P.O. Box 331, Millwood, VA 22646 (540) 837-1602



### June 29, 2012

BY HAND
Document Control Office Office ofPollutionPrevention and Toxics Room 6428 EnvironmentalProtection Agency 1200PennsylvaniaAvenue, NW Washington, D.C. 20460 1111111111111111111111111111111111111111111111111111111111111111 40120000075



### Re: Testing Consent Order for Ethylene Dichloride;

Dear Sirs: Study Plan for Extended One-GenerationDrinking Water Reproductive Toxicity Study ofEthylene Dichloride inRats (Docket No. OPPT-2003-0010) --_1.

C

.

.J



### Pursuantto Section IX oftheEnforceable ConsentAgreement for EthyleneDichloride, the HAP TaskForce submits the enclosed studyplan for an extended one-generation drinking waterreproductive toxicity study ofethylene dichloride 

### inrats. Six copies ofthe studyplan are enclosed.
Please do nothesitate to contact me ifthere are any questions.

Enclosures

cc: J. Schaeffer W. Caffey Norman, Esq. 

### CONTP.!NS NO CBI

3800151v1

### Extended One-Generation Drinking Water Reproductive Toxicity Study ofEthylene Dichloride in Rats Study Plan

I. Identity ofConsent Order
This study plan is submitted pursuant to the Testing Consent Order for Ethylene Dichloride, 68 Fed. Reg. 33125 (June 3, 2003), as amended 74 Fed. Reg. 36196 (July 22, 2009), as amendedby Addendum Modification signed by Maria J. Doaon November 23, 2011 and PeterE. Voytekon December 6, 2011, Docket No. EPA-HQOPPT-2003-0010. This plan contains the informationrequired by40 CFR § 

### 790.62(b).

II. Test Requirements Contained in Study Plan
This study plan contains the test requirements for an extended one-generation drinking waterreproductive toxicity study ofethylene dichloride in rats. Thetest methods are described in the attached protocol.

III. Principal Sponsor ofTest
HAPTaskForce P.O. Box 331 Millwood, VA 22646

IV. Administrative Official and ProjectManager for Principal Sponsor:
PeterE. Voytek, Ph.D. Manager HAPTaskForce P.O. Box 331 Millwood, VA 22646 (540) 837-1602

### V. Technical Contacts
Ann Davis EH&S Global ProductLeader The Dow Chemical Company 2020 Dow Center Midland, MI 48674 (989) 638-7409
David Goldsmith Principal Engineer Georgia GulfChemicals & Vinyls, LLC 26100 Highway 405 Plaquemine, LA 70764 (225) 685-2677
Heather Burleigh-Hayer, Ph.D. Manager, Environmental Health Sciences & Toxicology PPG Industries, Inc. 4325 Rosanna Drive Allison Park, PA 15101 (412) 492-5409 Lloyd H. Stone Corporate HSEManager The Westlake Group 2801 Post OakBlvd. Houston, TX 77056 (713) 585-2816
John Pastuck Environmental & Safety Manager FormosaPlastics Corporation, U.S.A. 9 PeachTree Hill Road Livingston, NJ 07039 (973) 716-7387
RobertS. Schuster Director

- HES Compliance Assurance Occidental Chemical Corporation P.O. Box 809050 Dallas, Texas 75380 (972) 404-2434

2

### VI. Testing Facility

- WIL Research 1407 George Road Ashland, Ohio 44805 Study Director: MelissaJ. Beck, Ph.D. AssistantDirector, Neurosciences ( 419) 289-8700
VII. Professional Qualifications
Curricula vitae providing the training and experience ofeach professional involved in this testing are attached as Appendix A to this study plan.

VIII. Identity ofTest Substance 1,2-Dichloroethane (CAS No. 107-06-2) CzH4Ch 

### MW=98.96 Clear, colorless, oily, highly flammable liquid BP = 83.5° C Soluble at 8490-9000 mg/L in water (20° C) LD50 

### orally in rats= 770-967 mg/kg
An analysis ofthe productused for the study is attached as Appendix B.

IX. StudyProtocols
Theprotocol for the extended one-generation drinking water reproductive toxicity study ofethylene dichloride in

### rats is attached as Appendix C. The outline ofan analytical validation and stability study of 1,2-dichloroethane in aqueous formulations is also attached in Appendix C.

X. Test Schedule Experimental Start Date: Experimental TerminationDate:
Final Report:

3738332 3 

### August 14, 2012
TBD
June 28, 2013 (unless second generation study is triggered)

### ATTACHMENT AFormalEducation:


### 2001

1995

ProfessionalExperience:



### 08/06/05

- Present

12/04/10- Present 

## MELISSAJ. BECK Neurosciences



### University ofMichigan Ann

### Arbor, MI PhD Toxicology
Ashland University BS Toxicology

WIL ResearchLaboratories, LLC Ashland, OH
Position: AssistantDirector Neurosciences CURRICULUM VITAE February21, 2012



### Responsible for assisting with the direction of all administrative and scientific functions within the discipline ofNeurosciences at WIL Research Laboratories, LLC. Support various pharmaceutical, agrochemical and veterinary medical product development programs. Assists in selecting, installing and validating new test systems into the Neurosciences Department. Assists in training personnel in the Neurosciences Department. Assists in the marketing of studies into the Neurosciences Department. Study Director, simultaneously oversee between 30- 40 developmental, reproductive and neurotoxicity studies. Responsible for the planning, conducting and successful reporting of assigned studies in accordance with FDA, EPA and various international regulatory requirements.
Position: Head ofJuvenile Toxicology
In addition to the above responsibilities, added the responsibility for assisting with the direction ofall administrative and scientific functions involving nonclinicaljuvenile toxicity safety assessment at WIL Research Laboratories, LLC. Assists in the marketing of nonclinicaljuenile toxicity studies. ProfessionalExperience (Continued):

### FormalEducation:
2001

1995



### ProfessionalExperience:

08/06/05

- Present

12/04110

- Present

10/01/01

- 08/05/05 

## MELISSAJ. BECK Neurosciences

University ofMichigan AnnArbor, MI PhD Toxicology
Ashland University BS Toxicology

WIL Research Laboratories, LLC Ashland, OH
Position: Assistant Director Neurosciences CURRICULUM VITAE February21, 2012

Responsible for assisting with the direction of all administrative and scientific functions within the discipline ofNeurosciences at WIL Research Laboratories, LLC. Support various pharmaceutical, agrochemical and veterinary medical product development programs. Assists in selecting, installing and validating new test systems into the Neurosciences Department. Assists in training personnel in the Neurosciences Department. Assists in the marketing of studies into the Neurosciences Department. Study Director, simultaneously oversee between 30- 40 developmental, reproductive and neurotoxicity studies. Responsible for the planning, conducting and successful reporting of assigned studies in accordance with FDA, EPA and various international regulatory requirements.
Position: Head ofJuvenile Toxicology

In addition to the above responsibilities, added the responsibility for assisting with the direction ofall administrative and scientific functions involving nonclinicaljuvenile toxicity safety assessment at WIL Research Laboratories, LLC. Assists in the marketing of nonclinicaljueniletoxicity studies.
Position: StaffToxicologist Developmental, Reproductive andNeurotoxicology

### Melissa

### J. Beck

ContinuingEducation: CURRICULUM VITAE



### Study Director for developmental, reproductive and neurotoxicity studies. Responsible for the planning, conduct and successful reporting of assigned studies. Participate in report review and generation process. Develop new capabilities for WIL Research Laboratories, Inc. Communicate with Sponsors as required.

Mechanisms, Biomarkers, Data Interpretation: An 

### Integrative Risk Assessment Using DevelopmentalNeurotoxicity as a Model. Teratology Society, 2002.
The Effects ofNon-Reproductive Hormones on the Reproductive System and the Implications for Toxicology. Society ofToxicology, 2003. Signaling Pathways and Tissue Interactions in Organ-System Development. Teratology Society, 2004.
Risk AssessmentinPharmaceutical Development. Society ofToxicology, 2005. Neuropathology for the Toxicologist. Society ofToxicology, 2006. Reproductive Toxicity Testing: Study Designs, Evaluation, Interpretation and Risk Assessment. Society ofToxicology, 2006.

ProfessionalActivities: 

### Toxicological Highlight Manuscriptfor Toxicological Sciences, Volume 62, Number 1, 

### 2001

SocietyAffiliations:


### 2003

- Present 2003

- Present 2009

- Present

MelissaJ. 

### Beck Member, Teratology Society Member, Neurobehavioral Teratology Society Member, Society forNeurosciences

Date

### FormalEducation:
1999
1990

1985



### ProfessionalExperience:

10/23/10

- Present

12/20/08- 10/22/10 

## DONALD G. STUMP DevelopmentalandReproductive Toxicology



### Diplomate American Board ofToxicology
VanderbiltUniversity Nashville, TN PhD Biochemistry PhiladelphiaCollege ofPharmacy and Science Philadelphia, PA BS Toxicology

WIL Research Laboratories, LLC Ashland, OH CURRICULUM VITAE January 4, 2012



### Position: Senior Director, Developmental and Reproductive Toxicology Responsibilities include directing the activities of Study Directors and managerial and technical personnel in the Developmental and Reproductive Toxicology, Juvenile Toxicology and Neuroscience Departments. Serves in a lead role in the conceptual design of studies for various product development programs, and serves as Study Director for developmental and developmental toxicology, adult neurotoxicology, and juvenile and developmental neurotoxicology studies. Provides consultation to clients on scientific issues, data interpretation and program development.
Position: Director, Developmental and Reproductive Toxicology
Provide senior guidance in the scientific and operational domains of the discipline. Directs the activities of Study Directors, managerial and technical personnel in a GLP-environment. Provide lead role in the conceptual design and internal ration of studies for various product developmentprograms and litigation

### Donald G. Stump CURRICULUM VITAE


### ProfessionalExperience (Continued):

11/10/00- 12/19/08

11/6/99 -11110/00

11108/97- 11/5/99 

### support. Responsible for the development of new business within the field. Study Director for various reproductive and developmental toxicology and adult neurotoxicology, juvenile and developmental neurotoxicology studies.
Position: Associate Director, Developmental and Reproductive Toxicology Responsible for managing junior level study directors. Study Director for developmental and reproductive toxicology studies. Responsible for the planning, conducting and successful reporting ofassigned studies in accordance with FDA, EPA and various international regulatory requirements. Responsible for assisting with the direction of all fmancial and scientific functions within the discipline of Developmental and Reproductive Toxicology at WIL Research Laboratories, LLC.
Position: Associate Director, Developmental, Reproductive and Neurotoxicology Responsible for managing a technical staffofapproximately 30 individuals. Study Director for developmental, reproductive and neurotoxicology studies. Responsible for the planning, conducting and successful reporting of assigned studies in accordance with FDA, EPA and various international regulatory requirements. Responsible for assisting with the direction ofall financial and scientific functions within the discipline of Developmental, Reproductive and Neurotoxicology at WIL ResearchLaboratories, Inc.
Position: Assistant Director, Developmental, Reproductive, andNeurotoxicology Responsible for assisting with the direction ofall financial and scientific functions within the discipline of Developmental, Reproductive and Neurotoxicology at WIL Research Laboratories, Inc. Support various pharmaceutical, agrochemical and veterinary medical product development programs. Study Director, simultaneously oversee between 30- 40 developmental, reproductive and neurotoxicity studies. Responsible for the planning, conducting and successful reporting of assigned studies in accordance with FDA, EPA and various international regulatory requirements.

### Donald G. Stump CURRICULUM VITAE


### ProfessionalExperience (Continued):



### 1985- 1990

1/84-6/84 Vanderbilt University School ofMedicine Nashville, TN
Position: Graduate Student
Performed graduate study for a Ph.D. in Biochemistry. Participated in laboratory instruction of graduate and medical students. In Dr. F. Chytil's laboratory, his thesis research focused on the characterization of the binding activity of rat cellular retinol-binding protein. He made extensive use of site-directed mutagenesis, bacterial protein expression and protein purification techniques in his research. Thesis title: Rat Cellular RetinolBinding Protein: Site-Directed Mutagenesis and Characterization ofthe Genomic Sequence.

Lederle Laboratories Pearl River, NY
Position: Industrial Traineeship, Department ofToxicology
Served as a study director for several acute studies following Good Laboratory Practices and internal standard operating procedures. He also assisted in the conduct ofsafety assessment studies.

### Donald G. Stump CURRICULUM VITAE
ContinuingEducation:


### Preclinical Developmental Toxicity Studies, Teratology Society Meeting, 2011. Postmarketing Surveillance and Communication ofRisk, Teratology Society Meeting, 2011.
Postnatal Drug Disposition and Teratogenesis. Teratology Society Meeting, 2010.
ICH Initiatives for Conducting Pharmaceutical Preclinical Safety Studies: New and Revised Guidelines and Challenges. Society ofToxicology, 2010. Epigenetic Mechanisms. Teratology Society Meeting, 2009. Bio-Imaging: New Approaches for Safety Assessment and Toxicology. American College of Toxicology Meeting, 2008.
CNS Functional Teratology. Teratology Society Meeting, 2008. Nanotoxicology: The Science of Developing a Safe Technology. Society of Toxicology Meeting, 2008. Principles in Teratology: Basic Concepts and Research Applications. Teratology Society Meeting, 2007.
The Biology and Toxicology of Peri- and Post-Natal Development. Society ofToxicology Meeting, 2007.
US FDA Critical Path Initiative: Building a New Toolkit to Transform Drug Development. Meeting ofthe American College ofToxicology, 2006.
Biomarkers as Predictive and Mechanistic Tools in Toxicology. The EUROTOX 2006/6 CTDC Congress, 2006.
Vertebrate Embryology. Teratology SocietyMeeting, 2006.
Non-traditional Animal Models in Toxicology. Meeting of the American College of Toxicology, 2005. Understanding Stem Cells: Current Research and Potential Clinical Applications. Teratology Society Meeting, 2005. Signaling Pathways and Tissue Interactions m Organ-System Development. Teratology Society Meeting, 2004. Understanding Lifespan Changes in Form and Function ofthe Female Reproductive System to Assess and Interpret Toxicity. Society ofToxicology Meeting, 2004.

### Donald G. Stump CURRICULUM VITAE



### ContinuingEducation (Continued): 

### Integration ofHuman and Animal Data in Understanding Mechanisms and Risk for Human Birth Defects. Teratology Society Meeting, 2003. Mechanisms, Biomarkers, Data Interpretation: An Integrative Risk Assessment usmg DevelopmentalNeurotoxicity as a Model. Teratology Society Meeting, 2002. Incorporation of Pharmacokinetic and Pharmacodynamic Data into Risk Assessments. Society ofToxicology Meeting, 2002. Principles of Teratology: Application of New Technologies Toward Understanding Abnormal Development. Teratology Society Meeting, 2001. Improving Risk Assessment for Human Developmental Defects: The Promise of Recent Advances in Developmental Biology and Genomics. Society ofToxicology Meeting, 2001. Signal Transduction: Pathways to Growth, Differentiation, and Morphogenesis. Teratology Society Meeting, 2000.
Advanced Neurotoxicity: Biomarkers and Mechanisms of Oxidative Stress-Induced Neurotoxicity. Society ofToxicology Meeting, 2000.
Evaluation ofMale Reproductive Toxicity: Sperm Markers and Epididymal Mechanisms of Toxicity. Society ofToxicology Meeting, 1999. Experimental Approaches for Studying Mechanisms ofAbnormal Development. Teratology Society Meeting, 1997.
Endocrine Control ofReproductive Development

- Normal and Abnormal Aspects. Society ofToxicology Meeting, 1997.
Risk Assessment in Developmental Toxicology. Teratology Society Meeting, 1996. Principles in Teratology II. Teratology Society Meeting, 1995.
New Endpoints in Developmental and Reproductive Regulatory Studies: Methods for Success. Society ofToxicology Meeting, 1995.

### Donald G. Stump CURRICULUM VITAE
Working GroupParticipation:


### International Life Sciences Institute, Risk Science Institute Project (2005-2007). Improving the Use of Toxicity Data in Constructing Statistically Based SAR Models for Developmental Toxicity.

JournalEditorialBoards: 

### Reproductive Toxicology (2008

- ) Congenital Anomalies (2009

- )

TeachingExperience: 

### Adjunctprofessorinthe Department ofBiology, AshlandUniversity, Ashland, OH. ProfessionalAffiliations:


### Middle Atlantic Reproduction andTeratology Association Society ofToxicology Teratology Society
Midwest Chapterofthe Society ofToxicology Japanese Society ofToxicology

Donald G. Stump Date

### FormalEducation:


### 2006

2003

1993

1985

1982 

## GARY D. COLEMAN AnatomicPathology

U.S. Army War College Carlisle Barracks, PA MS ofStrategic Studies CURRICULUM VITAE February I, 2012

Johns Hopkins Bloomberg School ofPublic Health Baltimore, MD PhD MolecularMicrobiology & Immunology

BostonUniversity Boston, MA MS Education
Auburn University College ofVeterinaryMedicine Aubum,AL DVM
Morehead State University Morehead, KY BS Biology



### BoardCertifications andLicensure: Diplomate, American College ofVeterinaryPathologists, 1998 Diplomate, American College ofVeterinaryPreventiveMedicine, 1994
Licensedto practice veterinarymedicine inAlabama

### Gary D. Coleman
ProfessionalExperience:



### 06/13/11 -Present

2008-2011 WIL Research Laboratories, LLC Ashland, OH
Position: Senior Pathologist Anatomic Pathology CURRICULUM VITAE



### Performs microscopic examination of material obtained from various in-house studies utilizing different species. These will include acute, subacute and chronic studies. Records the microscopic findings in the on-line computer system utilizing the pathology master list of terms or microscopic lesions established for the given species ororgan. Correlates the clinical pathology data with the microscopic observations. Writes the pathology summary, which includes organ weight, gross, microscopic and clinical pathology interpretations on various studies. Provides consultationto the necropsy group during the examination ofindividual animals sacrificed or which died on study. Provides direction and professional consultation for the department during the absence ofthe Director. Scientific and technical adviser for necropsy and histology labs. Maintains professional level ofexpertise by attending scientific meetings, reading appropriate professional journals, and participating in professional organizations. Special interests in neuropathology include brain morphometry in developmental toxicity studies.

Charles RiverLaboratories Frederick, MD
Position: Veterinary Pathologist Responsible for performing gross and microscopic evaluation of tissues, recording results, preparing reports delineating pathological findings, and assisting with necropsy supervision. May provide guidance to less experienced staff members. May assist with necropsy supervision and may conduct necropsies, including gross pathology interpretation, sample collection and fixation. Enter pathology findings in data capturing system(s). Write comprehensive report narratives detailing all test article effects. As needed, work with study directors to ensure data is accurately integrated into study reports. May serve as a study director orprincipal investigator. Provide scientific expertise for experimental design and other aspects ofresearch studies as needed.

### Gary D. Coleman CURRICULUM VITAE


### ProfessionalExperience (Continued):



### 2009-2011

2008-2011 Office ofthe Surgeon General ofthe Army Falls Church, VA
Position: Deputy Director Department ofDefense Veterinary Service Activity
Assist the Director in his role as principal veterinary staff officer to The Surgeon General, principal veterinary advisor to Health Affairs and the Chief, US Army Veterinary Corps on veterinary medical issues. Provide strategic planning, advocacy, oversight and policy guidance, maintain situational awareness and coordinate Veterinary Service with Department of Defense, Federal and state agencies and civilian entities. Oversee accomplishment of mission priorities of food safety, animal medicine and veterinary medical research throughout Department of Defense. Integrate the activities of the Army Veterinary Service with Department of Defense and Federal Agencies. Develop and disseminate veterinary regulatory and doctrinal publications for Department ofDefense food safety, veterinary public health, animal medicine and zoonotic diseases. Represent the Director in his absence to Office ofthe Surgeon General, Department ofthe Army and Department of Defense.

U.S. Army
Position: Consultantto the Surgeon General Veterinary Pathology Manage personnel assignments for U.S. Army veterinary pathologists supporting Department of Defense research institutions and the Armed Forces Institute of Pathology. Oversee career development for veterinary pathologists within the Army Veterinary Corps. Advise the Surgeon General and the Veterinary Corps Chief on the development and selection of veterinary officers for continuing education. Develop strategic policy for pathology support to the Department of Defense. Represent the veterinary pathology specialty to the larger military and civilian veterinary community and recruit incoming residents to the specialty.

### Gary D. Coleman CURRICULUM VITAE


### ProfessionalExperience (Continued):



### 2008-2009

2004-2008 Defense Threat Reduction Agency Fort Belvoir, VA
Position: Deputy Chief Chemical-Biological Medical Science & 

### Technology Division
Oversee research program development and execution addressing chemical, biological and radiologic medical countermeasures. Within a joint military and intergovernmental environment, guide a team of scientists and acquisition professionals with a $140 Million annual budget working to fill gaps in warfighter protection against chemical, biological and radiologic warfare and terrorism threats. Lead and coordinate technology transition efforts with the Joint Program Executive Office Advanced Developer for Medical Countermeasures. Assist Division Chief in daily operations and provide administrative oversight of, and support to, Science and Technology Managers. Represent the Medical Chem-Bio Division and Directorate at national and international chem-bio defense conferences and meetings. Provide responsive support to the Medical Chem-Bio Directorate, the Research and Development Enterprise and the Defense Threat Reduction Agency.

Walter Reed Army Institute ofResearch/ Naval Medical Research Command Silver Spring, MD
Position: Chair, Institutional Animal Care and Use Committee
As the Institutional Official's representative, oversee the administrative support of all animal research protocols within the two institutes to include animal use, approval, monitoring, and personnel qualifications in accordance with the Animal Welfare Regulations and Department of Defense policy. Responsible for maintaining Association of Assessment and Accreditation for Laboratory Animal Care (AAALAC) accreditation.

