Sanitized version 

roved. O.M.B. No. 2070-0012. Approval Expires 10-31-96. 

u. S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY 

&EPA PREMANUFACTURE 

NOTICE 

FOR NEW CHEMICAL SUBSTANCES 

Whcit­ -: 

I completed I 

send this : 

form to I 

, 

DOCUMENTCONTROLO~CER 

O~CE OF POLLUTION PREVENTION 

AND TOXIC SUBSTANCES, 7407 

U.S. E.P.A.401 M STREET, SW 

WASHINGTON D.C. 20460 

Enter the total number of pages 

in the Premanufacture Notice 

Date of receipt 

COMPANY SA"rm?~n 

~entconttolnuznber EPA case nuznber 

51010000 II 

GENERAL INSTRUCTIONS TS­ • You must provide all information requested In this form to the extent that II Is known to or reasoubly ascertainable by you. M.U:e rusonable estimates if you do 

not have adual data. ~ 

• Before you complete this form, you should rud the "Instructions Mmaal for Premanufadure Notificatioa" (the Instrudions M.anualls available from the Toxic 

Substances Contral Ad (TSCA) Informatloll Sen>ice by calling 2D2-S~1404, or faxing 202-~S603). 

·If a user fee ha been remitted for this aotice (40 CFR 70(US), Indic.ale In the boxes above the TS-...er fee identification number yoa have generated. 

Remember, your u~r fee ID number must also appeM on your corresponding fee remlltaAce, which 18 sent to: EPA, HQ Accounting Operations Branch <PM-266), 

P.O. 360399M, Pittsburgh, PA 1S2S1~99, Athl. TSCA User Fee. 

PmI-GE~L~ORMATION 

You must provide the cuxrently coaed Chemical Abslrads (eA) N.une of the 

new chemical substance, even If you dalm the identity as confidential. Yo'; may 

authorize ;mother person to submit chemical Identity information for you, but 

your submission will not be complete and the review will nol begin until EPA 

receives this Information. A letter in support of your submission should 

reference yourTS user fee identification number. You must submit m original 

and two copies of this notice including all test data. If you claimed my 

information as confidential. a single ~tized copy must also be submitted. 

Pm n - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

If there are several manufadure. processing. or use OperatiolUl to be described In 

Pm II. sections A and B of this notice, reproduce the sections .. needed. 

Pm ill- USTOFATTACHMENTS 

Attach additional sheets if there is nol enough sP'"""' to answer a question fully. 

Label each continuation sheel with the corresponding section heading. In Pm 

IlI, list these attachments, my test data or other data ;md any optional 

Information included In the notice. 

OPTIONAL INFORMATION 

You may include any Info~ation that you want EPA to consider in evaluating 

the new substance. On page 11 of this form, space ha been provided for you to 

describe pollution prevention and recycling information you may have regarding 

the new substance. 

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

your willingness to be bound to certain sbtements you make in this notice, such 

as use, production volume, protective equipment ••. This option is intended to 

reduce delays that routinely accompany the development of consent orderS or 

Significant New Use Rules. Except in the ase of exemption applications (such a 

"Th1EA, LYE, LOREX) where certain infocnation provided in such nolifiation is 

binding on the submitter when the Agency approves the exemption application, 

checldng a binding box In this notice.d.a.a..lul1 by itself prohibit the submitter 

from Ialerdevlatlng from the Information (except chemical identity) reported in 

the form. 

CONFIDENTIALITY CLAIMS 

You may dalm any Infomution In this notice a confidential. To assert a claim 

on the form, marie (X) the confidentw box next to the information that you dalm 

as confidentw. To assert a dalm in an attachment, drcIe or bradtet the 

information you claim a confidential. If yog dafm inhumation In thc Poticc as 

ronfidentla! yog must also providc j! unitized Ycajon of the notice lindudinc 

attachments). For addilionallnstructions on claiming Information as 

confidential, read the Instrudions Manual. 

W MMk 00 If any Information In this notice is claimed as confldenllal. 

TEST DATA AND OlHER DATA 

You are required to submit all test data in your possession or conttol 

;md to provide a description of all other data nown to or reasonably 

;ascertainable by you, if these data are related to the health and 

envlranmental effects of the manufadure, pracessing, distribution 

in commerce, use, or disposal of the new chemical substance. Standard 

Utenture dlations may be submitted for data In the open scientific 

UtentuJ:e. Complete test dab (written In EncUshl. not summaries of data 

must be submitted It they do Pot ilRxar In tbe oxn literature. You should 

clearly Identify whether test data is on the substance or on an analog. Also, 

the chemical compositloa of the tested material should be charaderized. 

Following are examples of test data and oIher data. Data should be 

submitted according to the requirements of §720.50 of the Premanufadure 

Notification Rule (40 CFR Pm 720). 

Test Data (Check Below any included in this notice) 

Environmental fate data D Yes • Otherdata 0 Yes 

• Health effects data I]] Yes Risk assessments 

• Environmental effects data D Yes StrudureJadivity relationshi ps 

PhysiallChemical Properties' mYes 	 Test data not in the possession 

or conttol of the submitter 

"A physical md chemical properties worksheells located on the last page 


of this form. 


TYPE OF NOTICE (Check Only One) 

[1] PMN (Premanufadure Notice) 	 '~::-:i 

"C rl 

D INTERMEDIATE PMN (submitted in sequence with flii;d prod~~) PMN> ...- ---; r'l 


D SNUN (Significant New Use Notice) '" 


C"~~ C:J 
 D !MEA (Test Marlceting Exemption Application)

D LVE (Low Volume Exem ption) @ ~ CFR 723.50 (c)(1) c..!1 

D 0) 

LOREX (Low ReleaselLow Exposure Exemption) @ 723.SO(c)(2) 

D LVE Modification D LOREX Modification 

IS nns A CONSOLIDATED PMN7 D Yes 

, of chemicals 

<PreBotice Communication t required, enter t on page 3) 

EPA FORM mO-25 (Rev. 5-95) / 	 Replaces previous editions of EPA Form 7710-25. 
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 an yother aspect of this coUection of information, including suggestions for reducing this burden, 

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

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

CERTIFICATION 


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

1. 	 The company named in Part I, section A, subsection 1a 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 §72050 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: 

[K] 	The Company named in Part I, Section A has remitted the fee of $2500 specified in 40 CFR 700.45 (b), or 

D The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined @ 40 CFR 700.43) in 

accordance with 40 CFR 7OO.45(b), or 

D 	The Company named in Part I, Section A is a small business concern under 40 CFR 700.43 and has remitted 


a fee of$100 in accordance with 40 CFR 700.45 (b). 


If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50 (c) (1) or a Low 

release and low exposure exemption (LoI,UnO application in accordance with 40 CFR 723.50 (c) (2), check the following 

certification statements: 

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


commercial purposes, other than in small quantities solely for research and development, under the termS 


of 40 CFR 72350. 


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 LVE in accordance with 40 CFR 723.50 (c){1), the manufacturer intends to commence 


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


30 day review period. . , 


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


substance dESa'lDed herein. Any knowing and willful misinterpretation is subject to criminal penalty pursuant to 18 USC 1001. 
 Confidential 

Signature and title of Authorized Official (Original Signature Required) Date 

Signature of agent. (if applicable) Date

FORM EPA 7110-25 (Rev. 5-95) 	 Page 2 
Part I - GENERAL INFORMATION 

Conii- f-...;S;..e;..;cti...;·..;.o.;.;n..;.A~-...;;S..;.UB~MI __I_I_ER_ID_ENTIFI ___CA_TI_O_N__----------------------1 dentid 

la. Person 

Submitting 

Notice 

(in US.) 

b. Agent(lf 

applicable) 

Mailing address (number and 

Oty, State, ZIP Code 

Name of authorized. oifid.al 

Not applicable 

Mailing address (number and street) 

Oty, State, Code 

Telephone 

Co If you are submitting this notice as part of a joint submission, mark 00 this box. -:----------------~ 

Joint Name of authorized offidal TItle 

SWmtitter Not applicable 

(If applicable) . --;~~;;y----------------'-----------:----:--------:---

Mailing address (number and street) 

2. Technical 

Contact 

em U.s.) 

aty, State, ZIP Code 

Name 

Company 

Mailing ad.o:b:ess (number and street) 

aty, State, ZIP Code 

Telephone 

TItle 

Telephone I 

x 

.1 

3. 	 If you have had a prenotia! oommunication (PC) conalrning this notice 

and EPA assigned. a PC NumDe:r to the notice. enter the number. Mark 00 

Unone 

4. 	 If you have submitted an exemption notice/application {or 

the chemicalsubstana! covered. by this notice, enter the exemption Mark 00 


number assigned. by EPA. If you have withdrawn a previously 
 if none 


submitted. PMN enter the PMN number.· >­

If you have submitted. a bona fide request (or the chemical substance Mark 00 oovered. by this notice, enter the bona fide request number assigned. -----:>~ [XJ if none ~~ > 

Manufacture 2. Type of Notice - Mark 00 1. D Only 	 0~ D 

D 

3. Both 

Bl.ndlng Option D BlndingOplion 

Mark Mark (x) . (x) 

FORM EPA 7710-25 (Rev. 1-91) 	 Page 3 
Part I - GENERAL INFORMATION ­ Continued 

Section'B - CHEl\1lCAL IDENTITY INFORMATION: You must provide a cu.rrently COlTect Chemical Abstracts (CA) name of the substance 

based on the ninth Collective Index (9CI) of CA nomenclature rules and conventions. 

Mark CO the 'Confidential" box next to any item you claim as confidential. 

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

If another person will submit chemical identity information for you (for either item 1 or Z), mark CO the box at the right. 0 

the and address of that in a continuation sheet. ::;.... 

1. Class 1 or 2 chemir::al substarttes (for definitions of class 1 and class Zsubstances, see the Instructions Manual) 

a. Class of substance ­ Mark 00 1 W Oass 1 or 2 0 Class 2 

b. o,emical name (Currently correct Chemical Abstracts (CA) Name that is consistent with T5CA Inventory listings for similar substances. 

For Qass 1substances a CA Index Name must be provided. For Qass Zsubstances either a CA Index Name or CA Preferred Name X 

be provided, whichever is appropriate based on CA 90 nomenclature rules and conventions.). 

c. Please identify which method you used to develop or obtain the sped.fl.ed chemical indentity information reported in this notice: 

(ched:: one). D Method 1 (CAS Inventory Expert Service - a copy of the Identification WMethod 2 (Other Source) 

report obtained from the CAS Inventory Expert Service must be 

submitted as an attachment to this notice) 

d. Molecular formula and CAS Registry Number (If a number already exists for the substance) 

CAS' 

e. For a cIass 1 substance, provide a complete and correct chemical structure diagram. For a class 2 substance - (1) List the immediate 

X 

X 

precursor substances with their respective CAS Registry Numbers. Q)Describe the nature of the reaction or process. (3) Indicate the X 

range of composition and the typical composition (where appropriate). (4-) Provide a rorrect representative or partial chemical 

structure diagram, as complete as can be known, if one can be reasonably ascertained. 

Mark CO this box if attach a continuation sheet. 

FORM EPA 7710-25 (Rev. 5-95) Page 4­
PartI - GENERAL INFORMATION - Continued 

~ Section B - CHEMICAL IDENTITY IN'FORMATION - Continued 

2. Polymen (For a definition of polymer, see the Instructions ManUllL) Confidential 

a. Indicate the number-average weight of the lowest mole<:Ul.ar weight composition of the polymer you intend to manufacture. 

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

SOO and below 1,000 absolute molecular weight of that composition. • 

Describe the methods of measurement or the basis for yow: estimates: GPC 0 Other0 : (Specify) 

i) lowest number avu:age molecular weight: 

ii) maximum weight 'l'io below sao molecular weight Not applicable 

iii) maximum weight 'l'io below 1000 moleeular weight

o Mark 00 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 00 the ~Confidentia1" box next to any item you clai.m as con.6.dential.. 

(1) - Provide the specific: chem.ic:a1 name and CAS Registry Number (if a on umber 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) - Marle 00 the identity column if you want a monomer or other reactant used at two weight percent or le~s to be listed as part of the 

polymer description on the TSCA Chemical SublJlance Inventory. 

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

(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 co=~ purposes. 

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

Monomer or other reactant and CAS Registry Number Confi- Typical Identity Confi- Maximum Confi· 

dential composition Mark (X) dential residual dential 

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

Not applicable 'l". ,. 

'r_ ,. 

,. 

,. 

,. ,. 

,. ,. 

,. <V. 

,. ,. 

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

Co Please identify which method you used to develop or obtain the specified chem.ic:a1 identity information reported in this notice. (check one). o Method 1 {CAS Inventory Expert Service - a copy of the identification report 0 Method 2 (other source) 

obtained from CAS Inventory Expert Service must be submitted as 

an attachment to this notice) 

d.. The currently correct Otem.ical Abstrad:9 (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers. 

Not applicable 

e. Provide a correct representative or partial chem.ic:a1 struc:tUIe diagram, as complete as can be known, if one can be reasonably ascertained. 

Not applicable 

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

FORM EPA mo.25 (Rev. 5-9S) PageS 
Part I - GENERAL INFORMATION - Continued 

~ Section B - CHEMICAL IDENTITY INFORMATION - Continued 

30 Impurities 

Ca) ­ Identify each impurity th.at may be reason.ably antidp.ated to be pr_t In the chemical subsLana lIS m.anufadured for cnmmerd.a.l purposes. 

Provide the CAS Registry Number if av.a.llable. If there are unidentified Impurities, enter "unidentified." 

(b) -Estimate the maximum weight 'I:. of each impurity. If there are unidentified impurities, estim.ate their toW weight 'l'.. 

Impurity and CAS Registry Number Maximum Conti­

(a) percent dential '" (b) 

, % X

% X 

% 

% 

r. 

% 

% 

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

4. Synonyms - Enter any chemical synonyms for the new chemical substance identified in subsection 1 or 2. ~::::1 

X 

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

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

YDC-1312 

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

6. Generic chemical name ­ [f you claim Ptemical identity as confidential. you must provide a generic chemical name for your substan~ 

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

Refer to the TSCA Otemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing 

generic names. 

Substituted phenylepoxide 

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

7. Byproducts ' n.--,.;Jow, any byproducts resulting from the manufacture, processing. use, or disposal of the new chemical substance. Provide the CAS 

RegIStry Nu.in~ if available. 

Byproduct CAS Registry Number Conti­

(1) (2) dential 

None 

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

FORM EPA 77l0-2S (Rev. 5-95) Page 6 
Part I -- GENERAL INFORMATION ­ Continued 

~ Section C - PRODUCTION, IM:PORT, AND USE INFORMATION: 

Mark C<f'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 years of production. Estimates should be on 

1000/0 new chemical substance basis. For a Low Volume Exemption applictztwn, if you cJwose to have your notice rf:Viewed at a lower produdwn 

volume than 10'{}(}o kg/yr, sper:ify 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) Confi- Binding 

dential Option 

(1000/0 new chemical substance basis) (1000/0 new chemical substance basis) Mark (x) 

X 

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

devoted to each category, the formulation of the new substance, and other use information. Mark (X) the ~~nfidential~ 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 (CBO. 

(3) - Indicate your willillgness to have the information provided in column (1) binding. 

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

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

(6) - EstiInate 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. m- Mark (X) this column if entry in column (6) is confidential business information (CBO. 

(8) -Indicate 'l'O 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 rolumn (8) is (are) confidential business information (CBO. 

category of use (1) CBl Binding Produc- CBr 'l'Oin 

CBI f;.0/ ru"""'~ """"'"" P'" ~ CDI 

(by function and application i.e. a dispersive dye for OptiOD tion'l'O Form­ (8) MMIc(x) ulation finishing polyester fibers) Con-' ICom- Binding 

(2) (3) (4) (5) (6) (7) ed sumer ~~erdal Option (9) 

X ~ X ~ X X 

• % 0/0 

% 0/0 

0/0 % 

0/0 0/0 

0/0 0/0 

'j­

% % 

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

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

consumer products and descn'be 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 If you claim any category of use description in subsection 2a as confidential, enter a generic description of that 

use category. Read the Instructions Manual for examples of generic use descriptions. 

description 

Electric device 

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

3. Hazard ~nfonna~on -Include in the notice a copy of reasonable facsimile of any hazard warning statement, label. material safety data sheet, Blacilns 

or o~er information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective Opdoa 

LX]eqwpment p~ces for the safe handling, transport, use, or disposal of the new substance. Ust in part mhazard information you include. M&d<(;jd 

X Mark (X) this box if YOU attach hazard information. 

FORMEPA771O-25 {Rev.5-95} Page 7 
Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER M":I'k (X) the 'Co~dentialff box next to any item you claim as confidential. 

Complel;j!.section A for each type of maJ.'tUiacture, processing, or use operation involving the,lJew 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 pmcessing or use operations alter import. You must desaibe these operations. See instructions manuaL 

1. Operation desaiption 

a. - Enter the identity of the site at which the oOE!ration will occur. 

Name 

Not applicable 

address (number and street) 

aty, County, State, ZIP Code 

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

additional sites on a continuation sheet, and if any of the sites have significantly different ~ 

i'roduction rates or operations, include all the information requested in this section for those ~ 

Con.6 

dential. 

o Manufacturing P Processing 

Hours/batch 

b. 	TypeMark CO o Use 

Batches/year 

1. Batch 

Maximum kg/day (100" _ c.hemlc.al Hours/day Days/year mbotanc:d 

2. 	Continuous 

d. 	Process desaiption 0 Mark 00 to indicate your willingness to have your process description binding. 

(1) 	Diagram the major urdt operation steps and chemical conversions. Include interim storage and transport containers (sped..fy-e.g. 5 gallon pails,55 

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

(2) 	Provide the identity, the appmximate weight (by kg!day or kg/batch on an 100% 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 intermittant releases, to the environment of the new chemical substance. 

Not applicable 

o Mark CO this box if you attach a continuation sheet. 

FORM EPA 7710-25 (Rev. 5-95) 	 PageS 
I Part II ~- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE ­ Continued I 

~ Section A - INDUSTRlAL SITES CONTROLLED BY THE SUBMI II t.K ­ Continued 

2. O<:rupational 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 filling. unloading drums, sampling. clea.ni..ng. etc.) in which workers may be exposed to the 

substance. 

(2) - Mark (X) this colw::n:n if entry in column (1) is confidential business information (CEl). 

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

(4) and (6) ­ Indicate you will1r].gness to have the information provided in column (3) or (5) binding. 

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

a mixture) at the time of exposure. ~ . 

(7) ­ ~~(X) this colw::n:n if entry in column (5) is confidential business information (CBl). 

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

(9) ­ Mark (X) this colw::n:n if entry in colw::n:n (8) is confidential business information (CBl). 

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

(12) - Mark (X) this colw::n:n if entries in colw::n:ns (10) and (11) are confidential business information (CEl) • 

Worker activity CBI Protective Eqllipmenll Binding .t'llYSlcal tormls) tBinding CEI II of CBr Maximum duration CBI 

(e.g. bag dumping. fiJ..J.i.ng drums) Engineering; ControLs Option (e.g.soUd:powder) pption Workers Mark (x) and \ID new ~lc.W Exposed HIs/day Days/yr 

(1) m m (4) subs~.gce ~(61 m {al 121 (10) (11) (12) 

Not appUcab e 

I 

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

3. Environmental Release and Disposal- ¥oumust make separate COnfidentiality claims for the release number and the amount of the new chemical 

substance released and other release and disposal. information. Mark (Xl the RConfidential" box next to each item you claim as confidential. . 

(1) - Enter the number of each release point identified in the process description, part Il, section A, subsection ld(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 colw::n:n if entries in columns (1) and (2) are confidential business information (CEl). 

(4) - Identify the media of release Le. stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or indneration, 

P01W, or other (please specify) to which the new substance will be released from that release point. 

(S) - a. Desaibe control technology, if any,,and control efficiency that will be used to limit the release of the new substance to the environment. 

ror releases disposed. of on land. characterize the disposal method and state whether it is approved for d.i.spo5al of RCRA hazardous waste. 

On a continuation sheet, for each site describe any additional disposal methods that will 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 (Xl this colw::n:n if entries in columns (4) and (5) are confidential business information (CEI). . 

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

dischargers or NPDES numbers of the P01W (Publidy Owned Treatment Works). Mark (Xl if the POTW name orNPDES.# is confidential 

business information (CBO. 

Release Amount of-new substance em Media of Control technology and efficiency (you may wish to optionally attach effidency data) CBl 

Number reJea5ed release 

e.f;.lItxXUr : Binding , (1) (23) ab) (3) (4) (Sa) Mulr.: ():) (5b) (6) . " I 

! I~Ul- cttJtJ.l..l.'-o.u.J..V I 

I 

I : I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

(7) Mark (Xl the o P01W provide name(s) below: CEI o Navigable 0 Other - Specify provide NPDES # CBI 

destination(s) of f- waterway 

releases to water. 

D Mark (Xl this box if you atbch a continll3tion sheet. 

FORM EPA 7710-25 (Rev. 5-95) Page 9 
I 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 rontrol.Importers do not have to 

complete this section for operations outside the US.; however. you must report any processing or use activities after import See the Instructions 

Manual. Compli:lI: a separau section B for t!ach I:ypt! of prrx:.essing, or use opt!TlI.tion inrolmng the nt!W chem:iaz1 subst.o.nCl!. If the same operation is performed 

at more than one site describe the typical operation common to these sites. IdentUy 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 (spediy- e.g. 5 gallon pails, 

55gallon drums, rail cars, tank"trucks, etc). On the d.ia.gram, identify by letter and briefly describe each worker activity. (2) ­ Provide the identity. 

the approximate weight (by kg/day or kg/batch, on an 100% new chemical 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 reieases, to the environment of the new chemical substance. 

(4) Please enter the # of sites (remember.to identUy tlIe locations of these sites on a continuation sheet); 

# of Sites 

< 

See the attachment at Page 15. 

D Mark (X) this box if you attach a contin~tion sheet. 

2. Worker Exposure/Environmental Release 

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

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

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

(6) ­ Describe physical form of exposure and % new chemical substance (if in mixture), and any protective equipment and engineering controls used 

to protect workers. 

(1) ­ 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. 

(10) Estimate the amount of the new sUbstance released (a) directly to the environment or (b) into control technology to the environment 

(in kg!day or kg/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, 

PO'IW, or other (specify) and control technology, if any, that will be used to limit the release of the new substance to the environment. 

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

(3), (5), (8), (11), (13) and (15). Mark 00 this column if any of the proceeding entries are confidential business information (CBD. 

Letter # of CBI Duration CBI Protective Equip. / 'fGin CBI Reiease Amount of CBI Media of Release CBI 

of Workers of Engineering Controls/ Form- Number New &reControl 

Act· Exposed Exposure Physical Form and 'fG ulation Substance Technology ivity new substance Released 

I 

(1) (2) (3) (4a) I (4b) (5) (6) (1) (8) (9) (10a) : nab) (11 (12) (13) 

I I 

I 

X I X I X I X X I 

I I 

I I 

I I 

I I 

I I 

I I 

I I 

I I 

I I 

(14) - Byproducts: None (15) 

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

FORM EPA 7710-25 (Rev. 5-95) Page 10 
OPTIONAL POLLUTION PREVENTION INFORMATION 


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

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

efforts to reduce or minimize potential risks associated with activities surrounding 

manufacturing, processing, use and disposal of the PMN substance. Please include new 

information pertinenuo 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 compl.!-ance 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 P:M::N" substance to potential alternatives may be provided. 

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

of this substance. See 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 a substitute for an existing substance that poses a greater overall risk to human health or the environment. 

Not applicable 

o Mart< (X) this box if you attach a continuation sheet. 

FORM EPA 7710-25 <Rev. 5-95) Page 11 
Part III - LIST OF ATTACHMENTS 

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

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

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

nUIllbers of each attachment. 

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

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

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

Attachment name Attachment Confipage nUIllber(s) dential 

Material Safety Data Sheet (MSDS) 1 6 1 7 

Physical and Chemical Properties Worksheet 1 3 

IR Chart 1 4 

Continuation sheet of Part 11, Section B.l 1 5 

Bacterial Reverse Mutation Study of YDC-1312 1 8 - 3 6 

Chromosome Aberration Study of YDC-1312 in Cultured Mammalian Cells 3 7 5 5 

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

FORM EPA mO-25 (Rev. 5-95) Page 12 
PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

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

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

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

confidential. The physical state of the neat substance should be provided.. These measured properties should be for the neat (100'l'. 

pure) chemical substance. Properties that are measured for mixtures or formulations should be so noted (0/., 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. 

Property Page Value M .....I'I!<! ContiMark 00 if or dential 

provided number Eotimat. 

(a) (b) (c) Mark 00 

(M or E) (d) 

Physical state of neat substance X X (s) __ (l) __ (sV 

Vapor pressure ·C 

@Temperature Torr 

20°C X 1. 12 M 

Density/relative density g/cm3 

Solubility 

@Temperature "C

Solvent g/L . 

"C X Insoluble E 

Solubility in water @Temperature "C 

X 141°C M 

Melting temperature ·c 

@ 2 

Boi!ing/sublimationtemperature torr pressure X 184°C M 

Spectra I R spectra X 14 M 

Dissociation constant 

Particle size distribution 

Octanol/water partition coefficient 

Henry's Law constant 

Volitalization from water 

Volilalization from soil 

pH @ concentration 

Flash point X 206°C 

Flammability 

Explodability 

Adsorption / coefficient 

Other ­ Specify 

PAGE 13 

o 

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a:l 

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a:l 

'tJ 

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S 

Q) 

s... 


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til 


a:l 

Q) 

S 

0:: 

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U:.. 

.::t 

Q) 

flO 

a:l 

0.... 
Continuation Sheet of Section B. 1 of PartII 

CONFIDENTIAL 

Page 15 

U.S. Department of Labor 

Occupational Safety and Health Administration 

(Non-Mandatory Form) 

Form Approved 

OMB No. 1218-007 

Note: Blank spaces are not pennitted. If any item is not applicable, or 

infonnation is available the s ace must be marked to indicate th, 

Section I 

Manufacturer's Name 

TOHTO KASEl CO. LTD. 

Emergency Telephone Number 

Address (Number, Street, City, State, and Zip Code) 

NihonbashiBakurocho l-chome, Chuo-ku, 

Telephone Number for Information 

+81-3-3665-1701 

__........:T::..;o:.:k::li~yo~".=J..=a:.A;:.:;Plan==-___________--IDate Prepared 

October 2000 

Signature of Preparer (Optional) 

Section n - Hazardous Ingredients/Identity Information 

Hazardous Components (Specific Chemicalldentiryj Common Name(s) OSHA PEL ACGIH TLV Other Limits % 

None 

Non-Hazardous Components 


YDC-1312 None None None 99% 


S ec f Ion m - Ph wSlca . I/ChemlcaI Ch aracterls'fICS 

Boiling Point Specific Gravity (H2O::1) 184't at 2 mmHg 1.12 

Vapor Pressure (mmHg) Melting Point 

Not determined 141't

Vapor Density (Air - 1) Evaporation Rate 

Not determined (Butyl Acetate:: 1) Not determined ., SolubIlity In Water 

Insoluble 

Appearance and Oder 

Transparent light yellow solid 

losion Hazard Data 

Flammable Limits UEL 206't N.A. 

Extinguishing Media Water spray, dry chemical, carbon dioxide, or chemical foam 

Special Fire Fighting Procedures Wear self-contained breathing apparatus in the case of fire 

at the close area. 

Unusual Fire and Explosion Hazards Not in particular. 

(Reproduce locally) OSHA 174, Sept 1985 

Page 16 
Section v.. Reactivitv Data ­ Stability Unstable Conditions to Avoid 

Strong oxidizer, strong acids and bases 

Stable X 

Incompatibility Materials to Avoid) None 

Hazardous Decomposition or Byproduc Carbon mono-oxide 

Hazardous 

Polymerization 

May Occur 

Will Not Occur 

X Conditions to Avoid Curing agents and high temperature 

C> 

Section VI.. Health Hazard Dau 

Route{s) of Entry: Inhalation? Skin? Ingestion? No No No 

Health Hazards (Acute and Chronic) Positive at Ames test but negative at chromosomal aberration 

test. Not known about other chronic effects. 

Carcinogenicity: NTP? No IARC Monograph? No OSHA Regulated? No 

Signs and Symptoms of Exposure Not known 

Medical Conditions 

Generally Aggravated by Exposure Not known 

Emergency and First Aid Procedures 

Eye contact: Immediately flush with plenty ofwater for at least 

15 minutes. Get medical aid. Skin contact: Wash with plenty of soap and water for at least 

15 minutes. Get medical aid, ifirritation develops or persists. Ingestion: Rinse out the 

Sweep and scoop up for disposal, avoiding 

dust genaration. Wash trace residues with water. Do not allow residues to enter waterways. 

Waste Disposal Method 

Waste material may be dumped or incinerated under conditions which meet 

all federal, state and local environmental regulations. 

Precautions to Be Taken in Handling and Storing Precautions must be taken to avoid contact with the 

mouse with plenty of water and get medical attention immediately. 

skin and eyes. Store in a cool, dry, ventilated place and in a tightly closed container. 

Other Precautions 

Not in particular 

Section V1II .. Control Measures 

Respiratory Protection (Specify Type) 

Seek professional advice prior to respirator selection and use. 

Ventilation Local Exhaust Recommended 

Mechanical (GeneraC Recommended 

Special Not required 

Other Not required 

Protective Gloves Rubber gloves Eye Protection Safety chemical goggles 


Other Protective Clothing or Equipment Recommended to wear chemical apron. 


Work/Hygienic Practices Wash thoroughly after handling, and avoid contact with chemical. 


Page 2 USGPO: 1966-491-529/45775 

Page 17 
4L134 


CONFIRMATORY STATEMENT OF THE STUDY REPORT 

General Testing Research Center, 


Japan Oilstuff Inspectors' Corporation 


Sponsor : TOHOTO Kasel CO., LTD. 

Title : Bacterial Reverse Mutation Study of YDC-1312 

Study No. : 4L134 

The said study has been conducted in General Testing Research Center, Japan 

Oilstuff Inspectors' Corporation. The original study report was written in 

Japanese language and translated into English language. I hereby declare that 

this report reflects faithfully the original study report as much as possible to 

my knowledge. 

/}~} .1/ i - .. Translator ~. ~~ Date: 1£1,.1 ZIffY V 

E i i ch i Mach i gak i, M. S. 


Scientist of Mutagenicity Testing Group 


2 
4L134 

Submi tted to: 


TOHOTO Kasei CO., LTD. 


(REPORT) 

Bacterial Reverse Mutation Study of YDC-1312 

(Study No. : 4L134) 

June 15. 1994 

GENERAL TESTING RESEARCH CENTER, JAPAN OILSTUFF INSPECTORS' CORPORATION 

i !' " 3 .A .. J 
41134 


S TAT EMEN T 

General Testing Research Center. 


