Ii6ilI·liUIUHUJUIU·Iftll!1IhfUUilt;lIU1ttli!Ii•illiUJIIt'lflJif;.Ji!1..:;iII..IlltIi . . .ItI0=.,.;illittillfllI':;;g.,c:•§.tlrh'.Jl'h111·1°=nmJ•erlr0=.htH!!Iltfif,lo:::::i:;a"ll!oo;;=-P,sft•'-.)ef:::1-1·iIll.I-f1!.tr....I!;IIllfIJi!.....l}iiJ""rli>tlft.f.•iJrJ'•·-1:n--"".111r·r•J·Ill'tlIJrl(nrr -r·IIrfIII;siiiIUJii,11. ,hJ!2..iODODiiDDi·...."p.J1i,l•.'flliO11I1JI{ll1IfihlJJilt!ih1···>li))I('...J·IthI.trillJlJlrJll.I·JJt1i·Iiti11ifirilli••ltr,''1J•n.'IEiIli.Jjrj·1ttJtrlfr!··(....f_fl•.-1fi_1..i(.·.l1fr)1s1.1l.mIt.•.I•rJffIJ...II.!11iL.._:.·•'•srffII1.frU1I,.,i'JrD .iJ,IItt!IIr .a·.I<I"C'Irl·r",.••8'p.2 MB08Tl
Public reporting burden for this collection ofinformation is estimated to average 110 hours per response, including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection ofinformation. Send comments regarding the burden estimate or any other aspect ofthis collection of information, including suggestions for reducing this burden, to Chief, Information-Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M St., S.W., Washington, D.C. 20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION I certify that to the best ofmy knowledge and belief: 1. The company named in Part I, section A, subsection 1 a ofthis notice form intends to manufacture or import for a commercial purpose, other thanin small quantities solely for research and development, the substance identified in Part I, Section B. 2. All information provided in this notice is complete and truthful as ofthe date ofsubmission. 3. I am submitting with this notice all test data in my possession or control and a description ofall other data known to or reasonably ascertainable by me as required by§720.50 ofthe Premanufacture Notification Rule. Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following user fee certification statement that applies: 

## [!] The Company named in Part I, Section A has remitted the fee of$2500 specified in 40 CFR700.45 (b), or 

# D The Company named in Part I, Section A has remitted the fee of$1000 for an Intermediate PMN (defined in accordance with 40CFR700.45(b), or 

# D The Company named in Part I, Section A is a small business concern under 40CFR700.43 and has remitted a fee of $1000 in accordance with 40CFR700.45(b) Ifyou are submitting a low volume exemption (LVE) application in accordance with 40CFR 723.50(c)(1) or a Low release and low exposure exemption (LoREX) application in accordance with 40CFR723.50(c)(2), check the following certification statements: 

# D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under the terms of40CFR723.50. 

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

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

# D Ifthis application is for an L VE in accordance with 40CFR723.50(c)(1), the manufacturer intends to commence ofthe exempted substance for commercial purposes within 1 year ofthe date ofthe expiration ofthe 30-dayreview The accuracy ofthe statements you make in this notice should reflect your best prediction ofthe anticipated facts regarding the chemical substance described herein. Any knowing and wilful misinterpretation is subject to criminal penalty pursuant to 18 

## usc 1001. Confidential Date
Signature ofagent- (ifapplicable) Date

FORM EPA 7710-25 (Rev. 5-95) Page2

## Part I- GENERAL INFORMATION

la. Person Submitting Notice Name ofauthorized official Position 

### Shintaro Vice President (in 

### U.S.)

b. Agent (ifapplicable) Company 

### MORIMURA BROS INC. Mailing address (number and street) 

### Parker Plaza 400 Street lOth Floor City, State, ZIP Code 

### Front NJ 07024 Name ofauthorized official 

### Susan D. M.S. Company 

### ENVIRON International Mailing address (number and street) 

### 4350 North Fairfax Suite 300 City, State, ZIP Code 

### Arlington, VA 22203-1619 Telephone 

### c. Ifyou are submitting this notice as Name ofauthorized official 

### oint submission, mark (x) this box.



### 2. Technical Contact (in U.S.) Company Mailing address (number and street) City, State, ZIP Code Name ofauthorized official 

### Susan D. M.S. Company 

### ENVIRON International Mailing address (number and street) 

### 4350 North Fairfax Suite 300 City, State, ZIP Code 

### Arlington, VA 22203-1619 3. Ifyou have had a prenotice communication (PC) concerning this notice and EPA assign a PC Number to the notice, enter the number. 4. Ifyou previously submitted an exemption application for the chemical substance coveredbythis notice, enterthe exemption number assigned by EPA. Ifyou previously submitted a PMN for this substance enter the PMN number EPA withdrawn or 5. Ifyou have submitted a notice ofBona fide intent to manufacture or import for the chemical substance covered by this notice, enter the notice number EPA. 6. Type ofNotice
- Mark (X)

FORM EPA 7710-25 (Rev. 5-95) ( ) Manufacture Only 1. ( ) Binding Option Mark(X)

Page3 Position

Telephone Position

Telephone

(X) Import Only 

### 2. ( ) Binding Option Mark p.3 MB08Tl

Mark(X) (X) ifnone Mark(X) (X) ifnone Mark(X) (X) Ifnone 

### 3. ( ) Bothp.4 MB08T1 

## Part I- GENERAL INFORMATION- Continued Section B- 

### CHEMICAL IDENTITY INFORMATION: You must correct 

### '-'ll'"lllJ'"'" Abstracts (CA) name ofthe substance based on the ninth Collective Index (9CI) ofCA nomenclature rules and conventions. Mark (X) the "Confidential" box next to any item you claim as confidential.

**Complet~** either item 1 (Class 1 or 2 substances) or 2 (Polymers) as appropriate. Complete all other items. Ifanother person will submit chemical identity information for you, mark (x) the box at the right. 

### [ ] Identify the name, and address ofthat person. 1. Class 1 or 2 chemical substances (for definitions ofclass 1 and class 2 substances, see the Instructions Manual)
a. Class ofsubstance
- Mark (X) 1 [X] Class 1 or 2 [ ] Class 2 b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substance. For Class 1 substance a CA Index Name must be provided. For Class 2 substances either a CA Index Name orCAPreferredName must beprovided, whichever is appropriate based on CA 9CI nomenclature rules and conventions.). 

### Titanate [Ti6013(2-)], dipotassium
c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice (check one): [ ] Method 1 (CAS Inventory Expert Service- a copy ofthe Identification [ ] Method 2 (Other Source) report obtained from the CAS Inventory Expert Service must be submitted as an attachment to this d. Molecular formula and CAS Registry Number (ifa number already exists for the substance).


### CAS # 

### 12056-51-8 e. For a class 1 substance, provide a complete and correct chemical structure diagram. For a class 2 substance- (1) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of reaction orprocess. (3) Indicate the range ofcomposition and the typical composition (where appropriate). ( 4) Provide a correct representative orpartial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. 

### / 

## "' /0 Ti 

### / 

# "' 0 0



# "' / Ti 

# /"'- 0 0



# ""- / Ti 

### / 

# "' /0 

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

FORM EPA 7710-25 (Rev. 5-95) 0 0 

# ""- / Ti 

# /"'- 0 0



# "' / Ti 

### / 

# " /0

Page4 Confidentialp.5 MB08Tl 

## Part I- GENERAL INFORMATION- Continued Section B- CHEMICAL IDENTITY INFORMATION- Continued a definition ofpolymer, see the Instructions Manual.) a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflowmolecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. t----1 Describe the method ofmeasurement or the basis for your estimates: GPC ( X ) Other ( ) : (Specify ___ ___, '-----1 i) Lowest number molecular weight: Daltons. ii) Maximum weight % below 500 molecular weight : ___ %. iii) Maximum weight % below 1000 molecular weight : % 

# D Mark (X) this box ifyou attach a continuation sheet. b. You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the "Confidential" box next to any item you claim as confidential. (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufacture ofthe polymer. (2) Mark (X) this column ifentry in column ( 1) is confidential. (3) Indicate the typical weight percent ofeach monomer or other reactant in the polymer. (4) Mark (X) the identity column ifyou want a monomer or other reactant used at two weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in column (3) and (4) are confidential. (6) Indicate the maximum weight percent ofeach monomer or other reactant that may be present as a residual in the polymer as manufactured for commercial purposes. (7) Mark (X) this column ifentry in column (6) is confidential. Monomers or other reactant and CAS Registry Number (1) Confi- Typical Identity Confi- Maximum Confidential composition Mark (x) dential Residual dential



# D Mark (X) this box ifyou attach a continuation sheet. c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice. [ ] Method 1 (CAS Inventory Expert Service - a copy ofthe Identification report obtained from the CAS Inventory Expert Service must be submitted as an attachment to this notice) [X] Method2 (Other Source) d. The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers.

e. Provide a correct representative orpartial chemical structure diagram, as complete as can be known, ifone can be reasonably be ascertained. 

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

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

### p.G MB08Tl 

## Part I- GENERAL INFORMATION- Continued Section B- CHEMICAL IDENTITY INFORMATION- Continued 3. Impurities a. Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercialpurposes. Provide the CAS RegistryNumberifavailable. Ifthere are unidentified impurities, enter ''unidentified. b. Estimate the maximum % ofeach · Ifthere are unidentified estimate their total %. Impurity and CAS Registry Number (a)



# D Mark (X) this box ifyou attach a continuation sheet. Maximum percent 

### % % % %

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

### Dipotassium hexatitanate 

# DMark (X) this box ifyou attach a continuation sheet. 5. Trade identification

- List trade names for the new chemical substance identified in subsection I or 2. 

### TOFIX-S 

# D Mark (X) this box ifyou attach a continuation sheet. ConfiConfidential

6. Generic chemical name- Ifyou claim chemical identity as confidential, you must provide a generic chemical name for your substance that reveals the specific chemical identity ofthe new chemical substance to the maximum extent possible. Refer to the TSCA Chemical Substance Inventory, 1985 edition, Appendix B for guidance on developing generic names. 