### Gary D. Coleman CURRICULUM VITAE


### ProfessionalExperience (Continued):

2004-2008

2002-2004

1997- 1999 

### Walter Reed Army Institute ofResearch Silver Spring, MD
Position: Director Division ofPathology
Lead the Division ofPathology at the largest medical research institute within the Department of Defense. Supervise 3 departments composed of approximately 69 military and civilian pathologists, Ph.D. scientists and technical and administrative staff, with a combined annual budget of approximately $1.3 Million. As the senior pathologist at WRAIR and the Naval Medical Research Center, direct medical research within the division and pathology services to the two institutes to support the development and testing of medical countermeasures against infectious agents and their toxins and other military-relevant diseases, health hazards and injuries. Responsible for resource planning and allocation to achieve efficient and effective utilization of fiscal, personnel and material resources inthe division.
Position: Chief Diagnostic Pathology
Guides and develops a professional staff for two laboratories providing Immunohistochemical and ultrastructural support to researchers at the Walter Reed Army Institute ofResearch, Walter Reed Army Medical Center and the Naval Medical Research Center. Develops and integrates advanced molecular pathology techniques for research and extramural collaborations. Provides comprehensive gross, histological and Immunohistochemical support of the pathology mission at the WRAIR, including professional consultation on the use oflaboratory animals, animal models of human disease, and research techniques. Serves as Associate Investigator on research protocols evaluating prophylactic and therapeutic candidates against agents ofmilitary significance.
Position: Chief Joint Immunohistochemistry Laboratory
Coordinates the joint laboratory effort between the Armed Forces Institute of Pathology and the Walter Reed Army Institute ofResearch. Conceives and develops protocols to

### Gary D. Coleman CURRICULUM VITAE


### ProfessionalExperience (Continued):

2004-2008

2002-2004

1997- 1999 Walter Reed Army Institute ofResearch Silver Spring, MD
Position: Director Division ofPathology
Lead the Division ofPathology at the largest medical research institute within the Department of Defense. Supervise 3 departments composed of approximately 69 military and civilian pathologists, Ph.D. scientists and technical and administrative staff, with a combined annual budget of approximately $1.3 Million. As the senior pathologist at WRAIR and the Naval Medical Research Center, direct medical research within the division and pathology services to the two institutes to support the development and testing of medical countermeasures against infectious agents and their toxins and other military-relevant diseases, health hazards and injuries. Responsible for resource planning and allocation to achieve efficient and effective utilization of fiscal, personnel and material resources in the division.
Position: Chief Diagnostic Pathology
Guides and develops a professional staff for two laboratories providing Immunohistochemical and ultrastructural support to researchers at the Walter Reed Army Institute ofResearch, Walter Reed Army Medical Center and the Naval Medical Research Center. Develops and integrates advanced molecular pathology techniques for research and extramural collaborations. Provides comprehensive gross, histological and Immunohistochemical support of the pathology mission at the WRAIR, including professional consultation on the use oflaboratory animals, animal models of human disease, and research techniques. Serves as Associate Investigator on research protocols evaluating prophylactic and therapeutic candidates against agents ofmilitary significance.
Position: Chief Joint Immunohistochemistry Laboratory
Coordinates the joint laboratory effort between the Armed Forces Institute of Pathology and the Walter Reed Army Institute ofResearch. Conceives and develops protocols to

### Gary D. Coleman

ProfessionalAffiliations: CURRICULUM VITAE



### support research and diagnostic efforts. Performs gross, histological, and Immunohistochemical evaluation and interpretation of laboratory tissues in support of the research mission. Provides comprehensive necropsy and biopsy reports to research scientists. Provides professional consultation and advice to scientists and investigators on the use of laboratory animals, animal models of human disease, and techniques to improve the quality and efficiency ofDepartment ofDefensedirected biochemical research. Periodically serves as Acting Chiefofthe Department ofDiagnostic Pathology.

Chair, Government Policy Committee, American College ofVeterinary Pathologists, 2009

- Present. Member, Public Visibility Committee, American College ofVeterinary Pathologists, 2009

- Present. Member, Role Delineation Task Force, American College ofVeterinary Pathologists, 2008- 2009. Member, Role Delineation Focus Group

- Anatomic Pathology, American College of Veterinary Pathologists, 2008. Member, Board ofTrustees, Association for the Assessment and Accreditation ofLaboratory Animal Care, International, Inc. (AAALAC), representing the American College of Veterinary Pathologists, 2002

- 2011. Representative, U.S. Army Medical Research and Materiel Command, to the Armed Forces Institute of Pathology Board of Governors Strategy Panel, Falls Church, Virginia, 2005.
Government Liaison, National Prion Research Program Scientific Merit Peer Review Meeting, Fairfax, Virginia, 2002. Member, American Society for Veterinary Clinical Pathology, 1998- Present. Member, American Veterinary Medical Association, 1985- Present.

Gary D. Coleman Date

## GEORGE ALLEN PARKER AnatomicPathology
FormalEducation: CURRICULUM VITAE November22, 2010



### 1999 University ofMedicine & 

### Dentistry ofNewJersey Newark, NJ

1977

1976

1972

1968 

### PhD
ArmedForces Institute ofPathology Washington, DC Residency Program
National Institute ofHealth Graduate School Bethesda, MD Postgraduate Courses
AuburnUniversity School ofVeterinaryMedicine Aubum,AL DVM University ofKentucky Lexington, KY Pre-Veterinary Curriculum

BoardCertifications andLicensure:


### 1977

- Diplomate, American College ofVeterinary Pathologists 2000- Diplomate, American Board ofToxicology North CarolinaVeterinary Medical Board (active) Ohio Veterinary Medical Board (inactive) KentuckyVeterinaryMedical Board (inactive) Virginia Veterinary Medical Board (inactive)

### George A. Parker
ProfessionalExperience:

10/23110

- Present

10/01/05

- 10/22/10

2009

- Present
10/01/2005- Present

1986- 1987 WIL Research Laboratories, LLC Ashland, OH
Position: Vice President, Pathology WIL Research Laboratories, LLC CURRICULUM VITAE



### Directs and coordinates pathology-related services of WIL Research Laboratories LLC. Responsible for administration of pathology department divisions in Ashland, OH and Hillsborough, NC, which includes 14 pathologists and approximately 70 technical and clerical personnel. Services include histology, clinical pathology, histopathology, flow cytometry, immunopathology, immunohistochemistry, morphometry and stereological analysis. Provides consultation to corporate management, sponsors, study directors and pathologists regarding issues related to pathology. Evaluates new methodology and technology, and develops new services when appropriate to meet research objectives. Interfaces with management of various scientific and support departments to insure the proper integration ofpathology services. Participates in business development activities as required to meet corporate objectives.
Position: Director, Pathology

AuburnUniversity College ofVeterinaryMedicine Auburn, AL
Position: Adjunct Professor, Pathology
Position: Laboratory Director
Serves as the local management of the histology and histopathology laboratory (Biotechnics LLC) located in Hillsborough, NC.

Virginia-Maryland Regional College ofVeterinaryMedicine Blacksburg, VA
Position: Adjunct Professor Department ofLarge Animal Clinical Sciences

### George A. Parker CURRICULUM VITAE


### ProfessionalExperience (Continued):



### 1980-2005

1978- 1980

1978

1977- 1980

1974- 1977

1975- 1976

1973- 1974 Biotechnics, LLC Hillsborough, NC
Position: President and Laboratory Director

Department ofVeterinary Pathology Armed Forces Institute ofPathology Washington, DC
Position: Adjunct StaffMember

Uniformed Services University ofthe Health Sciences Bethesda, MD
Position: Histology Instructor

Armed Forces Radiobiology Research Institute NationalNaval Medical Center Bethesda, MD
Position: Head, Comparative Pathology Division

Armed Forces Institute ofPathology Washington, DC
Position: Residency in VeterinaryPathology

National Zoological Park SmithsonianInstitution Washington, DC
Position: Prosecutor, Office ofPathology

Ubon Royal Thai Air Force Base Ubon, Thailand
Position: Base Veterinarian

### George A. Parker
ProfessionalExperience (Continued):



### 1972- 1973

ProfessionalAffiliations: 

### Macdill Air Force Base Tampa, FL
Position: Assistant Base Veterinarian

American College ofVeterinary Pathologists Society ofToxicologic Pathologists Society ofToxicology American College ofToxicology European Society ofToxicologic Pathology Japanese Society ofToxicologic Pathology American Society for Veterinary Clinical Pathology CURRICULUM VITAE

### FormalEducation:


### 1992

1988

1984

1980 

## CATHERINE A. PICUT Biotechnics



### Yale Law School New Haven, CT JD
Cornell University School ofVeterinary Medicine Ithaca, NY Diplomate, ACVP University ofPennsylvania School ofVeterinaryMedicine Philadelphia, PA VMD University ofRochester Rochester, NY BABiology

BoardCertificationsandLicensure:


### 2004 Board Certificationby American Board ofToxicology CURRICULUM VITAE January 27, 2011



### 2000 Registered Quality Assurance Professional, Good Laboratory Practices (RQAPGLP) 2000 North CarolinaVeterinary Medical Board, License Number 4759 2000 Quality Assurance Professional, Society ofQuality Assurance 1993 PatentAttorney Reg. No. 37,419 1993 District ofColumbiaBar (inactive status) 1992 Virginia State Bar (#34785) (inactive status) 1992 New Jersey State Bar (active status) 1988 Board Certificationby the American College ofVeterinary Pathologists 1984 Pennsylvania State Veterinary Medical License (inactive status) 1984 NewYork State Veterinary Medical License (inactive status)

### CatherineA. Picut



### ProfessionalExperience:

10/01/05- Present

01/95

- 09/05

01/99

- 09/05

11/93

- 12/95 WIL Research Laboratories, LLC/Biotechnics Hillsborough, NC
Position: Senior Pathologist CURRICULUM VITAE



### Provides GLP-compliant research pathology services to client laboratories engaged in the safety and efficacy evaluation ofnew drugs, biologics, medical devices and environmental chemicals. Specializes in on-site services at client facilities to ensure rapid and efficient completion of high profile projects. Interface extensively with study directors, histology supervisors, and quality assurance personnel to harmonize and expedite pathology support services.

Biotechnics, Inc. Hillsborough, NC
Position: Vice President and Pathologist
Provides GLP-compliant research pathology services to client laboratories engaged in the safety and efficacy evaluation ofnew drugs, biologics, medical devices and environmental chemicals. Specializes in on-site services at client facilities to ensure rapid and efficient completion of high profile projects. Interface extensively with study directors, histology supervisors, and quality assurance personnel to harmonize and expedite pathology support services.
Position: Quality Assurance Advisor/Legal Counsel

Hoffman-LaRoche Nutley, NJ
Position: Patent Attorney
50% Prosecution, 30% Collaborative Agreements, 20% Legal Opinions

### CatherineA. Picut

Pro(essionalExperience (Continued):

06/91

- 1 0/93 King & Spalding Washington, DC
Position: Attorney Food and Drug Law

Veterinary Labs ofAmerica/Biotechnics Sterling, VA


### 05190

- 09/90 Position: Diagnostic Pathologist

Veritas Laboratories, Inc. Burlington, NC
09/88

- 09/89 Position: SeniorToxicologistPathologist



### Pro(essionalAffiliations:

New Jersey Intellectual Property Law Association American College ofVeterinary Pathologists Society ofToxicologic Pathologists North Carolina Society ofQuality Assurance Society ofQualityAssurance

Catherine A. Picut Date CURRICULUM VITAEFormalEducation:


### 1988

1973

1971 

## ANN E. RADOVSKY AnatomicPathology



### Purdue University West Lafayette, IN PhD in Veterinary Pathology
Drexel University Graduate School Philadelphia, PA MS in Biomedical Engineering
Purdue University College ofVeterinaryMedicine West Lafayette, IN DoctorofVeterinary Medicine CURRICULUM VITAE January I0, 20II

BoardCertificationsandLicensure: 

### Diplomate, American College ofVeterinaryPathologists, 1991 Diplomate, AmericanBoard ofToxicology, 1996; Recertified for 2002-2011
Licensed to practice veterinary medicine inIndiana ProfessionalExperience:



### 03/23/02

- Present WIL ResearchLaboratories, LLC Ashland, OH
Position: Senior Pathologist Anatomic Pathology
Performs microscopic examination of material obtained from various in-house studies utilizing different species. These will include acute, subacute and chronic studies. Records the microscopic findings in the on-line computer system utilizing the pathology master list of terms or microscopic lesions established for the given species ororgan. Correlates the clinical pathology data with the microscopic observations.

### Ann E. Radovsky CURRICULUM VITAE


### ProfessionalExperience (Continued):

05/04/98

- 03/22/02 Writes the pathology summary, which includes organ weight, gross, microscopic and clinical pathology interpretations on various studies. Provides consultation to the necropsy group during the examination of individual animals sacrificed or which died on study. Provides direction and professional consultation for the department during the absence of the Director. Scientific and technical adviser for necropsy and histology labs. Maintains professional level of expertise by attending scientific meetings, reading appropriate professional journals, and participating in professional organizations. Special interests in neuropathology include brain morphometry in developmental toxicity studies.
Position: StaffPathologist Anatomic Pathology
Performs microscopic examination of material obtained from various in-house studies utilizing different species, including acute, subacute and chronic type (duration) studies. Records the microscopic findings in the on-line VAX system utilizing the pathology master list ofterms or microscopic lesions established for the given species or organ. Correlates the clinical pathology datawiththe microscopic observations. Writes the pathology summary, which includes gross, microscopic and clinical pathology on various studies 

### in a format acceptable to report writing and the study director. Provides consultation to the necropsy group during the examination of individual animals sacrificed or which died on study. Provides direction and professional consultation for the department during the absence of the Director. Maintains professional level of expertise by attending meetings andreading appropriate professionaljournals. Special interests in neuropathology include brain morphometry in developmental toxicity studies. Assists with the clinical care ofstudy animals.

### Ann E. Radovsky CURRICULUM VITAE


### ProfessionalExperience (Continued):

1993

- 2005

1993- 1998

1988- 1993 Safar Center for Resuscitation Research University ofPittsburgh
Position: Neuropathology Consultant Describes, evaluates and semi-quantitates histopathologic brain damage in experimental animals (rats and dogs) after transient cardiac arrest, shock or trauma in tests of therapeutic interventions.

Laboratory of Experimental Pathology National Institute of Environmental Health Sciences
Position: Veterinary Pathologist Responsible for toxicologic pathologist evaluations, interpreting and defending results ofchronic rodent bioassays for toxicity and carcinogenicity of administered environmental compounds and conducting research investigating characteristics ofreactions to foreign substances. Evaluated data for risk assessment. Consulted regularly in peer review on histopathology and data assessment. Conducted training sessions on basic histology and histopathology for biological scientists and technicians. Conducted reviews ofslides and written histopathology data and determine target organs and toxic reactions. Generated reports and presentations interpreting pathology fmdings, dose response and toxicokinetics.

Safar Center for Resuscitation Research University ofPittsburgh
Position: Assistant Professor
Conducted comparative and experimental neuropathology studies, evaluated the severity ofbrain damage in rats and dogs, semi-quantitative/computer-assisted methods. Supervised histotechnicians and trained research fellows.

### Ann 

### E. Radovsky CURRICULUM VITAE


### ProfessionalExperience (Continued):



### 1983-1988

1982- 1983

1981

- 1982

1979- 1981 Purdue University West Lafayette, IN
Position: Graduate Instructor
Combined Ph.D.

- Residency in Veterinary Pathology. Residency training in diagnostic pathology of all domestic species; necropsy and biopsy service, extensive experience in general and toxicologic pathology, electron microscopy and statistics. Ph.D. thesis correlated heart pathology with pacing threshold and electrical impedance.

Federated Medical Resources Honeybrook, PA
Position: Clinical Veterinarian for Laboratory Animal Facility
Clinical medicine for breeding colonies ofdogs, African Green Monkeys, sheep, pigs and rodents.

University ofPennsylvania Philadelphia, PA
Position: Research Associate Developed pre-clinical safety protocol for external electromagnetic bone stimulatorusing beagle dogs.

QuonsetAnimal Hospital East Greenwich, RI
Position: Clinical Veterinarian in small animal practice.

### Ann E. Radovsky CURRICULUM VITAE


### ProfessionalExperience (Continued):

1976- 1979

1974- 1979

1971

- 1973 University ofRhode Island Kingston, RI
Position: Research Associate and Lecturer Developed and taught a graduate course in rodent and rabbit experimental techniques. Assisted with acute surgical experiments in dogs (e.g. flowmeters around vessels).

Support from National Institute ofDental Research, University ofPennsylvania Philadelphia, PA
Position: Post-Doctoral Fellow
Assisted with the study of the clinical effects of electrical stimulation ofdentin in dogs.

Support from National Heart and Lung Institute, Drexel University, PA
Position: Post-Doctoral Fellow
Evaluated the gross, clinicopathologic and histopathologic effects of electrically-active biogalvanic power cells and correlated subcutaneous histopathology with measured electrical performance.

### Ann E. Radovsky CURRICULUM VITAE
ProfessionalAffiliations:


### 1991

- Present 1995 -Present 1996

- Present American College ofVeterinary Pathologists (Diplomate) Member, Society ofToxicologic Pathologists American Board ofToxicology (Diplomate)

ContinuingEducation: 

### Neuropathology. STPIIFSTP 2010 Joint Symposium, 2010. Experimental BiologyAnnual Meeting, 2007.

Second RTP Rodent Pathology Course, 2004.
Second Annual Workshop on the Pathology of Mouse Models for Human Disease 2003, Cornell University, 2003. Workshop onEvaluatingToxicityto the Male Reproductive Tract, 2002.
Annual Symposium ofthe Society ofToxicologic Pathologists, 1999- 2002, 2005, 2006, 2009
Annual Meeting ofthe American College ofVeterinary Pathologists, 1992-1997. Systemic PathologyofLaboratory Animals. International Life Sciences Institute, 1992-1995.
Mid-AmericaToxicology Course, 1996. Concepts inMolecularBiology, 1996.
MidwestAssociation ofVeterinary Pathologists: Attended and made oral presentations at annual summermeetings 1999, 2000, 2001

AnnE. Radovsky Date

### ATTACHMENT 8

### Attn: Peter Voytek

Date: 

### 6/19/2012

Sample: Composite of 4 - 4 Liter bottles of 1,2-Dichloroethane
EDC Purity



## RichardRhodes 1,2- Dichloroethane

DOW RESTRICTED

- For internal use only The Dow Chemical Company 2301 Brazosport Blvd Freeport, Texas 77541-3257



### ANALYZED

PPM/WT



### 99.97

### ATTACHMENT C

# WILR s RCt-1

Page I of53

PROTOCOL

AnExtended One-Generation DrinkingWater Reproductive Toxicity StudyofEthylene Dichloride in Rats

[OECD Guideline 443]

WIL StudyNumber: WIL-417005
Submitted To:
HAP TaskForce c/o PeterVoytek, PhD P.O. Box 331 Millwood, VA 22646

WILResearch 1407 George Road Ashland, OH 44805-8946 

### ATTACHMENT PROTOCOL

Wll-417005 June 25. 2012Page 2 of53 1 OBJECTIVE: WIL-417005 June 25, 2012

To evaluate the potential effects ofthe test substance on reproduction in an extended one-generation study covering life stages that are not covered by other types of toxicity studies. This will include determining the effects ofthe test substance on male and female reproductive processes including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and weaning, and on survival, growth, and development ofthe offspring. A minimum ofone litter will be produced.
As a part of this assessment, offspring will also be evaluated for effects on the nervous system, alterations in endocrine function (including thyroid perturbations), and effects on other systemic toxicityparameters. The extended one-generation reproductive toxicity study will serve as a substitute for the oral subchronic neurotoxicity and multigeneration reproductive studies described in Tier 2 testing for ethylene dichloride (EDC) under an enforceable consent agreement (U.S. EPA, 2003, U.S. EPA, 2009) between the HAP Task Force and the U.S. EPA.
This study will be conducted in accordance with the Organisation for Economic Cooperation and Development (OECD) Guideline for the Testing of Chemicals Guideline 443, Extended One-Generation Reproductive Toxicity Study, July 2011. This study will be conducted in compliance with the U.S. EPA Toxic Substances Control Act (TSCA; 40 CFRPart 792), the OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1 OECD ENV/MC/CHEM(98)17 Principles ofGood Laboratory Practice, and the European Community (EC)**Europ~an** Parliament and Council Directive 2004/10/EC (O.J. No. L 50/44, 02 February 2004).
2 PERSONNELINVOLVED IN THE STUDY:
2.1 Sponsor Representative:
PeterE. Voytek, PhD PO Box 331 1680 Bishop Meade Highway Millwood, VA 22646 Tel: (540) 837-1602 Email: pvoytekRSI@aol.comPage 3 of53 2.2 

### WIL Studv Director:
MelissaJ. Beck, PhD Associate Director, Neurosciences

and Head ofJuvenile Toxicology Tel: (419) 289-8700 Fax: (419) 289-3650 Email: melissa.beck@wilresearch.com
2.3 

### WIL Study Pathologist:
To be assigned at Study Initiation.


### 2.4 Contributing Scientist, Ovarian PrimordialFollicle Evaluation:
Catherine A. Picut, VMD, DACVP, DABT SeniorPathologist WILResearch 310 Millstone Dr. Hillsborough, NC 27278 Tel: (919) 245-3114 x106 Fax: (919) 245-3115 Email: catherine.picut@wilresearch.com


### 2.5 Principal Investigator (Ovarian Histology):
SirenaHudgins, HT (ASCP) Supervisor, Histology WIL Research 310 Millstone Dr. Hillsborough, NC 27278 Tel: (919) 245-3114 x114 Fax: (919) 245-3115 Email: sirena.hudgins@wilresearch.com WIL-417005 June 25,2012Page 4 of53 

### 2.6 Test Site Quality Assurance:
For histology conducted at WIL Research at Hillsborough:
Janice Jackson-Freeman Compliance Specialist, QualityAssurance WIL Research 310 Millstone Dr. Hillsborough, NC 27278 Tel: (919)245-3114xl19 Fax: (919) 245-3115 Email:janice.freeman@wilresearch.com 

### 2.7 WILDepartmentalResponsibilities: Pragati Sawhney Coder, PhD, DABT Senior Toxicologist, Developmentaland

Reproductive Toxicology 

### EmergencyContact Email: pragati.sawhney.coder@wilresearch.com
StevenD. Barkyoumb, DVM, PhD, DACVP SeniorVice President,

Global Operations- Safety Assessment
Donald G. Stump, PhD, DABT Senior Director, Developmental

andReproductive Toxicology
Eddie D. Sloter, PhD Assistant Director, Developmental and

Reproductive Toxicology
Bennett J. Varsho, MPH, DABT Director, Operations Gwendalyn M. Maginnis, DVM Clinical Veterinarian
CarolS. Wally, BA, SRS, RLATG GroupManager, Sample Processing Laboratory
Carol A. Kopp, BS, LAT Manager, Gross Pathologyand

Developmental ToxicologyLaboratory WIL-417005 June 25, 2012Pa e 5 of53 George A. Parker, DVM, PhD, DACVP, DABT Vice President, Pathology
Michael J. Schlosser, PhD, DABT Vice President, Analytical,

Metabolism and In Vitro Toxicology
Ronald E. Wilson, BS Director, Global Scientific Application Architecture
Heather L. Johnson, BS RQAP-GLP Manager, Quality Assurance
Robert A. Wally, BS Operations Manager, Reporting &

Technical Support Services


### 3 STUDY SCHEDULE: Proposed Experimental Starting (Animal Receipt) Date: Proposed Experimental Start (First DayofDosing) Date: Proposed Experimental Completion/TerminationDate: Proposed Audited Report Date: (without Optional Second Generation) July 31,2012

August 14, 2012

To be determined May 14,2013 WIL-417005 June 25,20124 TEST SUBSTANCE DATA:
4.1 Test Substance Shipment: Page 11 of53 WIL-417005 June 25, 2012

Test substance and applicable documentation, including a Certificate of Analysis, will be shipped under Sponsor'sresponsibility to:
Formulations Laboratory Attn: Terri Rafeld, Manager (WIL-417005; Melissa Beck) WIL Research 1407 George Road Ashland, Ohio 44805-8946 Tel: (419) 289-8700 Fax: (419) 289-3650 Email: terri.rafeld@wilresearch.com
4.2 Identification: Ethylene dichloride (EDC; 1,2-Dichloroethane)
4.3 CAS Number
107-06-2
4.4 Lot Number:
Not Applicable; there is no lot number assigned as the manufacturer has a continuous running plant.
4.5 Expiration/RetestDate:
NotApplicable.
4.6 Purity:
99.97% for the materialprovided.
4.7 Storage Conditions:
In a flammables cabinet in a climate controlled atmosphere.
4.8 Stability:
The test article is considered to be stable under the storage conditions provided bythe Sponsor.Page 7 of53 

### 4.9 PhysicalDescription:
To be documented by WIL Research.


### 4.10 Reserve Samples: WIL-417005 June 25,2012



### Reserve samples of the test substance will be taken in accordance with WIL Standard Operating Procedures (SOPs) and stored in the WIL Research Archives indefmitely, unless otherwise specified.