Japan Oi lstuff Inspectors' Corporat ion 


Sponsor : TOHOTOKasei CO.• LTD. 

Ti tie Bacterial Reverse Mutation Study of YDC-1312 

Study No. 4L134 

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

Laboratory Practice Standards. 

rJapanese GLP Standerds Applied to Industrial Chemicals CKanpogyo No. 39, 

Yakuhatsu No. 229. 59 Kikyoku No. 85, 1984) J 

rGLP Standards under the Japanese Industrial Saftey and Health Law 

(Notification No. 76. Ministry of Labor, 1988) J 

Management Osamu Ueno sealed Date: June 15. 1994 

Director of Microbiological Laboratory 

4 20 
4L134 


.. 


QUALITY ASSURANCE STATEMENT 

General Testing Research Center. 


Japan Oi lstuff Inspectors' Corporation 


Sponsor TOHOTO Kasei CO.. LTD. 

Title Bacterial Reverse Mutation Study of YDC-1312 

Study No. 4L134 

By the inspections and audits listed below. we hereby certify that this study 

was carried out in accordance with its protocol and with our Standard Operating 

Procedures. and that this report faithfully describes the methods and procedures 

employed, and that the reported results reflect accurately the raw data of the 

study. 

Inspection Date of inspection Date of reporting to 

or audit or audit the study director 

and to the management 

Study

protocol Apr i 1 27, 1994 April 27. 1994 

Study 

procedure 

June 1. 1994 June 1. 1994 

Study June 15, 1994 June 15, 1994 

report 

Quality Assurance Unit =Ma::..:sc.;;;:.a.::....;yo:...;:.s..:.::..h=--i..::..::K.:...;id:.::a'----=...:se::.:;a:...:.l.:::.;ed:.... Date: June 15, 1994 

5 
4L134 


OUTLINE OF THE STUDY 


1. Title : Bacterial Reverse Mutation Study of YDC-1312 (Study No. : 4Ll34) 

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

mutation assays. using Salmonella tyPhimurium and Escherichia coli 

strains. 

3. 	 Guideline : Japanese Guidelines on Industrial Chemicals(Kanpogyo No.70Q. 

Yakuhatsu No. 1039. 61 Kikypku No. 1014. 1986) and guidelines under 

the Japanese Industrial Safety and Health Law (Notification No. 77. 

Ministry of Labor. 1988) 

4. 	 G L P : Japanese GLP Standerds Applied to Industrial Chemicals (Kanpogyo. 

No. 39, kuhatsu No. 229. 59 Kikyoku No. 85. 1984) and GLP Standards 

unde~ the Japanese Industrial Saftey and Health Law (Notification 

No. 76, Ministry of Labor. 1988) 

5. 	 Sponsor : TOHOTO Kase i CO.. LTD. 

07-14 Higashikasai. 3-chome. Edogawa-ku, Tokyo) 

(Responsible person) Yoshiaki Nakamura 

6. 	 Contractor : Mitubichi Chemical Safety Institute Ltd. 

(1-30, 2-Chome, Shiba, Minato-ku, Tokyo) 

7. 	 Testing Facility: General Testing Research Center. 

Japan Oi lstuff Inspectors' Corporation 

(10-4, l-Chome, Mikagetsukamachi, Higashinada-ku, Kobe-shi, Hyogo) 

8. Managemen t : Osamu Ueno 

9. Study Director : Elichi Machigakl 

10. Other Contributors : Masato Nakamura, Manabu Kajihara. Mikiko Tatsumi 

9. 	 Study Date : (Initiation of the Study) April 27, 1994 

(Submission of the Final Report) June 15, 1994 

10. No statistical methods were used for data analysis. 

11. There was no unforeseeable circumstances that may have ill effects on test results. 

12. 	 Retention : All records will be retained in the safekeeping facility of General 

Testing Research Center for 10 years after the submission of the 

final report. Further retention wi II be discussed with the sponsor. 

6 
4Ll34 

Con ten t s 


Page 

Summary ......................... "............................................... 
 8 

Materials and Methods 

1. T est rna t e ria 1s ... . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . .. ... . . . . . . .. 9 


2. Bacterial tester strains ....................................... 10 


3. Media ..................................................................... 11 


4. S9 mix ................. ............................................. .... 11 


5. Test method ............................................................ 12 


Results and Conclusion ................................................ 14 


References .............................................................. .... 14 


Tab les .................................................................. 15 


Figures .................................................................. 18 


7 

4L134 


Summary 


YDC-1312 was examined for the mutagenicity in bacterial assays using five 

different strains, SaLmoneLla tyPhimurium TA9S, TAI00, TA1535, TA1537 and 

Escherichia coLi WP2 uvrA-. The numbers of revertants induced by the test 

substance were more than double of the solvent control value in S. typhimurium 

TAI00 and TA1535 wi th S9 mix. 

These findings led to the conclusion that YDC-1312 is mutagenic under the test 

conditions employed. 

S 

4L134 

Materials and Methods 

1. Test materials 

Confidential , 1. 1 Test substance 

1.2 Negative and positive controls 


The negative controls and the positive controls were as follows. 


Name Abbreviation Supplier Lot Pur ity I number (%) 

i Negative controls 

dimethylsulfoxide DMSO Wako Pure Chemical APE5938 min.99.5 

Industries. Ltd. I 

Positive controls I 

2-(2-furyl)-3-(5-nitro­ AF-2 Wa k 0 Pur e Chem i c a I . PDF0626 98"'-'102 

2-furyl)acrylamide Industries. Ltd. 

sodium azide NaN s Wako Pure Chemical DSG1561 

Industries. Ltd. 92. 2 

9-:-aminoacridine 9-AA SIGMA Chemical Company 80F-0186 ~ 98 

N- ethyl-N' -nitro-N­ ENNG SIGMA Chemical Company 56F-3651 ~ 97 

nitrosoguanidine

2-aminoanthracene 2-AA Wako Pure Chemical TSE4201 92.6 

Industries. Ltd. 

9 

--

--

--

4L134 


2. Bacterial tester strains 

2.1 Strains I} 

Genetically different five strains of Salmonella typhimurium TA98. TA100, 

TA1535. TA1537 and Escherichia coli WP2 uvrA- were employed. These strains 

were received from Mistubishi Chemical Safety Institute Ltd. on July 8, 1988. 

The genetic properties of the strains are listed in the following table. 

The genetic properties of the strains are listed in the following table. 

Strain 

Gene Additional mutations 

Affected. 

Repair LPS R-factor 

Mutation type 

S. tyPhimurium

TA98 his D uvr B ria pKM 101 Frameshift 

his G uvr B ria pKM 101 Base-pair change 


TA1535 


TA100 

his G uvr B Base-pair change
 TA1537 . 
 ria 

his C uvr B ria Frameshift 

E. 	 coli 

WP2 uvrA ­trP E uvr A + Base~pair change ! I 

2.2 Check on genotypic characteristics 

The tester strains were checked on their genotypic characteristics such as 

amino acid requirement, UV sensitivity. membrane mutation and R-factor before 

the study. 

2.3 Storage 

Eight ml of fresh bacterial suspension (cultured with shaking at 37°C for 

about 7 hours in a liquid growth medium) was mi~ed with 0.7 ml of DMSO and 

quickly frozen in a dry ice-acetone mixture. The stocks of the tester strains 

were stored below -80°C. 

2.4 Bacterial suspension 

Twenty /11 of stock cultures of tester strains were inoculated against the 

10ml liquid growth medium and grown with shaking at 37°C for about 7 hours. 

Densities of the fresh bacterial cultures were measured with spectrometer. 

1 0 
4L134 


3 Media 

3.1 Liquid growth medium 

Oxoid Nutrient Broth No.2 (Unipath Ltd. Lot NQ 19947080) was dissolved in 

distilled water (25g/1!.) and autoclaved at 121°C for 15 minutes. 

3.2 Minimal glucose agar plate 

The agar plate ( CLIMEDIA®AM-N MEDIUM) was purchased from Nisshln Flour 

Milling Co.. Ltd .. The dose finding tests were used lot No. AN090BJ prepared 

on February 21. 1994. and the main test were used lot No. ANlS0CJ prepared on 

March 28, 1994. 

3.3 Top agar 

Aqueous amino acid solution for S. tyPhimurium strains. which contained Lhistidine (Wako Pure Chemical Industries. Ltd.) and D-biotin (Wako Pure 

Chemical Industries, Ltd.) in 0.5 ruM respectively was prepared, sterilized by 

filtration (0.45tim pore size) and stored in a refrigerator. Aqueous amino 

acid solution for E. coli. which contained 0.5 ruM L-tryptophane (Wako Pure 

Chemical Industries. Ltd.) was prepared. sterilized by filtration (0.45 tim 

pore size) and stored in a refrigerator. O.S g of powdered agar ( Bact-Agar; 

Difco Laboratories) and 0.5 g of sodium chloride were dissolved in 100 ml of 

distilled water and autoclaved. The above mentioned amino acid solution was 

added to this agar solution in the ratio of 1 to 10 and kept at about 45°C 

until use. 

4 S9 mix 

4. 1 S9 

S9 from Kikkoman Corporation was the 9000Xg supernatant fraction of liver 

homoganate prepared from Sprague Dawley rats which were given i.p. injection 

of phenobarbital and 5.S-benzoflavone. The dose finding tests were used lot 

No. RAA-305 prepared on February 18. 1994. and the main tes t were used lot No. 

RAA-309 prepared on May 13. 1994. The S9 was stored below -80°C. 

1 1 
4L134 


4.2 	 S9 mix 

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

freshly "Prepared for each assay. and was stored in the ice. 

Components 

S9 


MgCh • 6H 2O 


KCI 


D-glucose 6-phosphate 
 /3 -NADPH 
 i 	 /3 -NADH 

Na-phosphate buffer (pH 7.4) 

Distilled water 

Amount in 1ml 

0.1 ml 

8 /l mo I 

33 /lmo I 

5 /l mo I 

4 /l mo I 

4 /l mo I 

100 /lmol

remainder 

5 	 Test method 2) 

5.1 	 Preparation of test substance solutions and positive controls 

The test substance was dissolved in DMSO. and diluted into each 

concentration. The test substance solution was prepared just before use. 

As for p~sitive controls. NaN 3 was dissolved in DW. the others were 

dissolved in DMSO. 

5.2 	 Test substance concentrations 

In the dose finding test and the main test. five dose levels was graded at 

nominal ratio of 2 (313 to 5000/lg per plate). 

5.3 	 Reverse mutation test 

The reverse mutation test was done by preincubation method. 

Point one ml of a test solution. a 0.1 ml aliquot of one of the bacterial 

suspensions and 0.5 ml of either Q 1 M sodium phosphate buffer (pH 7.4) for 

assay without metabolic activation or S9 mix for assay with metabolic 

activation were mixed in a sterilized test tube and incubated with gentle 

shaking at 37°C for 20 min. After preincubation, 2 ml of mol ten top agar was 

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

agar overlay solidified. these plates were incubated at 37°C for 48 hours. 

1 2 	 r. ,.... 

;;..() 
4L134 

The presence of the background lawn was observed using a stereoscopic 

microscope and microbial growth was confirmed. Revertant colonies were counted 

with eye or an automatic colony counter. Two plates per one dose level were 

used for each assay. Negative and positive control tests were also done in 

each assay. 

The positive controls and the concentrations were as follows. 

Strain I -S9 Mix Cttg/plate) tS9 Mix Cttg/plate) 

S. tyPhimllriWlt I 

TA98 

TA100. 

TA1535 

TA1537 

E. coLi 

WP2 llvrA -

AF-2 

AF-2 

NaN s 

9-AA 

ENNG 

0.1 

0.01 

0.5 

80 

2 

2-AA 

2-AA 

2-AA 

2-AA 

2-AA 

0.5 

1 

2 

2 

10 

5.4 Contamination test 

The highest dose suspensionon and S9 mix were tested for the bacterial 

contamination. 

5.5 Criteria for judging the results 

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

number of revertants is more than double of the negative control value and 

when it increases significantly dose-dependent. 

When the test substance is judged to be positive. specific activity was 

calculated as a induced revertants per Img test substance. 

1 3 
4Ll34 

Results and Conclusion 

The results are shown in Tables 1 ~ 2 and Figures 1 ~ 6. 

In the main test. the numbers of revertants induced by the test substance 

were more than double of the solvent control value in S. tyPhimurium TAI00 and 

TA1535 with S9 mix. The reproducibility of the these results was confirmed in 

the dose finding test. 

Maximum specific activity was 496 colonies/mg in TAI00 with 89 mix at 625u 

g/plate. 

Microbial growth inhibition was not observed. 

Precipitated test substance was observed on the agar plate. It was shown as 

" t" in the tables. 

In the positive control tests useing specific mutagBns. they induced 

significant revertants exceeding those of the negative control. 

No statistical methods were used for data analysis. 

The sponser confirmed that the lot of the test substance used in this study 

was stable during testing period. 

These results led to the conclusion that YDC-1312 is mutagenic under the 

test conditions employed. 

References 

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

mutagenici ty test, Mut. Res .. 113. 173-215. 

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

guideline and the GLP. edited by the Chemical Substance Investigation 

Division, Ministry of Labor of Japan. Japan Industrial Safety and Health 

Associat ion, Tokyo. 

1 4 r. r' . , 

L~ U 

I 
Table 4L134 1 Results of the Dose-finding Test 

Test substance . YDC-1312 

Testing periods From May 10. 

Number of revertants 

1994 to May 13. 1994 

Cnumberof colonies/plate) 

Wi th (+) or ITest material 

without( ) concentration Base-pair change type Frameshift type 

S9 Mix C/.lg /plate ) TA 100 TA1535 WP2 lLvrA- TA 98 TA1537 

Solvent 101 16 19 15 8. 

control 124 C113) 15 ( 16) 16 ( 18) 14 ( 15) 5 ( 7) 

t 105 15 29 11 10 

156 108 ( 107) 12 ( 14) 19 ( 24) 13 C 12) 9 ( 10) 

t 117 10 21 13 3 

313 98 ( 108) 9 ( 10) 19 C 20) 16 ( 15) 11 ( 7) 

- S 9mix t 107 16 23 12 7 

625 111 ( 109) 7 ( 12) 12 ( 18) 12 ( 12) 3 ( 5) 

t 138 15 11 11 6 

1250 112 ( 125) 11 ( 13) 10 ( 11) 12 ( 12) 8 ( 7) 

t 118 5 17 16 4 

2500 111 ( 115) 8 ( 7) 15 ( 16) 13 ( 15) 6 ( 5) 

t 117 5 15 10 6 

5000 123 C120) 6 ( 6) 14 ( 15) 15 C 13) 8 ( 7) 

I Solvent 98 10 18 25 14 

control 100 ( 99) 10 ( 10) 18 ( 18) 13 ( 19) 10 ( 12) 

140 10 19 24 6 

156 133 ( 137) 8 ( 9) 13 ( 16) 22 ( 23) 10 ( 8) 

192 76 20 

I 

19 12 

313 238 ( 215) 71 ( 74) 24 ( 22) 39 ( 29) 13 ( 13) 

+ S 9mix 373 211 22 24 13 

625 406 ( 390) 203 ( 207) 20 ( 21) 31 ( 28) 12 ( 13) 

t 451 248 22 29 10 

1250 452 ( 452) 266 ( 257) 24 ( 23) 25 ( 27) 7 ( 9) 

t I 465 237 23 25 10 

2500 439 ( 452) 241 ( 239) 27 ( 25) 25 ( 25) 7 ( 9) 

t 

~ro 194 10 16 12 

5000 343 ( 361) 230 ( 212) 22 C 16) 31 ( 24) 7 ( 10) 

Name. AF 2 NaN s ENNG AF-2 9-AA 

Posi ti ve 

control Concentration 0.01 0.5 2 0.1 80 

not (p.g /plate ) requiring

S9 Mix Number of 433 394 831 443 458 colonies/plate 431 ( 432) 382 ( 388) 843 ( 837) 441 ( 442) 498 ( 478) 

Name BP 2-AA 2-AA BP BP 

Positive 

control Concentration 5 2 10 5 5 requiring (p.g /plate ) 

I S9 Mix Number of 1096 263 

I 

924 283 190 colonies/plate 1043 (070) 264 ( 264) 946 ( 935) 287 ( 285) 194 ( 192) 

I 

• 

Af-2 : 2-(2:f~rYl)-3-(5-nitro~2-fur¥l~acrylamide. NaN 3 : sodium azide, ENNG : N-ethyl-N' nitro-NnltroSOguanldlne, 9-AA : 9-amlnoacrldlne. BP : benzo[a]pyran. 2-AA : 2-aminoanthracene. 

Notes () : Mean 

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

1 5 

Table 2 Results of the Main Test 8L134 

Test substance . I YDC-1312

Testing periods From May 31. 1994 to June 3, 1994 

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

without( ) concentration Base-pair change type Frameshift type S9 Mix (ttg ~plate ) TA 100 TA1535 WP2 uvrA- TA 98 TA1537 

Solvent 123 8 11 18 9 

control 100 ( 112) 7 ( 8) 10 ( 11) 20 ( 19) 8 ( 9) 

t 136 C 10 C 17 C 28 C 7 C 

156 94 ( 115) 10 ( 10) 9 ( 13) 24 ( 26) 5 ( 6) 

t 106 C 12 C 18 C 24 C 4 C 

313 126 ( 116) 9 ( 11) 15 ( 17) 19 ( 22) 6 ( 5) 

t 116 C 8 C 15 C 23 C 3 C 

S 9mix 625 122 ( 119) 11 ( 10) 22 ( 19) 22 ( 23) 8 ( 6) 

t 97 C 13 C 11 C 17 C 4 C 

1250 128 ( 113) 14 ( 14) 14 ( 13) 19 ( 18) 9 ( 7) 

t 111 C 11 C 14 C 23 C 5 C 

2500 119 ( 115) 11 ( 11) 14 ( 14) 19 ( 21) 13 ( 9) 

t 103 C 11 C 15 C 10 C 5 C 

5000 106 ( 105) 9 ( 10) 18 ( 17) 21 ( 16) 4 ( 5) 

Solvent 101 4 21 29 9 

control 101 ( 101) 8 ( 6) 17 ( 19) 28 ( 29) 7 ( 8) 

90 17 17 31 9 156 128 ( 109) 20 ( 19) 12 ( 15) 31 ( 31) 7 ( 8) 

248 110 29 28)~ 10 

313 230 ( 239) 99 ( 105) 27 ( 7 ( 9) 

+ S 9mix 452 247 21 25 625 369 ( 411) 272 ( 260) 21 ( 21) 37 ( 31) ) 

t 465 C 375 C 28 C 30 C 17 C 1250 484 ( 475) 369 ( 372) 19 ( 24) 30 ( 30) 10 ( 14) 

t 474 C 315 C 27 C 26 C 8 C 2500 452 ( 463) 315 ( 315) 21 ( 24) 24 ( 25) 14 ( 11) 

t 390 C 292 C 16 C 22 C 5 C 5000 381 ( 386) 273 ( 283) 18 ( 17) 39 ( 3D 7 ( 6) 

Name AF 2 

Positive NaN! ENNG AF-2 9-AA 

control Concentration 0.01 O. 5 2 0.1 80 

not (ttg ~plate ) requiring

S9 Mix Number of 469 290 1094 517 782 colonies~plate 500 ( 485) 281 ( 286) 1148 (121) 519 ( 518) 483 ( 633) 

Name BP 2-AA 2-AA BP BP Posi ti ve 

control Concentration 5 2 10 5 5 requiring (ttg ~plate ) S9 Mix 

Number of 951 218 870 268 159 colonles~plate 953 ( 952) 216 ( 217) 745 ( 808) 308 ( 288) 142 ( 151) 

Af-2 : 2-(2~f~ryl)-3-(5-ni tro-;-2-fur¥i!acry)amide. NaN s : sodium azide, ENNG : N-ethy)-N' ni tro-NnltroSOguanldlne. 9-AA : 9-amlnoacrldlne. BP : benzo[a]pyran. 2-AA : 2-aminoanthracene. 

Notes (): Mean 

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

1 6 

Results of Specific Activity 

Test substance YDC-1312 


/ Bacterial Wi tholJt S9 mix Wi th S9 mix 

Strains Specific I Concentration** Specific Concen tra tion** 

Acti vi ty* (,ug/plate) Acti vi ty * (,ug/plate) 

TAlOO 466 625 

TA1535 315 625 

Dose finding WP2 uvrAtest 

TA98 

TA1537 

I TAlOO 496 625 

TA1535 406 625 

Main test 

WP2 uvrATA98 

TA1537 

* : The number of induced revertants per 1 mg test substance. 

** 	: Maximum value of specific activity was mentioned at in the dose finding test and 

the main test. 

1 7 

600 ,-----~------,------,~-----,------~----_, 

; (TroD) 

Revertants 

per 

plate 

4 5 0-----­1..",:"", ....................................:.......................·....·..·....f.. ··.. ............,..................................................................... 

! i 

3 0 0 ....................................+....................................j..................................1....................................: ................................... .................................... 

iii i i 

OL-----~------~----~~----~------~-----

o 	 156 313 625 1250 2500 5000 

Concentration (.ug/plate) 

Fig. 	 1 Dose response curve of YDC-1312 (Main Test)

(0···0 : -S9mix. 0-0: +S9mix) 

40 ~----~------~----~,------~,------~-----, 

(W P 2 u v r A -,J 

3 0 ....................................+....................................j....................................+....................................j...................................+................................... 


Revertants 


per 


plate 


2O------l.::.~-::~~t~~::::T:::-:::.r ...______ 9_. _. _.' 


1 0 ·..·~·~.. ..~:... .. .. .. .. ..T..............................·..·!·..··..·......·..·..·....·........·r..................·..·..·..·..·:....·..................·....·..·..+................................. 


o L-____ ______ ____ ______ ______ ____~ 

o 	 156 313 625 1250 2500 5000 

Concentration (.ug/plate) 

Fig. 	 2 Dose response curve of YDC-1312 (Main Test) 


(0···0 : -S9mix. 0-0: +S9mix) 


1 8 

Revertants 


per


plate 


Revertants 


per


plate 


40 

30 


20 


10 


o 

400 


300 


200 


100 


o 

(T A 1 53 5 S9Mix J 

. ... ····0·····.. ,... '" .()....... .... 

o 	 156 313 625 1250 2500 5000 


Concentration (.u g/p la te)


Fig. 	 3 Dose response curve of YDC-1312 (Main Test) 
 . (0···0 : -S9mix) 


(T A 1 ~ 3 5 +~

'9M~>--. .n.·..·...····· ;V 

,/ r V 

o 156 313 625 1250 2500 5000 

Concentration (.ug/plate) Fig. 4 Dose response curve of YDC-1312 (Main Test) 

(0-0 : +S9mix) 

1 9 
40~----~------~----~------~----~-----, 

(T A 9 8) 

j 

Revertants 


per 


plate 


10i···_·····_·..·······..··.. ·..·+....·.. ·..··_--_·_..··.. ··I·..·.... ·....·..·............···+..·....·..··..........·..······i...... _·-_..·..·............. +............._.................... 


O~----~------~----~------~----~-----

o 	 156 313 625 1250 2500 5000 

Concentration (tLg/plate)

Fig. 	 5 Dose response curve of YDC-1312 (Main Test) (0···0 : -S9mix; 0-0: +S9mix) 

Revertants 

per

plate 

40,-----~------~----~------~----~-----, 

(T ! 1 5 3 7 .' 

301......·_..·..·....···.. _·..··...."..·......_..·····..··....·......·i...... · ..·· ..· ..· ........··..·f· ....··....······· ..·..........·j .... · .... · .......... · ........ · .. · ..·+........·..··......·_........·....1 

201..·....·....· ................·· .. ·t....·····....·......·.. ·· ....··....·I· ......·..........·· ....·.. ·......·,..............·..........··..·..···..1..·..·....··..·..·............··..+..·............·..·..............1 

lOR:._ 

156 313 625 1250 2500 5000 

Concentration (tLg/plate) Fig. 6 Dose response curve of YDC-1312 (Main Test)

(0"'0 : -S9mix, 0-0: +S9mix) 

2 0 

4L435 


CONFIRMATORY STATEMENT OF THE STUDY REPORT 

, 	 General Testing Research Center, 

Japan Oi lstuff Inspectors' Corporat ion 

Sponsor : TOHOTO Kasei Co., Ltd. 

Title Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells 

Study No. 4L435 

The said study has been conducted in General Testing Research Center. Japan 

Oilstuff Inspectors' Corporation. The original study report was written in 

Japanese language and translated into English language. I hereby declare that 

this report reflects faithfully the original study report as much as possible to 

my knowledge. 

Date: fiJ", Il, 11Yt r I 

Masato Nakamura, M.S. 


Scientist of Mutagenicity Testing Group 


2 

Translator 
4L435 


Submi tted to: 


TOHOTO Kasei Co., Ltd. 


(REPORT] 

Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells 

(Study N~ : 4L435) 

March 16, 1995 


GENERAL TESTING RESEARCH CENTER, JAPAN OILSTUFF INSPECTORS' CORPORATION 


3 
4L435 


S TAT EMEN T 


General Testing Research Center. 


Japan Oi lstuff Inspectors' Corporation 


Sponsor TOHOTO Kasei Co .. Ltd. 

Title Chromosoma1 Aberration Study of YDC-1312 in Cultured Mammalian Cells 

Study No. 4L435 

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

Laboratory Practice Standards. 

rJapanese GLP standards Applied to Industrial Chemicals CKanpogyo No. 39. 

Yakuhatsu No. 229. 59 Kikyoku No. 85. 1984) J 

rOECD Principles of Good Laboratory Practice (1981)J 

Management Osamu Ueno sealed Date: March 16. 1995 --~==~~------~~~ 

Director of Microbiological Laboratory 

4 
4L435 


QUALITY ASSURANCE STATEMENT 

General Testing Research Center. 


Japan Oi lstuff Inspectors' Corporat ion 


Sponsor : TOHOTO Kase i Co.. Ltd. 

Title Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian Cells 

Study No. 4L435 

By the inspections and audits listed below. we hereby certify that this study 

was carried out in accordance with its protocol and with our Standard Operating 

Procedures, and that this report faithfully describes the methods and procedures 

employed. and that the reported results reflect accurately the raw data of the 

study. 

Inspection Date of inspection Date of reporting to 

or audit or audit the study director 

and to the management 

Study 

protocol 

Oc tober 25, 1994 October 25. 1994 

Study 

procedure 

November 7. 1994 November 7. 1994 

Study March 16. 1995 March 16. 1995 

report 

Quality Assurance Unit Masayoshi Kida sealed Date: March 16. 1995 

5 
4L435 


OUTLINE OF THE STUDY 


1. 	 Title : Chromosomal Aberration Study of YDC-1312 in Cultured Mammalian 

Cells (Study No. : 4L435) 

2. 	 Purpose :To assess the clastogenicity of the test substance by 

the chromosomal aberration tests in cultured mammalian cells. 

3. 	 Guideline : Japanese Guidelines on Industrial Chemicals(Kanpogyo No. 700, 

Yakuhatsu No. 1039, 61 Kikyoku No. 1014, 1986) and EEC Guidelines 

(1992) . 

4. 	 G L P : Japanese GLP Standerds Applied to Industrial Chemicals (Kanpogyo 

No. 39, Yakuhatsu No. 229, 59 Kikyoku No. 85, 1984) and OECD GLP 

(1981) 

5. 	 Sponsor : TOHOTO Kasei Co., Ltd. 

(17-14 Higashikasai 3-chome. Edogawa-ku. Tokyo) 

(Responsible person) Yoshiaki Nakamura 

6. 	 Contractor : Mitsubishi Chemical Safety Institute Ltd. 

(1-30, Shiba 2-Chome. Minato-ku. Tokyo) 

7. 	 Testing Facility : General Testing Research Center. 

Japan Oilstuff Inspectors' Corporation 

(10-4. Mikagetsukamachi 1-Chome, Higashinada-ku. Kobe-shi, Hyogo) 

8. Management 	 : Osamu Deno 

9. Study Director 	 : Eiichi Machigaki 

10. Other Contributors 	 : Masato Nakamura 

11. Study Date : Onitiation of the Study) October 16. 1994 

(Initiation of Cell Growth Inhibition Test) October 28. 1994 

(Submission of Chromosomal Aberration Test) February 14. 1995 

(Submission of the Final Report) March 16. 1995 

12. There was no unforeseeable circumstances that may have ill effects on test results. 

13. 	 Retention : All records will be retained in the safekeeping facility of General 

Testing Research Center for 10 years after the submission of the 

final report. Further retention will be discussed with the sponsor. 

6 

4.1. 

4L435 


Contents 

Page 

Summary ..·····.. ···..···· .......... ..... ...... ............ ..... ....... ......... 8 


Materials and Methods 

1. Test materials ...................................................... 9 


2. Indicator cellI ine ................................................ 10 


3. Cu1ture.media ......................................................... 10 


4. S9 Mix ....... ..... ................ .... ......... ..... ...... ............... 10 


5. Preparation of test material solutions ..................... 11 


6. Cell growth inhibition test .................................... 11 


7. Chromosomal aberration test .................................... 13 


Resu 1ts and Cancl us ion ...... ..... .......... ......... ...... ............ 16 


References .............................................................. ..... 16 


Tables .................................................................. 17 


Figures .................................................................. 19 


7 

4L435 


Summary 


An in vitro chromosomal aberration study of YDC-1312 was conducted with an 

establ ished cell line (CHL!IU) from the lungs of a female Chinese hams ter. 

On the basis of the cell growth inhibition test. the chromosomal aberration 

test was set at 5000. 2500 and 1200.ug/ml in 24 and 48 hours treatment without 

metabolic activation and the metabolic activation. In the chromosomal aberration 

test. test substance did not significantly increaced cells with structural 

chromosomal and numerical aberration (polyploid cell) wi thand without metabalic 

act iva ti on. 

From these findings. it was concluded that YDC-1312 has no clastogenic 

potent ial in the CHL/IU ce 11 line. 

8 

4L435 

Materials and Methods 

1. 	 Test materials 

Confidential 

1. 1 Test substance 

1.2 Preparation of test substance 

Negative 	control material (solvent) 

1% sodium carboxymethylcellulose solution 

(l%-CMC-Na. Wako Pure Chemical Laboratory Co.. Ltd. Lot No. LKE1873) 

1.3 Positive material 

Positive controls 

Mi tomycin C (MMC, Kyowa Hakko Kogyo Co.. Ltd., Lot No.770AA!' puri ty 100%) 

Benzo[a]pyrene (BP. Tokyo Kasei Kogyo Co., Ltd, Lot No. AXOL puri ty 99.5%) 

9 
4L435 


2. Indicator cell line 

The CHL/IU cell line. originally derived from the lung of a female Chinese 

hamste~ was used. The cells were purchased from Dainippon Pharmaceutical Co.• 

Ltd( prepared on January 21, 1992). A cell suspension was mixed wi th one tenth 

volume of dimethylsulfoxide (DMSO). then frozen in 1 ml and stored in liquid 

nitrogen. The cells were used after thawing and cultivation through up to 5 

passages. Cells were cultured in plastic dishes (Becton Dickinson and Company; 

diameter of 6 cm or 10 cm) in an automated CO 2 incubator CTabai Espec Corp. 

model BNA-llIA. 121DA; CO 2 5%. temperature 37°C. humid condition). 

