TSCA NON-CONFIDENTIAL BUSINESS INFORMATION

DOCUMENT DESCRIPTION DOCUMENT CONTROL NUMBER DATE RECEIVED

COMMENTS:DE L S

DOES NOT CONTAIN CBI
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460

OFFICE OF

~PRO' CHEMICAL SAFETY AND

POLLUTION PREVENTION

Notice of Change in CBI Chemical Identity Status

Pre-Manufacture Notice (PMN) P-03-0645

Notice of commencement (NOC) received: 216/2004

Please be advised that the Confidential Business Information (CBI) claim for the

above referenced substance was dropped by the information submitter in the

NOC.

The identified non-confidential chemical name for this substance as provided in

the NOC is:

1 ,3-BENZENEDIMETHANAMINE, POLYMERS WITH EPICHLOROHYDRINPOLYETHLENE GLYCOL REACTION PRODUCTS*

The current TSCA Inventory name is

1 ,3-Benzenedimethanamine, polymers with epichlorohydrin-polyethylene glycol

reaction products

CASRN: 652968-34-8

This notice as submitted to the NCIC files on 9/1/201 1

Name of staffer: Jeffrey Santacroce

Position: Asst. NOC Coordinator

Branch: RDMB/IMD/OPPT/OCSPP

CONTAINS NO OI
PMN Page 1 ; Page 1 of 101

____________________________________________________ Form Approved. O.M.B. No. 2070-0012. Approval Exp s 10-31-96.

U.S. ENVIRONMENTAL PROTECTION AGENCV, - -~ GNY S NY_

PREMANIJFACTURE

NOTICE HOJ M, 1 8 AIN 101: 56

IL 111 ~ ~ II I I ii ompanY Sanitized

in the Premanufacture Notice

* Before you complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from the Toxic

Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603).

* If a user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Rememher,

your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, Washington Financial Management Center (3303), P 0

360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee.

Part I - GENERAL INFORMATION TEST DATA AND OTHER DATA

You must provide the currently correct Chemical Abstracts (CA) Name of the new You are required to submit all test data in your possession or control and t

chemical substance, even if you claim she identity as confidential. You may authorize provide a description of all other data known to or reasonably ascertainable by

another personi to submit chemical idensity information for you, but your submission will you, if these data are related to the health and envirornental effects on the

sot he complete and the review will not begin until EPA receives this information. A letter mauctrpoesndtibininomresoripslofhe w

in support of your submuission should reference your TS user fee identification number. mnfcue rcsig itiuini omre so ipslo h e

You must submit an original and two copies of this notice including all test data. If you ceia usac.Sadr ieauecttosmyh umte o ai h

claimed any information as confidential, a single sanitized copy must also be sbmittedl. open scientific literature. Complete test data (written in English). not surnaics

of data, must be submitted if they do not avnear in the open literature. Youshould

clearly identify whether test data is on the substance or on an analog. Als ,the

Pars 11 - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE chemical composition of the tested material should he characterized. Foilcwing

are examples of test data and other data. Data should be submitted according to

if there are several manufacture, processing, or use operations to be described in Pant 11, the requirements of §720.50 of the Premainufacture Notification Rule (40 GFR

sections A and B of tbis notice, repreduce the sections as needed. Part 720).

Part Ill - LIST OF ATI'ACITMENTS Test Dats (Check Below any included in this notice)

Attach additional sheets if there is not enough space so answer a question fully. Label each *Environmental fate data [) Yes *Other data []

continuation sheet with she corresponding section beading. in Pant ill, list these

attachments, any test data or other data and any optional information included in the notice. * Health effects data [] Yes Risk assessments

* Environmental effects data 5 Yes Structure/activity rela oshipsf

OPTIONAL INFORMATION Physical/Chemical Properties* ~ Yes Test data not in the po ssion

or control of the submi ter []

You may include any information that you want EPA so consider in evaluating the new

substance. On page I I of this, form, space has been provided for you so describe 'A physical and chemical propeities worksheet is located on the last page of thi fonrs.

pollution prevention and recycling information you may have regarding the new substance.

TYPE OF NOTICE (Check Only One)

So-called "binding" boxes are included throughout this form for you to indicate your

willingness to he bound so certain statements you make in this section, such as use, PMN (Premanufacture Notice)

production volume, protective equipment .. This option is intended to reduce delays that lJ

routinely accompany she development of consent orders or Significant New Use Rules.

Except in the case of exemption applications (such as TMEA, LVE, LOREX) where [J INTERMEDIATE PMN (submitted in sequence with final product

certain information provided in such notification is binding on the submitter when the

Agency approves the exemption application, checking a binding box in this notice does not SNUN (Significant New Use Notice)

by itself prohibit the submitter from later deviating from the information (except chemical E

idetiy) eprte i th frm ThIEA (Test Marketing Exemption Application)

CONFIDENTIALITY CLAIMS [] LYE (Low Volume Exemption) @ 40 CFR 723.50(c))r

You may claim any information in this notice as confidential. To assert a claim on the=

form, markc (X) the confidential box nest to the information that you claim as confidential. Li LOREX (Low Release/Lo Exposure Exemption) @ 40 CFR 7 c)22 "I To assert a claim in an attachment, circle or bracket the information you claim as E lI V)>C:

confidential. If you claim information in the notices as confidential. you mus also nrvde LE oiiainLRXMdfcto

asnitized version of she notice. ( including attachments). For additional instructions o LEMdicaon[] LRX oifCato

claiming information as confidential, rmad the Instructions Manual. ISTI ICNOIAEDPN e

[I Mark (x) if any information in this notice is claimed as confidential. # of chemicals or polymers 1

(Prenotice Communication #I required, enter # on page 3)___

EPA FORM 7710-2 Rv -5 Replaces previous editions of EPA Form, 710-25. -25 (Rev S-5
Page 2 of 101

Public reporting burden for this collection of information is estimated to average 1 10 hours per response, including time forrviwg

instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the coleion o

information. Send comments regarding the burden estimate or any other aspect of this collection of information, including suggestions Or

reducing this burden, to Director, Collection Strategies Division (2822), U.S. Environmental Protection Agency, 1.200 Pennsylvania A e., N.W.,

Washington, D.C. 20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20 03.

CERTIFICATION - A Printed copy of this signature page, with original signature, must be submittedI certify that to the best of my knowledge and beliefI. The company named in Part 1, section A, subsection Ila of this notice form intends to manufacture or import for a

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

Section B.

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

3. 1 am submitting with this notice all test data in my possession or control and a description of all other data known to or

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

Additional Certification Statements:

If you are submitting a PMN, Intermediate PM4N, Consolidated PMN, or SNUTN, check the following user fee certiflcati )n

statement that applies:

SThe Company named in Part 1, Section A has remitted the fee of $2500 specified in 40 CFR 700.45(b), or

El The Company named in Part I, Section A has remitted the fee of $ 1000 for an Intermediate PMN (defined @ 40 CF

700.43) in accordance with 40 CER 700.45(b), or

Dl The Company named in Part I Section A is a small business concemn under 40 CFR 700.43 and has remitted a fee o~ $ 100

in accordance with 40 CFR 700.45(b).

If you are submritting a low volume exemption (LVE) application in accordance with 40 CER 723.50(c)(1) or a Low re ease

and low exposure exemption (LoRex) application in accordance with 40 CER 723 .50(c)(2), check the following certi ~ation

statements:

F The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commerc al

purposes, other than in small quantities solely for research and development, under the terms of 40 CFR 723.50.

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

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

SIf this application is for an LVE in accordance with 40 CFR 723.50(c)(1), the manufacturer intends to commence

manufacture of the exempted substance for commercial purposes within I year of the date of the expiration of the 3 day

review period.

The accuracy of the statements you make in this notice should reflect your best prediction of the anticipated facts regarding the chemical substance

described herein. Any knowing and willful misinterpsretation Is subject to criminal penalty pursuant to 1s usc 1001. ____cfidential

Signa~tu d itie of A tborizoi Official (Ongina] signature Requwred) Date

Signat-6agh t- (isf plicabi - Date

EPA FORM 77 10-25 (Rev. 5-95) Page 2
PMN Page 3 Page 3of 101

Part I - GENERAL INFORMATION

Section A - SUBMITTER ILDENTIFICATION ConfiMark () the "Confidential" box next to any subsection you claim as confidential dential_

I a. Person Name of authorized official Position

NuitingU.. Mr.. 1(zuki Puj imoto Mathager -ChemicalsE ] Notce(inU .) CompanyF 1

Mitsubishi Gas Chemical America, Inc.

520 M~adison Avenue

b. Agent (if Name of authorized official Psto

applicable) Robert 7. .Reynolds consultant

Company

Regulatory Compiliance servicea

Mailing address (number and street)

22312 colony Woods Drive

City, State Postal Code Telephone (include area code)

Apex, NC 27523 (919) 303-5018

c. if you are submitting this notice as part of ajoint submission, mark (X) this box._________________________

Joint Submitter (if Name of authorized official Psto

applicable)[I

Company

2. Technical Name of authorized official Psto

Contact (in Robert J.. Reynolds Consultant

U.S.)

2312 Colony Woods Drive

3. If you have had a prenotice communication (PC) concerning this notce and

EPA asaigned a PC Number to the notice, enter the number. Mr X

4. If you previously submitted an exemption appl ication for the chemical

substance covered by this notice, enter the exemption number assigned by EPA If you previously submitted a PMN fur this substance enter the PMN fin number assigned by EPA (i.e. withdrawn or incomplete). - ifr none E :

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

for the chemical substance covered by this notice, enter the notice number Mr X

assigned by EPA. if none I

6.. 6. Typofotce-as(X Tp tr :2. I mf El ot fNtc ak()Only IOnly 1 3. Both [] Binding Option Lit Binding Option Mark (X) IMark (X) ____

EPA FORM 77 10-25 (Rev. 5-95) Page 3
PMN Page 4 Page 4of 101

Part I - GENERAL INFORMATION - Continued____

Sectin B iD -CHEMIAL TIT iNFOMATIO: YoumustproIde a currently conrect Chemical Abstracts (CA) name ofthe substance based othninth

ColcieIdx(9C0) of CA nomenclature rules and conventions. ________

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

Compilete either item 1 (Class I or 2 substances) or 2 (Polymers) as appropriate. Complete all other items.

If another person will submit chemical identity information for you (for either Item I or 2), mark (X) the box at the right. ConfiIdentify the name, company, and address of that person in a continuation sheet.L dential

1.Class 1 or 2 chemical substances (for definiitions of class I and class 2 substances, see the Istructions Manual)

a. Class of substance - Mark (X) I []CasI or 2 E ls

b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. For

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

which ever is appropriate based on CA 9C0 nomenclature rules and conventions).

c. Please identify which method you used to develop or obtain the specified chemical identit inormation reported in this notice: (cheek one). [IMethod I (CAS Inventory Expert Service - a copy of the Identification Method 2 (Other Source)

report obtained from the CAS Inventory Expert Services must be

submitted as an attachment to this notice)

exists for the substanceI

e. For a class I substance, provide a complete and correct chemical structure diagram. For a class 2 substance, provide a correct representative or

partial chemical structure diagram, as complete as can be ksnown, if one can be reasonably ascertained. Please see the E-PMN Instruction Manual For

discussion of "native format" diagram software which can be helpfu~l in reviewing your substance.X

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

EPA FORM 7710-25 (Rev. 5-95) Page 4
PMN Page 4a Page 5of 101

For a class 2 substance - (1) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of the recion or process.

(3) Indicate the range of composition and the typical composition (where appropriate)._____

e-(1) List the immediate precursor substances with their respective CAS Registry Numbers. GonfiName (CAS dential

e. (2) Describe the nature of the reaction or process.

This is an. imported material. The process is unknown.II

e. (3) Indicate the range of composition and the typical composition (where appropriate).

There is no detectable residual monomer in the PMN substance[I

10 Markc (X) this box if you attach a continuation sheet._______

EPA FORM 7710-25 (Rev. 5-95) Page 4a
PMN Page 5 ; Page 6of 101

Part I - GENERAL INFORMATION - Continued

Section B - CHEMICAL IDENTITY INFORMATION - Continued

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

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

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

below 1,000 absolute molecular weight of that composition.

Describe the methods of measurement or the basis for your estimates: GPC Li Other 1 ]: (Specify below)_____

(i) lowest number average molecular weight: 1,831 See Attachment 8

(ii) maximum weight % below 500 molecular weight:0

(iii) maximum weight % below 1000 molecular weight: 0

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

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

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

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

the polymer.

(2) - Mark (X) this column if entry in column (1) is confidential.

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

(4) - Choose "yes" from drop down menu if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer

description on the TSCA Chemical Substance Inventory.

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

(6) - Indicate the maximum weight percent of each monomer or other reactant that may be present as a residual in the polymer as manufa tured for

comamercial purposes.

(7) - Mark (X) this column if entry in column (6) is confidential.___________ _____

Monomer or other reactant and CAS Registry Number Confi- Typical Include in Confi- Maximum Confidential composition identity dential residual dential

_ _ _ _ _ _ _ _ _ _ _ _ _(4) % (_) (6) (7) T ._......

