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p.2 

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Public reporting burden for this collection of information is estimated to average 110 hours per response, including time for 

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

reviewing the collection of information. Send comments regarding the burden estimate or any other aspect of this collection of 

information, including suggestions for reducing this burden, to 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 of my knowledge and belief: 

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

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

I, Section B. 

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

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

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

Additional Certification Statements: 

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

certification 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$1 000 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) 

If you are submitting a low volume exemption (L VE) 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 of 40CFR723.50. 

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

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

D If this application is for an L VE in accordance with 40CFR723.50( c )(1 ), the manufacturer intends to commence 

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

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 wilful misinterpretation is subject to criminal penalty pursuant to 

18 usc 1001. 

Confidential 

Date 

Signature of agent- (if applicable) Date 

FORM EPA 7710-25 (Rev. 5-95) Page2 
Part I- GENERAL INFORMATION 

la. Person 

Submitting 

Notice 

Name of authorized official Position 

Shintaro Vice President 

(in U.S.) 

b. Agent 

(if applicable) 

Company 

MORIMURA BROS INC. 

Mailing address (number and street) 

Parker Plaza 400 Street lOth Floor 

City, State, ZIP Code 

Front NJ 07024 

Name of authorized 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. If you are submitting this notice as 

Name of authorized official 

oint submission, mark (x) this box. 

2. Technical 

Contact 

(in U.S.) 

Company 

Mailing address (number and street) 

City, State, ZIP Code 

Name of authorized 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. If you have had a prenotice communication (PC) concerning this 

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

4. If you previously submitted an exemption application for the chemical 

substance covered by this notice, enter the exemption number assigned 

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

the PMN number EPA withdrawn or 

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 EPA. 

6. Type of Notice - 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) if none 

Mark(X) (X) if none 

Mark(X) (X) If none 

3. ( ) Both 
p.4 

MB08T1 

Part I- GENERAL INFORMATION- Continued 

Section B- CHEMICAL IDENTITY INFORMATION: You must correct '-'ll'"lllJ'"'" 

Abstracts (CA) name of the substance based on the 

ninth Collective Index (9CI) of CA 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. 

If another person will submit chemical identity information for you, mark (x) the box at the right. [ ] 

Identify the name, and address of that person. 

1. Class 1 or 2 chemical substances (for definitions of class 1 and class 2 substances, see the Instructions Manual) 

a. Class of substance - 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 orCA Preferred Name must be provided, 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 of the 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 (if a 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 or process. (3) Indicate the range of composition and the typical composition (where appropriate). ( 4) 

Provide a correct representative or partial chemical structure diagram, as complete as can be known, if one can be 

reasonably ascertained. 

/ 

"' /0 

Ti 

0 / "' 0 

"' / Ti 

/"'-

0 0 

""- / Ti 

/0 / "' 

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

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

0 0 

""- / Ti 

/"'-

0 0 

"' / Ti 

/0 / " 

Page4 

Confidential 
p.5 MB08Tl 

Part I- GENERAL INFORMATION- Continued 

Section B- CHEMICAL IDENTITY INFORMATION- Continued 

a definition of polymer, see the Instructions Manual.) 

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 monomers, 

reactants, or solvents) below 500 and below 1,000 absolute molecular weight of that composition. 

t----1 

Describe the method of measurement 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 if you attach a continuation sheet. 

b. You must make separate confidentiality claims for monomer or other reactant identity, composition information, and 

residual information. 

Mark (X) the "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 

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

polymer description on the TSCA Chemical Substance Inventory. 

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

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

manufactured for commercial purposes. 

(7) Mark (X) this column if entry 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 if you 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 of the 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 or partial chemical structure diagram, as complete as can be known, if one can be 

reasonably be ascertained. 

D Mark (X) this box if you 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 

commercial purposes. Provide the CAS Registry Number if available. If there are unidentified impurities, enter ''unidentified. 

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

Impurity and CAS Registry Number 

(a) 

D Mark (X) this box if you 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 

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

ConfiConfidential 

6. Generic chemical name- If you claim chemical identity as confidential, you must provide a generic chemical name for your 

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

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

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

7. Byproducts - Describe any byproducts resulting from the manufacture, processing, use, or disposal of the new chemical 

substance. Provide the CAS · Number if available. 