3. Culture media 

a 1 Eagle's minimum essential medium 

Eagle's minimum essential medium (MEM; Eagle's MEM "NissuiCD", Nissui 

Pharmaceutical Co., Ltd.) 8.3gwere dissolved in 880 ml of distilled water and 

autoc1aved at 121°C for 15 min. This solution was supplemented with 8.8ml 

of sterilized 2.92% L-glutamine solution and 11.4 ml of sterilized 10% sodium 

bicarbonate solutio~ 

3.2 Growth medium 

Nine hundred ml of the above MEM solution was supplemented with 100 ml of 

heat-inactivated (56°C. 30 min.) calf serum (GIBCD BRL; Lot No. 38N3025). 

4. S9 mix 

4. 1 S9 

The S9 used. which was a preparation from the liver of male Sprague-Dawley 

rats treated with phenobarbital (PB; 30, 60 and 60 mg/kg respectively on 

three successive days, i. p.) and 5,6-benzoflavone (80 mg/kg v. p. at the third 

PB injection), was purchased from Kikkoman Corporation (Lot No. RAA-310. 

prepared on June 3, 1994). The S9 fraction was stored at below -80°C. 

1 0 
4L435 


4.2 S9 mix 

The following table shows the composition of 1 ml of S9 mix. The S9 mix was 

freshly prepared for each test and kept on ice until use. 

S9 0.3 ml 

MgCl 2 C6H2 0) 5 fl mo 1 

KCI 33 flmo I 

D-glucose 6-phosphate 5 fl mo I 

{3 -NADP+ 4 flmo 1 

HEPES buffer (pH 7.2) 4 fl mo I 

distilled water remaining 

& Preparation of test material solutions 

5.1 Solvent selection and preparation of test material solutions 

The test substance was insoluble in water. soluble more than 5% in DMSO. 

The test substance was most uniformalY suspended in l%-CMC-Na. Therefore 1%­

CMC-Na was used as the solvent for the test substance. 

The test substance suspension was diluted with 1%-CMC-Na to the required 

concentrat ions. 

5.2 Positive control solutions 

MMC was dissolved in saline (Otsuka Pharmaceutical) to a concentration of 

3 flg/ml before use. BP was dissolved in DMSO (Dojin Chemical Laboratory Co.. 

Ltd.) to 4 mglm1. frozen. and thawed at the room temperature before use. 

6 Cell growth inhibition test 

A cell growth inhibition test was conducted to determine appropriate 

concentrations for the chromosomal aberration tests. 

6.1 Test substance concentration 

Seven concentrations of 5000, 2500. 1250. 625. 313, 156 and 78fl g/ml 

solution were used in the tests. 

1 1 
4L435 


6.2 Treatment of cells 

In a 6 em plastic plate. 5 ml of cell suspension C4x10 3/ml) were incubated 

for 3 days. After removal of the culture medium from each plate. wi thout 

metabolic activation treatment of cells was performed for 24 or 48 hours using 

0.5 ml of each test substance suspension mixed separately with 4.5 ml of a 

fresh .growth medium. With metabolic activation. treatment of cells with 89 Mix 

was performed for 6 h using 0.3 ml of each test substance suspension mixed 

with 0.5 ml of S9 Mix and 2.2 ml of the growth medium. With metabolic 

-activation. treatment of cells without S9 Mix was performed for 6 h using 0.3 

ml of each test substance solution mixed with 2.7 ml of a growth medium. After 

6 h incubation, the cells were washed three times wi th MEM. and incubated in 

5 ml or a fresh growth medium for a further 18 h. 

In the negative control group. cells were treated wi th vehicle Cl%-CMC-Na). 

The test was performed using two plates for each concentration. 

6.3 Measurement of surviving cells 

Cells were washed with Dulbecco' s phosphate buffer solution CPB8(-); 

Dulbecco's PBS "Nissui", Nissui Pharmaceutical Co., Ltd.), fixed wi th 

methanol for 15 min., stained wi th 0.1 %crystal violet solution for 10 min., 

washed with water and dried. Cell density was measured with a cell density 

counter CMonocellator, Olympus Co., Ltd.). 

6.4 Measurement of 50% inhibition concentration of cell growth 

The ratio of the number of surviving cells in each treatment to the number 

surviving in its corresponding negative control was calculated, a survival 

curve drawn and 50% inhibition concentration of cell growth (IC 50 ) calculated. 

1 2 

4L435 


7. 	 Chromosomal aberration test 

7.1 	Test substance concentration 

In the cell growth inhibition tests, the test substance did not inhibit cell 

growth more than 50% at 24 hours without metabolic activation and with 

metabolic activation, the test substance concentrations resulting in 50% 

inhibition were 3154t1g/ml at 48 hours without metabolic activation. 

On the basis of the cell growth inhibition tests. the highest concentration 

in the chromosomal aberration test was set at 5000,ug/ml 24 and 48 hours 

, .' 

without metabolic activation. and with metabolic activation. 

In positive controls. final concentrations of MMC and BP were set at 0.03 

and 20,lig/ml. respectively. which are the concentration levels known to induce 

chromosomal aberration. 

7.2 	Treatment of cells 

The cultured cells were treated with the test substance using the same 

procedure as described in section 6.2. 

As positive controls. cells were treated with a mixture of 0.05 ml of MMC 

solution and 5 ml of th~ growth medium without metabolic activation. with a 

mixture of 2.5 ml of the growth medium. 0.5 ml 89 Mix and 0.015 ml of BP 

solution wi th metabol ic activation with S9 Mix. and with a mixture of 3 ml of 

the growth medium and 15,li1 of BP solution with metabolic activation without 

S9 Mix. 

7.3 Preparation of specimens 

In preparation of specimens. at 2 h before the end of treatment, colcemid 

was added to the medium in each plate to a final concentration of 0.1 ,lig/ml 

to arrest cells in metaphase. The cells were then washed with PBS(-). 

dissociated with 0.25% trypsin solution. and centrifuged ClOOO rpm, 5 min.; 

same below) for collection. After removal of the supernatant. 4 ml of 0.075 M 

potassium chloride solution was added for hYPotonic treatment (37°C. 15 min.). 

1 3 
4L435 

The cells were then fixed in 4 ml of cold methanol/acetic acid mixture 

(3/1. v/v). AfteFcentrifugation, the supernatant was removed and 4 ml of a 

fresh fixative added. This fixation procedure was repeated 3 to 4 times. 

Thereafter, the cells were suspended in a small amount of the fixative, and 

drops of the cell suspension were placed in two positions on a slide glass 

and dried. The cells were stained for 20 min. with 3% Giemsa solution diluted 

with 1/15 Mphosphate buffer (pH 6.8). The slides were then washed with water, 

dried, and coated with a mounting medium. Three slides were prepared for each 

plate. 

7.4 Observation 

(1) Structural aberration and numerical aberration 

Each specimen was coded, 100 metaphase cells for each plate (Le. 200 cells 

for each concentration) were observed in a blind manner. Metaphase cells with 

well-spread chromosomes were selected and obs~rved for structural aberrations. 

Cells without aberration but not having 25 2 chromosomes were omitted from 

the survey. Structural aberrations were classified as follows ll : 

gaps (g. including chromatid and chromosome type lesions) chromatid breaks (ctb) fchromatid exchanges (cte) chromosome breaks (csb) 1chromosome exchanges (cse, dicentric. ring, etc.) other aberrations (frg) 

A "gap" was distinguished from a "break" as follows. The "gap" is an 

achromatic region in a single chromatid which is equal to, or slightly larger 

than, the width of the chromatid, whose shape is clearly defined. 

(2) Numerical aberration (polyploid cell) 

Numerical aberration were observed and polyploid cells, including 

. endoredupl icated cells, were scored. 

1 4 

4L435 


7.5 Judgement " 

A cell having at least one structural chromosomal aberration was classified 

as an aberrant cell. The number of aberrant cells was counted in two ways. 

that is incr udin g eel Is wi t h no aberration other than gaps (+ gap) , and 

excluding such cells (-gap). 

The chromosomal aberration potential of the test substance was judged as 

follows. 

The number of aberrant cells was statistically compared with that of the 

control by the x2 -test. The number of polyploid cells was also analyzed in 

the same manner. 

The test substance was judged to induce chromosomal aberration when the number 

of aberrant cells is significantly different from tllat of "the solvent control 

and any of the following is observed: 

CD Significance at P < 0.001 


GD Obvious dose-dependent increase 


@ Reproducible increase at the significant concentration 


7.6 Collect of data 

Each of the sums and the incidence (I) of the chromosome aberration and 

numerical aberrations was shown. The chromosomeal aberration was shown 

dividing the number of cells in the type. 

When the test was positive. the "D 20 " values were calculated from result. 

The "D 20 " value is the concentration (mg/mD required to induce any aberration 

in 20% of metaphases. The microscopic photograph of a typical chromosomal 

aberration is appended. 

1:"":0 1 5 v 
4L435 


Results and Conclusion 

In the cell growth inhibition tests, the test substance did not inhibit cell 

growth more than 50% at 24 hours without metabolic activation and with 

metabolic activation. the test substance concentrations resulting in 50% 

inhibition were 3154,ug/ml at 48 hours without metabolic activation (Figure 

1. 2). 

On the basis of the cell growth inhibition tests. the chromosomal aberration 

test was set at 5000. 2500 and 1250 ,ug/ml 24 and 48 hours without metabolic 

aritivation and with metabolic activatio~ 

In the chromosomal aberration test. the test substance did not significantly 

increased cells with structural chromosomal aberrations and numerical 

aberration (polyploid cell) with and without metabolic activation. In the 

negative control. the incidences of aberrant cells were low. and in the 

positive controls, aberrant cells were increased significantly (Table L 2 

and Figure 3, 4). 

The sponser confirmed that the lot of the test substance used in this study 

was stable during testing period. 

From these findings, it was concluded that YDC-1312 does not have 

clastogenic potential in this CHL/IU cell I ine system. 

Reference 

1) Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of 

Japan: Atlas of chromosomal aberrations induced by chemicals, Asakura 

Press, TokYO, 1988. 

2) OECD guideline for testing of chemicals, Section 4 Health effects» 

GENETIC TOXICICOLOG" 473, (997). 

1 6 r' 

t...' 1 
TABLE 1 RESULTS OF THE CHROMOSOMAL ABERRATION TEST (WITHOUT METABOUC ACnVATION) 

Test substance: YDC-1312 

I--' 

--J 

("" C.,) 

Treatment Exposure Dose No. of Polyploids No. of cells with chromosomal structural aberrations 

time levels cells Judge- Gaps Chromatid type* Chromosomal type* Others* Total 

group (h) (J.l. g/mO al1alysed (%) ment** g ctb ' cte csb 

loo 0 0 0 0 

Negative 24 0 100 0 / 0 0 0 

control 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 

(l%-CMC-Na) 100 0 0 0 0 

48 0 100 0 / 0 0 0 

200 0(0.0) 0(0.0) OCO.O) 0(0.0) 0(0.0) 

100 0 0 0 0 

24 1250 100 0 - 0 0 0 

t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 

100 0 0 0 0 

24 2500 100 0 - 0 1 0 

t 200 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 

100 0 0 1 0 

24 5000 100 0 - 0 0 0 

Test t 200 0(0.0) 0(0.0) 1(0.5) 0(0.0) 0(0.0) 

substance 100 0 0 0 O. 

48 1250 100 0 - 0 0 0 

t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) HO.5) 

100 0 0 0 0 

48 2500 100 0 - 0 0 0 

t 200 O{O.O) 0(0.0) 0(0.0) 0(0.0) 0<0.0) 

toO 0 1 1 0 

48 5000 100 0 - 0 0 0 

t 200 0(0.0) 1(0.5) 1(0.5) 0(0.0) O(O.O} 

100 0 0 9 19 

Positive 24 0.03 100 0 - 2 10 15 

control 200 0(0.0) 2(1.0) 19(9.5) 34(17.0) 0(0.0) 

100 0 2 10 26 

(MMC) 48 0.03 100 0 - 1 12 26 

200 0(0.0) . 3(1.5)_ ~2(t 1.0) ~(~6.0) 3(1.5) . 

* ctb: chromatid break. cte : chromatid exchange. csb : chromosome break. cse : chromosome exchange. 

others: fragmentation(except pulverization) 

** X 2-test : significantly different from negative control: +++(p(O.OOl) 

l%-CMC-Na: 1% sodium carboxymethylcellulose solution. MMC : mitomycin C 

t : undissolved test substance was observed in medium at cell tratment time. 

cse -g +g 

0 0 0 0 0 

0 0 0 0 0 

0(0.0) 0(0.0) 0(0.0) 0(0.0) 

0 0 0 0 0 

0 0 0 0 0 

0(0.0) 0(0.0) 0(0.0) 0(0.0) 

() 0 0 0 0 

0 0 0 0 0 

0(0.0) 0(0.0) 0(0.0) 0(0.0) 

0 0 0 0 0 

0 0 0 1 1 

0(0.0) 0(0.0) 1(0.5) 1(0.5) 

0 0 0 1 1 

0 0 0 0 0 

0(0.0) . 0(0.0) 1(0.5) 1(0.5) .~ 

0 

.1 

0 0 0 0 

1 0 0 1 1 

0(0.0) 0(0.0) 1(0.5) 1(0.5) 

0 0 0 0 0 

0 0 0 0 0 

0(0.0) 0(0.0) 0(0.0) 0(0.0) 

0 0 0 1 2 

0 0 0 0 0 

0(0.0) 0(0.0) 1(0.5) 2(1.0) 

0 0 0 27 27 

0 0 0 25 27 

0(0.0) 0(0.0) 52(26.0) 54(27.0) 

0 1 1 34 36 

3 0 0 37 38 

1(0.5)_ ~(0.5) 71(35.§)_ ]1(37.0) 

Judgement**J 

I 

/ 

/ 

-

-

-

- i 

-

-

+++ 

+++ 

,.r.:.. 

r:­

,.r.:.. 

c...:> 

c..n 
TABLE 2 RESULTS OF TH IHROMOSOMAL ABERRATION TEST (WITH ME". IOUC ACTIVATION) 

Test substance: YDC-1312 

I-' 

co 

rn 

Treatment With or Dose No. of Polyploids No. of cells with chromosomal structural aberrations 

without levels cells Judge- Gaps Chromatid tvpe* Chromosomal type* 

group S9 Mix (11 g/mD analysed (%) ment** g ctb cte csb 

100 0 0 0 0 

Negative - 0 100 0 / 1 0 1 

control 200 0(0.0) 1(0.5) 0(0.0) 1(0.5) 0(0.0) 

(l%-CMC-Na) 100 1 0 0 1 . 

+ 0 100 0 / 1 , 0 0 

200 1(0.5) 1 (0.5) 0(0.0) 1(0.5) 0(0.0) 

100 0 0 1 0 - 1250 100 0 - 1 0 1 

t 200 0(0.0) 1(0.5) 1(0.5) 1(0.5) 0(0.0) 

100 0 1 1 0 

- 2500 100 0 - 0 0 0 

t 200 0(0.0) 1 (0.5) 1(0.5) 0(0.0) 0(0.0) 

100 0 0 0 0 

- 5000 100 0 - 0 0 1 • 

Test t 200 0(0:0) 0(0.0) 0(0.0) 1(0.5) 0(0.0) 

substance 100 0 0 0 0 

+ 1250 100 0 - 0 0 0 

t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 

100 0 1 0 1 

+ 2500 100 0 - 0 0 0 

0 

I 

t 200 0(0.0) 1(0.5) 0(0.0) 1(0.5) 0(0.0) 

100 0 0 0 0 

+ 5000 100 0 - 0 0 0 

t 200 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 

100 1 0 0 0 

Positive - 20 100 0 - 1 0 0 

control 200 1(0.5) 1(0.5) 0(0.0) 0(0.0) 0(0.0) 

100 0 1 5 29 

(BP) + 20 100 0 - 1 2 30 

200 0(0.0) . 2(1.0) 7(3.5) 59(28.5) 4(2.0)_ 

* ctb: chromatid break. cte : chromatid exchange. csb : chromosome break. cse : chromosome exchange. 

others: fragmentation(except pulverization) 

** X 2-test : significantly different from negative control; +++(p(O.OOl) 

Cells were treated with S9 (5% of final conc.) or without S9 for 6 hours and recovered for 18 hours. 

1 %-CMC-Na : 1" sodium carboxymethylcellulose solution. BP : benzo(a)pyrene 

t : undissolved test substance was observed in medium at cell tratment time. 

cse 

0 0 

0 0 

0(0.0) 

0 0 

0 0 

0(0.0) 

0: 0 

0 0 

0(0.0) 

0 0 

0 0 

0(0.0) 

0 0 

0 

0(0.0) 

0 0 

0 0 

0(0.0) 

0 0 

0 0 

0(0.0) 

0 0 

0 0 

0(0.0) 

0 0 

0 0 

0(0.0) 

2 1 

2 3 

4(2.0) 

Others* Total 

-g +g 

0 0 0 

0 1 2 

0(0.0) 1(0.5) 2(1.0) 

0 1 1 

0 0 1 

0(0.0) 1(0.5) . 2(1.0) 

0 1 1 

0 1 2 

0(0.0) 2(LO} 3(1.5) 

0 1 2 

0 0 0 

0(0.0) 1 (0.5) 2(1.0) 

0 0 0 

0 1 1 

0(0.0) 1 (0.5) 1(0,5) 

0 0 0 

0 0 0 

0(0.0) 0(0.0) 0(0.0) 

0 1 2 

0 0 0 

0(0.0) 1(0.5) 2(1.0) 

0 0 0 

0 0 0 

0(0.0) 0(0.0) 0(0.0) 

0 0 0 

0 0 1 

0(0.0) 0(0.0) 1 (0.5) 

0 36 37 

0 38 38 

0(0.0) 74(37.0) 75(3].5) 

Judgement** 

/ 

/ 

-

-

-

-

-

-

.... 

+++ 

.-

,j:::.. 

~ 

Li ,j:::.. 

v.:> 

en 
4L435 

• 24hours treatment 

- 0 - 48hours treatment 

100 ......... 


'-' "0 * 	 .. .. >< (1) .. .. "'0 .. J:: .. -.-4 .. 
 ...c:: .. 0 .. -+"' .. .. ~ 

0 50 .. .. .. Io-! .. .. .. .. bO .. .. .. r-1 


r-1 


(1) 

U 

o 

o 	 1000 2000 3000 4000 5000 

Concentration (J.L g/ml) 

Fig. 1 Cell growth inhbition treated with YDC-1312 

(Without metabolic activation) 

_With S9 mix 

100 

50 


o 

o 1000 2000 3000 4000 

Concentration (J.L g/mD 

Fig.2 Cell growth inhibition treated with YDC-1312 

(With metabolic activation) 

5000 

1 9 1:""7 

\.: '1 
4L435 


10.0 

8.0 

- 6.0 

-'* 

Q) 

u 

Q) 

=: 4.0 

"0 


U 


=: 

2.0 

0.0 

10.0 

8.0 

-. 

-'* Q) 6.0 u 

=: 

Q) 

"0 

U 

=: 4.0 

2.0 

0.0 

r • 24hours treatment 

- 0- 48hours treatment 

.. 0­ .­ ...... ~ - .. -. .. .. - .. .. .. J' .. .. .. ..;;. .. .. .. - o 	 1250 2500 5000 

Concentration (tlg/ml) 

Fig.3 	 Incedence of aberant cell treated with YDS-1312 

(without metabolic activation) 

- 0- Without S9mix 

• With S9mix 

.--0-_. . ... -- - ­ .. --- .. - -~.....-­ o 	 1250 2500 5000 

Concentration (Jl g/mD 

Fig.4 	 Incidence of aberrant cell treated with YDC-1312 


(with metabolic activation) 


2 0 
00'00,56 

--
000058 

Acute Oral Toxicity Test of YDC-1312 in Rats 

(English version) 

Study No. 4L437 

Study Title Acute Oral Toxicity Test ofYDC-] 3] 2 in Rats 

Sponsor Tohto Kasei CO" LTD 

This test was conducted in Kashima Laboratory. Mitsubishi Chemical Safety 

Inst.itute Ltd .. 14 Sunayama. Hasaki-machi. Kashima-gun. Ibaragi. Japan. 

This report is the English version of the original. which was written in Japanese. The 

undersigned hereby dpciare that. this version faithfully reflects the original to the best. of 

our knowledge. 

Translated by 

Takayoshi Hosono 

Manager for Technical Division 

Tokyo Head Office, Tohto Kasei CO., LTD 

Approved by 

I ~ , . I I =- .. r 7__ --.)..:..' / Dale: I 

Koyo Murakami 

n(meral Manager for 1\~ehnieal Division 

Tokyo Head Office. 'I'ohlo KaHPi CO.. LTD 

000.059 

COMPLIANCE WITH THE GLP STANDARDS 


Study No. 4L437 

Study Title Acut.e Oral Toxicity Test ofYDC-1312 in Rat8 

Sponsor Toht.o Kasei CO., LTD 

The study described in this report was conduct.ed in compliance with the Good 

Laborat.ory Pract.ice regulations recognized by the OECD GLP (1981). 

Facility Management. Sakae Koike Sealed date: March 14, 1995 

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

oooo~;o 
QUALITY ASSURANCE STATEMENT 

Study No. 4L487 

Study Title Acute Oral Toxicity Test ofYDC-l :312 in Rat.s 

Sponsor Tohto Kasei CO.. LTD 

We hereby cert.ify that. the above Rl.udy has been performed in accordance with the 

Gooel Laboratory Pract.ice (GLP) regulat.ions recognized by the OECD GLP (1981). 

Dates of inspect.ions and reportings to the Study Director and the Facility 

Managemenl are as rollows : 

In-progress study 

Final report 

Date of 

Inspection 

November 15. 1994 

January 11. 1995 

March 14, 1995 

Date 

Reporting 

November 16. 1994 

January 1:3, 1995 

March 14. 1995 

Qualit.y Assurance Personnel 

Kunihiko Ofuchi 

Mit.subishi Chemical Safet.y Institute Ltd. (M.s.l.) 

Sealed date: March 14, 1995 
STUDY PERSONNEL 


St.udy Personnel 

S1udy Director: 	 Masato Sudo Sealed 

Mayumi Kitami Sealed 

OOOOG2 

CONTENTS 


SYNOPSIS 

TEST SUBSTANCE AND ITS EVALUATION METHOD 

1 TEST SUBSTANCE 

2 EXPERIMENTAL ANIMALS 

a ANIMAL MANAGEMENT 

4 PREPARATION OF DOSAGE 

5 DOSE LEVEL 

G METHOD OF ADMINISTRATION 

7 CLiNICAL OBSRVATIONS OF RATS 

8 MEASURING BODY WEIGHTS OF RATS 

9 NECROPSY 

RESULTS AND CONCLUSION 

1 NUMBER OF DEAD RATS 

2 CLINICAL OBSERVATION 

:3 BODY WEIGHT 

4 NECROPSY 

5 CONCLUSION 

TABLE 1 INDIVIDUAL BODY WEIGHTS 

Page 

G 

7 

7 

7 

8 

8 

8 

8 

9 

9 

9 

10 

10 

10 

10 

10 

10 

11 

OOOOf;3 

SYNOPSIS 

(1) 	 YOC-IaI2 was dosed orally once to SO strain rals(SPF) of males and females with 

a dose of 2000mglkg and observations were made on the mort.ality and toxicological 

effect.s. 

(2) 	 No animalR died during 14 days test period. 

(a) 	 No abnormalities were obSt~rved in any animals up to the end of observation. 

(4) 	 Body weights of all animals increased normally. 

(5) 	 At necropsy. no nhnormalil.i0s were found in any animals. 

(6) 	 YOC-la12 gave no toxicological effecls to the rats by oral administ.ral.ion, and the 

lowest lethal dose ofYDC-l:312 was greater than 2000mg/kg. 

( i 

OOOOG4 
Test subst.ance and its evaluation method 

1. Test subst.ance 

---------------------~--------~---

2, Experimental animals 

Five males and females SD strain(Crj:CD) rats(SPF) were purchased from Charles 

River ,Japan Inc, on November 9, 1994. 

Aft.er quarantine and acclimatization for 6 days. it was confirmed that the rats were 

in goot! health condition. ancl the age of animals at administrat.ion was 5 week old. 

The rats were marked in their tails with oil base marker. so that. they were able to 

IlP identified individually. 

TIlt' cages which kepI the ahove animals were identified with a lahellisting stu(ly 

number. name or thl' test substance. animal numbers. sex, an(1 dose level. 

Their body weights at t.he first administration ranged from I:3G to 149 g in maiN;; 

and I()~ to 122 g in r('males, 

7 

0000';5 
:3 Animal management 

:3.1 Housing condit.ions 

Throughout. the study period. including t.he quarantine and acclimatization periods. 

t.he animal room was maint.ained at a temperature between 20·C and 25·C, and a 

relat.ive humidity betwe.en 40% and 70% with 12 fresh air ('hanges per hour and 

was lit for 12 hours per day (7:00-19:00). 

The animals were housed in polycarbonate cages(265W X 4260 X 200H m m, Tokiwa 

Kagaku Kikai Co .. Lt.d.) wit.h hard wood chip bedding for experiment.al animals 

(Beta.Chip. Charles River Japan Inc.). Five rats were housed in each cage. The 

cages were placed on a steel rack(4-tiered. Tokiwa Kagaku Kikai Co..Ltd.). 

Stainless steel feeders for pellet diet(rokiwa Kagaku Kikai Co.. Ltd.) and 

polycarbonate watering bott.los(700mL. Tokiwa Kagaku Kikai Co ..Lt.d.) were m;ed. 

The cages (including bedding). feeders. and watering bottles were autoclaved and 

replaced once a week. 

:3.2 Diet 

Pellte diet for experimental animals(MF. Oriental Yeast Co .. Ltd.) was given 

ad libitum and was renewed once a week. Chemical analysis of t.he diet for 

contaminants such as pesticide residue certified that their concentrations were 

lower than the limit values determined in our Standard Operat.ion Procedure. 

3.3 	 Drinking water 

Ta» water was given ad libitum and was renewed once a week. The water was 

filtrated through a 5- j.1 m filter and irradiated by UV. The wat.er was analyzed 

periodically in accordance with the Waterworks Law of Japan, and was cert.ified to 

meet the required standard. 

4 Preparat.ion of dosage 

YDC-l:312 which has been grounded with a mortar was dispersed in 0.5% 

aqueous CMC·Na solution to be an emulsion with a super high Hpeed homogenizer 

(Polyt.ron: Central Science Trading Co.. Ltd.). The emulsion waH Hlljust.ed to he a 

s(~lected concent.ration and t.hen dosed. 

5 OOl-:'e level 

Preliminary I.est.s wern carried out for two male rats with a dose of 2000mg/kg rat. 

The results indicat.ed t.hat there wnre no ahnormal obst'rvations and Hubl-;(~qlH'nt.ly 

further h~l-:'t with a dose of 2000mg/kg rat which is the upper limit df'f'inpd in the 

guideline was conducted for fivn mall' nIls and five f<'maln oneK 

The clost' lewl waH t'quivalpnt to 1.0 mil hody wl'ighl. WOg. 

() M<,t hod or adm inist rat ion 

All ralH wert' faHIt~d h('for<~ dOHe for ovprniJ.{ht. Oral dosage was given 10 their 

Hlom:whjo.; by usinJ,!; a stomneh tuhe. Tlw diet was wil.hlwld for thr('(' hoursaft,('r 

administ.ration. 

000 0.;r' <-. ~)".)·o 
7 Clinical observations of rats 

Clinical observat.ions of each rat. to be dosed were made 15. :30 min. and 1. :3, and 6 

hours later in the day when t.he dosage was given to the animal and subsequently 

once a day during 14 days. 

8 Measuring body weights of rats 

Body weights of all rat.s were measured by use of elect.ronic balances (ES·5000. EB· 

6200S Shimazu Corp.) in the int.ervals just before the dosage and on 8. 7. and 14 

days after administrat.ion. 

The day of administ.ration was designated as "0 day". 

9 Necropsy 

At 14 dny of test.. all rat.s were anest.hetized with a thiopent.al sodium 

anesthesia(Ravonal.Tanabe Seiyaku Co.. Ltd.) and t.hey were euthanized by severing 

t.he abdominal aorta and then subjected to necropsy. 

!) 
Results and Conclusion 

(1) No animals died during 14 days test period. 

(2) No abnormalities were observed in any animals up to the end or observation. 

(3) Body weight.s of all animals incrcaS0d normally. 

(4) At. necropsy. no abnormalities were found in any animals. 

(5) YDC-l:312 gave no t.oxicological effect.s to the rats by oral administration. and the 

. lowest lethal dose ofYOC·] :312 was greater than 2000mg/kg. 

10 

OOOGf;S 
(S tudy No. : 4L437) 

Table 1 Individual body weights 

unit g 

Sex : Days after administration 

Dose level Animal Gain 

emg/kg) No. 0 3 7 14 (Od--14d) 

Male : 1 135 184 223 300 165 

2 138 176 215 278 140 

2000 3 138 180 217 279 141 

4 149 197 243 316 167 

5 135 175 212 284 149 

mean 139 182 222 291 152 

S. D. 5.8 8.9 12.4 16.3 12.9 

N 5 5 5 5 5 

Fellale 1 109 139 158 192 83 

2 113 152 181 213 100 

2000 3 111 144 160 200 89 

4 122 157 183 215 93 

5 112 134 156 190 78 

mean 113 145 168 202 89 

S. D. 5.0 9.4 13.2 11.6 8.6 


N 5 5 5 5 5 


II OOOOG9 

BOI0234 


Translation Statement 


Kashima Laboratory 


Mitsubishi Chemical Safety Institute Ltd. 


Sponsor: Tohto Kasei Co., Ltd. 

Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study ofYDC-1312 in Rats 

Study number: 4lA36 

This study was conducted in Kashima Laboratory of Mitsubishi Chemical Safety Institute Ltd. The 

original final report was written in Japanese. I hereby declare that this report faithfully reflects the 

original final report as accurately as possible. 

Translator: Date: t8r:J 1, '}.-f'¢ l 

Kaname Takahashi 


Senior Research Scientist 


Toxicology Division of Kashima Laboratory 


Mitsubishi Chemical Safety Institute Ltd. 

4L436 


Submitted to: 


Tohto Kasei Co., Ltd. 


FINAL REPORT 

Twenty-Eight-Day Repeated Dose Oral Toxicity Study of 


YDC-1312 in Rats 


(Study No. 4L436) 


March 14, 1995 


Mitsubishi Chemical Safety Institute Ltd. 