# D Mark (X) this box ifyou attach a continuation sheet. 7. Byproducts

- Describe any byproducts resulting from the manufacture, processing, use, or disposal ofthe new chemical substance. Provide the CAS · Number ifavailable. Byproduct CAS Registry Number Confidential None 

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

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

### p. 7 MB08Tl 

## 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 maximumproductionvolume during the first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during the first three yeas ofproduction. Estimates should be on 100% new chemical substance basis. For a Low Volume Exemption application, 

### if you choose to haveyour notice reviewed at a lowerproduction volume than JO,OOOkg/yr, specify the volume and mark (x) in the binding box. 

### If granted, you are bound to this volume. Maximum first 12-month production (kg/yr) Maximum 12-month production (kg!yr) Confidential Binding Option (100% new chemical substance basis) ( 100% new chemical substance basis) Mark(x) 

# - - X 2. Use Information- You must make separate confidentiality claims for the description ofhe category ofuse, the percent of production volume devoted to each category, the formulation ofthe new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim as confidential. a. (1) Describe each intended category ofuse ofthe new chemical substance by function and application. (2) Mark (X) this column ifentry in column ( 1) is confidential business information (CBI). (3) Indicate your willingness to have the information provided in column (I) binding. (4) Estimate the percent oftotal production for the first three years devoted to each category ofuse (5) Mark (X) this column ifentry in column (4) is confidential business information (CBI). (6) Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7) Mark (X) this column ifentry in column (6) is confidential business information (CBI). (8) Indicate % ofproduct volume expected for the listed "use" sectors. Mark more than one box ifappropriate. Mark (X) to indicate your willingness to have the use type provided in (8) binding. (9) Mark (X) this column ifentry(ies) in column (8) is (are) confidential business information (CBI). Category ofUse (1) CBI Binding Produc- CBI %in CBI % ofsubstance expected per use (8) CBI (by function and application (2) Option tion% (5) Form- (7) Site- Con- Indus- Com- Binding (9)

(i.e. a dispersive dye for Mark(x) (4) ulation limited sumer trial mercia} Option finishing polyester fibers) (3) (6) Friction material for 

### 100% 10% Automotive Brake Pads 

### % % % % % % Ifyou have identified a "consumer use", please provide on a detailed description ofthe use(s) ofthis chemical substance in consumer products. In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product.



# D Mark (X) this box ifyou attach a continuation sheet. b. Generic use Ifyou claim any category ofuse description in subsection 2a as confidential, enter a generic description ofthat description category. Read the Instructions Manual for examples ofgeneric use descriptions.



# D Mark (X) this box ifyou attach a continuation sheet. 3. Hazard Information- Include inthe notice a copyofreasonable facsimile ofanyhazard warning statement, Binding Option label, material safety data sheet, or other information which will be provided to anyperson who is reasonably Mark(x) likely to be exposed to this substance regarding protective equipment or practices for the safe handling, transport, use or disposal ofthe new substance. List in part III hazard information you include. Please refer to the 

### MSDS. 

## [!] Mark (X) this box ifyou attach hazard information.

FORM EPA 7710-25 (Rev. 5-95) Page7p.8 MB08Tl 

## Part II- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Section A- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER 

# --- Complete section A for each type ofmanufacture, processing, or use operation involving the new chemical substance at industrial sites you control. Importers do nothave to complete this section for operations outside the U.S.; however, youmay still have reporting requirements ifthere are further industrial processing or use operations after import. You must describe these See instructions manual. 1. Operation Description Confia. Identity- Enter the 1dtanuy ofthe site at which the will occur. < dential Name 

### TOHO TITANIUM COMPANY, LIMITED Site address 

### 3-3-5 Chigasaki City, County, State, ZIP Code 

### Chigasaki-City, Kanagawa-Pref., Japan, 253-8510 Ifthe same operation will occur at more than one site, enter the number ofsites. Identify the additional sites #ofsites on a continuation sheet, and ifany ofthe sites have significantly different production rates or operations, include all the information requested in this section for those sites as attachments.



# D Mark (X) this box ifyou attach a continuation sheet. b. Type Mark(X) (X) Manufacturing ( ) Processing ( ) Use c. Amount and Duration l. Batch Maximum kg/batch (100% new chemical substance) Hours/batch Batches/year 2. Continuous Maximum kg/batch (100% new chemical substance)

**~ours/batch** Batches/year 

### X 2,000 

### 96 82.5 d. Process Description [ ] Mark (X) to indicate your willingness to have your process description binding. X (I) Diagram the majorunit operation steps and chemical conversions. Include interim storage and transport containers (specify e.g. 5 gallon pails, 55 gallon drum, rail car, tank truck, etc.). (2) Provide the identity, the approximate weight (by kg/day m kg/batch on a l00% new chemical substance basis), and entry point ofall starting materials and feedstocks (including reactants, solvents, and catalysts, etc), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily orper batch.). (3) Identify by number the points ofrelease, including small or intermittent releases, to the environment ofthe new(chemical substance.



# - 

# D Mark (X) this box ifyou attach a continuation sl.vv•.

FORM EPA 7710-25 (Rev. 5-95) Page 8p.9 MB08Tl 

## Part II
- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE --Continued Section A- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER- Continued 2. Occupational Exposure-You must make separate confidentiality claims for the description ofworker activity, physical form

ofthe new chemical substance, number ofworkers exposed, and duration ofactivity. Mark (X) the "Confidential" box next to any item you claim as confidential. (1) Describe the activities (e.g. bag dumping, tote filing, unloading drums, sampling, cleaning, etc) in which workers may be exposed to the substance. (2) Mark (X) this column ifentry in column (l) is confidential business information (CBI). (3) Describe any protective equipment and engineering controls used to protect workers. (4),(6) Indicate your willingness to have the information provided in column (3) or (5) binding. (5) Indicate the physical form(s) ofthe new chemical substance (e.g. solid: crystal, granule, powder, or dust) and% new chemical substance (ifpart ofa mixture) at the time ofexposure. (7) Mark (X) this column ifentry in column (5) is confidential business information (CBI). (8) Estimate the maximum number ofworkers involved in each activity for all sites combined. (9) Mark (X) this column ifentry in column (5) is confidential business information (CBI). (10), (11)Estimate the maximum duration ofthe activity for any worker in hours per day and days per year (12) Mark (X) this column ifentry in column (5) is '" •I information

**~CBI** Maxtmum

Worker activity Protective Equipment Optio; Physical Binding ,....,. # of ll""'nT llirano11 (e.g. bag dumping, CBI Option \..-Dlt..

**~** 1'-'o• (2) /Engineering Controls Mark Form(s)

**twnr~r**.... rsEXJ:IOsedl Hrs/ Days! filling drums) Mark (x) (7) 1

..
**~-~-·** (9) CBI (12) 

### (1) (3)

**~~~** 

### (5) (6) (8) day yr 

### --~~---------4~~--~~--~~----+-~(~110)~(~111)~~ Lumps& 

### X Dust mask, local powder 

### 2 2 400 ---+---1 

### X ventilation, n. 

### 2 2 Af\1\ 

### ---+:.::....j 

### powder form :uwuc• _-tvv 

### X ruwuc1 

### 1 1 200 

# 0 Mark (X) this box ifyou attach a continuation sheet. 3. Environmental Release and disposal-You must make separate confidentiality claims for the release number and the amount

ofthe new chemical substance released and other release and disposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. ( 1) Enter the number ofeach release point identified in the process description, Part II, section A, subsection 1 d(3). (2) Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology (in kg/day or kg/batch). (3) Mark (X) this column ifentries in columns (1) and (2) are confidential business information (CBI) (4) Identify the media ofrelease i.e. stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, POTW, or other (please specify) to which the new substance will be released from that release point. (5) a. Describe control technology, ifany, and control efficiency that will be used to limit the release ofthe new substance to the environment. Forreleases disposed ofon land, characterize the disposal method and state whetherit is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day). (6) Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CBI). (7) Identify the destination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct dischargers or NPDES numbers ofthe POTW (Publicly Owned Treatment Works). Mark (X) ifthe POTW name or NPDES# is confidential business information (CBil Release new



## ~oun~**~leased** CBI Number

(1) (2a) (2b) (3) (5) (7) 

### 1 kg/day (8) 

### 0 kg/day Media of release

(4) Control technology and efficiency

**~----------------------r-----,-------~CBI**



## ~~;) (Sb) (6) (Sa) 

### Incineration None '' ration None 0 kg/day 

### None 0 kg/day (7) Mark (X) the ( ) POTW provide name(s) below CBI ( ) Navigable waterway ( ) Other- specify Provide NPDES# CBI destination(s) of releases to water NA



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

FORM EPA 7710-25 (Rev. 5-95) Page9p.10 MB08T1 

## Part II- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE- Continued Section B- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section B for typical processing oruse operations involving the new chemical substance at sites you do not control. Importers do not have to complete this section for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate section B for each type ofprocessing, or use operation involving the new chemical substance. Ifthe same operation is performed at more than one site describe the typical operation common to these sites. Identify additional sites on a continuation sheet. 1. Operation Description- To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. (1) Diagram themajor unit operation steps and chemical conversions, including interim storage and transport containers (specify e.g. 5-gallon pails, 55-gallon drum, rail car, tank truck, etc.). On the diagram, identify by letter and briefly describe each worker activity. (2) Provide the identity, the approximate weight (by kg/day orkg/batch on a I00% new chemical substance basis), and entry point ofall feedstocks (including reactants, solvents, and catalysts, etc), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily orper batch.). (3) Identify by number the points ofrelease, including small or intermittent releases, to the environment ofthe new chemical substance. (4) Please enter the# ofsites (remember to identify the locations ofthese sites on a continuation sheet): 

### 2 #ofSites 

### A. Open bags ofthe product TOFIX-S and add TOFIX-S into mixer with other raw materials. B. Mix thoroughly and introduce the mixture into molds for forming brake pads. C. Press the mixture in the molds with heating and then remove the formed products from the mold. D. Attach the molded products to a basal plate for brake pads and then install the brake pads into a braking system. E. Install the braking system onto automobiles