### 4.11 Personnel Safety Data:
To be provided by the Sponsor. It 

### is the responsibility ofthe Sponsor to notifY the testing facility ofany special handling requirements for the test substance. A Material Safety Data Sheet (MSDS) should accompany the test substance upon arrival at the laboratory.


### 4.12 TestArticle Disposition:
With the exception ofthe reserve sample for each batch oftest substance, all neat test substance remaining at study completion will be returned to the Sponsor (storage conditions, contact, address, fax number and telephone to be provided). Alternatively, the test substance can be retained for subsequent studies.


### 5 TEST SYSTEM:
5.1 Species:
Rat


### 5.2 Breed/Strain: Sprague Dawley Crl:CD(SD)


### 5.3 Source:
Charles River Laboratories, Inc. (Facility to be documented in the studyrecords)


### 5.4 Number ofAnimals:
108 Males and 108 Females (maximum of 120 males and 120 females purchased; males and females will be ordered from separate barrier rooms to ensure avoidance ofsibling mating). Animals not assigned to the study will bePage Xof53 WIL-417005 June 25, 2012 

### transferred to the stock animal colony or will be euthanized by carbon dioxide inhalation and discarded.
The number of animals is based on the OECD Guideline for the Testing of Chemicals: Guideline 443, Extended One-Generation Reproductive Toxicity Study, July 2011, which recommends including a sufficient number ofmating pairs to yield at least 20 pregnant females per dose group. Given the possibility ofnon-gravid animals, unexpected deaths, total litter losses, ortreatment-related moribundity and/or mortality in each generation of the study, this is an appropriate number ofanimals to achieve the desired number ofanimals in each generation.


### 5.5 Body Weight Range:
At randomization:


### 5.6 Approximate Age:
At initiation ofdosing:


### 5.7 Identification System: Males 300-450 g. Females 175-300 g.

Males and Females: 90 days

The F 0 

### animals will be uniquely identified by a Monel® metal eartag displaying the animal number. Individual cage cards will be affixed to each cage and will display the animal number, group number, study number, dosage level and sex ofthe animal. Offspring will be identified by tattoo markings applied to the digits after parturition and by ear tag after weaning.


### 5.8 Justification for Selection:
This species and strain ofanimal is recognized as appropriate for reproduction and neurotoxicity studies. WIL Research has reproductive and neurotoxicity historical control data in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive and neurological toxicants.


### 6 SPECIFIC MAINTENANCE SCHEDULE:
6.1 Animal Housing:
Fa 

### animals will be housed individually in clean suspended wire mesh cages in an environmentally controlled room during acclimation and continuing until euthanasia (for males) or until mating (females). The cages will be elevated above cage board or other suitable material, which will be changed at least three times each week.Page 9 of53 WIL-417005 June 25,2012 During cohabitation, females will be paired (1: 1) with a male from the same treatment group in a suspended wire mesh cage (home cage of the male). following successful mating the females will be housed individually in a plastic maternity cage containing nesting material (Aspen BedTM1) and will remain in these cages until weaning ofthe offspring. Analyses for possible contamination
are conductedonrepresentative lots ofbedding and documented in the raw data. The F0 females will then be individually housed in wire-mesh cages until euthanasia. Aspen bedding has been shown to be a suitable bedding for reproduction studies; it does not affect pup survival (Burkhart, 1978) or significantly induce liver enzymes like soft-pine bedding, including heat-treated pine wood bedding (Weichbrod, 1988). Hardwood bedding, such as Aspen BedT"1, does not need to be heat treated because it does not release oils and resins like soft-pine bedding. Therefore, the use ofAspen bedding (non-heattreated) in the current study is not expectedto impact the outcome ofthe study.
All F1 offspring selected to remain on study after weaning will be housed (by
sex within litter) in plastic cages with nesting material until postnatal day (PND) 28. F1offspring not selected to continue on study will be necropsied on PND 21. Subsequently, the F1 males and females will be individually housed in clean suspended wire-mesh cages in an environmentally controlled room and willremain in these cages until euthanasia. Nylabone® (or a similar enrichment device) will be provided to each animal for environmental enrichment and to aid in maintaining the animal's oral health, beginning during acclimation and continuing throughout the course ofthe study. The cages will be subjected to routine cleaning at a frequency consistent with maintaining good animal health and WIL Standard Operating Procedures. The facilities at WIL Research are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The cage sizes and housing conditions employed in this study will be in compliance with the Guide for the Care and Use of Laboratory Animals (1996).
6.2 Environmental Conditions:
Controls will be set to maintain temperature at 71 ± 5°F (22 

### ± 3°C) andrelative humidity at 50 ± 20%. Temperature and relative humidity will be monitored continuously. Data for these two parameters will be scheduled for automatic collection on an hourly basis. Fluorescent lighting controlled by light timers will provide illumination for a 12-hour light/dark photoperiod (on at 6 a.m. and offat 6:00p.m). The ventilation rate will be set at a minimum of 10 room air changes perhour, 100% fresh air.Pa e I0 of53 

### 6.3 DrinkingWater: WIL-417005 June 25,2012



### Cage banks will not be connected to an automatic watering system. Municipal water (with test substance added during the treatment period for the animals assigned to Groups 2-4) will be available ad libitum 

### to all animals and will be provided from standard plastic water bottles (approximately 250 

### mL). Bottles will be checked daily for spillage and supplemented as necessary. The occurrence ofspillage will be documented. Filters servicing the water supply will be changed regularly according to WIL SOPs. The municipal water supplying the laboratory is analyzed according to WIL SOPs on a routine basis to ensure that contaminants are not present in concentrations that would be expectedto affect the outcome ofthe study.
6.4 BasalDiet:
Harlan Laboratories 2016CM

### Teklad Global 16% 

### Protein Rodent Diet will be offered ad libitum 

### during the study. The batch offeed used on this study must have a genistein-equivalent content (aglycone) of less than 300 

### ppm (Owens eta/., 2003). 

### Periodic analyses of the certified feed are performed by the manufacturer to ensure that heavy metals and pesticides are not present at concentrations that would be expected to affect the outcome of the study. Results of the analyses are provided to WIL Research by the manufacturer. Feeders will be changed and sanitized once per week. Samples (approximately 100 

### g each) of each batch ofthe diet used during the study will be retained frozen (approximately -20°C) 

### until issuance ofthe final report, in case the study results necessitate further analysis ofdiet ingredients.
7 EXPERIMENTALDESIGN:
7.1 Animal Receipt and Quarantine:
Each animal will be inspectedby a veterinarian orqualified technicianunder the direct supervision ofa laboratory veterinarian upon receipt. Ratsjudged to be in good health and suitable as test animals will be immediately placed in quarantine for a minimum of 10 

### days. During this time, the animals will also be acclimated to the water bottles that will be used during the study. All rats will be initially weighed, permanently identified with a metal ear tag and receive a clinical observation. During the quarantine period, each rat will be observed twice daily for changes in general appearance and behavior. Priorto the start of the in-life phase, those animals judged to be suitable test subjects will be identified.Pa e 11 of53 7.2 

### Randomization: WIL-417005 June 25, 2012

At the conclusion ofthe quarantine period, animals judged to be suitable test subjects and meeting acceptable body weight requirements will be assigned at random using a computer program. At that time, the animal numbers and corresponding body weights will be entered into the WIL Toxicology Data Management System (WTDMSTM). A printout containing the animal numbers and individual group assignments will be generated based on body weight stratification into a block design. Animals will thenbe arranged into the groups according to the printout. Body weights atrandomization will be within ±20% of the mean for each sex. The control group and three test substance groups will consist of27 males and 27 females. Any animal assigned to the study that is found dead, euthanized 

### in extremis or exhibits abnormal clinical signs, reduced food consumption or body weight losses prior to the start ofdosing may be replaced by an animal ofappropriate age when possible. Replacement animals will be arbitrarily assigned (not computer randomized) to the study based on comparable body weights (if possible) withrespect to the animal that was replaced.


### 7.3 Route and Rationale ofTest Substance Administration:
The route ofadministration will be oral via the drinking water as this route of exposure may be used to estimate equivalent inhalation exposures (Sweeney and Parker, 2010; see Section 7.4.1) and since this route ofadministration is a reliable and accurate method ofdelivering test substance in an extended onegenerationreproductive toxicity study. Historically, this routehas beenused for studies ofthisnature. 

### 7.4 Organization ofTest Groups, Dosage Levels and Treatment Regimen:
7.4.1 Rationale for Dose Selection:
The dosage levels were determined from results ofprevious studies and were provided bythe Sponsor Representative.
The primary exposure route ofconcern for EDC is inhalation. Based on a pharmacokinetic (PK) study (Saghir 

### etal., 2006) and a physiologically based PK (PBPK) model describing disposition in rats following oral or inhalation routes ofexposure (Sweeney and Parker, 2010), oral doses of 50, 150, and 300 mg/kg/daywere selected. The model willthenbe used to estimate equivalent inhalation exposures. A 300 mg/kg/day exposure to EDC (Z% in drinking water) would be expected to yield blood EDC peak and cumulative exposures that would produce some maternal toxicity. This selection is based onthe assumption that the narcotic-typePage 12 of53 WIL-417005 June 25, 2012 effects (central nervous systemdepression) may be attributed to elevated levels ofEDC rather than metabolite levels or GSH depletion. The low dose of50 mg/kg/day (X%) is predicted to identifY a clear no-observedadverse-cfiect level (NOAEL). Iftreatment-related effects are seen in the high-dose group, the mid dose of 150 mg/kg/day (Y%) is expected to provide dose-response data.
7.4.2 Organization ofTest Groups:
The following table presents the study group arrangement. Drinking Approximate Number of Group Test Substance Water Dosage Level Animals Number Concentration (%) (mg/kg/day)" Male Female 1 Control (Drinking_Water) 0 0 27 27 2 Ethylene Dichloride X 50 27 27 3 Ethylene Dichloride y 150 27 27 4 Ethylene Dichloride 

### z 300 27 27
a - Approxtmate dosage level based on htstoncal water consumptton values. Achteved dosage levels will be calculated at the conclusion of the study based on water concentration, recorded body weight, and water consumption values for each animal. 7.4.3 Vehicle (Drinking Water): Municipal water. 7.4.4 Treatment Regimen:
Untreated or test substance-treated drinking water formulations will be offered ad libitum via standard plastic water bottles (approximately 250 mL) with metal sipper tubes, and supplied for the duration ofthe study, as noted in Sections 7.4.4.1 and 7.4.4.2. Fresh vehicle and test substance drinking water formulations will be provided approximately weekly.
7.4.4.1 F0 Generation:
Males: Following randomization, F0 male animals will be offered the control or test substance-treated drinking water for approximately 28 days prior to mating, throughout mating (up to 14 days), and for approximately 49 additional days until euthanasia.
Females: Following randomization, F 0 female animals will be offered the control or test substance-treated drinking water for approximately 28 days prior to mating and continuing throughoutPage 13 of53 WIL-417005 June 25, 2012 

### mating (up to 14 days), gestation (approximately 21 days), and lactation (approximately 21 days) until euthanasia on lactation day(LD) 22

.. 7.4.4.2 F1 Generation (Fo Offspring):


### The F1 

### generation will potentially be exposed to the test substance in utero, 

### through nursing during lactation and via the drinking water from the time ofweaning through the scheduled euthanasia (as appropriate for the relevant Cohort).
7.4.5 Adjustment ofDoses: 

### Drinking water concentration adjustments are designed to provide a relatively constant mglkg body weight/day dose throughout all study phases.
The proposed drinking water adjustments during lactation and postweaning are presented below. During lactation, drinking water concentrations will be adjusted using historical control water consumption data for lactating females to account for the large and rapid increase in food consumption (2-3 fold) typical for rats in mid- to late lactation. Dams awaiting necropsy will receive drinking water containing the same concentration of EDC that was provided during breeding (i.e., 

### during LD 21-22).
To account for the large amount of water consumed per kg of body weight in weanling pups, from PND 21-35, male and female weanlings will receive drinking water containing the same concentration ofEDC that was provided to the 

### Fo females during the second week oflactation (half of the adult concentration). On PND 35, F1 

### male and female offspring will receive adult water concentrations until termination, unless an 

### Fz generation is required. Ifa second generation is produced, similar drinking water adjustments will be employed for the F1 

### dams during lactation.

Exposure Unadjusted Adjustment Adjusted TMI Increase• Concentration Concentration Period (%) Factor (%) LD 7-14 3.1X X,Y,Z 2 X, YzY, YzZ LD 14-21 3.8X X,Y,Z 3 X,YzY,YzZ PND 21-28b 2.4X X,Y,Z 2 X, YzY, YzZ PND 28-35c l.9X X,Y,Z 2 X,YzY,YzZ
a Test Matenal1ntake relative tononpregnant adult females b Assumes water intake mirrors food intake in young animals (Marty et a!., 2003) which is true at othertime points includedin this table. c After PND 35, pups return to unadjusted drinking water concentrations.Pa e 14 of53 WIL-417005 June 25, 2012 7.5 

### Preparation and Analysis ofTest Substance-Treated Drinking Water:
7.5.1 Preparation ofTest Substance-Treated Drinking Water:
The drinking water solutions will be prepared as a fixed percent EDC in water. Based on the physical characteristics of the test substance, appropriate methods will be used to ensure the best possible mixing of the test substance in the drinking water. Test substance formulations will be prepared approximately weekly. The test substance will be added to the drinking water and mixed to obtainthe appropriate drinking water concentrations, and the formulations will be mixed periodically throughout the studybased onstability data. Any special procedures required for formulation will be documented according to Good Laboratory Practice Standards and presented in the final report ofthis study.


### 7.5.2 Analysis ofTest Substance-Treated Drinking Water: Drinking water solutions will be analyzed at WIL Research using an analytical method that will be developed and validated under a concurrent study (see Appendix B).


### 7.5.3 Stability ofTest Substance-Treated Drinking Water: Stability ofthe test substance in the drinking water preparations will be established prior to the start of dosing, if possible. Representative batches ofthe drinking water at each ofthe selected dosage levels will be mixed, and duplicate sets ofsamples will be taken from the middle of the control formulation and each test formulation. One set ofsamples will be stored under laboratory conditions for up to 15 days after preparation. These samples will be analyzed for stability after eight and 15 days. The other set ofsamples will be stored frozen at approximately -20°C as backup samples.


### 7.5.4 Homogeneity and Concentration ofTest Substance in the Drinking Water:
Prior to the first exposures, duplicate sets ofsamples ofdrinking water will be collected from the top, middle, and bottom strata ofeach dosage formulation, and duplicate sets of samples from the middle of the controls. One set ofsamples from all dosage levels, including controls, will be analyzed for test substance concentration. Ifstability permits, analytical verification will be performed prior to introduction ofthe test substance-treated water to the animals. The other set ofsamples will be stored frozen at approximately -20°C and discarded at the end ofthe in-Page 15 of53 WIL-417005 June 25, 2012 life phase following acceptance of the analytical results by the Study Director. Samples will be taken from the middle of each dosing formulation prepared during the first 4 weeks of the dosing period and monthly thereafter for the remainder ofthe study. Any backup samples kept at WIL Research will be discarded following acceptance ofthe analytical results by the Study Director.
7.5.5 Reserve Samples: Samples (from each day ofpreparation will be collected from each dose level (including controls). These samples will be stored frozen at approximately -20°C in sealed vials in the WIL Research Archives and willbe discarded following acceptance ofthe studyresults.
7.6 

### .Eo Determination ofEstrous Cycles: Daily vaginal lavages will be performed to determine the stage of estrus beginning 14 days after the initiation oftest substance administration. Estrous cycles will be evaluated for 2 weeks prior to mating, and during cohabitation until evidence ofcopulation (vaginal copulatoryplug or evidence ofsperm in a vaginal lavage) is observed. Vaginal lavages will continue for those females with no evidence ofcopulation until termination ofthe 14-day mating period. On the day ofnecropsy, a vaginal lavage will also be performed to determine stage ofestrus. An analysis ofthe female cycle pattern will be reported.
7.7 

### Fo BreedingProcedures:
After a minimum of 28 days oftreatment, one female will be cohabited with one male rat of the same treatment group, avoiding sibling mating (Charles River Laboratories will supply non-litter mates). Detection of mating will be confirmed by the appearance ofa vaginal copulatory plug or by evidence of sperm in a vaginal lavage. After confirmation of mating, the female will be placed in an individual suspended plastic cage containing nesting materiaL and the day will be designated as day 0 ofgestation. Ifa breeding male dies or is removed from study, a second male (fromthe same dose group) that has already completed mating will be provided, ifavailable.
A maximum of 14 days will be allowed for mating. If no evidence of copulation is obtained after 14 days, the animals will be separated without further opportunity for mating, and the female will be placed in a plastic cage containing nesting material.Pa e \6 of53 7.8 F0 Parturition and Lactation and Ft Litters: WIL-417005 June 25,20\2

The day parturition is initiated will be designated as day 0 of lactation. Any difficulties at the time of parturition will be recorded. When parturition is judged complete, litters will be sexed, examined for gross malformations and the number of stillbirths and live pups will be recorded. Any changes or abnormalities in nesting and nursing behavior will be recorded. The dam and litter will remain together untilweaning on lactation day 21.
7.9 Standardization ofF1 LitterSize and Identification: Following completion of delivery, all pups will be individually identified by tattoo markings applied to the digits. To reduce variability among the litters, on PND 4, 10 pups ofequal sex distribution (ifpossible) from each litter will be randomly selected. For litters consisting offewer than 10 pups, adjustments for litter size will not be performed. Following selection and blood/thyroid collection for PND 4 thyroid hormone analysis and possible histopathology (See Section 7.10), any remaining culled pups will be euthanized by an intraperitoneal injection ofsodium pentobarbital, examined macroscopically, and discarded.
7.10 Thyroid HormoneAnalysis (PND 4 Culled Pups):
On PND 4, culled pups will be anesthetized by isoflurane inhalation and as much blood as possible will be collected via cardiac puncture and pooled by litter until a total of 15 samples/dose level (minimum of 1.5 mL each) has been obtained. Blood may be pooled by litter to ensure a sufficient volume for hormone analysis. To the extent possible, samples from the first 15 litters at each dose level with sufficient numbers ofculled pups will be used. Additional litters may be used if there is an insufficient number ofpups to obtain the desired blood volume. The following parameters willbe evaluated: Thyroxine (T4; Cobas e411) Thyroid stimulating hormone (TSH; Radioimmunoassay)
Blood will be collected into tubes without anticoagulant and immediatelyplaced on ice. Serum will be obtained as soon as possible following blood collection, and stored frozen (approximately -70°C) until analyzed. Ifthe volume ofserum samples in insufficient, additional samples may be collected and documented in the studyrecords.
While under deep anesthesia, culled pups will be examined macroscopically, blood will be collected (as described above), and the thyroid glands will be harvested for thyroid hormone analyses. The trachea, with the thyroid gland attached, will be removed, placed into cassettes labeled to match the pooledPage 17 of53 WIL-417005 June 25,2012 blood samples, and placed in 10% neutral-buffered formalin for possible future histopathological examination. Following thyroid gland removal, the animals will be euthanized by decapitation. If no histopathologic examination 1s required, these tissues will be discarded after acceptance ofthe fmal report. 7.11 Assignment ofF1Animals to Cohorts:
Prior to PND 21, 3 F1pups/sex/litter from all available litters (2:20 litters/group) will be selected to remain on study for further examinations. Pups will be selected randomly; only pups not expected to survive due to notable physical limitations and/or obvious runts (animals with body weight more than 2 standard deviations lower than the respective mean litter weight) will not be available for selection. A detailed evaluation of each pup excluded from selectionwill be recorded.
The selected pups will be randomly assigned to one ofthree cohorts. Cohorts 1A and 1B (containing a minimum of 20 pups/sex/group/Cohort) will be assigned to reproductive/developmental toxicity testing, and Cohort 2 (2A and 2B; containing a minimum of 10 pups/sex/group/Cohort and representing a minimumof20 litters) willbe assigned to developmental neurotoxicity testing.
The cohorts will be subdivided as follows:

Cohort Number ofAnimals PrimaryAssessments

lA l /sexilitter/group Primaryreproductive/developmental (>20/sexigroup) toxicitytesting lB l /sex/litter/group Follow-up reproductive assessment, if (>20/sex/group) required 2A l pup/litter (2:l0/sexigroup, Neurobehavioral testing representing 2':20 litters) 2B l pup/litter (2':10/sexigroup, PND 22 Neuropathology representing 2':20 litters) Assignment of same-sex littem1ates to a particular Cohort will be avoided whenever possible. Ifthere are insufficient numbers ofpups to fill all ofthe designated pup assignments, the following prioritization plan will be used (highest to lowest priority):
Cohort lA, Cohort 1B, Cohort 2A, Cohort 2B
Animals assigned to Cohort lB may be maintained on study with exposure to the test substance continuing beyond PND 90. Ifnecessary, these animals may be bred to produce an F2 generation (See Section 7.16).Page IK of53 7.12 Examination ofNonselected Pups:
7.12.1 Macroscopic Examination WIL-417005 June 25,2012



### Nonselected Ft pups will be weighed and anesthetized via isoflurane inhalation on PND 21. Blood will be collected from a subset of weanlings as described in Section 7.12.2, and all animals will be euthanized via decapitation and necropsied as described in Section 8.1 with an emphasis on evaluation of developmental morphology and organs ofthe reproductive system. The animals will not be fasted prior to euthanasia. All gross lesions will be collected and placed in 10% neutral-buffered formalin.
7.12.2 Blood Collection and Thyroid Hormone Analysis:


### Blood will be collected via cardiac puncture from a subset of nonselected Ft pups (15/sex/group) for thyroid hormone analysis. The site ofcollection, including right or left, will be documented for each animal. The following parameters will be evaluated: Thyroid stimulating hormone (TSH; RIA) Thyroxine (T4; Cobas e411)
Blood will be collected into tubes without anticoagulant and immediately placed on ice. Serum will be obtained as soon as possible following blood collection, and stored frozen (approximately -70°C) until analyzed. If 

### the volume of serum samples in insufficient, additional samples may be collected and documented in the study records. Following blood collection, all animals will be euthanized by decapitation.
7.12.3 Organ Weights:


### The following organs will be weighed and placed in 10% neutralbuffered formalin for possible future histopathologic examination. The brains, spleens, and thymuses of 10 randomly selected pups/sex/group (representing 20 litters/group, if possible) will be weighed, while the thyroids will be obtained from the same 15 pups/sex/group used for blood collection. Organ-to-fmal-body-weight ratios will be calculated.