0000 .1 
4L436 


Statement 

Kashima Laboratory 


Mitsubishi Chemical Safety Institute Ltd. 


Sponsor: TohlO Kasei Co., Ltd. 

Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study ofYDC-1312 in Rats 

Study number: 4IA36 

This study was conducted in compliance with the Good Laboratory Practice Standards applied to 

industrial chemicals of Japan. 

Management: Sakae Koike sealed Date: March 14, 1995 

2 

0000'72 
4L436 


Quality Assurance Statement 


Kashima Laboratory 


Mitsubishi Chemical Safety Institute Ltd. 


Sponsor: Tohto Kasei Co., Ltd. 

Title: Twenty-Eight-Day Repeated Dose Oral Toxicity Study of YDC-1312 in Rats 

Study number: 4LA36 

By the inspections and audits listed below, I hereby certify that this study was carried out in 

accordance with its protocol and the Standard Operating Procedures, that this report faithfully 

describes the methods and procedures employed, and that the reported results accurately reflect the 

raw data of the study. 

Inspection or audit Date of inspection or audit Date of reporting to the study 

director and management 

Study procedure 

Study report 

October 13, 1994 

October 21, 1994 

November 8, 1994 

November 11, 1994 

February 16, 1995 

March 14, 1995 

October 13, 1994 

October 21, 1994 

November 8, 1994 

November 11, 1994 

February 22, 1995 

March 14, 1995 

Quality Assurance Manager: 

Kunihiko Ofuchi sealed Date: March 14, 1995 

3 

0000'73 
4L436 


Study Outline 


L Title: 	 Twenty-Eight-Day Repeated Dose Oral Toxicity Study ofYDC-1312 in Rats 

(Study No. 4L436) 

2. 	 Purpose: The purpose of this study is to determine the toxicity of the test substance by 

observation of the changes in function and morphology in rats that received 

repeated oral administration of YDC-1312 for 28 days. 

3. 	 Guideline: Japanese Guidelines on Industrial Chemicals 

(Kanpogyo No.700, Yakuhatsu No.l039, 61 Kikyoku No. 1014, 1986) 

4. 	 GLP: Japanese GLP on Industrial Chemicals 

(Kanpogyo No.39. Yakuhatsu No. 229. 59 Kikyoku No.85, 1984) 

5. 	 Sponsor: (Name) Tohto Kasei Co., Ltd. 

(Address) 17-14, Higashikasai 3-chome, Edogawa-ku, Tokyo, Japan 

(Sponsor representative) 

Yoshiaki Nakamura 

6. 	 Organization under contract: 

(Name) Mitsubishi Chemical Safety Institute Ltd. 

(The company changed its name on October 1, 1994) 

(Address of head office) 

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

(Testing facility) 

Kashima Laboratory 

(Address of testing facility) 

14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, Japan 

4 	 0000'74: 
4L436 

7. Study dates: (Initiation of the study) September 8, 1994 

(Receipt of test animals) October 5, 1994 

(Start of administration) October 14,1994 

(Necropsy at the end of the dosing period) 

November 11, 1994 

(Necropsy at the end of the recovery period) 

November 25, 1994 

(Issue of final report) March 14, 1995 

8. 	 Study personnel: 

Study director Masato Sudo sealed March 14, 1995 

Study contributor Mayumi Kitami sealed March 14, 1995 

Pathologists Minoru Tsuchitani sealed March 14, 1995 

Yoshimasa Okazaki sealed March 14, 1995 

Clinical chemistry Naoto Toyota sealed March 14, 1995 

Katsuyo Takano sealed March 14, 1995 

9. 	 Unforeseeable circumstances that may have adverse effects on the reliability of the study: 

None. 

10. 	 Retention: 

After the submission of the final report, all records and documents concerning to 

this study will be retained for 10 years in the archives of the Kashima Laboratory. 

Further retention will be discussed with the sponsor. 

5 	 000075 

4L436 


Contents 


Summary ............................................................................................................................................. 8 


Materials and Methods ........................................................................................................................9 


1. Test substance .....................................................................................................................9 


2. Experimental animals ..........................................................................................................9 


3. AniInaI management ...........................................................................................................9 


4. Administration .................................................................................................................. 10 


5. Preparation of the dosing solution ..................................................................................... 11 


6. Recovery period ................................................................................................................ 11 


7. Constitution of treatment groups ....................................................................................... 12 


8. Clinical signs ..................................................................................................................... 12 


9. Body weight ...................................................................................................................... 12 


10. Food consumption ............................................................................................................. 12 


11. HeInatological examination .............................................................................................. 12 


12. Blood chemistry examination ........................................................................................... 13 


13. Urinalysis .......................................................................................................................... 14 


14. Pathological examination .................................................................................................. 14 


2.15 Statistical analysis ............................................................................................................. 15 


Results............................................................................................................................................... 16 


1. Dead animals ..................................................................................................................... 16 


2. Clinical signs ..................................................................................................................... 16 


3. Body weight ...................................................................................................................... 16 


4. Food consumption ............................................................................................................. 16 


5. Hell1atological examination .............................................................................................. 16 


6. Blood chemistry exatnination ........................................................................................... 16 


7. Urinalysis .......................................................................................................................... 17 


8. Organ weight ..................................................................................................................... 17 


9. Necropsy ........................................................................................................................... 17 


10. 	 Histopalhologicall·xaminalioJl...................... . . ................................................... 18 


6 


0000'76 
4L436 

Discussion......................................................................................................................................... 19 


References ......................................................................................................................................... 20 


Figure and Tables 

Figure 

Table 

Table 2 


Table 3 


Table 4,5 

Table 6 


Table 7 


Table 8 


Table 9 


Table 10 


Table 11 


" 

Appendices 


Appendix 


Appendix 2 


Appendix 3 


Appendix 4,5 


Appendix 6 


Appendix 7 


Appendix 8 


Appendix 9 


Appendix 10 


Appendix II 


PholOgraphs 

Body weight changes in rats treated orally with YDC-1312 for 28 days ....... I 


Incidence of clinical signs .............................................................................. 3 


Body weight - group mean values .................................................................. 7 


Food consumption - group mean values ......................................................... 9 


Hematology - group mean values ................................................................. 11 


Clinical chemistry - group mean values ....................................................... 19 


Urinalysis - group mean values .................................................................... 27 


Organ weight (absolute) - group mean values .............................................. 29 


Organ weight (relative: percentage of body weight) - group mean values ... 33 


Incidence of necropsy findings .................................................................... 37 


Incidence of histological findings ................................................................ 38 


Incidence of clinical signs .............................................................................. 1 


Body weight - individual values ..................................................................... 9 


Food consumption - individual values ......................................................... 17 


Hematology - individual values ................................................................... 25 


Clinical chemistry - individual values .......................................................... 49 


Urinalysis - individual values ....................................................................... 73 


Organ weight (absolute) individual values ................................................ 81 


Organ weight (relative: percentage of body weight) - individual values ..... 93 


Individual necropsy findings ...................................................................... 105 


Individual histological findings .................................................................. 108 


7 onon··"'i'·..., uU U I I

4L436 


Summary 


(1) 	 YDC-1312 was administered orally to male and female SD strain rats for 28 days. A 

recovery period of 14 days was also arranged for the control and 1000 mglkg groups. 

(2) 	 The test substance was suspended in a 0.5% sodium carboxymethylcellulose solution, and 

administered to the rats with a stomach tube. The dose levels were 20, 140, and 1000 mglkg, 

and the control group was administered the vehicle only. The dosing volume was set at 1.0 

mlJloog body weight in each group. 

(3) 	 No death was observed throughout the study period. 

(4) 	 No abnormalities were observed in clinical signs, body weight, food consumption, 

hematological examination, or urinalysis. 

(5) 	 In the blood chemistry examination, significantly increased albumin was observed in the 

males of the 1000 mglkg group. 

(6) 	 In the pathological examination at the end of the dosing period, significantly increased 

weight (relative weight) of the ovaries was observed in the females of the 1000 mglkg group. 

An increase in hyaline droplets in the proximal tubular epithelium in the kidneys was 

observed in the males of the 1000 mglkg group. 

After the end of the recovery period, significantly increased weight (absolute weight) of the 

kidneys was observed in males of the 1000 mglkg group. Basophilic change of the tubular 

epithelium accompanied with hyaline casts in the kidneys was also observed; however, 

incidence of the increase in hyaline droplets in the proximal tubular epithelium decreased. 

(7) 	 From the above results, it was estimated that the no observed effect level of YDC-1312 

under these study conditions was 140 mglkg in both sexes. 

8 	 000078 

4L436 


Materials and Methods 

I. 	 Test substance 

~--~---------------~ 2. 	 Experimental animals 

Thirty-six male and female SD strain (Crj:CD) rats (SPF) were purchased from Charles River 

Japan Inc. on October 5, 1994. 

After quarantine and acclimatization for 9 days, it was confirmed that the rats were in good 

general condition, and the administration was started at 5 weeks old. The rats were grouped 

by the stratified-by-weight randomization method one-day before the first administration. The 

animals were identified by the ear punch method and each cage was identified with a label 

listing study number, name of the test substance, animal numbers, sex, and dose level. 

Their body weights at the first administration ranged from 171 to 198 g in males and 133 to 

167 g in females. It was confirmed that the individual body weight was within mean body 

weight ± 20% 

3. 	 A limal management 

3.1 	 Housing conditions 

Throughout the study period, including the quarantine and acclimatization periods, the animal 

room was maintained at a temperature between 20· and 2ye. and a relative humidity between 

9 0000'79 
4L436 

40% and 70% with 12 fresh air changes per hour and was lit for 12 hours per day (7:00-19:00). 

The animals were housed in polycarbonate cages (265W x 4260 x 200H mm, TR-PC-2oo: 

Tokiwa Kagaku Kikai Co., Ltd.) with hard wood chip bedding for experimental animals (BetaChip, Charles River Japan Inc.). Two rats of the same sex were housed in each cage. The 

cages were placed on a steel rack (4-tiered, Tokiwa Kagab Kikai Co., Ltd.). Stainless steel 


feeders for pellet diet (Tokiwa Kagaku Kikai Co., Ltd.) and polycarbonate watering bottles 


(700 mL, T-564: Tokiwa Kagaku Kikai Co., Ltd.) were used. 


The cages (including bedding), feeders, and watering bottles were autoclaved and replaced 


once a week. 


3.2 	 Diet 

Pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.) was given ad libitum and 

was renewed once a week. Chemical analysis of the diet for contaminants such as pesticide 

residue certified that their concentrations were lower than the limit values determined in our 

Standard Operation Procedure. 

3.3 	 Drinking water 

Tap water was given ad libitum and was renewed once a week. The water was filtered through 

a 5-J.lm filter and irradiated by UV. The water was analyzed periodically in accordance with 

the Waterworks Law of Japan, and was certified to meet the required standard. 

4. 	 Administration 

4.1 	 Dose levels and basis for dose setting 

No abnormalities were found as the results of a 7-day repeated dose preliminary study 

conducted at dose levels of 500 and 1000 mg/kg. From the results of this preliminary study. 

the high dose level was determined as 1000 mg/kg, which was the highest dose described in 

the guideline, and the middle and low dose levels were set as 140 and 20 mg/kg. respectively. 

The control group was also arranged. 

The control group was received the vehicle only. 

10 	 000000 
4L436 


4.2 	Dosing period 

The administration was performed for 28 days, once a day in the morning. The start of the 

administration was designated as "0 day after commencement of the treatment" in this report. 

4.3 	 Method of administration 

The dosing solution was administered into the stomach by a syringe with a stomach tube. 

4.4 	 Dosing volume 

Dosing volume was set at 1.0 mUlOO g body weight. IndiVidual dosing volume was 

calculated by the most recent weighed individual body weight. 

5. 	 Preparation ofthe dosing solution 

An appropriate amount of the test substance was weighed and ground in a mortar. The dosing 

solutions were prepared by suspending the test substance in a 0.5% carboxymethylceUulose 

sodium aqueous solution. The preparation was performed once a week and the dosing 

solutions were kept in brown bottles and the bottles were stored in a refrigerator protected 

from light until just before dosing. 

The stability of the test substance in the dosing solution stored in a refrigerator for 8 days was 

confirmed by analysis of the dosing solutions. 

(Unit: mg/mL) 

r~ oday 

, 8 day • Concentration 

2 1.86 1.78 

100 95.4 I 101 i 

6. 	 Recovery period 

A recovery period of 14 days was arranged in the control and 1000 mg/kg groups after the 

dosing period. 

II 
4L436 


7. Constirution of treatment groups 


Constitution of treatment groups is described below. 


Group name Dose level 

(mglkg) 

Male 

Control group 0 6 6 

Low dose group 20 6 

Middle dose group 140 6 6

High dose group 1000 6 6 6 6 

Total number 24 24 12 12 

a Animals were necropsied on the day after the last administration. 

b Animals were necropsied after the recovery period of 14 days. 

8. 	 Clinical signs 

Observations of all rats on their fate, appearance, and behavior were made once a day. More 

detailed observations including palpation were also made once a week. 

9. 	 Body weight 

All rats were weighed on the day of the ftrst administration and once a week thereafter with an 

electronic balance (EB-5000, Shimadzu Corp.). 

10. 	 Food consumption 

All feeders including food was weighed on the day of the first administration and once a week 

thereafter with an electronic balance (EB-5000, Shimadzu Corp.). Daily consumption per rat 

was then calculated. 

11. 	 Hematological examination 

Hematological examinations were performed at each scheduled sacrifice. Blood samples were 

obtained from the inferior vena cava of the rat without fasting under intraperitoneal thiopental 

sodium anesthesia (Ravonal®, Tanabe Seiyak.'U Co., Ltd.). The items listed below were 

measured using the blood collected. As the anti-coagulant, 3.13 % sodium citrate aqueous 

solution was used for the measurement of the prothrombin time and activated partial 

thromboplastin time, and EDT A-2K was used for the other measurements. Items 1 7 and 10 

-12 of one male in the 140 mglkg group (MeOOS) could not be measured because of clotting. 

12 OOOO~i2 
4L436 

Items 8 and 9 of one female in the 140 mglkg group (FCOO1) also could not be measured 

because of clotting. 

Items Measurement/calculation 

1 Erythrocyte count Sheath flow DC impedance detection method 

2 Leukocyte count RFIDC impedance detection method 

3 Platelet count Sheath flow DC impedance detection method 

4 Hemoglobin concentration SLS hemoglobin method 

5 Hematocrit Cumulative pulse height detection method 

6 Differential leukocyte count Microscopic examination after Wright's staining 

7 Reticulocyte count Flow cytometry by argon laser method 

8 Prothrombin time Modified Quick method 

9 Activated partial thromboplastin time Activated cephaloplastin method 

10 Mean corpuscular volume Calculated from 1 and 5 

11 Mean corpuscular hemoglobin Calculated from 1 and 4 

12 Mean corpuscular hemoglobin Calculated from 4 and 5 

concentration 

Measuring instruments 

I - 5: Automated hematology analyzer (NE4500, Toa Medical Electronics Co., Ltd.) 

6: Auto cell analyzer (MICROX HEG-70A, Omron Tateishi Electronic) 

7: Automated reticulocyte analyzer (R-2000, Toa Medical Electronics Co., Ltd.) 

8, 9: Coagulometer (KC lOA, Amerung, GmbH) 

12. 	 Blood chemistry examination 

The blood samples simultaneously collected with those for the hematological examination 

were used. The serum samples were obtained after centrifugation at 3000 rpm for 10 min 

(maximum centrifugation gravity, 1570 - 2050 g) of blood which had been left to stand at 

room temperature for about 30 min and the measurements of each item listed below were 

performed. 

Items 

1 Total protein 

2 Albumin 

3 NG ratio 

4 Glucose 

5 Triglycerides 

6 Total cholesterol 

7 Urea nitrogen 

8 Creatinine 

9 Calcium 

10 Inorganic phosphorus 

1 I GOT (AST) 

12 GPT (ALT) 

13 y-GTP 

14 ALP 

15 Sodium (Na) 

16 Potassium (K) 

Measurement/calculation 

Biuret method 

BCG method 

Calculated from I and 2 

Enzymatic-UV method (GK-G6PDH method) 

Enzymatic method (LPL-GK-G3PO-POD method) 

Enzymatic method (CES-CO-POD method) 

Enzymatic-UV method (Urease-GLDH method) 

Jaffe method 

O-CPC method 


UV method 


UV-Rate method (modified SSCC method) 


UV-Rate method (modified SSCC method) 


y-glutamyl-p-nitroanilide substrate-method (modified SSCC 


method) 


p-nitrophenyJphosphale substrate method (modified GSCC 


method) 


lon-selective electrode method 


lOll-selective electrode method 

13 

000053 
4lA36 

17 Chlorine (CI) Ion-selective electrode method 

Measuring instrument: Automatic analyzer (Hitachi 736- 10, Hitachi Ltd.) 

13. 	 Urinalysis 

Twenty-five days after conunencement of treatment, fresh urine samples collected from all rats 

were examined for items listed below by a paper test method (Multistix 4!1, Miles-Sankyo Co., 

Ltd.) using a analyzer (Clinitek 10, Miles-Sankyo Co., Ltd.). 

I pH; 2 Occult blood; 3 Protein; 4 Glucose; 5 Ketone bodies; 6 Bilirubin; and 

7 Urobilinogen. 

14. Pathological examination 

14.1 Necropsy 

All surviving animals at each scheduled sacrifice were euthanized after blood collection by 

severing the abdominal aorta under thiopental sodium anesthesia (Ravonale, Tanabe Seiyaku 

Co., Ltd.) and then subjected to necropsy. 

14.2 Organ weight 

The following organs were weighed from all animals at each scheduled sacrifice with an 

electronic balance (ED-H60, Shimadzu Corp.), and the ratio of organ weight to the final body 

weight (relative organ weight) was calculated: the brain, liver, kidneys, adrenals and testes or 

ovaries. 

14.3 Histopathological examination 

The following organs of all animals at each scheduled sacrifice were removed and preserved in 

a 10% neutral phosphate-buffered formalin (except for the eyes and Harderian gland which 

were fixed in Davidson's fluid): the brain, pituitary, eyes (including Harderian glands), lungs, 

stomach, thyroids (including parathyroids), hean, liver, spleen, kidneys, adrenals, urinary 

bladder, testes or ovaries, and bone marrow (femur). 

Histopathological examination was performed on the heart, liver, spleen, kidneys, and adrenals 

of both sexes of the control and 1000 mglkg groups at the end of the dosing period. 

Additionally, the ovaries of the control and 1000 mglkg groups at the end of the dosing period 

14 
4U36 

was examined histopathologically since a change in organ weight was observed. Moreover, 

since gross lesions were observed at the necropsy, the following organs were examined: the 

pituitary of one male of the 20 mglkg group, the lung of one female of the 20 mglkg group, the 

liver of one male of the 140 mglkg group, and the lung 01 one male of the 1000 mglkg group. 

Since this histopathological examination revealed the changes attributable to the 

administration of the test substance in the kidneys of males, further examination in the kidneys 

of the males of the middle and low dose groups at the end of the dosing period and of all males 

at the end of the recovery period were carried out. Additionally, the liver of both sex of the 

middle and low dose groups at the end of the dosing period and of all animals at the end of the 

recovery period were also examined since hypertrophy of hepatocyte was suspected in the both 

sexes of the 1000 mglkg group at the end of the dosing period. 

15. 	 Statistical analysis 

Numerical data obtained in the study were analyzed by multiple comparison tests. They were 

first analyzed by Bartlett's test. When the group variance was determined to be homogeneous, 

"" 

groups were compared by a one-way analysis of variance. When intergroup differences were 

significant, Dunnett's multiple range test (for groups with equal number) or Scheffe's test (for 

groups with unequal number) were used. When Bartlett's test indicated heterogeneous group 

variance, comparisons were done by the Kruskal-Wallis test and when intergroup differences 

were significant, Dunnett's rank sum test (for groups with equal number) or Scheffe's test (for 

groups with unequal number) was used. The categorical data obtained by urinalysis was 

analyzed by Armitage's Chi-square test. 

15 

4L436 


Results 


I. 	 Dead animals 

No death was observed in either the dosing or recovery periods. 

2. 	 Clinical signs (fABLE 1) 

No changes considered caused by the administration of the test substance were observed. 

3. 	 Body weight (FIG. 1, TABLE 2) 

No changes considered caused by the administration of the test substance were observed. 

4. 	 Food consumption (fABLE 3) 

No changes considered caused by the administration of the test substance were observed. 

5. 	 Hematological examination (fABLE 4,5) 

Although significantly extended prothrombin time was observed in the females of the 1000 

mglkg group at the end of the recovery period, it was considered that this change was not 

caused by the administration of the test substance since this was not observed at the end of the 

dosing period and the prothrombin time of some control animals were almost the same as 

those of the 1000 mg/kg group. 

There were no significant changes in other items. 

6. 	 Blood chemistry examination (TABLE 6) 

At the end of the dosing period, significantly increased albumin in the males of the 1000 

mg/kg group and decreased AlG ratio in the males of the 20 mg/kg group were observed. In 

these changes, decreased AlG ratio in the males of the 20 mg/kg group was not considered 

related to the test substance because this change was not observed in the 140 and 1000 mg/kg 

groups. 

16 

000056 
4L436 


At the end of the recovery period, significantly increased total protein and inorganic 

phosphorus were observed in the males of the 1000 mglkg group and significantly increased 

albumin was observed in the females of the 1000 mglkg group; however, they were not 

considered caused by the administration of the test substance since they were not observed at 

the end of the dosing period. 

7. 	 Urinalysis (fABLE 7) 

In the examination 25 days after commencement of treatment, significantly increased urine 

protein was observed in the males of the 20 mglkg and increased urobilinogen was observed in 

the females of the 140 mglkg group; however, they were not considered related to the 

administration of the test substance since these changes were not observed in the 1000 mglkg 

group. 

8. 	 Organ weight (fABLE 8, 9) 

At the end of the dosing period, significantly increased weight ratio of the adrenals was 

observed in the males of the 20 mglkg group and increased weight ratio of the ovaries was 

observed in the females of the 1000 mglkg group. In these changes, increased relative adrenal 

weight of the males was not considered related to the administration of the test substance since 

this change was not observed in the 1000 mglkg group. 

At the end of the recovery period, significantly decreased weight ratio of the brain and 

increased weight ratio of the kidneys were observed in the males of the 1000 mg/kg group. In 

these changes, decreased relative brain weight was not considered related to the administration 

of the test substance since this change was observed at the end of the recovery period only. 

9. 	 Necropsy (TABLE 10) 

No changes attributable to the administration of the test substance were observed at the end of 

either the dosing or recovery periods. 

17 

00008'; 
4L436 

Incidental changes such as impression, yellow patch, and hepatodiaphragmatic nodule of the 

liver, white patch of the pituitary, and edema and red patch of the lungs were observed 

sporadically. 

10. 	 Histopathological examination (fABLE 11) 

Changes considered related to the administration of the tes; substance was observed in the 

kidneys of the males. 

At the end of the dosing period, increase in hyaline droplets in the proximal tubular epithelium 

was observed in five males of the 1000 mglkg group. 

At the end of the recovery period, increase in hyaline droplets in the proximal tubular 

epithelium was observed in one male of the 1000 mglkg group. Basophilic change of the 

tubular epithelium was enhanced in two males of the 1000 mglkg group. Hyaline cast was 

also observed in their tubular epithelium. These changes were not observed in the kidneys of 

the females. 

Although significantly increased weight ratio of the ovaries was observed in the females of the 

1000 mglkg group, no histopathological change was observed in the ovaries. 

The liver of both sexes of the middle and low dose groups at the end of the dosing period and 

of all animals at the end of the recovery period were additionally examined since hypertrophy 

of hepatocyte was suspected in the both sexes of the 1000 mglkg group at the end of the 

dosing period. After all examinations, it was concluded that hypettrophy of the hepatocyte in 

the liver did not occurr in any animals. 

In addition to the above changes, spontaneous changes frequently seen in rats were observed; 

however, there was no enhancement of these changes in the treatment groups. 

18 000088 
4L436 


Discussion 

YDC-1312 was administered orally to male and female SD rats for 28 days to investigate the 

changes on biological function and morphology. 

Changes considered related to the administration of the test substance were as follows: 

significantly increased serum albumin and increase in hyaline droplets in the proximal tubular 

epithelium in the kidneys in the males of the 1000 mglkg gwup and significantly increased 

weight ratio of the ovaries in the females of the I ()()() mglkg groups. At the end of the 

recovery period, changes of serum albumin and the ovaries were not observed. There were no 

histopathological changes in the ovaries. 

Increase in hyaline droplets in the proximal tubular epithelium in the kidneys was considered 

to be a similar change as alpha 2u globulin nephropathy [1, 2] because it was observed in 

males only and of its morphologic characteristics. 

This nephropathy was reported to be induced by a diverse group of hydrocarbons which was 

similar to the test substance and that only male rats developed this lesion [3]. The reversibility 

of this nephropathy was observed since this finding was reduced after the recovery period and 

basophilic change of the tubular epithelium, which was considered to be regeneration, was 

observed at the end of the recovery period. Significantly increased ratio of kidney weight of 

the males in the 1000 mg/kg group also considered to reflect the regeneration from the 

nephropathy. 

On the basis of the above, the non observable effect level of YDC-1312 was estimated to be 

140 mglkg in both sexes. 

19 

OOOOE'j9 
4L436 


References 

[I] 	 Alden, C.L. and Frith, C.H.: Urinary system, Inducible alpha 2u globulin nephropathy 


syndrome. In: Handbook of toxicologic pathology. W.M. Haschek and C.G. Rousseaux, 


eds., pp.293-294, Academic Press, San Diego, 1991 


[2] 	 Swenberg, J.A., et al.: The comparative pathobiology of a 2u-globulin nephropathy. Toxicol. 


Appl. Pharmacol., 97: 35-46, 1989. 


[3] 	 Alden, C.L.: A review of unique male rat hydrocarbon nephropathy. Toxicol. Pathol., 14, 


109-111, 1986. 


20 	 0000.:;0 

4L436 


Figure and tables 


OOOO:J.l 

BODY WEIGHT (G) 


500 


450 

400 

350 

300 

250 

200 

o MG/KG GROUP 

...... : 20 MG/KG GROUP 

•••• : 140 MG/KG GROUP 

150 	 ••_ •• : 1000 MG/KG GROUP 

100 


50 
 ADMINISTRATION -------------------------+ +-------- RECOVERY ------------+ 

o ~---------------------------------------------------------------------------

o 	 7 14 21 28 35 42 


o DAYS AFTER COMMENCEMENT OF TREATMENT


o 

o 	 FIG. 1 - M BODY WEIGHT CHANGES 

C)

! ~\ ."""

N 

IN RATS TREATED ORALLY WITH YDC-1312 FOR 28 DAYS. [MALE] 

- 1­
- - - -

500 

BODY WEIGHT (G) 

450 

)

400 

350 

300 

~.,..... .",.-.......... 250 

.".- o MG/KG GROUP 

...... : 20 MG/KG GROUP 

•••• : 140 MG/KG GROUP 

100 -_•• : 1000 MG/KG GROUP 

50 ADMINISTRATION RECOVERY 

0 

0 7 14 21 28 35 

o DAYS AFTER COMMENCEMENT OF TREATMENT 

o 

o FIG. 1 - F BODY WEIGHT CHANGES IN RATS TREATED ORALLY WITH YDC-1312 FOR 28 DAYS. [FEMALE] c.:,) 

(J:) 

W - 2­

200 

150 

42 
TABLE 1 - M - 1 STUDY NO. 4L436CEO 

INCIDENCE OF CLINICAL SIGNS 

TEST ARTICLES : YDC-1312 

SEX : MALE 

Dose Clinical Days After Commencement of Treatment 

Level Signs 

(mg/kg) N o 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 

20 6 No Abnormality 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 

N Number of Animals. 

o 

o 

o 

C) 

(;)

.. 

*'" 

3 - TABLE 1 - M - 1 

TABLE 1 - M - 2 STUDY NO. 4L436CEO 

INCIDENCE OF CLINICAL SIGNS 

TEST ARTICLES : YDC-1312 

SEX : MALE 

Dose Clinical Days After Commencement I Cessation of Treatment 

Level Signs 

(mg/kg) N 22 23 24 25 26 27 28/0 29/1 30/2 31/3 32/4 33/5 34/6 35/7 36/8 37/9 38/10 39/11 40/12 41/13 42/14 

o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/~ 6/6 6/6 6/6 6/6 

20 5 No Abnormality 6/5 6/6 6/6 6/6 6/6 6/6 6/6 

140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

N Number of Animals. 

o 

o 

o 

C)

r.:; 

(,ii 

4 - TABLE 1 - M - 2 
TABLE 1 - F - 1 STUDY NO. 4L436CEO 

INCIDENCE OF CLINICAL SIGNS 

TEST ARTICLES : YDC-1312 

SEX : FEMALE 

Dose Clinical Days After Commencement of Treatment 


level Signs 


(mg/kg) N o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

o 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 

20 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

140 6 No Abnormality 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

1000 12 No Abnormality 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 

N Number of Animals. 

o 

o 

o 

C

""""" \1:; 

0) 

5 - TABLE 1 - F - 1 
Dose 

level 

(mg/kg) N 

Clinical 

Signs 

o 12 No Abnormality 

20 6 No Abnormality 

140 6 No Abnormality 

1000 12 No Abnormality 

N Number of Animals. 

o 

o 

o 

C)

..... 

