ONLY

Date of receipt

Whl'n compktl'J**~end** t!ll'

.. torm tll 

### FOR NEW OIEMICAL SUBSTANCES

DOCUMENT CONTROL OmCER OFFICE OF POLLUTION PREVENTION AND TOXIC SUBSTANCES, 7407 U.S. E.P.A. 401 M STREET, SW

. WASHINGTON. D.C. 20460 

### Enter the total 

### number of pages in 

### the Premanufacture Notice '-· r 

## Company Sanitized

• You mut provide alllafoDIUitioa reqa..-..d Ia tlaia to- to the edeat tUt lt Ia kaoww. to or _.-,ly. ucertabaable by }'U'IL Make**~le** estimata If you do

not have actul. data. • Before yoa CXIIIlplete thia form. yoa llhoald lUd the "'utradiou Malul for Pftmauafac:tue Notificali011" (the lMtructiou M.u.ul ia n-ailable from the Toxic Substances Control Ad <TSCAl baformatioa Service 'by calling**2112~14CM,.** or fllmlr; 202·554-5603l. • If a user fee haa been temitied for thia notice (40 CFR ?011..'-5), indiate iD the biiXIS ahem! the TS-uer fee ldmtifiati011 a'IIJII.ber you hne genaated. Remember, your uer fee ID number mut abo appear Oil your mnapoadillr; fee nmiltace, which Ia aeat to: EPA, HQ Aa::ouatiDg Operatiou Brmch (PM-266), P.O. 360399M,.**Pitbb~** PA 15251-6399, Atm. TSCA UMrFPart I- GENERAL INFORMA noN
You mut provide the CII.Il'elltly cum!d Chemical Ab.t:rada (CAl Name of the
aew chemical•abiiQnce,. even If you c::J.al.Jil the ldeD.tlty • confldeatial. You.._,. authorlze aothu penon to nbmii chemka.lldatity iDfoDIUII:i011 foryoa. bat your •ubmiuioa 

### wm-t be cumplete and the review will aot bepa

.. 

### w EPA receiva tAla**illfo~oa.** A 1etlez: ia ••ppod of yav
.. .,__o• llhollld refereDCI! your TS uer fee ldeatifical:ioa aiUDbeL Yoa aut lllllnait • odp...l md two cupls of thia aotke illdadllls all tat data. If,_ dai.-1 aay IDformatioa • cunfid-tial. a .mgJ.e uaitlad mpy a..t ailo be nblllitt.d..
Part 

### n- HUMAN EXPOSUltE AND ENVIKONMENTAL**~SE** If there are ...vera! m.mafacture. piOCI!Uing, or ue operatiou to be dscrlbed Ia Part li, sediona A mel B of thia aotic:e,. repmclace the s.edla• • needed.
Part ID- UST OF ATTACHMENTS
Attach additional aheet8 If there ia not enoogh space to answer il question fully. Ubel each contlnaation sheet with the conapondillr; Ndioa headiJI.r;. In Part III. list thae attoK:hment., ;my tat d&a or other d&a ad ay optional information IJI.daded ill the notice.
OPTIONAL INFORMATION
You may illdade uy iDfol'JIUitioa tlUit yo• wul: EPA to CIIUici• b. enlutlaJ the aew •abstmce. 0. page 11 of thi8 fora. •!*Z U. b-. pzurided for yo• ID d-=ribe pollattoa pl'eYeDiioa md recydlaslafuz:aatiaR 

## JV•..., li-. npaillls the new sabRance.

So-c.alled "bindiJI.r;" box• are illdacloed tlmnaghollt tail &ma foryv• to ilacilca your willlnp- to be boaad to Cl!ltabl

.. .._.....,. yo11allk

...

b. thi8 aotlce, nell
as uae,. produdlon volume, prota::tive ecrulpmeal ••• TliJ8 optioa. iate.ded to reduce dela}'8**th~** matinely aa::ompany the deorelopaamt .of CDII8at ordta or Significant New UIIC Rules. EJuzpt ill the CIM of eumptioa appUatia• lluch • TMEA, L VE, LOREX> where certala lnfouaatioa provided Ia nch •otlfiatioa ia binding oa the Rbmil:ter whea tile AlleDCY appiDV• lila**DZ~Dptioa** appllcatioa. checldll.& a biDdillr; box ill thia aotice**~by** ll:8elf pmhlbtt the ll1lbmittllr from laer devtaill& &om the illfomutioa (except chaakalldatity) n:poned Ia the foiDI..

. -

CONFIDENnAUI'Y CLAIMS

You may c:laia my l:a.fomudioa Ia tiLia Bot:ice • CIOIIfidestW. To -at a c:l.ta on the form, Ja.uk 00 tbe coafideatial box .at ID**tile~** tlut yo• dai&
• cunfideD.tial. To ..- a daia Ia

- ettach•at, drde or bmck.et U.. IDfoanatioa yow. dalm • mafbieatial. If !pp da!m tvtnnnt!qn lp the pgtig: a· mnfldmtial YAP mplt abp mpyidc I vnHiv;d ycgiPP gf tllc pgtig; flpdgdt,nl i¢tlebmepgl. For addltio&al i..aadio

.. - d.liJaia& IJabvwKI- • coa.fid-tial. rud the 1a.RradiDBa Manu.al. 

# D Ma 00 If

### ~y infof'IIUitioa lD thia •oticz ia daimed • coafideatial.

EPA FORM 7710-25

. (Rev. 5-95}



# . I TEST DATA AND 01liEll. DATA
You aa required to nbmit all tat data iD your poueulon or 0011trol • and to proYiGe a cieKEipti- of all othB' de knowa 110 or -ably MC?Jta!nable by yoa.lf th- data ue re1Red to the ilealth ud m...UO...tal effec:D of the -afal:taa,.**~** diRdbatloa in CDIIIJIIell:a,.-.. or dlapoul of the •- c:beaic:alll1li:IRaD.CL Stadard lltentu. dtatloa..., 8e 1111bailted far- cia Ia the opa ldadflc iltantuJL CmgpJcte 

### tat d;U !writtrp lp Ep&!bbl pgt pmmaria gf dW. m!!!!l be nP•Ittcd If tbct dp PPf-.JPllCII' tp the gpcp l!tmtpz. Youllhoukl de.ady idatlfy wiletller a.t daa 18 oa the nbRaDa: or 011 aa aalog. Al8o, the chaalcal mmpadloa of the le*d JUtaialllhollld be ciYracterizecL FollowiJlr; ilft 

# -pi• of tat data ud other data. Data llhoald be •ubmitled 

### ac=mil.Jlll to the nquiremma of §7211.50 of the Premaaafac:tue Noti6atioa 1tule (40 0'11. Part '720).
Tat Data (Check Below my·indaded in thi8 aotice)

• E.a'rimammtal f&e data 

# [il Ys • Othudata 

# 0 y.,.

• Health effec:D data 

# [if Yes 

### IUI.k-en•

• E.avirimmeatallffe:ta data 

# D Ys Stmdareilldtrity rdationahip8

• PhyU:al/C'aeakal Ptapedia • 

# D Ys Tst data aot !.D. tlae**~-** or matrol of the .U.utter
•A phymai and clte.ic:al piOpCHs _...... ia loc:aled oa the lat pap

ofUU.fo-.**~** TYPfi OP NOTICE (a.eck Oaly 

### a. 

## S

# ~ PMN aor-..atadut

# ~**~** fg

# ~

0 IN'IEKMEDIATE PMN (aabaltteclla

## MCI"-~**~ad** PMN)

. .._- 

# 0**~** 

# n< SNUN (Sipifkaat Nellt U.e Notla=) 

### :X

- f"'1 <JO 

# 0 TMEA (fell MukeliaJEumptioa

### A~ 

# D LVE CI.-Vol-Eze.ptioll) ••

## CFR~C)(]) 

# 0 LOREX

### a-~ ExJ>o-re Ex-p&.) e 723.!0(c)Q.) 

# 0 L VE Modl.fkatloa 

# 0 LOREX Modifial:lc.

IS nns A coNsoLIDATED PMN7 

# 0 Y•
lofc:haak::Ua !Preaotica Camm..ucatioa I ""'!aiftd, alar t on**~e** 31 Replaces previOWI editions of EPA Form 7710-25

. 

# (oS 

# ?,ll}..•

Public reporting burden for this collection of inionnation is estimated to average 110 hours per response. including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed. and completing and reviewing the collection of information. Send
comments regarding the burden estimate or any other aspect of this collection of information,. including suggestions for reducing this burden.
to Otief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M St, S.W

... Washington. D.C. 20%0; and to the Office of**Mana~ent** and**Bud~t.** Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

## CERTIFICATION

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


### 1. The company named in 

### PaJ1 I, section 

### A, subsection 

### la of this notice 

### form intends to manufacture or import for 

### a commercial purpose, other than in small quantities solely 

### fur research and development, the substance identified in 

### Part I, Section 

### B.

2. All information provided in this notice is complete and truthful as of the date of submission.


### 3. I am submitting with this notice all test data in my possession or control and a**d~ption** 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 

## SNi.JN, check the following user fee certification statement that applies: 

# 0 The-Company named in 

### Part I, Section 

### A has remitted 

## the fee of 

# s2500 spe:ified in 

### 40 CFR 700.45 (b), or
- 

# 0 The Company named in 

### Part 

### I, Section 

### A has remitted the fee of $1000 for an 

### Intermediate 

### PMN (defined @ 

### 40 CFR 700.43) in accordance with 

### 40 CFR 

### 700.45(b), or · 

# 0 The Company named in 

### Part 

### I, Section 

### A is a small business concern under 

### 40 CFR 700.43 and has remitted
a fee of $100 in accordance with 

### 40 CFR 

### 700.45 (b).
If 

### you are submitting a low volume exemption (L VE) application in accordance with 

### 40 CFR 723..50 (c) (1) or a Low release and low exposure exemption (LoREX) applicatiOn in accordance with 

### 40 CFR 

### 723..50 (c) 

### (2), check the following certification statements: 

# D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for 

### research and development, under the terms of 4D CFR 723.50. · . . , 

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

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

# 0 If this application is for an 

### L VEin accordance with 

### 40 CFR 723.50 (c)(1), 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 accuracy**~f** the statements 

### you make in 

### this notice should 

### reflect 

### yow- best 

### predictial of 

### the anticipated fads 

### ieganfing the chemical

substance described herein. Any knowing and wiJlful misinletpn!tation is subject to aiminal 

### penalty 

### pursuant to 18 USC 1001. r-C-o-nfi-.d-en-, 

### -tW.--1 Signature and title of Authorized Official (Original Signature Required) Date

Signature of agent- (if applicable) Date

FORM EPA**771~25** (Rev. 5-95) Page2

## Part I- GENERAL INFORMATION

Coofi·

### ~~~~~~~~~~~----~~~~~~--------------------------------------------~dentiL
la. Person Submitting Notice
• (in U.S.)

b. Agent (if applicable) and street)
Cty, State, ZIP Code

Name oi authorized. official
Company
Mailing address (number and street)
Cty, State. ZIP Code Telepboae

c. If you are summtting this notice. part of a joint 

### submission. marie 

### 00**~box. ---------------~**  Joint Name of autbCllized official Submitter (if applicable)

2. Technical Contact (in U.S.} Company
Mailing addrell (number mci street)
Cty, State. ZIP Code

Name

Company
Mailing address (number and street)
Qty;State, ZIP Code

3. If you: have had. a prenotice .communicatioa (PC) amCI!I'Ding this notice and EPA assigned a PC Number to the notice. enter the number. Poailica

Telephone

Telephcae 1 I

Mark (X) if none

· 4. If you previously submitted an exemptKm applic::aticD foe the c:helnDi substaDce awered by tlUs-notioe. enter the exemptioa number l!!lllligned by EPA. If you previously submitted. PMN fot this 

## .w..nce enter the 

# L b 

# 2 Y)? Made 

### 00 PMN number llllligried by EPA (i.e. witbdzawn ar inalmplele).

. - 

### 0

- 'J () If nooe ·**~** If you have submitted a notice of Bona fide intalt to manufacture ar import foe the chemical substance covered by this notice, entl!r the notice number asaigued by EPA.

.... Type of Notice- Mark 00

# 1-D~- Optkm
FORM EPA 7710-25 CRev. 5-95) Pap3 

### MarkOO lineae 

### ----:)lo

.. 

# D

3. 

# DBoth

### Part I -GENERAL INFOR!viATION- Continued Section B - CHEMJCAL**ID~VITTY** 

### INFORMATION: You. •ut provide • Clln'entiy comet O!cmaJ. Abatrac:t3 !CAl name Oi the**~b.ta:na** bueoi em thnunth Collecuw lnoax (9(J) of CA nommcl..arun rules**an~ <:oriYeZ~tioN.** 
Uxltplete eitber item 1 (ClaA 1 or**l~~abttancea)** or l (Polymera) u appropriate. Complete all othEr itema. lf another**~** will submit chemic:al. identity informati011 for you (for either item 1 or 2), mark (X) 

### the box at the risllt 

# 0
and addres of that in a sheet. )II. 1. ClaN 1 or 2 c:bemicalmbiMa.Dces (for definiliona of cW8 1 and c:.la!e 2 substances, !lee the**Iru~c:tioa•** MamW)

a. Cl.a!IS of substance- Mark (X) 1 

# 0 ea.. 1

. or 2 

# D CJus 2

b. O.emical name (Currently·c:on-ec:t O.emiaal. Abstract3 (CA) Name that is c:oosistent with T5CA Inventory listings for similar subetanc:es. For 

### a- 1 substanc:e! a CA Index Name must be provided. Far 

### au. 2 substaiia!s either a CA Index Name or CA Preferred Name be provided, whichever is appropriate bued on CA 9C nome:ndature rules and conventions.). ·

c. Please identify which method you used ID dewlap ar obtliD tbe specified c:bemic:al indentity i:nfcanati011 repcrte:i in this notice:

(c:hec:k. one).

. . 

# 0 Method 1 (CAS Inventory Expert Service- a a:1py ri tbe

### Iden~tion 

# QMethod 2 (Other Sourt:e) report obtained. from the CAS Inventory 

### Expert Service must be submitted a an attachment to this notic:e) d. Molec:ular formula and CAS 

### Registrjr Number Of a number already ex1sls for tbe subatance)

CASt

e. For a dua 1 subltanc:e. provide a cxmpj* mel cmra:t**c:hemkal~inM:1:J.D'e** dlagrmL Fer • d.. 2 IUJ:wannr

- {1) IJit tbe immediate '\/ precunor Sl(u.taDces with their rapecttve CAS Repay Numbln. CZl Daaibe tbe Datare of the r-=tkla ar 

### prcaa (3) lndicate the 11.. range oi cxmpoation md the typlc:al. cxmpaatlaa (where apprapdale). (4) Provide a curect repraatlatlve ar pctial c:bemiCII. structure diagrmn. as cxmplae • em be kDown, If aae c:. be**~y** ...a:t.I:P&:l

/

7 •.

Mark (X) this box if you attach l ccnt!nuatioo sheet. FORM EPA m0-25 (Rev. 5-95) Page 4

## \ Part I -GENERAL INFORMATION- Continued**~** 

### Section B- CHEMICAL IDENTITY INFORMATION

- Continued

:z. Polymers (For a definition of polymer, see the lnatractiona ManuaL) Con: dent a. Indicate the number-average weight of the lowest molecular weight composition of the polymer you intend to-manufacture..**~** Indicate maximum weight percent of low molecular weight 

### speees (not inducting residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight of that composition. 

# OtherO :<Specify>**~** Describe the method• of meuun:ment or the 

### bam for your estimatu: GPC 

# 0 il lowest number average maiecular weight: Ul maximum weight "' below 500 molecular weight: iiD maximum weight"' below 1000 molecular weight: 

# D Mark (X) this box if

### yo~ attach a continuation sheet.

b. You must make separate confidentiality claims for monomer or other reactant identity, a:nnposition information, and residual information. Mark (X) the "Confidential" box next to any item you claim as confidential. (1)

- Provide the specific: c:hemic:al name and CAS Registry Number (if a number exists)· of eac:h monomer or other reactant used in the manufac:ture of the polymer. (2)

- Mark (X) this column if entry in column (1) is alnfidentiaL (3)

- Indicate the typical weight peram.t of eac:h monomer or other reactant in the polymer.

. (4)

- Mark 00 the identity column if yoa want a monomer or other reactant uaed at two weight percent or lea to be liated aa part of the polymer description on the TSCA Chemical Subatanc:e Inventory. · (5)

- Mark (X) this column if entries in columns (3) and (4) are confidential. (6)

- Indicate the maximum weight percent of eac:h mcn;tomer or other reac:tant that-may be present as a residual in the polymer as manufactured for c:cmmerdal 

### purposes. (7)

- Mark 00 this column if entry in column (6) is alnfidentiaL '
Monomer or other reactant and CAS Registry Number Confi- Typical Identity Con:.fi- Maximum Col dmtial c:ampositian 

### MarkOO dmtial residual der (1) (2) (3) (4) (5) (6) 

# . t
.,;, 

### r. 

# ·'" •%


# ,. .,;,


# .,. ,.

.,;, 

### r. 

# "· ,_


# ,. .,;, 

# 0 Mark (X) this box 

## u you attach a alntinuatian 

### sheet.

c. Please identify whic:h method you U!ed to develop or obtain the sped.fied chemical identity informatiOn reported in this noti.ce. (c:hec:k one).



# D Method. 1 (CAS Inventory Expert Service- a copy d the identification report 

# D Method. 2 (other source) · obtamed from CAS Invmtmy Expert Servia! must be submitted as an attac:hment to this notice) d. The omently correct <llemic:al Absttad:s (CA) lWl1e for the polymer that is amsistent with TSCA Inventory .l.istings far similar polymers.

.. 

# I

e. Provide a correct representative or 

### partial c:bemic:al structure diagram, as c:cmplete as c:an be known, 

## u one c:an be 

## reaaonably ucertained.



# ......



# .



# -

-



## .

-

I 

# 0 MarkOO this box 

## u you attach

### a~~~ sheet.
FORM EPA mG-25 (Rev. >95) PageS

## Part I -GENERAL INFORMATION- Continued

# ~ Section B - CHEMICAL IDENTITY INFORMATION

- Continued

3. 1m purities

(a.l- Identify each impu.Iity tha may be -!Ubly uticipaed to be prant In the chemic:.a.l nbtltaae • m.aaubaured far cnm.mad.a.l PurpoML Provide the CAS Regbtry NllDlber If available. U there 1ft IUiidmtified l.mp1Uitiea,. allu "'uidmtlfled. • (b) -&Um..te tM maximllDl weight'" of eaclllmpvity. U there are llllidaatified lmpuritiea, mtimllle their total weight'"·



# . Impurity and CAS Registry Number Maximum Confi-**~** (a) percent cient:ial jb) ' 'l'o 

## 'f.._

'l'o 

# '/...

'l'o 

# 'f.._

cr.

## "~-- '!'0



### .,..

cr. 

# 0 Mark 

### 00 this box if you attach a continuation sheet. 4. Synonym•- Enter any chemical synonyms for the new chemical substance identified in subsedian 1 or 2.



# .**~~** 

# X 

# 0 Mark 

### 00 this box if you attach a continuation sheet. 5. Trade idmt:ific:ati.OD

- List trade names for the new chemical subl!ltance identified in subeed:i.oo 1 or 2. 

# X 0 Mark 00 this box if you attach a continuation sheet. 6. Generic: c:hemic:al name
- If you claim chemical identity as 

### confidential. you·muat provide a generic chemical name far your substance that reveals the specific chemical identity of the new chemical substance to the maximum extent possible.**Refer~** the TSCA Chemical Substance Inventory, 1985 Edidan, Appendix B for guidance on**~eloping** genenc names.



# .



# l\ c y '/ 

# \C\.\**~** cf

# ~yo( 'rOK'j

# iW~',de_ 

# D Mark 00 _this box if you attach a c:cntinuatian sheet.
7. Byproduc:ts-...... my byprociucts resulting £ram the manufacture. processing. use, or 

### dispoul. ci the new dlemical subetanc2. Provide the CAS**R~~.** ·Number if available.

. . Byprociuc:t CAS Registry Number Confi- (1) Q) dent:ial



## . 

# x·

--

-



# D Mark 00 this box If you attach a mntinuaticn sheet.

. - FORM EPA 7710-25 (Rev. 5-95) Page6

## Part I -GENERAL INFORMATION- Contiriued**~** Section C- 

### PRODUCI'ION, IMPORT, AND 

### USE INFORMATION:
Mark 00 the "Confidential" box next to any item you claim as c:cmficlentiaL 1. Production volume- Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum production volume for any consecutive 12-month period during the first three years of production. Estimates should be 100% new chemical substance basis. For a Low Volw:zte Exemption application, if you choose to have your notice reviewed at a lowE production volume than 10,000 kg/yr, specify the volume and mark (x) in the binding box. 

### If granted, you are bound to this volum•
Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confi.. Biadiq dential Optioa (100% new chemical substance basis) (100% new chemical substance basis) MukW 

# X

2. Use Information- You must make separate c:on&ientiality claims for the dei!laiption of the category of use, the percent of production volume devoted to each category, the formulation of the new substance, and other use information. Mark 00 the "Confidential" Box next to any item you claim as (X)nfidentiaL a. (1) -Describe each intended category of use of the new c:hemical substance by function and applicatiCll\. 

### .. (2)

- Mark 00 this column if entry in (X)Wmn (1) is amfidential busineas information (CBD.

· (3)

- Indicate your willingneas to have the information provided in calumn (1) biDding.

(4) -Estimate the. percent of total production for the first three 

### years devoted to each c:ategary of UBe. (5)- Mark (X) this calumn if entry 

### 1n (X)lumn (4) is canfidmtial busineas information (CBD. 

# - (6)

- Estimate the percent of the new substance as formulated. in mixtures. suspealiaaa, emu1aions, solutions, or**~** as manufac:tured for commercial pmpoaes at sites under your control a-xi !!ted with each category of use. (7)

- Mark (X) this calumn if entry in CDlumn (6) is c:cmfidential busineas infarmatiaa (CBD. (8)

- Indicate " of prodw:t volume expected fa the listed "UIIe" sec:tan. Mark more than ane box if appropriate. Mark (X) to indicate your willingneas to have the Ulle type provided in (8) binding. (9)

- Mark 00 this column if entry(ies) in a:ilimm (8) is (are) c:onfldential bwlinls inft;aw.aioa (CBD. 

# .

Category of Wll! (1) 

## em 1liacUq Produc- CBI 'lit in 

## pn " of subltance expected per Ule

### ~ (by function and application i.e. ·a dispersive dye for Optillla 

## tioll" Foan- (8) Madt(x) ulation finishing polyester fibers) 

### 15Ue- ICeD-• iJldul.. Cam-**~inciing** (2) (3) (4) (5) (6) (7) limited**~er** trial merdal Option I 

# X 

# " )< 

# " ·'Y.. ·1 

## 1\

% %
% %
% %



## . % %

- 

# " " 

# " % •If you 

### have identified a "consumer" use, pleue provide on a continuation sheet a detailed description of the use(s) of this chemical substance in consumer products. In addition include estimates 

### of the concentration 

### of the new chemical substance 

### as expected in
consumer products and describe the chemical reactions 

### by which this substance loees 

### its identity in the consumer product. 

# D Mark 00 this box if you attach a tDDtinuatian sheet.
b. 

### Generic If you claim any category of uae delaiptian iD IUbaec:tica 2a • amfidential. enter a 

### geDeric dellaip&a of that
use category. Read the IDM:rllctiaaa Mamaal fa examples of gmtric. uae dac:riptimiL • desaiption

# ~**dk~~\U s:~**  D Mark 00 ttUa boxtfyouattac:h a tDDtinuatiaa sheet. 3. Hazard Infarmatioa

- Include in the notice a copy of re.caatM facw!mile ol. any hazard**warning~** label. material safety data 

### llbeet. lliBdb or other information which will be provided liD any perBDD wbo Ia**~ly** Watly liD be expcMCiiD thia suhMaDa! reprdiDg protec:tive OpbD 

# lD (pment or practic:es for tbe sate handling, transport. uae.or disposal of tbe new 11dwtanc:e 

### u.t tn put m**~** infmmatkm.yau mclude.

### ~ Mark (X) this box tf vou attach hazard information.

. 

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

## Part. II

- 

## HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

Complete section A for each type of manufacture, proceMing, IX' use operation involving the new chemical aubltaDCI! at inciuatriU sites you contrc Importen do not have to complete this 1ection for operation• outside the U.S. ; however, you may .till have reporting requirements if there are further induatrial"procesaing**oru~e** operation• after import. You mu1t deiCribe these operations. See.in1traction• manual.

of the site at which the willoa:ur. Name

address (number**~d** street)

City, County, State. ZIP Code

If the same operation will oa:ur at more than one site. enter the number of sites. Identify the additional sites on a continuation sheet, and if any of the sites have significmtly different · ....., production rates or operations, include all the information requested in this sec:tion for thoee

Mark 00 this box if b. TypeMark 00

1. Batch attach a continuation sheet:



# . D Manufacturing

2. Continuous 

# D ProcEMng

Hours/batch
Hours/day 

# D UseDays/yar

.d. Process desc::ription Mark 00 to iDclic:Ue yaar williDp- to bave·yaar pmcaa clucriptiaa biDcliDsCon dent

(1) Diagram the majarunit operation stepe and cbemical conversicna. Include interim storage and transport c:catainerl (specify-e.g. 5 gallon pails, gallon drum, rail car, tank truck,' etc.).