Brain Spleen Thymus Thyroids+

+ From the same 15 pups/sex/group used for blood collection. Tissue will be weighed after fixation in 10% neutral-buffered formalin.Page 19 of53 WIL-417005 June 25, 2012 Mammary tissue and any gross lesions will also be collected from all pups and placed in 10% neutral-buffered formalin for possible future histopathologic examination.
7.13 Euthanasia ofF0 Generation: Following completion ofweaning ofthe F 1 offspring, all Fo rats will be fasted overnight, and urine will be collected from the males as described in Section 7.18. On the following day, all animals will receive a detailed physical examination; all females will receive a vaginal lavage to determine the stage of estrus. The Fo animals will then be euthanized by carbon dioxide inhalation, blood will be collected as described in Section 7.18, the animals will be subjected to a gross necropsy, and tissues will be preserved as described in Section 8.1. Detailed evaluation of the process of spermatogenesis will be performed onall Fo males as described in Section 7.20. Any F0 rats not expected to survive to the next observation period (moribund) will be euthanized by carbon dioxide inhalation, subjected to a gross necropsy and tissues saved as described in Section 8. For females found dead or euthanized 

### in extremis during gestation, the number of corpora lutea and implantation sites will be recorded. Viable fetuses will be euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Recognizable fetuses will be examined externally and discarded. For females found dead or euthanized 

### in extremis during lactation, all pups will be examined externally and examined according to Section 7.14.7.
7.14 General Observations During The Experimental Period:
7.14.1 Parental Appearance and Behavior:
The parental rats will be observed twice daily for moribundity, mortality, and the availability of feed and water at least once in the morning and once in the afternoon from the day ofanimal receipt until euthanasia. This twice-daily observation will include cage-side examinations, while the animals are in their cages, to detect any significant clinical abnormalities that would be visible upon a limited examination, and to monitor the general health ofthe animals; only the presence ofadverse clinical signs will be recorded.
A detailed physical examination will be conducted weekly, beginning one week prior to the initiation oftest substance administration, on all parental animals. Detailed physical examinations will also be conducted on gestation days 0, 7, 14, and 20 for all mated (sperm- orplug-positive) females and on lactation days 0 (if possible), 1, 4, 7, 14 and 21. Mortality and all signs of overt toxicity will be recorded on the dayPage**~o** of53 WIL-417005 June 25,2012 observed. The observations shall include, but are not limited to, evaluation for changes in appearance of skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system function, somatomotor activity and behavior patterns. Special attention will be paid to the degree of salivation and lacrimation, presence or absence ofurination and defecation (including polyuria and diarrhea), pupil size, degree of palpebral closure, presence of convulsions, tremors or abnormal movements, presence ofposture and gait abnormalities, the presence of any unusual or abnormal behaviors and any repetitive actions (stereotypies). During the period ofexpected parturition, the dams will be observed twice daily for dystocia, prolonged labor, delayed labor or other difficulties at parturition (beginning on or around gestation day 20). Any abnormalities in nursing behavior will be recorded. All animals will also be observed on the day ofnecropsy and any findings willberecorded.
7.14.2 Detailed Clinical Observations:
All Fo animals will receive a Detailed Clinical Observation prior to the initiation of test substance administration, and weekly during the exposure period. The Detailed Clinical Observations utilized at WIL Research, are based on previously developed protocols (Moser 

### et a!., 1991; Irwin, 1968; Gad, 1982; Moser 

### et a!., 1988; Haggerty, 1989; O'Donoghue, 1989). Testing will be performed at approximately the same time each day by the same biologists, whenever possible, who will not know the animals' group assignments. Animals will be observed for the following:
Ease ofremoval from cage Lacrimationlchromodacryorrhea Piloerection Palpebral closure Red/Crustydeposits Eye prominence Mobility Convulsions/tremors Grooming Bizarre/Stereotypic behavior Pupillaryresponse Generalbodyposture Ease ofhandling animal in hand Salivation Fur appearance Respiratoryrate/character Mucous membranes/eye/skin color Muscle tone Gait Arousal Urination/defecation BackingPa e 21 of53 

### 7.14.3 ParentalBody Weights: WIL-417005 June 25,2012

All animals will have a final body weight recorded on the day on euthanasia.


### 7.14.3.1 Males:
Recorded individually on a weekly basis, beginning 1week prior tothe initiation oftest substance administration, until euthanasia.


### 7.14.3.2 Females:
Recorded individually on a weekly basis, beginning 1week prior to the initiation oftest substance administration, until evidence of copulation is observed, and on gestation days 0, 7, 14, and 20 and lactation days 1, 4, 7, 14, and 21.
Females with no evidence ofmating or that fail to deliver will continue to be weighed on a weekly basis until the scheduled euthanasia.


### 7.14.4 ParentalFood Consumption:
Individual food consumption measurement will be suspended during periods ofmating as the animals willbe cohabited.


### 7.14.4.1 Males:
Recorded individually on a weekly basis on the days of body weight collection, beginning 1 week prior to the initiation oftest substance administration, until euthanasia.


### 7.14.4.2 Females:
Recorded individually on a weekly basis on the days of body weight collection, beginning 1 week prior to the initiation oftest substance administration, until the start ofthe mating period and on gestation days 0, 7, 14, and 20 and lactation days 1, 4, 7, 14, and 21.
For females with no evidence ofmating or that fail to deliver, individual food consumption will continue to be weighed on a weekly basisuntilthe scheduled euthanasia.Pa e 22 of53 

### 7.14.5 ParentalWaterConsumption: WIL-417005 June 25, 2012

Individual water consumption measurement will be suspended during periods ofmating as the animals will be cohabited.


### 7.14.5.1 Males:
Recorded (by weight) individually ona weekly basis on the days of body weight collection, beginning 1 week prior to the initiation oftest substance administration, until euthanasia.


### 7.14.5.2 Females:
Recorded (by weight) individually ona weekly basis on the days of body weight collection, beginning 1 week prior to the initiation oftest substance administration, until the start ofthe matingperiod and ongestationdays 0, 7, 14, and 20 and lactation days 1, 4, 7, 14, and 21.
For females with no evidence ofmating or that fail to deliver, individual water consumption will continue to be recorded on a weeklybasisuntil scheduledeuthanasia.


### 7.14.6 Examination ofOffspring:
7.14.6.1 Appearance andBehavior:
All pups will be observed daily for general appearance, behavior and survival during lactation. A detailed physical examination willbe recorded for eachpup onpostnatal days 1, 4, 7, 14 and 21 oflactation. Any abnormalities in nesting and nursing behavior 

### willbe recorded. Thepupswillbe sexed onlactation days 0, 1, 4 (before culling), 7, 14 and 21. Following weaning, cage-side examinations (as described in Section 7.14.1) will continue to be conducted at least twice daily, and detailed physical examinations will be conducted weekly until euthanasia.


### 7.14.6.2 Body Weights:
Each pup will be weighed on PND 1, 4 (before culling), 7, 14, and 21, on a weekly basis following weaning, and on the day of euthanasia.Page 23 of53 7.14.6.3 Food Consumption: WIL-417005 June 25, 2012

Cage food consumption will be recorded on PND 21 and 28. Individual food consumption will be recorded on a weekly basis, beginning on PND 28 (when the animals are individually housed) until the dayprior to euthanasia.
7.14.6.4 Water Consumption: Cage water consumption (by weight) will be recorded on PND 21 and 28. Individual water consumption (by weight) will be recorded on a weekly basis, beginning on PND 28 (when the animals are individuallyhoused) until the daypriorto euthanasia.
7.14.6.5 Preweaning DevelopmentalLandmarks:
7.14.6.5.1 AnogenitalDistance:
The anogenital distance of all F1 pups will be measured on PND 1 (Gallavan 

### et al., 1999). Absolute and relative (to the cube root of body weight) will be reported.
7.14.6.5.2 Assessment ofAreolae/Nipple Anlagen:
On PND 12, all 

### F1 offspring (male and female) will be evaluated for the presence ofnipples/areolae in accordance with the methods described by Grey 

### eta!. (1990). A finding of'yes' and the number of nipples will be recorded ifnipples are present, and a finding of 'no' will be recorded if nipples are absent. Testing will be performed by biologists who will not know the animals' group assignments.
Ifthere is a statistically or biologically significant, treatment-related increase in retained nipples/areolae in males onPND 12, males assigned to Cohort 1A will also be examined at necropsy for nipple/areolae retention (upon written authorization by the Study Director). This examination will be similar to the PND 12 examination, with the additional requirement that the adult males will be shaved on the ventral surface to allow the accurate assessment ofnipple/areola number.Page 24 of53 WIL-417005 June 25,2012 7.14.6.6 Pup Detailed Clinical Observations: Beginning on PND 21 (after weaning), all offspring assigned to Cohort 2A will receive a weekly Detailed Clinical Observation (DCO). The DCOs will be conducted at approximately the same time each examination day. The DCO list utilized at WIL Research is based on previously developed protocols (Moser eta!., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; Haggerty, 1989; O'Donoghue, 1989). Testing will be performed by the same biologists, whenever possible, who will not know the animals' group assignments. Animals will be observed for endpoints listed below.

Arousal Backing Bizarre/Stereotypic behavior Convulsions/tremors Ease ofhandling animal in hand Ease ofremoval from cage Eye color Eye prominence Forelimb grip strength Fur appearance Gait General bodyposture Grooming Hind limb grip strength Lacrimation/

chromodacryorrhea Mobility Mucous membranes and skin color Muscle tone Palpebralclosure Red/Crusty deposits Piloerection Pupillary response Respiratoryrate/character Salivation Urination/defecation
7.14.6.7 Postweaning Developmental Landmarks:
All offspring will be evaluated for attainment ofthe following landmarks ofsexual maturity:
7.14.6.7.1 Balanopreputial Separation:
Each male pup will be observed for balanopreputial separation beginning on PND 35 (Korenbrot et a/, 1977). Examination of the males will continue daily until balanopreputial separation is present. Any abnonnalities ofgenital organs (e.g., persistent preputial threads) will be noted. The body weight of each male will be recorded on the day of attainment ofbalanopreputial separation.Pa e 25 of53 WIL-417005 June 25, 2012 7.14.6.7.2 VaginalPatency: Each female pup will be observed for vaginal patency on PND 25 (Adams et al, 1985). Examination ofthe females will continue daily until vaginal patency is present. Any abnormalities of genital organs (e.g., persistent vaginal threads) will be noted. The body weight ofeach female will be recorded on the day of attainment of vaginal patency.
7.14.6.8 Evaluation for FirstEstrus and Determination ofEstrous Cycles (F1 Cohort 1A): Beginning on the day ofvaginal patency, daily vaginal lavages willbe performed for all female offspring assignedto Cohort lA, and the vaginal lavages will be examined daily until the first sign ofestrus (cornifiedcells) is observed. Daily vaginal lavages will also be performed for all female offspring assigned to Cohort 1A to determine the stage ofestrus for 2 weeks from PND 75 through PND 88. On the day of necropsy, a vaginal lavage will also be performed to determine stage ofestrus.
7.14.7 Pup Deaths:
7.14.7.1 Pups 0 to 4 Days ofAge:
Moribund pups will be euthanized by an intraperitoneal injection of sodium pentobarbital. Stillborn pups, pups found dead between birth and PND 4, and any pups that are euthanized in extremis will be sexed, examined for the presence ofmilk in the stomach (PND 0 or 1 only), and dissected (including the heart, and the brain examined by a mid coronal slice) by a technique described by Stuckhardt and Poppe (Stuckhardt and Poppe, 1984). Ifa skeletal anomaly is suspected, the pups will be eviscerated, cleared and stained with Alizarin Red S as described by Dawson (Dawson, 1926) and examined. The cause of death will be determined, if possible. Representative specimens withmalformations may be preserved in 10% neutralbuffered formalin at the discretion ofthe study director.Pa e 26 of53 7.14.7.2 Pups 5 Days ofAge to Weaning: WIL-417005 June 25, 2012

Moribund pups will be sexed and euthanized by an intraperitoneal injection of sodium pentobarbital. A gross necropsy will be performed on pups found dead or euthanized in extremis, the sex will be determined, and the carcass ofall pups with gross lesions will be preserved in 10% neutral-buffered formalin for possible future histopathological examination. The cause ofdeath will be determined, ifpossible.
7.15 Developmental Neurotoxicity (Ft Cohort 2A):
F1 animals assigned to Cohort 2A will be assigned to the following tests: auditory startle response, functional observational battery, locomotor activity, and neuropathological and brain weight evaluations onPND 78.
Behavioral testing will be balanced across time ofday such that all groups are represented at each testing session. Body weights will be recorded on the days ofbehavioraltesting; these data will be maintained in the studyrecords.
7.15.1 Auditory Startle Response: Auditory startle response will be assessed on PND 24 using the StartleMonitor System (Kinder Scientific Company, LLC, Poway, CA) as described by WIL standard operating procedures. Auditory startle response testing will be performed in a sound-attenuated room equipped with a white noise generation system set to operate at 70 ±10dB.
The test sessions will consist ofa five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial will be a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 ms in duration). Responses will be recorded during the first 100 ms following onset ofthe startle stimulus for eachtrial.
The test session will consist of50 trials with an eight-second intertrial interval. Startle response data will be analyzed in five blocks of10 trials each. Startle response measures obtained are maximum response amplitude (MAX), average response amplitude (AVE) and latency to MAX (TMAx).
7.15.2 Locomotor Activity:
Locomotor activity will be monitored on PND 65. Locomotor activity will be measured using the Kinder Scientific Motor Monitor System (Kinder Scientific Company, LLC, Poway, CA) as described by WIL standard operating procedures. Locomotor activity sessions will bePa e 27 of53 WIL-417005 June 25,2012 

### performed in a room equipped with a white noise generator set to operate at 70 ± 10 dB.
The test session will be of60 minutes duration and will consist oftwelve 5-minute intervals. The 12 collection intervals will be presented in six 1 0-minute intervals for reporting purposes. Locomotor activity measures evaluated are total and ambulatory activity counts obtained for the 60-minute test session.
7.15.3 Functional Observational Battery (FOB):


### A functional observational battery will be conducted on all animals assigned to Cohort 2A on PND 65, prior to the locomotor activity assessment. If possible, at least 10 males and 10 females (from 20 litters) will be evaluated. The functional observational battery (FOB) utilized at WIL Research is based on previously developed protocols (Moser 

### et al., 1991; Irwin, 1968; Gad, 1982; Moser 

### et al., 1988; Haggerty, 1989; O'Donoghue, 1989). Testing will be performed in a sound-attenuated room equipped with a white noise generation system set to operate at 70 ±10 db. Testing will be performed by the same technicians, whenever possible, who will not know the animal's group assignment. Animals will be observed for the following as described below (see WIL SOP No. T1-112, as revised, for a description of scoring criteria):
7.15.3.1 Home Cage Observations:


### Posture Convulsions/tremors Feces consistency
7.15.3.2 Handling Observations:


### Ease ofremoval from cage Lacrimation/

chromodacryorrhea Piloerection Palpebral closure Red/crusty deposits Eye prominence Ease ofhandling animal inhand Biting Palpebral (eyelid) closure

Salivation Fur appearance Respiratoryrate/character Mucous membranes/eye/skin

color Muscle tonePage 2S of53 7.15.3.3 Open Field Observations: WIL-417005 June 25, 2012

Evaluated over a 2-minute observationperiod

Mobility Rearing Convulsions/tremors Grooming Bizarre/stereotypicbehavior Time to first step (seconds)
7.15.3.4 Sensory Observations: Approach response Startle response Pupil response Forelimb extension Airrighting reflex
7.15.3.5 NeuromuscularObservations:
Hindlimb extensor strength Hindlimb foot splay

7.15.3.6 Physiological Observations: Catalepsy Bodytemperature
7.16 Production ofa Second Generation: Gait Arousal Urination/defecation Gait score Backing

Touchresponse Tailpinch response Eyeblinkresponse Hindlimb extension Olfactory orientation

Grip strength

- hind and

forelimb Rotarodperformance

Bodyweight

Ifwarranted, the F1 animals may be mated to produce a second generation (with Sponsor approval, at additional cost). Reproductive and systemic toxicity data from the Fo generation will be used to assess whether a second generation will be needed. Males and females ofthe same dose group would be cohabited on or after PND 90, but not later than PND 120. Subsequent procedures will be similar to those outlined for the F0 generation/F1 litters unless otherwise specified in a protocol amendment. For example, based on a weight of evidence, it maybe sufficient to terminate the F2 litters onPND 4.Page 29 of53 7.17 Euthanasia ofF1 Reproductive/Developmental Toxicity Animals (Cohorts 1A and 1B): WJL-417005 June 25, 2012

On PND 91, all F1 males and females assigned to Cohort lA will be fasted overnight prior to euthanasia, and urine will be collected as described in Section 7.18. On the following day, all F1 Cohort lA animals will be weighed and receive a detailed physical examination. Ifa second mating is not required, 

### F1 male and female rats assigned to Cohort lB will be weighed and receive a detailed physical examination on PND 120. All 

### F, females in Cohorts lA and 1B will receive a vaginal lavage to determine the stage ofestrus prior to being transported to the Necropsy Department. The rats will be euthanized by carbon dioxide inhalation, and blood will be collected from Cohort 1A animals as described in Section 7.18. All Cohort 1 rats will be subjected to a gross necropsy, and tissues will be preserved as described in Section 8.1. Detailed evaluation of the process of spermatogenesis (Cohort lA only) will be performed as described in Section 7.20. Any F1 rats not expected to survive to the next observation period (moribund) will be euthanized by carbon dioxide inhalation and subjected to a gross necropsy and tissues saved as described in Section 8.
7.18 ClinicalPathology Evaluations (F0 andF 1 Cohort 1A):
Urine will be collected from the first 10 F 0 males that are available on studyday 91 and from the first 10 F1 Cohort 1A animals/sex/group that are available on PND 91. Urine will be collected overnight using metabolism cages. Water will be available during this procedure (animals will be fasted during the collection period). Urine will not be collected from F0 females.
Prior to the scheduled necropsy (LD 21 for the 

### Fo females, study day 91 for the 

### Fo males, and PND 90 for the Cohort lA males and females), all 

### Fo animals and 

### F1 animals assigned to Cohort 1A will be fasted overnight. Blood samples will be collected from the first 10 

### Fo and 

### F1 Cohort lAanimals/sex/group sentto the Necropsy Department. Blood samples for hematology and serum chemistry will be collected from the retro-orbital sinus from animals anesthetized with isoflurane. Blood samples for coagulation parameters will be collected by necropsy personnel from the inferior vena cava at the time ofeuthanasia from animals euthanized via C02 inhalation. In addition, blood smears will be prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for possible future evaluation. Ifneeded, additional samples may be collected to ensure the target sample size (n=lO/sex/group) is achieved. Any additional samples will be documented in the study file. Blood will not be obtained from animals that die or are euthanized in extremis priorto their scheduled necropsy.Page 30 of53 WTL-417005 June 25,2012 Anticoagulants used will be potassium EDTA for the hematology samples and sodium citrate for the coagulation test. Samples for serum chemistry will be collectedwithout anticoagulants. Any requirement for additional testing of serum or plasma samples must be determined prior to 90 days after the specimen has been tested. Consequently, any remaining samples may be discarded 90 days after testing is completed, unless directed otherwise by protocol.
7.18.1Urinalysis:
Bilirubin Color and clarity Glucose Ketones Leukocytes Microscopyofsediment
7.18.2Hematology:
Differential leukocyte counta Erythrocyte count Totalhemoglobin Hematocrit Mean corpuscular hemoglobin Mean corpuscularvolume Occult blood pH Protein Specifie gravity Urobilinogen Volume

Meancorpuscularhemoglobin

concentration Platelet count Reticulocyte count Total leukocyte count

a- Ifperformedmanually, results will include platelet estimates and RBC morphology.
7.18.3Serum Chemistry:
Alanine aminotransferase Albumin Aspartate aminotransferase Alkaline phosphatase Albumin/globulin ratio

(calculated) Calcium Chloride Creatinine Gamma glutamyltransferase Globulin (calculated) Glucose Phosphorus Potassium Sodium Totalbile acids Totalbilirubin Totalcholesterol Total protein Triglycerides Urea nitrogen Appearanceb

b -will include degree ofhemolysis, lipemia, and icterus (individual tables only)Page 31 of53 

### 7.18.4Coagulation Parameters:
Prothrombin time WIL-417005 June 25, 2012



### 7.19 Thvroid Hormone Analysis (F0 

### Males and F1 

### Cohort lAMales and Females):
Blood (approximately 1.5 mL) will be collected via the jugular vein from F0 males (15/group) on study day 85 and F1 males and females assigned to Cohort 1 A (15/sex/group) on PND 85 under isoflurane anesthesia to avoid the effects of fasting onthyroid hormone levels. The site ofcollection, including right or left, will be documented for each. The following parameters will be evaluated: Thyroid stimulating hormone (TSH; RIA) Thyroxine (T4; Cobas e411)
Blood willbe collected into tubes without anticoagulant and immediatelyplaced on ice. Serum will be obtained as soon as possible following blood collection, separated into two tubes containing approximately equal aliquots, and stored frozen (approximately -70°C) until analyzed. Ifthe volume ofserum samples is insufficient, additional samples may be collected and documented in the study records.
Blood for thyroid hormone analysis will not be collected from 

### Fo females as these animals are lactating and must provide care to the F1 offspring until overnight fasting andtermination onLD/PND 22.


### 7.20 Spermatogenesis Evaluation (F0 

### Males and F1 

### Cohort lAMales):
The following quantitative assessment oftheprocess ofspermatogenesis will be performed on all F0 males and all F1 males assigned to Cohort IA that survive to the scheduled euthanasia.
Ifthe left testis or epididymis is noted with a gross lesion, these organs will not be homogenized. Instead, the right testis and epididymis will be used for enumeration ofepididymal and testicular sperm and sperm production rate and the left testis and epididymis will be used for motility/viability and morphology assessments as well as for preservation in modified Davidson's solution for subsequent microscopic examination.