~ ....j 

TABLE 1 - F - 2 

INCIDENCE OF CLINICAL SIGNS 

TEST ARTICLES : YDC-1312 

SEX : FEMALE 

STUDY NO. 4L436CEO 

Days After Commencement I Cessation of Treatment 

22 23 24 25 26 27 28/0 29/1 30/2 31/3 32/4 33/5 34/6 35/7 36/8 

12/12 12/12 12/12 12/12 12/12 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

37/9 

6/6 

38/10 39/11 40/12 41/13 42/14 

6/6 6/6 6/6 6/6 6/6 

6/6 6/6 6/6 6/6 6/6 6/6 6/6 

6/6 6/6 6/6 6/6 6/6 6/6 6/6 

12/12 12/12 12/12 12/12 12/12 12/12 12/12 616 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 

6 - TABLE 1 - F - 2 
----------------------------------------------------------------------------------------------------------------------------------------

TABLE 2 - M- 1 

BODY WEIGHT - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

STUDY NO. 4L436CEO 

UNIT G 

DOSE LEVEL 

MG/KG o 7 14 

DAYS AFTER COMMENCEMENT 

21 281 0 351 7 

I CESSATION OF 

42114 

TREATMENT 

0 MEAN 184. 249. 311. 366. 405. 445. 479. 

S.D. 8.4 16.4 22.8 26.6 30.4 14.5 16.4 

N 12 12 12 12 12 6 6 

20 MEAN 185. 247. 311. 369. 411. 

S.D. 8.6 16.8 22.2 25.7 27.9 

N 6 6 6 6 6 

140 MEAN 184. 251. 317. 376. 417. 

S.D. 9.1 16.6 20.3 27.1 32.7 

N 6 6 6 6 6 

1000 MEAN 185. 249. 314. 371. 412. 445. 474. 

S.D. 7.2 11.5 15.4 21.2 25.8 21.0 24.0 

N 12 12 12 12 12 6 6 

o 

o 

o

C) 

(.t:; 

Q) 

7 - TABLE 2 -M- 1 

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 2 - F - 1 STUDY NO. 4L436CEO 

BODY WEIGHT - GROUP MEAN VALUES 

TEST ARTICLES: 

SEX: 

YDC-1312 

FEMALE 

UNIT G 

DOSE LEVEL 

MG/KG a 7 14 

DAYS AFTER COMMENCEMENT I 

21 261 0 351 7 

CESSATION OF TREATMENT 

42114 

0 MEAN 151. 184. 213. 240. 263. 276. 291. 

S.D. 7.5 10.2 14.2 21.4 20.6 13.7 17.5 

N 12 12 12 12 12 6 6 

20 MEAN 148. 180. 203. 235. 254. 

S.D. 9.2 9.4 16.2 16.4 21.0 


N 6 6 6 6 6 


140 MEAN 147. 174. 207. 234. 260. 

S.D. 6.1 10.1 13.9 16.7 20.3 

N 6 6 6 6 6 

1000 MEAN 152. 179. 207. 236. 255. 279. 291. 

S.D. 6.7 7.7 9.3 16.0 12.6 16.3 20.5 


N 12 12 12 12 12 6 6 


o 

o 

o 

C) 

(f) 

(.&) 

8 ­ TABLE 2 -F- 1 
----------------------------------------------------------------------------------------------------------------------------------------

TABLE 3 - M - 1 	 STUDY NO. 4L436CEO 

FOOD CONSUMPTION - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

UNIT G/ANIMAL/DAY 

DAYS AFTER COMMENCEMENT I CESSATION OF TREATMENT 

DOSE LEVEL 

MG/KG 7 14 21 25 351 7 38/10 


0 MEAN 22.6 26.1 27.9 28.5 28.1 28.3 

S.D. 1.07 1.39 1.27 1.61 1.15 0.72 

N 6 6 6 6 3 3 


20 MEAN 22.2 25.7 28.2 28.4 


S.D. 0.52 0.29 0.67 0.56 

N 3 3 3 3 


140 MEAN 22.9 26.5 28.5 28.7 


S.D. 1.54 1.89 2.39 1.86 

N 3 3 3 3 


1000 MEAN 22.6 26.2 28.2 28.5 27.4 26.1 


S.D. 	 1.03 1.20 1.94 2.21 1.50 0.98 


N 6 6 6 6 3 3 


c 

o 

o 

F 

~) 

C 

9 -	 TABLE 3 -M- 1 
----------------------------------------------------------------------------------------------------------------------------------------

TABLE 3 - F ­

FOOD CONSUMPTION - GROUP MEAN 

TEST ARTICLES: YDC-1312 

SEX: FEMALE 

1 

VALUES 

STUDY NO. 4L436CEO 

UNIT G/ANIMAL/DAY 

DOSE LEVEL 

MG/KG 7 14 21 

DAYS 

25 

AFTER COMMENCEMENT 

351 7 38/10 

I CESSATION OF TREATMENT 

) 

0 MEAN 17.0 18.7 20.0 20.7 20.1 20.2 

S.D. 0.91 1. 57 2.12 1.79 0.45 1.54 

N 6 6 6 6 3 3 

20 MEAN 16.8 18.1 20.0 20.0 

S.D. 0.31 0.74 0.32 0.23 

N 3 3 3 3 

140 MEAN 16.2 18.5 19.8 20.7 

S.D. 0.95 1.21 1.14 0.70 

.N 3 3 3 3 

1000 MEAN 16.8 18.3 19.5 20.0 21.8 20.3 

S.D. 0.77 1.13 1.02 1.22 1.50 3.00 

N 6 6 6 6 3 3 

o 

c::.> 

o 

!-~ 

o 

j-i;, 

10 - TARI 1= "Ii -1=- 1 

I 
----------------------------------------------------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 4 - FS - M- 1 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES : YDC-1312 


SEX: MALE 


ANIMALS KILLED ON SCHEDULE ( 4 \.lEEK) 


RBC H8 RETICULO-	 PLATELET 

DOSE LEVEL COUNT HT CONC. CYTE MCV MCH MCHC COUNT PT APTT 

MG/KG 	 COUNT 

(x 10},~U (X) (G/DU ( X. ) { (1M' ) (PG) (X) (x 10 Yp.U (SEC) (SEC) 

0 MEAN 742. 44.4 15.1 26. 59.8 20.3 33.9 102.3 12.4 17.5 

S.D. 20.2 1.17 0.35 3.5 1.61 0.50 0.22 9.24 0.41 1.02 

N 6 6 6 6 6 6 6 6 6 6 


20 MEAN 746. 44.6 15.1 30. 59.8 20.2 33.8 109.3 12.2 16.5 

S.D. 19.8 1.06 0.42 2.3 2.35 0.90 0.24 7.86 0.19 0.74 

N 6 6 6 6 6 6 6 6 6 6 


140 MEAN 742. 44.3 14.8 29. 59.7 20.0 33.5 101.3 12.0 17.2 

S.D. 	 25.4 0.46 0.23 2.7 1.96 0.44 0.47 6.73 0.26 0.49 


N 5 5 5 5 5 5 5 5 6 6 


1000 MEAN 730. 44.4 15.2 29. 60.S 20.8 34.2 101.4 12.0 16.7 

S.D. 	 35.4 1.58 0.50 5.0 1.63 0.45 0.29 5.56 0.21 1.39 


N 6 6 6 6 6 6 6 6 6 6 


o 

<:) 

o 

~ 

(;) 

N 

11-	 TABLE 4 -FS-M- 1 

----------------------------------------------------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 4 - FS - F - 1 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES : YDC-B12 

SEX : FEMALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

RBC HB RETICULO-	 PLATELET 

DOSE LEVEL COUNT HT CONC. CYTE MCV MCH MCHC COUNT PT APTT 

MG/KG 	 COUNT 

(x 10 rILL) (X) (G/DU (~ ) ( 11M' ) (PG) (X) (x 10 r~L) (SEC) (SEC) 

0 MEAN 705. 41.3 14.3 23. 58.7 20.3 34.6 94.4 13.0 15.5 

S.D. 23.5 1.60 0.43 3.8 1.90 0.63 0.45 6.58 0.46 1.47 

N 6 6 6 6 6 6 6 6 6 6 

20 MEAN 707. 41.9 14.4 24. 59.2 20.4 34.4 100.2 13.3 15.4 

S. O. 30.6 1.55 0.57 3.1 1.46 0.62 0.30 8.68 0.56 1.02 

N 6 6 6 6 6 6 6 6 6 6 

140 MEAN 710. 41.8 14.4 21. 58.9 20.3 34.4 88.3 13.6 14.8 

S.D. 28.1 2.06 0.73 2.6 1.69 0.70 0.25 20.43 0.54 1. 48 

N 6 6 6 6 6 6 6 6 5 5 

1000 MEAN 714. 43.0 14.8 23. 60.3 20.8 34.5 95.5 13.4 15.1 

S.D. 	 lS.7 1.41 0.48 2.6 1.90 0.64 0.38 9.56 0.42 0.67 

N 6 6 6 6 6 6 6 6 6 6 

o 

o 

o

1-;;, 

o 

(;..) 

12 -	 TABLE 4 -FS-F- 1 

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 4 - RS - M - 1 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 


RBe HB RETICULO- PLATELET 


DOSE LEVEL COUNT HT eONC. CYTE MCV MCH MCHC COUNT PT APTT 

MG/KG COUNT 

(x 10 i',uU (X) CG/DU (~) ( 144' ) (PG) (X) (X 10 rllu (SEC) (SEC) 

0 MEAN 756. 43.7 14.9 26. 57.8 19.7 34.1 98.7 13.1 16.7 

S.D. 32.0 1.61 0.58 3.2 0.72 0.23 0.32 9.74 0.19 2.02 

N 6 6 6 6 6 6 6 6 6 6 


1000 MEAN 768. 43.8 14.9 25. 57.1 19.5 34.1 99.8 13.1 16.9 


S.D. 19.3 1.09 0.42 2.2 2.04 0.73 0.22 6.05 0.16 1.30 

N 6 6 6 6 6 6 6 6 6 6 


o 

o 

o 

!-A 

o 

... M-< 

13- TABLE 4 -RS-M- 1 
TABLE 4 - RS - F - 1 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

RBC HB RETlCULO- PLATELET 

DOSE LEVEL COUNT HT CONC. CYrE MCV MCH MCHC COUNT PT APTT 

MG/KG COUNT 

(x 10,;s L> ex) (G/OL> ( %. ) (PM' ) (PG) (X) (X 10,;sL> (SEC) (SEC) 

0 MEAN 717. 41.1 14.2 27. 57.3 19.9 34.6 88.1 13.6 15.5 

S.D. 21.9 1.23 0.43 4.9 1.39 0.45 0.24 4.78 0.31 1.57 

N 6 6 6 6 6 6 6 6 6 6 


* 1000 MEAN 742. 42.1 14.6 24. 56.8 19.7 34.7 90.9 13.9 15.8 

S.D. 23.9 0.67 0.32 3.1 1. 78 0.55 0.39 6.32 0.17 0.87 

N 6 6 6 6 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05. 

o 

o 

o 

~ 

c' 

'-;~ 14 - TABLE 4 -RS-F- 1 
TABLE 5 - FS - M- 1 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

STUDY NO. 4L436CEO 

DOSE LEVEL 

MG/KG 

WBC 

COUNT 

(x 10 yp.L) 

LYMPHOCYTES 

DIFFERENTIAL COUNT OF 

NEUTROPHILS 

SEGMENTED BAND 

LEUKOCYTES 

EOSINOPHILS 

ex OF 

BASOPHILS 

TOTAL COUNTED 

MONOCYTES 

CELLS) 

0 MEAN 

S.D. 

N 

112. 

18.3 

6 

86. 

5.6 

6 

10. 

6.0 

6 

$ 

O. 

0.4 

6 

1­

1.3 

6 

O. 

$ 

0.0 

6 

3. 

2.2 

6 

20 MEAN 

S.D. 

N 

128. 

24.9 

6 

92. 

3.7 

6 

6. 

2.9 

6 

O. 

0.0 

6 

1. 

0.8 

6 

O. 

0.0 

6 

2. 

1.4 

6 

140 MEAN 

S.D. 

N 

119. 

20.7 

5 

94. 

2.4 

5 

4. 

2.8 

5 

O. 

0.4 

5 

1. 

0.8 

5 

O. 

0.0 

5 

1. 

1.0 

5 

1000 MEAN 

S.D. 

N 

131. 

26.4 

6 

91. 

4.4 

6 

6. 

2.7 

6 

O. 

0.4 

6 

1­

0.8 

6 

O. 

0.0 

6 

Z. 

2.1 

6 

$ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

1-4. 

o 

en 

15 - TABLE 5 -FS-M- 1 

TABLE 5 - FS - F ­ 1 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

STUDY NO. 4l436CEO 

DOSE LEVEL 

MG/KG 

\.JBC 

COUNT 

(x 10 y,.U 

LYMPHOCYTES 

DIFFERENTIAL COUNT 

NEUTROPHllS 

SEGMENTED BAND 

OF LEUKOCYTES 

EOSINOPHILS 

(X OF 

SASOPHILS 

TOTAL COUNTED 

MONOCYTES 

CELLS) 

0 MEAN 

S.D. 

N 

105. 

25.7 

6 

91. 

3.8 

6 

7. 

2.3 

6 

O. 

0.4 

6 

1. 

1.5 

6 

O. 

s 

0.0 

6 

2. 

2.0 

6 

20 MEAN 

S.D. 

N 

89. 

13.5 

6 

92. 

4.7 

6 

6. 

4.7 

6 

O. 

0.4 

6 

O. 

0.5 

6 

O. 

0.0 

6 

2. 

1.4 

6 

140 MEAN 

S.D. 

'N 

103. 

17.8 

6 

92. 

4.4 

6 

4. 

3.9 

6 

O. 

0.4 

6 

1. 

0.8 

6 

O. 

0.0 

6 

4. 

2.5 

6 

1000 MEAN 

S.D. 

N 

110. 

15.9 

6 

95. 

3.1 

6 

3. 

1.4 

6 

O. 

0.4 

6 

1. 

0.8 

6 

O. 

0.0 

6 

2. 

1.6 

6 

$ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

(:J 

o 

!w..,;. 

(..) 

~,j 

16 - TABLE 5 -FS-F- 1 

TABLE 5 - RS - M - 1 	 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

wac DIFFERENTIAL COUNT OF LEUKOCYTES (~OF TOTAL COUNTED CELLS)

DOSE LEVEL COUNT 

MG/KG 	 LYMPHO­ NEUTROPHILS EOSINO­ BASO­ MONO­

(x10Y/.Il) CYTES SEGMENTED BAND PHILS PHILS CYTES 

$ 


0 MEAN 81. 88. 8. 1. 1. O. 2. 


S.D. 23.6 6.0 4.5 0.5 1.0 0.0 1.9 

N 6 6 6 6 6 6 6 


1000 MEAN 94. 89. 9. 1. 1. O. 1. 


S.D. 	 18.3 4.2 4.9 0.5 0.8 0.0 0.8 


N 6 6 6 6 6 6 6 


$ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

1-& 

o 

()J 

17 -	 TABLE 5 -RS-M- 1 

TABLE 5 - RS - F - 1 STUDY NO. 4L436CEO 

HEMATOLOGY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

WBC DIFFERENTIAL COUNT OF LEUKOCYTES (X OF TOTAL COUNTED CELLS) DOSE LEVEL COUNT 

MG/KG LYMPHO­ NEUTROPHILS EOSINO­ BASO­ MONO­

(x 10 }II' U CYTES SEGMENTED BAND PHILS PHILS CYTES 

J S 


0 MEAN 77. 90. 7. 1. 1. O. 1. 


S.D. 16.9 2.8 3.6 0.8 1.0 0.0 0.8 

N 6 6 6 6 6 6 6 


1000 MEAN 86. 90. 8. O. 1. O. 2. 


S.D. 35.1 2.1 2.7 0.4 0.9 0.0 1.3 

N 6 6 6 6 6 6 6 


$ , STATISTICAL ANALYSIS IMPOSSIBLE. 


o 

o 

o 

I-~ 

o 

W 

18 ­ TABLE 5 -RS-F- 1 
TABLE 6 - FS - M- 1 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

STUDY NO. 4L436CEO 

DOSE LEVEL 

MG/KG 

GOT 

(lUlU 

GPT 

(lUlU 

r-GTP 

(lUlU 

ALP 

<lUlU 

UREA 

NITROGEN 

{MG/DU 

CREATININ 

(MG/DL) 

GLUCOSE 

(MG/DL> 

TOTAL 

CHOL. 

(MG/DL> 

TRIGLYCERIDE 

(MG/DU 

TOTAL 

oROTEIN 

{G/DU 

ALBUMIN 

(G/DU 

AlG 

RATIO 

o MEAN 

S.D. 

N 

100. 

21.2 

6 

35. 

6.2 

6 

s 

o. 

0.0 

6 

570. 

60.8 

6 

17.8 

2.34 

6 

0.4 

0.04 

6 

154. 

12.8 

6 

63. 

6.4 

6 

133. 

61. 7 

6 

6.60 

0.225 

6 

3.68 

0.109 

6 

1.27 

0.048 

6 

20 MEAN 

S.D. 

N 

90. 

11.9 

6 

31. 

1.8 

6 

O. 

0.0 

6 

545. 

64.8 

6 

15.5 

1.61 

6 

0.4 

0.05 

6 

156. 

10.6 

6 

70. 

7.1 

6 

172. 

35.1 

6 

6.70 

0.212 

6 

3.62 

0.111 

6 

1.18 ** 

0.027 

6 

140 MEAN 

S.D. 

N 

87. 

24.0 

6 

32. 

3.5 

6 

O. 

0.0 

6 

623. 

122.2 

6 

17.8 

1.53 

6 

0.4 

0.04 

6 

148. 

6.9 

6 

63. 

5.2 

6 

114. 

57.0 

6 

6.73 

0.209 

6 

3.77 

0.127 

6 

1.27 

0.043 

6 

1000 MEAN 

S.D. 

N 

n. 

17.0 

6 

34. 

5.2 

6 

O. 

0.0 

6 

642. 

100.3 

6 

16.9 

3.11 

6 

0.4 

0.05 

6 

150. 

12.4 

6 

63. 

14.3 

6 

153. 

35.3 

6 

6.84 

0.236 

6 

3.85 * 

0.074 

6 

1.29 

0.056 

6 

* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05 ; ** , P<O.Ol ; S , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

!-J:. It-, , 

o 

19 - TABLE 6 -FS-M- 1 

-----------------------------------------------------------------------------------------------------------------------------------------

TABLE 6 - FS - M- 2 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

INORGANIC 

DOSE LEVEL CALCIUM PHOS. NA K CL 

MG/KG 

(MG/DL) (MG/DL) (MEQ/L) (MEQ/L) (MEQ/L) 

0 MEAN 9.8 9.4 144. 4.5 100. 

S.D. 	 0.21 0.43 1.5 0.17 1.4 


N 6 6 6 6 6 


20 MEAN 9.9 9.2 143. 4.7 100. 

S.D. 0.20 0.41 0.8 0.08 1.2 

N 6 6 6 6 6 


140 '1EAN 10.1 9.3 144. 4.6 100. 

S.D. 	 0.31 0.31 1.0 0.15 1.4 


'N 6 6 6 6 6 


1000 MEAN 10.0 9.0 143. 4.6 100. 

S.D. 0.27 0.41 1.2 0.24 2.6 

N 6 6 6 6 6 


o 

o 

o 

l-'­

I---li. 

~ 

20 -	 TABLE 6 -FS-M- 2 

----------------------------------------------------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 6 - FS - F - 1 	 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES : YDC-1312 

SEX : FEMALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

UREA TOTAL TRI- TOTAL A/G

DOSE LEVEL GOT GPT T- GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO 

MG/KG 

CIU/U (LUlU (LUlL> (lUlL> (MG/DL> (MG/DL> (MG/DL> (MG/DL> (MG/DU (G/DU (G/DU 

S S 

0 MEAN 78. 27. O. 352. 18.8 0.5 150. 65. 83. 6.68 3.84 1.35 

S.D. 	 7.3 3.7 0.0 86.6 3.17 0.04 10.8 2.9 45.5 0.243 0.136 0.078 

N 6 6 6 6 6 6 6 6 6 6 6 6 

20 MEAN 86. 27. O. 364. 18.7 0.5 150. 69. 38. 6.62 3.79 1.34 

S.D. 	 13.6 7.8 0.4 100.4 2.99 0.06 9.7 7.5 11.2 0.121 0.096 0.049 

N 6 6 6 6 6 6 6 6 6 6 6 6 

140 MEAN 86. 27. O. 353. 17.1 0.5 150. 63. 105. 6.61 3.84 1.38 

S.D. 15.7 2.7 0.0 101.2 2.00 0.08 5.4 11.1 50.0 0.450 0.254 0.078 

N 6 6 6 6 6 6 6 6 6 6 6 6 

1000 MEAN 79. 29. O. 353. 18.0 0.5 141- 63. 38. 6.63 3.83 1.37 

S.D. 	 10.5 4.5 0.0 65.3 2.65 0.00 8.0 7.8 10.6 0.079 0.097 0.067 

N 6 6 6 6 6 6 6 6 6 6 6 6 

S , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

10-"::' 

t~!. 

N 

21 -	 TABLE 6 -FS-F- 1 

DOSE LEVEL 

MG/KG 

0 MEAN 

S.D. 

N 

20 MEAN 

S.D. 

N 

140 MEAN 

S.D. 

. 	N 

1000 MEAN 

S.D. 

N 

o 

o 

<:) 

l-Ji. 

r.', Vd 

CALCIUM 


CMG/DU 


9.8 

0.18 

6 


9.6 

0.14 

6 


9.9 

0.37 

6 


9.7 

0.43 


6 


INORGANIC 

PHOS. 

(MG/DU 

8.3 

0.57 

6 


8.2 

0.55 


6 


8.6 

0.87 

6 


8.1 

0.99 


6 


NA 

(MEG/U 

143. 

1.5 

6 


143. 

1.1 


6 


142. 

0.8 

6 


144. 

0.8 


6 


TABLE 6 - FS - F - . 2 	 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

K CL 

(MEG/U (MEG/L> 

4.2 102. 

0.20 2.1 

6 6 


4.0 103. 

0.29 	 0.8 


6 6 


4.2 101. 

0.17 	 1.7 


6 6 


4.1 104. 

0.21 	 1.4 


6 6 


22 -	 TABLE 6 FS-F- 2 

----------------------------------------------------------------------------------------------------------------------------------------

TABLE 6 - RS - M - 1 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

UREA TOTAL TRI- TOTAL AlG 

DOSE LEVEL GOT GPT T- GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO 

MGIKG 

<lUlU (lUlU ( lUlU (lUlU (MG/DU (MG/DU (MG/DU (MG/DU CMG/DL> (G/DL> CG/DL> 

$ 

0 MEAN 91. 27. O. 472. 20.3 0.5 162. 58. 178. 6.79 3.60 1.13 

S.D. 17.6 7.0 0.0 163.1 0.83 0.05 20.7 9.3 40.7 0.187 0.071 0.045 

N 6 6 6 6 6 6 6 6 6 6 6 6 


* 1000 MEAN 92. 29. O. 472. 19.1 0.5 148. 68. 178. 7.05 3.69 1.10 

S.D. 22.1 9.5 0.0 80.6 1.62 0.05 11.4 15.6 112.3 0.181 0.072 0.057 

N 6 6 6 6 6 6 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05 ; $ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

bJr. 

r··.-t. 

;;.:, 

23 - TABLE 6 -RS-M- 1 


I 
TABLE 6 - RS - M - Z STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

INORGANIC 

DOSE LEVEL CALCIUM PH OS • NA K CL 

MG/KG 

(MG/DU (MG/DU (MEG/L> CMEG/L) (MEG/L) 

0 MEAN 9.3 6.9 143. 4.5 102. 

S. D. 0.18 0.64 0.8 0.21 0.8 

N 6 6 6 6 6 

* 1000 MEAN 9.4 7.7 143. 4.6 101. 

S.D. 0.26 0.56 0.9 0.18 1.4 

N 6 6 6 6 6 

* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05. 

o 

o 

o

1-.;" 

t·..t 

24 - TABLE 6 -RS-M- 2 


C.ii 
----------------------------------------------------------------------------------------------------------------------------------------

TABLE 6 - RS - F - 1 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

rEST ARTICLES: YDC-1312 


SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

UREA 	 TOTAL TRI- TOTAL A/G 

DOSE LEVEL GOT GPT T- GTP ALP NITROGEN CREATININ GLUCOSE CHOL. GLYCERIDE PROTEIN ALBUMIN RATIO 

MG/KG 

(LUlL) (lUlU (lUlU (lUlU (MG/DU (MG/DU (MG/DU (MG/DU (MG/DU (G/DU (G/DL> 

$ 


0 MEAN 84. 21. O. 350. 23.5 0.5 150. 72. 56. 6.67 3.73 1. 27 


S.D. 	 11.7 3.0 0.0 78.9 5.79 0.05 9.5 5.8 15.4 0.208 0.111 0.045 


N 6 6 6 6 6 6 6 6 6 6 6 6 


* 1000 MEAN 87. 26. O. 315. 20.6 0.6 144. 75. 81. 6.99 3.92 1.28 

S.D. 	 12.4 5.8 0.4 83.2 3.05 0.04 7.0 8.4 25.6 0.349 0.156 0.049 


N 6 6 6 6 6 6 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05 ; $ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

~ 

t~~ 

(J'; 

25 -	 TABLE 6 -RS-F- 1 

TABLE 6 - RS - F - 2 STUDY NO. 4L436CEO 

CLINICAL CHEMISTRY - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

INORGANIC 

DOSE LEVEL CALCIUM PHOS. NA K CL 

MG/KG 

(MG/DU (MG/DU (MEG/U (MEGIll (MEGIll 

j 

0 MEAN 9.1 6.8 143. 4.0 103. 

S.D. 0.29 0.76 1.3 0.28 1.4 

N 6 6 6 6 6 


1000 MEAN 9.3 6.8 142. 4.0 101. 


S.D. 0.29 0.75 1.6 0.11 1.9 

N 6 6 6 6 6 

o 

o 

o 

l-.\ 

;'~.u6 

"' '-1 

26 - TABLE 6 -RS-F- 2 
-----------------

DOSE lEVEL 

MG/KG 

0 

20 

140 

1000 

o 

o 

o 

~ 

~_<a 

I ........
 ..", 

TABLE 7 - Cl - M - 1 

URINALYSIS - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

3 WEEKS AFTER COMMENCEMENT OF TREATMENT 

NUMBER PH PROTEIN GLUCOSE KETONES BILIRUBIN 

EXAMINED (MG/DU CG/DU CMG/DU 

7.5 8.0 8.5 9.0 - TR 30 100 ~300 - 0.1 0.25 0.5 ~1 - 5 15 40 ~80 - 1+ 2+ 3+ 

12 1 2 7 2 0 2 6 3 1 11 1 0 0 0 1 5 6 0 0 11 1 0 0 

* 6 0 1 5 0 0 0 0 6 0 6 0 0 0 0 0 1 4 1 0 6 0 0 0 

6 0 2 4 0 0 0 3 3 0 6 0 0 0 0 0 1 5 0 0 6 0 0 0 

12 0 3 5 4 0 0 4 8 0 12 0 0 0 0 0 6 6 0 0 12 0 0 0 

, NEGATIVE ; TR, TRACE ; 1+ , SLIGHT ; 2+, MODERATE ; 3+, SEVERE. * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<O.OS ; S , STATISTICAL ANALYSIS 

OCCULT BLOOD 

- TR 1+ 2+ 3+ 

12 0 0 0 0 

S 

6 0 0 0 0 

$ 

6 0 0 0 0 

S 

12 0 0 0 0 

IMPOSSIBLE. 

STUDY NO. 4L436CEO 


UROBILINOGEN 

CEU/DU 

0.1 1 2 4 8 

8 4 0 0 0 

2 4 0 0 0 

2 4 0 0 0 

6 6 0 0 0 

27 - TABLE 7 -C1-M- 1 

---------------------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------

TABLE 7 - Cl - F - 1 STUDY NO. 4L436CEO 

URINALYSIS - GROUP MEAN VALUES 

TEST ART! CLES : YDC-1312 

SEX : FEMALE 

3 WEEKS AFTER COMMENCEMENT OF TREATMENT 

NUMBER PH PROTEIN GLUCOSE KETONES BILIRUBIN OCCULT BLOOD UROBILINOGEN 

DOSE EXAMINED (MG/DL> (G/DL> (MG/DL> (EU/DU 

LEVEL ------------------------- ---------------- ----------------- -------------- ---------- ------------- -------------­ MG/KG 6.5 7.0 7.5 8.0 8.5 9.0 - TR 30 100 It 300 - 0.1 0.25 0.5 Itl - 5 15 40 1t80 - 1+ 2+ 3+ - TR 1+ 2+ 3+ 0.1 1 2 4 8 

0 12 0 0 0 1 6 5 1 7 4 0 0 12 0 0 0 0 7 5 0 0 0 12 0 0 0 12 0 0 0 0 8 4 0 0 0 

$ $ $ 

20 6 0 0 0 Z 2 2 0 5 0 1 0 6 0 0 0 0 1 5 0 0 0 6 0 0 0 6 0 0 0 0 2 4 0 0 0 

$ $ $ ** 1l.0 6 0 0 '1 0 4 1 0 1 5 0 0 6 0 0 0 0 1 5 0 0 0 6 0 0 0 6 0 0 0 0 0 6 0 0 0 

$ $ I 2 1000 12 .1 0 0 2 3 6 2 1 0 12 0 0 0 0 8 4 0 0 0 12 0 0 0 11 0 1 0 0 8 4 0 0 0 

, NEGATIVE ; TR, TRACE ; 1+ , SLIGHT ; 2+, MODERATE ; 3+, SEVERE. * SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , ** , P<O.Ol ; $ , STATISTICAL ANALYSIS IMPOSSIBLE. 

o 

o 

o 

lor';' 

p, 

<.D 

28 ­ TABLE 7 -Cl-F- 1 
DOSE LEVEL 

MG/KG 

0 MEAN 

S.D. 

N 

20 MEAN 

S.D. 

N 

140 MEAN 

S.D. 

N 

1000 MEAN 

S.D. 

N 

o 

o 

o 

}--1.t. 

t· ; 

o 

TABLE 8 - FS - M- 1 

ORGAN WEIGHT (ABSOLUTE) - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 

SEX: MALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

STUDY NO. 4L436CEO 

FINAL 

BODY 

WEIGHT 

(G) 

BRAIN 

(G) 

LIVER 

(G) 

KIDNEYS 

(G) 

ADRENALS 

(MG) 

TESTES 

(G) 

402. 

42.7 

6 

2.05 

0.101 

6 

15.78 

1.823 

6 

2.87 

0.320 

6 

59.3 

10.42 

6 

2.98 

0.253 

6 

410. 

27.8 

6 

2.04 

0.067 

6 

17.00 

1.185 

6 

2.83 

0.372 

6 

50.2 

2.90 

6 

3.10 

0.253 

6 

417. 

32.7 

6 

2.09 

0.057 

6 

16.68 

1.961 

6 

3.08 

0.279 

6 

55.9 

5.89 

6 

3.15 

0.312 

6 

414. 

33.7 

6 

2.08 

0.076 

6 

16.82 

2.173 

6 

2.85 

0.239 

6 

53.2 

5.48 

6 

3.11 

0.170 

6 

29 - TABLE 8 -FS-M- 1 
DOSE LEVEL 

MG/KG 

0 MEAN 

S.D. 

N 

20 MEAN 

S.D. 

N 

140 MEAN 

,S.D. 

N 

1000 MEAN 

S.D. 

N 

o 

o 

o 

p..;.. 

iJ 

~. 

