Form A roved. O.M.B. No. 2070-0012. A ires 10-31-96. 

u.s. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY ~D~.~te~o~f~r~~i~Pt~-----=~~~~~~--~~--~~~~----------------~ 

r: /.... A. 

PREMANUFACTURE 

C,'-) , 

ft

.., NOTICE ;ojr:'! " It::­ ,j 

Company Sanitized 

nter the tOtal nUll''''', v< ,,~o-­

in the Premanufacture Notice 

GENERAL INSTRUCTIONS 

You must provide all infonnation requested in this fonn to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates ifyou do 

not have actual data. 


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


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


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


your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM-226), P.O. 


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


Part I -- GENERAL INFORMATION TEST DATA AND OTHER DATA 

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

chemical substance, even if you claim the identity as confidential. You may authorize a description ofall other data known to or reasonably ascertainable by you, ifthese 

another person to submit chemical identity information for you, but your submission will data are related to the health and environmental effects on the manufacture, 
 not be complete and the review will not begin until EPA receives this information. A 
 processing, distribution in commerce, use, or disposal of the new chemical 

letter in support of your submission should reference your TS user fee identification substance. Standard literature citations may be submitted for data in the open number. You must submit an original and two copies of this notice including all test data. scientific literature. Complete test data (written in English) not summaries of data If you claimed any information as confidential, a single sanitized copy must also be must be submitted ifthev do not appear in the open literature. You should clearly submitted. identifY whether test data is on the substance or on an analog. Also, the chemical 

composition of the tested material should be characterized. Following are 

examples of test data and other data. Data should be submitted according to the Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 


requirements of §nO.50 ofthe Premanufacture Notification Rule (40 CFR Part 


If there are several manufacture, processing, or use operations to be described in Part II, nO). 


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


Part 1II -- LIST OF ATTACHMENTS Test Data (Check Below any included in this notice) 

Attach additional sheets if there is not enough space to answer a question fully. Label Environmental fate data 181 Yes Other data D Yes 


each continuation sheet with the corresponding section heading. In Part 1II, list these 


attachments, any test data or other data and any optional information included in the Health effects data 181 Yes Risk assessments 


notice. . 


Environmental effects data 181 Yes Structure/activity relationships 

Physical/Chemical Properties· 181 Yes Test data not in the possession 

OPTIONAL INFORMATION or control of the submitter 

You may include any information that you want EPA to consider in evaluating the new A physical and chemical properties worksheet is located on the last page of this form. 


substance. On page II of this form, space has been provided for you to described 


pollution prevention and recycling information you may have regarding the new TYPE OF NOTICE (Check Only One) 


substance. 


[8] PMN (Premanufacture Notice) So-called "binding" boxes are included throughout this form for you to indicate your 


willingness to be bound to certain statements you make in this section, such as use, 


production volume, protective equipment. . . This option is intended to reduce delays D INTERMEDIATE PMN (submitted in sequence 't'it!J final product PMN) 


that routinely accompany the development of consent orders or Significant New Use 
 = . 

= Rules. Except in the case of exemption applications (such as TMEA, L VE, LOREX) ..L.­ SNUN (Significant New Use Notice) where certain information provided in such notification is binding on the submitter when D :r­


the Agency approves the exemption application, checking a binding box in this notice 
 \:J Cl-ry --::) F;' does not by itself prohibit the submitter from later deviating from the information (except D TMEA (Test Marketing Exemption Application)-;':; -~Jn chemical identity) reported in the form. ~ '-Ir'q D LVE (Low Volume Exemption)@40CPR 723.50(cXI) . _: •._ 


CONFIDENTIALITY CLAIMS ~ (')r;;
 D LOREX (Low ReleaselLow Exposure Exemption)@40C:rii W.50(cX2)
 You may claim any information in this notice as confidential. To assert a claim on the CO.. form, mark (X) the confidential box next to the information that you claim as 

confidential. To assert a claim in an attachment, circle or bracket the information you 

claim as confidential. If you claim information in the notices as confidential you must 

also provide a sanitized version ofthe notice (including attachments). For additional 

instructions on claiming information as confidential, read the Instructions Manual. 

D Mark (x) if any information in this notice is claimed as confidential. 

D 

IS TH

LVE Modification 

IS A CONSOLIDATED PMN? 

D 

D 

LOREx..J)1odification 

U1 

Yes 

# of chemicals 

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

EPA FORM 7710-25 {Rev. 5-95) Replaces previous editions of EPA Fonn 7710-25. 

I 
Public reporting burden for this collection of information is estimated to average) 10 hours per response, including time for reviewing 

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

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

reducing this burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W., Washington, D.C. 

20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

I certify that to the best ofmy knowledge and belief: 

I. 	 The company named in Part I, section A, subsection I a ofthis 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 ofthe date of submission. 

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


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


Additional Certification Statements: 

Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following user fee certification 

statement that applies: 

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

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


700.43) in accordance with 40 CFR 700A5(b), or 


o 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). 

lfyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(cXI) or a Low release 

and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check the following certification 

statements: 

o 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 of40 CFR 723.50. 


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

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

o Ifthis application is for an L VE in accordance with 40 CFR 723 .s0(cXl), the manufacturer intends to commence 

manufacture ofthe exempted substance for commercial purposes within 1 year ofthe date ofthe expiration ofthe 30 day 

review period. 

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

described herein. Any knowinl! and willful misinterpretation is subiect to criminal penaltv pursuant to 18 USC 1001. Confidential 

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

Signature of agent - (if applicable) Date 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 2 

x 
x 

you the chemical 

PMN number assigned by EPA (i.e. withdrawn or incomplete). 

If you 

for the 

Type of Notice - Mark (X) 

J. 

Binding Option 

Telephone 

Position 

.. 

Name 

Submitting 

Notice (in U.S.) 

b. 	 Agent (if 


applicable) 


c. If you are 

Company 

City, State, ZIP Code 

Company 


Mailing address and street) 


mark box. 

Confidential 

Joint Submitter (if Name of 

applicable) 

street) 

City, 

Contact (in 

U.S.) 

Company 

Mailing address (number and street) 

City, State, Code 

Mark (X) 

if none 

substance covered by this notice, enter the exemption number assigned by Mark (X) EPA. Ifyou previously submitted a PMN for this substance enter the if none 

5. 

Mark (X) 

2. Import 6. Only 3. o Both 

o Binding Option 

Mark Mark 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 3 
1. 

a. 

b. name correct Abstracts that consistent listings similar 

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

be provided, which ever is appropriate based on CA 9CI nomenclature rules and conventions). 

c. Please identifY which method you used to develop or obtain the specified chemical identity information in this notice: (check 

o Method 1 (CAS Inventory Expert Service - a copy ofthe Identification 0 Method 2 (Other Source) 

report obtained from the CAS Inventory Expert Services must be 

d. 

e. For a class I a complete correct 

precursor substances their respective CAS Registry Numbers. (2) the nature ofthe reaction or process. (3) Indicate the 

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

diagram. as comolete as can be known. if one can be reasonablv ascertained. 

based on 

x 


X 


X 


X 


X 


attach a continuation sheet. 

EPA FORM 7710-25 (Rev. 5-95) Page 4 

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

Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and 

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement or the basis for your estimates: GPC 0 Other 0: (Specify) 

i) lowest number average molecular weight: 

ii) maximum weight % below 500 molecular weight: 

iii) maximum weight % below 1000 molecular weight: 

You must make separate monomer or other reactant composition information, and residual 

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

(1)· Provide the specific chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufacture of 

the polymer. 

Mark (X) this column ifentry in column (1) is confidential. 

Indicate the typical weight percent ofeach monomer or other reactant in the polymer. 

(2) -

(3) • 

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

description on the TSCA Chemical Substance Inventory. 

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

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

commercial purposes. 

Mark this column if 

(CAS Inventory Expert Service - a copy of the identification report 

obtained from CAS Inventory Expert Service must be submitted as 

as to this 

d. The currently correct name for the polymer 

% 

% 

% 

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

% 

% 

% 

polymers. 

attach a continutation sheet 

EPA FORM 7710-25 (Rev. 5-95) Page 5 
4-15-04; 3:00<"M;BaKer <"e1:rOII1:e HSE ;2812757385 # L,/

(n) 	 Identify each impurity that may be reasonably ~nticipated to be prescnt in the chemical subsmnce as manufactured for corrunercial purpose. Provide the 

CAS Registry Number if available. Ifthcre arc unidentified impurities, enter "unidentified." 

% of each . estimate their total 

1m 

% 

% 

% 

% 

% 

ConfiConfidential 

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

5. identified in subsection I or 2. 

o Mark (X) this box if you aunch a continuation sbeet. 

6. 	 you claim chemical liS con you mUSl provide a generic name for your subst:mce (hu( reveals 

the specific chemical identity of the new chemical substance to the ma)(imum extent possible. Refer to (he 

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

Quaternary ammonium hydroxide 

resulting from the manufacture. processing. use, or dispOSll I of the new chemical 

ct CAS 

.lIIach a continuation sheet. 

antiCipated 

EPA FORM 7710-25 (Rell.5-95) 	 I)age 6 
Part I -- GENERAL INFORMATION -- Continued 

Section C -- PRODUCTION, IMPORT, AND USE INFORMATION: 

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

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

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

substance basis. For a Low Volume EXemption application, if you choose to have your notice reviewed at a lower production volume than 10,000 

kg/yr, specifY the volume and mark (x) in the binding box. Ifgranted, you are bound to this volume 

Maximum fust 12-month production (kglyr) Maximum 12-month production (kg/yr) Conti- Binding 

(100% new chemical substance basis) (100% new chemical substance basis) dential Option 

Mark (x) 

X X 

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

devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item 

you claim as confidential. 

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

(2) -­ Mark (X) this column ifentry column (l) is confidential business information (CBI). 

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

(4) -­ Estimate the percent oftotal production for the first three years devoted to each category of use. 

(5) -­ Mark (X) this column ifentry in column (4) is confidential business information (CBI). 

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

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

(7) -­ Mark (X) this column ifentry in column (6) is confidential business information (CBI). 

(8) -­ Indicate % of product volume expected for the listed "use" sectors. Mark more than one box if appropriate. Mark (X) to indicate 

your willingness to have the use type provided in (8) binding. 

(9) -­ Mark (X) this column ifentr y(ies) in column (8) is (are) confidential business information (CBI). 

Category of use (I) CBI Binding Produc- CBI %in CBI % of substance expected per use CBI 

Option tion% Form­ (8) 

(by function and application i.e. a dispersive dye Mark (x) uIation Site- E Indus- Com- Binding 

for finishing polyester fiberS) (2) (3) (4) (5) 

± 

limited trial mercia! Option (9) 

X X X X 

% 

% % 

% % 

% % 

% % 

% % 

% % = .. If you have identified a "consumer" use, please provide on a continuation sheet a detailed description ofthe use(s) of this chemical substance in consumer products. 

In addition include estimates ofthe concentration ofthe new cllemical substance as expected in consumer products and describe the chemical reactions by which this 

substance loses its identity in the consumer product. 

DMark (X) this box ifyou attach a continuation sheet. 

b. Generic Ifyou claim any category of use description in subsection 2a as confidential, enter a generic description of that category. Read the 

use Instructions Manual for examples ofgeneric use descriptions. 

description Destructive use as a fuel additive. 

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

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

information wllich will be provided to any person wllo is reasonably likely to be exposed to this substance regarding protective equipment or practices Option 

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

D Mark (X) tllis box if you attach hazard information. X 

Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

EPA FORM 7710-25 (Rev. 5-95) Page 7 
x 

Section A you -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER 

Complete section A for each type of manufacture, processing, or use operation invo new at mdustrial 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 after' You must describe these See instructions manual 


1. Confiof the site at which the nnF'r"t,nn will occur. dential 

City, 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 


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


sites as attachments. 


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

Mark (X) 

b. 	 Type-­ x 

DProcessing 	 Duse 

x 

I. Batch 

Maximum kglbatch (100% new chemical Hourslbatch B atches/year 

substance) 

2. Continuous 

d. Process description Mark (X) to indicate your Willingness to have your process description binding. 

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


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


(2) 	 Provide the identity, the approximate weight (by kg/day or kglbatch on a 100"10 new chemical substance basis), and entry point of all starting materials and 

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

used daily or per batch.). 

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

this box if you attach a continuation sheet. 

Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

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

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

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

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

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

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

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

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

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

at the time of exposure. 

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

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

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

I 

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

(12) - Mark (X) this column ifentries in columns (IO) and (II) are confidential business information (CBI). 

i Worker activity eBl Protective Equipment! Binding Physical forms(s) Binding eBI # of eBI Maximu duration eBI 

m 

I 

(Le., bag dumping, filling drums) Engineering Controls Option and % new Option Workers Rrs/day Days/yr Mark (x) substance Marl< (x) Exposed 

(I) (2) (3) (4) (5) (6) (7) (8) (9) (IO) (II) (12) 

i Quality control sampling Gloves, face shield X X X X 

Drumming product Gloves, face shield X X X X 

Unit washout Gloves, safety X X X X 

aoaales 

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

3. Environmental Release and Disposal - You must make separate confidentiality claims for the release number and the amount of the new chemical substance 

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

(1) -- Enter the number of each release point identified in the process description, part 11, section A, subsection I d(3). 

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

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

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

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

(5) - a. Describe control technology, if any, and control efficiency that will be used to limit the release ofthe new substance to the environment. For releases 

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

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

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

(6) - Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CBl). 

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

NPDES numbers ofthe POTW (Publici Owned Treatment Works). Mark (X) if the POTW name or NPDES # is confidential business information (CBI). 

Release Amount of new substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CB] 

Number released release 

(1) (2a) (2b) e.g. stack air Binding 

(3) (4) (Sa) Mark (X) (5b) (6) 

X X X 

X X X 

(7) Mark (X) the U POTW provide name(s) below: CBI D Navigable D Other - Specify provide NPDES # CBI 

destination(s) of 7 waterway X releases to water. 

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

EPA FORM 7710-25 (Rev. 5-95) Page 9 
i Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers do not have to complete this 

section for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate 

section B for each type ofprocessing, or use operation involving the new chemical substance. If the same operation is performed at more than one site describe the 

typical operation common to these sites. Identify additional sites on a continuation sheet. 

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

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

drums, rail cars, tank trucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2) - Provide the identity, the approximate weight 

(by kg/day or kglbatch, on an 100% new chemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all 

products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not used daily or per batch). (3) --IdentifY by number the points of release, 

including small or intermittent releases, to the environment ofthe new chemical substance. (4) Please enter the # of sites (remember to identifY the locations of 

these sites on a continuation sheet): 

7 

# of sites 

[X] eBI 

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

2. Worker ExposnrelEnvironmental Release 

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

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

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

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

protect workers. 

(7) -- Estimate the percent ofthe 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 ofthe new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kglbatch). 

i (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, POTW, or other 

I 

(specifY) and control technology, ifany, that will be used to limit the release of the new substance to the environment. 

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

(3), (5), (8), (II), (131and (15) ­ Mark (X) this column if any of the proceediDlil entries are confidential business information (CBI). 

i Letter #of CBI Duration CBI Protective Equip. I %in CBI Release Amounlof CBI Media ofRelease CBI 

of Workers of Engineering Controlsl Form- Number New & Control i Act- EXPQSed Exposure Physical Form and % ulation Substance Technology 

ivily new substance Released 

(I) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (lOa) (lOb) (II) (12) (13) 

X X X X X 

i 

14) Byproducts: (15) 

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

EPA FORM 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 

ofthe PMN substance. Please include new information pertinent to pollution prevention, including source 

reduction, recycling activities and safer processes or products available due to the new chemical substance. 

Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological 

modification, process and procedure modification, product reformulation, raw materials substitution, and/or 

inventory controL Recycling refers to the reclamation ofuseful chemical components from wastes that would 

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

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

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

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 ofthe PMN substance to potential 

alternatives may be provided. 

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

substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and 

examples. 

Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in the 

volume manufactured; (3) a reduction in the generation of waste materials through recycling, source reduction or other means; (4) a 

reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in 

the cost ofproduction and/or improved operation efficiency ofthe new chemical substance in comparison to existing chemical 

substances used in similar application; 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. 

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

EPA FORM 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. Clearly identify the attachment and the section of the form to 

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

numbers of each attachment. 

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

page number(s) 

134-141 

Material Safety Data Sheet (MSDS) 

Inventory Expert Service Report 133 

Confidential 

Magnusson & Klingman Maximisation Study in the Guinea Pig 14-64 

Primary Skin Irritation - Rabbits 65-76 

Acute Oral Toxicity, LD50 - Rats 77-105 

Acute Toxicity to Photobacterium phosphoreum (Screen test) 106-113 

Acute Toxicity to Sheepshead Minnow 114-121 

Acute Toxicity to Fathead Minnow 122-129 

Raw Ready Biodegradability Closed Bottle Test data 130 

131-132 X 

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

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

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number 

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(g) 

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jM.easured or ConliEstimate dential 

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E 

M 

M 

ATTACHMENT 


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CONFIDENTIAL 


STUDY SPONSOR: 


ENV93 p1201 : 


MAGNUSSON &KLIGMAN MAXIMISATION 


STUDY IN THE GUINEA PIG 


PROJECT NUMBER: 654/1 


AUTHOR: ' R. Driscoll 

ISSUED BY: 


Safepharm Laboratories Limited 


P.O. Box No. 

DERBY 

DEI 2BT 

U.K. 

Telephone: 

Facsimile: 

Telex: 

45 

DERBY (0332) 792896 

(0332) 799018 

PAGE 1 OF 51 PAGES 
 377079 SAFPHM G
III 

• 

-2­

PROJECT NUMBER: 654/1 

la 

QUALITY ASSURANCE REPORT .It 

•'..... ~i' 

•. 

'" 

The routine inspection of short term studies at Safepharm Laboratories is 

~':.i\e ··· carried out as a continuous process designed to encompass all major phases of I

I each study type once per month~ Dates of the most recently completed series 

of monthly inspections relevant to the study type(s) in this report are given 

below. 


I 
 Date{s) of Inspection and Reporting: 


I 
 01, 09, 15, 16, 21, 24, 28 February 1994 


'1 This report has been audited by Safepharm Laboratories Quality Assurance Unit . 

.. It is considered to be an accurate account of the data generated and of the 

I procedures followed. 

I 


'3
 .•. Date of Report Audit: 

I 

~ 

~ 28 March 1994

I 

1 

I J.R. Paternan C. Biol., M.L Biol. 


FOR SAFEPHARM QUALITY ASSURANCE UNIT 


1 

DATE: ........................ ·······13D·O·O 1 5 

PROJECT NUMBER: 654/1 


GlP COMPLIANCE STATEMENT 

I, the undersigned, hereby declare that the objectives laid down in the 

protocol were achieved and as nothing occurred to adversely affect the 

quality or integrity of the study, I consider the data generated to be 

valid. This report fully and accurately reflects the procedures used and 

data generated. 

The work described was performed in compliance with the UK Principles of Good 

Laboratory Practice (The United Kingdom Compliance Programme, Department of 

Health 1989). These Principles are in accordance with GlP standards 

published by DECO in 1982; and are in conformity with, and implement, the 

requirements of Directives 87/18/EEC and 88/320/EEC. 

,These international standards are acceptable to the United States 

Environmental Protection Agency and Food and Drug Administration, and fulfil 

the requirements of 40 CFR Part 160, 40 CFR Part 792 and 21 CFR Part 58 (as 

amended) . 

2,-Li--Q l, . ........~~~~.~ .............. DATE: ..............\ .... \~ ....... ,. ..... 


R. Driscoll B.Tech. (Hons.) 

Study Director 

for Safepharm Laboratories 

000016 

PROJECT NUMBER: 654/1 

CON TEN T S 

PAGE 

SUMMARY 7

1. INTRODUCTION 9

2. TEST AND POSITIVE CONTROL MATERIALS 10 


2.1 Test Material 10 


2.2 Positive Control Material 10 


3. TEST SYSTEM 11 


3.1 Specification 11 


3.2 Husbandry 11 


4. PROCEDURE 11 


4.1 Selection of Concentrations for Main Study (Sighting Tests) 11 


4.2 Main Study 13 


5. EVALUATION OF DATA 18 


6. ARCHIVES 19 


7. RESULTS 20 


7.1 Intradermal and Topical Sighting Tests 20 


7.2 Main Study 20 


7.3 Bodyweight 23 


8. CONClUS ION 24 


TABLES 


TABLE 1 Individual Skin Reactions in Test Animals at Challenge: 


KEY TO TABLES 25 


26 

ENV 93 1201 Study ·····11 

'. 

'--, TABLE 2 Individual Skin Reactions in Control Animals at Challenge: 27 

ENV 93 1201 Study 

·.... TABLE 3 Individual Skin Reactions in Test Animals at Cha 11 enge: ......·.l 28
 ....! 

ONCS Study 

TABLE 4 Individual Skin Reactions in Control Animais at Challenge: 29 
 .-.•....•....~ .~,,' ONCS Study 

O 'J 
 ,"'I r"'I ," " -" 
 , I r I I, , ,) ....) J ; I 

-;)­

PROJECT NUMBER: 654/1 

CON TEN T S (continued) 

- PAGE 

30 APPENDICES 

31 KEY TO APPENDICES 

APPENDIX I Intradermal Sighting Test - Summary of Results: 32 

ENV 93 1201 Study 

APPENDIX II Intradermal Sighting Test - Summary of Results: 33 

DNCS Study 

APPENDIX III Topical Sighting Test For Induction Application - 34 

(48-hour Exposure) - Individual Skin Reactions: 

ENV 93 1201 Study 

APPENDIX IV Topical Sighting Test for Induction Application - 35 

(48-hour Exposure) - Individual Skin Reactions: 

DNCS Study 

APPENDIX V Topical Sighting Test For Challenge Application - 36 

(24-hour Exposure) - Individual Skin Reactions: 

ENV 93 1201 Study 

APPENDIX VI Topical Sighting Test For Challenge Application - 37 

(24-hour Exposure) - Individual Skin Reactions; 

ONCS Study 

APPENDIX VII Intradermal Induction· Individual Si<in Reactions 39 

in Test Animals: ENV 93 1201 Study 

APPENDIX VIII Intradermal Induction - Individual Si<in Reactions 40 

in Control Animals: ENV 93 1201 Study 

APPENDIX IX Intradermal Induction - Individual S~in Reactions 41 

in Test Animals: ONeS Study 000018 
-6­

PROJECT NUMBER: 654/1 

CON TEN T S (continued) 

PAGE 


APPENDICES (contd) 

APPENDIX X Intradermal Induction - Individual Skin Reactions 42 

in Control Animals: ONCS Study 

APPENDIX XI Topical Induction - Individual Skin Reactions 43 

in Test Animals: ENV 93 1201 Study 

APPENDIX XII Topical Induction - Individual Skin Reactions 44 

in Control Animals: ENV 93 1201 Study 

APPENDIX XIII Topical Induction - Individual Skin Reactions 45 

in Test Animals: ONCS Study 

APPENDIX XIV Topical Induction - Individual Skin Reactions 46 

in Control Animals: ONCS Study 

APPENDIX XV Individual Sodyweights and Sodyweight Gains 47 

of Test Animals: ENV 93 1201 Study 

APPENDIX XVI Individual Bodyweights and Bodyweight Gains 48 

of Control Animals: ENV 93 1201 Study 

APPENDIX XVI I Individual Bodyweights and Bodyweight Gains 49 

of Test Animals: DNCS Study 

APPENDIX XVIII Individual Bodyweights and Bodyweight Gains 50 

of Control Animals: ONeS Study 

APPEND IX XIX GLP Statement of Compliance "'1 -,,,, 

c(,-.r-,49

I ~J UU l ... 
i 

i 

-7­

PROJECT NUMBER: 654/1 

SUMMARY 

-

STUDY SPONSOR 	 PETROLITE CORPORATION 

, 

PROJECT NUMBER 	 654/1 

TEST 	 MATERIAL ENV 93 1201 

1. 	 A study was performed to assess the skin contact sensitisation potential 

of the test material in the albino guinea pig. The method used followed 

that described in the OECD Guidelines for Testing of Chemicals No. 4Q6 

"Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission 

Directive 92/69/EEC (which constitutes Annex V of Council Directive 

67/548/EEC) . 