### 7.20.1 MotilityNiability Assessment: Immediately after euthanasia, the reproductive tract ofthe male will be exposed via a ventral mid-line incision. The right testis and epididymis will be excised and weighed separately. An incision will be made in the distal region ofthe right cauda epididymis, and the cauda will be placedPage 32 of53 WIL-417005 June 25, 2012 in a beaker containing Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/ml Bovine Serum Albumin (BSA). A small sample will be loaded into a 100-f.lm chamber for determination of motility. Because sperm motility can be affected by temperature shock, all slides and diluents will be warmed prior to use in an incubator, and motility determinations will be performed under constant temperature (approximately 37°C) using the Hamilton-Thome HTM-IVOS Version 12.3 computer-assisted sperm analysis (CASA) system. Analysis ofat least 200 motile and nonmotile spermatozoa per animal, if possible, from all dose groups will be performed by the analyzer. The motility score will be reported as the total percentage of motile sperm (showing any motion) and the total percentage of progressively motile sperm (showing net forward motion). The right testis and epididymis will then be preserved in accordance with Section 8.
7.20.2 Morphology Assessment:
For all F0 males in the control and high dose groups and all F1 males assigned to Cohort lA in the control and high dose groups, sperm morphology will be evaluated by a modification of the wet-mount evaluation technique (Linder 

### et al, 1992) by trained biologists who do not know the animals' group assignments. Abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa (if possible) per animal in all groups will be recorded. Ifa treatment-related effect is observed, males from the lower dose groups will be evaluated (with Sponsor approvaL at additional cost).
7.20.3 Enumeration ofEpididymal and Testicular Sperm and Sperm Production Rate (SPR):
For all F 0 males in the control and high dose groups and all F1 males assigned to Cohort lAinthe control and high dose groups, the left testis and epididymis will be weighed, homogenized and evaluated for sperm numbers and the sperm production rate will be calculated (Blazak 

### eta!, 1985) utilizing the Hamilton-Thome CASA system. The homogenized sample will be mixed with a DNA-specific fluorescent dye to allow identification of the sperm under fluorescent illumination. If a treatment-related effect is observed, males from the lower dose groups will be evaluated (with Sponsor approval, at additional cost).Page 33 of53 8 ANATOMIC PATHOLOGY: WIL-417005 June 25, 2012

8.1 Macroscopic Examination**(Fo~ 1**  Cohorts 

### lA and 

### lB, and Unscheduled Deaths):
A complete necropsy will be conducted on all Fo and F1post-weaning animals dying spontaneously, euthanized 

### in extremis (including animals with dystocia), or at the scheduled euthanasia for F0 animals and F1 animals assigned to Cohorts lA and 1 B, This will include examination ofthe external surface, all orifices, the cranial cavity, the external surface ofthe brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention will be paid to the organs ofthe reproductive system. In addition, the eyes will be examined 

### in situ by application of a moistened microscope slide to each cornea. For F0 females that were mated, the number of former implantation sites and corpora lutea will be recorded.Pa e 34 of53 WIL-417005 June 25,2012 At the time ofnecropsy, the following tissues and organs will be collected and placed in 10% neutral-buffered formalin (except as noted):
Adrenals (2) Aorta (1) Bone with marrow

(sternebrae) Brain (cerebrum, brainstem,

cerebellum) (3) Coagulating glands (2t Eyes with optic nerve (2)b Gastrointestinaltract

Esophagus Stomach Duodenum Jejunum Ileum Cecum Colon Rectum Heart Kidneys (2) Lacrimal/Harderian

glands (2) Larynx (1) Liver (sections oftwo lobes)(3) Lungs (including bronchi,

fixed by inflation with fixative) (5) Lymph nodes

Mediastinal (1) Mesenteric (1) Mediastinal tissues (1) Mesenteric tissues (1) Nasaltissues/pharynx (2) Oraltissues (2) Ovariesc,d and oviduct (2t Pancreas (1

# t Peripheral nerve (tibial) Pituitary (l)a Prostate (1

# t Mandibular salivary

glands (1) Seminal vesicles (2t Skeletal muscle (rectus femoris)(1) Skin with mammary gland and subcutis(1 

# t Spinalcord (cervical,

thoracic, lumbar) (3) Spleen (1) Testes with epididymides

and vas deferens (1

### t.f,g Tongue (0) Thymus Thyroids withTracheah

[withparathyroids if present (1)

# t Urinarybladder Uterus with cervix and

vagina (4t All gross lesions

(all groupst

The munber in parentheses is the minimum number ofsections to be examined ifthetissue is chosen for histopathologic examination. All tissues will be collected from animals that are found dead or euthanized in extremis. a- May also be examined for F1Cohort 18 animals ifCohort lAresults are equivocal orifreproductive or endocrine toxicity is suspected. b- Eyes with optic nerves will be fixed in Davidson's solution.
c- Ovaries will be fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries will be transferred to 70% Ethanol. d

- An additional section will be taken from Fo females for qualitative evaluation; additional sections (See Section 8.3) will betaken from F1Cohort lAfemales for quantitative evaluation ofprimordial follicles. e - For females; a corresponding section ofskin will be taken from the same anatomic area for males. f- Testis and epididymis (right only) will be fixed in modified Davidson's solution. Both testes and epididymides from males found dead or euthanized in extremis will be fixed in modified Davidson's solution. Care will be taken to ensure separation between the left andright organs. g- Ifthe left testis or epididymis is noted with a gross lesion, the left testis and epididymis will be fixed in modified Davidson's solution and the right testis and epididymis will be homogenized according to section 7.20.3. h

- Thyroids and parathyroids (ifpresent) will be removed from the trachea following fixation to ensure tissue structure is maintained during prosection.Page 35 of53 8.2 Organ Weights: WJL-417005 June 25,2012

The following organs will be weighed from Fo animals and F1 animals assigned to Cohorts lA and lB at the scheduled termination as noted below. Organ-tofinal-body-weight ratios will be calculated.
Adrenalsa Braina Epididymides*

(total and cauda)b Hearta Kidneysa Livera Ovariesb Pituitary+b Prostate"b Seminal vesicles with coagulating glands

(with accessory fluids)b Spleena Testes*b Thyroids with parathyroidsa+ Thymusa Uterus (with oviducts and cervix)b

a- Collected from F0 and F1Cohort IA animals only b

- Collected from F0 andF1 Cohort 1 A and 1 B animals 

### *

-These pairedorgans will be weighed separately. +-Tissuewill be weighed after fixation in 10% neutral-buffered formalin "

- If there is a treatment-related effect on prostate weights, the dorsolateral and ventral segments willbe dissected (after fixation) and weighed separately.
8.3 Microscopic Examination (F0 and Ft Cohorts lA and lB): Microscopic examination will be performed on the tissues listed in Section 8.1 from Fo animals and F1 animals assigned to Cohort lAin the control and highdose groups. These tissues will also be examined from all parental animals dying spontaneously or euthanized in extremis. For F1 animals assigned to Cohort lB, these indicated tissues will be processed to the block stage (in paraffin) and will be examined (upon Sponsor approvaL at additional cost) if results from Cohort lA are equivalent or in cases ofsuspected reproductive or endocrine toxicity.
Tissues and organs fixed in 10% neutral-buffered formalin, or other fixatives (as noted in Section 8.1) will be embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic evaluation. Processing ofthe testes, epididymides, and ovaries will be performed as notedbelow.
Sections of2-4 microns ofthe testis (transverse) and epididymis (longitudinal) will be stained with Periodic Acid-Schiffs stain (PAS) and hematoxylin in addition to routine hematoxylin and eosin (H&E) staining. Testes from males that are found dead or euthanized in extremis will be stained with H&E only. The following regions of the epididymis will be paraffin embedded: caput, corpus, and cauda; the vas deferens will be examined when possible. Histopathological examination ofthe testis will include a qualitative assessment of the stages of spermatogenesis. For males that survive to the scheduledPage 36 of53 WIL-417005 June 25, 2012 euthanasia, microscopic evaluation will include a qualitative assessment ofthe relationships between spermatogonia, spermatocytes, spermatids, and spennatozoa seen in cross-sections of the seminiferous tubules. The progression ofthese cellular associations defines the cycle ofspermatogenesis. In addition, sections of both testes will be examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections ofthe rete testis will be examined in the F1 Cohort lA males.
Five (5) sections will be taken approximately 100 J..lm apart from the inner third of each ovary from 10 F1 Cohort IA females in the control and high-dose groups at scheduled termination and for any F0 females suspected ofreduced fertility. In addition, a single sectionwill be takenfrom l0 F 0 and 10 F1 females per dose level for a qualitative bilateral evaluation of each ovary. For all F1 females in the control and high-dose groups at scheduled termination, and for any females suspected of reduced fertility, a quantitative histopathologic evaluation of multiple sections will be conducted. This examination will include enumeration ofthe total number ofprimordial follicles according to the methods ofBolon eta/. (1997), Bucci eta/. (1997) and Picut eta/. (2008).
For the F1 Cohort 1A females, a tiered approach will be used for the evaluation ofovarian follicle counts. A quantitative evaluation ofprimordial and growing follicles in at least 1% ofthe ovary will be performed as a screening measure for potential adverse effects. If the results are within 10% of controls, no further action will be required. If results are less than 90% of controls, a quantitative evaluation ofprimordial and growing follicles in at least 5% ofthe ovary will be performed as a definitive measure for potential adverse effects. If the results are equivocal, a quantitative evaluation ofprimordial and growing follicles in at least 5% ofthe ovary may be conducted on F1 females assigned to Cohort lB.
If treatment-related effects are seen, qualitative (Fo) or quantitative (F1; following mating to produce a second generation [see Section 7.16]) ovarian assessments will be performed on animals from the low- and mid-dose groups. Uterine and ovarian histopathology will be considered in light ofthe terminal estrous stage.
For females found dead or euthanized in extremis, a single section from each ovarywill be qualitatively evaluated.
If a target organ is identified in the high-dose group, that organ will be examined from all animals in the low and mid-dose groups (after the approval ofthe Sponsor and at additional cost). In addition, reproductive organs ofall animals suspected ofreduced fertility, e.g., those that failed to mate, conceive,Page 37 of53 WIL-417005 June 25,2012 sire or deliver healthy offspring, or for which estrous cyclicity or spermnumber, motility or morphology were affected, will be subjected to histopathological evaluation.
Ovarian tissues will be shipped to the attention of the Principal Investigator (Ovarian Histology) at the address provided in Section 2. This is a WILdesignated test site. Histology slide preparation will be conducted following the Standard Operating Procedures of WIL Research at Hillsborough, and in accordance with the Protocol and applicable GLPs. The Principal Investigator (Ovarian Histology) will be responsible for all delegated-phase activities. Any deviations from the GLPs or Protocol and any study specific SOP deviations will be reportedto the StudyDirector.


### 8.4 F1 

### Offspring Scheduled for Neuropathology/Brain Weight Measurements (Cohorts 2A and 2B):
8.4.1 PND 21 Neuropathology and Brain Weights (Cohort 2B)
8.4.1.1 Neuropathology- Macroscopic Examination:
On PND 21, F1 animals assigned to Cohort 2B (2:10/sex/group) will be macroscopically examined for neuropathology. The pups will be deeply anesthetizedby intraperitoneal injection ofsodium pentobarbital and perfused in situ as described in Section 8.4.2.1. The brains will be removed (including olfactory bulbs), weighed and the size (length [excluding olfactory bulbs] and width) will be recorded. Any abnormal coloration or lesions ofthe brain and spinal cord will be recorded. The brain, head, and any relevant gross lesions from all animals will be placed in 10% neutralbuffered formalin for possible future histopathological examination.


### 8.4.1.2 Neuropathology- Microscopic Examination: 

### F, pups assigned to Cohort 2B will be prepared for microscopic neuropathologic examination as described in Section 8.4.2.2. Brains for all groups willbe embedded in paraffin; however, only pups in the control and high-dose groups will be initially evaluated. The brains will be prepared for a qualitative histopathological examination by embedding in paraffin, sectioning, and staining with hematoxylin and eosin. Sections from all major brain regions (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, brainstem and cerebellum) will be examined. In addition,Pa e38of53 WIL-417005 June 25, 2012 sections will be prepared from thetrigeminal ganglion and nerve, and the pituitary gland.
Based on the results of the qualitative examination and the professional judgment of the pathologist, selected sites and cellular components may be further evaluated by the use of additional methods (at additional cost). Ifno neuropathological alterations are observed in samples from the high-dose group, subsequent analysis will not be performed. If a lesion is observed in the high-dose group, then animals from the low- and mid-dose groups will be examined (at additional cost) by the pathologist without knowledge of the animals' group assignments.


### 8.4.2 PND 78 Neuropathology and Brain Weights (Cohort 2A):
8.4.2.1 Neuropathology- Macroscopic Examination:
F1 offspring assigned to Cohort 2A will be fasted overnight and weighed on PND 78 prior to being transported to the Necropsy Department. The 

### F1 Cohort 2A animals will be deeply anesthetized by intraperitoneal injection ofsodium pentobarbital and perfused in situ as described in WIL Standard Operating Procedure T3-034, as revised. The central and peripheral nervous system tissues will be dissected and preserved as described in WIL Standard Operating Procedure T3-035, as revised. The whole brains shall be removed (including olfactory bulbs), weighed, and the size (length [excluding olfactory bulbs] and width) recorded. Any abnormal coloration or lesions ofthe brain or spinal cord will be recorded. Nervous system tissues will beretained from all animals.


### 8.4.2.2 Neuropathology
- Microscopic Examination:
The offspring perfused in situ on PND 78 will be processed for neuropathological evaluation. Brains for all groups will be embedded in paraffm, however, only rats in the control and highdose groups willbe initially evaluated.Page 39 of53 WIL417005 June 25, 2012 The following tissues willbe examined microscopically:
Brain

- olfactory bulbs, cerebralcortex, hippocampus, basal

ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata (including nucleus gracilis/cuneatus) Spinal cord

- at cervical swellings C3-C7 and at lumbar swellings

Tl3-L4 Trigeminal ganglia/nervesa Lumbar dorsalroot ganglia at Tl3-L4• Lumbar dorsalroot fibers at T13-L4• Lumbar ventralroot fibers at Tl3-L4• Cervical dorsalroot ganglia at C3-C7' Cervical dorsalroot fibers at C3-C7' Cervical ventral root fibers atC3-C7• Pituitary gland Caudal nerves < 2l+ Sciatic nerves<2)+ Suralnerves<2)+ Tibial nerves<2)+ Peroneal nerves<2l+ Nasal tissue witholfactory epithelium Optic nervesa Eyes (retinat Skeletal muscle (anterior tibial and gastrocnemius)

a * + Both processed and evaluatedmicroscopically, 4-6 tissues will be collected at necropsy; 2 will be evaluatedmicroscopically, Oneprocessed for microscopic examination, (2) Indicates that two sections (one cross and one longitudinal) ofthe tissue will be evaluated from the right hind leg. The tissues from the left hind leg and both forelimbs will be collected and preserved for possible future evaluation.
The centralnervous systemtissues listed above willbe embedded in paraffin. The peripheral nervous system tissues will be embedded in plastic. Tissues will be prepared for a qualitative histopathological evaluation by sectioning and staining with hematoxylin and eosin. The histopathological examination will include a simple morphometric analysis.
Based on the results of the qualitative examination and the professional judgment of the pathologist, selected sites and cellular components may be further evaluated by the use of additional methods (at additional cost). Ifno neuropathological alterations are observed, subsequent analysis will not be performed.Pa e 40 of53 

### 8.4.3 SubjectiveDiagnosis: WIL-417005 June 25, 2012

If any evidence of neuropathological alterations is found in the qualitative examination ofpups euthanized on PND 22 (Cohort 2B) or PND 78 (Cohort 2A), then a subjective diagnosis will be performed for the purpose of evaluating possible dose-response relationships. All regions of the nervous system exhibiting any evidence of neuropathological changes shall be included in this evaluation. Sections from all dose groups from each region will be examined in random order, without knowledge of the group assignment. The type and severity ofeach lesion will be recorded. Each type ofdose related lesion (ifpresent) and normal areas for comparison will be photographed.
9 DURATION OFSTUDY:
The two generations to be studied (parental animals and first generation offspring) will be termed F 0 and F1 , respectively. The conduct of this study will require approximately six months for acclimation, mating, gestation, and lactation of the Fo generation, and the growth and/or behavioral testing of the Ft generation until euthanasia.


### 10 STATISTICALMETHODS:
All analyses will be two tailed for minimum significance levels of5% and 1%. All means will be presented with standard deviations and/or standard errors ofthe mean. Coefficients ofvariation will also be presented for brain weights and morphometric measurements. All statistical tests will be performed by a computer with appropriate programming as referenced below. Following completion ofthe mating period, data from nongravid females, females with resorptions only, and/or females found to be gravid following staining ofthe uteri will be excluded from calculation ofmeans and from comparative statistics. The litter, rather than the pup, will be considered as the experimental unit.


### 10.1 In Life Data:
Continuous data variables (mean body weights, body weight gains, and food, water, and test substance consumption at each interval), pre-coital intervals, mean estrous cycle**length~** gestation length, former implantation sites, unaccounted-for sites, mean days of attainment of developmental landmarks (balanopreputial separation and vaginal patency) and the body weight on the day ofattainment, anogenital distance (absolute and relative to the cube root of body weight), numbers ofnipples/areola, and time from vaginal patency to first estrus, will be subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup difference. If the results of the ANOVA are significant (p<0.05), Dunnett's test (Dunnett,Page 41 of53 WIL-417005 June 25,2012 

### 1964) will be applied to the data to compare the treated groups to the control group. Fo male and female mating, fertility, copulation, and conception indices, and F1

### nipple retention ofthe treated groups will be compared to the control group using the Chi-square test with Yates' correction factor (Hollander and Wolfe, 1999).
10.2 Litter Data:
The mean litter proportions (% 

### per litter) ofpup viability during the postnatal period and sex ratio at birth will be subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup difference. Iftheresults ofthe ANOVA are significant (p<0.05), the Dunn's Test (Dunn, 1964) will be applied to compare the treated and control groups. Mean litter weights will be subjected to the parametric ANOVA test (Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above withthe litterrepresenting the experimentalunit.
10.3 Histopathology, Clinical Patholoey, Thyroid Hormone, and Orean Weight Data: Histopathologic fmdings ofthe treated groups will be compared to those ofthe control group by the Fisher's Exact Test (Steel and Torrie, 1980). The mean number ofovarian primordial follicles, clinical pathology and thyroid hormone (T4 

### and TSH) values, organ weights (absolute and relative to body weights) and mean brain lengths and widths will be subjected to a parametric ANOVA test (Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above.
10.4 Spermatogenic Endpoint Evaluations:
The percentage of motile (showing any motion) and progressively motile (showing net forward movement) spermatozoa and percentage of sperm with normal morphology will be subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) followed by the Dunn's test (1964) as described above. Slides containing less than 200 sperm will be excluded from analysis. Epididymal and testicular sperm counts and sperm production rates will be analyzed by a parametric ANOVA test (Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above.Page 42 of53 10.5 Detailed ClinicalObservation, FunctionalObservationalBattery, LocomotorActivity, and Auditory Startle Data: WIL-417005 June 25, 2012

Repeated Measures Analysis will be conducted by BioSTAT Consultants, Inc.
10.5.1 Detailed ClinicalObservation and Functional ObservationalBattery Data:

Continuous Detailed Clinical Observation (DCO) and Functional Observational Battery (FOB) data (including the average of 3 grip strength trials and 3 landing foot splay trials) will be subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. Ifsignificant differences are indicated by the ANOVA, Dunnett's test (1964) will be used to compare the control and treated groups. FOB and DCO parameters which yield scalar and descriptive data will be analyzed by the Fisher's Exact Test (Steel and Torrie, 1980).
10.5.2 LocomotorActivity Data:
Total and ambulatory activity counts will be analyzed, by sex and session, with a repeated measure analysis of variance (RMANOVA). Factors in the model will include treatment group (TRT), time interval (TIME), and the interaction of time interval and treatment group (TRT*TIME). The SAS® procedure PROC MIXED will be used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time will be selected by comparing Ak:aikc's Information Criterion (AIC).
The monotonic dose response relationship will be evaluated using sequential linear trend tests based onordinal spacing ofdose levels. The linear dose by time interaction (LinTrt*Time) will be evaluated and, if significant at the 0.05 level, trend tests on treatment means will be performed at the 0.05 level for each time interval. Ifthe linear dose by time interaction is not significant, the trend test will be conducted across the pooled time intervals ofthe entire session only.
Nonmonotonic dose responses will be evaluated whenever no significant linear trends arc detected but TRT and/or TRT*TIME interaction is significant at the 0.01 level. Within the framework ofthe RMANOVA, pairwise comparisons will be made for each individual treated group with the control group through linear contrasts. If TRT*TIME is significant, the comparisons will be conducted for each time interval. If only the TRT effect is significant, the comparisons will be conducted across the pooled time intervals of the entire session. ThesePage 43 of53 WJL-417005 June 25,2012 nonmonotonic dose response compansons will be conducted at the 0.01 significance level.
All statistical analyses will be conducted using SAS version 9.2 (SAS Institute, Inc., 2002-2008), orhigher, software.
10.5.3 Auditory Startle Response Data:
MAX and Tmax will be analyzed, by sex and session with a RMANOVA. Factors in the model will include TRT, trial block (TRIAL), and the interaction oftrial block and treatment group (TRT*TRIAL). The SAS® procedure PROC MIXED will be used for analysis with the random effect ofanimal included as the repeated measurement. The covariance structure across time will be selectedby comparing AIC.
The monotonic dose response relationship will be evaluated using sequential linear trend tests based onordinal spacing ofdose levels. The linear dose by trial interaction (LinTrt*Trial) will be evaluated and, if significant at the 0.05 level, trend tests on treatment means will be performed at the 0.05 level for each block oftrials. Ifthe linear dose by trial interaction is not significant, the trend test will be conducted across the pooledtrials ofthe entire session only.
Nonmonotonic dose responses will be evaluated whenever no significant linear trends are detected but TRT and/or TRT*TRIAL interaction is significant at the 0.01 level. Within the framework ofthe RMANOVA, pairwise comparisons will be made for each individual treated group with the control group through linear contrasts. If TRT*TRIAL is significant, the comparisons will be conducted for each block oftrials. Ifonly the TRT effect is significant, the comparisons will be conducted across the pooled trials ofthe entire session. These nonmonotonic dose response comparisons will be conducted at the 0.01 significance level.
All statistical analyses will be conducted using SAS version 9.1 (SAS Institute, Inc., 2002-2003), orhigher, software. Average response will be subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup difference. If significant differences are indicated by the ANOVA, Dunnett's test (1964) willbe used to compare the control and treated groups. 10.6 Brain Morphometric Data {Cohort 2A):
Brain morphometric data will be analyzed by the parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. IfPa c44of53 WIL-417005 June 25,2012 significant differences are indicated by the ANOVA, Dunnett's test (1964) will be used to compare the control and treated groups. 11 QUALITYASSURANCE:
The study will be audited by the WIL Quality Assurance Department while in progress to assure compliance with Good Laboratory Practice Regulations, adherence to the protocol and to WIL Standard Operating Procedures. The protocol, protocol amendments, raw data, draft report and final report will be audited by the WIL Quality Assurance Department prior to submission to the Sponsor to assure that the fmal report accurately describes the conduct and the findings ofthe study. This study will be conducted in compliance with the EPA TSCA (40 CFR Part 792), OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number I OECD ENV/MC/CHEM(98)17 Principles of Good Laboratory Practice, and the European Community (EC) European Parliament and Council Directive 2004/10/EC (O.J. No. L 50/44, 02 February 2004) and OECD ENV/MC/CHEM(98)17 Principles ofGood LaboratoryPractice. Histology slide preparation for ovarian primordial follicle evaluation will be conducted following the Standard Operating Procedures of WIL Research at Hillsborough, and in accordance with the Protocol and GLPs. Quality Assurance monitoring of these analyses for Protocol, SOP and GLP compliance is the responsibility ofWILResearch at Hillsborough. Inspectionreports will be supplied to the Study Director. Upon completion ofthe prescribed activities, WIL Research at Hillsborough will provide a signed QualityAssurance Statement to the StudyDirector which will be included in the fmal report.
Data requmng RMANOVA statistical analysis will be analyzed by BioSTAT Consultants, Inc. following the current procedural guidelines of BioSTAT Consultants, Inc. BioSTAT Consultants, Inc. will provide a statistical analysis report, which will be included as an appendix to the fmal report. Quality Assurance auditing ofthe statistical report (for internal consistency and consistency with the studyreport) will be conducted under the direction ofthe Quality Assurance Department ofWIL Research.
This studywill be included onthe WIL master list ofregulated studies.
12 RECORDS TO BEMAINTAINED:
All original raw data records, as defined by WIL SOPs and the applicable GLPs, will be stored as described in Section 13 in the Archives at WIL Research.
13 WORKPRODUCT:
The Sponsor will have title to all documentation records, raw data, specimens and other work product generated during the performance ofthe study. Any remainingPa e45 of53 WJL-417005 June 25,2012 samples (e.g. formulation or blood/plasma/serum) will be discarded after issuance of the Final Report. All work product generated at WIL Research Hillsborough will be returned to WIL Research Ashland. All work product, including raw paper data, pertinent electronic storage media and specimens, will be retained at no charge for six months following issuance of the final report in the WIL Research Archives. Thereafter, WIL Research will charge a monthly archiving fee for retention of all work product. All work product will be stored in compliance with regulatory requirements. Any work product, including documents, specimens, and samples, that are required by this protocol, its amendments, or other written instructions ofthe Sponsor, to be shipped by WIL Research to another location will be appropriately packaged and labeled as defined by WIL's SOPs and delivered to a common carrier for shipment. WIL Research will notbe responsible for shipment following delivery to the common carrier. Appropriate supporting documentation for statistical analyses conducted and reported byBioSTAT Consultants, Inc. will be maintained in the WIL Research Archives.
14 REPORTSAND STUDYPROGRESS UPDATES:
The Study Director will provide periodic updates of study progress to the Sponsor. Draft summary tables ofunaudited electronicallyrecorded data may accompany these updates.
The final report will contain a summary, test substance information, methods and procedures, appropriate individual animal and summary data tables, WIL Historical Control Data, supporting sub-reports (e.g. analytical chemistry report, etc.), a copy of the protocol and amendment(s), if any, and an interpretation and discussion ofthe studyresults.
In addition, the final report will also include a graphical depiction* ofmeandata for:

a. Maternalbody weights during gestation b. Maternalbody weights during lactation c. Litterbody weights during lactation d. Male and female bodyweights ofpostweaning animals c. Locomotor activity interval data at eachage f. Auditory startle peak and latencyto peakresponse databytrial block at each age g. Forelimb and hindlimb grip strength values across each age tested 