. · (2) Provide the identity, the approximate weight (by kg/ day or kg/batch on a 1001fo new chemical substance bm), and entry point of all starting materials and feecistockB (including reactants, solvents, and catalysts, etc.), and oi all products, recycle streams, and wutes. lndude cleaning chemicals (note frequency if not used daily or per batch.). (3) Identify by number the points of releue, including small <r intermittant rel-. to the environment of the new chemical substance.



## -.

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

## Part II

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE- Continued**~** Sedion A- INDUSTRIAL SITES CONTROLLED. BY THE SUBMIII EK- Continued

2. Occupational E.xpo8ure -You must make separate mnfidentiality c:laims for the delaiption of worker activity, physical form of the new chemical substance, number of workers expoeed, and duration of activity. Mark (X) the "Confidential" box next to any item you claim u c:onfidential. (1)

- Desc:ribe the activities (i.e. bag dumping, tote filling, unloading clrums, sampling, deaning, etc.) in which workers may be expoaeci to the substance. (2) -Mark 00 this column if entry in column (1) is ccnfidential business informatiOI\ (CBD. (3)

- Desc:ribe an 'I protective equipment and engineering controls used to protect workers. (4) and (6)

- Indicate you willingness to have the information provided in cclumn (3) or (5) binding. (5}

- Indicate the physical form(s) and. 'ro new chemical substance (if in mixture) of the new c:hemical substance at the time of 

### exposure (ie., solid: crystal, granule, powder, or dust) and ,. new chemical substance (if in mixture). (1)- Mark (X) this column if entry in cohmm·(S) is CXInfidential business information (CBD. (8)

- Estimate the maximum number of worke:rs involved in each activity for all sites c:cmbined. (9) -Mark (X) this column if entry in column (8) is mnfidential business information (CBD. (10) and (11)- Estimate the maximum duration of the activity for any worker in hours per day and days per year. (12)- Mark (X) this column if entries in columns (10) and (11) are amfidential business information CCBD. Worker activity CBI Protective Equipment/ Bmdms Physical farm(s BIDdias CBI jof CBI Maxim_llll duration CB (i.e.. bag dumping, filling drums) Engineering Contzola OptiDa and,. new OptiCIIl Workers Hrs/day M.k(xl substance M.k(x) Exposed. Days/yr (1) (2) (3) (4) (5) (6) (1) (8) (9} (10} (11}**(1~**

....



# .

. 

# D Mark (X) this box if you attach a amtinuatioll sheet. 3. "Enviroamentallleleue ancl Diapoal- You m'Uit make separate amfidentiaUty claims for tbe releae nUIIlbK md. tbe amount of tbe new c:hemic substance rel.eued and other release ed disposal infarmatiOil. Mark (X) tbe "Confidential• box next to -=h item you claim • confidential. (1}

- Enter the number of each releae point identified in**the~** dieeaiptilm. part 

### II. !edian A, subeectim 1d(3). (2}

- Estimate the amount of tbe new substance rel..-d (a) directly to the eDYiramDIDt ar (b) into amtrol technology (in kg/ day or kg/batc:h). (3)

- Mark 00 this c:alumn if entries in columns (1) and (2) are amfldi!Dtlal buslnees infarmatioll 

### <CBD. (4)

- Identify the media (stack air, .fugitive air (optional-see lnstructlon Manual), surface water, an-site ar off-site 1aNi or inc:ineratian, P01W, or other (specify}) to which the new substaDc:e will be released froiD that rel.eue point. (5)

- a. Describe control technology, if any, ,and control efficiency that will be used ta limit the release of tbe new substance to the environment. For releases dlsposed of on land, c:harac:terlze the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet , for each site describe any additional disposal methods that will be used and whether tbe waste is subject to secondary or tertiisry on-site treatment. 

## Jl. Estimate tbe amount releaaed to the environment after control technology (in kg/ day). (6)

- Mark (X) this c:olumn if entries in columns (4) and (5) are amfidential business infarmatloD 

### (CBD. 

# m
- Identify the destination(s) of releases to water. Please supply NPDES (Naticmal Pollutant Dlac:harge Elimination System) numbers for direc1

. dischargers or NPDES numbers of tbePOI'W (Publldy Owned Treatment Works). Mark 00 If. tbe P01W DIIJDe or NPDES 41 i8 confidential business information (CBD. Release Amount of new subetanc:e 

## em 

# IM= CantroltecbnoJogy and effideDc:y (you may willh to optiooally attac:b effic:il!ncy data) 

## c Number I Jli8dllls (1} (2a) (2b} 

# ... 

# --- (Sa)· : Wuk (X) (Sb} (3) (4) I I I I ·I I
I 

# ' I·



## - I I ' I I

., I

(1) Marx 00 the 

# . D Po1w provide 

## name
 below: CBI 

# DNnipble 0 OtlMI'- Specify provide NPDES • destinatian(s) of 

# - waterway releases to water.

I 

# 0 Mark 00 this box if you attach a cxmt:inuaticm sbeet. FORM EPA 7710.25 (Rev. 5-95)

# ______ ---u

...**~** 

# ............ , • .a.AVL'ILYL.r:.J.'(J.AL 

### RELEASE- Continued**~.Section** 

### B-. INDUSTRIAL SITES CONTROLLED BY OTHERS· : · .·· , ·, ., · · · · · .· · , ..**~.** 

### . . Complete sedian B fer typ!.cal procemng**or~** operatiaaa inval.vtng tbe new chemic:al Sllbstana It si.te5 you cio not**aml:rtll.lm~** del net have to· eomplete this aed:i.on fa: ope:ralicm oul3icle tbe U.S.; howwK, you must npcrt my prcx:eSng ar \lie adivi.tles after iinpcrt. $ee the Instructicm 

### ManuaL**~** 

### • 

# ,..,.a: JldXm B 

### far Md!type af.**~g,** ar IlK opntim bu:IOI'IIir&g Usc MID c1aUc&l**ndls~.** If the 

### same <ipeiatian is pe:r:formecl at men thm**~e** Ste delaibe the typicil operatlan ccmman to thale**!i~** Identify add:itiCDal. lites an a cantiDuation INlet. · . - ..... · . .. :

. . . · .

. .

Letter ' oi 

## an Dllriltiat

. 

## ICBJ PJ...-.idi:N !quip./.

. 

# I,.. I CBi 11--. AmauDt**~** .CBI

. Media of &e.. : CBl -d. · 0 Workm r;Jl · . · 

## ltV ("I 

### CoaaiaW rc..,. · · · . , .. _ N.w .' '.- &t Cmtzal

. · · o Ad.- Expo.d**-~.** · Pb.,.alllaraadS 'lllllllaa

. . 0 S.Jblaaal .· ·. Tedm...J......;. · ivity

. .. . . !:1ft''.' 7

..**~** . ..._,. 

### n> CZl 

## ·a> ·**c~** 

## ·_.**~** 

## ..**~-** (5)

. ,. ·: C6) : ·· · (i') (I) (9) 

## n.W 

# 1 n·llt). 

# cit

... 02)

0 03) I 'A I

# X·-~- . ;

# ~ - ---- . r 'f·o 

# r**-~.** 

# 'f ' 

# otx

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Y·**_,;~** I ·· I l ·:·'1' ·. . . I 

## -.

. · .. I ·. . I

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. ,

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### . -I

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### ·.·· . .-**(1~)** :- Byproducts:

_ . . .. •

. . (15)

### FORMEPAi1l~~· (Rft

. .5-95). ·. 0 '
·- . PaplO

'o

## OPTIONAL POLLUTION PREVENTION INFORMATION

To claim information 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 this 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**~sure)** 

## .. In 

## addition,. information on the relative cost or performance characteristics of the PM:N substance to potential alternatives may 

## be provided. All information provided in 

## this section 

## will be taken into consideration during the review of thiS substance. See Instructions 

## Manual and Pollution Prevention GUidance 

## manual for · guidance and examples.

. Describe the expected net benefits, such as (1) 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 inaeue in product performance, a decrease in the cost of production 

### ard./ or improved 

### operatioll: efficiency of the new 

### c:hemical substance in comparison to existing chemical substance used in similar applications; 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

.



## .



# .



# -



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

### ..

fURM EPA 7710-25 <Rev. 5-95) Papll

## Part III-- LIST OF ATTACHMENTS

Attach continuation sheets for sections of the form and test data and other data (including physical/chemical properties and structure/activity information), and optional information after this page. Oearly 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 (X) the "Confidential" box next to any attachment name you claim as confidential. Read the lnsbuctions Manual for guidance on how to claim any information 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.
Attachment name Attachment Confi· page number(s) dentia 

## Material Safety Data Sheet (MSDS) 

# 'x X



# 0 Mark (X) this box if you attach a continuation sheet. Enter the attachment name and number. FORM EPA 7710-25 (Rev. 5-95) Page 12

# I


# Material Safety Data Sheet

.**~** 

### . 

# - Effective Date· '11-Mar

-**'~000** 

### SECTION I MATERIAL IDENTIFIC ACTION

- 

### Trade name: . -**~** .... ....

- - - -



# - -------- __ ..., -s::

- - 

### Generic name : --,

-- .**~** .-.

- --**~-·----** .. 

## -**---~** _;..;..:,;,;: -""';**----~** -**~---"** ::-

-- - ... - 

# -

- , ..**_,.I-~-' --.:.;-=_-_:::__.:.~.:;**  ._:.._
---**~:., ~**  

## --· ,.. 

## -· ··---·

.J -,-.., ..... J

... ...**~u...;.i..** 

### Chemical family : Acrylic monomer CAS No.:**~** .... 

## ,..._- -

.



### . - - -.: 

### TSCA: 

## - Manufacturer :**':;::.;:;.t;;:..~-"** 

### vu.,**.w~.u.** 

### address(Number, Street, City, State, and ZIP Code) :



## -- --- "I

. .. ....... -- • , . ., • -:-: - - ·-- ·--**·---~~-** 

### __ .:.J '--

.. .,;_:. 

## ----· 

# ___ ... 

## _ ----- . -·

--- 

# - __ ....... -...:.,:.-__ 

## ----

- 

## - Phone:**~** .. - _·.,;..·- '-'V-. ·-""'·"· 

### Facsimile : r uJ.·u•viJv 1 

## I--- SECTION 

### 11 HAZARDOUS INGREDIENTS pp.emic_al family : Acry_li_c monomer Molecular weight: 

### Mw 249.29 Content % : 

### 88'"'-'98 wt%

.. 

## -

-- 

### -- ... 'l- : ;I I ;: 

# - Rational formula : 

# -

Other components hazardous to health COMPONENTS CM:i No. 

### % 

### ACGIH : TLV TWA Toluene 108-88-3 < 1 

### 50ppm impurities 1'""'-'11


### SECTION JI[ 

### PHYSICAL/CHEMICAL DATA Appearance : solid Color : light yellow Specific gravity N.D. Odor: toluene's odor Solubility in water Insoluble Boiling point : 

### SECTION IV STABILITY AND REACTMTY This product is considered to be stable under normal and anticipated storage and handling condition. A spontaneous polymerizing reaction may occur under the condition
as follows-heat, shock, open flame, ultraviolet, radiation, direct sunlight radiation, oxidizing conditions, free radical initiators, inert gas atmosphere. 

### SECTION 

# v FIRE AND EXPLOSION Flash point : N.D. 

### 'C Flammability limits in air : N.D. Ignition temperature : N.D. Extinguishing media

Carbon Dioxide, Dry Chemical. Sand, Foam,

### Wate~· Fog. Hazardous decomposition products : (Products causing hazardous decomposition)

Carbon Monoxide. Carbon Dioxide. Organic Acids. S12ecial fire-fighting j2rocecl ures :

· Move container from fire areas 

### if it can be done without risk.

· Firefighters· should wear proper protective equipment.

· Keep personnel removed from and upwind of fire. Unusual fire and exnlosion hazards :



### NIA



## 13•, 

### SECTION 

### VT HEALTH HAZARD INFORMATION Acute toxicity

Acute oral toxicity (LD50 in rat) : N.D. Skin irritation (in Rabbits) : N.D.
Chronic toxicity

.Al.VIES test negative Primary skin Irritation Index(PII) 0.0 (classified as non-irritant) First aid

· Eye contact : Gently rinse the affected eyes with clean water for at least 15minutes. Arrange for transport to the nearest medical facility for examination and treatment by a physician
as soon as possible.

· Skin contact : Wash the affected area with water using a mild soap.

· Inhalation : Rinse mouth with water. Give the person water. Do not induce vomiting as this may mcrease the risk of aspiration of the liquid into the lungs causing chemical pneumonia

. Consumption of non-oily liquids(such as milk) may assist in delaying absorption of the liquid. Personal Erotection/E;sEosure control

Respiratory protection : Chemical respirator with an organic absorbent in a cartridge. Hand, skin; and body protection : Gloves Eye protection : Safety goggles Other : Use with local exhaust ventilation. 

### SECTION 

### VJJ SPILLS, LEAKS, AND DISPOSAL PROCEDURES Accidental release measures

Evacuate nonessential personnel. Shut off all sources of ignition; No flares, smoking or flames in area. Absorb spill with inert material (e.g., dry sand or earth), then place in a chemical waste container with covers for disposal, using nonsparking tools. Remove leaking containers to a safe place, iffeasible. DisQosal consideration



### This combustible material may be burned in 

### a chemical incinerator equipped with an afterburner and scrubber. 

### SECTION VI!/ 

### STORAGE 

### & HANDLING / TRANSPORT Storage 

### & handling

Keep tightly sealed and store indoors out of direct sunlight in a cool place. Keep away from sources of ignition

- No smoking. TransQort

IMDG code: N.D. 

### SECTION 

### !X GENERAL N.D. means No Data Available N/A means NOT Available CAS means Chemical Abstract Service(Registry number)
To the best· of our knowledge, the information contained in this sheet lS 

### correct. However, we also disclaim all responsibility for any consequences arising from its use.

End ofPage



# 1'+-·In1puritiesI-IllandthierCASRegistryNu1nberI1npurityI""'-I1npurityIIIn1purityIIIrChe111icalstructures\\CASNo.IINoneNoneNonei:'..........,,Maxinu1n1Percent5.01.05.0ConfidentialXXX(

### PHYSIQ\,L AND CHEMICAL PROPERTIES WORKSHEET

·To assist EPA's review of physical and chemical propertieS data, please complete the following worksheet for data you provide

I 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 substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. Properties that are measured for mixtures or formulations should be so 11oted (% PMN substallce in_ ).You are .11ot required to stibmit this worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure

· that amfidential information is properly protected. You shOuld submit this worksheet as a supplement to yoilr submission of test

data. This worksheet is not a substitute for submission of test data.

I Meumcl Confi- : Property Mark (X) if Page Value or dential provided number Eltilmle

. (a) (b) (c) Mark (X) (MorE)· (d)



### . \ Physical state of neat substance _(s)_Q)_Ut) 

# X
Vapor pressure

. @Temperature "C Torr

Density/relative density g/an3 

# X

SOlubility "C 

# X

@Temperature

Solvent _g/L

Solubility in water. @ Temperature · 

## oc "C 

# )(

Meltjng temperature 

## oc 

# X

Boiling/sublimation temperatUre @ torr pressure 

# X

.**s~** 

# )(

Dissociation constant ' I

Particle size distribution

Octanol/water partition coefficient I

I Henry;s Law constant '

Volitalization from water

Volitalizallon from soil

pH @ concenl:raticin

Flammability

Explodabllity

Adsorption/ coefficient

Other

- Specify


# /b

# 1'\

# It<;•,



## FINAL REPORT


## M.S.I. REPORT No.9B374G

Ready Biodegradability Test of

.

Subrni tted to

Prepared by



### Mitsubishi Chemical Safety Institute Ltd.

November 12, 1999


# t9 

# 7

## ) COMPLIANCE WITH THE GLP STANDARDS

9B374G Study No. Study Title Ready Biodegradability Test of Sponsor**a,~-.:..~-**



### The study described in this report was conducted in compliance with the Good Laboratory Practice {GLP) regulations recognized by the Basic Industries Bureau of the Japanese Ministry of International Trade and Industry.



### Facility Management

# 7a,~~**a~E!EL** Date

# Tadayo~hi Shigeoka,

# Ph~

Facility Management Head of Yokohama Laboratory



### Study Director

# Ju~:KOY:Su: B.Sc.

Study Director Environmental Science Division



### ::toStudy No. Study Title Sponsor 

### QUALITY ASSURANCE STATEMENT

9B374G Ready Ei odegradabili ty Test of •
-**-~** ·---


### -,,

We hereby certify that the above study has been**perfo~ed** in accordance with the Good Laboratory Practice (GLP) regulations recognized by the Basic Industries Bureau of the Japanese**Minis~ry** of International Trade and Industry.

Dates of inspections and reportings to the Study Director and the Facility Management are as follows :

Date of Date of Inspection Reporting In-progress study September 28, 1999 September 28, 1999 October 26, 1999 October 26, 1999 Final report November 12, 1999 November 12, 1999



### Quality Assurance Personnel

Date Yoshihiro Miura, M.Sc.

Date Tsuneki Susuki, B.Sc.

## ) I, the undersigned, declare that the work was performed according to the procedures herein described, and that this report provides a true and correct record of the results obtained.

Date Rie Yamaguchi Environmental Science Division1 2 3 

### 4
5
6 7
8 9 SUMMARY STUDY TITLE PURPOSE TEST GUIDELINES GLP COMPLIANCE STUDY PERIOD SPONSOR TESTING FACILITY 

### CONTENTS

STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS TEST SUBSTANCE 10 CONFIRMATION OF IDENTITY AND STABILITY

10 .1 IDENTITY 10.2 STABILITY 11 TEST METHODS

11.1 ACTIVATED SLUDGE 11.2 EXPOSURE CONDITIONS
11.3 11.4 11.5
11. 6 11.7 BOD MEASUREMENT pH MEASUREMENT DOC MEASUREMENT MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION EQUATIONS FOR CALCULATION OF DEGRADABILITY 12 RESULTS

12.1 OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD 12.2 12.3 12.4 12.5 12.6

13 pH MEASUREMENT ACTIVITY OF SLUDGE DEGRADABILITY BASED ON BOD DEGRADABILITY BASED ON DOC DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE CONCENTRATION CONCLUSIONS TABLES AND FIGURES 

### Page
6 7 7 7
7 7
7 7
B 9 10 10 10
11 11 11 12 12 12
13 14 15 15 15 15 15 15

16 16 17-34'.**···;~~** 

## SUMMARY.
STUDY TITLE

Ready Biodegradability Test of


### METHOD The method described in the Order, which prescribes tne procedure of testing

new chemical substances as required by 

### the Chemical Substances Control L·aw {Japanese Law 117, 1973). ("Ready biodegradability, Modified MITI test" in OECD Guidelines for Testing of Chemicals).

Test bottle Bottle 1 

### aniline 

### + sludge 

### + basal medium Bottle 2 

### sludge 

### + basal medium Bottles 

### 3

....... 5 test substance (ARONIX 

### T0-1534)+ sludge 

### + basal medium Bottle 6 : 

### test substance (ARON!X 

### T0-15 3 4 ) 

### + water
Measurements Biochemical oxygen demand (BOD) (for 28 

### days) Dissolved organic carbon (DOC) (at day 28) 

### Residual test substance concentration (at day 28) 

### [measured by high performance**~iquid** 

### chromatography} 

### RESULTS

Measured values (day 28)



### BOD,· mg DOC, mg/1 14.1 

### 43.5 8.7 14.1 43.6 44.5 

### Theoretical 

### 6 value 0. 0 60.7 62.7 62. 6 

### Test substance, mg/1 <1 <1 <1 107.5 100.0 

### *1 value corrected with BOD or DOC value of Bottle 2. Degradabilities (%) 

### Bottle No. 3 4 5 

### average BOD 23 14 

### 23 20 

### DOC 31 30 29 30 

### Test substance >99 >99 >99 >99


## CONCLUSION

From the degradability results based on the BOD and DOC measurements, it can be concluded that the test substance is not read.i,ly biodegradable under the conditions of the Modified MITI test.

Although the degree of degradability based on the residual test substance concentration was calculated to be >99, the degree of degradability based on the BOD was**20~** 

### in average, DOC was 30!t. in average. Th.:=.refore, it was suggested that the test substance was transformed, and transformed product(s)

was remained in the test solution.

### l STUDY TITLE Ready Biodegradability Test of _- •



### 2 

### PURPOSE

This study was conducted to evaluate the ready biodegradability of the test substance (ARONIX T0-1534) for notification under the Chemical Substances Control Law of Japan.

3 

### TEST GUIDELINES

The test was conducted according to the method described in the o=der, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese Law 117, 1973)

. This method .is also known as the "Ready biodegradability, Modified MITI test" in the OECD Guidelines for the Testing .of Chemicals No. 301C.

4 GLP COMPLIANCE

The test was conducted according to the Good Laboratory Practice (GLP) regulations recognized by the Basic 'Industries Bureau of the Japanese Ministry of International Trade and Industry.



### 5 STUDY PERIOD

September 24, 1999 to November 12, 1999 (BOD measurement : September 28, 1999 to October 26, 1999)



### 6 SPONSOR

:-

.. '**-~!'""-**

7 

### TESTING FACILITY

Yokohama Laboratory Mitsubishi Chemical Safety Institute Ltd. (M.S.I.)

1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan

Head Office: Mitsubishi Chemical Safety Institute Ltd. (M.S. I

.. )

1-30 Shiba 2-chome, Minato-ku, Tokyo 105-0014, Japan

# -**·.~~;~~;**



## ) B STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS The following records, the raw data, and a small sample of the test substance will be stored. in the archives of M.S.I. for 10 years after the submission of the final report. After this period, the sponsor will be contacted for the approval of the disposal of data. Data can be stored in the archives for a further specified period at the sponsor's request with an additional fee. 1) Protocol and its amendment. 2) Final report. 3) Raw data. 4) Quality assurance reports. 5) Test substance (approx. 

### 2 g). 6) Other documents required under the MITI GLP regulations.

# ) 9 TEST SUBS'r.ANCE 

### l)Name*

2)Chemical name*

3)Structural formula*

4)Molecular formula 5)Molecular weight* 6)Elemental composition J

e



### _,

. 249.29

C 62.6%, H 6.1%, 0 

### 25.7%, N 5.6% (Calculated by M.S.I.) 7) Physico-chemical properti·es*

I 

### Solubility

Melting point B) 

### Batch* 9) 

### Purity* Water insoluble 

### DMSD, Acetone, 

### DMF, toluene, MIBK, Acetonitrile oo 

### 61 

# oc Lot No. NH-9301-01

. 94.1% Component of impurities (Dimpuri ty 1

@impurity 2

\

....) 2. 7% (Q)+@)10)Appearance 11)Supplied quantity* 12) Supplier @impurity · 3

/• 

# .. ',

) white powder 0.1 kg (net).

10 CONFr.RMATION OF IDENTITY AND STABILITY·

10 . 1 IDENTITY 3.2%

I 

### \-,..



### * provided by the sponsor.

For the identification of the test substance, the infrared absorption spectrum of the test substance was measured. The measured spectrum was consistent with.that supplied by the sponsor.

1)Apparatus Perkin-ELmer model 1640 infrared spectrophotometer. (Figure 1 (P-21))

1 0

. 2 STABILITY

After the exposure period, the infrared absorption spectrum of the test substance that had been stored in a desiccator during the course of the ) study was measured. The spectrum was consistent with that shown in Fig. 1, indicating that the test substance was stable under the storage condition.

1) Apparatus Perkin-Elmer model 1640 infrared spectrophotometer. [Figure 2 (P-22))ll TEST METHODS The test was performed according to the method described in the Order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese Law 117, 1973}. This method is also known as the '!Ready biodegradability, Modified MITI test" in OECD Guidelines for Testing of Chemicals No.301C (1992}.

The test substance was exposed to the activated sludge in a closed-syst;em
oxygen consuntption measuring apparatus. The_ biochemical oxygen demand (BOD l

was measured over a 2 8 day period. After this period, the concentrations of the dissolved organic carbon (DOC.) and the r'esidual test substance in the
test bottles were measured. The biodegradability of the test substance was evaluated from these results.

11.1 ACTIVATED SLUDGE

1)Mixed liquor suspended solid (MLSS) : 3700 mg/1 2}Source

3)Date of receipt Chemical Biotesting Center, Chemical Inspection and Testing Institute, Japan September 10, 1999

11.2 EXPOSURE CONDITIONS

1) Temperature 25 

### ± 1 

### "C 2)Exposure period 3)Test volume 28 days 300 ml 4)Concentration test substance (bottles 3-6) aniline*l(bottle 1) 100 mg/1 100 mg/1

30 mg/1 activated sludge (bottles 1-5) *l Reference substance : KANTO CHEMICAL Co., INC. Lot No.103Gl134 5)Test bottle contents Bottle 1

Bottle 2

Bottles 3-5

Bottle 6 Aniline + activated sludge + basal medium**29~(30** mg) of aniline was added to the basal medium* 2

, then activated sludge was added. Activated sludge 

### + basal medium Activated sludge was added to the basal medium*=. Test substance 

### + activated sludge 

### + basal medium 30 mg of the test substance· was added to the basal medium* 2

, then activated sludge was added.
Test substance 

### + ultra-pure water 30 mg of the test substance was added to 300 ml of ultra-pure water (purified by Milli-Q, >lOMohm-cm).