______ _[___EiOL EZ [Ii _ _ L

_ V0i

LI %] _L

0 cninaio ak X ti bxifyu het tac E ]____

EPA 5-9)ORM771025 Rev Pge%

6%
PMN Page 5a; Page 7 of 101

c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice (check one). CBI [E] Method I (CAS Inventory Expert Service - a copy of the identification report [] Method 2 (other source)

obtained from CAS Inventory Expert Service must be submitted as an attachment to this notice) L]

d. The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers.

CAS Registry Number (if a number already exists for the tubstance)li ] e. Provide a correct representative or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertained.. Plea! e

see the E-PMN Instruction Manual for discussion of "native format" diagram software which can be helpful in reviewing your substance.[ J

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

EPA FORM 77 10-25 (Rev. 5-95) Page Sa
PMN Page 6 Page 8ofl101

Part I - GENERAL INFORMATION - Continued ___

Section B - CHEMICAL IDENTITY INFORMATION - Con~tinued

3. Impurities

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

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

(b) - Estimate the maximum weight% of each impurity. If there are unidentified impurities, estimate their total weight%.4

Impurity and CAS Registry Number Maximum Confipercent dential

(a) _b

0

0

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

4. Synonyms - Enter any chemical synonyms for the new chemical identified in subsection I or 2. Confidential

L~x]

E] Mar. (X) this box if you attach a continuation sheet.

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

Memory Green Crystal[I

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

6. Generic chemical name - If you claim chemical identify as confidential, you must provide a generic name for your substance that reveals

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

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

Polyether polyol

fl Mark (X) this box if you attach a continuation theet_ ____

7. Byproducts - Describe any byproducts resulting from the rmufacture, processing, use, or disposal of the new chemical substance. Provide the CAS Registry

N umber if available. _______________ ____

Byproduct CAS Registry Number Confi-

(1) (2) dential

None_

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

EPA FORM 77 10-25 (Rev. 5-95) Page 6
PMVN Page 7 Page 9of 101

Part I - GENERAL INFORMATION - Continued

Section C - PRODUCTION, IMPORT, AND USE INFORMATION:

Mark (X) the "Confidential" box next to any item you claim as confidential. ____

1. Production volume - Estimate the maximum production volume during the first 12 months of production. Also estimate the max nurn

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

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

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

Maximum first 12-month production (kg/yr) Maximum 1 2-month production (kglyr) Cot i- Binding

( 0 % n w c e i a su sa c ba i)(100% new chem ical substance basis) den ial Option~

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

category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim as confident a].

a. (1) --Describe each intended category of use of the new chemical substance by funtction and application. (2) -Mark (X) this column if entry col mn(I) is

,onlldential business information (CB1). (3) --Indicate your willingness to have the infonmation provided in column (1) binding. (4) --Estimate the percent of total

rToduction for the first three years devoted to each category of use. (5) -Mark (X) this column if entry in column (4) is confidential business information Q RBI). (6) --

Estimate the percent of the new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes al sites under

(our control associated with each category of use. (7) --Mark (X) this column if entry in column (6) is confidential business information (CB1). (8) -- Indic kte % of

3roduct volume expected for the listed "use" sectors. Mark more than one box if appropriate. Mark (X) to indicate your willingness to have the use type p ovided in (8)

iinding. (9) --Mark (X) this column if entry(ies) in column (8) is (ae) onfidential business information (C 1)

Category of use (1) (by function and application i.e. a CR1 &-ng Prod- CBI % in CBI % of substance expected peru! e CR!

dispersive dye for finishing polyester fibers) Mark~ %uc tion Fon(8

(2) (3) (4) (5) (6) (7) S11. i-ldn cors- liding (9) boartd mmrtot mria S0on __

water retention Aid for orrianental %]F~

plAnts [ 1 10010 EL 1 100%00

_______[II[II[]%[IHI

*If you have identified a "consumer" use, please provide on a continuation sheet a detailed description of the use(s) of this chemical substance in consui e 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 loses its identity in the consumer product.

[X1Mark (X) this box if you attach a continuation sheet.

b. Generic use If you claim any ctitegory of use description in subsection 2a as confidential, enter a generic description of that category. Read t e Instruction

description Manual for examples of generic use descriptions.

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

3. Hazard Information - Include in the notice a copy of reasonable facsimile of any hazard warning statement, label, material safety data sheet, or othei Biding

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or practices for Mwkto)

the safe handing, transport, use, or disposal of the new substance. List in part III hazard information you include.

M~ Mark (X) this box if you attach hazard information.[1 1

EPA FORM 77 10-25 (Rev. 5-95) Page 7
PMN Page 8 Pagel10of 101

Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X) the "Confidential" box next to ary item you claim as confidential

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

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

-there are further industrial processing or use operations after import. You must describe these operations. See instructions manual

1. Operation description Confla. Identity -- Enter the identity of the site at which the operation will occur. dential

Name[I

Site address (number and street)

City, County, State, ZIP code

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

production rates or operations, include all the information requested in this section for those[X

Mak()0 Manufacturing 0I Processing 1 [Zs] c. Amount and Duration -- Complete 1 or 2 as appropriate_______________ _____________

Maximum kg/batch tiooA.cwc ciicai Hours/batch Batches/year

1. Batch btac)[i]

Maximum kg/day (I OVA amchanical Hours/day Days/year

2. Continuous substancc) [ii

d. Process description [] Mark (X) to indicate your willingness to have your process description binding. [ I

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

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

(2) Provide the identity , the approximate weight (by kg/day or kg/batch on a 100% new chemnical substance basis), and entry point of all starting mtrials and

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

used daily or per batch.).

(3) Identity by number the points of release, including small or intermittent releases, to the environment of the new chemical substance. If releasing o two media

at the same step, assign a second release number for the second medium.

Product will be imported to the U.S. and stored at strategically located public warehouses fr

distribution to retail outlets. Submitter may import t Ihe PM'N substance in bulk, and contrac with

adomestic facility for repackaging None of these facilities 3.s under control of the subsj ea'.

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

EPA FORM 7710-25 (Rev. 5-95) Page 8
PMN Page 8a; Page 11 of 101

Confidential

EPA FORM 7710-25 (Rev. 5-95) Page 8a
PMN Page 9 ; Page12 ofl101

Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMHTTER - Continued

2. Occupational Exposure - You must make separate confidentiality claims for the description of worker activity, physical form of the new chemical SubaCe, number of

works exposed, and duration of activity. Mark (X) the "Confidential" box next to any itemn you claim as confidential.

(1) - Describe the activities (i.e. bag dumping, tote filling, unloading drums, sam~pling, cleaning, etc.) in which workers may be exposed to the substance

(2) - Mark (X) this column if entry in column (1) is confidential business information (CBI).

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

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

(5) - Indicate the physical form(s) of the new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance (if part a mixture) at the

time of exposure.

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

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

(9) -Mark (X) this column if entry in column (8) is confidential business information (C131).

(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 colwnmin if etries in columns (10) and (11) are confidential business infrmtion_(C131). ____ ____ __

Worker activity CBI Protective Equipment/ Bindig Physical Bawidg C~l # of CB1 Maximum duration CBI

forms(s)

(i.e., bag dumping, filling drums) Engineering Controls Opt

ion and % new Option Workers Hrs/day Days/yr

Mark WX substance Mark W Exposed

(2) (4) (5) (6) (7) (8) (9) (10) (1I) (12)

Mark L ZXE _ -_s _Hf

___ (Rv __A 5-95 9[]R Zr12 
PMN Page 9a; Page 13 of 101

3. Environmental Release and Disposal -- You must make separate confidentiality claims for the release numnber and the amount of the new chemical sub -,ace

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

(1) -- Enter the number of each release point identified in the process description, part It, section A, subsection Ild(3).

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

(3).-- Mark (X) this column if entries in coluims (1) and (2) are confidential business information (C131).

(4) - Identify' the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, POTW, or other (! peci1f')) to

which the new substance will be released from that release point.

(5) -- a. Describe control technology, if any, and control efficiency that will he used to limit the release of the new substance to the environment. For rel-ases

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

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

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

(6) - Mark (X) this column if entries in columns (4) and (5) are confidential business information (CB I).

(7) -I1dentify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct dischages or

NPDES numbers of the POT (Publicly Owned Treatment Works). Mak(X) if the POTW name or NPDES # is confidential business informatio (C2BI).

Release Amount of new C131 Medium of Control technology and efficiency (you may wish to optionally attach Cal

Number substance released release efficiency data) ___ _______

(1) (2a) (2b) cg. 9. Binding

(3) (4) (5a) Mr()(5b) (6)

El IL I

_E D _E El_________Li]d (7)[I Mak_)th_ eesstowtr _ __tinsf N D S B

Li __Li] FII

()Mark (X) the desf.ata otinuation sheeto t. N D S

AF M1 (evs) 5-5)Pge9
PMN Page 10; Page 14 of 101

Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued

Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. importers do not have to comp] -e this section for

operations outside the U.S.; however, you must report any processing or use activities after impost See the Instructions Manual. Complete a separate section Bfor each type of

processing, or mve operation involving the new chemical subsiance. If the same operation is performed at more than one site describe the typical operation counon to these

sites. Identify additional sites on a continuation sheet.

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

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

cars, tank trucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2) -- Either in the diagram or in the text field 1(b) below, provide the

identity, the approximate weight (by kg/day or kg/batch. on an 100% new chemical substance basis), and entry point of aUl feedstocks (including reactant solvents and

catalysts, etc) and all products, recycle steams, and wastes. Include cleaning chemicals (note frequency if not used daily or per bateh). (3) -- Either in th, diagram or in the

text field I1(b) below, identify by number the points of release, including small or intermittent releases, to the environment of the new chemical substance. (4) Please enter

the l# of sites (remember to identify the locations of these sites on a continuation sheet): ______

#I of sitesCB

Diagram the major unit operation steps and chemical conversions...

(b). (Optional) This space is for a text description to clarify the diagram above. [E1 ]

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

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

/Y
PMN Page 10a Page15 of 101

2. Worker Exposure/Environmental Release

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

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

(4) - Estimate the typical duration of exposure per worker in (a) hours per day and (b) days per year.

(6) - Describe physical fonm of exposure and % new chemical substance (if in mixture), and any protective equipment and engineering controls, if any, usCd to protect

workers.

- (7) - Estimate the percent of the ne~w substance as formulated when packaged or used as a final product.

(9) -- From the process diagram above, enter the numnber of each release point. Complete 9-13 for each release point identified.

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

(12) - Describe media of release i.e. stack air, fuigitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, o ther (specify)

and control technology, if any, that will be used to limit the release of the new substance to the environmsent.

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

_______ ~(3), (5), (8) (11), (13) and (15) -- Mark (X) this column if any of the proceeding entries are confidential business information (CBI)____

Lett # o CR1 DuraionrcOf CB1 Protective Equip. /Engineering Controls/Physical Form and! % new substance i B

of Workers Epsur FormActi- Exposed ulat ion

vity

(1) (2) (3) (a (4) (5) (6) (7) (8)

Release Amiount of BIMedia of Release & Control Technology CR!

Number New

Substance

Reteased

(9)L (10a) (10b) (i)(12) (13)

(14)- Byproducts: (15)

EJ Mark (X) this box if you attach a continuation sheet,_______

OPTIONAL POLLUTION PREVENTION INFORMATION

To claim information in the following 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 patetial risks

associated with activities surrounding manufacturing, processing, use and disposal of the PMN substance. Please include new* formation

pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due t4 the new

chemical substance. Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological mad fcation,

process and procedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refe sto the

reclamation of useful chemical companents from wastes that would otherwise be treated or released as air emissions or water & -harges, or

land disposal. Descriptions of pollution prevention, source reduction and recycling should emphasize potential risk reduction su )Sequent to

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

assess over-all net reductions in toxicity or environmental releases and exposures, not the shifting of risks to other environmental media or nonenvironmental areas (e.g., occupational or consumer exposure). In addition, information on the relative cost or performance cha acteristics of

the PMN 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 PMN Instructions

1Manual and Pollution Prevention Guidance manual for guidance and examples. __ ____

EPA FORM 7710-25 (Rev. 5-95) Page 10a
PMN Page 11 ; Page 16 of 101

Optional Pollution Prevention Information (Continued) Describe the expected net benefits, such as (1) an overall redudion in risk to

human health or the environment (2) a reduction in the volume manufactured; (3) a reduction in the generation of waste mn terials

through recycling, source reduction or other means; (4) a reduction in potential toxicity or human exposure and/or environn ental

release; (5) an increase in product performance, a decrease in the cost of production and/or improved operation efficiency othe new

chemical substance in comparison to existing chemical substances used in similar application; or (6) the extent to which tht new

chemical substance may be a substitute for an existing substance that poses a greater overall risk to human health or the en% ironment.