Byproduct CAS Registry Number Confidential 

None 

D Mark (X) this box if you 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 maximum production volume during the first 12 months of production. Also estimate the 

maximum production volume for any consecutive 12-month period during the first three yeas of production. Estimates 

should be on 100% new chemical substance basis. For a Low Volume Exemption application, if you choose to have your 

notice reviewed at a lower production volume than 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 of he category of use, the percent of 

production volume devoted 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 confidential. 

a. ( 1) Describe each intended category of use of the new chemical substance by function and application. 

(2) Mark (X) this column if entry 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 of total production for the first three years devoted to each category of use 

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

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

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

(7) Mark (X) this column if entry in column ( 6) is confidential business information (CBI). 

(8) Indicate % of product 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 provided in (8) binding. 

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

Category of Use (1) CBI Binding Produc- CBI %in CBI % of substance 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 

% % 

% % 

% % 

If you have identified a "consumer use", please provide on a detailed description of the use(s) of this chemical substance in 

consumer products. In addition include estimates of the concentration of the new chemical substance as expected in consumer 

products and describe the chemical reactions by which this substance loses its identity in the consumer product. 

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

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

description category. Read the Instructions 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, Binding Option 

label, material safety data sheet, or other information which will be provided to any person 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 of the new substance. List in part III hazard information you include. 

Please refer to the MSDS. 

[!] Mark (X) this box if you attach hazard information. 

FORM EPA 7710-25 (Rev. 5-95) Page7 
p.8 MB08Tl 

Part II- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

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

See instructions manual. 

1. Operation Description Confia. Identity- Enter the 1dtanuy of the 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 

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

on a continuation sheet, and if any of the 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 if you 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 major unit 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 l 00% new chemical substance basis), and entry point of all 

starting materials and feedstocks (including reactants, solvents, and catalysts, etc), and of all products, recycle streams, and wastes. 

Include cleaning chemicals (note frequency if not used daily or per batch.). 

(3) Identify by number the points of release, including small or intermittent releases, to the environment of the new( chemical substance. 

-

D Mark (X) this box if you attach a continuation sl.vv •. 

FORM EPA 7710-25 (Rev. 5-95) Page 8 
p.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 of worker activity, physical form 

of the new chemical substance, number of workers exposed, and duration of activity. 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 if entry 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) of the new chemical substance (e.g. solid: crystal, granule, powder, or dust) and% new chemical 

substance (if part of a mixture) at the time of exposure. 

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

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

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

(10), (11)Estimate the maximum duration of the activity for any worker in hours per day and days per year 

(12) Mark (X) this column if entry in column (5) is '" •I information ~CBI 

Maxtmum 

Worker activity Protective Equipment Optio; Physical Binding ,....,. # of ll""'nT lli rano11 (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 if you attach a continuation sheet. 

3. Environmental Release and disposal-You must make separate confidentiality claims for the release number and the amount 

of the new chemical substance released and other release and disposal information. Mark (X) the "Confidential" box next to 

each item you claim as confidential. 

( 1) Enter the number of each release point identified in the process description, Part II, section A, subsection 1 d(3). 

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

(3) Mark (X) this column if entries in columns (1) and (2) are confidential business information (CBI) 

(4) Identify the media of release 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, if any, and control efficiency that will be used to limit the release of the new substance to the 

environment. For releases disposed of on land, characterize the disposal method and state whether it is approved for disposal ofRCRA 

hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will be used and whether the 

waste is subject to secondary or tertiary on-site treatment. 

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

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

(7) Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for 

direct dischargers or NPDES numbers of the POTW (Publicly Owned Treatment Works). Mark (X) if the 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 if you attach a continuation sheet. 

FORM EPA 7710-25 (Rev. 5-95) Page9 
p.10 

MB08T1 

Part II- 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 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 of processing, or use operation involving the new chemical substance. If the 

same operation is performed at more than one site describe the typical operation common to these sites. Identify additional sites on a 

continuation sheet. 

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

you claim as confidential. 

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

pails, 55-gallon 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 or kg/batch on a I 00% new chemical substance basis), and entry point of all 

feedstocks (including reactants, solvents, and catalysts, etc), and of all products, recycle streams, and wastes. Include cleaning chemicals 

(note frequency if not used daily or per batch.). 

(3) Identify by number the points of release, including small or intermittent releases, to the environment of the new chemical substance. 

(4) Please enter the# of sites (remember to identify the locations of these sites on a continuation sheet): 

2 

#of Sites 

A. Open bags of the 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 if you 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 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 form of exposure and% new chemical substance (if in mixture}, and any protective equipment and engineering controls 

used to protect workers. 