# D Mark (X) this box ifyou attach a continuation sheet. 2. Worker Exposure/Environmental Release (I) From the diagram above, provide the letter for each worker activity. Complete 2-8 for each worker activity described. (2) Estimate the number ofworkers exposed for all sites combined. (4) Estimate the typical duration ofexposure per worker in (a) hours per day and (b) days per year. (6) Describephysical form ofexposure and% new chemical substance (ifin mixture}, and any protective equipment and engineering controls used to protect workers. (7) Estimate the percent ofthe new substance as formulated when packaged orused as a final product. (9) From the process diagram above, enter the number ofeach release point. Complete 9-13 for each release point identified. (I0) Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day orkg/batch). (12) Describe media ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POWT, or other (specify) and control technology, ifany, that will be used to limit the release ofthe new substance to the environment. (14) Identify by-products, which may result from the operation. (3}, (5}, (8), (II), (13) and (15): Mark (X) this column ifany ofthe proceeding entries are confidential business information (CBI). Duration of Protective Equip./ Amount of Media of Letter #of %in New Substance

of Workers CBI Exposure CBI Engineering Form- CBI Release Released CBI Release& CBI Activity Exposed (3) hours days/ (5) Controls/ ulation (8) Number (ll) Control (13)

(I) (2) /day year Physical Form and (7) (9) (lOa) (lOb) Technology (4a) (4b) % new substance (12) Dust mask, local 0.5 lnciner- A 2 2 200 ventilation, 10 A kg/day ation powder form (14) By-products: CBI**~** 

# D Mark (X) this box ifyou attach a continuation sheet.
FORM EPA 7710-25 (Rev. 5-95) Page 10

### p. 11 MB08Tl 

## OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle orbracket 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 minimise potential risks associated with activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new information pertinent to pollutionprevention, including source reduction, recycling activities and saferprocesses or products available due to the new chemical substance. Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological modification, process and procedure modification, product reformulation, raw materials substitution, and /or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions ofpollution prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance with existing regulatory requirements and canbe either quantitative or qualitative. The EPA is interested in this information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting ofrisks to other environmental media or non-environmental areas (e.g., occupational orconsumerexposure). In addition, information onthe relative cost orperformance characteristics ofthe PMN substance to potential alternatives may be provided. All information provided in this section will be taken into consideration during the review ofthis substance. See the revised Instructions Manual that includes a Pollution Prevention manual for guidance and examples. Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in the volume manufactured; (3) a reduction in the generation ofwaste materials through recycling, source reduction or other means; (4) a reduction in potential toxicity orhuman exposure and/or environmental release; (5) an increase in product performance, a decrease in the cost ofproduction and/or improved operation efficiency ofthe new chemical substance in comparison to existing chemical substances used in similar applications; or (6) the extent to which the new chemical substance maybe substitute for an existing substance that poses a greater overall risk to human health or the environment. Currently, needle-shaped fibers (e.g., whiskers) ofpotassium titanium oxide (potassium octatitanate) are used in automobile brake pads. In 

### 2006, the World Health Organization (WHO) reviewed potential health effects ofpotassium octatitanate fibers and advised that respirable potassium octatitanate fibers are likely to pose a high hazard to humans after inhalation exposure. Because ofthe WHO advisory, "TOFIX-S" was developed to reduce and eventually eliminate such risks. This product "TOFIX-S" is potassium hexatitanate and is in the form of a powder or a stick shape, both ofwhich are different from potassium octatitanate fibers or whiskers.



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

FORM EPA 7710-25 (Rev. 5-95) Page IIp.12 MB08Tl 

## Part III -LIST OF ATTACHMENTS Attach continuation sheets for sections ofthe form and test data and other data (including physicaVchemical properties and structure/activity information), and optional information after this page. Clearly identify the attachment and the section ofthe form to which it relates, ifappropriate. Number consecutively the pages ofthe attachments. In the column below, enter the inclusive page numbers ofeach attachment. Mark (x) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for

**guidanc~** on how to claim any information in an attachment as confidential. You must include with the sanitized copy ofthe notice form a sanitized version ofany attachment in which you claim information as confidential. 

### Attachment Confi- Attachment Name Page dential Number(s) Physical and Chemical Properties Worksheet 13 Material Safety Data Sheet (MSDS) 14-17 Properties ofMatter Data ofSample TOFIX-S 18 Research Report "Biological Effects ofTOFIX-S Using Intratracheal Instillation" 19



# D Mark (X) this box ifyou attach a continuation sheet. Enter the attachment name and number.

FORM EPA 7710-25 (Rev. 5-95) Page 12p.13 MB08Tl 

## PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA'sreview ofphysical and chemicalproperties data, please complete the following worksheet for data youprovided and include it in the notice. Identify the propertymeasured, the page ofthe notice onwhich the property appears, the value ofthe property, the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties shouldbe for the neat ( 100%) chemical substance. Properties that are measured for mixtures or formulations should be noted (% PMN substance in -). You are not required to submit this worksheet; however, EPA stronglyrecommends that you do so, as it will simplify review and ensure that confidential information is properly protected. You should submit this worksheet as a supplement to your submission oftest data. This worksheet is not a substitute for submission oftest data. Page Measured Confidential Property Mark(x) Number Value or Mark(x) (a) ifprovided (c) Estimated (b) (MorE) (d) Physical state ofneat substance Solid Vapor pressure @ temperature 25°C Density 

### I relative density 

### 3.6 glmL 

### M Solubility in organic solvents Insoluble 

### E

**@temperature~** Solubility in water 

### <0.1

### o/o M @ temperature 20 25°C Melting temperature 

### 1347°C M Boiling/sublimation temperature _Torr pressure Spectra (IR, UV-vis) Dissociation constant Average particle size: Particle size distribution S.S micrometer, by 

### M laser light diffraction Octanol/waterpartition coefficient Henry's Law constant Volatilization from water Volatilization from soil pH @ concentration 

### 8-10 E Flammability- Flash point None 

### E Explodability None 

### E Adsorption/coefficient Other

- Specify



# 0 Mark (X) this box ifyou attach a continuation sheet. Enter the attachment name and number.

FORM EPA 7710-25 (Rev. 5-95) Page 13p.14 MB08Tl



### TOFIX·S Page 1/4 MSDS No. : TTC·0002·2



## MATERIAL SAFETY DATA SHEET 

# •
!.CHEMICAL PRODUCT IDENTIFICATIONAND OOMPANY IDENTIFICATION MSDSNumber Issue Date Product Name Manufacturer

Production Method : TTC·0002·2 :Aucuat 27, 2007 : TOFIX·S Potaeaium Titanate Powder(at.ick abape) : TOHO TITANIUM CO.,LTD. Reeemlh & DevelopmentDiviaion 3·3·5,Chigaaaki,Cbipeaki·City,Ka.napwa·Pref.253·8510,Japan Phone : +81·467·87·2813 : Calcination method 2.COMPOSITION I INFORMATION ONINGREDIENTS Chemical Name Chemical Formula CAS Number wt% Potaaaium h exatitanate K!TieOu 12056·51·8 >98 Titanium dioxide 

### TiOt 18463·67·7 <7 3.HAZARDS IDENTIFICATION Emergency Overview : Odorleea white powder with stick shapeAvoid 

### breathinc this product. TOFIX·S contain a small amount ofrespirable fibers which may become airborne during handline. Potential Health Effects : Prolonged inhalation ofexeeeaive concentration ofrespirable fibers may cause deposition ofquantities offibers inlungs sufficient to cause a tissue response which could be associated with injury. 4.FIRSTAID MEASURES Inhalation
Skin Contact :Gargle throat. If there is exposure to large amounts 

### ofrespirable fibers, , vacate premises immediately and ifneceaaary seek medical aaaistance. : Ifthere is exeeaaive exposure ofakin to fibers, wash with soap and water. 

### Page 14TOFIX·S Pace 214 MSDS No. : TTC-0002·2 Eye Contact : Wash immediatelywith clean water or eye-wash fiuid and ifnecessary seek medical assistance for examination and treatment. Ingestion : No adverae effects anticipatedby swallowing. 5.FIRE FIGHTING MEASURES Nonflammable Flammable Properties

Flash Point : None Flammable Limits Method Used : Not applicable

Lower Flammable Limit : Not applicable Ext.inguishinr Media : Not applicable · Fire & Explosion Hazards : Not applicable Fire· Fighting Equipment :Not applicable &.ACCIDENTAL RELEASE MEASURES Upper Flammable Limit : Not applicable

Note : Use appropriate PERSONAL PROTECTIVE EQUIPMENT during clean up. Wash, Bhovel or sweep and place in solid waste containers. Clean up dusts and fibers with high efficiency particulate air (HEpA) filters vacuum equipment. 7.HANDIJNGAND STORAGE Use routine protective measures to avoid inhalation ofmateriale. S.EXPOSURE CONTROLS I PERSONAL PROTECTION Engineering Controls : Use local exhaust ventilation to control airborne levels bellows the exposure guidelines. Personal Protective Equipment

Respiratory Protection : Recommend use ofNIOSHIMSHA approved respiratoryprotection against dust. Eye Protection Skin Protection Exposure Guideline

TLV<ACGIH) : Recommend use ofsafety rlasees : Recommend use ofprotective rlovea

: None Established Page IS p.15p.16
MB08Tl


### TOFIX-S
PEL(OSHA) : None Establiahed Page 314 MSDS No. : 'M'C-0002·2

9.PHYSICALAND CHEMICAL PROPERTIES Physical State : Solid Form : Stick shape powder Odor :Odorless Color :white Melting Point : 1847 degrees C (typical data) Boiling Point : Not applicable Vapor Pressure : Not applicable VaporDenaity : Not applicable Water Solubility : Insoluble in water Denaity (g/cmB) 

## :a.a (typical data) Bulk Denaity (g/cml) :o.9 (typical data) pH :s-10 lO.STABILITYAND RECTMTY This productis stable andhazardous polymerization will not occur. Incompatibility with Other Substances : None Hazardous Decomposition Products : None ll.TOXICOLOGICAL INFORMATION Acute Toxicity

Local Effects Long Term 'lb:xicity : Short-term harmful etrecta are not expected from skin contact. inhalation or swallowinr. The acute oralLDeo value is·more than 5000mr/kg in mice. The acute dermal LDao value is more than 2000mg/kg in rata The acute inhalation LCeo value is more than 2()()()mglkg in rata. :May cause eye irritation ifget dust into eyes. May irritate to the akin. in suaoeptible individuals. : Prolonged or repeated inhalation exposure to excessive concentrations ,

orreapriable fibers may induze irreversible lung injury.
Page 16p.17
MB08Tl 

### TOFIX·S

12. ECOLOGICAL INFORMATION No information is available. 18.DISPOSAL CONSIDERATIONS Page 4/4 MSDS No. : TTC·0002·2

Buryin landfill in accordance with all applicable reculations.Any disposal practice mustbe in compliance with local,state, andfederal laws and regulations (contact local or state environmental agency for specific rulee). Waste such ae dustfrom air filters may contain high depoaite ofrespirable fibers. Bag and dispose ae nonhazardousindustrialwaete. 14.TRANSPORT INFORMATION Not a hazardous material for DOT shipping. 15.REGULATORY INFORMATION OSHA :Thisproductis not hazardous under the criteria ofthe Federal OSHAhazard Communication Standard 29 CFR 1910.1200. 16.0THERINFORMATION To the beat our knowledge, the information containedherein is accurate. However, we cannot aeaume anyliability whatsoeverfor the accuracy orcompleteneaa ofthe information contained herein. Final determination ofsuitability ofany material ia the eole responsibility ofthe user. All materials maypreaant unknown hazards and should be used with caution. Although certain hazards are deacn'bed herein, we cannotguarantee that these are the only hazards that exist.