### * Graphs should depict the smnmary data presented after statistical analysis (i.e. data should be combined unless statisticallyanalyzed separatelybysex)Pa e46of53 WIL-417005 June 25,2012 The final report will be comprehensive and shall defme Ievel(s) inducing toxic effects as well as no-effect Icvcl(s) under the conditions ofthis investigation. The report will contain all information necessary to conform with current regulatory specifications.
WIL Research will submit an electronic copy (PDF and MS Word copy ofthe report text for editing and comments) ofthe audited draft report in a timely manner upon completion ofdata collection prior to issuance ofthe final report. It is expected that the Sponsor will review the draft report and provide comments to WIL Research within a two-month time frame following submission. WIL Research shall provide a revised draft report that incorporates the Sponsor's reasonable revisions and suggestions. One revision will be permitted as part ofthe cost ofthe study; additional changes orrevisions may be made, at extra cost. WIL Research shall submit the final report within two weeks of receiving authorization to finalize the report from the Sponsor. Ifthe Sponsor's comments and/or authorization to finalize the report have not been received at WIL Research within one year following submission ofthe draft report, WIL Research may elect to finalize the report following appropriate written notification to the Sponsor. An electronic copy (bookrnarked and hyperlinked PDF) ofthe final report will be provided; requests for paper copies ofthe fmal report may result in additional charges.
15 ANIMALWELFAREACT COMPLIANCE:
This study will comply with all applicable sections ofthe Final Rules ofthe Animal Welfare Act (AWA) regulations (9 CFR). The protocolwill be reviewedbythe WIL Institutional Animal Care and Use Committee (IACUC) prior to commencement of study specific activities. The Sponsor should make particular note ofthe following:

• The Sponsor Representative's signature on this protocol documents for the Study Director the Sponsor's assurance that the study described in this protocol does not unnecessarily duplicate previous experiments.

• Whenever possible, procedures used in this study have been designed to avoid or minimize discomfort, distress or pain to animals. All methods are described in this studyprotocolor in written laboratoryStandard Operating Procedures.

• Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized as deemed appropriate by the veterinary staff and Study Director. The Sponsor will be advised by the Study Director of all circumstances which could lead to this action in as timely a manner as possible.

• Methods of euthanasia used during this study are in conformance with the above-referenced regulation.

• The Sponsor/Study Director has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals and hasPage 47 of53 WIL-417005 June 25, 2012 provided a written nanative description (AW A covered species) ofthe methods and
sourcesusedto determine that alternatives arc not available.
16 PROTOCOLMODIFICATION:
Modification of the protocol may be accomplished during the course of this investigation. However, no changes will be made in the study design without the verbal or written permission ofthe Sponsor. In the event that the Sponsor verbally requests or approves a change in the protocol, such changes will be made by appropriate documentation in the form ofa protocol amendment. All alterations of the protocol and reasons for the modification(s) will be signed by the Study Director and the Sponsor Representative.
17 REFERENCES: Adams, J.; Buelke-Sam, J.; Kimmel, C.A.; Nelson, C.J.; Reiter, L.W.; Sobotka, T.J.; Tilson, H.A.; Nelson, B.K. Collaborative behavioral teratology study: protocol design and testing procedure. Neurobehavioral Toxicology and Teratology 1985, 7, 579-586. Biel, W.C. Early age differences in maze performance in the albino rat. Journal of GeneticPsychology 1940, 56, 439-453. Blazak, W.F.; Ernst, T.L.; Stewart, B.E. Potential indicators ofreproductive toxicity: testicular sperm production and epididymal sperm number, transit time and motility in Fischer 344rats. FundamentalandAppliedToxicology 1985, 5 (6Pt. I), 1097-1103. Bolon, B., Bucci, T.J., Warbritton, A.R., Chen, J.J., Mattison D.R. and Heindel, J.J. Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: Results from continuous breeding bioassays. Fundam. Appl. Toxicol. 1997,39, 1-10. Bucci, T.J., Bolon, B., Warbritton, A.R., Chen, J.J., and**Heinde~** J.J. Influence of Sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies. Reprod Toxicol. 1997, II(5), 689-696. Burkhart, C.A. and Robinson, J.L. High rat pup mortality attributed to the use of cedar-wood shavings asbedding. LaboratoryAnimals 1978, I2, 221-222. Dawson, A.B. A note onthe staining ofthe skeleton ofcleared specimens with Alizarin RedS. Stain Technology 1926, I, 123-124. Dunn, O.J. Multiple comparisons using rank sums. Technometrics 1964, 6(3), 241-252. Dunnett, C.W. New tables for multiple comparisons with a control Biometrics 1964, 20, pp. 482-491.Page 48 of53 WIL-417005 June 25, 2012 Gad, S. A neuromuscular screen for use in industrial toxicology. Journal ofToxicology andEnvironmentalHealth 1982, 9:691-704. Gallavan, R.H., Jr., Holson, J.F., Stump, D.G., Knapp, J.F., and Reynolds, V.L. Interpreting the toxicological significance ofalterations in anogenital distance: potential for confounding effects of progeny body weights. Reprod. Toxicol. 1999, 13(5), 383-390. GrayJr., L. E., Ostby, J., Monosson, E., Kelce, W. R. Environmental antiandrogens: low doses ofthe fungicide vinclozolin alter sexual differentiation ofthe male rat. Toxicology and Industrial Health. (1990) 15, 48-64. Haggerty, G.C. Development of Tier 1 neurobehavioral testing capabilities for incorporation into pivotal rodent safety assessment studies. Journal ofthe American College Toxicology 1989, 8:53-69. Hollander, M.; Wolfe, D.A. Nonparametric StatisticalMethods, 2nd ed.; Hollander, M., Wolfe, D.A., Eds.; John Wiley and Sons, Inc.: NewYork, NY, 1999; p 468. !twin, S. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse. Psychopharmacologia 1968, 13:222-256. Korenbrot, C.C.; Huhtaniemi, LT.; Weiner, R.W. Preputial separation as an external sign of pubertal development in the male rat. Biology ofReproduction 1977, 17, 298-303. Kruskal, W.H.; Wallis, W.A. Use ofranks in one-criterion variance analysis. Journal**~/the**American StatisticalAssociation 1952,47, 583-621. Linder, R.E.; Strader, L.F.; Slott, V.L.; Suarez, J.D. Endpoints ofspermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants. Reproductive Toxicology 1992, 6, 491-505. Marty, M.S., Johnson, KA., Carney, E.W. Effect offeed restriction onHershberger and pubertal male assay endpoints. Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 2003, 68, 363-374. Moser, V.C. McCormick, J.P.; Creason, J.P. and MacPhail, R.C. Comparison of Chloridimeform and Carbaryl Using a Functional Observational Battery. Fundamental and AppliedToxicology 1988, 11, 189-206. Moser, V.C.; McDaniel, K.L.; Phillips, P.M. Rat strain and stock comparisons using a functional observational battery: Baseline values and effects ofAmitraz. Toxicology andAppliedPharmacology 1991, 108, 267-283.Page 49 of53 WIL-417005 June 25, 2012 O'Donoghue, J.L. Screening for neurotoxicityusing a neurologically based examination and neuropathology. Journal of theAmerican College Toxicology 1989, 8:97-116. Owens, W.; Ashby, J.; Odum, J. and Onyon, L. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses. Environmental Health Perspectives 2003, 111(12), 1159-67. Picut, C.A.; Swanson, C.L.; Scully, K.L.; Roseman, V.C.; Parker, RF.; Remick, A.K. Ovarian Follicle Counts Using Proliferating Cell Nuclear Antigen (PCNA) and Semi-AutomatedImage Analysis in Rats. ToxicolPathol. 2008 May8. Saghir, S.A., Clark, A.J., Gilles, M.M. et a!. 1,2-Dichloroethane (EDC): Limited pharmacokinetics and metabolism study in Fischer 344 rats. The Dow Chemical Company, Midland, MI, 2006.
SAS® Proprietary Software, Version 9.2; SAS Institute, Inc.: Cary, NC, 2002-2008. Snedecor, G.W.; Cochran, W.G. One Way Classifications; Analysis ofVariance. In Statistical Methods, 7th ed.; The Iowa State University Press: Ames, IA, 1980; pp. 215-237. Steel, RG.D.; Torrie, J.H. Principles and Procedures of Statistics, A Biometrical Approach, 2nded.; McGraw-HillBookCompany: NewYork, NY, 1980; pp. 504-506. Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination ofrat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis 1984, 4, 181-188. Sweeney, L. M., and Parker, A. Internal dosimetry considerations for dose selection for
an extended one-generation reproductive toxicity study of 1,2-dichloroethane in the rat. ReportofToxicologyExcellence forRiskAssessment, Cincinnati, Ohio, 2010.
U.S. EPA. 1,2-Ethylene Dichloride: Final Enforceable Consent Agreement and Testing Consent Order, Fed. Reg. 2003,68: 33125-33129.
U.S. EPA. 1,2-Ethylene Dichloride: Completion ofEPA Program Review, Fed Reg. 2009, 74: 36196-36198. Weichbrod, RH.; Cisar, C.F.; Miller, J.G.; Simmonds, RC.; Alvares, A.P.; Ueng, T.H. Effects ofCage Beddings on Microsomal Oxidative Enzymes in Rat Liver. Laboratory AnimalScience 1988; 38(3), 296-298.II • II a II 

# ••••• I II II

II • 

# ••••• 

# ••••• 6/25/2012 7:4S AH FRml: 5032419078 TO: 14192893650 PAGE: 001 OF 002**W!l.-·117WJ~**

·-··**~----·** ----- Jli)t!L'**-~-~~-~~-----** .----···--··---**----·--~-~-··· ·-·•·•~·----~~-~~~~~~~-~-~--**  lH PROTOCOLAPPHOVAL: Sponsor

## <~pprov~ll reeei\·ed

## via_£1Cl'~L__________

## on_..!:!_/cUi/JO I'd,

IIi\P Task Force



# __lthUu_**~-e~j_wal_** Peter E. VoyJt.- PhD Sponsor Representative
WIL**Rc:~carch**



# ___

# :):x_L<-l~Qadi_ir_d.==---------- 1vlelissa J. Beck, PhD Study Director Dmc

### ,}fi_~.,, 

## d.Or**~~~.** Date27 F0 Males/Group

All F1Pups Anogenital Distance on PND 1 Nipple/ Areola Retention on PND 12 F1Culled Pups (PND 4} Thyroid Hormone Assessment (Minimum of 15 Samples/Group)

Tissues for Possible Thyroid Histopathology Pa e 51 of53 APPENDIX A
STUDY DESIGN

F0 GENERATION

Exposure to Test Substance for 28 Days Prior to Mating WeeklyDetailed Clinical Observations

beginning Prior to Initiation ofTest Substance Administration Determination ofEstrous Cycles Beginning 2 weeks after Initiation of Test Substance Administration MATE TO PRODUCE F1 LITTERS

· 1Male x 1Female Mate 14 days Exposureto Test Substance Throughout

Gestation and LactationPeriods (Fo Males and Females)

Urinalysis Conducted on F 0 Males; Hematologyand Serum Chemistry Conducted on all F0 Animals prior to the Scheduled Necropsy

F0 adults euthanized after F1 weaning WIL-417005 June 25,2012

27 F0 Females/Group



### .E

2 Males Thyroid Hormone Assessment I Week Priorto NecropsyPage 52 of53 WIL-417005 June 25, 2012

F0 Histopathology Fo Gross Pathology Spermatogenesis Evaluation Thyroid Hormone Assessment (15 Males/Group) 27 rats/sex (Control & High)

PND 22Nonselected F 1Weanlings

Thyroid Hormone Assessment 15/sex/group F1 WEANLINGS
At Weaning, F 1Pups from 2:20 Litters/Group Assigned to Cohorts (2/Sex/Litter) Weekly DetailedClinical Observations Conducted Weekly beginning on PND21



### I 

# I Cohort 1 (Reproductive and Developmental Toxicology) 

# I Cohort 2 (Developmental Neurotoxicology)
Cohort IA 2:20/sex/group

(1/sex/litter) Sexual Developmental Landmarks Day ofFirst Estms DailyEstrous Cycle (PND 75-88) Thyroid Hormone Assessment (-PND 85) Spermatogenesis

Evaluations Clinical Pathology Assessments 1Week

Prior to Necropsy

Euthanasia on

-PND 91 Histopathology Cohort IB 2:20/sex/group

(!/sex/litter) Sexual Developmental Landmarks Maintained for Potential F1Mating and/or Additional Histopathology Euthanasia on -PND 120 Cohort2A 10/sex/group (representing 20 litters, ifpossible) Sexual Developmental Landmarks Neurobehavioral

Assessments Euthanasia on -PND 78 Neuropathology Cohort 2B 1 0/sex/group (representing 20 litters, ifpossible) Sexual Developmental Landmarks Euthanasia on PND 21 NeuropathologyPage 53 of53 

### APPENDIXB WIL-417005 June 25, 2012

AnalyticalValidation and Stability Studyof 1,2-Dichloroethanein Aqueous Formulations

P99e 1of 1 Analytical Validation and Stability Study of 1.2-Dichloroethan<! in Aqueous Formulations CompiJanc.e· F::ll>.' GLP
OeVIIIopm&nl and validation ol a msthod lor the detarmrnation of analyle concentration in aqueous formulations: MeL"od del'eiopmeM usua!y incU:es tbu! rs r-ot limiied to) the following activitiea: (1 i ;,-,,-estigation ol po;en!ia! so'ubility 'imrtations; (2) lhl analysis of slzndaros prepared in an approprillle solwn! to es!ablisb**cllromalogt~~phy,** inc""'ding re:errtion times, resolution, ;and to check propcrtbr.-lily of respon!l!; (3) the 11nlliysisa' the anlllyte prepared in the m11trix io confirm the presence or ab..<erce of interferences, to ellliiUi!l!e potential •tabilrty**lim~atior:s,** and to evaiua:e response proportionafrty. Method development will be bile<! a! a rate of $25JI'hour and will not exceed !he amO!Jm propcsed 'Nill'o!1 sponsorllill-"'lCVaL Validabon wiil be conducted using the currem Wtl SOP guidelines for the auenment of system suitabil!y. method speeficltylselectivity, in1ra- 11nd mler·eesSIOn method oalibratior acoepteb<lrty, irnr&· and inter-session melhod accuracy are precision, ruggednes.s, and processed sample stabilty. A mnmum of three V1!1ida!ioo sessions win be conducted. All laboratory wort; associated with vartdatioos will be conducted in accordaoce wilh applicable GLP regu\aOOns. Unl1orm11y and stabllttyu
.. nrMnt of analyte in aqu.ous formulations:

Quallly Assuranea:

CONFIDENTIAL Testing itlcludu the assessment of tes! article uniformity in formulations**spam~** the range of concentration arlioipaled an t<..rture studoes. In adddion, statlilrty will be IISM!SMd tallowtng a single**stora~** auratrorr Additional stabiwty lime-colnts can be added for an acdilional fee. Afl laboratory work. eS50Ciated with sample analysis viii be conducted in aooc«la!"Ce with applicable GLP regu:ations. The study will be**cond~cted** ir co!"1pfiaN:I! with Good LaooratD!j' Practice (GlPl standards ar.d wi'l bemorr.cred bythe Qu;a!ilyAssurance Unit

We have listening down to a science.

### ATTACHMENT ECA
Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33125
Region VII, 901 N. 5th Street, Kansas City, Kansas 66101. SUPPLEMENTARY INFORMATION: This proposed settlement is intendedto resolve the liabilityofSaveway Petroleum at the Great Lakes Container Corporation Superfund Site in St. Louis, Missouri.

Great Lakes ContainerCorporation is a former drum reclamationcompany who operatedatthe Site from 1976 to 1985. The same businesswas operated as NorthwesternCooperage from the 1950'sto 1976 and then operated as Great Lakes ContainerCorporation. EPA conducteda time-critical removal completed in 1998 thatconsisted primarilyofsoil and drum removal. The EPA incurredcosts ofapproximately $9,127,244.30. Thehazardous substances atthis Siteconsisted primarily oflead andpolychlorinated biphenyls. Liability is based onthe theorythat de minimis parties arranged for disposal ofhazardous substances at the Siteby shipping drums for reclamation coatedwithpaint containing lead. The de minimisparties either admittedthatthey sent drums for reclamation to the Site orEPA had separate evidencetoprovethat de minimisparties sent drums for reclamationto the Site.

This settlement isbeing offered to Savewaybecause itis liable for nomore than one quarter a percent (.25%) of EPA's pastcosts atthe Site. The majority ofde minimisparties are each requiredto pay $4,839.44 or $5,133.72 depending onwhetherthe partywas requiredto pay prejudgment interest. Other settlements made for six de minimisparties variedfrom $3,794.19 to $22,856.56 because more volumespecific informationwas available for them allowingEPAtorefine the calculation. The amount andtoxicity of hazardous substances contributedby Savewaywasminimal as comparedto otherparties' shares ofhazardous substances. TheEPA determinedthis amountto be Saveway's fair share of liabilitybased onthe amount of hazardous substancesgenerated and disposed ofatthe Site andthe volume ofwaste contributed. However,because Savewayhas demonstrated an inability to pay, itwill notbe requiredto pay any ofEPA's pastcosts atthe Site. As a result, Savewayhas agreed to provide access to EPA andmaintainrecords for five (5) years.

The settlement also includes contributionprotection from lawsuits by other potentiallyresponsible parties as provided for under section 122(g)(5) of CERCLA, 42 U.S.C. 9622(g)(5). The de minimis settlement provides that EPA covenants notto sue Saveway for response costs at the Site or for injunctive reliefpursuant to sections 106 and 107 ofCERCLA and section 7003 ofthe Resource Conservation and Recovery Act of 1976, as amended (RCRA), 42 U.S.C. 6973. The settlement contains a reopener clause which nullifies the covenantnotto sue ifany information becomes knownto EPA that indicatesthat Saveway no longer meets the criteria for a de minimis settlement setforth in section 122(g)(l)(A) of CERCLA, 42 U.S.C. 9622(g)(l)(A). The United States maintains the abilityto bring an action inthe eventthatthe financial informationprovidedby Saveway was false. Thecovenantnotto sue does not applytothe following matters:

(a) Claimsbased onthe future arrangement for disposal ortreatment of anyhazardous substance, pollutant, or contaminantat the Site after the effective date ofthe de minimis settlement;

(b) Criminal liability; or (c) Liability for damages cir injuryto, destruction of, or loss ofthe natural resources and for the costs ofany naturalresource damage assessments.

The de minimissettlementwill becomeeffective uponthe date which theEPA issues a writtennoticeto Savewaythatthe statutorypublic comment periodhas closed andthat commentsreceived, ifany, do not require modification, of orEPA withdrawal from the settlement.

Dated: May 22, 2003. James B. Gulliford, RegionalAdministrator,Region VII. [FR Doc. 03-13565 Filed 6-2-Q3; 8:45am] BILLING CODE 6560-511-P

ENVIRONMENTAL PROTECTION AGENCY [OPPT -2003-0010; FRL-7300--6] 1,2-Ethylene Dichloride; Final Enforceable ConsentAgreement and Testing Consent Order
AGENCY: EnvironmentalProtection Agency (EPA). ACTION: Notice.
SUMMARY: Under section 4 oftheToxic

.SubstancesControl Act (TSCA), EPA has issued a testing consentorder (Order) thatincorporates anenforceable consent agreement (ECA) withthe Dow Chemical Company, Vulcan Materials Company; Occidental Chemical Corporation, Oxy Vinyls, LP, Georgia GulfCorporation, Westlake Chemical Corporation, PPG Industries, Inc., and Formosa Plastics Corporation, U.S.A. TheCompanies are members ofthe Hazardous Air Pollutant (HAP) Task Force, which represents the manufacturers of 1,2-ethylene dichloride (EDC). The Companies have agreed to: Conducttoxicity testing, develop pharmacokinetics and mechanistictest data, and develop a computational dosimetry model for quantitativeroute-to-route extrapolations ofdose-response for EDC for acute, subchronic, developmental, reproductive and neurotoxicity effects that were identified in a proposed test rule for hazardous airpollutants. This notice announcesthe ECA and Order for EDC and summarizes the terms ofthe ECA.
DATES: Theeffective date oftheECA and Order is May 13, 2003. FOR FURTHER INFORMATION CONTACT: For general information contact: Barbara Cunningham, Acting Director, Environmental Assistance Division (7408M), Office ofPollution Prevention andToxics, Environmental Protection Agency, 1200PennsylvaniaAve., NW., Washington, DC 20460-0001; telephone number: (202) 554-1404; e-mail address: TSCA-Hotline@epa.gov.