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

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

(I 0) Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in 

kg/day or kg/batch). 

(12) Describe media of release 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, if any, that will be used to limit the release of the 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 if any of the 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 if you 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 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 minimise 

potential risks associated with activities surrounding manufacturing, processing, use and disposal of the PMN substance. Please 

include new information pertinent to pollution prevention, including source reduction, recycling activities and safer processes or 

products available due to the new chemical substance. Source reduction includes the reduction in the amount or toxicity of 

chemical wastes by technological modification, process and procedure modification, product reformulation, raw materials 

substitution, and /or inventory control. Recycling refers to the reclamation of useful chemical components from wastes that 

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

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

regulatory requirements and can be either quantitative or qualitative. The EPA is interested in this information to assess overall 

net reductions in toxicity or environmental releases and exposures, not the shifting of risks to other environmental media or 

non-environmental areas (e.g., occupational or consumer exposure). In addition, information on the relative cost or performance 

characteristics of the PMN substance to potential alternatives may be provided. 

All information provided in this section will be taken into consideration during the review of this 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 of waste materials through recycling, source reduction or other 

means; (4) a reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product 

performance, a decrease in the cost of production and/or improved operation efficiency of the new chemical substance in 

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

may be substitute for an existing substance that poses a greater overall risk to human health or the environment. 

Currently, needle-shaped fibers (e.g., whiskers) of potassium titanium oxide (potassium octatitanate) 

are used in automobile brake pads. 

In 2006, the World Health Organization (WHO) reviewed potential health effects of potassium 

octatitanate fibers and advised that respirable potassium octatitanate fibers are likely to pose a high 

hazard to humans after inhalation exposure. 

Because of the 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 

of which are different from potassium octatitanate fibers or whiskers. 

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

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

MB08Tl 

Part III -LIST OF ATTACHMENTS 

Attach continuation sheets for sections of the 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 of the 

form to which it relates, if appropriate. Number consecutively the pages of the attachments. In the column below, enter the 

inclusive page numbers of each attachment. 

Mark (x) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for 

guidanc~ 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 

Attachment Confi- Name Page dential Number(s) 

Physical and Chemical Properties Worksheet 13 

Material Safety Data Sheet (MSDS) 14-17 

Properties of Matter Data of Sample TOFIX-S 18 

Research Report "Biological Effects of TOFIX-S Using Intratracheal Instillation" 19 

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

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

MB08Tl 

PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

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

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

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

The physical state of the neat substance should be provided. These measured properties should be for the neat ( 100%) 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 strongly recommends 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 of test 

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

Page Measured 

Property Mark(x) Confidential 

Number Value or Mark(x) (a) if provided (c) Estimated (b) (MorE) (d) 

Physical state of neat substance Solid 

Vapor pressure 

@ temperature 25°C 

Density I relative density 3.6 glmL M 

Solubility in organic solvents 

@temperature~ Insoluble E 

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/water partition 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 if you attach a continuation sheet. Enter the attachment name and number. 

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

MB08Tl 

TOFIX·S Page 1/4 

MSDS No. : TTC·0002·2 

MATERIAL SAFETY DATA SHEET 

• 

!.CHEMICAL PRODUCT IDENTIFICATION AND 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 & Development Diviaion 

3· 3·5,Chigaaaki,Cbipeaki·City,Ka.napwa·Pref.253·8510,Japan 

Phone : +81·467·87·2813 

: Calcination method 

2.COMPOSITION I INFORMATION ON INGREDIENTS 

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 shape Avoid breathinc this product. 

TOFIX·S contain a small amount of respirable fibers which may become 

airborne during handline. 

Potential Health Effects : Prolonged inhalation of exeeeaive concentration of respirable fibers 

may cause deposition of quantities of fibers in lungs sufficient to cause 

a tissue response which could be associated with injury. 

4.FIRST AID MEASURES 

Inhalation 

Skin Contact 

:Gargle throat. If there is exposure to large amounts of respirable fibers, , 

vacate premises immediately and ifneceaaary seek medical aaaistance. 

: If there is exeeaaive exposure of akin to fibers, wash with soap and 

water. 

Page 14 
TOFIX·S Pace 214 

MSDS No. : TTC-0002·2 

Eye Contact : Wash immediately with clean water or eye-wash fiuid and if necessary 

seek medical assistance for examination and treatment. 