Page 17

### MB08Tt 

# Properties ofmatter 

# data ofsampleTOFIX-S TOHO TITANIUM CO.,LTD. 

## TO FIX -S 

## is a 

## titanic acid potassium particle shown in

## LO/6Ti02. 

## TOFIX-S 

## is superior heat resistance, insulation characteristics, a minute particle having

## low Mobs hardness. Properties ofmatter ofTOFIX-S 

## CAS No.12056-51·8 SAMPLE NAME TOFIX·S 

### An analysis method 

### Chemical composition 

### K20 • 6Ti0a XRD 

### Tomof

## mind I shape 

## WHITE/ POWDER VIeWing



### Particle 

### diameter 5.27Micron Laser difrraction method 

### 'li'Uth specific gravity 

## 3.5 Documents value 

### Solubility in water

**~0.1%** 

### Weight loss method Morse hardness 4 

### Documents value 

## PH 9.44 

## pH METER Meltingpoint 

### L300-1,350CC Documents value Electrical

### resistance 3.3Xl0150cm Documents value



# SEM Photo.
Page 18 p.18p.19



### MB08TJ



# Research Report
Biological Effects ofTOFIX-S Using Intratracheal Instillation June 8, 2007



# Professor Isamu



# Tanak~ Institute of Industrial Ecological Sciences University of Occupational and Environmental Health, Japan Page 19··-.-·· p.20 MB08Tl 

# Table of Contents Chapter 1 Research Project and Research Team Chapter 2 Research Objectives Chapter 3 Research Design and Methods Chapter 4 TOFIX-S Particle Characteristics 4.1 Contents of fibrous materials 4.2 Fiber diameters and lengths Chapter 5 

### The Recovery of TOFIX-S from the Animal Tissue 5. 

### 1 Recovery procedures 5.

### 2 Recovery rates Chapter 6 · Intratracheal Instillation 6.1 Instillation study 6.2 Protocol Chapter 7 Results ofthe Intratracheal Instillation 7.1 Rat growth curves 7.2 Wet weights ofthe organs at each time point of sacrifices 7.3 Quantification of lung burdens of TOFIX-S 7.4 Biopersistence of TOFIX-S from the lungs (biological

**ha~f** 

### time) 7.5 Formation of 8-hydroxydeoxyguanosine in lung tissue 7.6 Expression of gene related to inflammation and fibrosis in lung tissue Page 20· ..-.,.. p.21 MB08TI 

### 7.7 Histopathological examination Chapter 8 

### Discussion 8.1 

### Rat growth curves



### .8.2. . W e.t organ weights of the rats at each time point .of
. 

### sacrifices 8.3 

### Biopersistence ofTOFIX-S 8.4 

### 8-hydroxydeoxyguanosine 8.5 

### TIMP-2 8.6 Histopathological changes Chapter 9 Conclusion

8 Figures
· 3 Tables

7 Micrographs

Page 21

### Chapter 

### 1 Research Project 

### and Research Team Research Project 

### Biological Effects ofTOFIX-S Using Intratracheal Instillation Research Team Lead 

### investigator: ProfessorIsamu Tanaka, Dr. Eng.,

# vJ.~ Department of Environmental Health Engineering, Institute 

### of Industrial Ecological Sciences, University ofOccupational and Environmental**Heal~** 

### Japan (Graduated 

### from the 

### Department ofEngineering, Kyushu 

### University in 1967) 

### Researchers involved in the project: 

### Professor Yasuo Morimoto, MD, OMS, Department of Occupational Pneumology, Institute 

### of Industrial Ecological Sciences, University ofOccupational and Environmental**Heal~** 

### Japan {Graduated 

### from K.agoshima 

### University School ofMedicine in 1986) Associate

### Pro&asorToshibiko Myojo, 

### l.k Eog., 

# 1**~** Department of Environmental Health Engineering, 

### Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, 

### Japan (Graduated from Department ofEngineering, Kanazawa University in 1977) 

### Associate ProfessorAkira 

### Ogami, MD, OMS, Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan p.22
MB08Tl



### {Graduated from University ofOccupational and Environmental Health 

### Graduate School Page 22**··~--··** 

### ofMedicine in 1997) Assistant 

### Professor Thblro 

### Oyabu. 

## OMS. 

# Z

**~E_u** Department of Environmental Health Engineering,

- 

### - . 

### Institute of 

### Industrial Ecological Sciences, University ofOccupational and Environmental Health, Japan (Graduated from the Department ofScience, University ofKyushu in 

### 1983)

Page 23 

### p.23
MB08Tlp.24
MB08Tl 

### Chapter 2 Research Objectives The aim oftbis study 

### is to assess the potential biological hazards ofTOFIX-S when they 

### enter the body, especially into the lung, by determining the biopersistence of TOFIX-S in the lung and

. 

# - . 

### - . . . 

### biological reactions including histopathological examination and expression of genes 

### which are related to fibrogenesis and carcinogenesis. It is known that certain 

### types of dust cause respirato.ry 

### diseases when 

### enter the alveolar region where gas exchange takes place but the effects and degree ofthe diseases vary 

### greatly according to the dust type. For pulmonary fibrosis (pneumoconiosis) and 

### canoers in the 

### lungs,-it is 

### postulated 

### ' · that the longer retention of dust in the alveolar region increases the length of time of dust 

### contacting the 

### body,

**re~ulting** 

### in the induction ofvarious kinds ofbiological reactions 

### that may progress to fibrosis and cancers.

,__.... 

### In 

### this study, examination of TOFIX-S with electron microscopy is 

### first 

### made to determine the shapes of TOFIX-S. Then, we 

### conducted an experiment in 

### which TOXIF-S are intratracheally instilled in rat lungs and 

### the amounts ofTOFIX

# -s retained in rat lungs are quantified by means of serial 

### sacrifices to determine the rate 

### oflungclearance ofTOFIX-S. Histopathological examination and 

### cytokines which are responsible for fibrotic and 

### carcinogenic changes in the lung are examined to assess the biological effects ofTOFIX-S.

Page 24

### p.25 MB08TJ


### Chapter 3 Research Design and Methods The biopersistence ofchemicals that are introduced into 

### the lung via airways is vital to 

### assess their health effects or biological effects in humans. The fate ofchemical substances in the lung depends largely on their physiochemical characteristics, dimension and especially biopersistance in the lun& over time, that is involved directly with the length oftime required 

### for the 

### initiation ofbiological responses and the 

### effects ofsuch responses. The significance ofthis understanding 

### has begun to 

### be appreciated in recent 

### years and, therefore, biopersistence offibrous dust 

### which contributes especially to 

### fibrogenesis and carcinogenesis in the lung have being investigated as the most important parameter. M. 

### gene expression of cytokines which 

### is 

### an obvious potential for mediating fibrosis and carcinogenic changes is related to 

### the response mechanisms by which the chemical produces final 

### conditions, the measurement of gene expression of cytokine is 

### a widely used tool to determine fibrotic and carcinogenic changes that occur in the lungs. On 

### this basis, we planned and conducted an experiment according to 

### the following procedures to 

### obtain 

### our objective: 1. 

### Identify the geometric shapes ofTOFIX-S by 

### electron microscopy 2. Conduct an animal experiment in which

**TOFIX~S** 

### is instilled intratracheally into rats 

### 3. Decide which method to 

### use for quantification ofTOFIX-S entered in the animal lung 4. Quantify TOFIX-S retained in the lungs to 

### determine the rate oflung clearance by 

### means of serial sacrifices ofa given number ofrats 

### at 

### certain time points 5. 

### Measure the amount ofcytokine which is 

### deeply involved in fibrotic or carcinogenic changes in the lungs 6. Histopathological examinations ofthe lungs

Page 25p.26



### MB08Tl 

### Chapter 4 TOFIX-S Particle Characteristics 4.1 Contents offibrous materials Filters containing TOFIX-S and deposited platinum on them 

### were prepared 

### for scanning electron microscopy assessment ofTOFIX-S lengths and diameters; and. subsequently digital micrographs 

### were made (Micrograph 1 ).

Micrograph 1 A scanning electron micrograph ofTOFIX-S There are particulate matter and fibrous 

### shaped particles fibrous shaped 

### particles having a length-to-diameter ratio greater than 

### 3:1. Particles with 

### a diameter of more than 0.2fJ.Dl in 

### the randomly selected 20 fields ofview ofthe scanning electron microscope were counted. The 

### number offibrous particles and 

### non-fibrous particles quantified by 

### the scanning 

### electron microscopy were 124 and 

### 1,136, respectively. This 

### indicates that 

### the number offibrous particles accounts for 9.8% oftotal number ofparticles counted. Since a product that 

### contains asbestos exceeding 0.1 % 

### by weight which asbestos bears 

### to the whole product is defined as an asbestos product according

# to the 

### Ordinance on the 

### Prevention ofHazards due to Asbestos, TOFIX-S can be defined 

### as an fibrous product, therefore, 

### we though that it is relevant to assess the potential hazards ofTOFIX-8. Page 26

### ···-- p.27

MB08Tl 

### 4.2 Fiber diameters and lengths Diameter and 

### length are the basic 

### determinants offibrous 

### materials. 

### TOFIX-S 

## was collected on the filter and, then. platinum was 

### deposited on the 

### filter and an image 

## was taken using a scanning

.. . . - 

# -

. . 