TABLE 8 - FS - F ­ 1 STUDY NO. 4L436CEO 

ORGAN WEIGHT (ABSOLUTE) TEST ARTICLES: - GROUP MEAN 

YDC-1312 

VALUES 

SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

FINAL 

BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES 

WEIGHT 

(G) CG) (G) (G) (MG) (MG) 

266. 1. 94 10.14 1.87 76.2 107.2 

27.8 0.044 1.343 0.160 9.02 10.96 

6 6 6 6 6 6 

255. 1. 91 9.49 1.89 75.9 118.5 

21.4 0.129 1.184 0.151 10.20 18.14 

6 6 6 6 6 6 

260. 1.92 9.67 1.80 67.5 106.3 

19.9 0.065 0.916 0.088 6.00 7.31 

6 6 6 6 6 6 

25I. 1.97 9.10 1.84 73.4 120.2 

10.5 0.058 0.300 0.113 6.44 9.82 

6 6 6 6 6 6 

'1:1'\ _ TARI ~ R -1="-1=- 1 
TABLE 8 - RS - M - 1 STUDY NO. 4L436CEO 

ORGAN WEIGHT (ABSOLUTE) - GROUP MEAN VALUES 

TEST ARTICLES: YOC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

FINAL 

DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES 

MG/KG WEIGHT 

(G) (G) (G) (G) (MG) (G) 


0 MEAN 479. 2.16 19.38 2.97 56.3 3.32 


S.D. 16.4 0.071 1.781 0.173 2.51 0.172 

N 6 6 6 6 6 6 


1000 MEAN 474. 2.05 19.52 3.20 57.S 3.21 


S. D. 23.8 0.116 1.338 0.246 6.32 0.245 

N 6 6 6 6 6 6 


o 

o 

o 

l-" 

\', " t ',,' 

(\) 

31 - TABLE 8 -RS-M- 1 

...., 

:"~ 

TABLE 8 - RS - F - 1 STUDY NO. 4L436CEO 

ORGAN WEIGHT (ABSOLUTE) - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

FINAL 

DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES 

MG/KG WEIGHT 

(G) (G) (G) (G) (MG) (MG) 


0 MEAN 291. 1. 97 10.45 1.82 77.2 106.0 


S.D. 17.3 0.048 1.244 0.071 11.05 16.37 

N 6 6 6 6 6 6 


1000 MEAN 291. 1.95 10.79 1.85 79.1 103.8 


S.D. 20.6 0.029 1.232 0.165 11.88 12.06 

N 6 6 6 6 6 6 


o 

o 

o 

1-1c. 

{";J 

c:.J 

32 - TABLE 8 -RS-F- 1 

TABLE 9 - FS - M- 1 	 STUDY NO. 4L436CEO 

ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT) - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 


ANIMALS KILLED ON SCHEDULE ( 4 WEEK) 

FINAL 

DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES 

MG/KG WEIGHT 

(G) 	 (x 10 ") 

0 MEAN 402. 0.51 3.92 0.72 14.7 0.74 

S.D. 	 42.7 0.034 0.104 0.035 1.56 0.019 


N 6 6 6 6 6 6 


* 20 MEAN 410. 0.50 4.15 0.69 12.3 0.76 

S.D. 27.8 0.038 0.108 0.066 1.05 0.060 

N 6 6 6 6 6 6 


140 MEAN 417. 0.50 4.00 0.74 13.5 0.76 

S.D. 32.7 0.030 0.208 0.063 1. 78 0.089 

N 6 6 6 6 6 6 


1000 MEAN 414. 0.50 4.05 0.69 12.9 0.75 

S.D. 33.7 0.045 0.238 0.062 1.00 0.079 

N 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05. 

o 

o 

o 

~ \.-- \ 

~ 

33 -	 TABLE 9 -FS-M- 1 

IV 
----------------------------------------------------------------------------------------------------------------------------------------

TABLE 9 - FS - F - 1 	 STUDY NO. 4l436CEO 

ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT) - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: FE"ALE 


ANI"ALS KILLED ON SCHEDULE ( 4 WEEK) 


FINAL 


DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS OVARIES 

MG/KG WEIGHT 

(G) 	 (X 10 ") (X 10 ") 

J 

0 MEAN 266. 0.74 3.82 0.70 28.8 40.5 

S.D. 	 27.8 0.073 0.268 0.048 3.02 3.87 


N 6 6 6 6 6 6 


20 MEAN 255. 0.76 3.72 0.74 30.1 46.7 

S.D. 21.4 0.092 0.166 0.034 5.80 7.10 

N 6 6 6 6 6 6 


140 MEAN 260. 0.74 3.71 0.69 26.1 40.9 


S.D. 19.9 0.079 0.145 0.039 3.45 2.31 

'N 6 6 6 6 6 6 


* 1000 MEAN 251. 0.79 3.63 0.74 29.4 48.0 

S.D. 10.5 0.033 0.146 0.071 2.55 3.90 

N 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05. 

o 

o 

o 

~ 

~.,) 

Cil 

34 -	 TABLE 9 -FS-F- 1 

TABLE 9 - RS - M- 1 STUDY NO. 4L436CEO 

ORGAN WEIGHT (RELATIVE : PERCENTAGE OF BODY WEIGHT) - GROUP MEAN VALUES 

TEST ARTICLES: YDC-1312 


SEX: MALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

fINAL 

DOSE LEVEL BODY BRAIN LIVER KIDNEYS ADRENALS TESTES 

MG/KG WEIGHT 

(G) (x 10 ") 


0 MEAN 479. 0.45 4.04 0.62 11.8 0.69 


S.D. 16.4 0.008 0.294 0.029 0.56 0.033 

N 6 6 6 6 6 6 


* * 1000 MEAN 474. 0.43 4.12 0.68 12.3 0.68 

S.D. 23.8 0.016 0.247 0.052 1.56 0.041 

N 6 6 6 6 6 6 


* SIGNIFICANTLY DIFFERENT FROM CONTROL VALUE , * , P<0.05. 

o 

o 

o 

~ "',",

..... 

r:J) 

35 - TABLE 9 -RS-M- 1 

DOSE LEVEL 

MG/KG 

0 MEAN 

S.D. 

N 

1000 MEAN 

S. O. 

N 

o 

o 

o 

~ " , t ..... 

'j 

ORGAN WEIGHT 

TABLE 9 - RS - F - 1 

(RELATIVE : PERCENTAGE OF BODY WEIGHT) - GROUP MEAN 

TEST ARTICLES: YDC-1312 

SEX: FEMALE 

ANIMALS KILLED ON SCHEDULE (RECOVERY) 

VALUES 

STUDY NO. ~L436CEO 

FINAL 

BODY 

WEIGHT 

(G) 

BRAIN LIVER KIDNEYS ADRENALS 

ex 10 ") 

OVARIES 

ex 10 ") 

291. 

17.3 

6 

0.68 

0.038 

6 

3.59 

0.287 

6 

0.62 

0.042 

6 

26.7 

4.67 

6 

36.S 

6.11 

6 

291­

20.6 

6 

0.67 

0.047 

6 

3.71 

0.309 

6 

0.63 

0.029 

6 

27.3 

3.84 

6 

35.6 

1.89 

6 

36 - TABLE 9 -RS-F- 1 

TABLE 10 STUDY NO. 4L436CEO 

INCIDENCE OF NECROPSY FINDINGS 

TEST ARTICLES : YDC-1312 

Period 28 days Recovery 

Sex Male Female Male Female 

Organ

Findings 

Dose level(mg/kg) Number of animals 

o 

6 

20 140 1000 

666 

o 

6 

20 

6 

140 

6 

1000 

6 

o 

6 

1000 

6 

o 

6 

1000 

6 

Liver 

Hepatodiaphragmatic nodule 

Impression

Yellow patch 

0 

1 

1 

0 

0 

0 

1 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Lung

Edema 0 0 0 0 0 1 0 0 0 0 0 0 

Red patch 0 0 0 1 0 0 0 0 0 0 0 0 

Pituitary

White patch 0 1 0 0 0 0 0 0 0 0 0 0 

o 

o 

o 

~ 

,

~'. 

...., 

00 

- 37 - TABLE 10 

TABLE 11 - 1 STUDY NO. 4L436CEO 

INCIDENCE OF HISTOLOGICAL FINDINGS 

TEST ARTICLES : YDC-1312 

Period 28 days Recovery 

Sex Male Female Male Female 

Organ Dose level(mg/kg) o 20 140 1000 o 20 140 1000 o 1000 o 1000 

Findings Number of animals 6 666 6 666 6 6 6 6 •••••••••••• _ ••••••••.•••••••••••• ~ ••• ~ •• _.~ ••••• ~ •••••• _ ••• _~ .......... _.~ •• _~~._"._.~ •••• _ .... ~ ..... _.~ ..... " .... * ......................... ~ ...... ~_ ..... ~ ... _ .. _~ •• ~ •• __ w. __ ........ _ .......... ~ .. _~._* ..•. _._


Heart (Grade) Inflammatory cell infiltration, focal (+) 2 2 o 1 * * * * * * * * 

Liver 

Bile duct hyperplasia (+) o o 1 o o o o o o o o o 

Fibrosis, focal (+) 1 o 1 o o o o o o o o o 

Inflammatory cell infiltration, focal (+) 2 1 3 1 2 2 3 1 1 2 2 o 

Microgranuloma (+) 6 3 2 5 6 2 3 5 3 4 11 5 

Necrosis,·focal (+) o o 1 1 o o o o o o o o 

Vacuolation, hepatocyte, focal (+) o o o o o 1 o 1 o o o o 

Kidney

Basophilic change, tubular epithelium (+) 5 4 5 6 4 4 5 3 * * * * (++ ) o o o o o o o 2 * * * * Calcification, corticomedullary junction (+) o o 1 o o o o o * * * * Cast, hyaline (+) o o o o o * * o o 2 * * Cyst (+) o o o o o * * 1 o o * * Increase of hyaline droplet, proximal tubular epithelium (+) o o o 4 o o o 1 * * * * (++ ) o o o 1 o * * o o o * * Inflammatory cell infiltration, focal (+) 2 2 1 1 o 1 o 1 * * * * 

Adrenal 

o Vacuolation, cortical cell (+) 1 * * 1 o * * * o * * * 

o 

(~ 

Spleen NO * NO NO NO * * * * * * * ~ 

~\,j + Slight, ++ : Moderate. 

<.D * Not examined, NO : No abnormalities were detected. 

38 - TABLE 11 - 1 
TABLE 11 - 2 STUDY NO .. 4L436CEO 

INCIDENCE OF HISTOROGICAL FINDINGS 

TEST ARTICLES : YDC-1312 

Period 28 days Recovery 

Sex Male Female Male Female .. '" ... ~ ... ~ '" " ." .......... " ................ " .......... . ...................................................... ....................... . .................. 

Organ Dose level(mg/kg) 0 20 140 1000 a 20 140 1000 a 1000 0 1000 

Findings Number of animals 6 6 6 6 6 6 6 6 6 6 6 6 .. . ............ ".. . ... ........ ........... " .. " .. ~ ........................ , .................. " ...................................... " .......................... " ........................................ " ........ , .. .. .... .. ............ " .. -...... ~ ... .. .. .... .. ....... " ........... " ......... '" ..... " .. "


(Grade) 

Ovary NO * * NO * * 

Lung

Accumulation of foam cell (+) * * * l/U * O/U * * * * * * 

Edema, focal (+) * * * 011* * 1/U * * * * * * 

Pituitary

Cyst. anterior lobe (+) * l/U * * * * * * * * * * 

+ Slight. * : Not examined. # Number of animals with lesion / number of animals examined, NO No abnormalities were detected. 

o 

" o 

~ t' • V! 

o 

39 - . TABLE 11 - 2 
Ready Biodegradability Test of YDC-1812 

(English version) 

StUf1y No. 4FOO8(:3)G 

Study Title Ready Biodegradability Test ofYDC-l:312 

Sponsor Tohto Kasei CO., LTD 

This test was conducted in Kitakyusyu Laboratory, Mitsubishi-Kasei Institute of 

Toxicological and Environmental Sciences LTD, I-I Kurosakishiroishi. Yahatanishi-ku, 

Kitakyusyu-shi, Fukuoka 806, Japan. 

This report is the English version of the original, which was written in Japanese. The 

undersigned hereby declare that this version faithfully reflects the original to the best. of 

our knowledge. 

Translated by 

/.2 , 2... C' ,') I 

Takayoshi Hosono 

Manager for Technical Division 

Tokyo Head Office Tohto Kasei CO., LTD 

Approved by 

-"1 )\::1 

--~.J-'[_"1-..'__X-_-'__!:J_""_-,-lJr.-LI7J"'--___ Da te . 

Koyo Murakami 

General Manager for Technical Division 

Tokyo Head Office, 'Ibhto Kasei CO.. LTD 

0001:3.1 

COMPLIANCE WITH THE GLP STANDARDS 


St.udy No. 4 F008(:3)G 

St.udy Tit.le Ready Biodegradability Test.ofYDC·1812 

Sponsor Toht.o Kaflei CO., LTD 

The st.udy described in this report was conduct.ed in compliance with t.he Good 

L~lborat.ory Practice (GLP) regulations recognized by the Basic Indust.ries Bureau of t.he 

Japanose Minist.ry of Intklrnat.iona\ Trade and Indust.ry. 

Facility Management 

Tadashi Entlo Sealed dat.e : June 22, 1994 

Mitsubishi-Kasei Institute of Toxico\ogical and Environmental Sciences LTD(MITES) 

0001.,32 

QUALITY ASSURANCE STATEMENT 


Study No. 4F008(:3)G 

St.udy Tit.le Ready Biodegradabilit.y Test of YDC·l :312 

Sponsor Tohto Kasei CO.. LTD 

We hereby cert.ify t.hat. the above study has been performed in accordance with the 

Good Laboratory Practice (GLP) regulations recognized by t.he Basic Industries Bureau 

of the Japanese Minist.ry of International Trade and Industry. 

Dat.es of inspections and reporting~ to t.he Study Director and t.he Facility 

Management. are as follows: 

Jn·progress study 

Final report 

Date of 

Inspection 

May 13. 1994 

June 10. 1994 

June 22, 1994 

Date of 

Report.ing 

May 1:3. 1994 

June 10, 1994 

June 22. 1994 

Quality Assurance Personnel 

Takeshi Miura Sealed date: June, 22 1994 
STUDY PERSONNEL 

Study Personnel 

St.udy Supervisor: Deputy Facility Management Katsumi Inoue sealed 

St.udy Director: Shiro Furukawa sealed 

Yoshihiro Kusumoto sealed 

0001.34 

CONTENTS 

Page 

THE TEST PROGRESS 	 6 

SYNOPSffi 	 7 

] TITLE. PURPOSE 8 

1.1 TITLE OF STUDY 8 

1.2 AIM OF THE STUDY 8 

1.:3 TEST METHOD 8 

1.4 	 COMPLIANCE WITH GLP 8 

1.5 TEST PERIOD 8 

].6 STORAGE OF THE DOCUMENTS AND SAMPLE 8 

2 TEST SUBSTANCE 	 8 

2.1 	 CHEMICAL NAME, CHEMICAL STRUCTURAL. PHYSICAL 8 

AND CHEMICAL PROPERTIES 

2.2 SAMPLE TO BE SUPPLIED 9 

2.:3 IDENTIFICATION OF THE SAMPLE 9 

2.4 	 CONFIRMATION OF SAMPLE'S STABILITY 9 

3 TEST METHODS 10 

:3.1 TEST CONDITIONS FOR BIODEGRADABILITY MEASUREMENT 10 

3.2 	 BOD MEASUREMENT 11 

3.3 	 PRETREATMENT PROCEDURE FOR THE TEST SOLUTION 11 

AFTER ITS BOD MEASUREMENT 

3.4 pH MEASUREMENT 12 

:3.5 DOC MEASUREMENT 12 

:3.6 RESIDUAL YDC·1312 MEASUREMENT 12 

3.7 	 CALCULATION FOR BIODEGRADABILITY ]3 

4 RESULTS AND DISCUSSION 	 14 

4.1 	 ENVIRONMENTAL FACTORS WHICH HAVE AFFECTED 14 

TEST RESULTS RELIABILITY 

4.2 	 OBSERVATIONS OF TEST SOLUTIONS DURING 14 

BOD MEASURING TESTS 

4.:3 RESULTS OF pH MEASUREMENT 14 

4.1 	 BIODEGRADABILITY PERFORMANCE BY ACTIVATED SLUDGE 14 

4.5 	 BOD BASIS BIODEGRADABILITY ] 4 

4.6 	 DOC BASIS BIODEGRADABILITY 15 

4.7 	 RESIDUAL YDC·]:312 CONCENTRATION SASIS 15 

BIODEGRADABILITY 

Ij CONCLUSION 	 11) 

TABLES AND FIGURES 	 16·:32 

OOO:1as 

THE TEST PROGRESS 


The biodegradability t.est. of YDC-l:312 was at. first started as test. number of 

4FOO8G on April 12th. 1994 and measurement.s of BOD values start.ed on April 18t.h. 

1994. 

However. on day 7 of BOD measurement, April 25t.h. BOD basis decomposition of 

aniline as reference substance of t.he decomposition did not exceed 40 % and 

subsequently t.he decomposit.ion test was terminat.ed on the above same day. 

Furt.hermore, t.he second biodegradabilit.y t.est of YDC-1312 as t.est number of 

4FOO8(2)G was start.ed on April 26th, 1994 and measurements of BOD values started on 

April 27th, 1994. 

However. on day 7 of BOD measurement, May 4th, BOD basis decomposit.ion of 

aniline as reference substance of the decomposition was did not. exceed 40% and 

subsequently the decomposit.ion test was terminated on the above same day. 

The study with test number of 4FOO8(3)G for biodegradabilit.y test of YDC-1312 

was carried out by use of t.he same test met.hod as the test number of 4FOO8G and the 

same lot number of test sample as the study. 

Results concerning the identification of test substance YDC·1312, the calibrat.ion 

and recovery test of YDC-1312 described in this report were obtained from test number 

of 4FOO8G and included in this study report. 

(; 

SYNOPSIS 


Title of Study 

Ready biodegradability test ofYDC·l:31 2(Test No. : 4F008(:3)G) 

Test method 

The test.s were conducted in accordance wit.h t.he test. met.hod concern:ng Chemical 

Substances Cont.rol Law (: the not.ice defined by Japanese Law 117, 197:3). 

(Content.s of test.s) 

No.1 : Confirmative test of biodegradabilit.y by using a system of aniline. 

activated sludge and basal medium 

No.2 : Basic respirat.ion test by using a system of acHvated sludge and basal 

medium 

No.:3........ 5 : Biodegradabilit.y tests ·1,2,and3 ofYDC·1312 by using a syst.em YDC13] 2, activat.ed sludge and basal medium 

No.6 : Stabilit.y of YDC-1312 in water by using a system ofYDC·] 312 and water 

(Items to be measured) 

(a) Biochemical oxygen demand(BOD) (for 28days) 

(b) Dissolved organic carbon(DOC) (After 28day test) 

(c) Residual YDC·1312 content. by HPLC (After 28day test) 

[measured by high performance liquid chromatography] 

28day-Test results "'" 

Measured Results(A

Measurement 

fter 2&lay test) 

Biodegrad

1 

abilit.y 

2 

Test* 

3 

Stabilit.y 

in water 

Theoretical 

value 

BOD, mg 3.1 -0.9 -1.7 0.8 73.2 

DOC, mg/l 0.0 3.2 2.4 1.] 73.9 

YDC·1312, mg/l 102.1 102.2 103.5 100.:3 100.0 

*) Calibrated values to have compensate basic respiration basis value for each t.est 

Biodegradahility aft.er 28 day test. 

Degradabj)ity Biodegradability Test 

BOD basis value 4 -1 .:2 

DOC basis value No calculat.ion du(~ to its difficult solubility in waIN 

YDC·]:312 basis value -:2 -:2 

(~oncl usion 

. II has come to a conclusion that. YDC-l:312 waF> not. wadily biod<'gradahle because 

it.s 28 day BOD hasis <!egradahilily is 4% or less and r(>si(\ual YDC·]3]2 basis one 

is -2% or Ipss. 

7 
1. TITLE, PURPOSE 

1.1 Title of study 


Biodegradability te8t ofYDC-1812 


1.2 Aim of the study 

The study was performed t.o obtain data on biodegradability and stabilit.y in water 

required for the application ofYDC·1:312 as new chemical substance in accordance 

with t.he Chemical Substance Control Law. 

1.:3 Test method 


The t.e8tS were carried out in accordance with the test method concerning the 


Chemical Substance Cont.rol Law (Japanese Law] 17, 197:3). 


1.4 Compliance wit.h GLP 

The tests were conducted in compliance with Good Laboratory Practice (GLP) 

defined by t.he above mentioned Law. 

1.5 Test period 


May 13, to June 22, 1994 


(Where BOD measurements were made from May 13, to June 10, 1994) 


1.6 Storage of the documents and samples 

The documents and sample which will be archived at the Kitakyushu Laboratory 

of Mitsubishi chemical Safety Institute Ltd. For ten years are described in the 

following: 

1) Protocol. 


2) Test report. 


:3) Raw data. 


4) Quality assurance reports. 


5) Sam])le (about 2g) 


6) Miscellaneous 


2. Test substance 

H 

0001.38 
~\a~~ ~C\~~ , 

000:139 

8. Test Met.hods 

The test.s were carried out. in accordance with t.he test. method as requ,jred hy the 

Chemical Subst.ance Cont.rol Law (Japanese Law 117. 197:3). 

The test. subst.ance was exposed to the act.ivated sludge in a closed-system oxygen 

consumpt.ion measuring apparatus. The biochemical oxygen demand (BOD) was 

measured over a 28 day period. After this period, the concen:.rations of the dissolved 

organic carbon (DOC) and the residual test subst.ance in t.he test. bottles were measured. 

The biodegradability of t.he test subst.ance was evaluated from t.hese result.s. 

8.1 Test conditions for biodegradabilit.y measurement. 

Biodegradability tests were conduct.ed in the following conditions. 

(Sbmtiard Act.ivat.ed Sludge) 

l)Mixed liquor suspended solid (MLSS) : 5700mg/l 

2)Source oCthe sludge Chemical Biotesting Center. Chemical Inspect.ion and 

Testing Institut.e. Ja})an 

:3) Date of receipt. May 10, 1994 

(rest condit.ion) 

1) Temperature 25±1 °C 

2) Exposure period 28 days 

8) Test volume 300ml 

4) Concentrations YDC-1312 (test substance) 100mgIl 

Aniline*1 100m gil 

Activated sludge 30mgIl 

*1 : Aniline: Reference substance for the biodegradabilit.y for YDC-1312. 

KANTO CHEMICAL Co., INC. Special grade. Lot No. 503E1288) 

(Contents of test.s and chemicals) 

Test No.1 : Confirmative test for biodegradability in the system of aniline. 

act.ivated sludge and hasal medium. 

Procedure for No.1 

(1) Take baf.:al medium*2 into a culturing bottIe. 

(2) Add weighed 29,u1(30 mg) aniline to the medium and mix 

t.hem. 

(8) Add standard activated sludge to t.he mixture. 

TtH;l No.2 : Basic I'ef.:piration test in t.he system of act.ivated sludge and basal 

medium. 

Procedure for No.2 

(1) 1'Hke basal medium*2 into a culturing bottle. 

(2) Ad(l st.andard aetivat.ed sludge to the mixt.ure. 

Test No.:)- No.5: Biodpgradabilily test. for YDC-l:312 in tlw system of YDC-1 :312. 

activat.ed sludge and basal medium. 

Pl'oeedure for No.a 10 No.5 

(J) Take hasal mp(lium*2 into a culturing boull' , 

(2) Ad(l w('iglwd:30 mg YDC-l:312to Ill!' hasal mC'dium and 

10 

0001-::0 
mix t.hem. 


(:3) Add s1.amhml activated sludge fo the mixture. 


Test No.6 : Stability ofYDC·l:312 in water by using a system ofYDC-l:312 


ami water 

Procedure for NO.6 

:30mg ofYDC·l:312 was aclded to 300 ml of double-disfillecl 

water (purified by Milli.Q. >10 Mohm·cm). 

*2 Quantjty of basal medium is adjust.ed to be the one of which is equal fo 300ml 


volume of act.ivated sludge to be added. 


3.2 BOD measurement. 

BOD was measured by use of the following equipment and also observations were 


made for t.he content inside t.he culturing bottle during t.he measurement. 


1) BOD measurement unit: closed type of oxygen consuming measuring unit 


Ohkura Electric Co.. Model OM·2001 (MITES 10 No.B) 

3.3 Pretreatment. procedure for the t.est solution after its BOD measurement 

Procedures for the pret.reatment were made in order to measure pH. DOC, 


residual YDC·l:312 in the test. solution in accordance with the following scheme. 


300ml t.est. solutjon 

Sample for DOC measurement 


Procedure for DOC measurement. 


(1) Take 10m) the test solution into lOml centrifuge tube by use of lOmlllipet. 

and centrifuge it for lOminutes. 

(2) Take 5ml centrifuged solution into Saml»)e bottle and inject it into DOC 

meter. 

Sample for pH measurement 


Procedure for DOC measurement 


(1) Take 20m) test solut.ion to a suit.able size of beaker a nel measure it wit.h pii 

me1.er. 

(2) Transfer t.he solution to t.he funnel forYDC·]:312 measurement. 

Sample for residual YDC-l:312 measurement. 


Procedure for residual YDC-1312 measurement. 


(1) 	Mix the above centrifuged test solution and activafed sludge and the 

residual test. solution before it.s centrifuge. 

(2) 	Transfer the mixture into 500ml liquid separation f'unnpJ and ext.ract it 

wit.h IOOml chloroform for 5 minut{'s shaking twice. 

(:3) 	Filtrate the chluruform lay<'r lind dewau>r it through a 11G:3type giasH filter 

muunted wit.h 40g of sodium f>ulfate anhydrous. 

(4) Transfer the filtrate 10 a rotary evaporalor and concentrate to dryness at 

b'mIWrnluf(l lowol' than 4ctC using wal.er hath. 


(fi) Dissolve t1w re!-;idue with ac(>lonitril<' to 25 In\. 


11 

0001. :1 
(5) Take acetonitrile solution t.o 2ml samilling bottle and inject it to HPLC t.o 

measure YDC·1812. 

:3.4 pH measurement 

pH value was measured t.o confirm pH change during BOD measurement: 

Apparat.us : pH met.er . Table type pH meter (fOA Denpa Co.. Model 

HM·:30S) 

:3.5 DOC measurement 

The measuring unit. for DOC is described t.ogether with it'> operat.ing condibons 

in t.he following. 

(I)Apparatu8 Tot.al Organic Carbon analyzer. Shimazu Corp.(Japan) 

Model TOC-5000 

(2)Measuring Furnace temperat.ure 680"(; (fC). 

Conditions Air flow rate 150mllmin. 

Measurement. sensitivity level xa 

Injection quantity lOttl 

(:3) Calibrat.ion curve 

The following st.andard solutions were injected int.o the TOC analyzer. 

The calibrat.ion curve was prepared by t.he data processor of the analyzer. 

St.andard solutions 

TC(t.otal carbon) 	 20 and 50 mg CII aqueous solutions of potassium 

biphthalate. 

IC(inorganic carbon) 	 omg CII deionized water (purified by Milli-Q) and 10 

mg cn aqueous solution of sodium hydrogen 

carbonate and sodium carbonate. 

(4) Measurement. 

Pretreated test solution in accordance with § a.:3 was used for DOC 

measurement. 

8.6 Residual YDC·1312 measurE'ment 

Residual YDC·131 2 after its biodegradability test was determined by a HPLC 

system which is de-scribed in t.he following: 

(1) Apparat.us 

HPLC 

Pump Yokogawa Electric Co., Model HP79851A 

Ultra viol(>t det.ect.or Yokogawa Electric Co., Model HP79853C 

Automatic sampler System I nst.rument Co.. Model 2:3 

LC work !-:ilalion Yokogawa Electric Co., Model WSI050 

(2) M(>asuring ('onditions 

Column YMC Co.. YMC·Pack ODS·A A·302, 

4.6mmLd. x 150mm 

Eluent Acelonitrile/double·distilled wa1.er = 80/20 

Flow nth~ Iml/min. 

D('h>clion wav('I(>ngth 280nm 

Allpnualioll (deh>el.or) 2 AU/FS 

I~ 

0001/12 
Inject.ion quantity 20.£11 

(:3) Calibrat.ion curve 

The st.andard solutions of t.he test substance wit.h O. 500. 1000. and 2000 mg/l 

in Acetonit.rile were prepared and analyzed. The peak area of t.he YDC·1812 was 

plotted against t.he concentration. The correlation coefficient. was calculated to be 

1.0 by the method described in Japanese Indust.rial Standards Z9041-1968. 

Concentration of the YDC·1812 for each Test No. was calculated from ratio of 

peak area for t.he sample t.o t.hat. for the standard solution wit.h 1000.0 mg/l in 

Acet.onitrile. 

[Figure :3 (P ·21) and Figure 4 (P·22-2:3)] 

(4) Recovory t.est 

Each test bottle (described in § 3.1) were kept in a closed-syst.em oxygen 

consumptjon measuring apparat.us for 30 minut.es. 

Each test. solut.ion was treated by the procedures deRCribed in § a.:3. The 

concent.ratjon of t.h(~ YDC·l:312 was measured by HPLC. The average recovery of 

the YDC·1312 was 96%. 

The concent.ratjon of the residual YDC·l:312 were corrected with the average 

recovery value. 

[Table :3 (P ·18) and Figure 5 (p·24-25)] 

(I) Det.ectjon limit 

The detection limit of t.he test substance was calculated to be 1 mgll based on 

t.he minimum detectable peak area of 10 mAU . sec for the peak in the HPLC 

chromatogram. 

[Table 3 (P-18) and Figure 5 (P·24-25)] 

(6) Measurements 

Pretreated test solution in accordance with § :3.3 was used for residual 

YDC·l:312 measurement.. 

:3.7 Calculation for biodegradability 

Three different kinds of calculation formulas are used for the biodegradability in 

the following: 

1) BOD basis biodegradability (for 7.14,21, and 28 <lays t.est.s) 

Biodegradability(%) = (BODs - BOOb) I ThoD x 100 

where BODs Oxygen commmp1.ion (mg) for t.he confirmat.iv(> test or for 

YDC·l:312 biodegradahility. 

BOOb Oxygen commmpt.ion (mg) for the basic re8pirat.ion of 

act.ivated sludge. 

ThaD TheorC'lical oxygen opmlmd (mg) for aniline or YDC·1312. 

Calculat.ion for 1.heoret.ical oxygl>n demand(ThoD) 

Aniline : nO.2 mg O~I :30 mg·aniline 

Aniline iR at;sumed In 1)(' oxidized in acconlanc(> wilh tilt-! following 

0001/13 
chemical react.ion. 


C.,H7N + 85/402 ~ 6COz + 7/211. 20 + N02 


YDC-1812 : 7:3.2 mg O2 /80 mg-YDC-1312 

YDC-l :312 is assumed to be oxidized in accordance with the following 

C2oH3/J04 + 511202 ~ 20CO;z + 15H20 

2) DOC basis biodegradation for 28 days test. 