Ingestion : No adverae effects anticipated by 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.HANDIJNG AND STORAGE 

Use routine protective measures to avoid inhalation of materiale. 

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 of NIOSHIMSHA approved respiratory protection 

against dust. 

Eye Protection 

Skin Protection 

Exposure Guideline 

TLV<ACGIH) 

: Recommend use of safety rlasees 

: Recommend use of protective rlovea 

: None Established 

Page IS 

p.15 
p.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 

Vapor Denaity : 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.STABILITY AND RECTMTY 

This product is stable and hazardous 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 oral LDeo 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 if get dust into eyes. May irritate to the akin. 

in suaoeptible individuals. 

: Prolonged or repeated inhalation exposure to excessive concentrations , 

or reapriable fibers may induze irreversible lung injury. 

Page 16 
p.17 

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TOFIX·S 

12. ECOLOGICAL INFORMATION 

No information is available. 

18.DISPOSAL CONSIDERATIONS 

Page 4/4 

MSDS No. : TTC·0002·2 

Bury in landfill in accordance with all applicable reculations. Any disposal practice must be in 

compliance with local,state, and federal laws and regulations (contact local or state environmental 

agency for specific rulee). 

Waste such ae dust from air filters may contain high depoaite of respirable fibers. Bag and dispose 

ae nonhazardous industrial waete. 

14.TRANSPORT INFORMATION 

Not a hazardous material for DOT shipping. 

15.REGULATORY INFORMATION 

OSHA :This product is not hazardous under the criteria of the Federal OSHA hazard 

Communication Standard 29 CFR 1910.1200. 

16.0THER INFORMATION 

To the beat our knowledge, the information contained herein is accurate. 

However, we cannot aeaume any liability whatsoever for the accuracy or completeneaa of the 

information contained herein. Final determination of suitability of any material ia the eole 

responsibility of the user. All materials may preaant unknown hazards and should be used with 

caution. Although certain hazards are deacn'bed herein, we cannot guarantee that these are the 

only hazards that exist. 

Page 17 
MB08Tt 

Properties of matter data of sample TOFIX-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 of matter of TO FIX -S CAS No.12056-51·8 

SAMPLE NAME TOFIX·S An analysis method 

Chemical composition K20 • 6Ti0a XRD 

Tom of 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 

Melting point L300-1,350CC Documents value 

Electrical resistance 3.3Xl0150cm Documents value 

SEM Photo. 

Page 18 

p.18 
p.19 

MB08TJ 

Research Report 

Biological Effects of TOFIX-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 of the Intratracheal Instillation 

7.1 Rat growth curves 

7.2 Wet weights of the 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 of TOFIX-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: 

Professor Isamu Tanaka, Dr. Eng., vJ.~ Department of Environmental Health Engineering, 

Institute of Industrial Ecological Sciences, 

University of Occupational 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 of Occupational and Environmental Heal~ Japan 

{Graduated from K.agoshima University School ofMedicine in 1986) 

Department 

Associate Pro&asor 

of Environmental 

Toshibiko Myojo, 

Health 

l.k 

Engineering, 

Eog., 1 ~ 

Institute of Industrial Ecological Sciences, 

University of Occupational and Environmental Health, Japan 

(Graduated from Department ofEngineering, Kanazawa University in 1977) 

Associate Professor Akira Ogami, MD, OMS, 

Department of Occupational Pneumology, 

Institute of Industrial Ecological Sciences, 

University of Occupational and Environmental Health, Japan 

p.22 

MB08Tl 

{Graduated from University of Occupational 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 of Occupational and Environmental Health, Japan 

(Graduated from the Department of Science, University ofKyushu in 1983) 

Page 23 

p.23 

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p.24 

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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 of the 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 of various kinds of biological 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 of TO FIX -s retained in rat lungs are quantified by means of 

serial sacrifices to determine the rate oflung clearance 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 of chemicals that are introduced into the lung via airways is vital to assess their 

health effects or biological effects in humans. 

The fate of chemical 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 of time required for the initiation of biological responses and the effects of such responses. 