*2 The total volume of the basal medium and the sludge was held fi:,ed at 300 ml.**~.·.** 11 . 3 BOD MEP.SUREMENT The BOD was measured for 28 days. 1)Apparatus closed system oxygen consumption measuring apparatus Ohkura Electric Co., Model OM-2001 (M.S.I. ID No.F).

11.4 pH MEASUREMENT

After the exposure period, 20 ml of the test solution in each test bottle

was transferred into a 20-ml glass beaker for pH**mea~urement.**

After pH measurement, the test solution was returned to each bottle for
measurement of residual test substance concentration. 1)Apparatus : ·pH meter, ORION Research, Model 720A

11

. 5 DOC MEASUREMENT**,~:::~** The concentration of the DOC was measured as follows.

1)Apparatus 2)Conditions TOC analyzer, Shimadzu Co., model TOC-5000 Furnace temperature Air flow rate Sensi ti vi.ty Injection volume 3)Calibration curve '680" C (TC) 150. ml/min. X 5**50~**

The following standard solutions were injected into the TOC analyzer. The calibration curve was prepared by the data processor ·of the analyzer. standard solutions

TC (total carbon) 20 and 50 mg C/1 aqueous solutions of potassium biphthalate. IC(inorganic carbon) 0 mg C/1 Ultra-pure water (purified by Milli-Q) and 10 mg C/1 aqueous solution of sodium hydrogen carbonate and sodium carbonate.

· .) 4) Measurement of DOC in the bottles·.
Ten milliliter of the test solution in each bottle were transferred into

a 10-ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes. Five milliliter of· supernatant were used for the measurement of DOC. The remaining supernatant and the precipitate were returned to each bottle for the measurement of the residual test substance concentration.11.6 MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION

. The concentration of the residual test substance was measured by high performance liquid chromatography (HPLC) as follows:
1) Appara t\ls HPLC Shimadzu Co., Model LC-10AWS-3 Pump Shimadzu Co. 1 Model LC-10AD Auto Injecor Shimadzu Co., Model SIL-l OA.'<L 

## uv-vrs Detector: Shimadzu Co. 1 Model SPD-M10AVP**Col~** Oven Shimadzu Co., Model CTO-lOAC Work Station Shimadzu Co. 1 Model CLASS-LC10 Degasser Shimadzu Co. 1 Model DGU"-14A Communication bus module : Shimadzu Co., Model CBM-lOA 2)Conditions Column Mobile phase Flow rate Wavelength GL sciences Inc. Inertsil ODS-3V, 4. 6 rnrn i. d. X 150 mm Acetonitrile : Water = 60 : 40 1 ml/rnin. 230 nm Attenuation [Detector] -10 "' BOO AU/FS Injection volume: 20**~** Oven Temperature :. 25 

### OC · 3)Calibration curve

The standard solutions of the test· substance with 0, 25, 50, and 100 mg/1 in 40% acetonitrile 

### I water. were prepared. and analyzed. The total peak area of the test .substance was calculated by adding the area of the peaks with retention time of about 3 .1, 3. 7, 4. 4 and 6. 9 minutes. The total peak area of the test substance was plotted against the concentration. The correlation coef.ficient was calculated to be 1. 00 by the method described in Japanese Industrial Standards Z9041-1968. Concentration of the test substance for each bottles was calculated from ratio of total peak area for the sample to that for the standard solution with 50 mg/1 in 40% acetonitrile 

### I water. [Figure.3 (P-23) and Figure 4 (P-24...,..·25)] 4)Recovery test

Duplicate**t~st** bottles (identical to the test bottles 3-5 described in 

### § 11.2) were kept in a closed-system oxygen consumption measuring apparatus
at 25±1t for 30 minutes.

Each test solution was treated by the procedures described in 

### § 11.6. 6)

. The concentration of the test substance was measured by HPLC. The average recovery of the test substance was 98 %. [Table 3 (P-19) and Figure 5 (P-26...,..,27)] The concentration of the residual test substance measured for bottles 3

.......... 6 were corrected with the average recovery value. 

# ·3\,· 

### 5)Detection limit The detection limit of the test substance was calculated to be 1 mg/1 based on the minimum detectable peak area of 1000 rnAbs • sec for the (retention time of about 

### 4.4 minutes) in the HPLC largest peak chromatogram. (Table 3 {P-19) and Figure 5 (P-26

....... ·27) 

### J 6)Measurement of the test substance in test bottles

The content in each bottle was separately transferred into a 500-ml glass volumetric flask, which was the brought up to 500 rnl with acetonitrile. Ten milliliter of the test solution was centrifuged at 3000
rpm for 10 minutes. The supernatant was analyzed by HPLC. The test substance concentration was analyzed by HPLC under the conditions described in 

### § 11. 6. 2)

.

11.7 EQUATIONS 

### FOR CALCULATION 

### OF DEGRADABILITY

Equations for calculation of degradability based on BOD, DOC, and residual**te~t** 

### substance concentration are as follows: 1)Degradability based on BOD Degradability {%) = 

### {BODs- BODb)/ThOD X 100

where BODs BODb ThOD

ThOD aniline Oxygen consumption {mg) in bottles 1, 3, 

### 4 and 5. Oxygen consumption (rng) in bottle 2. Theoretical oxygen demand (mg) of aniline and the test substance.

C6H1N 

### + 35/ 40z -+ 6COz 

### + 7 /2Hz0. 

### + NOz 

### ThOD 90.2 mg Oz/30 

### rng-aniline
test substance C13H1sO,N 

### + 63/ 40z

- 

### 13COz 

### + 15/2Hz0 

### + NO: ThOD : 60.7 rng Oz/30 

### rng-test substance ) 2)Degradability based on DOC Degradability (%) 

## = {1- (DOCs-DOCb)/DOCc}X100

where DOCs DOCb DOCc DOC (mg/1) in bottles 3, 4 

### and 5. DOC (mg/1) in bottle 

### 2. DOC (mg/1) in bottle 6. 3)Degradability based on residual test substance concentration Degradability (%) = 

### (1

- Cs/Cc) X 100

where Cs Cc concentration (mg/1) in bottles 3, 4 

### and 5. concentration (mg/1) in bottle 6.**·~.**

j


### -·· 12 RESULTS There was no specific circumstances which might have affected the reliability of the test results.

12.1 OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD

The solution in bottle 1 was·cloudy yellow. The solutions in bottles 2, 3, 4, 5 and 6 were colorless. Growth of the sludg.e was observed in bottle 1 in
contrast with control (bottle 

### 2). No growth was observed in bottles 3, 4 and 5.

12 . 2 

### pH MEASUREMENT

After 28 

### days of exposure, the pH was determined to be 6.6, 6.6, 6.5 and 5.6 for bottles 3, 4, 5 and 6, respectively. (Table 1 (P-17))

12. 

### 3 ACTIVITY OF SLUDGE

The degradabili ty of aniline, based on the ·BOD measurements was 55 % 

### after 7 days. The activity of the sludge was thus shown to be satisfactory. [Table 1 (P-17) 

### and Figure 6 (P-28

........ 31))

12

. 

### 4 DEGRADABILITY BASED ON BOD

BOD* 1 

### in bottles 3, 4, and 5 (as corrected with the value in 

### bottle 2) 

### were 14.1, 8.7 

### and 14.1 

### mg, respectively. BOD in bottle 6 was 0.0 mg. (* 1 

### Maximum theoretical value= 60.7 mg) The degree of degradability based on. the BOD measurements were 23, 14 

### and 23 

### % for bottles 3, 

### 4, and 5. [Table 1 (P-17} 

### and Figure 6 (P-28----31)]

12.5 

### DEGRADABILITY BASED ON DOC

DOC*2 in bottles 3, 4 and 5 (as corrected with the value in bottle 2) 

### were 43.5, 43.6, 

### and 44.5 mg/1, respectively. DOC in**b~ttle** 6 

### was 62.7 

### mg/1. (* 2 

### Maximum theoretical value= 62.6 

### rng/1) The degree of degradability based on the DOC measurements were 31, 

### 30 and 29 

### % for bottles 3, 4, and 5. (Table 1 (P-17) and Table 2 

### (P-18)]•,

_ .. 

### - 

### ) 12.6 DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE CONCENTRATION
The test substance*J was not detected in bottles 3, 4 and 5, and detected
107.5 mg/1 in bottle 6. (*3 Initial concentration= 100 mg/l}

The**deg~ee** of degradability based on the residual test substance concentration were >99, >99 and >99 

### % for bottles 3, 4 and 5, respectively. [Table 1 (P-17), Table 4 

### (P-20), and Figure 7**(P-32~34))**

13 

### CONCLUSIONS

From the degradability results based on the BOD and DOC measurements, it can be concluded that the test substance is not readily biodegradable under the conditions of the Modified MIT! test.

Although the degree of degradabili ty based on the residual test substance concentration was calculated to be >99, the degree of degradability based on the BOD was 20% in average, DOC was 30% in average. Therefore, it was suggested that the test substance was transformed, and transformed product(s)

was remained in the test solution.'Table1SummaryofthetestresultsDegradabilitieswerecalculatedbytheequationsshowninSec.11.7.) D;raegdabilitvbasedBODBottleTestThODday7day14No.substancemgBOD%Degra-BOD%DegraillI!dai.Jilitvrugdabilitv1aniline90.250.65563.2672--------1.3---2.9---3,.60.77.31014.619--"'.-41_·-&-.60.75.8 711.6145--60.76.6 914.619----------6•r'60.70.0---___----·-.L_______:___._)D-·radabilitvbased..DOCBottleOrganiccarbonTCICDOC%DegraNo.addedwg/lwg/lwg/1wg/1dabiiity2-----1.60.01.6----362.645.10.045.131462.645.20.045.230562.646.10.046.129662.662J-____Q.Q -62.7----J-·-----·----b)PH-·..----..da21da'28BottleIHIBOD%Degra-BOD%Degra-No.day0day281llj:!dabilitvmgdabilitv65.46965.4691---7.9'3.2---3.2---27.07.217.32317.3233---6.611.91411.9144---6.616.02111.3235---6.50.0---0.0---67.85.6]d)D---dabilitvbased..'dualtestsubstanceBottleConcentrationDegradabilityNo.wg/l%2<1---3<1)994<1)995<1>9961Q7.5---J
_. Table 2 Re.aulta of DOC**mea~urement**
Standard TC: 

### &o; o •elL vIa. I : al



### MN f area 1 2 3 24849 24809. 24829 24829

Bottl.e 2 TC:
f area TC: 20 .o •elL vla.l :a2

liN

RMK f· area 1 10174 2 10149 3 10110

. 10144

B o t t I e S TC
f area IC: 10.0 ar/L vial :a3 •

liN • a.rea 1 2 3 4973 6062 6111 &048

RIIK**-----------------~---------- -------------------~--------** 

loiN 1 2 3 827 782 72.4 771 1. 89 1. SB 1.48 1. 58 liN

1 

### c rc
# area lle/1." .RMK 1 0 0.00 2 0 

### o.oo 3 0 

### o.oo 1 22188 2 :noos 3 22054 22075

# area 1 0 2 0 3 0 46.21 44.95 45 

### .os· 45.09

aefl.



### o.oo o.oo 0.00 RIIK



# ---------------------------- ---------------------------- MN 0

Bottle 6 TC: 0.00 MN 0

B o t t l e B TC: 

### :o.oo



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

MN

IC

loiN # area 1 2 3 22451 22682 22624 22552

# area 1 2 3 0 0 0
0

TOC-5000 DATA mc/1. 45.88 46.13 46.21 46.07

me/L

0.00 0.00 0.00 0.00

RE.PORT RMK

KN

IC: RMK



### MN # area 1 30478 2 30708 3 30838 306U

# area
1 2 3 0 .0 0
0 me/1.
62.28 62.73 82.99 62.66

me/1.



### o.oo 0.00 0.00**0.0~**



## ---------------------------------------------------- SPL # TC,u/L RWK IC.me/L RWK TOC,mr/L



## ---------------------------------------------------- 2 1. 58 0.00 1. 68 3 46.09 0.00 46.09 4 45 .1 

### e o.oo 46.16 5 46.07 0.00 46.07 6 62.66 0.00 82.66 RMK

RMK



### <---- 

## <---- <----**<---~** 

### <---- B B B 

### B B IC: 0.0 ae/L vial :a4
# area 1 2 3 0 0 0 

## ------:--------

B o t t l e 4 TC

MN


### IC # area "1 22012 2 21993 3 22317 22107

# area 1 0 2 0 3 0 ae/L 44.98 44.93 45.59 45.18

1u/L

0.00 0.00 0.00 0

RMK

RMK



# ---------------------------- liN 0 0. QQ.

0 t t l e 2 0 t t I e 8 0 t t l e 4 0 t t I e 

### 5 0 t t I e 8w_...)Table3CalculationofrecoveryanddetectionlimitConcentrationinPeaksolutionmdlRecoveredAddedRecoveryDetectionareaforHPLCinbottlemassmasslimitmAbs·secanalysismgmg%mg/l(A)(B)(C)(D)(E)(F)(G)Recover}':test1409692758.898.1.29.430.098----Recoverytest2407867758.697.6.'29.330.098----Blank<2000<O.1<O.1-------Q.O----1----------Averagerecovery:50mg/l98%StandardsolutionConcentrationPeakareaVolumeofsolutionforHPLCanalysis·VolumeoftestsolutionforHPLCanalysisVolumeoftestsolutioninbottle(H):(I):(J):(K):(L):3481229mAbs·sec0.5001o,30010.3001Equation:B=HXA+IC=HXA+IXJ+KD=CXLF=D+EX100Calculationofdetectionlimit50mg/1std.BlankRetentionPeakareaRateofpeakPeakareatimeminmAbs·secarea%mAbs·sec·3.1146520.4----3.7.411841.2----4.4333433095.8<10006.9910632.6----_3481229100.0<2000------Theminimumdetectablepeak.areaofthetestsubstancewascalculatedbythefollowingequationbasedonthemi.nimumdetectablepeak.areaof1000UL'\bs·secforthelargestpeak(retentiontime4.4min.)inHPLCchromatogram.Minimumdetectablepeakarea=(mAbs·sec)3481229(mAbs·sec)1000(mAbs·sec)X----------3334330(mAbs·sec)G:raisedecimalfractionstounit.1044(mAbs·sec)<2000(IDA.bs·sec)w0\J··....·:.!'--Table4Residual.testsubstanceconcentrationintestbottlesBottleNo.23456ConcentrationinPeaksolutionmg/lforHPLCinbottle·mAbs·secanalysis(A)(8)(C)<2000(0.1<1(2000.(0.1<1(2000<O.1·<1<2000<O.1<1344701362.2107.5StandardsolutionConcentration(D):Peakarea(E):VolumeofsolutionforHPLCanalysis(F):Volumeofsolutionfor.HPLCanalysis(G):Averagerecovery(H):Equation:B=DXA+E100.50mg/12772249mAbs·sec0.50010."29519896

### Figure 1 Infrared absorption spectra oftJ:te test substance
Measured by the. !!J)onsor

100

10

70

eo

ISO

3D

2000 • 1511D' 1000 500 w.......oor. 1cm-1J



### Measured.by M.S.I

X:**~000.0. ~00.00**  cm-1;**0.2~.~5.39 ~T**  99/09/03 10:18**~** scans; mace ratlo; r-esol 4.00 cm-1: apoc str-ong smooth 32.0 cm-1; flat; 9837 .o!G {lfll'/vr 1 ot. no
. NH-930 1-01 R. Y lllllaguch 1 

### ) thr-eshold 3.00X; bane · cm-1 X cm-1 X cm-1 X cm-1 X 3451.4 30.28 2942.8 16.89 1708.2 0.23 1633.5 16.75 1401.5 3.68 1368.2 1:5.19 1:3:30.1 17.2:3 1298.9 15.20 1187.5 2.25 1073A z2.'os 1018.2 15:76 965.5 11.27 945.3 27.50 900.7 30.39 B11.B 22.54 744.5 28.78 716.7 26.22 669.4 35.40 548.3 .34.98 462.6 32.99 409.4 33.19 21 paeks 1ound

<16 .<17 IT


# _.I

C-H stretch

·o.&:-f00-0 ---J-,5U-0---11--rO-o-,**~~-~-0** --.--\ !SUD C=O stretch 

# \**~---~;-** C-N stretch C-0-C stretch

### ) Figure 2 

### Infrared absorption spectrum 

### of the test substance stored in desiccator

x:**~ooo.o,** 40o.oo cm-1; 0.10,63.53 XT 4 scans; 110de r-atio: r-esol 4 .co cm-1: smooth 11.:3 cm-1: flat: · 96374G : :**~** ."

_ ,'1ot..no .HH-9301-01 thr-eehold e.oox: band.

cm-i X cm-1 l cm-1 3449.2 '49.65 2943.7 21.46 1711.6 1-437,0 10.22 1398 ,B 1.:35 1370.1 1:301.9 19.17 1187.2 0.21 1077.1 986.5 6.81 946.0 36.56 902.4 832.6 40.22 812.3, 23.42 748.0 670.5 50.37 550.5 52.29 48:3.5 24 peak= fo!Jnd

73.RS IT

### ~ C-H stretch 99/10/28 09:53 IPOC Str-ong

.

l 0.09 12.37 :33.99 42.36 37.45 47.59

.. cm-1 1634.1 1331.5 1017.7 861.7 713.4 H:3.9 X 21.56 16.75 14.60 44.50 34.76 49.8:3

··l.ID.f---.,---.,---.,....--,.---+-'--,--==f-.....;...---r""c-----,- 4DDD 3SOO 31100 Z:SDD 20110

. co"' 5DD C=O stretch C-N stretch C-0-C stretch

### ) 

## Figure 3 Calibration Curve of 

## the Test Substance



## Curve Fitting [Least Square Method] 

# J!!P..Y.:LDa_g;_

----- Concentration Peak Area**--1'1..9.~.----** 

# ... X.**~II!&LJ.L** 

## Y**.~!!'Ab~..§~c;:l__** 

## · 1 0 0 2 25

. 1781328 3 50 3580618

### -~-~------- 100 70825§3.· Y 

### = 1. 2138E+04 · 

# + 7. 0834E+04 X 

### X r 

## = 0. 99998



## 8000000



## "' u Q) 

## 7000000
6000000**~** 

## 5000000
..c < e 

## '-" 4000000 C"d**~** 

## ::: 3000000**~** 0.... 

## 2000000
1000000 Calibration Curve·

0**~--~----~--~----~--~--~** 

### 0 

## 20 40 60 80 -100 120 Concentration (mg/1)



# 'I-I\.Figure4HPLCchromatogramsofthetestsubstance---Calibrationcurve1.\SS-LC!OYer.=I.6011:'r'-I=STDO.KOl15:68:54!:·J": . ....;!J:!18374G.ii:Jdi::20i.i/11:111:SI'IHI!OAYJ>I,·1-14>:R.YAIIAGUCJU•hf':f,·:ICIIRIIl.IIOl·••tni1'7.J.-IBOOI0.2230nll600-(::("40020024'06.0..•.1!t'-tlllh!Jll:ith-!IStd.0mg/1in40%Ac.etonitri1e/Water.8alna..ASS-ti:lOVer.=1.60"*'-··-IJ=2r'-I=ST025.KOl99/08/2716:10:25:...1D:118374G.lt.AJI::20.!*Hill.ISPIHIJOAYJ>H;f;IR.JAJIAGIJOII:..n,•·:IO!RIIt.IIOtIIAh•.2:loq116004002QOn0--t'-?"*'-·-:e!1lll...ID__rl_______ci_24·6PKNOO.MlTill&AREAJllf«)ax«:III!IGIITNAIIRI23.116223.6983.2426.939743218701171387841317Tc!.al.Atea1781328111425902019543365I00.0000209023Std.25mg/1in40IIWater!cd8ll!n'--·Figure4(Continued)l"USS-LCLOVer.=1.60$-.Xj'.Lfi.JJ-=199/08/'1:116:21:58tn·":.-.·-JD:98374Gilo)..JI:20fiilflIt·:SPIHIIOATJ1R.YAXAGUCHI11,1'1.:IO!RJII.IIOI....?117},.7,1....I600...11;t.200olI\.!I. ' '·8'0.......0!1/0TillEI23.12022 .3.7023 24.396t26.946'2'AREAIDNO14197387643«104986608TotalArea35806184cooclEIGHTNAliE2l7862584011W56891100.ooOo4111172Std.50mg/1in40'AcetonitrileIWater8lllila.ASS-LC10Yer.=l.60$-.X;'.t..+:l.l-t"-11flt=4f'-1"5111100.K0199/08/'1:118:33:27r.n·.,·:<..•IP:9B374G::.10..II:·SI'IHIIDAYJ1H:fi1II.YAIIAGVOII111r:fi!IOIRII1.11Df......a\bs800_,..,""a.:':2:"·-::o230n=-.....·1100400200.Iol0...t'...I2PK1«J01.'11TIIIBAIII!AI23.12828!1115z23.70978343324.4028799328428.955.176019Total.7082683!§!!2.21\I48IDI«JOK:IIEIGI!fNA11B.439010113&787214.13873100.0000816113Std.100mgllin40IAcetonitrileIlaterii2.,8llln.,.Figure5HPLCchromatogramsofthetestsubstance---RecoveryanddetectionlimitTherecoveriesanddetectionlimitareshowninTable3.Yor.=1.6oI'J.fJ.Jfft-:1K0199/09/2811:48:1Z, :r•:.fWUJr•·.II:98374Gi'.ldi:20:·:R:YAIIAGUOIJ·1,1'<S·:·ICJ!RIIt.IID1.•J•T•r1.1.-.BOOICh2230o•IhOO-F200CDl!lOJI§.!I1\I;IIo;!•..a8,,..t'-JU'-l'liNOOtNOTillEI2.3.Ill2 23.69632.4.31!8426.9-14ARPAIDNO14652..411843334330·.91063TotalArea3481229CONCHEIctn'NAlll!21)8li284:1617086851100.OQOO381959.Std.50mg/lin:AcetonitrileIWater·llf.n.<USS-lC10Yer.=i.60vJ.f!JI'-Jt=l-tJUt=2·f'-I=R_BL11:01·I)IJ/09/28·12:00:-Ht:rr•·:ID. :98374G.·!tAll::20.l*f:lUI··:f"R.YAIIAGIJOII·.·.n,r:·IOIRJit.Jllll..;.,..,.,.,..._iiAbs'...80010i'2230o•·1100-400.200n•·02.--t'-JW-·- .II!!·Blank4fi..8•InFigure5(Continued)i.AS5-LCIOVer,=1.60#-.J=X1«!1.Jl0199/09/2812:ll:te,:7·.,:{Wrm--f-IU:9B3HG.tAli,:20··;;uua.·:SPIHIIO.Aopr·,·ItYAIIAGUOir:IOIRIII.IIOt..•'"•r71·-""b•BOOI0!2.·...•..I600\I01o•o.}\1!·,I0....o.NOTill!!I2.!1.1182 2:i.'70232.f.39726.9652AREAJDNO'16587464983935782119060TotalArea4096927Recoverytestl..aN;6IIEIGifl'NAliE2361'6816.4376007547.100.-0000·453323·8•in.-:lUSS-t.ClOVer.=-LeOi'$'-f.lJ=oif'-l=ftH02.Jt:01·99/09/2812:23:18t!lr•:-:JD. :'98374G.U:J..8:·:20:.lllfltfiSI'IHW1Aopf'-.It-If.:R.WIAGUCIIln,•·<15.:10111111.1101....,....10\bssoot012230n•11100.41100»1Ol"ciIo·2-t'-11-$'...- .a.NO·rnmARI!ATim.'1.23.123.2 23.709'. 3 2(.40lt426.975"15707..439903922745.96235TotalArea4078677Recoverytest2...0....!;i04rnNC•6JIBIGHTNAllS22936674·436688..7264·100,0000451898!;;'.!oli.8aln·Figure6BODchart1\)1\)1\)1\)IllI)0_.Nc.l.eL-Nta1....01-·---.-.-75-i.!-----------!"••--·-..!...so+-·---,-.--....6---+--+-·-:----..!!·lleasurementofBOD•······-···•····!·······i•-···'·-·..:-:..•---_,-·--llodelOlof-2001(I.D,.II:F)-:.I-·-........--•··•·----.-··r·--7"1-------·--·--..1........Cfuiit'speed··'··!'....·_,;;___.£;:}it-'--:-·-·-3··--AROIIIX·T1>71634·..·-.;.,.30-.-yJ----:::·_.-.!:.:.:_:__1--.;_·:--:-_:_T__r-;·.·_;·:6 .J.ROHIXTO-II'i34...'iOO.:.L<.::+-:-. '_,__;___________..ji--...f:.81i.•--.:.-·bo...;-:::;':r;ccl;-:---f':,0fday,..1:,:;.I.,..I7l;:..!....L'.....:;·_·_;__l_:_i__;.:..:.!.................!..l..............!'_,...1..(168)LlC!;f!:CII.\"1:-,;-1·1tiD.F'teAC1l.'5k'O!I.SS..\5HIFigure6(Continued);.01(I-lCDU)...6NColIll01Is:Ji;---1-:t=i"·l-.......r···!·+!............,!•i:-:'·t--r-·1-f·-··:.1--!·:··:·-;···1I:I·:--f---.H:!..·:·..,=Bottla6J_iBotte5..··,...i..-;..!:.:.1....:...Ji:i:)·i.:..;._.:-...::.-:.:...;:·-L.!.·::.1...:·::..::....:J&tti&4..,·-·---,i-;'ottle'i!..J.;.••.:.-!:...+-t··l;_;_:..!(1·'-··'i·:... :..•!',....-;--:--7-H-+i-'··i;:.......·.j..-.l·t:!·..1..:-ii.r.:.,·cJ·,Botti•<.-•·-·..,_.j....I.,_,__-'r:,+···J__,.,..•--.'-i-'1-._,..','------.,..•--•..,-•·'+·,_''·'-·-.....'__,_.J:-j+-----•j....7f(168)! .:-;I.:...1•j··••••·-'-,•I•I,•CHARTtt0.P"1!'RCfOC:.!·•KO<i-\YA7':1•..1t.Ot.'!:-i-r.....\i.....ilCf!.:..(SS&)..··-..-.:CM'.I.fl!liO2

23

22 I J ••
i i I I 

## I**~** .. 

### I. 

# I· i i

. 