Biodegradabilit.y(%) = [1 - (DOCs - DOCb) 1DOCc] x 100 


DOCs DOC (mg/l) for YDC-1:312 


DOCl> DOC (mgll) for 1J1e basic respiration 


DOCc DOC (mg/l) for t.he stability t.est: in water 


:3) YDC-1812 basis biodegradability for 28 days t.est 

Biodegradability(%) =(1 - Cs 1Cc ) x 100 


Cs YDC-1312 concentrat.ion (mgll) aft.er its biodegradability test 


Cc : YDC·1312 concentrat.ion (mgll) after its stabilit.y test in water 


4 	 Results and Discussion 

4.1 	 Environmental factors which have affected test results reliability 

Any environment.al phenomena which have affected test. results qualit.y were not 


observed. 


4.2 	 Observations of test solut.ions during BOD measuring tests 


1) Color of test solution was pale yeJJow for No.1 confirmat.ive test. in t.he 


system of aniline, activated sludge and basal medium. 


2) The test solution was colorless and transparent for No.2 test for basic 

respiration by activated sludge, No.3, 4, 5 t.ests for YDC-1812 biodegradability 

and No.6 test for the stabilit.y t.('.st ofYDC-1312 in the water. 

:3) 	 Growth of the sludge was observed in confirmatjve test bott.le compared 10 

t.he basic respiration system(No.2). No growth of t.he sludge was observed in 

YDC- 1 312 degradability systems, No.3,4,5. 

4.3 	 Result.s of pH measurement 


The pH values were 6.4, G.8. and 7.1 for No.3. No.4. and No.5 tests. The 


pH values was 7.2 after the st.ability test in the water. 

ITable 1 (P-16) 1 

4.4 	 Biodegradability )lPrformance by activated l;lu<lge 

BOD basif.; biodegradability was 60% based on 7 days confirmative 1£':;;1. 

u!-;ing anilinp. The result indicated I.lulI l.Iw f.;ludge hl-ld good peri'orml-lnc(' f()r 

aniline de!{radability. 

ITHbl<> 1 (P·16) l-lnd Figun' G (P·2G--2D)] 

4.5 	 BOD baHi!> biodegradability 

(1) 	 Tlworptical vahH' (lfBOJ) tl8 <h!y!') for YDC·1312 is n.2 mg. 

(2) 	 Act.ual non valu<~ ohtainpd frum tlw h'st.s fur YDC·l:312 were :~.I, 

11 

0001.'14 
5 

·0,9, and -1.7 mg after calibration using sludge basic respiration value, 

(:3) BOD value for the stability test using YDC-1312 was 0,8 mg, 

(4) BOD basis biodegradabiliti(ls were calculated to be 4 ..1. and -2 % by 

use of the above valu(l, 

[Table 1 (P ·16) and Figure 6 (P·26-29)] 

4,6 DOC basis biodegradability 

(1) DOC of sample to be used was 78,9 mg/I. 

(2) 	 On the other hand, DOC for biodegradable tests for YDC·l:312 was 0,0, 

:3,2, and 2.4 mg/l after t.he same calibration as the above one, 

(3) 	 DOC for the stability t.est ofYDC-1312 in water was measured to be 1.1 

mglJ, (28 days test) 

(4) However, DOC basis biodegradability was not calculaled because 

YDC-1312 was almost insoluble in water, 

[Table I (P·16) and Table 2 (P-I7)] 

4,7 Residual YDC·1312 concentration basis biodegradability 

(1) YDC·1312 concentratjon to be used for the test was 100 mg/1. 

(2) 	 Remaining YDC·l:3l2 concentration after the tests were 102.1, 102,2, 

and 103.5 mgll, 

(3) 	 Remaining YDC-] 312 after the stability test was measured to be 100,:3 

mgll. 

(4) 	 YDC-13l2 basis degradability was calculated to be -2, ·2, and -3 %. 

" [Table 1 (P·16), Table 4 (p·18) and Figure 7 (P·30-32)} 

Conclusion 

It has come t.o a conclusion that YDC·1312 was not readily biodegradable because 

its 28 day BOD basis degradabilit.y is 4% or less and residual YDC·1312 basis one is 

-2% or less. 

I;' 

0001/15 
----

----

Table 1 Test results 


Test No. Test systems Test samples ThOD BOD basis dee-adability 

added. mg 7 days 14 days 

mg BOD Degrad- BOD Degradmg ability " mg ability " 

No.1 Confirmative test aniline 90.2 55.6 60 67.9 68 

of biodegradabilitv 30.0 

No.2 Basic respiration ---­ -­ 1.9 -­ 6.5 -­ test of activated sludf:es 

No.3 Biodegradability YDC-1312 73.2 1.9 0 6.9 1 

tests-l of YDC-1312 30.0 

No.4 Biodegradability YDC-1312 73.2 0.5 -2 5.0 -2 

tests-2 of YDC-1312 30.0 

No.5 Biodegradability YDC-1312 73.2 1.9 a 6.0 -1 

test8-3 of YDC-1312 30.0 

No.6 Stability of YDC-1312 73.2 0.0 -­ 0.2 -­ _ 21>Q=-1312 _____ 30.0 -

21 days 28 days 

BOD 

ma­ . 

70.0 

7.3 

8.4 

5.4 

6.2 

0,3 

--

Degrad- BOD Degradability " mg abilitv " 

69 73.7 71 

-­ 10.0 -­

2 13.1 4 

-3 9.1 -1 

-2 8.3 -2 

-­ 0.8 -­

-

pH I 

Start Rnish 

-­ 6.9 

7.0 7.1 

-­ 6.4 

-­ 6.8 

-­ 7.1 

6.2 7.2 

Test sample: aniline for No.1, YDC-1312 for No.3-No.6 


Where % degradability was calculated to be negative. this value is shown in brackets. 


Test No. 

No.2 

No.3 

No.4 

No.5 

No.6 

Test systems 

Basic respiration 

test of activated sludges 

Biodegradability 

tests-1 of YDC-1312 

Biodegradability 

tests-2 of YDC-1312 

Biodegradability 

tests-3 of YDC-1312 

Stability of 

YDC-1312 

Origanic carbon 

added mg/l 

73.2 

73.2 

73.2 

73.2 

DOC basis degradability 

TC DOC' 

mg/l 

IC 

mg/l mg/I 

1.1 0.0 1.1 

1.1 0.0 1.1 

4.3 0.0 4.3 

3.5 0.0 3.5 

1.1 0.0 1.1 

1 

Degradability " 1 

. 

No calculation 

, 

No calculation j 

I 

No calculation 

. 

Residual YDC-1312 basis degradabilitv I 

concentration degradability I 

mf:/1 % 

<1 

102.1 -2

102.2 -2

103.5 -3

100.3 

'-­ -_._....... _--­

o DOC basis biodegradability was not calculated due to the insolubility of YDC-1312 in water. Where % degradability was calculated to be negative,

o this value is showt. in brackets.

o 

~ 

~ 

a·~ 
----------------

----------------

----------------

----------------

Table 2 DOC measurements (28 days) 


standard solution No.2 test system No.4 test system No.6 test system 

TC 50mg/l TC TC TC 


STD-HI 50.0 St1PL01 sr1PL03 SMPU35 


TC 01 010016 TC 01 000000 1.1 TC 01 000741 4.7 TC 01000000 1.1 

TC e2 010249 TC 02 e00ea0 1.1 TC 02 000&14 4.1 TC 02 000000 1.1 

TC 03 009861 TC 03 000000 1.1 TC 03 e00611 4.1 TC 03 000008 1.1 

i1 Ehi-I 0 -H3 eJ 2 MEAN 080088 1.1 MEAN 000&55 4.3 tlEAN e0008e 'J • -I 

T C ZOn/) I C I C I C 


;:TCI-LO 20.e 
 ::: r-1 F'L0 -1 SHPL03 Sr-tPL05 

TC 01 0039'14 IC 01 0aB00a 0.8 IC 01 000000 0.0 IC 01 90000e 0.0 ;c ~:2 012:!03 IC 02 000000 0.8 IC 02 000000 0.0 IC 02 000000 0.8 rc 03 (H33945 IC 03 000000 0.0 IC 03 0000000.0 IC 03 000000 8.8 TC+i.34 003770 

_t:j", No use due- to 


....J '.'" . abnorma 1 value. MEAN ee0000 0.0 MEAN 00000e 0.0 MEAN 000880 a.e 


i'1EM1 00387E. 

I C lOmg/l No.3 test system NO.5 test system Final results (mg/l) 

ST(I-HI -1 €I • '3 TC TC SMPL01 iC '1. ")' No.2 Tes~ systel!l . o baslc resplratlon SHPL02 SNPL04 IC by sludge IC 81 OB44JB TOC 1 • 1 

IC 02 084188 TC 0 -1 000000 1.1 TC e1 000467 3.4 SMPLC12 TC L 1'; No.3 Test system TC 02 000000 1. '1 TC 02 000530 3.7 e YDC-1312 degradability IC BJ 00415B IC 

TC 03 0eaaee 1 • -1 TC 03 000506 3.6 TOC -1. 1 

~tEAN 0134256 SMPL03 TC 4.3; No.4 Test system MEAN 000000 1. 1 MEAN 000501 3.5 IC o YDC-1312 degradability

I C Omg/ I I C I C TOC 4.3

SMPL04 SMPL..04 TC 3.S\- No.5 Test system STO-LO e.0 SMPL02 Ie 3.~~ YDC-1312 degradability 

IC 01 00ee0e a.e TOC IC 01 000000 0.0 IC 01 00Beee IS 92 ~B99"e BieT sr1PL05 TC No.6 Test system o Ie 82 (H3eeee IC 02 000000 0.0 . o'tJSe ue to L-t, Stability of YDC-1312 -02 abnormal value • IC 8 o Ie 03 000000 0.0 Ie 03 (I(1eB00 in water

o Ie 92 000000 0.0 TOC "'-1 ---------------­ .~ IC 03.000000 0.0 MEAN 088880 0.8 ttErHl 013130£10 ~ 

..,1 MEAN 800000 0.0 
Table 3 Calculation -of recovery and detection limit 


Test systems Peak area 

mAU·sec 

Concentration in solution 

m til 

Recovered 

mass 

mg 

(D) 

Added 

mass 

mg 

(E) 

Recoverry 

% 

(F) 

Detection 

limit 

mgll 

(A) (G)

for HPLC 

analysis 

(8) 

in culturing 

bottle 

(0) 

Recovery test 1 10063.03 1172.5 97.7 29.3 30.0 98 --I 

Recovery test 2 9816.39 1143.8 95.3 28.6 30.0 95 - Blank <10.00 <1.2 <0.1 -­ 0.0 -­ 1 

·Average recovery; 100% 

Standard solution Concentration (H) : l000mgll 

Peak area (I) : 8582.61 mAU •sec 

Volume of solution for HPLC analysis (J) : 0.0251 

Volume of test solution for HPLC analysis (K) : 0.300 I 

Equation :B=HxA+I C=HXA+tXJ+K O=B x J F= 0 + EX 100 G=C raise decimal fractions unit. 

Table 4 Residual YDC-1312 

::x: 

Test No. Test systems Peak ar.ea Concentration in solution 

mAU·sec mu'l 

for HPLC in culturing 

analysis bottle 

(A) 18) (C) 

No.2 Basic respiration test of activated sludges <10 <1 <1 

No.3 Biodegradability tests-l of YDC-1312 11486.75 1156.9 102.1 

No.4 Biodegradability tests-2 of YDO-1312 11500.23 1158.3 102.2 

No.5 Biodegradability tests-3 of YDC-1312 11643.54 1172.7 103.5 

No.6 Stability of YDC-1312 11280.56 1136.1 I­ 100.3 

Standard solution Concentration (D) : 1000mg/l 

Peak area (E) : 

Volume of solution for HPLO analysis (F) : 

Volume of test solution for HPLC analysis (G) : 

Average recovery (H) : 

c Equation : B=A x D+E C=AxO+Ex F+G+H x100 

o 

<::> 

~ 

..;.::. 

CX; 

9928.84mAU· sec 

0.0251 

0.295 I 

96% 
0001/19 


"2.1 

.----­

0001~2 
.---­
0001.:;4: 

,"! r r- o{) 0 :.t..)::> 
Figure 6 BOD chart 

.,.. ;::j .,,! .. ...., ..... ~ .;:; tV CD ~ w ~ ... ~ 

l_t:~:I..... 75 ""'7f"; .. !!.. ....., 1-:'f::1~ ·'::CC::. '. Measurement of BOD i . !...' ';. : ., i i . I ~i-. '-i·:-~~.:" Test substance YDC-1312 r :' . ~.; -.. ~ ,. ":·~·;··T·;':';·· ~!.': .. 1 . .~~--!---i-- ,_. .. .1 ;. :•. ~ .• : . -~ --!-..... ,.- ; ....: ..... -i . ... L .. . l 1 c. ' J __L-.!...._...;. Study number 4F008(3)G . ,-. '.' .. :. r"'" ·I-t-,,· ;'-1' ... 1 _. i-r··.. : : ... L _I I '.'~'''''. . ~ .~. it:: '~~ I j' ~~~e. r-""r··;--~ -1 .."!. it" .... :. ,.,I.. • I. , t .1 •• I , "":"60 : - ; •. , . ,-... ,.. ,_.,- _., _.. _.• ' :..._,. 1--, .... _.-t .. ! .... 

I' 

' .. • • J-f-±: ""'\'-'. Period 5/13/1994 to 6/10/1994 -l'..;.. J:i:.': .".. :~~:-i:-L_~=,=:".·r-.:·j-·-t.... -+ :·l·l···-f",+·~-~·:·· +-1-1- =;:::'-j . T t !- j I I I . t J ... r- ....... ....., r ·.. ·i~.. ·. -. -,-....-.. .. ... . i ~ ~ :: -I . 

~H- i'-,'" empera ure 25·C :.._~i··I-:-:I:· .;.r.:.....1:'.-1 t·..· -!--',-'''r-i-''r-: ·t··;·; ... -t. ;_ ...; ... . '17 afit /1.) .,. ­

..t=. -'---j~':- Apparatus Ohkura Electric Co .. t 1·!-t· ;.~ ...+ T.. ·: ..· ..-1- ;.- -i-·· r '" " ._' '.- ..... 

·ti . : : '. "1:--."~" '-j-'-"-r-'I-I-,' ; ...,-, !·--t-i-",-"-: 

· . ' .'........,--1 ... ,....,-- 'b-'" ..1-. - . /, ,! • ' 
 ~.Mt!I: -Fit ~-4-;~: aaUi!i/1.) Chart speed ~:'~o~-2001 (I.D. #:8) : .. q-- r:~r ,:;': L. :.;=':=~-::;: =l~..'~':+'(~ 1) .~Q.:l ~!~~~~~ 1 _, . -t-+- :·II....:!:.;::~..:;:~:.T.::J:· 1-1+ I t:++':~-I-;-'----!-'j......,- ..--.. ( 55.S

'~-EP · , . !~--:-.. No. Substance Cone. C' Sludge =1=t:"="::::;_i:.:;:-- , ..+:t::~TI·::::t=r-!-!: r-r-i' __ '~_'/~_1 . l'T"~. c::::t:._.-r , ! T"'-'- -. -~:.-t=~:;-:. I--.~""" .tF -;-. one. ! 1.__ ._ .. 1 ;!-"....~ .- ! +'- I' f- .:...i. -I---,---,--;""""'--+...ij.....!'1 

:,=~ j:'C:;:: 1 No, 1 test system Aniline 1~/~ ~8/~ ~:::I:::::: ,+ ::'i:::~ t:-!='.--'-t :ttt::±i'. =ff!:::1:$:-j-:';:-': : : 7 , 

: , -7'30 2 No.2testsystem 30'0 --t :-- . ", .. _.:-_h : ,=4:l--l+- . ;-'+11" -;"'i-'::-" 'IT 

~~ .,......, ... 3 No.3testsystem YDC-1312 1000 30'0 --+-r"l- ...!._: ...... l.-l....~ l-+/i' "-+.-' !_..

~ 

~LI--l !..:;:~ 4 No.4 test sysyem YOC-1312 100:0 30:0 ':~:F r=1- .-;:.~-~: ...~:+.:....;. . ~ -t:.~.::t:._~-+=- L.~ _.- .. i' 5 No.5 test system YDC-1312 1000 30 O·I·-;..r----·-:· .. ··, :":- -'-. .J_.+. ---rf-+·;"·-i-i-:--· 4-' , .. M 

~. -';_1::. 6 No.6 test system YOC-1312 100:0 . .J=l:.:;:= _,,;.. ' .:.... ::.::. .. :".__,.. 1+_;.-+-..:._-;_...j_.! :.!_+ .-: .. ~ .. ;.:. :J ._. -1-' '-- •• IT .-.--:----+-.... f-i....... _.-!._J_ -t...:..... :..... ...'.-LL 

~ -I-~'-i--- --'''-'' .... "'-~ ;----1'" , . -'-15 H ." ' ..-.-+-- - .......--".~ ,_.... , -.....:=-r-- ,--:' ·....,-:~.. i-r1..~·-!----;-· .. I±, ..:........ I 't =til i'. , -F· -t,'- .-:- .! : .""1'- --j", 1- .~. . , : .. :.....!_:__ ;~:: _, Operator ; .1) IV" I\r 'j ,.:t,.. - ......... - 'r ._, --'---+-+-.. -,:1: '. ' .' 
 ., 0';-_+_ ; '-~;""'_~..:.: ..> ___ 1... : .L_.i , '" .1. 'J..' ~'. . L..C. ;.:;'''' -1.: ~:". :.~~"'.~-'.. -".:'.:.' : _.~:-t- '(N\~!) NO.~ test System' ';--'-i . , I, '.". 

i 

. • , "'! -:--r:'-, .1-!-",.--.-- "'-";" ...-' ~-I- •. 1 '....,·(Nt.3) No.3 test system . 


~=r::r-- -": ;- :....-.. ::::--~::i--r ~I ;.1=:::.. 1_ '_~... ~ .I--=r l-+-~-::--l"+-::._ -I--._.:_=_~--.- .. " . No.6 testd:(Nt.5) !i0'§. t~t.~_~tern . .,.-!·....;::-_·_·_:-+-t....;·;t- t
 -'-+-t-----r-t: ___....-------r-1 --t::::r--i--+- .. i'-i·~---t-'-H-l-...l---:- ... -+~-~I- _.-+--;. -:-..,',.,... .-:-+-.(lh8) system rt=::::JjNt.4} ijp..:..:C'!r§~~.Y~tellJ.. ..... ::'J.. . '. -.' ..­ --t-'- ,----', -,.- ...----;-1";-; .-!-i--- ---'---, - ".::I:l I "1' I....d: ~ I I I ! r J. I. ',-!...-- -1-. -," """ -:. -: ..­ .~. o 

l - .- ., 

..• - -Inl 0 .• :,. I l' I '...1 -+--!....L._ I· .----~ !::lli'±-!±l·' - I ' 'llilti1 .. ·-,--H- ... ' ' .. l·...;.. I -~ ..... ,--,

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0001.60 

0001~J:l 

Bioconcentration Study of 


YDC·1312 with Carp 


(Englisb version) 

Sponsor Tohto kasei Co.,Ltd. 

Study Title Bioconcentration study of YDC-1312 with Carp 

Study No. AOI0070 

This report is the English version of the original, which 


was written in Japanese. The undersigned hereby declare that 


this version faithfully reflects the original report to the 


best of our knowledge. 


Translated by 

.~.~~ /) ~ -".1 (p .1.O~ ! ......~~~.......---.---. Da te : __ ~.f!.~~..___ .______ L.____ ..___ . _..... 

Motoshi MASUDA, M.Sc. 

Ecochemistry Division of Yokohama Laboratory, 

Mitsubishi Chemical safety Institute Ltd. 

Approved by 

..-.~..tSJ;....-.....------.--.------...--------.....-Date:.--AP-t~-g...._LJ..:...1::..~-~-!-- .. - Yasuo SATO 

Research Scientist. 


Ecochemistry Division of Yokohama Laboratory, 


Mitsubishi Chemical Safety Institute Ltd. 


0001.53 

Report No. 4B376G 

Bioconcentration Study of 


YDC-1312 with Carp 


Submitted to: 


Tohto Kasei Co.,Ltd. 


Prepared by: 


Mitsubishi Chemical Safety Institute Ltd. 


February 21, 1995 


000:1G4 
Compliance with the GLP Standards 

Yokohama Laboratory. 


Hitsubishi Chemical Safety Institute Ltd. 


Sponsor: Tohto Kasei Co.,Ltd. 

Study Title: Bioconcentration Study of YDC-1312 with Carp 

Study No.: 4B376G 

The study described in this report was conducted in 

compliance with the Good Laboratory Practice (GLP) 

regulations recognized by the Basic Industries Bureau of the 

Japanese Ministry of International Trade and Industry. 

Facility Management: 

Jun TORIYA, B.Sc. Sealed date: February 21, 1995 

Facility Management 

Head of Yokohama Laboratory 

2 

0001G5 
Quality Assurance Statement 

Yokohama Laboratory. 

Mitsubishi Chemical Safety Institute Ltd. 

Sponsor: Tohto Kasei Co.,Ltd. 

Study Title: Bioconcentration Study of YDC-1312 with Carp 

Study No.: 4B376G 

We hereby certify that the above study has been performed 

in accordance with the Good Laboratory Practice (GLP) 

regulations recognized by the Basic Industries Bureau of the 

Japanese Ministry of International Trade and Industry (MITI) . 

Dates of inspections and findings reported to the Study 

Director and the Facility Management are as follows: 

Date of Date of 

Inspection Reporting 

In-progress study: November 28, 1994 December 1, 1994 

December 26, 1994 December 28, 1994 

January 9, 1995 January 9, 1995 

Final report: February 21, 1995 February 21, 1995 

Quality Assurance Personnel: 

Junko KATOH, Ph.D. 

Yoshihiro MIURA, M.Sc. 

Sealed date: 

Sealed date: 

February 21, 

February 21, 

1995 

1995 

3 
Introduction 

Study Title: 

Bioconcentration Study of YDC-1312 with Carp 

Purpose: 

This study was conducted to evaluate the bioconcentration 

potential of the test substance, YDC-1312, for notification 

under the Chemical Substances Control Law of Japan. 

Test Guideline: 

The study was conducted according to the testing method for 

New Chemical Substances, which prescribes the procedure of 

testing new chemical substances as required by the Chemical 

Substances Control Law of Japan. 

GLP Compliance: 

The study was conducted according to the Good Laboratory 

Practice (GLP) standards applied to industrial chemicals under 

the Chemical Substances Control Law of Japan. 

Sponsor: 

Tohto Kasei Co.,Ltd. 

Testing Facility: 

Yokohama Laboratory, 

Mitsubishi Chemical Safety Institute Ltd., 

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

Study Period: 

November 22, 1994 to February 21, 1995 

4 
The person in charge of the study 

Tadayoshi SHIGEOKA, Ph.D. Sealed date: February 21, 1995 

Study Management, 

Chief Research Scientist of Environmental Science Division 

Ayao NISHIKAWA, Sealed date: February 21, 1995 

Study Director, 

Research Scientist of Environmental Science Division 

Eiichiro MURAKAMI Sealed date: February 21, 1995 

Environmental Science Division 

(Experimental work and Reporting) 

Storage and Retention of Test Substance and Records 

The following records, the raw data, and a small sample of 

the test substance will be stored in the archives of the 

Yokohama Laboratory for 10 years after the submission of the 

final report. After this period, the sponsor will be contacted 

for the approval of the disposal of data. The data can be 

stored in the archives for a further specified period at the 

sponsor's request with an additional fee. 

1) Protocol and its amendment 

2) Final report 

3) Raw data 

4) Quality assurance reports 

5) Test substance (approx. 2 g in freezer) 

6) Other documents required under the GLP standards 

0001~8 
Content 

page 

SUMf.IARY 	 8 

1. Test Substance ................................................................................................. 9 


1.1 	Identification 9 

1.2 	Source 9 

1.3 	Confirmation of Identity 10 

1.4 	Confirmation of Stability 10 

2. Outlines of Bioconcentration Test .................................................11 


3. Acute Toxicity Tes 	 1 

3.1 	Test Fish 11 

3.2 	Test Conditions 12 

3.3 	preparation of Test Water 12 

3.4 	Results 13 

4. Analytical Methods " ......................................................................................14 


4.1 	Apparatus and Reagents 14 

4.2 	HPLC System and Conditions 15 

4.3 	Calibration Curve 15 

4.4 	Analytical Method of Test Substance in Test Water 16 

4.5 	Analytical Method of Test Substance in Test Fish 17 

4.6 	Blank and Recovery Test 18 

5. Bioconcentration Test Methods ............................................................19 


5.1 	Test Fish 19 

5.2 	Test Conditions 20 

5.3 	Test System 20 

5.4 	Preparation of Test Water 21 

5.5 	Stability under Test Conditions 22 

5.6 	Introduction of Fish. Observations and Environmental 


Control 22 


5.7 	Measurement of Test Substance 23 

5.8 	Bioconcentration Factor 23 

6 

0001~~9 
6. 	Results and Discussion of Bioconcentration Test..................... 24 


6.1 	Circumstances That May Have Affected the Reliability 


of the Test Results 24 


6.2 	Monitoring of Test Conditions 24 


6.3 	Concentration of Test Substance in Test Water 24 


6.4 	Concentration of Test Substance in Test Fish and BCF 24 


Tables 1-9 25-31 


Figures 1-14 28,29,32-42 


Appendixes 

1. 	Infrared absorption spectrum of the test substance 

(Data provided by the sponsor) 2 pages 

2. HPLC chromatograms (analysis of test water) 18 pages 

3. HPLC chromatograms (analysis of test fish) 9 pages 

7 


00011'0 
SUMMARY 


Sponsor: 	 Tohto Kasei Co.,Ltd. 

Study tittle: 	 Bioconcentration Study of YDC-1312 with Carp 

Stuiy number: 	 4B376G 

Stuly period: 	 Nov~mber 22, 1994 to February 21. 1995 

Methods: 	 The study was conducted according to the testing 

methods. for New Chemical Substances (1974), 

which prescribes the procedure of testing new 

chemical substances as required by the Chemical 

Substances Control Law of Japan. 

* 	Supply of test water: Continuous flow-through diluter 


system (800 L/day) 


* 	Exposure level: 0.1 Ilg/mL (High exposure level) 


(nominal concentration) O.Olllg/mL (Low exposure level) 


* 	Analytical method: High performance liquid 


chromatography (HPLC) 


* 48hr-LC50 to Medaka: 	 91 Ilg/mL 

* Fat content: 	 4.6\(n-4. 3.8 - 5.5\) 

Results 

Bioconcentration factors (BCFs) obtained in this study were as 

follows: 

Exposure period (weeks) 	 2 4 6 8 

High Mean conc. 

(llg/mL) 

in water 0.097 0.100 0.099 0.099 

exposure 

level 

BCF 

« 6) 

No.1 

No.2 

4 

2 

2 

2 

2 

1 

3 

6 

Low Mean conc. 

( 11 g/mL) 

in water 0.0094 0.0097 0.0097 0.0097 

exposure 

level 

BCF 

« 11) 

No.1 

No.2 

<10 

<11 

< 9 

<10 

< 9 

<10 

<11 

<11 

Discussion 

On the basis of the BCFs obtained. it can be concluded that 


the test substance is not bioaccumulative in carp under the 


prescribed test conditions. 


8 

OOOA! I~,"~ .A. 	1 ..IL.. 
9 

000:1."72 

2. OUTLINES OF BIOCONCENTRATION TEST 


The study was conducted in accordance with the testing methods 

for New Chemical Substances, which prescribes the procedure for 

testing new chemical substances, as required by the Chemical 

Substances Control Law of Japan. 

The bioconcentration test system shown in Fig.4 (p.34) was set 

up in triplicate. In addition to the control, two groups of carp 

were exposed to the test substance at different concentration 

levels.During the exposure period, the concentrations of the test 

substance in water and fish were measured periodically. The 

bioconcentration factors (BCF) calculated from the results were 

used to evaluate the potential for the test substance to 

accumulate in the tissues of carp. 

Before starting the bioconcentration test, an acute toxicity 

test was conducted for 48hours using Medaka and a 50\ lethal 

concentration (LC50) was determined. The bioconcentration test 

was carried out at two exposure levels not exceeding a hundredth 

and a thousandth of the 48hour LC50. 

3. ACUTE TOXICITY TEST 

The acute toxicity test of the test substance to f ish was 

conducted according to the Japanese Industrial Standard Method 

"JIS K0102-1986, Industrial Waste Water Testing Method, 71, Acute 

toxicity study using fish". 

3.1 TEST FISH 

l)Test fish 


Species Medaka (Oryzias latipes) 


Source Sankyo Suisan Co. 


(1-1 Ichigayatamachi, Shinjuku ku, Tokyo) 


Date of purchase: November 7, 1994 


Lot Nu 94·H-1107 


Body length approx. 2 cm 


II 
Body weight approx. 0.2 g 

2)Acclimation 

Aquarium No. : 0-1 

Wa ter tempera ture : 25 ± 2t 

Period over 1 week 

Feeding Feed (Tetramin, Tetra Werke, Germany) of 

approx. 2 ... of the body weight was given to 

fish everyday except holidays and for 

48 hours before testing. 

Mortality of fish during a week before testing < 0 ... 

3.2 	 TEST CONDITIONS 

Test substance conc. : 0 (control), 10. 18, 32, 58, 100#g/mL 

The number of fish: 10 per aquarium 

Volume of test water 2 L 

Water temperature : 25 ± 2t 

Renewal of test water: every 24 hours 

Aeration continuous 

Dissolved oxygen > 4.0 mg/L 

3.3 	 PREPARATION OF TEST WATER 

l)Exposure Level 

The test substance, 1.00g, was dissolved in 100mL of dimethyl 

sulfoxide. This solution was added dropwise to 1500mL of 20 

w/v\ HCO- 30 in Millipore water to disperse the test substance 

and diluted to 2000mL with Millipore water. Each of 40, 72, 128, 

232 and 400mL from the solution (concentration of test 

substance: 500#g/mL) was diluted to 2000mL with dilution water 

(to produce this water, Yokohama municipal tap water was 

treated with active charcoal and dechrolinated), resulting in 

test concentration with each of 10, 18, 32, 58 100 # g/mL test 

substance. 

2)Control Level 

Twenty milliliters of dimethyl sulfoxide was dissolved with 

300mL of 20\ HCO·30 in Millipore water and diluted to 2L with 

dilution water. But. concentrations of HCO-30 and dimethyl 

12 

0001'75 
sulfoxide are the same of concentration in the highest exposure 

level. 

3.4 RESULTS 

The 48hr-LCso value of the test substance was 91 Jl g/mL as 

follows : 

Test substance conc. r

(Jlg/mL) 

Number of 

dead fish 

Mortali ty ! 