The significance of this understanding has begun to be appreciated in recent years and, therefore, 

biopersistence of fibrous 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 of a given number of rats at certain time points 

5. Measure the amount of cytokine which is deeply involved in fibrotic or carcinogenic changes 

in the lungs 

6. Histopathological examinations of the lungs 

Page 25 
p.26 

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Chapter 4 TOFIX-S Particle Characteristics 

4.1 Contents of fibrous materials 

Filters containing TOFIX-S and deposited platinum on them were prepared for scanning electron 

microscopy assessment of TOFIX-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 of view of the scanning electron microscope were counted. The number 

of fibrous particles and non-fibrous particles quantified by the scanning electron microscopy were 

124 and 1,136, respectively. This indicates that the number of fibrous particles accounts for 9.8% 

of total number of particles 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 of Hazards 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 of fibrous 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 of the 321 TOFIX-S. The results were plotted on a 

logarithmic-normal probability paper as shown in the Figures 1 and 2. 

98 

85 

--'#. 80 

.._, 

r 

80 

70 

80 

l 50 

40 

-~ 30 

-a 20 

s 10 

5 

2 

0.1 

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 of the 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 28 
p.29 

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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 of samples), and then a method to recover the given amount of TOF1X-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 of either 0, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 mg was added to each 1g of homogenized 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 of TOFIX-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 of TOFIX-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 29 
p.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 of 50 m1 

r 

Quantify Ti amount (ICP-AES analysis) 

Figure 3 Procedures for the quantification ofTOFIX-S lung burdens 

Page 30 
p.31 

MB08Tl 

Table 1 The results of recovery rates 

Slmpla Pbmii!Mric cpulities 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 of chemical 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 of test 

materials in the animal lung. A given amount of test 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 of the method, intratracheal instillation administers an 

accurate dosage of materials but it has the disadvantages that large amount of dose 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 of introduction into body is an unnatural route of exposure in human 

exposure, but the method does provide useful information on how the test particles behave in the 

lungs. 

The main theme of this 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 of rats 

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 of physiological 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. 

- 1 week after instillation, 5 rats per group were sacrificed. 

- 1 month 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 of the 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 of sacrifices 

The results of the measurement of the wet weights of rat lung, liver, kidney and spleen at each time 

point of sacrifices are shown in Table 2. 

Page 34 
-- ~ ... · 

p.35 

MB08Tl 

Table2 Wet weights (g) of rat lung, liver, kidney and spleen at each time point of sacrifices. 

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 in the two groups. 

7.3 Quantification oflung burdens ofTOFIX-S 

1) Method for quantification ofTOFIX-S in the 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 of TOFIX-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 of measurements are shown in Table 3. 

Table 3 Lung burden of'IDFIX·S at each time point of sacrifices 

3 days after instillation 

1 week 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 

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Ti amounts in rat lungs of the control groups were found to be extremely low (less than 1 J.Lg) when 

compared with to those in rat lungs of the 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 of them 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 of approximately 3 X 108 alveoli of tiny 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 of inhaled 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 36 
p.37 

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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 of TOFIX-S at each given time point after instillation were 

plotted on semi-logarithmic graph to estimate the rate of TOFJX-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 

I Biological halftime: 12 months 

• 

I Biological half time: 2.2 months 

OPT1 

•TOAX-S 

0 

<> 

• 

0 2 3 4 5 6 

Time points after instillation (month) 

Figure 5 Changes oflung burden 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 obtained by lung burdens of TOFIX-S measured at three -days, 1 

Page 37 
p.38 

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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 of the 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 of particles is cleared from the lung. The T 112 is given by using the following 

Equation (4): 

T 112 = 0.693/k (4) 

The biological half time of the lung burden of TOFIX-S calculated by using the measured lung 

burdens was 2.2 months. The T 112 can also be assumed from the gmdient of the line shown in 

Figure 3. T 112 for PT1 (potassium octatinate wisker) is 12 months. 

7.5 Formation of 8-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 
·---··· 

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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 1 ml 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 39 
p.40 

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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 of sodium acetate buffer solution, 4 !J.l of Smw'ml nuclease 

P1, and 2 !J.l of phosphoric 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 of 80HdG can be determined. 

7.5.2 Measurements of 8-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 
.. _ ... 

·· .. -· 

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3.0 0 Control group 

• TOFIX-S instil1ated group 

2.5 

2.0 

~ 

~ 1.5 ..... 

-

1.0 

0.5 

0.0 

3 days after iDstiDation 6 months after instiDation 

Figure 6 The levels of 80HdG in the lungs of rat 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 of 80HdG both at 3 days and 6 months after instillation. 

7.6 Expression of gene 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 of collagen. 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 of collagen. 

7.6.1.1 Excessive production of collagen 

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 of excessive production of collagen 

could be determined from examination of whether gene expression ofType I collagen increased. 