## 1·:' 21 I,

.... · !·' 

### ' L

.. I

17 .. 

## ,:.!$..!

..Figure7HPLCchromatogramsofthetestsubstance---MeasurementofresidualtestsubstanceconcentrationTheconcentrationofresidualtestsubstanceinbottleisshowninTable4.CLASS-l.CIOVor.=1.601-J:f.lJI'-ij-=j"'*"-t!l"Jt=l7"-J:f_STD.I(OJ99/10/2616:07:55t:.-7·,:ID:98374G.!V-A::20. :SPIHIIOAypt..,·R.YMIAGUOUn,1-.1;:IOIR.IIt.v01..-.1•1H"'.J.-IIAbsIaooIa.z230o•600200ol20-t"-·-PKNOO.NO.TillEAREAIz3.22511316.223.880282503z4.6542672910{27.51969173TotalArea2772249In""""ciIDNO··f8ci'.f........"'1100.0000I6HEIGHTNAVE171339783287836070•339'!4IStd.50mg/1inAcetonitrile/Water"'ti8alna..ASS-l.CJO·Yer."'1.601-J:f.l.Jf=lf"-J=T_N02•.KOI99/10/26.15:19:27·t/t,:ID:98374GU:.A:R:20tlii:JS.:SPIHilOAYpt":.R.YAIIAGIJOIIl:l7t"4'.:IOlRIIl.IIOl-sooIChZ230n•I600.400.201!OlI'-.I2l680aln--!IIIBottle2(blanktestofsludge)Figure7(Continued)CLASS-LCIOVer.•'i.60t"-1=1_1103.KOt99/10/26·15:31;00t;,:rt.:W"nJv-F.·I D :llll374Gli:J-Qi20:SPIHIIDAYpt''l--145:·R.:Y,a.JIACUOllJ:l,f"4',:DOlRIIL.IIOl...,,.,.,.,...-m.\bs800IOt2230n•I600200024.68•In""*!!IIBottle3(sludge+testsubstance).:_'..'!·fCUSS-LCIOVer."'L.60'U'-).JJ-=13r·-t=T_IIm.KOI99/10/2616:42:33lr:Hf"Tifl-·-'fID:98374GfV..fi:20ISPIHIIOAvpf-"R.YAllAGUOU.·J;f,rif.:IOIRIII.IIOt-I•T)I"'-'.....ilba.. . . . . . . , . .·..aooIa.z23011•I61)0•.400ill...l.0.2'-468•ln. -t"-1V*"-)-·Pmla.NOTI!IF.AREAtom.aJNC.IEIGHI'HAllE1 21.35213H419J49994131Hl9'100.000014!1994Bottle4(sludge+testsubstance)Figure7(Continued)Cl.ASS-L.CIOVor.=l:60J-"-I=T_N05.JI:Ol99/10/2816:54:0•t!l7"-":ID. :91l374G·ltJ,.Jl·:20.l;t.:SPIHIIOA..-p..t""lr-1-1,:LYAliAGIJOUn1r·:IOIRIII.IIOl-1•'1',.'-"-II>Ab.s.•'••••1BOOI012"230iuo600if)400f"200,._.,n0'02468•In--PliNOCbNOTTII!lAmIllli"OlEIGHTtwiEI 2I.337. I207!>901601311207090100.0000.150131Bottle5(sludge+testsubstance)/J:ftl;•.•.tUSS-I.CIOYer.=t.605-A'I'J..JJ%1t:l;"f"NOll;KOl·99/10/21116:05:37f"/J"ft:(WT!JI-t-ID·.:98374G·tJ:J,.t:20.lltlflllISPIHIIOAwpI.YIJIAGUOIIJM•:IOIRIII.1101-,..,.,.,1-I!Abs,18001Cb2'600_,.00·200J./\0..-t"-11-*"-·-·PKNOO.NOTillEI23.2252 2.3.8813 24.656427.62:1d.I2AIIEA.ID.'II1579728958332285979399TotalArea3447013·!§idI4eo«:100.0000Bottle6(water+testsubstance)aci6811lnlElGHTNMie2166"3990416658676242911fi8-=t

# !

' CONFIDENTIAL

Sponsor

'

. I• J

.. ::
.. T 

### TSI 173/992799/SE

T0-1534


## SKIN IRRITATION TO THE RABBIT

Research Laboratory Huntingdon Life Sciences Ltd 

### P.O. Box2 Huntingdon Cambridgeshire PE18 6ES ENGLAND Report issued 1 June 1999

# l l TSI 173/992799/SE
CONTENTS Page


### COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS..................... 3

QUALITY ASSURANCE STAT'EMENT

......................................................................... 4

RESPONSIBLE PERSONNEL

.................... ...... ....................................................... .. ...... 5



### SUlvll\1AR Y . . . .

.. . . . .. . . . . . . . . . . .. . . .. . . . . . ... ..... .. .. . ... . . .. . . . . ... . .. .... .. ...... .. . . . .. .. .. . .. . .. . . ........ .. . ............. 6

INTRODUCTION

............................................................................................................ 7

TEST SUBSTANCE......................................................................................................... 8

EXPER.llv1ENT AL PROCEDURE..................................................................................... 9

RESULTS......................................................................................................................... 12

CONCLUSION................................................................................................................. 12

TABLE

1. Dermal reactions

. . . . . . . . . . . . . . . . . . . . . ... . . ... ... . . .. . ... . . . . . . .. . .. ..... .. . . . . . . . . . ... .. . . . . .. . . .. .. . .. . .. ....... .. . . . . . . . 13

### TSI 173/992799/SE
COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

The study described in this 

### report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data 

### generated to 

### be valid.
The 

### UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654).
OECD Principles of Good Laboratory Practice (as revised in 1997), 

### ENV /MC/CHEM(98) 17.
EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15129), and from 1 May 1999 EC Commission Directive 1999/11/EC of8 March 1999 (Official Journal No L 77/8)

.



# ......**~0.~** 

# ................ . Stephen J. Mason, B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. 

# . .. l

..**J:~.I~lf.'········** Date

# r
I 

### TSI 173/992799/SE QUALITY ASSURANCE STATEMENT

The following have been inspected or audited in relation to 

### this study

Study Phases Inspected Date of Inspection Date ofReporting
Protocol Audit 10 March 1999 10 March 1999

Process Based Inspections Husbandry 19 March 1999 19 March 1999 Housing/Environment 19 March 1999 19 March 1999 Test Material:- Control 19 March 1999 19 March 1999 Treatment Procedure 19 March 1999 19 March 1999 Scoring 19 March 1999 19 March 1999 Records Audit 19 March 1999 19 March 1999 Report Audit 18 April 1999 20 April 1999

Protocol Audit: An 

### audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above.

Process based inspections: At or about the time 

### this study was 

### in progress inspections and audits of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above. Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data

.



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

# ......**!3.~:::.~** ................... .. Margaret Blows, Quality Assurance Group Leader, Department of Quality Assurance, Huntingdon Life Sciences Ltd. 

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

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

# .. i.':l.:i:;. Date

### Stephen 

### J. Mason, B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. TSI 173/992799/SE


### RESPONSffiLE PERSONNEL

### f. 

### f 

## l ' TSI 173/992799/SE


### SUMMARY

A study was performed to assess the skin irritation potential ofT

- · _._;,t 

### to the rabbit. The method followed
was that 

### described in the Code of Federal Regulations, Consumer Product Safety Commission, Subchapter C

- Federal Hazardous Substances Act Regulations, Title 16, Section 1500.41. Method of testing primary irritant substances, 1 January 1994.
Six rabbits were each administered a dermal dose of0.5 ml ofthe test 

### substance to one intact and one abraded skin site and observed for three days.
A single occlusive application of' · - ( to 

### intact and abraded rabbit skin for twenty-four hours elicited no dermal irritation.
The Primary Irritation Index (PII) 

### was calculated to be 0.0 and· 

## ->-, ·is classified as non-irritant.

### TSI 173/992799/SE


### INTRODUCTION

The study was designed to assess skin irritation potential of':

__ :....... following a single dermal application to rabbits. The test substance may come into contact with 

### skin during handling or use. The study was conducted in 

### compliance with 

### the Code of Federal Regulations, Consumer Product Safety Commission,**Subcbapt~r** 

### C - Federal Hazardous Substances Act Regulations, Title 16, Section 1500.41. Method of testing primary irritant substances, 1 January 1994.
The albino rabbit 

### was chosen as it has 

### been shown to be a suitable model for skin irritation studies and is the animal recommended in the test guideline.
The amount oftest substance administered was 

### chosen in 

### compliance with 

### the guideline.
The protocol 

### was approved by Huntingdon Life Sciences Management on 25 February 1999, by the Sponsor
on 3 March 1999 and by the Study Director on 4 March 1999.
The experimental phase ofthe study was 

### undertaken between 9 and 12 March 1999.

# ..

· TSI 173/992799/SE


### TEST SUBSTANCE

Identity: 

## __ _,.,.

Chemical name: -.i ---' ,



### Intended use: 

### Coating

Appearance: Clear liquid

Storage conditions: Room temperature 

### in the dark

Lot number: 80622

Expiry: 22 February 2000

Purity: >95%

Sample received: 25 February 1999

### TSI 173/992799/SE**EXPE~NTALPROCEDURE**



### ANIMAL MANAGEMENT
Animals for this study were selected from a stock supply ofhealthy adult rabbits of the New Zealand White Strain, obtained from Charles River 

### UK Ltd, Margate, Kent, England. They were in the weight range of2.1 to 3.3 kg and at least 10 weeks of age, prior to treatment (Day 1). All rabbits were acclimatised to the experimental environment for a period of six days prior to the start 

### of the study.
The rabbits were selected without conscious bias for the study. They were housed individually in metal cages with perforated floors in Building Rl4 Room 5.
A standard laboratory rabbit diet (Special Diet Services STANRAB (P) 

### SQC pellet) and drinking water were provided ad libitum. 

### The batch of diet used for the study 

### was analysed by the supplier for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if 

### in excess of specified amounts, might have an undesirable effect on the test system. The animals were fed hay on arrival and subsequently three times a week.
The water supplied to Huntingdon Life Sciences by Anglian Water was 

### potable water for human consumption. Anglian Water takes its guidelines on water quality from the EEC directive relating to water for human consumption (80/778/EEC) and conforms to 

### the United Kingdom Water Act 1989 and subsequent amendments. Results of routine physical and chemical examination of drinking water at Consumers· taps. as conducted by the supplier, are made available to Huntingdon Life Sciences Ltd as quarterly summaries.
Animal room temperature was maintained at 

### 19 to 20°C and relative humidity at 35 to 52%. These environmental parameters were recorded daily. Lighting was controlled by means of a time switch to give 12 hours of artificial light (0700- 1900 hours) in 

### each 24 hours period.
Each animal was identified by a numbered aluminium tag 

### placed through the edge of one ear. This 

### identification was unique within the Department throughout the duration of the study. Each cage was identified by 

# a coloured label displaying the study number, animal number and initials of the Study Director and Home Office licensee.



### TEST SUBSTANCE PREPARATION
T0-1534 was administered as supplied by the Sponsor.
The absorption of the test substance was not determined.
Characterisation of the homogeneity, stability and purity ofthe test substance was 

### not undertaken as part of this study and remains the responsibility of the Sponsor.



# &r

### TSI 173/992799/SE
TREATMENT PROCEDURE Approximately 24 hours prior to application of the test substance, hair 

### was removed with electric clippers from the dorso-lumbar region of each rabbit exposing an area of skin approximately 100 

### mm x 100 

### mm. Immediately prior to application of the test substance two areas of skin each approximately 

### 25 

### mm x 

### 25 

### mm were identified in the clipped dorso-lumbar region, one to 

### the left and one 

### to the right of the spine. The area on the right 

### was abraded using the tip of a scalpel blade to 

### make minor incisions through the stratum 

### corneum. These were not deep enough to 

### reach the dennis or produce bleeding. The area 

### on the left remained intact. Approximately 

### 0.5 

### mi of the test substance was applied under 

### 25 

### mm x 

### 25 

### mm porous gauze pads to one intact and one abraded skin site.
The treatment sites were covered with "Elastoplast" elastic adhesive dressing backed with "Sleek" waterproof strapping for twenty-four hours. The animals were not restrained during the exposure period and were returned to their cages immediately after treatment.
At the end of the 24-hour exposure period, the occlusive dressing and gauze pads were removed and the treatment sites were washed with warm water (38°C) to remove any residual test substance. The treated areas were blotted dry with absorbent paper.

OBSERVATIONS
Clinical signs


### All animals were observed daily for signs of ill health or toxicity.
Dermal responses
Examination of the treated skin 

### was made on Day 2 (ie 

### approximately 60 

### minutes after removal of the dressings) and on Day 4 (equivalent to 72 hours after exposure).
Local dermal irritation was assessed using the prescribed numerical system: Erythema and eschar formation:
No erythema Very slight erythema (barely perceptible) Well-defined erythema Moderate to severe erythema Severe erythema (beet redness) or eschar fonnation (injuries in depth) preventing erythema reading 0 1 2·

.., .)
4

### TSI 173/992799/SE
Oedema fonnation:
No oedema Very slight oedema (barely perceptible) Slight oedema (edges of area 

### well-defined by definite raising) Moderate oedema (edges raised approximately 1 

### millimetre) Severe oedema (raised more than 1 

### millimetre and extending beyond the area of exposure)

CALCULATION OF PRIMARY IRRITATION INDEX 0 1 

### 2

., ,;) 

### 4



### The numerical scores for erythema and oedema at both the 24 and 72 hour readings for both 

### intact and abraded skin sites were added together and divided by 24 to give the Primary Irritation Index (PII).


### The PIT 

### was used to 

### classify the test 

### substance as follows:


### PII

0 

### >0- 2.0 2.1-5.0 5.1-6.0

>6.0 Classification
Non-irritant Mildly irritating Moderate irritant Moderate to severe irritant Severe irritant
Based on 

### Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials ofthe United States. 1959, p 47.

ARCHIVES
All raw data and study related documents generated during the course of the study at Huntingdon, together with a copy of the final report will be lodged 

### in the Huntingdon Life Sciences Ltd Archive, Huntingdon.
Such records will be retained for a minimum period offive 

### years from the date of issue ofthe final report. At the end of the five year retention period the client will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the client's lmowledge.

DEVIATIONS FROM PROTOCOL
There were no deviations from the protocol.

### TSI 173/992799/SE
RESULTS

CLINICAL SIGNS
There were no signs of toxicity or 

### ill health 

### in any rabbit during the observation period.



### DERMAL 

### RESPONSES
The numerical values given to the dermal reactions elicited b} : ,. are shown 

### in Table l.

No dermal irritation 

### was observed 

### in any 

### a.rllmal throughout the study.
The 

### Primary Irritation Index (PII) was calculated to be 0.0.



### CONCLUSION

A single occlusive application of to intact and abraded rabbit skin for twenty-four hours elicited no dermal irritation and is classified as non-irritant.

12 :

### TSI 173/992799/SE 

## TABLE I


### Dermal reactions

Rabbit no. & 

### E=Erythema 24 hours 72 

### hours sex 0= Oedema Intact 

### Abraded Intact 

### Abraded 2098Male E 0 0 0 0 

### 0 0 0 0 0 2099Male E 0 0 0 0 

### 0 0 0 0 

### 0 2100Male E 0 0 

### 0 0 

### 0 0 0 0 0 2101 Male E 0 0 0 0 

### 0 0 0 

### 0 0 2102Male E 0 0 

### 0 0 

### 0 0 0 0 0 

### 2103 Male E 

### 0 0 0 0 

### 0 0 0 0 0 Primary 

### Irritation Index= 0.0



### 13 :

### F i 

### n. 

### a 1 Report



### Mutagenic ty Study of

in Bacterial Reverse Mutation Assay
(StudyNumber 5739)

BML. 

### INC.

# AUTHENTICATION

I, the undersigned, hereby dedare that this report is an accurate translation of the original report written in Japanese, No. 5739, issued on Msuch 20, 1998, by BltfL, 

### Inc.

Title of Report: A mutagenicity study of.'- : .:-.- --'- \"' 

### .... ·· 

### .. , ethyl ester in Bacterial Reverse Mutation Assay

Report Number: 5739

Signature: 

# T Yoshihiro Oguma Study Director, Division of Safety Evaluation 8c Reagent BML, Inc. Date: 

# f-'1 C( 

# r. 3 

# I

# STATEMENT OF STUDY DIRECTOR

Title of Study. A mutRgenicity study**~** __ .• ' 

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

### _., ethyl ester 

### in Bacterial Reverse MutRtion Assay

Study Number: 5739

This is to certify that the this study was conducted 

### in accordance with "Standards to be observed by Mutagenicity Testing**Institution~'** {Notification No. 76, Ministry of Labor, Japan, September 1, 1988).



### sprure:

# 1~ 

# UJ- tft 'l4L-"'"-
Yoshihiro Ogui: Study Director, Division of Safety Evaluation & Reagent BML, Inc.

# QUALITY ASSURANCE STATEMENT

Tide of Study: A mutagenicity study of ' ""' • ethyl ester in Bacterial Reverse Mutation Assay

Study Number: 5739

This study 

### was conducted in accorda.nce with "Standards to be observed by Mutagenicity Testing Institutions" (Notiiication No. 76,; Ministry of Labour, japan, September 1, 1988). The circumstances of audits a.nd inspections are as follows.

Date of inspection
February 12, 1998

February 23, 1998

March 19, 1998

March 12, 1998

March 18, 1998

March 

### 23, 1998

March 31, 2000 Phases

Review of draft protocol and SOP

Review of protocol and SOP

Review of amendment
to the protocol

General inspection

Review of draft report and audit of mw data

Review of fina.l report (in japanese)

Review of .5na.I report (in English) Date of Report to Management
February 12, 1998

February 24, 1998

March 20, 1998

March 12, 1998

March 19, 1998

March 23, 1998

March 31, 2000

I certify that the methods and procedures used in the test are described precisely in the final report,

. and that the reported results re.Dect the mw data accurately.

Signature:

Masahiro Kurar.now Manager, Qulliity Assumnce Quality Management Division B.M.L, Inc. Date:**~tudy** 

### number : 5739
Title : A mutagenic! ty study of

.**-~· ~-**  k 

### in bacterial reverse mutation assay

Purpose of the test: To assess the effect of the test substance in bacterial reverse mutation assay.
This study was performed for Mutagenicity Tests using in accordance with the guidelines set forth in "Standards Microorganisms" (Notification No. 67, June 2, 1997,. Ministry o f L abo u r , J ap an)

. And it was conducted in compliance with "Standards to be Observed by Mutagenicity Testing Institutions" (Notification No. 76, Ministry of Labour, Japan, September 1, 

### 1988).

Sponsor
Name Address Testing facility
Name Address
Date of study
Initiation Completion
Persons in charge Study Director ·-..:;:;.- _.**~** - '' 

### . 

# . 

# . '



### Section of Safety Evaluation, 1361-1, 

### Matoba, Kawagoe-shi,

February 10, 1998 March 20, 

### 1998 .. :":.::.::::--·:: --·. . -.r-

... - ,.



### Division of Biocellology,

Saitama 350-1101 Japan

Yoshihiro Oguma BML, INC.

Persons in charge of study Miko Sarada, Hiroshi Seki, Fumio Yokota

Name of the person who prepared the final report, and date of preparation
Yoshihiro Oguma
Title : Study Director
Date : March 20 

### 1998**----~~~~--~~---** 

### certify that this study was conducted under the specified protocoL

K



### iyosh i 

### Och i


### Title :Administrator 

# '70 Date : March 20 

### 1998**----~~~~~~;_--**

### I. ' 

### Summary
Mutagenicity potential of - - L

--
l' 

### was assessed by using Salmonella typhimurium 

### strains TAlOO, TA1535, TA98, TA1537 and Escherichia coli 

### WP2 uvrA. 

### In this study, neither increase in the number of revertant
as many as that of the negative control in any strains of colonies more than twice base-pair substitution type or frameshift type, with or without metabolic activation, nor dose-related response
was observed. Thus, it is considered· that has no mutagenicity to bacteria under the conditions of this study.



# ?!

### 2. Date of test
Investigation of the solubility February 6, 1998 Initiation of the dose-finding test February 25, 1998 Initiation of the main test March 11, 1998 Completion of the main test March 14, 1998

3. Test substance

(1) Name{Another name}

(2) BML-CODE (3) Lot No.
(4) 

### Structural formula

(5) Purity n ""'

-- . -- 

### "**~'** - -


### BML-3845
71107

Over 99wt%
(6) 

### Name and concentration of impurities Toluene:under lwt%
(7) 

### CAS No. (8) Molecular weight 249. 27 (9) Melting point 50"C (10) Boiling point (11) Vapor pressure (12) Partition coefficient (13) Appearance at room temperature Wb 

### it 

### e so I i 

### d (14) Stability (15) Degree of solubility

(16) Stability in solvent (17) Condition of storage (18) Notice in handling Stable at room temperature
Water: insoluble Dimethyl sulfoxide(DMSO): over 50mg/ml Acetone: soluble Toluene: soluble
Stable in DMSO, acetone and toluene
Room temperature in shade
Wear the gloves in order to prevent the rash
The test substance was analysed and all the data are retained by the sponsor. The solubility in DMSO was tested by BML, INC. 

## _1( 

# 2**~ ~ester**  

### strains

(1) 

### Strains used 1 ' · 2 ' · 3 ' 

### The following 5 strains were used. Base-pair substitution type : Salmonella typhimurium 

### TA!OO, TA!535 Escherichia coli 

### WP2 uvrA 

### F r arne s h i f t 

### type : Salmonella typhimurium 

### TA98, TAI537
(2) Supplier and date obtained

S. 

### typhimurium 

### TA100 The Division of Mutagenesis, National Institute of Japan (The Division of Genetics and Mutagenesis, Research Center National Institute of Hygienic Science, ; April 15, 1982 Other S. typhimurium 

### TA strains Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 E. coli 

### WP2 uvrA 

### Hygienic Biological Sciences, Safety Japan at present)

Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 

### 1987
(3) Characterization date

S. 