The results may be used as a basis for classification and labelling 

under Annex VI of Council Directive 67/548/EEC (as adapted to technical 

progress by Commission Directive 91/325/EEC).. 

2. 	 Twenty test and ten control animals were used for the main study. 

Based on the results of sighting tests, the concentrations of test 

material for the induction and challenge phases were selected as 

fa 11 ows: 

Intradermal Induction 	 0.1% w/v in distilled water 

Topical Induction 10% v/v in distilled water 

Topical Challenge 5% and 1% v/v in distilled water 

3. 	 The test material produced a 0% (0/20) sensitisation rate and was 

classified as a non-sensitiser to guinea pig skin. The test materiai 

was also classified as a non-sensitiser according to EC labelling 

regulations. No risk phrase is required. 

000J20 
-0­

PROJECT NUMBER: 654/1 


SUM MAR Y {continued 

4. 	 Aconcurrent study using ten test and five control animals was performed 

with the known positive contact sensitiser, 2,4-Dinitrochlorobenzene 

(ONCS). Based on the results of sighting tests the concentrations of 

positive control material for the induction and challenge phases were 

selected as follows: 

Intradermal Induction 0.1% w/v in arachis oil B.P. 

Topical Induction 0.75% w/v in absolute ethanol 

Topical Challenge 0.75% and 0.5% w/v in absolute ethanol 

The positive control material, ONCS, produced a 100% (10/10) sensitisation rate. The positive reactions seen with ONCS were consistent 

with the known contact ~ensitisation potential of the material. 

000021 

-9­

PROJECT NUMBER: 654/1 


ENV 93 1201: 


MAGNUSSON &KLIGMAN MAXIMISATION 


STUDY IN THE GUINEA PIG 


1. INTRODUCTION 

This study was performed to assess the skin contact sensitisation 

potential of the test material (Safepharm Definitive Protocol Number 

654/1). The method used followed the recommendations of the OECD 

Guidelines for the Testing of Chemicals No. 406 "Skin Sensitisation" 

(adopted 17 July 1992) and Method 86 of Commission Directive 92/69/EEC 

(which constitutes Annex V of Council Directive 67/548/EEC). 

The results may be used as a basis for classification and labelling 

under Annex VI of Council Directive 67/548/EEC (as adapted to technical 

progress by Commission Directive 91/325/EEC). 

The test system was chosen because the guinea pig has been shown to be a 

suitable species for this type of study and is recommended in the test 

method. The strain used in these laboratories has been shown to produce 

a satisfactory sensitisation response using known positive sensitisers. 

The results of the study are believed to be of value in predicting the 

likely contact sensitisation potential of the test material to man. 

The study was conducted in accordance with the internationally accepted 

general principles of Good Laboratory Practice and Safepharm Standard 

Operating Procedures. 

The study was performed between 7 February 1994 and 12 March 1994. 

000022 
-.I.VPROJECT NUMBER: ~54/1 

2. 	 TEST AND POSITIVE CONTROL MATERIALS 

2.1 	 Test Material 

a} Description. Identification and Storage Conditions 

The test material was supplied by the study sponsor as follows: 

Sponsor's identification ENV 93 1201 


Batch number 93-73731 and 93-73732 


Activity 70% 


Date received 26 January 1994 


Description brown slight1y viscous liquid 


Container c1ear glass bottle 


Storage conditions room temperature 


Data relating to the identity. purity and stability of the test 


material are the responsibility of the sponsor. 


b) Method of Preoaration 


For the purpose of this study the test material was freshly pre­


pared as follows: 


Intradermal Induction 	 0.1% wlv in distilled water 

0.1% w/v in a mixture of Freund's 

Complete Adjuvant plus distilled water 

(l: 1) 

Topical Induction 10% v/v in distilled water 

Topical Challenge 5% and 1% v/v in distilled water 

The concentration, homogeneit~ and stability of the formulations 

were not determined by analysis. 

2.2 	 Positive Control Material 

The positive control material 2,4-dinitrochlorobenzene (ONCS) was 

supplied by Sigma, Poole, Dorset, U.K. 

For the purpose of the study the positive controi material was 

freshly prepared as follows: 

000023 
-11­

PROJECT NUMBER: 654/1 


2.2 	 Positive Control Material (contd) 

Intraderma 1 Induct ion 0.1% w/v in arachis oil 

0.1% w/v in a mixture of Freund's 

Complete Adjuvant plus distilled water 

(1: 1) 

Topical Induction 0.75% w/v in absolute ethanol 

Topical Challenge 0.75% and 0.5% w/v in absolute ethanol 

3. 	 TEST SYSTEM 

3.1 	 Specification 

Sixty-four female, albino Dunkin-Hartley guinea pigs were supplied 

by David Hall limited, Burton-an-Trent, Staffordshire, U.K. At the 

start of the main study the animals weighed 337 - 444g, and were 

approximately eight to twelve weeks old. After a minimum acclimatisation period of five days, each animal was selected at random 

and given a number unique within the study which was written on a 

small area of clipped rump using a black indelible marker-pen. 

3.2 	 Husbandry 

The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodf1akes. Free access to mains tap 

water and food (Guinea Pig FDI Diet, Special Diets Services 

limited, Witham, Essex, U.K.) was allowed throughout the study. 

The animal room was maintained at a temperature of 20 - 23~C and 

relative humidity of 46 - 67%. The rate of air exchange was 

approximately 15 changes per hour and the lighting was controlled 

by a time switch to give 12 hours light and 12 hours darkness. 

4. 	 PROCEDURE 

The method used for assessing the sensitising properties of the test 

material was based on the Guinea Pig Maximisation test of Magnusson B. & 

Kligman A.M., J. Invest. Dermatol. (1969) 52: 268 - 276. 

4.1 	 Selection of Concentrations for Main Study (Siahtino Tests) 

The concentrations of test material and ONeS to be used at each 

stage of the main study were determined by 'sighting tests' in 

which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows: OI I~l n ~ I I 0'. ~.J ~.J . 
-12­

PROJECT NUMBER: 654/1 


4.1 Selection of Concentrations for Main Study (Sighting Tests) 

4.1.1 Test Material 

a) Selection of Concentration for Intradermal Induction 

Four animals were intradermally injected with preparations of test 

material (O.l%, 0.5%, 1% or 5% w/v in distilled water). The degree 

of erythema at the injection sites was assessed approximately 24, 

48 and" 72 hours and 7 days after injection according to the Draize" 

scale shown in Section 4.2.1a. The degree of oedema was not 

evaluated. Any evidence of systemic toxicity was also recorded. 

The highest concentration that did not cause local necrosis, 

ulceration, severe irritation or systemic toxicity, was selected 

for the intradermal induction stage of the main study. 

b) Selection of Concentration for Topical Induction 

Two guinea pigs (intradermally injected with Freund's Complete 

Adjuvant 9 days earlier) were treated with four preparations of the 

test material (10%, 5%, 2% and 1% v/v in distilled water). 

Two more guinea pigs (intradermally injected with Freund's Complete 

Adjuvant twelve days earlier) were treated with two concentrations 

of the test material (50% and 25% w/v in distilled water). These 

animals were killed for humane reasons immediately after dressing 

removal due to severe dermal necrosis. 

The h.ighest concentration producing only mild to moderate dermal 

irritation after a 48-hour occlusive exposure, was selected for the 

topical induction stage of the main study. 

c) Selection of Concentration for Topical Challenae 

Four preparations of the test material (10%, 5%, 2~' and 1% v/v in 

distilled water) were applied occlus;vely to the flanks of two 

guinea pigs for a period of 24 hours. These guinea pigs did not 

form part of the main study but had been treated identically to 

the control animals of the main study, up to Day 14. The highest 

non-irritant concentration of the test material and one lower 

concentration were selected for the topical challenge stage of the 

main study. 000025 
-13­

PROJECT 'NUMBER: 654/1 


Induction 

4.1 Selection of Concentrations for Main Stud 

4.1.2 Positive Control Material 

a) Selection of Concentration for Intradermal 

Three animals were intradermally injected with preparations of test 

material (1%, 0.5% or 0.1% w/v in arachis oil B.P.). The injection 

sites were evaluated as in Section 4.1.1a. The highest concentration that did not cause 10ca1necrosis, ulceration~1 ~evere 

irritation or systemic toxicity, was selected for the intradermal 

induction stage of the main study. 

b) Selection of Concentration for Topical Induction 

Two guinea pigs (intraderma1ly injected with Freund's Complete 

Adjuvant 9 days earlier) were treated with four preparations of the 

test material (3%, 1.5%, 0.75% and 0.3% w/v in absolute ethanol). 

The highest concentration producing only mild to moderate dermal 

irritation after a 48-hour occlusive exposure, was selected for the 

topical induction stage of the main study. 

c} Selection of Concentration for Topical Challenge 

Four preparations of the test material (0.75%, 0.5%, 0.25% and 0.1% 

w/v in absolute ethanol) were applied occ1usively to the flanks of 

two guinea pigs for a period of 24 hours. TheSe guinea pigs did 

not form part of the main study but had been treated identically to 

the control animals of the main study, up to Day 14. Two more 

guinea pigs, which had been intradermally injected with Freund's 

Complete Adjuvant 11 days.,earlier, were treated with four 

concentrations of the test material (2%, 1.5%, 1% and 0.75% w/v in 

absolute ethanol). The highest non-irritant concentration of the 

test material and one lower concentration were selected for the 

topical challenge stage of the main study. 

~ 

I 

, ···'· ' 4.2 Main Study 

A total of forty-five guinea pigs were used for the study: twenty 

test and ten control animals were used to determine the skin 

sensitisation potential of the test material and a further ten test 

I 

B and five control animals comprised the concurrent positive contro' ..".·.·. r study. n n n ';) f~ 
-14­

PROJECT NUMBER: 654/1 

4.2 	 Main Study (contd) 

4.2.1 Test Material 

a) Induction of the ENV 93 1201 Test Animals 

Shortly before treatment on Day a the hair was removed from an area 

approximately 40 mm x 60 mm on the shoulder region of each animal 

with veterinary clippers. A row of three injections (0.1 ml each) 

was made on each side of the mid-line. The injections were: 

. i) 'Freund's Complete Adjuvant'plus distilled wah~~in'~fhe 

ratio 1:1 

ii) a 0.1% w/v dilution of test material in distilled water 

iii) a 0.1% w/v dilution of test material in a 1:1 mixture of 

Freund's Complet~ Adjuvant plus distilled water 

Approximately 24 and 48 hours after intradermal injection the 

degree of erythema at the test material injection sites (i.e. 

injection site ii) was evaluated according to the following scale 

from Draize J.H., (1977) "Dermal and Eye Toxicity Tests ll In: 

Principles and Procedures for Evaluating the Toxicity of Household 

Substances, National Academy of Sciences, Washington D.C. p.31: 

I 

EVALUATION OF SKIN REACTIONS 

Erythema and Eschar Formation Value 

No erythema ................................................... 0 

Very slight erythema (barely perceptible) ..................... 1 

We 11 -defi ned erythema ......................................... 2 

Moderate to severe erythema ................................... 3 

Severe erythema (beet redness) to slight eschar formation 

(injuries in depth) .......................................... 4
 I 

Oedema Formation

I No oedema ..................................................... 0 

Very slight oedema (barely perceptible) ....................... 1 

, 

, Slight oedema (edges of area well-defined by definite 

ra1 slng) ..................................................... 2 

Moderate oedema (raised approximately 1 millimetre) ........... 3 

I 

Severe oedema (raised more than 1 millimetre and extending 

beyond the area of exposure) ............. , ................ _.. 4 

000027 
(contd) 

-15­

PROJECT, NUMBER: 654/1 


4.2.1 Test Material (contd) 

a) Induction of the ENV 93 1201 Test Animals 

One week later (Day 7), the same area on the shoulder region used 

previously for intradermal injections was clipped again and treated 

with a topical application of the test material formulation (10% 

,;i,v/v'in distilled water). The test material formulation was applied 

to saturation on filter paper (WHATMAN No.4: approximate size 40 mm 

x 20 mm) which was held in place by a strip of surgical adhesive 

tape (BLENOERM: approximate size 50 mm x 30 mm) and covered with an 

overlapping length of aluminium foil. The patch and foil were 

further secured by a strip of elastic adhesive bandage 

(ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double 

layer around the torso of each animal. This occlusive dressing was 

kept in place for 48 hours. 

The degree of erythema and oedema was quantified one and twenty~ 

four hours following removal of the patches using the Draize scale 

shown previously. 

Any other reactions were also recorded. 

b) Induction of the ENV 

Intradermal injections 

93 1201 Control Animals 

were administered using an identical 

procedure to that used for the test animals, except that the 

injections were: 

;) Freund's Complete Adjuvant plus distilled water in the 

ratio 1:1. 

ii) distilled water. 

iii) 50% w/v distilled water in a 1:1 mixture of Freund's 

Complete Adjuvant plus distilled water 

The topical applications followed the same proceaure as for the 

test animals except that the vehicle alone was applied to the 

filter paper. Skin reactions were quantified as for the test 

animals. rU' ('; Ii I, .--:, 0 

'.) 'J i.,.; L. u 
-16­

PROJECT ~UMBER: 654/1 

4.2.1 Test Material (contd) 

c) Challenge of ENV 93 1201 Test and Control Animals 

Shortly before treatment on Day 21, an area, approximately 50 mm x 

70 mm on both flanks of each animal, was clipped free of hair with 

veterinary clippers. 

A quantity of 0.1 - 0.2 ml of the test material formulation (5% v/v 

in distilled water) was applied to the shorn right flank of each 

animal on a square of filter paper (WHATMAN No.4: approximate size 

20 mm x 20 mm) which was held in place by a strip of surgical 

adhesi ve 'tape (BLENDERM: approximate size 40 mm x 50mm). To ensure 

that the maximum non-irritant concentration was used at challenge, 

the test material at a concentration of 1% v/v in distilled water 

was also similarly applied to a skin site on the left shorn flank. 

The patches were occluded with an overlapping length of aluminium 

foil and secured by a strip of elastic adhesive bandage 

(ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double 

layer around the torso of each animal. 

After 24 hours, the dressing was carefully cut using blunt-tipped 

scissors, removed and discarded. The challenge sites were swabbed 

with cotton wool soaked in diethyl ether to remove residual 

material. The position of the treatment sites was identified by 

using a black indelible marker-pen. 

Prior to the 24-hour observ~tion the flanks were clipped using 

veterinary cl ;ppers to remove regrown hair. 

d) Evaluation of Skin Reactions 

The degree of erythema and oedema was quantified approximately 24 

and 48 hours after challenge dressing removal using the Draize 

scale shown in Section 4.2.1a. 

Any other reactions were also recorded. 

000029 

I 


I 

7 

-17­

PROJECT NUMBER: 654/1 


4.2.2 Positive Control Material 

a) Induction of ONCS Test Animals 

Shortly before treatment on Day 0 the hair was removed from an area 

approximately 40 mm x 60 mm on the shoulder region of each animal 

with veterinary clippers. A row of three injections (0.1 ml each) 

was made on each side of the mid-line. The injections were: 

i) Freund's Complete Adjuvant plus distilled water in the 


ratio 1:1 


ii) a 0.1% w/v dilution of ONCS in arachis oil B.P. 


iii) a 0.1% w/v dilution of ONCS in a 1:1 mixture of Freund's 


Complete Adjuvant plus distilled water 


Approximately 24 and 48 hours after intradermal injection the 

degree of erythema at the test material induction site (i.e. 

injection site ii) was evaluated using the Oraize scale shown in 

Section 4.2.1a. 

The topical applications for induction of the ONC8 test animals 

followed the same procedure as for the ENV 93 1201 animals except 

that ONCS at a concentration of 0.75% w/v in absolute ethanol was 

applied. 

b) Induction of ONCS Control Animals 

Intradermal injections were administered using an identical 

procedure to that used for the ONCS test animals, except that the 

injections were: 

i) Freund's Complete Adjuvant plus distilled water 

ii) arachis oil B.P. 

iii) 50% w/v arachis o"il B.P. in a 1:1 mixture of Freund's 

Complete Adjuvant plus distilled water 

The topical application for the ONCS control animals followed the 

same procedure as the ONCS test animals except that absolute 

ethanol was applied. 
-18­

PROJECT ,NUMBER: 654/1 


4.2.2 Positive Control Material (contd) 

c) Challenge of DNCS Test and Control Animals 

The challenge applications for the ONCS animals fallowed the same 

procedure as the ENV 93 1201 animals except that 0.1 - 0.2 ml of 

DNCS at concentrations of 0.75% and 0.5% w/v in absolute ethanol 

was applied to the skin. 

d) Evaluation of Skin Reactions 

The degree of erythema and oedema was quantified approximately 24 

and 48 hours after challenge dressing removal using the Draize 

scale shown in Section 4.2.1a. 

Any 	 other reactions were also recorded. 

5. 	 EVALUATION OF DATA 

The percentage of test animals that showed a more severe reaction at the 

test material challenge site than the most severe reaction seen in the 

control animals, was compared with the following scale: 

~ of animils 	 Classification of 

sensitiseg 	 sensitisation Qotential 

a non-sensitiser 

> a 8 weak sensitiser 

> 8 - 28 mild sensitiser 

> 28 - 64 moderate sensitiser 

> 64 - 80 strong sensitiser 

> 80 - 100 extreme sensitiser 

The data obtained may be used to classify the test material under the 

Council Directive 67/548/EEC, as amended by Commission according to 

Directive 91/325/EEC, on the classification, packaging and labelling of 

dangerous substances. These classification criteria are also in the UK 

Approved Code of Practice "Classification and Labell ing of Substances 

Dangerous for Supply~. 

A response in at least 30% of the animals is considered positive and the 

risk phrase R 43 "MAY CAUSE SENSITISATION BY SKIN CONTACT" is required. 

000031 

-19­

PROJECT NUMBER: 654/1 

I:o \...I/!;, 

-..t L,1',{! lJ 

6. 	 ARCHIVES 1./ (.!:!J 

Unless instructed otherwise by the sponsor, all original data and61J~pp' ' 

4...J," ~ l 	 . 

of the final report will be retained in the archives of Safepharm(J~ 

Laboratories for a period of ten years. After this period, the 

sponsor's instructions will be sought. 
-20­

654/1 

7. 	 RESULTS 

7.1 	 Intradermal and Topical Sightino Tests 

A summary of the results of the intradermal sighting test and~e 

individual skin reactions observed in the topical sighting tests , 

are given in Appendices I to VI. 

Based on these results the following concentrations were selected 

for the main study. 

Test Material [NV 93 1201 

Intradermal Induction 0.1% w/v in distilled water 

Topical Induction 10% v/v in distilled water 

Topical Challenge 5% and 1% v/v in distilled water 

Positive Control Material ONCS 

Intradermal Induction 0.1% w/v in arachis oil B.P. 

Topical Induction . 0.75% w/v in absolute ethanol 

Topical Challenge 0.75% and 0.5% w/v in absolute ethanol 

7.2 	 Main Study 

7.2.1 Test Material 

a) Skin Reactions Observed After Intradermal Induction 

Individual reactions observed at the intradermal induction sites of 

test and control group animals are given in Appendix VII and VIII. 

Very slight to well-defined erythema was noted at the intradermal 

induction sites of all test group animals at the 24-hour observation and in nineteen test group animals at the 48-hour 

observation. 

No skin reactions were noted at the intradermal induction sites of 

control group animals at the 24 and 48-hour observations. 

b) Skin Reactions Observed After Topical Induction 

Individual reactions observed at the topical induction sites of the 

test and control group animals are given in Appendices XI and XII. 

000033 
-21­

PROJECT NUMBER: 654/1 

7.2.1 Test Material (contd) 

",", . , b) Skin Reactions Observed After Topical Induction (contd) ."1....:, 

Very slight to moderate to severe erythema with or without very 

slight to slight oedema was noted at the induction sites of all 

test group animals at the l-hour observation. Very slight to well­

"defined erythema with or without very slight oedema was noted at 

the induction sites of seventeen test group animals at the 24-hour 

observation. Other skin reactions noted were bleeding caused by 

animal scratching intradermal induction site, dried blood, 

desquamation, hardened dark coloured scab and small scattered 

scabs. Skin reactions prevented accurate evaluation of erythema at 

the induction sites of three test group animals at the 24-hour 

observation. 

j. • "~ ( 

Very slight erythema with or without very slight oedema was noted 

at the treatment sites of seven control group animals at the l-hour 

observation. No skin reactions were noted at the treatment sites 

of control group animals at the 24-hour observation. 

c) Skin Reactions Observed After Topical Challenae 

Individual reactions at the challenge sites of test and control 

group animals are given in Tables land 2. 

No skin reactions were noted at the challenge sites of the test or 

control group animals at the 24 and 48-hour observations. 

7.2.2 Positive Control Material (DNCB) 


a) Skin Reactions Observed After Intradermal Induction 


Individual reactions observed at the intradermal induction sites of 


test and control group animals are given in Appendices rx and X. 


Very slight to well-defined erythema was noted at the intradermal 

induction sites of all ONe8 test group animals at the 24 and 48­

hour observations. 

000034 
654/1 

Induction (contd) 

-22­

Positive Control Material (ONCSl (contd) 

a) Skin Reactions Observed After Intradermal 

Very slight erythema was noted at the intradermal induction sites 

of all control group animals at the 24-hour observation and in four 

control group animals at the 48-hour observation. 

b) Skin Reactions Observed After TODical Induction 

Individual reactions observed at the topical induction sites of 

test and control group animals are given in Appendices XIII and 

XIV. 

Yellow-coloured staining caused by the ONCS was noted at the 

induction sites of all ONCS test group animals at the 1 and 24-hour 

observations. The staining did not affect evaluation of skin 

responses. 

Well-defined to moderate to severe erythema and slight oedema were 

noted at the induction sites of all ONCS test group animals at the 

I-hour observations with well-defined erythema and slight oedema at 

the 24-hour observation. 

Very slight erythema was noted at the treatment site of one control 

group animal at the I-hour observation. No skin reactions were 

noted at the treatment sites of control group animals at the 24­

hour observation. 

c) Skin Reactions Observed After Topical Challenge 

Individual reactions at the challenge sites of test and control 

group animals are given in Tables 3 and 4. 

Pale yellow-coloured staining caused by the ONeS was noted at the 

challenge sites of all test and control group animals at the 24 and 

48-hour observations. The staining did not affect evaluation of 

skin responses. 

n '0 ':; r.: OI O'...;' '-"-..)
-23­

PROJECT NUMBER: 654/1 


7.2.2 Positive Control Material (ONCS} (contd) 

c) Skin Reactions Observed After Topical Challenoe (contdJ 

0.75% w/v in Absolute Ethanol 

Positive skin responses, well-defined erythema (grade 2) with or 

without very slight to slight oedema were noted at the challenge 

sites of nine test group animals at the 24 and 48-hour 

observations. Other incidents of skin reactions noted were dark 

brown-coloured scab, small· superficial scabs and well-defined 

erythema surrounding other skin reactions. Skin reactions prevented 

accurate evaluation of erythema and oedema at the challenge site of 

one test group animal at the 24 and 48-hour observations. 