For technical information contact: Richard Leukroth or John Schaeffer, Chemical Control Division (7405M), Office ofPollutionPrevention and Toxics, EnvironmentalProtection Agency, 1200PennsylvaniaAve., NW., Washington, DC 20460-0001; telephone number: (202) 564-8157; fax number: (202) 564-4765; e-mail address: ccd.citb@epa.gov.
SUPPLEMENTARY INFORMATION: I. GeneralInformation A.Does thisActionApplyto Me?

This announcement is directedto the public ingeneral. However, as described inUnitIV., this ECA and Ordermay affect others in thatEPA has initiated rulemaking underTSCA section 12(b) (62 FR 67038, December 23, 1997) (FRL-5762-8). When finalized, that rulemaking willrequire all persons who export or intendto exportEDC to complywith the export notification regulations at 40 CFR part 707, subpart D. Although others maybe affectedby subsequentactions relatedto this announcement, this ECA andOrder only applies to those Companies that are specificallynamed inthis ECA and Order. Ifyou have any questions regarding the applicability ofthis action to a particular entity, consultthe technical person listedunder FOR FURTHER INFORMATION CONTACT.33126 Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices

B. How Can I Get Copies of this Documrmtand OtherRelated Information?
1. Docket. EPA has established an official public docket for this action under docket identification (ID) number OPPT-2003-0010. The official public docket consists ofthe documents specificallyreferencedinthis action, any public commentsreceived, and other informationrelated to this action. Althougha partofthe official docket, the public docket does not include Confidential Business Information (CBI) orother informationwhose disclosure is restrictedby statute. The official public docket is the collectionofmaterials that is available for publicviewing atthe EPA Docket Center, Rm. B102-Reading Room, EPA West, 1301 Constitution Ave., NW., Washington, DC. TheEPA DocketCenter is openfrom 8:30a.m. to 4:30p.m.,MondaythroughFriday, excluding legal holidays. TheEPA Docket CenterReading Room telephone numberis (202) 566-1744 andthe telephone number for the OPPTDocket, whichis located intheEPA Docket Center, is (202) 566-0280.

2. Electronicaccess. You may access this Federal Register document electronicallythroughtheEPA Internet underthe "FederalRegister" listings at http:/ /www.epa.gov/fedrgstr/. Anelectronicversion ofthe public docket is available throughEPA's electronic public docket and comment system, EPA Dockets. You mayuse EPA Dockets athttp://www.epa.gov/edocket/ to submit or viewpubliccomments, access the index listing ofthecontents ofthe official public docket, andto access those documents inthe public docketthat are available electronically. Althoughnotall docket materials may be available electronically, youmay still access any ofthepublicly available docket materialsthroughthe docket facility identifiedinUnitI.B.l. Once in the system, select "search,"then key in the appropriate docket ID number. II. Background A. What isEDC?

EDC is used as a chemical intermediateprincipally inthe productionofvinyl chloride,but also vinylidenechloride, 1,1,1- trichloroethane, trichloroethylene, tetrachloroethylene, aziridines, and ethylene diamines. It is also used as a solvent. Anestimated 77,111 workders are exposedto EDC (Ref. 1). The ChemicalAbstract ServiceRegistry Number (CAS No.) for EDC is 107-06- 2. B. WhyDoes EPA NeedHealth Effects Data on EDC?

EPA proposed health effects testing underTSCA section4(a) for a number ofhazardous air pollutants ("HAPs" or "HAPchemicals"), includingEDC (61 FR 33178,June 26, 1996) (FRL-4869-1). as amended at 62 FR 67466, December 24, 1997 (FRL-5742-2) and 63 FR 19694, April21, 1998 (FRL-5780-6). In the original HAPs proposal, theAgency made preliminaryfindings for EDC (61 FR 33178, 33190, 33191; andRef. 1) that:

1. EDC may present anunreasonable risk ofinjuryto health.

2. EDC is orwill beproduced in substantial quantities, andthere is or maybe substantial humanexposures to the chemical.

3. There are insufficient data to determine or predictthe effects of activities onhumanhealth involving EDC.

4. Testing is necessary to develop healtheffects data for EDC.

The HAPs rule, as amended, proposed testing ofEDC for acute toxicity, subchronic toxicity, developmental toxicity, reproductive effects toxicity, and neurotoxicity (61 FR 33178, 33198, June 26, 1996; 62 FR 67466, 67483, December 24, 1997). III. ECA Development and Conclusion A.HowisEPA Going to Obtain Health Effects Testing on EDC?

Inthe proposedHAPs testrule, as amended, EPA invitedthe submission ofproposals regardingtheperformance· ofpharmacokinetics studiesthat would permitextrapolation from oral datato predictrisk from inhalationexposure. Suchproposals couldprovidethe scientificbasis for alternativetesting to thetestingproposed and form thebasis for developing needed HAPs datavia ECAs (61 FR 33178, 33189, June 26, 1996; 62 FR 67466, 67474, December 24, 1997). EPA uses ECAs to accomplish testing where a consensus exists among EPA, affected manufacturers and/or processors, and interested members of the public concerningtheneed for and scope oftesting (40 CFR 790.1(c)).

The procedures for ECA development are described at 40 CFR 790.22(b).

In response to EPA's request for proposals for ECAs, theHAP Task Force submitted a proposal for alternative testing that includedphysiologicallybased pharmacokinetics (PBPK) studies and computational modeling to inform route-to-route extrapolations ofdoseresponse for EDC onNovember 22, 1996 (Ref. 2). EPA respondedto this proposal onJune 26, 1997 (Ref. 3). indicatingthat the HAP Task Force alternative approach offered sufficient merit to proceed with discussions for developing an ECA for EDC. Consequently, EPA issued a document which was published inthe FederalRegister of December 19, 1997 (62 FR 66626) (FRL5763-1), soliciting interestedpartiesto monitor orparticipate inthese discussions.

EPA held a public meeting to develop anECA for EDC onJanuary 12, 1998. Representatives .ofthe Companies and other interestedparties attendedthis meeting. The participants reached consensus onthe general scope ofthe testing to berequiredunderthe ECA. Followingthe public meeting, the HAP TaskForce submitted (March 19, 1999) a revised proposal for a testing program (Ref. 4). OnFebruary 13, 2001, EPA respondedto the HAP TaskForce with comments onthe revised proposal and byinitiating a draft ECA for considerationbytheHAP Task Force (Ref. 5). A final version oftheECA was latercirculatedto theHAP Task Force for signature, andreturnedto EPA for signature. OnFebruary 3, 2003, EPA received theECA signedbythe Companies. OnMay 13,2003,EPA signedtheECA and accompanying Order (Ref. 6). B. What TestingDoes the EGAforEDC Require?

TheEDC ECA alternative testing programhas four segments as follows: TierIHAPs Testing, TierIProgram ReviewTesting, EPA Program Review, andTierIT Testing. This is described in Table 1 inthis unitandincludes the following testing, reporting, andreview activities:

1. TierIHAPs Testing. This testing consists ofendpointtesting, conducted byinhalationexposure, thatEPA deemednecessaryto meetcertain data needsidentifiedintheproposedHAPs testrule, as amended, and includes acute toxicitywithbronchoalveolar lavage (BAL) andhistopathology, and acute neurotoxicitytesting. Thesetests willbe conducted under a combined protocol as described inAppendixD.1 oftheECA.

2. TierIProgram Review Testing. Underthis segment oftheEDC ECA alternativetestingprogram, the Companies will conduct studies to extendthecomputational dosimetry model ofD'Souza etal. (1987; 1988; Refs. 7 and 8) in orderto applythe model to the specifichealth effects endpoints for EDC listed intheECA, validate themodel, and verify the model's abilityto perform quantitative route-to-route extrapolations ofdoseresponse. In addition,the Companies will sponsor development ofFederal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33127
pharmacokinetics andmechanistic (PK/ MECH) data to supportthe application ofthe model for the endpoints listed underTier II oftheEDC ECA. Specifically, thePK/MECH testing will develop data to inform:

i. Oral-to-inhalation extrapolation of subchronic toxicity datareportedby Daniel, etal. (1994; Ref. 9) relevant to corn oil gavage.

ii. Oral-to-inhalationextrapolationof 'subchronic neurotoxicity datarelevant to drinking waterexposure ofa studyto · be conducted underTierII Testing.

iii. Oral-to-inhalationextrapolation of reproductive effects testing conducted underTierII Testing and each dosing paradigm ofstudies reportedbyAlumot etal. (1976; Ref. 10), Rao, etal. (1980; Ref. 11) and Lane etal. (1982; Ref. 12).

In addition, the Companies will providemodel simulations withpoint anduncertainty estimates ofinternal dose metrics (parent chemical peak and area underthe curve (AUC) concentrations inblood andbrain, and 24-hourtotal glutathione-dependent metabolism) inrats andhumans to inform quantitativeroute-to-route extrapolations ofdose-response. These simulationswillbe usedto evaluatethe acceptability of: Subchronic neurotoxicitytesting oforal exposure via drinking water inrats, extant oral subchronic toxicity data ofDaniel etal. (1994; Ref. 9) inrats via corn oil gavage, andreproductive toxicitytesting oforal e,?Posure via drinking water inrats.

3l**~PA** Program Review. Model

·'ffi:!velopment and data from Tier I Program Review Testing are subject to an EPA Program Review. Itis essential to the success oftheEDC ECA alternative testing program for EPA to ensure that the model andthe PK/ MECH data used to supporttherouteto-route extrapolations ofdose-response are ofthehighest quality. The purpose oftheEPA Program Review willbe to determine:

i. Whether it is feasible and ' appropriate to applyTier IProgram :Review Testing data anddata from other

studies acceptable to EPA to support 'computationalroute-to-route extrapolations ofdose-response for endpoints listedinthe TierII Testing segment ofthe ECA.

ii. Whetherthe data from theTier I Program Review Testing segment provide a sufficientbasis for conducting theendpointtesting and/orthe computational route-to-route extrapolations for the dose-responses specified intheTierII Testing segment.

iii. Thenature and scope ofany additional work thatmayberequired beforeTier II Testingand application of theEDC model for route-to-route extrapolation ofdose-response reporting (e.g., development ofadditional PK/ MECH data, modification to theEDC model).

4. TierII Testing and/orExtrapolation Reporting. This segment ofthe EDC ECA alternative testing program consists of endpointtesting by drinking water exposure for subchronic neurotoxicity andreproductive toxicity. The reproductive effects toxicity testing is intendedto confirm studiesreportedby Alumot etal. (1976; Ref. 10), Rao etal. (1980; Ref. 11), and Lane etal. (1982; Ref. 12), andprovide data needed on fertility index, gestation index, gross necropsy, organ weight, histopathology, estrous cycle, sperm evaluation, vaginal opening, andpreputial separation as described inthe ECA.

This segment will also include application oftheEDC model for quantitativeroute-to-route extrapolation reporting (oral to inhalation) for Tier II endpointtesting (subchronic neurotoxicity andreproductive toxicity) and similarcomputational extrapolation reporting for extant subchronictoxicity reportedbyDaniel etal. (1994; Ref. 9).

Testing conductedunderthisECA will allowEPA to characterize certain potentialhealthhazardsresulting from inhalation exposureto EDC. The following Table 1 sets forth therequired testing, test standard, andreporting requirements undertheECA for EDC.
TABLE 1.-REQUIRED TESTING, TEST STANDARD, AND REPORTING REQUIREMENTS FOR EDC
Testing Segment Required testing Test standard Deadline for final report1 (Months)

'• Tier I HAPs Testing. Acute toxicity, with BAL and histopathology 40 CFR 799.9135 (as annotated in ECA 1& (inhalation). Appendix D.1). Acute neurotoxicity (inhalation). 40 CFR 799.9620 (as annotated in ECA 18 Appendix D.1). Tier I Program Review PKIMECH data to support model validation and ECA Appendix C (1-4). 21

Testing. verification of oral-to-inhalation extrapolation of dose-response for the following data needs in the F344 rat: a. Subchronic toxicity. b. Subchronic neurotoxicity. c. Reproductive toxicity. PBPK model simulations. ECA Appendix C (1-5). 21 Tier II Testing and/or Ex- Subchronic toxicity route-to-route extrapolation of ECA Appendix C.2 and C.6. 36

trapolation Reporting. dose-response (oral Tier II Testing to inhalation) ofa study reported by Daniel eta/. (1994). Subchronic neurotoxicity (oral). 40 CFR 799.9620 (as annotated in ECA 42 Appendix D.2). Subchronic neurotoxicity route-to-route extrapo- ECA Appendix C.3 and C.6. 52

lation of dose-response (oral Tier II Testing to inhalation). Reproductive toxicity (oral). 40 CFR 799.9380 (as annotated in ECA 42 Appendix D.3).33128 Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices

TABLE 1.-REQUIRED TESTING, TEST STANDARD, AND REPORTING REQUIREMENTS FOR EDC-Continued
Testing Segment Required testing Test standard Deadline for final report' (Months) Reproductive toxicity route-to-route extrapolation EGA Appendix C.4 and C.6. 52

of dose-response (oral data to inhalation, including Tier II Testing and extant studies reported by Alumot eta/. (1976), Rao et al. (1980), and Lane eta/. (1982)). ' Number of months after the effective date of the Order that incorporates this EGA when the final report is due. In addition, every 6 months from the effective date of the Order until the end of the EGA testing program, interim reports describing the status of all testing to be performed under this EGA must be submitted by the Companies to EPA.

C. What are the Usesfor the Test Data forEDC? EPA would usethe data obtained from testing to implement several provisions ofsection 112 ofthe Clean AirAct (CAA), includingthe determination ofresidual risk, the estimation ofthe risks associated with accidental releases ofchemicals, and otherHAP riskassessments. EPA will also usethe data from this ECA to fulfill part oftheTier ITesting portionofthe Voluntary Children's Chemical EvaluationProgram (VCCEP). (For more information about VCCEP, see: http:// www.epa.gov/chemrtk/vceep/.) In addition, the data will beusedby other Federal agencies (e.g., theAgency for Toxic Substances andDisease Registry (ATSDR), the National Institute for Occupational Safety and Health (NIOSH), the Occupational Safety and HealthAdministration (OSHA), andthe ConsumerProduct SafetyCommission (CPSC)) in assessing chemical risks and intaking appropriate actions within their programs (see the proposed HAPs testrule at 61 FR 33178, 33179, June 26, 1996). D. Does theEGAforEDCMeet all the TestingRequirementsforEDCthat were Containedin theProposed TestRule?

Intheproposed HAPs testrule, as amended, EPA proposedtesting ofEDC for acute, subchronic, developmental, and reproductive effects and neurotoxocitybythe inhalationroute of exposure. TheECA alternativetesting program for EDC requires inhalation testing for acutetoxicity and acute neurotoxicity, and oral drinking water testing for subchronicneurotoxicity and reproductive effects toxicity. TheECA requires the development ofPK/MECH data to supportcomputationalPBPK modelingto inform quantitative routeto-route extrapolations ofdose-response (oral to inhalation) for theendpoints of subchronic toxicity, subchronic neurotoxicity, andreproductiveeffects toxicity as described intheECA.

During discussions to develop this ECA, EPA concludedthatthe developmental toxicity studies reported by Rao etal. (1980; Ref. 11), inrabbits, and Payan etal. (1995; Ref. 13), inrats, adequately fulfill the HAPs rulemaking testing requirement for developmental toxicitytesting for EDC and, therefore, theECA does notrequire, andthe final HAPs test rule will notrequire this testing. In addition, theECA does not require, andthe final HAPs testrule will not require, macrophage function testing (a component ofEPA's acute toxicity test gudeline 40 CFR 799,g135) because EPA considers existing databy Sherwood etal. (1987; Ref. 14) adequate to fulfill this aspect ofthe acute testing need. Furthermore, theTierI HAPs Testingendpoints (acute toxicity and acute neurotoxicity) willnotbe includedinthe final HAPs testrule because theroute oftestingtobe conductedunderthis ECA is identical to that specifiedintheHAPs rulemaking. Finally, depending onthe outcome ofEPA's Program Review, the Agency anticipates thatthebalance of thetesting for EDC thatwas contained intheproposedHAPs testrule, as amended, will also notbe included in thefinal HAPs test rulebecause the Companies will conductequivalent testing as TierII Testing and Extrapolation Reportingunderthis ECA alternative testingprogram for EDC. Therefore, EPA anticipates the fulfilling ofall ofthe healtheffects testing requirements, identifiedintheHAPs proposedrule, as amended,by implementingtheECA and Order.

The issuance oftheECA and Order constitutes final EPA action for purposes of5 U.S.C. 704. E. What if EPA ShouldRequire AdditionalHealth Effects Testing on EDC?

IfEPA decides inthe future thatit requires additionalhealtheffects data onEDC, theAgency will initiate a separate action. IV. OtherImpactsoftheECA for EDC

The issuance ofthe ECA and Order underTSCA section4 subjects the Companies that signedtheECA to export notificationrequirements under TSCA section 12(b)(1), as setforth at 40 CFR part 707, subpartD, iftheyexport or intendto exportEDC.

Inthe 12(b) proposal published inthe Federal Register ofDecember 23, 1997 (62 FR 67038) (FRL-5762-8), EPA proposedto amend40 CFR 799.5000by adding EDC to the list ofchemicals subject to testing consent orders. The listing ofa chemical substance at40 CFR 799.5000 serves as notificationto all persons who exportor intendto exportthechemical substancethat:

1. The chemical substance is the subject ofanECA and Order.

2. EPA's export notification regulations at 40 CFR part 707, subpart D, applyto those exporters whohave signedtheECA, as well as those exporters whohave not signedtheECA (40 CFR 799.1g),

When a final rulebased onthe proposedruleis published inthe FederalRegister, all persons who export orwhointendto exportEDC willbe subjectto export notification requirements. V. PaperworkReductionAct

TheECA and Order announced inthis notice do notcontain anyinformation collectionrequirements thatrequire additional approvalbythe Office of Management andBudget (OMB) under the Paperwork Reduction Act (PRA), 44 U.S.C. 3501 etseq. The information collectionrequirementsrelatedto test rules andECAs issuedunderTSCA section4 have alreadybeen approvedby OMB underOMB control number 2070- 0033 (EPA ICR No. 1139). The one-time publicburden for this collectionof information is estimatedto be approximately 3,364 hourstotal (Ref. 15). Underthe PRA, "burden" means thetotal time, effort, or financial resources expendedby personsto generate, maintain, retain, or disclose or provideinformation to or for a Federal agency. Forthis collection it includes thetime neededto review instructions; complete andreviewthecollection of information; andtransmit or otherwise disclose the information. An agencyFederal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33129

maynot conduct or sponsor, and a person is not required to respond to a collection ofinformationunless it displays a currentlyvalid OMB control number. The OMB control number for EPA's regulations, after initial display in the final rule, are listed in40 CFR part 9. VI. References 1. U.S. EPA, OPPT. I. Ethylene Dichloride (107-06-2). Pp 24-27 In: "TSCA Section4 Findings for 21 Hazardous AirPollutants: A Supporting Document for ProposedHazardousAir Pollutants (HAPs) Test Rule." (June 25, 1996).

2. The HAP Task Force. Letter from Peter E. Voytekto Charles M. Auer with attachment entitled: "Proposal for Pharmacokinetics Study ofEthylene Dichloride, November 22, 1996." (November 22, 1996).

3. U.S. EPA. Letter from Charles M. AuertoPeterE. Voytek with attachment entitled: "PreliminaryEPA Technical Analysis ofProposedIndustry Pharmacokinetics (PK) Strategyfor EthyleneDichloride, June, 1997." (June 26, 1997).

4. The HAP Task Force. Letter from PeterE. Voytek to Charles M. Auer, U.S. EPA. (March 19, 1999).

5. U.S. EPA. Letter from Charles M. AuertoPeterE. Voytek, HAP Task Force, Re: ECA Developmentof Ethylene Dichloride (EDC) (OPPTS 42197C, with attachment: "EDC ECADRAFT, datedFebruary, 2001." (February 13, 2001).

6. FinalEnforceable Consent Agreement for EthyleneDichloride and AccompanyingTesting Consent Order, signedbyEPA onMay 13, 2003.

7. D'Souza, R.W., Francis, W.R., Bruce R.D., andAndersen, M.E. Physiologicallybasedphamacokinetic model for ethylenedichloride andits application in riskassessment. Pp 286- 301, In: Pharmacokinetics inRisk Assessment. National AcademyPress. Washington, D.C. (1987).

8. D'Souza, R.W., Francis, W.R., and Andersen, M.E. Physiologicalmodel for tissueglutathione depletion and increasedresynthesis afterethylene dichloride exposure. Journal of Pharmacology andExperimental Therapeutics 245(2):563-568. 1988.

9. Daniel, F.B., Robinson, M., Olson, G.R., York, R.G., andCondie, L.W. Ten andninety-daytoxicitystudies of1,2- dichloroethane in Sprague-Dawleyrats. Drug andChemical Toxicology 17: 463- 477. 1994.

10. Alumot, E., Nachtomi, E., Mandel, E., Holstein, P., Bondi, A., and Herzberg, M. Tolerance andacceptable daily intake of chlorinated fumigants in the rat diet. Food, Cosmetics and Toxicology 14: 105-110. (1976).

11. Rao, K.S., Murray, J.S., Deacon, M.M., John, J.A., Calhoun, L.L., and Young, J.T. Teratogenicityand reproduction studiesin animals inhalingethylenedichloride. Banbury Report 5: 149-166. (1980).

12. Lane, R.W., Riddle, B.L., and Borzelleca,J.F. Effects of1,2- dichloroethane and 1,1,1- trichloroethane in drinking wateron reproduction anddevelopmentin mice. Toxicology andAppliedPharmacology 63: 409-421. 1982.

13. Payan, J.P., Saillenfait, A.M., Bonnet, P., Fabry, J.P., Langonne, I.,and SabateJ.P. Assessmentofthe developmental toxicityandplacental transfer ofthe 1,2-dichloroethanein rats. Fundamental andApplied Toxicology 28: 187-198. 1995.

14. Sherwood, R.L., O'Shea, W., Thomas, P.T., Ratajczak, H.V., and Aranyi, C. Effects of inhalation of ethylene dichloride onpulmonary defenses of miceandrats. Toxicology andAppliedPharmacology 91: 491-496. 1987.

15. U.S. EPA, OPPTS. "Burden Estimates for the Enforceable Consent Agreement for Ethylene Dichloride." (January 31, 2002). List ofSubjects

Environmental protection, Hazardous chemicals.