Page 41 
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7.6.1.2 Regulation of collagen 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 of collagen 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 of TIMPs than of M:MPs 

may contnbute to the downregulation of degradation resulting in the deposition of collagen. 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 of collagen. This is seen in an early 

stage or lung injury and inflammation in the process of fibrosis 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 of collagen can be elucidation by means of quantification and analysis of the results. 

It seems a reasonable procedure to first conduct a screening for expression of these genes, then, if 

significant changes are found in any of these three genes, expression of the other two genes are 

further examined. We decided to first measure the activation of TIMP and, if significant differences 

in the levels of TIMP 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 of Solution 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 of 2M Sodium Acetate (pH4.0), 1.2 ml of Phenol DEPC 

Page 42 
p.43 

MB08Tl 

H20 and 120 J.1l of Chloroform-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 of isopropanol 

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} of SolutionD to the pellet, 

8) Add 360 J1} of isopropanol and hold at -80°C for 15 minutes, 

9) Centrifuge at 15,000 rpm for 10 minutes at 4°C, 

10) Discard the supernatant, mix with 0.5 ml 75% ethanol and centrifuge at 15,000 rpm for 10 

·~···· minutes at 4°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 extracting the RNA, gene expression ofTIMP was measured by perform reverse-transcription 

polymerase chain reaction (RT-PCR). 

Page 43 
~-· ' 

p.44 

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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 eppendoif tube, 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 44 
p.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 l 0 J.L). of ethidium 

bromide. 

6) Electrophoreses at 100 volts for 1 hour. 

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 of the bands is measured 

using NIH image. 

Page 4S 
.;. 

~--· 

p.46 

MBOSTI 

9) The ratio of the densities of the target genes to J3-actin genes as a control (internal standard). 

7.6.3 TIMP2 measurements 

The results of the measurements of TIMP2 in association with gene expression that is related to 

fibrosis are shown in Figure 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 of rats at 3 days and 6 months after instillation. 

·--~ Figure 7 shows that no differences were found in gene expression of TIMP 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 of examination at 3 days after instillation 

In the TOFIX-X instillated group, the degree of inflammation was only minimal and no significant 

infiltration of inflammatory cells was observed. No specific aggregation of particles was observed 

in the lung fields and lymph nodes, whereas 2 cases of spontaneous alveolitis were observed in the 

control group. Micrographs i - 4 represent the examples of histopathological micrographs. 

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

Page 47 
p.48 

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

·-·-·; 

Micrograph 4 Control group (3 days after instillation) 

Page 48 
p.49 

7. 7.2.2 Results of examination at 6 months after instillation 

In the TOFIX-S instillated group, the degree of inflammation 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 of histopathological 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 

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Micrograph 7 PTl instillated group(6 months after instillation) 

7.7.2.3 Inflammation point counting (degree of inflammation) 

In the conventional histopathological analysis, the presence or absence of findings of tumors and 

fibrosis and qualitative description of morphologic changes have been of major 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 mm2 of cut surfaces on the preparations and 

tlie total area of the images of 3.9 mm2 that was 2 % of the cut surface of the 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 of inflammatory areas in the pulmonary parenchyma was calculated and group mean 

was obtained. 

2) ·Results ·of the ·inflammation degree eVaiuation · 

The degrees of inflammation 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 or no inflammation was observed. 

0.05 

0.04 

0.03 

0 ccatrol 

• TOFIX 

0.02 

0.01 

0 

3d 

Figure 8 Degrees of inflammation 

Page Sl 
' . • • 

p.52 

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Chapter 8 Discussion 

8.1 Rat growth curves 

. . - - ~-

No differences were found in the growth curves of the body weight between two groups. 

This suggests that exposure to TOFJX-S did not have an effect on the growth of the rats. 

8.2 Wet organ weights of the rats at each time point of sacrifices 

No significant differences were found in the weight of any of the lung, liver, kidney or spleen of the 

rats between two groups. 

8.3 Biopersistence ofTOFIX-S 

The biological half time (TIll) of the lung bmden of TOFJX-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 of time of contacting the 

body, and induces various kinds of biological reactions at higher degrees. It has been assumed that 

the clearance half time of asbestos is on the scale of years, its hazardous potential is much greater. 

Since the biological half time of 2.2 months obtained in this study is similar to that of particulate 

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 

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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 of TIMP-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 53 
I • 

' . 