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

EPA FORM 7710-25 (Rev. 5-95) Page I1I
PMN Page 12 ; Page 17 of 19

Part III -- LIST OF ATTACHMENTS___

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

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

which it relates, if appropriate. Number consecutively the pages of any paper attachments. In the colurmi below, enter the inclusive page

numbers of each attachment. Electronic attachments can be identified by filename.

Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions 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 Filename Attachment Confi-

________________________________________________page_ number(s) dential

1 Consumer Use continuation (19 L

2 1 11

3 Material Safety Data Sheet I ________ 21-25 11

4 Bacterial Reverse Mutation Study j_ ________j26-47jj

5 Skin Sensitization Study j_ ________j48-64 _ El

6 Primary Skin Irritation Study j ________ 65-79 L

7 Acute Oral Toxicity Study [B__________ O-100 0I

811

L

L

L

L

El

n () hi see. bx f ntr ouatac heatacmet acotiuaio am ad umer

EPA_ FOM71-5(ev -5 _ _ _ _ _ ae1 _ 
PMN PageI13 ; Page18 ofl101

PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET__

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

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

the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. If the attachments

are electronic, give the attachment number (found on page 12) at (b). The physical state of the neat substance should be provided.

These measured properties should be for the neat (100/6 pure) chemical substance. Properties that are measured for mixtures or

formulations should be so noted (%/ PMN substance in -J. You are not required to submit this worksheet; however, EPA strongly

recommends that you do so, as it will simplify' review and ensure that confidential informnation is properly protected. You should submit

this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission of test data.

Property Mark (X PaeValue Measured ConSiif jnumer Euiate dendal

(a) provided (b) (c) ( NrE)

Physical state of neat substance [Ii[J (s) L( Ig M [j]

Vapor pressure[I

@ Temperature 0C ElTonr E:_]

Density/relative density [E ] g/cm3 ____ I

Solubility

@ Temperature ______C[] ED Solvent___ __ __ g/L _

Solubility in water @Ternperature _____ 111.1/L ::

Melting_ tepeatr _ _ o _ 

Boiling / sublimation temperature@_ -_tonf pressure [I1I0 E::]

Spectra EI] [I

Dissociation constant[ ] ]

Particle size distribution L ]__ I

Octanol / water partition coefficient FI] I]J

Henry's L.aw constant E l ____________ i

Volatilization from water EI] [Fi1

Volatilization from soil 0 1] [I

pH @ concentration [ED ____________ __

Flammability LI E:1_ L

Explodability 17] ____________3 ___ I

Adsorption / coefficient Lii ED11

Other - Specify EZ________________ I]-

Other - Specify[I_________ _ ___1 ]

[J Mark~ (X) this box Nf you attach a continuation sheet Enter the attachment name and number.

ATTACHMENT

PAGE 13

49
Page 19 of 101

Continued From: Part 1, Section C2a, pg 7

Continuation Title: Consumer Use continuation

Insert narrative continuation here Cni

The PMN substance will be supplied as small, irregularly-shaped pellets that are about 0.1-1 cT L

in diameter. The pellets, though dry in appearance and feel, will be about 80% water. They will

be packaged in containers up to 20 kg capacity per package and sold to consumers through

garden supply outlets. The PMN substance may also be shipped in bulk or large capacity

containers to nurseries.

The PMN substance may also be shipped in bulk to a domestic, contract packaging facility.

The PMN substance will be mixed 1:1 with potting soil for use.

The PMN substance does not react, dissolve, degrade or decompose during normal use.

Insert any picture here (PDF) Confiiential

Check here to add another continuation sheet

Page 19 of 19
---- - --------- Page 20 of 1011wVJ

CI~3 Inventory Expert Se ice

A division of the American Chemical Sociecy Phone: 800-631-1 884, 614-447-36 (e60- 2308)

FaX:6 4-447-3747*

PoWstj Fax Note 7671 DaeE-mail: ans rs@casorg

Phy I 0 5/8 jPhane

Fax * - #_ _ _ _ _ _ _ _ _ _

INVENTORY EXPERT SERVICE REPORT

IES-ORTIER NUMBER: 69691

REGISTRY NUMBER.- Not Registered

CA INDEX NAME

Please print the above CA Index Namue on the appropriate page of your PMN.

Ifthis box is checked, CAS has made correction(s) marked in red to

your IES ordex. -Please make the same correction(s) to your PXM1 before

submitting it to the EPA.

2W406lnngy "dvr Road PO. 09 3343 Caleanb"g Ohio 43210-034

FEB-4-1999 THU 12:55AM ID: PAIE: 1
Page 21 of 101

MITSUBISHI GAS CHEMICAL COMPANY, INC. Page 1 f 5

MATERIAL SAFETY DATA SHEET

Product Name: Memory Green Crystal Preparation date: 2/2C/03

_SECTION 1 -CHEMICALPOUTADCMAYIETFATN ]

MSDS REVISION #: New

PRODUCT NAME: Memory Green Crystal

SYNONYMS: None

DISTRIBUTED BY: Mitsubishi Gas Chemical America, Inc.

520 Madison Avenue, 17th Floor

New York, NY 10022, USA

PHONE NUMBERS: Inquiries - (212) 752-4620 (business hours)

CHEMTREC - (800) 424-9300 (transportation emergencies,

ON h'R.E.0 b 1E .-,]

Component % CAS No. Exposure Limits

Polyether polyol 20 None Not established

Water 80 7732-18-5 Not established

***********************EMERGENCY OVERVIEW * ***' *V

Product consists of odorless, yellow-green, irregularly-shaped beads. There are no known

or expected hazards associated with handling this product. Avoid contact with eyes. Wash

thoroughly after handling.

POTENTIAL HEALTH EFFECTS

EYES:

No adverse health effects known or expected, although small particles may cause

mechanical irritation.

SKIN:

No adverse health effects known or expected.

Section 3 continued on next page
Page 22 of 101

MITSUBISHI GAS CHEMICAL COMPANY, INC. Page 2 cf 5

MATERIAL SAFETY DATA SHEET

Product Name: Memory Green Crystal Preparation date: 2/2C/03

INGESTION (swallowing):

No adverse health effects known or expected.

INHALATION (breathing):

No adverse health effects known or expected.

CHRONIC EFFECTS/CARCINOGENICITY:

This product (or component) is not listed in IARC Monographs, the NTP N nth

Annual Report or the ACGIH TLVs as a carcinogen or potential carcinogen. 0,%- HA

does not regulate it as a carcinogen.

EYE CONTACT: Flush with water, lifting upper and lower lids occasionally.

FLASH POINT: Not applicable FLASH POINT METHOD: Not applicable

UPPER EXPLOSION LIMIT: Unavailable

LOWER EXPLOS ION LIMIT: Unavailable

AUTOIGNITION TEMPERATURE: Unavailable

EXTINGUISHING MEDIA:

Water spray, carbon dioxide, dry chemical or foam

FIRE AND EXPLOSION HAZARDS:

None known

FIRE-FIGHTING EQUIPMENT:

Wear full protective clothing and self-contained breathing apparatus with full facepiece. Combustion products may include toxic fumes.
Page 23 of 101

MITSUBISHI GAS CHEMICAL COMPANY, INC.Pae3 5

MATERIAL SAFETY DATA SHEET

Product Name: Memory Green Crystal Preparation date: 2/20)03

Shovel spilled material into containers. Thoroughly sweep up residual material.

Keep in closed or covered containers when not in use. Store in cool dry place with

adequate ventilation.

SCTIO 8 - EXPOSURE CONTROLSIPROA PRTC -N

RESPIRATORY PROTECTION:

Not required under normal conditions of use.

SKIN PROTECTION:

Not required under normal conditions of use.

EYE PROTECTION:

Protective eye wear or safety glasses are recommended.

ENGINEERING CONTROLS:

Not required under normal conditions of use.

APPEARANCE: Yellow-green beads @ 770 F (250 C)

SPECIFIC GRAVITY: Unavailable

VAPOR PRESSURE (mm Hg): Nil

VAPOR DENSITY (Air =1): Heavier than air

INITIAL BOILING POINT: Decomposes

EVAPORATION RATE: Nil

(Ethyl Ether = 1)

SOLUBILITY in WATER: Insoluble, although product absorbs significant amoL nts

of water

BULK DENSITY: Unavailable

VOLATILE %: 80%

SOFTENING POINT: Unavailable
Page 24 of 101

MITSUBISHI GAS CHEMICAL COMPANY, INC. Page 4of 5

MATERIAL SAFETY DATA SHEET

Product Name: Memory Green Crystal Preparation date: 2/20,103

STABILITY (conditions to avoid):

Stable under normal conditions.

INCOMPATIBILITIES (materials to avoid):

Avoid contact with acids, and oxidizing agents.

HAZARDOUS DECOMPOSITION PRODUCTS:

Thermal decomposition products include: carbon monoxide, carbon dioxide rnd

noxious gases

HAZARDOUS POLYMERIZATION:

Not known to occur

Acute Oral LD5o (rat) - greater than 2,000 mg/kg

Skin irritation - Not irritating

Mutagenicity (Ames test) - negative

No data available

Incineration is the recommended disposal method for all chemical wastes; however,

this material may be deposited in a landfill in accordance with applicable local, slate

and Federal regulations. This material is not considered a hazardous waste under

current RCRA definitions.

Not regulated under current DOT, IMO or ICAO regulations.
Page 25 of 101

MITSUBISHI GAS CHEMICAL COMPANY, INC. Page 5 f 5

MATERIAL SAFETY DATA SHEET

Product Name: Memory Green Crystal Preparation date: 2/20/03

SECTIO 15 -REG1JATORY777777"nN

TSCA INFORMATION:

This product is not, to the best of our knowledge, listed on the TSCA Inventory. se

of this material is restricted to technically qualified individuals for research ind

development purposes only.

SARA 313 INFORMATION:

SARA requires submission of annual reports of release of toxic chemicals hat

appear in 40 CFR 372. This information must be included in all MSDS that are

copied and distributed for this material.

Components present in this product at a level that could require reporting under the

statute are: None

HAZARD RATING:

HEALTH 0 0 -LEAST

FIRE 1 1 - SLIGHT

REACTIVITY 0 2 - MODERATE

OTHER -3 -HIGH

4- EXTREME

HAZARD RATING METHOD: HMIS

REASON FOR REVISION:

New MSDS

The product information contained herein is believed to be accurate as of the date of the Material Safety )ata

Sheet, and is provided without warranty, expressed or implied, as to the results of use of this information o-the

product to which it relates. Recipient assumes all responsibility for the use of this information and the use (E lone

or in combination with any other product), storage or disposal of the product, including any resultant personal

injury or property damage.

~END OF REPORT
Page 26 of 101

B0205 14

Confirmation Statement

General Testing Research Center,

Japan Oistuff Inspectors' Corporation

Sponsor Mitsubishi Gas Chemical Company, Inc.

Title Bacterial Reverse Mutation Study of MEMORY GREEN CRYSTAL

Study No. B020514

The study had been conducted in the General Testing Research Center, Japan Oilstuff

Inspectors' Corporation. The original study report was written in Japanese and

translated into English. I hereby declare that this report faithfully raflects the

original study report as accurately as possible to my knowledge.

Translator _________ _____ Date:

Masato Nakamura, M. S.

Scientist of Mutagenicity Testing Group

2
Page 27 of 101

B020514

Submitted to:

Mitsubishi Gas Chemical Company, Inc.

Re p or t

Bacterial Reverse Mutation Study of MEMORY GREEN CRYSTAL

(Study No. :B020514)

June 25, 2002

GENERAL TESTING RESEARCH CENTER, JAPAN OILSTUFF INSPECTORS' CORPOZATION

-I3
Page 28 of 101

B020514

Stat ement

General Testing Research Center,

Japan Oistuff Inspectors' Corpor tion

Sponsor Mitsubishi Gas Chemical Company, Inc.

Title :Bacterial Reverse Mutation Study of MEMORY GREEN CRYSTAL

Study No. B 020514

The study in this report was conducted in compliance with the followirg Good

Laboratory Practice Standards.

Japanese GLP standards Applied to Industrial Chemicals (Kanpogyo No. 39,

Yakuhatsu No.229, 59 Kikyoku No.85, 1984; revised in 1988 and 2000)

GLP Standards under the Japanese Industrial Safety and Health Law (N tification

No.76, Ministry of Labor, 1988 revised in 2000)

Management Masakatsu Usami sealed Date: June 25, 2002

Study Director : Eiichi Machigaki sealed Date: June 25, 2002

4
Page 29 of 101

B020514

Quality Assurance Statement

General Testing Research Center,

Japan Oilstuff Inspectors'I Corporation

Sponsor Mitsubishi Gas Chemical Company, Inc.

Title :Bacterial Reverse Mutation Study of MEMORY GREEN CRYSTAL

Study No. B 020514

By t he inspec t ions and aud it s i s ted belIow, I hereby c ert if y that th e s tud rwas carri ed

out in accordance with its protocol and with our Standard Operating Pro- edures, and

that the report faithfully describes the methods and procedures applied, and that the

reported results reflect the raw data of the study accurately.