### typhimurium 

### TA100 December 25, 1997 S. 

### typhimurium 

### TA1535 February 16, 1998 E. coli 

### WP2 uvrA 

### December 2, 1997 S. 

### typhimurium 

### TA98 December 25, 1997**~** 

### typhimurium 

### TA1537 February 16, 1998
(4) 

### Reason for choice

These strains are very sensitive to mutagens and used most commonly in mutagenicity study in bacterial reverse mutation assay.
(5) Condition of storage

The cell suspension and DMSO (spectrophotometric grade) were mixed 0. 8 ml to 0. 07 ml. The mixture was subdivided into 0. 3 ml aliquots,
were frozen and stored at -80l:. in a and ratio of then they

### 5. Con t ro I compounds

(1) 

### Negative control Negative control plates were treated with only DMSO.
(2) Positive controls

Strain Without S9 (/.L g/plate) With S9(/.Lg/plate)

TA100 AF-2 (0. 0 1) 

### B [a] P ( 5. 0) TA1535 NaN3 (0. 5 ) 2AA ( 2. 0) WP2 uvrA 

### AF-2 (0. 0 1) 

### 2AA (1 0. 0) TA98 AF-2 (0. 1 ) B [a] P ( 5. 0) TA1537 ICR-191 (1. 

### 0 ) B [a] P ( 5. 0)

AF-2 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (Wako Pure Chemical Industries, Ltd.; Lot No. SAJ0748; Special grade of JIS; Purity, more than 95%) NaN3 Sodium azide (Wako Pure Chemical Industries, Ltd.; Lot No. DCJ6398; Special grade of JIS; Purity, more than 95%) ICR-191 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine ·2HCI (Polysc'iences, Inc. ; Lot No. 52300; Pur.i ty, more than 95%) 2AA 2-Aminoanthracene (Wako Pure Chemical Industries, Ltd.; Lot No. KPQ0892; Special grade of JIS; Purity, more than 95%) B [a] P Benzo [a] pyrene (Wako Pure Chemical Industries, Ltd.; Lot No. KCF4188; Special grade of J!S; Purity, more than 95%)
1) 

### Solvents

AF-2, ICR-191, 2AA and B[a]P were dissolved in DMSO (Wako · Pure Chemical Industries, Ltd.; Lot No. TPK5055). NaN3 was dissolved in distilled water for injection (Hikari Pharmacy Co., Ltd.; Lot No. 9708ST). These solutions were subdivided, stored at -20't: and thawed just prior to use. 2) Reason for choice of the positive control compounds

These compounds were chosen in accordance with the guideline of the Industrial Safety and Health**La~**

### 6. Materials

(1) 

### S9 mix

1) S9 Name Manufacturer Lot No. Manufacture date Purchase date Storage Materials for S9 S9 Fraction Kikkoman Corporation RAA-375 January 9, 1998 January 20, 1998 Stored at below -80 \: in freezer Fraction Species and strain Sprague-Dawley rat Male Sex Age Weight 7 weeks 183- 245g Inducing substance Phenobar bit a I (PB) and 5, 

### 6-benzo flavone (BF) i.p. injection Administration Dose
2) 

### Cofactor Name Manufacturer Lot No. Storage 3) Composition of Water S9 Fraction MgCI2 KCl G-6-P NADPH NADH PB=30t60t60+60 mg/kg (4 

### days); BF=80 mg/kg(at the third day)

Cofactor Boehringer Mannheim 

## L L 725 Stored at below 5 'C 

### in refrigerator S9 mix (per 1ml) 0. 9 ml 0. 1 ml 8. 0 tL 

### mo I 33. 0 tL 

### mo I

5. 0 tL 

### mo I 4. 0 tL 

### mol 4. 0 tL 

### mo I Na-Phosphate buffer (pH 7. 4) 100. 0 tL 

### mol
(2) Top agar Used agar Name Manufacturer Lot No. Bacto-Agar Difco Laboratories 99703JA

### · (3) Minimal glucose agar plate
Name Manufacturer Lot No. Manufacture date Purchase date Storage

7. Method

(1) 

### Solvent
Name Manufacturer Lot No. Purity Minimal glucose agar medium M ORIENTAL YEAST CO. , LTD. BM010AN January 28, 1998 February 10, 1998 Stored at room temperature

Dimethyl sulfoxide (DMSO) Wako Pure Chemical Industries, Ltd. TPK5055 More than 99% (Special grade of JIS)
(2) Preparation of the test substance solution
Based on the information from the sponsor that the test substance was insoluble the test in water, the solubility test substance dissolved at 50 mg/ml was performed in in DMSO. DMSO was DMSO. From the result, solvent for preparation of the test substance solutioa The preparation was used as carried out just prior to use.
(3) Condition of pre-culture

I) 

### Nutrient Broth Name Nutrient Broth No. 2 Manufacturer Oxoid Limited Lot No. 105 59702 2) Incubator Model Cool Bath Shaker ML-10 Manufacturer Taitech Co., Ltd. 3) Pre-culture procedure

Bacterial cells of each strain (20 111 

### of S. typhimurium 

### TA strains, 5 111 of E. coli 

### WP2 uvrA) 

### were inoculated into a L-form cui ture tube (capacity of 35 ml) containing 10 ml of Nutrient Broth. This culture tube was left at 4 'C 

### for 7 hours and 45 minutes and incubated shaking (IOO rpm) at 37 'C 

### for 8 hours. After incubation, optical density was measurel The bacterial cultures were stored at room temperature and used within 2 hours after incubation.

## 4) Number of bacteria at the end of pre-culture Number of bacteria in cui ture was estimated from optical density at the end of pre-culture.

S. typhimurium 

### TA100 S. typhimurium 

### TA1535 E. coli 

### WP2 uvrA**~** 

### typhimurium 

### TA98 S. typhimuri um 

### TA1537
(4) Dose selection dose-finding test

1. 

### 48X 10 9 

### /ml 1.88Xl0 9 

### /ml 4.59Xl0 9 

### /ml 2.14X10 9 

### /ml 1.59X10 9 

### /ml main test

1. 

### 46X 10 9 

### /ml 1. 

### 80Xl0 9 

### /ml 4. 57Xl0 9 

### /ml 2. lOX 10 9 

### /ml 1. 

### 49 X 10 9 

### /ml

The dose range was determined from the dose-finding test using the following
seven dose levels: 1. 

### 2, 4. 9, 20, 78, 313, 1250 and 5000 ug/plate. In the dose-finding test, growth inhibition was observed at 1250 ug/plate and more in S.typhimurium 

### TAstrains without metabolic activation and S.typhimurium 

### TA1537 with metabolic activation, and 5000 ug/plate in E.coli 

### WP2 uvrA 

### with or without metabolic activation and in S.typhimurium 

### TAIOO, TA1535 and TA98 with metabolic activation. Precipitate by the test substance on the plate was not observed with or without metabolic**activatio~** 

### Therefore, as the highest dose level of the test substance in the main test, 1250 ug/plate was selected for 

## s. typfdmurium 

### TA strains without metabolic activation and 

# s. typhimurium 

### TA1537 with metabolic activation, 5000 ug/plate was selected for E. coli 

### WP2 uvrA 

### with or without metabolic activation and S. typhimurium 

### TAlOO, TA1535 and TA98 with metabolic activation. These highest doses were diluted 5 times (using a common ratio of 2) to provide a total of 6 dose levels for all strains with or without metabolic**activatio~·** 

### (5) Test procedures (the preincubation method) 41

· 51

· 61

· 71



### For the tests wi thou! metabolic activation, 0. 5 ml of 0. 1M sodium-phosphate buffer (pH7. 4) and 0. 1 ml of each bacterial cul lure were added to a tube containing 0. 1 ml of the test substance solution. For the tests with metabolic activation, 0. 5 ml of S9 mix instead of 0. 1M sodium-phosphate buffer was added to each tube. The mixture
was preincubated at 37 

## t: for 20 min shaking horizontally. Then 2. 0 ml of top agar was added to the mixture, and the contents in each tube were poured over ·the surface of the minimal glucose agar plate. For the sterility test, 0.1 ml of the test solution at the highest dose and 0.1 ml of S9 mix were put into each tube, 2. 0 ml of top agar was then added, and the contents in each tube were poured
over the surface of each minimal glucose agar plate.

After incubation at 37 

## t: for 48 hours, the revertant colonies on the plate were counted and the bacterial cells were observed by a stereoscopic microscope in order to check whether or not growth of the tester strains was inhibited. As top agar, soft agar solution (0.6% Bacto-agar and 0.5% NaCl) with 1/10 volume of 0.5 mM biotin

- 0. 5 mM L-histidine for the S. typhimurium 

### TA strains or with 1/10 volume of 

### 0. 5 mM tryptophan for the E coli 

### strain was used. Two minimal glucose agar plates were used for each dose I eve I. 

# 7'l

### (6)' Counting procedure
The number of revertant colonies was counted by colony counter.

1) 

### Colony counter Model Manufacturer 2) 

### Correct ions Correction with 3) 

### Coefficient data - I 

### 00 101

- 400 401

-

8. 

### Judging Criteria s> 

### the Colony Analyzer System Science

coefficient

coefficient

I. 

### II I. 

### I6 I. 

### 25 

### CA-7TI Co., Ltd.

In the dose-finding test and the main the test plates increased significantly test, if the number of revertant colonies on in comparison with that on the control plates (more than twice as many as that of the negative control), and dose-response and reproducibility were observed, the test substance was judged positive. Statistical analysis was not done.

9. Results and discussion

The results were shown in the Table In both the dose-finding rest and of revertant colonies more than twice I, 

### the 2 

### and the Figure I, 

### 2. 

### main test, nei !her increase
as many as that of the in any strains of base-pair substitution type or frameshift type, metabolic activation, nor dose-related response was observed. in the number negative control

with or without

The revertants of the positive controls showed increase more than twice as many
as that of the negative controls, indicating that this study was performed properly.

From these results, mutagenicity of the test substance was judged negative. The growth inhibition was observed at doses marked (*) 

### in the Table 1 and 2, but the precipitate by the test substance was not observed on the plates with or without metabolic activation. In the sterility test on test solution and S9 mix, no growth of bacteria was observed.

10. Deviation from protocol, etc. During the study period, there was no environmental factor that was thought to have affected the credibility of the test, or there was no deviation from the protocol.

### 11. Cone l us ion

From the results above, it was concluded**that~~** 

### . ------::· "•

..

-- 

## --- .. 

### was non-mutagenic in bacterial reverse mutation assay under the experimental condi lions.

12. 

### Storage of records and test substance

All records and test substance generated during this study (listed below) will be stored in the GLP archival area of BML, INC. for 10 

### years after the application of permission. At the end of the period, the sponsor wi 11 

### be consul ted regarding the handling of them.
Stored records and test substance

1) 

### Protocol and Amendments 

### Z) Records concerning test substances 3) 

### Raw data 4) 

### Test substance 5) 

### Records on activities of Quality Assurance Unit 6) 

### Draft of final report 7) 

### Final report

13. 

### References
1) 

### B. N. 

### Ames, F. D. 

### Lee, and W. 

### E. Durston: An 

### Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Nat!. Acad. Sci. USA Vol. l.Q., 

### No.3, pp. 782-786, 

### March 1973
2) 

### J. 

### McCann, N. E. 

### Spingarn, J. 

### Kobor i, and B. N. 

### Mutagens: Bacterial Tester Strains with R Factor USA Vol. 

## 11. No.3, pp. 979-983, 

### March 1975 

### Ames: Detection of Plasmids, Proc. Carcinogens Natl. Acad. as sci.

3) M.H.L. 

### Green and W. 

### J. 

### Muriel: Mutagen Testing using Trpt Reversion in Escherichia coli, 

### Mutation Res. Vol. 38, pp. 3-32, 1976 4) 

### T. Yahagi, M. 

### Nagao, Y. 

### Seino, T. Matsushima, T. Sugimura and M. 

### Okada of N-nitrosamines on Salmonella, 

### Mutation Res. Vol. 48, 

### pp. 121-130, 

### Mutagenic i t,i 

### e s 1977 5) D. 

### M. Maron and B. N. 

### Ames: Revised Methods 1983 

### for the Salmonella 

### Mutagenic! ty Test, Mutation Res. Vol.ffi, pp. 173-215, 6) A 

### guidebook for Mutagenicity Studies using Bacteria, New Edition, Ed. by Chemical Substances of Labor, Investigation Division, Industrial Safety and Health Department, Ministry

7) 

### Testing Tamura, Japan Industrial Safety and Health Association, 1986


### Methods for Environmental Mutagens : Ed. by Taj ima, Koudan-sha Co., pp. 56-68, 1980 

### Kada, Kondo andTable 1 Table of Dose-Finding Test Results

Name of test substanct.: ·· --·" ac

--- - .... : :.;':..1;.;. -.. ; :. ' :**~;:.-J.** .No. 5 7 3 9 A term of test February 25. !998


## - February 28, 1998 Number of revertants (number of colonies/plate) With(+) or Test substance without(-) concentration Base-pair substitution type Frameshi f t type S9mix ( t.L g/pl at e) TAlOO TA1535 WP2uvrA Solvent

. 109 23 21 control 142 ( 12 6) 22 ( 23) 20 ( 143 25 18 l. 2 124 ( !34) 20 ( 23) 27 ( 145 18 19 4. 9 141 ( 143) 20 ( 19) 23 ( 159 22 19 2 0 122 ( 141) 22 ( 22) 19 ( -89 mix 124 27 19 7 8 156 ( 140) 20 ( 24) 18 ( 146 20 19 3 1 3 134 ( 140) 17 ( 19) 17 ( 118* 11* 22 1 2 5 0 102* ( 11 O) 1* ( 9) 17 ( 0* 0* 13* 50 0 0 0* ( O) 0* ( 0) 20* (

Solvent 146 17 38 control 141 ( 144) 20 ( 19) 33 ( 146 22 29 1. 2 !50 ( 148) 17 '• ( 20) 24 ( 147 13 28 4. 9 143 ( 145) 14 ( 14) 29 ( !58 25 26 2 0 118 ( 138) 25 ( 25) 27 ( +S9 mix 152 25 28 7 8 147 ( 150) 24 ( 25) 27 ( 141 22 28 3 1 3 156 ( 149) 20 ( 21) 20 ( 133 26 20 1 2 5 0 !51 ( 142) 21 ( 24) 19 (
70* 2* 17* 5 0 0 0 61* ( 66) 6* ( 4) 9* ( Name AF-2 'I NaN, •2 AF-2 Positive control Concentration not (ug/plate) 0. 0 1 0. 5 0. 0 1 requiring S9mix Number of 514 412 136 colonies/plate 555 ( 535) 400 ( 406) !55 ( Name B [a] P 

## •• 2AA 

## •• 2AA Positive control Concentration requiring (t.Lg/plate) 5. 0 2. 0 1 0- 0 S9mix Number of 870 310 407 colonies/plate 841 ( 856) 306 ( 308) 454 ( #1 :2-(2-Furyl)-3-(5-ni tro-2-funl)acryl amide #2:Sodium azide f3:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropy!amino)acridine·2HC1 #4:Benzo[a)pyrene #5:2-Aminoanthracene *:Growth inhibition of tester strains was observed. 21)
23)
21)
19)
19)
!8)
20)
17)
36)
27)
29)
27)
28)
24)
20)
13)

146)

431) TA98 TA1537 27 13 21 ( 24) 12 ( 13) 17 9 23 ( 20) 14 ( 12) 23 11 22 ( 23) 13 ( 12) 17 12 23 ( 20) 8 ( 1 O) 22 8 26 ( 24) 7 ( 8) 24 7 20 ( 22) 7 ( 7)

8* 6* 13* ( 11) 2* ( 4) 0* 0* 0* ( O) 0* ( O) 30 22 30 ( 30) 17 ( 20) 28 14 28 ( 28) 20 ( 17) 31 19 23 ( 27) 17 ( 18) 24 17 29 ( 27) 18 ( 18) 28 19 29 ( 29) 17 ( 18) 30 17 30 ( 30) 19 ( 18) 31 6* 28 ( 30) 12* ( 9) 11* 3* !H ( 11) 8* ( 6) AF-2 ICR-191"'

0. 1 1- 0 613 1482 646 ( 630) 1551 ( 1517)

B [a] p B [a 

### J P

5. 0 5. 0 210 61 205 ( 208) 69 ( 65)T-a b 1 e 2 Table of Test Results

Name of test**substance:~** . No. 5 7 3 9 A term of test March II. 1998


# - March 14, 1998 Number of revertants (number of colonies/plate) With(+) or Test substance without(-) concentration Base-pair substitution type S9mix (.ug/p!ate) TA100 TA1535 WP2uvrA Solvent !51 17 20 control 147 ( 149) 14 ( 16) 21 ( 154

. 15 3 9 142 ( 148) 17 ( 16) 152 13 13) \ 7 8 !56 ( !54) 12 ( 146 II 22 1 5 6 !50 ( 148) I2 ( 12) 23 ( -S9 mix !54 I3 26 3 1 3 154 ( I 54) !0 ( 12) 22 ( 163 7 22 6 2 5 148 ( 156) !3 ( I O) 26 ( 131 

## * H 16 1 2 5 0 123* ( 127) 10* ( 9) 22 ( 19* 2 5 0 0 16* ( 

### D* 5 0 0 0 

### D* ( Solvent 160 13 13) 1 36 control 163 ( 162) 13 ( 37 (

3 9
7 8 176 15 29 1 5 6 !59 ( I68) 16 ( !6) 36 ( 168 18 28 3 1 3 !51 ( 160) 12 ( !5) 3! ( +S9 mix !55 21 34 6 2 5 172 ( 164) 14 ( 18) 38 ( 175 12 36 1 2 5 0 171 ( 173) 16 ( 14) 26 ( 69* 16* 

### 21* 2 5 0 0 65* ( 67) 10* ( !3) 21* ( 56* 8* 9) 

# I 16* 5 0 0 0 65* ( 6!) 10* ( 17* ( Name AF-2 

## ., NaN, 

## ., AF-2 Positive control Concentration not 

### ( .u g/p I at e) 0. 0 1 0. 5 0. 0 1 requiring S9mix Number of 558 421 166 colonies/plate 516 ( 537) 410 ( 416) 134 ( Name B (a] P 

# . ' 2AA 

# . ' 2AA Positive control Cone en t ration requiring (.LL g/plate) 5. 0 2. 0 1 0. 0 S9mix Number of 949 322 428 colonies/plate 966 ( 958) 289 ( 306) 437 ( #!:2-(2-Fury1)-3-(5-nitro-2-fury!)acry1amide #2:Sodium azide #3:2-Methoxy-6-chloro-9-[3-(2-chloroethy!)aminopropy!amino)acrldine·2HCI #4:Benzo [a]pyrene #5:2-Amlnoanthracene
*:Growth inhibition of tester strains was observed.

# ~~ 21)

23)
24)
24) !9) !8) 

### 0)
37)

33) 30) 36)
3!)
24)
17)

!50)

433) Frameshift type TA98 TA1537 27 11 20 ( 24) 11 ( II) 20 8 20 ( 20) 11 ( 1 0) 19 9 2I ( 20) 6 ( 8) 27 8 18 ( 23) 10 ( 9) 2I 7 19 ( 20) 8 ( 8) 22 8 25 ( 24) 8 ( 8) 16* 

### D* 6* ( II) 0* ( O)

41 21 32 ( 37) 16 ( 19) 19 15 ( 17) 15 19 ( 17) 36 15 31 ( 34) I6 ( I 6) 28 II 31 ( 30) 18 ( 15) 34 14 33 ( 34) 9 ( 12) 31 8* 31 ( 31) 9* ( g) 18* 18* ( 18) 12* II* ( 12)

AF-2 ICR-191"'

0. 1 1 . 0 627 1505 588 ( 608) 1574 ( 1540)

B [a 

### J p B [a] P

5. 0 5. 0 258 61 265 ( 262) 68 ( 65).a. ..._0 • ..L 

### (Colonies/Plate) D o 

## s e - r e s p 

### o 

## n s e

,,, '20'0

180



### 160

140

120



### 100

80

60

40

20

0 0 

### 39 78 156 313 625

Fig. 

### 2 (Colonies/Plate) 

## D o s e - r e s p 

## o n s e

200

180

160



### 140



### 120

100



### 80

60



### 40


### 20

0 0 

### 39 78 156 

### 313 625

# .::O'Jc u r v e



### 1250 2500



### c u r v e

1250 

### 2500 No.5739 

# ( s 9 - )



### oTA100 eTA1535 OWP2uvrA 

### *TA98 +TA1537

5000 ( 11 

### g/plate)

No.5739 

# ( s 9 

## + )

oTAlOO eTA1535 OWP2uvrA 

### *TA98 +TA1537

5000 ( 11 

### g/plate)

### VERIFICATION OF TRANSLATION



## This is to certify that English translation listed below conforms to the Japanese original report.

Title : Acute Oral Toxicity Study of lmidoacrylate in Rats
Study No. : 10101



# Ut dwk i, 

# t}JJ**r;ti~k~** Hideaki Hiratsuka, Ph.D. Study Director Panapharm Laboratories Co., Ltd.

-83- 

## Date 

# lPPL .t<'J.N.P..L !lliPUl{l': lUlUl



### Acute Oral Toxicity Study of Imidoacrylate 

### in Rats

- Final Report

-

Aprill2, 2001

Sponsor : ·'

-· :::: ::·

..



### Organization under contract

: Mitsubisbi Chemical Safety Institute Ltd.

1-30, Shiba 2-cbome, Minato-ku, Tokyo 105-0014, Japan

Test Facility : Panapbarm Laboratories Co., Ltd. 1285, Kurisaki-machi, Uta, Kumamoto 869-0425, Japan



### -84--

## QUALITY ASSURANCE STATEMENT
Study No. : 10101 Title :Acute Oral Toxicity Study of Imidoacrylate 

## in Rats

Personnel of the Quality Assurance Unit inspected each phase of 

## this study. The phases, dates of inspection and dates of the QA report to the study director and the management are listed below.
Phase

·Protocol

· Animal receipt and body weight measurement

· Information, receipt, storage, and preparation of test substance mixture

· Administration and clinical observation ·Necropsy ·Raw data (Draft report) and draft 

### report

·ditto (reinspection)

· Return document oftest substance

· Final report and 

### all records Date of inspection Jan.24,2001 Jan.30,2001 Feb.6,2001
Feb.6,2001 Feb.20,2001 Mar. 21, 2001

- Mar. 22, 2001 Mar. 23, 2001 Mar. 24, 2001 Apr. 12, 2001 Date of QAreport Jan.24,2001 Jan.30,2001 Feb.6,2001
Feb.6,2001 Feb.20,2001 Mar. 22, 2001

Mar. 23,2001 Mar. 24, 2001 · Apr.12, 2001


### I 

### assure that this study was conducted 

## in compliance 

### with OECD Principles of Good Laboratory Practice (recommended by OECD, 1997) and this report accurately reflects the methods and raw data of 

## this study.

Manager of Quality Assurance 

## Unit

April 12, 2001 Hi detosbj Ogata sealed Panapharm Laboratories Co., Ltd.

-85-.-J:>J:>L .l:<lNAL .liliJ:>U.lU: lUlUl



## Signature

Study Director



### April 12, 2001 Hjdeaki Hiratsuka sealed Panapharm Laboratories Co., Ltd.



### -86-- - i -

### Study Schedule
1. 

### Initiation of Study : 2. Animal Receipt : 3. Initiation of Experiment : 4. Administration: 

### 5. Necropsy:
6. End of Experiment : 

## 7. Ena of Study :

Conduct of Study
1. Guideline**rrLrll'V~LKCrU.tU:** .LU.LUl



### January 24,2001 January 30, 2001 February 6, 2001 February 6, 2001 February 20, 2001 February 20, 2001 April12, 2001

OECD Guideline for Testing of Chemicals " Acute Oral Toxicity " ( 401, Adopted by the Council

on 24th February 1987)
2.GlP
OBCD Principles of Good Laboratory Practice (recommended by OBCD, 1997)

Unforeseeable Circumstances that may have Adverse Effects on the Reliability of the Study and Deviations
from the Protocol
This study was carried out in accordance with its protocol, and there were no unforeseeable
circumstances that might have adverse effects on the reliability of the study.

Kinds of Records and Location of Storage
1. 

### Protocol 

### 2. Records of animals 

### 3. Records of animal room condition
4. Records of test substance 

### 5. Records of study 

### 6. CD-R 

### 7. Reports

-87--

- ll

-.I:".I:"L .I:'J.NAL .K.J:;.I:"UK!': lUlUl



### 8. Other data concerning with 

### study All 

### records described above 

### will be retained for ten years after the submission of the final report (Aprill3, 2001

- Apri112, 2011) in archive of PPL. After ten years, transaction of records 

### will be
discussed with the sponsor.

Responsible Personnel 1. Study Director : Hideaki Hiratsuka
2. 

### Technician in 

### Charge : Kumiko Honda 

### 3. Pathologist : Masao Hamamura
4. 

### Veterinarian (Quarantine) : Emiko Kuwasaki 5. 

### Test Substance Management

: Hiroyuki Izumi
6. Preparation of Test Substance

7. Animal Receipt 8. Animal Husbandry

9. Grouping 10. Administration 11. Clinical Observation : Mikako Sukita

: Ryuji Yoshida

: Hideaki Hirll.tsuka, Eiji Murata, Yasuo Nakaoka, Ryuji Yoshida,

Hidehiro Ogata, Kumiko Honda, Kenji Ichimura, Takeshi Higuchi, Terohiko Enoshima

: Kumiko Honda

: Hideaki Hiratsuka

: Hideaki Hiratsuka, Eiji Murata, Yasuo Nakaoka, Ryuji Yoshida,

Hidehiro Ogata, Kumiko Honda, Kenji Ichimura, Takeshi Higuchi, Teruhiko Enoshima 12. Measurement of Body Weight

13. Necropsy : Hideaki Hiratsuka, Ryuji Yoshida, Hidehiro Ogata, Kumiko Honda, K.enji Ichimura

: Hideaki Hiratsuka 14. Pathological Examination : Masao Hamamura 15. Management of Computer System

: Ryoichi Tagami, Miho Iwanaga

-88-_

-iii-**J.J.L....l'.L.L"'ru....~J.Vl'\..1,** J..VJ.\!J.