Dated: May 13, 2003. StephenJohnson, AssistantAdministrator forPrevention, PesticidesandToxicSubstances. [FR Doc. 03-13721 Filed 6-2-03; 8:45am] BILLING CODE 6560-50--S
ENVIRONMENTAL PROTECTION AGENCY [OPPT-2002-0067; FRL-7287-4] TSCA Section S(e); Notification of Substantial Risk; Policy Clarification and Reporting Guidance
AGENCY: Environmental Protection Agency (EPA). ACTION: Notice.
SUMMARY: EPA is hereby finalizing revisions to certainparts ofEPA's "Statement ofInterpretation and Enforcement Policy; Notification of Substantial Risk" (policy statement) issuedMarch 16, 1978, concerning the reporting of "substantialrisk" information pursuantto section 8(e) of theToxic Substances Control Act (TSCA). EPA is making these revisions after having consideredpublic commentsthatwere solicited in 1993 and 1995. Specifically, therevisions address thereporting ofinformation on the release ofchemical substances to, andthe detection ofchemical substances in, environmental media, the reporting deadline for written "substantial risk" information, andthe circumstances under which certain information neednotbereported to EPA undersection8(e) ofTSCA. EPA is republishing thepolicy statementinits entiretyinthis document, including boththoseportions ofthepolicy statementthat are revised andthose portionsthatare not affectedbyany revisions. Sincethepolicy statement was publishedin 1978, this republication is intendedto ensurethat a single reference sourcefor theTSCA section 8(e) policy and guidance is easily available to theregulated community and otherinterestedparties. FOR FURTHER INFORMATION CONTACT: For general information contact: Barbara Cunningham, Director, Environmental Assistance Division (7408M), Office of PollutionPrevention andTaxies, EnvironmentalProtectionAgency, 1200 PennsylvaniaAve., NW., Washington, DC 20460-Q001; telephone number: (202) 554-1404; e-mail address: TSCAHotline@epa.gov.

Fortechnicalinformation contact: RichardHefter, Chief, High Production Volume Chemicals Branch, Risk AssessmentDivision, OfficePollution PreventionandTaxies, Environmental ProtectionAgency, 1200Pennsylvania Ave., NW., Washington, DC 2046Q0001; telephone number: (202) 564- 7649; e-mail address: hefter.richard@epa.gov. SUPPLEMENTARY INFORMATION: I. GeneralInformation A. Does thisActionApplyto Me?

You maybe potentiallyaffectedby this action ifyou manufacture, process, import, or distribute incommerce chemical substances andmixtures. Potentially affected entities may include, butare notlimitedto:

• Chemical manufacturers, processors, and distributors (NAICS 325)

• Petroleumrefiners and distributors (NAICS 324)

• Manufacturers ofplasticparts and components (NAICS 325211)

• Paints and coatings and adhesive manufacturing (NAICS 3255)

• Cleaning compounds and similar products manufacturing (NAICS 3256)

• Electronics manufacturing (NAICS 334 and 335)

• Automobiles manufacturing (NAICS 3361)36196 Federal Register IVol. 74, No. 139IWednesday, July 22, 2009 INotices
(Amendment) (Final Rule); in 40 CFR part 1042, subparts C, D, G and H; was approved 07/08/2009; OMB Number 2060-0287; expires 07/31/2012.

EPA ICR Number 1745.06; Criteria for Classification of Solid Waste Disposal facilities and Practices (Renewal); in 40 CFR part 257, subpart B; was approved 07/10/2009; OMB Number 2050-0154; expires 07/31/2012.

EPA ICR Number 2027.04; NESHAP for Flexible Polyurethane Foam Fabrication (Renewal); in 40 CFR part 63, subpartMMMMM; was approved 07/10/2009; OMB Number 2060-0516; expires 07/31/2012.

EPA ICR Number 2056.03; NESHAP for Miscellaneous Metal Parts and Products (Renewal); in 40 CFR part 63, subpart MMMM; was approved 07/10/ 2009; OMB Number 2060-0486; expires 07/31/2012.

EPA ICR Number 1656.13; Risk ManagementProgram Requirements and Petitions to Modify the List ofRegulated Substances under Section 112(r) ofthe Clean Air Act (Renewal); in 40 CFR part 68; was approved 07/10/2009; OMB Number 2050-0144; expires 07/31/2012.

EPA ICR Number 2317.01; Generator Standards Applicable to Laboratories Owned by Eligible Academic Entities (Final Rule); in 40 CFR part 262, subpart K; was approved 07/10/2009; OMB Number 2050-0204; expires 07/31/2012. OMB Comments Filed

EPA ICR Number 2355.01; Restructuring ofStationary Source Audit Program (Proposed Rule); OMB filed comment on 06/30/2009.

EPA ICR Number 1975.06; NESHAP for Stationary Reciprocating Internal Combustion Engines (40 CFR part 63, subpart ZZZZ) (Proposed Rule); OMB filed comment on 07/04/2009.

EPA ICR Number 2348.01; NESHAP for Paints and Allied Products Manufacturing (40 CFR part 63, subpart CCCCCCC) (Proposed Rule); OMB filed comment on 07107/2009.

EPA ICR Number 2332.01; NESHAP for Aluminum, Copper, and Other Nonferrous Foundries (Proposed Rule); OMB filed comment on 07/10/2009.

Dated: July 16, 2009. John Moses, Director, Collection Strategies Division. [FR Doc. £9-17397 Filed 7-21-09; 8:45am]
BILLING CODE 6560-50-P ENVIRONMENTAL PROTECTION AGENCY
[EPA-HO-OPPT -2003-0010; FRL-8426-6] 1,2-Ethylene Dichloride; Completion of EPA Program Review
AGENCY: Environmental Protection Agency (EPA). ACTION: Notice.
SUMMARY: EPA issued a testing consent orderthat incorporated an enforceable consent agreement (ECA) for 1,2- Ethylene Dichloride (EDC) in June 2003, using authorities under section4 ofthe Toxic Substances Control Act (TSCA). The companies subject to the ECA agreed to conduct toxicity testing in a tiered testing program that included development ofpharmacokinetics and mechanistic data and a computational dosimetry model for route-to-route extrapolations. Thetesting program was designed to satisfy the toxicological data needs for EDC identified in a TSCA section4 proposedtestrule for a number ofhazardous air pollutant chemicals. The modeling is intended to allow toxicological studies conducted using oral exposures to be interpreted so that they could also be used to predict the effects ofinhalation exposures. This notice announces the completion ofthe program review component ofthe ECA for EDC. This notice also states EPA's findings and conclusion regarding the adequacy ofthe derived models to perfprm satisfactory route-to-route extrapolations, responds to comments on the Tier IProgram Review Testing, and establishes revised deadlines for completion ofTier IItesting and computational route-to-route dosimetry modeling for extrapolations listed under Tier II ofthe ECA for EDC. FOR FURTHER INFORMATION CONTACT: For general information contact: Colby Lintner, Regulatory Coordinator, Environmental Assistance Division (7408M), Office ofPollution Prevention and Taxies, Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460-0001; telephone number: (202) 554-1404; e-mail address: TSCA-Hotline@epa.gov.

For technical information contact: John Schaeffer, Chemical Control Division (7405M). Office Pollution Prevention and Taxies, Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460- 0001; telephone number: (202) 564- 8173; e-mail address: schaeffer.john@epa.gov. SUPPLEMENTARY INFORMATION: I. General Information A. Does thisActionApplyto Me?

This action is directed to the public in general, and may be ofparticular interestto those persons who are or may be required to conduct testing of chemical substances underTSCA. Since other entities may also be interested, the Agency has not attempted to describe all the specific entities that may be affected by this action. Ifyou have any questions regarding the applicability ofthis action to a particular entity, consultthe technical person listed under FOR FURTHER INFORMATION CONTACT. B. How Can I Get Copies ofthis Document and OtherRelated Information?

1. Docket. EPA has established a docket for this action under docket identification (ID) numberEPA-HQOPPT-2003-0010. All documents inthe docket are listed in the docket index available at http://www.regulations.gov. Although listed in the index, some information is not publicly available, e.g., Confidential Business Information (CBI) or other information whose disclosure is restricted by statute. Certain other material, such as copyrighted material, will be publicly available only in hard copy. Publicly available docket materials are available electronically at http:// www.regulations.gov, or, ifonly available in hard copy, at the OPPT Docket. The OPPTDocket is located in the EPA Docket Center (EPA/DC) at Rm. 3334, EPA West Bldg., 1301 Constitution Ave., NW., Washington, DC. The EPA/DC Public Reading Room hours ofoperation are 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding Federal holidays. The telephone number ofthe EPA/DC Public Reading Room is (202) 566-1744, and the telephone number for the OPPTDocket is (202) 566-0280. Docket visitors are required to show photographic identification, pass through a metal detector, and sign theEPA visitor log. All visitorbags are processed through an X-ray machine and subjectto search. Visitors will be provided an EPA/DC badgethat mustbe visible at all times in thebuilding and returned upon departure.

2. EleCtronic access. You may access this Federal Register document electronically through the EPA Internet under the "Federal Register" listings at http://www.epa.gov/fedrgstr.Federal Register/Val. 74, No. 139/Wednesday, July 22, 2009/Notices 36197

II. Background A. What is the EPA Program Reviewfor EDC?

In the Federal Register ofSeptember 5, 2006 (71 FR 52329) (FRL-8088-3), EPA announced that it was conducting the program review component ofthe ECA for the EDC alternative testing program, and solicited public comment on data received under the Tier I Program Review testing segment ofthe ECA for EDC (CAS No. 107-06-2). Comments were to inform EPA's decision on whether or not additional data and/or model development are needed before Tier II testing and computational route-to-route dosimetry modeling extrapolations could proceed for the Tier II endpoints listed in the ECA for EDC. Details ofthe testing program for EDC are available in the ECA and in the Federal Register ofJune 3, 2003 (68 FR 33125) (FRL-7300-6), in which EPA announced it had entered into an ECA and issued a testing consent order for EDC. The ECA for EDC was developed inresponse to EPA's request for ECA proposal for health effects testing ofa number ofhazardous air pollutants (HAPs or HAP chemicals), including EDC (see the proposed test rule in the Federal Register ofJune 26, 1996 (61 FR 33177) (FRL-4869-1), and the proposed test rule, as amended, in the Federal Register ofDecember 24, 1997 (62 FR 67466) (FRL-5742-2); February 5, 1998 (63 FR 5915) (FRL5769-3); and April21, 1998 (63 FR 19694) (FRL-5780-6)). The HAPs rulemaking proposed testing for health effects by the inhalation route of exposure. In the proposed rule, EPA also invited the submission ofproposals that included pharmacokinetics studies and model developmentthat would permit the extrapolation oftesting administered by other exposure routes, such as the oral route, to predict for inhalation exposures. On November 22, 1996, Dow Chemical Company, Vulcan Materials Company (no longer in existence), Occidental Chemical Corporation, Oxy Vinyls, LP, Georgia GulfCorporation, Westlake Chemical Corporation,PPG Industries, Inc., and Formosa Plastics Corporation, U.S.A. (the "Companies"), under the auspices ofthe HAP Task Force, submitted a proposal for alternative testing ofEDC that included physiologically based pharmacokinetics (PBPK) and model developmentto support route-to-route extrapolation oftesting to be conducted under the ECA by the oral route. EPA considered this proposal sufficient to enter into ECA negotiations with the Companies and other interested parties (62 FR 66626; December 19, 1997) (FRL-5763-1). The ECA for EDC that resulted was announced in the Federal Register ofJune 3, 2003 (68 FR 33125). Since the route-to-route extrapolation of test results was a new approach, EPA and the Companies included a program review step within the testing program. The testing program consists ofTier I HAPs Testing; Tier I Program Review Testing; EPA Program Review; and Tier II Testing.

Tier I HAPs Testing consisted of endpoint testing conductedby inhalation exposure for acute toxicity, with bronchoalveolar lavage (BALl and histopathology, and acute neurotoxicity. The Tier IProgram Review Testing consisted ofstudies to develop PK/ MECH data, analyze glutathione metabolism, and perform model simulation. These studies were conducted to extend the computational dosimetry model ofD'Souza eta!. (1987, 1988; Refs. 1 and 2) to improve the model's application to the specific health effects endpoints for EDC listed in the ECA. The studies also enabled EPA to validate the model and verify the model's ability to adequately perform quantitativeroute-to-route extrapolations ofdose response. Further description ofthe Tier I HAPs Testing and the Tier I Program Review Testing is provided in the Federal Register of September 5, 2006 (71 FR 52329). That notice, as well as the final study reports, can be accessed in the docket (EPAHQ-OPPT-2003-0010) as explained in Unit LB.

As specified in the ECA, the EPA program review is required before the Tier II Testing segment is undertaken. In the Federal Register ofSeptember 5, 2006 (71 FR 52329), EPA announced that it was conductingthe program review component ofthe ECA for EDC, and solicited public comment on data received under theTier I Program Review Testing segment oftheECA. Comments were to inform EPA's decision on whether or not additional data and/or model development were needed before Tier II Testing and computational route-to-route dosimetry modeling extrapolations can proceed for the Tier II endpoints listed in the ECA for EDC. B. What were the Public Comments on the TierIProgram Review Testingfor EDC?

EPA received two public comments in response to its solicitation for comments on the Tier IProgram Review Testing. Comments from People for theEthical Treatment ofAnimals (PETA) were on behalfofthemselves and the following organizations: The Physicians' Committee for Responsible Medicine, the Humane Society ofthe United States, the Doris Day Animal League, and Earth Island Institute. PETA expressed support for the use ofPBPK modeling to limit additional animal testing through the use ofroute-to-route extrapolation to existing studies. However, PETA also stated that they disagreed that additional Tier II tests (i.e., for reproductive effect or subchronic neurotoxicity) are needed; contending that existing studies for these effects are adequate. EPA disagrees, and its basis for requiring this additional testing is discussed in previous Federal Register documents, cited in Unit II.A. A second comment, from a private citizen, was focused on opposition to the manufacture of chlorine compounds in general, including EDC, and not the testing program. C. What are the Conclusions ofthe EPA TierIProgram Review Testingfor EDC?

The companies have completed the Tier I and Tier I Program Review testing segments ofthe ECA for EDC. The companies have also examined additional PBPK models and other available information in order to more fully updatethe model developed by D'Souza eta!., 1987, 1988; as specified in theECA agreement. The results of this work, the updated model, and model simulations have been discussed with EPA (Refs. 3 through 6) and have also been recently published as a peerreviewed article in the scientific literature (Ref. 7). It is EPA's conclusion that the PBPK model developed by the test sponsors under the EDC ECA is acceptable for route-to-route extrapolations and that Tier II testing and extrapolation reporting can proceed as per the schedule set forth below (Ref. 8). Specifically, EPA concludes that:

1. The PK/MECH data report and Tier I toxicity studies have been conducted in accordance with the protocols and specifications as described in Appendix C ofthe ECA.

2. The available study records are sufficient to allow an evaluation ofthe quality ofthe studies performed.

3. The EDC PBPK model is appropriately chemical-specific, and suitably based on the current understanding ofthe kinetics ofEDC.

4. The species, dose level, exposure regimens, and vehicles used are relevant for the toxicity data that are the object ofthe Tier II extrapolations.

5. The Tier IProgram Review PK/ MECH data, along with additional data, show that periodicity was demonstrated and that the various data sets bearing on the issue ofperiodicity can be properly36198 Federal Register/Val. 74, No. 139/Wednesday, July 22, 2009/Notices
interpreted and managed in the studies that support the model.

6. Refinements ofthe model related to absorption, tissue distribution, and metabolism were accomplished, or suitably explained, including the role of extrahepatic metabolism as it impacts the model dose metrics and route-toroute extrapolation; appreciably improving prior PBPK models ofEDC. It is EPA's decision that the HAP Task Force can proceed with the Tier II Testing under the schedule set forth in Table 1. ofthis Federal Register document.
TABLE 1.-REOUIRED TESTING, TEST STANDARDS, AND REPORTING REQUIREMENTS FOR EDC
Testing segment Required testing Test standard Deadline for final report' (months)

Tier II testing andfor extrapolation reporting Subchronic toxicity route-to-route extrapolation of dose-response (oral Tier II testing to inhalation) of a study reported by Daniel, et al., (1994) EGA appendix C.2 and C.6 12

Subchronic neurotoxicity (oral) 1 40 CFR 799.9620 (as annotated in 18 EGA appendix D.2) Subchronic neurotoxicity route-to- EGA appendix C.3 and C.6 21

route extrapolation of dose-response (oral Tier II testing to inhalation) Reproductive toxicity (oral) 40 CFR 799.9380 (as annotated in 25 EGA appendix D.3) Reproductive toxicity route-to-route EGA appendix C.4 and C.6 28

extrapolation of dose-response (oral data to inhalation, including Tier II testing and extant studies reported by Alumot, et al.. (1976), Rao, et al., (1980), and Lane, et al., (1982)) 'Number of months after the date of publication of this Federal Register document, which announces that EPA has concluded the EPA Program Review, when the final report is due. In addition, every 6 months from the effective date of the Order until the end of the EGA testing program, interim reports describing the status of all testing to be performed under this EGA must be submitted by the Companies to EPA.

III. References

1. D'Souza, R.W., Francis, W.R., Bruce R.D., and Andersen, M.E. Physiologically based pharmacokinetic model for ethylene dichloride and its application in risk assessment. P. 286- 301, In: Pharmacokinetics in Risk Assessment. National Academy Press. Washington, D.C. (1987).

2. D'Souza, R.W., Francis, W.R., and Andersen, M.E. Physiological model for tissue glutathione depletion and increased resynthesis after ethylene dichloride exposure. Journal of Pharmacology and Experimental Therapeutics. 245(2):563-568. (1988).

3. EPA, Office ofPrevention, Pesticides and Toxic Substances, Chemical Control Division. Letter from Jim Willis, Director, CCD to Dr. Peter Voytek, HAP TaskForce. RE: EPA Tier I Program Review for EDC. January 10, 2007.

4. EPA and HAP Task Force. Technical Consultation Meeting on 1, 2- Ethylene Dichloride Program Review. February 12, 2007.

5. HAP Task Force. Letter from Peter E. Voytek, Manager, HAP Task Force to Jim Willis, Director, Chemical Control Division, Office ofPollution Prevention andTaxies, with Enclosure: Response to Issues Raised in EPA's Tier 1 Data Evaluation Meeting. May 23, 2007.

6. Sweeny, L. M. and Gargas, M.I. Physiologically based pharmacokinetic model development and simulations for ethylene dichloride (1,2-dichlorethane) in rats. Preparedby the Sapphire Group, Dayton Ohio for the HAP Task Force, Millwood, Virginia. Revised Draft Report. March 11,2009.

7. Sweeny, L. M., Saghir, S. A., and Gargas, M.I. Physiologically based pharmacokinetic model development and simulations for ethylene dichloride (1 ,2-dichlorethane) in rats. Regulatory Toxicology and Pharmacology. 51:311- 323. (2008).

8. EPA. Email from Rob Dewoskin, PhD, DABT, US EPA/NCEA (National Center for Environmental Assessment) to John Schaeffer. Review ofFinal report

- EDC ECA Program Review Completion. April 22, 2009. List ofSubjects

Environmental protection, 1,2- Ethylene Dichloride, EDC, Hazardous chemicals. Dated: July 10, 2009. Jim Willis, Director, Chemical ControlDivision, Office of

Pollution Prevention and Taxies. [FR Doc. E9-17170 Filed 7-21-09; 8:45am] BILLING CODE 656()..5()-S

ENVIRONMENTAL PROTECTION AGENCY [EPA-HQ-ORD; FRL-8930-3] Flexible Approaches to Environmental Measurement-The Evolution ofthe Performance Approach
AGENCY: Environmental Protection Agency (EPA). ACTION; Notice.
SUMMARY: Assuring the quality of environmental measurements is essential to the implementation ofthe Environmental Protection Agency's (EPA's or the Agency's) environmental programs, both regulatory and voluntary. In an October 6, 1997, Notice ofIntent (FRL-5903-2), the Agency outlined a "Performance Based Measurement System (PBMS)" concept12!06/2011 12.08 FAX 202 457 8315 PATTOH BOGGS

FROM :RS! FAX NO. :5408371602

Novemberl7.2011

AddendumModification To**.Enfor~eable** ConsentAgreement For 1,2-Ethylene Dichloride

(CASNo. 107-06-2) 

### Tier/HAPs Tutittg



### . DocketNo. OPPT-2003-0010 141002/003

Dec. e6 2811 01;32PM P2

12/0S/2011 TIJE 12:03 (TX/RX HO 8030] lfl]00212/08/2011 12:08 FAX 202 457 8315 PATTON BOGGS f4l 003/0o:::

FR0f'1 : F:S I FAX NO. :5408371602**D~c.** 06 2011 01:34PM Pl

Wetheundersigned agreeto thechange

### to the Enforceable .Consent

### Agreement

### for 1**,2~Bthylene.** bichloride (CAS No. 107-06-2) as descri.bed inthetablebelow: Original Language (\n Table 1 oftheECAas amended ·OD July22, 1.009 (74 FR36196) nod by appro-val letter ofMay 12,2010)
Tier IJ Testing and/or Program Review ExtnJpolationReporting

Suhchronic neurotoldcity**(o~al)**

0

.....**~-:!--.,.**....;.

.........**~~** ...**~-v-:**..J • .._•.,....,_ ...u JO.

..n

..H.\.o extrapolationofdose-response (oral TierITtestitig toinhalation) Reproductive> rvxicity (om)

Reproductivetoxicity rome-to route <-xtrapolation of**doso-r~ponse** (oral data to inhalation, including Tier 11 testing**~d** e:ll:ttlrttswdics reported by Alumot, c;l a!., ( 1976},lU!o,**'!~**!''·· (1980), and Lane. ct

## at,,**(1~~2))** TestStandard

40 CFR799.9620 (as annowed In ECA appendix0.2)

40 CF'R799.9380 (ns m!llotatect in ECA**at~pendlx** 0.3)

f.:cA npl'OIIdixC.4 and C.6 DeadlldtforFmalRl!pori

To bedetennincd follmving. EPA review and IIPProval of COmbined**.R~productlve Effocts/N~rotoxlcity**  protocol
1Jlft:Y montrtS IOllOWtngnnar .f6j;Orl

.. - ..···-"" "

......... for Combined ReproductiveEffilct$1 Neurotoxic:itySIUdy
To bedetermined followiog EPA reviewand approval of Combined Reproductive Effeet!i/Neurototiciry protoeo!**Th~ee** months followinifino.!**~ort** for CombinedReproductive**Effc~** Neurotoxi.,ity sruciy

Amended, !..anguj;\ge (clumgeinTable 1·oftheECA ·as amended ou No-vember 17, 2011)

Tiet< II Testing and/orProgram Review**Extrap<~latlon·**ReportiDI! Reproouctivc tox:lclcy/Neurotoxicity (oral)

Rcpn)du.ctive tox:icity/Neuro>oxicity raute·to-rout.: extrapolation ofdosc-rosponse (oral <1ata toinhalation, inoluding Ti"r U tesling and extant studies**~pon:ed** by Alumot, ct aL (1976), Rao, et<).l., (1980). lllld LMe, 1:ta!., (1**<;)~2))** ''

Director Chemicai Control Division Test Standard DeadlineforFinalRJ!port

ComblaedReproductive: Effects /NeurotoxicityProtoool

ECA appendix C.4. an(! C.6 June: 28, 2013 (New study plill'l due February**2~,** 2012) Septernbvr 21,2013

# ~**PeterE:YOYtek~.D:** 

# ;yW
bate Manager HAP TaskForce

12/08/2011 TUE 12:05 [D(!RX NO 8031] @001