Inspctin Dte o inpecion Date of reporting to

InspecitionrDate ofi ns e t o the study director

or aditor aditand to the management

Prtocol May 8, 2002 May 8, 2002

Srtdyl

Procdur June 4, 2002 June 4, 2002

Draft June 19, 2002 June 19, 2002

Report

Report June 25, 2002 June 25, 2002

Quality Assurance Unit: Sadayoshi Ohtubo sealedDate: June 25, 2002
Page 30 of 101

B0205 14

Outline of the Study

1. Title :Bacterial Reverse Mutation Study of MEMORY GREEN CRYSTAL

(Study No. : B020514)

2. Purpose: To assess the mutagenicity of the test substance in bacter al

mutation assays, using Salmonella typhimui-iuv and

Eschez-ichia coli strains.

3. Guideline: Guidelines under the Japanese Industrial Safety and Heal ;h Law

(1997) and Guidelines under the Japanese Industrial Safetj and Health

Law (1988 and revised in 1997).

4.GLP :GLP under the Japanese Industrial Safety and Health Law 1984;

revised 1988 and 2000) and GLP under the Japanese Industria LSafety and

Health Law (1988 and revised in 2000).

5. Sponsor: Study ordered by

Mitsubishi Gas Chemical Company, Inc.

5-2, Marunouchi 2-chome Chiyoda-ku, Tokyo, Japan

(Responsible person) Masanori Hataya

Study ordered

Mitsubishi Chemical Safety Institute Ltd.

1-30, Shiba 2-chome, Minato-ku, Tokyo

6. Organization in: Testing Facility

Contract

General Testing Research Center,

Japan Oilstuff Inspectors' Corporation

10-4, 1-Chome, Mikagetsukamachi, Higashinada-ku,

Kobe-shi, Hyogo

6
Page 31 of 101

B020514

7. Study Contributors

Management Masakatsu Usami

Data Recorder :Etsurou Fujiwara

Study Director :Ejichi Machi gaki sealed Date: Jn 2,O00

Other Contributors Masato Nakamura, Osamu Iwaihara, Chikako Tsutsufti,

Eriko Iwasaki, Naomi Ikeda

8. Study Date :(Initiation of the Study) May 8, 2002

(Initiation of the Test) May 27, 2002

(Submission of the Test) June 6, 2002

(Submission of the Final-Report) June 25, 2002

9. Data analysis: No statistical methods were used.

10. There were no unforeseeable circumstances that had affected t st results

undesirably.

11. Retention The protocol, all raw data, the test substance sample, documents and

the final report will be retained in the safekeeping facility of

-General Testing Research Center. The materials described hbove except

for the test substance sample will be retained for ten years after

submission of the application for Japanese Industrial Safety and

Health Law, section 57-3-1 or the notification of coming ur der sect ion

4-1-1, 2, or 3 of Industrial Chemicals. The test substance and specimen

will be retained for a shorter period of the following two periods:

10 years after receiving notification according to the Industrial

Chemicals or making notification according to the Industrial Safety

and Health Law, or a period that allows stable storage without

deterioration in quality. Further retention will be discus 3ed with the

sponsor.

7
Page 32 of 101

B020514

Contents

Page

Summary ............................................................ ...... 9

Materials and Methods............................................................10

1. Test materials ............................................................ 10

2. Bacterial tester strains. ............................................. 11

3. Media.... ............................................................... 12

4. S9 mix ................................................................. 13

5. Test method ......................... I...................... ............ 13

Results and Discussion ......................................................... 16

References .................................................................. 17

Tables...... ................................................................ 18

Figures ................................................................... 20

Background data .............................................................. 23

8
Page 33 of 101

B0205 14

Summary

MEMORY GREEN CRYSTAL was examined for the mutagenicity in bacterial ass a s using five

different strains, Salmonella typhimur-ium TA98, TA1QO, TA1535, TA1537 and

Esche-ric-hia coli WP2 uvrA.

In both the dose-finding test and the main test, the number of revertant colonies

induced by the test substance was less than twice that in the corresponcling solvent

control value for all tester strains both the presence and absence of $9 mix.

Based on these findings, it was concluded that MEMORY GREEN CRYSTAL is nt mutagenic

under the test conditions employed.

9
Page 34 of 101

B020514

Materials and Methods

1. Test materials

1 .1 Test substance

Name of the new chemical

substance (IIJPAC

nomenclature)

Trade name MEMORY GREEN CRYSTAL

Structural formula or

rational formula

(or outline of

manufacturing method,

in case both

are unknown)

Purity of the new Impossible Lot No. of the new chemica1

chemical substance to measure substance to be tested MG-R02

tested

Name and concentration

of impurities ________ ____

CAS No. Vprpesr

Molecular weight Impossible Partition____

Melting point Appearance at Lgtylo lsi

Boiling point ambenLihtyelowelati

Solubility in Sovn ouiiyS ability in

several solvents SovnSolblt lvent

Water (water holding ability) tbe2

Water suspended at 50 mg/mL1)tbe)

DMSO suspended at 50 mg/mL '

Acetone suspended at 100 mg/niL

others (-)

DMS0: diniethyl sulfoxide

Storage condition: kept at a room temperature and in desiccator

1): Based on the results of the vehicle selection test conducted in the testing facility.

2): Heat, discoloring and foam formation were not observed in the test substance

preparation.

10
Page 35 of 101

B020514

1. 2 Control Substance

The negative control and the positive controls were as follows.

Name Abbreviation Supplier Lot No. Purity(%)

Negative control Wk ueCeia W92

Sterilized distilled DW Wand ures Ltd.a DG72

Water IdsreLd

Positive controls Wk ueCeia

2-(2--Furyl)-3-(5-nitro AF-2 WaoPr hmcl WTH1046 8. 1

-2-furyl)acrylamide Industries, Ltd.

Sodium azide NaN3 Wako Pure Chemical DM78 9 Sodium az___________ Industries, Ltd. DW45 98

9-Aminoacridine 9-A Aldrich Chemical 03024JR 8. 8

hydrochloride CompanyII

NAEthyl-jN-nitro-AN- ENNG Aldrich Chemical 12219AS 97

nitrosoguanidine Company_____

2-Aminoanthracene 2-AA Wake Pure Chemical ELJ6826 7. 4

_______________________________Industries, Ltd. _____ __

2. Bacterial tester strains

2. 1 Strains1

Five genetically different strains of Salmonella typhimui-ium TA98, TA .00, TA1535,

TA1537 and Esche-richia coi WP2 uvrA- were employed. These strains w re received

from Japan Bioassay Research Center on the date of May 13, 1998. rhe genetic

properties of the strains are listed in the following table.

Gene Additional mutations Stan eai emrne Rfatenetai tp

Affectedmutation

TA98 his D uvi- B rfa pkMl01 Frameshift

TA1535 his G u v2- B .rfa pk11 Base-pair change

TAIS37 his C u P-- B rfa Frameshift

WP2 uvrA- ti-p E uvi- A + Base-pair change

2. 2 Reason for selection

The tester strains were widely used in a bacterial reverse mutatiar study and

were recommen~ded in Guideline the Japanese Industrial Safety and iealth Law

(Notification No.77, Ministry of Labor, September 1, 1988; revised Jume 2, 1997)

and Japanese Guidelines on Industrial Chemicals (Kanhoan No. 287, Eis i NO, 127,

Heisei 09. 10. 31 Kikycku No. 2, October 31 1997) and Guideline th Japanese
Page 36 of 101

B020514

Indus trialI Safety and Health Law (Not if ication No. 77, Minis try of Labo ,September

1, 1988; revised June 2, 1997).

2.3 Check of genotypic characteristics

The tester strains were checked for their genotypic characteristics such as amino

acid requirement, UV sensitivity, membrane mutation(rfa) and R-factor before the

study.

2.4 Storage

Eight milliliters of fresh bacterial suspension (cultured with shaking at 370C

for 8 hours in a liquid growth medium) was mixed with 0. 7 mL of DMSO and quickly

frozen in a dry ice-acetone mixture. The stocks of the tester Strains were stored

below -80*C.

2.5 Bacterial suspension

Twenty microliters of stock culture of each tester strain was mo mulated into

10 mL liquid growth medium and grown with shaking at 37 "C for 8 hours. Densities

of the fresh bacterial cultures were measured with a spectrometer. (ell numbers

for the suspensions were calculated by the 10-fold dilution method.

3. Media

3. 1 Liquid growth medium

Oxoid Nutrient Broth 'No. 2 (Unipath Ltd. lot No. 02859365) was dissolved in

distilled water (25 g/L) and autoclaved at 121 'C for 15 minutes.

3.2 Minimal glucose agar plate.

The agar plate (TESMEDIADAN MEDIUM, lot No. ANI190DR prepared on Ap -il 2, 2002)

used for this study was purchased from Oriental Yeast Co. , Ltd.

3.3 Top agar

Aqueous amino acid solution for S. typhimurium strains, which contained

0. 5 mmol/L each of L-histidine and D-biotin was prepared and sterilized by

filtration (0. 45 u m pore size), and then stored in a refrigerator. Aqueous amino

acid solution for E. coli, which contained 0. 5 mmol/L L-tryptophane Vas prepared

and sterilized by filtration (0. 45 g m pore size), and then stored in a refrigerator.

12
Page 37 of 101

B020514

Powdered agar (0. 6g Bacto-Agar; Difco Laboratories) and 0. 5 g of sod urn chloride

were dissolved in 100 mL of distilled water and autoclaved. The abo- e-mentioned

amino acid solution was added to this agar solution in the ratio of l.to 10 and

kept at about 45 00 until use.

4. S9 mix

4. 1 S9

The S9, which was prepared from the liver of rats treated with phenobarbital

(30 mg/kg i.p. on the first day and 60 mg/kg i.p. on the second to fourth day)

and 5,6-benzoflavone (80 mg/kg i.p. at the third phenobarbital injection), was

purchased from Kikkoman Corporation. The S9 was stored below -80 'C and was used

in both the dose-finding test and the main test.

Details of the S9 used were as follows:

Lot No. RAA-459

Prepared on February 22, 2002

Using animal SrgeDwe a

Species, Strain SrgeDwe a

Sex, Age (in weeks) Male, 7 weeks old

Weight 214 - 239 g

4. 2 S9 mix

The composition of the S9 mix 'is listed in the following table. Th( S9 mix was

freshly prepared for each assay, and was stored in ice.

Components Amount in 1 mL

S9 0. 1 mL

MgClz 6H20 8 u mol

KCl 33 Atmol

D-glucose-6-phosphate 5 A mol

/3-NADPH 4 g mol

/-NADH 4 u mol

Na-phosphate buffer (pH 7.4) 100 'Umol

Sterilized distilled water Remainder13
Page 38 of 101

B020514

5. Test method2

5. 1 Preparation of the test substance solutions and the positive contro s

According to the information from the sponsor, the test substance inc uded water.

The examination of vehicle was performed with sterilized distilled wat r(DW), DM30

and acetone. The test substance suspended in DW(S0mg/mL), DMSO(50 mg/ni) and

acetone (100 mg/mL).

Thus, DiV was selected as the vehicle for the test substance. The test substance

was suspended in DiV and was diluted into each concentration by 2-fold dilution.

The test substance suspensions were prepared just before use.

As for positive control, NaN3 was dissolved in DW, and others were issolved in

DM30. One milliliter aliquots of these solutions were dispensed into tubes for

freezing and stored below -20 00. Froze aliquots were thawed at room temperature

before use.

5. 2 Test substance concentrations

In the dose-finding test, six dose levels ranging from 5 to 5000 Ug /plate were

graded ata common ratio of 4.

Microbial growth inhibition was not observed in any tester strains

Precipitation of the test substance was observed on the agar plate at

313 u g/plate or more in the absence of 39 mix and at 1250 g g/plate or more in

the presence of 39 mix

In the main test, five dose levels ranging 313 to 5000 p g/plate were graded

at the common ratio of 2 based on the results of the dose-finding test.

5. 3 Rev~rse mutation test

The reverse mutation test was done by the preincubation method.

For one concentration, 0. 1 mL, of the test substance solution, 0. 1 mL of each

bacterial suspension and 0. 5 aL, of either 0. 1 mol/L sodium phosp iate buffer

(PH 7. 4) for the assay without metabolic activation or 39 mix for thE assay with

metabolic activation were mixed in a sterilized test tube and incubated by gentle

shaking at 37 'C for 20 minutes. After preincubation, 2 mL of molten op agar was

14
Page 39 of 101

B020514

added to this mixture and poured onto a minimal glucose agar plate. l en the agar

overlay solidified, these plates were incubated at 37 'C for 48 hourt. The level

of microbial toxicity was confirmed by observation of backgrounc lawn with

stereoscopic microscope. Revertant colonies were counted manually or with an

automatic colony counter.

Two plates per dose level were used for each assay. The test was perf rmed twice.

Negative and positive control tests listed below were also done in each assay.