### electron microscope. Computer-assisted measurements using NIH image were 

### made from 

### scanned SEM micrograph for diameters and lengths ofthe 321 TOFIX-S. The 

### results were 

### plotted on a logarithmic-normal probability paper as shown in

### the Figures 1and 2.
98 85



# -- '#. 80 

### .._, 

# r 80 70 80 

# l 50 40



# -~ 30 

# -a 20 

# s 10 5 20.1

98 85 '0'

**~** 

### .._, 80



# I 80 70 80 

### 4::1 50



### ~ 40 

## "i 30 

### '$ 20 

# s 10 5 

## • 2 1' 

# • 

# • •· 

## • 

# • • 

# • • • • 

# • • • 

# • • • 

# • • •Fiber diameter (J.un) 

### Figure 1

### Fiber diameter distribution



# • 

# • • • • • 

# • • • • • 

# • • • • •

10 

# • •

Fiber length (J.llll) 

### Figure 2 Fiber length distnbution Page 27 10...

# _. p.28

MB08TI



### Both the distribution of diameter and length 

### were found to be 

### a logarithmic-normal probability distribution fitted 

### in 

### an approximate 

### linear curve on the logarithmic-normal probability paper. For fiber 

### diameter, the 

### geometric mean diameter and geometric standard 'deviation ofthe fibers 

### were -·· ...... - ·- 

### - . . . -. 

### -- . . 

### 1.4 J.LIIl 

### and 1.5, respectively. For fiber length, the geometric mean length and 

### geometric standard deviation of the fibers 

### were 6.2 J.LIIl 

### and 1.8, respectively. Approximately fifteen 

### percent of the 

### fibers 

### were found to be 

### less than 

### 0.8 J.11I1 

### in 

### diameter but 

## were shorter than 20 J.UD. It is 

### well known that 

### for asbestos the thinner and 

### longer the fibers 

### (over 20 J.1D1 

### in 

### length), the 

### greater the 

### hazardous potential; no fibers longer than 20 J.11I1 were 

### found for TOFIX-S
.

Page 28p.29

MB08Tl 

### Chapter 5 

### The Recovery ofTOFIX-S from the

### Animal Tissue 5.1 Recovery procedures

. . . . -· 

### In order to 

### measure TOFIX-S satisfactorily: to 

### obtain excellent recovery rates, we 

### examined procedures, including pretreatment, conditions and instrument suitable for quantitative analysis of TOFIX-S present 

### in the animal tissue, to 

### determine lung burdens. The samples to 

### obtain

**~very** 

### rates were 

### prepared by 

### first 

### taking a precise amount ofTOFIX-S · and adding 

### it to 

### the lung tissue (liver 

### was used 

### in the recovery procedures because it provides us a larger amount ofsamples), and then a method to 

### recover the 

### given amount ofTOF1X-S from each 

### sample by using several ashing techniques 

## Was studied 

### A quantification method used 

### in this 

### study 

### has the combined advantages of suitability of pretreatment 

### and analytical conditions and instruments and, therefore, will provide us with excellent recovery rates. The 

### procedures are shown inFigure3. 5.2 Recovery rates 

### TOFIX-S ofeither 0, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 mg 

### was added to 

### each 1g ofhomogenized liver and each placed in a glass bottle. Three preparations each were made, making 21 samples in total. 

### For the quantitative analysis of TOFIX-S in the lung tissue samples 

### was made following the

**·-~·** 

### procedmes, as shown in Figure 3, to 

### obtain recovery 

### rates. The amount ofTOFIX-S in 

### each sample which contained 50.1 % 

### Ti 

### was calculated by converting measured Ti concentrations. The results are shown in Table 1. The results showed that 

### almost 1 000/o ofTOFIX-S added to 

### each sample 

### was recovered, therefore, we 

### concluded that our quantification procedmes could be used 

### as a method to 

### quantify the 

### lung burden ofTOFlX-S in this intratracheal instillation study.

Page 29p.30
MB08Tl



### Lungs 

# +

.. 

### Freeze drying · 

# + Low-temperature ashing



# ~ Filtering ( 2.Sml 

### Acid soln.• 

## ... l.Oml

**HN~ ~**  

### 2.0ml 

### H2<h 

# J

**~**

# .. Microwave wet 

### ashing (Milestone Ethos 

### 1Ype 1600) Digesting conditions ( 2SOW 3min 

# soow 20min 

# J

**~r** 

### Prepared to 

### constant volume of50 

### m1

r Quantify 

### Ti amount (ICP-AES analysis)
Figure 3 Procedures for the quantification ofTOFIX-S lung burdens Page 30p.31



### MB08Tl 

### Table 1 The 

### results 

### ofrecovery rates Slmpla Pbmii!Mriccpulities TOFIX-8 quauli&d TOFIX-8 added

**~lUll** (mg) (mg) (%) C-1 427200 <0.001 C-2 416000 <0.001 C-3 416000 <0.001 C+O.OSmg-1 1469000 

# o.osos 0.05 101.1 C+O.OSmg-2 1463000 0.0503 0.05 100.5 C+O.OSmg-3 1466000 0.0504 0.05 100.8 100.8 C+0.1mg-1 2410000 0.0956 0.10 95.6 C+0.1mg-2 2573000 0.1034 0.10 103.4 C+0.1mg-3 2539000 0.1018 0.10 101.8 100.3 C+O.lmg-1 4792000 0.2098 0.20 104.9 C+O.lmg-2 4645000 0.2027 0.20 101.4 C+O.lmg-3 4716000 0.2061 0.20 103.1 103.1 C+O.Smg-1 11130000 0.5134 

# o:so 102.7 C+0.5mg-2 9850000 0.4521 

# o.so 90.4· C+0.5mg-3 11140000 0.5139 0.50 102.8 102.8 C+l.Omg-1 21650000 1.0175 1.00 101.8 C+1.0mg-2 21530000 1.0118 1.00 101.2 C+l.Omg-3 21920000 1.0304 1.00 103.0 102.0

... ,_. *Exduded from the data due to an error in measurement

Page 31**'~** .../ 

### p.32
MB08Tl


### Chapter 

### 6 Intratracheal Instillation 6.1 

### Instillation study

. . . . - 

### . . 

### Potential hazards ofchemical substances in human differ significantly with the 

### routes of

### exposure (ora4 skin 

### and respiratory tracts), 

### biological effects of such chemicals in humans need to be 

### assessed in animal 

### studies by using routes through which human 

### exposure occur. Human contact with TOFIX-S generally takes the 

### following form: they are 

### inhaled with air and 

### may 

### possibly enter 

### the body through respiratory 

### tracts. For this 

### reason, biological 

### effects should be available from 

### animal studies in which 

### inhalation would be the most 

### practical study design for this purpose. However, since it requires certain 

### equipment and 

### considerable cost 8J)d 

### time, intratracheal instillation whose 

### route or exposure is 

### similar to inhalation has 

### been widely used 

### as an alternative exposure procedure. A 

### method 

### was developed to administer accmate intratracheal instillation oftest 

### materials in the animal 

### lung. A given amount oftest particles suspended in a solution is 

### injected by 

### syringe through the cannula into 

### the trachea, delivering the 

### test particles directly into 

### the 

### lungs. Regarding the advantages and disadvantages ofthe method, 

### intratracheal 

### instillation administers an accurate 

### dosage ofmaterials but it has the disadvantages that 

### large amount ofdose in quantity is 

### delivered at one time and a solution in which 

### test materials are 

### suspended is also administered in the lungs. However, the route ofintroduction into 

### body is an 

### unnatural route ofexposure in human exposure, but the method does provide useful information on how the test 

### particles behave in the 

### lungs. The 

### main theme ofthis Study is to estimate the biological effects ofTOFIX-S. For this purpose, we 

### conducted 

### an intratracheal instillation experiment to examine the biopersistence ofTOFIX-S in the lungs in relation to its lung clearance. 6.2 Protocol 6.2.1 

### Experimental animals Nine-week-old male Wistar rats were 

### used. 6.2.2 

### Number ofrats
Page 32

### Seventy rats 

### were randomly divided 

### into two similarly sized groups: Intratracheal instillation group: TOFIX-S exposing group Con1rol group: Physiological saline exposing group

. . .. 