### Signature Study Schedule Conduct of Study 

# Contents

e I. 0 0 0 ° 0 I I 0 I 0 I 0 I 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 0 I 

### Unforeseeable Circumstances that may have Adverse Effects Page



### ii
ii

on the Reliability of the Study and Deviations from the Protocol · · · · · · · · · · · · · · · · · · · · · · · · · 

### ii
Kinds of Records and Location of Storage Responsible Personnel

Summary 

### Objective
Materials and Methods
Results
Discussion ii 

### iii

1 

### 2
2


### 4
5



### Fig. 1 Body weight changes in 

### rats after a single oral administration of Imidoacrylate · · · · · · · 6

Table 1 Mortality and approximate lethal dose in rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · · · · 7 Table 2 Clinical signs in rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · 8 Table 3 Body weights in rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · 9 Table 4 Necropsy findings in rats after a single oral administration of Imidoacrylate · · · · · · · · · · 10

Appendix (Individual data)

-89-- -iv-L L J.., .L'.Ll. 'lru..,. J.'\o..lj.L VJ.\.1. J.VJ.VJ.



# Summary

lmidoacrylate was administered once orally to Crj: CD (SD) IGS rats at the dose levels of 1000 and 2000 mglkg. Five males and five females were used in each group. Obtained results are shown below.

No males and females died in any groups. Hypoactivity, salivation, loose stool, and lacrimation were observed on the day of administration, however, these signs disappeared by 6 hour after administration. No abnormal signs were observed up to the end of observation. Body weights in all animals increased normally. Necropsy revealed no abnormal findings in any animals. These results suggested that approximate lethal dose of Imidoacrylate was greater than 2000 mg!kg for male and female in the present study condition.

-90-- - l -PPL .t<'lNAL .t<.bPU.I:U: lUlUl



# Objective

Imidoacrylate was administered orally to rats, and its acute toxicity and approximate lethal dose were investigated.



# Materials and Methods

1. 

### Test substance and Vehicle Imidoacrylate (Lot No. NL-9301-02) supplied from Mitsubishi Chemical Safety Institute Ltd. was used for this study. This test substance was a yellowish solid, and its purity was 95.4%. The remaining test substance was returned to the sponsor from Panapharm Laboratories 

### Co., Ltd. for chemical analysis, and the stability of the test substance during the period of use in the study was confirmed (The analytical data were :filed in the raw 

### data). Corn oil (Nacalai Tesque, Inc., Lot No. VOR1096) was used as vehicle. The test substance was stored in a locker in 

### Test Substance Room at

room temperature shielded from light and the vehicle was stored in 

### a locker in 

### Test Substance Room at

room temperature.

2. Animal and Animal Husbandry Twenty-one male and 21 female Crj: CD (SD) IGS rats aged 5 weeks were purchased from Charles River Japan, Inc. The animals of both sexes were quarantined and acclimatized for 6 days. In this period, all animals were examined for clinical signs and weighed in order to confinn the health conditions. One day before administration, 10 male and 10 female rats were selected by the stratified sequential randomization method and used for this study at age of 6 weeks. Body weights on the day of administration were 161.5

- 172.1 g for males and 111.2

- 125.8 g for females. Individual body weight was within ± 

### 20% of the mean body weight. Animal room No. 03 in banier system A Area

was used for this study. The animal room was controlled at temperature 24 ± 

### 2°C (permissible range: 21-27°C), humidity 55 ± 

### 10% (permissible range: 35-75%), 12-hour light/dark cycle (lighting from 7:00A.M. to 7:00P.M.) and ventilation 13-15 changes/hour. The animals were housed in stainless steel cage (W260xH200xD380 mm) at one animal per cage. Actual ranges of temperature and humidity in the animal room during the study were 21

- 26°C and 47

- 54%, respectively. The animals were allowed to receive an autoclaved pellet diet (CRF-1, Oriental Yeast Co., Ltd.) and to drink well water supplemented sodium hypochlorite (approximately 2 ppm) ad libitum. 

### The diet containing impurities below the permissible limit, which was verified by Japan Food Research

-91-- - 2 -t't'L .l"lNAL Kbt'U!U: !U!Ul



### Laboratories (a juridical foundation) at the request of Oriental Yeast Co., 

### Ltd. Water was analyzed at K.akujo Nankyukaken Center 

### Co., Ltd., and it was verified that impurities were below the permissible limit. All housing equipments were sterilized by autoclaving before use. The animal cage and rack

were changed once at group assignment, and the sanitary tray was changed twice a week. The floor of the animal room was cleaned and wiped every day with a disinfectant-soaked mop.



### 3. Group Constitution, Reason for Determination of Dose Levels, and Group Assignment Group constitution was shown below.

Dose level Concentration Dose volume Number of Animal Group Sex (mgfkg) (w/v %) 

### (mlJkg) animal Number 

### cf' 5 101-105 Low-dose 

### 1000 5 20**~** 

### 5 201-205 

### r:J 5 106-110 High-dose 

### 2000 10 20**~** 

### 5 206-210
Because the toxicity of the test substance was thought to be weak, 2000 mg!kg, which is the maximum dose described 

### in OECD Guideline for Testing of Chemicals 

### 401, was set as the high dose. 

### In ad,dition, 1000 mgfkg was set according to a common ratio of 2. The number of animals per group was set at 

### 5 each for males and females. The animals were allocated to each group one-day before administration by the stratified sequential randomization method on the basis of their body weights. After group allocation, the animals were identified individually by tail number with oily felt pen and ear punch. Cage card listing the study number, animal number, dose level, and sex were attached to each cage. Finally, the remaining animals were excluded from the study.



### 4. Route and Method of Administration Oral route was chosen to evaluate the safety of the test substance when exposed by oral route. All animals were fasted for about 

### 17 hours, and the test substance was orally administered with a stomach tube. Dose volume was 20 ml)kg. Individual dose volume was calculated from the body weight on the day of administration. The animals were given diet 

### 3 hours after administration.



### 5. Method of Preparation of Test Substance Mixture On the day of administration, Imidoacrylate was suspended in corn 

### oil at the concentrations of 

### 5 and 

### 10 (w/v) 

### %.

-92-- - 3 -

### 6. Method and Frequency of Qinical Observation, Examination, and Measurement 1) Clinical Observation Observation period was for 14 days after administration. Animals were observed frequently for clinical signs and mortality until 6 hours after administration and twice daily (AM and PM) 

### up
to 13 days after administration. Animals were also observed once 

### in the morning on Day 14. 2) Body Weight Body weights were measured before the administration (Day 0), and Days 1, 3, 7 and 14. 3) Necropsy All animals were eutbanized by bloodletting under ether anesthesia and then subjected 

### to
necropsy.

7. Statistical Analysis Mean and standard deviation of body weights were calculated in each group.



# Results

1. Mortality Mortality and approximate lethal dose are shown in Table 

### 1. No males and females died in any groups. Therefore, approximate lethal dose was greater than 2000 mglkg for male and female.

2. Clinical Observation Results of the clinical observation are shown 

### in Table 2 and Appendix 1, 2. Hypoactivity, salivation, and loose stool were observed from 15 min after administration in males and females in the 

### 1000 mg!kg group. 

### In addition to these signs, lacrimation was observed in males and females in the 2000 mg!kg group. These signs disappeared by 6 hours after administration, and no abnormal signs were observed up to the end of observation.

3. Body Weight Body weight changes are shown in Fig.l, Table 3, and Appendix 3, 4. Body weights in all animals increased normally.

-93- - 4

-

### 4. Necropsy Necropsy findings are shown in Table 4. No abnonnal findings were observed in 

### any animals.



# Discussion

Imidoacrylate was administered orally to male and female rats 

### with the dose levels of 1000 and 2000 mglkg, and its acute toxicity and approximate lethal dose were investigated. No males and females died in the 1000 and 2000 mg!kg groups. Hypoactivity, salivation, loose stool, and lacrimation were observed on the day of administration. However, these signs disappeared by 6 hours after administration, and no abnormal signs were observed up to the end of observation. Body weights in all animals increased normally. Necropsy revealed no abnormal findings in any animals. These results suggested that approximate lethal dose of Imidoacrylate was greater than 2000 mglkg for male and female in the present study condition.

-94-

- :::> -0\I\0UlII,..--.,b.O'---'.....cbJJ·v;>-,-o00)350-,300250200150Male1000mg/kg--<>--2000mg/kg.--TIIIIIIIIIT--T--I.I012 3 456 7 8 91011121314Daysafteradministration250200.......c::a.o......150"'00co10050StudyNo.10101Female--o---1000mg/kg---<>--2000mg/kgIIIIIIII1IIIIIl0I2 3 456 7 8 91011121314DaysafteradministrationFig.lBodyweightchangesinratsafterasingleoraladministrationofImidoacrylate'\0-._]0\'ITable1SexMaleFemaleMortalityandaEEroximatelethaldoseinratsafterasingleoraladministrationofImidoacrllateGroupNumberTimeafteradministrationandofMinuteHourDaydoseanimals015304513 61234567"-'141000mg/kg5000000000000002000mg/kg5000000000000001000mglkg5000000000000002000mg/kg500000000000000StudyNo.10101TotalnumberApproximateoflethaldosedeadanimals(mglkg)00>200000>2000IXl\0'IIITable2Clinicalsignsinratsafterasingleoral_administrationofImidoacrvlateSexGroupMaleFemaleanddose1000mglk:g2000mg/kg1000mglk:g2000mg/kgClinicalsignNumberofanimalsNoabnormalityHypoactivitySalivationLacrimationLoosestoolNumberofanimalsNoabnormalityHypoactivitySalivationLacrimationLoosestoolNumberofanimalsNoabnormalityHypoactivitySalivationLacrimationLoosestoolNumberofanimalsNoabnormalityHypoactivitySalivationLacrimationLoosestoolrMinute0153045.5 5 5 55 3000255012 3000.o00005 5 5 554000055012 3002100005 5 5 554 300005012 2000000005 5 5 55200015 5024 400110000StudyNo.10101TimeafteradministrationHour1 3 65 5 50355002000000205 5 505 55003001000005 5 504550020000001055 505 5500100000000Day123 4565500005500005 5 55 5 5000000000000555 500000000555 5 55 55 5 50000000005 5 50000000000000005 5 5 5 5 55 5 5 5 5 50000000000000000000000005 5 5 5 5 55 5 5 5 5 500000000000000000000000050000550000550000Table3BodyweightsinratsafterasingleoraladministrationofImidoacrylateStudyNo.10101GroupBodyweight(g)ondaySexanddose0I3714Male1000mg/kgN55555MeanI69.0182.I206.6256.4326.8S.D.±4.3±4.9±4.6±1.2±1.52000mg/kgN55555Mean167.9179.8198.5245.0315.1S.D.±3.1±6.6±4.9±7.2±12.1Female1000mg/kgN5555 5Mean117.0127.1142.9163.1189.4S.D.±5.8±6.8±5.3±6.7±10.12000mg/kgN5. 5555Mean1I8.3128.1141.9161.5189.3S.D.±5.8±7.3±12.3±13.9±15.1I\()00Table4NecropsyfindingsinratsafterasingleoraladministrationofImidoacrylateOrgansandAllorgansandtissuesNR:Noremarkablechanges.I\0(\0(IISexGroupanddoseNumberofanimalsMale1000mg/kg2000mg/kg1000mg/kg555NRNRNRFemale2000mg/kg5NRStudyNo.10101.....:...........t'.t'L .t'.U"'AL lU:J:"U.K.J.: lUJ.Ul



### Acute Oral Toxicity Study 

### of Imidoacrylate 

### in Rats 

# Appendix (Individual data)

-100-rrL r.u'l.M.L .1.\..CrU.l\.J.:**~u~uJ.**



# Content

Page Appendix 1 Individual clinical signs in male rats after a single oral administration of lmidoacrylate · · · · · · · · · · · · · · · · · · · · · · · 1 Appendix 2 Individual clinical signs in female rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · 2 Appendix 3 Individual body weights in male rats after a single oral administration of lmidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · 3 Appendix 4 Individual body weights in female rats after a single oral administration of Imidoacrylate · · · · · · · · · · · · · · · · · · · · · · · · 4

-101--I......C)NIAppendix1Individualclinicalsignsinmaleratsafterasingle_QraJadministrationofImidoacrylateGroupAnimalTimeafteradministrationandNo.MinuteHourdose01530451361000mglkg101-aa,ba,ba d102-a,ba,b1031041052000mglkg106107108baaaa,b,ca,b,caa,ba,ba,ba,baaaaa,b,ca,b,ca,ba,baa109--aaa,bd110--aa,baAbbreviations:-,noabnormality;a,bypoactivity;b,salivation;c,lacrimation;d,loosestoolStudyNo.10101Day123456 7.........14I......No(.))IAppendix2Individualclinical signsinfemaleratsafterasingleor-aladministrationofImidoacrylateGroupAnimalTimeafteradministrationandNo.MinuteHourdose01530451 3 611000mg/kg2012000mg/kg202203204205206207208209210bbbabba,b,ca,baa,ba,baaa,ba,baaa,ba,baada,b,caa,baaaa,baa,ba,bAbbreviations:-,noabnormality;a,hypoactivity;b,salivation;c,lacrimation;d,loosestoolStudyNo.l0101Day234567"""'-'14Appendix3Groupanddose1000mg/kgZOOOmg/kgI1-'0""'IIndividualbodyweightsinmaleratsafterasingleoraladministrationofImidoacrylateStudyNo.lOIOlAnimalBodyweight(g)ondayNo.013714l01161.5174.0200.4245.8313.9102171.3186.5211.0264.0328.0103170.5182.8208.6259.7328.4104169.4181.9209.8259.7330.5I05I72.lI85.4203.I252.6333.0106I63.II73.7192.9238.7302.8107170.7182.7203.1249.5324..3108167.2171.7193.5236.9301.3109170.3184.4200.9246.0326.4110168.3186.4202.2254.0320.8Appendix4Groupanddose1000mg/kg2000mg/kgIf-'0UlIIndividualbodywefghlsinfemaleratsafterasingleoraladministrationofImidoacrylateStudyNo.10101AnimalBodyweight(g)ondayNo.013114201125.8138.2152.3173.1204.6202119.0129.0141.5164.5188.2203Ill.4I21.9140.6160.7187.5204116.3123.1140.9162.8190.4205I I2.5123.2139.3154.6116.2206l23.7137.0158.3177.7204.3207124.9134.8151.4174.1205.2208115.9121.8132.0146.217Z.O209115.8125.4137.2156.0187.3210111.2121.7130.6153.1177.7. :,•· 

# .LGa

FINAL REPORT

... - -::.::...-:':"-

.. - -=--- fl 

## ,, 

## Induction of micronuclei in the bone marrow of treated mice
Study Number: S277



### Sponsor**~** _, 

### .. 

## . .**-~.** -· -::-: 

### ·- ...**~** --
'II



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

-106-. 

### Test Facility
Toxicology Center LGCI, Life Sciences R&D 

### 1 04-1 Munji-dong Yuseong-gu Daejeon 305-380 

### KOREA

## lo study Number. 

### S277



### . Report



## CONTENTS
Page


### CONTENTS

................................................................................................... 1
STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT

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

## .4
QUALITY ASSURANCE STATEMENT

...................................................**~** 

### ...... 6
RESPONSIBLE PERSONNEL

....................................................................... 6
ARCHIVE STATEMENT

................................................................................ 6
1. SUMMARY

.............................................................................. ;

..................... 7
2. INTRODUCTION

........................................................................................... 8 2. 1 Study title

....................................................................................................... 8 2.2 Study Objective

.............................................................................................. 8

3. STUDY DATES

.............................................................................................. 8
4. TEST ARTICLE AND FORMULATION

............................................................ 8 4.1 Test article

........................................................................................ , ............ 8 4.2 Formulation

..................................................................................................... 8
5. CONTROL ARTICLES

................................................................................... 9 5.1 Negative control article

.................................................................................... 9 5.2 Positive control article

.................................................................................... 9
6. TEST SYSTEM

.............................................................................................. 9 6.1 Animals: supply, acclimatization and allocation

.............................................. 9 6.2 Animals: housing, diet and water supply

....................... :

.............................. 1 0
7. TREATMENT

............................................................................................... 10 7.1 Preliminary dose finding experiment

............................................................ 10 7.2 Justification of dose levels

............................................................................. 11 7.3 Dose groups and animal numbers of micronucleus test.

................................ 11 7.4 Route and duration of administration

........................................................... 11
8. OBSERVATIONS

........................................................................................ 11 8.1 Mortality

....................................................................................................... 11 8.2 Clinical signs

................................................................................................ 11 8.3 Body weight

.. · ............................................................................................... 12
9. NECROPSY AND SLIDE PREPARATION

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

### 12

### study Number: S277 

### Report



### 10. SCORING MI.CRONUCLEI

........................................................................... 12


### 11. ANALYSIS OF RESULTS

............................................................................ 12 

### 11 .1 Treatment of data

........................................................................................ 12 11 .2 Acceptance criteria

...................................................................................... 13 11.3 Evaluation criteria

........................................................................................ 13
12. RESULTS

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

### 13 12.1 Mortalities

.................................................................................................... 13 12.2 Clinical findings

............................................................................................ 13 12.3 Body weights

............ :

............................................................ :

..................... 13 12.4 Micronuclei findings

........................................**~..........................................** 

### 13

13. CONCLUSION

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

### 13

14. REFERENCES

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

### 14
15. PROTOCOL DEV.IATIONS

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

### 14.


### 16. TABLES

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

### 15


### 17. APPENDICES

............................................................................................. 20

### study 

### Number: 

# s2n Report



### LISt OFT 

# ABLES
Table 

### 1.

- - __ . - .**~** 

### _ :; Summary of MNPCE frequency and PCEI(PCE+NCE) ratio

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

### 15 Table 2. /

..**~ ~,.**  

### - = :- • - /: 

### Summary of group mean body weight changes

............... 16 

### Table 3. ·- _. ::· · -::. :Summary of Mortality

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

### 17 Table 4.**:-r--~·** 

# ···--:-:: ·**-~** 

# .: Summary of group mean body weight changes (Preliminary range f1nding)

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

### 18 Table 5. , .. _**=-~** 

# ·· - -:- = :

_ :.: Summary of Mortality (Preliminary range finding)

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

### 19

LIST OF APPENDICES Appendix 

### 1. . _ , __ ,,-- , _ ·: PCE, NCE, and MNPCE on each animal

.................. 20 Appendix 2

.. - __ ...... _ : .: Individual Body weights

............................................ 23 Appendix 3.**~-** 

### _. • · : Individual clinical signs

............................................... 24 Appendix 4.

_**~:":'~:.-_** 

### . _. : Individual Body weights (Preliminary range finding)

.. 25 Appendix 5

... 

# >.-:..: ::·:-:-

__ , _**~:** 

### Individual clinical signs (Preliminary range finding)

... 26 Appendix 6. Certificate of Analysis

.......................................................................... 27

### Study Number: S277



## STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT Report



### Induction of micronuclei in the bone marrow of treated mice
Study Number: S277

I, the undersigned,**hereb~** declare that the work described in this report was performed under 

### my supervision, as Study Director, and that the report provides a true and accurate record of the results obtained.
The study was performed in accordance with the agreed protocol, unless otherwise stated, and the study objectives were achieved. The study was also performed in accordance with LCCI Standard Operating Procedures and the principles of the following codes of Good Laboratory Practice:
Korea National Institute of Environmental Research Good Laboratory Practice Regulations for the Testing of Chemicals KNIER Notification No.1998-41, 23 December 1998 Organization for Economic Co-operation and Development Principles. on Good Laboratory Practices (as revised in 1997) Paris 1998 

# D'-**d~ P~.JL**  ).O 

### 0 

## I ' 

# f-l. t.u;:;y. ' '.L... 2..- l Du Hee Park, DVM, MSc. Study Director Toxicology Center LGCI/Llfe Sciences R&D

Confirmed by:

Sangk 

### n Lee, Ph.D. Management Toxicology Center LGCI/Life Sciences R&D Date 1;

Date •



### Pa.-110- 

# :n

### Study Number; Sf:!J Report



## QUALITY ASSURANCE STATEMENT Study Number: S277 Study**Title:~---** 

## ·... ;:Induction of micronuclei in the bone marrow of treated mice

The study described in this report was subject to audit by the independent Quality Assurance Unit as indicated below.
The findings of 

### each audit were reported to the Study Director and Management as prescribed by Standard Operating Procedures

.



### .. The report audit was designed to confirm that as far as can be reasonably establi-shed the methods described and results incorporated in the report accurately reflect the raw data produced during the study.

Audit Type

Study-based

Ha-Kang Kim Section of GLP Quality Assurance LGCI, Life Sciences R&D Audit Phase
Study protocol

In process

Raw data Study report Audit Date

27 Jun 2001

11 Jul 2001 13 Jul 2001 26 Jul2001

20 Aug 2001
20 Aug 2001

Page 5 of27

-111- 

### Report to Study Report to Director Management

28 Jun 2001 29 Jun ·2001

11 JLil 2001 12 Jul2001 13 Jul 2001 14 Jul2001 26 Jul 2001 30 Jul2001

22 Aug 2001 23 Aug 2001
22 Aug 2001 23 Aug 2001

Date

### Study Number: S277 

### Report



## ·RESPONSIBLE PERSONNEL

Induction of micronuclei in the bone marrow of treated mice

The following staffs were responsible for key elements of the study.

Director of Toxicology: SangKyun Lee, PhD Study Director: Du Hee Park, DVM MSc Quality Assurance Unit Ha-kang Kim, MSc

'The following staffs were responsible for 'key elements of the study:

Responsible Scientist:

Formulation: Yun Wha Jung, MSc Ok Youn Kim, BSc Eun-Kyeu Son, MSc



## ARCHIVE 

# STATEMENT ·

All primary data or authenticated copies thereof, specimens and the final report will be retained in the Archives and the Document Control Center (DCC) of LGCI/Life Sciences R&D for five years after submission of the final report. At this time the Sponsor will be contacted to determine whether data should be returned, retained or destroyed on their behalf... ,··

.**~** . . 

### Study Number: S277 

### Report



## 1. SUMMARY
The potential of

.**~** 1 to induce micronuclei was determined 

## in vivo in bone marrow cells of mice. For the micronucleus test, preliminary dose range finding experiment was done. In the preliminary experiment, 2000 mg/kg dosing resulted in all animal death and two consecutive 1000 mg/kg dosing was thought to be around LD 50 •

In the micronucleus experiment, test article groups of six male of ICR mice were dosed once daily on two consecutive days by intraperitoneal route with · ·-**:=::-:L~.** 

## ·**~ ~**  

## _ • at dose levels of 125, 250 and 500 mglkg/day.

Animals in the negative control groups were dosed with vehicle via the same route following the same treatment schedule as that in the test article groups. Animals in the positive control group were dosed once by intraperitoneal injection with Mitomycin C at 2 mg/kg.

.

Bone marrow samples were collected from animals in the test article and negative control groups at 24 hours after the second dosing, while they were collected from positive control animals at 24 hours after the first dosing.
'. In the micronucleus test, mortality case was not observed. After administration, hypoactivity, rough fur, ataxia, abdominal distension were·observed in the highest test article groups (500 mg/kg). Mean body weights of all groups were not decreased by the administrations.

Bone marrow smear slides from all groups were analyzed. The ratio of polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE) and the total number of micronucleated polychromatic erythrocytes (MNPCE) were counted for each animal.

All animals dosed with ;

.. :·_ ·-.:::- -**~** ;. - ' exhibited similar PCEJ(PCE+NCE) ratios and MNPCE frequencies to those of historical negative control animals. MNPCE numbers of negative control group fell. within historical range, but in the statistical analysis, it was different with test article groups. It was not considered meaningful value of the statistical difference 

### (p=D.02). It is concluded that ::**~';-,;>** 

### ;_· . - , did not induce micronuclei in the mouse bone marrow cells under the conditions employed for this test.



### P -113- '7

ag --.

### study Number: 

### S277 

### Report



### 2. INTRODUCTION
2.1 Study title

;· ·--"

---

. Induction of micronuclei in the bone marrow of treated mice


### 2.2 Study objective



## 3.

4. The objective of this study was to assess the ability of the test article, .'.**~** 

### =--
_ , to induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of treated mice. 

### STUDY DATES Study initiation: 26 June 2001 Experimental phase initiation: 28 June 2001
Animal arrival: 28 June and 6 July 2001
Treatment: 11 July and 12 July 2001
Slide preparation: 13 July 2001 Experimental phase complete: 07 August 2001 Study c9mpletion: 22 August. 2001



### TEST ARTICLE AND FORMULATION

The Sponsor provided the test article and a Certificate of Analysis (COA). The COA was added in this report (Appendix 6).


### 4.1 Test article

Test article identity: Chemical name: Supplied by: Lot number: Purity: Test article received and quantity: Storage conditions: Appearance: Stability of test article:

Safety precautions: Current expiry date:


### 4.2 Formulation - ' r not applicable
NE-1301-07 above 95% 15 June 2001, 50 g under 25'C White solid stable in air and water, unstable to light and heat This article can be polymerized by UV light, heat and oxidation Not provided Not provided

Preliminary solubility assessment was made. Test article was insoluble in water and corn oil and suspension status was not a satisfying one to injection. 5% Pluronic F-68 in saline was chosen as a solvent to improve suspension status. Formulations were prepared in solution with a solvent::·.:·.