Strain -S9smix (Mug! plate) +S9mix (gug/plate)

TA98 AF-2 0.1 1 2-AA 0.5

TA100 AF-2 0.01 2-AA 1

TA1535 NaN3 0. 5 2-AA - 2

TAIS37 9-AA 80 2-AA 2

WP2 uvrA- ENNG 2 2-AA 10

5. 4 Contamination test

The highest dose solution and S9 mix was mixed with molten top agar and the mixture

was overlaid on a minimum glucose agar plate. No bacterial contamination was

observed.

5.6 5judgment criteria

The test substance was judged to be positive in this assay when the mean number

of revertant colonies is more than double of the negative control va ue and when

it showed significantly dose-dependent increase in a reproduci le manner.

Otherwise, the mutagenicity of the test substance was judged to be negative.

No statistical methods were used for data analysis.
Page 40 of 101

B020514

Results and Discussion

The results are shown in Tables 1, 2 and Figs. 1 - 5.

In the main test, the number of revertant colonies induced by the test substance

was less than twice of that in the corresponding solvent control value for all test

strains both the presence and absence of S9 mix. The same results were obtained

in the dose-finding test. The reproducibility of these negative iesults was

confirmed.

Neither bacterial nor fungal contamination were observed in the resalts of the

contamination test.

Microbial growth inhibition was not observed.

Precipitated test substance was observed on the agar plate.

The viable bacteria counts were as follows.

Baceril tste Stain -Base pair exchange type Frameshift type

TAlOO TA1535 WP 2 uvrA- TA98 TA1537

Number of dose-finding 4. 46452412

viable test 4 . . . .

bacteria Main test 29 4 . . . (X109 /mL)2. 47492613

In the positive control tests using specific mutagens with and with ut S9 mix,

significant revertant colonies more than double of the solvent control value were

induced. For solvent- control and positive control, the number of reverta. t colonies

in all the tester strains were within the normal range of our 1aboratory' s

background data. These results confirmed that the tests were performed correctly.

Based on these results, it was concluded that MEMORY GREEN GRYSrAL is not

mutagenic under the test conditions employed.

16
Page 41 of 101

B020514

References

1) Maron, D. M. and Ames, B. N. (1983) Revised methods for the Salmon( la

mutagenicity test, Mut. Res. , 113, 173-215.

2) Mutagenicity test in Industrial Safety and Health Law (1991) The t st

guideline and the GLP, edited by the Chemical Substance Investigat: on

17
Page 42 of 101

B020614

Table 1 Results of the Dose-finding Test

Test substance :MEMORY GREEN CRYSTAL

Testing periods From May 27, 2002 to May 30, 2002

With (+) or Test material Number of revertants (number of clonies/plate)

without -) concentration Base-pair change type craiseshift type

S9 nix (Mg/plate) TA 100 TA 1535 WP2 uvrA- TA 98 TA 1537

75 9 27 11 4

Solvent control 88( 82) 5 7) 25( 26) 10( 1) 0 7)

89 9 24 13 8

5 79( 84) 8( 9) 25( 25) 8( 1 5( 7)

87 7 30 8 6

S 9nix 20 97 ( 92) 5 6 ) 30( 30) 8( 8) 8( 7)

79 6 29 13 3

-)78 78 ( 79) 7( 7) 33( 31) 11( 12) 4( 4)

go9 5 18 9 6

313 89(90) 4( 5) 22(20) 5 7) 8( 7)

t104 8 18 15 10

1250 78( 91) 7( 8)28(23) 9(12) 5 8)

t 90 5 30 16 6

5000 84( 87) 7 6) 26 (28) 9(13) 3 5)

92 7 26 27 9

Solvent control 106( 99) 10 9) 25 ( 26) 26( 27) 7 8)

100 29 15 10

5 97(101) 9 7) 34(32) 21(18) C 9)

97 6 32 15 9

S 9aix 20 124(111) 9 8 8) 32(32) 21(18) 5 7)

98 8 29 16 7

()78 134 (116) 9 9 ) 28( 29) 19( 18) 6( 7)

105 6 31 20 6

313 115 ( 110) 6( 6 ) 26(29) 13C 17) 9 8)

t100 7 28 16 4

1250 112 ( 106) 6 C 7) 24( 26) 14( 15) 7 6)

t 98 9 24 13 5

5000 97 (98) 8( 9) 29(27) 16(15) 5 5)

Name and A F- 2 NaN3 ENNG AF-2 9 -AA

Positive concentration 0. 01 0. 5 2 0. 1 80

control (u g/plate) _________________

not requiring Number of revertant 418 351 633 569 164

S9 ii colonies per plate 457 ( 438 ) 354 ( 353 ) 648 C 641 ) 585 ( 577 )170 C167

Nameand 2-AA 2-AA 2-AA 2-AA 2-AA

Positive concentration 12 10 0. 5 2

control (m a/plate) ______________________________________

requiring Number of revertant 633 350 564 532 161

S9 mix colonies per plate ,664 C649 )359 ( 355 )665 C 615 ) 51 ( 517 )L 92 C177

AF-2 2-(2-furyl)-3-(5-nitro-2-furyl)acrylaaide, NaN 3 sodium azide,

ENNG A-ethyl-V' -nitro-N-ntorosoguani dine, 9-AA :9-aminoacridine hydrochloride, 2-AA 2-aninoanthracene

):Mean

Precipitated test substance was observed on the agar plate.
Page 43 of 101B051

Table 2 Results of the Main Test

Test substance: MEMORY GREEN CRYSTAL

Testing periods From June 3, 2002 to June 6, 2002

With (+) or Test material Number of revertants (number of colonies/plate) ________

without H- concentration Base-pair change type Frameshift type

S9 mix (g g/plate) TA 100 TA 1535 WP2 uvrA- TA 98 TA 1537

85 11 12 12 6

Solvent control 98( 92) 5( 8)26(19) 18 15) 9 8)

t 78 8 21 14 8

313 117( 98) 5( 7)20(21) 13( 14) 10 9)

ill1 12 19 11 9

S 9mix 625 92(102) 5 9)23(21)14(13) 5( 7)

t100 10 18 23 6

C)1250 91( 96) 8( 9) 18( 18 ) 10( 17) 5 6)

t 75 10 27 15 7

2500 99( 87) 6 8) 15( 21) 13( 14) 5 6)

T 101 10 17 17 9

5000 117(109) 5 .8)23(20) 16( 17) 7 8)

132 9 20 18 12

Solvent controll109(121 8( 9)21(21)31(25-)] 10( 11)

T 130 14 30 24 15

313 128(129) 11( 13) 22( 26) 23 24) 13( 14)

T 128 7 23 24 16

S 9mix 625 115(122) 8( 8)22(23)26(25) 1 16(16)

t124 10 26 18 11

()1250 110(117) 14( 12) 26( 26) 28 23) 9(10)

'118 11 35 15 10

2500 105(112) 8(10) 19(27)24(20) 16(13)

t114 4 22 18 13

5000 102(108) 6( 5)28(25)29(24) 7(10)

Name and AF-2 N a N3 ENNG AF-2 9 -AA

Positive concentration 0. 01 0. 5 2 0. 1 s0

control Cu a/plate)

not requiring Number of revertant 378 378 658 508 144

S9 mix colonies per plate 377 ( 378 )329 ( 354 )625 ( 642 )494 ( 501 )160 (152

Name and 2 -AA 2-AA 2-AA 2 -AA 2-AA

Positive concentration 1 2 10 0. 5 2

control(u/ t) ________ _______ ________ _______ ____ ___

reuring Number of revetant 609 361 6014528

S9 mix colonies per plate 584 ( 597 )342 ( 352 )635 (621 )404 ( 430 1 74 (151

AF-2 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, NaN3 sodium azide,

ENNG A-ethyl-A" -nitro-N-ntorooguanidine, 9-AA : -aminoacridine hydrochloride, 2-AA 2-aminoanthracene

C :Mean

t:Precipitated test substance was observed on the agar plate.

19
Page 44 of 101B051

.200

[TA 100]

150

0.

0.

5 0

0

40

40

020
Page 45 of 101

B020514

*40

30

aT

CL

.~20

0

0

C

*~10

0

O 313 625 1250 2500 5000

Consentration ( gg/plate)

Fig.3 Dose-response curve of MEMORY GREEN CRYSTAL

(Main Test)

0 -S9mix .0- .+s9rix

40

CTA981

30

0.E

0

0

~ 10
Page 46 of 101

B020614

[TA 1537 1

30

CL

LD 20

C

0

0

0

0 313 625 1250 2500 5000

Consent rat ion (pu g/p I ate)

Fig.5 Dose-response curve of MEMORY GREEN CRYSTAL

(Main Test)

--S9mix -0- .S9mix

22
Page 47 of 101

B020514

Background Data

WlBackground data of the negative (solvent) control value

Strain ______ -S9mix _____+S9mix ___

AMerage Average± Number Average Average:± umber

______________ 2SD _____2SD ___

TA100 90 72 - 107 60 ill 89 - 133 59

TA1535 8 4 -12 58 8 4 -12 _56

VWP2 uvzA- 20 14 - 26 53 22 16 - 28 _52

TA98 16 11 - 21 61 21 16 - 27 60

TA1537 7 5 -10 58 10 7 -13 58

(2)Background data of the positive control value

Strain -S9mix _____+S9mix ___

Average Average±t Number Average Average-± Number

_______ 2SD _____ _____ 2SD ___

TA100 468 346 - 589 48 666 542 - 789 __48

TA1535 382 337 - 427 43 313 266 - 359 42

WP2uvrA- 614 471 - 756 40 723 600 - 846 __40

TA98 552 458 - 645 49 447 349 - 546-] 49

TA1537 193 91 -294 43 158 134 - 181 43

Background data was collected from September 4, 2001 to, March 27, 2002

23
Page 48 of 101 PPL FINAL REPORT: P020071

VERIFICATION OF TRANSLATION

This is to certify that the English translation presented here conforms to the origina. Japanese

report.

Study No.: P020071

Title: Skin Sensitization Study of MEMORY GREEN CRYSTAL in Guinea Pigs

According to Buehler Test Method

Masami Kokubu Date

Panapharm Laboratories Co., Ltd.
Page 49 of 101 PPL FINAL REPOF T: P020071

Skin Sensitization Study of MEMORY GREEN CRYSTAL in Guinea Pigs

According to Buehler Test Method

FINAL REPORT -

August 16, 2002

Sponsor: Mitsubishi Gas Chemical Company, Inc.

2-5-2, Marunouchi, Chiyoda-ku,

Tokyo 100-8324, Japan

Organization under Contractor:

Mitsubishi Chemical Safety Institute Ltd.

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

Test Facility: Safety Assessment Laboratory

Panapharm. Laboratories (PPL) Co., Inc.

1285, Kurisaki-machi, Uto-shi,

Kumamoto 869-0425, Japan
Page 50 of 101 PPL FINAL REPORT: P020071

SIGNATURE PAGE

Study Director

Signed in the original Seal affixed August 16, 2002

Masarni Kokubu Date

Panapharnn Laboratories Co., Ltd.
Page 51 of 101 PPL FINAL REPORT: P 20071

STUDY SCHEDULE

1. Study inception: May 2, 2002

2. Animal receipt: May 9, 2002

3. Sensitization period: May 21, 2002 - June 4, 2002

4. Challenge date: June 18, 2002

5. Observation period: June 19, 2002 - June 20, 2002

6. Study completion: August 16, 2002

RETAINED MATERIALS AND THEIE REPOSITORY

1.- Records concerning the protocol

2. Records concerning the animals

3. Records concerning the animal room conditions

4. Records concerning the test article

5. Records concerning the results of the study

6. Final reports

7. Other materials concerning the study

The above materials shall be kept in the archives of Panapharm, LaboratorieE Co.,

Ltd. for 5 years after completion of the study (from August 16, 2002 to August 16,

2007). The handling of the above materials after 5 years shall be determined

through discussion with the sponsor.
Page 52 of 101 PPL FINAL REPORT: P( 2007 1

CONTENTS

Page

SIGNATURE PAGE...............

STUD SCHDULE...........................................i

RETAINED MATERIALS AND THEIR REPOSITORY

SUMMARY...........................................1

OBJECTIVE...........................................2

MATERIALS AND METHODS....................................2

RESULTS............................................6

DISCUSSION...........................................6

REFERENCES..........................................7

Table 1 Body weights of guinea pigs in skin sensitization study of

MEMORY GREEN CRYSTAL ............................ 8

Table 2 Skin scores of guinea pigs in skin sensitization study of

MEMORY GREEN CRYSTAL ............................ 9

Photographs I and2....................................... 10

Appendix (individual data)
Page 53 of 10 1 PPL FINAL REPORT: P02 071

SUMMARY

The skin sensitivity of MEMORY GREEN CRYSTAL was investigated using H-artley

guinea pigs according to the Buehler Test method.

In the case that the MEMORY GREEN CRYSTAL sensitized group was elicited by

MEMORY GREEN CRYSTAL, no skin responses were seen. The positive rates were 0 %

and the mean scores of skin response were 0 at each observation time after 24 and 48 ho

respectively.