(III) 

0 (control) 0 

I 

0 i 

10 0 0 

18 0 0 

32 0 0 

58 0 0 

100 6 60 

From this result, the nominal concentrations to be used in the 

bioconcentration test were set at 0.1 and O.OlJlg/mL in high and 

low exposure levels, lower than a hundredth and a thousandth of 

the 48hour LC50, respectively. And measured water temperature and 

dissolved oxygen. they are appropriate during the exposure period. 

test water 24.9 - 25. ot 

dissolved oxygen 6.0 - 7.8mg/L 

13 

000j.'76 
4. ANALYTICAL METHODS 


4.1 APPARATUS AND REAGENTS 

1) Apparatus 

Even electrobalance Mettler Model AE163 (No.1 ) 

Automatic even Balance Mettler Model PL1200 (No.1) 

Homogenizer Nihon Seiki Co. Model AM·S (No.1 ) 

Aspirator Yamato Kagaku Co.Model BP·51 (No.2) 

Evaporator Buchi Model RE-111 (No _ 1) 

Shaker Taiyokagaku Co. Model SR-ll (No.1) 

2) Reagents 

Acetonitrile Kokusan Chemical Co., Guaranteed Reagent 

n-Hexane Kishida Chemical Co., Guaranteed Reagent 

Ethyl acetate 

: Kokusan Chemical Co., Guaranteed Reagent 

IN-Soduim Hydroxide Solution 

Kishida Chemical Co. 

Sodium Chloride Kishida Chemical Co., Guaranteed Reagent 

Anhydrous Sodiu~ Sulfate 

: Kokusan Chemical Co .. Guaranteed Reagent 

14 
4.2 	 HPLC SYSTEM AND CONDITIONS 

1) Apparatus Shimadzu Co. , Model LC-10AWS (No.3) 

(with column switching apparatus) 


Pump Shimadzu Co. , Model LC-10AD (2 sets) 


Detector Shimadzu Co. , Model SPD-10AV 


Data Processor Shimadzu Co. , Model CLASS-LC10 


six course valve: GL-Science Inc., Model SPV-N-6 (No.4) 


2)Condition 

Column (for analysis): 

GL-Science Inc., Inertsil C8, 5~m 4.6 mm i.d.X150 mm 

Column (for concentration) : 

GL-Science Inc., Inertsil ODS-2, 4.6 mm i.d.X 1 cm 

Mobile phase (for analysis) : acetonitrile / water • 62 : 38 

Mobile phase (for concentration) : Millipore water 

Flow rate (for analysis) : 1 mL/min. 

Flow rate (for concentration) : 2 mL/min. 

Wavelength : 285 run 

Attenuation : 2 

Injection volume 

40 ~L (except test water and test fish analysis ) 

4.3 CALIBRATION CURVE 

The solution of the test substance with 100 ~ g/mL in 

acetoni trile was diluted with acetoni trile to prepare standard 

solution of three concentrations 0.5, 1.0, 2.0 ~ g/mL. These 

standard solutions were analyzed by HPLC. The total peak area 

( ~ V' sec) for the test substance in each solution was plotted 

against its concentration. The calibration curve yielded a 

straight line passing through the origin and its correlation 

coefficient was calculated to be 1.000 by the least square method. 

[Fig.5 - 6 (p.35 . 36)] 

At each measurement of the test substance, peak area for the 

standard solution (conc. 1. 0 ~ g/mL) was measured and a 

concentration of the test substance was determined from a ratio 

of peak area of sample to the standard solution. 

15 

4.4 ANALYTICAL METHOD OF THE TEST SUBSTANCE IN TEST WATER 

The test water was analyzed by HPLC with column switching 

apparatus. Test water sample was pumped to concentration column 

and the test substance concentrated in concentration column was 

pumped to analytical column by switching of valve and analyzed. 

It is checked that the HPLC system is running 

under the conditions given in Section 4 2 

(the switching valve must being the "analysis" position 

I 

4000 Il L (400 Il L)' of test water is taken wi th a 

mjcrosyringe 

I 

Immediately after the valve is rotated to 

the "concentration" posjtjon, the sample is injected 

I 

The HPliC system is run for about 3 minutes to trap the test 

substance in concentratjon column 

I 

The valve is returned to the "analysis" posjtion to start 

HPliC anal ysi s 

* : For the testing at high exposure level. 

16 

0001.'79 
4.5 	 ANALYTICAL METHOD OF THE TEST SUBSTANCE IN TEST FISH 

Prior to HPLC analysis, test fish was treated according to the 

following 	procedure : 


One fish of ahout 2Sg. weighted 


Cut 	 jnto pieces with scissors 

- Repeated once again 

I-ace tonitril e SOmL 

Homogenized for 5 mjn at about 10000rpm 

Filtered with suction though a sheet of S.60 filter paper 

on a Buchner funnel 

I 

Acetonitrile layer 	 Residue 

-Millipore water 500mL 


Discarded
 -Sodium Chloride 25g 

-IN·Soduim Hydroxide Solution 5mL 

Shaken for 10 mjn for extraction with n·Hexane (JOOmI.) 

(SR·IT Shaker, Setting the dial at 6) 

I 	 I 

n·Hexane layer 	 Water. acetonitrjle layer 

I 

Dehydrated and filtered 	 Discarded 

(by passing through a glass-filter plugged 

with 50g of anhydrous sodium sulfate) 

I 

Djluted to 200m1. with n.Hexane 

Collect 4mL (Sampling factor' 50) 

passed through a SEP-PAK FlorisiJ cartridge 

(then 6mL of n-Hexane was passed and discarded) 

BmI. of n-Hexane/ethyl acetate - 95/5 mjxture was passed 

I 

Eluate 

I 

Evaporated to dryness(at 40t: by rotary evaporator) 

I~Acetonitrile/water=62/38 mixture, 5mL 

dissolved 

Collected 200~L and analyzed by HPLC 

17 

000:1.0 
4.6 BLANK AND RECOVERY TEST 

The minimum detectable peak area was set at 1500 11 V • sec. 

which could be recognized visually. This value corresponded to 

about 0.06ll1g/mL on the calibration curve. 

l)Test water 

• Blank test 

Twice in the exposure period. control water was analyzed by 

the method of low exposure level shown in §4.4. The absence of 

interfering peaks in the HPLC analysis was confirmed and 

detection limit concentration of the test substance in control 

water was O.OOll1g/mL. [Table 1 (p.2S), Fig.? (p.3?)] 

• Recovery test 

The standard solution (the test substance conc. : 1.0 I1g/mL 

in acetonitrile) of 0.5 mL was dispersed in control water and 

diluted to 100 mL. The resul ting concentration was O. 0111 g/mL, 

the same value as the nominal concentration in test water of 

low exposure level. This solution was analyzed by the method of 

low exposure level shown in §4.4 to evaluate recovery. Recovery 

was measured twice and averaged 99.6 \. Values analyzed for 

test water were corrected by this factor. 

[Table 2 (p.25)' Fig.8 (p.38)] 

2)Test fish 

• Blank test 

Two control fish were analyzed by the method shown in §4.5 at 

the beginning and the termination of the exposure period. 

respectively. The absence of interfering peaks in the HPLC 

analysis was confirmed and detection limit concentration of the 

test substance in control fish was below 0.14I1g/g. 

[Table 3 (p.26), Fig.9-l0 (p.39-40)) 

• Recovery tes t 

One milliliter of standard solution (conc. 100 11 g/mL in 

acetonitrile) was added to one control fish. This fish was 

analyzed by the method shown in §4.5 to evaluate recovery. When 

the fish weight is 25 g. the test substance concentration in 

the fish body will be 2 11 gig, corresponding to 20 times the 

nominal value in test water of high exposure level. Recovery 

was measured in two individual fish and averaged 94.8 %. Values 

analyzed for test fish were corrected by this factor. 

[Table 4 (p.26), Fig.ll (pAl)) 

18 

5. BIOCONCENTRATION TEST METHODS 

5.1 TEST FISH 

l)Test fish 


Species Carp (Cyprinus carpio) 


Source Sankyo Suisan Co. 


(1·1 Ichigayatamachi, Shinjuku-ku, Tokyo) 

Date of receipt: August 17, 1994 

Lot No. 94-K-0817 

Body length approx. 10 cm (measured with the eye) 

Body weight 20-40 g (measured with the eye) 

2) Acclimation 

Aquarium No. : B-3 

Water temperature : 25±2 t 

Period over one week 

Feeding Feed (Minipet, Kyorin Co., Ltd., Japan) of 

approx. 2 , of the body weight was given 

everyday except holidays. 

Mortality of fish during the week before use 0\ 

19 

5.2 TEST CONDITIONS 

l)Nominal concentrations of the test substance in water 


High exposure level 0.1 tlg/mL 


Low exposure level O.Oltlg/mL 


Control o tl g/mL 


2)Flow rate of dilution water : aoo L/day 

(Turnover rate :10 times/day) 

3)Flow rate of feed solution : 160 mL/day 

4)Population density (at the beginning of the exposure period) 

High exposure level 15 fish / aO-L test water 

Low exposure level 15 fish / aO-L test water 

Control 12 fish / aO-L test water 

5)Water temperature : 25±2 't: 


6)Dissolved oxygen > 4 mg/L 


7)Exposure period a weeks 


5.3 	 TEST SYSTEM 

The outline is shown in Fig.4 (p.34). 

20 

5.4 	 PREPARATION OF TEST WATER 

l)Preparation of feed solutions 

The feed solution of each exposure level was prepared 

according to the following procedures : 

(High exposure level) 


Test substance 1.00g 


I-Dimethyl sulfoxide 100mL 


Disso]ved 


mixed with 20 w/v' HCO-30 jn Millipore water (1500 mI.) to disperse 


the test substance 


I-Millipore water 


Diluted to 2000mL 


Concentration of test substance 0.50mg/mL 


Concentration of HCO-30 150mg/mL 


(Low exposure level) 


Feed solution of hi gh exposure 1 eve] (200mL) 


I-Millipore water 


Dil uted to 2000mL 


Concentration of test substance 0.050mg/mL 


Concentration of HCO-30 15mg/mL 


(control) 


MiJ 1 jpore water of 20 w/V' HCO- 30 (J 500 mI.) 


I-Dimethyl sulfoxide (lOOmL) 


Djssolved 


mjx and djsperse 

I-Millipore water 


Diluted to 2000mI. 


Concentration of HCO-30 	 150mg/mL 

Each of these solution was renewed at about lO-day 


intervals. 


2) Preparation of test water 

The feed solutions prepared in §S.4 1) were supplied to their 

respective mixing tubes by pumps to be mixed with dilution 

water so that concentrations of the test substance could reach 

their respective nominal values. The test water thus prepared 

were continuously supplied to their respective test aquarium. 

[Fig.4 (p.34)] 

21 

000:1. ,1 
5.5 STABILITY UNDBR THB TBST CONDITIONS 

1)Stability in feed solution 


The feed solution of low exposure level concentration of 


the test substance 50 /.1 g/mL in aqueous solution (see § 5.4 


1» was used as a test solution. A portion of the tes~ solution 


left at room temperature was diluted 50 fold on days 0, 1, 


and 14. Each sample was analyzed by HPLC. and variation of 


concentration with time was evaluated. On day 14. 93.8 \ of the 


initial concentration was detected (48.2/.1g/mL on day 0 - 45.2 

/.1 9 / mL on day 14) . 

On the basis of this resul t, it is concluded that the test 


substance in the feed solution is sufficiently stable to allow 


approx. 14 days use. 


[Table 5 (p.21). Fig.12 (p.42») 


2)Particle size 


The 1 /.1 g/mL water dispersion prepared in § 5.5 1). was 

filtered with a membrane filter of pore size 0.4S/.1m. The test 


substance in the filtrate was analyzed by the HPLC. The 


particle size of the dispersion was determined from the 


concentration of the test substance in the fil trate. On the 


basis of the result that particles below 0.45/.1m were 91.5 \. it ""\ 

was concluded that the dispersion was satisfactory for the test 


solution. 


5.6 	 INTRODUCTION OF FISH. OBSBRVATIONS AND BNVIRONMBNTAL CONTROL 

The test system was operated for over 4 days prior to the 

introduction of fish. It was confirmed that the test substance in 

water was kept at nominal concentrations during this period. Then 

fish were introduced into the test system. Feeding, observation 

of fish and control and measurement of water quality parameters 

were carried out as follows : 

1) Feeding 

Feed (Minipet, Kyorin Co.Ltd.) of approx. 2\ of the body weight 

were given to fish everyday except holidays. 

2) Observation of fish 

Conditions of fish were observed. 

3) Dissolved oxygen concentration and temperature : 

Dissolved oxygen concentration and temperature were measured and 

recorded twice a week. 

4) Measurement of fat content : 

Fat content of 4 test fish in the control aquarium were measured 

during the exposure period. 

22 

0001.55 
5.7 	 MEASUREMENT OF THE TEST SUBSTANCE 

l)water 

Test water was analyzed twice a week, regularly, as described 

in § 4 .4 . 

2) fish 

Three fish were collected and two of tilem were analyzed on 

weeks 2, 4, 6 and 8 of exposure period as described in §4.5. 

5.8 	 BIOCONCENTRATION FACTOR 

l)Calculation of bioconcentration factors (BCFs) 

Bioconcentration Factors (BCF) were calculated from the 

following calculation equation. 

BCF - (Cf - Cfo) I Cw 

where 	Cf : Concentration in treated fish body (Ug/g) 

Cfo: Concentration in control fish body (Ug/g) 

Cw Mean concentration in treated test water(Ug/mL) 

Cfo equals 0 in this study because blanks confirmed the 

absence of interfering peaks. 

2)Detection limit 

As described in Section 4.6.2, the test substance in fish is 

detectable down to 0.14 U gig, indicating that bioconcentration 

factor can be obtained when of 0.1 and 0.01 U g/mL are 

concentrated by factors of about 1 and 14, respectively. 

23 

6. 	RESULTS AND DISCUSSION OF BIOCONCENTRATION TEST 

6.1 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE RELIABILITY OF THE 

TEST RESULTS 

'There was no significant matter that may have affected the 

reliability of the test ~esults during the expusure period of 8 

weeks. 

6.2 MONITORING OF TEST CONDITIONS 

There was no dead fish or abnormal condition during the 

exposure period of 8 weeks. 

Condition of fish There was no abnormality in shape of the body 

or in swimming and feeding behavior. 

Body weight 22.36 - 37.34 g 

Body length 9.5 - 11.4 cm 

Water temperature 23.1 - 25.3 'C 

Dissolved oxygen 5.3 - 7.9 mg/L 

Fat content 4.6 \ (n-4. 3.8 . 5.S \) 

6.3 	 CONCENTRATIONS OF THE TEST SUBSTANCE IN WATER 

The mean concentrations of the test substance during the 

exposure period of 8 weeks were 0.099 11 g/mL for high exposure 

level and 0.0097 11 g/mL for low exposure level. which almost 

corresponded to the nominal values. The coefficients of variation 

were calculated to be 8.6 \ for high exposure level and 6.1 \ for 

low exposure level. 

[Table 6 ·7 (p.28 ·29), Fig.13 . 14 (p.28 - 29), Appendix-2] 

6.4 	CONCENTRATIONS OF THE TEST SUBSTANCE IN FISH AND BCF 

The concentrations of the test substance in fish were 

calculated to be below 0.57I1g/g at high and below 0.11I1g/g low 

exposure level. The bioconcentration factor (BCF) were determined 

to be below 6 for high exposure level and below 11 for low 

exposure level. 

[Table 8 - 9 (p.30 31), Appendix-3] 

On the basis of the resul ts, it was concluded that the test 

substance was not bioaccumula tive in carp under the prescribed 

test conditions. 

24 

000:1 
Tab. 1 Blank test for water 

A B C 

sample 

Na. 

peak area 

J.L V· sec 

conc. 

in water 

1 

samEle 

<1500 . std. 

24395 

/.1.glmL 

<0.001 

2 <1500 24395. <0.001 

Concentration of std. solution (Cstd) 

Injection volume of std. (Vst) 

Injection volume of sample (Vsa) 

Calculations: C=A+BxCstdxVst+Vsa 

1. 0 /.1. g/mL 

40 J.LL 

4000 J.LL 

Tab. 2 Recovery test for water 

A B C D R 


sample test peak area measured recovery 

No. substance amount 

added J.LV·sec 

/.1.g samEle std. /.1.g % 

1 1.0 24313 24395 0.997 99.7 

2 1.0 24266 24395 0.995 99.5 

Average,n=2 	 99.6 

Concentration of std. solution (Cstd) 1.0 J.Lg/mL 

Amount of test water (W) 100 ml 

Injection volume of std. (Vst) 40 /.1.L 

Injection volume of sample (Vsa) 4000 /.1.L 

Calculations: 	D=B+CxCstdxVst+VsaxW 

R=(B+CxCstdxVst+VsaxW)+Ax 100 

25 

000:158 
Tab. 3 Blank test for fish 

W A B C D 

sample fish 

weight peak area 

tJ. V· sec 

cone. 

in final 

cone. 

in fish 

solution 

No. g samE Ie std. jj.g{aL jj.gLg 

1 22.36 <1500 24380 <0.012 <0.14 

2 23.03 <1500 24380 <0.012 <0.13 

3 25.40 <1500 24230 <0.012 <0.12 

4 28.75 <1500 24230 <0:012 <0.11 

Concentration of std. solution (Cstd) 1.0 tJ.g/mL 

Injection volume of std. (Vst) 40 tJ.L 

Injection volume of sample (Vsa) 200 jj.L 

Final volume (FV) 5mL 

Sampling factor (SF) 50 

Calculations: C=A+BxCstdxYst+Vsa 

D=(A+ Bx Cstd x Vst-:-Ysa) x FY x SF -:-W 

Tab. 4 Recovery test for fish 

W A B C 

sample fish test peak area 

weight substance tJ. V· sec 

added 

No. g ,u.g samEle 

1 29.70 50.0 24019 

2 26.81 50.0 22207 

std. 

24380 

24380 

D 

conc. 

in final 

solution 

,u.gLmL

0.1970 

0.1822 

Concentration of std. solution (Cstd) 

Injection volume of std. (Vst) 

Injection volume of sample (Vsa) 

Final volume (FV) 

Sampling factor (SF) 

Calculations: D=B-:-CxCstdxVst+Vsa 

Average,n=2 

1.0 tJ.g/mL 

40 jj.L 

200 tJ.L 

5 ml 

50 

R=(B+C xCstdx Vst-:- Vsa) x FVx SF-:-Ax 100 

E 

measured 

8.IIount 

,u.g 

49.3 

45.5 

R 

recovery 

% 

98.5 

91.1 

94.8 

26 

000:189 
Tab.5 Stability of the test substance in feed solution 

A B C D 

period peak area conc. 

in feed 

ratio 

to 

day 

0 

J,J.V·sec s3Jll~le std. 

23210 24071 

solution 

JJ.gLmL 

48.2 

initial 

conc. % 

100 

7 23183 24395 47.5 98.6 

14 22033 24369 45.2 93.8 

Concentration . of std. solution (Cstd) 1.0 J,J.g/mL 

Dilution ratio (DR) 50 

Cal cuI ations : C=A-:-B x Cstd x DR, D=C i -:- Co x 100 

i : Number of analysis 

27 

0001. 0 
Tab.6 Analysis of the test substance in water - High exposure level 

i A B C D 

samplirur period number peak area conc. mean conc. 

date of /.J. V· sec in water in water 

dal week anallSis samI!le std. Il:.gimL Il:.gl..mL 1994/11/28 0 0 1 22863 24563 0.093 0.093 

1994/12/01 3 2 23404 24395 0.096 0.095 

1994/12/05 7 1 3 26543 24623 0.108 0.099 

1994/12/08 10 4 23041 24369 0.095 0.098 

1994/12/12 14 2 5 22932 25017 0.092 0.097 

1994/12/15 17 6 25097 24231 0.104 0.098 

1994/12/19 21 3 7 26961 24420 0.111 0.100 

1994/12/22 24 8 25216 24301 0.104 0.100 

1994/12/26 28 4 9 22979 24517 0.094 0.100 

1994/12/29 31 10 23914 24756 0.097 0.099 

1995/01/02 35 5 11 28254 24632 0.115 0.101 

1995/01/05 38 12 23887 24725 0.097 0.101 

1995/01/09 42 6 13 20228 24408 0.083 0.099 

1995/01/12 45 14 26309 24567 0.108 0.100 

1995/01/16 49 7 15 22622 24814 0.092 0.099 

1995/01/19 52 16 26003 24307 0.107 0.100 

1995/01123 56 8 17 22822 24650 0.093 0.099 

Standard deviation 0.009 J..l(!./mL

Coefficient of variation : 8.6 ~ 

Concentration of std. solution (Cstd.) 1.0 /.J.g/mL Injection volume of std. (Vst) 40 /.J.L 

Injection volume of sample (Vsa) 400 /.J.L

Recovery (R) 99.6~ 

Calculations: C=A+BXCstdxVst+Vsa+(R+lOO). D=kCi+i 

Fig.13 Concentration of the test substance in water - High exposure level 

concentration (ttg/aL) 

0.200 ----.-- analyzed value - .. - - - Iminal value I. 

0.150 

0.050 

0.000 -I----_+_--+----+-------;O--,---+----+----+--__+_­

o 7 14 21 28 35 42 49 56 

exposure period (day) 

28 

000.1:3.1 
Tab. 7 Analysis of the test substance in water - Low exposure level 

i A B C D 

saDlpliM period number peak area conc. mean conc. 

date of j.J. V' sec in water in water 

dal week anallsis saDl:Rle std. j.J.gLmL Il:.gLmL 

1994/11/28 0 0 1 22579 24563 0.0092 0.0092 

1994/12/01 3 2 21393 24395 0.0088 0.0090 

1994/12/05 7 1 3 20639 24623 0.0084 0.0088 

1994/12/08 10 4 25146 24369 0.0104 0.0092 

1994/12/12 14 2 5 25542 25017 0.0103 0.0094 

1994/12/15 17 6 26081 24231 0.0108 0.0097 

1994/12/19 21 3 7 24519 24420 0.0101 0.0097 

1994/12/22 24 8 22265 24301 0.0092 0.0097 

1994/12/26 28 4 9 24042 24517 0.0098 0.0097 

1994/12/29 31 10 22988 24756 0.0093 0.0096 

1995/01/02 35 5 11 24304 24632 0.0099 0.0097 

1995/01/05 38 12 24242 24725 0.0098 0.0097 

1995/01/09 42 6 13 24092 24408 0.0099 0.0097 

1995/01/12 45 14 24484 24567 0.0100 0.0097 

1995/01/16 49 7 15 23488 24814 0.0095 0.0097 

1995/01/19 52 16 23644 24307 0.0098 0.0097 

1995/01/23 56 8 17 23923 24650 0.0097 0.0097 . Standard deviation . 0.0006 J.1f!./mL

Coefficient of variation : 6.1 % 

Concentration of std. solution (Cstd.) 1. 0 j.J. g/mL Injection volume of std. (Vst) 40 j.J.L 

Injection volume of sample (Vsa) 4000 j.J.L 

Recovery (R) 99.6 % 

Calculations: C==A-:-BXCstdxVst-:-Vsa-:-(R-:-100). D=2:Ci-:-i 

Fig.14 Concentration of the test substance in water - Low exposure level 

concentration (tLg/aL) 

0.0200 • analyzed value - - - - - roIIinal value 

0.0150 

0.0050 

0.0000 l-----+-----4----t----I------t--_--+----+-­

o 7 14 21 28 35 42 49 56 

exposure period (day) 

29 

Tab.8 Analysis of the test substance in fish and bioconeentration factor (BCF) 

- High exposure level 

W A B C D E F 

exposure fish peak. area cone. cone. me~ BCF

period weight in final in fish cone. 

j.1V· sec solution body in water 

week No. g sample std. ttg/mL /.J,g/g /.J,g/mL 

2 1 30.31 5333 24450 0.044 0.38 0.097 4 

2 2 28.52 2906 24450 0.024 0.22 0.097 2 

4 1 34.47 3295 24562 0.027 0.21 0.100 2 

4 2 33.53 3745 24562 0.030 0.24 0.100 2 

6 1 32.34 2473 24460 0.020 0.16 0.099 2 

". 	 6 2 30.45 1827 24460 0.015 0.13 0.099 1 

8 1 30.66 4826 24230 0.040 0.34 0.099 3 

8 2 32.40 8548 24230 0.071 0.57 0.099 6 

Concentration of std. solution (Cstd.) 1. 0 j.1g/mL 


Injection volume of std. (Vst) 40 ttL 


Injection volume of sample (Vsa) 200 j.1L 


Final volume (FV) 	 5IlL 

Sampling factor (SF) 	 50 

Recovery (R) 	 94.8 ,; 

Calculations: 	C=A7B xCstdx Vst7Vsa 

D=(A7B X.QstdXVst7Vsa) XFVXSF7(R7100)7W. F=D7E 

30 
Tab. 9 	 Analysis of the test substance in fish and bioconcentration factor (BeF) 

- Low exposure level 

W A B C D E F 

exposure fish peak area conc. ronco mean BCF 

period weight in final in fish conc. 

j.J. V· sec 	 solution JQdy in water 

week 	 No. g sample std. /J.g/IIL Ltg!g /J.g!mL 

2 1 35.52 <1500 24450 <0.012 <0.09 0.0094 <10 

2 2 31.62 <1500 24450 <0.012 <0.10 0.0094 <11 

4 1 36.76 <1500 24562 <0.012 <0.09 0.0097 <9 

4 2 33.37 <1500 24562 <0.012 <O.lD 0.0097 <10 

6 1 34.64 <1500 24460 <0.012 <0.09 0.0097 <9 

6 2 33.21 <1500 24460 <0.012 <0.10 0.0097 <10 

8 1 30.58 <1500 24230 <0.012 <0.11 0.0097 <11 

8 2 30.79 <1500 24230 <0.012 <0.11 0.0097 <11 

Concentration of std. solution (Cstd.) 1. 0 j.J. g/mL 

Injection volume of std. (Vst) 40 j.J. L 

Injection volume of sample (Vsa) 200 j.J.L 

Final volume (FV) . 5IlL 

Sampling factor (SF) 50 

Recovery (R) 94.8 % 

Calculations: 	C=A-:-BxCstdxVst-:-Vsa 

D=(A-:-B x Cstd x Vst-:-Vsa) x FVx SF -:- (R -:-100) -:-W, F=D-:-E 

31 

ooo ~ c~r­ J' _L . .l,.:} 

Fig.4 Bioconcentration test system 

tap water 

air pump pump

feed solution o 	 activated charcoal 

(dech lorina t ion) 

dilution water 

(25'"'C) 

mixing tube 

to waste treatment 

system 85L-glass aquarium 


(80L water) 


(Apparatus) 

Pump (dilution water supply) : 


Exposure : FMI Co.,QD-2CSC (No.l,2) 


Control : FMI Co.,RP-D 2CKC (No.1) 


Pump (feed solution supply) : 

Exposure : Nihon Seimitu Kagaku Co.,NP-FX-l(02) (N~I,2) 

Control : FMI Co.,QG 6-RHOOSTY (No.1-i) 

34 

Fig. 6 Calibration curve of the test substance 

Curve fitting (Least square method) 

concentration peak area 

,ug/ml ,uV-sec 

o o 

0.5 	 12357 

1.0 	 24293 

2.0 	 50136 

R: 1.000 


60000 

,. 50000 

,/ 

,/ -(.) ,/ 

,/ ~ 40000 ,/ 

>­ ,/

,/ ::t ,/ 

:; 30000 Q) 

<: 

~ A

..

,/ 

./ 

,/ 

,/ 

,/ 

7J 20000 ,/

Q) 

~ 

,/ 


,/ 


10000 

,/ 

,/ o ______ ______ ______L­____~ 

o 	 0.5 1 1.5 2 

Concentration (.l1g/mL) 

36 

OO~Q9 '- ..;\• •.. 1 
),"/ 
--.------­
3; 


L{-I 


APPENDIX-l 

Infrared absorption spectrum of the test substance 

(Data provided by the sponsor) 

(2 pages) 
B,C~" \Z. lA S<L.

-------. 


L 

APPENDIX-2 

HPLC chromatograms (analysis of test water) 

(18 pages) 

000208 
000209 


000:2"1:1 

O()O",-:> ..~ r> 

U '".. ~ ."-' 
0002t



/0 


0002

I( 

r')

/' 

l7 

Or. 0 ........-., 
 1:' ••,' ",. f, ~ ,,"-¥ .. ..., 
'--------­
APPENDIX-3 


HPLC chromatograms (analysis of test fish) 

(9 pages) 
~L\6y..5 'L2')- L ') ~ 

~\c~\\.~ 




I I 

0002;~.::; 


~1/~1 

", 

.: J I 

April 27, 200 1 TD p~ 0 1,- II c.f 

SANITIZED 1 5DO (000 37Cfh

Ms. Geraldine Hilton 

Office of Pollution Prevention 

and Toxic Substances, 7407 

U.S.B.P.A 

401 M Street, S.W. 

Washington, D.C. 20460 

Re: Selection of Regulatory Option and Submission of Additional Information 

Our Ref: TS-600212 

Your Ref: P-0l-114 

Dear Ms. Hilton: 

Pursuant to my recent telephone message, attached please fmd a completed Selection of 

Regulatory Option form for the above-identified case, as well as additional information together 

with four translations of test reports which were recently discovered (acute oral toxicity test in rats, 

twenty-eight day repeated dose oral toxicity study in rats, ready biodegradability test, and 

bioconcentration study with carp). The Selection of Regulatory Option form authorizes a 6O-day 

suspension, based on your indication. Since the submitter wishes to import the new chemical 

substance as soon as possible, expedited consideration by EPA is respectfully requested, as is the 

revocation of any remaining suspension time once the PMN is found by BP A to be acceptable, 

If you have any questions, please do not hesitate to contact us. 

Very truly yours, 

I 

C:l 

Technical Contact 

Company Sanitized 

? 2 ('

p<-- / ) 
-7­

: .. ',) ~;"-; ~ 

• ' t " " -­

SELECTION OF REGULATORY OPTION 

FOR PMN # P-01-114 


I have reviewed the Agency's letter dated ~~.2$, ?-DD I , 

which outlines the regulatory options for the ab~ferenced 

PMN, and I select the following option: 

[ J~ Option 1 	 upfront submission of additional information 

before commencement of manufacture 

(suspension as appropriate) 

Option 2 upfront ,subm.ission of specified data b~fore'commencEilnerit;of manufacture 

,CsuspEmsi'orfas'appropitiate) ..... ', : ~~ .•'\~. ~,'.' ,'":" '"\ "\ ~-f"-' ~ ..•~., 

(,:".;;-~,: t .<).::,'/'" " ' d f ~ ~: 

Option 3 withdrawal of PMN per 40 CFR 720.75(e) 


(no suspension necessary) 


To provide time for implementation of the selected option, I 

request, pursuant to 40 CFR 720.75(b), a suspension of the PMN 

review period for be days or until the following date: 

'-7,~1 Dat