### 6.2.3 Administration ofTOFIX-S p.33


### MB08Tl



### TOFIX-S 

### was suspended in physiological saline at the rate 

### of2 mg per 0.4 ml 

### and administered to 35 rats by a single intratracheal instillation into 

### the under ether anesthesia. Rats in the control group were 

### given only the 0.4 ml ofphysiological saline. 6.2.4 Time points for sacrifices after 

### instillation Rats were 

### sacrificed at

### the following time points after 

### intratracheal instillation: - 3 days after instillation 10 rats 

### per 

### group were sacrificed. - 1week after 

### instillation, 5 

### rats per group were 

### sacrificed.

- 1month after 

### instillation - 3 months after instillation - 6 months after instillation 5 

### rats per group were sacrificed. 5 

### rats per 

### group were sacrificed 10 rats 

### per group 

### were sacrificed.

Page 33

# -- p.34



### Chapter 7 Results of the Intratracheal Instillation 7.1 Rat 

### growth curves

- - - 

### The growth curves ofthe rats were 

### recorded every 

### week throughout the 

### experiment The results 

### are shown in Figure 4. 700 600 

## 'bO '-" 

# i 500 

# "i 400 

# i 300 

# f lOO 

# < 100 0 ! 10 ___. Saline

### instilled group/Control group

- TOFIX-S instilled group

1! 30 

### Time (age 

### in weeks) 

### Figure 4 Growth curves. 3!



### No 

### difference was 

### found in rat growth 

### curves (weight changes) between two 

### groups. · --- Behaviors and health conditions of rats 

### in 

### the cages were 

### also observed every day; there was 

### nothing particular

### to be 

### noted. 7.2 Wet weights of the 

### organs at each time point ofsacrifices The results ofthe measurement ofthe wet 

### weights ofrat 

### lung, liver, kidney and spleen at each time point ofsacrifices 

### are shown in

### Table 2.

Page 34--

**~**...· p.35



### MB08Tl


### Table2 Wet weights 

### (g) of

### rat lung, liver, 

### kidney and spleen at eachtime point ofsacrifices. 

### Lung Liver 

### Kidney Spleen After3 

### days

... . - . . -

**~** Coolrol group 

### 1.26+0.07 15.3+1.3 2.34+0.15 0.85+0.12 TOFIX-S iDIIil1a1l!d 8JOUP 

### 1.29+0.22 14.4+1.3 2.30+0.18 0.82+0.12 After 1 

### week Coolrol group 

### 1.30+0.09 15.8+1.3 2.48±0.27 0.78+0.14 lUFIX-S ioslillated 8JOUP 

### 1.28+0.06 15.7+0.7 2.50+0.14 0.86+0.08 After 1 month Coolrol group 

### 1.36+0.05 18.0+1.7 2.87+0.19 0.97+0.08 TOFIX-S ioslillated 8JOUP 

### 1.36+0.04 17.3+0.8 2.74+0.06 0.91+0.08 After 3 months Coolrol group 

### 1.39+0.12 15.8+1.7 2.75+0.28 0.85+0.06 TOFIX-S ioslillated 8JOUP 

### 1.50+0.09 17.2+1.6 2.81±0.13 0.91±o.16 After 6 months Coolrol group 

### 1.61 +0.06 19.4+2.5 3.32+0.27 0.95+0.13 TOFIX-S iDstillated 8JOUP 

### 1.53±0.10 19.7±3.0 3.24+0.37 0.96+0.12
No significant difference in wet 

### weights of lung, liver, 

### kidney and spleen at each time point of

. sacrifices were found 

### inthe two 

### groups.

7.3 Quantification oflungburdens ofTOFIX-S 1) Method for quantification ofTOFIX-S inthe lungs Five rats 

### in each group were 

### sacrificed at 

### given time points to 

### quantify the amount of TOFIX-S retained in the lungs following the 

### procedures descnbed 

### in Figure 3. 

### The amount ofTOFIX-S 

### in each sample which contained 50.1 % 

### Ti 

### was calculated by converting measured Ti amounts.

Page 35

### 2) Lung burden ofTOFIX-S The results ofmeasurements are shown in 

### Table 3. Table 3 Lung burden of'IDFIX·S at each time point ofsacrifices

3 days after 

### instillation 1week after 

### instillation 1 month after 

### instillation 3 months after 

### instillation 6 months after 

### instillation Lung burden ofTOFIX-S (mg) 1.21 

## + 0.28 0.73 

## + 0.22 0.71 

### + 0.15 0.32 

## + 0.11 

### 0.17 

### + 0.06 p.36
MB08Tl



### Ti amounts in rat 

### lungs ofthe control groups were found to 

### be extremely low (less than 1 J.Lg) when compared with to 

### those in 

### rat lungs ofthe TOFIX-S instil1ated group, those concentrations were 

### regarded as zero in 

### quantifying the amount ofTOFIX-S in

### the lungs. It

### was found that the lung burden ofTOFIX-S decreased with time after instillation. The dose of TOFIX-S 

### was 2 mg and it decreased to 

### an average of 1.21 mg 

### at three days after instillation, suggesting that particles deposited in

### the tracheobronchial airways were cleared. It 

### is known that particles deposited in 

### the tracheobronchial airways are borne away by the continuously upward heaving mucous layer, most ofthem are then exhaled in about one day. Other particles retained in 

### the lung are thought to 

### be penetrated more deeply get into 

### the pulmonary alveoli, which is a collection ofapproximately 3 X 

### 108 

### alveoli oftiny air sacs with a surface are of about 200 J.Uil, 

### having a total surface area of about 100 square milimeters. 

### Plllmonary alveoli are surrounded on the outside with fine blood vessels. It is in 

### the alveoli in the lungs that gas 

### exchange takes place as the vessels absorb oxygen from the alveolus through its thin-wall. Since the alveoli in 

### the lungs are lined by epithelial cells without cilia, in 

### the case ofinhaled foreign substanCes the alveoli dissolve the substances by its fluid and clear them or produce macrophages to 

### phagocyte and digest such substances to 

### clear. Such substances phagocytosed by these m.acropbages are cleared as some transported to 

### bronchial regions lined by 

### ciliated epithelial .cells or some get into Page 36p.37

MB08Tl


### the lymphatic system. The values shown in Table 3 

### may indicate that TOFJX-S deposited in the alveolar region was 

### cleared from the lungs by 

### these clearance mechanisms.

0 ·- •• 

### 7.4 Biopersistence ofTOFIX-S from the lungs (biological halftime) Relationship between the lung burden ofTOFIX-S at 

### each given time point after instillation were 

### plotted on semi-logarithmic graph to 

### estimate the 

### rate ofTOFJX-S pulmonary clearance from the measurements shown in Table 3. The results are indicated in the form of a :6.1led 

### rhombus mark 

### (+).
10.0

0.1 

# IBiological halftime: 12 months


# • 

# IBiological halftime: 2.2 months OPT1 

### •TOAX-S
0 

### <> 

# •

0 2 3 4 5 6 

### Time points after 

### instillation (month) Figure 5 

### Changes oflungburden ofTOFIX-S and PTl with time The 

### results obtained in our laboratory for the changes of lung burden of potassium 

### octatitanate whisker (PTl) with time are indicated in Figure 5 

### in the form of an open rhombus 

### mark (0) 

### for reference. The lung clearance rate 

### of TOFIX-S can be 

### expressed as the 

### biological half 

### time of TOFIX-S retained in 

### the rat lungs that can be obtainedby lung burdens ofTOFIX-S measured at three 

### -days, 1 Page 37p.38


### MB08Tl


### week, 1 month, 3 months and 6 months after 

### instillation (Co). 

### This 

### can be 

### summarized by the 

### formula: dC/dt=-kC

. . 

### Initial condition: t 

## =0, C 

## =Co 

### (1) 

### (2) where 

### the lung clearance rate 

### of TOFIX-S is 

### assumed to 

### be 

### proportional to 

### the lung burden of TOFIX-S (C) and k is 

### a lung clearance mte constant (1/day) which 

### can be 

### obtained by using the 

### results 

### ofthe experiment 

### Then, the following equation is obtained when

### Equation (1) is solved using the initial condition (2): C/Co= EXP( 

### -kt) (3) 

### The term 

### biological half time (Tta) 

### refers to 

### (is 

### defined as) the time by which half of the 

### administered amount ofparticles is cleared from the 

### lung. The T112 

### is given by using the following Equation (4): 

### T112

## =0.693/k (4) 

### The biological half 

### time ofthe lung burden of TOFIX-S calculated by using the measured lung burdens was 

### 2.2 months. The T112 

### can also be 

### assumed from the 

### gmdient of the 

### line shown in Figure 

### 3. T112 

### for PT1 (potassium octatinate wisker) is 12 months. 7.5 Formation of8-hydroxydeoxyguanosine in lung tissue 8-hydroxydeoxyguanosine which is formed when hydroxylation of deoxyguanosine at the C-8 

### position occurred is a major product 

### of oxidative DNA damage and may induce lung injury and 

### carcinogenesis. Up to 

### the 

### present time, 8-hydroxydeoxyguanosine was found in the cases in which lung injury was produced 

### due to environmental 

### exposure to substances such 

### as tobacco, asbestos 

### and 

### diesel exhaust emissions; and a relationship between 

### 8-hydroxydeoxyguanosine and lung cancer has been Page 38·---··· p.39


### MB08Tt 

### reported. 7.5.1 

### Measurement method 1) Flush 

### a 1.5 ml centrifuge tube 

### with nitrogen, 2) Put

### the lung tissue in

### the tube 

### and add 

### 1ml Lysis solution, 3) 

### Hold the tube on the ice for homogenization, 4) Centrifuge at 10,000 

### g 

### for 20 

### seconds, 5) 

### Discard the 

### supernatant and add 

### 1ml Lysis solution again, 6) 

### Centrifuge 

### at 10,000 

### g 

### for 20 seconds, 

### 7) 

### Discard the supernatant and add 180 

### J.Ll Enzyme Reaction solution, 8) 

### Add 2 ml RNase 

### and allow the 

### reaction mixture to react 

### in 

### a constant-temperature bath 

### at 50°C 

### for 10 

### minutes,

**·~·** 

### 9) Add 10 J,.L1 

### Proteinase solution and store in the 

### constant-temperature bath 

### at 50°C 

### for 60 

### minutes, 10) Centrifuge 

### at 10,000 g 

### for 10 

### minutes, 11) 

### Discard the 

### supernatant, 12) 

### Add 0.3ml Nal 

### solution, 13) Add O.Sml isopropyl alcohol, 14) Centrifuge 

### at 10,000 g 

### for 10 

### minutes, Page 39p.40

MB08Tt



### 1

5) Discard the supernatant, 16) Add 1 ml A (wash fluid) and 

### wash by 

### centrifugation, 11) Add 1 ml B 

### (wash fluid) and 

### wash by 

### centrifugation, 18) Drying 

### 19) Add 0.1ml 1mM EDTA, 11J.l 2M ofsodium acetate 

### buffer solution, 4 

## !J.l of

### Smw'ml nuclease P1, and 

### 2 

## !J.l ofphosphoric acid; and incubate at 

### 37 °C for 30 

### minutes, 20) Perform ion-exchange resin and 

### centrifuge, 21) Transfer the solution 

### to a tube 

### with a filter, centrifuge and extract the solution, 22) Inject 20 

### ,.U DNA solution in the HPLC detector (electrochemical detector, ESA, Coulochem, 

### USA), 23) Use the 80HdG/dG ratio to quantify 80HdG . With the 80HdG/dG ratio, the amount of80HdG canbe 

### determined. 7.5.2 Measurements of8-hydroxydeoxyguanosine The levels of 8-hydroxydeoxyguanosine measured in the lungs of rats 

### sacrificed at 3 days 

### and 6 months after 

### instillation are shown in Figure 6.

Page 40..

# _ ...

··..