·~.,.. . 

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 of asbestos, fibers longer than 20 J.Lin were of potential . . . . 

health concern. No such fiber lengths were found for TOFIX-S. 

The assessment of the biopersistence or dmability in the lmtg is known to be an index to predi£t the 

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 of the measurements of TIMP2 in association with gene expression that are indices for 

inflammation and fibrogenesis were not those predictive of fibrogenesis. This was consistent with 

the patholO"gic features. 

For 8-hydroxydeoxyguanosine, no formation was observed. No significant changes were observed 

in the histopathological examination of the lungs. 

Page 54 
Jul-08-2008 10:57 From-ENVIRON CORP 7035162304 T-371 P. 005/005p. ~~ 27 

MJSUITl 

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(3) Indica= )'OUT willmp$ ID have me information provicW in colunm (I) binding. 

(4) Estimare me~ ofwml produaioa foT me tim~ ye&~no devOied to eadl ~of &Jse 

(5) Marit (X) du:t coJ~o~n~R ifemry m c:oJWM (4) is confidential bi&Sinca intbmlariou (CBI). 

(6) £stima(c me J'd'"fl' of" ncw ~ :tS tbn'nuWea in~ ~ons, emulsions. solwion», or gd:; as manufac:aan:d for 

comm~al puJIX*i at sites under )'OlU" coniJ'Ol ~ad Widii:I!Cb ~ of~ 

(7) Mlllk (X) this 1:0)~ •f eJJQ')' in col~ (6) is conficit;utial bu~:~ infonDIIIion (CBI). ....._ -. 

(8) lndi~% ofprodl.lct \lolume ~pec:Jed fof dle li~ad i.ISC"' SgQors. Mark mon; !han one boA if~ M~) 

w mdic:aw your williagnc» JO h~J\1~ Qh; UK 1ypc providCld m ( 8) ~ <::.'.:> 

(9) Mark (X) dtis column lfQia}(ld) in column (I) is (are) confidential busitless infonnanon (C81). ·~ -- __ 

Category of Use ( 1) CBI 

(by funcliou II$Kl applic;atioD (2) 

(i.~:. a dispc:nivc dye: for 

fini .. hm~ po)ycb'1Ct tibet's) 

Frietiou material for 

Automotin Bl'm Pad$ 

BUldiug 

Optiop 

Mark(x} 

(3) 

Prodqc. CBI 

uon o/o (5) 

(4) 

100 11/e 

%in 

Fonnulaliou 

(6) 

10% 

~br* Pad 

% % 

% % 

CBI 

(7) 

%of subsuuu:c: cxpecred pa ~ {8) - cal 

Si• Cou- ludus.- Cc:iQ: BU:i.dUJg (9) 

linUJ.ed SQtnet" vial mercia! ~ 

. __ } 

If you ~ve ideutifi...d a "co~ ...se", pl~ provide OD a deW.led description of~ ~s) of this cbelnical s\lbSQilCe in 

COD:IWlCT pfOduciS. 1o additiou iDe tude C"!timarcs of the c~p of the new chemical subSfiDCe' as expeciCid iu c~ 

pr~ llD4 ckSCribe tbe chemical reactiPQS by which dUs ~ loses iu; ideoliey iD lhe coasumer product. 

0 Mluk (X) du:i box if you ~b a comillualiou ~-

b. Geuenc U$C: If you clainl any ~gory of use dellcripnoD in mb~ 2a llll ccmfidential, emer a senertc description of that 

ckscriptiou cafeSoty. Read lbe IusuuctiOQj Mmwal fur examples of gcqeric use descriptious. 

3. 8a2atd lnformadoa -laclude iD We-~ a copy o!reasouable &csuDi1e of my hazud ~ ~~. 

label, material safety data ~ oro~ Udbtwati011 which will be provided TO auy perso11 wbo is ~ly 

hkely TO be ~ to du:l sub:iJance re~ proleC'Iive equipment or pnctioe5 for Jbe safe handling, 

~use or disposal of~ uew aubslaoce. List iD pan m buard iuf'onMtiop ~ U.:l~. 

Please refer to tbe MSDS. 

~ Mark (X) thb box lfyo" ~ ~ UlfoJmation. 

Binding Opaon 

MU'k(ll.) 

liD PI~~ PIIJI! Ill! ~II PI ~III,WI,illl,lll,~ I~J II Ul 

~8·-<0:>(0 ~/\~