Very slight erythema was noted at the challenge site of one control 

group animal at the 24 and 48-hour observations. 

O.5~ w/v in Absolute Ethanol 

Positive skin responses, well-defined erythema (grade 2) with or 

without very slight to slight oedema were noted at the challenge 

sites of all test group animals at the 24-hour observation and at 

the challenge sites of six test group animals at the 48-hour 

observation. Very slight erythema with or without very slight 

oedema was noted at the challenge sites of four test group animals 

at the 48-hour observation. Isolated incidents of small superfical 

scabs were also noted. 

Very sl ight erythema was noted at the challenge sites of t'l~O 

control group animals at the 24 and 48-hour observations. 

7.3 	 Bodvweight 

Individual bodyweights and bodyweight gains of test and control 

group animals of the ENV 93 1201 and ONeS studies are given in 

Appendices XV to XVIII. 

Bodyweight gains of guinea pigs in the test groups, between Day 0 

and 	 Day 24, were comparable with those observed in the concurrent 

control group animals over the same period. 

000036 
-24­

654/1 

8. CONCLUSION 

The test material, ENV 93 1201, produced a 0% (0/20) 


and was classified as a NON-SENSITISER to guinea pig skin. 


The test material was also classified as a non-sensitiser according to 

EC labelling regulations. No risk phrase is therefore required. 

The positive control material, ONCS, produced a 100% (l0/10) sensitisation rate. The positive reactions seen with ONCS were consistent 

with the known contact sensitisation potential of the material. 

000037 
-25­

KEY TOT A 8 L E S 


PROJECT NUMBER: 654/1 


Er = erythema 


Oe = oedema 


- = no other skin reactions 


Ss = small superficial scabs 


St = dark brown-coloured scab 


STA = yellow-coloured staining at challenge site not affecting 

evaluation of skin responses 

We = well-defined erythema surrounding other skin reactions 

?e =evaluation of erythema precluded by other skin reactions 

?Od = evaluation of oedema precluded by other skin reactions 

000038 

[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

TAO t E I JNOIVIOlJAL SKIN REACTIONS IN TEST ANIMALS AT CHALLENGE: ENV 93 1201 STUDY 

CIlAU[NGE CONCENTRATIONS: 5% AND 1% v/v VEtIICLE: DISTILLED WATER 

Skin Reactions (Hours After Removal of Dressing) 24 hours 48 hours Animal Number 1% 5% 1% 5% 

Er Oe Other Er Oe Other Er Oe Other Er Oe Other 

1 0 0 0 0 0 0 0 0 

2 0 0 0 0 0 0 0 0 

3 0 0 0 0 0 0 0 0 N 

I 

0'1 4 o . 0 0 0 0 0 0 0 I 

o . 5 0 0 0 0 0 0 0 

. 0 6 0 0 0 0 0 0 0 


7 0 0 0 0 0 0 0 0 


8 0 0 0 0 0 0 0 0 


9 0 0 0 0 0 0 0 0 


10 0 0 0 0 0 0 0 0 

11 0 0 0 0 0 0 0 0 

12 0 0 0 0 0 0 0 0 '"1;l

;;c 13 0 0 0 0 0 0 0 0 ~ 14 0 0 0 0 0 0 0 0 r'T1 

15 0 0 0 0 0 0 0 0 -~

16 0 0 0 0 0 0 0 0 :z 

c: 17 0 0 0 0 0 0 0 o b-. :I:

0 -f'i t= 18 0 0 0 0 0 0 0 o .~ r'T1 

0 19 0 0 0 0 0 0 0 o .~ ..

:;0 

0 

0 

20 0 0 0 0 0 0 0 o c-~] 01 

0'1 

(J) -'"' 11< ­

...... ill 
ENV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

T A 8 L. [ 2 INDIVIDUAL. SKIN REACTIONS IN CONTROL ANIMALS AT CIIALLENGE: ENV 93 1201 STUDY 

CIIALL.ENGE CONCftHRATIONS: 5% AND 1% v/v VEIIICLE: DISTILLED WATER 

Skin Reactions (ltours After Removal of Dressing) 

24 hours 48 hours Animal Number 1% 5% . 1% 5% 

Er Oe Other Er Oe Other Er Oe Other Er Oe Other 

21 0 0 0 0 0 0 0 0 N 

I 

"-J 

22 0 0 0 0 0 0 0 0 I 

23 0 0 0 0 0 0 0 0 

24 0 0 0 0 0 0 0 0 

25 0 0 0 0 0 0 0 0 

26 0 0 0 0 0 0 0 

27 0 0 0 0 0 0 0 -0 

28 0 0 0 0 0 0 0 2 

29 0 0 0 0 0 0 0 ~ ...,.j 

:iii!: 30 0 0 0 0 0 0 0 c::: 

:J: .=:;' CD -' 

0 ~ 

C) 

C) 0\

tn 

.,D ....... 


I-' Cl 
•• 

ENV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

TAD l E 3 INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CllALLENGE: DNCO STUDY 

CIiAlLENGE CONCENTRATIONS: 0.75% AND 0.5% w/v VEIlICLE: ADSOLUTE ETIlANOl 

Skin Reactions (Hours After Removal of Dressing) 24 hours 48 hours Animal Number 0.5% 0.75% 0.5% 0.75% 

Er Oe Other Er Oe Other Er Oe Other Er Oe Other 

31 2 0 STA 2 0 STA 2 0 STA 2 0 STA N

I 

(Xl 

32 2 1 STA 2 1 STA 1 1 STA 2 1 STA I 

33 2 1 STA 2 2 STA 2 1 STA 2 1 STA 

34 2 1 STA ?e ?Od StWeSTA 1 0 STA ?e ?Od StWeSTA 

35 2 1 STA 2 2 STA 2 1 STA 2 1 STA 

36 2 1 STA 2 1 STA 2 1 STA 2 1 STA 

37 2 2 STA 2 2 STA 2 1 STA 2 1 STA 

;;.a " 

30 2 2 STASs 2 2 STASs 2 I STASs 2 0 STASs E 

,." 

39 2 2 STA 2 2 STASs 1 1 STA 2 1 STASs n 

-I 

­ ;z: 40 2 1 STA 2 2 STA 1 0 STA 2 2 c::

:I: 

OJ 

,."

:;a 

0 

0 0'\ 

STA·6 

.g lT1 CJ ~ 

......... 


jooo& .r:::. 

~ 
ENV 93 1201 : HAGNUSSON &KLIGMAN MAXIHISATION STUDY IN TilE GUINEA PIG 

T A [} L E 4 INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS AT CIIALlENGE: DNCD STUDY 

CIIAttl:NG[ CONC[NTIU\TIONS: 0.75% ANn 0.5% w/v VEIlICLE.: ADSOlUTE. ETHANOL 

Skin Reactions (Hours After Removal of Dressing) 24 hours . 48 hours Animal Number 0.5% 0.75% 0.5% 0.75% 


Er Oe Other Er Oe Other Er Oe Other Er Oe Other 
 I 

N 

1.0 

I 

41 0 0 STA 0 0 STA 0 0 STA 0 0 STA 

42 0 0 STA 0 0 STA 0 0 STA 0 0 STA 

43 0 STA 0 0 STA 1 0 STA 0 0 STA 

44 0 STA 1 0 STA 1 0 STA 1 0 STA 

" 45 0 0 STA 0 0 STA 0 0 STA 0 0 STA .... ' 

;0 

~ m 

n 

-1 

z 

c:: 

0 co ~.' , m 

0 1'1 3: 

/'" ... :;0 

0 

C:J en ,t-, 01 

!~ t:," f 

(,,- ""­ r,) #' 
-30­

PROJECT NUMBER: 654/1 


A P .p END ICE S 

000043 

-31­

PROJECT NUMBER: 654/1 

KEY T 0 

Er :: erythema 

Oe :: oedema 

- :: no other skin reactions 

Ss = small scattered scabs 

St :: hardened dark-coloured scab 

STA :: yellow-coloured staining not affecting evaluation of 

skin responses 

?e = evaluation of erythema precluded by other skin reactions 

?s :: yellow-coloured staining prevented accurate evaluation 

of erythema 

Cr = superficial cracking of skin 

Bs :: bleeding caused by animal scratching intradermal induction site 

Bd :: dried blood 

D= desquamation 

000044 

[NV 93 1201 : HAnmSSON & KLIGMAN MAXIMISATION STUDY IN TIlE GUINEA PIG 

A P P f N 0 I X I INTlMOfRHAl SIGHTING TEST - SUMMARY OF RESULTS: ENV 93 1201 STUDY 

VEIIICl£: nISTILlED WATER 

An Concentration of ima1 Time of Grade of Eviden'ce Evidence of 


Test Material Erythema at . of local Systemic
 Identification Observation (% w/v) Inject ion SHe Necrosis Toxicity 

24 hours 2 - 4 Necrosis None 

A 48 hours 3 - 4 Eschar None

72 hours 3 - 4 Eschar None 

7 days 2 - 4 Eschar None 

I 

w 

N 

I 24 hours 4 Eschar None 

B 48 hours 4 Eschar None 5 72 hours 4 Eschar None 

7 days 4 Eschar None 

24 hours 2 - 3 None None 


E 48 hours O. 0-1 None None
 I '"C 72 hours 0 None None ;;0 

7 days 0 None None ~ E 

I !'1"\ 

n 

-1 

24 hours 2 - 4 Necrosis :z 

c::: None 0 x F 48 hours 0.5 2 - 4 Necrosis None c:l 

0 !'1"\ 72 hours 2 - 4 Necrosis None' J :;0 C) 7 days 4 Eschar None .. CO) 

0'1 

U1 .r:.. l ~ 

G"lThe ........
 concentration of the test material selected for the intradermal induction stage of the main study waif::­ ....... 


0.1% w/v in distilled water [r 
fNV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TUE GUINEA PIG 

A P P f N D I X I I INTRADERMAL SIGIITING TEST - SUMMARY OF RESULTS: ONes STUDY 

VftlIClf: ARACIIIS OIL D.P. 

Anima 1 Time of Concentration of Grade of Evidence Evidence of 

Test Material Erythema at of Local Systemic Identification Observation I njec ti on Site Necrosis Toxicity (% w/v) 

24 hours 2 None None 

C 	 40 hours 2 None None 0.1 72 hours 2 None None .... I

. 7 days - 2 None None w 

w 

I 

24 hours 2 - 3 None None 

48 hours 3 None None 0 	 0.5 72 hours 3 - 4 Eschar/focal eschar None 

7 days 2 - 4 Eschar/focal eschar None 

I 24 hours 2 - 4 Necrosis None -c

;0 

G 48 hours 1 2 - 4 Eschar None E 

72 hours 	 2 - 4 Eschar None rrt

n 7 days 1 - 4 Eschar None 	 --4 

:z

c::

:::r. 

C) 	 The cOllcentral ion of the test material selected for the intradermal induction stage of the main study was ~~_ rrt

;0

.. 0 0.1% w/v ill arachis ojl B.P. C) 	 01 :,1 01

OJ 

C) 	 ~-. j .:.. 

r, . ""­ .r::a 	 ..... C0 

[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN 0 J X I I I TOPICAL SIGIITING TEST FOR INDUCTION APPLICATION 

(48-1I0UR EXPOSURE) - INDIVIDUAL SKIN REACTIONS: ENV 93 1201 STUDY 

V[lIIClE: IHSTILLED UATEn 

Sk in Reactions Concentration of Anilllal (liours After Removal of Patches) Test Material ldent ificatlon 1 24 48 (% v/v) Er Oe Other Er Oe Other Er Oe Other 

10 

II 5 

2 

1 

;' 

t 

I 

W 

.;.. 

I 

'"'0 

E 


,." 

n 

:z 

c: 

:J: 

D:I 

,." 

;;0

.. 


~ 

U'1 

.;.. 

.......


-


10 

5 

2 

1 

CJ 

c=) 

c=) 

o 

P 50 

25 

1 0 

0 0 

0 0 

0 0 

1 0 

1 0 

1 0 

0 0 

Animal 

2 1 2 1 

0 0 0 0 

0 0 0 0 

0 0 0 0 

2 1 2 1 

0 0 0 0 

0 0 0 0 

0 0 0 0 

killed immediately after dressing removal 

due to severe necrosis 

-J () 50 Animal killed immediately after dressing removal 

25 due to severe necrosis 

The concentration of the testlnaterial selected for the main study topical induction was 10% v/v 1n distilled 

water 

-i 
[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TItE GUINEA PIG 

A P PEN 0 I X I V TOPICAL SIGIITING TEST FOR INDUCTION APPLICATION 

(48-1I0UR EXPOSURE) - INDIVIDUAL SKIN REACTIONS: ONCB STUDY 

VEIHCLE: ABSOLUTE ETlIANOl 

Skin Reactions 

Concentration of Animal . (Hours After Removal of Patches) Test Mater; a 1 

Identi fi cation ('Yo v/v) 1 24 48 

Er Oe Other Er Oe Other Er Oe Other 

3 I 2 2 STA 2 2 STA 1e 2 STA W 

l) 1.5 2 1 STA 2 1 STA 1 1 STA en 

0.75 2 0 STA 1 0 STA 0 0 STA I 

0.3 1 0 STA 0 0 STA 0 0 STA 

3 2 2 STA 1e 2 STACr 1e 2 STASt 

STA 

STA 

in absolute 

K 1.5 2 1 STA 2 2 STA 1 1 STA 

0.75 2 1 STA 1 0 STA 0 0 ", 

0.3 2 0 STA 0 0 STA 0 0 ," 

~ 

~ 

The concentral1on of the test material selected for the main study topical induction was 0.75% w/v ~ 

z 

ethanol ~ CD 

~ o 

o 01 

o 01

.j:Io 

o .......


I-' 

..t::lI 

(X) 
ENV 93 1201 : MAGNUSSON & KLIGMAN MAXIHISATION STUDY IN TilE GUINEA PIG 

API}ENOIX V TOPICAL SIGIITING TEST FOR CHALLENGE APPLICATION 

(24-IIOUR EXPOSURE) - INDIVIDUAL SKIN REACTIONS: ENV 93 1201 STUDY 

V[JtICL( : OISTlll.EO WATER 

Skin Reactions Concentratlon of Anlmal (H~urs After Removal of Patches) Test Material Identification (% v/v) 1 24 48 

Er De Other Er De Other Er De Other 

L 

10 

5 

0 

0 

0 

0 

1 

0 

0 

0 

1 

0 

0 

0 

I 

W 

0'1 

I 

2 0 0 0 0 1 0 

1 0 0 0 0 0 0 

10 1 0 1 0 0 0 

~1 5 0 0 0 0 0 0 

2 0 0 0 0 0 0 

1 0 0 0 0 0 0 ""0 

E 

m 

~ 

Tile concentrations of the test material selected for the main study topical challenge were 5~ and 1% v/v in :z 

i distilled water a:'l 

m 

:;c;l

.. o 

0'1 o .~! 01 

~ 

o -I-' 

a 

.r.::. 

ill 
fNV 93 1201 MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN 0 I X V I TOPICAL SIGIITING TEST FOR CIiAllENGE APPLICATION 

(24-1I0UR EXPOSURE) - INDIVIDUAL SKIN REACTIONS: DNCO STUDY 

VEIIICLE: ABSOLUTE ETIIANOL 

Skin Reactions Concentration of Animal (Hours After Removal of Patches) Test Material Identification w 

I 

1 24 48 (% v/v) Er Oe Other Er Oe Other Er Oe Other I 

0.15 0 0 STA 0 0 STA 0 

N 0.50 0 0 STA 0 0 0 

0.25 0 0 STA 0 0 STA 0 

0.10 0 0 STA 0 0 0 

'"'C 

;::0 0.15 0 0 STA 0 0 STA 0 0 0 

c..... o 0.50 0 0 STA 0 0 0 0 f"T1 

n 0.25 0 0 STA 0 0 0 0 -4 

0.10 0 0 STA 0 0 0 0 z: 

c::: 

3: 

co 

f"T1 

:;a

.. o 

o 01 '" .$00 o ........ 


..... C~) 

Ul 

CJ 
[NV 93 1201 : MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN TIlE GUINEA PIG 

A P P [ N D I X V I TOPICAL SIGIITING TEST fOR CttAllENGE APPLICATION 

(contd) .'\ 

(24-IIOUn EXPOSURE) - INDIVIDUAL SKIN REACTIONS: ONCS STUDY 

V[IIICLE: ABSOLUTE ETIIANOL. 

Skin Reactions Concentration of An ima 1 . (llours After Removal of Patches) Test Material 

Ident i ficat ion (% v/v) 1 24 48 

Er Oe Other Er Oe Other Er Oe Other I 

W 

I 2 1s 1 2 1 STA 2 1 STA 00 

p l.5 1s 1 2 1 STA 1 1 STA 

1 15 0 2 1 STA 1 1 STA 

0.75 1s 0 0 0 STA 0 0 STA 

2 1s 1 2 1 STA 1 1 

Q 1.5 1s 1 2 1 STA 1 0 

1 1s 0 2 0 STA 1 1 

0.75 1s 0 0 0 STA 0 0 

The 

STASs 

STA 

STA .-.,

, 

STA i-.C

""" . 

, -, ......... 

:~~>... 

t. 

.. ~ 

~ -l 

2: 

c:: 

X 

OJ 

.. 

~ 

Ol 

01 

.+:0-

....... 

...... 

concentrations of the test material selected for the main study topical challenge were 0.75% and 0.5% w/v (i'~ 

absolute ethanol 

o 

o 

C:> 

o 


01 


........:L 

'I 


fNV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN 0 I X V I I INTRADERMAL INDUCTION - INDIVIDUAL SKIN REACTIONS 

IN TEST ANIMALS: ENV 93 120) STUDY 

INTRADERMAL INDUCTION CONCENTRATION: 0.1% w/v VEIII elE : D I STI llED WATER 

Animal Number Skin Reactions at Observation Time:

24 hours 48 hours 

left Side Right Side left Side Right Side 

1 2 2 1 1 

2 2 2 1 1 

3 2 2 1 1 W 

I 

1.0 4 1 1 1 1 I 

5 2 2 1 1 

6 2 2 1 1 

7 1 2 1 1 

8 2 2 1 2 

9 1 1 1 1 

10 2 2 1 0 

11 1 1 1 0 

12 2 2 1 1 } -0 . ;;0

13 1 1 1 1 E 14 1 1 1 2 J'TI 

15 2 2 0 1 -i 

n 

, 

16 2 2 0 0 :z

c:: 17 2 2 2 1 :I: 

0 18 2 1 0 1 J'TI 

:;;d 0 19 2 2 2 2 •• 

C) 20 2 2 0 1 

.. 

CD 

<:) 0"1 

U1 

{:ft- ....... 


..... N 
., 

[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P P [ N 0 I X V I I I INTRADERMAL INDUCTION - INDIVIDUAL SKIN REACTIONS 

. IN CONTROL ANIMALS: [NV 93 1201 STUDY 

VEIIICLE: lHSTIlL[1l WATEn 

Animal Number Skin Reactions at Observation Time:

24 hours 48 hours 

Left Side Right Side. Left Side Right Side 

21 0 0 0 0 

I 

22 0 0 0 0 0 

I 

23 0 0 0 0 

24 0 0 0 0 

25 0 0 0 0 

26 0 0 0 

27 0 0 0 

"'l:l 28 0 0 0 0 {' 'J 

29 0 0 0 0 ;;: Ii E

m 

n ( -f 30 0 0 0 0 '"" _ :z: 

c:

::r: 

CD 

m 

.. C) 0\ 

01 U1 ~ 

.......
 CD ...... 
fNV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN 0 I X I X INTRADERMAL INDUCTION - INDIVIDUAL SKIN REACTIONS 

IN TEST ANIMALS: DNCB STUDY 

INTllAOERHAl INDUCTION CONCENTRATION: 0.1% w/v VEtIlClE: ARACIHS OIL B.P. 

Animal Number Skin Reactions 

24 hours 

at Observation Time: 

48 hours 

Left Side Right Side left Side Right Side 

31 2 2 1 0 	 I 

.Po 

..... 32 2 2 2 2 I 

33 1 1 1 1 

34 1 1 1 

35 2 2 2 2 

36 1 1 1 2 

37 2 2 2 2 

, 

-c 38 2 2 	 2 2 l:::: ' ;;c 

E 

"., 39 1 1 	 2 2 n {r:.::. .' /' -; 

40 2 2 1 1 r' . z 

c: 

3: 

CD 

"., 0 	 ~~, ;;c

.. 0 

CJ 	 0'1 

U1 01 	 (;1 .Po

........
 .l:::!i 	 ..... 
fNV 93 1201 MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 


A P P f N D I X X INTRADERMAL INDUCTION - INDIVIDUAL SKIN REACTIONS 

IN CONTROL ANIMALS: DNCO STUDY 

VEHICLE: ARACIIIS OIL D.P. 

Animal Number Skin Reactions at Observation Time:

24 hours· 46 hours 

Left Side Right Side Left Side Right Side 

I 

.l;Ia 

41 1 1 1 I

N 

42 1 1 1 1 


43 0 1 0 1 


44 1 1 1 


45 1 0 U 
 ~J '"C 

,." 

~ 

n 

-I 

::z:

c: 

3: 

rr1 

~ 

0 fIJ to 

.. 0 

C~ m 

01 

0-1 .l;Ia I -) ........
 <.n ....... 

fNV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P P f N 0 I X X I TOPICAL INDUCTION - INDIVIDUAL SKIN REACTIONS IN 

TEST ANIMALS: ENV 93 1201 STUDY 

INDUCTION CONCENTRATION: 10% v/v VEIHCLE: DISTILLED WATER 

Skin Reactions (Hours After Removal of Dressing) 

Animal Number 1 hour 24 hours 

Er Oe Other Er Oe Other 

1 2 1 Bs 2 1 BdSt 

2 2 1 1 1 OSt 

3 2 1 1 1 5tO 

4 2 1 1 1 StSsO I 

-Po 

5 2 1 Bs ?e 1 StOBd w 

I 

6 3 2 ?e 1 StOBd 

7 2 1 1 0 

8 2 1 Bs 1 0 

9 2 1 Bs 2 1 5tOBd 

10 2 1 Bs 2 1 StDlJd 

II 3 2 Bs ?e 1 StO 

12 2 1 1 1 

13 3 2 2 1 

14 2 1 1 1 

15 2 1 1 0 

16 2 1 1 0 

17 2 2 1 0 

18 2 1 Bs 1 I 

0 19 1 0 1 0 

CJ 20 1 1 1 0 

:1 

0 

01 

0) 

0 

DSt 

05t 

5t 

"'C 

E 

P'-l -~ 

:z 

c: 

:J: 

c::l 

I'T1 

:;0

.. 

0"1 

tn

.:.. 

....... 

~ 
ENV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN D I X X I I TOPICAL INDUCTION - INDIVIDUAL SKIN REACTIONS IN 

CONTROL ANIMALS: [NV 93 1201 STUDY 

VEIIIClE: DISTILLED WATER 

Skin Reactions (Uours After Removal of Dressing) 

Animal Number 1 hour 24 hours 

Er Oe Other . Er Oe Other 

21 1 1 0 0 t 

I 

I 

22 0 0 0 0 


23 1 0 0 0 


24 0 0 0 0 


25 1 0 0 0 


26 1 0 0 0 


27 1 0 0 0 '"'C 

28 1 0 0 0 2

,." 

n

29 1 0 0 0 -I 

:z 

c:: 30 0 0 0 0 0 :J: 

c:I 

0 ,." 