### -· p.41



### MB08TJ



### 3.0 

## 0 Control group • TOFIX-S instil1ated group 

### 2.5 2.0

**~**



## ~ 1.5 

# - ..... 1.0 0.5 0.0 3 days afteriDstiDation 6 months after instiDation 

### Figure 6 The levels of80HdG 

### in

### the lungs ofrat 

### sacrifices 

### at 3 days and 6 months 

### after instillation No significant differences were found between the 

### instilled group and the control group for the levels of80HdGboth 

### at 3 days 

### and 6 months 

### after instillation. 7.6 Expression ofgene related to inflammation and fibrosis 

### in lung tissue 7.6.1 Fibrosis-related gene Type I collagen, 

### Matrix metalloproteinases (MMPs) 

### and Tissue inhibitors of metalloproteinses (TIMPs) are 

### the genes involved 

### in the production and metabolism ofcollagen. Collagen is deeply involved in pulmonary fibrosis. Extensive deposition of collagen is thought to 

### be 

### the most significant condition for pulmonary fibrosis and presumably depends on a balance between synthesis and degradation ofcollagen. 7.6.1.1 Excessive production ofcollagen Collagen 

### in lung tissue is primarily of

### Type I collagen and it 

### has been postulated that the extensive deposition of

## 1YPe I collagen induces fibrosis. The occurrence ofexcessive production ofcollagen couldbe determined from examination ofwhether gene expression ofType I collagen increased. Page 41p.42



### MB08Tl 

### 7.6.1.2 Regulation ofcollagen degradation Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (MMPs) are matrix-degrading enzymes. Deposition of collagen is regulated by 

### a balance between 

### MMPs and

. . . . . . . . . 

### - TIMPS. Abnormal degradations ofcollagen frequently 

### occur when there is 

### an imbalance 

### between MMP and 

### TIMP. An imbalance of MMPs and 

### TIMPs has been reported 

### to result in 

### metalloproteinase activation or inactivation, and relatively higher levels ofTIMPs than 

### ofM:MPs may contnbute 

### to the downregulation ofdegradation resulting in 

### the deposition ofcollagen. A 

### late stage 

### or 

### fibrotic stage 

### in 

### the process 

### of fibrosis, the gene 

### expression of 

### TIMP is elevated Conversely, when 

### the level of MMP activation is higher 

### than that 

### of 

### TIMP, the degradation of collagen is facilitated, which in tum 

### results in 

### lesser deposition ofcollagen. This is 

### seen in 

### an early stage or lung injury and inflammation in 

### the process offibrosis and also 

### seen in 

### pulmonary lung masses 

### that 

### can be attnbuted 

### to cigarette smoking. The mechanism of fibrosis caused whether by increased production of collagen or by regulated degradation ofcollagen can be 

### elucidation by means ofquantification and analysis ofthe results. It seems a reasonable procedure 

### to first 

### conduct a screening for expression ofthese genes, then, 

### if 

### significant changes are found in any ofthese three 

### genes, expression ofthe other two 

### genes are further examined. We decided to first 

### measure the activation ofTIMP and, if

### significant differences in the levels ofTIMP were 

### found compared to the control group, then further measure the levels of
,_ ·' 1yPe I collagen and 

### MMP. 7.6.2 Measurement method 7.6.2.1 RNA extraction procedures Lung tissue was 

### homogenized with 1.2 

### ml ofSolution D. RNA was 

### extracted from the lung tissue by using a guanidium chiocyanate-phenol-chloroform extraction technique. RNA extraction from lung tissue: 1) Add 1.2 m1 

### of Solution D, 120 

## J1l of2M Sodium Acetate (pH4.0), 1.2 

### ml ofPhenol DEPC Page 42p.43
MB08Tl


### H20 

### and 120 

### J.1l ofChloroform-Isoamyl alcohol (49:1) to 

### the homogenized solution, 2) The mixture is 

### agitated for 10 

### seconds and held on the ice.

. . . - . . . 

### - . . . . . . . 

### 3) Centrifugate 

### at 15,000 rpm 

### for 20 minutes 

### at 4°C, 4) Inject the supernatant in the PHASE 

### LOCK GEL(PLG) 

### tube and centrifugate at 

### 8,000 

### rpm for 5 

### minutes, 5) Transfer the resulting supernatant to 

### a new centrifuge 

### tube, mix with 1.2 

### ml ofisopropanol and 

### hold at -20°C for 

### 15 

### minutes, 6) Centrifugate 

### at 15,000 

### rpm for 20 minutes 

### at 4°C, 7) Discard the supernatant and add 360 J1} 

### ofSolutionD to 

### the pellet, 8) Add 360 J1} 

### ofisopropanol and hold at -80°C for 

### 15 minutes, 9) Centrifuge 

### at 15,000 rpm 

### for 10 minutes 

### at4°C, 10) Discard the supernatant, 

### mix with 0.5 

### ml 75% ethanol and 

### centrifuge at 

### 15,000 

### rpm for 10

**·~····** 

### minutes at4°C, 11) 

### Again mix with 0.5 m1 

### of75% ethanol and centrifuge at 

### 15,000rpm for 10 minutes at 4°C, 12) Discard the supernatant and centrifuge for 

### 5 minutes while 

### sucking, 13) Sufficiently dry the RNA pellet 

### and add TE 

### 20-50 

### J.1l to 

### dissolve the pellet, 14) Adjust the concentration to 

### 2.2 

### J.L81J.1l by using spectrometer, After 

### extractingthe RNA, gene expression ofTIMP 

### was measured by perform reverse-transcription polymerase chain reaction (RT-PCR). Page 43**~-·** ' p.44
MB08Tl



### 7.6.2.2 RT (Reverse transcriptase) 1) Mix 5 

### x RT buffer, d-NTP, R.andam 

### hexamer(RH), RNAse inhibitor (RI), 

### Riverse 

# transcripmse. (RT) to 

### 75.5 J.L1 

### in 

# total in-

### a 

# t.5 m1 

# eppendoiftube, 

# ad<i a sample 

### of 

# io

**~of** tissue (contains 22 J.l8 

### ofRNA), and dilute 

### the mixture 

### with 114.5 

### J.Ll ofDEPC H20 to 

### 200 

### J.Ll in 

### total. RT-mixture: 5XRTbuffer 40 

### J.Ll d-NTP 

### 20 

### J.Ll RI 

### 0.5 

### J.Ll RT 5 

### J.Ll RH 

### 10 

### J.Ll Sample 10 

### J.Ll DEPCH20 114.5 

### J.Ll 2) Place the sample in

### a thermal cycler and incubated at the following 

### settings: (1) 

### 25 

# oc (2) 42 

# oc (3) 

### 95 

# oc (4) 5

# oc 10min 

### 59 min

5min 5min 3) After the processes from these (1)-(4) are completed, store the sample at-20 °C.

Page 44p.45


### MB08TI


### 7.6.2.3 PCR (polymerase chain 

### reaction) 1) Add 

### 5 ml ofPCR. 

### mixture to the RT sample PCR mixture: MgCh 

### 1 

### J.Ll 10 X

### PCR. Buffer 2 

### J.Ll ddH20 16.4 

### J.Ll primer AS 

### 0.25 

### J.Ll primer 

# s 0.25 

### J.Ll Total19.9 

## f.Ll per sample 2) Place the sample in the thermal cycler and add 

### a drop of

### mineral oil. 3) Program the thermal cycler, for 20
- 40 cycles as 

### required, for gene amplification · The 

### thermal cycler 

### was programmed 

### as follows: 94 °C 

### 45 

## sec 60°C 

### 45 

## sec 72 

# oc 2 

### min

,_. 4) After amplification, extract 10 

## f.Ll from the sample and add 

### 1 J.L). 

### of Loading 

### Buffer to the fragments. 5) 

### The PCR 

### product is resolved 

### in 110 g of 2 % 

### agarose gel containing l0 J.L). 

### of ethidium 

### bromide. 6) Electrophoreses at 100 volts for 1hour.
' 

### 7) 

### The gel is illuminated 

### from underneath with UV light and photographed with Polaroid 

### film. 8) The bands from the Polaroid photograph are scanned and the density ofthe bands is measured using NIH image. Page 4S.;. **~--·** p.46

MBOSTI


### 9) The ratio ofthe densities ofthe target genes 

### to 

## J3-actin genes as a control (internal standard). 7.6.3 TIMP2 

### measurements The results ofthe 

### measurements of

### TIMP2 in association 

### with gene 

### expression 

### that is 

### related to 

### fibrosis 

### are shown inFigure 7. 1.50 

## 0 Control group 

# l • TOFIX-S instillated group 

### s 1.00 

# 4

....... 

# ·I 0.50 

# I



# ·~ 0.00 

### After 3 days After 6 months Figure 7 Gene expression ofTIMP in the lungs ofrats 

### at 3 days and 

### 6 months after instillation.

**·--~** 

### Figure 7 

### shows that no 

### differences were found in gene expression ofTIMP 

### in the instilled group and the control group at 3 days and 6 months after instillation. 

### 7.7 Histopathological examination 7.7.1 

### Lung tissue preparation The lungs of 

### 5 rats per group 

### were sacrificed at 3 

### or 6 

### months after instillation of TOFIX-S 

### and examined histopathologically. The hmg 

### was fully inflated by 

### intratracheal infusion of 4% 

### paraformaldehyde and fixed. After fixation, the left lung 

### was sectioned along the sagittal plane 

### to 

### include hilar surface so as to 

### make the cut surfaces largest. The lung 

### sections were 

### then embedded in paraffin. 

### Three J.LDl-thick sections 

### were then stained 

### with hematoxylin and examined Page 46' 

# . p.47



### hitopathologically. 7.7.2 Results ofhistopathological examination

. . . . . . . . -4 

### 7.7.2.1 Results ofexamination at 3 days after 

### instillation In the 

### TOFIX-X instillated group, the degree 

### ofinflammation 

### was only minimal 

### and 

### no significant infiltration ofinflammatory cells 

### was observed. No specific aggregation ofparticles 

### was observed in the lung fields and lymph nodes, whereas 2 cases ofspontaneous alveolitis were 

### observed in 

### the control group. Micrographs 

# i
- 4 represent the examples ofhistopathological micrographs.

Micrograph 2 Alveolitis observed in the con1rol group (3days after 

### instillation)

Page 47p.48



### MB08Tl

Micrograph 3 TOFIX-S instillated group (3 days after instillation)

·-·-·;