·,(.**·~·** 

### study Number: 

### S277 

### Report

on the day of dosing. The formulations were handled at the room temperature during the dosing period. All treatment solutions were prepared on the day of administration and dosed within approximately 1 hour of the initial formulation of the test article. Details on treatment solutions are shown below:

Concentration of Dose levels of**~periment** treatment solution Administered

Dose range finding

Micronucleus

experiment



### 5. CONTROL ARTICLES

5.1 Negative control article (mg/ml) 100 200
12.5 25.0 50.0 (mg/kg) 1000 2000

125 250 500

Negative (5% Pluronic F-68 in saline) control treatments were administered the vehicle alone with the same dosing volume (1 0 ml!kg), once daily for two consecutive days.
5.2 Positive control article
Positive control treatment was administered the Mitomycin 

## c· (MMC, Sigma Chemical Co.). It· was freshly prepared in water for injection at concentration of 0.2 mg/ml and dosed via intraperitoneal route at a dose level of 2 mg/kg. MMC was administered once, 24 hours prior to necropsy. 

### 6. TEST SYSTEM

6.1 Animals: supply, acclimatization and allocation


### 6.1.1

6.1.2

6.1.3 Animals Species 

### I Strain: Age ordered: Number of animals ordered: Weight range ordered: Supplier: Justification of test system:

Acclimatization Duration: Husbandry conditions:
Allocation to treatment groups Allocation: Method: Mouse /ICR(SPF) 7 weeks. 20 males for preliminary toxicity study 40 males for the micronucleus study within an 20% of the overall mean Daehan Biolink Co. Ltd., Korea. The mouse was selected because it was a rodent species recommended for the micronucleus test by other various regulatory authorities and background data were available.

5 days prior to dosing to allow for acclimatization. Refer to Section 6.2.

One day prior to treatment. Random.



### Pa 

### -115- _27

,_ -

### Study Number: S277

6.1.4 Identification Numbering: Method: Cage labels: Unique for each animal within study. Tail marking method. 

### Report

Colored label displaying the dose level, study number, animal number
6.2 Animals: housing, diet and water supply
6.2.1 Environmental control Rodent facility: Air supply: Temperature: Relative humidity: Lighting:
6.2.2 Animal accommodation Animals per cage: Cage material and flooring:

6.2.3 Diet and water supply
Diet supply Diet name: Diet type:. Availability: Certification: ·'":

.. ·· Limited access (Room no. 1 PB71) Hepa-filtered, not recirculated Was maintained within the range of 19.47 to 23.22"C. Was maintained within the range of 57.34 to 69.88 

### %. 12 hours light : 12 hours dark.

Five or Six of the same sex, unless reduced by mortality or isolation. Polycarbonate cages {W260 x 0280 x H180 mm) with bedding.

Rodent feed (Purina, Korea) Pellet. Non-restricted. Before delivery, each batch .of diet was analyzed by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates were scrutinized and approved before any batch of diet was released for use. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Water supply Supply: Availability:
Certification:



## 7. TREATMENT Filtered and UV -treated tap water. Non-restricted via polycarbonate bottles with sipper tubes. The water was routinely analyzed for the specific contaminants at Daejeon Regional Institute Health and Environment. Certificates of analysis were received and archived.

7.1 Preliminary dose finding experiment
Test article was administered by intraperitoneal route for two consecutive: days with 6 males in each dose group. At 2000 mg/kg, all animals were dead in 30 minutes after test article administrations. At the dose level of 1000 mg/kg, three of six animals were dead in 3 hours after the second test article administration( day 2). Clinical signs of dead animals were severe in the both dose groups(see appendix 5). From the results of this experiment, the highest dose level of micronucleus study was determined as 500 mg/kg/day.

### Pag-n~=~-?7'


## . . ,.

.....

.;_. 

### study Number: S277 

### Report

7.2 Justification of dose levels Preliminary dose finding experiment was performed. Regarding the results of preliminary dose range finding study, high dose level of micronucleus test was determined.



### 7.3 Dose groups and animal numbers of micronucleus test After randomization, 6 mouse were assigned a cage and were grouped to 3 treatment, 1 vehicle control and 1 positive control groups. A color-coded card that had labeled study number, group assignment, and animal numbers was placed on the front of each cage. Control animals were received vehicle only. Individual animals doses was calculated using a individual body weight. A constant dose volume of 1 0 ml/kg body weight was used.

Dose 24 hour Sampling_iMale) Group Test article (mg/kg) Total Number of Number of treated animals Treated animals**N~ative** Vehicle 0 

### 6 M01**~** M06 Test Low (125) 6 M07- M12



## -

- Middle(250) 6 M13- M18 Article 

# -

. - High (500) 6 M19- M24 Positive MMC 2 

### 6 M25- M30



### 7.4 Route and duration of administration
7.4.1 

### 7.4.2 Route: Justification:



### 7.4.3 7.4.4 Administration: Frequency:



### 8. OBSERVATIONS

8.1 Mortality
Debilitated animals:
Premature sacrifice:

8.2 Clinical signs
Animals:

Deviations from normal:
recorded at the time in respect of · Additional observations: Intraperitoneal route The Intraperitoneal route was chosen for this study because it was recommended ·exposure route by the various authorities for micronucleus test.· Was administered by a syringe with 22

- 26G needle. Dosed once daily two consecutive days

Observed carefully, may be isolated to prevent cannibalism. Animals may be killed on humane grounds or 

### if considered 

## in extremis.

Individually observed after dosing hourly during the first 4 hours with special attention, and daily for a total of 3 days. Nature and severity.
Date and time of onset. Duration and progress of the observed condition. Changes in skin and fur, eyes and mucous membrane, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and

### study Number: 

### 8277

8.3 Body weight
Individual bodyweight recording: behavior pattem.

At the first dosing (Day 1 ). At the second dosing (Day 2). At necropsy (Day 

### 3).
The day of first dosing was designated as Day 

### 1.



## 9. NECROPSY AND SLIDE PREPARATION Report

Test article-and vehicle-treated mice were killed in groups, 24 hours after the second administration. MMC-treated mice were killed 24 hours after the single dose administration. Mice were killed by cervical dislocation in the same sequence used for the dosing. · Both femurs from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks, Using a syringe and needle, bone marrow was flushed from the marrow cavity with 0.5 ml fetal bovine serum into appropriately labeled centrifuge tubes (1 per animal). The tubes were centrifuged at 1250 x 'g' for 2-3 minutes; the serum was aspirated to leave 1 or 2 drops and the cell pellet. The pellet was mixe'd into this small volume of serum in each tube, and from each tube 1 drop(about 6 pl) of suspension was place on the end of each of 2 slides labeled with the appropriate study number, sampling time, date of preparation. A smear was made from the drop by drawing the end of a clean slide along the labeled slide. Two slides were prepared .each animal. Slides were allowed to**.~ir-dry** and then**fix~d** for 5 minutes in absolute methanol before being stained. After ·air-drying, slides were stained for 30 minutes in '5% Giemsa stain in Sorensen's buffer,· pH6.8. Stained slides were rinsed for 4- 5 seconds in 0.004% citric acid solution and then were rinsed 3 times in distilled water. After air-drying, randomized numeric numbers was labeled which serve as the code so analysis can be conducted "blind". Slide was examined under microscope 

### (x 1 000). ·



## 1 D. 

## SCORING MICRONUCLEI

The slides from all groups were arranged by randomized numeric number order. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or purple stained enucleate cells, and normochromatic erythrocytes (NCE), seen as pink-stained enucleate cells, was determined until a total of at least 500 cells (PCE plus NCE) has been analyzed and counting will continue until at least 1000 PCE per slide have been examined. All micronucleated polychromatic erythrocytes (MNPCE) observed during counting were recorded. Total2000 PCE(1000 per each slide) per animal was examined. 

## 11. ANALYSIS OF RESULTS

11.1 Treatment of 

### data
After completion of microscopic analysis and decoding of the data, the ratio of PCE/NCE for each animal and the mean for each group was calculated, and the frequency of MNPCE for each animal and the mean for each group will also be determined. The PCE/NCE ratio was examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity. The number of MNPCE in each treated group will then be compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p :5 0.05 was accepted as significant.



### P -118-:27



### ag_**~·**::':' 

### Study Number: S277 

### Report

11 ;2 Acceptance criteria
This micronucleus test was considered valid because the following criteria were met: 1) The frequencies of MNPCE in vehicle control groups fell within or close to the historical vehicle control range, and 2) The positive control chemical (MMC) induced a statistically significant increase in the frequency of MNPCE, and 3) At least 5 animals out of each group were available for analysis.
11.3 Evaluation criteria
The test article was considered as positive in this assay if: 1) The assay was valid (see acceptance criteria). 2) Statistically significant increase in the frequency of MNPCE at one or more concentrations, and 3) The frequency of MNPCE at such a point exceeded the historical control range. 

## 12. RESULTS

12.1 Mortalities (Table 3, Appendix 3) Any treat-related death was not observed in test article groups.
12.2 Clinical findings (Table 3, Appendix 3}
At the highest dose level (500 mg/kg}, animals showed hypoactivity, rough fur, ataxia, abdominal distension. Abdominal distension seemed to be caused by intestinal distension (enlarged and 

### full of food)
12.3 Body weights (Table 2, Appendix 2)
Mean body weights of all groups were not decreased by the administrations. In the highest dose, body weight was increased after one day after test article administration (day 2) due to abdominal distension.
12.4 Micronuclei findings {Table 1, Appendix 1)



### 13. MNPCE numbers of negative control group fell within historical range, but in the statistical analysis, it was different with test article groups (p=0.02). It was considered due to unexpected low frequency numbers of MNPCE in the negative control group that could often affect the statistical results. It was not considered meaningful value of the statistical difference, because MNPCE numbers of test article groups were all similar with historical negative control (saline). All animals dosed with .· -- · · - exhibited similar PCE/(PCE+NCE) ratios to those of negative control groups. MNPCE frequencies of all animals dosed with were also considered similar to those of historical negative control groups. The positive control group exhibited clear increases in the frequencies of MNPCE (p:;; 0.001 ).



### CONCLUSION
It is concluded· that did not exhibit clastogenic activity in the polychromatic erythrocytes in the bone marrow of mice when tested under the conditions employed for this study.

### study Number: S277 

### Report



### 14. REFERENCES

Wei L Y, Chao 

### J S and Hong C C (1997) Assessment of the ability of propoxur, methomyl and aldicarb, three carbamate insecticides to induce micronuclei in vitro in cultured chinese hamster ovary veils and in vivo in BALB/c mice. Environmental and Molecular Mutagenesis 29, 386-393.
Crebelli R, Carere A, Leopardi P, Conti L, Fassio F, Raiteri F, Barone D, Ciliutti P, Cinelli Sand Vericat 

### J A (1999) Evaluation of 1 0 aliphatic halogenated hydrocarbons in the mouse bone marrow micronudeus test. Mutagenesis 14, 207-215. Higashikumi N and Sutou S (1995) An optimal, generalized sampling time 30± 6 h after double dosing in the mouse peripheral blood micronucleus test. Mutagenesis 10, 313-319
Proudlock R 

### J, Statham 

### J and Howard W (1997) Evaluation of the rat bone marrow and peripheral blood micronucleus test using monocrotaline. Mutation Res 392, 243-249.
Holden H E, Majeske 

### J B and Studwell D (1997) A direct comparison of mouse and rat bone marrow and blood as target tissues in the micronucleus assays. Mutation Res 391, 87-89.
Chao 

### J S, Wei L Y •. Huang M C, Liang S C and Chen H H C (1999) Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo. Mutation Res 444, 167-174.
Asano N, Katsuma 

### Y, Tamura H, Higashikuni N and Hayashi M (1998) 

### An automated new technique for scoring the rodent micronucleus assay: computerized image analysis of acridine orange. supravitally stained**periph~ral** blood cells. Mutation Res 404 149-154.
Lovell D P, Anderson D, Albanese R, Amphlett G E, Clare G, Ferguson R, Richold M, Papworth D G, and Savage 

### J R K (1989) Statistical analysis of in vivo cytogenetic assays. In "Statistical evaluation of mutagenicity test data" Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part Ill. Ed D 

### J Kirkland. Cambridge University Press, pp 184-232. 

### 15. PROTOCOL DEVIATIONS

1. Acclimatization period was stated 6 days in the protocol, but in this study, acclimatization period was 5 days which was marginal period of regulations.



### -120-.**Pag~** 

### -- · 27

### study Number: S277 

### Report



## 16. Tables

Table 1.

- - · - ::. -:.: Summary of MNPCE 

### frequency and PCE/(PCE+NCE) 

### ratio

Group mean Group mean frequency of MNPCE Dose group PCE PCE+NCE (per 1000)

Vehicle 0.58 0.33* 10 ml/kg**-~** 

### ·- ----·



### . ,, . - 

# - ' ,. 0.66 0.83 125 mg!kg



# - '-

..**~** ,., . 0.60 1.08 250 mg/kg j 

### -.**~** ... 0.42 1.25 500 mg/kg Mitomycin C 0.33 81.5**

2 mg/kg 

### *: Statistical significance was observed (P=0.02). MNPCE numbers of negative control group fell within historical range, but in the statistical analysis, 

### it was different with test article groups. It was not considered meaningful value of the statistical difference.

**:Statistical significance was observed (P<0.01).



### P -12l-_r27**a~:,.** 

### --

### Study Number: S277 Report

Table 2.**"'~ ·~:**  

### Summary of group mean body weight changes 

### (unit: Q) Dose group**Gro~** 

### mean body weight Day 1 Day2 Day3 Vehicle 34.13 33.95 34.70 10 ml/kg

.' .. 

### 33.68 34.33 34.22 125 mg/kg

-

. . 

## - 34.13 33.88 34.17 250 mg/kQ



## -

. 

## -**·.-~** . '\-. 

### 34.37 36.10* 34.17 500 mg/kp Mitomycin C



# - 33.78 34.68

2 mg/kg 

# l j -: Not applicable 

### 1 "': Statistical significance was observed (P<0.05).



### \:**Pa;_ 22-~27**  

# jL..... :•·. 

### study Number: S277

Table 3.

.. : Summary of Mortality



### Dose group Day 1

Vehicle 0 

### 10 ml/kg

I



### .. 0 125 mg/kg

t

.. 

## .. 

# - 0 

### 250 mg/kg



### ..

.. 

### 0 

### 500 mg/kg

Mitomycin C



# -

2 mg/kg

Sa: Sacrifice for bone marrow sampling

- : Not applicable Mortality Day2

-123- Parre 1 / -;r'27 

### 0

0

0

0

0 

### Report

Day3

Sa

Sa

Sa

Sa

Sa

### Study Number: 

# s2n Report

Table 4

.. '**~:** 

### Summary of group mean body weight changes {Preliminary range finding)


### (unit:_g_l
Dose group Day 1 Day2

1 •••• ..._- ___ ... _ 

### . 

## - 32.7 33.1 1000 mg/kg



# . ' '· 

### .**~** . -

. ' 33.7 

# - 2000 mg/kg

-:Not applicable



### Pag-124<27!·+ 

# t study Number: 

### S277 

### Report

'\,' t

>: 

# f 6 ,, 

### Table 5.. ._;.:Summary of Mortallty{Preliminary range finding) 

### f;: 

# I**~:**

## ~· w

## ~:**~-**

# ~- €·**~'·** g, 

### F; m' 

# t '" (' 

### i:".**~1:** 1': '-



## fi: 

# f

- : Not applicable


## f.

# ~ li i.'· },t )j 

### Dose group

1000 mg/kg

- 

### 2000 mg/kg

-125- 

### Page

. ::_, "27 

### Number of dead animal/Total animal Day 1 Day2 Day3

0/6 3/6


# -

6/6 

# - -i**:~' ~·.** 

'·• 

### study Number: S277



## 17. Appendices

Appendix 1. 

### 1-

. •·'·

Dose group Animal Number M1 

### d'

M2 Vehicle 

### d'
10 ml/kg M3 

### d'

M4 

### d'

M5 

### d'

M6 

### d'

M7 

### d'

MB

: 

### d'

. '


## .,.., . ..


### 125 mg/kg M9 

### d'

M10



### d'

M11 

### d'

M12



### d' 

### Report



# -. ·; =:-= , PCE, NCE, and MNPCE on each animal 

### Slide PCE:NCE PCE PCE MNPCE Counted PCE+NCE Counted counted .per1000 

### A 230:270 0.46 1000 0 0 B 241 :259 0.48 1000 1 1 Total 471:529 0.47 2000 1 0.5



### A 290:219 0.57 1000 0 0 B 283:217 0.57 1000 0 0 Total 573:436 0.57 2000 0 0



### A 395: 105 0.79 1000 1 1 B 354:146 0.71 1000 0 

## o· Total 749:251 0.75 2000 1 0.5



### A 259:242 0.52 1000 0 0 B 275:226 0.55 1000 0 0 Total 534:468 0.53 2000 0 0



### A 302: 199 0.60 1000 0 0 B 309: 193 0.62 1000 0 0 Total 611 : 392 0.61 2000 0 0



### A 264:239 0.52 1000 0 0 B 264:236

.. 0.53 1000 2 2

.. 

### Total 528:475 '· > 

### 0.53 2000' 2 1



### A 302:202 0.60 1000 0 0 B 307: 193 0.61 1000 3 3 Total 609:395 0.61 2000 3 1.5



### A 294:206 0.59 1000 1 1 B 344: 156 0.69 1000 1 1 Total 638:362 0.64 2000 2 1



### A 326: 174 0.65 1000 0 0 B 270: 130 0.68 1000 0 0 Total 596:304 0.66 2000 0 0



### A 285:239 0.54 1000 0 0 B 301: 199 0.60 1000 0 0 Total 586:438 0.57 2000 0 0



### A 470: 139 0.77 1000 1 1 B 564: 173 0.77 1000 0 0 Total 1034: 312 0.77 2000 1 0.5



### A 358: 157 0.70 1000 2 2' B 360: 175 0.67 1000 2 2 

### Total 718:332 0.68 2000 4 2

Pa:_126-.tj27

# r

.. 1;,•

:.:

!': i.' 

### study Number: 

### S277 

### Report

Appendix 1. ,: PCE, NCE, and MNPCE on each animal each animal (continued)
Dose group Animal Slide Number

M13 

### A**~** 

### 8 Total M14 

### A**~** 

### 8



## ..**~** Total
250 mg/kg M15 

### A**~** 

### 8 Total M16 A**~** 

### 8 Total M17 

### A**~** 

### 8 Total M18 

### A**~** 

### 8 Total M19 

### A**~** 

### 8 Total M20 

### A**~** 

### B**~** '**~** Total
500 mg/kg M21 

### A**~** 

### 8 Total M22 

### A**~** 

### 8 Total M23 

### A**~** 

### B Total M24 

### A 6' 

### 8 Total PCE:NCE PCE Counted PCE+NCE 330:275 0.55 249:280 0.47 579:555 0.51 514: 302 0.63 464:291 0.61 978:593 0.62 281 : 223 0.56 362:272 0.57 643:495 0.57 457:229 0.67 339:214 0.61 796:443 0.64 364: 177 0.67 362: 174

. 0.68 726: 351 0.67 305: 198 0.61 309:229. 0.57 614·: 427 0.59 272:275 0.50 275:322 0.46 547:597 0.48 531 : 555 0.49 302:270 0.53 833:825 0.50 184: 375 0.33 208:431 0.33 392: 806 0.33 250:273 0.48 375:538 0.41 625: 811 0.44 316: 668 0.32 344:596 0.37 660 : 1264 0.34 380: 260 0.59 223:277 0.45 603: 537 0.53



### -127- 

### Page**~J-** 

### of27 PCE MNPCE Counted counted per1000

1000 0 0 1000 0 0 2000 0 0 1000 1

. 1 1000 3 3 2000 4 2 1000 0 0 1000 2 2 2000 2 1 1000 3 3 1000 0 0 2000 3 1.5 1000 2 2 1000 2 2 2000 4 2 1000 0 0 1000 0 0 2000 0 .. ., 0 1000 0 0 1000 2 2 2000 2 1 1000 2 

### 2 1000 1 1 2000 3 1.5 1000 0 0 1000 1 1 2000 1 0.5 1000 3 3 1000 1 1 2000 4 

### 2 1000 3 3 1000 0 0 2000 3 1.5 1000 2 

### 2 1000 0 0 2000 2 

### 1i:. :'i' i.,

,',.··**~·.** 

### Study Number: 

### S277

Appendix 1 

### continued)

Dose group Animal Slide Number

M25 

### A**~** 

### 8 Total M26 

### A 

## MMC**~** 

### 8 Total 2 mg/kg M27 

### A**~** 

### 8 Total M28 

### A**~** 

### 8 Total M29 

### A**~** 

### 8 Total M30 

### A



### 6' 

### 8 Total 

### Report

:: PCE, 

### NCE, and MNPCE 

### on each animal

PCE:NCE 

### EQ£ PCE MNPCE Counted PCE+NCE Counted counted per 1000 219:291 0.43 1000 94 94 198:305 0.39 1000 83 83 417: 596 0.41 2000 177 88.5 243:376 0.39 1000 93 93 138:364 0.27 1000 74 74 381 :740 0.34 2000 167 83.5 166: 334 0.33 1000 77 77 156:344 0.31 1000 75 75 322:678 0.32 2000 152 76 125: 385 0.25 1000 71 71 127:379 0.25 1000 76 76 252:764 0.25 2000 147 73.5 271 :330 0.45 1000 100 100 182: 360 0.34 1000 85 85 453:690 0.40 2000 185 92.5 148: 361 0.29 1000 78 78 135: 369 0.27 1000 72 72 283:730 0.28 2000· 150 75.

..... ·:·.··'

### Study Number: 

### S2n

Appendix2.



### Dose group

Vehicle
10 ml/kg

I



## - r

" 

### 125 mg/kg**~:. ~-~.** 



## ·. -·**~·** ,._


### 250 mg/kg

- ··-·



### 500 mg/kg

Mitomycin C

2 mg/kg 

## t

!:•



### - : Not applicable 

### : Individual Body weights



### Animal Number M1 M2 M3 M4 M5 M6 M7 MS M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 Day 1
32.7 32.7 33.4 35.0 35.7 35.3 32.6 32.4 35.2 32.8 34.7 34.4 33.6 33.3 33.0 35.0 33.5 36.4 33.5 33.8 33.9 34.5 34:3 36.2



# -

-

-

-

-

-

-129- 

### Pagc

-- (27 

### Report

1unlt: fl) Day2 Day3
32.7 33.8 31.9 32.1 33.2 34.1 34.2 34.4 35.5 36.3 36.2 37.5 32.8 32.6 33.0 33.4 34.6 33.6 34.7 34.6 35.2 35.5 35.7 35.6 33.3 33.7 33.6 33.9 33.5 33.7 34.3 34.7 32.6 33.3 36.0 35.7 35.2 32.6 34.9 35.0 36.1 33.1 34.8 34.0 38.0 33.0 37.6 37.3 31.5 32.6 33.6 34.3 33.4 33.7 34.4 34.5 33.9 35.7 35.9 37.3

# ,f

;,**~·.**
:

## ~:=

(**~···** o'**~** ·. :.

: ..

'l_'· 

### Study Number: 

### S277

Appendix 3. .-- --A: Individual clinical si_gns



### Dose group Animal Day 1 Number M1 N M2 N Vehicle M3 N 10 ml/kg M4 N M5 N M6 N M7 N M8 N



## -

. 

### -· - . -·. 

### M9 N 125 mg/kg M10 N M11 N M12 N M13 N M14 Hypoactivity

--- p ..... ---· 

### M15 N 250 mg/kg M16 N M17 Hypoactivity M18 N M19 Ataxia;**hyPoac~ivity** 

### M20 Ataxia, hypoactivity

--



# -- M21 N 500 mg/kg M22 Ataxia, hypoactivity M23 Hypoactivity M24 Ataxia, Hypoactivity M25 

# - M26



# - Mitomycin C M27 

# -

2 mg/kg M28



# - M29



# - M30 

# -

N: Normal A* : Common clinical signs (Abdominal distension, rough fur) -:Not applicable 

### Report

Day2

N N N N N N N N N N N N N N N N N N A*,.ataxiq, hypoactivity, dyspnea A* A* A*, hypoactivity A* A*, h_ypoactiv!!Y N N N N N N

### Study Number: 

### S277 

### Report

Appendix4. .: Individual 

### Body weights (Preliminary range finding} (unit: Q\ 

### Dose group Animal Day 1 Day2 Number

M13 32.7 33.3 M14 32.6 32.5

.. :..·-- : ._. .**~** 

### M15 33.9 33.8 1000 mg/kg M16 33.3 33.9 M3 32.6 33.4 M4 31.1 31.4 M9 32.1 

# - M10 32.5


# -

: .. . ; ... 

### M11 34.3 

# -. 2000 mg/kg M12 34.3


# - M2 34.1


# - M1 35.0


# -
-:Not applicablei. I.**:~ ~·**  

## ,, I' 

## c

.,, ,, f· i 

## t**~~·** 

### study Number: S277

Appendix 5.