In the case that non-sensitized group was applied MEMORY GREEN CRYSTAL, no s in

responses were seen on any of the applied sites, and no skin irritation could be recogniize

From the above mentioned, skin sensitivity for MEMORY GREEN CRYSTAL could o

be recognized under the present study conditions. f
Page 101PPL 54of FINAL REPORT: P 20071

OBJECTIVE

As a part of non-clinical studies of the safety evaluation of MEMORY GREEN CRY TAL,

the skin sensitivity in guinea pigs was investigated according to the Buehler Test methol .

MATERIALS AND METHODS

1. Test Article and the Vehicle

MEMORY GREEN CRYSTAL ( Lot No. MG-R02) provided by Mitsubishi Gas

Chemical Company, Inc. was used as the test article. MEMORY GREEN CRYST

was yellow powder. Japanese pharmnacopoeia water for injection (Lot No. I E90, 0 suka

Pharmaceutical Factory, Inc.) was used as the vehicle. The test article was preserved in a

desiccator in the test article room at room temperature. The vehicle was preserved ina

locker in the test article room at room temperature.

2. Preparation of Test Article

MEMORY GREEN CRYSTAL and water for injection was mixed at the rate of 0 5 g of

MEMORY GREEN CRYSTAL to 3 ml, of water for injection, and the mixture was ellkneaded to prepare the gel like material immediately before use. 0.05 g of the mix e to

be used was weighed on a piece of lint cloth of 2.0 X 2.0 cm.

3. Animals and Husbandry

Eighteen female guinea pigs (Cij:Hlartley, Charis River, Japan, Inc.) were pur hased

at an age of 5 weeks. During the quarantine and acclimation periods for 11I days, all

animals were examined for clinical signs and weighed to confirm that they were

healthy. Fifteen animals at an age of 7 weeks were used for the study. Body

weights on the first day of application were 373.1-432.6g. The animals were h used

in an aluminum cage (Wx H xD: 430x360x700 mm) with 1 or 2 animals per ca e in

the animal room (No. 16) in the rabbit and guinea pig area of the pharmacology

laboratory, which was controlled at a temperature of 24±2'C (permissible range: 21 -

27'C), relative humidity 55±10% (permissible range: 35-75%), 12-hour light/da -k

cycle (lighting from 7:00A.M. to 7:00P.M.) and 13-15 changes/hour ventilation. The

animals were allowe d to receive a pellet diet (RC4, Oriental Yeast Co., Ltd.) ani to

drink well water supplemented with NaCIO (about 0.2 ppm) ad libitum. The aimal

cages were changed at frequency of once or more per two weeks. The animalioom

was washed with water, drained and cleaned with a disinfectant- soaked mop every day.

-2-
Page 55 of 101 PPL FINAL REPORT 0~7

4. Constitution of Test Groups, Reason for Selection of Dose Levels and Selection of

Animals

Constitution of the test groups is as shown below.

Sensitization Challenge Am sf

Group Sex

Article for DoeArticle for Dose (Animal os.)

sensitization Doechallenge

MEMORY MEMORY 10 -

I GREEN 0.05 g/site X 3 times RSEN 0.05 glsite (Nos. 286 295)

CRYSTALCRSA

MEMORY5

TI Non-treated GREEN 0.05 glsite (Nos. 2 9 -300)

_______________CRYSTAL ____

0.05 glsite x 3 times of MEMORY GREEN CRYSTAL was applied to the sensitiz tion

group and 0.05 glsite of MEMORY GREEN CRYSTAL was applied to the challenge

group. The usual dosage level for the Buheler Test method was selected. After th

health condition and body weight of the animals a day before the start of the sensitiz tion

was confirmed, 15 animals were selected. A study animal number was given to the

individual animals, in the order of younger quarantine numbers. The study animals were

identified by ear tag number, and a label was attached each cage bearing the study n mber,

animal number, study group number and sex. After group assignment, the remaini g

animals were excluded from the study.

5. Route and Method for Dosing

The method, frequency and period of dosing were determined according to the B ehler

Test method'), of which is described in the guidelines for toxicity studies of skin

sensitization to drugs.

5.1. Sensitization Method

The hair of the animals was clipped (about 4 x 5 cm) on the back of the ne k

region with a clipper (DC-5, Shimizu Denki Kogyo Co. Ltd.) and was shaved ith

an electric shaver (BS 5 597, BRAUN). The clipped skin area was wiped of with a

piece of sanitary cotton containing ethanol. 0.05 g of article for sensitizatio was

loaded onto a 2 x, 2 cm. piece of lint cloth (Yamato Kogyo Co. Ltd.). The ii t cloth

was affixed on the skin area and covered with a sheet of laboratory film

(PARAFILM'M, American National Can TM). Elastic adhesive bandage

(NICHIBAN -ELASTOPORE, No.50-5) was bound around the neck to hold he skin

-3-
Page 56 of 101 PPL FINAL REPORT: P0 0071

application for 6 hours. The first dosing day was determined as-day 0.- The

procedure of 6 hour occlusive application a day was performed a total of three t mes

on day 0, day 7 and day 14 for two weeks at frequency of once per week (at the

interval of 7 days). The non-sensitized group was free from this sensitization

procedure.

5.2. Challenge

Two weeks after the final sensitization (day 28), the hair of the animals was

clipped on the back of the neck region with a clipper and was shaved with an el ctric

shaver. One square part measuring 2 x 2 cmn was obtained from theletfak A

piece of 2 x 2 cm lint cloth loaded with 0.05 g of article for challenge, was affix. d on

the square part, and was covered with a sheet of laboratory film. After 6 hour , the

animals were free from the occlusive application, and the article was wiped off..

Group 1, 11

Anterior

LMEMORY GREEN CRYSTAI,

Posterior

*2x2 cm

-4-
Page 57 of 101 PPL FINAL REPORT: P02 071

6. Method and Frequency of Observation and Evaluation of the Applied Skin

The applied skin was scored for the grade of erythema or edema 24 and 48 hours

after the removal of the occlusive application according to the standards for evalua ion

of Draize') as shown below.

(1) Erythema and eschar formation

0 No erythema

I Very slight erythema (barely perceptible)

2 Well defined erythema

3 Moderate to severe erythema

4 Severe erythema (beet redness) to slight eschar formation

(injuries in depth)

(2) Edema formation

0 No edema

1 Very slight edema (barely perceptible)

2 Slight edema(edges of area well defined by definite

raising)

3 Moderate edema (raised approximately I mm)

4 Severe edema (raised more than 1 mm and extending

beyond area of exposure)

Mean score =X~)()

Number of tested animals

Positive rate _Number of positive animals*X10

M% Number of tested animals

*Number of animals showing skin response of a score of one or more in (1) r (2)

items.

The mean score of skin response and the positive rate were calculated at each

observation time of 24 and 48 hours after the removal of the occlusive applicatio

The skin sensitivity of test article was evaluated based on these parameters.

7. Clinical Observation and Body Weight Measurement

Clinical observation was performed twice at pre- and post-application of the

sensitization and the challenge, respectively. The animals were weighed on th first

sensitization day, the challenge day and the observation day 48 hours after remo al of

the occlusive application.

-5 -
Page 58 of 101 PPL FINAL REPORT: P0 0071

8. Photography

The photography was performed 24 and 48 hours after the challenge.

9. Statistical Analysis

Mean value and standard deviation of body weight for each group were calculate

Statistical analysis was not performed.

RESULTS

1. Clinical Observation

No abnormal clinical signs were observed in each group during the sensitization eriod.

2. Body Weight

Body weights of each group are shown in Table 1.

No abnormal changes to the body weight were observed in any group.

3. Skin Reaction

The score of skin response and the positive rate are shown in Table 2 and Appen ~x1,

and photographs of the skin are shown in Photographs I and 2.

In MEMORY GREEN CRYSTAL sensitized animals (Group I), no skin reaction

induced by MEMORY GREEN CRYSTAL challenge was seen on any of the applie sites

of 10 animals during the observation times of 24 and 48 hours after the challenge. e

positive rate was 0 %, and the mean scores of skin response were 0 at each observat on

time, respectively.

In non-sensitized animals(Group HI), no skin reaction induced by MEMORY G EN

CRYSTAL application was seen on any of the applied sites of 5 animals during the

observation times of 24 and 48 hours after the challenge. The positive rate was 0 0,and

the mean score of skin response was 0 at each observation time, respectively.

DISCUSSION

The potency of skin sensitivity for MEMORY GREEN CRYSTAL was investigated using

guinea pigs according to the Buehler Test method ').

In the case that MEMORY GREEN CRYSTAL was applied on the skin of the non-

-6-
Page 59 of 101 PPL FINAL REPORT: P-02 71

sensitized group, no skin reactions were seen on any of the applied sites at each observatio

time and the skin response by irritation of MEMORY GREEN CRYSTAL could not be

recognized. In the case that MEMORY GREEN CRYSTAL was challenged on the skin f

the MEMORY GREEN CRYSTAL sensitized group, no skin reaction was seen on any of he

applied sites at each observation time and the skin response by sensitivity of MEMORY

GREEN CRYSTAL could not be recognized.

From the above mentioned, the skin sensitivity of MEMORY GREEN CRYSTAL coul

not be recognized under the present study conditions.

REFERENCES

1) Buehler E.V. (1965) :Delayed contact hypersensitivity in the guinea pig. Arch.

Dermatol., 91, 171.

2) Draize, J.H. (1959): "Dermal toxicity", Appraisal of the safety of chemicals in foo s,

drugs and cosmetics, p4.6-59, Association of Food & Drug Officials of the United St tes,

Topeka, Texas.

-7 -
Page 60 of 101

Study &o.P020071

Table I Body weights of guinea pigs in skin sensitization study of MEMORY GREEN CRYSTAL

Group Animal Body weights (g)

number Day 0') Day 28 b) Day 30 c

286 397.1 592.5 593.2

287 374.5 516.6 516.4

288 412.4 582.8 582.9

289 432.6 666.9 667.2

290 374.3 530.4 531.6

1291 402.7 578.6 577.2

292 390.1 559.6 562.1

293 392.9 603.1 601.9

294 377.9 583.3 583.4

295 373.1 546.9 546.6

Mean 392.8 576.1 576.3

±S.D. 19.4 42.3 42.1

296 357.9 569.8 567.2

297 388.9 531.5 534.7

298 391.0 605.0 607.1

UI 299 400.5 636.4 638.5

300 393.3 553.4 555.4

Mean 386.3 579.2 580.6

:±S.D. 16.5 41.7 41.8

a) Day of the start of the sensitization

b) Day of the challenge

c) Day of the final observation
Page 61 of 101

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Page 62 of 101 PPL FINAL REPORT :P020071

Photograph 1

The skin response of the sensitized guinea pigs

24 hours after application of MEM )RY GREEN

CRYSTAL.

Animal No. :288

24hr MEMORY GREEN CRYSTAL I eft side

No.2S8No skin reaction is seen.

Phoorp2

The skin response of the non-sensil zed guinea pigs

24 hours after MEMORY GREEN CRYSTAL

application.

Animal No. :300

P020071 MEMORY GREEN CRYSTAL /Left side

24hr No skin reaction is seen.

No.300

10-
Page 63 of 101 PPL FINAL REPORT: P 20071

Skin Sensitization Study of MEMORY GREEN CRYSTAL in Guinea Pigs

according to Buehler Test Method

(Individual data)

Appendix I Individual skin scores of guinea pigs in skin sensitization study of MEM RY

GREEN CRYSTAL
Page 64 of 101

SuyNo.P020071

Appendix 1 Individual skin scores of guinea pigs in skin sensitization study of MEMORY GREEN CR)STAL

Score of skin response ____

Article for Article for Animal 24 h?) 48 hr?

GOP sensitization challenge number E em d Edema E~hm d Ed-ma

eschar c) eschar

frainb) formation fomtinb formation'c

286 0 0 0D

287 0 0 0

288 0 0 0

289 0 0 0

MEMORY MEMORY 290 0 0 0

GREEN GREEN

CRYSTAL CRYSTAL 291 0 0 0

292 0 0 0

293 0 0 0

294 0 0 0

295 0 0 0 ____

296 0 0 0

MEMORY 297 0 0 00

HI GREEN 298 0 0 0 0

CRYSTAL 299 0 0 0 0

300 0 0 0 0

a) Time after removal of occlussive application.

b) Score of erythema formation: 0; No erythema

1; Very slight erythemna (barely perceptible)

2; Well defined erythema

3; Moderate to severe erythema i

4; Severe erythema (beet redness) to slight eschar formation (injuried i depth)'

c) Score of edema formation: 0-; No edema

I1; Very slight edema (barely perceptible)

2; Slight edema (edges of area well defined by definite raising)

3; Moderate edema (raised approximately I mm)

4; Severe edema (raised more than I mm and extending beyond area of exposure)
Page 65 of 101 PPL FINAL REPORT: P20070

VERIFICATION OF TRANSLATION

This is to certify that the English translation presented here conforms to the ogna

Japanese report.