### Micrograph 4 Control group (3 days after 

### instillation) Page 48p.49



### 7.7.2.2 Results ofexamination at 

### 6 months after instillation In 

### the 

### TOFIX-S instillated group, the degree ofinflammation was 

### only minimal 

### and little changes were 

### observed compared with 

### the control group. No findings including alveolitis were observed in both 

### groups. Micrographs 

### 5 and 

### 6 represent the examples ofhistopathological micrographs. The micrograph of PTl case is represented for reference.

Micrograph 

### 5 

### TOFIX-S instillated group (6 months after instillation)

Micrograph 6 Control group (6 months after 

### instillation) Page 49' 

### ' p.50 

### MB08Tt



### Micrograph 7 PTl instillated group(6 months after 

### instillation) 7.7.2.3 Inflammation point counting (degree ofinflammation) In 

### the conventional histopathological analysis, the presence or absence offindings oftumors and fibrosis and qualitative description ofmorphologic changes have been ofmajor description. Certain time course is required to 

### progress to 

### pulmonary fibrosis and tumors, however, changes in the 

### degree of inflammation in lung tissue in the process needs to be 

### assessed quantitatively. 

### In this ·,__ study, an attempt was 

### made to analyze changes in the inflammation areas semiquantitatively under an optical microscope. 1)Measurement method Six images per specimen were 

### randomly captured digitally under an optical microscope of 100 magnifications. To capture mainly lung fields, blood vessels and large bronchi were excluded. By placing a 300-point grid over each image on the PC screen, the inflammatory areas in each image were 

### examined using the point counting method (PCM). Six fields per section were examined; 1,800 points in total, an average pulmonary area of200 mm 2 

### ofcut surfaces on the preparations and tlie total area ofthe images of3.9 mm 2 

### that 

### was 2 % 

### ofthe cut surface ofthe lung surface area were examined. Excluding blood vessels and large 

### bronchi, the 

### point on the 

### inflammatory areas Page Set' 

# . ' 

# .

__:.. p. 51


### MB08TI 

### including alveolar walls, 

### alveolar ducts, terminal 

### bronchioles and airspaces. Excluding airspaces, 

### the percentage ofinflammatory areas in

### the pulmonary parenchyma was 

### calculated and group mean was 

### obtained. 2) ·Results ·ofthe ·inflammation 

### degree eVaiuation · The degrees ofinflammation were evaluated and the results are shown in

### Figure 8. No significant 

### differences were found both at 

### 3 days and 6 months after 

### instillation compared to 

### the 

### control group. The percentage 

### of the inflammation resulted from TOFIX-S instillation was 

### averaged less than 

### 1 %, 

### only a minimal orno inflammation was 

### observed.

0.05
0.04
0.03 

### 0 ccatrol • TOFIX 0.02
0.01
0 3d 

### Figure 8 Degrees ofinflammation

Page Sl' 

# . • • 

### p.52
MB08TI 

### Chapter 8 Discussion 8.1 Rat growth curves

. . - 

### -

**~-** 

### No differences were found in the 

### growth curves ofthe body weight between two groups. This suggests that exposure to TOFJX-S did not have an effect on the growth ofthe 

### rats. 8.2 Wet organ weights ofthe rats at each time point ofsacrifices No significant differences were found in the weight ofany ofthe lung, liver, kidney or spleen ofthe 

### rats 

### between two 

### groups. 8.3 Biopersistence ofTOFIX-S The biological half time (TIll) ofthe lung bmden ofTOFJX-S calculated by using the measured lung burdens was 

### 2.2 months. TIll for PTl (potassium octatinate wisker) that has 

### been stated earlier for reference is 12 months. It is postulated that 

### the 

### longer retention in the 

### lung increases the length oftime ofcontacting the 

### body, and induces various kinds 

### ofbiological reactions at higher degrees. It has 

### been assumed that the clearance halftime ofasbestos is 

### on the scale of

### years, its hazardous potential is much 

### greater. Since the 

### biological half

### time of2.2 months obtained in this study is 

### similar to that ofparticulate materials obtained in our laboratory, TOF1X-S are predicted to have little biological effect 8.4 8-hydroxydeoxyguanosine No significant formation of 8-hydroxydeoxyguanosine 

### was observed compared to the control group. Significant increase in the 

### formation of 8-hydroxydeoxyguanosine occurred as a major product of oxidative damage caused by active oxygen species, indicating the 

### substance's higher po1ency of hazard Therefore, the substance can be 

### considered to have higher hazardous potential. Page 52·' 

# . . ..

··...... ·· p.53


### MB08Tl



### In 

### this study, however, no production of 8-hydroxydeoxyguanosine 

### was observed following TOFIX-S instillation, it can be 

### predicted that significant pulmonary injury and carcinogenesis induced by 

### active oxygen species will not occur.

. . 

### 8.5 11lv.DP-2 No significant differences in 

### gene expression ofTIMP-2 between two groups were 

### found From this, it can be 

### predicted that TOFIX-S has 

### little effect on inflammation and fibrosis These 

### results are consistent with the 

### histopathological examination in

### this study. 

### 8.6 Histopathological changes Histopathological examination showed no significant differences between two groups and the biological halftime ofTOFIX-S was 

### 2.2 months which 

### was quite short These results are consistent with the postulate that 

### TOFIX-S bave little biological effect

Page 53I • ' 

# .

**·~.,..** . p.54
MB08Tl 

### Chapter 9 Conclusion Particles constitute most ofTOFIX-S, but it 

### also contains fibrous particles as well. When there 

### had been 

### concern about potential health effects ofasbestos, fibers longer than 

### 20 J.Lin 

### were 

### ofpotential

. . . . 

### health concern. No such fiber lengths were found for TOFIX-S. The assessment ofthe biopersistence or dmability in the lmtg is known 

### to be 

### an index 

### to predi£tthe 

### level of biological effects of fibrous materials. The 

### biological half 

### time or biopersistence of TOFIX-S 

## was 2.2 months 

### that is 

### consistent with 

### that of particulate materials obtained 

### in our laboratory which have little biological effect 

### The results 

### ofthe measurements ofTIMP2 in association with 

### gene expression 

### that are indices for inflammation and fibrogenesis were 

### not those predictive offibrogenesis. This 

## was consistent with the patholO"gic features. For 8-hydroxydeoxyguanosine, no formation was 

### observed. No significant changes were 

### observed in the 

### histopathological examination ofthe lungs.

Page 54

### Jul-08-2008 10:57 

### From-ENVIRON CORP 7035162304 T-371 P. 005/005p.**~~** 

### 27 MJSUITl 

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### (CBJ). -·· {6) EsrilllafC the; p=:cslJ oflbc; Dc;W**lO~Cc;** Ill>**fonnwJ~** in ffi1ll.ll.lrl;::o.,**lO~siOM, .:;ID~,J)l>iDnS,**  soluuon;, Drgds Ill>**man~** for 

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### (are) cont'id=t.al**bus1~** 

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### CaJ¢gory ofU$ (1) CBJ BJndwg Produc- 

### CBI %Jll 

### CBI %ofsubstaDoe 

### expected per

### use (8) CBI 

### (by fuxn.'1ion IWd**~li~n** 

### (2) Option tion% (S) 

### Fonn- (7) Site- Con- IndQs- 

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### (i.e. a dispersive 

### dye for 

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### Opa.on finishulg**pol~** fibm) (3} (6) 

### FriClion maten.l for 

### 10% AutomQtiw Brake Pacb 

### 100 o/o iu. brake o.ad 

### % % 

### % % Ifyou 

### have**id~c!d** 

### a ..**consumer~",** 

### please proVide on a detailed**~ptiou** 

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### consumer**prod~** 

### 8.Pd 

### describe lb:

### chemical**~tions** 

### by which 

### this subsmnce loses its identitY in 

### the consumer product.



# 0 Mark (X) 

### tllis box if

### you auacb a coiUilluatiOll sbeet. 

### b. Generic use Ifyou clainl any category ofux

### de:scnpnon m**~on** 

### 2a u 

### collfidential, enter a gellCric**~ption** 

### ofthat**~ption** 

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# 0 Ma,rl( (X) this box if

### you aRaeh a continuatioQ 

### sheet. 3. 

### H.autdJDfo.-madon -lucll.Uk in

### The notice a copy ofreasonable facsimile ofany hazard waming

### swemem. 8i11clin& Option 

### label, 

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### pJt>vidcd lO**any~** wbo lS**~ly** Manc(:ll.) 

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# 00 Mark (X) 

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FORM

# ~OMPANY'SANlTlZED Page7

# 11

# ~~-.~'~JMJJJ(ilfllll**~~d** SDg-Q)b

### Aur-28-2008 09:27 

### From-ENVIRON CORP 7035162304 T-372

**P.004/004P·~.fij42**

**~YUM'Qll** 

## Part 1- GENERAL INFORMATION
- Continued Section C- PRODUCTION, IMr.uKl.·. AND USE INFORMATION: 1. PrcMiu(riou

**vol~- fsJiQlA~**  

### ·...: **~~~~~** 

### 12 monlbs ofproducl;ion. Alsoestimme the maximloAll 

### ptodU'lion volume for 

### auy



### ~nve 1'7-



### :~**~lllfirsl** three yeas ofproduction. Est:imateli sbo!Ud be onlOO% DCW 

### cbdmcll

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### basis. Fur a Low YolJmW

**&emp~ion** 

### applicarion. 

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### wlumeandmark (x) in Jhe binding 

### box. 

### if granred. you an bowul 

### tu tlus vol...,e. 

### Maxiwum 6r.n 12-mootb production (ksfyr) 

### (100% new chemical subst.ulcc

**~)** MaxiWimJ 12ftmourb

**~tion** (q/yr) (100010 

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**CCl~,** 

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### tbe "'Confidelltial"

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**~of**use of

## me new chcmiatl mbsmncc by fUnction and appliCidiOP. (2) Matt (X)lbis column 

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(4) 

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(6) 

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- 

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**~**

.

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3. 

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**~ oro~**  Udbtwati011

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### pan

# m buard 

### iuf'onMtiop

**~ U.:l~.**  

### Please refer to tbe MSDS.

**~** Mark (X) thb box lfyo"



### ~ **~** UlfoJmation. BindingOpaon MU'k(ll.) 

# liD



# PI~~PIIJI!Ill!



# ~II

# PI

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