Dose group

__ ..j.


### 1000 mg/kg

2000 mg/kg

N: Normal D: dead 

### Report

: Individual clinical signs (Preliminary range finding)

Animal Day 1 Day2 Number M9 N D M10 N 

### D M11 N Hypoactivity, tremor M12 N Hypoactivlty, tremor, dyspnea, ataxia M2 N Hypoactivity, ataxia, tremor M1 Hyt:mactivity 

### D M13 

### D



# - M14 

### D 

# - M15 

### D



# - M16 D



# - M3 

### D 

# - M4 

### D


# -

(showed severe clinical signs and dead 10 to 120 minutes after administration) (Major clinical signs of dead animals were dyspnea, convulsion, ataxia, and lateral position)
- : Not applicable

### Study Numper: 

### S277



### 18. Certificate of Analysis

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### 7 --133-

### I 

## o**~**



## . B001286



## Submitted to:



# ( R 

# e p 

# o· r 

# t 

# J

Chromosomal Aberration Study**of~. ''"~**  

## in Cultured Mammalian Cells

(Study No. : B001286)



## May 22, 2001



# GENERAL TESTING RESEARCH CENTER. JAPAN DILSTUFF INSPECTORS' CORPORATION

-134-. 

### ,-

### B001286 

# S t 

# a t 

# e m e n t

General Testing Research Center. Japan Oilstuff Inspectors' Corporation

Sponsor Title Chromosomal Aberration Study of • in 

## Cultured Mammalian Cells. Study No. B001286

The study for the report has been conducted in compliance with the following Good Laboratory Practice Standard.

rJapanese 

# GLP standards Applied to Industrial Chemicals (Kanpogyo No.39, Yakuhatsu No.229. 59 Kikyoku No. 85. 1984; revised in 1988 and 2000 )J



## Management Masakatsu Usami

Study Director Masato 

## Nakamura

-135-

## B001286 

# Quality Assurance Statement

General Testing Research Center, Japan Oils tuff Inspectors' Corporation

Sponsor Title Chromosomal Aberration Study of in Cultured Mammal·ian Cells .Study No. B001286



## .By the inspections and audits listed below, we hereby certify that the study

was carried out in accordance with its protocol and with our Standard Operating Procedures, and that the report faithfully describes the methods and procedures applied, and that the reported results reflect the raw data of the study accurately.
Inspection or audit
St.udy protocol Study procedure

Draft report Study report

Quality Assurance Unit Date of inspection or audit

March 21. 2001


## April 6, 2001



## May 18, 2001


## ·May 22, 2001



## Sadayoshi Ohtubo Date of reporting to the study director and to the management March 21. 2001


## Ap·r i 1 6, 

## 2001

May 18, 2001


## May 22, 2001



## Date: 

# M/l.Jf. ))., :zoo/ (!

## 1. Title



## 2. Purpose



## 3. Guideline

4. G 

## LP

5. Sponsor

6. Organization in Contractor B001286 

# Outline of the Study
:Chromosomal Aberr.a:t ion Stu.dy of

-- _ · in Gu 1 

## tured Mammal ian Cells. (Study No.: B001286)

:To assess the clastogenicity of the test substance by the

chromosomal aberration tests using cultured mammalian cells.

:Guidelines under the Japanese Industrial Safety and Health Law 

## (1997)

: GLP under the Japanese Industrial Safety and Health Law 

## (1984;

revised 

## 1988 and 2000)

' 1 

## (RespGnsible person)

:Mitsubishi Chemical Safety Institute Ltd. (1-30, 2-Chome, Shiba, Minato-ku, Tokyo)
7. Testing Facility:Gene-ral Testing Research Center, Japan Oilstuff Inspectors' Corporation (10-4. 1-Chome. Mikagetsukamachi. Higashinada-ku, Kobe-shi, Hyogo)

8. Study Contributors Management :Masakatsu Usami Responsibility person of data keepi.ng:Etsurou Fujiwara
Study Director 

## Date: 

# %.y l2 1 

# lw/
Masato Nakamura Other Contributors :Osamu Iwaihara. Sumiyo Mitsui

9. Study Date :(Initiation of the Study) March 21, 2001 (Initiation of the Test) March 23. 2001 (Submission of the Test) May 10, 2001 (Submission of the Final Report) May 22. 2001

10. 

## Data analysis :No statistical methods were used.


## 11. There was no unforeseeable circumstances that had affected test results undesirably.
12. Retention :All records will 

## be retained in the safekeeping facility of General Testing Research Center for 10 years after the submission of the final report. Furt-137-._:ention will be discussed with the sponsor. 4

### B001286 

# Contents Page Summary

........................................................................ 6

Materials and Methods

................................................... · 7 1. Test materials

...................................................... 7 2. Indicator cell line

................................................. 9 3. Culture media

.......................................................... 9 4. 89 

## mix

.................................................................. 9 5. Test method

.................. ,......................................... 10

Resu Its and Dis cuss ion ·················· ··· ··· ························ 16

References · · .. ·· · .. · ·· ··· ... · · · · .. · · · · · · · ·· · · · · · · .. · .. · · .............. ·· · ·.. 17

Tables

.................................................................. 18

Figures

.................................................................. 19

Photograph

.................................................................. 22

The attached sheet l(Structural formula)

........................ 23

The attached sheet 2 (Mitotic Index)

........................... 24

-138- 

## 5

## B001286 

# Summary

A 

## chromosomal aberration study of · in vitro 

## was made with an established cell line (CHL/IU) from the lungs of a female Chinese hamster. The pulse treatment test was made and the incidences of structural chromosomal aberration was 65.5% at 12.5 t.tg/ml without metabolic activation and· the incidences of structural chromosomal aberration was 10.5% at 150 fl. 

## g/ml and 31.5% at 200 {.l.g/ml with metabolic activation. The incidences of numerical aberration were less than 5% in all treatment groups both of without and with metabolic activation. From these findings, it has come to conclusion that potential in the CHL/IU ce 11 1 

## in e.

-139-;- 

### 6 

## -- " has a clas togenic

## B001286 

# Materials·and Methods
1. 

## Test materials 1. 

## 1 Substance to be tested

Name of the new chemical substance - - ,"J ··-. - - ...**~** 

# -. - - . - 

## - 

## (IUPAC nomenclature) Trade name Structural formula or rational formula (or outline of [Refer-to the sheet attached 1) manufacturing method, in case both are unknown) Purity of the new Lot No. of the new chemical substance 95.4% 

## chemical substance to NL-9301-02 tested [GPC area] be tested Name and concentration of impurities

CAS. No. Vapor pressure Molecular weight 249.29 Partition coefficient

'" 

## Melting point 61 "C 

## Appearance at ambient ligbtyellow· temperatures so 1 i d Boi 1 ing 

## point Stab i·l 

## i ty Solvent Solubilities Stability in Solvent Water insoluble So 1 ubi 1 i ties in DMSO soluble several solvents Acetone soluble at 500 mg/ml 1 > 

## stable 2 ) 

## others( ) 

## DMSO : dimethylsulfoxide Storage condition : kept at a room temperature, darkplace, s·hield the 1 ight
1) : 

## According to examination of vehicle. 2) : Heat of dissolution, discoloring and foam formation were not observed during preparation for dissolving the test substance to be tested.

-140- 

### 7

## B001286 1.2 Preparation of test 1) 

## Negative control mater1al .(solvent) Ace tone ( Lot No. KSH4520, Wa.ko Pure Chemica 1 Labor a tory Co. , Ltd. ) 2) Solvent selection and preparation for making solution of test substance Based on the information in which the test substance was not dissolved in
water and was dissolved in DMSO and acetone, the examination of vehicle was per'formed at sal ine(50 mg/ml), DMSO(lOOO mg/ml), l%CMC-:Na solution(50 mg/ml) and acetone(500 mg/ml). The test substance was insoluble in saline, l%CMCNa solution and DMSO. However, the test substance was dissolved in acetone. Therefore, acetone was used as solvent for the test substance. The test substance solution was diluted with acetone to the required concentrations. 1.3 Positive material 1) Positive controls Mi Urni,ycin C 

## CMMC, Lot No. KSM4520, purity 99. 8%, Wak6 Pure Chemical Laboratory Co., Ltd.) Benzo[a]pyrene CBP, Lot No. GGOL 

## purity 95. 6%, Tokyo Kasei Kogyo Go., Ltd.) 2) Positive control solutions MMC was dissolved in saline (Otsuka Pharmaceutical Factory, Inc.) to a concentration of 

## 1.5 ,ug/ml before use. BP was dissolved in DMSO (Dojin Chemical Laboratory Co., Ltd.) to 4 mg/ml, frozen, and thawed at the room temperature before use.

-141- --.1- 

### 8

## B001286 2. Indicator cell line The CHL/IU cell line, originally derived from the lung of 

## a female Chinese hamster, was used. The cells were purchased f.rom Dainippon Pharmaceutical Co., Ltd. A cell suspension was mixed with one tenth volume of DMSO, and then frozen in 1 ml and stored in liquid nitrogen. The suspended cells were used

. 

### . 

## after thawing and cultivation through up to 

## 5 passages. The cel'ls were cultured in plastic dishes (Becton Dickinson and Company; diameter of 6 em 

## or 10 em) in an automated C02 incubator (Tabai Espec Corp. model BNA-111A, 121DA; C02 5%, temperature 37°C, humid condition). The final doubling time of the cell used for the test was about 16.7 hours.

3. Culture media 3.1 Eagle's minimum essential medium Eagle's minimum essential medium CMEM; 

## Eagle's MEM 

## "Nissui(i)", Nissui Pharmaceutical Co., Ltd·.) of 8. 3g ·was dissolved i·n 880 ml.of distilled water and autoclaved at 121 

# oc for 15 min. The solution was supplemented with'· 8. 8ml of sterilized 2. 92% L-glutamine solution and 11.2 ml of sterilized 10% sodium bicarbonate solution.· 3.2 Growth medium Nine hundred ml of the above 

# MEM solution was supplemented with 100 ml of heat-inactivated 

## (56 °C, 30 min.) calf serum (GIBCO 

## BRL; Lot No. 296130).



## 4. S9 mix 

## 4. 1 89 89 from Kikkoman Corporation was a supernatant fraction of liver homogenate prepared from rats which were ,given i.p. injection of phenobarbital at 30, 60, 60 and 60 mg/kg every day and 5, 6-benzoflavone at 80 mg/kg at the third PB injection. The S9 was stored below -BO"C.

-142-_ 

### 9

## Source of 89 lot No. : RAA-440 

## Prepared on : February 16. 2001. 

## Using animal Species, Strain : Sprague-Dawley Rat Sex, Age (in weeks) : Male. 7 weeks old Weight : 211

- 245 

## g Final concentration of the S9 was 5%. protein of 89 was 1. 341 

## mg/ml. 4.2 

## S9 mix B001286



## The following table shows the composition of 1 ml of 89 mix. Each of 89 mix

was prepared freshly for each assay and**wa~** 

## stored in the ice. S9 MgCl2 C6H2D) KCl D-glQcose-6-phosphate /3 

## -NADP+ HEPES buffer (pH 7.2) 

## Sterilized distilled water

5. Test Method 5.1 

## Pulse treatment test 1) Cell growth inhibition test (1) 

## Test substance concentration 0.3 ml 5 11- 

## mo 1 33 11- 

## mo 1

5 11- 

## mo 1 .4 11- 

## mo 1 4 11- 

## mo 1 remainder

The highest concentration was set at 5000 

## ,ug/ml. Then, following concentrations were graded at a common ratio of 4. (2) 

## Treatment of cells In a 6 em plastic plate, 5 ml of cell suspension (4X10 8 

## /ml) were incubated for 3 days. After removal of the cui ture medium from each plate, the following table shows the composition,· treatment of cells was performed for 6 hours and the tests were made using two plates for each concentration. After 6 hr. incubation, the cells were washed three times with MEM. and incubated in 5 ml of a fresh growth medium for a further 18 hr.

-143-_ 

## 1 0

## B001286



## In the negative control group, cells were treated with vehicle.

test substance solutions S9 mix the growth

and negative control medium without metabolic 0.03 

## ml 3. 0 

## ml



# - activation with metabo 1 i c 0.03 

## ml 0. 5 

## ml 2. 5 

## ml activation

(3) 

## Measurement of surviving cells Cells were washed with Dulbecco's phosphate**buff~red** 

## saline·CPBS(-); Dulbecco' s PBS uNissui", Nissui Pharmaceutical Co., Ltd.), fixed with methanol for 15 

## min., stained with 0.1% 

## crystal violet solution for 10 

## min,, and washed with water and dried. Cell density was measured with a cell density
counter (Monocellator, Olympus Co., qd.). (4) 

## Measurement of 50% 

## inhibition concentration of cell growth The ratio of surviving cells in treatment groups were calculated in comparison with negative control. Then, a survival curve was drewed, and 50% 

## inhibition concentration of cell growth (IC5o) was calculated. 2) Chromosomal aberration test (1) 

## Test substance concentration Fifty percent inhibition concentration of cell growth was calculated to be a 13.9 

## pg/ml without metabolic activation and 136 

## ,ug/ml with metabolic
act iva t i on. Without metabolic activation concentration negative 0. 08 0.3 1. 

## 25 5 2"0 78 313 1250 5000 

## (,ug/ml) control cell growth 100 

## 96 85 77 

## 73 38 0 0 0 

## 0

-144-_ 

### 1 1

## B001286 With metabolic activation concentration negative 0.08 0

.. 3 1. 25 5 20 78 313 1250 5000**(~/ml)** 

## control cell growth 100 95 88 87 93 88 81

. 12 0 0

The concentration was set at 50, 25, 12.5, 6.3 and 3.1 f.lg/ml without metabolic activation and 300, 200, 150, 100, 50 and 25 f.lg/ml with metabolic activation on the result of cell growth inhibition tests. In positive controls, final concentrations. of MMC and BP were set at 0.15 and 20 ,ug/ml. respectively, which are the concentration levels as· known to induce chromosomal aberration. (2) 

## Treatment of cells The cultured cells were treated with the test substance using the same procedure as described above. For positive controls, the cells were treffted with a mixture of 2. 7 ml of the growth medium an.d 0.3 ml MMC solution in the treatment without metabolic activation and treated with a mixture of 2. 5 ml of the growth medium and 0.5 ml of 89 mix and 15 f..ll 

## BP solution in 

## the treatment with metabolic activation. (3) 

## Preparation of specimens and measurement of surviving cells Cell density was measured in the preparation of specimens. The cultured cells were treated with the test substance using the same procedure as described above. Before 2 hours from the end of the treatment, colcemid was added to the medium in each plate to a final concentration of 0.1/.lg/ml to arrest- cells in metaphase during preparing specimen. The cells were then washed with 

## PBS(-), dissociated with 0.25% trypsin solution, and centrifuged (1000 rpm, 5 min.; same below) for collecting them.

-145-- 

## 1 2

### B001286 

## After removal of the supernatant, 4 ml of 0. 075 M potassium chloride solution was added for hypotonic treatment (37 QC, 

## 15 min.). The cells were then fixed in 0. 5 ml of cold methanol/acetic acid mixture (3/1. v/v). After their centrifugation, the supernatant was removed and 4 ml of a fresh fixative added. The fixation procedure was repeated 3 to 4 times. Thereafter. the cells were susp.ended in a small amount of the fixative, and drops of the cell suspension were placed in two positions on a slide glass and dried. The ce·lls were stained for 20 min. with 3% Giemsa' s solution and then diluted with 1/15 M phosphate buffer (pH 6.8). The slides were then washed with water, dried, and coated with a mounting medium. Three slides were prepared for each plate. (4) 

## Observation Q) 

## PreliminarY. observation Each specimen was observed first to confirm that it has an adequate (50 cells for one plate) number of mitotic**·cells~** 

## F-or po·sitive. and nega:tive controls, the incidence of structural aberrations was confirmed to be appropriate. 

# aD Structural aberration and numerical aberration Each specimen was coded, 100 metaphase cells for each plate (i.e. 200 cells for each concentration) were observed in a blind manner. Metaphase cells of which chromQsomes are spread well were selected and observed for structural aberrations. Cells without aberration but not having 25+2 chromosomes were omitted from the survey. Structural aberrations were classified as follows 1 l 

# J

chromatid breaks (ctb) chromatid exchanges (cte) 

# 1

chromosome breaks (csb) chromosome exchanges Ccse, other aberrations (frg) dicentric, ring, etc.)

-146-;_ 

## 1 3

## B001286 A**~gap"** 

## was distinguished from a**~break"** 

## as follows. The**~gap"** 

## is an achromatic region in a single chromatid which is narrower than the width of the chromatid. Gaps are not included to the chromosomal aberration. The recorded gaps were distinguished from that of the other structural aberration. Numerical aberration was observed and polyploid cells, which include endoreduplicated cells, were scored. @ 

## Mitotic index One thousand cells from each plate (i.**~** 

## 2000 cells for Bach concentration)

were observed to calculate mitotic index. (5)J 

## udgemen t A cell with at least one structural chromosomal aberration was classified as aberrant cell.. The number pf

.. aberra.n.t cells was counted in different kinds of
two ways, one includes cells with no aberration other than**ga~s** 

## (tgap), and another excludes such above cells (-gap). The chromosomal aberrati6ri**potenti~l** 

## of the test substance was judged as follows. When both of incidence of -gap aberrant cells and that of numerical aberrations are less than.5%. the substance is negative (-). When either of incidences is 5% or more and less**t~an** 

## 10%. the test substance is inconclusive (+). 

## When either of incidences is 10% or more, the test substance is positive (+). 

## When the chromosomal aberration test is inconclusive or positive, the reproducibility of these results or dose-dependent confirms. Treatment concentration of confirmation examination is decided according to the incidence of the chromosome aberration. According to case, examine by the equal ratio. It is 

## judged positive in the case where reproducibility is admitted.

-147-



# ·- 1 4

### B001286 

## 5.2 Taking data Each of the sums and the incidence (%) 

## of the**chro~osome** 

## aberration and numerical aberrations was shown. The chromosomal aberration was shown dividing the number of cells in the type. Each of Cell growth index in cerl growth inhibition tests and chromosome aberration .tests were shown.

When the test wa·s positive, the "D2o" 

## values were calculated from the·test results. The**"D~o"** 

## value is the concentration (mg/ml) required to induce any aberration in**20~** 

## of metaphases. The microscopic photograph of a typical chromosomal aberration is appended.

-148-_

## B001286 

# Results and Discussion

Jn the cell growth inhibition tests of pulse treatment test, fifty percent inhibition concentration of cell growth was calculated to be 13.9**~/ml** 

## without metabolic activation and 136**~/ml** 

## with metabolic activation. On the basis of the cell growth inhibition tests, the concentration was set
at 50, 25, 12. 5, 6. 3 and 3.1 ,Llg/ml without metabolic activation and ·300, 200, 150. 100, 50 and 25 ,Llg/ml with metabolic activation(Figure 

## 1. 2). In the cell growth inhibition test of pulse treatment more than 1250 ,Ltg/ml, precipitated test·substance was observed in the medium before incubation, but

was not observed after incubation. In the chromosomal aberration test of pulse treatment test, the incidences of structural chromosomal.aberration was 65.5% at 12.5 11 

## g/ml without metabolic activation and the incidences of structural chromosomal aberration

was 10.5% at 150 11 

## g/ml and 31.5% at 200 11 

## g/ml with metabolic activation. The incidences of numerical aberration were less than 5% in all treatment
groups both of without and with metabolic activation. The incidences of structural aberrations in the positive control groups were significantly higher than ones in negative control groups (Table 1 and Figure 3, 4, 5 and 

## 6).

D2o 

## value was calculated from the treatment group with the increase in structural chromosomal aberration cell. and was 0. 005 mg/ml without metabolic activation and 0.17 mg/ml with metabolic activation. Microscopic photographs of normal cell for the negative control and structural cell in w"ithout metabolic activation are shown in photo. 1, 2.

No statistical method was used for the analysis of data. From these findings, it has come to conclusion that potential in this CHL/IU cell 

## line system.

-149-_ 

### 1 f)**~-** 

## have clastogenic

## B001286 

# Reference

1) Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan : Atlas of chromosomal aberrations induced by chemicals. Asakura Press. Tokyo, 1988.

-150--

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### 8001286 

# Pulse treatment test
100 

# • Cell growth inhibition test



## . o- Chromosomal aberration test

,-..... 

## ?!2-

.._....

P< CD "'0 

# = •1"'"1 

## " ..t:: 

## 50 '\ 

# ..., 13: '\ 0 

## D**~** t:lLJ 

### ' r--1 

## ' r--1 

### ' CD 

### u 

## ' ' ' 

# b

.. 

# .. ..
.. .. 0 0 

## 25 50 

## 75 100 

## Concentration ( Jl 

## g/ml) Fig. 1 Cell· growth inhbition treated with without metabolic activation

100 

# • Cell growth inhibition test



## - o- Chromosomal aberration test

----. 

## ?!2- ... 

# "

........... 

# "

><: CD "'0 

# = •.-I 

### ..c: 

## 50


# ..., 13:: '\ 0 /-; '\ bD \. r--1 '\ r--1 CD 

# o

.. u



# .. 

# .. .. .. 

# ..
0 0 100 200 300 400 500 Concentration ( J.L 

## g/ml) Fig.2 Cell growth inhibition treated with with metabolic activation
-152-_**1~**

### 100.0 

# I
80. 0

,.-..., '?fi. 

## 60. 0 '-' (].) 0 .t:: (].) "'CC •r-1 0 

## 40.0 .t:: H



## 20. 0

0.0 

## 0

10.0

8.0

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## cf2- 6. 0 "-"

(].) 0 .t:: (].) "'CC 

### 4. 0 •r-1 

### o· .t:: H


### 2. 0



## 0.0 0 B001286 

# Pulse treatment test

_.,_Without metabolic act1 vat ion 

# I

) 

# 7 /_ _/

-



# - 3. 1 

## 6. 3 12. 5

Concentratin ( JJ. 

### g/ml) F.ig. 3 Incidence of structural aberrent cell treated with



# I**~Without** 

### metabolic activation 

# I



# ..

# ~ - 3. 1 6. 3 

### Concentration ( J.l. 

### g/ml)
Fig.4 Incidence of numerical aberrant cell treated 

## with

_

-153-. 

## 12. 5

## 40.0

30.0

,-._ ?Je. 

### ......, Cll c.J 

## § 

## 20.0 ""0 •.-I c.J 

### c::**~**

,-._**~** 0

........ Cll c.J 

### c:: Cll '1j •.-I c.J 

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### 10.0

0. 0



## 10.0

8. 0



### 6. 0

4.0



## 2. 0

0. 0 

# I

0



# I

0 B001286 

# Pulse treatment test



# - -o- With metabolic activation 

# I

I I 

## , I 

# ,
I 

## ,
I 

# ,
I 

## n 

## ,.



# ..


# ..
.. ,. 

# ..

- 0

- 

# - 

# - - -0 0 

# - - 

# - -


# - 

# -
- - - 25 50 100 

## 150 

## 200 Concentratin ( /.1 

### g/ml) Fig. 

## ·s Incidence of structural aberrent cell treated with



# - o- With metabolic activation 

# I

,.0-



# - - .. 

# -

- 0- 

# .. 

# - .. ..

.. 25 50 1 DO 

## 150 Concentration ( /.1 

### g/ml)
Fig. 6 Incidence of numerical aberrant cell treated with ·

-154-- 

# - 

# - - 200

## Photo. 1 Normal cell for the negative control (Without metabolic activation)

Photo. 2 Structural aberration cell for the treated group

(Without metabolic activation: 12.5 ,ug/ml)

-155-.
? ? 

## B001286



# ·r·



# t I, 1! 

# j**'J**

. 1 i

1 

# i ":.:**~** 

# 11 

# I

.

.

. ' 

# I

2:

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## Stractural fomula of 

## To-1534


# CD Main chemical substance 

## (Lot. No. NL-9301-02)



# ® Impurity 1



# ® Impurity 

## 2

/

/



# @ Impurity 3

\ 

# '

-156-- 

## Purity 

## CD 95.4% 

## B001286

# ~

(not separate 

## GD and 

## QD)

Purity
l. 

### 6%(®+@)



## Purity

@3.0%/- ()



## B001286 (The attached sheet 2) Mitotic Index (1) 

## Pulse treatment tests Without metabolic activation With metabolic activation ( 6-18hr.) ( 6-18hr.) Treatment Number Mitotic Treatment Number Mitotic Concentration of cells index Concentration of cells index

(,Lt&/ml) (%) 

## (.u&/ml) (%) Negative Negative control 2000 9. 3 control 2000 

## 14.3 (Acetone) (Acetone) 

# I

3. 1 2000 9. 8 25 2000 15. 7

.. 

## 6.3 2000 8.2 50 2000 

## 15.2 12.5 2000 1.6 

## 100 2000 8. 7 

## I 25 

## r TOX J 

## r TOX J 

## 150 2000 8.4 50 

## r TOX J 

## r TOX J 

## 200 2000 

## 1.2 300 

## r TOX J 

## r TOX J 

## Positive 2000 

## 5.4 con t ro-1 (MMC) 

## Positive. 2000 6. 0 con t ro 1 

## (BP) MMC : Mitomycin C, BP : Benzo (a) pyrene 

## r TOX J : 

## Metahase cell had not obtained more than 50 cells

-157-. 

### 2 4