Study No.: P020070

Title: Primary Skin Irritation Study of MEMORY GREEN CRYSTAL in Rabbits

A/'

Masami Kokubu Date

Panapharm Laboratories Co., Ltd.

1 /& /A2 
Page 66 of 101 PPL FINAL REPORT: P)20070

Primary Skin Irritation Study of

MEMORY GREEN CRYSTAL in Rabbits

FINAL REPORT -

June 27, 2002

Sponsor: Mitsubishi Gas Chemical Company, Inc.

2- 5-2, Marunouchi, Chiyoda-ku,

Tokyo 100-8324, Japan

Organization under Contractor:

Mitsubishi Chemical Safety Institute Ltd.

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

Test Facility: Safety Assessment Laboratory

Panapharm Laboratories (PPL) Co., Ltd.

1285, Kurisaki-machi, Uto-shi,

Kumamoto 869-0425, Japan
Page 67 of 101 PPL FINAL REPORT: P(20070

SIGNATURE PAGE

Study Director

Signed in the original Seal affixed June 27, 2002

Masami Kokubu Date

Panapharmn Laboratories Co., Ltd.
Page 68 of 101 PPL FINAL REPORT: P(20070

STUDY SCHEDULE

1. Study inception: May 2, 2002

2. Animal receipt: May 7, 2002

3. Administration: May 14, 2002

4. Observation period: May 15, 2002 - May 17, 2002

5. Study completion: June 27, 2002

RETAINED MATERIALS AND THEIR REPOSITORY

1. Records concerning the protocol

2. Records concerning the animals

3. Records concerning the animal room conditions

4. Records concerning the test article

5. Records concerning the results of the study

6. Final reports

7. Other materials concerning the study

The above materials shall be kept in the archives of Panapharm Laboratorie Co.,

Ltd. for 10 years after completion of the study (from June 27, 2002 to June 26,-2012).

The handling of the above materials after 10 years shall be determined throug

discussion with the sponsor.
Page 69 of 101 PPL FINAL REPORT: 1020070

PARTICIPATING PERSONNEL IN THE STUDY

1. Study director: Masami Kokubu

2. Responsible technician: Shigenari Okumura

3. Person responsible for test article management:

Hiroyuki Izumi

4. Person responsible for quarantine (veterinarian):

Emiko Kuwasaki

5. Preparatioii of test article, etc.:

Chikako Tokumitsu and Naoko Kimura

6. Animal receipt: Shigenari Okumura

7. Animal care: Shigenari Okumura, Kazuaki Sasaki, Tatsumi In i,

Hironori Shigekane and Misuzu Ueba

8. Group assignment: Shigenari Okumura

9. Clinical observation: Shigenari Okumura, Toshihiro Katsuta, Kazuaki Sasaki and

Tatsumi Inui

10. Body weight measurement: Shigenari Okumura, Toshihiro Katsuta ai d Hironori

Shigekane

11. Administration: Toshihiro Katsuta

12. Scoring: Masami Kokubu. and Toshihiro Katsuta

13. Photography: Shigenari Okumura and Toshihiro Katsuta
Page 70 of 101 PPL FINAL REPORT: P 20070

CONTENTS

Page

SIGNATURE PAGE...............

STUDY SCHEDULE.......................................i

RETAINED MATERIALS AND THEIR REPOSITORY

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

PARTICIPATING PERSONNEL IN THE STUDY

SUMMARY 1...........

OBJECTIVE ........... .... 2

MATERIALS AND METHODS....................................2

RESULTS ............ ... 6

DISCUSSION .......... ..... 6

REFERENCES .......... ..... 6

Table 1 Body weights of rabbits in primary skin irritation study of

MEMORY GREEN CRYSTAL

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

Table 2 Skin scores of rabbits in primary skin irritation study of

MEMORY GREEN CRYSTAL

Photograph I......................................... .9
Page 71 of 101 PPL FINAL REPORT: P(20070

SUMMARY

The primary skin irritation for MEMORY GREEN CRYSTAL was investigated using ma e

New Zealand White rabbits.

No skin reaction was observed on any of intact and abraded skin from 24 hours to 72 hours

after MEMORY GREEN CRYSTAL occlusive application. The primary irritation index (...

was 0. MEMORY GREEN CRYSTAL was evaluated as mildly irritants.

From the above mentioned, the primary irritation of MEMORY GREEN CRYSTAL coul Inot

be recognized under the present study conditions.
Page 72 of 101 PPL FINAL REPORT: P20070

OBJECTIVE

As a part of non-clinical studies of the safety evaluation of MEMORY GREEN CRYSTA L, the

primary skin irritation study was carried out using rabbits.

MATERIALS AND METHODS

1. Test Article

MEMORY GREEN CRYSTAL (Lot No.MG-R02) provided by Mitsubishi Gas

Chemical Company, Inc. was used as the test article. Japanese pharmacopoeia wat r for

injection (Lot No.1I E90, Otsuka Pharmaceutical Factory, Inc.) was used as the vehicle

The test article was preserved in a desiccator in the test article room at room temper Lture.

The vehicle was preserved in the test article room at room temperature.

2. Preparation of Test Article

MEMORY GREEN CRYSTAL and water for injection was mixed at the rate of 0 5g of

MEMORY GREEN CRYSTAL to 3 mL of water for injection, and the mixture was ellkneaded to prepare the gel like material. These preparations were conducted

immediately before use.

3. Animals and Husbandry

Five male New Zealand White (Kbs:NZW) Rabbits aged 4 months were purc ased

from Kitayama Labes Co., Ltd. The weights of some animals at the receipt were less

than the minimum value (3.0 kg) of weights at the time of receipt in a protocol. The

reduction in body weights of the animals, was judged to be due to the stress duriv g the

transportation, because no abnormal clinical signs were observed in any of the animals

at the receipt of animals. The deviation of protocol was judged not to affect the study,

since there were no clinical sign of change in the animals and the body weights

increased smoothly after the receipt. The animals were quarantined and acclim ted

for six days. In this period, all animals were examined for clinical signs and weighed

to confirm that they were healthy. Three animals were selected and used for the study.

Body weights on the application day were 2.497-2.986 kg. The animals were hused

in an aluminum cages (Wx H xD: 430x360x700 mm) at one per cage in the animal

room (No. 14) in the rabbit and guinea pig area of the metabolism-pharmacology

research institute, which was controlled at a temperature of 24±2'C (permissible range:

21-27C), relative humidity 55±1 0% (permissible range: 3 5-75%), 12-hour lighqdark

cycle (lighting from 7:00A.M. to 7:00P.M.) and 13-15 changes/hour ventilation.

-2-
Page73 o 101PPL FINAL REPORT: P)20070

Actual ranges of temperature and relative humidity in the animal room during th study

were 23-25 'C and 54-65 %, respectively. The animals were allowed to receiv a

pellet diet (RC4, Oriental Yeast Co., Ltd.) and to drink well water supplemented with

NaCIO (about 0.2 ppm) ad libitum. The animal cages were changed once at gr up

assignment. The animal room was washed with water, drained and cleaned wit a

disinfectant-soaked mop every day.

4. Constitution of Test Group, Reason for Selection of Dose Levels and Selection f

Animals

Three animals of one group were used in this study.

Test article Dose Sex Number of Animal Nos. _____________(glsite) animals

MEMORY

GREEN 0.5 6" 3 No. 1-3

CRYSTAL _________ ____

The hair of the animals on the back of their necks was gently clipped with a clipper

(DC-6, Shimiizu Denki Kogyo Co. Ltd.) a day before the start of application. The ;kin

condition was observed, and three healthy animals, which had slight island skin and good

skin condition for the application, were selected, and a study animal number was given to

the individual animals, in the order of younger quarantine numbers. The animals ere

identified by numbers on the inside of their left ear, marked with a black oily felt-ti pen,

and each cage had cage cards attached with study number, animal number and sex. The

remaining animals after the animal selection were excluded from the study.

The dose of 0.5 glsite MEMORY GREEN CRYSTAL was selected as the genera dose

level for the primary skin irritation study.

5. Route and Method for Dosing

Percutaneous route by occlusive application was selected because of the usua

administration route of primary skin irritation study. The hair clipped on their backs

was divided into two parts and a 2.5 X 2.5 cm square dotted by means of a blac oily

felt-tip pen was marked in each part. A # -shaped abrasion was inflicted with 23G

needle in one of the square sites on either the right or left side. Non-injured (iritact)

skin and injured (abraded) skin was one set, and the test article was applied on I ese

squares.

-3-
Page 74 of 101 PPL FINAL REPORT: P)20070

A 0.5 g of the gel-like test article was loaded uniformly onto a piece of 2.5 X 2.5 -m

square lint cloth. The lint cloth was affixed to each square of the clipped skin ith a

piece of 4 X4 cm mesh bandage (NICHIBAN -MESHPOREO, No.50-10). The mnal

was bound around the body with an net bandage (NABIS -NET BANDAGE No f4,

AO-2424-04) to maintain the 24 hour occlusive application. The animal was

equipped with a protective collar. The lint cloth was removed from the applied skin

after 24 hours. At the same time, the dosing article was removed and the skin s juare

was wiped with a piece of sanitary cotton containing distilled water.

< Application pattern >

Animal No. Animal No.

1,3 2

Anterior Anterior

Memory Memory Memory I Memory

Green I Green Green Green

Crystal Crystal Crystal I Crystal

2.5 I2.5

cm cm

2.T5 cm 2.5 cm

Posterior Posterior

6. Observation, Evaluation and Measurement

The skin reaction on the applied skin of all animals was scored according to t ie

procedure for evaluation of skin reactions by Draize') 24 hours, 48 hours and 72 ours

after the application.

The scores of reactions (erythema, eschar and edema formation) of the intact kin

and abraded skin in each animal were summed up 24 hours and 72 hours after t

application. The sum of the scores was divided with 4 to obtain mean score.

-4-
JPage 75 of 101 PPL FINAL REPORT: P(20070

6.1. Erythema and eschar formation

Score Skin reaction

O No erythema

1 Very slight erythema (barely perceptible)

2 Well defined erythema

3 Moderate to severe erythema

4 Severe erythema (beet redness) to slight eschar

____formation (injuries in depth)

6.2. Edema formation

Score Skin reaction

0 No edema

1 Very slight edema (barely perceptible)

2 Slight edema (edges of area well defined by

2 definite raising)

3 Moderate edema (raised approximately I mm)

4 Severe edema (raised more than 1 mmn and

extending beyond area of exposure)

The mean score of all animals was calculated to obtain primary irritation index

(P.1.1., The maximum 8 points). The irritation grade was evaluated on the b sis of

scale shown below. Photography of the application skin was carried out r the

typical sample at each following time.

o : P.M. 5 2 Mildly irritants

2 < P.M.. :_ 5 Moderately irritants

5 < P.M.. !- 8 Severe irritants

The animals were observed once a day and weighed at pre-application on the

application day and the final observation day.

7. Statistical Analysis

Mean value and standard deviation of body weight were calculated. Statistical

analysis was not performed.

-5-
* PPL FINAL REPORT: Pi 120070 Page 76 of 101

RESULTS

1. Clinical Observation

No abnormal clinical signs were observed in any animal.

2. Body Weight

Body weights are shown in Table 1.

All animals showed favorable weight transition.

3. Skin Reaction

Skin reactions are shown in Table 2.

For MEMORY GREEN CRYSTAL occlusive application, no skin reactions on either the

intact or abraded skin were observed at observation times of 24 hours, 48 hours and 2

hours after the occlusive application. The P.1.I. was 0.

DISCUSSION

After the gel-like article and water for injection was absorbed in MEMORY GREEN

CRYSTAL, it was applied occiusivelly for 24 hours, the skin condition of the applied are was

observed. No skin reaction on any of the sites of the intact or abraded skin area was

observed at any observation time. The P.1.1. was 0. MEMORY GREEN CRYSTAL was

evaluated as a mildly irritant from the scale of P.1.1. by Draize.

From the above mentioned, primary skin irritation of MEMORY GREEN CRYSTAI could

not recognized.

REFERENCES

1) Draize, J.H. (1959) :"Dermal toxicity", Appraisal of the safety of chemicals in foods,

drugs and cosmetics, p46-59, Association of Food & Drug Officials of the United States,

Topeka, Texas.

-6-
Page 77 of 101

-0 cn W) C

>1cr ON C) Ci 1=

0- 4r4"C

in.H

I-.4
Page 78 of 101

0.

ccl

c,0 00

*00

00

z 0 'd I

.0C- e

C< . ~- .~cc

~ ~0c0

~ cc

cuU

~ 0 0 0

~~8
ray%= Ivui iuu YrL rNAL KJrUKI: ruz/-UU

Page 79 of 101

Photogrph I

~ Skin of 24 hours after the application

:r >~'Animal No.2.

~ MEMORY GREEN CRYSTAL:

24wNo reaction at sites ofmitact and

abraded skins.

9
Page80 o 101PPL FINAL REPORT: P 0069

VERIFICATION OF TRANSLATION

This is to certify that the English translation presented here conforms to the orignal

Japanese report.