:;cI 

0 

0 0'1 !Ii U1

.. 

01 .( ,

_I .fa 

.......
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[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN 0 I X X I I I TOPICAl. INDUCTION - INDIVIDUAL SKIN REACTIONS IN 

TEST ANIMALS: DNCD STUDY 

INnUCTION CONCENTHATION: o. 75~ w/v VEIIICLE: ADSOLUTE ETIIANOL 

Skin Reactions (Hours After Removal of Dressing) 

Animal Number 1 hour 24 hours 

Er Oe Other Er Oe Other 

..&k 31 2 2 STA 2 2 STA I 

lit 

32 2 2 STA 2 2 STA I 

33 3 2 STA 2 2 STA 

34 2 2 STA 2 2 STA 

35 3 2 STA 2 2 STA 

36 3 2 STA 2 2 STA 

37 3 2 STA 2 2 STA 

38 2 2 STA 2 2 STA 

39 3 2 STA 2 2 STA 

40 3 2 STA 2 2 STA 

c::I. 0 

'~.: 

,." 

:;t;l 

I 

'----" .. 0 

(71 0 In 

{J1 ..&k 

........... 


...... (X) 

,;' % ¢,; t,: C 

3: 
[NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P P [ N 0 I X X I V TOPICAL INDUCTION - INDIVIDUAL SKIN REACTIONS IN 

CONTROL ANIMALS: DNC8 STUDY 

VEIlICLE: ABSOLUTE HIIANOL 

Skin Reactions (liours After Removal of Dressing) 

Animal Number 1 hour 24 hours 

Er Oe Other Er Oe Other 

.,.I 

Q Q Q 0'1 41 0 I 

42 o 0 0 Q 

43 o 0 0 Q 

44 Q 0 Q 

45 Q Q 0 Q .~l "'C 

;;;0 

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-4 

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0:1 

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:.0

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0"1 

01

.,. 


........ 


..... 
[NV 93 1201 : MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN TilE GUINEA PIG 


A P P [ N D I X X V 


INDIVIDUAL 80DYWEIGIITS AND 80DYWElGIlT GAINS OF TEST ANIMALS: ENV 93 1201 STUDY 


Bodyweight (g) Bodyweight (g) 

Animal Number Day 0 Day 24 Increase 

0 

0 

1 


2 


3 


4 


5 


6 


1 


8 


9 


10 


11 


12 


13 


14 


15 


16 


17 


18 


19 


20 


368 


402 


358 


371 


381 


398 


386 


404 


391 


364 


361 


318 


312 


409 


427 


371 


399 


407 


384 


367 


551' 


601 


492 


555 


565 


594 


622 


608 


613 


539 


558 


578 


565 


537 


575 


551 


606 


574 


567 


506 


183 


199 


134 


184 


184 


196 


236 


204 


222 


175 


191 


200 


193 


128 


148 


180 


207 


167 


183 


139 


I 


.$0­

........ 


I 


" 2 


/'Tl

n 

-t 

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c 

:J: 

m 

/'Tl

:;0

.. 
 0 0'1 

m U1

.,.. 0 ..... """ 
ENV 93 1201 : MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN TIlE GUINEA PIG 

A P PEN D I X X V I 

INDIVIDUAL BODYWEIGIITS AND BOOYWEIGIIT GAINS OF CONTROL ANIMALS: ENV 93 1201 STUDY 

Bodyweight (9) Bodywelght (g) 

Animal Number Day 0 Day 24 Increase 

21 396 553 155 

22 366 532 164 I 

23 375 581 206 I 

24 ,~ 414 596 182 

25 387 555 168 

26 375 530 155 

27 427 575 148 

26 430 606 176 

29 415 576 161 f~ '"'C 

c'I. E 

30 370 541 171 f'T1 

-.

n 

:z 

t:: 0 C··, :J:

co 0 1::: . f'T1 

"IC :;a 0 ~ .. D 

m 0\

01 I:: ./ .j::Io ......... ,-' 
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...... 

_______________________...,,"'...._ ..... """'''''''_'''''''~~L:..::_ ".~.....~~":~~"!_ __:--""""""""~;~~~_::~E::':m ,.-~_"~"'. ..,........ ~,_,~:r..'~~

.. -."'~~-~'~.~~'--'---------~-----~-----~--
ENV 93 1201 : MAGNUSSON &KLIGHAN MAXIMISATION STUDY IN TilE GUINEA PIG 

A P PEN D I X X V I I 

INDIVIOlJAl OODYWEIGIITS AND BODYWEIGIiT GAINS OF TEST ANIMALS: DNCO STUDY 

Bodyweight (g) Oodyweight (g) 

Animal Number Day 0 Day 24 Increase 

31 366 581 215 

I 32 385 579 194 ~ 

1.0 

33 381 569 188 I 

34 352 526 174 

35 363 550 187 

36 411 572 161 

37 341 509 168 

38 444 545 101 -0 

39 337 471 134 .t,

...... , ". 2 ... iTI 40 421 596 175 n 

-1 

;z

c: 

3: 

C:I 

iTI 

;0

0 .".~ .. 0 0'1 

0 01 /.I ........
 0 ~ 

1-4 

(j) 

N 
£NV 93 1201 : MAGNUSSON &KLIGMAN MAXIMISATION STUDY IN TII£ GUIN£A PIG 


A P PEN 0 I X X V I I I 


INIHVIOUAL 800YW£IGlITS AND BODYWEIGIIT GAINS OF CONTROL ANIMALS: ONCB STUDY 

Bodyweight (g) Bodyweight (g)
 Animal Number 
 Day 0 Day 24 Increase I 


U1 


CI 

I 


41 420 576 156 


42 380 594 214 


43 380 549 169 


44 398 580 182 
 .'­ ""C

45 386 570 184 ::.:; 2 


/"1"1

n

--I 

:z

c: 

:J: 

CD o /"1"1 

o .. o 

0'\ c-:::> ~ ()) I-' 0-) ,', - -

01 

., 
-51­

PROJECT NUMBER: 654/1 


A P PEN D I X X I X 


TIIE DEPARTMENT OF HEALTH OF TIm GOVERNMENT 


OF TIm UNITED KINGDOM 


GOOD IABORATORY PRACI1CE 

STATE~NTOFCOMP~CE 

IN ACCORDANCE WITH DIRECTIVE 88/320 EEC 

LABORATORY 

SafePharm Laboratories Limited 

POBox No 45 


Derby 


DEl 2ET 


DATE OF INSPECI10N 

31 January 1994 

A general inspection for compHance with [he Principles of Good Laboratory PraC'"oice 

was carried out at [he above laboratory as part of the UK GLP Compliance Programme. 

At the time of the inspection no deviations were found of sufficient magnitude to affect 

the vaiidicy of studies performed at these facilities. 

_ .. n00064 

D. 1:". Moori!' 

Direaor 

UK GLP Monitoring Unit 
~- . . , , "1·' 

BIOSEARCH INCORPORATED 

P.o. Box 8598, Philadelphia, Pennsylvania 19101 ' ; Telephone: (215) 739-4499 It····· .. ··~.··'.'·:.···.····'.··. Fax: (215) 423-9210 :1 . . 


:1'·.··.···. 


.··1 

l' '~ 

Project Number: 93-7958A 

SUM:MARY OF RESULTS OF PRIMARY SKIN IRRITATION STUDY 

Conducted for: 

Test Article: ENV931201 

Primary Skin Irritation: The test article, when administered as supplied, is corrosive to 

the skin of albino rabbits. 

1 


1 


1 

t 

i 

r 

, 

; 

000065 


1 
Study Title 


Primary Skin Irritation ~ Rabbits 


Test Article 


ENV931201 


Sponsor 


Author 


George E. Moore 


Study.Completion Date 

03/21/94 

Performing Laboratory 

Biosearch ll1corporated 


3408-50 "B" Street 


Philadelphia, Pennsylvania 19134 


BiQsearch Project Number 

93-7958A 

Page 1 of 11 

000066 

Page 2 of 11 

,­

....,i 

PAGE RESERVED FOR CONFIDENTIALITY STATEMENT 

- " 

. .J 

j 

J 


J 


-J 


J . 

.-r 

J 

'1 

.J 

] 

'J 000067 

"] BIOSEARCH, L"lCORPORATED· P.O. BOX 8598 - PHIUDELPHIA, PE'J:-JSYLVANlA 19101 (215) 739-4499 
, Page 3 of 11 

BIOSEARCH INCORPORATED 

P.o. Box 8598, Philadelphia, Pennsylvania 19101 


Telephone: (215) 739-4499 


Fax: (215) 423-9210 


..,..." .J:;) _, 

GOOD LABORATORY PRACTICE COMPLIANCE .~T~~f~NT ,,~ }~/! , 

"'4, I';~ .', r....... 


rv .. 

Project Number: 93-7958A 	 ''<", ..!, J.,...,.t;~ . "', 

,"" . 	 Sponsor: 

- .... ; 

i Test Article: ENV931201 i .", 

j .,j Test: 	 Primary Skin Irritation - Rabbits (fSCA) 

...., 	 Protocol Number: PSI-B06 (Rev. 02193)

.J 

. ..., 

~J This study was carried out in compliance with the 40 CFR 792 and OECD Regulations for 

"Good Laboratory Practice". 

] 


] 


] 


~reC:;.s.~ 3-;;2. J- 9'-1

Date 

Study DirectorlLaboratory Director 

01110/94 93 040 7958 116 	 Form QA-700A 000068 
Project Number: 


Sponsor: 


Test Article: 


Test: 


, Protocol Number: 

... Study Initiated: 

Study Audit: 

] 

''''I 

i -wi 

-, 

! 

J 

.! 

1jlU~bAl,(L.t1 ll~LUl'(YUl{Al hV 

P.O. Box 8598, Philadelphia, Pennsylvania 19101 


Telephone: (215) 739-4499 


Fax: (215) 423-9210 


QUALITY ASSURANCE STATEMENT 

ENV931201 


Primary Skin Irritation - Rabbits (TSCA) 


Sponsor 

PSI-806 (Rev. 02193) Signed Protocol: 12122193 

01102194 Study Completed: 03/21194 

Critical phases for this study or this type of study were inspected on: 

Individual Study Audits: 

Shaving 

Test Article Admin. 

Final Report 

01/03/94 

01104194 

03121/94 

Study Type Audits: 

Test Article Preparation 01125194 

Any findings 

protocol were 

of deviation from standard operating procedure 

immediately reported to the Study Director and 

or 

to 

management. 

The QAU Study Inspection Report to the Study Director and 


management was submitted by 02111/94.. 


The results shown in this report accurately reflect the raw data records. 


02128/94930407958 116 Form QA~700B 

000069 

Project Number: 93~7958A P,age 5 of 11 

TABLE OF CONTENTS 

Title Page ................................................................................................... l' 


Confidentiality Statement .............................................................................. 2 


Good Laboratory Practice Compliance Statement .............................................. 3 


"I 

,

i Quality Assurance Statement ......................................................................... 4 


Table of Contents .......................................................................................... 5 
 ......., 


,J 	 Objective of Test .....................................................,.......................... :......... 6 


Re ference .................................................................................................... 6


~] Justification for Test System .......................................................................... 6 


J Animals ...................................................................................................... 6 


Husbandry Conditions ................................................................................. 7 


J Procedure ......................................................:............................................ 7 


Data Retention ............................................................................................. 7
 ] 

] 


Test Article Characterization .......................................................................... 7 


Results ....................................................................................................... 8 


Conclusion .................................................................................................. 8 


J Signatures ................................................................................................... 8 


Table 1 ~ Primary Skin Irritation Scores ........................................................... 9
 J 

] 


Appendix A - Draize Scale for Scoring Skin Reactions ..................................... 10 


Appendix B ~ Protocol Deviation ................................................................... 11 


] 


] 


J 


J 


] 


000070 

BIOSEARCH, L"ICORPORATED· P.O. BOX 8598· PHILADELPHIA, PEJ'<NSYLVANLA 19101 (215) 739-1499 

u 

BIOSEARCH INCORPORATED 


Proj ect Number: 

frotocol Number: 

Testing Facility: 

Sponsor: 

Test Article: 

Test Article 

Description: 

Test Article 

Received: 

In-Life 

Study Initiated: 

Objective of Test: 

Reference: 

Justification for 

Test System: 

Animals: 

P.O. Box 8598, Philadelphia, Pennsylvania 19101 


Telephone: (215) 739-4499 


Fax: (215) 423-9210 


93-7958A 

PSI-806 (Rev. 02/93) 

Biosearch Incorporated 


3408-50 liB" Street 


Philadelphia, PA 19134 

ENV931201 

Amber liquid 

12123/93 

In-Life 


01/04194 Study Completed: 01106/94 


Primary Skin Irritation - Rabbits 

To determine the degree of irritation, if any, which the test article 

may produce when applied to the skin of albino rabbits. 

The methods employed in this study were similar to those described 

in 40 CPR Part 798, EPA Health Effects Testing Guidelines - Subpart 

E - Specific Organffissue Toxicity, September 1985. 

The laboratory albino rabbit was the system of choice since it is 

required in the federal guideline for this type of study and has a 

long history of use in dermal irritation studies. 

The New Zealand White rabbit was used for this study. The 

animals were obtained from Davidson Mill Farm, Jamesburg, NT 

08831 (U.S.D.A. License #22-A-7). 

The animals were individually housed and maintained in 

accordance with standards set forth in the Guide for the Care and 

Use of Laboratory Animals (NIH Publication No. 86-23). 

The rabbits were acclimated to the laboratory for at least 5 days 

prior to the initiation of the study. The animals were individually 

identified by an ear tag and each cage was identified with a cage 

card, displaying the project number, animal number, sex, date 

initiated and responsible technician's initials. 

000071 


BfOSEA.RCH. u'ICORPORATED . P.O. BOX 8598· PHILADELPHJA. PE"INSYLVANIA 19101 (215) 7394499 
Project Number: 93-7958A 	 Page 7 of 11 

Wayne 15% Rabbit Ration was provided as a maintenance diet 

Based on our current 

. ENV931201 

Primary Skin Irritation - Rabbits 

Hllsbandry .. Conditions: 	 Temperature: 21.2°C - 21.8°C 

Relative Humidity, 	%: 41 - 47 

Light: 12-hour light/dark cycle 

Diet: 

and tap water was provided ad libitum. 

knowledge, no contaminants are known to be in this diet or 

water which might be expected to interfere with the objectives

i 	 of the study. 1 

Caging: Stainless steel elevated wire mesh flooring, 1 rabbit/cage. 

Procedure: 	 A group of six rabbits was clipped over a wide area on their backs 

approximately 24 hours prior to application. Animals with healthy intact 

skin were used. 

The test article was applied dermally as supplied. 

A 0.5 m1 portion of the test article was applied dermally to the test site 

(approximately 6 crn 

2

). An adjacent area of untreated skin served as a 

control for the test. Gauze patches on non-irritating tape were placed 

over the treated and control areas. A suitable semi-occlusive dressing 

(gauze) was wrapped around the trunk of the animal and secured with 

elastic tape. 

The wrapping was removed at the end of the four-hour skin contact 

period and the test sites were wiped with deionized water. The treated 

areas were then examined for signs of erythema and edema, using the 

Draize method of scoring (Appendix A), within 30 to 60 minutes after 

patch removal. Readings were also made after 24 and 48 hours. 

Due to the necrosis produced by the test article, the study was 

terminated after the 48 hour readings and the animals were euthanized 

for humane reasons . 

. ' Data Retention: 	 The ra:w, da!a,aIld a copyof this report are filed in the archives of 

Biosearch, Incorporated, Philadelphia, Pennsylvania. 

Test Article 

Characterization: The Sponsor was responsible for test article characterization and stability 

determination. 

Details regarding the identity, strength, stability, purity, and 

composition of the test article are in the possession of the Sponsor. 

Information supplied to Biosearch, Incorporated regarding 

characterization of the test article is on file in the archives of Biosearch, 

Incorporated, Philadelphia, Pennsylvania. 

000072 


BIOSEARCH. lNCORPORATED· po, BOX 8598· PHILADELPHIA. PEN'NSYLVANIA 19101 (215) 739-4499 
Project Number: 93-7958A 	 Page 8 of 11 

ENV931201 


.... 
 Primary Skin Irritation - Rabbits 

.... l Results: See Table 1 . 

CQnclusion: 	 The test article, when administered as supplied, is corrosive to fhe ,~idn 

of albino rabbits. .. '''~, ] 

Signatures: 	 Submitted by Biosearch, Incorporated: 

] 

] 	 ~kC?/~ 3h.) J9'~ 

Laboratory 

G;orgeRoore, 

Director/Study 

B.S. 

Director 

~ 

] 

Reviewedf Audited by: 

J 

] 

J 	

Manager, 

pa~Quality Assurance 

Approved by: ] 

es ong, Sc.D. Date ?#;If~

.I].' ... Vice Pr sident, Laboratory Operations 

& Regulatory Affairs 

000073 


B[OSEARCH, INCORPORATED - P.O. BOX 8598· PHlL>\DELPHlA, PENNSYLVANIA 19101 (215)739-4499 
...., Project Number: 93-7958A 	 Page 9 of 11 

- ENV931201, applied dermally as supplied. 

-, Primary Skin Irritation - Rabbits 

TABLE 1 

Primary Skin Irritation Scores 

-, 

i 

Rabbit Number 

1 2 3 .4 5 .6 1 

.J 5..ex:t M M M E E E 

Initial ] Body Weight (kg): 	 2.71 2.49 2.38 . 2.63 2.57 2.85 

EI)!thema and. Reading 1 

J 	 Eschar Formation (Hours) 

Intact Skin "'1 N N N N N N

] Intact Skin 	 24 N N N N N N 

Intact Skin 	 48 N N N N N N ] 

Intact Skin 	 ""'72 

1 

Edema Formation 

Intact Skin "'1 o 2 3 3 2 3 

Intact Skin 24 3 3 3 3 4 4 

Intact Skin 48 3 3 3 3 4 4 

Intact Skin 

Primary Irritation Index: Corrosive 

'" 	 Readings were made 30 to 60 ininutes after patch removal, after a 4-hour exposure. 

Due to the necrosis of the test sites, the animals were euthanized for humane reasons 

after the 48-hour readings. 

t M = Male F = Female 

000074 

BIOSEARCH. INCORPORATED - P.O. BOX 8598 - PHILADELPP.IA. PENNSYLVA,"llA 19101 (215) 739-1499 
Project Number: 93-7958A Appendix A - Page 10 of 11 

( - ENV931201 

Primary Skin Irritation - Rabbits 

,....

l 

Draize Scale for Scoring Skin Reactions» 

l

..... 

Erythema and Eschar Formation Value 

No erythema o 

Very slight erythema (barely perceptible) 1 

Well defined erythema 2 

Moderate to severe erythema 3 

Severe erythema (beet redness) to slight eschar formation 

J 
 (injury in depth) 4 


Edema Formation Value 

No edema 0 

Very slight edema (barely perceptible) 1 

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

Moderate edema (raised approximately 1 mm) 3 

;:;1,', Severe edema (raised more than 1 mm and extending beyond the .,J area of exposure) 4 

:J 

+"'~ 

'-J: 

i, , 

J 


] 


,.. Association of Food and Drug Officials of the United States, APPRAISAL OF THE J SAFETY OF o-rEMICALS IN FOODS, DRUGS AND COSNIETICS, p. 48. Bureau of Foods 

and Drugs, Texas State Department of Health, Austin, Texas, 1959. 

] 

J 

00007b 

B[OSEARCH. J.NCORPORATED· P,O, BOX 3598 PHILADELPHIA. PENNSYLVANIA 19101 (215) 739-4499 
Appendix B- Page 11 of 11 

Project Number: 


Protocol Number: 


Sponsor 


Test Article: 


Test: 


GLP Compliance: 


Deviation: 


Submitted by: 

Approved by: 

Acknowledged by: 

BIOSEARCH INCORPORATE.D 

P.O. Box 8598, Philadelphia, Pennsylvania 19101 

Telephone: (215) 739-4499 


Fax: (215) 423-9210 


DEVIATION FROM PROTOCOL #1 

93-7958A 

PSI-806 (Rev. 02/93) 

ENV931201 

Primary Skin Irritation - Rabbits (TSCA) 

40 CFR 792 OECD 

Due to the necrosis produced by the test article, the study was terminated 

·after the 48-hour readings and the' animals were euthanized for humane 

reasons. 

This deviation is not thought to affect the integrity of this study. 

;11:1.t/q~ 

Date 


Laboratory Director/Study Director 


J/I/rCt 

() 

Date 


Manager, Quality Assurance 


Jle5 E~g:s;D. Vice President, Laboratory Operatios 


& Regulatory Affairs 


Sponsor RepresentL . 

Feb 2, 1994 93 040 7958 116 Form QA-500A 

000076 
: ... 

Studv, Title 

Acute Oral Toxicity, LD - Rats 

50 

Test Article 

ENV931201 

Sponsor 

J 


1 


;] Author 

David Gabriel j 

Study Completion Date 

1] 05112194 

J 

I:erforrning Laboratory 

J Biosearch Incorporated 

3408-50 "B" Street 

Philadelphia, Pennsylvania 19134 -1.:J 

-Jl!III!I 

J

! Biosearch Project Number 

93-7958A 

Page 1 of 28 

000078 
Page 2 of 28 


PAGE RESERVED FOR CONFIDENTIALn-V STATEMENT 

000079 

Page 3 of 28 

BIOSEARCH INCORPORATED 

P.o. Box 8598, Philadelphia, Pennsylvania 19101 

Telephone: 	(215) 739-4499 

Fax: (215) 423-9210 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

Project Number: 93-7958A 

, . Sponsor: 


T est Article: ENV931201 


Test: 	 Acute Oral Toxicity, LD - Rats (TSCA) so 

Protocol Number: AOT-800 (Rev. 02193) 

This study was carried out in compliance with the 40 CFR 792 and OECO Regulations for 

"Good Laboratory Practice". 

i.-_,4) 

D-=: < bC.. ~ ~~ 5'-12-'1Lf 

David Gabriel, B. A. Date 

Study Director 

.....1 

-, 

J 

; 	 000080 

03/28/94 93 040 7958 1485 	 Form QA-700A 
12122193 

05112194 

.t)lU~hAl{LH lNCORPORATED 

P.O. Box 8598, Philadelphia, Pennsylvania 19101 


Telephone: (215) 739-4499 


Fax: (215) 423-9210 


QUALITY ASSURANCE STATEMENT 

Project Number: 93-7958A 

Sponsor: 

Test Article: ENv931201 

Test: Acute Oral Toxicity, LD50 - Rats (TSCA) 

Sponsor 

Protocol Number: AOT-800 (Rev. 02193) Signed Protocol: 

Study Initiated: 12129/93 Study Completed: 

Study Audit: Critical phases for this study were inspected on: 

Individual Study Audits: 

Dose Calculation 02110/94 

Test Article Admin. 02110/94 

Initial Weights 02110194 

Euthanasia 03/03/94 

Necropsy/Gross Path. 03/03/94 

Necropsy/Gross Path. 03/08/94 

Observations 03121/94 

Final Report 05113/94 

Any findings of deviation from standard operating procedure or 

protocol were immediately reported to the Study Director and to 

management. 

The QAU Study Inspection Report to the Study Director and 

management was submitted by 04108/94. 

The results shown in this report accurately reflect the raw data records. 

05/13/94 93 0407958 1485 Form QA-700B 

000081 

.. 
. 

Project Number: 93-7958A rage 5 of 28 

TABLE OF CONTENTS 

... .,"''' 

Title Page ...................................................................................... ~.:.: :'..j7)';';;., 1 


Confidentiality Statement .......................................................................:.::::: ·2:,
 .' ..-/./:.>~ 

Good Laboratory Practice Compliance Statement .............................................. 3 • 


Quality Assurance Statement .......................................................................... 4 


Table of Contents ......................................................................................... 5 


Objective of Test .......................................................................................... 6 


.... Reference ..................................................................................................... 6 
 ,

, 

Justification for Test System .......................................................................... 6 


Animals ...................................................................................................... 6 


Husbandry Conditions ................................................................................. 7 


., 

~ Procedure ................................................................................................... 7 


Data Retention ..............................:............................................................. 8 


Test Article Characterization .......................................................................... 8 


Results ....................................................................................................... 8 


Conclusion .......................................................................:.......................... 8 


Signatures ................................................................................................... 9 


Table 1 - Mortality & Average Body Weights - Males ....................................... 10 


Table 2 - Observations - Males ...................................................................... 11 


.. -j Table 3 - Gross Pathology - Males ................................................................. 13 


~ 

Table 4 - Mortality & Average Body Weights - Females .................................... 14 


Table 5 - Observations - Females ................................................................. 15 


Table 6 - Gross Pathology - Females'.............................................................. 17 


Table 7 - Individual Body Weights - Males ..................................................... 19 


Table 8 Individual Body Weights - Females .................................................. 23 


Appendix A - Deviation from Protocol .......................................................... 28 


000082 

...., 

BlOSEARCH. INCORPORATED· PO. sox 8598 - PHIL'\DElPHIA. P!::''-.'NSYLVAMA 19101 • (215) 739--499 
Project Number: 

Protocol Number: 

Jesting Facility 

Sponsor: 

.-,.• 

Test Article: 

Jest Article 

Description: 

Jest Article 

Received: 

In-Life 


Study Initiated: 


Ie.s.:t: 


J Objective of Test: 


Reference: 

Justification for J Test System: 

J 

Animals: 

] 


J 


] 

BIOSEARCH INCORPORATED 


P.O. Box 8598, Philadelphia, Pennsylvania 19101 


Telephone: (215) 739-4499 


Fax: (215) 423-9210 

93-7958A 

AOT-800 (Rev. 02193) 

Biosearch Incorporated 

3408-50 "B" Street 

Philadeluhia, PA 19134 J. 

ENV931201 

Amber liquid 

12123/93 

In-Life 

01128/94 Study Completed: 03/23/94 

Acute Oral Toxicity, LD50 - Rats 

To determine the acute oral LD of the test article in rats. 

50 

The methods employed in the testing of this test article are similar 

to those described in 40 CFR Part 798, EPA Health Effects Testing 

Guidelines - Subpart B - General Toxicity Testing, September 1985. 

The laboratory rat was the system of choice since it is required in 

the federal guideline for this type of study and has a long history of 

use in acute oral toxicity studies. 

The outbred Sprague-Dawley rat, weighing 200 to 300 grams, was 

used for this study. The animals were obtained from Buckshire 

Corp., Perkasie, PA 18944 (U.S.D.A. License #23-BL). 

000083 


I3[OSEARCH. [NCORPORATED· ?o. GOX 3593· PH[L-'.OELPH['\, PENNSYLVANfA 19101 . (215) 739-+199 
t:NV931201 

Acute Oral Toxicity, LD50 - Rats 

'j 

Animals: L 	 i ,~,

The animals were housed and maintained in accordance with standards' , ".,;­ (continued) 

set forth in the Guide for the Care and Use of Laboratory Animals (NIH 

Publication No. 86-23). The rats were acclimated to the laboratory for at 

least 5 days prior to the initiation of the study. 

Animals were individually identified by ear punch. Each cage was 

identified with a cage card, displaying the number of animals, sex, 

project number, date administered and responsible technician's initials. 

Husbandry 


Conditions: Temperature: 20.5°C - 24.0°C 


Relative Humidity, %: 32 - 60 

Light: 	 12-hour light/dark cycle 

Diet: 	 Teklad Rodent Diet (W) 8604 and tap water were provided ad 

libitum. Based on our current knowledge, no contaminants 

are known to be in this diet or water which might be expected 

to interfere with the objectives of the study. 

J 

_..J ! 	 Caging: Stainless steel elevated wire mesh flooring,S rats/cage by sex.

'" 	 Procedure: The test article was administered as supplied. 

J A group of 85 (35 male & 50 female) albino rats was used in this study. 

The animals were deprived of food but not water overnight prior to 

] 	 dosing. Each animal was weighed and dosed by direct administration of 

the test article into the stomach by gavage. 

J 
 The following dose levels were administered: 


Males: 	 Females: 

] 	 2.00 g/kg 2.00 glkg 

LOO glkg* 1.00 glkg 

0.944 glkg 0.794 glkg* ] 0.891 glkg* 0.631 glkg* 

0.794 glkg* 0.562 	 g/kg 

0.631 glkg 0.50 glkg* J 0.50 glkg 0.473 glkg 

0.447 glkg 

0.398 g/kg* J 	 0.355 glkg 

* 	Levels used to calculate LD. and 95% confidence limits employing the Thompson moving 

averages and interpolation -~ethod. The fourth level of 1.12 g/kg used to calculate the 

LDso for the males was inferred. 

000084 

3ICSEARCH. INCORPORATED. P,O. [lOX 8598· PH!L'.DELPI-'JA. PE.NNSYLVANIA \9101 (215) 739-4499 

.........
l ­ ENV931201 

Acute Oral Toxicity, LDso - Rats 

Procedure: 

(continued) Following administration, the animals were allowed food and water ilii 

libitum for the 14-day observation period during which time, the rats 

were observed for signs of toxicity and mortality. Animals were 

observed frequently on the day of dosing, and twice per day (morning 

and afternoon) on weekdays. On weekends and holidays, the animals 

were observed once per day. Individual weights were recorded on the 

day of dosing, weekly thereafter, and prior to sacrifice. 

The animals were euthanized with carbon dioxide at the conclusion of 

the observation period. Gross necropsies were performed on all 

animals. 

The LD and its 95% confidence interval were calculated employing the 

Thompi8n, W.R., Use of moving averages and interpolation, to estimate 

median effective dose, Bacteriological Rev. 11: 115-145, 1947. 

Data Retention: 	 The raw data and a copy of this report are filed in the archives of 

Biosearch, Incorporated, Philadelphia, Pennsylvania. 

Tes", Article 

Characterization: The Sponsor was responsible for· test article characterization and stability 

determina tion. 

Details regarding the identity, strength, stability, purity and composition 

of the test article are in the possession of the sponsor.. Information 

supplied to Biosearch, Incorporated regarding characterization of the test 

article is on file in the archives of Biosearch, Incorporated, Philadelphia, 

Pennsylvania. 

Results: 	 See Tables 1 through 8. 

Conclusion: 	 The test article, when administered as supplied to male albino rats, has 

an acute oral LD of 0.901 gfkg with 95% confidence limits from 0.852 to 

0.953 g/kg. 50 

The test article, when administered as supplied to female albino rats, has 

an acute oral LD of 0.562 glkg with 95% confidence limits from 0.494 to 

50

0.640 glkg. 

000085 


S[OSE.-\RCC1, [NCORPORATED - P.O. [lOX 8598 - PHILADELPH[A. ?ENNSYL'/A,'1IA t 9101 - (215) 739-4499 
- ENV931201 

Acute Oral Toxicity, LD - Rats 

50 

Signatures: Submitted by Biosearch, Incorporated: 

~- hc:::c-~ $'·\2-1'\ 

David Gabriel, B.A. Date 

Study Director 

1 

f 

I 

Reviewed!Audited by: ~ 

.1 ___ if 

~ Manager, Quality Assurance 

Approved by: 

"~ <"~/?k_ P.!i?s~~D. Vice President, Laboratory Operations 

~. 1 

.J & Regulatory Affairs 

l

...... 

J 

J 


000086 


BlOSEARCH, fNCORPORATED - P.o. BOX 8598 - PHrL.... OElPHlA, ?ENNSYL,,'ANIA 19101 - (215) 739-1499 
.I. ""DC:: .l.V Vi ':"'U 7,J-j 7")U1-'" !JroJect l'lUmuer: 

_ENV93120l, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 1 


Mortality & Average Body Weights - Males 


Density: 0.93 glml 

Dose Levels, glkg: 2.00 1.00 0.944 0.891 0.794 0.631 0.50 

Animals:· Number 5 5 5 5 5 5 5 

1 

i 

.­

~ 

1 

J 

.., 

J 

Mortality: 

14-Day Totals: 

Day 0 

Day 1 

Day 2 

Day 3 

Day 4 

Day 5 

Day 6 

Day 7 

Day 8 

Day 9 

Day 10 

Day 11 

Day 12 

Day 13 

Day 14 

Dead 

Live 

1 

4 

5 

0 

0 

0 

2 

,2 

1 

5 

0 

0 

0 

0 

0 

2 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

2 

3 

0 

0 

0 

1 

1 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

2 

3 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

5 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

5 

0 

0 

0 

0 

0 

0 

0 

a 

0 

0 

0 

0 

0 

0 

0 

0 

5 

"1 

i 

j 

,

, 

Average Body 

Weights, g: 

Initial 

7th Day 

Final 

290 

NA 

NA 

295 

NA 

NA 

249 

259 

331 

262 

265 

304 

214 

247 

297 

207 

260 

316 

262 

311 

356 

i 

... NA = Not Applicable 

..., 

000087 

1lIOSEARCH, !NCORPORAfED - p,O gOX 3598 - PHILADELPHIA. PE.'1NSYLVN~:,\ 19101 - (215) 739-4499 
Page 11 of 28 

. - ENV931201, administered as supplied. 

-.., I Acute Oral Toxicity, LD50 - Rats 

TABLE 2 

Obserya tions - Males 

l

. .J 

2.00 glkg: Immediate - All animals appeared to be nonna!. 


1 1 Hour All animals appeared to be lethargic and exhibited
 j 

ruffled fur. 

4 Hours 5/5 animals appeared to be lethargic, and exhibited 

yellow anogenital staining and ruffled fur. 

5 Hours 115 animals found dead. 

Day 1 415 animals found dead. 

1 1.00 glkg: Immediate - All animals appeared to be nonnaL 

4 Hours 2/5 animals appeared to be lethargicr 2/5 animals 

j appeared to be lethargic and exhibited gasping, and 

115 animals appeared to be lethargiC and exhibited .--I 

gasping and brown oral staining. 

Day 1 	 2/5 animals appeared to be lethargic and exhibited 

brown oral and anogenital staining and ruffled fur. 

3/5 animals appeared to be lethargic and exhibited 

brown oral and anogenital staining, ruffled fur 

and gasping. 

Day 2 2/5 animals found dead. 3/5 animals exhibited brown 

body and anogenital staining and piloerection. 

Day 3 2/5 animals found dead. 115 animals exhibited brown

- body and anogenital staining and piloerection. 

Day 4 115 animals found dead. 

0.944 glkg: 	 Immediate - All animals appeared to be normal. 

4 Hours All animals appeared to be normal. 

Day 1 All animals exhibited dark anogenital staining and 

ruffled fur. 

Day 2 All animals exhibited dark anogenital staining. 

Day 3 All animals appeared to be normal. 

Day 4 2/5 animals found dead. 3/5 animals exhibited ruffled 

fur. 

Days 5&6 - 3/5 animals exhibited ruffled fur. 

Days 7-14 - 3/5 animals appeared to be normal. 

000088 


B[OSE....RCH. t."1CORPORATED P.o. BOX 8598· PHILADELPHIA. PENNSYLVAJ"1A 19101 • (215) 739-4499 
- ENV931201, administered as supplied. 

Acute Oral Toxicity, LD50 

0.891 glkg: 	 Immediate ­

4 Hours 

Day 1 

Day 2 ~., " 

Day 3 

Day 4 

DayS 

Day 6 

Day 7 

, -	 Days 8-10 ­

Days 11-14 ­

......, 0.794 glkg: 	 Immediate4 Hours 

Day 1 

Days 2&3 

Day 4 

Days 5-14 

- Rats 

TABLE 2 (continued) 

Qbservations - Males (continued) 

All animals appeared to be normal. 

All animals appeared to be normal. 

All animals appeared to be normal. 

All animals exhibited dark body and anogenital staining. 

1/5 animals found dead. 4/5 animals exhibited dark 

body and anogenital staining. 

115 animals found dead. 3/5 animals exhibi ted dark 

body and anogenital staining. 

3/5 animals exhibited dark body and anogenital staining. 

3/5 animals exhibited dark anogenital staining. 

215 animals exhibited dark anogenital staining. 115 

animals exhibited dark anogenital staining and appeared 

thin. 

215 animals appeared to be normaL 1/5 animals 

exhibited dark anogenital staining and appeared thin. 

3/5 animals appeared to be normaL 

A loss of body weight (60 grams) was exhibited by 115 


animals (#75) between Days 0 and 7. This animal did not 


regain to its initial weight by 14 days. 


All animals appeared to be normal. 


All animals appeared to be normal. 


All animals appeared to be normal. 


All animals exhibited ruffled fur and anogenital staining. 


All animals exhibited ruffled fur. 


All animals appeared to be normal. 


0.631 glkg: 	 All animals appeared to be normal throughout the 14-day observation 

period. 

0.50 g/kg: 	 All animals appeared to be normal throughout the 14-day observation 

period. 

,

-

000089 


afOSEi\RCH, fNCORPORATED· P.O. flOX 5595· PH!LAOELPHrA, PENNSYLVANrA 19101 . (215) 739-4G9 
- ENV931201, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 3 


2.00 glkg: 

__I 

1.00 glkg: 

0.944 glkg: 

0.891 glkg: 

...., 

. ; 

.J 

0.794 g/kg: ~ ~ 

-: 0.631 glkg: 

-' 

0.50 glkg: 

.-J 

Gross Pathology - Males 

Yellow anogenital staining, ocular opacity, ruffled fur, amber liquid in the 

stomach and intestines, and an irritated stomach lining (dark red) were 

observed in the one animal found dead at five hours. Yellow anogenital 

staining, amber liquid in the stomach and intestines, and an irritated stomach 

(50% dark red or red spots) were observed in the first two animals necropsied 

on Day 1. Yellow anogenital staining, amber liquid in the stomach and red 

liquid in the intestines, and an irritated stomach (50% red spots) were 

observed in the third animal found dead on Day 1. Yellow anogenital staining, 

amber liquid in the stomach and intestines were observed in the fourth animal 

found dead on Day 1. 

Red-brown muzzle and yellow-brown anogenital staining, and dark liquid in 

the stomach and lower gastrointestinal tract were observed in the first animal 

found dead on Day 2. Red-brown muzzle and yellow-brown anogenital 

staining, and dark material and gas in the stomach,' and gas and yellow liquid 

in the lower gastrointestina! tract were observed in the second animal found 

dead on Day 2. Red-brown muzzle and genital staining, and dark liquid in 

the stomach and lower gastrointestinal tract were observed in the 2J5 animals 

found dead on Day 3. Red-brown muzzle and genital staining, and dark 

liqUid in the stomach and lower gastrointestinal tract were observed in the one 

animal found dead on Day 4. 

Tan, dry anogenital staining was observed in the two animals found dead on 

Day 4. No gross abnormalities were observed in any of the three animals 

necropsied at the conclusion of the 14-day observation period. 

Dark muzzle and body staining, dark liquid in the stomach and lower 

gastrointestinal tract were observed in the one animal found dead on Day 3. 

Dark muzzle, body and anogenital staining, and clear liquid in the stomach 

and lower gastrointestinal tract were observed in the one animal found dead 

on Day 4. No gross abnormalities were observed in any of the three animals 

necropsied at the conclusion of the 14-day observation period. 

No gross abnormalities were observed in any of the five animals necropsied at 

the conclusion of the 14-day observation period. 

No gross abnormalities were observed in any of the five animals necropSied at 

the conclusion of the 14-day observation period. 

No gross abnormalities were observed in any of the five animals necropsied at 

the conclusion of the 14-day observation period . 

- ' ••::1 

000090 


I3[OSEARCH, INCORPORATED P.O. cox 8598 . PHIL~DELPHlA. PE."fNSYlVAN!A :9101 . (215i 739-1499 
E!'-JV931201, administered as supplied. 

Acute Oral Toxicity, LD 50 ~ Rats 

TABLE 4 

Mortality & Average Body Weights ~ Females 

Density: 0.93 glmi 

Dose Levels, glkg: 2.00 l.00 0.794 0.631 0.562 0.50 0.473 0.447 0.398 0.355 

Animals: 	 Number 5 5 5 5 5 5 5 5 5 5 

Mortality: 	 Day 0 1 1 0 0 0 a a a a 0 

Day 1 3 2 2 0 a a a a 0 a 

Day 2 1 1 2 a c' 3 0 0 0 0 

Day 3 1 1 1 0 1 a 0 0 0 

Day 4 0 0 0 0 a a a 

Day 5 a a 0 0 a a a 

Day 6 0 a 0 0 a 0 0 

Day 7 a a 0 0 0 a 0 

Day 8 0 a 0 0 0 a a 

Day 9 0 a a a a a a 

Day 10 0 a a a 0 0 a 

Day 11 a a a 0 0 0 a 

Day 12 0 0 0 a 0 0 0 

Day 13 a 0 a a 0 a a 

Day 14 0 a a a 0 a 0 

14-Day 

Totals: Dead 5 5 5 1 0 4 0 a a a 

Live 0 a a 4 5 1 5 5 5 5 

... Average Body 

Weights, g: 

Initial 223 235 206 223 218 221 213 237 218 216 

7th Day NA NA NA 240 233 235 236 254 238 242 

Final NA NA NA 255 251 238 247 266 246 256 

NA = Not Applicable 

-


.. 

000091 


[lIOSEARCH. lNCORl'ORATED· P.o. !lOX 5598· PHlw\DELPHfA. PENNSYLVANIA 19101 (215) 739-4499 
. ENV931201, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 5 

Qbservations - Females 

2.00 glkg: Immediate ­

1 Hour 

4 Hours 

5 Hours 

Day 1 

Day 2 

LOa glkg: Immediate ­

~ 

4 Hours 

.~ 

. ," 

Day 1 

,-I 

Day 2 

Day 3 

0.794 glkg: Immediate2 Hours 

4 Hours 

Day 1 

J 	 Day 2 

Day 3 ! ; 

1 ..... 

J 

1 

J 

All animals appeared to be normaL 


All animals appeared to be lethargic and exhibited 


ruffled fur. 


All animals appeared to be lethargic and exhibited 


ruffled fur and yellow anogenital staining. 


115 animals found dead. 


3/5 animals found dead. 115 animals appeared to be 


lethargic and exhibited ruffled fur and yellow 


anogenital staining. 


115 animals found dead. 


115 animals exhibited audible breathing. 415 animals 

appeared to be normal. 

215 animals appeared to be lethargic. 115 animals 

exhibited gasping and red-brown nasal staining. 115 

animals appeared lethargic and exhibited gasping 

and red-bro;wn nasal staining. 115 animals appeared 

to be lethargic and exhibited gasping and red-brown 

nasal staining, and was found dead at 5 1/2 hours. 

115 animals found dead. 115 animals appeared to be 

lethargic and exhibited ruffled fur and brown oral 

and anogenital staining in the morning and was 

found dead in the afternoon. 215 animals appeared to 

be lethargic and exhibited gasping, ruffled fur, brown 

oral and anogenital staining, and red-brown nasal 

staining. 

1/5 animals found dead. 115 animals exhibited piloerection 

and brown body and anogenital staining. 

1/5 animals found dead. 

All animals appeared to be normal. 


All animals appeared to be lethargic. 


All animals appeared to be lethargic, and exhibited 


ruffled fur. 


215 animals found dead. 3/5 animals appeared to be 


lethargic~ and exhibited ruffled fur and dark body 


staining. 


2/5 animals found dead. 1/5 animals appeared to be 


lethargic and exhibited ruffled fur and dark body staining. 


115 animals found dead. 


8[OSEARCH, [NCORPORATED· P.O. BOX 8598 - PHILADELPHIA, PENNSYLVANIA 1910J· (21S) 739-4499 

000092 

PrQject Number: 93-7'35(5A 	 yage Ib or L.1:5 

. ENV931201, administered as supplied. 

Acute Oral Toxicity, LD - Rats 

50 

IABLE 5 (continued) 

Observations - Females (continued) 

0.631 g/kg: 	 Immediate- All animals appeared to be normaL 

4 Hours All animals appeared to be normal. 

Days 1&2 - 415 animals exhibited ruffled fur, and brown body 

staining. 1/5 animals exhibited ruffled fur, and 

brown body and anogenital staining. 

Day 3 115 animals found dead. 415 animals exhibited ruffled 

fur and brown body staining. 

Day4 415 animals exhibited ruffled fur and brown body 

staining. . 

Days 5&6 415 animals exhibited ruffled fur. 

Days 7-14 - 415 animals appeared to be normaL 

"'".\ 0.562 glkg: 	 Immediate- All animals appeared to be normal. 


4 Hours All animals appeared to be normal. 
 --1 , 

Days 1-4 415 animals exhibited brown body staining and 

ruffled fur. 115 animals exhibited brown body and 

anogenital staining and ruffled fur. 

Days 5&6 - 415 animals exhibited ruffled fur. 

.-. , 	 Days 7-14 - 415 animals appeared to be normaL 1 

0.50 glkg: 	 Immediate - All animals appeared to be normal. 

,­, 4 Hours All animals appeared to be normal. 

1 Day 1 All animals appeared to be lethargic and exhibited 

- yellow anogenital staining. 


Day 2 3/5 animals found dead. 215 animals appeared to be 
 ..... 

J lethargic and exhibited yellow anogenital staining. 

Day 3 115 animals found dead. 115 animals appeared lethargic 

and exhibited yellow anogenital staining. 

Day 4 115 animals appeared to be lethargic and exhibited 

yellow anogenital staining. 

Days 5-14 - 115 animals appeared to be normal. 

0.473 glkg: 	 Immediate- All animals appeared to be normal. 

4 Hours All animals appeared to be normal. 

-; Day 1 All animals exhibited ruffled fur. 

J Days 2-14 - All animals appeared to be normal. 

-­, 0.447 glkg: All animals appeared to be normal throughout the 14-day observation 

j period. 

0.398 g/kg: All animals appeared to be normal throughout the 14-day observation - period. J 

0.355 glkg: 	 All animals appeared to be normal throughout the 14-day observation 

period. 

000093 

J OIOSEARCH, [NCORPORATED - P.o. BOX 8598 PHILADELPHIA, PENNSYLVAN!A 19101 (215) 739-4499 
- ENV931201, administered as supplied. 

Acute Oral Toxicity, LD - Rats 

50 

2.00 g/kg: 

, . 

1.00 glkg: 

0.794 glkg: 

0.631 glkg: 

0.562 g/kg: 

TABLE 6 


GrQSS PatholQgy - Females 


Ruffled fur, yellow anogenital staining, ocular opacity, brown liquid in the 

stomach and intestines, and an irritated stomach lining (dark red) were 

. observed in the one animal found dead at five hours. Yell<?w anogenital 

staIning, amber liquid in the stomach and intestines were observed in the 

first animal found dead on Day 1. Yellow anogenital staining, corneal opacity 

and amber liquid in the stomach and intestines were observed in the second 

animal found dead on Day 1. Yellow anogenital staining, brown liquid in the 

stomach and intestines, and an irritated stomach (50% dark red spots) were 

observed in the third animal found dead on Day 1. Yellowfbrown anogenital 

staining, yellowfbrown liquid in the stomach and intestines, and an irritated 

stomach (50% dark red spots) were observed in the one animal found dead 

on Day 2. 