Study No.: P020069

Title: Single Oral Administration Toxicity Study of MEMORY GREEN CRYSTAL in

Rats (Limit Test)

Kumiko Honda Date

Panapharm Laboratories Co., Ltd.
Page 81 of 101

PPL FINAL REPORT: PC 20069

Single Oral Administration Toxicity Study of

MEMORY GREEN CRYSTAL in Rats

(Limit Test)

FINAL REPORT -

June 25, 2002

Sponsor: Mitsubishi Gas Chemical Company, Inc.

2-5-2, Marunouchi, Chiyoda-ku,

Tokyo 100-8324, Japan

Organization under Contract:

Mitsubishi Chemical Safety Institute Ltd.

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

Test Facility: Safety Assessment Laboratory

Panapharm Laboratories (PPL) Co., Ltd.

1285, Kurisaki-machi, Uto-shi,

Kumamoto 869-0425, Japan
Page 82 of 101

PPL FINAL REPORT: P0' 0069

SIGNATURE PAGE

Study Director

Signed in the original Seal affixed June 25, 2002

Kumiko Honda Date

Panapharm Laboratories Co., Ltd.
Page 83 of 101

PPL FINAL REPORT: P0K 0069

STUDY SCHEDULE

1. Study inception: April 25, 2002

2. Animal receipt: May 7, 2002

3. Administration: May 14, 2002

4. Necropsy: May 28, 2002

5. Study completion: June 25, 2002

ADVENTITIOUS CIRCUMSTANCES AFFECTING THE RELIABILITY 0

THLE STUDY AND DEVIATION FROM THE PROTOCOL

There were no adventitious circumstances affecting the reliability of the stud

and no deviation from the protocol.

RETAINED MATERIALS AND THEIR REPOSITORY

1. Records concerning the protocol

2. Records concerning the animals

3. Records concerning the animal room conditions

4. Records concerning the test article

5. Records concerning the results of the study

6. CD-R

7. Final reports

8. Other materials concerning the study

The above materials shall be kept in the archive of Panapharm Laboratod es

Co., Ltd. for 10 years after completion of the study (from June 26, 2002 to. une

25, 2012). The handling of the above materials after 10 years shall be

determined through discussion with the sponsor.

PARTICIPATING PERSONNEL IN THE STUDY

1. Study director: Kumiko Honda

2. Responsible technician: Eiji Murata

3. Person responsible for pathological examination:

Masao 'Hamamura

4. Person responsible for test article management:

Hiroyuki Izumi

5. Person responsible for quarantine (veterinarian):

Emiko Kuwasaki

6. Preparation of test article mixture, etc.:

Noriko Kojima, and Naoko Kimura
Page 84 of 101

PPL FINAL REPORT: PO 0069

7. Animal receipt: Yasuo Nakaoka

8. Animal care: Eiji Murata, Teruhiko Enoshima, Yasuo Nakaok

Tomohiro Ikeda, Hidehiro Ogata, Kumiko Hond

Sayumi Tominaga, Misako Ueba, Noriko, Kojima.

Akemi Goto and Yukari Douki

9. Group assignment: Eiji Murata

10. Administration: Eiji Murata

11. Clinical observation: Eiji Murata, Teruhiko Enoshima, Yasuo Nakaok;t

Tomohiro Ikceda, Hidehiro Ogata, Kumiko Hond;t

Sayumi Tominaga, Misako Ueba and Noriko Ko. inia

12. Body weight measurement: Eiji Murata, Teruhiko Enoshima, Yasuo Nakaokk

Kumiko Honda, Akemi Goto and Yukari Douki

13. Necropsy: Takafumi Oshikata, Yutaka Nakahara and Hideyaki

Watanabe

14. Management of computer system:

Ryoichi Tagami and Miho Watanabe
Page 85 of 101

PPL FINAL REPORT: P 0069

CONTENTS

SIGNATURE PAGE ........

STUDY SCHEDULE......... i

ADVENTITIOUS CIRCUMSTANCES AFFECTING THE RELIABILITY OF

THE STUDY AND DEVIATIONS FROM THE PROTOCOL

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

RETAINED MATERIALS AND THEIR REPOSITORY...................iii

PARTICIPATING PERSONNEL IN THE STUDY......................iii

SUMMARY ......... 1

OBJECTIVE ......... 2

MATERIALS AND METHODS ......... 2

RESULTS ......... 4

DISCUSSION ......... 5

Figure 1 Body weight changes.................................6

Table 1 Clinical signs 7

Table 2 Body weights .. .......... 9

Table 3 Necropsy findings . ........... 10

Appendices

-iv -
Page 86 of 101

PPL FINAL REPORT: PO 069

SUMM4ARY

MEMORY GREEN CRYSTAL was orally administered to SD-IGS rats (5 male ind

5 female Crj:CD rats) at a dose level of 2000 mg/kg.

No death occurred in any animal during the observation period. There were no

changes ascribable to administration of the test article in any of the clinical observa -ion,

body weight measurement or necropsy.

In conclusion, under the conditions of this study, it was considered that the

approximate lethal dose in rats of both sexes was above 2000 mg/kg.
Page 87 of 101

PPL FINAL REPORT: P02 069

OBJECTIVE

As a part of non-clinical studies of the safety evaluation, MEMORY GREEN

CRYSTAL was orally administered to rats to evaluate its acute toxicity.

MATERIALS AND METHODS

1. Test Article

MEMORY GREEN CRYSTAL (lot No. MG-R02) provided by Mitsubishi Gas

Chemical Company, Inc. was used as the test article. Corn oil (lot No. V1P5345

Nacalai Tesque Co., Ltd.) was used as a vehicle. The test article was stored ina

desiccator, and the corn oil was stored in a locker in the test article room at room

temperature.

2. Animals and Husbandry

S even male and female rats (Crj :CD(SD)IGS) aged 5 weeks old were purchas d

from Charles River Japan, Inc. The animals were quarantined and acclimatize for

6 days. During this period, all the animals were examined for clinical signs anl

weighed in order to confirm the in health status. Finally, 5 males and 5 femnales

were selected by the randomization method on the day before dosing and emplo red

in this study at the age of 6 weeks. The body weights on the day of dosing wer

165.5-169.6 g for the males and 117.3-121.5 g for the females. The animals were

housed in an animal room (No. 09) in barrier system area a controlled at a

temperature of 24t20C (permissible range: 21-27'C) and relative humidity of

55±10% (permissible range: 35-75%) with a 12-hour light/dark cycle (lighting fom

7:00 A.M. to 7:00 P.M.) and 13-15 ventilation changes/hour. The animals wer.

housed individually in stainless steel wire bottom cages (WxHxD: 260xH200xD380

mm). The actual temperature and relative humidity of the animal room during the

study were 22-26'C and 53-64%, respectively. The animals were given access to

pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and well water

supplemented with sodium hypochlorite (approximately 2 ppm) ad libitum fro an

automatic dispenser or feeding bottle. Diet containing impurities below the

permissible limit, as verified by Japan Food Research' Laboratories (a juridical

foundation, Tokyo, Japan) at the request of Oriental Yeast Co. Ltd., was purchased.

The water was analyzed at Kakujo Nankyukaken Center Ltd,. (Kumamoto, Japan)

and verified as satisfying the requirements of the Water Quality Standard

(Waterworks Law). All housing equipment and diet were sterilized by autoel ving

-2-
Page 88 of 101

PPL FINAL REPORT: P 069

before use. Cages and cage racks were changed once at group assignment, and the

sanitary trays under the cages were changed 2 times a week. The animal room was

cleaned and wiped with a disinfectant-soaked mop every day.

3.. Constitution of Test Group, Reason for Determination of Dose Levels and

Group Assignment

Constitution of the test groups is tabulated below.

Group Dose Concentration Dose volume Sex No. of Anij al (mg/kg) (mg/niL) (niL/kg) animals numer

M 5 101-05 2000 mg/kg 2000 100 20F 5 20-5

Based on the information on the test article analysis provided by the sponsor, the

test article was considered to be hypotoxic. The dose level was set at 2000 mg kg,

which was the highest dose of the toxicity guidelines.

The numbers of animals were 5 males and 5 females. On the day before do ing,

the animals were divided into groups by the randomization method on the basis f

body weights. After the animal assignment, the animals were identified

individually by tail number with an oily felt pen and ear punch. A cage card 11 ting

the study number, animal number, dose level and sex was attached to each cage.

The animals that remained after grouping were excluded from the study.

4. Route and Method for Administration

The oral route was chosen based on the expected route of human exposure. All

the animals were fasted overnight (for about 18 hours). The test article mixtui e

was administered to the rats once by oral gavage. The amount of the dosing

solution was calculated based in the individual body weight on the day of

administration. The rats were given food about 3 hours after the administraticn

5. Method of Preparation of The Test Article Mixture with The Vehicle and

Frequency of Preparation

Just before dosing, the test article was suspended in corn oil at a concentrati n of

100 mg/mL. The containers of the test article mixture were labeled with the udy

number, test article name, date of preparation and name of the preparing perso nel.

-3-
Page 89 of 101

PPL FINAL REPORT: P920069

6. Method and Frequency of Observation, Examination and Measurement

1) Clinical Observation

The observation period was for 14 days after administration. All the animals

were observed for clinical signs and mortality until 6 hours after administration

on the day of administration (Day 0) and once daily thereafter.

2) Body Weight Measurement

The body weights were measured before administration (Day 0) and on D. LYS 1,

3, 7, 10 and 14.

3) Necropsy

All the animals were anesthetized with either and necropsied at the end of the

observation period for 14 days.

7. Statistical Analysis

The mean and standard deviation of body weights were calculated.

RESULTS

1. Clinical Observation

The clinical signs are presented in Table 1 and Appendix 1.

Diarrheal stool was observed in 2 males and 3 females from 1 to 3 hours alter

administration. In addition, soiled periproctal was observed in 1 female at 1.hour

after administration. No abnormal signs were observed in any animals thereafter.

2. Body Weight

The body weight changes are presented in Figurel, Table 2 and Appendix 2.

The body weights of all the animals increased normally during the observation

period.

3. Necropsy

The results are presented in Table 3 and Appendix 3.

No abnormal findings were observed in any animal.

-4-
Page 90 of 101REOT 206

DISCUSSION

MEMORY GREEN CRYSTAL was orally administered to male and female rats at a

dose level of 2000 mg/kg.

No death occurred in any animal during the observation period. Diarrheal stool was

observed in both sexes from 1 to 3 hours after administration. Soiled periproctal ias

observed in 1 female from 1 to 3 hours after administration. These signs had

disappeared by 6 hours after administration. In addition, there were no changes

ascribable to administration of the test article in the body weight measurement or

necropsy. The diarrheal stool and soiled periproctal observed in this study were

considered to have been induced by corn oil and not related to administration of th( test

article.

In conclusion, under the conditions of this study, it was considered that the

approximate lethal dose in rats of both sexes was above 2000 mg/kg.

-5-
Page 91 of 101

Study No P020069

-- 2000 mg/kg 

M~

300-

S200-

100

0 1 3 7 10 14

Days

400 F

--0- 2000 mg/kg

'~300-

200-

0 1 3 7 10 14

Days

Figue 1 Body weight changes

6
Page 92 of 101

0-

).o -x m

=ll

ucD u

w C13 -- --

-n s :v

w- w c 0u

0 ./ 0 0
Page 93 of 101

0,

0~

C

C.-,

0- ~0 U~ U0 ~0

0- ~0 uo uo

0- .~0 U0 U~ ~J0

0- tO

0-

0- C.~0 = *.tO UO =

0- UO ~.tO ~t0C.tO ~

00

0- UO ~.t0 U0 C.tO ~

C.-

0- CO ~.tO ~0

0- UO UO UO UO

tO

C - C -~

OC .0-0 . 0-00

C ~-0 ~-0~-

00 0 tO.- tO.C.

.0.) 00 t.t-Qt ~4Cfl

QtO C)CV S

tO 2.- tO-O~

- tO C... tO C.- tO C. tOO) - 000) 000)

tO 0.0 0.0C

- L) C...fl~ C.-0)

0 -~ tO .0 tO

.-. tO tOO.- tO0.-'0

- ~ ~

*0

0.) 00 00

C -~

C ~ ~

00 00 tO tO

C C. o 0 0) 0) 0 -~ C~

~CO

I C.~tO~C -.

0)

0) 0) tO

.0 - tO O 0) tO 0) C-,

8
Page 94 of 101

02-

r,-

_d cm~

Co m;-

cu0

CD

ce

Eo

C49
u Page 95 of 101

CCM

C.)D

o o 

01
Page96 o 101PPL FINAL REPORT: F020069

CONTENTS

Appendix 1 Individual clinical signs - Male, Female.......................1

Appendix 2 Individual body weights - Male, Female.......................3

Appendix 3 Individual necropsy findings - Male, Female, Day 14 ............... 4
Page 97 of 101

o r~ < It II I I

*0

0

I III III

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liii I I I I

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Page 98 of 101

0

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Page 99 of 101

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Page 101 of 101