Red-brown nasal staining, 25% of the stomach lining colored with dark red 

spots, and yellow liquid in the stomach and intestines were observed in the 

one animal found deag at 5 1/2 hours. Nasal and body staining, gas in the 

stomach and yellow liquid in the lower gastrointestinal tract were observed in 

the first animal found dead on Day 1. Nasal, body and genital staining, gas 

in the stomach and yellow liquid in the lower gastrointestinal tract were 

observed in the second animal found dead on Day 1. Red-brown muzzle and 

yellow-brown anogenital staining, dark liquid in the stomach and pink liquid 

in the lower gastrointestinal tract and no formed feces were observed in the 

one animal found dead on Day 2. Red-brown muzzle and genital staining, 

dark liquid in the stomach and lower gastrointestinal tract were observed in 

the one animal found dead on Day 3. 

Dark muzzle and genital staining and dark liquid in the lower gastrointestinal 

tract were observed in the two animals found dead on Day 1. Dark muzzle 

and genital staining and dark liquid in the lower gastrointestinal tract were 

observed in the two animals found dead on Day 2. Dark muzzle and genital 

staining, dark liquid in the lower gastrointestinal tract were observed in the 

one animal found dead on Day 3. 

Brownish anogenital staining and dark liquid in the lower gastrointestinal 

tract were observed in the one animal found dead on Day 3. No gross 

abnormalities were observed in any of the four animals necropsied at the 

conclusion of the 14-day observation period. 

No gross abnormalities were observed in any of the five animals necropsied 

at the conclusion of the 14-day observation period. • 

000094 


B[OSEARCH, INCORPORATED· P.O. BOX 8598 • PHILJ\DELPHlA. PENNSYLVANIA 19101 • (215) 739-+499 
ENV931201, administered as supplied. 

Acute Oral Toxicity, LD - Rats 

so 

TABLE 6 (continued) 

Gross Pathology - Females (continued) 

O.SO g/kg: 	 Dark muzzle, body and genital staining and dark liquid in the stomach and 

lower gastrointestinal tract were observed in the three animals found dead on 

Day 2. Dark muzzle and genital staining, and dark liquid in the stomach and 

lower gastrointestinal tract were observed in the one animal found dead on 

Day 3. No gross abnormalities were observed in the one animal necropsied at 

the conclusion of the 14-day observation period. 

0.473 g/kg: 	 No gross abnormalities were observed in any of the five animals necropsied 

at the conclusion of the 14-day obserration period. 

0.447 glkg: 	 No gross abnormalities were observed in any of the five animals necropsied 

at the conclusion of the 14-day observation period. 

0.398 glkg: No gross abnormalities were observed in any of the five animals necropsied 

at the conclusion of the 14-day observation period. 

0.355 glkg: No gross abnormalities were observed in any of the five animals necropsied 

at the conclusion of the l~day observation period. 

000095 


UlOSEARCH, INCORPORATED· PO. BOX 3598· PHILADELPHIA, PENNSYLVANIA 19101 . (215) 739--\499 
1. lY)C,\",L 1'fUlllJJC1. l:'age 19 of 28

- ENV93120l, administered as supplied. 

.. Acute Oral Toxicity, LDso - Rats 

TABLE 7 


Individual Body Weights 


MALES 


Dose Level: 2.00 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

I2.ay: Intery:al: 

Animal#···0 7 14 Animal# (0-7) (7-14) (0-14) 

16 278 FD FD 16 FD FD FD 

1.7 290 FD FD 17 FD FD FD 

18 293 FD FD 18 FD FD FD 

;;1 19 294 FD FD 19 FD FD FD 

20 296 FD FD 20 FD FD FD 

Mean 290 NA NA Mean NA NA NA 

S.D. 7 NA NA S.D. NA NA NA 

N 5 NA NA N NA NA NA 

Dose Level: 1.00 glkg 


Individual Body Weights (g) Individual Body Weight Change (g) 


~: InterYal: i (ur. 

! Animal# 0 7 14 Anirnal# (0-7) (7-14) (0-14) 

..J 

1 288 FD FD 1 FD FD FD 

2 291 FD FD 2 FD FD FD 

3 299 FD FD 3 FD FD FD 

4 299 FD FD 4 FD FD FD 

5 300 FD FD 5 FD FD FD 

Mean 295 NA NA Mean NA NA NA 

S.D. 6 NA NA S.D. NA NA NA 

N 5 NA NA N NA NA NA 

FD = Animal found dead. - NA Not applicable. 

- 000096 

[JIOSEARCH, INCORPORATED· P.O. BOX 8598· pHIL-\DELPH!A, PENNSYl.'/ . .1.,"'_-\ 19101 . (llS) 739-4499 
ENV931201, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 7{continued) 

Individual BQd¥ Weights " .~ 

.......
 , . MALES 

Dose Level: 0.944 glkg 

Individual Body Weights (g) Individual BQdy Weight Change (g) 

~: Interval: 

. AniriuH#: 0 7 14 Animal#: (0-7) (7-14) (0-14) 

31 240 251 321 31 11 70 81 

32 254 FD FD 32 FD ill FD 

33 242 FD FD 33 FD FD FD 

}- .• 34 234 237. 306 34 3 69 72 j 

35 275 288 366 35 13 78 91 

Mean 249 259 331 .Mean 9 72 81 

S.D. 16 26 31 S.D. S 5 10 

N S 3 3 N 3 3 3

J 

Dose Level: 0.891 glkg ] Individual Body Weights (g) Individual BQdy Weight Change (g) 

J 


I&y: Interval: 


Animal#: 0 7 14 Animal# (0-7) (7-14) (0-14) 


71 274 FD FD 71 FD FD FD 

72 266 FD FD 72 FD FD FD 

J 

J 

73 265 305 351 73 40 46 86 

74 218 264 316 74 46 52 98 

75 285 225 244 75 -60 19 41 

Mean 262 265 304 Mean 9 39 48 

S.D. 26 40 55 S.D. 60 18 77 .] N ., ,.., 5 3 3 N 3 ..) " 

J -, FD = Animal found dead. 

000097 

srOSEARCH. INCORPORATED - P.O. BOX 8598· PH!LADELPHIA. PENNSYLVANIA 19101 (115) 739-499 
..L J.V)>'>J. J. 'f'rl..j,L~y ..... ,.L. ..,...., . "" ...... -...... - -'0 --.. -- -­

- ENV931201, administered as supplied. 

Acute Oral Toxicity, LD 50 - Rats 

IABLE 7(continued) 

Individual Body Weights 

MALES 

Dose Level: 0.794 glkg 

Individual Body W eights (g) Individual Body Weight Change (g) 

1J..B¥: InteIYial: 

" Animal# 0 7 - 14 Animal# (0-7) (7-14) , (0-14) 

21 235 269 322 21 34 53 87 

22 205 216 279 22. 11 63 74 " 

23 219 265 311 23 46 46 92 

:" 24 200 220 275 24 20 55 75 

25 209 263 297 25 54 34 88 

Mean 214 247 297 Mean 33 50 83 

S.D. 14 26 20 S.D. 18 11 8 ."0 N 5 5 5 N 5 5 5 

.• Dose Level: 0.631 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

-' 

r:la}!: InteIYal: -i Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) ~ 

1 207 253 311 1 46 58 104 

- 2 218 264 316 2 46 52 98 

3 211 283 3"'''' .;X)3 72 50 122

4 200 260 315 4 60 55 115 

5 200 239 306 5 39 67 106 

Mean 207 260 316 Mean 53' 56 109 

S.D. 8 16 10 S.D. 13 7 9 r- N 5 5 5 N :J 5 5 

000098 


BIOSEARCH, INCORPORATED - P,O. BOX 8598 - PHILADELPHIA, PENNSYLVANIA 19101 - (2!5) 739,4499 
Project Number: 93-7958A Page 22 of 28 

- E:Nv931201, administered as supplied. 

Acute Oral Toxicity, LD50 - Rats 

TABLE 7(continued) 


Individual Body Weights 


MALES 


Dose Level: 0.50 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

D.e.y: InteIval: . 

Animal# a 7 14 Animal# (0-7) (7-14) (0-14) 

51 255 302 346 51 47 44 91 

52 273 325 378 52 52 53 105 

53 262 318 362 53 56 44 100 

54 272 322 360 54 50 38 88 

55 247 287 334 55 40 47 87 

Mean 262 311 356 Mean 49 45 94 

S.D. 11 16 17 S.D. 6 5 8 

N 5 5 5 N 5 5 5 

000099 


1l10SEARC:l:, INCORJ'ORATED· po. nox 3598 PHILADELPHIA, PENNSYLVANIA 19101· (215) 739.4499 
l-'TOJect NumQer: 'j;j-/'j:JOA 

- ENV931201, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 8 

Individual Body Weights 

FEMALES 

Dose Level: 2.00 glkg 

Individual Body Weights (g) Individual Body Weight Cbange (g) 

. p.g.y: Interval: 

Animaltf 0 7 14 Ariimaltf (0-7) (7-14) (0-14) 

11 216 FD FD 11 FD FD FD 

12 234 FD FD 12 FD FD ill 

13 218 FD FD 13 FD FD FD 

14 225 FD FD 14 FD FD FD 

15 222 FD FD 15 FD FD FD 

Mean 223 NA NA Mean NA NA NA 

S.D. 7 NA NA S.D. NA NA NA 

N 5 NA NA N NA NA NA 

Dose Level: 1.00 glkg 

Individual Body Weights (g) Iudividual Body Weight Change (g:) 

J::2gy: Intex:vgl: 

Animaltf 0 7 14 Animal# (0-7) (7-14) (0-14) 

6 218 FD FD 6 FD FD FD 

7 234 FD FD 7 FD FD FD 

8 243 FD FD 8 FD FD FD 

9 247 FD FD 9 FD FD FD 

10 234 FD FD 10 FD FD FD 

Mean 235 NA NA Mean NA NA NA 

S.D. 11 NA NA S.D. NA NA NA 


N 5 NA NA N NA NA NA 


FD = Animal found dead. 

NA - Not applicable. 

... 

000100 


13rOSEARCH. INCORPORATED· P.O. BOX 8598· PHILADELPHIA. PENNSYL'/AN[A 1910t . (215) 739.4499 
PrQject Numbe.r: 93-7958A Page 24 of 28 

- ENV931201, administered as supplied. 

Acute Oral Toxicity, LD - Rats 

50 

TABLE 8(cQntinued) 


Individual BQdy Weights 


FEMALES 


Dose Level: 0.794 glkg 

Individual BQdy Weights (g) Individual BQdy Weight Change (g) 

Day: InterYs:!l: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 

71 207 FD FD 71 FD FD FD 

72 200 FD FD 72 FD FD FD 

73 201 FD FD 73 FD FD FD 

74 208 FD FD 74 FD FD FD 

75 215 FD FD 7S FD FD FD 

Mean 206 NA NA Mean NA NA NA 

S.D. 6 NA NA S.D. NA NA NA 

N 5 NA NA N NA NA NA 

Dose Level: 0.631 glkg 


Individual BQdy W eights (g) Individual BQdy Weight Change (g) 
 ........ 


I

- 129~D Interval: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 

16 226 FD FD 16 FD FD FD 

17 217 236 242 17 19 6 2S 

18 218 240 243 18 22 3 2S 

19 228 232 252 19 4 20 24 

,....., 20 228 2S0 281 20 22 31 -J.:J 

Mean ?~ 223 240 _:J:J Mean 17 15 32 

S.D. 5 8 18 S.D. 9 13 14 

- N 5 4 4 N 4 4 4 

FD = Animal found dead. 

NA = Not applicable.

-

000101 


llrOSC:ARCH, INCORPORATED· P.O. BOX 8598 . PHilADELPHIA, ?S'\INSYLVANfA 19101 . (215) 739.<1499 
--

A 1 VJ el. L 1'1 UH (Vel. 

- ENV931201, administered as supplied. 

Acute Oral Toxicity, LDso - Rats 

TABLE 8(continued) 


Individual Body Weights 


FEMALES 


Dose Level: 0.562 glkg 

Indiyidual Body Weights (g) Individual Body Weight Change (g) 

~: InteIYsl: .. -Anim"al#' 0 7 14 Animal# (0-7) (7-14) (0-14) 

11 225 226 242 11 1 16 17 

12 205 230 238 12 25 8 33 

13 214 232 250 13 18 18 36 

14 221 233 258 14 12 25 37 

15 223 246 266 15 23 20 43 

Mean 218 233 251 Mean 16 17 33 

S.D. 8 8 11 S.D. 10 6 10 

N 5 5 5 N 5 5 5 

Dose Level: 0.50 glkg 

Individual Bod:y W eights (g) Individual Body Weight Change (g) 

Day: InteIYal: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 

56 235 FD FD 56 FD FD FD 

57 214 235 238 57 21 3 24 

58 212 FD FD 58 FD FD FD 

59 214 FD FD 59 FD FD FD 

60 232 FD FD 60 FD FD FD 

Mean 221 NA NA Mean NA NA NA 

S.D. 11 NA NA S.D. NA NA NA 

N 5 NA NA N NA NA NA 

FD = Animal found dead. 


NA = Not applicable. 


000102 


mOSEARCH. INCORPORATED - PO. flOX 8598· PHILADELPHIA. PENNSYLVANIA 19101 . (215) 739-+;99 
Project Number: 93-7958A Page 26 of 28 

. E"N'V931201, administered as supplied. 

Acute Oral Toxicity, LD50 - Rats 

TABLE 8(continued) 

Individual Body Weights 

FEMALES 

Dose Level: 0.473 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

D..gy: InteIYal: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 


36 204 229 235 36 25 6 31 


37 215 235 239 37 20 4 24 


38 215 239 . 259 38 24 20 44 


39 229 260 277 39 31 17 48 


40 200 216 225 40 16 9 25 


Mean 213 236 247 Mean 23 11 34 

S.D. 11 16 21 S.D. 6 7 11 


N 5 5 5 N 5 5 5 


Dose Level: 0.447 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

~: InteIYal: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 

76 224 239 253 76 15 14 29 

77 237 248 262 77 11 14 25 

78 238 257 277 78 19 20 39 

79 239 254 260 79 15 6 21 

80 245 274 279 80 29 5 34 

Mean 237 254 266 Mean 18 12 30 '7 S.D. S 13 11 S.D. I 6 7 

N 5 5 5 N 5 5 5 

000103 


B£OSEARCH. INCORPORATED - P.O. BOX 8598 - PHIL·\DELPHIA. PENNSYLVANIA 19101 - (215) 739-4499 
Project Number: ~J-'/~.JtiA t'age L.I or L.~ 

- ENV931201, administered as supplied. 

Acute Oral Toxicity, LD50 - Rats 

TABLE 8(continued) 


Individual Body Weights 


FEMALES 


Dose Level: 0.398 glkg 

Individual Body Weights (g) Individual Body Weight Change (g) 

I2ay: Intl:!nml: 

Animal# 0 7 14 Animal# (0-7) (7-14) (0-14) 

26 207 225 238 26 18 13 31 

27 220 242 251 27 22 9 31 

28 228 245 254 28 17 9 26 

29 212 238 239 29 26 1 27 

30 223 240 249 30 17 9 26 

- Mean 218 238 246 Mean 20 8 28 

S.D. a 8 7 S.D. 4 4 3 

~ N 5 5 5 N 5 5 5 

Dose Level: 0.355 glkg 


Individual Body Weights (g) Individual Body Weight Change (g) 


Day: InterY!ill: 

j 

,'. 

Animal# 0 7 14 Animal:t (0-7) (7-14) (0-14) , 

6 230 236 257 6 6 21 27 

- 7 207 236 249 7 29 13 42 

8 214 238 252 8 24 14 38 

9 211 257 272 9 46 15 61 

10 217 241 248 10 24 7 31 

Mean 216 242 256 Mean. 26 14 40 

~ 

S.D. 9 9 10 S.D. 14 5 13 

N 5 5 5 N 5 5 j 

000104 


8!OSEARCH. INCORPORATED P.O. 80X 3598· PH!LADELPHIA. PENNSYLVANIA 19101 . (215) 739-4499 
Project Number. 


Protocol Number. 


Sponsor 


Test Article: 


Test: 


GLP Compliance: 


Deviation: 


•...i 

SubmiHed by: 

'-t 

-

I 

'''''"l 

J 

Approved by:

-

-i 

.... , 

Acknowledged by: 

.... 

Appendix A - Page 28 of 28 

BIOSEARCH INCORPORATED 

P.o. Box 8598, Philadelphia, Pennsylvania 19101 ..-" Telephone: (215) 739-4499 I 

Fax: (215) 423-9210 

DEVIATION FROM PROTOCOL #1 

93-79S8A 

AOT-800 (Rev. 02/93) 

ENV931201 

Acute Oral Toxicity, LDSO - Rats (TSCA) 

40 CFR 792 OECO 

Due to a technician error, afternoon observations for males in the 6.31 glkg 

dose level were made but not recorded on Day 9. In addition, due to 

technician error, morning observations on Day 10 for these animals were 

made but again not recorded . 

This deviation"is not thought to affect the integrity of this study. 

C;::;/";\ 

David Gabriel, B. A. 

~ 

, . S 

I ality Assurance 

Vice President, Laboratory Operations 

& Regulatory Affairs 

Jf~~' o;::~ 

~13-4~ Date 

t;-;:; -ii 

Date 

'!f

te 

J

I

/ 1Y 

-f/?~ Date 

5----Ie! 

, -/;, c: )

Dare 

lvfay 12, 1994 93 040 7958 1485 Form QA-500A 

000105 

=1 
,...; 

Acute Toxicity of 

to Photobacterium phosphoreum 

(Screen Test) 

,..... 

July 1995 

by 

-, 

000106 

Title: Acute T oxicityof to Photobacterium phosphoreum 

-. Issue Date: July 1995 

Report Preparation: 

Skl,\).,1 l. srt$'u..·-rrod 

Sheryl L. Spotswood 

Study Director 

Environmental Services Group 

l i Report Approval: 

] 


J 
 Report and Study Inspection: 

Date: ['2- - 5u.b.. - q IS 

Date: Il-1~-/47S

I 

Date: a.:::rc.,~ f {'

I 

Date of testing: 

Protocol: 

Data/Archive 

Reference: 

Date Protocol 

Approved: 

;JI22~·)r~ WIlliam J. Kuc 

Study Inspector 

Environmental Services Group 

December 2, 1993 


Microtox Basic Test Procedure (Microbics Corp., 1991) 


SX-2081 


May 30, 1991 (Microbics Corp., 1991) 


000107 

i 

:.. .' ! ill, ',,­

'-~·l;., 

Test Material: 

Batch/Lot: 93-72733 

Test Date: December 2, 1993 

Summary: The 15 minute ECSO of SX-2081 to Photobacterium phosphoreum is estimated 

to be 1 - 10 mg/L. 

Species: Photobacterium phosphoreum 

Source: Petrolfte Corporation 

15 Minute EC50: 1 - 10 mg/L (estimation) 

Phenol Standard Test: 5 minute EC50 = 23 mg/L 

(Acceptable range =13 - 26 mg/L) 

. Control 10: 89 

Temperature: 15°C 

000108 

Introduction 

This Microtox bioassay was performed by the 

The Microtox test system determines toxicity by measuring the decrease in light production of the 

luminescent marine bacterium, Photobacterium phosphoreum, following a timed exposure to a test 

material. During these organisms' normal cellular respiration, about ten percent of their metabolic 

energy is converted into visible light. Changes in this metabolic process cause a change in light output 

of this species. A reduction in cell metabolism, caused by exposure to a test sample, is directly 

proportional to the measured reduction in bioluminescence (Bulich and Isenberg. 1981). The 

concentration of test sample which produces a 50% reduction in bioluminescence, following a tiined 

exposure, is considered the effective concentration (EC50). Microtox bioassay EC50s are usually 

calculated following 5 and 15 minute exposures, however 30 minute exposure times are sometimes 

required. 

This study followed the Basic Test protocol which was published by Microbics Corporation (Microbics, 

1991). All original raw data is retained in the' archives. 

Methods and Materials 

Test Organism 

A Microtox reagent comprised of a lyophilized (freeze-dried) preparation of Photobacterium 

phosphoreum was rehydrated with Microtox Reconstitution Solution. This bacterial suspension was 

maintained in a separate well of the analyzer at 5°C and allowed to equilibrate a minimum of 30 

minutes prior to testing. 

Test Substance and Stock Preparation 

A sample of. . batch 93-72733, was obtained from I , 

facility. is a brown, water soluble liquid. A stock of was prepared by 

weight to a preCision of one milligram and diluted to volume in volumetric glassware with deionized 

water . 

• 


Test Conditions 


This toxicity bioassay was performed using a Microtox Model 500 analyzer and software manufactured 

by Microbics Corporation (Carlsbad, CAl. The software was loaded onto an IBM-compatible computer 

which interfaced with the analyzer. All supplies used were those obtained from or recommended by 

... Microbics Corporation. . 

Unused cuvettes were added to rows A and B of the analyzer. Micratox Osmotic Adjustment Solution 

and Diluent (a 2% saline solution) were added to the cuvettes as required by protocol for a test with a 

dilution factor of 10. All were maintained at a constant temperature of 15°C for 15 minutes. 

Subsequently, 10 pi of the bacterial suspension were pipetted into row B cuvettes containing 500 pi of 

diluent and allowed to equilibrate for 15 minutes. 

000109 


7 
Four dilutions of the I stock were made in row A with Microtox Diluent while the bacteria were 

equilibrating. Following the equilibration time, light emissions of the bacteria in row B were recorded 

as initial values (10)' Immediately, 500 pi from each cuvette in row A were transferred into the 

... corresponding row B cuvettes. At 5 and 15 minutes following the sample dilution, light emissions were 

recorded (15 and 115)' . 

Statistical Analvsis 

The ratio of light lost to the light remaining in each of the cuvettes in row B is used to calculate the 

EC50. A ratio for each concentration is expressed as a gamma value. That concentration of sample 

which was calculated to produce 50% reduction in light or a gamma of 1.0, following a given timed 

"expos'ure,"Was designated as the EC50 value. EC50 values are expressed in mg/L, with lower EC50 

values corresponding to higher toxicity levels. Ninety-five percent confidence limits were calculated for 

each EC50. 

Results and Discussion 

Upon addition of the product to the test cuvette it was observed that appeared soluble in the .,-! test media . 

I 

The 15 minute EC50 of to Photobacterium phosphoreum is estimated to be 1 - 10 mg/L. The 

phenol standard test which was performed simultaneously with the " Basic Test resulted in an 

acceptable EC50 value for the 5 minute exposure. 

, 


This assay was a range-finding or screen' test. A definitive test was not performed. 


:..J 

References 

J BUlich, A. A. and Isenberg, D.L, 1981, "Use of the Luminescent Bacterial system for the Rapid 

Assessment of Aquatic Toxicity." ISA Transactions, Vol.20, NO.1. 

Microbics Corporation, 1991, "Microtox Model 500 Toxicity Test System Manua'" (Preliminary 

-i Release), Microbics Corp., Carlsbad, CA. 

~ 

.J 

.J 000110 
----- ------------ ---------- ---------

TEST DATE: 

TEST TIME:---------­

MICROTOX DATA REPORT 

FILE NAME: .K15 

Investigator: C:;;. see ~u..rr:-c(J 10 

~ 

";-' -~~ Approved by:_______ 

Sample Descriptidn: Test date;\.",12/2/93, i (93-72733) . 

Procedure: BASIC '" .. __/ Osmotic Adjustment: MOAS 

Initial Concentration: 9.876 MG/L Dilution Factor: 10 

.... Test Time: 15 minutes Concentration Units: MG/L

NUMBER IO/IT CONC. GAMMA 

1 82.53/ 74.66 0.0099 0.05284# 

2 81.11/ 76.08 0.0988 0.01541# 

3 85.30/ 69.30 0.9876 0.,17234# . ". :,,,~, 

4 " 89.55/ 0.06 ., 9: 8760 > 999 * 

, CONTROL IT/IO = 84.31/ 88.52 ......1 CORRECTION FACTOR = 0.9524 

.IC25 0.5602 (95% CONFIDENCE RANGE EXCEEDS LIMITS) 

~jECOMMEND: RERUN ASSAY AT HIGHER CONCENTRATIONS 

~ # Used for calculations 

* Invalid data or controls 

... I 

..J 

-.lIIIj , , 

2 .~ 

- 000111 

-
• STATISTICAL REPORT FOR DATA FILE - _. K15 

.. ESTIMATING EQUATION: LOG C = 0.9338 x LOG +0.1939 


'.f! NUMBER OF DATA PAIRS USED: 3 


RESIDUAL VARIANCE: 1.52054 

95% CONFIDENCE FACTOR: %9999.00000 FOR IC25 

CORRELATION COEFFICIENT: R =: 0.48962 

.,. 

COEFFICIENT OF DETERMINATION: RU2 =: 0.23973 

INHIBITING CONCENTRATIONS (95% CONFIDENCE RANGE) : 

ICSO =: 1.5627 MG/L ( 0.0000 TO % 628.6954E+26)*** 

IC20 =: 0.4282 MG/L ( 0.0000 TO % 446.7338E+14) 

ICI0 = 0.2008 MG/L ( 0.0000 TO % S30.4764E+08) 

ICOl =: 0.0214 MG/L ( 0.0000 TO % 746.4709E+13) 

*** EXTRAPOLATED RESULTS 

'""'I 

J 

I 

i 

.J 

-


-


000112 

------------

TEST TIME: -----...:.~~ 

-----­

Osmotic Adjustment: MOAS 

MICROTOX DATA REPORT 

TEST DATE: FILE NAME: .. K5 -" ­

Investigator: <;-: s-r±tw'1~J ~ ~Approved by:
 date,,- '-1.2/2/93 I (93-72733). Sample Descriptio: Test 

.Procedure: BASIC ~. 

: 9.876 MG/L Dilution Factor: 10 Initial Concentration 

Test Time: 5 minutes 

NUMBER IO/IT 

1 82.53/ 74.15 

2 81.11/ 73.91 

3 85.30/ 75.74 

4 89.55/ "0.44 

CONTROL IT/IO = 81.03/ 88.52 

i CORRECTION FACTOR = 0.9154 . \ 

Concentration Units: MG/L 

CONC. 

0.0099 

0.0988 

0.9876 

"9.8760 

GAMMA 

0.01884 

0.00456* 

0.03093 

185.30194* 

...........NO TOXICITY AT CONCENTRATIONS TESTED 


* Invalid data or controls 

.. "", 

i 

'.. 

.­

, ...... 

.­, 

-


000113 

ACUTE TOXICITY OF 

TO THE SHEEPSHEAD MINNOW k 

Cyprinodon variegatus 

'. 

July 1995 

by 

-


000114 

''''I 

1 


....! 

. 

'-

1-' 

J

! 

, 

I..... 

-, 

J 

I 

-
, 

.J 

TITLE: Acute Toxicity of 

variegatus. 

ISSUE DATE: July 1995 

REPORT PREPARED BY: 

1I1£~~1~v Date: 11::) IF;.).-/ 

Scientific Investigator 

REPORT APPROVED BY: 

Date: 17:S~ /7i~

DATES OF TESTING: January 17 - 21, 1994. 

PROTOCOL NO: FM-01-5A 

Quality Assurance Statement 

This study was not conducted according to Good Laboratory Practices (40CFR792). 

The test data was reviewed on January 21, 1994 by the Quality Assurance Officer 

to assure that Standard Operating Procedures and the protocol used for this study 

were adhered to. This report is an accurate reflection of the raw data. 

Date: 

Sheryl L. Spotswood 

Designated Quality 

Assurance Officer 

2 

000115 
-. i 

MATERIAL: 

BATCH/LOT No.: (93-72731 & 93-72732) 

-

i TEST DATE: January 17 - 21, 1994. 

-, 

...l 

SUMMARY: 

l

.­

SPECIE: 

SOURCE: 

The 96 hour static LCso of , in synthetic seawater to the 


sheepshead minnow is 2.2 mg/l. 


Sheepshead minnow, Cyprinodon variegatus. 


Aquatic Research Organisms, Hampton, New Hampshire 


96 HOUR LCso AND 95% CONFIDENCE LIMITS: 

2.2 (1.0 - .3.2) mg/l 

-J 96 HOUR NO OBSERVED EFFECT CONCENTRATION (NOEC): 

] 

'""l 

.J 

WATER TYPE: 

TEMPERATURE: 

- pH: 

- SALINITY: 

-

-

1.0 mg/I 

Synthetic seawater 

15.5 -17.5 0 C 

8.3·· 

31 parts-per-thousand 

3 


000116 

INTRODUCTION 

. )."-, 

This static acute bioassay was performed by the -	 ,. '.\'­

during the period of January 17 - 21, 1994. 	 'J.""~"".'" ;~... "'~ 

Bioassay procedures described below are patterned after those described by the " . 

American Public Health Association (1), American Society of Testing and Materials 

(2), Office of Pesticides and Toxic Substances (3), the US EPA (4) and the

., 	 Organization for Economic Cooperation and Development (5). Test procedures 

J 	 follow I. • Standard Operating 

] 

Procedures which were compiled following the intent of Good Laboratory Practices 

(6). All original raw data is retained in the archives. 

METHODS AND MATERIALS 

.] The sheepshead minnows, Cyprinodon variegatus, used for this study were 

obtained from Aquatic Research Organisms, Hampton, New Hampshire. Test fish 

were cultured in an activated carbon filtered, recirculating synthetic seawater ] system for at least ten days prior to testing. All test fish were maintained 

according to the procedures outlined by Brauhn et a/ (7) on a 16 hour daylight 

photoperiod. Fish were fed dry commercial fish food or live Artemia nauplius larva 

on a daily basis (Monday thru Friday) during holding. Fish appeared in good health 

and mortalities during the 48 hours immediately preceding the test were less than 

3%. Fish used in this test were randomly selected from the stock culture. 

Twenty-four hours before initiating the test, fish were taken off feed. Ten fish 

were introduced at random into the control and each exposure vessel. The fish had 

a mean (10 organism) total length of 1.9 cm and a mean weight of 0.09 grams. 

Biological loading was 0.06 g/1. 

Artificial seawater used during this study was prepared by dissolving Hawaiian ] 	 Marinemix sea salts in deionized water; deionized water test was prepared by 

passing St. Louis municipal water through multiple deionization beds. Test water 

had a salinity of 31 parts-per-thousand and a pH of 8.3. Salinity was determined 

with a YSI Model 33 SCT meter, pH with a CORNING pHI/on Meter Model 135 and 

dissolved oxygen with a YSI Model 54 oxygen meter. Temperature was determined 

with a handheld glass thermometer. 

Environmental control was achieved through the use of a Heinicke HC 982M walkin environmental chamber. Thermostatic controls were adjusted to achieve a 

nominal environmental chamber test temperature of 17 .±. 1°C. Temperature range 

in the test tanks was 15.5 - 17.5 °C while the photoperiod consisted of a 16 hour 

daylight regime. 

The study employed two separate experiments, a preliminary or rangefinding study 

and a definitive study. The rangefinding study delineated a narrower range of test 

concentrations which were utilized for the definitive study. 

4 

000117 
L' 

\,,::.., , 

. ~ . 

A sample of fa 50/50 composite sample of batches 93-72731 ~~3 72732) was obtained from Technology Department, <'. '>b 

was received in a clear glass bottle and stored at roo~ 

temperature until use. The test material, , , is a brown, water soluble liquid:-'"" 

Seven concentrations and a control were tested to determine the toxicity of ~ 

to the sheepshead minnow. A 10 mgtml stock solution of was 

prepared by weight and diluted to volume with deionized water. Test tank position 

was randomized in the test chamber using random number generating program 

(RAND). The study was initiated by adding the toxicant to test vessels containing 

15 liters of dilution water, thoroughly mixing, and then randomly introducing the 

fish. 

Dissolved oxygen, pH and temperature were determined initially and every 24 hours 

thereafter for the control and all concentrations tested. Salinity in the control tank 

was determined initially and at 96 hours. Dead organisms were removed from test 

tanks every 24 hours; behavioral observations of surviving fish were performed at 

this time. 

Mortality data were used to calculate the median lethal concentration (LC ) and so

their respective 95% confidence limits for the appropriate exposure periods. 

Calculations were performed using a computer program developed by Stephan st al 

(8). 

RESULTS AND DISCUSSION 

Test solutions of - were completely soluble in all concentrations tested. 

......, The 96 hour LCso with 95 % confidence limits for to the sheepshead

i minnow is 2.2 (1.0 - 3.2) mg/l. The 96 hour No Observed Effect Concentration 

{NOECI is 1.0 mgt!. Mortalities and LCso values with asso.ciated 95% confidence 

limits are presented in Tab/es 1 and 2. Behavioral observations conducted during 

the study are presented in Table 3. Chemical and physical parameters monitored 

during the test are presented in Tables 4, 5, and 6. 

DEVIATIONS FROM PROTOCOL 

The following temperature deviations from the specified 17.0 ± 1.0°C occurred: 

1.0 mgtl at 0 hours (15.5°C) and 0.56 mgtl at 24 hours (15.8°C). 

It is not felt that these' deviations 'should effect the outcome of the study. 

"'­

""'':v 

'-. 

5 


000118 

... 


... 


. -


.~

, 

; 

"' 
j 

...: 

..,. 

I 

. ,J 

, .., 

,J 

.] 

" "J. 

. " 

, 

I 

,0IIi 

", 

J 

i 

....... 
 J 

j ..... 

...., 

.. 

1 

! 

... 


... 


REFERENCES 

1. American Public Health Association. 1992. Standard Methods for the 


Examination of Water and Wastewater, 18th ed. Washington, D.C. 


2. American Society of Testing and Materials. 1980. Standard Practice for 

Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians. 

E-729, Philadelphia, PA. 

3. U.S. E.P.A. 1982 - Environmental Effects Tests Guidelines (EG-9), EPA 


560/6-82-002. 


4. Office of Pesticides and Toxic Substances. 1980. Fish Acute Toxicity Test 


Standard Toxic Substances Control Act. U.S. E.P.A., Washington, D.C; 


5. OECD Guidelines for Testing of Chemicals. 1984. Organization for Economic 


Co-operation and Development, Paris . 


6. U.S. E.P.A. July 1, 1986. Good Laboratory Practice Standards. Code of Federal 

Regulations. 40 CFR Part 792. Washington, D.C. 

7. Brauhn, J. L., R. A. Schoettger, and L. H. Mueller. 1975. Acquisition and Culture 

of Research Fish. Rainbow Trout, Fathead Minnows, Channel Catfish and Bluegills. 

EPA-660/3-75-001. . 

8. U.S. E.P.A. 1978 - Stephan, C.E. and R.W. Andrews. A Computer Program for 

Calculating an LCso, Duluth, MN. 

6 


000119 


.... 

m 
~. 

: .... 

.' ,: 

. :' i... ... 

,.... 

Table(s) 1, 2, 3: Toxicity to the Sheepshead Minnow,". 

Cyprinodon variegatus ., ,:'.. '" 

Mortality Observations, LC50 Calculations and Behavioral Observations/ 

j

A# 

/'! 

,f 

" ./ 

Table 1: Mortality Observations (#Dead out of 10) 

'-',

, 

", ........;.;.:..<;.::;:; :::;. 


!i:4~:i)fm::::m o o o o 2 10 10 10 .;.; .::::=;::::: :;:;:;:::;:::.;. 

:::::::i??Wflf::::}:: o o o o 2 10 10 10

"] o o o o 2 10 10 10 

Table 2: LCso Calculations ,] 

1.8 - 3.2 Binomial 

1.0 - 3.2 Binomial 

1.0 - 3.2 Binomial 

1.0 - 3.2 .Binomial 

Table 3: Behavioral Observations 

1. GENERAL 

a. quiescent 

b. hyperexcitable 

c. irritated 

d. surfacing 

e. sounding 

f. twitching 

g. tetanus 

h. flaccid 

i. normal 

2. SWIMMING 3. PIGMENTATION 

a. ceased a. light discolored 

b. erratic b. dark discolored 

c. gyrating c. varidiscolored 

d. skittering 

e. inverted 

f. on side 4. INTEGUMENT 

a. mucus shedding 

b. mucus coagulation 

c. hemorrhagic 

.- Data not collected after 100% mortality occurs. 

5. RESPIRATION 

a. rapid 

b. slow 

c. irregular 

d. ceased 

e. gulping air 

f. labored 

7 


000120 

Table(s) 4,5,6: Toxicity to the Sheepshead Minnow, 

Cyprinodon variegatus 

Chemical and Physical Parameters 

. pH 

.., 


J 


-, 
j? 

---" 

J 


I 

-" 

8.3 8.3 

8.2 8.2 8.2 8.2 8.2 

8.2 8.2 8.2 8.2 8.2 

8.2 8.2 8.2 8.2 8.2 

8.2 8.2 8.2 8.2 8.1 

Dissolved Oxygen, ppm 

7.9 7.9 • 7.9 8.0 8.0 

7.7 7.S 7.8 7.7 7.9 

7.7 7.8 8.0 7.9 8.0 

7.7 7.9 8.0 7.9 7.8 

Temperature °C 

16.0 16.0 15.8 16.0 16.1 

16.3 16.2 16.3 16.3 16.2 

16.3 16.4 16.2 16.4 16.5 

16.3 1'6:5 16.3 16.5 16.3 

Control Tank Salinity, parts-per~thousand 

30.5 

31.0 

- Data not collected after 100% mortality. 

000121 

8 
] 

ACUTE TOXICITY OF 

] TO THE FATHEAD MINNOW, 

Pimephales promelas 

oJ 

,-l 

.J 

-

'-' ! 

..J 

-

-


-


July 1995 

- by 

000122 


-

, 


TITLE: Acute Toxicity of co the Fathead MinnowI Pimephales 

promelas. 

ISSUE DATE: July 1995 

REPORT PREPARED BY: 

1 	 ?y~~~. I;::~ - . .-J 	 William J. Kuc t:1t 

Scientific Investigator 

] 

REPORT APPROVED BY: 

..J 

] 

DATES OF TESTING: January 31 - February 4, 1994. 

] PROTOCOL NO: FF-01-3A 

",- . /"" 

Date: 17.Jv2r l1Y'~ 

Date: '9:.r~ 177 S 

Quality Assurance Statement 

This study was not conducted according to Good laboratory Practices (40CFR792). 

The test data was reviewed on February 2, 1994 by the Quality Assurance Officer 

to assure that Standard Operating Procedures and the protocol used for this study 

, were adhered to. This report is an accurate reflection of the raw data. ....-; 

Date: 

Sheryl L. Spotswood 

Designated Quality 

Assurance Officer 

2 

000123 

MATERIAL: 

...... BATCH/LOT No.: (93-72731 & 93-72732) 

...... TEST DATE: January 31 - February 4, 1994. 

"SUMMARY': ' The 96 hour static LCso of in reconstituted freshwater to 

the fathead minnow is 0.80 mg/l. 

SPECIE: Fathead minnow, Pimephales promelas 

SOURCE: Aquatic Research Organisms, Hampton, New Hampshire 

96 HOUR LCso AND 95% CONFIDENCE LIMITS: 

0.80 (0.56 : 1.00) mg/l 

J 96 HOUR NO OBSERVED EFFECT CONCENTRATION (NOEC): 

0.32 mg/l 

WATER TYPE: Reconstituted freshwater 

TEMPERATURE: 15.5 - 17.4 0 C 

pH: 8.3 

HARDNESS: 160 mg/I as CaCOa " .r ,,., , 

ALKALINITY: 11 3 mg/l as CaCOa 

3 

-

INTRODUCTION 

This static acute bioassay was performed by the 

during the period of January 31 - February 4, 1994. 

Bioassay procedures described below are patterned after those described by the 

American Public Health Association (1), American Society of Testing and Materials 

(2), Office of Pesticides and Toxic Substances (3), the US EPA (4) and the 

Organization for Economic Cooperation and Development (5). Test procedures ~.:': . i, .. "< r' .

f()lIo.w. . . .... . . Standard Operating 

'ProCecll.ireswhich were compiled following the intent of Good Laboratory Practices 

(6). All original raw data is retained in the archives. 1 

,....I 

METHODS AND MATERIALS 

The fathead minnows, Pimephales promelas, used for this study were obtained 

from Aquatic Research Organisms, Hampton, New Hampshire. Test fish were held 

in recirculating, 29 gallon glass aquariums for five days prior to testing. Holding 

water was reconstituted hard water prepared from deionized St. Louis municipal 

water. All test fish were maintained according to the procedures outlined by 

Brauhn et al (7) on a .16 hour daylight photoperiod. Fish were fed dry commercial 

fish food or live Artemia nauplius larvae on a daily basis (Monday thru Friday) during 

holding. Fish appeared in good health and mortalities during the 48 hours 

immediately preceding the test were less than 3%. Fish used in this test were 

] 	 randomly selected from the stock culture. Twenty-four hours before initiating the 


test, fish were taken off feed. Ten fish were introduced at random into each 


control and exposure vessel. The fish had a mean (10 organism) total length of 2.2 


] 	 cm and a mean weight of 0.08 grams. Biological loading was 0.05 gil. 

J 

Dilution water for the test was prepared by the carbon filtration and deionization of 

municipal water. Treated water was then reconstituted according to procedures 

outlined by Marking and Dawson (8). The static fish bioassays were conducted in 

21 liter glass-silicone aquaria containing 15 liters of reconstituted water. , -1 Reconstituted water was characterized as having a pH of 8.3, total hardness of 1 .­ 160 mg/l as CaC03, total alkalinity of 113 mg/l as CaC03, and a specific 

conductance of 410 pmhos/cm. Hardness and alkalinity were determined according 

to standard analytical procedures APHA (1), pH with a CORNING pHllon Meter 

Model 135, conductivity with a YSI Model 33 conductivity meter, dissolved oxygen 

with a YSI Model 54 oxygen meter, and temperature with a handheld thermometer. 

-- Environmental control was achieved through the use of a Heinicke HC 982M walk­


in environmental chamber. Thermostatic controls were adjusted to achieve a 


nominal environmental chamber test temperature of 17 ± 1ac. Temperature range 


in the test tanks was 15.5 . 17.4 ac while the photoperiod consisted of a 16 hour 


daylight regime. 


4 

000125 
-


The study employed two separate experiments, a preliminary or rangefinding study 

and a definitive study. The rangefinding study delineated a narrower range of test 

concentrations which were utilized for the definitive study. 

A sample of (a 50/50 composite sample of batches 93-72731 & 93-72732) 

was obtained from' . 

was received in a clear glass bottle and stored at room temperature until use. 

The test material, I, is a brown, water soluble liquid. Eight concentrations and 

a control were tested to determine the toxicity of to the fathead minnow. 

A 10 mg/ml stock solution of was prepared by weight and diluted to volume 

,~,i~h,d.eionized wat~r. Test tank position was randomized in the test chamber using 

random number generating program (RAND). The study was initiated by adding the 

toxicant to test vessels containing 15 liters of dilution water, thoroughly mixing, and 

then randomly introducing the fish. 

Dissolved oxygen, pH and temperature were determined initially and every 24 hours 

thereafterfor the control and all concentrations tested. Dead organisms were removed 

from test tanks every 24 hours; behavioral observations of surviving fish were 

performed at this time. 

Mortality data were used to calculate the median lethal concentration (LC ) and their so 

respective 95 % confidence limits for the appropriate exposure periods. Calculations 

were performed using a computer program developed by Stephan et at (9). 

RESULTS AND DISCUSSION 

Test solutions of were completely soluble in all concentrations tested. 

The 96 hour LCso with 95 % confidence limits for , to the fathead minnow is 

0.80 (0.56 - 1.00) mg/1. The 96 hour No Observed Effect Concentration (NOEC) is 

0.32 mg/I. Mortalities and LCso values with associated 95% confidence limits are 

presented in Tables 1 and 2. Behavioral observations conducted during the study are 

presented in Table 3. Chemical and physical parameters monitored during the test are 

presented in Tables 4, 5, and 6. 

DEVIATIONS FROM PROTOCOL 

Test organisms were held for 5 days rather than 10 days as specified in the protocol. 

The following temperature deviation from the specified 17.0 ± 1.0°C occurred: 0.56 

mg/l at 0 hours (15.5°C). ' 

It is not felt that these deviations should effect the outcome of the study, since the 

fish appeared in excellent health during holding and no mortalities or aberrant behavior 

was evident in the control tank during testing. 

5 

000126 

..., REFERENCES 

1. American Public Health Association. 1992. Standard Methods for the Examination .... of Water and 	Wastewater, 18th ed. Washington, D.C . 

2. American Society of Testing and Materials. 1980. Standard Practice for Conducting ..... ! 	 Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians. E-729, 

Philadelphia, PA. 

3. U.S. E.P.A. 1982 - Environmental Effects Tests Guidelines IEG-9), EPA 

560/6-82-002. 

4. Office of Pesticides and Toxic Substances. 1980. Fish Acute Toxicity Test Standard ! 

J Toxic Substances Control Act. U.S. E.P.A., Washington, D.C. 

5. OECD Guidelines for Testing of Chemicals. 1984. Organization for Economic 

Co-operation and Development, Paris. 

.-, 6. U.S. E.P.A. July 1, 1986. Good Laboratory Practice Standards. Code of Federal 

J Regulations. 40 CFR Part 792. W~shington, D.C. 

..... 7. Brauhn, J. L., R. A. Schoettger, and L. H. Mueller. 1975. Acquisition and Culture 

.J of Research Fish. Rainbow Trout, Fathead Minnows, Channel Catfish and Bluegills. 

EPA-660/3-75-001. 

8. Marking, L.L. and V. K. Dawson. 1973. Toxicity of Quinaldine Sulfate to Fish. Fish 

Control No. 48. U. S. Fish Wildlife Service, Washington, D. C. 

9. U.S. E.P.A. 1978 - Stephan, C.E. and R.W. Andrews. A Computer Program for 

Calculating an LCso, Duluth, MN. 

6 

000127 
~< 

«/~ 

. ... /. I~J-:-:! r":"" .".' 

Table{s} 1, 2, 3: Toxicity to the Fathead Minnow, Pimephalespromefas 

Mortality Observations, LC50 Calculations and Behavioral Observatforis"~i<; < 

Table1: Mortality Observations I#Dead out of 10) .•, 

-! 

..J• 

J 


] 


<"!'"'i ««J 


., 


J 


< -r ! 

-

~ 

.~ 

-


o 

o 

o o o 6 10 

o 0< 8 10 

o o o 9 10 

Table 2: LCso Calculations 

10 10 10 

10 10 10 

10 10 10 

Table 3: Behavioral Observations 

0.93 

0.83 

0.80 

1.00 ­ 1.80 

0.56 ­ 1.80 

0.56 - 1.80 

0.56 ­ 1.00 

Binomial 

Binomial 

Binomial 

Binomial 

li li 1ad.2b 

3b.5f 

1i 1a.3b 1ad,3b 

Sf 

1i 1ad,3b 1ad,3b, 

5f 

1i 1a,3b 1ad,3b 

Sf 

1. GENERAL 2. SWIMMING 3. PIGMENTATION 

a. quiescent a. ceased a. light discolored 

b. hyperexcitable b. erratic b. dark discolored 

c. irritated c. gyrating c. varidiscolored 

d. surfacing d. skittering 

e. sounding e. inverted 

f. twitching f. on side 4. INTEGUMENT 

g. tetanus a. mucus shedding 

h. flaccid b. mucus coagulation 

i. normal c. hemorrhagic 

-- Data not collected after 100% mortality occurs. 

7 

5. RESPIRATION 

a. rapid 

b. slow 

c. irregular 

d. ceased 

e. gulping air 

f. labored 

000128 


i 
Table(s) 4,5,6: Toxicity to the Fathead Minnow, Pimephales promelas 


Chemical and Physical Parameters 


pH 

..., 

! 

-1 

-, 

8.3 

8.3 

8.3 

8.3 

8.3 

8.3 

8.3 8.3 8.3 8.3 

8.3 8.3 8.3 8.3 

8.3 8.3 8.3 8.3 

8.3 8.3 8.3 8.3 

, 

, ~-.j 

Dissolved Oxygen (ppm) .-, 

I 

-, 

f •. 1 

9.8 

9.2 

9.3 

9.2 

9.8 

9.8 9.8 9.8 9.9 

9.5 9.4 9.6 9.6 

9.7 9.6 9.6 9.7 

9.4 9.3 9.3 9.4 

Temperature. °e 

~ 

- , 7.1 

17.4 

17.4 

17.3 

16.0 

, 6.7 16.5 16.2 16.7 

16.5 16.8 16.8 16.5 

16.5 16.8 16.3 16.6 

16.5 16.8 16.8 16.8 

- Data not collected after 100% mortality. 

00012'3 

8 
Protocol'90-0'.-01 !'est Sn~l~ I Batch/tot' IDate Started I Investigator 

RfAOY BIOVfGRADAB'lr,Y 

CLOSED BOrTLE TfST 

Innoculum Data Buffers Added 

• ...,1. C__...tI......... ,,,,.,, I I"VI._.fAl 'fI."" I - Medium High Form' 005a Medium High lOM . A Issue Date: July 1990 5.0 10.0 D,~~ 

VotlJlll! Used ~r 300 ml BotHe 

Reference Standard S..,le Of lot fon Ueter 

Date seed Collected: 

lyp!! Seed Collt'Cted: ~$tlel 

Seed location: "SO-Sissell Poi 

Seed concentration: O. o·~' 

trial 

1 mill 6.53% Potassfum Phosphate 

1 mIll 2.25% Magnesium ~ulfate 

0.6 mg/l Ma "<:etate Source: lab trade delonfzed 

Filtrat(on: 0.22 _(eron 

Data 

Botth!

, 

'nit lal 

00, II'I!lIl 

5 Day 

DO, 1119/1 

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seeded 

controls 

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Acetate 

Standard 

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1 mill 0.025% Ferric Chloride 

8 leN: 

linseed«! 

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Standard 

S...,Ie 

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81och","Ical Oxygen Ol!ft8nd 

Z.O RJ'II 5.0 RJm IfO.O pp!I 

Samp'e Sample Sample 

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POG)E? \30 

AQUATIC TOXICOLOGY 

Static Acute Freshwater Toxicity 

PARAMETERS 

Specie: Fathead minnow 

Temperature: 17 degrees C 

Duration: 96 hours 

Static Acute Saltwater Toxicity 

PARAMETERS 

Specie: Sheepshead minnow 

Temperature: 17 degrees C 

Duration: 96 hours 

Specie: Photobacterium phosphoreum 

Test Type: Microtox 

Duration: 15 minutes 

ENVIRONMENTAL FATE 

Oxygen Demand 

PARAMETERS 

Test Concentration: 100 ppm 

Ready Biodegradability 

PARAMETERS 

Media: Freshwater 

Inoculum Acclimation Per: 0 days 

Test Concentration: 2 ppm 

RESULTS 

Lethal Concentration 50% 

No Effect Concentration ­

RESULTS 

Lethal Concentration 50% 

No Effect Concentration ­

- 96 hours 

96 hours 

- 96 hours 

96 hours 

Effective Concentration 50% - 15 minutes 

RESULTS 

Chemical Oxygen Demand 

RESULTS 

Biodegradability, 5 days 

Biodegradability, 14 days 

Biodegradability, 20 days 

Biodegradability, 28 days 

VALUES 

0.8 mg/l 

0.32 mgtl 

VALUES 

2.2 mgtl 

1.0 mg/l 

1 - 10 mgtl 

VALUES 

192 mg 

VALUES 

26% 

39% 

57% 

78 % 

NOTE: Percent biodegradability is based on a chemical oxygen demand (COD) value of 192 mg per 100 ppm product. 

April 13, 2004 Page 1of 2

Disclaimer of Liability: , warranls to purchaser, but nO third parties 0( others Ihe specifICations fO( the product shall fa" within a generally recognized range for typiCal physical 

properties eslablished by_when the product departs -.point of origin and thai any services shall be performed in accordance with applicable written documenls, ~AKES NO OTHER WARRENTY OR 

GUARANTEE OF ANY KIND, EXPRESS OR IMPUED, INCLUDING NO IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, REGARDING ANY SERVICES 

PERFORMED OR PRODUCT SUPPUED. _will give purchaser Ihe benellt of _best judgemenl in making interpretations of data, but does not guarantee the accuracy of correctness of such interpretations, 

_ recO(Omendations contained herein are adviSO(y only and without representafions as to Ihe resufts, ..shall not be liable for any ind~ect, special, punitive, exemplary 0( consequential damages or losses frO(O any 

cause whatsoever including but not limited to ils negligence, 
MAMMALIAN TOXICOLOGY 

Acute Oral Toxicity 

PARAMETERS 

Specie: Rat-female 

Observation Period: 14 days 

Specie: Rat-male 

Observation Period: 14 days 

Primary Skin Irritation 

PARAMETERS 

Specie: Rabbit 

Scoring Method: Draize 

Dermal Sensitization 

PARAMETERS 

Specie: Guinea Pig 

Method: Magnussen-Kligman Maximization 

RESULTS 

Lethal Dose 50% 

Lethal Dose 50% 

RESULTS 

Skin Irritation Index 

RESULTS 

Sensitization 

VALUES 

0.56 g/kg 

0.90 g/kg 

VALUES 

Corrosive 

VALUES 

Non-sensitizer 

April 13, 2004 Page20f 2

Disclaimer of Uability: ,warrants to purchaser, but no Ihird parties or others the specifICations for the product shall fall within a generally recognized range for Iypical physical 

properties established by"when Ihe product departs _poinl of origin and that any services shall be performed in accordance with applicable written documents, ___MAKES NO OTHER WARRENTY OR 

GUARANTEE OF ANY KIND. EXPRESS OR IMPLIED. INCLUDING NO IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARllCULAR PURPOSE. REGARDING ANY SERVlCES 

PERFORMED OR PRODUCT SUPPLIED. _ will give purchaser the benefil of _ best judgement in making interprelalions of dala. but does not guarantee the accuracy of correctness of such inlerprelatiOIlS, 

--"ecommendations conlained herein are advisory only and without representations as to the resu~s, _ shall not be liable for any indirect, special, punitive, exemplary or consequential damages or losses from any 

cause whatsoever including but not limited 10 its negligence, 
A division of the American Chemical Society 

Phone: 800-631-1884. 614-447-3870 

Fax: 614-447-3747 

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Internet http://www.cas.org/Supportlclienthtml 

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

If this box is checked, CAS has made correction{s) marked in red to 

your lES order. Please make the same correction(s} to your PMN before 

submitting it to the EPA. 

2540 Olen tangy River Road P.O. Box 3343 Columbus, Ohio 43210·0334 
Material Safety Data Sheet 


Product Name 

Supplier 

Material Uses 

24 Hour 

Emergency 

Numbers 

CANUTEC 613-996-6666 (Canada 24 hours) 

CHEMTREC Int'l 01-703-527-3887 (International 24 hour) 

National Fire Protection 

Association (U.S.A.) 

~ amme.bllity 

HeaD. 3 ~ 0 Reactivi" 

Speclnc Hazard 

Code 

Version 1.0 

Effective Date 411312004 

Print Date 4114/2004 

Quaternary ammonium hydroxide Trade secret. 30-60 

Fatty amine Trade secret 10-30 

Isopropanol 67-63-0 30-60 

Ethylene Glycol 107-21-1 1-5 

Not available. 

Not available. 

ACGIH (United States). 

TWA: 490 mg/m3 

STEL: 980 mg/m3 

TWA: 200 ppm 

STEL: 400 ppm 

OSHA (United States). 

TWA: 400 ppm 

STEL: 500 ppm 

TWA: 980 mg/m3 

STEL: 1225 mg/m3 

ACGIH (United States). 

CEIL: 100 mg/m3 Form: Aerosol only. 

OSHA (United States). 

CEIL: 50 ppm 

CEIL: 125 mg/m3 
Not applicable. 

skin and tract. 

CERCLA Reportable 

Qua 

Hazard Summary 

Routes of 

Potential Acute Health 

Effects 

Medical Conditions 

aggravated by 

Exposure 

See Toxicol 

Additional Hazard 

Identification Remarks 

DANGER. May cause chronic effects. Flammable liquid. Vapors can form an ignitable or 

explosive mixture with air. Can form explosive mixtures at temperatures at or above the flash 

point. Vapors can flow along surfaces to a distant ignition source and flash back. Static 

discharges can cause ignition or explosion when container is not bonded. May be corrosive to 

cause central nervous effects if inhaled. 

Eyes May be corrosive to the eyes. May cause eye burns and permanent eye injury. 

Skin May be corrosive. Skin contact may produce burns. 

Inhalation May cause central nervous system (CNS) effects if inhaled. May be severely irritating to the 

lungs. 

Ingestion Not considered a likely route of exposure, however, may be corrosive if swallowed. 

Exposure to this product may aggravate medical conditions involving the following: kidneys, 

nervous system, respiratory tract, skin/epithelium, eyes. 

May be harmful if ingested. This product may be aspirated into the lungs during swallowing or 

vomiting of swallowed material. Aspiration into the lungs may produce chemical pneumonitis, 

pulmonary edema, and hemorrhaging. 

Eye Contact 

Skin Contact 

Inhalation 

Ingestion 

Additional First Aid 

Remarks 

Immediately flush eyes with plenty of water for at least 15 minutes, occaSionally lifting the 

and lower Get medical attention imm 

Remove contaminated clothing and shoes immediately. Wash affected area with soap and mild 

detergent and large amounts of water until no evidence of chemical remains (apprOXimately 

15-20 mi Get medical attention if irritation occurs. 

Remove to fresh air. Oxygen may be administered if breathing is difficult. If not breathing, 

administer artificial respiration and seek medical attention. Get medical attention if symptoms 

Get medical attention immediately. If swallowed, do not induce vomiting unless directed to do 

so by medical personnel. Wash out mouth with water if person is conscious. Never induce 

vom or mouth to a victim who is unconscious or havi convulSions. 

Not 

Not available. 
Flammability of the 

Product 

OSHA Flammability 

Class 

Autoignition 

ra 

Flash Points 

Flammable Limits 

Products of 

Combustion 

Fire Hazards in 

Presence of Various 

Substances 

Fire Fighting Media 

and Instructions 

Protective Clothing 

Special Remarks on 

Fire Hazards 

Flammable liquid. Vapors can form an ignitable or explosive mixture with air. Can form 

explosive mixtures at temperatures at or above the flash point. Vapors can flow along surfaces 

to a distant ignition source and flash back. Static discharges can cause ignition or explosion 

when container is not bonded. 

IC 

Not available. 

Closed 

L.E.L. Not available. U.E.L. Not available. 

These products are carbon oxides (CO, C02) nitrogen oxides (NO, N02...). 

Open Flames/Sparks/Static. Heat. 

In case of fire, use foam, dry chemicals, or C02 fire extinguishers. Evacuate area and fight fire 

from a safe distance. Water spray may be used to keep fire-exposed containers cool. Keep 

water run off out of sewers and public waterways. Note that flammable vapors may form an 

;~~,;+..,Ikl" mixture with air. V travel considerable distances and flash back if innitari 

Do not enter fire area without proper personal protective equipment, including NIOSH approved 

self-contained breath 

Not available. 

Spill 

Other Statements 

Put on appropriate personal protective equipment. Keep personnel removed and upwind of 

spill. Shut off all ignition sources; no flares, smoking, or flames in hazard area. Approach 

release from upwind. Shut off leak if it can be done safely. Contain spilled material. Keep out 

of waterways. DIke large spills and use a non-sparking or explosion-proof means to transfer 

material to an appropriate container for disposal. For small spills add absorbent (soil may be 

used in the absence of other suitable materials) scoop up material and place in a sealed, 

liquid-proof container. Note that flammable vapors may form an ignitable mixture with air. 

Vapors may travel considerable distances from spill and flash back, if ignited. Waste must be 

of in with federal state and local environmental control ulations. 

If RQ (Reportable Quantity) is exceeded, report to National Spill Response- Office at 

1-800-424-8802. 

Additional Accidental Not available. 

Release Measures 

Remarks 
Handling and Storage 	 Put on appropriate personal protective equipment. Avoid contact with eyes, skin, and clothing. 

Avoid breathing vapors or spray mists. Use only with adequate ventilation. Store in a dry, cool 

and well ventilated area. Keep away from heat, sparks and flame. KE!ep away from 

incompatibles. Keep container tightly closed and dry. To avoid fire or explosion. ground 

container ment and before handl 

Additional Handling and Not available. 

Storage Remarks 

or to ns 

vapors or particles below their respective threshold limit value. Ensure that eyewash stations 

and showers are to the work-station location. 

Personal Protection 

Personal Protective Equipment recommendations are based on anticipated known manufacturing and use conditions. 

These conditions are expected to result in only inCidental exposure. A thorough review of the job tasks and conditions by a 

safety professional is recommended to determine the level of personal protective equipment appropriate for these job 

tasks and conditions. 

Eyes Chemical safety goggles. Use full face shield if splashes could occur. 

Body Wear long sleeves and chemical resistant apron to prevent repeated or prolonged skin contact. 

Respiratory Respirator use is not expected to be necessary under normal conditions of use. In poorly 

ventilated areas, emergency situations or if exposure levels are exceeded, use NIOSH 

approved full face respirator. 

Hands Chemical resistant gloves. 

Feet Chemical resistant boots or overshoes. 

Other information Nitrile or neoprene gloves. 

Protective Clothing 

(Pictograms) 

Additiofuil Exposure 

Control Remarks 

Not available. 

Color Li ht Amber. 
Pour Point 

Solu 

Boilin Point 

Physical Chemical 

Not available. 

Not available. 

Soluble 


Not available. 


Not available. 


ments 

Stability and Reactivity ..:.T.:..:.he:::....t:.p:....:ro:..:::d~uc::..:t;..:;is:::...;;;..;st::::;;a;;;.;bl.=..e:...._________________________ 

Conditions of Instabil Not available. 

Incompatibility with 

Various Substances 

Oxidizing material. Acid. 

Hazardous 

Decomposition 

Products 

Not applicable. 

Hazardous Hazardous polymerization is not expected to occur. 

Special Stability & Not available. 

Reactivity Remarks 

Component Toxicological Information 

Acute Animal Toxicity 

Quaternary ammonium hydroxide 

Fatty amine 

Isopropanol 

Ethylene Glycol 

Chronic Toxicity Data 

1) Quaternary ammonium hydroxide 

Not available. 

2) Fatty amine 

Not available. 

3) Isopropanol 

Not available. 

ORAL (LD50): Acute: 3000 mg/kg [Mouse]. 

ORAL (LD50): Acute: 5045 mg/kg [Rat]. 3600 mg/kg 

[Mouse]. DERMAL (LD50): Acute: 12800 mg/kg [Rabbit]. 

VAPOR (LC50): Acute: 16000 ppm 8 hour(s) [Rat]. 

ORAL (LD50): Acute: 5500 mg/kg [Mouse]. 4700 mg/kg 

[Rat]. 4000 mg/kg [Female rat]. DERMAL (LD50): Acute: 

10600 mg/kg [Rabbit]. 
Isopropanol, a component of this product. Ingestion has produced hyperglycemia (high blood sugar) in humans 

(Lacouture, P, et ai, 1983, "American Journal of Medicine" and Chan K-M, et ai, 1993, "Clinical Chemistry"). 

In a four month study, inhalation of isopropanol vapors for 20 hours per week by laboratory animals produced bronchitis, 

pneumonia, and blood effects (International Program of Chemical Safety, 1990, Environmental Health Criteria 103: 

2-propanol, World Health Organization). Ataxia (a jerky or shaky movement that occurs during voluntary muscle 

movement) and microscopic hyaline droplets (fungal or branched structures) in the kidneys were seen in rats exposed tc 

isopropanol at concentrations up to 5000 ppm for 6 hours per day, 5 days per week, for 13 weeks (Burleighflayer et ai, 

1994). Inhalation of high levels of isopropanol (4,000 and 8,000 ppm for 8 hours) has produced congestion in the liver, 

lungs, and spleen of laboratory animals (Laham S, et al. 1980, "Drug and Chemical Toxicology). 

In addition, isopropanol has shown to be a fetotoxin in tests on laboratory animals [Nelson, BK et al (1988), Food and 

Chemical Toxicology, 26(3), pps 247-254]. 

4) Ethylene Glycol 

Ethylene glycol (EG) is a component of this product. Chronic ingestion has shown to cause adverse kidney, liver, bladder, 

and blood effects in laboratory animals (NTP Technical Report, 1993; Fund. Appl. Toxicol. 7:547-65; FD Cosmet Toxico!. 

Vol. 3:229-34; Drug and Chern ToxicoI13(1):43-70). 

EG is an animal teratogen at doses which produced mild toxicity to the mother. EG given at doses up to 5,000 mg/kg/day 

to pregnant rats or up to 3,000 mg/kg/day to mice induced a wide variety of fetal malformations, including those of the 

musculoskeletal, bone marrow, and spleen (RTECS. 1996). 

Ethylene glycol is used to cryopreserve embryos of many mammalian species, including pigs, goats, cows and horses 

(Otoi et ai, 1995; Fieni et ai, 1995; Hochi et ai, 1994). This makes it unlikely that ethylene glycol itself is the active teratogen 

in whole animal studies. The EG metabolite, glycolic acid, was active in contrast to EG itself for inducing developmental 

defects in whole rat embryos in culture (Carney et ai, 1996). EG inhibited metabolic cooperation of Chinese hamster cells 

in vitro, a finding which may have implications for its mechanism of teratogeniCity (Loch-Caruso et ai, 1984). 

Product Toxicological Information 

Not 

nervous ..,,,,,,,rern 

Other Adverse Effects This product results from a reaction involving ethylene oxide (EtO) (CAS No 75-21-8). It 

may, therefore contain residual EtO (less than 0.1 %) which can accumulate in the container 

headspace and be released to the ambient environment when opened. This release of EtO 

would tend to increase when the product is agitated during unloading or blending operations. 

The Occupational Safety and Health Administration (OSHA) has set the worker exposure 

level for EtO at 1 ppm (8-hour time weighted average). The STEL is 5 ppm (15 minutes). This 

standard regulates occupational exposure to EtO from sources including products containing 

residual EtO. It is the responsibility of the employer to comply with OSHA EtO Standard (29 

CFR 1910.1047). The OSHA action level TWA for EtO is 0.5 ppm. The OSHA excursion level is 

5 ppm as averaged over a sampling period of 15 minutes. 

The International Agency for Research on Cancer (IARC) has identified EtO as a "human 

carcinogen (Group 1); human evidence is limited; animal evidence is sufficient". EtO has been 

listed as an "anticipated carCinogen" by the National Toxicology Program (NTP). Eto has also 

been designated as a carcinogen and reproductive toxin under the State of California's Safe 

Drinking Water and Toxic Enforcement Act (Proposition 65). A no significant risk level of 2 

meg/day (an acceptable intake level is 20 meg/day) was established under Proposition 65. 
8005 and COD Not available. 

Biodegradable/OECD Not available. 

Special Remarks Not available. 

Toxicity of the Products Not available. 

ofB 

Responsibility for proper waste disposal rests with the generator of the waste. Dispose of any waste material in 

accordance with all applicable federal, state and local regulations. Note that these regulations may also apply to empty 

containers, liners and rinsate. Processing, use, dilution or contamination of this product may cause its physical and 

chemical erties to 

Additional Waste Not available. 


Remarks 


DOT Reportable 

Marine Pollutant 

Additional DOT 

information 

Emergency Response 

Guide Page Number 

U.S. Federal 

Regulations 

Environmental 

Regulations 

CORROSIVE LIQUID, FLAMMABLE, N.O.S. (Contains: 

Quaternary ammonium hydroxide, Isopropanol), 8(3), 

UN2920, " 

Not applicable. 

Not icable. 

Not available. 

132 

Extremely Hazardous Substances: Not applicable to any components in this product. 

SARA 313 Toxic Chemical Notification and Release Reporting: Ethylene Glycol; 

SARA 3021304 Emergency Planning and Notification substances: Not applicable to any 

components in this product. 

Hazardous Substances (CERCLA 302): Not applicable to any components in this product. 

SARA 311/312 MSDS distribution - chemical inventory - hazard identification: fire; immediate 

health hazard; 
Clean Water Act (CWA) 307 Priority Pollutants: Not applicable to any components in this 


product. 


Clean Water Act (CWA) 311 Hazardous Substances: Not applicable to any components in this 


product. 


Clean Air Act (CAA) 112(r) Accidental Release Prevention Substances: Not applicable to any 


components in this product. 


Threshold Not applicable. 

Planning 

Quantity (TPQ) 

TSCA Inventory This product or its components, if a mixture, are not listed on the TSCA inventory. 

Status 

This product is manufactured and distributed in compliance with the United States Toxic 

Substance Control Act (TSCA) Research and Development (R&D) Exemption Regulation, 40 

CFR 720.36. It is intended for R&D use only and may contain one or more ingredients that are 

not listed on the TSCA Inventory. This product must be handled by a technically qualified 

(trained) individual or under the supervision of a trained individual. 

This product does not contain any components that are subject to the reporting requirements of 

TSCA Section 12(b) if exported from the United States. 

State State s ...,.....,·+O" information is available u on from

International 

Regulations 

Canada 
 ",n,,...n,,.n~C! are included on the Canadian Domestic Substances List. 

WHMIS(Canada) ~~~~~__________________________________________________~ 

European Union Not all components are included on the European Inventory of Existing Commercial Chemical 

or the Euro List of Notified Substances. 

International inventory status information is available upon request from i for the 

fol countries: Austral Korea Phil 

Harmonized Tariff Code Not available. 

Other Regulatory No further regulatory information is available. 

Information 

Other Special Not available. 

Considerations 

Disclaimer 

NOTE: The.'informatioifdrfthis MSDS is based on data which is considered to be accurate: _. . however, 

makes no guarantees or warranty, either expressed or implied of the accuracy or completeness of this information. 

The conditions or methods of handling, storage, use and disposal ofthe product are beyond our control and may be 

beyond our know/edge. For this and other reasons, we do not assume responsibility and expressly disclaim liability for 

loss, damage or expense arising out of or in any way connected with the handling, storage, use or disposal of this product. 

This MSDS was prepared and is to be used for this product. If the product is used as a component in another product, this 

MSDS information may not be applicable.